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diff --git a/43227-0.txt b/43227-0.txt index 6cee3ca..2c84400 100644 --- a/43227-0.txt +++ b/43227-0.txt @@ -1,34 +1,4 @@ -The Project Gutenberg EBook of The Fundamentals of Bacteriology, by -Charles Bradfield Morrey - -This eBook is for the use of anyone anywhere at no cost and with -almost no restrictions whatsoever. You may copy it, give it away or -re-use it under the terms of the Project Gutenberg License included -with this eBook or online at www.gutenberg.org - - -Title: The Fundamentals of Bacteriology - -Author: Charles Bradfield Morrey - -Release Date: July 16, 2013 [EBook #43227] - -Language: English - -Character set encoding: UTF-8 - -*** START OF THIS PROJECT GUTENBERG EBOOK THE FUNDAMENTALS OF BACTERIOLOGY *** - - - - -Produced by Jennifer Linklater, Jason Isbell and the Online -Distributed Proofreading Team at http://www.pgdp.net - - - - - +*** START OF THE PROJECT GUTENBERG EBOOK 43227 *** Transcriber’s Note: @@ -13811,360 +13781,4 @@ The following changes have also been made: End of the Project Gutenberg EBook of The Fundamentals of Bacteriology, by Charles Bradfield Morrey -*** END OF THIS PROJECT GUTENBERG EBOOK THE FUNDAMENTALS OF BACTERIOLOGY *** - -***** This file should be named 43227-0.txt or 43227-0.zip ***** -This and all associated files of various formats will be found in: - http://www.gutenberg.org/4/3/2/2/43227/ - -Produced by Jennifer Linklater, Jason Isbell and the Online -Distributed Proofreading Team at http://www.pgdp.net - - -Updated editions will replace the previous one--the old editions -will be renamed. - -Creating the works from public domain print editions means that no -one owns a United States copyright in these works, so the Foundation -(and you!) can copy and distribute it in the United States without -permission and without paying copyright royalties. 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You may copy it, give it away or -re-use it under the terms of the Project Gutenberg License included -with this eBook or online at www.gutenberg.org - - -Title: The Fundamentals of Bacteriology - -Author: Charles Bradfield Morrey - -Release Date: July 16, 2013 [EBook #43227] - -Language: English - -Character set encoding: ISO-8859-1 - -*** START OF THIS PROJECT GUTENBERG EBOOK THE FUNDAMENTALS OF BACTERIOLOGY *** - - - - -Produced by Jennifer Linklater, Jason Isbell and the Online -Distributed Proofreading Team at http://www.pgdp.net - - - - - - -Transcriber's Note: - -Formatting and non-latin characters are indicated thus: - - _italic_ - =bold= - ^{superscript} - {subscript} - #Greek transliteration# - - - - -[Illustration: PLATE I - -ANTHONY VON LEEUWENHOEK - -Who first saw bacteria] - - - - - THE FUNDAMENTALS - OF - BACTERIOLOGY - - BY - CHARLES BRADFIELD MORREY, B.A., M.D. - - PROFESSOR OF BACTERIOLOGY AND HEAD OF THE DEPARTMENT - IN THE OHIO STATE UNIVERSITY, - COLUMBUS, OHIO - - - ILLUSTRATED WITH 171 ENGRAVINGS AND 6 PLATES - - _Second Edition, thoroughly Revised_ - - [Illustration: Publisher's logo] - - LEA & FEBIGER - PHILADELPHIA AND NEW YORK - 1921 - - COPYRIGHT - LEA & FEBIGER - 1921 - - - TO - GRACE HAMILTON MORREY - AMERICAN PIANIST - - - - -PREFACE TO SECOND EDITION - - -The first edition seems to have fulfilled a need for a general -text-book on the subject of bacteriology. The original method of -presentation is preserved. The text-book idea is adhered to, so that -the individual instructor may have full liberty to expand on topics in -which he is especially interested. A number of illustrations have been -added, the text has been improved in many instances by the addition -of further explanatory matter and the most recent general advances in -the Science. Examples are the System of Classification of the Society -of American Bacteriologists, which is used throughout the text, their -Key to the Genera of Bacteria, a discussion of the H-ion concentration -method of standardization, the selective action of anilin dyes, the -mechanism of entrance of pathogenic organisms into the body, a more -detailed explanation of the origin of antibodies, the nature of -antigens and a table of antigens and antibodies. - -Professor Vera McCoy Masters has assisted in the revision by aiding -in the preparation of manuscript and the reading of proof and in the -making of the index, for which services the author's thanks are hereby -expressed. - - C. B. M. - Columbus, Ohio, 1921. - - - - -PREFACE TO FIRST EDITION - - -An experience of nearly twenty years in the teaching of Bacteriology -has convinced the author that students of this subject need a -comprehensive grasp of the entire field and special training in -fundamental technic before specializing in any particular line of work. -Courses at the University are arranged on this basis. One semester is -devoted to General Bacteriology. During the second semester the student -has a choice of special work in Pathogenic, Dairy, Soil, Water, or -Chemical Bacteriology. A second year may be devoted to advanced work in -any of the above lines, to Immunity and Serum Therapy, or to Pathogenic -Protozoa. - -This text-book is intended to cover the first or introductory -semester's work, and requires two classroom periods per week. Each -student is compelled to take two laboratory periods of three hours per -week along with the class work. The outline of the laboratory work is -given at the end of the text. Results attained seem to justify this -plan. A text-book is but one of many pedagogical mechanisms and is not -intended to be an encyclopedia of the subject. - -The author makes no claim to originality of content, since the facts -presented are well known to every bacteriologist, though the method -of presentation is somewhat different from texts in general. During -the preparation of this work he has made a thorough review of the -literature of Bacteriology, covering the standard text-books as well -as works of reference and the leading periodicals dealing with the -subject. Thus the latest information has been incorporated. - -No attempt has been made to give detailed references in a work of this -character. - -The photomicrographs are original except where otherwise indicated -and are all of a magnification of one thousand diameters where no -statement to the contrary appears. These photographs were made with -a Bausch & Lomb Projection Microscope fitted with a home-made camera -box. Direct current arc light was used and exposures were five to ten -seconds. Photographs of cultures are also original with a few indicated -exceptions. All temperatures are indicated in degrees centigrade. - -For use of electrotypes or for prints furnished the author is indebted -to the following: A. P. Barber Creamery Supply Company, Chicago, Ill.; -Bausch & Lomb Optical Company, Rochester, N. Y.; Creamery Package -Manufacturing Company, Chicago, Ill.; Davis Milk Machinery Company, -North Chicago, Ill.; Mr. C. B. Hoover, Superintendent of Sewage -Disposal Plant, Columbus, O.; Mr. C. P. Hoover, Superintendent of Water -Filtration Plant, Columbus, O.; The Hydraulic Press Manufacturing -Company, Mt. Gilead, O.; Loew Manufacturing Company, Cleveland, O.; -Metric Metal Works, Erie, Pa.; Sprague Canning Machine Company, -Chicago, Ill.; U. S. Marine Hospital Service; Wallace and Tiernan -Company, New York City, N. Y. - -For the preparation of many cultures and slides, for great assistance -in the reading of proof and in the preparation of the index, Miss Vera -M. McCoy, Instructor in Bacteriology, deserves the author's thanks. - -The author trusts that the book will find a place in College and -University courses in Bacteriology. - - C. B. M. - - - - -CONTENTS - - - Historical Introduction--Spontaneous Generation--Causation - of Disease--Putrefaction and Fermentation--Study of - Forms--Chronological Table 17 - - CHAPTER I. - - Position of Bacteria--Relationships to - Algæ--Yeasts--Molds--Protozoa 37 - - - PART I. - - MORPHOLOGY. - - CHAPTER II. - - Cell Structures--Cell Wall--Protoplasm--Plasmolysis - --Plasmoptysis--Nucleus--Vacuoles--Capsules--Metachromatic - Granules--Flagella--Spores 41 - - CHAPTER III. - - Cell Forms--Coccus--Bacillus--Spirillum--Involution Forms 52 - - CHAPTER IV. - - Cell Groupings 55 - - CHAPTER V. - - Classification--Migula's--Society of American - Bacteriologists'--Key to the Latter 59 - - - PART II. - - PHYSIOLOGY. - - CHAPTER VI. - - Occurrence--General Conditions for - Growth--Moisture--Temperature--Light--Oxygen-- - Osmotic Pressure--Electricity--X-rays and Radium - Emanations--Pressure--Mechanical Vibration 71 - - CHAPTER VII. - - Chemical Environment--Reaction of Medium--Chemical - Composition 81 - - CHAPTER VIII. - - Chemical Environment (Continued)--General Food - Relationships--Metabolism of Elements 86 - - CHAPTER IX. - - Physiological Activities--Fermentation of - Carbohydrates--Splitting of Fats 93 - - CHAPTER X. - - Physiological Activities (Continued)--Putrefaction of - Proteins--Cycles of Nitrogen, Carbon, Sulphur, Phosphorus 102 - - CHAPTER XI. - - Physiological Activities (Continued)--Production of - Acids, Gases, Esters, Alcohols, Aldehydes, Aromatic - Compounds--Phosphorescence--Chromogenesis--Reduction-- - Oxidation--Production of Heat--Absorption of Free - Nitrogen--Nitrogen Nutrition of Green Plants 110 - - CHAPTER XII. - - Physiological Activities (Continued)--Production of - Enzymes--Discussion on Enzymes--Toxins--Causation of - Disease 121 - - CHAPTER XIII. - - Disinfection--Sterilization--Disinfectants--Physical - Agents--Pasteurization 130 - - CHAPTER XIV. - - Disinfection and Sterilization (Continued)--Chemical - Agents--Anilin Dyes 156 - - CHAPTER XV. - - Disinfection and Sterilization (Continued)--Choice - of Agent--Standardization of Disinfectants--Phenol - Coefficient--Practical Sterilization and Disinfection 164 - - - PART III. - - THE STUDY OF BACTERIA. - - CHAPTER XVI. - - Culture Media--Broth, Milk, Gelatin, Agar, Potatoes, Blood - Serum--Standardization of Media--H-ion Concentration - Method--Synthetic Media 171 - - CHAPTER XVII. - - Methods of Using Culture Media--Culture Tubes--Plates-- - Anaërobic Cultures--Vignal Tubes--Fermentation Tubes-- - Deep Culture Tubes--Novy Jars--Inoculation of Culture Media 184 - - CHAPTER XVIII. - - Isolation of Bacteria in Pure Culture--Dilution - --Plating--Streaking--Barber Apparatus--Aids in - Isolation--Heat--Selective Antiseptics--Selective - Food---Indicators--Animal Inoculation 194 - - CHAPTER XIX. - - Study of the Morphology of Bacteria--Bacteriological - Microscope--Hanging Drop Slides--Staining--Gram's - Method--Spores--Acid-fast Bacilli--Capsules-- - Flagella--Metachromatic Granules 200 - - CHAPTER XX. - - Study of the Physiology of Bacteria--Temperature - --Incubators--Thermal Death Point--Oxygen Relationships - --Study of Physiological Activities--Appearance of Growth - on Culture Media--Appearance of Molds on Plate Cultures 213 - - CHAPTER XXI. - - Animal Inoculation--Material for Bacteriological - Examination 227 - - - PART IV. - - GENERAL PATHOGENIC BACTERIOLOGY. - - CHAPTER XXII. - - Introduction--Infection--Acute Infection--Chronic - Infection--Specific--Non-specific--Koch's - Postulates--Virulence--Susceptibility 231 - - CHAPTER XXIII. - - Pathogenic Bacteria Outside the Body--As Saprophytes--As - Facultative Saprophytes--Latent--Carriers--Universal - Carriers--Accidental Carriers--Necessary Intermediate Hosts 237 - - CHAPTER XXIV. - - Channels of Infection--Skin--Mucosæ--Respiratory Tract - --Alimentary Tract--Mechanism of Entrance of Organisms - --Dissemination in the Body--Paths of Elimination-- - Specificity of Location 243 - - CHAPTER XXV. - - Immunity--Natural--Artificial--Active--Passive-- - Production of Immunity--Vaccine--Antiserum--Practical - Applications of Immunity Reactions 250 - - CHAPTER XXVI. - - Theories of Immunity--Pasteur--Chauveau--Baumgärtner - --Metchnikoff--Ehrlich--Principles of Ehrlich's Theory 256 - - CHAPTER XXVII. - - Ehrlich's Theory (Continued)--Receptors of the First - Order--Antitoxin--Antienzyme--Preparation of Antitoxins - --Units 261 - - CHAPTER XXVIII. - - Ehrlich's Theory (Continued)--Receptors of the Second - Order--Agglutinins--Agglutination Reaction--Precipitins - --Precipitin Test 265 - - CHAPTER XXIX. - - Ehrlich's Theory (Continued)--Receptors of the Third - Order--Cytolysins--Amboceptor--Complement--Anti-amboceptors - --Antisnake Venoms--Failure of Cytolytic Serums in - Practice--Complement-fixation Test 271 - - CHAPTER XXX. - - Phagocytosis--Opsonins--Opsonic Index--Bacterial - Vaccines--Preparation of--Use of--Lipovaccines - --Aggressins 280 - - CHAPTER XXXI. - - Anaphylaxis--Author's Theory--Tuberculin Test--Table of - Antigens and Antibodies--Summary of Immunity as Applied to - Protection from Disease 289 - - - - -BACTERIOLOGY. - - - - -HISTORICAL INTRODUCTION. - - -Bacteriology as a science is a development of the latter half of the -nineteenth century. It may be said to have begun in the decade between -1870 and 1880, due largely to the wide circulation given to Koch's -work in proving that _Bacillus anthracis_ is the cause of Anthrax in -1876, in devising new culture methods and in demonstrating that wound -infections are due to microörganisms, 1878. Associated with this -work were the great improvements in the microscope by Abbé and the -introduction of anilin dyes for staining bacteria by Weigert. These -results attracted workers throughout the world to the "new science." -Nevertheless, this work of Koch's was preceded by numerous observations -and experiments which led up to it. Certainly the most important -discoveries immediately responsible were those of Pasteur. He must be -considered as the greatest of the pioneer bacteriologists since he -worked in all fields of the subject. Some of the antecedent work was -done in attempting to disprove the old "spontaneous generation" theory -as to the origin of organisms; some in searching for the causes of -disease and some in the study of fermentation and putrefaction. - - -SPONTANEOUS GENERATION. - -Speculation as to the first origin of life is as old as history and -doubtless older. Every people of antiquity had its own legends, as for -example, the account in Genesis. This question never can be definitely -settled, even though living matter should be made in the laboratory. - -The doctrine of the "spontaneous origin" of particular animals or -plants from dead material under man's own observation is a somewhat -different proposition and may be subjected to experimental test. The -old Greek philosophers believed it. Anaximander (B.C. 610-547) taught -that some animals are derived from moisture. Even Aristotle (B.C. -384-322) said that "animals sometimes arise in soil, in plants, or -in other animals," _i.e._, spontaneously. It can be stated that this -belief was general from his day down through the Dark and Middle Ages -and later. Cardano (A.D. 1501-1576) wrote that water gives rise to -fish and animals and is also the cause of fermentation. Van Helmont -(1578-1644) gives directions for making artificial mice. Kircher -(1602-1680) describes and figures animals _produced under his own eyes_ -by water on plant stems. - -However, many thinkers of the seventeenth century doubted the truth -of this long-established belief. Francesco Redi (1626-1698) made a -number of experiments which tended to prove that maggots did not -arise spontaneously in meat, as was generally believed, but developed -only when flies had an opportunity to deposit their eggs on the -meat. It seems that by the latter part of this century the idea that -organisms large enough to be seen with the naked eye could originate -spontaneously was generally abandoned by learned men. - -The work of Leeuwenhoek served to suspend for a time the subject of -spontaneous generation, only to have it revived more vigorously later -on. He is usually called "The Father of the Microscope," though the -compound microscope was invented probably by Hans Zansz or his son -Zacharias, of Holland, about 1590. Leeuwenhoek used a simple lens, -but his instruments were so much more powerful that they opened up an -entirely new and unknown world. (Fig. 1.) - -Anthony van Leeuwenhoek (1632-1723) was apprenticed to a linen draper -and accumulated a comfortable fortune in this business. He became -interested in the grinding of spectacle lenses, then an important -industry in Delft, Holland, where he lived, and did a great deal of -experimental work in this line, mainly for his own enjoyment. Finally -he succeeded in making a lens so powerful that he could see in water -and various infusions very minute living bodies never before observed. -Leeuwenhoek contributed 112 papers to the Royal Society of Great -Britain, the first in 1673, many of them accompanied by such accurate -descriptions and drawings, for example a paper submitted September -12, 1683, that there is no doubt that he really saw bacteria and was -the first to do so (Fig. 2). Rightly may he be styled "The Father -of Bacteriology," if not of the microscope. He says in one paper: -"With the greatest astonishment I observed that everywhere through -the material I was examining were distributed _animalcules_ of the -most microscopic dimness which moved themselves about in a remarkably -energetic way." Thus he considered these living objects to be animals, -from their motion, and this belief held sway for nearly two hundred -years. - -[Illustration: FIG. 1.--Leeuwenhoek's Microscope. A is the simple -bi-convex lens held firmly in place. In front of this is the small -table, B, with the support, C, on the tip of which the object to be -examined was held. This support could be brought nearer to or removed -further away from the lens and held firmly in place by the screw, D. -E is a second screw for raising or lowering the entire table. A concave -mirror that Leeuwenhoek sometimes used to focus more light on the -object under examination, is shown at the right.] - -Leeuwenhoek was a pure observer of facts and made no attempt at -speculation, but his discoveries soon started the theorists to -discussing the origin of these minute organisms. Most observers, as -was probably to be expected, believed that they arose spontaneously. -Needham, in 1749, described the development of microörganisms -around grains of barley in water. Bonnet, in 1768, suggested that -probably Needham's animalcules came from ova in the liquid. The Abbot -Spallanzani, in 1769, called attention to the crudeness of Needham's -methods and later, in 1776, attempted to disprove spontaneous origin -by heating infusions of organic material in flasks and then _sealing_ -them. His critics raised the objections that heating the liquids -destroyed their ability to support life, and that sealing prevented -the access of fresh air which was also necessary. The first objection -was disproved by the accidental cracking of some of the flasks which -thereafter showed an abundant growth. This accident seemed also to -support the second objection, and Spallanzani did not answer it. Though -Spallanzani's experiments failed to convince his opponents, they led to -important practical results, since François Appert, in 1810, applied -them to the preserving of fruits, meats, etc., and in a sense started -the modern canning industry. - -[Illustration: FIG. 2.--The first drawings of bacteria by Leeuwenhoek. -The dotted line _C-D_ indicates the movement of the organism.] - -[Illustration: FIG. 3.--Schultze's experiment. The set of bulbs next to -the face contained KOH and the other set concentrated H{2}SO{4}. Air was -drawn through at frequent intervals from May until August but no growth -developed in the boiled infusion.] - -From Spallanzani to Schultze, there were no further experiments to -prove or disprove spontaneous generation. Schultze, in 1836, attempted -to meet the second objection to Spallanzani's experiment, _i.e._, -the exclusion of air, by drawing air through his boiled infusions, -first causing it to bubble through concentrated sulphuric acid to -kill the "germs" (Fig. 3.). His flasks fortunately showed no growths, -but his critics claimed that the strong acid changed the properties -of the air so that it would not support life. This experiment of -Schultze's, though devised for a different purpose, was really the -first _experiment_ in the use of _chemical disinfectants_, though -Thaer (page 31) had used chemicals in a practical way. Schwann, in -1837, modified this experiment, by drawing the air through a tube -heated to destroy the living germs (Fig. 4). His experiments were -successful but the "spontaneous generation" theorists raised the same -objection, _i.e._, the change in the air by heating. This was the first -_experiment_ in which the principle of "_dry heat_" or "_hot air_" -sterilization was used. Similar arguments were brought forward, also -to the use of _cotton plugs_ as filters by Schroeder and Dusch in 1859 -(Fig. 5). This was the first use of the principle of _sterilization by -filtration_. It remained for Chevreuil and Pasteur to overcome this -objection in 1861 by the use of flasks with long necks drawn out to a -point and bent over. These permitted a full access of air by diffusion -but kept out living germs, since these cannot fly but are carried -mechanically by air currents or fall of their own weight (Fig. 6.). -Hoffman, the year before (1860), had made similar experiments but these -remained unnoticed. The Pasteur flasks convinced most scientists that -"spontaneous generation" has never been observed by man, though some -few, notably Dr. Charlton Bastian, of England, vigorously supported the -theory from the early seventies until his death in November, 1915. - -[Illustration: FIG. 4.--Schwann's experiment. After boiling, as shown -in the diagram, and cooling, air was drawn into the flask by aspiration -while the coiled tube was kept hot with the flame.] - -[Illustration: FIG. 5.--Schroeder and Dusch's experiment. The -aspirating bottle drew the air through the flask after it had been -filtered by the cotton in the tube.] - -[Illustration: FIG. 6.--Pasteur's flask.] - -[Illustration: FIG. 7.--Tyndall's box. One side is removed to show -the construction. The bent tubes at the top are to permit a free -circulation of air into the interior. The window at the back has one -corresponding in the front (removed). Through these the beam of light -sent through from the lamp at the side was observed. The three tubes -received the infusion and were then boiled in an oil bath. The pipette -was for filling the tubes. (Popular Science Monthly, April, 1877.).] - -John Tyndall, in combating Bastian's views showed that boiled infusions -left open to the air in a closed box through which air circulated -did not show any growth of organisms provided the air was so free of -particles that the path of a ray of light sent through it from side to -side could not be seen (Fig. 7). Or if such sterilized infusions were -exposed to dust-free air, as in the high Alps, the majority showed -no growth, while all infusions in dusty air did show an abundance of -organisms. Tyndall's experiments confirmed those of Pasteur and his -predecessors and showed that the organisms developed from "germs" -present in the air falling into the liquids and not spontaneously. - -While Tyndall's experiments were of great value as indicated, they -probably were harmful in another way. These "germs in the air" were -considered by bacteriologists as well as laymen to include necessarily -many _disease germs_ and to indicate the very general, if not -universal, presence of these latter _in the air_. This idea led to many -erroneous practices in sanitation and disinfection which even to this -day are not eliminated. - - -CAUSATION OF DISEASE. - -The transmission of disease from person to person was recognized by the -ancients of European and Asiatic countries. Inoculation of smallpox was -practiced in China and India probably several thousand years ago and -was introduced by Lady Mary Wortley Montague into England in 1721, from -Constantinople. These beliefs and practices do not seem to have been -associated with any speculations or theories as to the cause of the -disease. - -Apparently the first writer on this subject was Varo, about B.C. 70, -who suggested that fevers in swampy places were due to invisible -organisms. The treatment of wounds during the thirteenth and fourteenth -centuries by hot wine fomentations and by the application of plasters -was based on the theory that the _air_ brought about conditions in the -wounds which led to suppuration. These practices were indeed primitive -antisepsis, yet were not based on a _germ theory_ of the conditions -which were partially prevented. Fracastorius (1484-1553), in a work -published in 1546, elaborated a theory of "disease germs" and "direct -and indirect contagion" very similar to modern views, though based -on no direct pathological knowledge. Nevertheless Kircher (mentioned -already) is usually given undeserved credit for the "contagium vivum" -theory. In 1657 by the use of simple lenses he observed "worms" in -decaying substances, in blood and in the pus from bubonic plague -patients (probably rouleaux of corpuscles in the blood, certainly not -bacteria in any case). Based on these observations and possibly also on -reading the work of Fracastorius, his theory of a "living cause" for -various diseases was published in 1671, but received little support. - -The discoveries of Leeuwenhoek which proved the existence of -microscopic organisms soon revived the "contagium vivum" idea of -Kircher. Nicolas Andry in a work published in 1701 upheld this view. -Lancisi in 1718 advanced the idea that "animalcules" were responsible -for malaria, a view not proved until Laveran discovered the malarial -parasite in 1880.[1] Physicians ascribed the plague which visited -Southern France in 1721 to the same cause, and many even went so far as -to attribute all disease to animalcules, which brought the theory into -ridicule. Nevertheless the "contagium vivum" theory survived, and even -Linnaeus in his _Systema Naturæ_ (1753-6) recognized it by placing the -organisms of Leeuwenhoek, the contagia of diseases and the causes of -putrefaction and fermentation in one class called "Chaos." - -Plenciz, a prominent physician and professor in the Vienna Medical -School, published in 1762 a work in which he gave strong arguments for -the "living cause" theory for transmissable diseases. He taught that -the agent is evidently transmitted through the air and that there is -a certain period of incubation pointing to a multiplication within -the body. He also believed that there was a specific agent for each -disease. His writings attracted little attention at the time and the -"contagium vivum" theory seems to have been almost lost sight of for -more than fifty years. Indeed, Oznam, in 1820, said it was no use to -waste time in refuting hypotheses as to the animal nature of contagium. - -Isolated observers, were, however, keeping the idea alive, each in -his own locality. In 1787 Wollstein, of Vienna, showed that the pus -from horses with glanders could infect other horses if inoculated -into the skin. Abilgaard, of Copenhagen, made similar experiments at -about the same time. In 1797 Eric Viborg, a pupil of Abilgaard's, -published experiments in which he showed the infectious nature not -only of the pus but also of the nasal discharges, saliva, urine, etc., -of glandered horses. Jenner in 1795-98 introduced vaccination as a -method of preventing smallpox. This epoch-making discovery attracted -world wide attention and led to the overcoming of this scourge which -had devastated Europe for centuries, but contributed little or nothing -to the question of the causation of disease. Prevost's discovery of -the cause of grain rust (_Puccinia graminis_) in 1807 was the _first -instance of an infectious disease of plants_ shown to be _due to a -microscopic plant organism_, though not a bacterium in this case. - -Doubtless one reason why the work on glanders and grain rust attracted -little attention among the practitioners of human medicine was owing to -the prevalent belief in man's complete separation from all lower forms -of life. The evolutionists had not yet paved the way for experimental -medicine. - -In 1822 Gaspard showed the poisonous nature of material from infected -wounds by injecting it into animals and causing their death. Tiedemann -(1822), Peacock (1828) described "little bodies" in the muscles of -human cadavers which Hilton (1832) considered to be parasitic in -nature. Paget (1835) showed that these bodies were round worms and -Owen (1835) described them more accurately and gave the name _Trichina -spiralis_ to them. Leidy (1846) found organisms in the muscles of -hogs which he considered to be the same as Owen's Trichina and paved -the way for the work of Zenker (1860) in showing the pathological -relation between the Trichina of pork and human Trichinosis. Bearing -on the "contagium vivum" theory was the rediscovery of the "itch mite" -(_Sarcoptes scabiei_) by Renucci (1834), an Italian medical student. -This had been declared several hundred years before but had been lost -sight of. Chevreuil and Pasteur, in 1836, showed that putrefaction did -not occur in meat protected from contamination, and suggested that -wound infection probably resulted from entrance of germs from without. -Bassi, investigating a disease of silkworms in Italy, demonstrated -that a certain mold-like fungus (_Botrytis bassiana_) was the cause in -1837. This was the _first instance of a microscopic vegetable organism_ -proved to be capable of _causing disease in an animal_. - -Boehm, in 1838, observed minute organisms in the stools of cholera -patients and conjectured that they might have a causal connection -with the disease. Dubini of Milan in 1838 discovered the _Ankylostoma -duodenale_ which later was further described by Omodei in 1843 and -shown to be the cause of Egyptian chlorosis by Griesinger (1851). -The fungous nature of favus, a scalp disease, was recognized by -Schönlein in 1839, and the organism was afterward called "_Achorion -schoenleinii_." Berg, in 1839-41, showed that thrush is likewise due to -a fungus, "_Oidium albicans_." - -These discoveries led Henle, in 1840, to publish a work in which -he maintained that all contagious diseases must be due to living -organisms, and to propound certain postulates (afterward restated -by Koch and now known as "Koch's postulates" p. 233) which must be -demonstrated before one can be sure that a given organism is the -specific cause of a given disease. The methods then in vogue and the -instruments of that period did not enable Henle to prove his claims, -but he must be given the credit for establishing the "contagium vivum" -theory on a good basis and pointing the way for men better equipped to -prove its soundness in after years. - -[Illustration: PLATE II - -SIR JOSEPH LISTER] - -In 1842-43 Gruby showed that Herpes tonsurans, a form of ringworm, is -due to the fungus _Trichophyton tonsurans_. Klencke, in 1843, produced -generalized tuberculosis in a rabbit by injecting tuberculous material -into a vein in the ear, but did not carry his researches further. -In 1843, Doctor Oliver Wendell Holmes wrote a paper in which he -contended that puerperal fever was contagious. Liebert identified the -_Peronospora infestans_ as the cause of one type of potato rot in 1845. -The skin disease Pityriasis (tinea) versicolor was shown to be due to -the _Microsporon furfur_ by Eichstedt in 1846. In 1847 Semmelweiss of -Vienna recommended disinfection of the hands with chloride of lime by -obstetricians because he believed with Holmes in the transmissibility -of puerperal fever through poisons carried in this way from the -dissecting room but his theories were ridiculed. - -[Illustration: PLATE III - -ROBERT KOCH] - -Pollender, in 1849, and Davaine and Rayer, in 1850, independently -observed small rod-like bodies in the blood of sheep and cattle -which had died of splenic fever (anthrax). That Egyptian chlorosis, -afterward identified with Old World "hookworm disease," is caused by -the _Ankylostoma duodenale_ was shown by Greisinger in 1851. In the -same year the _Schistosomum hematobium_ was shown to be the cause -of the "Bilharzia disease" by Bilharz. Küchenmeister discovered the -tapeworm, _Tænia solium_, in 1852, Cohn, an infectious disease of -flies due to a parasitic fungus (_Empusa muscæ_) in 1855, and Zenker -showed the connection between trichinosis of pork ("measly pork") -and human trichinosis (1860) as indicated above. The organisms just -mentioned are, of course, not bacteria, but these discoveries proved -conclusively that _living things of one kind or another, some large, -most of them microscopic, could cause disease in other organisms_ and -stimulated the search for other "living contagiums." In 1863 Davaine, -already mentioned, showed that anthrax could be transmitted from -animal to animal by inoculation of blood, but only if the blood -contained the minute rods which he believed to be the cause. Davaine -later abandoned this belief because he transmitted the disease with -old blood in which he could find no rods. It is now known that this -was because the bacilli were in the "spore" form which Davaine did not -recognize. He thus missed the definite proof of the bacterial nature -of anthrax because he was not familiar with the life history of the -organism which was worked out by Koch thirteen years later. In 1865 -Villemin repeatedly caused tuberculosis in rabbits by subcutaneous -injection of tuberculous material and showed that this disease must be -infectious also. In the same year Lord Lister introduced antiseptic -methods in surgery. He believed that wound infections were due to -microörganisms getting in from the air, the surgeon's fingers, etc., -and without proving this, he used carbolic acid to kill these germs -and prevent the infection. His pioneer experiments made modern surgery -possible. In this year also, Pasteur was sent to investigate a disease, -Pebrine, which was destroying the silkworms in Southern France. He -showed the cause to be a protozoan which had been seen previously by -Cornalia and described by Nägeli under the name _Nosema bombycis_ and -devised preventive measures. This was the _first infectious disease_ -shown to be _due to a protozoan_. In 1866 Rindfleisch observed small -pin-point-like bodies in the heart muscle of persons who had died -of wound infection. Klebs, in 1870-71, published descriptions and -names of organisms he had found in the material from similar wounds, -though he did not establish their causal relation. Bollinger, in -1872, discovered the spores of anthrax and explained the persistence -of the disease in certain districts as due to the resistant spores. -In 1873 Obermeier observed in the blood of patients suffering from -recurrent fever long, flexible spiral organisms which have been named -_Spirochæta obermeieri_. Lösch ascribed tropical dysentery to an ameba, -named by him _Amoeba coli_, in 1875. Finally, Koch, in 1876, isolated -the anthrax bacillus, worked out the life history of the organism -and reproduced the disease by the injection of pure cultures and -recovered the organism from the inoculated animals, thus establishing -beyond reasonable doubt its causal relationship to the disease. This -was the _first instance of a bacterium_ proved to be the cause of a -_disease in animals_. Pasteur, working on the disease at the same time, -confirmed all of Koch's findings, though his results were published -the next year, 1877. Bollinger determined that the _Actinomyces bovis_ -(_Streptothrix bovis_) is the cause of actinomycosis in cattle in -1877. Woronin in the same year discovered a protozoan (_Plasmodiophora -brassicæ_) to be the cause of a disease in cabbage, the _first proved -instance of a unicellular animal causing a disease in a plant_. In 1878 -Koch published his researches on wound infection in which he showed -beyond question that microörganisms are the cause of this condition, -though Pasteur in 1837, had suggested the same thing and Lister had -acted on the theory in preventing infection. - -These discoveries, especially those of Koch, immediately attracted -world-wide attention and stimulated a host of workers, so that within -the next ten years most of the bacteria which produce disease in -men and animals were isolated and described. It is well to remember -that the first _specific_ disease of man proved to be caused by a -_bacterium_ was _tuberculosis_, by Koch in 1882. - -Progress was greatly assisted by the introduction of anilin dyes as -suitable stains for organisms by Weigert in 1877, by Koch's application -of special technic and gelatin cultures for isolation and study, 1881, -and the great improvements in the microscope by Prof. Abbé, of Jena. - -Laveran's discovery of the malarial parasite in 1880 turned attention -to protozoa as the causes of disease and led to the discovery of the -various piroplasmoses and trypanosomiases in man and the lower animals. - -Pasteur's protective inoculations in chicken cholera and anthrax -directed attention to the possibility of using bacteria or their -products as a specific protective or curative means against particular -diseases. This finally led to the discovery of diphtheria antitoxin by -Behring, and independently by Roux, in 1890, a discovery which opened -up the wide field of immunity which is so persistently cultivated at -the present time. - -[Illustration: PLATE IV - -LOUIS PASTEUR] - -While the causation of disease by bacteria has probably attracted -most attention, especially in the popular mind, it should not be -forgotten that this is but one of the numerous ways in which these -organisms manifest their activities, and in a sense it is one of -their least-important ways, since other kinds are essential in many -industries (dairying, agriculture) and processes (sewage purification) -and are even _indispensable for the very existence of all green plants -and hence of animals, including man himself_. - - -PUTREFACTION AND FERMENTATION. - -The idea that there is a certain resemblance between some infectious -diseases and the processes of putrefaction and fermentation seems to -have originated during the discussion on spontaneous generation and -the "contagium vivum" theory which followed Leeuwenhoek's discoveries. -Plenciz (1762) appears to have first formulated this belief in writing. -He considered putrefaction to be due to the "animalcules" and said that -it occurred only when there was a coat of organisms on the material -and only when they increased and multiplied. Spallanzani's experiments -tended to support this view since his infusions did not "spoil" when -boiled and sealed. Appert's practical application of this idea has been -mentioned. - -Thaer, in his _Principles of Rational Agriculture_, published in the -first quarter of the nineteenth century, expressed the belief that the -"blue milk fermentation" was probably due to a kind of fungus that -gets in from the air, and stated that he had prevented it by treating -the milk cellars and vessels, with sulphur fumes or with "oxygenated -hydrochloric acid" (hypochlorous acid). - -In 1836 Chevreuil and Pasteur showed that putrefaction did not occur -in meat protected from contamination. In 1837 Caignard-Latour, in -France, and Schwann, in Germany, independently showed that alcoholic -fermentation in beer and wine is due to the growth of a microscopic -plant, the yeast, in the fermenting wort. C. J. Fuchs described the -organism which is commonly called the "blue milk bacillus" in 1841 and -conjectured that the souring of milk was probably bacterial in origin. -It remained for Pasteur to prove this in 1857. During the following -six or seven years Pasteur also proved that acetic acid fermentation, -as in vinegar making, butyric acid fermentation (odor of rancid butter -and old cheese) and the ammoniacal fermentation of urea, so noticeable -around stables, were each due to different species of bacteria. Pasteur -also, during the progress of this work, discovered the class of -organisms which can grow in the absence of free oxygen--the anaërobic -bacteria. There is no question that Pasteur from 1857 on did more to -lay the foundations of the science of bacteriology than any other -one man. Influenced by Pasteur's work von Hesseling, in 1866, stated -his belief that the process of cheese ripening, like the souring of -milk, was associated with the growth of fungi, and Martin also, in -1867, stated that cheese ripening was a process which was akin to -alcoholic, lactic and butyric fermentations. Kette, in 1869, asserted -the probability of Pasteur's researches furnishing a scientific basis -for many processes of change in the soil. In 1873 Schlösing and Müntz -showed that nitrification must be due to the action of microörganisms, -though the discovery of the particular ones remained for Winogradsky -in 1889. Thus the belief that fermentation and putrefaction are due to -microörganisms was as well established by the early eighties of the -last century as that similar organisms are the causes of infectious -diseases. - - -STUDY OF FORMS. - -An important part of the scientific knowledge of living organisms is -dependent on a study of their forms and relationships. As has been -stated, Leeuwenhoek considered bacteria to be "animalcules" because -they showed independent movement. But little attention was paid to -the natural history of these animalcules for nearly a hundred years -after Leeuwenhoek. During the last quarter of the eighteenth century, -however, workers busied themselves chiefly with the discovery and -description of new forms. Among these students were Baron Gleichen, -Jablot, Lesser, Reaumur, Hill and others. Müller, of Copenhagen, in -1786 published the first attempt at classification, a most important -step in the study of these organisms. Müller introduced the terms -Monas, Proteus and Vibrio, which are still in use. Ehrenberg, in his -work on _Infusoria_, or the organisms found in infusions, published -in 1838, introduced many generic names in use at present, but still -classed the bacteria with protozoa. Joseph Leidy, the American -naturalist, considered that the "vibrios" of previous writers were -plants and not "animalcules." He seems to have been the first to have -made this distinction (1849). Perty (1852) recognized the presence of -spores in some of his organisms. Ferdinand Cohn (1854) classed the -bacteria among plants. Nägeli (1857) proposed the name "Schizomycetes" -or "fission fungi," which is still retained for the entire class of -bacteria. Cohn in the years 1872-1875 established classification on -a modern basis and added greatly to the knowledge of morphology and -natural history of bacteria. He described spore formation and the -development of spores into active bacteria, and showed the close -relationships as well as differences between the bacteria and the lower -algæ. Robert Koch was a pupil of Cohn. - -An examination of the accompanying chronological table will show how -the investigations and discoveries in connection with "spontaneous -generation," the "contagium vivum" theory and putrefaction and -fermentation must have been mutually suggestive: - - 1546. Fracastorius, disease germs theory and direct and indirect - contagion. - - 1671. Kircher, "contagium vivum" theory. - - 1675. Leeuwenhoek, first saw bacteria, "animalcules." - - 1701. Andry, "animalcules" cause of diseases. - - 1718. Lancisi, "animalcules" cause of malaria. - - 1749. Needham, described development of organisms in water around - barley grains. - - 1762. Plenciz, arguments for "living cause" theory and that - "animalcules" cause putrefaction. - - 1768. Bonnet, suggested that probably Needham's organisms came from - germs in the liquid. - - 1776. Spallanzani, boiled and sealed infusions. - - 1786. Müller, first classified "animalcules." - - 1787. Wollstein, glanders pus infectious. - - 1795-1798. Jenner, vaccination against smallpox. - - 1797. Viborg, transmitted glanders repeatedly. - - 1807. Prevost, grain rust, _Puccinia graminis_. _The first instance - of a microscopic plant organism shown to be the cause of a disease in - a higher plant._ - - 1810. Appert, directions for "canning." - - 1822. Gaspard, infectiousness of material from wounds. - - 1834. Renucci, itch--itch mite (_Sarcoptes scabiei_). - - 1835. Paget and Owen, _Trichina spiralis_. - - 1836. Schultze, air through acid to kill "germs." - - 1837. Chevreuil and Pasteur, protected meat did not putrefy; - suggested wound infection due to entrance of germs from without. - - 1837. Caignard-Latour, Schwann, alcoholic fermentation--yeast. - - 1837. Schwann, air through heated tubes to kill germs. - - 1837. Bassi, muscardine of silkworms, _Botrytis bassiana_. _The first - instance of a microscopic plant organism shown to be the cause of a - disease in an animal._ - - 1838. Boehm, cholera, saw organisms in stools (not the cause). - - 1838. Dubini discovered _Ankylostoma duodenale_. - - 1838. Ehrenberg, study of forms. - - 1839. Schönlein, Favus, _Achorion schoenleinii_. - - 1839-41. Berg, Thrush, _Oidium albicans_. - - 1840. Henle, theory of contagious diseases. - - 1841. Fuchs, bacterial cause of blue milk. - - 1842-43. Gruby, Herpes tonsurans, _Trichophyton tonsurans_. - - 1843. Klencke, inoculations of tuberculous material into rabbit. - - 1843. Holmes, puerperal fever contagious. - - 1845. Liebert, a potato rot, _Peronospora infestans_. - - 1846. Leidy, Joseph (American Naturalist), _Trichina spiralis_ in - pork. - - 1846. Eichstedt, Pityriasis versicolor, _Microsporon furfur_. - - 1847. Semmelweiss, recommended disinfection to prevent puerperal - fever. Not followed. - - 1849. Leidy, considered "vibrios" to be plants. - - 1849. Pollender, Anthrax, saw rods in blood. - - 1850. Davaine and Rayer, Anthrax, saw rods in blood. - - 1851. Griesinger, Egyptian chlorosis, _Ankylostoma duodenale_. - - 1851. Bilharz, Bilharzia disease, _Schistosomum hematobium_. - - 1852. Kückenmeister, tapeworm, _Tænia solium_. - - 1852. Perty, saw spores in bacteria. - - 1854. Cohn, classed bacteria as plants. - - 1855. Cohn, disease of flies, _Empusa muscæ_. - - 1857. Nägeli, named bacteria, Schizomycetes. - - 1857. Pasteur, lactic, acetic, butyric acid fermentation. - - 1860. Zenker, Trichinosis, _Trichinella spiralis_. - - 1861. Pasteur, disproof of spontaneous generation. - - 1863. Davaine, transmitted anthrax by blood injections. - - 1865. Pasteur, Pebrine of silkworms, _Nosema bombycis_. _The first - instance of a protozoan shown to be the cause of a disease in a - higher animal._ - - 1865. Villemin, repeatedly transmitted tuberculosis to rabbits. - - 1865. Lister, introduced antisepsis in surgery. - - 1860. Rindfleisch, Pyemia, organisms in the pus. - - 1866. Von Hesseling, cheese ripening. - - 1867. De Martin, cheese ripening akin to alcoholic fermentation. - - 1869. Kette, Pasteur's researches scientific basis for many processes - in the soil. - - 1871. Klebs, Pyemia, organisms in the pus. - - 1872. Bollinger, spores in anthrax. - - 1872-75. Cohn, definite classification. - - 1873. Obermeier, recurrent fever, _Spirochæta obermeieri_. - - 1873. Schlösing and Münz, nitrification due to organisms. - - 1875. Lösch, amebic dysentery, _Amoeba coli_. - - 1875-76. Tyndall, germs in the air. - - 1876. Robert Koch, anthrax, _Bacillus anthracis_. _The first instance - of a bacterium shown to be the cause of disease in an animal._ - - 1877. Bollinger, actinomycosis, _Actinomyces bovis_ (_Streptothrix - bovis_). - - 1877. Weigert, used anilin dyes for staining. - - 1877. Woronin, cabbage disease, _Plasmodiophora brassicæ_. _The first - instance of a protozoan shown to be the cause of a disease in a - plant._ - - 1878. Koch, wound infections, bacterial in origin. - - 1881. Koch, gelatin plate cultures, Abbé, improvements in the - microscope. - - - - -CHAPTER I. - -POSITION--RELATIONSHIPS. - - -Bacteria are considered to belong to the plant kingdom not because of -any one character they possess, but because they most nearly resemble -organisms which are generally recognized as plants. While it is not -difficult to distinguish between the higher plants and higher animals, -it becomes almost, if not quite, impossible to separate the lowest, -forms of life. It is only by the method of resemblances above mentioned -that a decision is finally reached. It has even been proposed to make a -third class of organisms neither plants nor animals but midway between -in which the bacteria are included, but such a classification has not -as yet been adopted. - -In many respects the bacteria are most nearly related to the lowest -_algæ_, since both are unicellular organisms, both reproduce by -transverse division and the forms of the cell are strikingly similar. -The bacteria differ in one important respect, that is, they do not -contain _chlorophyl_, the green coloring matter which enables all -plants possessing it to absorb and break up carbon dioxide in the -light, and hence belong among the fungi. Bacteria average much smaller -than even the smallest algæ. - -Bacteria are closely connected with the _fission yeasts_ and the -_yeasts_ and _torulæ_. All are unicellular and without chlorophyl. The -bacteria, as has been stated, reproduce by division but the others -characteristically by budding or gemmation, though the fission yeasts -also by division. - -There is a certain resemblance to the _molds_ in their absence -of chlorophyl. But the molds grow as branching threads and also -have special fruiting organs for producing spores as a means of -reproduction, neither of which characteristics is found among the -_true_ bacteria. The higher thread bacteria do show true branching -and rudimentary fruiting bodies (Streptothrix) and appear to be a link -connecting the true bacteria and the molds. - -[Illustration: FIG. 8.--A thread of blue-green algæ.] - -[Illustration: FIG. 9.--A thread of small blue-green algæ.] - -[Illustration: FIG. 10.--A thread of bacteria. Compare with Figs. 8 -and 9.] - -[Illustration: FIG. 11.--A chain of spherical blue-green algæ.] - -[Illustration: FIG. 12.--A chain of spherical bacteria.] - -[Illustration: FIG. 13.--A pair of spherical blue-green algæ.] - -Further the _chemical composition_ of bacteria is more like that of -other fungous plants than of any of the forms classed as animals. - -[Illustration: FIG. 14.--Spherical bacteria. Several pairs are shown.] - -[Illustration: FIG. 15.--Yeast cells. Some show typical budding.] - -The food of bacteria is always taken up in solution by diffusion -through the outer covering of the cell as it is in all plants. Plant -cells never surround and engulf particles of solid food and digest them -within the cell as many single-celled animals do, and as the leukocytes -and similar ameboid cells in practically all multicelled animals do.[2] - -[Illustration: FIG. 16.--A portion of the mycelium of a mold. Note the -large size and the branching.] - -One of the most marked differences between animals and plants is with -respect to their energy relationships. Plants are characteristically -storers of energy while animals are liberators of it. Some bacteria -which have the power of swimming in a liquid certainly liberate -relatively large amounts of energy, and in the changes which bacteria -bring about in the material which they use as food considerable heat is -evolved ("heating" of manure, etc.). Nevertheless the evidence is good -that the bacteria as a class store much more of the energy contained -in the substances actually taken into the body cell as food than is -liberated in any form. - -Bacteria do show some resemblance to the protozoa, or single-celled -animal forms, in that the individuals of each group consist of one cell -only and some bacteria have the power of independent motion from place -to place in a liquid as most "infusoria" do, but here the resemblance -ceases. - -Bacteria are among the smallest of organisms, so small that it requires -the highest powers of the microscope for their successful study, and -the use of a special unit for their measurement. This unit is the -one-thousandth part of a millimeter and is called the micro-millimeter -or micron. Its symbol is the Greek letter _mu_ (µ). - -The size varies widely among different kinds but is fairly constant in -the same kind. The smallest described form is said to be only 0.18µ -long by 0.06µ thick and is just visible with the highest power of the -microscope, though it is possible and even probable that there are -forms still smaller which cannot be seen. Some large rare forms may -measure 40µ in length, but the vast majority are from 1µ to 4µ or 5µ -long, and from one-third to one-half as wide. - -From the above description a bacterium might be said to be a -_microscopic, unicellular plant, without chlorophyl, which reproduces -by dividing transversely_. - - - - -PART I. - -MORPHOLOGY - - - - -CHAPTER II. - -CELL STRUCTURES. - - -The _essential_ structures which may by appropriate means be -distinguished in the bacterial cell are _cell wall_ and _cell -contents_, technically termed _protoplasm_, cytoplasm. The cell wall is -not so dense, relatively, as that of green plants, but is thicker than -the outer covering of protozoa. It is very similar to the cell wall -of other lower fungi. Diffusion takes place readily through it with -very little selective action on substances absorbed as judged by the -comparative composition of bacteria and their surrounding medium. - -=Cytoplasm.=--The cytoplasm according to Bütschli and others is -somewhat different and slightly denser in its outer portion next to the -cell wall. This layer is designated the _ectoplasm_, as distinguished -from the remainder of the cell contents, the _endoplasm_. When bacteria -are suddenly transferred from a given medium into one of decidedly -_greater_ density, there sometimes results a contraction of the -_endoplasm_, due to the rapid diffusion of water. This phenomenon is -designated _plasmolysis_ (Fig. 17), and is similar to what occurs in -the cells of higher plants when subjected to the same treatment. This -is one of the methods which may be used to show the different parts of -the cell just described. - -If bacteria are suddenly transferred from a relatively dense medium -to one which is of decidedly _less_ density, it occasionally happens -that water diffuses into the cell and swells up the endoplasm so much -more rapidly than the cell wall that the latter ruptures and some of -the endoplasm exudes in the form of droplets on the surface of the cell -wall. This phenomenon is called _plasmoptysis_. Students will seldom -observe the distinction between cell wall and cell contents, except -that in examining living bacteria the outer portion appears more highly -refractive. This is chiefly due to the presence of a cell wall, but is -not a proof of its existence. - -[Illustration: FIG. 17.--Cells of bacteria showing plasmolysis. The -cell substance of three of the cells in the middle of the chain has -shrunk until it appears as a round black mass. The cell wall shows as -the lighter area.] - -[Illustration: FIG. 18.--Vacuoles in the bacterial cell. The lighter -areas are vacuoles.] - -=Nucleus.=--Douglas and Distaso[3] summarize the various opinions with -regard to the nucleus in bacteria as follows: - -1. Those who do not admit, the presence of a nucleus or of anything -equivalent to it. (Fischer, Migula, Massart). - -2. Those who consider that the entire bacterial cell is the equivalent -of a nucleus and contains no protoplasm. (Ruzicka). - -3. Those who admit the presence of nuclein but say that this is not -morphologically differentiated from the protoplasm as a nucleus. -(Weigert). - -4. Those who consider the bacterial protoplasm to consist of a central -endoplasm throughout which the nuclein is diffused and an external -layer of ectoplasm next to the cell wall. (Bütschli, Zettnow). - -5. Those who say that the bacterial cell contains a distinct nucleus, -at least in most instances. These authors base their claims on staining -with a Giemsa stain. (Feinberg, Ziemann, Neuvel, Dobell, Douglass and -Distaso). - -That nucleoproteins are present in the bacterial cell in relatively -large amounts is well established. Also that there are other proteins -and that the protoplasm is not all nuclein. - -Some workers as noted above have been able to demonstrate collections -of nuclein by staining, especially in very young cells. In older cells -this material is in most instances diffused throughout the protoplasm -and can not be so differentiated. - -The following statement probably represents the generally accepted view -at the present time: - -A nucleus _as such_ is not present in bacterial cells, except in a few -large rare forms and in very young cells. _Nuclein_, the characteristic -chemical substance in nuclei, which when aggregated forms the nucleus, -is scattered throughout the cell contents and thus intimately mingled -with the protoplasm, and cannot be differentiated by staining as in -most cells. - -The close association of nuclein and protoplasm may explain the rapid -rate of division of bacteria (Chapter VIII, p. 91). - -The chemical composition of the bacterial cell is discussed in Chapter -VII. - -In addition to the _essential_ parts just described the bacterial cell -may show some of the following _accidental_ structures: _vacuoles_, -_capsules_, _metachromatic granules_, _flagella_, _spores_. - -=Vacuoles.=--_Vacuoles_ appear as clear spaces in the protoplasm when -the organism is examined in the living condition or when stained very -slightly (Fig. 18). During life these are filled with liquid or gaseous -material which is sometimes waste, sometimes reserve food, sometimes -digestive fluids. Students are apt to confuse vacuoles with spores (p. -47). Staining is the surest way to differentiate (Chapter XIX, p. 209). -If vacuoles have any special function, it is an unimportant one. - -[Illustration: FIG. 19.--Bacteria seen within capsules.] - -[Illustration: FIG. 20.--Metachromatic granules in bacteria. The dark -round spots are the granules. The cells of the bacteria are scarcely -visible.] - -=Capsule.=--The _capsule_ is a second covering outside the cell wall -and probably developed from it (Fig. 19). It is usually gelatinous, -so that bacteria which form capsules frequently stick together -when growing in a fluid, so that the whole mass has a jelly-like -consistency. The term _zoögloea_ was formerly applied to such masses, -but it is a poor term and misleading (zoön = an animal) and should -be dropped. The masses of jelly-like material frequently found on -decaying wood, especially in rainy weather, are in some cases masses -of capsule-forming bacteria, though a part of the jelly is a product -of bacterial activity, a gum-like substance which lies among the -capsulated organisms. When these masses dry out, they become tough -and leathery, but it is not to be presumed that capsules are of this -consistency. On the contrary, they are soft and delicate, though they -certainly serve as an additional protection to the organism, doubtless -more by selective absorption than mechanically. Certain bacteria -which cause disease form capsules in the blood of those animals which -they kill and not in the blood of those in which they have no effect -(_Bacterium anthracis_ in guinea pig's blood and in rat's blood). The -presence of capsules around an organism can be proved only by staining -the capsule. Many bacteria when stained in albuminous fluids show a -clear space around them which appears like a capsule. It is due to the -contraction of the fluid away from the organism during drying. - -=Metachromatic Granules.=--The term "_metachromatic_" is applied to -granules which in stained preparations take a color different from -the protoplasm as a whole (Fig. 20). They vary widely in chemical -composition. Some of them are glycogen, some fat droplets. Others are -so-called "granulose" closely related to starch but probably not true -starch. Others are probably nuclein. Of many the chemical composition -is unknown. They are called "Babes-Ernst corpuscles" in certain -bacteria (typhoid bacillus). Since they frequently occur in the ends -of cells the term "polar granules" is also applied. Their presence is -of value in the recognition of but few bacteria ("Neisser granules" in -diphtheria). - -=Flagellum.=--A _flagellum_ is a very minute thread-like process -growing out from the cell wall, probably filled with a strand of -protoplasm. The vibrations of the flagella move the organism through -the liquid medium. Bacteria which are thus capable of independent -movement are spoken of as "motile bacteria." The actual rate of -movement is very slight, though in proportion to the size of the -organism it may be considered rapid. Thus Alfred Fischer determined -that some organisms have a speed for short periods of about 40 cm. per -hour. This is equivalent to a man moving more than 200 miles in the -same time. - -It is obvious that bacteria which can move about in a liquid have an -advantage in obtaining food, since they do not need to wait for it to -be brought to them. This advantage is probably slight. - -[Illustration: FIG. 21.--A bacterium showing a single flagellum at the -end--monotrichic.] - -[Illustration: FIG. 22.--A bacterium showing a bundle of four flagella -at the end--lophotrichic.] - -An organism may have only one flagellum at the end. It is then said -to be monotrichic (Fig. 21) (#monos# = alone, single; #trichos# = hair). -This is most commonly at the front end, so that the bacterium is drawn -through the liquid by its motion. Rarely it is at the rear end. Other -bacteria may possess a bundle of flagella at one end and are called -_lophotrichic_ (Fig. 22) (#lophos# = tuft). Sometimes at approaching -division the flagella may be at both ends and are then _amphitrichic_ -(Fig. 23) (#amphi# = both). It is probable that this condition does not -persist long, but represents the development of flagella at one end -of each of a pair resulting from division of an organism which has -flagella at one end only. In many bacteria the flagella arise from -all parts of the surface of the cell. Such bacteria are _peritrichic_ -(Fig. 24) (#peri# = around). The position and even the number of the -flagella are very constant for each kind and are of decided value in -identification. - -[Illustration: FIG. 23.--A bacterium showing flagella at each -end--amphitrichic.] - -[Illustration: FIG. 24.--A bacterium showing flagella all -around--peritrichic.] - -Flagella are too fine and delicate to be seen on the living organism, -or even on bacteria which have been colored by the ordinary stains. -They are rendered visible only by certain methods which cause a -precipitate on both bacteria and flagella which are thereby made thick -enough to be seen (Chapter XIX, p. 210). The movement of liquid around -a bacterium caused by vibrations of flagella can sometimes be observed -with large forms and the use of "dark-field" illumination. - -Flagella are very delicate and easily broken off from the cell body. -Slight changes in the density or reaction of the medium frequently -cause this breaking off, so that preparations made from actively motile -bacteria frequently show no flagella. For this reason and also on -account of their fineness the demonstration of flagella is not easy, -and it is not safe to say that a non-motile bacterium has no flagella -except after very careful study. - -The motion of bacteria is characteristic and a little practice in -observing will enable the student to recognize it and distinguish -between motility and "Brownian" or molecular motion. Dead and -non-motile bacteria show the latter. In fact, any finely divided -particles suspended in a liquid which is not too viscous and in which -the particles are not soluble show Brownian motion or "pedesis." This -latter is a dancing motion of the particle within a very small area -and without change of place, while motile bacteria move from place to -place or even out of the field of the microscope with greater or less -speed. There is a marked difference in the character of the motion of -different kinds of bacteria. Some rotate around the long axis when -moving, others vibrate from side to side. - -Among the higher thread bacteria there are some which show motility -without possessing flagella. Just how they move is little understood. - -=Spores.=--Under certain conditions some bacterial cells undergo -transformations which result in the formation of so-called _spores_. -If the process is followed under the microscope, the changes observed -are approximately these: A very minute point appears in the protoplasm -which seems to act somewhat like the centrosome of higher cells as a -"center of attraction" so that the protoplasm gradually collects around -it. The spot disappears or is enclosed in the collected protoplasm. -This has evidently become denser as it is more highly refractive than -before. In time all or nearly all of the protoplasm is collected. A new -cell wall is developed around it which is thicker than the cell wall of -the bacterium. This thickened cell wall is called the "spore capsule." -Gradually the remnants of the former cell contents and the old cell -wall disappear or dissolve and the spore becomes "free" (Fig. 25). - -[Illustration: FIG. 25.--The smaller oval bodies in the middle of the -field are free spores.] - -If the spore is placed in favorable conditions the protoplasm absorbs -water, swells, the capsule bursts at some point, a cell wall is formed -and the bacterium grows to normal size and divides, that is, it is an -active growing cell again. This process is called "germination" of the -spore. The point at which the spore capsule bursts to permit the new -cell to emerge is characteristic for each kind of bacterium. It may be -at the end when the germination is said to be _polar_ (Fig. 26). It may -be from the middle of one side which gives _equatorial_ germination -(Fig. 27). Rarely it is diagonally from a point between the equator and -the pole, which type may be styled _oblique_ germination. In one or -two instances the entire spore swells up, lengthens and becomes a rod -without any special germination unless this type might be designated -_bi-polar_. - -[Illustration: FIG. 26.--Spores showing polar germination. The lighter -part of the two organisms just below A and B is the developing -bacterium. In the original slide the spore was stained red and the -developing bacterium a faint blue.] - -[Illustration: FIG. 27.--A spore showing equatorial germination. -The spore in the center of the field shows a rod growing out of it -laterally. In the original slide the spore was stained red and the -developing bacterium blue.] - -[Illustration: FIG. 28.--Spores in the middle of the rod without -enlargement of the rod. The lighter areas in the rods are spores.] - -[Illustration: FIG. 29.--Spores in the middle of the rod with -enlargement of the rod around them. The lighter areas in the rods are -spores.] - -Spores are most commonly oval or elliptical in shape, though sometimes -spherical. A spore may be formed in the middle of the organism without -(Fig. 28) or with (Fig. 29) a change in size of the cell around it. -If the diameter through the cell is increased, then the cell with -the contained spore becomes spindle-shaped. Such a cell is termed a -"_clostridium_." Sometimes the spore develops in the end of the cell -either without (Fig. 30) or with enlarging it (Fig. 31). In a few -forms the spore is placed at the end of the rod and shows a marked -enlargement. This is spoken of as the "_plectridium_" or more commonly -the "drumstick spore" (Fig. 32). The position and shape of the spore -are constant for each kind of bacteria. In one or two instances only, -two spores have been observed in a single organism. - -[Illustration: FIG. 30.--Spores in the end of the rod with no -enlargement of the rod around them. The lighter areas in the rods are -spores.] - -[Illustration: FIG. 31.--Spores in the end of the rod with enlargement -of the rod, _A_, _A_, _A_, _A_.] - -[Illustration: FIG. 32.--Drumstick spores at the end of the rod.] - -The fact that the protoplasm is denser and the spore capsule thicker -(the percentage of water in each is decidedly less than in the growing -cell) gives the spore the property of much greater resistance to all -destructive agencies than the active bacterium has. For example, all -actively growing cells are destroyed by boiling in a very few minutes, -while some spores require several hours' boiling. The same relation -holds with regard to drying, the action of chemicals, light, etc. That -the coagulation temperature of a protein varies inversely with the -amount of water, it contains, is shown by the following table from -Frost and McCampbell, "General Bacteriology": - - Egg albumin plus 50 per cent. water coagulates at 56° - " " " 25 per cent. " " " 74-80° - " " " 18 per cent. " " " 88-90° - " " " 6 per cent. " " " 145° - " " dry " " " 160-170° - -This resistance explains why it happens that food materials boiled -and sealed in cans to prevent the entrance of organisms sometimes -spoil. The spores have not been killed by the boiling. It explains -also in part the persistence of some diseases like anthrax and black -leg in pastures for years. From the above description it follows that -the spore is to be considered as _a condensation of the bacterial -protoplasm surrounded by an especially thick cell wall_. _Its function -is the preservation of the organism under adverse conditions._ It -corresponds most closely to the encystment of certain protozoa--the -ameba for example. Possibly the spore represents a very rudimentary -beginning of a reproductive function such as is gradually evolved in -the higher thread bacteria, the fission yeasts, the yeasts, the molds, -etc. Its characteristics are so markedly different, however, that the -function of preservation is certainly the main one. - -It must not be supposed that spores are formed under adverse conditions -only, because bacteria showing vigorous growth frequently form spores -rapidly. Special conditions are necessary for their formation just as -they are for the growth and other functions of bacteria (Chapters VI -and VII). - - - - -CHAPTER III. - -CELL FORMS. - - -Though there is apparently a wide variation in the shapes of different -bacterial cells, these may all be reduced to _three_ typical _cell -forms_. These are: first and simplest, the round or _spherical_, -typified by a ball and called the _coccus_ form, or _coccus_, plural -cocci[4] (Fig. 33). The coccus may be large, that is, from 1.5µ to 2µ -in diameter. The term _macrococcus_ is sometimes applied to these large -cocci. If the _coccus_ is less than 1µ in diameter, it is sometimes -spoken of as a _micrococcus_; in fact, this term is very commonly -applied to any coccus. When cocci are growing together, many of the -cells do not appear as true spheres but are more or less distorted -from pressure of their neighbors or from failure to grow to full size -after recent division. Most cocci divide into hemispheres and then each -half grows to full size. A few cocci elongate before division and then -appear oval or elliptical. - -The second cell form is that of a _cylinder_ or rod typified by a -section of a lead-pencil. The name _bacillus_, plural _bacilli_, is -applied to this type (Fig. 34). The bacillus may be short (Fig. 35), -1µ or less in length, or long, up to 40µ in rare cases. Most bacilli -are from 2µ to 5µ or 6µ long. The ends of the rod are usually rounded, -occasionally square and very rarely pointed. It is evident that a very -short rod with rounded ends approaches a coccus in form and it is not -always easy to differentiate in such cases. Most bacilli are straight, -but some are slightly curved (Fig. 36). - -The third cell form is the _spiral_, typified by a section of a -cork-screw and named _spirillum_, plural _spirilla_ (Fig. 37). A very -short spiral consisting of only a portion of a turn is sometimes called -_vibrio_ (Fig. 38). Vibrios when seen under the microscope look like -short curved rods. The distinction between the two can be made only by -examining the organism alive and moving in a liquid. The vibrio shows a -characteristic spiral twisting motion. Very long, flexible spirals are -usually named _spirochetes_ (Fig. 39). The spirochetes are motile but -flagella have not been shown to be present. - -[Illustration: FIG. 33.--Cocci.] - -[Illustration: FIG. 34.--Bacilli.] - -[Illustration: FIG. 35.--Short bacilli.] - -[Illustration: FIG. 36.--Curved bacilli. Only the one in the center of -the field is in focus. The others curve out of focus.] - -Besides the three typical cell forms bacteria frequently show -very great irregularities in shape. They may be pointed, bulged, -club-shaped or even slightly branched. These peculiar and bizarre -forms practically always occur when some of the necessary conditions -for normal growth, discussed in Chapters VI and VII, are not fulfilled. -They are best regarded as _involution_ or _degeneration_ forms for this -reason (Fig. 40). In a very few cases it is not possible to obtain the -organism without these forms (the diphtheria group). It is probable -that these cell forms are normal in such cases, or else conditions -suitable for the normal growth have not been obtained. - -[Illustration: FIG. 37.--Spirilla.] - -[Illustration: FIG. 38.--Vibrio forms of spirilla. Compare with Fig. -36.] - -[Illustration: FIG. 39.--Spirochetes.] - -[Illustration: FIG. 40.--Involution forms. The organisms are tapering -and branched at one end.] - - - - -CHAPTER IV. - -CELL GROUPINGS. - - -It has been stated that bacteria reproduce by transverse division, that -is, division across the long axis. Following repeated divisions the new -cells may or may not remain attached. In the latter case the bacteria -occur as separate isolated individuals. In the former, arrangements -characteristic of the particular organism almost invariably result. -These arrangements are best described as _cell groupings_ or _growth -forms_. - -[Illustration: FIG. 41.--Streptospirillum grouping.] - -[Illustration: FIG. 42.--Diplobacillus grouping.] - -In the case of spiral forms it is obvious that there is only one -possible grouping, that is, in chains of two or more individuals -adherent end to end. A chain of two spirilla might be called -a _diplospirillum_ (#diplos# = double); of three or more, a -_streptospirillum_ (#streptos# = necklace, chain) (Fig. 41). These terms -are rarely used, since spirilla do not ordinarily remain attached. -Likewise the bacillus can grow only in chains of two or more, and -the terms _diplobacillus_ (Fig. 42), bacilli in groups of two, and -_streptobacillus_ (Fig. 43), bacilli in chains are frequently used. -Still the terms _thread_, _filament_, or _chain_ are more common for -_streptobacillus_. - -[Illustration: FIG. 43.--Streptobacillus grouping.] - -[Illustration: FIG. 44.--Typical diplococcus grouping. Note that the -individual cocci are flattened on the apposing sides.] - -[Illustration: FIG. 45.--Long streptococcus grouping.] - -[Illustration: FIG. 46.--Short streptococcus grouping.] - -Since the coccus is spherical, _transverse_ division may occur in any -direction, though in three planes only at right angles to each other. -Division might occur in _one plane only_ as in spirilla and bacilli, -or in _two planes only_ or in _all three planes_. As a matter of fact -these three methods of division are found among the cocci, but only one -method for each particular kind of coccus. As a result there may be a -variety of cell groupings among the cocci. When division occurs in one -plane only, the possible groupings are the same as among the spirilla -or bacilli. The cocci may occur in groups of two--_diplococcus_ -grouping (Fig. 44), or in chains--_streptococcus_ grouping (Figs. 45 -and 46). When the grouping is in _diplococci_, the individual cocci -most commonly appear as hemispheres with the plane surfaces apposed -(Fig. 44). Sometimes they appear as spheres and occasionally are even -somewhat elongated. The individuals in a streptococcus grouping are -most commonly elongated, either in the same direction as the length of -the chain, or at right angles to it. The latter appearance is probably -due to failure to enlarge completely after division. Streptococci -frequently appear as chains of diplococci, that is, the pair resulting -from the division of a single coccus remain a little closer to each -other than to neighboring cells, as a close inspection of Fig. 45 will -show. - -If division occurs in _two planes only_, there may result the above -groupings and several others in addition. The four cocci which result -from a single division may remain together, giving the _tetracoccus_ -or _tetrad_ grouping. Very rarely all the cocci divide evenly and the -result is a regular _rectangular flat mass_ of cells, the total number -of which is a multiple of four. The term merismopedia (from a genus of -algæ which grows the same way) is applied to such a grouping. If the -cells within a group after a few divisions do not reproduce so rapidly -(lack of food), as usually happens, the number of cells becomes uneven -or at least not necessarily a multiple of four and the resultant _flat -mass_ has an _irregular_, _uneven outline_. This grouping is termed -_staphylococcus_ (#staphylos# = a bunch of grapes) (Fig. 47). It is the -most common grouping among the cocci. - -When division occurs in all three planes, there is in addition to all -the groupings possible to one- and two-plane division a third grouping -in which the cells are in _solid packets_, _multiples of eight_. The -name _sarcina_ is applied to this growth form (Fig. 48). The individual -cells in a sarcina packet never show the typical coccus form so long as -they remain together, but are always flattened on two or more sides. - -The above descriptions indicate how the method of division may be -determined. If in examining a preparation the _sarcina_ grouping -appears, that shows _three-plane division_. If there are no sarcina, -but _tetrads_ or _staphylococci_ (rarely merismopedia), then the -division is in _two planes_. If none of the foregoing is observed but -only _diplo-_ or _streptococci_, these indicate _one-plane division_ -only. Cocci show their _characteristic_ groupings only when grown in a -liquid medium, and such should always be used before deciding on the -plane of division. - -[Illustration: FIG. 47.--Staphylococcus grouping. The large flat masses -are staphylococcus grouping. Diplococcus grouping, tetrads and short -streptococci are also evident.] - -[Illustration: FIG. 48.--Sarcina grouping.] - -As the above description shows, these terms which are properly -adjectives describing the cell grouping, are quite generally used as -nouns. Thus the terms a diplococcus, a tetrad, a streptococcus, etc., -are common, meaning a bacterium of the cell form and cell grouping -indicated. - - CELL FORM. CELL GROUPING. - - coccus-- {diplococcus--in 2's. - round or spherical. {streptoccus--in chains. - {tetracoccus, tetrads--in 4's. - {staphylococcus--irregular flat masses. - {sarcina--regular, solid packets, multiples of 8. - - bacillus-- {diplobacillus--in 2's. - rod-shaped {streptobacillus--in chains. - or cylindrical. - - spirillum-- {diplospirillum--in 2's, little used. - spiral-shaped. {streptospirillum--in chains, little used. - - - - -CHAPTER V. - -CLASSIFICATION. - - -The arrangement of living organisms in groups according to their -resemblances and the adoption of _fixed names_ is of the greatest -advantage in their scientific study. For animal forms and for the -higher plants this classification is gradually becoming standardized -through the International Congress of Zoölogists and of Botanists -respectively. Unfortunately, the naming of the bacteria has not as -yet been taken up by the latter body, though announced as one of the -subjects for the Congress of 1916 (postponed on account of the war). -Hence there is at present no system which can be regarded as either -fixed or official. - -[Illustration: FIG. 49.--Illustrates the genus Streptococcus. Typical -chains, no staphylococcus grouping, no sarcina grouping, no flagella.] - -[Illustration: FIG. 50.--Illustrates the genus Micrococcus. -Diplococcus, tetrads short chains and staphylococcus; no sarcina, no -flagella.] - -[Illustration: FIG. 51.--Illustrates the genus Sarcina. Sarcina -grouping, no flagella.] - -[Illustration: FIG. 52.--Illustrates the genus Bacillus. A bacillus -with peritrichic flagella. (Student preparation.)] - -Since Müller's first classification of "animalcules" in 1786 numerous -attempts have been made to solve the problem. Only those beginning with -Ferdinand Cohn (1872-75) are of any real value. As long as bacteria -are regarded as plants it appears that the logical method is to follow -the well-established botanical principles in any system for naming -them. Botanists depend on morphological features almost entirely in -making their distinctions. The preceding chapters have shown that -the minute plants which are discussed have very few such features. -They are, to recapitulate, _cell wall_, _protoplasm_, _vacuoles_, -_metachromatic granules_, _capsules_, _flagella_, _spores_, _cell -forms_ and _cell groupings_. Most bacteria show not more than three -or four of these features, so that it is impossible by the aid of -morphology alone to distinguish from each other the large number of -different kinds which certainly exist. In the various systems which -are conceded to be the best these characteristics do serve to classify -them down to genera, leaving the "species" to be determined from their -_physiological_ activities. One of these systems was adopted by the -laboratory section of the American Public Health Association and by the -Society of American Bacteriologists and was practically the standard -in this country until superseded by the Society's own classification. -It is that of the German Bacteriologist Migula and is given below for -comparison. Since practically the entire discussion in this book is -concerned with the first three families the generic characteristics -in these only will be given. The full classification as well as a -thorough discussion of this subject is given in Lafar's _Handbuch_, -whence the following is adopted: - -[Illustration: FIG. 53.--Illustrates the genus Pseudomonas. A bacillus -with flagella at the end only.] - -[Illustration: FIG. 54.--Illustrates the genus Microspira. It is -(though the photograph does not prove it) a short spiral with one -flagellum at the end.] - -[Illustration: FIG. 55.--Illustrates the genus Spirillum. Spiral -bacteria with more than three, in this case four, flagella at the end.] - -[Illustration: FIG. 56.--Illustrates the genus Spirochæta.] - -[Illustration: FIG. 57.--Illustrates the genus Chlamydothrix. Fine -threads with a delicate sheath.] - -[Illustration: FIG. 58.--Illustrates the genus Crenothrix. The -thickness of the cell walls is due to deposits of iron hydroxide. -(After Lafar.)] - -[Illustration: FIG. 59.--Illustrates the genus Beggiatoa. The filament -_A_ is so full of sulphur granules that the individual cells are not -visible. _B_ has fewer sulphur granules. In _C_ the granules are -nearly absent and the separate cells of the filament are seen. (After -Winogradsky, from Lafar.)] - - -ORDER I. Eubacteria. - -Cells without nuclei, free from sulphur granules and from -bacteriopurpurin (p. 112); colorless, or slightly colored. - -1. Family: COCCACEÆ (Zopf) Migula, all cocci. - - {Genus 1. _Streptococcus_ Billroth: - { division in one plane only (Fig. 49). - Non-flagellated, { " 2. _Micrococcus_ (Hallier) Cohn: - Non-motile { division in two planes only (Fig. 50). - { " 3. _Sarcina_ Goodsir: - { division in three planes only (Fig. 51). - - { " 4. _Planococcus_ Migula: - Flagellated, { division in two planes only. - motile { " 5. _Planosarcina_ Migula: - { division in three planes only. - -2. Family: BACTERIACEÆ Migula, all bacilli. - - Genus 1. _Bacterium_ (Ehrenberg) Migula: no flagella; non-motile. - " 2. _Bacillus_ (Cohn) Migula: flagella peritrichic (Fig. 52). - " 3. _Pseudomonas_ Migula: flagella at the end: - monotrichic, lophotrichic, amphitrichic (Fig. 53). - -3. Family: SPIRILLACEÆ Migula, all spirilla. - - {Genus 1. _Spirosoma_ Migula: - { non flagellated; non-motile. - { " 2. _Microspira_ (Schroeter) Migula: - Cells stiff { flagella one to three at the end (Fig. 54). - { " 3. _Spirillum_ (Ehrenberg) Migula: - { flagella more than three - { at the end (Fig. 55). - - Cell flexible { " 4. _Spirochæta_ Ehrenberg: - { motile; no flagella (Fig. 56). - -4. Family: CHLAMYDOBACTERIACEÆ. - -Cells cylindrical in long threads and surrounded by a sheath. -Reproduction also by gonidia formed from an entire cell. - - Genus 1. _Chlamydothrix_ Migula (Fig. 57). - " 2. _Crenothrix_ Colin (Fig. 58). - " 3. _Pragmidiothrix_ Engler. - " 4. _Spherotilus_ (including Cladothrix). - - -ORDER II. THIOBACTERIA: SULPHUR BACTERIA. - -Cells without a nucleus, but containing sulphur granules, may be -colorless or contain bacteriopurpurin and be colored reddish or violet. - -1. Family BEGGIATOACEÆ. - - Genus 1. _Thiothrix_ Winogradsky. - - " 2. _Beggiatoa_ Trevisan. Of interest since it is without a - sheath, is motile, but without flagella (Fig. 59). - -2. Family RHODOBACTERIACEÆ. - -This has five subfamilies and twelve genera, most of which are due to -the Russian bacteriologist Winogradsky who did more work than anyone -else with the sulphur bacteria. - - -THE CLASSIFICATION OF THE SOCIETY OF AMERICAN BACTERIOLOGISTS. - -The Committee on Classification of the Society of American -Bacteriologists at the meeting held in December, 1919, submitted its -final report. This report has not been formally adopted as a whole, -but in all probability will be substantially as outlined below. This -outline does not attempt to give the detailed characterizations of the -different groups as defined by the committee, but does show the names -to be applied to the commoner organisms. These organisms are included -in the 4th and 5th orders. Details of the first three orders have not -been worked out. They are listed merely for completeness. - -CLASS SCHIZOMYCETES. - -Unicellular, chlorophyl-free plants, reproducing by transverse division -(some forms by gonidia also). - -ORDERS: - - A. Myxobacteriales--Cells united during vegetative stage into - a pseudo-plasmodium which passes over into a highly developed - cyst-producing resting stage. - - B. Thiobacteriales--Sulphur bacteria. - - C. Chlamydobacteriales--Iron bacteria and other sheathed bacteria. - - D. Actinomycetales--Actinomyces, tubercle and diphtheria bacilli. - - E. Eubacteriales--All the other common bacteria. - -GENERA OF ORDERS D AND E. - - D. ACTINOMYCETALES-- - FAMILY I. ACTINOMYCETACEÆ Buchanan, 1918. - Genus 1. _Actinobacillus_, Brampt, 1900. - Type species, _Actinobacillus lignieresi_ Brampt, 1900. - Genus 2. _Leptotrichia_ Trevisan, 1879. - Type species, _Leptotrichia buccalis_ (Robin, 1847) Trevisan. - Genus 3. _Actinomyces_ Harz, 1877. - Type species, _Actinomyces bovis_ Harz. - Genus 4. _Erysipelothrix_ Rosenbach, 1909. - Type species, _Erysipelothrix rhusiopathiæ_ (Kitt, 1893) - Rosenbach, swine erysipelas. - FAMILY II. MYCOBACTERIACEÆ Chester, 1897. - Genus 1. _Mycobacterium_ Lehmann and Neumann, 1896. - Type species, _Mycobacterium tuberculosis_ (Koch, 1882) L. - and N. - Genus 2. _Corynebacterium_ Lehmann and Neumann, 1896. - Type species, _Corynebacterium diphtheriæ_ (Loeffler, 1882) - L. and N. - Genus 3. _Fusiformis_ Hoelling, 1910. - Type species, _Fusiformis termitidis_ Hoelling. Vincent's - angina. - Genus 4. _Pfeifferella_ Buchanan, 1918. - Type species, _Pfeifferella mallei_ (Loeffler, 1896) Buchanan. - Glanders bacillus. - - E. EUBACTERIALES - FAMILY I--NITROBACTERIACEÆ--Proto- or autotrophic for N - or C and sometimes for both (except Acetobacter). - TRIBE I--NITROBACTEREÆ--autotrophic for C. - Genus 1. _Hydrogenomonas_ Jensen, 1909. - Type species, _Hydrogenomonas pantotropha_ (Kaserer, 1906) - Jensen; oxidizes free H. - Genus 2. _Methanomonas_ Jensen, 1909. - Type species, _Methanomonas methanica_ (Söhngen) Jensen; - oxidizes CH{4}. - Genus 3. _Carboxydomonas_ Jensen, 1909. - Type species, _Carboxydomonas oligocarbophila_ (Beijerinck - and Van Delden, 1903) Jensen; oxidizes CO. - Genus 4. _Acetobacter_ Fuhrman, 1905. - Type species, _Acetobacter aceti_ (Thompson, 1852) Fuhrman; - oxidizes alcohol to acetic acid. - Genus 5. _Nitrosomonas_ Winogradsky, 1892. - Type species, _Nitrosomonas europoea_ Winogradsky; oxidizes - ammonia or ammonium salts to nitrous acid, - hence nitrites. - Genus 6. _Nitrobacter_ Winogradsky, 1892. - Type species, _Nitrobacter Winogradskyi_ Committee of 1917; - oxidizes nitrous acid (nitrites) - to nitric acid (nitrates). - TRIBE II--AZOTOBACTEREÆ--prototrophic for N. - Genus 7. _Azotobacter_ Beijerinck, 1901; large, free-living, - aerobic N absorbers. - Type species, _Azotobacter chroococcum_ Beijerinck. - Genus 8. _Rhizobium_ Frank, 1889. - Type species, _Rhizobium leguminosarum_ Frank; root tubercle - bacteria of legumes. - FAMILY II--PSEUDOMONADACEÆ, Committee of 1917. - Genus 1. _Pseudomonas_ Migula, 1894. - Type species, _Pseudomonas violacea_ (Schroeter, 1872) Migula. - FAMILY III--SPIRILLACEÆ Migula, 1894--all spiral bacteria. - Genus 1. _Vibrio_ Müller, 1786, emended by E. F. Smith, 1905. - Type species, _Vibrio choleræ_ (Koch, 1884) Schroeter, 1886. - Genus 2. _Spirillum_ Ehrenberg, 1830, emended Migula, 1894. - Type species, _Spirillum undula_ (Müller, 1786) Ehrenberg. - FAMILY IV--COCCACEÆ Zopf, 1884, emended Migula, 1894--all cocci. - Tribe I--NEISSEREÆ. - Genus 1. _Neisseria_ Trevisan, 1885. - Type species, _Neisseria gonorrhoeae_ Trevisan. - Tribe II--STREPTOCOCCEÆ Trevisan, 1889. - Genus 2. _Diplococcus_ Weichselbaum, 1886. - Type species, _Diplococcus pneumoniae_ Weichselbaum. - Genus 3. _Leuconostoc_ Van Tieghem, 1878. - Type species, _Leuconostoc mesenterioides_ (Cienkowski) Van - Tieghem. - Genus 4. _Streptococcus_ Rosenbach, 1884; emended Winslow - and Rogers, 1905. - Type species, _Streptococcus pyogenes_ Rosenbach. - Tribe III--MICROCOCCEÆ Trevisan, 1889. - Genus 5. _Staphylococcus_ Rosenbach, 1884; animal parasites. - Type species, _Staphylococcus aureus_ Rosenbach. - Genus 6. _Micrococcus_ Cohn, 1872, emended Winslow and Rogers, - 1905. Facultative parasites or saprophytes. - Type species, _Micrococcus luteus_ (Schroeter, 1872) Cohn. - Genus 7. _Sarcina_ Goodsir, 1842, emended Winslow and - Rogers, 1905. - Type species, _Sarcina ventriculi_ Goodsir. - Genus 8. _Rhodococcus_ Zopf, 1891, emended Winslow and - Rogers, 1905; cocci with red pigment. - Type species, _Rhodococcus rhodochrous_ Zopf. - FAMILY V--BACTERIACEÆ Cohn, 1872, emended by Committee of 1917; - bacilli without spores not above included. - Tribe I--CHROMOBACTEREÆ Committee of 1919; producing red or - violet pigment, mainly water forms. - Genus 1. _Erythrobacillus_ Fortineau, 1905. - Type species, _Erythrobacillus prodigiosus_ - (Ehrenberg, 1848) Fortineau. - Genus 2. _Chromobacterium_ Bergonzini, 1881. - Type species, _Chromobacterium violaceum_ Bergonzini. - Tribe II--ERWINEÆ Committee 1919; plant pathogens. - Genus 3. _Erwinia_ Committee 1917. - Type species, _Erwinia amylovora_ (Burrill, 1883) Committee - 1917. - Tribe III--ZOPFEÆ Committee of 1919; Gram +, no pigment, - non-carbohydrate-fermenting. - Genus 4. _Zopfius_ Wenner and Rettger, 1919. - Type species, _Zopfius zopfii_ (Kurth) Wenner and Rettger. - Tribe IV--BACTEREÆ Committee of 1919; Gram -, carbohydrate - fermenters. - Genus 5. _Proteus_ Hauser, 1885; liquefy gelatin. - Type species, _Proteus vulgaris_ Hauser. - Genus 6. _Bacterium_ Ehrenberg, 1828, emended Jensen, 1909; - liquefy gelatin rarely. - Type species, _Bacterium coli_. - Tribe VI--LACTOBACILLEÆ Committee of 1919; Gram +, high acid, - thermophils. - Genus 7. _Lactobacillus_ Beijerinck, 1901. - Type species, _Lactobacillus caucasicus_ (Kern?) Beijerinck; - Bulgarian bacillus. - Tribe VI--PASTEURELLEÆ Committee of 1919; organisms of - hemorrhagic septicemia. - Genus 8. _Pasteurella_ Trevisan, 1888. - Type species, _Pasteurella cholerae-gallinarum_ - (Flügge, 1886); Trevisan. - Tribe VII--HEMOPHILEÆ Committee of 1917; require hemoglobin for - growth. - Genus 9. _Hemophilus_ Committee of 1917. - Type species, _Hemophilus influenzae_ (Pfeiffer, 1893) - Committee of 1917. - FAMILY VI--BACILLACEÆ Fischer, 1895. Spore forming rods. - Genus 1. _Bacillus_ Cohn, 1872; aerobic, no change of form - around the spore. - Type species, _Bacillus subtilis_ Cohn. - Genus 2. _Clostridium_ Prazmowski, 1880; anaërobic, frequently - enlarged around spore. - Type species, _Clostridium butyricum_ Prazmowski. - -As compared with Migula's classification it is to be noted that there -are 38 genera listed by the Committee instead of 13 in the same general -groups. - -The following list of _Genera conservanda_ submitted by the Committee -was formally adopted by the Society and these are therefore its -official names for the organisms included in these genera. - - _Acetobacter_ Fuhrman - _Actinomyces_ Harz - _Bacillus_ Cohn - _Bacterium_ Ehrenberg - _Chromobacterium_ Bergonzini - _Clostridium_ Prazmowski - _Erythrobacillus_ Fortineau - _Leptotrichia_ Trevisan - _Leuconostoc_ Van Tieghem - _Micrococcus_ Cohn - _Rhizobium_ Frank - _Sarcina_ Goodsir - _Spirillum_ Ehrenberg - _Staphylococcus_ Rosenbach - _Streptococcus_ Rosenbach - _Vibrio_ Müller - -_It is greatly to be desired that the Society's Classification when -finally completed shall become the standard in the United States at -least._ - -_Such names as have been adopted by the Society are used throughout -this work._ - -The Committee also submitted the following artificial key for -determining the genera in the two orders _ACTINOMYCETALES AND -EUBACTERIALES_: - - A--Typically filamentous forms _Actinomycetacae_ - B--Mycelium and conidia formed _Actinomyces_ - BB--No true mycelium - C--Cells show branching - D--Gram negative _Actinobacillus_ - DD--Gram positive _Erysipelothrix_ - CC--Cells never branch. Gram positive threads later fragmenting - into rods _Leptotrichia_ - AA--Typically unicellular forms (though chains of cells may occur) - B--Cells spherical--_COCCACEÆ_ - C--Parasitic forms (except Leuconostoc), cells generally grouped - in pairs or chains, never in packets, generally active - fermenters. - D--Cells in flattened coffee-bean-like pairs, gram -. - _Neisseria_ - DD--Not as D - E--Saprophytes in zoögloea masses in sugar solutions. - _Leuconostoc_ - EE--Not as E. Gram +. - F--Cells in lanceolate pairs or in chains. Growth on - media not abundant. - G--Cells in lanceolate pairs. Inulin generally fermented. - _Diplococcus_ - GG--Cells in chains. Inulin not generally fermented. - _Streptococcus_ - FF--Cells in irregular groups. Growth in media fairly - vigorous. White or orange pigment. - _Staphylococcus_ - CC--Saprophytic forms. Cells in irregular groups or packets, - not in chains. Fermentative powers low. - D--Packets _Sarcina_ - DD--No packets. - E--Yellow pigment _Micrococcus_ - EE--Red pigment _Rhodococcus_ - BB--Rods: - C--Spiral rods - D--Short, comma-like rods. One to three flagella. - _Vibrio_ - DD--Long spirals. Five to twenty flagella. _Spirillum_ - CC--Straight rods. - D--No endospores. - E--Rods of irregular shape or showing branched or filamentous - involution forms. - F--Cells irregular in shape. Staining unevenly. Animal - parasites. - G--Acid fast _Mycobacterium_ - GG--Not acid fast. - H--Cells elongated, fusiform _Fusiformis_ - HH--Cells not elongated, sometimes branching. - I--Gram positive. Slender, sometimes club-shaped. - _Corynebacterium_ - II--Gram negative. Rods sometimes form threads. - Characteristic honey-like growth on potato. - _Pfeifferella_ - FF--Cells staining unevenly but with branched or filamentous - forms at certain stages. Never acid fast. - Not animal parasites. - G--Metabolism simple, growth processes involving oxidation - of alcohol or fixation of free N (latter in symbiosis - with green plants). - H--Cells minute. Symbiotic in roots of legumes. - _Rhizobium_ - HH--Oxidizing alcohol. Branching forms common. - _Acetobacter_ - GG--Not as G. Proteus-like colonies. - H--Not attacking carbohydrates _Zopfius_ - HH--Fermenting glucose and sucrose at least. - _Proteus_ - EE--Regularly formed rods. - F--Metabolism simple, growth processes involving oxidation - of C, H, or their simple compounds or the fixation - of free N.--_NITROBACTERIACEÆ._ - G--Fixing N or oxidizing its simple compounds. - H--Fixing N, cells large, free in soil _Azotobacter_ - HH--Oxidizing N compounds. - I--Oxidizing NH{4} compounds _Nitrosomonas_ - II--Oxidizing nitrites _Nitrobacter_ - GG--Not as G. - H--Oxidizing free H _Hydrogenomonas_ - HH--Oxidizing simple C compounds, not free H. - I--Oxidizing CO _Carboxydomonas_ - II--Oxidizing CH{4} _Methanomonas_ - FF--Not as F. - G--Flagella usually present, polar--_PSEUDOMONADACEÆ_ - _Pseudomonas_ - GG--Flagella when present peritrichic--_BACTERIACEÆ_ - H--Parasitic forms showing bi-polar staining. - _Pasteurella_ - HH--Not as H. - I--Strict parasites growing only in presence - of hemoglobin - _Hemophilus_ - II--Not as I. - J--Water forms producing red or violet pigment. - K--Pigment red _Erythrobacillus_ - KK--Pigment violet _Chromobacterium_ - JJ--Not as J. - K--Plant pathogens _Erwinia_ - KK--Not plant pathogens. - L--Gram +, forming large amount of acid - from carbohydrates, sometimes CO{2}, - never H _Lactobacillus_ - LL--Gram -, forming H as well as CO{2} if - gas is produced _Bacterium_ - DD--Endospores present--_BACILLACEÆ_ - E--Aerobes, rods not swollen at sporulation. _Bacillus_ - EE--Anaërobes, rods swollen at sporulation. _Clostridium_ - - - - -PART II. - -PHYSIOLOGY. - - - - -CHAPTER VI. - -GENERAL CONDITIONS FOR GROWTH. - - -OCCURRENCE. - -Bacteria are probably the most widely distributed of living organisms. -They are found practically everywhere on the surface of the earth. -Likewise in all surface waters, in streams, lakes and the sea. They -occur in the air immediately above the surface, since they are carried -up mechanically by air currents. They cannot fly of themselves. There -is no reason to believe that any increase in numbers occurs to an -appreciable extent in the air. The upper air, for example, on high -mountains, is nearly free from them. So also is the air over midocean, -and in high latitudes. As a rule, the greater the amount of dust in -the air, the more numerous are the bacteria. Hence they are found more -abundantly in the air in cities and towns than in the open country. -The soil is especially rich in numbers in the upper few feet, but they -diminish rapidly below and almost disappear at depths of about six -feet unless the soil is very porous and open, when they may be carried -farther down. Hence the waters from deep wells and springs are usually -devoid of these organisms. In the sea they occur at all levels and have -been found in bottom ooze dredged from depths of several miles. It is -perhaps needless to add that they are found on the bodies and in the -alimentary tract of human beings and animals; on clothing, utensils; -in dwellings, stables, outhouses, etc. From one-fourth to one-half of -the dry weight of the feces of animals and men is due to the bacteria -present. The urine is practically free from them in health. - -While bacteria are thus found nearly everywhere, it is an entirely -mistaken idea to suppose that all are injurious to man. As a matter -of fact, those which are dangerous are relatively few and are for the -most part found only in close association with man. Most bacteria are -harmless and the vast majority are beneficial or even essential to -man's existence on the earth. These facts must be constantly borne in -mind, and it is hoped that the pages which follow will make them clear. - -In order that any organism may thrive there are a number of general -environmental conditions which must be fulfilled. These conditions -vary more or less for each kind of organism. Bacteria are no exception -to this general rule. These conditions may be conveniently considered -under the general heads of _moisture_; _temperature_; _light_; _oxygen -supply_; _osmotic pressure_; _action of electricity_; of _Röntgen_ -and _radium rays_; _pressure_; _mechanical vibration_; and _chemical -environment_, including the _reaction of the medium_, _the effect -of injurious chemicals_, and especially the _food requirements of -bacteria_. For each of these conditions there is a _maximum_, meaning -the greatest amount of the given condition which the organism can -withstand, a _minimum_, or the least amount, and an _optimum_ or that -amount which is most favorable for development. Further, there might be -distinguished a maximum for _mere existence_ and a lower maximum for -_development_; also a minimum for _mere existence_ and a higher minimum -for _development_. These maxima, minima, and optima for bacteria have -been determined with exactness for only a very few of the general -conditions and for comparatively few kinds. - - -MOISTURE. - -The _maximum_ moisture is absolutely pure water, and no organism can -thrive in this alone owing to the factor of too low osmotic pressure -and to the further factor of absence of food material. There are many -bacteria which thrive in water containing only traces of mineral salts -and a large class whose natural habitat is surface water. These "water -bacteria" are of great benefit in the purification of streams. They are -as a class harmless to men and animals. Some of the disease-producing -bacteria like _Bacterium typhosum_ (of typhoid fever) and _Vibrio -choleræ_ (of Asiatic cholera) were undoubtedly originally water -bacteria, and it is rather striking that in these diseases conditions -are induced in the intestine (diarrheas) which simulate the original -watery environment. The _minimum_ moisture condition is absolute -dryness, and no organism can even exist, not to say develop, in such -a condition since water is an essential constituent of living matter. -Some bacteria and especially most spores may live when dried in the -air or by artificial means for months and even years, while some are -destroyed in a few hours or days when dried (typhoid, cholera, etc.). -The optimum amount of moisture has not been determined with any great -accuracy and certainly a rather wide range in percentage of water is -permissible with many, though a liquid medium is usually most favorable -for artificial growth. The "water bacteria" have been mentioned. In the -soil a water content of 5 to 15 per cent. seems to be most suitable for -many of the organisms which aid in plant growth. In animals and man the -organisms infecting the intestinal tract prefer a high percentage of -moisture as a rule, especially those causing disease here. Those found -on the surface of the body (pus cocci) need a less amount of water, -while those invading the tissues (tuberculosis, black-leg, etc.) seem -to be intermediate in this respect. In artificial culture media a water -content of less than 30 per cent. inhibits the growth of most bacteria. - -As a general rule those bacteria which require the largest percentage -of water are most susceptible to its loss and are most readily killed -by drying. The typhoid and cholera organisms die in a few hours when -dried, while pus cocci and tubercle bacilli live much longer. - - -TEMPERATURE. - -The temperature conditions for bacterial existence and growth have been -determined more accurately than any of the other general conditions. -The maximum for existence must be placed at or near 100° since it is -known that all bacteria including spores may be killed by boiling in -time. Nevertheless, certain forms have been reported as thriving in hot -springs where the water temperature was 93°. This is the highest known -temperature for development. The minimum for existence lies at or near -the absolute zero (-273°) since certain organisms have been subjected -to the temperature produced by the sudden evaporation of liquid -hydrogen (-256° to -265°) and have remained alive. Whether they could -withstand such temperatures indefinitely is not known. The minimum for -development is near the freezing-point of water, since reproduction -by division has been observed in the water from melting sea-ice at -a temperature of -1.5°. Thus bacteria as a class have a range for -existence of about 373° (-273° to +100°) and for development of 94.5° -(-1.5° to +93°) certainly much wider ranges than any other group of -organisms.[5] - -The optimum temperature for development varies within rather wide -limits for different organisms. In general it may be stated that the -optimum temperature is approximately that of the natural habitat of -the organism, though there are exceptions. The optimum of the "hot -spring" bacteria just mentioned is apparently that of the springs (93° -in this case). Many soil organisms are known whose optimum is near -70° (a temperature rarely, if ever, attained in the soil), _but only -when grown in air or oxygen_; but is very much lower when grown in the -_absence of oxygen_. Many other soil organisms exhibit very little -difference in rate or amount of growth when grown at temperatures which -may vary as much as 10° or 15°, apparently an adaptation to their -normal environment. The disease-producing organisms show much narrower -limits for growth, especially those which are difficult to cultivate -outside the body. For example, the bacterium of tuberculosis in man -scarcely develops beyond the limits of 2° or 3° from the normal body -temperature of man (37°), while the bacterium of tuberculosis in birds -grows best at 41° to 45°, the normal for birds, and the bacterium of -so-called tuberculosis of cold-blooded animals at 14° to 18°. - -Those bacteria whose optimum temperature is above 40° are sometimes -spoken of as the "_thermophil_" bacteria. The fixing of the "thermal -death-point" that is, the minimum temperature at which the bacteria -are killed is a matter of great practical importance in many ways -and numerous determinations of this have been made with a great many -organisms and by different observers. The factors which enter into such -determinations are so many and so varied that unless all the conditions -of the experiment are given together with the time of application, -the mere statements are worthless. It may be stated that all _young, -actively growing_ (non-spore-containing) _disease-producing bacteria, -when exposed in watery liquids and in small quantities are killed at -a temperature of 60° within half an hour_. It is evident, that this -fact has very little practical application, since the conditions stated -are rarely, if ever, fulfilled except in laboratory experiments. (See -Sterilization and Pasteurization, Chapter XIII.) - - -LIGHT. - -Speaking generally, it can be said that light is destructive to -bacteria. Many growing forms are killed in a few hours when properly -exposed to direct sunlight and die out in several days in the diffuse -daylight of a well-lighted room. Even spores are destroyed in a -similar manner, though the exposure must be considerably longer. -Certain bacteria which produce colors may grow in the light, since -the pigments protect them. Some few kinds, like the sulphur bacteria, -which contain a purplish-red pigment that serves them to break up -H{2}S, need light for their growth. Since disease-producing bacteria -are all injuriously affected by light, the advantage of well-lighted -habitations both for men and animals is obvious. - - -OXYGEN SUPPLY. - -Oxygen is one of the constituents of protoplasm and is therefore -necessary for all organisms. This does not mean that all organisms -must obtain their supply from _free oxygen_, however, as animals and -plants generally do. This fact is well illustrated by the differences -among bacteria in this respect. Some bacteria _require free oxygen_ for -their growth and are therefore called _aërobic_ bacteria or _aërobes_ -(sometimes _strict aërobes_, though the adjective is unnecessary). -Others _cannot grow in the presence of free oxygen_ and are therefore -named _anaërobic bacteria_ or _anaërobes_ (strict is unnecessary). -There are still other kinds which may grow either in the presence of -free oxygen or in its absence, hence the term _facultative anaërobes_ -(usually) is applied to them. The distinction between _facultative -aërobe_ and _facultative anaërobe_ might be made. The former means -those which grow best in the absence of free oxygen, though capable of -growing in its presence, while the latter term means those which grow -best in the presence of free oxygen, but are capable of growing in its -absence. The amount of oxygen in the atmosphere in which an organism -grows may be conveniently expressed in terms of the oxygen pressure, -_i.e._, in millimeters of mercury. It is evident that the maximum, -minimum and optimum oxygen pressures for anaërobic bacteria are the -same, namely, 0 mm. Hg. This is true only for natural conditions, -since a number of anaërobic organisms have been gradually accustomed -to increasing amounts of O, so that by this process of training they -finally grew in ordinary air, that is, at an oxygen pressure of about -150 mm. Hg. (Normal air pressure is 760 mm. Hg. and oxygen makes up -one-fifth of the air.) The minimum O pressure for facultative anaërobes -is also 0 mm. Hg. Some experiments have been made to determine the -limits for aërobes, but on a few organisms only, so that no general -conclusions can be drawn from them. To illustrate: _Bacillus subtilis_ -(a common "hay bacillus") will grow at 10 mm. Hg. pressure but not at 5 -mm. Hg. It will also grow in compressed oxygen at a pressure of three -atmospheres (2280 mm. Hg.), but not at four atmospheres (3040 mm. Hg.), -though it is not destroyed. - -Parodko has determined the oxygen limits for five common organisms as -follows: - - Minimum - Maximum. Vol. Mm. - In atmospheres. Mm. Hg. per cent. Hg. - - _Bacterium 1.94 to 2.51 1474 to 1908 0.00016 = 0.0012 - fluorescens_ - _Sarcina lutea_ 2.51 to 3.18 1908 to 2417 0.00015 = 0.0011 - _Proteus vulgaris_ 3.63 to 4.35 2749 to 3306 0 0 - _Bacterium coli_ 4.09 to 4.84 3108 to 3478 0 0 - _Erythrobacillus 5.45 to 6.32 3152 to 4800 0 0 - prodigiosus_ - -These few instances do not disclose any general principles which may -be applied either for the growth or for the distinction of aërobes or -facultative anaërobes. - -It has been shown that compressed oxygen will kill some bacteria but -this method of destroying them has little or no practical value. Oxygen -in the form of ozone, O{3}, is rapidly destructive to bacteria, and this -fact is applied practically in the purification of water supplies for -certain cities where the ozone is generated by electricity obtained -cheaply from water power. The same is true of oxygen in the "nascent -state" as illustrated by the use of hypochlorites for the same purpose. - -It was stated (p. 74) that certain thermophil bacteria in the soil have -an optimum temperature for growth _in the air_ which is much higher -than is ever reached in their natural habitat and that they grow at a -moderate temperature under _anaërobic_ conditions. It has been shown -that if these organisms are grown with aërobes or facultative anaërobes -they thrive at ordinary room temperature. These latter organisms by -using up the oxygen apparently keep the tension low, and this explains -how such organisms grow in the soil.[6] - - -OSMOTIC PRESSURE. - -Like all living cells bacteria are very susceptible to changes in -the density of the surrounding medium. If placed in a medium less -concentrated than their own protoplasm water is absorbed and they -"swell up"; while if placed in a denser medium, water is given off and -they shrink (plasmoptysis or plasmolysis). Should these differences -be marked or the transition be sudden, the cell walls may even burst -and the organisms be destroyed. If the differences are not too great -or if the transition is made gradually, the organisms may not be -destroyed, but will either cease to grow and slowly die out, or will -show very much retarded growth, or will produce abnormal cell forms. -This is illustrated in the laboratory in attempting to grow bacteria on -food material which has dried out. A practical application of osmotic -effects is in the use of a high percentage of sugar in preserving -fruits, etc., and in the salting of meats. Neither the cane-sugar nor -the common salt themselves injure the bacteria chemically, but by the -high concentration prevent their development. In drying material in -order to preserve it there are two factors involved: first, the loss of -water necessary for growth and second, the increased osmotic pressure. - -In a medium of greater density diffusion of water is outward from the -cell and this will continue until an equilibrium is established between -cell contents and medium. Food for the organism _must be in solution -and enter the cell by diffusion_. Therefore, growth ceases in a medium -too dense, since water to carry food in solution does not enter the -cell. - - -ELECTRICITY. - -Careful experimenters have shown that the electric current, either -direct or alternating, has no direct destructive effect on bacteria. -In a liquid medium the organisms may be attracted to or repelled -from one or the other pole or may arrange themselves in definite -ways between the poles (galvanotaxis), but are not injured. However, -electricity through the _secondary_ effects produced, may be used to -destroy bacteria. If the passage of the electric current _increases the -temperature_ of the medium sufficiently, the bacteria will be killed, -or if _injurious chemical substances_ are formed (ozone, chlorine, -acids, bases, etc.), the same result will follow (see Ozone, pp. 77 and -157). - - -RADIATIONS. - -Röntgen or _x_-rays and radium emanations when properly applied to -bacteria will destroy them. The practical use of these agents for -the direct destruction of bacteria in diseases of man or animals is -restricted to those cases where they may be applied directly to the -diseased area, since they are just as injurious to the animal cell -as they are to the bacteria, and even more so. Their skilful use as -_stimuli to the body cells_ to enable them to resist and overcome -bacteria and other injurious organisms or cell growths is an entirely -different function and will not be considered here. - - -PRESSURE. - -Hydrostatic pressure up to about 10,000 pounds per square inch is -without appreciable effect on bacteria as has been shown by several -experimenters and also by finding living bacteria in the ooze dredged -from the bottom of the ocean at depths of several miles. - -Pressures from 10,000 to 100,000 pounds show variable effects. Some -bacteria are readily killed and others, even non-spore formers, -are only slightly affected. The time factor is important in this -connection. The presence of acids, even CO{2}, or organic acids, results -in the destruction of most non-spore formers. - - -MECHANICAL VIBRATION. - -Vibrations transmitted to bacteria in a liquid may be injurious to them -under certain circumstances. Some of the larger forms like _Bacillus -subtilis_ may be completely destroyed by shaking in a rapidly moving -shaking machine in a few hours. Bacteria in liquids placed on portions -of machinery where only a slight trembling is felt, have been found -to be killed after several days. Reinke has shown that the passing of -strong sound waves through bacterial growths markedly inhibits their -development. - - - - -CHAPTER VII. - -CHEMICAL ENVIRONMENT. - - -REACTION OF MEDIUM. - -Most bacteria are very susceptible to changes in the degree of acidity -or alkalinity of the medium in which they grow. Some kinds prefer a -slightly acid reaction, some a slightly alkaline, and some a neutral -(with reference to litmus as indicator). The organism which is the -commonest cause of the souring of milk thrives so well in the acid -medium it produces that it crowds out practically all other kinds, -though its own growth is eventually stopped by too much acid. Acid -soils are usually low in numbers of bacteria and as a consequence -produce poor crops. The disease-producing bacteria as a class grow best -in a medium which is slightly alkaline. - -Accurate determination of limits have been made on but few organisms. -The reaction is a most important factor in growing bacteria on -artificial media (see Making of Media, Chapter XVI). - - -INJURIOUS CHEMICAL SUBSTANCES. - -(SEE DISINFECTION AND DISINFECTANTS, Chapter XIII.) - - -CHEMICAL COMPOSITION. - -The chemical composition is subject to wide variation chiefly for -two reasons: First, the cell wall in most instances seems to exert -only a slight selective action in the absorption of mineral salts -so that their concentration within the cell is very nearly that of -the surrounding medium. Second, the chief organic constitutents vary -remarkably with the kind and amount of food material available--a -rich protein pabulum increases the protein, a plentiful supply of -carbohydrates or of fat results in the storing of more fat, especially -and _vice versa_. These facts must be borne in mind in considering the -chemistry of bacteria. - -Of the chemical elements known, only the following seem to be essential -in the structure of bacteria: carbon, hydrogen, oxygen, nitrogen, -sulphur, phosphorus, chlorine, potassium, calcium, magnesium, iron, -manganese. Other elements, as sodium, iodine, silicon, aluminum, -lithium, copper, etc., have been reported by different analysts, but -none of them can be regarded as essential, except possibly in isolated -instances. - -These elements exist in the bacterial cell in a great variety of -combinations of which the most abundant is _water_. The amount of water -varies in different species from 75 to 90 per cent. of the total weight -in growing cells, and is less in spores. The amount of _ash_ has been -shown by different observers to vary from less than 2 per cent. to as -much as 30 per cent. of the _dry weight_. The following table compiled -from various sources will give an idea of the relative abundance of the -different elements in the ash. - - S as SO{3} 7.64 per cent. (much more in sulphur bacteria) - P as P{2}O{5} 18.14 " to 73.94 per cent. - Cl 2.29 " - K as K{2}O 11.1 " to 25.59 " - Ca as CaO 12.64 " to 14.0 " - Mg as MgO 0.7 " to 11.55 " - Fe as Fe{2}O{3} 1.0 " to 8.15 " (iron bacteria) - Mn traces - -As to the form in which the last six elements in the table exist in -the cell, little is known. The sulphur and phosphorus are essential -constituents of various proteins. The high percentage of phosphorus -points to nuclein compounds as its probable source. - -The carbon and nitrogen, together with most of the hydrogen and oxygen -not united as water, make up the great variety of organic compounds -which compose the main substances in the bacterial cell. - -It has already been stated that the essential structures in the -bacterial cell are cell wall and protoplasm, including the nuclein. -These differ markedly in chemical composition. It is well known that -the cell walls of green plants consist largely of cellulose and closely -related substances.[7] _True cellulose_ has been recognized in but -very few bacteria. (_Sarcina ventriculi_, Migula; _Mycobacterium -tuberculosis_, Hammerschlag, Dreyfuss, Nishimura; _Bacillus -subtilis_, Dreyfuss; _Acetobacter xylinum_, Brown; _Acetobacter -acidi oxalici_, Banning; and a few others.) It is certainly not an -important constituent of the cell wall in many. On the other hand, -_hemicellulose_ and _gum-like_ substances have been identified in -numerous organisms of this class as important constituents of the cell -wall and of the capsule which is probably an outgrowth from the latter. -Practically always associated with these substances are compounds -containing nitrogen. One of these has been certainly identified as -_chitin_ or a closely similar substance. Chitin is the nitrogenous -substance which enters largely into the composition of the hard parts -of insects, spiders and crustaceans. It is an interesting fact to find -this substance characteristic of these animals in bacteria, as well as -other fungi. - -Though it is extremely difficult to separate the cell wall of bacteria -from the cell contents, in the light of our present knowledge it can be -stated that the cell walls are composed of a carbohydrate body closely -related to cellulose, though not true cellulose, probably in close -combination with chitin. - -Of the organic constituents of the cell contents the most abundant are -various proteins which ordinarily make up about one-half of the dry -weight of the entire cell. The "Mycoproteid" of Nencki, 1879, and other -earlier workers is deserving of little more than historical interest, -since these substances were certainly very impure and probably -consisted of mixtures of several "proteins" in the more recent sense. - -From later studies it seems probable that substances resembling -the albumin of higher forms do not occur in bacteria, at least in -appreciable quantities. Globulin has been reported by Hellmich in an -undetermined bacterium, but is certainly not commonly found. The larger -portion of the protein is of a comparatively simple type, in fact, -consists of protamins most of which are in combination with nucleic -acid as nucleoprotamins. Practically all recent workers find a high -percentage of nuclein, both actually isolated and as indicated by the -amounts of purin bases--xanthin, guanin, adenin--obtained, as well as -by the abundance of phosphorus in the ash, already mentioned. Some of -these nucleins have been shown to have poisonous properties. - -Closely related to but not identical with the proteins are the enzymes -and toxins which are formed in the cell and exist there as endo-enzymes -or endo-toxins respectively. These substances will be discussed later -under the heading "Physiological Activities of Bacteria" (Chapter XII). - -Carbohydrates are not commonly present in the cell contents, though -glycogen has been observed in a few and a substance staining blue -with iodine in one or two others. This latter substance was at first -considered to be starch "granulose," but is probably more closely -related to glycogen. - -Fats seem to be very generally present. The commoner fats--tri-olein, -tri-palmitin, tri-stearin have been found by many analysts. The -"acid-fast bacteria" are particularly rich in fatty substances, -especially the higher wax-like fats. Lecithins (phosphorized fats) and -cholesterins (not fats but alcohols) have been repeatedly observed and -probably occur in all bacteria as products of katabolism. - -Organic acids and esters occur as cell constituents but will be -discussed in connection with their more characteristic occurrences -as products of bacterial activity, as will also pigments which may -likewise be intracellular in some instances. - -The following analysis of tubercle bacilli, from de Schweinitz and -Dorset, while not intended as typical for all bacteria, still -illustrates the high percentage of protein compounds which undoubtedly -occurs in most, as well as showing the large amount of fatty substance -in a typical "acid-fast" organism: - - { 8.5 per cent. tuberculinic acid - { 24.5 " nucleoprotamin } - In the dried { 23.0 " neucleoprotein } 55.8 per cent. - organisms { 8.3 " proteinoid } protein. - { 26.5 " fat and wax - { 9.2 " ash - - - - -CHAPTER VIII. - -CHEMICAL ENVIRONMENT (CONTINUED). - - -GENERAL FOOD RELATIONSHIPS. METABOLISM. - -The foregoing brief review of the chemical composition of the bacterial -cell illustrates the variety of compounds which necessarily occurs, -but affords no definite clue as to the source of the elements which -enter into these compounds. These elements come from the material -which the organism uses as food. Under this term are included elements -or compounds which serve as building material, either for new cell -substance or to repair waste, or as sources of energy. - -An organism which is capable of making use of an element in the free -state is said to be _prototrophic_ for that particular element. -Thus aërobes and facultative anaërobes are prototrophic for O. The -"root-tubercle bacteria" of leguminous and other plants and certain -free living soil organisms are prototrophic for N.[8] - -On the other hand, if the element must be secured from compounds, then -the organism is _metatrophic_ in respect to the element in question. -Should the compound be inorganic, the term _autotrophic_ is applied -to the organism and _heterotrophic_ if the compound is organic. It is -very probable that anaërobes, exclusive of a few nitrogen absorbers, -are metatrophic for all the elements they utilize. With the exception -of the anaërobes it seems that all bacteria are _mixotrophic_, that is, -prototrophic for one or two elements and auto- or heterotrophic for the -others.[9] - -Those bacteria whose food consists of dead material are spoken of as -_saprophytes_, while those whose natural habitat, without reference to -their food, is in or on other living organisms are called _parasites_. -The _host_ is the organism in or on which the parasite lives. -Parasites may be of several kinds. Those which neither do injury nor -are of benefit to the host are called _non-pathogenic_ parasites or -_commensals_; many of the bacteria in the intestines of man and other -animals are of this class. Those which do injury to the host are -called _pathogenic_ or disease-producing, as the organisms causing the -transmissible diseases of animals and plants.[10] Finally, we have -those parasites which are of benefit to and receive benefit from the -host. These are called _symbionts_ or _symbiotic parasites_ and the -mutual relationship _symbiosis_. Certain of the intestinal bacteria in -man and especially in herbivorous animals are undoubted _symbionts_, as -are also the "root-tubercle bacteria" already mentioned. - -It is evident that all parasites that may be cultivated outside -the body are for the time _saprophytic_, hence the terms _strict_ -parasites and _facultative_ parasites, which should require no further -explanation. - -The changes which the above-mentioned types of food material undergo -in the various anabolic and katabolic processes _within the cell_ are -as yet but very slightly known. Nevertheless there are a number of -reactions brought about by bacteria acting on various food materials, -partly within _but largely without the cell_ which are usually -described as "physiological activities" or "biochemical reactions." -Some of these changes are to be ascribed to the utilization of certain -of the elements and compounds in these materials as tissue builders, -some as energy-yielding reactions and still others as giving rise to -substances that are of direct benefit to the organism concerned in its -competition with other organisms. - -Though all of the twelve elements already mentioned are essential for -the growth of every bacterium, two of them are of especial importance -for the reason that most of the "physiological activities" to be -described in the next chapters are centered around their acquisition -and utilization. These elements are _carbon_ and _nitrogen_. Some few -of the special activities of certain groups have to do with one or the -other of the remaining nine, as will be shown later. But generally -speaking _when a bacterium under natural conditions secures an adequate -supply of carbon and nitrogen, the other elements are readily available -in sufficient amount_. - -Carbon is necessary not only because it is an essential constituent -of protoplasm but because its oxidation is the chief source of the -energy necessary for the internal life of the cell, though nitrogen -and sulphur replace it in this function with a few forms. This -latter use of carbon constitutes what may be called its _respiratory -function_. Bacteria like other organisms in their respiration utilize -oxygen and give off carbon dioxide. The amount of the latter given off -from the cell in this way is very small as compared with that which -is frequently produced as an accompaniment of other reactions (see -Fermentation, next chapter). But there is no doubt of its formation and -it has been determined by a few investigators. On account of this use -of carbon, bacteria require relatively large amounts of this element. -One group of bacteria concerned in the spontaneous heating of coal -seems to be able to use free carbon from this material. Another group -is said to be able to oxidize marsh gas, CH{4}, and use this as its -source of carbon. The nitrite, nitrate and sulphur bacteria mentioned -later utilize carbon dioxide and carbonates as their carbon supply, -and one kind has been described which uses carbon monoxide. With these -few exceptions bacteria are dependent on _organic compounds_ for their -carbon and cannot use CO{2} as green plants do. - -The oxygen requirement is high partly for the same reason that -carbon is, _i.e._, respiration. Oxygen is one of the constituents of -protoplasm, and combined with hydrogen forms water which makes up such -a large part of the living cell. Anaërobic bacteria are dependent on -so-called "molecular respiration" for their energy. That is, through -a shifting or rearrangement of the atoms in the compounds used as -food the oxidation of carbon is brought about. Enzymes are probably -responsible for this action. Carbon dioxide is produced by anaërobes -as well as by aërobes, and frequently in amounts readily collected. A -carbohydrate is usually though not always essential for the growth of -anaërobes and serves them as the best source of energy. - -Nitrogen is the characteristic element of living material. Protoplasm -is a chemical substance in unstable equilibrium and nitrogen is -responsible for this instability. No other of the commoner elements -is brought into combination with such difficulty, nor is so readily -liberated when combined (all commercial explosives are nitrogen -compounds). Bacteria, like other forms of protoplasm, require nitrogen. -More marked peculiarities are shown by bacteria with reference to the -sources from which they derive their nitrogen than for carbon. Some can -even combine the free nitrogen of the air and furnish the only natural -means of any importance for this reaction. Some few forms (the nitrite -and nitrate formers, Chapter XI) obtain their energy from the oxidation -of inorganic nitrogen compounds, ammonia and nitrites respectively, and -not from carbon. These latter bacteria use carbon from carbon dioxide -and carbonates. A great many bacteria can secure their nitrogen from -nitrates but some are restricted to organic nitrogen. Many bacteria -obtain their carbon from the same organic compounds from which their -nitrogen is derived. - -Sulphur serves mainly as a constituent of protein compounds in the -protoplasmic structure. In some of the _sulphur_ bacteria it is a -source of energy, since either free sulphur or H{2}S is oxidized by -them. Some of these bacteria can obtain their carbon from CO{2} or -carbonates, and their nitrogen from nitrates or ammonium salts. - -Whether the _iron_ bacteria, belonging to the genus _Crenothrix_ of the -higher, thread bacteria, use this element or its compounds as sources -of energy is still a disputed question. The evidence is largely in -favor of this view. - -Free hydrogen has been shown to be oxidized by some forms which obtain -their energy in this way. - -Whether there is a special class of _phosphorus_ bacteria remains to be -discovered. That phosphorus is oxidized during the activity of many -bacteria is undoubted, but whether this represents a source of energy -or is the accidental by-product of other activities is undetermined. - -Practically nothing is known about the metabolism of the other elements -as such. - -From the preceding brief review of the relation of certain bacteria -to some of the elements in the free state and from the further fact -that there is scarcely a known natural organic compound which cannot -be utilized by some kind of bacterium, it is evident that this class -of organisms has a far wider range of adaptability than any other -class, and this adaptability helps to explain their seemingly universal -distribution. - -As to the metabolism _within the cell_, no more is known than is the -case with other cells, nor even as much. The materials used for growth -and as sources of energy are taken into the cell, built up into various -compounds some of which have been enumerated and in part broken down -again. Carbon dioxide and water are formed in the latter process. What -other katabolic products occur it is not easy to determine. Certainly -some of the substances mentioned in the next chapters are such products -but it is not always possible to separate those formed _inside_ the -cell from those formed _outside_. Perhaps most of the latter should be -considered true metabolic products. It would seem that on account of -the simplicity of structure of the bacterial cell and of the compounds -which they may use as food they would serve as excellent objects -for the study of the fundamental problems of cell metabolism. Their -minuteness and the nearly impossible task of separating them completely -from the medium in or on which they are grown makes the solution of -these problems one of great difficulty. - -When all of the environmental conditions necessary for the best -development of a given bacterium are fulfilled, it will then develop -to the limit of its capacity. This development is characterized -essentially by its reproduction, which occurs by transverse division. -The rate of this division varies much with the kind even under good -conditions. The most rapid rate so far observed is a division in -eighteen minutes. A great many reproduce every half-hour and this may -be taken as a good average rate. If such division could proceed without -interruption, a little calculation will show that in about sixty-five -hours a mass as large as the earth would be produced. - - Starting with 1 coccus, 1µ in diameter, - its volume = 0.0000000000005 cc. - - 1/2 hour = 2 - 1 hour = 4 - 2 hours = 16 - 4 hours = 256 - 5 hours = 1024 = 10^{3}+ - 15 hours = 1,000,000,000 = 10^{9} = 0.5 cc. - 35 hours = 10^{21}+ = 500.0 cu.m. - About 65 hours = 2 × 10^{42}+ = 5 × 10^{20} cu.m. = a mass as large - as the earth. - -Such a rate of increase evidently cannot be kept up long on account of -many limiting factors, chief of which is the food supply. - -The foregoing calculation is based on the assumption that the organism -divides in one plane only. If it divides in 2 or 3 planes, the rate is -much faster, as is shown by the following formulæ, which indicate the -theoretical rate of division: - - S = number of bacteria after a given number of divisions. - a = number at the beginning, and n = number of divisions. - 1 plane division S = 2^{_n_}a - 2 " " S = 2^{2_n_}a - 3 " " S = 2^{3_n_}a - -With two-plane or three-plane division, assuming that each organism -attains full size, as was assumed in the first calculation, the "mass -as large as the earth" would be attained in about thirty-two and -twenty-two hours respectively. - -This extraordinary rate of increase explains in large measure why -bacteria are able to bring about such great chemical changes in so -short a time as is seen in the rapid "spoiling" of food materials, -especially liquids. The reactions brought about by bacteria on -substances which are soluble and diffusible are essentially "surface -reactions." The material diffuses into the cell over its entire surface -with little hindrance. The bacteria are usually distributed throughout -the medium, so that there is very intimate contact in all parts of -the mass which favors rapid chemical action. The following calculation -illustrates this: - - The volume of a coccus 1µ in diameter is 0.5236 × 10^{-13} cc. - The surface of a coccus 1µ in diameter is [pi] × 10^{-8} sq. cm. - -It is not uncommon to find in milk on the point of souring -1,000,000,000 bacteria per cc. - -Assuming these to be cocci of 1µ diameter the volume of these bacteria -in a liter is only 0.05 cc. or in the liter there would be 19999 parts -of milk and only 1 part bacteria. The surface area of these bacteria is -3141.6 sq. cm. With this large surface exposed, it is not strange that -the change from "on the point of souring" to "sour" occurs within an -hour or less. - -Although large numbers of bacteria can and do cause great chemical -changes the amount of material actually utilized for maintenance of -the cell is very slight, infinitesimal almost, and yet is fairly -comparable to that required for man, as is illustrated by the following -computations: - -E. Kohn has shown that certain water bacteria grew well in water to -which there was added per liter 0.000002 mg. dextrose, 0.00000007 mg. -(NH{4}){2}SO{4} and 0.0000000007 mg. (NH{4}){2}HPO{4}. The bacteria -numbered about 1000 per cc. Taking the specific gravity at 1 (a little -too low) the mass of the bacteria in the liter was about 0.001 mg. -Hence the bacteria used 0.002 of their weight of carbohydrate and -0.00007 of ammonium sulphate. A 150-pound (75-kilo) man can live on 375 -g. of sugar (0.005 of his weight) and 52.5 g. of protein (0.0007 of his -weight). From these figures it can be calculated that the man utilizes -about two and a half times as much carbohydrate and about seven times -as much nitrogen as the bacterium, relatively speaking. - - - - -CHAPTER IX. - -PHYSIOLOGICAL ACTIVITIES. - - -The physiological activities of motion, reproduction and metabolism -within the cell have been discussed in previous chapters. The -objects in view in the discussion of the "physiological activities" -(sometimes spoken of as "biochemical" activities) of bacteria in -this and subsequent chapters are to familiarize the student to some -extent with the great range of chemical changes brought about by these -minute organisms, to show their usefulness, even their necessity, and -to impress the fact that it is chiefly by a careful study of these -"activities" that individual kinds of bacteria are identified. It -should always be borne in mind that the bacteria, in bringing about -these changes which are so characteristic in many instances, are simply -engaged in their own life struggle, in securing the elements which -they need for growth, in liberating energy for vital processes, or -occasionally in providing conditions which favor their own development -and hinder that of their competitors. - - -FERMENTATION OF CARBOHYDRATES. - -By this is meant the changes which different carbohydrates undergo when -subjected to bacterial action.[11] - -These changes are marked chiefly by the production of gas or acid. The -former is called "gaseous fermentation" the latter "acid fermentation." -The gases commonly produced are carbon dioxide (CO{2}) hydrogen and -marsh gas (CH{4}). Other gases of the paraffin series may also be -formed as ethane (C{2}H{6}), acetylene (C{2}H{2}), etc. CO{2} and -H are the ones usually formed from sugars by the few gas-forming -bacteria which produce disease, though even here some CH{4} is present. -The common _Bacterium coli_ forms all three, though the CH{4} is in -smallest quantity. - -[Illustration: FIG. 60.--Cylinder to show the formation of gas by -bacteria. The gauge shows 265 pounds. It went beyond 500 pounds.] - -[Illustration: FIG. 61.--A burning natural gas well at night. From a -photograph colored.] - -In the fermentation of the polysaccharids--starch and especially -cellulose and woody material--large amounts of CH{4} occur, particularly -when the changes are due to anaërobic bacteria. This phenomenon may be -readily observed in sluggish streams, ponds and swamps where vegetable -matter accumulates on the bottom. The bubbles of gas which arise when -the mass is disturbed explode if a lighted match is applied to them. - -The author has conducted a number of experiments to demonstrate this -action as follows: Material taken from the bottom of a pond in the -fall after vegetation had died out was packed into a cylinder five -feet long and six inches in diameter, water was added to within about -2 inches of the top. After leaving them open for a few days to permit -all the dissolved oxygen to be used up by the aërobes, the cylinders -were tightly capped and allowed to stand undisturbed. Pressure gauges -reading to 500 lbs. were attached (Fig. 60). At the end of six months -the gauge showed a pressure beyond the limits of the readings on it. -Most of the gas was collected and measured 146 liters. An analysis -of portions collected when about one-half had been allowed to escape -showed the following composition, according to Prof. D. J. Demorest of -the Department of Metallurgy: - - CO{2} 18.6 per cent. - CH{4} 76.1 " - H 1.0 " - N 4.3 " - -In the author's opinion natural gas and petroleum have been formed in -this way[12] (Figs. 61 and 62). - -[Illustration: FIG. 62.--A "flowing" oil well.] - -One of the very few practical uses of the gaseous fermentation of -carbohydrates is in making "salt rising" bread. The "rising" of the -material is due not to yeasts but to the formation of gas by certain -bacteria which are present on the corn meal or flour used in the -process (Fig. 63). - -[Illustration: FIG. 63.--A loaf of "salt rising" bread. The porous -structure is due to the gas formed by bacilli and not by yeasts.] - -Another is in the formation of the "holes" or "eyes" so characteristic -of Swiss and other types of cheese (Fig. 64). - -[Illustration: FIG. 64.--Ohio Swiss cheese. The "eyes" are due to gas -formed by bacteria during the ripening of the cheese.] - -A great many organic acids are formed during the "acid fermentation" -of carbohydrates by bacteria. Each kind of bacterium, as a rule, forms -several different acids as well as other substances, though usually one -is produced in much larger amounts, and the kind of fermentation is -named from this acid. One of the commonest of these acids is lactic. -The "lactic acid bacteria" form a very large and important group and -are indispensable in many commercial processes. In the making of butter -the cream is first "ripened," as is the milk from which many kinds of -cheese are made (Fig. 65). The chief feature of this "ripening" is the -formation of lactic acid from the milk-sugar by the action of bacteria. -A similar change occurs in the popular "Bulgarian fermented milk." The -reaction is usually represented by the equation: - - Milk-sugar. Lactic acid. - C{12}H{22}O{11} + H{2}O + (bacteria) = 4C{3}H{6}O{3} - -It is not probable that the change occurs quantitatively as indicated, -because a number of other substances are also formed. Some of these -are acetic and succinic acids and alcohol. Another industrial use of -this acid fermentation is in the preparation of "sauer kraut." These -bacteria are chiefly anaërobic and grow best in a relatively high salt -concentration. They occur naturally on the cabbage leaves. - -[Illustration: FIG. 65.--A cream ripener. In this apparatus cream is -"ripened," _i.e._, undergoes lactic acid fermentation, preparatory to -making it into butter.] - -In the formation of ensilage (Fig. 66) the lactic acid bacteria play -a very important part, as they do also in "sour mash" distilling, and -in many kinds of natural "pickling." In fact, whenever green vegetable -material "sours" spontaneously, lactic acid bacteria are always present -and account for a large part of the acid. This property of lactic acid -formation is also taken advantage of in the preparation of lactic acid -on a commercial scale in at least one plant in this country. - -[Illustration: FIG. 66.--Filling a silo on the University farm.] - -Acetic acid is another common product of acid fermentation. However, in -vinegar making the acetic acid is not formed directly from the sugar in -the fruit juice by bacteria. The sugar is first converted into alcohol -by yeasts, then the alcohol is _oxidized_ to acid by the bacteria (Fig. -67). The reaction may be represented as follows: - - Dextrose. Ethyl alcohol. Acetic acid. - C{6}H{12}O{6} = 2C{2}H{5}OH + 2CO{2} - - C{2}H{5}OH + O{2} + (bacteria) = CH{3}COOH + H{2}O. - -Butyric acid is generally produced where fermentation of carbohydrates -occurs under _anaërobic_ conditions. Some of the "strong" odor of -certain kinds of cheese is due to this acid which is formed partly -from the milk-sugar remaining in the cheese. Most of it under these -conditions comes from the proteins of the cheese and especially from -the fat (see page 101). - -As has been indicated alcohol is a common accompaniment of most acid -fermentations, as are the esters of acids other than the chief product. -Bacteria are not used in a commercial way to produce alcohol, however, -as the yield is too small. There are some few bacteria in which the -amount of alcohol is prominent enough to call the process an "alcoholic -fermentation" rather than an acid one. In brewing and distilling -industries, _yeasts_ are used to make the alcohol, though molds replace -them in some countries ("sake" and "arrak" from rice). - -[Illustration: FIG. 67.--A vinegar ripener. The tank shown opened at -the side is filled with a special type of beech shavings which thus -provide a very large surface. The apple juice which has been previously -fermented with yeast, which converts the sugar into alcohol, is allowed -to trickle through the openings at the top over the shavings. The -acetic acid bacteria on the shavings rapidly oxidize the alcohol to -acetic acid. The vinegar is drawn off below.] - -Under ordinary conditions the carbohydrate is never completely -fermented, since the accumulation of the product--acid--stops -the reaction. If the acid is neutralized by the addition of an -alkali--calcium or magnesium carbonate is best--then the sugar -may all be split up. Where such fermentation occurs under natural -conditions, the products are further split up, partly by molds and -partly by acid-destroying bacteria into simpler acids and eventually -to carbon dioxide and water, so that the end-products of the complete -fermentation of carbohydrate material in nature are carbon dioxide, -hydrogen, marsh gas, and water. - -In all of these fermentations the bacteria are utilizing the _carbon_ -both as building material and for oxidation and the fermentations -are incidental to this use. As a rule, the acid-forming bacteria can -withstand a higher concentration of acid than the other bacteria that -would utilize the same material, and in a short time crowd out their -competitors or inhibit their growth, and thus have better conditions -for their own existence, though finally their growth is also checked by -the acid. - - -SPLITTING OF FATS. - -The _splitting of fats_ into glycerin and the particular acid or -acids involved may be brought about by bacteria. An illustration -is the development of rancidity in butter at times and the -"strong" odor of animal fats on long keeping and of many kinds of -cheese--"limburger"--in this country. Generally speaking, however, fats -are not vigorously attacked, as is illustrated by the difficulties due -to accumulation of fats in certain types of sewage-disposal works. The -chemical change is represented by the equation: - - Fat. Glycerin. - C{3}H{5}(C{_n_}H{2}{_n_-1}O{2}){3} + 3 H{2}O = C{3}H{5}(OH){3} - Fatty acid. - + 3 (C{_n_}H{2}{_n_}O{2}). - - - - -CHAPTER X. - -PHYSIOLOGICAL ACTIVITIES (CONTINUED). - - -PUTREFACTION OF PROTEINS. - -The word "_putrefaction_" is now restricted to the action of bacteria -on the _complex nitrogen-containing substances_, proteins, and their -immediate derivatives. The process is usually accompanied by the -development of foul odors. - -Bacteria make use of proteins chiefly as a source of nitrogen, but also -as a source of carbon and other elements. Proteins contain nitrogen, -carbon, hydrogen, oxygen, sulphur and frequently phosphorus. Some of -the metals--potassium, sodium, calcium, magnesium, iron and manganese -and the non-metal chlorine--are nearly always associated with them more -or less intimately. Since these bodies are the most complex of natural -chemical substances it follows that the breaking up of the molecule to -secure a part of the nitrogen gives rise to a great variety of products. - -There are marked differences among bacteria in their ability to -attack this class of compounds. Some can break up the most complex -natural proteins such as albumins, globulins, glyco-, chromo-, and -nucleoproteins, nucleins and albuminoid derivatives like gelatin. The -term _saprogenic_ (#sapros# = rotten) is sometimes applied to bacteria -which have this power. These proteins are large-moleculed and not -diffusible, so that the first splitting up that they undergo must occur -outside the bacterial cell. The products of this first splitting may -diffuse into the cell and be utilized there. The bacteria of this class -attack not only these proteins in the natural state or in solution, -but also in the coagulated state. The coagulum becomes softened and -finally changed into a liquid condition. The process when applied to -the casein of milk is usually called "digestion," also when coagulated -blood serum is acted on. In the latter case the serum is more commonly -said to be "liquefied" as is the case when gelatin is the substance -changed. Most of these bacteria have also the property of coagulating -or curdling milk in an alkaline medium, and then digesting the curd. -A second class of bacteria has no effect on the complex proteins just -mentioned but readily attacks the products of their first splitting, -_i.e._, the proteoses, peptones, polypeptids and amino-acids. They are -sometimes called _saprophilic_ bacteria. - -Other bacteria derive their nitrogen from some of the products of the -first two groups, and still further break down the complex protein -molecule. Under normal conditions these various kinds of bacteria -all occur together and thus mutually assist one another in what is -equivalent to a symbiosis or rather a metabiosis, a "successive -existence," one set living on the products of the other. The result -is the complete splitting up of the complete protein molecule. A part -of the nitrogen is built up into the bodies of the bacteria which are -using it as food. A part is finally liberated as _free nitrogen_ or as -_ammonia_ after having undergone a series of transformations many of -which are still undetermined. - -One class of compounds formed received at one time much attention -because they were supposed to be responsible for a great deal of -illness. These are the "ptomaines," basic nitrogen compounds of -definite composition--amines--some few of which are poisonous, most of -them not. The basic character of ptomaines may be understood if they be -regarded as made up of one or more molecules of ammonia in which the -hydrogen has been replaced by alkyl or other radicals. Thus ammonia -(NH{3}) may be represented as - - /H - / - N--H - \ - \H. - -The simplest ptomaine is - - /CH{3} - / - N--H - \ - \H, - -in which one H is replaced by methyl, methylamine, a gaseous ptomaine. -With two hydrogens replaced by methyl, - - /CH{3} - / - N--CH{3} - \ - \H, - -dimethylamine, also a gas at ordinary temperature, is formed. -Trimethylamine, - - /CH{3} - / - N--CH{3} - \ - \CH{3}, - -a liquid, results when three hydrogens are similarly replaced. -All three of these occur in herring brine and are responsible -for the characteristic odor of this material. Putrescin and -cadaverin--tetramethylene--diamine, and pentamethylenediamine -respectively--occur generally in decomposing flesh, hence the names. -They are only slightly poisonous. One of the highly poisonous ptomaines -is neurin C{5}H{13}NO or C{2}H{3}N(CH{3}){3}OH = trimethyl-vinyl -ammonium hydroxide. This is a stronger base than ammonia, liberating -it from its salts. Numerous other ptomaines have been isolated and -described. These bodies were considered for a long time to be the -cause of various kinds of "meat poisoning," "ice cream poisoning," -"cheese poisoning," etc. It is true that they may sometimes cause these -conditions, but they are very much rarer than the laity generally -believe. Most of the "meat poisonings" in America are due, not to -ptomaines, but to infections with certain bacilli of the _Bacterium -enteritidis_ group. Occasionally a case of poisoning by the true toxin -(see Chapter XII) of _Clostridium botulinum_ occurs, and in recent -years has become entirely too common due to insufficient heating of -canned goods. _The boiling of such material will destroy this toxin. -The safest rule to follow is not to eat any canned material that shows -any departure from the normal in flavor, taste or consistency._ - -As ptomaines result from the putrefaction of proteins, so they are -still further decomposed by bacteria and eventually the nitrogen is -liberated either as free nitrogen or as ammonia. - -Another series of products are the so-called aromatic compounds--phenol -(carbolic acid), various cresols, also indol and skatol or methyl indol -(these two are largely responsible for the characteristic odor of human -feces). All of these nitrogen compounds are attacked by bacteria and -the nitrogen is eventually liberated, so far as it is not locked up in -the bodies of the bacteria, as free nitrogen or as ammonia. - -The carbon which occurs in proteins accompanies the nitrogen in many of -the above products, but also appears in nitrogen-free organic acids, -aldehydes and alcohols which are all eventually split up, so that the -carbon is changed to carbon dioxide or in the absence of oxygen partly -to marsh gas. - -The intermediate changes which the sulphur in proteins undergoes are -not known, but it is liberated as sulphuretted hydrogen (H{2}S) or as -various mercaptans (all foul-smelling), or is partially oxidized to -sulphuric acid. Some of the H{2}S and the sulphur of the mercaptans -are oxidized by the sulphur bacteria to free sulphur and finally to -sulphuric acid. - -Phosphorus is present especially in the nucleoproteins and nucleins. -Just what the intermediate stages are, on whether there are any, so -far as the phosphorus is concerned, in the splitting up of nucleic -acid by bacterial action is not determined. The phosphorus may occur -as phosphoric acid in such decompositions, or when the conditions are -anaërobic, as phosphine (PH{3}), which burns spontaneously in the air to -phosphorus pentoxide (P{2}O{5}), and water.[13] - -The hydrogen in proteins appears in the forms above indicated: H{4}C, -H{3}N, H{3}P, H{2}S, H{2}O and as free H. The oxygen as CO{2} and H{2}O. - -In the breaking down of the complex protein molecule even by a single -kind of bacterium there is not a perfect descending scale of complexity -as might be supposed from the statement that there result proteoses, -peptones, polypeptids, amino-acids. These substances do result, but at -the time of their formation simpler ones are formed also, even CO{2}, -NH{3} and H{2}S. It appears that the entire molecule is shattered in -such a way that less complex proteins are formed from the major part, -while a minor portion breaks up completely to the simplest combinations -possible. A more complete knowledge of these decompositions will aid -in the further unravelling of the structure of proteins. The presence -or absence of free oxygen makes a difference in the end-products, as -has been indicated. There are bacteria which oxidize the ammonia to -nitric acid and the H{2}S to sulphuric acid. (See Oxidation, Chapter -XI.) Bacteria which directly oxidize phosphorus compounds to phosphoric -acid have not been described. It does not seem that such are necessary -since this is either split off from nucleic acid or results from the -spontaneous oxidation of phosphine when this is formed under anaërobic -conditions. - -Not only are proteins decomposed as above outlined, but also their -waste products, that is, the form in which their nitrogen leaves the -animal body. This is largely urea in mammals, with much hippuric acid -in herbivorous animals and uric acid in birds and reptiles. These -substances yield NH{3}, CO{2} and H{2}O with a variety of organic acids -as intermediate products in some cases. The strong odor of ammonia -in stables and about manure piles is the everyday evidence of this -decomposition. - -Where the putrefaction of proteins occurs in the soil with moderate -amounts of moisture and free access of air a large part of the products -is retained in the soil. Thus the ammonia and carbon dioxide in the -presence of water form ammonium carbonate; the nitric, sulphuric and -phosphoric acids unite with some of the metals which are always present -to form salts. Some of the gases do escape and most where the oxygen -supply is least, since they are not oxidized. - -The protein-splitting reactions afford valuable tests in aiding in -the recognition of bacteria. In the study of pathogenic bacteria the -coagulation and digestion of milk, the digestion or liquefaction -of blood serum, the liquefaction of gelatin and the production of -indol and H{2}S are those usually tested for. In dairy bacteriology -the coagulation of milk and the digestion of the casein are common -phenomena. Most bacteria which liquefy gelatin also digest blood serum -and coagulate and digest milk, though there are exceptions. In soil -bacteriology the whole range of protein changes is of the greatest -importance. - -[Illustration: - - +----<----------------------+ - | | - | | - v | - _Nuclein of | - animal cells_ | - | | - | | - v | - | | - _Decomposition | - bacteria_ | - | \ | - | \ ^ - v \ | - _Unknown _PH{3} | - P oxidizes | - compounds_ spontaneously | - | to_ | - | / | - v / | - _Phosphoric | - acid_ | - | | - | | - v | - _Phosphates | - in the | - soil_ | - | ^ - | | - v | - _Green | - plants_ | - | | - | | - v | - _Nuclein of | - plant | - cells_ | - | | - | | - v | - _Animals_----->----------------+ - -FIG. 68.--Diagram to illustrate the circulation of phosphorus through -the agency of bacteria.] - -[Illustration: - - _Fats and - various C <---------------------------+ - compounds_ | - | | - | | - v | - _Decomposition | - bacteria_ | - | | - | | - v | - _Various | - C | - compounds | - eventually ^ - to_ | - | | - | | - v | - _CO{2} <---------------+ | - in | | - the air_<-+ | | - | | | | - | | _Plant respiration_ | - v | | | - _Green | | | - plants_---+ | | - | | | - | | | - v | | - _Carbohydrates, | | - fats and | | - other C compounds_ | | - | | | - | | | - v | | - _Animals_---->_Animal respiration_ | - | | - | | - +---->----------------------------+ - -FIG. 69.--Diagram to illustrate the circulation of carbon through the -agency of bacteria.] - -[Illustration: - - +---------------<-----------------+ - | | - _Dead animal | - protein_ | - | ^ - | | - v | - _Free N taken <-----_Decomposition <-------------+ | - up by free living bacteria_ <----------+ | | - N absorbers and | | | | - root tubercle | | | | - bacteria_ v | | | - | _NH{3} compounds | | | - | in soil_ | | | - | | | | | - | | | | | - | v | | | - | _Nitrite bacteria_ | | | - | | | | | - | | | | | - | v | | | - | _Nitrites_ | | | - | | | | | - | | | | | - | v | | | - | _Nitrate bacteria_ | | | - | | ^ ^ ^ - | | | | | - | v | | | - | _Nitrates in soil_ | | | - | | | | | - | | | | | - | v | | | - | _Green plants_ | | | - | | | | | - | | | | | - | v _Dead plant | | - +------->_Plant protein_----> protein_ | | - | | ^ - | | | - v _Animal waste, | - _Animals_---------->urea, etc._ | - | | - | | - +-------->------------------------+ - -FIG. 70.--Diagram to illustrate the circulation of nitrogen through the -agency of bacteria.] - -[Illustration: - - +--------------<---------------+ - | | - | | - v | - _Dead animal | - protein_ | - | | - | | - v | - _Decomposition bacteria_ | - | | | - | | | - v ^ | - _H{2}S_ | | - | | | - | | | - v | | - _Sulphur bacteria_ | | - | | | - | | | - v | | - _Free S_ | ^ - | | | - | | | - v | | - _Sulphur bacteria_ | | - | | | - | | | - v | | - _Sulphates | | - in the | | - soil_ | | - | | | - | | | - v | | - _Green plants_ ^ | - | | | - | | | - v | | - _Plant protein_----->_Dead plant | - | protein_ | - | | - v | - _Animals_ | - | | - | | - +-------------->---------------+ - -FIG. 71.--Diagram to illustrate the circulation of sulphur through the -agency of bacteria.] - -The three physiological activities already discussed explain how -bacteria break down the chief complex, energy-rich substances ---carbohydrates, fats and proteins which constitute the bulk of the -organic material in the bodies of plants and animals, as well as -the waste products of the latter--into energy-free compounds like -carbon dioxide, water, ammonia, nitric, sulphuric and phosphoric -acids--mineralize them, as is frequently said. By so doing the bacteria -act as the great scavengers of nature removing the dead animal -and vegetable matter of all kinds which but for this action would -accumulate to such an extent that all life, both on land and in the -water, must cease. It is further to be noted that not only is all this -dead organic matter removed; but it is converted into forms which are -again available for plant growth. Carbon dioxide forms the source of -the carbon in all green plants, hence in all animals; the sulphates and -phosphates are likewise taken up by green plants and built up again -into protein compounds; the ammonia is not directly available to green -plants to any large extent but is converted by the nitrifying bacteria -(Chapter XI) into nitrates which is the form in which nitrogen is -assimilated by these higher types. Even the free nitrogen of the air -is taken up by several kinds of bacteria, the symbiotic "root-tubercle -bacteria" of leguminous and other plants, and some free-living forms, -and made available. Hence bacteria are indispensable in nature, -especially in keeping up the circulation of nitrogen. They are also of -great service in the circulation of carbon, sulphur and phosphorus. -Though some few kinds cause disease in man and animals, if it were not -for the saprophytic bacteria above outlined, there could be no animals -and higher plants to acquire these diseases. - - - - -CHAPTER XI. - -PHYSIOLOGICAL ACTIVITIES (CONTINUED). - - -PRODUCTION OF ACIDS. - -The production of organic acids has been sufficiently discussed in -preceding chapters. It should be noted that not only these in great -variety are produced by bacteria but that under certain conditions -mineral acids, such as nitric, sulphuric and phosphoric may be formed -(see Oxidation, p. 114). Acid production is of great value in the -identification of bacteria in dairy and soil work and in connection -with certain types of pathogenic bacteria. - - -GAS PRODUCTION. - -It will be sufficient merely to enumerate collectively the various -gases mentioned in preceding paragraphs and to state that those -commonly observed in the study of pathogenic bacteria are the first six -mentioned. Most of them come in in dairy work either in the study of -bacteria causing milk and cheese "failures" or as affecting the flavors -of butter or cheese. In the study of soil organisms, any or all of -them are liable to be of importance. The gases are: CO{2}, H, CH{4}, -N, NH{3}, H{2}S, gaseous mercaptans, gaseous ptomaines, volatile fatty -acids, ethereal salts or esters and others, both of pleasant and of -foul odor, but of unknown composition. - - -PRODUCTION OF ESTERS. - -The production of esters, as mentioned in Chapters IX and X, of various -alcohols and aldehydes are activities which are sometimes of value in -the study of bacteria, but need not be further discussed. - - -PRODUCTION OF "AROMATIC" COMPOUNDS. - -These have been mentioned in discussing the putrefaction of proteins, -as indol, skatol, phenol and various cresols. Of these only the first -is ordinarily tested for in the study of bacteria, though others of the -group become of value in certain special cases. - -[Illustration: FIG. 72.--Culture of phosphorescent bacteria in an -Ehrlenmeyer flask photographed by their own light. Time of exposure -twelve hours. (Molisch, from Lafar.)] - - -PHOSPHORESCENCE OR PHOTOGENESIS. - -This is a most interesting phenomenon associated with the growth of -some bacteria. The "fox fire" frequently seen on decaying wood which -is covered with a slimy deposit is most commonly due to bacteria, -though also to other fungi. Phosphorescent bacteria are very common -in sea water, hence they are frequently found on various sea foods, -especially when these are allowed to decompose, such as fish, oysters, -clams, etc. The light is due to the conversion of the energy of -unknown easily oxidizable compounds directly into _visible_ radiant -energy through oxidation without appreciable quantities of heat. -The light produced may be sufficient to tell the time on a watch in -absolute darkness, and also to photograph the growths with their own -light, but only after several hours' exposure (Fig. 72). None of the -phosphorescent bacteria so far discovered produce disease in the higher -animals or man. - - -PRODUCTION OF PIGMENT OR CHROMOGENESIS. - -One of the most striking results of bacterial activity is this -phenomenon. The particular color which results may be almost any one -throughout the range of the spectrum, though shades of yellow and of -red are of more frequent occurrence. - -In the red sulphur bacteria the "bacteriopurpurin" which they contain -appears to serve as a true respiratory pigment in a manner similar to -the chlorophyl in green plants, except that these bacteria oxidize -H{2}S in the light as a source of energy instead of splitting up CO{2}. -The red pigment produced by certain bacteria has been shown to have -a capacity for combining with O resembling that of hemoglobin, and -some investigators have believed that such bacteria do store O in this -way for use when the supply is diminished. With these few exceptions -the pigments seem to be merely by-products of cell activity which are -colored and have no known function. - -The red sulphur bacteria above mentioned and one or two other kinds -retain the pigments formed within the cell. Such bacteria are called -_chromophoric_ as distinguished from the _chromoparic_ bacteria whose -pigment lies outside the cell. - -The chemical composition of no bacterial pigment has been determined -up to the present. Some are soluble in water, as shown by the -discoloration of the substances on which they grow. Others are not -soluble in water but are in alcohol, or in some of the fat solvents -as ether, chloroform, benzol, etc. These latter are probably closely -related to the _lipochromes_ or "fat colors" of higher plants and -animals. Attempts have been made to render the production of pigments -a still more reliable means of identification of species of bacteria -through a careful examination of the spectra of their solutions, but -such study has not as yet led to any valuable practical results. - -The production of pigment depends on the same general factors which -determine the growth of the organism but does _not necessarily run -parallel_ with these. It is especially influenced by the oxygen -supply (only a very few organisms are known which produce pigment -anaërobically--_Spirillum rubrum_ is one); by the presence of -certain food substances (starch, as in potato, for many bacteria -producing yellow and red colors; certain mineral salts, as -phosphates and sulphates, for others); by the temperature (many -bacteria cease to produce color at all if grown at body temperature, -37°--_Erythrobacillus prodigiosus_--or if grown for a longer time at -temperatures a few degrees higher). - - -REDUCING ACTIONS. - -Reduction of nitrates to nitrites or to ammonia or even to free -nitrogen is brought about by a great many different kinds of bacteria. -In many instances this phenomenon is due to a lack of free oxygen, -which is obtained by the bacteria from these easily reducible salts. -In other cases a portion of the nitrogen is removed to be used as food -material in the building up of new protein in the bacterial cell. This -latter use of the nitrogen of nitrates by bacteria might theoretically -result in considerable loss of "available nitrogen" in the soil as has -actually been shown in a few experiments. The reduction of nitrates as -above mentioned would also diminish this supply, but probably neither -of these results has any very great practical effect on soil fertility. -The building up of protein from these mineral salts by bacteria in the -intestines of herbivorous animals has been suggested by Armsby as a -considerable source of nitrogenous food, and this suggestion appears -possible. - -The liberation of nitrogen from nitrates or nitrites, either as free -nitrogen or as ammonia, is spoken of as "dentrification," though this -term was formerly applied to such liberations, from compounds of -nitrogen generally even from proteins. - -Certain bacteria may also reduce sulphates and other sulphur compounds -to H{2}S, a phenomenon frequently observed in sewage and likewise of -importance in the soil. It is possible that phosphates may be similarly -reduced.[14] Further and more careful study of the reducing actions of -bacteria is needed. - - -OXIDATION. - -As has been stated in discussing the respiration of bacteria (Chapter -VIII) most of these organisms gain their energy through the oxidation -of carbon in various forms, chiefly organic, so that CO{2} is a product -of the activity of nearly all bacteria. Some few oxidize CO to CO{2}, -others CH{4} and other paraffins to CO{2} for this purpose. One class of -bacteria even oxidizes H in small amounts for its energy and uses the -carbon dioxide of the air or traces of organic carbon in the air as a -source of carbon for "building" purposes. - -One of the familiar oxidations of organic carbon is that of the acetic -acid bacteria in the making of vinegar. These oxidize the alcohol -which results from the action of yeast to acetic acid according to the -formula CH{3}CH{2}OH + O{2} = CH{3}COOH + H{2}O (see Fig. 67). - -Of the various phenomena of oxidation due to bacteria, the formation of -nitrites and nitrates has the greatest practical importance, since it -is by this means that the ammonia which results from the decomposition -of animal and vegetable tissue and waste products is again rendered -available to green plants as food in the form of nitrates. Practically -all the nitrates found in nature, sometimes in large quantities, are -formed in this way. There are two distinct kinds of bacteria involved. -One, the nitrous bacteria, oxidizes the ammonia to nitrous acid -which forms nitrites with bases, and the other, the nitric bacteria, -oxidizes the nitrous to nitric acid, giving nitrates with bases. A -striking peculiarity of these two classes of organisms is that they -may live entirely on inorganic food materials, are proto-autotrophic, -prototrophic for oxygen (aërobic) and autotrophic for the other -elements. Their carbon is derived from CO{2} or carbonates. The -importance of such organisms in keeping up the supply of nitrates in -the soil can scarcely be overestimated. - -[Illustration: FIG. 73.--Sprinkling filters of the Columbus -sewage-disposal plant--devices which provide a good supply of oxygen -for the bacteria that oxidize the organic matter in the sewage.] - -The oxidation of the H{2}S, which is formed in the putrefaction -of proteins, to free S by the sulphur bacteria and the further -oxidation of this free S to sulphuric acid, and of the phosphorus, so -characteristic of the nucleins, to phosphoric acid have been referred -to. These activities of bacteria are of great value in the soil. -Doubtless the commercial "phosphate rock" owes its origin to similar -bacterial action in ages past. - -The oxidation of H{2}S to free S may be an explanation of the origin of -the great deposits of sulphur which are found in Louisiana and along -the Gulf coast. These deposits occur in the same general regions as -natural gas and oil. The sulphur might have been derived from the same -organic material carried down by the Mississippi which yielded the oil -and gas.[15] - -A purposeful utilization of the oxidizing power of bacteria is in -"contact beds," "sprinkling filters" and "aërated sludge tanks" in -sewage disposal works. In these instances the sewage is thoroughly -mixed with air and brought in contact with large amounts of porous -material so as to expose an extensive surface for oxidation (Fig. 73). - -[Illustration: FIG. 74.--One of the University hot beds.] - - -PRODUCTION OF HEAT. - -A direct result of the oxidizing action of bacteria is the production -of heat. Under most conditions of bacterial growth this heat is not -appreciable. It may become well marked. The "heating" of manure is one -of the commonest illustrations. The temperature in such cases may reach -70°. The heating of hay and other green materials is due chiefly to -bacterial action. This heating may lead to "spontaneous combustion." -The high temperatures (60° to 70°) favor the growth of thermophil -bacteria which cause a still further rise. The heat dries out the -material, portions of which are in a state of very fine division due -to the disintegrating action of the organisms. The hot, dry, finely -divided material oxidizes so rapidly on contact with the air that it -ignites. - -A practical use of heat production by bacteria is in the making of "hot -beds" for forcing vegetables (Fig. 74). - - -ABSORPTION OF FREE NITROGEN. - -[Illustration: FIG. 75.--Root tubercles on soy bean. × 3/7.] - -This is likewise one of the most important practical activities of -certain types of bacteria present in the soil. The ability of plants -of the legume family to enrich the soil has been known and taken -advantage of for centuries, but it is only about thirty years since -it was demonstrated that this property is due to bacteria. These -plants, and several other kinds as well, have on their roots larger or -smaller nodules (Fig. 75) spoken of as "root tubercles" which are at -certain stages filled with bacteria. When conditions are favorable, -these bacteria live in symbiotic relationship with the plant tissues, -receiving carbonaceous and other food material from them and in return -furnishing nitrogenous compounds to the plant. This nitrogenous -material is built up from free nitrogen absorbed from the air by the -bacteria. The utilization of this peculiar property through the proper -cultivation of clover, alfalfa, soy beans and other legumes is one of -the best ways of building up and maintaining soil fertility in so far -as the nitrogen is concerned. The technical name of these bacteria is -_Rhizobium leguminosarum_. - -[Illustration: FIG. 76.--Free-living nitrogen absorbing bacteria -"Azotobacter." Note their large size as compared with other bacteria -shown in this book.] - -There are also types of "free-living," as distinguished from these -symbiotic, bacteria which absorb the free nitrogen of the air and aid -materially in keeping up this supply under natural conditions. One -of the most important of these types is the aërobic "Azotobacter" -(Fig. 76), while another is the anaërobic _Clostridium pasteurianum_. -The nitrogen which is absorbed is built up into the protein material -of the cell body and this latter must in all probability be "worked -over" by various types of decomposition bacteria and by the nitrous -and nitric organisms and be converted into utilizable nitrates just -as other protein material is, as has been discussed in Chapter X. At -any rate there is as yet no definite knowledge of any other method of -transformation. Up to the present no intentional practical utilization -of this valuable property of these free-living forms has been made. - -=Nitrogen Nutrition of Green Plants.=--It is the belief of botanists -that green plants obtain their nitrogen chiefly in the form of -nitrates, though ammonium salts may be utilized to some extent by -certain plants at least. Exceptions to this general rule are those -plants provided with root tubercles (and the bog plants and others -which have mycorrhiza?). These plants obtain their nitrogen in the -form of organic compounds made for them by the bacteria growing in -the tubercles. That nitrogen circulates throughout the structure of -plants in organic combination is certain. There does not appear to -be any reason why similar compounds which are soluble and diffusible -(amino-acids?) should not be taken up through the roots of plants and -utilized as such. _It seems to the author that this is very probably -the case._ Arguments in favor of this view are: (1) The nitrogen -nutrition of leguminous and other plants with root nodules. (2) The -close symbiosis between "Azotobacter" and similar nitrogen-absorbing -bacteria and many species of algæ in sea water at least. (3) The -vigorous growth of plants in soils very rich in organic matter, which -inhibits the production of nitrates by the nitrous-nitric bacteria -when grown in culture, and possibly (?) in the soil, so that nitrates -may not account for the vigorous growth. (4) The effect of nitrate -fertilizers is to add an amount of nitrogen to the crop much in excess -of the amount added as nitrate. (5) The most fertile soils contain -the largest numbers of bacteria. The doctrine that nitrates furnish -the only nitrogen to plants was established before the activities of -bacteria in the soil were suspected, and, so far as the author is -aware, has not been supported by experiments under conditions rigidly -controlled as to sterility. - -It would seem that one of the chief functions of soil bacteria is to -prepare soluble organic compounds of nitrogen for the use of green -plants and thus to make a "short cut" in the nitrogen cycle (p. 107), -as now believed in, direct from the "decomposition bacteria" to green -plants. - -Experiments have been made by different observers in growing seedling -plants of various kinds in water culture with one or in some cases -several of the amino-acids as sources of nitrogen. Most of these -experiments were disappointing. Plant proteins are not so different -from animal proteins, or plant protoplasm (apart from the chlorophyl -portions of plants) from animal protoplasm as to lead one to suppose -that it could be built up from one or two amino-acids any more than -animal protoplasm can. The author is strongly convinced that this -subject should be thoroughly investigated. It will require careful -experimentation and perhaps rather large funds to provide the amounts -of amino-acids that would probably be needed, but might result in a -decided change in our ideas of soil fertility, and especially in the -use of nitrogen fertilizers. - - - - -CHAPTER XII. - -PHYSIOLOGICAL ACTIVITIES (CONTINUED). - - -PRODUCTION OF ENZYMES. - -Most of the physiological activities of bacteria which have been -discussed are due to the action of these peculiar substances, so that -a knowledge of their properties is essential. This knowledge cannot as -yet be exact because no enzyme has, up to the present, been obtained in -a "pure state," though it must be admitted that there are no certain -criteria which will enable this "pure state" to be recognized. It was -formerly thought that they were protein in nature, but very "pure" and -active enzymes have been prepared which did not give the characteristic -protein reactions, so this idea must be abandoned. That they are large -moleculed colloidal substances closely related to the proteins in many -respects must still be maintained. There are certain characteristics -which belong to enzymes, though no one of them exclusively. These may -be enumerated as follows: - -1. Enzymes are _dead_ organic chemical substances. - -_Dead_ is used in the sense of non-living, never having lived, not in -the sense of "ceased to be alive." - -2. They are always produced by _living_ cells: - -Sometimes as active enzymes, sometimes as _pro-enzymes_ or _zymogens_ -which are converted into enzymes outside the cell by acids, other -inorganic substances or other enzymes. - -3. They produce very great chemical changes without themselves being -appreciably affected. - -Enzymes will not continue to act indefinitely, but are used up in the -process (combination with products?). The amount of change is so great -in proportion to the amount of enzyme that the above statement is -justified in the relative sense. Thus a milk-curdling enzyme has been -prepared that would precipitate 100,000,000 times its own weight of -caseinogen. - -4. Their action is specific in that each enzyme acts on one kind of -chemical substance only, and the products are always the same. - -The substance may be combined with a variety of other chemical -substances so that the action appears to be on several, but in reality -it is on a definite group of molecules in each instance. For example, -emulsin attacks several different glucosides but always sets free -dextrose from them. - -5. The action is inhibited and eventually stopped, and in some cases -the enzyme is destroyed by an accumulation of the products of the -action. If the products are removed, the action will continue, if the -enzyme is not destroyed. This effect is explained partly because the -enzyme probably combines with some of the products, since it does -not act indefinitely, and partly because of the reversibility of the -reaction. - -6. Like many chemical reactions those of enzymes are reversible, that -is, the substance broken up may be reformed by it from the products -produced in many instances. Thus: - - maltose + maltase <-- glucose + glucose + maltase. - --> - fat + lipase <-- glycerin + fatty acid + lipase. - --> - -7. The presence of certain mineral salts seems to be essential for -their action. These and other substances which are necessary are -sometimes called _co-enzymes_. A salt of calcium is most favorable for -a great many. - -8. They may be adsorbed like other colloids by "shaking out" with -finely divided suspensions like charcoal or kaolin, or by other -colloids like aluminum hydroxide or proteins. - -9. When properly introduced into the tissues or blood of an animal, -they cause the body cells to form _anti-enzymes_ which will prevent the -action of the enzyme (see Chapter XXVII). - -10. Though inert, they show many of the characteristics of living -organisms, that is - -(_a_) Each enzyme has an optimum, a maximum and a minimum temperature -for its action. - -All chemical reactions have such temperature limits, the distinction -is that for enzymes as for living substance the _range_ is relatively -narrow. - -(_b_) High temperatures destroy enzymes. All in water are destroyed -by boiling in time and most at temperatures considerably below the -boiling-point. When dry, many will withstand a higher degree of heat -than 100° before they are destroyed. - -(_c_) Temperatures below the minimum stop their action, though they are -not destroyed by cold. - -(_d_) Many poisons and chemical disinfectants (Chapter XIV) which kill -living organisms will also stop the action of enzymes, though generally -more of the substance is required, so that it is possible to destroy -the living cells by such means and yet the action of the enzyme will -continue. - -(_e_) Most enzymes have an optimum reaction of medium either acid, -alkaline or neutral, depending on the particular enzyme, though some -few seem to act equally well within a considerable range on either side -of the neutral point. - -_The final test for an enzyme is the chemical change it brings about in -the specific substance acted on._ - -The most prominent characteristic of enzymes is that they bring about -very great chemical changes without themselves being appreciably -affected. This property is also shown by many inorganic substances -which are spoken of as "catalytic agents" or "catalyzers" so that -enzymes are sometimes called "organic catalyzers." The function of -catalytic agents seems to be to hasten the rate of a reaction which -would occur spontaneously, though in a great many cases with extreme -slowness. - -Just how enzymes act is not certain and probably will not be until -their composition and constitution are known. Most probably they form a -combination with the substance acted on (_the substrate_) as a result -of which there is a rearrangement of the atoms in such a way that new -compounds are formed, nearly always at least two, and the enzyme is at -the same time set free. It is rather remarkable that chiefly optically -active substances are split up by enzymes and where two modifications -exist it is usually the dextro-rotatory one which is attacked. No -single enzyme attacks both. This probably means that the structure of -the enzyme corresponds to that of the substrate, "fits it as a key fits -a lock," as Emil Fischer says. - -The production of enzymes is by no means restricted to bacteria since -all kinds of living cells that have been investigated have been shown -to produce them and presumably _all_ living cells do. Hence the -number of different kinds of enzymes and of substances acted upon -is practically unlimited. Nevertheless they may be grouped into a -comparatively few classes based on the general character of the change -brought about by them. - -I. Class I is the so-called _"splitting" enzymes_ whose action is -for the most part hydrolytic, that is, the substance takes up water -and then splits into compounds that were apparently constituents of -the original molecule. As examples may be mentioned _diastase_, the -enzyme first discovered, which changes starch into a malt-sugar, hence -is more commonly called _amylase_[16] (starch-splitting enzyme); -_invertase_,[16] which splits cane-sugar into dextrose and levulose: -C{12}H{22}O{11} + H{2}O = C{6}H{12}O{6} + C{6}H{12}O{6}. _Lipase_[16] -or a fat-splitting enzyme, which decomposes fat into glycerin and fatty -acid: - - C{3}H{5}(OC{n}H{2}{n-1}O){3} + 3H{2}O = C{3}H{5}(OH){3} - Fat Glycerin - - + 3C{n}H{2}{n}O{2}. - Fatty acid - -_Proteases_, which split up proteins into proteoses and peptones. - -Other classes of "splitting enzymes" break up the products of complex -protein decomposition, such as proteoses, peptones and amino-acids. A -variety of the "splitting enzymes" is the group of - -_"Coagulases" or coagulating enzymes_ as the rennet (lab, chymosin) -which curdles milk; fibrin ferment (thrombin, thrombase) which causes -the coagulation of blood. These apparently act by splitting up a -substance in the fluids mentioned, after which splitting one of the -new products formed combines with other compounds present (usually a -mineral salt, and in the cases mentioned a calcium salt) to form an -insoluble compound, the curd or coagulum. - -_Another variety is the "activating" enzymes or "kinases"_ such as the -enterokinase of the intestine. The action here is a splitting of the -_zymogen_ or mother substance or form in which the enzyme is built up -by the cell so as to liberate the active enzyme. - -Of a character quite distinct, from the splitting enzymes are - -II. The _zymases_. Their action seems to be to cause a "shifting on -rearrangement of the carbon atoms" so that new compounds are formed -which are not assumed to have been constituents of the original -molecule. Most commonly there is a closer combination of the carbon -and oxygen atoms, frequently even the formation of CO{2} so that -considerable energy is thus liberated. Examples are the _zymase_ or -_alcoholase_ of yeast which converts sugar into alcohol and carbon -dioxide; C{6}H{12}O{6} = 2C{2}H{6}O + 2CO{2}: also _urease_, which -causes the change of urea into ammonia and carbon dioxide. Another -common zymase is the _lactacidase_ in lactic acid fermentation. - -III. _Oxidizing enzymes_ also play an important part in many of the -activities of higher plants and animals. Among the bacteria this action -is illustrated by the formation of nitrites, nitrates and sulphates and -the oxidation of alcohol to acetic acid as already described. - -IV. _Reducing enzymes_ occur in many of the dentrifying bacteria and -in those which liberate H{2}S from sulphates. A very widely distributed -reducing enzyme is "catalase" which decomposes hydrogen peroxide. - -As previously stated, most of the physiological activities of -bacteria are due to the enzymes that they produce. It is evident -that for action to occur on substances which do not diffuse into the -bacterial cell--starches, cellulose, complex proteins, gelatin--the -enzymes must _pass out_ of the bacterium and consequently may be -found in the surrounding medium. Substances like sugars, peptones, -alcohol, which are readily diffusible, may be acted on by enzymes -_retained within_ the cell body. In the former case the enzymes are -spoken of as extra-cellular or "_exo-enzymes_," and in the latter as -intra-cellular or "_endo-enzymes_." The endo-enzymes and doubtless also -the exo-enzymes may after the death of the cell digest the contents -to a greater or less extent and thus furnish substances that are -not otherwise obtainable. This process of "self-digestion" is known -technically as "_autolysis_." - -A distinction was formerly made between "organized" and "unorganized -ferments." The former term was applied to the minute living organisms, -bacteria, yeasts, molds, etc., which bring about characteristic -fermentative changes, while the latter term was restricted to enzymes -as just described. Since investigation has shown that the changes -ascribed to the "organized ferments" are really due to their enzymes, -and that enzymes are probably formed by all living cells, the -distinction is scarcely necessary at present. - - -PRODUCTION OF TOXINS. - -The injurious effects of pathogenic bacteria are due in large part to -the action of these substances, which in many respects bear a close -relationship to enzymes. The chemical composition is unknown since -no toxin has been prepared "pure" as yet. It was formerly thought -that they were protein in character, but very pure toxins have been -prepared which failed to show the characteristic protein reactions. It -is well established that they are complex substances, of rather large -molecule and are precipitated by many of the reagents which precipitate -proteins. Toxins will be further discussed in Chapter XXVII. It will -be sufficient at this point to enumerate their chief peculiarities in -order to show their marked resemblance to enzymes. - -1. Toxins are _dead_ organic chemical substances. - -2. They are always produced by _living_ cells. - -3. They are active poisons in _very small quantities_.[17] - -4. Their action is specific in that each toxin acts on a particular -kind of cell. The fact that a so-called toxin acts on several -different kinds of cells, possibly indicates a mixture of several -toxins, or action on the _same substance_ in the cells. - -5. Toxins are very sensitive to the action of injurious agencies such -as heat, light, etc., and in about the same measure that enzymes are, -though as a rule they are somewhat more sensitive or "labile." - -6. Toxins apparently have maxima, optima, and minima of temperature for -their action, as shown by the destructive effect of heat and by the -fact that a frog injected with tetanus toxin and kept at 20° shows no -indication of poison, but if the temperature is raised to 37°, symptoms -of poisoning are soon apparent. Cold, however, does not destroy a toxin. - -7. When properly introduced into the tissues of animals they cause the -body cells to form antitoxins (Chapter XXVII) which are capable of -preventing the action of the toxin in question. - -8. _The determining test for a toxin is its action on a living cell._ - -It is true that enzymes are toxic, as are also various foreign -proteins, when injected into an animal, but in much larger doses than -are toxins. - -A marked difference between enzymes and toxins is that the former may -bring about a very great chemical change and still may be recovered -from the mixture of substances acted on and produced, while the toxin -seems to be permanently used up in its toxic action and cannot be -so recovered. _Toxins seem very much like enzymes whose action is -restricted to living cells._ - -Just as enzymes are probably produced by all kinds of cells and not by -bacteria alone, so toxins are produced by other organisms. Among toxins -which have been carefully studied are _ricin_, the poison of the castor -oil plant (_Ricinus communis_); _abrin_ of the jequirity bean (_Abrus -precatorius_); _robin_ of the common locust (_Robinia pseudacacia_); -poisons of spiders, scorpions, bees, fish, snakes and salamanders. - -It has been stated that some enzymes are thrown out from the cell and -others are retained within the cell. The same is true of toxins, hence -we speak of _exo-toxins_ or toxins excreted from, and _endo-toxins_ -or toxins retained within the cell. Among the pathogenic bacteria -there are very few which secrete toxins when growing outside the body. -_Clostridium tetani_ or lockjaw bacillus, _Corynebacterium diphtheriæ_ -or the diphtheria bacillus, _Clostridium botulinum_ or a bacillus -causing a type of food poisoning, _Pseudomonas pyocyanea_ or the blue -pus bacillus are the most important. Other pathogenic bacteria do not -secrete their toxins under the above conditions, but only give them up -when the cell is disintegrated either within or outside the body. For -the reason that endotoxins are therefore difficult to obtain, their -characteristics have not been much studied. The description of toxins -as above given is intended to apply to the _exo-toxins_ of bacteria, -sometimes spoken of as _true toxins_, and to the vegetable toxins -(phytotoxins) which resemble them. - -The snake venoms and probably most of the animal toxins (zoötoxins) are -very different substances. (See Chapter XXIX.) - - -CAUSATION OF DISEASE. - -This subject belongs properly in special pathogenic bacteriology. It -will be sufficient to indicate that bacteria may cause disease in one -or more of the following ways: (_a_) blocking circulatory vessels, -either blood or lymph, directly or indirectly; (_b_) destruction of -tissue; (_c_) production of non-specific poisons (ptomaines, bases, -nitrites, acids, gases, etc.); (_d_) production of specific poisons -(toxins). - - -ANTIBODY FORMATION. - -Bacteria cause the formation of specific "antibodies" when properly -introduced into animals. This must be considered as a physiological -activity since it is by means of substances produced within the -bacterial cell that the body cells of animals are stimulated to form -antibodies. (See Chapters XXVI-XXIX.) - - -STAINING. - -The reaction of bacteria to various stains is dependent on their -physico-chemical structure and hence is a result of physiological -processes, but is best discussed separately (Chapter XIX). - - -CULTURAL CHARACTERISTICS. - -The same is true of the appearance and growth on different culture -media. (Chapter XX.) - - - - -CHAPTER XIII. - -DISINFECTION--STERILIZATION--DISINFECTANTS. - - -The discussion of the physiology of bacteria in the preceding chapters -has shown that a number of environmental factors must be properly -correlated in order that a given organism may thrive. Conversely, it -can be stated that any one of these environmental factors may be so -varied that the organism will be more or less injured, may even be -destroyed by such variation. It has been the thorough study of the -above-mentioned relationships which has led to practical methods for -destroying bacteria, for removing them or preventing their growth when -such procedures become necessary. - -The process of killing all the living organisms or of removing them -completely is spoken of as _disinfection_ or as _sterilization_, -according to circumstances. Thus the latter term is applied largely in -the laboratory, while the former more generally in practice outside the -laboratory. So also disinfection is most commonly done with chemical -agents and sterilization by physical means, though exceptions are -numerous. The original idea of disinfection was the destruction of -"infective" organisms, that is, organisms producing disease in man or -animals. A wider knowledge of bacteriology has led to the application -of the term to the destruction of other organisms as well. Thus the -cheese-maker "disinfects" his curing rooms to prevent abnormal ripening -of cheese, and the dairy-worker "disinfects" his premises to avoid bad -flavors, abnormal changes in the butter or milk. _Sterilization_ is -more commonly applied to relatively small objects and _disinfection_ to -larger ones. Thus in the laboratory, instruments, glassware, apparatus, -etc., are "sterilized" while desks, walls and floors are "disinfected." -The surgeon "sterilizes" his instruments, but "disinfects" his -operating table and room. The dairy-workers mentioned above sterilize -their apparatus, pails, milk bottles, etc. Evidently the object of the -two processes is the same, removing or destroying living organisms, the -name to be applied is largely a question of usage and circumstances. -Any agent which is used to destroy microörganisms is called a -"disinfectant." Material freed from _living_ organisms is "sterile." - -The process of _preventing the growth_ of organisms without reference -to whether they are killed or removed is spoken of as "_antisepsis_," -and the agent as an _antiseptic_. Hence a mildly applied "disinfectant" -becomes an "antiseptic," though it does not necessarily follow that -an "antiseptic" may become a disinfectant when used abundantly. Thus -strong sugar solutions prevent the development of many organisms, -though they do not necessarily kill them. - -_Asepsis_ is a term which is restricted almost entirely to surgical -operations and implies the taking of such precautions that foreign -organisms are _kept out_ of the field of operation. Such an operation -is an _aseptic_ one, or performed _aseptically_. - -A "deodorant or deodorizer" is used to destroy or remove an odor and -does not necessarily have either antiseptic or disinfectant properties. - -The agents which are used for the above-described processes may be -conveniently divided into _physical agents_ and _chemical agents_. - - -PHYSICAL AGENTS. - -=1. Drying.=--This is doubtless the oldest method for _preventing the -growth_ of organisms, and the one which is used on the greatest amount -of material at the present time. A very large percentage of commercial -products is preserved and transported intact because the substances -are kept free from moisture. In the laboratory many materials which -are used as food for bacteria (see Chapter XVI) "keep" because they -are dry. Nevertheless, drying should be considered as an _antiseptic_ -rather than as a _disinfectant_ process. While it is true that the -_complete_ removal of water would result in the death of all organisms -this necessitates a high temperature, in itself destructive, and does -not occur in practice. Further, though many pathogenic bacteria are -killed by drying, many more, including the spore formers, are not. -Hence drying alone is not a practical method of _disinfecting_. - -[Illustration: FIG. 77.--A small laboratory hot-air sterilizer.] - -=2. Heat.=--The use of heat in some form is one of the very best -means for destroying bacteria. It may be made use of by combustion, -or burning, as direct exposure to the open flame, as dry heat (hot -air), or as moist heat (boiling water or steam). Very frequently in -veterinary practice, especially in the country, occasionally under -other conditions, the infected material is best burned. This method is -thoroughly effective and frequently the cheapest in the end. Wherever -there are no valid objections it should be used. Exposure to the open -flame is largely a laboratory procedure to sterilize small metallic -instruments and even small pieces of glassware. It is an excellent -procedure in postmortem examinations to burn off the surface of the -body or of an organ when it is desired to obtain bacteria from the -interior free from contamination with surface organisms. - -_Dry Heat._--Dry heat is not nearly so effective as moist heat as a -sterilizing agent. The temperature must be higher and continued longer -to accomplish the same result. Thus a dry heat of 150° for thirty -minutes is no more efficient than steam under pressure at 115° for -fifteen minutes. Various forms of hot-air sterilizers are made for -laboratory purposes (Fig. 77). On account of the greater length of -time required for sterilization their use is more and more restricted -to objects which must be used dry, as in blood and serum work, for -example. In practice the use of hot air in disinfecting plants is now -largely restricted to objects which might be injured by steam, as -leather goods, furs, and certain articles of furniture, but even here -chemical agents are more frequently used. - -_Moist Heat._--Moist heat may be applied either by boiling in water -or by the use of steam at air pressure, or, for rapid work and on -substances that would not be injured, by steam under pressure. -Boiling is perhaps the best household method for disinfecting all -material which can be so treated. The method is simple, can always -be made use of, and is universally understood. It must be remembered -that all pathogenic organisms, even their spores, are destroyed by a -few minutes' boiling. The process may be applied to more resistant -organisms, such as are met with in canning vegetables, though the -boiling must be continued for several hours, or what is better, -repeated on several different days. This latter process, known as -"_discontinuous sterilization_," or "_tyndallization_," must also be -applied to substances which would be injured or changed in composition -by too long-continued heating, such as gelatin, milk, and certain -sugars. In the laboratory such materials are boiled or subjected to -steaming steam for half an hour on each of three successive days. In -canning vegetables the boiling should be from one to two hours each -day. The principle involved is that the first boiling destroys the -growing cells, but not all spores. Some of the latter germinate by the -next day and are then killed by the second boiling and the remainder -develop and are killed on the third day. Occasionally a fourth boiling -is necessary. It is also true that repeated heating and cooling is more -destructive to bacteria than continuous heating for the same length of -time, but the development of the spores is the more important factor. -Discontinuous heating may also be used at temperatures below the -boiling-point for the sterilization of fluids like blood serum which -would be coagulated by boiling. In this case the material is heated at -55° to 56° for one hour, but on each of seven to ten successive days. -The intermittent heating and cooling is of the same importance as the -development of the spores in this case. (Better results are secured -with such substances by collecting them aseptically in the first place.) - -[Illustration: FIG. 78.--The Arnold steam sterilizer for laboratory -use.] - -[Illustration: FIG. 79.--Vertical gas-heated laboratory autoclave.] - -[Illustration: FIG. 80.--Horizontal gas-heated laboratory autoclave.] - -_Steam._--Steam is one of the most commonly employed agents for -sterilization and disinfection. It is used either as "streaming steam" -at air pressure or confined under pressure so that the temperature is -raised. For almost all purposes where boiling is applicable streaming -steam may be substituted. It is just as efficient and frequently -more easily applied. The principle of the numerous forms of "steam -sterilizers" (Fig. 78) is essentially the same. There is a receptacle -for a relatively small quantity of water and means for conducting the -steam generated by boiling this water to the objects to be treated, -which are usually placed immediately above the water. Surgical -instruments may be most conveniently sterilized by boiling or by -steaming in especially constructed instrument sterilizers. If boiled, -the addition of carbonate of soda, about 1 per cent., usually prevents -injury. - -[Illustration: FIG. 81.--A battery of two horizontal autoclaves in one -of the author's student laboratories. Steam is furnished direct from -the University central heating plant.] - -_Steam under pressure_ affords a much more rapid and certain method of -destroying organisms. Fifteen to twenty pounds pressure corresponding -to temperatures of 121° to 125° is commonly used. Variations depend on -the bulk and nature of the material. Apparatus for this purpose may -now be obtained from sizes as small as one or two gallons up to huge -structures which will take one or two truckloads of material (Figs. -79-91). The latter type is in common use in canning factories, dairy -plants, hospitals, public institutions, municipal and governmental -disinfecting stations. Very frequently there is an apparatus attached -for producing a vacuum, both to exhaust the air before sterilizing, -so that the steam penetrates much more quickly and thoroughly and for -removing the vapor after sterilizing, thus hastening the drying out of -the material disinfected. - -[Illustration: FIG. 82.--A "process kettle" (steam-pressure sterilizer) -used in canning. Diameter, 40 inches; height, 72 inches.] - -The smaller types of pressure sterilizers are called "autoclaves" -and have become indispensable in laboratory work. Fifteen pounds -pressure maintained for fifteen minutes is commonly sufficient for a -few small objects. For larger masses much longer time is needed. The -author found that in an autoclave of the type shown in Fig. 81 it -required ten minutes for 500 cc. of water at 15 pounds pressure to -reach a temperature of 100°, starting at room temperatures, 20° to -25°. Autoclaves may be used as simple steam sterilizers by leaving the -escape valves open so that the steam is not confined, hence they have -largely replaced the latter.[18] - -[Illustration: FIG. 83.--Horizontal steam chest used in canning. -Height, 32 inches; width, 28 inches; length, 10 feet.] - -[Illustration: FIG. 84.--A battery of horizontal rectangular steam -chests in actual use in a canning factory.] - -[Illustration: FIG. 85.--A battery of cylindrical process kettles in -actual use in a canning factory.] - -A process closely akin to sterilization by heat is _pasteurization_. -This means the heating of material at a temperature and for a time -which will destroy the actively growing bacteria but not the spores. -The methods for doing this vary but are essentially two in principle. -1. The material in small quantities in suitable containers (bottles) is -placed in the apparatus; the temperature is raised to 60° to 65° and -maintained for twenty to thirty minutes and then the whole is cooled -(beer, wine, grape juice, bottled milk) (Figs. 92, 93 and 94). - -[Illustration: FIG. 86.--A steam chamber used in government -disinfection work. Size, 4 feet 4 inches × 5 feet 4 inches × 9 feet.] - -[Illustration: FIG. 87.--Circular steam chamber used in government -disinfection work, 54 inches in diameter.] - -[Illustration: FIG. 88.--Portable steam chamber used in government -disinfection work.] - -[Illustration: FIG. 89.--Steam chambers on deck of the U. S. quarantine -station barge "Defender."] - -[Illustration: FIG. 90.--Steam chambers in hold of U. S. quarantine -station barge "Protector." Disinfected space.] - -[Illustration: FIG. 91.--Municipal disinfecting station, Washington, -D. C.] - -[Illustration: FIG. 92.--A pasteurizer for milk in bottles.] - -[Illustration: FIG. 93.--A pasteurizer for grape juice, cider, etc., in -bottles.] - -2. Pasteurizing machines are used and the fluid flows through -continuously. In one type the temperature is raised to 60° and by -"retarders" is kept at this temperature for twenty to thirty minutes -(Figs. 95 to 98). In another type the temperature is raised to as -high as 85° for a few seconds only, "flash process" (Fig. 99), and then -the material is rapidly cooled. It is certain that all pathogenic -microörganisms, except the very few spore formers in that stage, are -killed by proper pasteurization. The process is largely employed in the -fermentation and dairy industries. - -[Illustration: FIG. 94.--A pasteurizer for beer in bottles.] - -[Illustration: FIG. 95.--A continuous milk pasteurizer.] - -[Illustration: FIG. 96.--A pasteurizer for cream to be used in making -ice-cream.] - -[Illustration: FIG. 97.--A continuous milk pasteurizer with holder; -capacity 1500 pounds per hour. _A_, pasteurizer--the milk flows in -tubes inside of a jacket of water heated to the proper temperature; -_B_, holder; _C_, water cooler; _D_, brine cooler.] - -[Illustration: FIG. 98.--A continuous pasteurizing plant in operation. -Similar to Fig. 97 but larger. Capacity, 12,000 pounds per hour. _A_, -pasteurizer; _B_, seven compartment holder; _C_, _D_, coolers.] - -=3. Cold.=--That _cold_ is an excellent _antiseptic_ is illustrated -by the general use of refrigerators and "cold storage." Numerous -experiments have shown that although many pathogenic organisms of a -given kind are killed by temperatures below freezing, not all of the -same kind are, and many kinds are only slightly affected. Hence cold -cannot be considered a practical means for _disinfection_. - -[Illustration: FIG. 99.--A "flash process" pasteurizing outfit, with -holder. _A_, flash pasteurizer; _B_, holder; _C_, cooler.] - -=4. Light.=--It has been stated (p. 75) that light is destructive to -bacteria, and the advisability of having well-lighted habitations -for men and animals has been mentioned. The practice of "sunning" -bedclothing, hangings and other large articles which can scarcely be -disinfected in a more convenient way is the usual method of employing -this agent. Drying and the action of the oxygen of the air assist -the process to some extent. Undoubtedly large numbers of pathogenic -organisms are destroyed under natural conditions by the combined -effects of drying, direct sunlight and oxidation, but it should not -be forgotten that a very slight protection will prevent the action of -light (Figs. 100 and 101). - -[Illustration: FIG. 100.--Effect of light on bacteria. × 7/10. The -plate was inoculated in the usual way. A letter _H_ of black paper was -pasted on the bottom. The plate was then exposed for four hours to the -sun in January outside the window and then incubated. The black paper -protected the bacteria. Outside of it they were killed except where -they happened to be in large masses. Hence the letter shows distinctly. -(Student preparation.)] - -=5. Osmotic Pressure.=--Increase in the concentration of substances -in solution is in practical use as an _antiseptic_ procedure. -Various kinds of "sugar preserves," salt meats and condensed milk -are illustrations. It must be remembered that a similar increase in -concentration occurs when many substances are dried, and is probably -as valuable in the preservative action as the loss of water. That the -process cannot be depended on to _kill_ even pathogenic organisms is -shown by finding living tubercle bacilli in condensed milk. The placing -of bacteria in water or in salt solution in order to have them die and -disintegrate (greatly aided by vigorous shaking in a shaking machine) -("autolysis," p. 126) is a laboratory procedure to obtain cell -constituents. It is not a practical method of disinfection, however. - -[Illustration: FIG. 101.--Effect of light on bacteria. × 7/10. This -plate was treated exactly as the plate in Fig. 100, except that the -letter is _L_, and that it was exposed inside the window and wire -screen. The window was plate glass. It is evident that few of the -bacteria were killed, since the letter _L_ is barely outlined. The -exposure was at the same time as the plate in Fig. 100. (Student -preparation.)] - -=6. Electricity.=--Electricity, though not in itself injurious to -bacteria, is used as an indirect means for destroying bacteria in a -practical way. This is done by electrical production of some substance -which is destructive to bacteria as in ozone water purification -(Petrograd, Florence, and elsewhere), or the use of ultra-violet rays -for the same purpose (Marseilles, Paris) and for treatment of certain -disease conditions. Electricity might be used as a source of heat for -disinfecting purposes should its cheapness justify it. It has also -been used in the preservation of meats to hasten the _penetration of -the salt_ and thus reduce the time of pickling. Electrolyzed sea water -has been tried as a means of flushing and disinfecting streets, but -it is very doubtful if the added expense is justified by any increased -benefit. A number of electric devices have been put forth for various -sterilizing and disinfecting purposes and doubtless will continue to -be, but everyone should be carefully tested before money is invested in -it.[19] - -[Illustration: FIG. 102.--An electric milk purifier (pasteurizer). The -milk flowing from cup to cup completes the circuit when the current is -on. The effect is certainly a heat effect. Sparking occurs at the lips -of the cups.] - -[Illustration: FIG. 103.--One of the ten filter beds of the Columbus -water filtration plant with the filtering material removed. Sand is -the filtering material. All of the beds together have a capacity of -30,000,000 gallons daily.] - -[Illustration: FIG. 104.--Suction filtration. _A_, Berkefeld filter in -glass cylinder containing the liquid to be filtered; _B_, sterile flask -to receive the filtrate as it is drawn through; _C_, water pump; _D_, -manometer, convenient for detecting leaks as well as showing pressure; -_E_, bottle for reflux water.] - -[Illustration: FIG. 105.--Pressure filtration. _A_, cylinder which -contains the filter candle; _B_, cylinder for the liquid to be -filtered; _C_, sterile flask to receive the filtrate; _D_, air pump to -furnish pressure.] - -=7. Filtration.=--Filtration is a process for rendering fluids sterile -by passing them through some material which will hold back the -bacteria. It is used on a large scale in the purification of water for -sanitary or manufacturing reasons (Fig. 103). Air is also rendered -"germ free" in some surgical operating rooms, "serum laboratories" and -breweries by filtration. In the laboratory it is a very common method -of sterilizing liquids which would be injured by any other process. -The apparatus consists of a porous cylinder with proper devices for -causing the liquid to pass through either by suction (Fig. 104) where -the pressure will be only one atmosphere (approximately 15 pounds per -square inch), or by the use of compressed air at any desired pressure -(Fig. 105). The two main types of porous cylinders ("filter candles," -"bougies") are the Pasteur-Chamberland (Fig. 106) and the Berkefeld. -The former are made of unglazed porcelain of different degrees of -fineness, the latter of diatomaceous earth (Fig. 107) The Mandler -filter of this same material is now manufactured in the United States -and is equal if not superior to the Berkefeld. The designs of complete -apparatus are numerous. - -[Illustration: FIG. 106.--Pasteur-Chamberland filter candles about -one-half natural size.] - -=8. Burying.=--This is a time-honored method of disposing of infected -material of all kinds and at first thought might not be considered -a means of _disinfection_. As a matter of fact, under favorable -conditions it is an excellent method. The infected material is -removed. Pathogenic organisms tend to die out in the soil owing to an -unfavorable environment as to temperature and food supply, competition -with natural soil organisms for what food there is, and the injurious -effects of the products of these organisms. Care must be taken that -the burial is done in such a way that the _surface_ soil is not -contaminated either directly or by material brought up from below by -digging or burrowing animals, insects, worms, or movement of ground -water to the surface. Also that the underground water supply which is -drawn upon for use by men or animals is not contaminated. Frequently -infected material, carcasses of animals, etc., are treated in some -way so as to aid the natural process of destruction of the organisms -present, especially by the use of certain chemical agents, as quicklime -(see p. 158). - -[Illustration: FIG. 107.--Berkefeld filter candles about one-half -natural size.] - - - - -CHAPTER XIV. - -DISINFECTION AND STERILIZATION (CONTINUED). - - -CHEMICAL AGENTS. - -A very large number of chemical substances might be used for destroying -bacteria or preventing their growth either through direct injurious -action or by the effect of concentration. Those which are practically -useful are relatively few, though this is one of the commonest methods -of disinfecting and the word "disinfectant" is frequently wrongly -restricted to chemical agents. - -Chemical agents act on bacteria in a variety of ways. Most commonly -there is direct union of the chemical with the protoplasm of the cell -and consequent injury. Some times the chemical is first precipitated -on the surface of the cell without penetrating at once. If removed -soon enough, the organism is not destroyed. This is true of bichloride -of mercury and formaldehyde. If bacteria treated with these agents in -injurious strength be washed with ammonia or ammonium sulphate, even -after a time which would otherwise result in their failure to grow, -they will develop. Some chemicals change the reaction of the material -in a direction unfavorable to growth, and if the change is enough, -may even kill the bacteria. Some agents remove a chemical substance -necessary to the growth of the organism and hence inhibit it. Such -actions are mainly preventive (antiseptic) and become disinfectant only -after a long time. - - -ELEMENTS. - -=Oxygen.=--Oxygen as it occurs in the air is probably not injurious to -living bacteria but aids them with the exception of the anaërobes. In -the nascent state especially as liberated from ozone (O{3}) hydrogen -peroxide (H{2}O{2}) and hypochlorites (Ca(ClO){2}) it is strongly -bactericidal. - -=Chlorine.=--Chlorine is actively disinfectant and is coming into use -for sterilizing water on a large scale in municipal plants (Fig. 108). - -[Illustration: FIG. 108.--Apparatus for sterilizing water with liquid -chlorine.] - -_Iodine_ finds extended use in aseptic surgical operations and -antiseptic dressings. Bromine, mercury, silver, gold, nickel, zinc -and copper are markedly germicidal in the elemental state but are not -practical. - - -COMPOUNDS. - -=Calcium Oxide.=--Calcium oxide (CaO), _quick lime_, is an excellent -disinfectant for stables, yards, outhouses, etc., where it is used -in the freshly slaked condition as "white wash;" also to disinfect -carcasses to be buried. It is very efficient against the typhoid -bacillus in water, where it is much used to assist in the softening. - -=Chloride of Lime.=--Chloride of lime, _bleaching powder_, which -consists of calcium hypochlorite, the active agent, and chloride and -some unchanged quicklime is one of the most useful disinfectants. It -is employed to sterilize water for drinking purposes on a large scale -and to disinfect sewage plant effluents. A 5 per cent. solution is the -proper strength for ordinary disinfection. Only a supply which is fresh -or has been kept in air-tight containers should be used, as it rapidly -loses strength on exposure to the air. The active agent is nascent -oxygen liberated from the decomposition of the hypochlorite. - -=Sodium Hypochlorite.=--Sodium hypochlorite prepared by the -electrolysis of common salt has been used to some extent. - -=Bichloride of Mercury.=--Bichloride of mercury, _mercuric chloride, -corrosive sublimate_ (HgCl{2}), is the strongest of all disinfectants -under proper conditions. It is also extremely poisonous to men and -animals and great care is necessary in its use. It is precipitated by -albuminous substances and attacks metallic objects, hence should not be -used in the presence of these classes of substances. - -It is used in a strength of one part HgCl{2} to 1000 of water for -general disinfection. Ammonium chloride or sodium chloride, common -salt, in quantities equal to the bichloride, or citric acid in one-half -of the amount should be added in making large quantities of solution or -for use with albuminous fluids to prevent precipitation of the mercury -(Fig. 109). - -None of the other metallic salts are of value as practical -disinfectants aside from their use in surgical practice. In this -latter class come boric acid, silver nitrate, potassium permanganate. -The strong mineral acids and alkalies are, of course, destructive to -bacteria, but their corrosive effect excludes them from practical use, -except that "lye washes" are of value in cleaning floors and rough -wood-work, but even here better _disinfection_ can be done more easily -and safely. - -[Illustration: FIG. 109.--Tanks for bichloride of mercury, government -quarantine disinfecting plant.] - - -ORGANIC COMPOUNDS. - -=Carbolic Acid or Phenol.=--Carbolic acid or phenol (C{6}H{5} OH) is one -of the commonest agents in this class. It is used mostly in 5 per -cent. solution as a disinfectant and in 0.5 per cent. solution as an -antiseptic. For use in large quantities the crude is much cheaper and, -according to some experimenters, even more active than the pure acid, -owing to the cresols it contains. The crude acid is commonly mixed with -an equal volume of commercial sulphuric acid and the mixture is added -to enough water to make a 5 per cent. dilution, which is stronger than -either of the ingredients alone in 5 per cent. solution. - -=Cresols.=--The cresols (C{6}H{4}CH{3}OH, ortho, meta and para), -coal-tar derivatives, as phenol, are apparently more powerful -disinfectants. A great number of preparations containing them have -been put on the market. _Creolin_ is one which is very much used in -veterinary practice and forms a milky fluid with water, while _lysol_ -forms a clear frothy liquid owing to the presence of soap. Both -of these appear to be more active than carbolic acid and are less -poisonous and more agreeable to use. They are used in 2 to 5 per cent. -solution. - -=Alcohol.=--Ordinary (ethyl) alcohol (C{2}H{5}OH) is largely used as a -_preservative_, also as a disinfectant for the body surface, hands, and -arms. Experiments show that alcohol of 70 per cent. strength is most -strongly bactericidal and that absolute alcohol is very slightly so. - -=Soap.=--Experimenters have obtained many conflicting results with -soaps when tested on different organisms, as is to be expected from -the great variations in this article. Miss Vera McCoy in the author's -laboratory carried out experiments with nine commercial soaps--Ivory, -Naphtha, Packer's Tar, Grandpa's Tar, Balsam Peru, A. D. S. Carbolic, -German Green, Dutch Cleanser, Sapolio--and obtained abundant growth -from spores of _Bacillus anthracis_, from _Bacterium coli_ and from -_Staphylococcus pyogenes aureus_ in all cases even when the organisms -had been exposed twenty-four hours in 5 per cent. solutions. From -these results and from the wide variations reported in the literature -it is clear that _soap solutions alone cannot be depended on_ as -disinfectants. Medicated soaps do not appear to offer any advantages in -this respect. The amount of the disinfectant which goes into solution -when the soap is dissolved is too small to have any effect. - -=Formaldehyde.=--Formaldehyde (HCHO) is perhaps the most largely used -chemical disinfectant at the present time. The substance is a gas -but occurs most commonly in commerce as a watery solution containing -approximately 40 per cent. of the gas. This solution is variously known -as formalin, formol, and formaldehyde solution. The first two names -are patented and the substance under these names usually costs more. -It is used in the gaseous form for disinfecting closed spaces of all -kinds to the exclusion of most other means today. A great many types -of formalin generators have been devised. The gas has little power of -penetration and all material to be reached should be exposed as much as -possible. The dry gas is almost ineffective, so that the objects must -be moistened or vapor generated along with the gas. A common method -in use is to avoid expensive generators by pouring the formaldehyde -solution on permanganate of potash crystals placed in a vessel removed -from inflammable objects on account of the heat developed which -occasionally sets the gas on fire. The formalin is used in amounts -varying from 20 to 32 ounces to 8-1/2 to 13 ounces of permanganate to -each 1000 cubic feet of space. This method is expensive since one pint -(16 ounces) of formalin is sufficient for each 1000 cubic feet, and -since the permanganate is an added expense. Dr. Dixon, Commissioner of -Health of Pennsylvania, recommends the following mixture to replace the -permanganate, claiming that it works more rapidly and is less expensive -and just as efficient: - - 1. Sodium bichromate, ten ounces. - 2. Saturated solution of formaldehyde, sixteen ounces. - 3. Common sulphuric acid, one and a half ounces. - -Two and three are mixed together and when cool are poured on the -bichromate which is placed in an earthenware jar of a volume about ten -times the quantity of fluid used. The quantities given are for each -1000 cubic feet of space. - -A very simple method is to cause the formalin, diluted about twice -with water to furnish moisture enough, to drop by means of a regulated -"separator funnel" on a heated iron plate. The dropping should be so -regulated that each drop is vaporized as it falls. The plate must -have raised edges, pan-shaped, to prevent the drops rolling off when -they first strike the plate. Formaldehyde has no corrosive (except on -iron) or bleaching action, and is the most nearly ideal closed space -disinfectant today. In disinfecting stations it is made use of in -closed sterilizers such as were described under steam disinfection -particularly in connection with vacuum apparatus. It is also used -in solution as a preservative and as a disinfectant. The commonest -strength is 2 or 3 per cent. of formalin or 0.8 to 1.2 per cent. of -the formaldehyde gas. As an _antiseptic_ it is efficient in dilutions -as high as 1 to 2000 of the gas. It is very irritant to mucous -membranes of most individuals. - -=Anilin Dyes.=--Some of the anilin dyes show remarkable selective -disinfectant and antiseptic action on certain kinds of bacteria with -little effect on others. This has been well shown by Churchman in his -work on Gentian Violet. This dye inhibits the growth of _Gram positive_ -organisms up to a dilution of one part in 300,000 while for _Gram -negative_ organisms it is without effect even in saturated solution. -This is nicely shown in the accompanying illustration. This inhibiting -effect of anilin dyes is taken advantage of in several methods of -isolating bacteria (Chapter XVIII). - -[Illustration: FIG. 110.--The lower half of the plate is plain agar -medium, the upper half the same medium plus gentian violet to make one -part in 300,000. The Gram positive organism is on the right and the -Gram negative on the left. Streak inoculations were made across both -media.] - -In addition to the above-discussed disinfectants a large number of -substances, particularly organic, are used in medicine, surgery, -dentistry, etc., as more or less strong antiseptics, and the list is a -constantly lengthening one. - -In the laboratory chloroform, H{2}O{2}, ether and other volatile or -easily decomposable substances have been used to sterilize liquids -which could not be treated by heat or by filtration. The agent is -removed either by slow evaporation or by exhausting the fluid with -an air pump. The method is not very satisfactory, nor is absolute -sterilization easily accomplished. It is much better to secure such -liquids aseptically where possible. - - - - -CHAPTER XV. - -DISINFECTION AND STERILIZATION (CONTINUED). - - -CHOICE OF AGENT. - -The choice of the above-described agents depends on the conditions. -Evidently a barn is not to be disinfected in the same way that a -test-tube in the laboratory is sterilized. Among the factors to be -considered in making a choice are the thing to be disinfected or -sterilized, its size and nature, that is, whether it will be injured by -the process proposed, cost of the agent, especially when a large amount -of material is to be treated. Among the conditions which affect the -action of all agents the following should be borne in mind particularly -when testing the disinfecting power of chemical agents: - -1. _The kind of bacterium_ to be destroyed, since some are more readily -killed by a given disinfectant than others, even though no spores are -present. - -2. _The age of the culture._ Young bacteria less than twenty-four hours -old are usually more readily killed than older ones since the cell wall -is more delicate and more easily penetrated, though old growths may -be weakened by the accumulation of their products and be more easily -destroyed. - -3. _Presence of spores_, since they are much more resistant than the -growing cells. - -4. Whether the organism is a _"good" or "bad" growth_, _i.e._, whether -it has grown in a favorable environment and hence is vigorous, or under -unfavorable conditions and hence is weak. - -5. _The number of bacteria present_, since with chemical agents the -action is one of relative masses. - -6. _Nature of the substance in which the bacteria are._ Metallic salts, -especially bichloride of mercury, are precipitated by albuminous -substances and if employed at all must be used in several times the -ordinary strength. Solids require relatively more of a given solution -than liquids. - -7. _State of the disinfectant_, whether solid, liquid or gas, and -whether it is ionized or not. Solutions penetrate best and are -therefore more quickly active and more efficient. - -8. _The solvent._ Water is the best solvent to use. Strong alcohol (90 -per cent. +) diminishes the effect of carbolic acid, formaldehyde and -bichloride of mercury. Oil has a similar effect. The action is probably -to prevent the penetration of the disinfectant. - -9. _Strength of solution._ The stronger the solution, the more rapid -and more certain the action, for the same reason as mentioned under 5. -In fact, every disinfectant has a strength below the lethal at which it -stimulates bacterial growth. - -10. _Addition of salts._ Common salt favors the action of bichloride -of mercury and also of carbolic acid. Other salts may hinder by -precipitating the disinfectant. - -11. _Temperature._ Chemical disinfectants, as a rule, follow the -general law that chemical action increases with the temperature, up to -the point where the heat of itself is sufficient to kill. - -12. _Time of action._ It is scarcely necessary to point out that a -certain length of time is necessary for any disinfectant to act. One -may touch a red hot stove and not be burned. All the above-mentioned -conditions are influenced by the time of action. - - -STANDARDIZATION OF DISINFECTANTS--"PHENOL COEFFICIENT." - -Many attempts have been made to devise standard methods for testing -the relative strengths of disinfectants. The one most widely used in -the United States is the so-called "Hygienic Laboratory" method of -determining the "phenol coefficient" of the given substance and is a -modification of the method originally proposed by Rideal and Walker -in England. In this method as proposed by Anderson and McClintic, -formerly of the above laboratory, the strengths of the dilution of the -disinfectant to be tested which kills a culture of _Bacterium typhosum_ -in 2-1/2 minutes is divided by the strength of the dilution of carbolic -acid which does the same; and the dilution which kills in 15 minutes is -likewise divided by the corresponding dilution of carbolic acid. The -two ratios thus obtained are averaged and the result is the "phenol -coefficient." For example - - Phenol 1:80 killed in 2-1/2 minutes - Disinfectant "A" 1:375 " " " " - Phenol 1:110 " " 15 " - Disinfectant "A" 1:650 " " " " - 375 ÷ 80 = 4.69 - 650 ÷ 110 = 5.91 - ----- - 2)10.60 - ----- - Average = 5.30 = "phenol coefficient." - -Standard conditions of temperature, age of culture, medium, reaction, -etc., and of making the dilutions and transfers are insisted on. -Details may be found in the Journal of Infectious Diseases, 1911, 8, p. -1. - -This is probably as good a method as any for arriving at the relative -strengths of disinfectants and in the hands of any given worker -concordant results in comparative tests can usually be attained. -Experience has shown that the results obtained by different workers -with the same disinfectant may be decidedly at variance. This is to -be expected from a knowledge of the factors affecting the action of -disinfectants above stated and from the known specific action of -certain disinfectants on certain organisms (compare anilin dyes, p. -162). - -It seems that the only sure way to test the action of such a substance -is to try it out in the way it is to be used. It is scarcely wise to -adopt the "phenol coefficient" method as a legal standard method as -some states have done. - - -PRACTICAL STERILIZATION AND DISINFECTION. - -The methods for sterilizing in the laboratory have been discussed and -will be referred to again in the next chapter. - -In practical disinfection it is a good plan always to _proceed as -though spores were present_ even if the organism is known. Hence use an -_abundance of the agent_ and _apply it as long as practicable_. Also -it is best to secure the _chemical substances used as such_ and _not -depend on patented mixtures purporting to contain them_. As a rule the -latter are _more expensive_ in _proportion to the results secured_. - -_Surgical instruments_ may be sterilized by boiling in water for -fifteen minutes, provided they are clean, as they should be. If dried -blood, pus, mucus, etc., are adherent, which should never be the case, -they should be boiled one-half hour. The addition of sodium carbonate -(0.5 to 1 per cent.) prevents rusting. Surgeons' sterilizers are to -be had at reasonable prices and are very convenient. Whether the -instruments are boiled or subjected to streaming steam depends on -whether the supporting tray is covered with water or not. The author -finds it a good plan to keep the needles of hypodermic syringes in a -small wire basket in an _oil bath_. The oil may be heated to 150° to -200° and the needles sterilized in a very few minutes. The oil also -prevents rusting. - -_Rooms_, _offices_ and all spaces which may be readily made practically -gas-tight are best disinfected by means of formaldehyde by any of the -methods above described (Figs. 111 and 112). - -_Stables_ and _Barnyards_ (Mohler): "A preliminary cleaning up of all -litter is advisable together with the scraping of the floor, mangers -and walls of the stable with hoes and the removal of all dust and -filth. All this material should be burned since it probably contains -the infective agent. Heat may be applied to the surfaces, including -barnyard, by means of a 'cyclone oil burner.' When such burning is -impracticable, the walls may be disinfected with one of the following: - - 1. Whitewash 1 gallon + chloride of lime 6 ounces. - - 2. Whitewash 1 gallon + crude carbolic acid 7 ounces. - - 3. Whitewash 1 gallon + formalin 4 ounces. - -The same may be applied with brushes or, more rapidly, sprayed on with -a pump; the surface soil of the yard and surroundings should be removed -to a depth of 5 or 6 inches, placed in a heap and thoroughly mixed -with quicklime. The fresh surface of soil thus exposed may be sprinkled -with a solution of a chemical disinfectant as above described. - -[Illustration: FIG. 111.--Formaldehyde generator used in city work for -room disinfection.] - -[Illustration: FIG. 112.--Government formaldehyde generator.] - -"Portions of walls and ceiling not readily accessible may be -disinfected by chlorine gas liberated from chloride of lime by crude -carbolic acid. This is accomplished by making a cone of 5 or 6 pounds -of chloride of lime in the top of which a deep crater is made for the -placement of from 1 to 2 pints of crude carbolic acid. The edge of -the crater is thereupon pushed into the fluid, when a lively reaction -follows. Owing to the heat generated, it is advisable to place the -chloride of lime in an iron crucible (pot), and to have nothing -inflammable within a radius of two feet. The number and location of -these cones of chloride of lime depend on the size and structure -of the building to be disinfected. As a rule it may be stated that -chlorine gas liberated from the above sized cone will be sufficient for -disinfecting 5200 cubic feet of air space." - -_Liquid manure_, _leachings_, etc., where collected are thoroughly -disinfected by chloride of lime applied in the proportion of 2 parts to -1000 of fluid. - -[Illustration: FIG. 113.--Chamber used in government work for -formaldehyde disinfection. The small cylinder at the side is the -generator.] - -_Vehicles_ may be thoroughly washed with 2 per cent. formalin solution, -or if closed space is available, subjected to formaldehyde gas -disinfection, after cushions, hangings, etc., have been removed and -washed with the disinfectant. - -_Harness_, _brushes_, _combs_ should be washed with a solution of -formalin, carbolic acid, or creolin as given under these topics. - -_Washable articles_ should be boiled, dropped into disinfectant, -solutions as soon as soiled, and then boiled or steamed. - -_Unwashable articles_--burn all possible. Use formaldehyde gas method -in a closed receptacle (Fig. 113). - -_Stock cars_--the method described for stables is applicable here. - -_Animals, large and small_, may have the coat and surface of the body -disinfected by washing with 1 to 1000 bichloride or strong hot soapsuds -to which carbolic acid has been added to make a 5 per cent. solution; -they should then be given a good warm bath. - -Frequently time and money are saved by a combination of steam and -formaldehyde disinfection. This is a regular practice in municipal and -quarantine disinfection (Fig. 114). - -[Illustration: FIG. 114.--Chamber in actual use at government -quarantine station for disinfecting baggage and dunnage with steam -or formaldehyde or both. The small cylinder at the side is the steam -formaldehyde generator.] - -Persons engaged in disinfection work should wear rubber boots, coats -and caps which should be washed in a disinfectant solution and the -change to ordinary clothing made in a special room so that no infective -material will be taken away. - - - - -PART III. - -THE STUDY OF BACTERIA. - - - - -CHAPTER XVI. - -CULTURE MEDIA. - - -The study of bacteria may be taken up for the disciplinary and -pedagogic value of the study of a science; with the idea of extending -the limits of knowledge; or for the purpose of learning their -beneficial or injurious actions with the object of taking advantage of -the former and combating or preventing the latter. - -Since bacteria are classed as plants, their successful study implies -their cultivation on a suitable soil. A growth of bacteria is called -a "_culture_" and the "soil" or material on which they are grown is -called a "_culture medium_." In so far as the culture medium is made up -in the laboratory it is an "artificial culture medium" as distinguished -from a natural medium. A culture consisting of one kind of bacteria -only is spoken of as a "pure culture," and accurate knowledge of -bacteria depends on obtaining them in "pure culture." After getting -a "pure culture" the special characteristics of the organism must be -ascertained in order to distinguish it from others. The discussion of -the _morphology_ of bacteria in Chapters II, III, and IV shows that -the morphological structures are too few to separate individual kinds. -They serve at best to enable groups of similarly appearing forms to be -arranged. Hence any further differentiation must be based on a study -of the _physiology_ of the organism as discussed in the chapters on -Physiological Activities of Bacteria. - -The thorough study of a bacterium involves, therefore: - -1. Its isolation in pure culture. - -2. Its study with the microscope to determine morphological features -and staining reactions. - -3. Growth on culture media for determining its physiological activities -as well as morphological characteristics of the growths themselves. - -4. Animal inoculations in certain instances. - -5. Special serum reactions in some cases. - -Since isolation in pure culture requires material for growing the -organism, the first subject to be considered is culture media. - -A culture medium for a given bacterium should show the following -essentials: - -1. It must contain all the elements necessary for the growth of the -organism except those that may be obtained from the surrounding -atmosphere. - -2. These elements must be in a form available to the organism. - -3. The medium must not be too dry, in order to furnish sufficient -moisture for growth and to prevent too great a concentration of the -different ingredients. - -4. The reaction must be adjusted to suit the particular organism dealt -with. - -5. There must be no injurious substances present in concentration -sufficient to inhibit the growth of the organism or to kill it. - -Ordinarily, more attention must be paid to the sources of the two -elements N and C than to the others, for in general the substances -used to furnish these two and the water contain the other elements in -sufficient amount. For very exact work on the products of bacteria, -_synthetic media_ containing definite amounts of chemicals of known -composition have been prepared, but for most of the work with bacteria -pathogenic to animals such media are not needed. - -Culture media may be either _liquid_ or _solid_, or for certain -purposes may be liquid at higher temperatures and solid at lower, as -indicated later. Liquid media are of value for obtaining bacteria for -the study of morphology and cell groupings and for ascertaining many of -the physiological activities of the organisms. Solid media are useful -for studying some few of the physiological activities and especially -for determining characteristic appearances of the isolated growths -of bacteria. These isolated growths of bacteria on solid media are -technically spoken of as "_colonies_," whether they are microscopic in -size or visible to the unaided eye. - -It is clear that the kinds of culture media used for the study of -bacteria may be unlimited but the undergraduate student will need to -use a relatively small number, which will be discussed in this section. - -=Meat Broth (Bouillon).=[20]--This itself is used as a medium and as -the basis for the preparation of other solid and liquid media. - -Finely ground _lean_ beef is selected because it contains the necessary -food materials. Fat is not desired since it is a poor food for most -bacteria and in the further processes of preparation would be melted -and form an undesirable film on the surface of the medium. The meat is -placed in a suitable container and mixed with about twice its weight of -_cold_ water (not distilled) and allowed to soak overnight or longer. -The cold water extracts from the meat water-soluble proteins, blood, -carbohydrates in the form of dextrose (occasionally some glycogen), -nitrogenous extractives and some of the mineral salts. The fluid is -strained or pressed free from the meat. This "meat juice" should now -be thoroughly boiled, which results in a coagulation of a large part -of the proteins and a precipitation of some of the mineral salts, -particularly phosphates of calcium and magnesium, both of which must be -filtered off and the water loss restored by adding the proper amount of -distilled water. The boiling is done at this point because the medium -must later be heated to sterilize it and it is best to get rid of the -coagulable proteins at once. The proteins thus thrown out deprive -the medium of valuable nitrogenous food material which is replaced -by adding about 1 per cent. by weight of commercial peptone. It is -usual also (though not always necessary) to add about 0.5 per cent. by -weight of common salt which helps to restore the proper concentration -of mineral ingredients lost by the boiling. The chlorine is also an -essential element. The reaction is now determined and adjusted to the -desired end point, "standardized," as it is called. The medium is -again _thoroughly_ boiled and filtered boiling hot. The adjusting of -the reaction and the boiling ordinarily cause a precipitate to form -which is largely phosphates of the alkaline earths with some protein. -The filtered medium is collected in suitable containers, flasks or -tubes, which are plugged with well-fitting non-absorbent cotton plugs -and sterilized, best in the autoclave for twenty minutes at 15 pounds -pressure, or discontinuously in streaming steam at 100°. If careful -attention is paid to _titration_ and to _sufficient boiling_ where -indicated, the meat broth prepared as above should be clear, only -faintly yellowish in color and show no precipitate on cooling. - -The conventional method for standardizing an acid medium is as follows: -Take 5 cc of the medium, add 45 cc of distilled water and 1 cc of -_phenolphthalein_ as indicator. Boil the solution and while still hot -run in from a burette N/20 NaOH solution until a faint pink color -appears. From the number of cc of N/20 NaOH used to "neutralize" the -5 cc of medium it is calculated how many cc of N/1 NaOH are necessary -to give the desired end reaction to the volume of medium which is to -be standardized. The resulting reaction is expressed as % _acid or -alkaline to phenolphthalein_. If it is necessary to add to each 100 cc -of the medium 1 cc of N/1 NaOH to make it neutral to phenolphthalein, -the reaction is called 1% acid: if to each 100 cc of medium there is -added 1 cc of N/1 alkali in addition to the quantity necessary to -neutralize, the reaction is called 1% alkaline. - -In order to obtain a pink color when titrating with this indicator -not only must the "free acid" be neutralized by the alkali but also -loosely combined acid and any other substances present which will -combine with the alkali rather than with the indicator so that in many -media _more alkali_ is added than is necessary to neutralize the "free -acid," _i.e._, the free H ions present. - -It is well established that the controlling factor in the growth of -bacteria in so far as "reaction" is concerned is not the _titratable -substances_ present but only the "free acid," _i.e._, the _number of -free H ions_, consequently it is better to determine the concentration -of H ions and to _standardize to a definite H ion concentration_. -Phenolphthalein as shown above is not a good indicator for this purpose. - -The H ions present can be determined accurately in all cases only by -electrolytic methods. The apparatus necessary is usually relatively -expensive and scarcely adapted to the use of large classes of students. -There are a number of indicators each of which will show color changes -within rather narrow ranges of H ion concentration. Standardization by -the use of these indicators, the "colorimetric method," is recommended -by the Society of American Bacteriologists and is coming into general -use. - -The H ion concentration is ordinarily _indicated_ by the conventional -symbol P{H}, _e.g._, the concentration in pure water which is regarded -as neutral is expressed as P{H} 7; of normal HCl, P{H} 0; of normal -NaOH, P{H} 14. The figure after P{H} does not in reality represent the -concentration of H ions in the solution. This, like the concentration -of acids, is expressed on the basis of normality, _i.e._, as compared -with the concentration of a normal solution (1 g. equivalent) of H -ions. Concentration of H ions in pure water is N/10,000,000, _i.e._, -is 1/10,000,000 of the concentration in a normal solution of H ions. -Expressed in other words, it is the concentration in a normal solution -of H ions diluted ten million times. 10,000,000 = 10 to the 7th power -= 10^{7}. Hence the figure after the P{H} indicates the _logarithm of -the number of times the solution is diluted_. Therefore this number -_increases with the dilution_, and the larger the figure after the -P{H}, the _less acid the solution is_. - -Most saprophytic organisms and many parasitic ones grow within a wide -range of H ion concentration so that titration with phenolphthalein -gives sufficient accuracy for media for such organisms. On the -other hand, many organisms grow within a very narrow range of H ion -concentration, hence accurate standardization to a definite H ion -concentration is necessary. It is also evident that for comparative -work, such standardization is essential because this reaction can be -reproduced in other media and by other workers.[21] - -Broth may be prepared from Liebig's or Armour's meat extract by adding -5 grams of either, 10 grams peptone and 5 grams NaCl to 1000 cc of -water, boiling to dissolve, then titrating and filtering as above. - -The author after much experience finds _meat juice_ preferable to meat -extract for broth and other media for pathogenic bacteria, and has -abandoned the use of meat extracts for these organisms. - -=Glycerin Broth.=--Glycerin broth is made by adding 4 to 6 per cent. of -glycerin to the broth just previous to the sterilization. The glycerin -serves as a source of carbon to certain bacteria which will not grow on -the ordinary broth--as _Mycobacterium tuberculosis_. - -=Sugar Broths.=--Sugar broths are used for determining the action of -bacteria on these carbohydrates, since this is a valuable means of -differentiating certain forms, especially those from the intestinal -tract. Broth _free from sugar_ must first be made. This is done by -adding to broth prepared as already described, _just previous to final -filtering and sterilization_, a culture of some sugar-destroying -organism (_Bacterium coli_ is ordinarily used), and then allowing the -organism to grow in the raw broth at body temperature for twenty-four -hours. Any carbohydrate in the broth is destroyed by the _Bacterium -coli_. This mixture is then boiled to kill the _Bacterium coli_, -restandardized and then 1 per cent. by weight of required sugar is -added. Dextrose, saccharose and lactose are the most used, though -many others are used for special purposes. After the sugar is added -the medium must be sterilized by _discontinuous heating_ at 100° for -three or four successive days, because long boiling or heating in the -autoclave splits up the di- and polysaccharids into simpler sugars and -may even convert the simple sugars (dextrose) into acid. - -Various other _modified broths_ are frequently used for special -purposes but need not be discussed here. - -=Dunham's peptone solution=, frequently used to determine indol -production, is a solution of 1 per cent. of peptone and 0.5 per cent. -of salt in tap water. It does not need to be titrated, but should be -boiled and filtered into tubes or flasks and sterilized. - -=Nitrate Broth.=--Nitrate broth for determining nitrate reduction -is 1 per cent. of peptone, 0.2 per cent. of C. P. potassium nitrate -dissolved in distilled water and sterilized. - -=Milk.=--Milk is a natural culture medium much used. It should be -fresh and thoroughly skimmed, best by a separator or centrifuge to -get rid of the _fat_. If the milk is not fresh, it should be titrated -as for broth and the reaction adjusted. The milk should be sterilized -discontinuously to avoid splitting up the lactose as well as action on -the casein and calcium phosphate. - -_Litmus Milk._--Litmus milk is milk as above to which litmus has been -added as an acid production indicator. The milk should show blue when -the litmus is added or be made to by the addition of normal NaOH -solution. It should be sterilized discontinuously. Frequently on -heating litmus milk the blue color disappears due to a reduction of -the litmus. This blue color will reappear on shaking with air or on -standing several days, due to absorption of O and oxidation of the -reduced litmus, provided the heating has produced no other change in -the milk, as proper heating will not. - -=Gelatin Culture Medium.=--Gelatin to the extent of 10 to 15 per -cent. is frequently added to broth and gives a culture medium of -many advantages. It is solid at temperatures up to about 25° and -fluid above this temperature, a property which is of great advantage -in the isolation of bacteria. (See Chapter XVIII.) Further gelatin -is liquefied (that is digested, converted into gelatin proteose and -gelatin peptone, which are soluble in water and do not gelatinize) -by many bacteria and not by others, a valuable diagnostic feature. -The gelatin colonies of many bacteria are very characteristic in -appearance, as is the growth of many on gelatin in culture tubes. - -Gelatin medium may be prepared by adding the proper amount of gelatin -(10 to 15 per cent. by weight) broken into small pieces (powdered -gelatin in the same proportion may be used) to broth, gently warming -until the gelatin is dissolved, standardizing as for broth, filtering -and sterilizing. It is usually cleared before filtering by stirring -into the gelatin solution, cooled to below 60°, the white of an egg -for each 1000 cc., and then thoroughly boiling before filtering. The -coagulation of the egg albumen entangles the suspended matter so that -the gelatin filters perfectly clear, though with a slight yellowish -color. The filtering may be done through filter paper if the gelatin -is well boiled and filtered boiling hot, but is more conveniently done -through absorbent cotton, wet with boiling water. - -Or, the gelatin may be added to _meat juice before it is boiled_, then -this is heated to about body temperature (not too hot, or the proteins -will be coagulated too soon) until the gelatin is dissolved. Then -the material is standardized and thoroughly boiled and filtered. The -proteins of the meat juice coagulate and thus clear the medium without -the addition of egg white. Commercial gelatin is markedly acid from the -method of manufacture, hence the medium requires careful titration, -even when made from a standardized broth. - -Gelatin should be sterilized by discontinuous heating at 100° on three -successive days, because long boiling or heating above 100° tends to -hydrolyze the gelatin into gelatin proteose and peptone and it will -not gelatinize on cooling. It may be heated in the autoclave for -ten to fifteen minutes at 10 pounds' pressure and sometimes not be -hydrolyzed, but the procedure is uncertain and very resistant spores -may not be killed. The medium should be put into the culture tubes in -which it is to be used as soon as filtered, and sterilized in these, -since, if put into flasks these must be sterilized, and then when -transferred to tubes for use, it must be again sterilized unless great -care is taken to have the tubes plugged and sterilized first, and in -transferring aseptically to these tubes. These repeated heatings are -very apt to decompose the gelatin, so it will not "set" on cooling. The -prepared and sterilized tubes of gelatin should be kept in an ice-box -or cool room, as they will melt in overheated laboratories in summer or -winter. - -=Agar Medium.=--Agar agar, usually called agar, is a complex -carbohydrate substance of unknown composition obtained from certain -seaweeds along the coast of Japan and Southeastern Asia. It occurs -in commerce as thin translucent strips or as a powder. It resembles -gelatin only in the property its solutions have of gelatinizing when -cooled. Gelatin is an albuminoid closely related to the proteins, -agar a carbohydrate. Agar is much less soluble in water, 1 or 1.5 -per cent. of agar giving a jelly as dense as 10 to 15 per cent. of -gelatin. It dissolves only in water heated to near the boiling-point -(98° to 99°) and only after much longer heating. This hot solution -"jells," "sets" or gelatinizes at about 38° and remains solid until -again heated to near boiling. Hence bacteria may be grown on agar at -the body temperature (37°) and above, and the agar will remain solid, -while gelatin media are fluid above about 25°. No pathogenic bacteria -and none of the saprophytes liable to be met with in the laboratory are -able to "liquefy" agar. - -An agar medium is conveniently prepared from broth by adding 1 or -1.5 per cent. of the finely divided agar to the broth and boiling -until dissolved, standardizing, clearing, filtering, and sterilizing. -The agar must be thoroughly boiled, usually for ten to fifteen -minutes, and the water loss made up by the addition of distilled -water before titration. Agar is practically neutral so that there is -little difference between the titration of the dissolved agar and -the original broth. The agar solution should be kept hot from the -beginning to the end except the cooling down to below 60°, when the -egg white for clearing is added. Though filtration through paper is -possible as with gelatin, if the agar solution is thoroughly boiled -and filtered boiling hot, it is more satisfactory for beginners to -use absorbent cotton wet with boiling water and to pour the hot agar -through the same filter if not clear the first time. The solidified -agar medium is never perfectly clear, but always more or less -opalescent. The agar medium may be sterilized in the autoclave for -fifteen minutes at 15 pounds pressure as the high temperature does not -injure the agar. - -=Potato Media.=--Potatoes furnish a natural culture medium which is -very useful for the study of many bacteria. The simplest, and for most -purposes the best, way to use potatoes is in culture tubes as "potato -tube cultures" (No. 8, Fig. 119). These are prepared as follows: -Large tubes are used. Large healthy potatoes are selected. Each end -of the potato is sliced off so as to have parallel surfaces. With a -cork-borer of a size to fit the tubes used, cylinders about one and -one-half inches long are made. Each cylinder is cut diagonally from -base to base. This furnishes two pieces each with a circular base and -an oval, sloping surface. The pieces are then washed clean and dropped -for a minute into boiling water to destroy the oxidizing enzyme on the -surface which would otherwise cause a darkening of the potato. (The -darkening may also be prevented by keeping the freshly cut potatoes -covered with clean water until ready to sterilize.) A bit of cotton -one-fourth to one-half inch in depth is put into each of the test-tubes -to retain moisture and a piece of potato dropped in, circular base -down. The tubes are then plugged with cotton and sterilized in the -autoclave at 15 pounds pressure for not less than twenty-five minutes, -since potatoes usually harbor very resistant spores, and it is not -unusual for a few tubes to spoil even after this thorough heating. - -Potatoes are sometimes used in "potato plate cultures." The term "plate -culture" is a relic of the time when flat glass plates were used for -this and other "plate cultures." Now glass dishes of the general -form shown in Fig. 115, called "Petri dishes," or plates are used for -practically all plate culture work. For "potato plates" slices from -potatoes are cut as large and as thick as the relative sizes of potato -and dish permit (Fig. 116). The slices should be thin enough not to -touch the lid and thick enough to be firm. - -[Illustration: FIG. 115.--Petri dish with the lid partly raised. × 1/2] - -[Illustration: FIG. 116.--A potato plate. × 1/2] - -It is a good plan to wrap each dish separately in paper to retain the -lid securely, then sterilize as for potato tubes, and leave plates -wrapped until wanted. - -It sometimes happens that the natural acidity of potatoes is too great -for the growth of many organisms. The acidity is sufficiently corrected -by soaking the pieces of potato in a 1 per cent. solution of sodium -carbonate for an hour before they are put into the tubes or plates. - -_Glycerinized potato tubes_ are conveniently prepared by covering the -potato in the tube with glycerin broth, sterilizing and pouring off the -excess broth immediately after sterilizing, taking care that the tubes -do not become contaminated which is not very probable if the work is -quickly done while the tubes are still hot. - -=Blood Serum Media.=--Blood serum, usually from the larger, domestic -animals on account of convenience in securing it in quantity, is used -in the study of the bacteria causing disease in man and animals. Most -commonly the serum is collected from the clotted blood after it has -well separated (usually about forty-eight hours is required for this). -It is then run into tubes which are plugged with cotton and placed in -an apparatus for coagulating the serum by heat. A copper water bath -with a tightly closed air compartment or the horizontal autoclave (Fig. -81) is sufficient for this purpose, though special forms of apparatus -are to be had. It is important that the temperature be raised slowly so -that the blood gases escape gradually. Three to five hours or longer -should be allowed for the temperature to reach the boiling-point. If -the tubes are heated too rapidly, the serum is filled with bubbles and -badly torn since the gases are driven off suddenly. _Löffler's serum_ -is made by adding one part of dextrose broth to three parts of serum -and then coagulating as above. The solidified serum in either case is -best sterilized discontinuously, though with care the autoclave at 15 -pounds pressure may be used for a single sterilization. This is very -apt to cause a greater darkening of the serum and frequently also a -laceration of the solid mass by escaping gases. - -Blood serum is also used in the liquid state. For this purpose it is -best to collect it aseptically; or it may be sterilized discontinuously -at a temperature of 55° or 56° on seven to ten consecutive days. Novy -has recently suggested dialyzing the serum to free it from salts and -thus prevent its coagulation when heated. Whether the removal of the -various "extractives" which diffuse out with the salts deprives the -serum of any of its advantageous properties remains to be ascertained. - -From the discussion of the physiological activities of bacteria in -Chapters IX-XII it is apparent that a very great variety of culture -media other than those described is necessary for the study of special -types of bacteria, but such media are beyond the scope of the present -work. - -The ideal culture media are without a doubt the _synthetic media_, that -is media of definite known chemical composition, so that the various -changes due to the growth of bacteria can be accurately determined -and thus a means of sharply differentiating closely related organisms -be secured. Such media have been prepared and every bacteriologist -believes strongly in their future usefulness when media of wider -application shall have been devised. An example of this type of culture -media is Uschinsky's synthetic medium, of which the following is one of -the modifications: - - Distilled-water 1000 parts - Asparagin 4 " - Ammonium lactate 6 " - Disodium phosphate 2 " - Sodium chloride 5 " - -A criticism of this medium is that the elements K, Ca, Mg, Fe, Mn, and -S which have been shown to be essential are not present if chemically -pure salts are used in the preparation. - - - - -CHAPTER XVII. - -METHODS OF USING CULTURE MEDIA. - - -The way in which culture media shall be used depends on the purpose -in view. By far the larger part of bacteriological work is done with -cultures in "bacteriological culture tubes." Various laboratories have -their own special types but all are more or less after the "Board of -Health" form. They differ from ordinary chemical test-tubes in that -they are usually longer, have no "lip" and have much thicker walls to -prevent breakage and consequent loss of the culture as well as danger -from pathogenic organisms. The author finds two sets of tubes most -serviceable for student use--one size 15 cm. long by 19 mm. outside -diameter (No. 9, Fig. 119), the other 15 cm. long by 13 mm. (Nos. 1 to -7, Fig. 119). Culture tubes are conveniently used in "wire baskets" -circular or square in section and of a size to correspond with the -length and number of tubes used. These baskets are light, do not break, -and if made of good galvanized wire netting do not readily rust (Figs. -117 and 118). - -Liquid media such as broth, milk, litmus milk, indol and nitrate broths -are used in the above-mentioned tubes when small quantities only are -to be worked with. The tubes are filled approximately one-third full, -then plugged with _non-absorbent_ cotton and sterilized. _Cotton plugs_ -are used so much in bacteriological work because they permit a free -circulation of air and gases and at the same time act as filters to -keep out the bacteria of the air. - -Sugar broths or other media in which gas may be produced are used in -fermentation tubes (Smith tubes) of the type shown in Fig. 120 so that -the gas may be collected in the closed arm of the tube, measured (Fig. -121) and tested if desired. - -[Illustration: FIG. 117.--Round wire basket.] - -[Illustration: FIG. 118.--Square wire basket.] - -[Illustration: FIG. 119.--Culture tubes with media in them. × 2/3. _1_ -to _7_ are the smaller tubes mentioned in the text; _9_ the larger -tube; _8_ is extra large for potato tubes; _1_, plain broth; _2_, plain -milk; _3_, litmus milk; _4_, gelatin for "stab" or "puncture" culture; -_5_, agar for "stab" or "puncture" culture; _6_, agar for "slope" or -"slant" culture; _7_, blood serum; _8_, potato tube; _9_, agar for -plating. Note the transparency of the broth and gelatin and the slight -opalescence of the agar.] - -One method of using gelatin and also agar is as "puncture" or "stab" -cultures. The tubes (the narrower tubes are to be preferred for most -"stab" cultures) are filled one-third full of the medium while it is -still fluid, plugged, sterilized and allowed to cool in the vertical -position. The medium is then "inoculated" with a _straight_ platinum -needle by plunging this into the center of the surface down to the -bottom of the tube (Fig. 119, Nos. 4 and 5). - -[Illustration: FIG. 120.--Fermentation tubes. _1_, filled ready for -use; _2_, shows a cloudy growth and the development of gas in the -closed arm.] - -Agar and blood serum are frequently used in the form of "slope" or -"slant" cultures. That is, the medium solidifies with the tubes lying -on their sides which gives a long, sloping _surface_ on which the -bacteria are inoculated (Fig. 119, Nos. 6 and 7). - -[Illustration: FIG. 121.--Method of estimating percentage of gas in a -fermentation tube by means of the "gasometer", the reading is 45 per -cent.] - -[Illustration: FIG. 122.--A toxin flask showing a large surface -growth.] - -Potato tubes are likewise used for "slant" or "slope" cultures (Fig. -119, No. 8). Potatoes as "plate cultures" have been referred to. Agar -and gelatin are very largely used in the form of "plate cultures" -also. For this purpose Petri dishes are first sterilized, then the -melted agar or gelatin poured into them and allowed to "set" while the -plates are kept horizontal. The melted media may be "inoculated" before -they are poured, or a portion of the material to be "plated" may be -placed in the dish, then the melted medium poured in and distributed -over the dish by tilting in various directions, or the medium after -solidifying may be inoculated by "strokes" or "streaks" over its -surface, according to the purpose in view in using the plate. The -larger sized tubes should be used for making plates in order to have -sufficient medium in the plate (No. 9, Fig. 119). - -For using large quantities of medium, Florence flasks, Ehrlenmeyer -flasks, special toxin flasks (Fig. 122) or various other devices -(Vaughan and Novy's "mass cultures," Figs. 123 and 124) have been -employed. - -For growing _anaërobic organisms_ it is evident that some method for -removing and excluding the oxygen of the air must be used. A very great -variety of appliances have been devised for these purposes. Some are -based on the principle of the vacuum, exhausting the air with an air -pump; some on replacing the air with a stream of hydrogen; others on -absorbing the oxygen by chemical means, as with an alkaline solution -of pyrogallic acid, or even by growing a vigorous aërobe in the -same culture or in the same container with the anaërobe, the aërobe -exhausting the oxygen so that the anaërobe then develops, or finally -by excluding the air through the use of deep culture tubes well filled -with the medium, or in the closed arm of fermentation tubes. For many -purposes a combination of two or more of the above methods gives good -results. - -In any event the culture medium should have been _freshly sterilized_ -just before use, or _should be boiled_ in order to drive out the -dissolved oxygen. For most, anaërobes the presence in the medium of -about 1 per cent. of a carbohydrate, as dextrose, is advisable. - -A description of all the various devices is unnecessary in this work, -but the following have answered most of the purposes of general work in -the author's laboratories. - -[Illustration: FIG. 123.--Tank with raised lids. (Vaughan.)] - -[Illustration: FIG. 124.--Tank with lids lowered. (Vaughan.) - -FIGS. 123 and 124.--Vaughan and Novy's mass culture apparatus.] - -_A._ "_Vignal tubes_" of the style shown (Fig. 125) are made from -glass tubes of about 6 to 8 mm. outside diameter, sealed at the small -end, plugged with cotton above the constriction and sterilized. The -medium, agar or gelatin, which has been previously inoculated with the -anaërobic culture, is then drawn up into the tube, after breaking off -the tip, as far as the constriction. The tube is then sealed in the -flame at the small end and also at the constriction. Since it is full -of the medium and sealed, access of air is prevented. This forms an -excellent means for "isolation" (Chapter XVIII); the tube needs merely -to be cut with a file at the point where colonies appear, then these -may be readily transferred. - -[Illustration: FIG. 125.--Vignal tubes. × 1/3 _1_, the sterile tube -ready for inoculation; _2_, fourth dilution tube showing a few isolated -colonies, one near the figure; _3_, third dilution showing colonies -isolated but numerous; _4_, second dilution tube showing colonies still -more numerous; _5_, first dilution tube showing colonies so numerous -and small as to give a cloudy appearance to the tube. In use tube _2_ -would be filed in two at the colony and inoculations made from it.] - -_B._ "_Fermentation tubes_" form a simple means for growing liquid -cultures of anaërobes, the growth occurring in the closed arm only, -while with facultative anaërobes, growth occurs both in the closed arm -and in the open bulb. A little "paraffin oil" (a clear, heavy petroleum -derivative) may be poured on the fluid in the open bulb as a very -efficient seal, though it is not usually necessary. - -_C._ "_Deep culture tubes._"--The medium, agar, gelatin or a liquid is -poured into tubes until they are approximately one-half full, a little -paraffin oil is poured on the surface (not essential always), then the -tubes are plugged and sterilized. Inoculation is made to the bottom -and anaërobes grow well (Fig. 126). - -[Illustration: FIG. 126.--Deep tubes showing anaërobic growth. _1_, -shows a few small gas bubbles; _2_, shows the medium broken up by the -excessive development of gas.] - -_D._ For slope or plate, or any type of surface cultures the Novy jar -(Fig. 127) is the most practical device. It is good practice to combine -the vacuum method, the hydrogen replacement method and the oxygen -absorption method in using these jars. In operation a solution of 20 -per cent. NaOH is poured on the bottom of the jar to a depth of 1 or 2 -cm., the cultures are placed on glass supports above the alkali and a -short wide tube of strong pyrogallol is set in on the bottom in such a -way that it may be easily upset and mixed with the alkali when it is -desired to do so. The cover is now clamped in position with all joints -well vaselined. Then the outlet tube is connected with a suction pump -and the air drawn out. Meanwhile the inlet tube has been connected -with a hydrogen generator, and after the jar is exhausted hydrogen is -allowed to flow in, and this process is repeated until one is satisfied -that the air is replaced. The suction exhausts the air from the tubes -or plates so that much less time is required to replace the air with -hydrogen. Finally the stop-cock is closed, and the pyrogallol solution -is gently shaken down and mixed with the alkali so that any remaining -oxygen will be absorbed. - -[Illustration: FIG. 127.--Novy jars.] - -It must be remembered that facultative anaërobes as well as anaërobes -will grow under any of the above conditions, so that cultures of -organisms so obtained must be further tested aërobically in order to -determine to which group the organisms belong. - -Reference has been made above to the "inoculation" of culture media, -which means introducing into the medium used the desired material in -the proper way. For small quantities this is most conveniently done -with platinum "needles," that is, pieces of platinum wire inserted -into the ends of glass rods. The "straight" needle is a piece of -heavy platinum wire of about 0.022 inch in diameter (Fig. 128). It is -used most frequently to inoculate all forms of _solid media_. The -platinum loop is of lighter wire, 0.018 inch. The loop in the end is -conveniently made by twisting the wire around the lead of an ordinary -lead-pencil. The "loop needle" (Fig. 129) is most used in transferring -liquid media. On account of the high price of platinum, the author -has substituted "nichrome" wire for student use. This is stiffer, not -so easily made into loops and breaks out of the rods more easily. -The latter defect is remedied to some extent, by imbedding the wire -only slightly for about one-fourth of an inch on the side of the end -portion of the rod. The low cost, less than one-twentieth of platinum, -justifies its use. - -[Illustration: FIG. 128.--Straight needle.] - -[Illustration: FIG. 129.--Straight and loop needles.] - -[Illustration: FIG. 130.--Pasteur flask--"ballon pipette."] - -Sterile graduated pipettes varying in capacity from 1 cc graduated in -hundredths, upward, permit the transfer of definite amounts of liquids. -Large quantities are conveniently transferred by means of Pasteur -flasks (Fig. 130). The details of inoculation are best derived from -laboratory practice. - - - - -CHAPTER XVIII. - -ISOLATION OF BACTERIA IN PURE CULTURE. - - -As has been stated, the thorough study of a bacterium depends on -first getting it in pure culture. In the early days of bacteriology -supposedly pure cultures were obtained by (1) _dilution in liquid -media_. A series of tubes or flasks containing sterile liquid media was -prepared. Number one was inoculated with the material to be examined -and thoroughly mixed. A small portion of the mixture was transferred -to number two, and mixed; from this to number three, and so on until a -sufficient number were inoculated, the last three or four in the series -receiving the same amounts of a very high dilution of the original -material. If one or two of these latter showed a growth and the others -not, it was assumed that the dilution had been carried so far that only -a single organism was transferred and therefore the culture obtained -was "pure." The method in this crude form is too uncertain to be of -value today and recourse is had to more exact means. The procedure most -widely used is that of (2) "_plating out_" by means of gelatin or agar -plates. The material to be plated out is diluted by transferring to -three or more tubes of melted gelatin or agar as in the first method -and then all the tubes are poured into Petri dishes and grown under -suitable conditions. By proper mixing in the tubes the bacteria are -well scattered through the medium which holds the individual organisms -separate when it solidifies. On some of the plates a sufficient -dilution will be reached so that the colonies developing from the -bacteria will be so few that they are separate and pure cultures may -be obtained by inoculating from one of these a tube of the appropriate -medium (Figs. 131 to 134). The chief uncertainty with this method is -that occasionally two kinds of bacteria stick together so closely that -even the separate colonies contain both organisms. This is not common, -however. The plate colonies frequently develop from groups of bacteria -which were not separated, but as these are of the same kind the culture -is essentially pure. - -[Illustration: FIG. 131.--Dilution plates. × 3/10. _1_, shows the -first dilution, the colonies are so numerous and small that they -are invisible (compare Fig. 132); _2_, shows fewer and hence larger -colonies, but too crowded to isolate (compare Fig. 133); _3_, shows the -colonies larger and well separated, so that it is easy to isolate from -them (compare Fig. 134).] - -[Illustration: FIG. 132.--A portion of plate _1_ in Fig. 131 as seen -under the low-power objective. × 100. Very small, closely crowded -colonies.] - -Another method which is frequently applicable with material from human -or animal sources is to (3) _rub the material over the surface_ of a -slope tube or of medium solidified in a Petri dish with a sterile heavy -platinum needle, glass rod, or cotton swab. If the bacteria are not -too numerous, pure cultures may frequently be obtained. A modification -of this method is to make a series of (4) _parallel streaks on a -slope tube or plate of medium_ with a needle inserted _but once_ into -the material to be plated. On the first streak most of the bacteria -are rubbed off and a continuous growth results, but usually on the -last of a series only isolated colonies appear, which are presumably -pure. The ideal method for securing pure cultures is to be absolutely -certain that the culture starts from a single organism. This may be -accomplished by means of the (5) _apparatus and pipettes devised by -Professor Barber_ of the University of Kansas (Figs. 135 and 136). With -this instrument a single organism is picked out under the microscope -and isolated in a drop of culture medium and observed until it is -seen to divide, thus proving its viability. Transfers are then made -to the proper media. The method requires much practice to develop the -necessary skill in the making of pipettes, determining the proper -condition of the large cover-glasses used over the isolating box, and -in manipulation, but the results fully compensate. - -[Illustration: FIG. 133.--From the thinnest part of plate _2_, Fig. 131 -as seen under the low-power objective. × 100. Colonies much larger than -on plate _1_, but still crowded.] - -[Illustration: FIG. 134.--The smallest colony on plate _3_, Fig. 131, -as seen under the low-power objective. × 100. Large, single, isolated -colony.] - -Professor W. A. Starin of the author's department, a former student of -Professor Barber, has done some excellent work with this apparatus. - -[Illustration: FIG. 135.--Diagram of Barber's isolation apparatus. _b_, -moist chamber; _ms_, large cover-glass over moist chamber; _p_, small -pipette drawn out to a fine point; _k_, _r_, _g_, pipette holder; _f_, -screw for raising and lowering _k_, _r_, _g_; _s_, screw for lateral -motion of _k_, _r_, _g_; _n_, screw for clamp on pipette which allows -it to be moved in or out; _m_, mechanical stage of microscope; _t_, -rubber tube held in the mouth and used to move the liquid culture -medium in the pipette. (Journal of Infectious Diseases, October 20, -1908, vol. 5, No. 4, p. 381.)] - -[Illustration: FIG. 136.--Photograph of microscope with Barber's -isolation apparatus set up to use.] - -A number of procedures may be used to greatly facilitate the above -methods of isolation by taking advantage of the different physiological -properties of different organisms in a mixture such as ability -to form spores, different resistance to antiseptics, special food -requirements, and pathogenic properties. (_a_) If material contains -resistant spores, it may be _heated to temperatures high_ enough to -kill all of the organisms except the spores (80° for half an hour, for -example) and then plated out. Or (_b_) _an antiseptic which restrains -the growth_ of some organisms and not others may be placed in the -culture media (carbolic acid, various anilin dyes, (p. 162), excess -acid, or alkali, ox bile, etc.), when the more resistant organisms grow -on the final plates, the others not. (_c_) _Special food substances_ -(various carbohydrates) from which the organism desired forms special -products (acids, aldehydes) that may be shown on the plates by various -indicators, is one of the commonest means. Or media in which certain -organisms thrive and others not, so that the former soon "crowd out" -the latter (unsterilized milk for lactic acid bacteria, inorganic -media in soil bacteriology) may be used. A combination of the general -methods (_b_) and (_c_) is much used in the separation of the organisms -of the "intestinal group" in human practice. (_d_) _The inoculation -of a susceptible animal_ with a mixture suspected to contain a given -pathogenic bacterium frequently results in the development of the -latter in pure culture in the body of an animal, from which it may be -readily recovered. In all of the above methods (except Barber's) the -first "pure culture" obtained should be "purified" by replating in a -series of dilution plates to make sure that it is pure. - - - - -CHAPTER XIX. - -STUDY OF INDIVIDUAL BACTERIA--STAINING. - - -When an organism has been obtained in pure culture by any of the -methods described in the preceding chapter the next step is the study -of its morphology as discussed in Chapters II--IV. This involves the -use of the microscope, and since bacteria are so small, objectives -of higher power than the student has presumably used will be needed. -Doubtless only the two-thirds inch or 16 mm. and the one-sixth inch or -4 mm. objectives are all that have been used in previous microscopic -work, while for examining bacteria a one-twelfth inch or 2 mm. is -necessary. It will have been observed that the higher the power of -the objective the smaller is the front lens or object glass and -consequently the less is the amount of light which enters. With the use -of the one-twelfth inch or 2 mm. objective it is necessary to employ -two devices for increasing the amount of light entering it, with which -the student is probably not familiar. One of these is to place a drop -of cedar oil between the front lens and the object and to immerse the -lens in this oil--hence the term "oil-immersion objective;" the other -is the substage or Abbé condenser. The latter is a system of lenses -placed below the stage and so constructed as to bring parallel rays of -light--daylight--from an area much larger than the face of the front -lens of the objective to a focus on the object to be examined, thus -adding very greatly to the amount of light entering the objective. -Since the condenser brings _parallel_ rays to a focus on the object, -the _flat-mirror_ is always used with the condenser when working with -daylight. With _artificial light close_ to the microscope, the concave -mirror may be used to make the divergent rays more nearly parallel and -thus give better illumination. - -The function of immersion oil is to prevent the dispersion of -considerable light that would otherwise occur owing to refraction -as the light passes up through the slide and into the air. The -accompanying diagram will help to make this clearer (Fig. 137). A ray -of light (_A B_) coming through the slide will be refracted in the -direction _B C_ if the medium has a lower refractive index than the -slide, as air has, and hence will not enter the objective _O_. If, -however, there is interposed between the objective and the slide a -medium which has the same refractive index as the slide, as immersion -oil has, then the ray will continue in the same direction (_B D_) at -the point _B_ and hence enter the objective. Evidently the immersion -oil causes much more light to enter the front lens and makes the field -brighter and at the same time prevents considerable refraction and -dispersion of light from the object seen and hence this appears more -distinct and sharply defined. The Abbé condenser and the oil-immersion -objective are practically always used in the microscopic study of -bacteria (Fig. 138). - -[Illustration: FIG. 137.--Diagram of use of immersion oil.] - -[Illustration: FIG. 138.--Diagram of paths of rays of microscope.] - - -HANGING DROP SLIDE. - -It is sometimes necessary to examine living bacteria and for this -purpose the device known as the "hanging drop slide" is used (Fig. -139). The slide has a slight concave depression ground in the middle -of one face. A ring of vaseline is placed around this depression with -the loop needle. On a clean cover-glass, large enough to fit over the -ring of vaseline, several drops of a broth culture, or of material -from a solid culture suspended in broth or physiological normal salt -solution are placed. The slide is inverted on the cover-glass in such -a way that the ring of vaseline seals the latter to the slide. When -the whole preparation is quickly turned cover side up, the drops are -seen "hanging" to the under side of the cover over the depression in -the slide. In examining such a preparation with the microscope great -care is necessary in order to focus on the bacteria, without breaking -the cover. To see the organisms distinctly the _lower iris diaphragm of -the condenser must be nearly closed_, so that the light coming through -consists mainly of parallel vertical rays, otherwise the transparent -bacteria themselves refract and diffract the light and appear blurred -and indistinct. By studying living bacteria with this device it can -be determined whether they are motile or not. The motility should not -be confounded with the familiar "Brownian movement" of all minute -insoluble inert particles which non-motile living bacteria and -also dead bacteria show. The hanging drop slide is of value in the -measurement of bacteria, since this is properly done on the living -organism. Measurement is done with a calibrated ocular micrometer as in -other kinds of measurement with the microscope with which the student -is presumably familiar. The direct effect of various agents on living -bacteria as light, electricity, heat, etc., in the study of "tropisms" -and "taxes" has been investigated on various modifications of the -above-described hanging drop slide. - -[Illustration: FIG. 139.--Hanging drop slide.] - -Cell forms and cell groupings may be studied in the same way but -these features are best determined on _stained_ preparations in many -instances. - -"Dark field" illumination and the ultramicroscope are of great value in -the study of living bacteria and other minute objects, but apparatus -of this type would scarcely be used by the student in an introductory -course, so that they will not be discussed in the present volume. - - -STAINING. - -The main use of the microscope in bacteriology is in the study of -_stained preparations_ of the organisms. Staining makes bacteria opaque -and hence more easily seen than the transparent unstained forms. Some -methods of staining also show morphological structures which are either -imperfectly recognized in the unstained cell, spores, or are not -visible at all--capsules, metachromatic granules, flagella. Finally -certain bacteria are colored by special methods of staining which do -not affect others, so that under proper conditions these bacteria may -be recognized by staining methods alone--tubercle bacilli in the organs -of animals. - -The phenomena of staining are essentially chemical, though sometimes -the chemical union is a very weak one, even resembling an absorption of -the dye rather than true chemical union--most watery stains. In other -cases the chemical compounds formed are decidedly stable and are not -decomposed even by strong mineral acids--staining of tubercle bacilli -and other "acid-fast" organisms. In still other cases the principal -action is a precipitation on the surface of the object stained--methods -for staining flagella. - -In many methods of staining in addition to the dyes used other -substances are added to the solution which assist in fixing the dye in -or on the organism stained. Such substances are called _mordants_. The -principal mordants used are alkalies, anilin, carbolic acid, iodine, -metallic salts, tannic acid. - -While it is true that some bacteria may be stained by that standard -histological nuclear dye, hematoxylin, it is of little value for this -purpose. Practically all bacteriological stains are solutions of the -_anilin dyes_. These dyes, as is well known, are of nearly every -conceivable color and shade but relatively very few are used in -bacteriological work. The beginning student will rarely use solutions -of other than the three dyes _fuchsin_ (red), _methylene blue_ and -_gentian violet_ for staining bacteria, with occasionally Bismarck -brown, or eosin, or safranin as tissue contrast stains. - -The bacteriological dyes are kept "in stock" as saturated solutions in -95 per cent. alcohol which are _never used as stains_, but merely for -convenience in making the various staining solutions. - -The approximate percentages of the three common dyes in such solutions -are indicated in the following table adapted from Woods _Chemical and -Microscopical Diagnosis_, Third Edition, 1917, Appendix: - - Fuchsin 3.0% - Gentian Violet 4.8% - Methylene Blue 2.0% - -The stains made from these dyes which are in most common use are the -following: - - 1. Aqueous (watery) gentian violet solution. - - Saturated alcoholic solution of gentian violet 1 part - Distilled water 20 parts - Mix well and filter. - - 2. Anilin gentian violet. - - Saturated alcoholic solution of gentian violet 1 part - Anilin water (see below) 10 parts - Mix well and filter. - - 3. Anilin Fuchsin. - - Saturated alcoholic solution of fuchsin 1 part - Anilin water (see below) 10 parts - Mix and filter. - -These stains rarely keep longer than ten days in the laboratory (unless -kept in the ice-box) and must be made fresh on the first sign of a -deposit on the glass of the container. - -=Anilin Water.=--Anilin water is made by putting 3 or 4 cc of anilin -"oil" in a 120 cc. flask, adding 100 cc of distilled water, shaking -vigorously for a minute or so and filtering through a wet filter, in -other words, a saturated solution of anilin in water. - - 4. Löffler's (methylene) blue. - - Saturated alcoholic solution of methylene blue 3 parts - Aqueous solution of NaOH (or KOH), 1 to 10,000 10 " - Mix and filter. - - 5. Carbol-fuchsin (Ziehl's solution). - - Saturated alcoholic solution of fuchsin 1 part - 5 per cent. aqueous solution of carbolic acid 10 parts - Mix and filter. - - 6. Gabbet's (methylene) blue (solution). - - Dry methylene blue 4 parts - Concentrated H{2}SO{4} 25 " - Distilled water 75 " - Dissolve the dry dye in the acid and add the solution to the - distilled water and filter. - -[Illustration: FIG. 140.--Author's staining set. Square bottles are set -in square holes in the block. The capacity of each bottle is 30 cc.] - -Staining solutions are conveniently kept in square dropping bottles -inserted in a block as shown in Fig. 140. This form of holder -necessitates the use of _one hand only_ in securing the stain and -dropping it on the preparation. - -The actual staining of bacteriological preparations can be learned only -by repeated laboratory practice, yet the following methods have given -such uniform results in class work that it is felt they are not out of -place in a text-book. - -=Preparation of the "Film."=--The author learned to stain bacteria, -on the "cover-glass" but does not recall having used this method in -fifteen years and does not teach it to his students. All staining is -done on the slide. To prepare a film from a solid culture medium the -procedure is as follows: - -First, be sure the slide is clean and _free from grease_. This is -accomplished most readily by scouring a few minutes with finely -ground pumice stone and a little water, then washing and drying with -a grease-free cloth, handkerchief, or piece of cheese-cloth. With the -"loop" needle place in the middle of the slide a small loop of water. -This is best done by filling the loop by dipping in water, then tapping -it gently so that all that remains is the water that just fills the -loop level full, and this amount is placed on the slide by touching -the flat side of the loop to the glass. Then the _straight needle_ is -sterilized, dipped into the culture and just touched once into the -small drop of water on the slide. The remainder of the culture on the -straight needle is then burned off and the needle is used to spread the -drop of water containing the bacteria into a thin even film, which will -result, provided the slide is free from grease. This is dried and then -"fixed" by passing three times through the Bunsen flame at intervals of -about one second, passing through slowly for thick slides and a little -more rapidly for thin ones. If the culture is in a liquid medium, the -use of the loop of water is unnecessary; a loop of the fluid from the -surface, middle or bottom as the culture indicates is spread out to a -thin film, dried and fixed. - -After the film is fixed the stain desired is dropped on, allowed -to act for the proper time, which will depend on the stain and the -preparation, washed in water, dried thoroughly and examined with the -oil-immersion lens, without a cover. If it is desired to preserve the -preparation it may then be mounted in balsam. This is not necessary, as -they keep just as well, provided the immersion oil is removed. To do -this, fold a piece of filter paper so that at least three thicknesses -result. Lay this on the slide and press firmly several times, when the -surplus oil will be taken up by the paper. Slides not mounted in balsam -are more apt to become dusty than those that are. This is the only -disadvantage. - -=Gram's Method of Staining.=--It has been ascertained that some -bacteria contain a substance, possibly a protein, which forms a -compound with gentian violet and iodine, which compound is insoluble in -alcohol, and other bacteria do not contain this substance. Consequently -when bacteria are stained by Gram's method (given below), those that -contain this chemical remain colored, while if it is not present the -dye is washed out by the alcohol and the bacteria are colorless and may -be stained by a contrast stain. The bacteria which stain by this method -are said to "take Gram's" or to be "Gram-positive," while those that -decolorize are called "Gram-negative." The method is: - -1. Prepare the film as above given. - -2. Stain with fresh anilin gentian violet 1 minute. - -3. Wash in tap water. - -4. Cover with Gram's solution 1 minute. - -5. Wash in tap water. - -6. Wash with 95 per cent. alcohol three times or until no more color -comes out. - -7. Dry and examine. - -Gram's solution is: - - I 1 part - KI 2 parts - H{2}O 300 " - -This method is excellent for differentiating Gram-positive and -Gram-negative organisms on the same slide. First stain by this -method and after washing with alcohol stain with a counter-stain, -carbol-fuchsin diluted ten to fifteen times with water is excellent. -The Gram-positive bacteria are violet and the Gram-negative are red. - -It is also of great value in staining Gram-positive bacteria in -tissues, but the sections should be stained about five minutes in -the anilin gentian violet and be left about two minutes in the Gram's -solution. Sections are to be counter-stained in Bismarck brown, dilute -eosin or safranin solutions and cleared in oil of bergamot, lavender or -origanum and not in clove oil or carbol-xylol, as these latter dissolve -out the dye from the bacteria. - -=Staining of Spores in the Rod.=--Prepare the films as usual. Cover -with carbol-fuchsin, using plenty of stain so that it will not dry on -the slide; heat until vapor arises, not to boiling; cool until the -stain becomes cloudy and heat again until the stain clears, and repeat -once more; wash in tap water and then wash in 1 per cent. H{2}SO{4} -three times, dropping on plenty of acid, tilting and running this -over the slide three times and then pour off and use fresh acid and -repeat this once. Wash thoroughly in _distilled_ water, then stain with -Löffler's blue one to three minutes. Wash, dry and examine. The spores -should be bright red in a blue rod. - -This method will give good results if care is taken to secure cultures -of the right age. If the culture is too old the spores will all be free -outside the rods, while if too young they will decolorize with the -acid. For _Bacillus subtilis_ and _Bacillus anthracis_, cultures on -agar slants forty-eight hours in the 37° incubator are just right. For -the spores of _Clostridium tetani_, the culture should be three days -old, but may be as old as a week. - -=Staining of "Acid-fast" Bacilli.=--_Mycobacterium tuberculosis, -Mycobacterium of Johne's disease, "grass" and "butter bacilli," -Mycobacterium lepræ, Mycobacterium smegmatis._ - -_Gabbet's method_: - - 1. Prepare the film as usual. - - 2. Stain with carbol-fuchsin as given above for spores. - - 3. Wash with tap water. - - 4. Decolorize and stain at the same time with Gabbet's blue, two or - three minutes. - - 5. Wash, dry and examine. - -The sulphuric acid in Gabbet's blue removes the carbol-fuchsin from -everything except the "acid-fast" bacteria, which remain red, and the -blue stains the decolorized bacteria and nuclei of any tissue cells -present. - -_Ziehl-Neelson method_: - - 1, 2, 3, as in Gabbet's method. - - 4. Decolorize with 10 per cent. HCl until washing with water shows - only a faint pink color left on slide. - - 5. Wash thoroughly. - - 6. Stain with Löffler's blue one or two minutes. - - 7. Wash, dry and examine. - -The results are the same as with Gabbet's method. - -=Staining of Capsules.=--_Räbiger's Method._--Films of the organism -to show capsules should be _freshly prepared, dried but not fixed_. -Material is usually obtained from milk or blood. A drop of the fluid -is placed on the middle of a slide about one-fourth of the distance -from one end. The narrow edge of another clean slide is placed in this -drop and then drawn lengthwise across the slide with firm pressure. -This gives a _thin layer_ which is necessary if good results are to -be expected. The preparation is covered with a _freshly prepared_ -saturated solution of gentian violet in formalin and this allowed -to stain for 30 seconds. Then wash _lightly_, dry and examine. The -organisms appear deeply violet and much larger than with ordinary -stains and capsules are well stained and show well. - -_Welch's Method._--Prepare films as in the above method. Cover with -glacial acetic acid for 10 to 20 seconds. Wash off the acid with -carbol-fuchsin. Wash the stain off with physiological normal salt -solution (0.85 per cent.) until all surplus stain is removed. Dry and -examine. Capsules and bacteria are red. - -=Staining of Flagella.=--The rendering of flagella visible is -considered one of the most difficult processes in staining. Experience -of a number of years during which whole classes numbering from one -hundred to three hundred students accomplish this result shows that it -is no more difficult than many other staining processes. The essentials -are: (1) clean slides, (2) young cultures on agar slopes, (3) freshly -prepared mordant and stain which are kept free from precipitate, -(4) gentle heating. The author's students are furnished only stock -materials and make their own cultures, mordants and stains. - -The slides are cleaned with pumice in the usual way. An agar slope -culture of the organism to be stained from six to twenty-four hours -old is selected. A bit of the culture is removed and placed in a -watch-glass of water. The bacteria are allowed to diffuse of themselves -without stirring. After several minutes a loop of this water is removed -and three streaks are made across the slide, one in the middle and one -on each side of this about one-quarter of an inch from it. This gives -well scattered bacteria in one of the three streaks at least and very -little other material on the slide to cause precipitates. The slide is -carefully dried and fixed and then covered with an abundance of the -mordant by filtering through a small filter onto the slide so that the -mordant shows transparent on the slide. The preparation is then gently -warmed and cooled three times, adding mordant if necessary. _Do not -heat to steaming._ After mordanting for about five minutes the excess -is washed off under the tap. It is a good plan to hold the slide level -and allow the water to run into the center of the mordant and flow it -off. Inclining the slide is apt to cause the film on the surface of the -mordant to settle down on the slide and spoil the preparation. After -the mordant is washed off and all traces of it removed with a clean -cloth if necessary the stain is applied and gently heated and cooled -the same way for from three to five minutes. The preparation is then -washed, dried and examined. - -The mordant used is a modification of Löffler's which is somewhat -simpler in preparation since the stock solution of FeCl{3} is more -permanent than FeSO{4} solution. - -Mordant sufficient for one student: - - 5 per cent. solution of FeCl{3} 20.0 cc - 25 per cent. solution of tannic acid 20.0 cc - Anilin fuchsin 4.0 cc - Normal NaOH 1.5 cc - -The solution of FeCl{3} is made up in the cold and must be perfectly -clear. The tannic acid solution must be thoroughly boiled and filtered -until clear. The iron and the acid are carefully mixed, boiled and -filtered clear. The anilin fuchsin must be added slowly with constant -stirring and the mixture boiled and filtered. The NaOH is added in the -same way and this mixture boiled and filtered. The final mordant should -not leave a film on a clean slide when poured on and allowed to run -off. Unless the mordant is in this condition and perfectly clear, it -should not be used, but a new one must be made up. Time and care in the -preparation of the mordant are essential. - -The stain to follow this mordant is anilin fuchsin. - -=Staining of Metachromatic Granules.=--_Neisser's Method._ Prepare the -film in the usual way. Stain with Neisser's stain a few seconds only. -Wash and stain with Bismarck brown a few seconds only. - - _Neisser's Stain_: - - Sat. alcoholic solution of methylene blue 1.0 part - Glacial acetic acid 2.5 parts - Distilled water 50.0 parts - - _Bismarck Brown_: - - Bismarck brown (dry dye) 2 parts - Distilled Water 1000 parts - -By the use of the hanging drop slide and the methods of staining just -described all the various morphological features of the bacterial cell -may be ascertained. - -It is necessary when _cell groupings_ as characteristic of definite -modes of division are to be determined to make slides from a liquid -culture, as broth. Place a drop of the material, preferably from the -bottom of the tube in most instances, from the top in case a pellicle -or scum is formed on the surface, on the slide and allow this to dry -_without spreading it out_, fix, wash gently with water, then stain -lightly with Löffler's blue. Such slides also show characteristic -_cell forms_ as well. Slides should be made from solid media to show -variations in form and size and involution forms. These latter are -especially apt to occur on potato media. - - - - -CHAPTER XX. - -STUDY OF THE PHYSIOLOGY OF BACTERIA. - - -Of the environmental conditions influencing the growth of bacteria the -following are the chief ones ordinarily determined: - -_A._ Temperature.--The optimum temperature for growth is usually -about the temperature of the natural environment and ordinarily one -determines merely whether the organism grows at body temperature (37°) -and at room temperature (20°) or not. For exact work the maximum, -minimum and optimum temperature must be ascertained by growing in -"incubators" with varying temperatures. - -A bacteriological incubator is an apparatus for growing bacteria at a -constant temperature. This may be any temperature within the limits for -bacterial growth. If temperatures above that of an ordinary room are -desired, some source of artificial heat is needed. Electricity, gas or -oil may be used. A necessary adjunct is some device for maintaining -the temperature constant, a "thermoregulator" or "thermostat." For -lower temperatures a cooling arrangement must be installed. For the -great part of bacteriological work only two temperatures are used, 20° -so-called "room temperature" (this applies to European "rooms" not to -American) and 37° or body temperature. Incubators for 37° of almost any -size and style desired may be secured from supply houses and need not -be further described. Figs. 141 and 142 illustrate some of the types. - -For use with large classes "incubator rooms" are to be preferred. The -author has one such room for 37° work with 200 compartments for student -use which did not cost over $60 to install. - -[Illustration: FIG. 141.--Small laboratory incubator, gas heated.] - -[Illustration: FIG. 142.--Electric incubator.] - -The styles of incubators for lower temperatures, 20° and below, are not -so numerous nor so satisfactory. The author has constructed a device -which answers every purpose for a small class. The diagram, Fig. 143, -explains it. - -[Illustration: FIG. 143.--Diagram of fittings for a cold incubator. -_1._ small tank for constant head, about 1 foot in each dimension. _a_, -inflow; _b_, overflow; _c_, lead pipe. _2_, refrigerator. _a'_, ice; -_b'_, flat coil under ice; _c'_, outflow to incubator. _3_, incubator. -_a"_, cold water inflow; _b"_, overflow; thermometer and burner -omitted. The diagram explains the construction. The water cooled to -about 14° with artificial ice by flowing through the lead coil under -the ice, flows into the incubator which may be heated and regulated in -the usual way.] - -The thermal death-point is determined by exposing the organisms in -thin tubes of broth at varying temperatures for ten-minute periods and -then plating out to determine growth. The effect of heat may also be -determined by exposing at a given temperature, _e.g._, 60°, for varying -lengths of time and plating out. - -_B._ Oxygen relations--whether the organism is aërobic, anaërobic, or -facultative is determined by inoculation in gelatin or agar puncture or -stab cultures and noting whether the most abundant growth is at the -top, the bottom or all along the line of inoculation. - -_C._ Reaction of the medium--acid, alkaline or neutral as influencing -the rate and amount of growth. - -_D._ The kind of medium on which the organism grows best. - -_E._ The effect of injurious chemicals, as various disinfectants, on -the growth. - -_F._ Osmotic pressure conditions, though modifying decidedly the growth -of bacteria, are not usually studied as aids in their recognition, nor -are the effects of various forms of energy, such as light, electricity, -_x_-rays, etc. - -Among the "Physiological Activities" discussed in Chapters IX-XII those -which, in addition to the staining reactions described, are of most -use in the identification of non-pathogenic bacteria are the first ten -listed below. For pathogenic bacteria the entire thirteen are needed. - -1. Liquefaction of gelatin. - -2. Digestion of blood serum. - -3. Coagulation and digestion of milk. - -4. Acid or gaseous fermentation in milk, or both. - -5. Acid or gaseous fermentation of various carbohydrates in -carbohydrate broth, or both. - -6. Production of indol in "indol solution." - -7. Production of pigments on various media. - -8. Reduction of nitrates to nitrites, ammonia, or free nitrogen. - -9. Production of enzymes as illustrated in the above activities. - -10. Appearance of growth on different culture media. - -11. Production of free toxins as determined by injection of animals -with broth cultures filtered free from bacteria. - -12. Causation of disease as ascertained by the injection of animals -with the bacteria themselves, and recovery of the organism from the -animals. - -13. Formation of specific antibodies as determined by the -proper injection of animals with the organism or its products -and the subsequent testing of the blood serum of the inoculated -animals. - -For special kinds of bacteria other activities must be determined -(oxidation, nitrate and nitrite formation, action of sulphur and iron -bacteria, etc.). - -The first nine activities are determined by inoculating the different -culture media already described and observing the phenomena indicated, -making chemical tests where necessary. - - -APPEARANCE OF GROWTH ON DIFFERENT CULTURE MEDIA. - -In addition to those changes that are associated with the manifestation -of different physiological activities, many bacteria, show -characteristic appearances on the various culture media which are of -value in their identification. - -Too much stress should not be laid on these appearances alone, however, -since slight variations, particularly in solid media due especially to -the age of the medium, may change decidedly the appearance of a colony. -This is true of variations in the amount of moisture on agar plates. -Colonies which are ordinarily round and regular may assume very diverse -shapes, if there chance to be an excess of moisture on the surface. - -Also in slope and puncture cultures on the various solid media much -variation results from the amount of material on the inoculation needle -and just how the puncture is made, or the needle drawn over the slope. -These variations are largely prevented by the use of standard media and -by inoculating by standard methods. The Laboratory Committee of the -American Public Health Association has proposed standard methods for -all culture media and tests and for methods of inoculation, and these -have been generally adopted in this country for comparative work. - -Likewise the Society of American Bacteriologists has at different times -(1904, 1914, 1917) adopted "descriptive charts" for detailing all the -characteristics of a given organism. A committee is at present working -on a revision of the 1917 chart to be presented at the 1920 meeting. -One of the earlier charts which includes a glossary of descriptive -terms is inserted in this chapter. - -Among the cultural appearances the following are of most importance: - -[Illustration: FIG. 144.--Broth cultures × 2/3. _1_ uninoculated -transparent broth; _2_, broth cloudy from growth of organisms; _3_, -broth slightly cloudy with a deposit in bottom; _4_, broth slightly -cloudy with a heavy membrane at the surface.] - -[Illustration: FIG. 145.--A filiform stab or puncture culture. × 3/5.] - -[Illustration: FIG. 146.--A beaded stab or puncture culture. × 1/2.] - -[Illustration: FIG. 147.--A villous stab or puncture culture. × 1/2.] - -In broth cultures the presence or absence of growth on the surface -and the amount of the same. Whether the broth is rendered cloudy or -remains clear, and whether there is a deposit at the bottom or not -(Fig. 144). An abundant surface growth with little or nothing below -indicates a strict aërobe, while a growth or deposit at bottom and a -clear or nearly clear medium above, an anaërobe. These appearances are -for the first few days only of growth. If the broth is disturbed, or -after the culture stands for several days many surface growths tend to -sink to the bottom. So an actively motile organism causes in general -a cloudiness, especially if the organism is a facultative anaërobe, -which tends to clear up by precipitation after several days when the -organisms lose their motility. Non-motile facultative anaërobes -usually cloud the broth also, but settle out more rapidly than the -motile ones. - -In gelatin and agar punctures the oxygen relationship is shown by -surface growth for aërobes, growth near the bottom of the puncture -for anaërobes, and a fairly uniform growth all along the line of -inoculation for facultative anaërobes. In the case of these last -organisms, a preference for more or less oxygen is indicated by the -approach to the aërobic or anaërobic type of growth. - -[Illustration: FIG. 148 FIG. 149 FIG. 150 FIG. 151 - -FIG. 148.--Crateriform liquefaction of gelatin. × 1/2. - -FIG. 149.--Funnelform liquefaction of gelatin. × 1/2. - -FIG. 150.--Saccate liquefaction of gelatin. × 1/2. - -FIG. 151.--Stratiform liquefaction of gelatin. × 1/2.] - -Along the line of puncture the commonest types are _filiform_ (Fig. -145), which indicates a uniform growth; _beaded_ (Fig. 146), or small -separate colonies; _villous_ (Fig. 147), delicate lateral outgrowths -which do not branch; _arborescent_, tree-like growths branching -laterally from the line. In agar these branchings are usually short and -stubby, or technically, _papillate_. - -[Illustration: FIG. 152.--Filiform slope culture. × 1/2.] - -[Illustration: FIG. 153.--Filiform, slightly spreading, slope culture. -× 1/2.] - -[Illustration: FIG. 154.--Beaded slope culture. × 1/2.] - -Further, in the gelatin puncture the liquefaction which occurs is -frequently characteristic. It may be _crateriform_ (Fig. 148), a -shallow saucer at the surface; or _funnel-shaped_ (Fig. 149); or it -may be of uniform width all along the puncture, _i.e._, _saccate_ (Fig. -150); or it may be _stratiform_, (Fig. 151), _i.e._, the liquefaction -extends to the sides of the tube and proceeds uniformly downward. - -[Illustration: FIG. 155 FIG. 156 FIG. 157 FIG. 158 - -FIG. 155.--Effuse slope culture. × 1/2. - -FIG. 156.--Rhizoid slope culture. × 1/2. - -FIG. 157.--Rugose slope culture. × 1/2. - -FIG. 158.--Verrucose slope culture. × 1/2.] - -On agar, potato and blood serum slope tubes the amount of growth, its -form and elevation, the character of the surface, and the consistency -should be carefully noted, and in some few cases the character of the -edge. Figures 152 to 158 show some of the commoner types. - -[Illustration: FIG. 159.--Punctiform colonies on a plate. × 1/2.] - -[Illustration: FIG. 160.--A rhizoid colony on a plate. Natural size.] - -[Illustration: FIG. 161.--Ameboid colonies on a plate. × 1/2.] - -[Illustration: FIG. 162.--Large effuse colony on a plate. The edge is -lacerated. Incidentally the colony shows the rate of growth for six -successive days. × 2/3.] - -[Illustration: FIG. 163.--Colony with edge entire as seen under the -low-power objective. × 100.] - -[Illustration: FIG. 164.--Colony with edge coarsely granular as seen -under the low-power objective. × 100.] - -[Illustration: FIG. 165.--Colony with edge curled as seen under the -low-power objective. × 100.] - -[Illustration: FIG. 166.--Colony with edge rhizoid as seen under the -low-power objective. × 100.] - -[Illustration: FIG. 167.--A small deep rhizoid colony as seen under the -low-power objective. × 100.] - -On agar and gelatin plates made so that the colonies are well isolated, -the form of the latter, the rate of their growth, the character of the -edge and of the surface, the elevation and the internal structure -as determined by a low-power lens are often of almost diagnostic -value. Also in the case of the gelatin plates, the character of the -liquefaction is important. Figs. 159 to 167 show some of the commoner -characteristics to be noted. - -[Illustration: FIG. 168.--A small mold colony natural size as viewed by -transmitted light.] - -[Illustration: FIG. 169.--The same colony as viewed by reflected light.] - -[Illustration: FIG. 170.--A portion of the thin edge of the same colony -as seen with the lower-power objective. × 100.] - -[Illustration: FIG. 171.--A single fruiting body (sporangium) from the -same colony as seen under the lower-power objective. × 100.] - -Colonies of mold frequently appear on plates. These are readily -differentiated from bacterial colonies after a little experience. With -the naked eye usually the fine radiations of the edge of the colony -are apparent. The surface appears duller and by reflected light more -or less "fuzzy." With the low-power objective the relatively large, -branching threads of the mold (mycelia) show distinctly. Also the large -fruiting bodies (sporangia) are easily distinguished. Figs. 168 to 171 -illustrate a common black mold (_Rhizopus nigricans_). - - - - -CHAPTER XXI. - -ANIMAL INOCULATION. - - -Animal inoculation has been referred to (1) as a method of assisting -in the preparation of pure cultures of pathogenic organisms; (2) as a -means of testing the poisonous properties of substances produced in -bacterial cultures; (3) in order to test the ability of an organism to -cause a disease; (4) for the production of various antibodies; it may -be added (5) that some bacteria produce in the smaller experimental -animals lesions which do not occur in animals naturally infected, but -which nevertheless are characteristic for the given organism. The best -illustration is the testicular reaction of young male guinea-pigs to -intraperitoneal injections of glanders bacilli. Experimental animals -are also inoculated (6) to test the potency of various bacterial and -other biological products, as toxins, antitoxins, etc. - -Guinea-pigs are the most widely used experimental animals because they -are easily kept and are susceptible to so many diseases on artificial -inoculation. Rabbits are used very largely also, as are white mice. For -special purposes white rats, pigeons, goats and swine are necessary. -For commercial products horses (antitoxins) and cattle (smallpox -vaccine) are employed. In the study of many human diseases the higher -monkeys and even the anthropoid apes are necessary, since none of the -lower animals are susceptible. - -The commonest method of animal inoculation is undoubtedly the -_subcutaneous_. This is accomplished most readily with the hypodermic -needle. The skin at the point selected (usually in guinea-pigs the -lateral posterior half of the abdominal surface, in mice the back near -the root of the tail) is pinched up to avoid entering the muscles and -the needle quickly inserted. Clipping the hairs and washing with an -antiseptic solution should precede the inoculation as routine practice. -Frequently a small "skin pocket" is all that is needed. The hair is -clipped off, the skin pinched up with small forceps and a slight snip -with sharp scissors is made. The material may be inserted into this -pocket with a heavy platinum needle. _Cutaneous_ inoculation is made -by shaving the skin and rubbing the material onto the shaved surface -or scratching with a scalpel or special scarifier, but without drawing -blood, and then rubbing in the material to be inoculated. - -_Intravenous_ injections are made with larger animals. In rabbits the -posterior external auricular is a convenient vein. In larger animals -the external jugular is used. - -_Intraperitoneal_, _-thoracic_, _-cardiac_, _-ocular_, _-muscular_ -injections, and injections into the parenchyma of internal organs are -accomplished with the hypodermic needle. In the case of the first two, -injury to contained organs should be carefully avoided. Intracardiac -injection, or aspiration of the heart to secure blood, requires -considerable practice to be successful without causing the death of the -animal at once through internal hemorrhage. In _subdural_ injections -into the cranial cavity it is necessary to trephine the skull first, -while such injections into the spinal canal may be accomplished between -the vertebra with needles longer and stronger than the usual hypodermic -needle. Occasionally animals are caused to _inhale_ the organisms, or -are _fed_ cultures mixed with the feed. - - -SECURING AND TRANSPORTING MATERIAL FROM ANIMALS FOR BACTERIOLOGICAL -EXAMINATION. - -If the site of the lesion is readily accessible from the exterior, -material from the _living animal_ should be collected with sterile -instruments and kept in sterile utensils until the necessary tests can -be made. Testing should be done on material as soon after collection -as possible, in all cases, to avoid the effects of "decomposition" -bacteria. - -If the blood is to be investigated it may be aspirated from a -peripheral vein with a sterile hypodermic syringe of appropriate size -or allowed to flow through a sterile canula into sterile receptacles. -The site of the puncture should be shaved and disinfected before the -instrument is introduced. - -Discharges of whatever kind should likewise be collected in sterile -receptacles and examined as soon as may be. - -If internal organs are to be examined it is best to kill a moribund -animal than to wait for death, since after death, and in severe -infections even sometimes before, the tissues are rapidly invaded by -saprophytic bacteria from the alimentary and respiratory tracts which -complicate greatly the isolation of the specific organism. Hence the -search for specific bacteria in carcasses or organs several hours after -death is frequently negative. Animal inoculation with such material is -very often followed by sepsis or septicemia in a few hours, so that the -specific organism has no opportunity to manifest itself. - -In securing material for cultures from internal organs it is a good -plan to burn the surface of the organ with a gas or alcohol flame, or -to sear it with a hot instrument to kill surface organisms, then make -the incision or puncture through the burned area and secure material -from the interior of the organ. Such punctures made with a stiff -platinum needle frequently give pure cultures of the organism sought. -Slides may be made from such material and culture media inoculated at -once. - -Since a bacteriological diagnosis depends most commonly on growing the -organisms, it is evident that material sent for examination must _never -be treated with an antiseptic or preservative_. If decomposition is to -be feared the only safe procedure is to _pack the material in ice_ and -forward in this way. - -_Tuberculous material from the parenchyma_ of internal organs may be -forwarded in a preservative (not _formalin_, since this makes it very -difficult to stain the bacteria) as _in this special_ case a very -positive diagnosis may be made by staining alone. Even here it is -better to _pack in ice_ in order that the diagnosis by staining may be -confirmed by inoculating the living organisms into guinea-pigs. - -In the case of material _from a rabid animal_ and many protozoal -diseases the rule against preservatives is not absolute, since staining -is a reliable diagnostic means. Even in these cases it is often -desirable to inoculate animals, hence, as before stated, it is best to -make it a uniform practice to _pack material for examination in ice and -use no preservatives_. - - - - -PART IV. - -GENERAL PATHOGENIC BACTERIOLOGY. - - - - -CHAPTER XXII. - -INTRODUCTION. - - -Pathogenic Bacteriology treats of the unicellular microörganisms which -are responsible for disease conditions, _i.e._, pathological changes -in other organisms. Hence not only are bacteria considered, but also -other low vegetable forms, as yeasts and molds, likewise protozoa in -so far as they may be pathogenic. For this reason the term pathogenic -"Microbiology" has been introduced to include all these organisms. It -is largely for the reason that the methods devised for the study of -bacteria have been applied to the investigation of other microörganisms -that the term "bacteriology" was extended to cover the entire field. -The general discussion in this chapter is intended to include, -therefore, microörganisms of whatever kind pathogenic to animals. - -The term pathogenic as applied to an organism must be understood in a -purely _relative_ sense, since there is no single organism that can -cause disease in all of a certain class, but each is limited to a more -or less narrow range. Some form of tuberculosis attacks nearly all -vertebrates, but no other classes of animals and no plants. Lockjaw or -tetanus attacks most mammals, but not any other vertebrates naturally. -Typhoid fever affects human beings; hog cholera, swine, etc. This point -is more fully discussed in Chapter XXIII but can not be too greatly -insisted upon. - - "The greatest enemy to mankind is man." - -Exceptions to this statement do occur and are important and must be -considered in efforts to protect completely human beings from disease -(tuberculosis from cattle, glanders from horses, poisoning from spoiled -canned goods, anthrax from hair, hides, wool, of animals dead of the -disease), but the most common human diseases are derived from other -human beings directly or indirectly. - -Diseases which are due to unicellular pathogenic microörganisms are -called _infectious_ diseases, while if such diseases are transmitted -under natural conditions from organism to organism they are spoken of -as _contagious_ diseases. Most infectious diseases are contagious but -not all. Tetanus is a good illustration of a non-contagious infectious -disease. There are very few such diseases. - -When a unicellular microörganism gains entrance into the body and -brings about any pathological changes there, the result is an -_infection_. Undoubtedly many pathogenic organisms get into the body -but never manifest their presence by causing disease conditions, hence -do not cause an infection. It is the pathological conditions which -result that constitute the infection, and not the mere _invasion_. - -The time that elapses between the entrance of the organism and the -appearance of symptoms is called the _period of incubation_ and varies -greatly in different diseases. - -The term _infestation_ is used to denote pathological conditions due to -_multicellular_ parasites. Thus an animal is _infested_ (not infected) -with tapeworms, roundworms, lice, mites, etc. Many of these conditions, -probably all, are contagious, _i.e._, transmissable naturally from -animal to animal. The word _contagious_ has been used in a variety -of ways to mean _communicated by direct contact_, communicated by a -living something (_contagium_) that might be carried to a distance -and finally _communicable_ in any manner, transmissable. The agency -of transmission may be very roundabout--as through a _special tick_ -in Texas fever, a _mosquito_ in malaria, etc.,--or by direct personal -contact, as generally in venereal diseases. After all, though exactness -is necessary, it is better to learn all possible about the _means of -transmission of diseases_, than quibble as to the terms to be used. - -An infectious disease may be _acute_ or _chronic_. An acute infection -is one which runs for a relatively short time and is "self-limited," -so-called, _i.e._, the organisms cease to manifest their presence after -a time. In some acute infections the time is very short--German measles -usually runs five or six days. Typhoid fever may continue eight to -ten weeks, sometimes longer, yet it is an acute infectious disease. -It is not so much the time as the fact of _self-limitation_ that -characterizes acute infections. - -In chronic infections there is little or no evidence of limitation of -the progress of the disease which may continue for years. Tuberculosis -is usually chronic. Leprosy in man is practically always so. Glanders -in horses is most commonly chronic; in mules and in man it is more apt -to be acute. - -Many infections begin acutely and later change to the chronic type. -Syphilis in man is a good illustration. - -The differences between acute and chronic infections are partly due -to the nature of the organism, partly to the number of organisms -introduced and the point of their introduction and partly to the -resistance of the animal infected. - -An infectious disease is said to be _specific_ when one kind of -organism is responsible for its manifestations--as diphtheria due -to the _Corynebacterium diphtheriæ_, lockjaw due to _Clostridium -tetani_, Texas fever due to the _Piroplasma bigeminum_, etc. It is -_non-specific_ when it may be due to a variety of organisms, as -_enteritis_ (generally), _bronchopneumonia_, _wound infections_. - -Henle, as early as 1840, stated certain principles that must be -established before a given organism can be accepted as the cause of a -specific disease. These were afterward restated by Koch, and have come -to be known as "Koch's postulates." They may be stated as follows: - -1. The given organism must be found in all cases of the disease in -question. - -2. No other organism must be found in all cases. - -3. The organism must, when obtained in pure culture, reproduce the -disease in susceptible animals. - -4. It must be recovered from such animals in pure culture and this -culture likewise reproduce the disease. - -These postulates have not been fully met with reference to any disease, -but the principles embodied have been applied as far as possible -in all those infections which we recognize as specific, and whose -causative agent is accepted. In many diseases recognized as infectious -and contagious no organism has been found which is regarded as the -specific cause. In some of these the organism appears to be too small -to be seen with the highest powers of the microscope, hence they are -called "_ultramicroscopic_" organisms. Because these agents pass -through the finest bacterial filters, they are also frequently called -"_filterable_." The term "_virus_" or "_filterable virus_" is likewise -applied to these "ultramicroscopic" and "filterable" agents. - -The term _primary infection_ is sometimes applied to the first -manifestation of a disease, either specific or non-specific, while -_secondary_ refers to later developments. For example, a _secondary_ -general infection may follow a _primary_ wound infection, or _primary_ -lung tuberculosis be followed by _secondary_ generalized tuberculosis, -or _primary_ typhoid fever by a _secondary_ typhoid pneumonia. The -terms _primary_ and _secondary_ are also used where the body is -invaded by one kind of an organism and later on by another kind; thus -a _primary_ measles may be followed by _secondary_ infection of the -middle ear, or a _primary_ influenza may be followed by a _secondary_ -pneumonia, or a _primary_ scarlet fever by a _secondary_ nephritis -(inflammation of the kidney). Where several organisms seem to be -associated simultaneously in causing the condition then the term _mixed -infection_ is used--in severe diphtheria, streptococci are commonly -associated with the _Corynebacterium diphtheriæ_. In many cases of -hog-cholera, mixed infections in the lungs and in the intestines are -common. Wound infections are usually _mixed_. _Auto-infection_ refers -to those conditions in which an organism commonly present in or on the -body in a latent or harmless condition gives rise to an infectious -process. If the _Bacterium coli_ normal to the intestine escapes into -the peritoneal cavity, or passes into the bladder, a severe peritonitis -or cystitis, respectively, is apt to result. "Boils" and "pimples" -are frequently autoinfections. Such infections are also spoken of as -_endogenous_ to distinguish them from those due to the entrance of -organisms from without--_exogenous_ infections. _Relapses_ are usually -instances of autoinfection. - -Those types of _secondary infection_ where the infecting agent is -transferred from one disease focus to another or several other points -and sets up the infection there are sometimes called _metastases_. Such -are the transfer of tubercle bacilli from lung to intestine, spleen, -etc., the formation of abscesses in internal organs following a primary -surface abscess, the appearance of glanders nodules throughout various -organs following pulmonary glanders, etc. - -The characteristic of a pathogenic microörganism which indicates -its ability to cause disease is called its _virulence_. If slightly -virulent, the effect is slight; if highly virulent, the effect is -severe, and may be fatal. - -On the other hand, the characteristic of the host which indicates -its capacity for infection is called _susceptibility_. If slightly -susceptible, infection is slight, if highly susceptible, the infection -is severe. - -Evidently the degree of infection is dependent in large measure on -the relation between the _virulence_ of the invading organism and the -_susceptibility_ of the host. High virulence and great susceptibility -mean a severe infection; low virulence and little susceptibility a -slight infection; while high virulence and little susceptibility or -low virulence and great susceptibility might mean a moderate infection -varying in either direction. Other factors influencing the degree of -infection are the number of organisms introduced, the point where they -are introduced and various conditions. These will be discussed in -another connection (Chapter XXV). - -The study of pathogenic bacteriology includes the thorough study -of the individual organisms according to the methods already given -(Chapters XVIII-XXI) as an aid to diagnosis and subsequent treatment, -bacteriological or other, in a given disease. Of far greater -importance than the _treatment_, which in most infectious diseases -is not specific, is the _prevention_ and _ultimate eradication_ of -all infectious diseases. To accomplish these objects involves further -a study of the _conditions under which pathogenic organisms exist -outside the body_, _the paths of entrance into and elimination from -the body_ and those _agencies within the body itself_ which make it -_less susceptible to infection or overcome the infective agent after -its introduction_. That condition of the body itself which prevents any -manifestation of a virulent pathogenic organism after it has been once -introduced is spoken of as _immunity_ in the modern sense. Immunity is -thus the opposite of susceptibility and may exist in varying degrees. - -That scientists are and have been for some years in possession of -sufficient knowledge to permit of the prevention and eradication -of most, if not all, of our infectious diseases can scarcely be -questioned. The practical application of this knowledge presents -many difficulties, the chief of which is the absence of a public -sufficiently enlightened to permit the expenditure of the necessary -funds. Time and educative effort alone can surmount this difficulty. It -will probably be years yet, but it will certainly be accomplished. - - - - -CHAPTER XXIII. - -PATHOGENIC BACTERIA OUTSIDE THE BODY. - - -Pathogenic bacteria may exist outside the body of the host under a -variety of conditions as follows: - - I. In or on inanimate objects or material. - (_a_) As true saprophytes. - (_b_) As facultative saprophytes. - (_c_) Though obligate parasites, they exist in a latent - state. - II. In or on other animals, or products from them: - A. Susceptible to the disease. - (_a_) Sick themselves. - (As far as human beings are concerned these are - mainly: - 1. Other human beings for most diseases. - 2. Rats for plague. - 3. Dogs for rabies. - 4. Horses for glanders. - 5. Cattle, swine, parrots for tuberculosis). - (_b_) Recovered from illness. - (_c_) Never sick but "carriers." - B. Not susceptible. - (_d_) Accidental carriers. - (_e_) Serving as necessary intermediate hosts for certain - stages of the parasite--this applies to _protozoal_ - diseases only, as yet. - - -I. - -(_a_) The bacilli of tetanus, malignant edema and the organisms of -"gas gangrene" are widely distributed. There is no evidence that their -entrance into the body is at all necessary for the continuation of -their life processes, or that one case of either of these diseases -ever has any connection with any other case; they are true saprophytes. -Manifestly it would be futile to attempt to prevent or eradicate such -diseases by attacking the organism in its natural habitat. _Clostridium -botulinum_, which causes a type of food poisoning in man, does not even -multiply in the body, but the disease symptoms are due to a soluble -toxin which is produced during its growth outside the body. - -(_b_) Organisms like the bacterium of anthrax and the bacillus of -black-leg from their local occurrence seem to be distributed from -animals infected, though capable of a saprophytic existence outside the -body for years. These can no more be attacked during their saprophytic -existence than those just mentioned. Doubtless in warm seasons of the -year and in the tropics other organisms pathogenic to animals may live -and multiply in water or in damp soil where conditions are favorable, -just as the cholera organism in India, and occasionally the typhoid -bacillus in temperate climates do. - -(_c_) Most pathogenic organisms, however, when they are thrown off -from the bodies of animals, remain quiescent, do not multiply, in fact -always tend to die out from lack of all that is implied in a "favorable -environment," food, moisture, temperature, light, etc. Disinfection is -sometimes effective in this class of diseases in preventing new cases. - - -II. A. - -(_a_) The most common infectious diseases of animals are transmitted -more or less directly from other animals of the same species. Human -beings get nearly all their diseases from other human beings who are -sick; horses, from other horses; cattle, from other cattle; swine, -from swine, etc. Occasionally transmission from one species to another -occurs. Tuberculosis of swine most frequently results from feeding -them milk of tuberculous cattle or from their eating the droppings of -such cattle. Human beings occasionally contract anthrax from wool, -hair and hides of animals dead of the disease or from postmortems -on such animals; glanders from horses; tuberculosis (in children) -from tuberculous milk; bubonic plague from rats; rabies practically -always from the bites of dogs and other rabid animals, etc. The -mode of limiting this class of diseases is evidently to isolate the -sick, disinfect their discharges and their _immediate_ surroundings, -sterilize such products as must be handled or used, kill lower animals -that are dangerous, and disinfect, bury properly, or destroy their -carcasses. - -Classes of the sick that are especially dangerous for the spread of -disease are the mild cases and the undetected cases. These individuals -do not come under observation and hence not under control. - -(_b_) This class of carriers offers a difficult problem in the -prevention of infectious diseases since they may continue to give off -the organisms indefinitely and thus infect others. Typhoid carriers -have been known to do so for fifty-five years. Cholera, diphtheria, -meningitis and other carriers are well known in human practice. -Carriers among animals have not been so frequently demonstrated, -but there is every reason for thinking that hog-cholera, distemper, -roup, influenza and other carriers are common. Carriers furnish the -explanation for many of the so-called "spontaneous" outbreaks of -disease among men and animals. - -It is the general rule that those who are sick cease to carry the -organisms on recovery and it is the occasional ones who do not that are -the exceptions. In those diseases in which the organism is known it -can be determined by examination of the patient or his discharges how -long he continues to give off the causative agent. In those in which -the cause is unknown (in human beings, the commonest and most easily -transmitted diseases, scarlet fever, measles, German measles, mumps, -chicken-pox, small-pox, influenza), no such check is possible. It is -not known how long such individuals remain carriers. Hence isolation -and quarantine of such convalescents is based partly on experience -and partly on theory. It is highly probable that in the diseases just -mentioned transmission occurs in the _early stages only_, except -in small-pox and chicken-pox where the organism seems to be in the -pustules and transmission by means of material from these is possible, -though only by direct contact with it. - -The fact that such individuals are _known to have had the disease_ is a -guide for control. The methods to be used are essentially the same as -for the sick, (_a_), though obviously such human carriers are much more -difficult to deal with since they are well. - -(_c_) Another class of carriers is those who have never had the -disease. Such individuals are common and are very dangerous sources -of infection. Many of them have _associated with the sick or with -convalescents_ and these should always be suspected of harboring the -organisms. Their control differs in no way from that of class (_b_). -Unfortunately a history of such association is too often not available. -Modern transportation and modern social habits are largely responsible -for the nearly universal distribution of this type of carrier. Their -detection is probably the largest single problem in the prevention -of infectious diseases. A partial solution would be universal -bacteriological examination. In our present stage of progress this is -impossible and would not detect carriers of diseases of unknown cause. - -The various classes of carriers just discussed are in a large part -responsible for the continued presence of the commoner diseases -throughout the country. The difficulties in control have been -mentioned. A complete solution of the problem is not yet obtained. The -army experience of the past few years in the control of infectious -diseases shows what may be done. - -There is another class of carriers which might be called the "universal -carrier," _i.e._, there are certain organisms which seem to be -constantly or almost constantly present in or on the human body. These -are _micrococci_, _streptococci_ and _pneumococci_, all _Gram positive_ -organisms. They are ordinarily harmless parasites, but on occasion may -give rise to serious, even fatal, infection. Infected wounds, pimples, -boils, "common colds," most "sore throats," bronchitis, pneumonia are -pathological conditions that come in this class. Such infections are -usually autogenous. There is a constant interchange of these organisms -among individuals closely associated, so that all of a group usually -harbor the same type though no one individual can be called _the_ -carrier. Whenever, for any reason, the resistance of an individual (see -Chaps. XXV et seq.) is lowered either locally or generally some of -these organisms are liable to gain a foothold and cause infection. It -sometimes happens that a strain of dangerous organisms may be developed -in an individual in this way which is passed around to others with its -virulence increased and thus cause an epidemic. Or, since all of the -group are living under the same conditions the resistance of all or -many of them may be lowered from the same general cause and an epidemic -result from the organism common to all (pneumonia after measles, -scarlet fever and influenza in camps). Protection of the individual is -chiefly a personal question, _i.e._, by keeping up the "normal healthy -tone" in all possible ways: The use of protective vaccines (Chap. XXX) -appears to be advisable in such instances (colds, pneumonia after -measles and influenza, inflammation of throat and middle ear following -scarlet fever and measles). Results obtained in this country during -the recent influenza epidemic have been conflicting but on the whole -appear to show that preventive vaccination against _pneumonia liable to -follow_ should be practiced. - -It would seem that among groups of individuals where infection may be -expected the proper procedure would be to prepare autogenous vaccines -(Chapter XXX) from members of the group and vaccinate all with the -object of protecting them. - - -II. B. - -(_d_) In this class come the "accidental carriers" like flies, fleas, -lice, bed-bugs, ticks, and other biting and blood-sucking insects, -vultures, buzzards, foxes, rats, and carrion-eating animals generally; -pet animals in the household, etc. Here the animals are not susceptible -to the given disease but become contaminated with the organisms -and then through defilement of the food or drink or contact with -individuals or with utensils pass the organisms on to the susceptible. -Some biting and blood-sucking insects transmit the organisms through -biting infected and non-infected animals successively. The spirilloses -and trypanosomiases seem to be transmitted in this way, though there -is evidence accumulating which may place these diseases in the next -class. Anthrax is considered in some instances to be transmitted by -flies and by vultures in the southern United States. Transmission -of typhoid, dysentery, cholera and other diseases by flies is well -established in man. Why not hog-cholera from farm to farm by flies, -English sparrows, pigeons feeding, or by turkey buzzards? Though this -would not be easy to prove, it seems reasonable. - -Preventing contact of such animals with the discharges or with the -carcasses of those dead of the disease, destruction of insect carriers, -screening and prevention of fly breeding are obvious protective -measures. - -(_e_) In this class come certain diseases for which particular -insects are necessary for the parasite in question, so that certain -stages in its life history may be passed therein. The surest means -for eradicating such diseases is the destruction of the insects -concerned. Up to the present no _bacterial_ disease is known in which -this condition exists, unless Rocky Mountain spotted fever and typhus -fever shall prove to be due to bacteria. Such diseases are all due to -protozoa. Among them are Texas fever, due to _Piroplasma bigeminum_ -in this country which has been eradicated in entire districts by -destruction of the cattle tick (_Margaropus annulatus_). - -Piroplasmoses in South Africa among cattle and horses, and in other -countries are transmitted in similar ways. Probably many of the -diseases due to spirochetes and trypanosomes are likewise transmitted -by _necessary_ insect intermediaries. In human medicine the eradication -of yellow fever from Panama and Cuba is due to successful warfare -against, a certain mosquito (_Stegomyia_). So the freeing of large -areas in different parts of the world from _malaria_ follows the -destruction of the mosquitoes. The prevention of typhus fever and -of trench fever by "delousing" methods is familiar from recent army -experience though for typhus this method has been practiced in Russia -for more than ten years to the author's personal knowledge. The -campaign against disease in animals and man from insect sources must be -considered as still in its infancy. The full utilization of tropical -lands depends largely on the solution of this problem. - - - - -CHAPTER XXIV. - -PATHS OF ENTRANCE OF PATHOGENIC ORGANISMS, - -OR - -CHANNELS OF INFECTION. - - -_A._ =The Skin.=--If the skin is healthy there is no opportunity for -bacteria to penetrate it. It is protected not only by the stratified -epithelium, but also in various animals, by coats of hair, wool, -feathers, etc. The secretion pressure of the healthy sweat and oil -glands acts as an effective bar even to motile bacteria. Nevertheless a -very slight injury only is sufficient to give normal surface parasites -and other pathogenics, accidentally or purposely brought in contact -with it, an opportunity for more rapid growth and even entrance -for general infection. Certain diseases due to higher fungi are -characteristically "skin diseases" and rarely become general--various -forms of favus, trichophyton infections, etc. A few disease organisms, -tetanus, malignant edema, usually get in through the skin; others, -black-leg, anthrax, quite commonly; and those diseases transmitted -by biting and blood-sucking insects, piroplasmoses, trypanosomiases, -spirilloses, scarcely in any other way. Defective secretion in the skin -glands from other causes, may permit lodgment and growth of bacteria -in them or in the hair follicles. "Pimples" and boils in man and local -abscesses occasionally in animals are illustrations. Sharp-edged and -freely bleeding wounds are less liable to be infected than contusions, -ragged wounds, burns, etc. The flowing blood washes out the wound and -the clotting seals it, while there is less material to be repaired -by the leukocytes and they are free to care for invading organisms -(phagocytosis). Pathogenic organisms, especially pus cocci, frequently -gain lodgment in the _milk glands_ and cause local (mastitis) or -general infection. - -_B._ =Mucosæ directly continuous with the skin and lined with -stratified epithelium= are commonly well protected thereby and by the -secretions. - -(_a_) The external auditory meatus is rarely the seat even of local -infection. The tympanic cavity is normally sterile, though it may -become infected by extension through the Eustachian tube from the -pharynx (_otitis media_). - -(_b_) The conjunctiva is frequently the seat of localized, very rarely -the point of entrance for a generalized infection, except after severe -injury. Those diseases whose path of entrance is generally assumed to -be the respiratory tract (see "Lungs" below) might also be admitted -through the eye. Material containing such organisms might get on the -conjunctiva and be washed down through the lachrymal canal into the -nose. Experiment has shown that bacteria may pass in this way in a -few minutes. In case masks are worn to avoid infection from patients -suffering with these diseases, the eyes should therefore be protected -as well as the nose and mouth. - -(_c_) The nasal cavity on account of its anatomical structure retains -pathogenic organisms which give rise to local infections more -frequently than other mucosæ of its character. These may extend from -here to middle ear, neighboring sinuses, or along the lymph spaces of -the olfactory nerve into the cranial cavity (meningitis). Acute coryza -("colds" in man) is characteristic. Glanders, occasionally, is primary -in the nose, as is probably roup in chickens, leprosy in man. The -meningococcus and the virus of poliomyelitis pass from the nose into -the cranial cavity without local lesions in the former. - -(_d_) The mouth cavity is ordinarily protected by its epithelium -and secretions, though the injured mucosa is a common source of -_actinomycosis_ infection, as well as thrush. In foot-and-mouth disease -no visible lesions seem necessary to permit the localization of the -unknown infective agent. - -(_e_) The tonsils afford a ready point of entrance for ever-present -_micrococci_ and _streptococci_ whenever occasion offers (follicular -tonsillitis, "quinsy"), and articular rheumatism is not an uncommon -sequel. The diphtheria bacillus characteristically seeks these -structures for its development. Tubercle and anthrax organisms -occasionally enter here. - -(_f_) The pharynx is the seat of localized infection as in -_micrococcal_, _streptococcal_ and diphtherial "sore throat" in human -beings, but both it and the esophagus are rarely infected in animals -except as the result of injury. - -(_g_) The external genitalia are the usual points of entrance for -the venereal organisms in man (gonococcus, _Treponema pallidum_, and -Ducrey's bacillus). The bacillus of contagious abortion and probably -the trypanosome of dourine are commonly introduced through these -channels in animals. - -_C._ =Lungs.=--The varied types of pneumonia due to many different -organisms (tubercle, glanders, influenza, plague bacilli, pneumococcus, -streptococcus, micrococcus and many others) show how frequently these -organs are the seat of a localized infection, which may or may not be -general. Whether the lungs are the actual point of entrance in these -cases is a question which is much discussed at the present time, -particularly with reference to tuberculosis. The mucous secretion -of the respiratory tract tends to catch incoming bacteria and other -small particles and the ciliary movement along bronchial tubes and -trachea tends to carry such material out. "Foreign body pneumonia" -shows clinically, and many observers have shown experimentally that -microörganisms may reach the alveoli even though the exchange of -air between them and the bronchioles and larger bronchi takes place -ordinarily only by diffusion. The presence of carbon particles in the -walls of the alveoli in older animals and human beings and in those -that breathe dusty air for long periods indicates strongly, though it -does not prove absolutely, that these came in with inspired air. On the -other hand, experiment has shown that tubercle bacilli introduced into -the intestine may appear in the lungs and cause disease there and not -in the intestine. It is probably safe to assume that in those diseases -which are transmitted most readily through close association though -not necessarily actual contact, the commonest path is through the -respiratory tract, which may or may not show lesions (smallpox, scarlet -fever, measles, chicken-pox, whooping-cough, pneumonic plague in man, -lobar and bronchopneumonias and influenza in man and animals, some -cases of glanders and tuberculosis). On the other hand, the fact that -the _Bacterium typhosum_ and _Bacterium coli_ may cause pneumonia when -they evidently have reached the lung from the intestinal tract, and the -experimental evidence of lung tuberculosis above mentioned show that -this route cannot be excluded in inflammations of the lung. - -_D._ =Alimentary Tract.=--The alimentary tract affords the ordinary -path of entrance for the causal microbes of many of the diseases of -animals and man, since they are carried into the body most commonly and -most abundantly in the food and drink. - -(_a_) The stomach is rarely the seat of local infection, even in -ruminants, except as the result of trauma. The character of the -epithelium in the rumen, reticulum and omasum in ruminants, the -hydrochloric acid in the abomasum and in the stomachs of animals -generally are usually sufficient protection. Occasionally anthrax -"pustules" develop in the gastric mucosa. (The author saw nine such -pustules in a case of anthrax in a man.) - -(_b_) The intestines are frequently the seat of localized infections, -as various "choleras" and "dysenteries" in men and many animals, -anthrax, tuberculosis, Johne's disease. Here doubtless enter the -organisms causing "hemorrhagic septicemias" in many classes of animals, -and numerous others. These various organisms must have passed through -the stomach and the question at once arises, why did the HCl not -destroy them? It must be remembered that the acid is present only -during stomach digestion, and that liquids taken on an "empty stomach" -pass through rapidly and any organisms present are not subjected to -the action of the acid. Also spores generally resist the acid. Other -organisms may pass through the stomach within masses of undigested -food. The fact that digestion is going on in the stomach of ruminants -practically all the time may explain the relative freedom of _adult_ -animals of this class from "choleras" and "dysenteries." - - -MECHANISM OF ENTRANCE OF ORGANISMS. - -In the preceding chapters statements have been made that "bacteria -enter" at various places or they "pass through" different mucous -membranes, skin, etc. Strictly speaking such statements are -incorrect--bacteria do not "enter" or "pass through" of themselves. -It is true that some of the intestinal organisms are motile, but most -of the bacteria which are pathogenic are non-motile. Even the motile -ones can not make their way against fluids secreted or excreted on free -surfaces. Bacteria cannot pass by diffusion through membranes since -they are finite particles and not in solution. - -In the case of penetrating wounds bacteria may be carried mechanically -into the tissues, but this is exceptional in most infections. Also -after gaining lodgment they may gradually grow through by destroying -tissue as they grow, but this is a minor factor. Evidently, there -must be some mechanism by which they _are carried_ through. The known -mechanisms for this in the body are ameboid cells, especially the -phagocytes. It is most probable that these are the chief agents in -getting bacteria into the tissues through various free surfaces. The -phagocytes engulf bacteria, carry them into the tissues and either -destroy them, are destroyed by them, or may disgorge or excrete them -free in the tissues or in the blood. - - -DISSEMINATION OF ORGANISMS. - -Dissemination of organisms within the tissues occurs either through -the lymph channels or the bloodvessels or both. If through the lymph -vessels only it is usually much more restricted in extent, or much more -slowly disseminated, while blood dissemination is characterized by the -number of organs involved simultaneously. - - -PATHS OF ELIMINATION OF PATHOGENIC MICRÖORGANISMS. - -I. Directly from the point, of injury. This is true in infected -wounds open to the surface, skin glanders (farcy), black-leg, -surface anthrax, exanthemata in man and animals (scarlet fever (?), -measles (?), smallpox; hog erysipelas, foot-and-mouth disease): -also in case of disease of mucous membranes continuous with the -skin--from nasal discharges (glanders), saliva (foot-and-mouth -disease), material coughed or sneezed out (tuberculosis, influenza, -pneumonias), urethral and vaginal discharges (gonorrhea and syphilis -in man, contagious abortion and dourine in animals), intestinal -discharges (typhoid fever, "choleras," "dysenteries," anthrax, -tuberculosis, Johne's disease). Material from nose, mouth and lungs -may be swallowed and the organisms passed out through the intestines. - -II. Indirectly through the secretions and the excretions where the -internal organs are involved. The _saliva_ of rabid animals contains -the ultramicroscopic virus of rabies (the sympathetic ganglia -within the salivary glands, and pancreas also, are affected in this -disease as well as the cells of the central nervous system). The -_gall-bladder_ in man is known to harbor colon and typhoid bacilli, -as that of hog-cholera hogs does the virus of this disease. It may -harbor analogous organisms in other animals, though such knowledge -is scanty. The _kidneys_ have been shown experimentally to excrete -certain organisms introduced into the circulation within a few minutes -(micrococci, colon and typhoid bacilli, anthrax). Typhoid bacilli occur -in the urine of typhoid-fever patients in about 25 per cent. of all -cases and the urine of hogs with hog cholera is highly virulent. Most -observers are of the opinion, however, that under natural conditions -the kidneys do not excrete bacteria unless they themselves are infected. - -The _milk_ both of tuberculous cattle and tuberculous women has been -shown to contain tubercle bacilli _even when the mammary glands are not -involved_. Doubtless such bacteria are carried through the walls of the -secreting tubules or of the smaller ducts by phagocytes and are then -set free in the milk. - - -SPECIFICITY OF LOCATION OF INFECTIVE ORGANISMS. - -It is readily apparent that certain disease organisms tend to locate -themselves in definite regions and the question arises, Is this due -to any specific relationship between organism and tissue or not? -Diphtheria in man usually attacks the tonsils first, gonorrhea and -syphilis the external genitals, tuberculosis the lung, "choleras" the -small intestine, "dysenteries" the large intestine, influenza the -lungs. In these cases the explanation is probably that the points -attacked are the places where the organism is most commonly carried, -with no specific relationship, since all of these organisms (Asiatic -cholera excepted) also produce lesions in other parts of the body _when -they reach them_. On the other hand, the virus of hydrophobia attacks -nerve cells, leprosy frequently singles out nerves, glanders bacilli -introduced into the abdominal cavity of a young male guinea-pig cause -an inflammation of the testicle, malarial parasites and piroplasms -attack the red blood corpuscles, etc. In fact, most _pathogenic -protozoa_ are specific in their localization either in certain tissue -cells or in the blood or lymph. In these cases there is apparently a -real chemical relationship, as there is also between the _toxins_ of -bacteria and certain tissue cells (tetanus toxin and nerve cells). -Whether "chemotherapy" will ever profit from a knowledge of such -chemical relationships remains to be developed. It appears that a -search for these specific chemical substances with the object of -combining poisons with them so that the organisms might in this way be -destroyed, would be a profitable line of research. - - - - -CHAPTER XXV. - -IMMUNITY. - - -Immunity, as has already been stated, implies such a condition of the -body that pathogenic organisms after they have been introduced are -incapable of manifesting themselves, and are unable to cause disease. -The word has come to have a more specific meaning than resistance in -many instances, in other cases the terms are used synonymously. It is -the opposite of susceptibility. The term must be understood always in a -relative sense, since no animal is immune to all pathogenic organisms, -and conceivably not entirely so to anyone, because there is no question -that a sufficient number of bacteria of any kind might be injected -into the circulation to kill an animal, even though it did it purely -mechanically. - -Immunity may be considered with reference to a single individual or to -entire divisions of the organic world, with all grades between. Thus -plants are immune to the diseases affecting animals; invertebrates to -vertebrate diseases; cold-blooded animals to those of warm blood; man -is immune to most of the diseases affecting other mammals; the rat to -anthrax, which affects other rodents and most mammals; the well-known -race of Algerian sheep is likewise immune to anthrax while other sheep -are susceptible; the negro appears more resistant to yellow fever than -the white; some few individuals in a herd of hogs always escape an -epizoötic of hog cholera, etc. - -Immunity within a given species is modified by a number of -factors--age, state of nutrition, extremes of heat or cold, fatigue, -excesses of any kind, in fact, anything which tends to lower the -"normal healthy tone" of an animal also tends to lower its resistance. -Children appear more susceptible to scarlet fever, measles, -whooping-cough, etc., than adults; young cattle more frequently have -black-leg than older ones (these apparently greater susceptibilities -may be due in part to the fact that most of the older individuals -have had the diseases when young and are immune for this reason). -Animals weakened by hunger or thirst succumb to infection more readily. -Frogs and chickens are immune to tetanus, but if the former be put -in water and warmed up to and kept, at about 37°, and the latter be -chilled for several hours in ice-water, then each may be infected. -Pneumonia frequently follows exposure to cold. The immune rat may -be given anthrax if first he is made to run in a "squirrel cage" -until exhausted. Alcoholics are far less resistant to infection than -temperate individuals. "Worry," mental anguish, tend to predispose to -infection. - -The following outlines summarize the different, classifications of -immunity so far as mammals are concerned for the purposes of discussion. - -Immunity. - - { {1. Inherited through - { { the germ cell or cells. - { { {(_a_) By having the - {A. Congenital {2. Acquired { disease _in utero_. - I. Natural { { _in utero_. {(_b_) By absorption - { { { of immune - { { substances - {B. Acquired by { from the mother. - { having the disease. - - II. Artificial--acquired through human agency by: - 1. Introduction of the organism or its products. - 2. Introduction of the blood serum of an immune animal. - -Immunity. - - I. Active--due to the introduction of the organism or due to the - introduction of the products of the organism. - A. Naturally by having the disease. - B. Artificially. - 1. By introducing the organism: - {1. Passage through another animal. - {2. Drying. - (_a_) Alive and virulent. {3. Growing at a higher temperature. - (_b_) Alive and virulence {4. Heating the cultures. - reduced by {5. Treating with chemicals. - (_c_) Dead. {6. Sensitizing. - {7. Cultivation on artificial media. - 2. By introducing the products of the organism. - - II. Passive--due to the introduction of the blood serum of an actively - immunized animal. - -Immunity present in an animal and not due to human interference is to -be regarded as _natural_ immunity, while if brought about by man's -effort it is considered _artificial_. Those cases of natural immunity -mentioned above which are common to divisions, classes, orders, -families, species or races of organisms and to those few individuals -where no special cause is discoverable, must be regarded as instances -of true _inheritance_ through the germ cell as other characteristics -are. All other kinds of immunity are _acquired_. Occasionally young are -born with every evidence that they have had a disease _in utero_ and -are thereafter as immune as though the attack had occurred after birth -("small-pox babies," "hog-cholera pigs"). Experiment has shown that -immune substances may pass from the blood of the mother to the fetus -_in utero_ and the young be immune for a time after birth (tetanus). -This is of no practical value as yet. It is a familiar fact that with -most infectious diseases recovery from one attack confers a more or -less lasting immunity, though there are marked exceptions. - -=Active Immunity.=--By active immunity is meant that which is due -to the actual introduction of the organism, or in some cases of its -products. The term active is used because the body cells of the animal -immunized perform the real work of bringing about the immunity as -will be discussed later. In _passive_ immunity the blood serum of an -actively immunized animal is introduced into a second animal, which -thereupon becomes immune, though its cells are not concerned in the -process. The animal is _passive_, just as a test-tube, in which a -reaction takes place, plays no other part than that of a passive -container for the reagents. - -In _active_ immunity the organism may be introduced in what is to -be considered a natural manner, as when an animal becomes infected, -has a disease, without human interference. Or the organism may be -purposely introduced to bring about the immunity. For certain purposes -the introduction of the products of the organism (toxins) is used to -bring about active immunity (preparation of diphtheria and tetanus -antitoxin from the horse). The method of producing active immunity by -the artificial introduction of the organism is called _vaccination_, -and a _vaccine_ must therefore contain the organism. _Vaccines_ for -_bacterial_ diseases are frequently called _bacterins_. The use of -the blood serum of an immunized animal to confer passive immunity on -a second animal is properly called _serum therapy_, and the serum so -used is spoken of as an _antiserum_, though the latter word is also -used to denote any serum containing any kind of an antibody (Chapters -XXVII-XXXI). In a few instances both the organism and an antiserum are -used to cause both active and passive immunity (_serum-simultaneous -method_ in immunizing against hog cholera). - -In producing active immunity the organism may be introduced (_a_) -_alive and virulent_, but in very small doses, or in combination with -an immune serum, as just mentioned for hog cholera. The introduction -of the live virulent organism alone is done only experimentally as -yet, as it is obviously too dangerous to do in practice, except under -the strictest control (introduction of a _single tubercle_ bacillus, -followed by gradually increasing numbers--Barber and Webb). More -commonly the organisms are introduced (_b_) alive but with their -_virulence reduced_ ("attenuated") in one of several ways: (1) By -passing the organism through another animal as is the case with -_smallpox vaccine_ derived from a calf or heifer. This method was -first introduced by Jenner in 1795 and was the first practical means -of preventing disease by _vaccination_. This word was used because -material was derived from a cow--Latin _vacca_. (2) By drying the -organism, as is done in the preparation of the vaccine for the _Pasteur -treatment of rabies_, where the spinal cords of rabbits are dried for -varying lengths of time--one to four days, Russian method, one to three -days, German method, longer in this country. (It is probable that the -passage of the "fixed virus" through the rabbit is as important in this -procedure as the drying, since it is doubtful if the "fixed virus" is -pathogenic for man.) It would be more correct to speak of this as a -_preventive vaccination against rabies_, since the latter is one of the -few diseases which is not amenable to _treatment_. The patient always -dies if the disease develops. (3) The organism may be attenuated by -growing at a temperature above the normal. This is the method used in -preparing _anthrax vaccine_ as done by Pasteur originally. (4) Instead -of growing at a higher temperature the culture may be heated in such -a way that it is not killed but merely weakened. _Black-leg_ vaccines -are made by this method. (5) Chemicals are sometimes added to attenuate -the organisms, as was formerly done in the preparation of black-leg -vaccine by Kruse's method in Germany. The use of toxin-antitoxin -mixtures in immunizing against diphtheria and in the preparation -of diphtheria antitoxin from horses is an application of the same -principle, though here it is the _product_ of the organism and not the -organism whose action is weakened. (6) Within the past few years the -workers in the Pasteur Institute in Paris have been experimenting with -vaccines prepared by treating living virulent bacteria with antisera -("sensitizing them") so that they are no longer capable of causing -the disease when introduced, but do cause the production of an active -immunity. The method has been used with typhoid fever bacilli in man -and seems to be successful. It remains to be tried out further before -its worth is demonstrated (the procedure is more complicated and the -chance for infection apparently much greater than by the use of killed -cultures). The term _sero-bacterins_ is used by manufacturers in this -country to designate such bacterial vaccines. (7) Growing on artificial -culture media reduces the virulence of most organisms after a longer -or shorter time. This method has been tried with many organisms in the -laboratory, but is not now used in practice. The difficulties are that -the attenuation is very uncertain and that the organisms tend to regain -their virulence when introduced into the body. - -In producing active immunity against many bacterial diseases the -organisms are introduced (_c_) dead. They are killed by heat or by -chemicals, or by using both methods (Chapter XXX). - -When the products of an organism are introduced the resulting immunity -is against the products only and not against the organism. If the -organism itself is introduced there results an immunity against it -and in some cases also against the products, though the latter does -not necessarily follow. Hence the immunity may be _antibacterial_ or -_antitoxic_ or both. - -Investigation as to the causes of immunity and the various methods by -which it is produced has not resulted in the discovery of specific -methods of treatment for as many diseases as was hoped for at one -time. Just at present progress in serum therapy appears to be at a -standstill, though vaccines are giving good results in many instances -not believed possible a few years ago. As a consequence workers in all -parts of the world are giving more and more attention to the search for -_specific chemical substances_, which will destroy invading parasites -and not injure the host (_chemotherapy_). Nevertheless, in the study -of immunity very much of value in the treatment and prevention of -disease has been learned. Also much knowledge which is of the greatest -use in other lines has been accumulated. Methods of _diagnosis_ of -great exactness have resulted, applicable in numerous diseases. Ways -of _detecting adulteration_ in foods, particularly foods from animal -sources, and of _differentiating proteins_ of varied origin, as well -as means of establishing _biological relationships_ and differences -among groups of animals through "immunity reactions" of blood serums -have followed from knowledge gained by application of the facts or the -methods of immunity research. Hence the study of "immunity problems" -has come to include much more than merely the study of those factors -which prevent the development of disease in an animal or result in -its spontaneous recovery. A proper understanding of the principles of -immunity necessitates a study of these various features and they will -be considered in the discussion to follow. - - - - -CHAPTER XXVI. - -THEORIES OF IMMUNITY. - - -Pasteur and the bacteriologists of his time discovered that bacteria -cease to grow in artificial culture media after a time, because of -the exhaustion of the food material in some cases and because of the -injurious action of their own products in other instances. These facts -were brought forward to explain immunity shortly after bacteria were -shown to be the cause of certain diseases. Theories based on these -observations were called (1) "_Exhaustion Theory_" of _Pasteur_, and -(2) "_Noxious Retention Theory_" of _Chauveau_ respectively. The fact, -soon discovered, that virulent pathogenic bacteria are not uncommonly -present in perfectly healthy animals, and the later discovery that -immunity may be conferred by the injection of dead bacteria have led -to the abandonment of both these older ideas. The (3) "_Unfavorable -Environment_" theory of _Baumgartner_, _i.e._, bacteria do not grow -in the body and produce disease because their surroundings are not -suitable, in a sense covers the whole ground, though it is not true as -to the first part, as was pointed out above, and is of no value as a -working basis, since it offers no explanation as to _what the factors -are_ that constitute the "_unfavorable environment_." Metchnikoff -brought forward a rational explanation of immunity with his (4) -"_Cellular or Phagocytosis Theory_." As first propounded it based -immunity on the observed fact that certain white blood corpuscles, -_phagocytes_, engulf and destroy bacteria. Metchnikoff has since -elaborated the original theory to explain facts of later discovery. -Ehrlich soon after published his (5) "_Chemical or Side-chain Theory_" -which seeks to explain immunity on the basis of _chemical substances_ -in the body which may in part destroy pathogenic organisms or in -part neutralize their products; or in some instances there may be -an absence of certain chemical substances in the body cells so that -bacteria or their products _cannot unite_ with the cells and hence can -do no damage. - -[Illustration: PLATE VI - -PAUL EHRLICH] - -At the present time it is generally accepted, in this country at -least, that Ehrlich's theory explains immunity in many diseases as -well as many of the phenomena related to immunity, and in other -diseases the phagocytes, frequently assisted by chemical substances, -are the chief factors. Specific instances are discussed in _Pathogenic -Bacteriologies_ which should be consulted. It is essential that the -student should be familiar with the basic ideas of the chemical -theory, not only from the standpoint of immunity, but also in order to -understand the principles of a number of valuable methods of diagnosis. - -The chemical theory rests on three fundamental physiological -principles: (1) the response of cells to stimuli, in this connection -_specific chemical stimuli_, (2) the presence within cells of _specific -chemical groups_ which combine with chemical stimuli and thus enable -them to act on the cell, which groups Ehrlich has named _receptors_, -and (3) the "_over-production_" activity of cells as announced by -Weigert. - -1. That cells respond to stimuli is fundamental in physiology. These -stimuli may be of many kinds as mechanical, electrical, light, thermal, -chemical, etc. The body possesses groups of cells specially developed -to _receive_ some of these stimuli--touch cells for mechanical stimuli, -retinal cells for light, temperature nerve endings for thermal, -olfactory and gustatory cells for certain chemical stimuli. _Response_ -to chemical stimuli is well illustrated along the digestive tract. That -the chemical stimuli in digestion may be more or less specific is shown -by the observed differences in the enzymes of the pancreatic juice -dependent on the relative amounts of carbohydrates, fats, or proteins -in the food, the specific enzyme in each case being increased in the -juice with the increase of its corresponding foodstuff. The cells of -the body, or certain of them at least, seem to respond in a specific -way when substances are brought into direct contact with them, that is, -without having been subjected to digestion in the alimentary tract, -but injected directly into the blood or lymph stream. Cells may be -affected by stimuli in one of three ways: if the stimulus is too weak, -there is no effect (in reality there is no "stimulus" acting); if the -stimulus is too strong, the cell is injured, or may be destroyed; if -the stimulus is of proper amount then it excites the cell to increased -activity, and in the case of _specific chemical stimuli_ the increased -activity, as mentioned for the pancreas, shows itself in an _increased -production of whatever is called forth by the chemical stimulus_. In -the case of many organic chemicals, the substances produced by the -cells under their direct stimulation are markedly specific for the -particular substance introduced. - -2. Since chemical action always implies at least two bodies to react, -Ehrlich assumes that in every cell which is affected by a chemical -stimulus there must therefore be a chemical group to unite with this -stimulus. He further states that there must be as many different -kinds of these groups as there are different kinds of chemicals which -stimulate the cell. Since these groups are present in the body cells to -_take up_ different kinds of chemical substances, Ehrlich calls them -_receptors_. Since these groups must be small as compared with the cell -as a whole, and must be more or less on the surface and unite readily -with chemical substances he further speaks of them as "side-chains" -after the analogy of compounds of the aromatic series especially. The -term _receptors_ is now generally used. As was stated above, the effect -of _specific chemical stimuli_ is to cause the production of _more of -the particular substance_ for which it is specific and in the class -of bodies under discussion, the _particular product is these cell -receptors_ with which the chemical may unite. - -3. Weigert first called attention to the practically constant -phenomenon that cells ordinarily respond by doing more of a particular -response than is actually called for by the stimulus, that there is -always an "overproduction" of activity. In the case of chemical stimuli -this means an _increased production of the specific substance_ over and -above the amount actually needed. - -The student will better understand this theory if he recalls his -fundamental physiology. Living substance is characterized, among other -things, by irritability which is instability. It is in a constant, -state of unstable equilibrium. Whenever the equilibrium becomes -permanently stable the substance is dead. It is also continually -attempting to restore disturbances in its equilibrium. Whenever a -chemical substance unites with a chemical substance in the cell, a -receptor, the latter is, so far as the cell is concerned, _thrown out -of function_ for that cell. The chemical equilibrium of the latter -is upset. It attempts to restore this and does so by making a _new_ -receptor to take the place of the one thrown out of function. If this -process is continued, _i.e._, if the new receptor is similarly "used -up" and others similarly formed are also, then the cell will prepare -a supply of these and even an excess, according to Weigert's theory. -Whenever a cell accumulates an excess of products the normal result -is that it excretes them from its own substance into the surrounding -lymph, whence they reach the blood stream to be either carried to -the true excretory organs, utilized by other cells or remain for a -longer or shorter time in the blood. Hence the excess of receptors -is _excreted from the cell that forms them_ and they become _free_ -in the blood. These free receptors are termed _antibodies_. _They -are receptors_ but instead of being retained in the cell are _free -in solution in the blood_. One function of the free receptor, the -antibody, is _always to unite with the chemical substance which caused -it to be formed_. _It may have additional functions._ The chemical -substance which caused the excess formation of receptors, antibodies, -is termed an _antigen_ for that particular kind of antibody. - -To recapitulate, Ehrlich's theory postulates _specific chemical -stimuli_, which react with _specific chemical substances in the body -cells, named receptors_, and that these _receptors_, according to -Weigert, are _produced in excess_ and hence are excreted from the -cell and become _free receptors_ in the blood and lymph. These _free -receptors_ are the various kinds of _antibodies_, the kind depending -on the nature of the stimulus, antigen, the substance introduced. Any -substance which when introduced into the body causes the formation of -an antibody of any kind whatsoever is called an _antigen_,[23] _i.e._, -anti (body) former. - -The foregoing discussion explains Ehrlich's theory of immunity. -According to this theory the _manner of formation of all antibodies_ is -the same. The _kind of antibody_ and the _manner of its action_ will -differ with the _different kinds of antigens_ used. - -The succeeding chapters discuss some of the kinds of antibodies, the -theory of their action and some practical applications. It must be -borne in mind throughout the study of these, as has been stated, that -_every antibody has the property of uniting with its antigen whether it -has any property in addition or not_. - -Just what antibodies are chemically has not been determined because -no one has as yet succeeded in isolating them chemically pure. To the -author they appear to be enzymes. - -Antigens were considered by Ehrlich to be proteins or to be related to -proteins. Most workers since Ehrlich have held similar views. Dr. Carl -Warden of the University of Michigan has been doing much work in recent -years in which he is attempting to show that the antigens are not -proteins but are fats or fatty acids. Mr. E. E. H. Boyer, in his work -(not yet published) in the author's laboratory for the degree of Ph.D., -received in June, 1920, succeeded in producing various antibodies from -_Bacterium coli_ antigens. In these antigens he could detect only fatty -acids or salts of fatty acids. If the work of these men is confirmed, -it will open up a most interesting and extremely important field in -immunity and in preventive medicine. It is not apparent that the nature -of the antigen would affect Ehrlich's theory of the formation of -antibodies. - -The author has no doubt that eventually the formation of antibodies and -the reactions between them and their antigens will be explained on the -basis of physical-chemical laws, but this probably awaits the discovery -of their nature. - - - - -CHAPTER XXVII. - -RECEPTORS OF THE FIRST ORDER. - - -ANTITOXINS--ANTIENZYMES. - -The general characteristics of toxins have been described (Chapter -XII). It has been stated that they are more or less specific in -their action on cells. In order to affect a cell it is evident that -a toxin must enter into chemical combination with it. This implies -that the toxin molecule possesses a chemical group which can combine -with a receptor of the cell. This group is called the _haptophore_ or -combining group. The toxic or injurious portion of the toxin molecule -is likewise spoken of as the _toxophore_ group. When a toxin is -introduced into the body its _haptophore_ group combines with suitable -_receptors_ in different cells of the body. If not too much of the -toxin is given, instead of injuring, it acts as a chemical stimulus -to the cell in the manner already described. The cell in response -produces more of the specific thing, which in this instance is more -receptors which can combine with the toxin, _i.e._, with its haptophore -group. If the stimulus is kept up, more and more of these receptors -are produced until an excess for the cell accumulates, which excess is -excreted from the individual cell and becomes free in the blood. These -free receptors have, of course, the capacity to combine with toxin -through its haptophore group. When the toxin is combined with these -free receptors, it cannot combine with any other receptors, _e.g._, -those in another cell and hence cannot injure another cell. These free -receptors constitute, in this case, _antitoxin_, so-called because -they can combine with toxin and hence neutralize it. Antitoxins are -specific--that is, an antitoxin which will combine with the toxin of -_Clostridium tetani_ will not combine with that of _Corynebacterium -diphtheriæ_ or of _Clostridium botulinum_, or of any other toxin, -vegetable or animal. - -When a toxin is kept in solution for some time or when it is heated -above a certain temperature (different for each toxin) it loses its -poisonous character. It may be shown, however, that it is still capable -of uniting with antitoxin, and preventing the latter from uniting with -a fresh toxin. This confirms the hypothesis that a toxin molecule has -at least two groups: a combining or _haptophore_, and a poisoning or -_toxophore_ group. A toxin which has lost its poisonous property, its -toxophore group, is spoken of as a _toxoid_. The theory of antitoxin -formation is further supported by the fact that the proper introduction -of _toxoid_, the _haptophore_ group, and hence the real stimulus, can -cause the production of _antitoxin_ to a certain extent at least. - -The close relationship between toxins and enzymes has already been -pointed out. This is still further illustrated by the fact that when -enzymes are properly introduced into the tissues of an animal there -is formed in the animal an _antienzyme_ specific for the enzyme in -question which can prevent its action. The structure of enzymes, -as composed of a _haptophore_, or uniting, and a _zymophore_ or -_digesting_ (or other activity) group, is similar to that of toxins, -and _enzymoids_ or enzymes which can combine with the substance acted -on but not affect it further, have been demonstrated. - -These free cell receptors, antitoxins or antienzymes, which are -produced in the body by the proper introduction of toxins or enzymes, -respectively, have the function of _combining_ with these bodies -_but no other action_. As was pointed out above, this is sufficient -to neutralize the toxin or enzyme and prevent any injurious effect -since they can unite with nothing else. Since these receptors are the -simplest type which has been studied as yet, they are spoken of by -Ehrlich as _receptors of the first order_. Other antibodies which are -likewise free receptors of the first, order and have the function of -combining only have been prepared and will be referred to in their -proper connection. They are mainly of theoretical interest. - -Ehrlich did a large part of his work on toxins and antitoxins -with _ricin_, the toxin of the castor-oil bean, _abrin_, from the -jequirity bean, _robin_ from the locust tree, and with the toxins and -antitoxins for diphtheria and tetanus. Antitoxins have been prepared -experimentally for a large number of both animal and vegetable poisons, -including a number for bacterial toxins. The only ones which, as yet, -are of much practical importance are _antivenin_ for snake poison, (not -a true toxin, however, see p. 275), _antipollenin_ (supposed to be -for the toxin of hay fever) and the antitoxins for the true bacterial -toxins of _Corynebacterium diphtheriæ_ and _Clostridium tetani_. - -The method of preparing antitoxins is essentially the same in all -cases, though differing in minor details. For commercial purposes -large animals are selected, usually horses, so that the yield of -serum may be large. The animals must, of course, be vigorous, free -from all infectious disease. The first injection given is either a -relatively small amount of a solution of toxin or of a mixture of -toxin and antitoxin. The animal shows more or less reaction, increased -temperature, pulse and respiration and frequently an edema at the -point of injection, unless this is made intravenously. After several -days to a week or more, when the animal has recovered from the first -injection, a second stronger dose is given, usually with less reaction. -Increasingly large doses are given at proper intervals until the animal -may take several hundred times the amount which would have been fatal -if given at first. The process of immunizing a horse for diphtheria or -tetanus toxin usually takes several months. Variations in time and in -yield of antitoxin are individual and not predictable in any given case. - -After several injections a few hundred cubic centimeters of blood -are withdrawn from the jugular vein and serum from this is tested -for the amount of antitoxin it contains. When the amount is found -sufficiently large (250 "units" at least for diphtheria per cc.)[24] -then the maximum amount of blood is collected from the jugular with -sterile trocar and cannula. The serum from this blood with the addition -of an antiseptic (0.5 per cent. phenol, tricresol, etc.) constitutes -"antidiphtheritic serum" or "antitetanic serum," etc. All sera which -are put on the market must conform to definite standards of strength -expressed in "units" as determined by the U. S. Hygienic Laboratory. -In reality a "unit" of diphtheria antitoxin in the United States is -an amount equivalent to 1 cc. of a given solution of a _standard_ -diphtheria _antitoxin_ which is kept at the above-mentioned laboratory. -This statement, of course, gives no definite idea as to the amount -of antitoxin actually in a "unit." Specifically stated, a "unit" of -antitoxin contains approximately the amount which would protect a 250 -gram guinea-pig from 100 minimum lethal doses of diphtheria toxin, or -protect 100 guinea-pigs weighing 250 grams each from one minimum lethal -dose each. The minimum lethal dose (M. L. D.) of diphtheria toxin is -the least amount that will kill a guinea-pig of the size mentioned -within four days. Since toxins on standing change into toxoids to a -great extent, the amount, of antitoxin in a "unit," though protecting -against 100 M. L. D., in reality would protect against about 200 M. L. -D. of toxin containing no toxoid. - -The official unit for tetanus antitoxin is somewhat different, since it -is standardized against a _standard toxin_ which is likewise kept at -the Hygienic Laboratory. The unit is defined as "ten times the amount -of antitoxin necessary to protect a 350 g. guinea-pig for 96 hours -against the _standard test dose_" of the standard toxin. The standard -test dose is 100 M. L. D. of toxin for a 350 g. guinea-pig. To express -it another way, one could say that a "unit" of tetanus antitoxin -would protect one thousand 350 g. guinea-pigs from 1 M. L. D. each of -standard tetanus toxin. - -Various methods have been devised for increasing the amount of -antitoxin in 1 cc. of solution by precipitating out portions of the -blood-serum proteins and at the same time concentrating the antitoxin -in smaller volume. It is not considered necessary in a work of this -character to enter into these details nor to discuss the process of -standardizing antitoxin so that the exact amount of "units" per cc. may -be known. - - - - -CHAPTER XXVIII. - -RECEPTORS OF THE SECOND ORDER. - - -AGGLUTININS. - -Charrin and Rogers appear to have been the first (1889) to observe the -clumping together of bacteria (_Pseudomonas pyocyanea_) when mixed -with the blood serum of an animal immunized against them. Gruber and -Durham (1896) first used the term "agglutination" in this connection -and called the substance in the blood-serum "agglutinin." Widal (1896) -showed the importance of the reaction for diagnosis by testing the -blood serum of an infected person against a known culture (typhoid -fever). - -It is now a well-known phenomenon that the proper injection of cells -of any kind foreign to a given animal will lead to the accumulation in -the animal's blood of substances which will cause a clumping together -of the cells used when suspended in a suitable liquid. The cells settle -out of such suspension much more rapidly than they would otherwise -do. This clumping is spoken of as "agglutination" and the substances -produced in the animal are called "agglutinins." If blood cells are -injected then "hemagglutinins" result: if bacterial cells "bacterial -agglutinins" for the particular organism used as "glanders agglutinin" -for _Pfeifferella mallei_, "abortion agglutinin" for _Bacterium -abortus_, "typhoid agglutinin" for _Bacterium typhosum_, etc. - -The phenomenon may be observed either under the microscope or in small -test-tubes, that is, either _microscopically_ or _macroscopically_. - -In this case the cells introduced, or more properly, some substances -within the cells, act as stimuli to the body cells of the animal -injected to cause them to produce more of the specific cell receptors -which respond to the stimulus. The substance within the introduced -cell which acts as a stimulus (_antigen_) to the body cells is called -an "_agglutinogen_." That "agglutinogen" is present in the cell has -been shown by injecting animals experimentally with extracts of cells -(bacterial and other cells) and the blood serum of the animal injected -showed the presence of agglutinin for the given cell. It will be -noticed that the receptors which become the free agglutinins have at -least _two functions_, hence at least _two chemical groups_. They must -combine with the foreign cells and also bring about their clumping -together, their agglutination. Hence it can be stated technically that -an agglutinin possesses a _haptophore group_ and an _agglutinating -group_. - -It is probable that the agglutination, the clumping, is a secondary -phenomenon depending on the presence of certain salts and that the -agglutinin acts on its antigen as an enzyme, possibly a "splitting" -enzyme. This is analogous to what occurs in the curdling of milk -by rennet and in the coagulation of blood. This probability is -substantiated by the fact that suspensions of bacteria may be -"agglutinated" by appropriate strengths of various acids. - -The formation of agglutinin in the body for different bacteria does -not as yet appear to be of any special significance in protecting the -animal from the organism, since the bacteria are not killed, even -though they are rendered non-motile, if of the class provided with -flagella, and are clumped together. The fact that such bodies are -formed, however, is of decided value in the diagnosis of disease, and -also in the identification of unknown bacteria. - -In many bacterial diseases, agglutinins for the particular organism -are present in the blood serum of the affected animal. Consequently -if the blood serum of the animal be mixed with a suspension of the -organism supposed to be the cause of the disease and the latter be -agglutinated, one is justified in considering it the causative agent, -provided certain necessary conditions are fulfilled. In the first place -it must be remembered that the blood of normal animals frequently -contains agglutinins ("normal agglutinins") for many different -bacteria when mixed with them in full strength. Hence the serum must -always be diluted with physiological salt solution (0.85 per cent.). -Further, closely related bacteria may be agglutinated to some extent by -the same serum. It is evident that if they are closely related, their -protoplasm must contain some substances of the same kind to account for -this relationship. Since some of these substances may be agglutinogens, -their introduction into the animal body will give rise to agglutinins -for the related cells, as well as for the cell introduced. The -agglutinins for the cell introduced "chief agglutinins," will be -formed in larger quantity, since a given bacterial cell must contain -more of its own agglutinogen than that of any other cell. By _diluting -the blood serum_ from the animal to be tested the agglutinins for the -related organisms (so-called "coagglutinins" or "partial agglutinins") -will become so much diminished as to show no action, while the -agglutinin for the specific organism is still present in an amount -sufficient to cause its clumping. _Agglutinins are specific for their -particular agglutinogens_, but since a given blood serum may contain -many agglutinins, the _serum's specificity for a given bacterium_ -can be determined only by diluting it until this bacterium alone -is agglutinated. Hence the necessity of diluting the unknown serum -in varying amounts when testing against several known bacteria to -determine for which it is specific, _i.e._, which is the cause of the -disease in the animal. - -The agglutinins in the serum may be removed from it by treating it with -a suspension of the cells for which agglutinins are present. If the -"chief" cell is used all the agglutinins will be absorbed. If related -cells are used, only the agglutinins for this particular kind are -removed. These "absorption tests" furnish another means of determining -specificity of serum, or rather of determining the "chief agglutinin" -present. - -Just as an unidentified _disease_ in an animal may be determined by -testing its serum as above described against _known_ kinds of bacteria, -so _unknown bacteria_ isolated from an animal, from water, etc., may -be identified by testing them against the _blood sera_ of different -animals, each of which has been properly inoculated with a different -kind of _known bacteria_. If the unknown organism is agglutinated -by the blood of one of the animals in high dilution, and not by the -others, evidently the bacterium is the same as that with which the -animal has been inoculated, or _immunized_, as is usually stated. This -method of identifying cultures of bacteria is of wide application, -but is used practically only in those cases where other methods of -identification are not readily applied, and especially where other -methods are _not sufficient_ as in the "intestinal group" of organisms -in human practice. - -The diagnosis of disease in an animal by testing its serum is also a -valuable and much used procedure. This is the method of the "Widal" or -"Gruber-Widal" test for typhoid fever in man and is used in veterinary -practice in testing for glanders, contagious abortion, etc. In some -cases a dilution of the serum of from 20 to 50 times is sufficient for -diagnosis (Malta fever), in most cases, however, 50 times is the lowest -limit. Evidently the greater the dilution, that is, the higher the -"titer," the more specific is the reaction. - - -PRECIPITINS. - -Since agglutinins act on bacteria, probably through the presence of -substances within the bacterial cell, it is reasonable to expect that -if these substances be dissolved out of the cell, there would be some -reaction between their (colloidal) solution and the same serum. As -a matter of fact Kraus (1897) showed that broth cultures freed from -bacteria by porcelain filters do show a precipitate when mixed with -the serum of an animal immunized against the particular bacterium and -that the reaction is specific under proper conditions of dilution. -It was not long after Kraus's work until the experiments were tried -of "immunizing" an animal not against a bacterium or its filtered -culture, but against (colloidal) solutions of proteins, such as white -of egg, casein of milk, proteins of meat and of blood serum, vegetable -proteins, etc. It was ascertained that in all these cases the animal's -serum contains a substance which causes a _precipitate_ with solutions -of the protein used for immunization. The number of such precipitating -serums that have been made experimentally is very large and it appears -that protein from any source when properly introduced into the blood -or tissues of an animal will cause the formation of a precipitating -substance for its solutions. This substance is known, technically as a -"_precipitin_." The protein used as antigen to stimulate its formation, -or some part of the protein molecule (haptophore group), which acts -as stimulus to the cell is spoken of as a "precipitinogen," both -terms after the analogy of "agglutinin" and "agglutinogen." In fact -the specific precipitation and agglutination are strictly analogous -phenomena. Precipitins act on proteins in (colloidal) _solution_ and -cause them to settle out, agglutinins act on substances within cells -which cells are in _suspension_ in a fluid and cause the cells to -settle out. Ehrlich's theory of the formation of precipitins is similar -to that of agglutinins, and need not be repeated. Substitute the -corresponding words in the theory of formation of agglutinins as above -given and the theory applies. - -The precipitin reaction has not found much practical use in -bacteriology largely because the "agglutination test" takes its place -as simpler of performance and just as accurate. The reaction is, -however, generally applicable to filtrates of bacterial cultures and -could be used if needed. The so-called "mallease" reaction in glanders -is an instance. - -Precipitins find their greatest usefulness in legal medicine and -in food adulteration work. As was noted above, if animals, rabbits -for example, are immunized with the blood of another animal (human -beings) precipitins are developed which are specific for the injected -blood with proper dilution. This forms an extremely valuable means -of determining the _kind of blood_ present in a given spot shown by -chemical and spectroscopic tests to be blood and has been adopted as -a legal test in countries where such rules of procedure are applied. -Similarly the test has been used to identify the different kinds of -meat in sausage, and different kinds of milk in a mixture. An extract -of the sausage is made and tested against the serum of an animal -previously treated with extract of horse meat, or hog meat, or beef, -etc., the specific precipitate occurring with the specific serum. Such -reactions have been obtained where the protein to be tested was diluted -100,000 times and more. Biological relationships and differences have -been detected by the reaction. Human immune serum shows no reaction -with the blood of any animals except to a slight extent with that of -various monkeys, most with the higher, very slight with the lower Old -World and scarcely any with New World monkeys. - -It is a fact of theoretical interest mainly that if agglutinins -and precipitins themselves be injected into an animal they will -act as _antigens_ and cause the formation of _antiagglutinins_ or -_antiprecipitins_, which are therefore receptors of the first order -since they simply combine with these immune bodies to neutralize their -action, have only a combining or haptophore group. Also if agglutinins -or precipitins be heated to the proper temperature they may retain -their combining power but cause no agglutination or precipitation, -_i.e._, they are converted into agglutinoid or precipitinoid -respectively after the analogy of toxin and toxoid. - -Precipitins like agglutinins possess at least two groups--a combining -or _haptophore_ group and a _precipitating_ (sometimes called -zymophore) group. Hence they are somewhat more complex than antitoxins -or antienzymes which have a combining group only. For this reason -Ehrlich classes agglutinins and precipitins as _receptors of the second -order_. - - - - -CHAPTER XXIX. - -RECEPTORS OF THE THIRD ORDER. - - -CYTOLYSINS. - -Before Koch definitely proved bacteria capable of causing disease -several physiologists had noted that the red corpuscles of certain -animals were destroyed by the blood of other animals (Creite, 1869, -Landois, 1875), and Traube and Gescheidel had shown that freshly drawn -blood destroys bacteria (1874). It was not until about ten years -afterward that this action of the blood began to be investigated in -connection with the subject of immunity. Von Fodor (1885) showed that -saprophytic bacteria injected into the blood are rapidly destroyed. -Flügge and his pupils, especially Nuttall in combating Metchnikoff's -theory of phagocytosis, announced in 1883, studied the action of the -blood on bacteria and showed its destructive effect (1885-57). Nuttall -also showed that the blood lost this power if heated to 56°. Buchner -(1889) gave the name "alexin" (from the Greek "to ward off") to the -destroying substance and showed that the substance was present in -the _blood serum_ as well as in the whole blood, and that when the -serum lost its power to dissolve, this could be restored by adding -fresh blood. Pfeiffer (1894) showed that the destructive power of the -blood of animals immunized against bacteria (cholera and typhoid) was -markedly specific for the bacteria used. He introduced a mixture of -the blood and the bacteria into the abdominal cavity of the immunized -animal or of a normal one of the same species and noted the rapid -solution of the bacteria by withdrawing portions of the peritoneal -fluid and examining them ("Pfeiffer's phenomenon"). Belfanti and -Carbone and especially Bordet (1898) showed the specific dissolving -action of the serum of one animal on the blood corpuscles of another -animal with which it had been injected. Since this time the phenomenon -has been observed with a great variety of cells other than red blood -corpuscles and bacteria--leukocytes, spermatozoa, cells from liver, -kidney, brain, epithelia, etc., protozoa, and many vegetable cells. - -It is therefore a well-established fact that the proper injection of -an animal with almost any cell foreign to it will lead to the blood of -the animal injected acquiring the power to injure or destroy cells of -the same kind as those introduced. The destroying power of the blood -has been variously called its "cytotoxic" or "cytolytic" power, though -the terms are not strictly synonymous since "cytotoxic" means "cell -poisoning" or "injuring," while "cytolytic" means "cell dissolving." -The latter term is the one generally used and there is said to be -present in the blood a specific "cytolysin." The term is a general one -and a given cytolysin is named from the cell which is dissolved, as a -_bacteriolysin_, a _hemolysin_ (red-corpuscle-lysin), _epitheliolysin_, -_nephrolysin_ (for kidney cells), etc. If the cell is _killed_ but -not _dissolved_ the suffix "cidin" or "toxin" is frequently used as -"bacteriocidin," "spermotoxin," "neurotoxin," etc. - -The use of the term "cytolysin" is also not strictly correct, though -convenient, for the process is more complex than if _one substance -only_ were employed. As was stated above, the immune serum loses its -power to dissolve the cell if it is heated to 55° to 56° for half an -hour, it is _inactivated_. But if there be added to the heated or -inactivated serum a small amount of _normal serum_ (which contains only -a very little cytolytic substance, so that it has no dissolving power -when so diluted) the mixture again becomes cytolytic. It is evident -then that in cytolysis there are _two distinct substances_ involved, -one which is _present in all serum, normal or immune_, and the other -_present only in the immune cytolytic_ serum. This may be more apparent -if the facts are arranged in the following form: - - I. Immune serum dissolves cells in high dilution. - - II. Heated immune serum does not dissolve cells. - - III. Normal serum in high dilution does not dissolve cells. - - II. + III., _i.e._, Heated immune serum plus diluted normal serum - dissolves cells. - -Therefore, there is something in heated immune serum necessary for -cell dissolving and something different in diluted normal serum which -is necessary. This latter something is present in unheated immune -serum also, and is destroyed by heat. Experiment has shown that it is -the substance present in all serum both normal and immune that is the -true dissolving body, while the immune substance serves to unite this -body to the cell to be destroyed, _i.e._, to the antigen. Since the -immune body has therefore _two uniting groups_, one for the dissolving -substance and one for the cell to be dissolved, Ehrlich calls it the -"_amboceptor_." He also uses the word "_complement_" to denote the -dissolving substance, giving the idea that it completes the action of -dissolving after it has been united to the cell by the amboceptor, thus -replacing Buchner's older term "alexin" for the same dissolving body. - - -AMBOCEPTORS. - -The theory of formation of amboceptors is similar to that for the -formation of the other types of antibodies. The cell introduced -contains some substance, which acts as a chemical stimulus to some of -the body cells provided with proper receptors so that more of these -special receptors are produced, and eventually in excess so that they -become free in the blood and constitute the free amboceptors. It will -be noticed that these free receptors differ from either of the two -kinds already described in that they have _two uniting groups_, one for -the antigen (cell introduced) named _cytophil-haptophore_, the other -for the complement, _complementophil haptophore_. Hence amboceptors -are spoken of as _receptors of the third order_. They have no other -function than that of this double combining power. The action which -results is due to the third body--the complement. It will be readily -seen that complement must possess at least two groups, a combining or -_haptophore group_ which unites with the amboceptor, and an active -group which is usually called the _zymophore_ or _toxophore_ group. -Complements thus resemble either toxins, where the specific cell -(antigen) is injured or killed, or enzymes, in case the cell is -likewise dissolved. This action again shows the close relation between -toxins and enzymes. Complement may lose its active group in the same -way that toxin does and becomes then _complementoid_. Complement is -readily destroyed in blood or serum by heating it to 55° to 56° for -half an hour, and is also destroyed spontaneously when serum stands for -a day or two, less rapidly at low temperature than at room temperature. - -Amboceptors appear to be _specific_ in the same sense that agglutinins -are. That is, if a given cell is used to immunize an animal, the -animal's blood will contain amboceptors for the cell used and also for -others closely related to it. Immunization with spermatozoa or with -epithelial or liver cells gives rise to amboceptors for these cells -and also for red blood corpuscles and other body cells. A typhoid -bactericidal serum has also some dissolving effect on colon bacilli, -etc. Hence a given serum may contain a chief amboceptor and a variety -of "coamboceptors," or one amboceptor made up of a number of "partial -amboceptors" each specific for its own antigen ("amboceptorogen"). -Amboceptors may combine with other substances than antigen and -complement, as is shown by their union with lecithin and other -"lipoids," though these substances seem capable of acting as complement -in causing solution of blood corpuscles. - - -COMPLEMENTS. - -As to whether complements are numerous, as Ehrlich claims, or there -is only one complement, according to Buchner and others, need not be -discussed here. In the practical applications given later as means of -diagnosis it is apparent that all the complement or complements are -capable of uniting with at least two kinds of amboceptors. - -If complement be injected into an animal it may act as an antigen and -give rise to the formation of _anticomplement_ which may combine with -it and prevent its action and is consequently analogous to antitoxin. -If amboceptors as antigen are injected into an animal there will be -formed by the animal's cells _antiamboceptors_. As one would expect, -there are two kinds of antiamboceptors, one for each of its combining -groups, since it has been stated that it is always the combining group -of any given antigen that acts as the cell stimulus. Hence we may have -an "anticytophil amboceptor" or an "anticomplementophil amboceptor." -These antiamboceptors and the anticomplements are analogous to -antitoxin, antiagglutinin, etc., and hence are receptors of the first -order. - - -ANTISNAKE VENOMS. - -A practical use of antiamboceptors is in antisnake venoms. Snake -poisons appear to contain only _amboceptors_ for different cells of the -body. In the most deadly the amboceptor is specific for nerve cells -(cobra), in others for red corpuscles, or for endothelial cells of the -bloodvessels (rattlesnake). The complement is furnished by the blood -of the individual bitten, that is, in a sense the individual poisons -himself, since he furnishes the destroying element. The antisera -contain antiamboceptors which unite with the amboceptor introduced and -prevent it joining to cells and thus binding the complement to the -cells and destroying them. With this exception these antibodies are -chiefly of theoretical interest. - - -FAILURE OF CYTOLYTIC SERUMS. - -The discovery of the possibility of producing a strongly bactericidal -serum in the manner above described aroused the hope that such sera -would prove of great value in passive immunization and serum treatment -of bacterial diseases. Unfortunately such expectations have not been -realized and no serum of this character of much practical importance -has been developed as yet (with the possible exception of Flexner's -antimeningococcus serum in human practice. What hog cholera serum is -remains to be discovered). - -The reasons for the failure of such sera are not entirely clear. -The following are some that have been offered: (1) Amboceptors do -not appear to be present in very large amount so that relatively -large injections of blood are necessary, which is not without risk -in itself. (2) Since the complement is furnished by the blood of the -animal to be treated, there may not be enough of this to unite with a -sufficient quantity of amboceptor to destroy all the bacteria present, -hence the disease is continued by those that escape. (3) Or the -complement may not be of the right kind to unite with the amboceptor -introduced, since this is derived from the blood of a _heterologous_ -("other kind") species. In hog-cholera serum, if it is bactericidal, -this difficulty is removed by using blood of a _homologous_ ("same -kind") animal. Hence Ehrlich suggested the use of apes for preparing -bactericidal sera for human beings. The good results which have been -reported in the treatment of human beings with the serum of persons -convalescing from the same disease indicate that this lack of proper -complement for the given amboceptor is probably a chief factor in the -failure of sera from lower animals. (4) The bacteria may be localized -in tissues (lymph glands), within cavities (cranial, peritoneal), in -hollow organs (alimentary tract), etc., so that it is not possible to -get at them with sufficient serum to destroy all. This difficulty is -obviated by injecting directly into the spinal canal when Flexner's -antimeningococcus serum is used. (5) Even if the bacteria are dissolved -it does not necessarily follow that their _endotoxins_ are destroyed. -These may be merely liberated and add to the danger of the patient, -though this does not appear to be a valid objection. (6) Complement -which is present in such a large excess of amboceptor may just as -well unite with amboceptor which is not united to the bacteria to be -destroyed as with that which is, and hence be actually prevented from -dissolving the bacteria. Therefore it is difficult to standardize the -serum to get a proper amount of amboceptor for the complement present. - - -COMPLEMENT-FIXATION TEST. - -Although little practical use has been made of bactericidal sera, -the discovery of receptors of this class and the peculiar relations -between the antigen, amboceptor and complement have resulted in -developing one of the most delicate and accurate methods for the -diagnosis of disease and for the recognition of small amounts of -specific protein that is in use today. - -This method is usually spoken of as the "complement-fixation" or the -"complement-deviation test" ("Wassermann test" in syphilis) and is -applicable in a great variety of microbial diseases, but it is of -practical importance in those diseases only where other methods are -uncertain--syphilis in man, concealed glanders in horses, contagious -abortion in cattle, etc. A better name would be the "Unknown Amboceptor -Test" since it is the amboceptor that is searched for in the test by -making use of its power to "fix" complement. - -The principle is the same in all cases. The method depends, as -indicated above, on the ability of complement to combine with at least -two amboceptor-antigen systems, and on the further fact that if the -combination with one amboceptor-antigen system is once formed, it -does not dissociate so as to liberate the complement for union with -the second amboceptor-antigen system. If an animal is infected with a -microörganism and a part of its defensive action consists in destroying -the organisms in its blood or lymph, then it follows from the above -discussion of cytolysins that there will be developed in the blood of -the animal amboceptor specific for the organism in question. If the -presence of this _specific amboceptor_ can be detected, the conclusion -is warranted that the organism for which it is specific is the cause of -the disease. Consequently what is sought in the "complement-fixation -test" is a _specific amboceptor_. In carrying out the test, blood -serum from the suspected animal is collected, heated at 56° for half -an hour to destroy any complement it contains and mixed in definite -proportions with the specific antigen and with complement. The -antigen is an extract of a diseased organ (syphilitic fetal liver, -in syphilis), a suspension of the known bacteria, or an extract of -these bacteria. Complement is usually derived from a guinea-pig, -since the serum of this animal is higher in complement than that -of most animals. The blood of the gray rat contains practically as -much. If the specific amboceptor is present, that is, if the animal -is infected with the suspected disease, the complement will unite -with the antigen-amboceptor system and be "fixed," that is, be no -longer capable of uniting with any other amboceptor-antigen system. No -chemical or physical means of telling whether this union has occurred -or not, except as given below, has been discovered as yet, though -doubtless will be by physico-chemical tests, nor can the combination -be seen. Hence an "indicator," as is so frequently used in chemistry, -is put into the mixture of antigen-amboceptor-complement after it has -been allowed to stand in the incubator for one-half to one hour to -permit the union to become complete. The "indicator" used is a mixture -of sheep's corpuscles and the heated ("inactivated") blood serum of -a rabbit which has been injected with sheep's blood corpuscles and -therefore contains a _hemolytic amboceptor specific_ for the corpuscles -which is capable also of uniting with complement. The indicator is -put into the first mixture and the whole is again incubated and -examined. If the mixture is _clear_ and _colorless_ with a _deposit -of red corpuscles_ at the bottom, that would mean that the complement -had been bound to the first complex, since it was not free to unite -with the second sheep's corpuscles (antigen)--rabbit serum (hemolytic -amboceptor) complex--and destroy the corpuscles. Hence if the -complement is bound in the first instance, the _specific amboceptor_ -for the first antigen must have been present in the blood, that is, the -animal was infected with the organism in question. Such a reaction is -called a "positive" test. - -On the other hand, if the final solution is _clear_ but of a _red_ -color, that would mean that complement must have united with the -corpuscles--hemolytic amboceptor system--and destroyed the corpuscles -in order to cause the _clear red_ solution of hemoglobin. If complement -united with this system it could not have united with the first system, -hence there was no _specific amboceptor_ there to bind it; no specific -amboceptor in the animal's blood, means no infection. Hence a _red -solution_ is a "negative test." - -The scheme for the test may be outlined as follows: - - Antigen + Patient's Serum, heated + Complement - (specific for (unknown amboceptor) (derived from - the amboceptor guinea pig's serum) - sought) - -Incubate one-half hour in a water bath or one hour in an incubator. - -Then add the indicator which is - - Antigen + Amboceptor - (red blood corpuscles) (for corpuscles, serum of - a rabbit immunized against - the red corpuscles) - -Incubate as above. - -In practice all the different ingredients must be accurately tested, -standardized and used in exact quantities, and tests must also be run -as controls with a known normal blood of an animal of the same species -as the one examined and with a known positive blood. - - It should be stated that in one variety of the Complement-Fixation - Test, namely, the "Wassermann Test for Syphilis" in human beings, - an antigen is used which is not derived from the specific organism - (_Treponema pallidum_) which causes the disease nor even from - syphilitic tissue. It has been determined that alcohol will extract - from certain tissues, _human or animal_, substances which _act - specifically_ in combining with the syphilitic amboceptor present - in the blood. Alcoholic extracts of beef heart are most commonly - used. Details of this test may be learned in the advanced course in - Immunity and Serum Therapy. - -The complement-fixation test might be applied to the determination -of unknown bacteria, using the unknown culture as antigen and trying -it with the sera of different animals immunized against a variety -of organisms, some one of which might prove to furnish _specific -amboceptor_ for the unknown organism and hence indicate what it is. The -test used in this way has not been shown to be a practical necessity -and hence is rarely employed. It has been used, however, to detect -traces of unknown proteins, particularly blood-serum proteins, in -medico-legal cases in exactly the above outlined manner and is very -delicate and accurate. - - - - -CHAPTER XXX. - -PHAGOCYTOSIS--OPSONINS. - - -It has been mentioned that Metchnikoff, in a publication in 1883, -attempted to explain immunity on a purely cellular basis. It has -been known since Haeckel's first observation in 1858 that certain of -the white corpuscles do engulf solid particles that may get into the -body, and among them bacteria. Metchnikoff at first thought that this -engulfing and subsequent intracellular digestion of the microörganisms -were sufficient to protect the body from infection. The later -discoveries (discussed in considering Ehrlich's theory of immunity) -of substances present in the blood serum and even in the blood plasma -which either destroy the bacteria or neutralize their action have -caused Metchnikoff to modify his theory to a great extent. He admitted -the presence of these substances, though giving them other names, -but ascribed their formation to the phagocytes or to the same organs -which form the leukocytes--lymphoid tissue generally, bone marrow. It -is not within the province of this work to attempt to reconcile these -theories, but it may be well to point out that Ehrlich's theory is one -of _chemical substances_ and that the _origin_ of these substances is -not an _essential_ part of the theory, so that the two theories, except -in some minor details, are not necessarily mutually exclusive. - -[Illustration: PLATE V - -ELIE METCHNIKOFF] - -Sir A. E. Wright and Douglas, in 1903, showed that even in those -instances where immunity depends on phagocytosis, as it certainly does -in many cases, the phagocytes are more or less inactive unless they are -aided by chemical substances present in the blood. These substances -_act on the bacteria, not on the leukocytes_, and change them in such -a way that they are more readily taken up by the phagocytes. Wright -proposed for these bodies the name _opsonin_, derived from a Greek -word signifying "to prepare a meal for." Neufeld and Rimpau at about -the same time (1904), in studying immune sera, observed substances of -similar action in these sera and proposed the name _bacteriotropins_, -or bacteriotropic substances. There is scarcely a doubt that the two -names are applied to identical substances and that Wright's name -_opsonin_ should have preference. - -The chemical nature of opsonins is not certainly determined, but they -appear to be a distinct class of antibodies and to possess two groups, -a combining or haptophore and a preparing or opsonic group and hence -are similar to antibodies of Ehrlich's second order--agglutinins and -precipitins. Wright also showed that opsonins are just as specific as -agglutinins are--that is, a micrococcus opsonin prepares micrococci -only for phagocytosis and not streptococci or any other bacteria. - -Wright showed that opsonins for many bacteria are present in normal -serum and that in the serum of an animal which has been immunized -against such bacteria the opsonins are _increased_ in amount. Also -that in a person infected with certain bacteria the opsonins are -either increased or diminished, depending on whether the progress of -the infection is favorable or unfavorable. The _opsonic power_ of a -serum normal or otherwise is determined by mixing an emulsion of fresh -leukocytes in normal saline solution with a suspension of the bacteria -and with the serum to be tested. The leukocytes must first be washed in -several changes of normal salt solution to free them from any adherent -plasma or serum. The mixture is incubated for about fifteen minutes -and then slides are made, stained with a good differential blood -stain, Wright's or other, and the average number of bacteria taken up -by at least fifty phagocytes taken in order in a field is determined -by counting under the microscope. The number so obtained Wright calls -the _phagocytic index_ of the serum tested. The phagocytic index of a -given serum divided by the phagocytic index of a normal serum gives -the _opsonic index_ of the serum tested. Assuming the normal opsonic -index to be 1, Wright asserts that in healthy individuals the range -should be not more than from 0.8 to 1.2, and that an index below 0.8 -may show a great susceptibility for the organism tested, infection with -the given organism if derived from the individual, or improper dosage -in case attempts have been made to immunize by using killed cultures, -vaccines, of the organism. - -On the occasion of the author's visit to Wright's clinic (1911) he -stated that he used the determination of the _opsonic index_ chiefly as -a _guide to the dosage_ in the use of vaccines. - -Most workers outside the Wright school have failed to recognize any -essential value of determinations of the opsonic index in the use -of vaccines. Some of the reasons for this are as follows: The limit -of error in phagocytic counts may be as great as 50 per cent. in -different series of fifty, hence several hundred must be counted, which -adds greatly to the tediousness and time involved; the variation in -apparently healthy individuals is frequently great, hence the "normal" -is too uncertain; finally the opsonic index and the clinical course of -the disease do not by any means run parallel. Undoubtedly the method -has decided value in the hands of an individual who makes opsonic -determinations his chief work, as Wright's assistants do, but it can -scarcely be maintained at the present time that such determinations -are necessary in vaccine therapy. Nevertheless that opsonins actually -exist and that they play an essential part in phagocytosis, and hence -in immunity, is now generally recognized. - - -BACTERIAL VACCINES. - -Whether determinations of opsonic index are useful or not is largely -a matter of individual opinion, but there is scarcely room to doubt -that Wright has conferred a lasting benefit by his revival of the -use of _dead cultures of bacteria_, _bacterial vaccines_, both for -protective inoculation and for treatment. It is perhaps better to use -the older terms "vaccination" and "vaccine" (though the cow, _vacca_, -is not concerned) than to use Wright's term "opsonic method" in this -connection, bearing in mind that the idea of a vaccine is that it -contains the _causative organism_ of the infection as indicated on p. -253. - -As early as 1880 Touissant proposed the use of dead cultures of -bacteria to produce immunity. But because injections of such cultures -were so frequently followed by abscess formation, doubtless due to -the _high temperatures_ used to kill the bacteria, the method was -abandoned. Further, Pasteur and the French school persistently denied -the possibility of success with such a procedure, and some of them -even maintain this attitude at the present time. The successes of -Wright and the English school which are being repeated so generally -wherever properly attempted, leave no doubt in the unprejudiced of the -very great value of the method and have unquestionably opened a most -promising field both for preventive inoculation and for treatment in -many infectious diseases. That the practice is no more universally -applicable than are immune serums and that it has been and is still -being grossly overexploited is undoubted. - -The use of a vaccine is based on two fundamental principles. The first -of these is that the cell introduced must not be in a condition to -cause serious injury to the animal by its multiplication and consequent -elaboration of injurious substances. The second is that, on the other -hand, it must contain antigens in such condition that they will act as -stimuli to the body cells to produce the necessary antibodies, whether -these be opsonins, bactericidal substances, or anti-endotoxins. In the -introduction of living organisms there is always more or less risk -of the organism not being sufficiently attenuated and hence of the -possibility of its producing too severe an infection. In using killed -cultures, great care must be exercised in destroying the organisms, -_so that the antigens are not at the same time rendered inactive_. -Hence in the preparation of bacterial vaccines by Wright's method the -_temperature and the length of time used to kill the bacteria are most -important factors_. This method is in general to grow the organisms -on an agar medium, rub off the culture and emulsify in sterile normal -salt solution (0.85 per cent. NaCl). The number of bacteria per cc. -is determined by staining a slide made from a small volume of the -emulsion mixed with an equal volume of human blood drawn from the -finger and counting the relative number of bacteria and of red blood -corpuscles. Since the corpuscles are normally 5,000,000 per c.mm., -a simple calculation gives the number of bacteria. The emulsion of -bacteria is then diluted so that a certain number of millions shall -be contained in each cc., "standardized" as it is called, then heated -to the proper temperature for the necessary time and it is ready for -use. A preservative, as 0.5 per cent. phenol, tricresol, etc., is added -unless the vaccine is to be used up at once. The amounts of culture, -salt solution, etc., vary with the purpose for which the vaccine is to -be used, from one or two agar slant cultures and a few cc. of solution, -when a single animal is to be treated, to bulk agar cultures and liters -of solution as in preparing antityphoid vaccine on a large scale. - -Agar surface cultures are used so that there will be as little -admixture of foreign protein as possible (see Anaphylaxis, p. 289 _et -seq._). Normal saline solution is isotonic with the body cells and -hence is employed as the vehicle. - -=Lipovaccines.=--The suspension of bacteria in neutral oil was first -used by Le Moignac and Pinoy who gave the name "lipovaccines" (#lipos# -= fat) to them. It was claimed that the reaction following injection -of these vaccines was less severe than with saline vaccines in many -instances; also, that the bacteria were much more slowly absorbed. For -these two reasons it was hoped that much larger numbers of bacteria -could be injected at one dose and one injection would suffice instead -of three or more as ordinarily used. The technique of preparation, -standardization and killing of the organisms has not as yet been -sufficiently well established to warrant the general substitution of -lipovaccines for ordinary saline suspensions. - -Vaccines are either "_autogenous_" or "_stock_." An "autogenous" -vaccine is a vaccine that is made from bacteria derived from the -individual or animal which it is desired to vaccinate and contains -not only the particular organism but the particular strain of that -organism which is responsible for the lesion. Stock vaccines are -made up from organisms like the infective agent in a given case but -derived from some other person or animal or from laboratory cultures. -Commercial vaccines are "stock" vaccines and are usually "polyvalent" -or even "mixed." A "polyvalent" vaccine contains several strains of the -infective agent and a "mixed" contains several different organisms. - -Stock vaccines have shown their value when used as preventive -inoculations, notably so in typhoid fever in man, anthrax and black-leg -in cattle. The author is strongly of the opinion, not only from the -extended literature on the subject, but also from his own experience -in animal, and especially in human cases, that stock vaccines are -much inferior and much more uncertain in their action when used in -the _treatment_ of an infection, than are autogenous vaccines. This -applies particularly to those instances in which _pneumococci_, -_streptococci_, _micrococci_, and _colon bacilli_ are the causative -agents but to others as well. The following are some of the reasons for -this opinion: The above organisms are notoriously extremely variable in -their virulence. While there is no necessarily close connection between -virulence and antigenic property, yet since virulence is so variable, -it is rational to assume that antigenic property is also extremely -variable. Individuals vary just as much in susceptibility and hence in -reactive power, and generally speaking, an individual will react better -in the production of antibodies to a stimulus to which he has been more -or less subjected, _i.e._, to organisms derived from his own body. - -In the preparation of a vaccine great care must be used in heating so -that the organisms are killed, but the _antigens_ are not destroyed. -Many of the enzymes present in bacteria, especially the proteolytic -ones, are not any more sensitive to heat than are the antigens, hence -are not destroyed entirely. Therefore a vaccine kept in stock for a -long time gradually has some of its antigens destroyed by the uninjured -enzymes present with them, and so loses in potency. Therefore in -treating a given infection it is well to make up a vaccine from the -lesion, use three or four doses and if more are necessary make up a new -vaccine. - -If the above statements are borne in mind and vaccines are made and -administered accordingly, the author is well satisfied that much better -results will be secured. - -In accordance with the theory on which the use of vaccines is based, -_i.e._, that they stimulate the body cells to produce immunizing -antibodies, it is clear that they are especially suitable in those -infections in which the process is _localized_ and should not be of -much value in _general_ infections. In the latter case the cells of -the body are stimulated to produce antibodies by the circulating -organisms, probably nearly to their limit, hence the introduction of -more of the same organisms, capable of stimulating though dead, is apt -to overtax the cells and do more harm than good. It is not possible to -tell accurately when this limit is reached, but the clinical symptoms -are a guide. If vaccines are used at all in general infections they -should be given in the early stages and in small doses at first with -close watch as to the effect. In localized infections only the cells in -the immediate neighborhood are much stimulated, hence the introduction -of a vaccine calls to their aid cells in the body generally, and much -more of the resulting antibodies are carried to the lesion in question. -Manifestly surgical procedures such as incision, drainage, washing -away of dead and necrotic tissue with normal saline solution, not -necessarily antiseptics, will aid the antibodies in their action and -are to be recommended where indicated. - -In the practical application of any remedy the _dosage_ is most -important. Unfortunately there is no accurate method of determining -this with a vaccine. Wright recommended determining the number of -the organisms per cc. as before mentioned, and his method or some -modification of it is still in general use. From what was said with -regard to variation, both in organisms and in individuals, it can -be seen that the number of organisms is at least only a very rough -guide. This is further illustrated by the doses of micrococcus -(staphylococcus) vaccines recommended by different writers, which -vary from 50,000,000 to 2,000,000,000 per cc. The author is decidedly -of the opinion that _there is no way of determining the dosage of -a vaccine in the treatment of any given case except by the result -of the first dose_. Hence it is his practice to make vaccines of a -particular organism of the same approximate strength, and to give a -dose of a measured portion of a cubic centimeter, judging the amount -by what the individual or animal can apparently withstand, without too -violent a reaction. If there is no local or general reaction or if it -is very slight and there is no effect on the lesion, the dose is too -small. If there is a violent local reaction with severe constitutional -symptoms clinically, and the lesion appears worse, the dose is too -large. There should be some local reaction and some general, but not -enough to cause more than a slight disturbance, easy to judge in human -subjects, more difficult in animals. In cases suitable for vaccine -treatment no _serious_ results should follow from a properly prepared -vaccine, though the process of healing may be delayed temporarily. -Wright claimed, and many have substantiated him, that always following -a vaccination there is a period when the resistance of the animal is -diminished. This is called the "negative phase," and Wright considered -this to last as long as the opsonic index remained low, and when this -latter began to increase the stage of the "positive" or favorable phase -was reached. As has been stated the opsonic index is pretty generally -regarded as of doubtful value, though the existence of a period of -lowered resistance is theoretically probable from the fact that -antibodies already present in the blood will be partially used up in -uniting with the vaccine introduced and that the body cells are called -upon suddenly to do an extra amount of work and it takes them some time -to adapt themselves. This time, the "negative phase," is much better -determined by the clinical symptoms, general and especially local. It -is good practice to begin with a dose relatively small. The result -of this is an indication of the proper dosage and also prepares the -patient for a larger one. The second dose should follow the first not -sooner than three or four days, and should be five to seven days if the -first reaction is severe. These directions are not very definite, but -clinical experience to date justifies them. It is worth the time and -money to one who wishes to use vaccines to learn from one who has had -experience both in making and administering them, and then to remember -that each patient is an individual case, for the use of vaccines as -well as for any other kind of treatment. - - -AGGRESSIN. - -Opsonins have been shown to be specific substances which act on -bacteria in such a way as to render them more readily taken up by the -leukocytes. By analogy one might expect to find bacteria secreting -specific substances which would tend to counteract the destructive -action of the phagocytes and bactericidal substances. Bail and his -co-workers claim to have demonstrated such substances in exudates in -certain diseases and have given the distinctive name "aggressins" to -them. By injecting an animal with "aggressins," antiaggressins are -produced which counteract their effects and thus enable the bacteria to -be destroyed. The existence of such specific bodies is not generally -accepted as proved. The prevailing idea is that bacteria protect -themselves in any given case by the various toxic substances that they -produce, and that "aggressins" as a special class of substances are not -formed. - - - - -CHAPTER XXXI. - -ANAPHYLAXIS. - - -Dallera, in 1874, and a number of physiologists of that period, -observed peculiar skin eruptions following the transfusion of blood, -that is, the introduction of foreign proteins. In the years subsequent -to the introduction of diphtheria antitoxin (1890) characteristic -"serum rashes" were not infrequently reported, sometimes accompanied by -more or less severe general symptoms and occasionally death--a train of -phenomena to which the name "serum sickness" was later applied, since -it was shown that it was the horse serum (foreign protein) that was -the cause, and not the antitoxin itself. In 1898 Richet and Hericourt -noticed that some of the dogs which they were attempting to immunize -against toxic eel serum not only were not immunized but suffered even -more severely after the second injection. They obtained similar results -with an extract of mussels which contain a toxin. Richet gave the name -"anaphylaxis" ("no protection") to this phenomenon to distinguish it -from immunity or prophylaxis (protection). - -All the above-mentioned observations led to no special investigations -as to their cause. In 1903, Arthus noticed abscess formation, -necrosis and sloughing following several injections of horse serum -in immediately adjacent parts of the skin in rabbits ("Arthus' -phenomenon"). Theobald Smith, in 1904, observed the death of -guinea-pigs following properly spaced injections of horse serum. This -subject was investigated by Otto and by Rosenau and Anderson in this -country and about the same time von Pirquet and Schick were making a -study of serum rashes mentioned above. The publications of these men -led to a widespread study of the subject of injections of foreign -proteins. It is now a well-established fact that the injection into an -animal of a foreign protein--vegetable, animal or bacterial, simple or -complex--followed by a second injection after a proper length of time -leads to a series of symptoms indicating poisoning, which may be so -severe as to cause the death of the animal. Richet's term "anaphylaxis" -has been applied to the condition of the animal following the first -injection and indicates that it is in a condition of supersensitiveness -for the protein in question. The animal is said to be "sensitized" -for that protein.[25] The sensitization is specific since an animal -injected with white of chicken's egg reacts to a second injection of -chicken's egg only and not pigeon's egg or blood serum or any other -protein. The specific poisonous substance causing the symptoms has -been called "anaphylotoxin" though what it is, is still a matter of -investigation. It is evident that some sort of an antibody results from -the first protein injected and that it is specific for its own antigen. - -A period of ten days is usually the minimum time that must elapse -between the first and second injections in guinea-pigs in order that a -reaction may result, though a large primary dose requires much longer. -If the second injection is made within less time no effect follows, -and after three or more injections at intervals of about one week the -animal fails to react at all, it has become "immune" to the protein. -Furthermore, after an animal has been sensitized by one injection and -has reacted to a second, then, if it does not die from the reaction, it -fails to react to subsequent injections. In this latter case it is said -to be "antianaphylactic." - -It must be remembered that proteins do not normally get into the -circulation except by way of the alimentary tract. Here all proteins -that are absorbed are first broken down to their constituent -amino-acids, absorbed as such and these are built up into the proteins -characteristic of the animal's blood. Hence when protein as such gets -into the blood it is a foreign substance to be disposed of. The blood -contains proteolytic enzymes for certain proteins normally. It is -also true that the body cells possess the property of digesting the -proteins of the blood and building them up again into those which are -characteristic of the cell. Hence it appears rational to assume that -the foreign proteins act as stimuli to certain cells to produce more -of the enzymes necessary to decompose them, so that they may be either -built up into cell structure or eliminated as waste. If in this process -of splitting up of protein a poison were produced, then the phenomena -of "anaphylaxis" could be better understood. As a matter of fact -Vaughan and his co-workers have shown that by artificially splitting -up proteins from many different sources--animal, vegetable, pathogenic -and saprophytic bacteria--a poison _is produced_ which appears to be -the same in all cases and which causes the symptoms characteristic of -anaphylaxis. On the basis of these facts it is seen that anaphylaxis -is simply another variety of immunity. The _specific antibody_ in -this case is an _enzyme_ which decomposes the protein instead of -precipitating it. The enzyme must be specific for the protein since -these differ in constitution. Vaughan even goes so far as to say that -the poison is really the central ring common to all proteins and that -they differ only in the lateral groups or side chains attached to this -central nucleus. The action of the enzyme in this connection would be -to split off the side chains, and since these are the specific parts of -the protein, the enzyme must be specific for each protein. The pepsin -of the gastric juice and the trypsin of the pancreas split the native -proteins only to peptones. As is well known, these when injected in -sufficient quantity give rise to poisonous symptoms, and will also -give rise to anaphylaxis under properly spaced injections. They do not -poison normally because they are split by the intestinal erepsin to -amino-acids and absorbed as such. Whether Vaughan's theory of protein -structure is the true one or not remains to be demonstrated. It is -not essential to the theory of anaphylaxis above outlined, _i.e._, -a phenomenon due to the action of specific _antibodies_ which are -enzymes. On physiological grounds this appears the most rational of the -few explanations of anaphylaxis that have been offered and was taught -by the author before he had read Vaughan's theory along the same lines. - -On the basis of the author's theory the phenomena of protein immunity -and antianaphylaxis may be explained in the following way which the -author has not seen presented. The enzymes necessary to decompose the -injected protein are present in certain cells and are formed in larger -amount by those cells to meet the increased demand due to injection -of an excess of protein. They are retained in the cell for a time at -least. If a second dose of protein is given before the enzymes are -excreted from the cells as waste, this is digested within the cells in -the normal manner. If a third dose is given, the cells adapt themselves -to this increased intracellular digestion and it thus becomes normal -to them. Hence the _immunity_ is due to this increased intracellular -digestion. - -On the other hand, if the second injection is delayed long enough, then -the _excess_ enzyme, but not all, is excreted from the cells and meets -the second dose of protein in the blood stream and rapidly decomposes -it there, so that more or less intoxication from the split products -results. This uses up _excess_ enzyme, hence subsequent injections are -not digested in the blood stream but within the cells as before. So -that "antianaphylaxis" is dependent on the exhaustion of the excess -enzyme in the blood, and the condition is _fundamentally_ the same as -protein immunity, _i.e._, due to _intracellular_ digestion in each case. - -As has been indicated "serum sickness" and sudden death following -serum injections are probably due to a sensitization of the individual -to the proteins of the horse in some unknown way. Probably hay fever -urticarial rashes and idiosyncrasies following the ingestion of certain -foods--strawberries, eggs, oysters, etc., are anaphylactic phenomena. - -In medical practice the reaction is used as a means of diagnosis in -certain diseases, such as the tuberculin test in tuberculosis, the -mallein test in glanders. The individual or animal with tuberculosis -becomes sensitized to certain proteins of the tubercle bacillus and -when these proteins in the form of tuberculin are introduced into the -body a reaction results, local or general, according to the method of -introduction. The practical facts in connection with the tuberculin -test are also in harmony with the author's theory of anaphylaxis -as above outlined. Milder cases of tuberculosis give more vigorous -reactions because the intracellular enzymes are not used up rapidly -enough since the products of the bacillus are secreted slowly in such -cases. Hence excess of enzyme is free in the blood and the injection of -the tuberculin meets it there and a vigorous reaction results. In old, -far-advanced cases, no reaction occurs, because the enzymes are all -used in decomposing the large amount of tuberculous protein constantly -present in the blood. The fact that an animal which has once reacted -fails to do so until several months afterward likewise depends on the -fact that the _excess_ enzyme is used in the reaction and time must -elapse for a further excess to accumulate. - -The anaphylactic reaction has been made use of in the identification of -various types of proteins and is of very great value since the reaction -is so delicate, particularly when guinea-pigs are used as test animals. -Wells has detected the 0.000,001 g. of protein by this test. It is -evident that the test is applicable in medico-legal cases and in food -examination and has been so used. - - -A TABULATION OF ANTIGENS AND ANTIBODIES AS AT PRESENT RECOGNIZED. - - CLASS OF - ANTIGEN ANTIBODY ACTION OF ANTIBODY RECEPTOR - - Toxin Antitoxin Combines with toxin and I. - hence prevents toxin - from uniting with a - cell and injuring it, - _i.e._, neutralizes toxin. - - Enzyme Antienzyme Combines with enzyme I. - and thus prevents enzyme - from uniting - with anything else and - showing its action, _i.e._, - neutralizes enzyme. - - Solution of Precipitin Unites with its antigen II. - protein and causes its precipitation - from solution. - - Solution of ? Causes phenomenon of (?) - protein anaphylaxis(?) - - Suspension of Agglutinin Unites with its antigen II. - cells causes its clumping together - and settling out - of suspension. - - Suspension of Opsonin Unites with its antigen II. - cells and makes the cells (?) - more easily taken up - by phagocytes. - - Suspension of Amboceptor Unites with its antigen III. - cells and also with complement - which latter then - dissolves the antigen. - - Precipitin Antiprecipitin Neutralizes precipitin. I. - - Agglutinin Antiagglutinin Neutralizes agglutinin. I. - - Opsonin Antiopsonin Neutralizes opsonin. I. - - Amboceptor Antiamboceptor Neutralizes amboceptor. I. - (two kinds) - - Complement Anticomplement Neutralizes complement. I. - - -SUMMARY OF IMMUNITY AS APPLIED TO PROTECTION FROM DISEASE. - -The discussion of "immunity problems" in the preceding chapters serves -to show that protection from disease either as a condition natural to -the animal or as an acquired state is dependent on certain properties -of its body cells or fluids, or both. The actual factors so far as at -present known may be summarized as follows: - -1. _Antitoxins_ which neutralize true toxins; shown to exist for very -few diseases. - -2. _Cytolytic substances_ which destroy the invading organism: in -reality two substances; amboceptor, which is specific, and complement, -the real dissolving enzyme. - -3. _Phagocytosis_ or the destruction of the invading organisms within -the leukocytes. - -4. _Opsonins_ which render the bacteria more readily taken up by the -phagocytes. - -5. _Enzymes_ other than complement possibly play a part in the -destruction of some pathogenic organisms or their products. This -remains to be more definitely established. - -6. It is possible that in natural immunity there might be no receptors -in the body cells to take up the organisms or their products, or the -receptors might be present in certain cells but of a very low chemical -affinity, so that combination does not occur. It is even highly -probable that many substances formed by invading organisms which might -injure specialized cells, such as those of glandular, nervous or muscle -tissue, have a more rapid rate of reaction with, or a stronger affinity -for, lower unspecialized cells, such as connective and lymphoid tissue, -and unite with these so that their effects are not noticed. - -The importance of these different, factors varies in different diseases -and need not be considered in this connection. - -The question "which of the body cells are engaged in the production -of antibodies" is not uncommonly asked. On physiological grounds it -would not seem reasonable that the highly specialized tissues above -mentioned could take up this work, even though they are the ones which -suffer the greatest injury in disease. Hence it is to be expected that -the lower or unspecialized cells are the source, and it has been shown -that the antibodies are produced by the phagocytes (though not entirely -as Metchnikoff maintained), by lymphoid tissue generally, by the bone -marrow and also by connective-tissue cells, though in varying degrees. - -Since immunity depends on the activity of the body cells it is evident -that one of the very best methods for avoiding infectious diseases is -to keep these cells up to their highest state of efficiency, to keep in -"good health." Hence good health means not only _freedom from disease_ -but also _protection against disease_. - - - - -LIST OF LABORATORY EXERCISES GIVEN IN CONNECTION WITH THE CLASS WORK -INCLUDED IN THIS TEXT-BOOK. - - Exercise 1. Cleaning glassware. - - Exercise 2. Preparation of broth medium from meat juice. - - Exercise 3. Preparation of gelatin medium from broth. - - Exercise 4. Preparation of agar medium from broth. - - Exercise 5. Potato tubes. - - Exercise 6. Potato plates. - - Exercise 7. Plain milk tubes. - - Exercise 8. Litmus milk tubes. - - Exercise 9. Sugar broth media. - - Exercise 10. Blood-serum tubes. - - Exercise 11. Inoculation of tubes. Action on complex proteins. - - Exercise 12. Production of gas from carbohydrates. - - Exercise 13. Production of indol. - - Exercise 14. Reduction of nitrates. - - Exercise 15. Chromogenesis: Illustrates nicely the variation with - environment. - - Exercise 16. Enzyme production. - - Exercise 17. Making of plate cultures; isolation in pure culture. - - Exercise 18. Stain making and staining. - - Exercise 19. Cell forms and cell groupings. - - Exercise 20. Hanging drop slides. - - Exercise 21. Staining of spores. - - Exercise 22. Staining of acid-fast bacteria. - - Exercise 23. Staining of capsules. - - Exercise 24. Staining of metachromatic granules. - - Exercise 25. Staining of flagella. - - Exercise 26. Study of individual species. - - Exercise 27. Determination of thermal death-point. - - Exercise 28. Action of disinfectants on bacteria. - - Exercise 29. Action of sunlight on bacteria. - - - - -DESCRIPTIVE CHART--SOCIETY OF AMERICAN BACTERIOLOGISTS. - -_Prepared by Committee on Methods of Identification of Bacterial -Species.--F. D. Chester, F. P. Gorham, Erwin F. Smith._ - -_Endorsed by the Society for general use at the Annual Meeting, -December, 1907._ - - -GLOSSARY OF TERMS. - -AGAR HANGING BLOCK, a small block of nutrient agar cut from a pour -plate, and placed on a cover-glass, the surface next the glass having -been first touched with a loop from a young fluid culture or with a -dilution from the same. It is examined upside down, the same as a -hanging drop. - -AMEBOID, assuming various shapes like an ameba. - -AMORPHOUS, without visible differentiation in structure. - -ARBORESCENT, a branched, tree-like growth. - -BEADED, in stab or stroke, disjointed or semiconfluent colonies along -the lines of inoculation. - -BRIEF, a few days, a week. - -BRITTLE, growth dry, friable under the platinum needle. - -BULLATE, growth rising in convex prominences, like a blistered surface. - -BUTYROUS, growth of a butter-like consistency. - -CHAINS, - Short chains, composed of 2 to 8 elements. - Long chains, composed of more than 8 elements. - -CILIATE, having fine, hair-like extensions, like cilia. - -CLOUDY, said of fluid cultures which do not contain pseudozoogleæ. - -COAGULATION,[22] the separation of casein from whey in milk. This may -take place quickly or slowly, and as the result either of the formation -of an acid or of a lab ferment. - -CONTOURED, an irregular, smoothly undulating surface, like that of a -relief map. - -CONVEX surface, the segment of a circle, but flattened. - -COPROPHYL, dung bacteria. - -CORIACEOUS, growth tough, leathery, not yielding to the platinum needle. - -CRATERIFORM, round, depressed, due to the liquefaction of the medium. - -CRETACEOUS, growth opaque and white, chalky. - -CURLED, composed of parallel chains in wavy strands, as in anthrax -colonies. - -DIASTASIC ACTION, same as DIASTATIC, conversion of starch into -water-soluble substances by diastase. - -ECHINULATE, in agar stroke a growth along line of inoculation, with -toothed or pointed margins; in stab cultures growth beset with pointed -outgrowths. - -EFFUSE, growth thin, veily, unusually spreading. - -ENTIRE, smooth, having a margin destitute of teeth or notches. - -EROSE, border irregularly toothed. - -FILAMENTOUS, growth composed of long, irregularly placed or interwoven -filaments. - -FILIFORM, in stroke or stab cultures a uniform growth along line of -inoculation. - -FIMBRIATE, border fringed with slender processes, larger than filaments. - -FLOCCOSE, growth composed of short curved chains, variously oriented. - -FLOCCULENT, said of fluids which contain pseudozoogleæ, _i.e._, small -adherent masses of bacteria of various shapes and floating in the -culture fluid. - -FLUORESCENT, having one color by transmitted light and another by -reflected light. - -GRAM'S STAIN, a method of differential bleaching after gentian violet, -methyl violet, etc. The + mark is to be given only when the bacteria -are deep blue or remain blue after counter-staining with Bismarck brown. - -GRUMOSE, clotted. - -INFUNDIBULIFORM, form of a funnel or inverted cone. - -IRIDESCENT, like mother-of-pearl. The effect of very thin films. - -LACERATE, having the margin cut into irregular segments as if torn. - -LOBATE, border deeply undulate, producing lobes (see _Undulate_). - -LONG, many weeks, or months. - -MAXIMUM TEMPERATURE, temperature above which growth does not take place. - -MEDIUM, nutrient substance upon which bacteria are grown. - -MEMBRANOUS, growth thin, coherent, like a membrane. - -MINIMUM TEMPERATURE, temperature below which growth does not take place. - -MYCELIOID, colonies having the radiately filamentous appearance of mold -colonies. - -NAPIFORM, liquefaction with the form of a turnip. - -NITROGEN REQUIREMENTS, the necessary nitrogenous food. This is -determined by adding to _nitrogen-free_ media the nitrogen compound to -be tested. - -OPALESCENT, resembling the color of an opal. - -OPTIMUM TEMPERATURE, temperature at which growth is most rapid. - -PELLICLE, in fluid bacterial growth forming either a continuous or an -interrupted sheet over the fluid. - -PEPTONIZED, said of curds dissolved by trypsin. - -PERSISTENT, many weeks, or months. - -PLUMOSE, a fleecy or feathery growth. - -PSEUDOZOOGLEÆ, clumps of bacteria, not dissolving readily in water, -arising from imperfect separation, or more or less fusion of the -components, but not having the degree of compactness and gelatinization -seen in zoogleæ. - -PULVINATE, in the form of a cushion, decidedly convex. - -PUNCTIFORM, very minute colonies, at the limit of natural vision. - -RAPID, developing in twenty-four to forty-eight hours. - -RAISED, growth thick, with abrupt or terraced edges. - -RHIZOID, growth of an irregular branched or root-like character, as in -_B. mycoides_. - -RING, same as RIM, growth at the upper margin of a liquid culture, -adhering more or less closely to the glass. - -REPAND, wrinkled. - -SACCATE, liquefaction the shape of an elongated sac, tubular, -cylindrical. - -SCUM, floating islands of bacteria, an interrupted pellicle or bacteria -membrane. - -SLOW, requiring five or six days or more for development. - -SHORT, applied to time, a few days, a week. - -SPORANGIA, cells containing endospores. - -SPREADING, growth extending much beyond the line of inoculation, -_i.e._, several millimetres or more. - -STRATIFORM, liquefying to the walls of the tube at the top and then -proceeding downward horizontally. - -THERMAL DEATH-POINT, the degree of heat required to kill young fluid -cultures of an organism exposed for ten minutes (in thin-walled -test-tubes of a diameter not exceeding 20 mm.) in the thermal -water-bath. The water must be kept agitated so that the temperature -shall be uniform during the exposure. - -TRANSIENT, a few days. - -TURBID, cloudy with flocculent particles; cloudy plus flocculence. - -UMBONATE, having a button-like, raised centre. - -UNDULATE, border wavy, with shallow sinuses. - -VERRUCOSE, growth wart-like, with wart-like prominences. - -VERMIFORM-CONTOURED, growth like a mass of worms or intestinal coils. - -VILLOUS, growth beset with hair-like extensions. - -VISCID, growth follows the needle when touched and withdrawn, sediment -on shaking rises as a coherent swirl. - -ZOOGLEÆ, firm gelatinous masses of bacteria, one of the most typical -examples of which is the _Streptococcus mesenterioides_ of sugar vats. -(_Leuconostoc mesenterioides_), the bacterial chains being surrounded -by an enormously thickened, firm covering inside of which there may be -one or many groups of the bacteria. - - -NOTES. - -(1) For decimal system of group numbers see Table I. This will be found -useful as a quick method of showing close relationships inside the -genus, but is not a sufficient characterization of any organism. - -(2) The morphological characters shall be determined and described -from growths obtained upon at least one solid medium (nutrient agar) -and in at least one liquid medium (nutrient broth). Growths at 37° C. -shall be in general not older than twenty-four to forty-eight hours, -and growths at 20° C. not older than forty-eight to seventy-two hours. -To secure uniformity in cultures, in all cases preliminary cultivation -shall be practised as described in the revised Report of the Committee -on Standard Methods of the Laboratory Section of the American Public -Health Association, 1905. - -(3) The observation of cultural and biochemical features shall cover -a period of at least fifteen days and frequently longer, and shall be -made according to the revised Standard Methods above referred to. All -media shall be made according to the same Standard Methods. - -(4) Gelatin stab cultures shall be held for six weeks to determine -liquefaction. - -(5) Ammonia and indol tests shall be made at end of tenth day, nitrite -tests at end of fifth day. - -(6) Titrate with N/20 NaOH, using phenolphthalein as an indicator; make -titrations at same time from blank. The difference gives the amount of -acid produced. - -The titration should be done after boiling to drive off any CO{2} -present in the culture. - -(7) Generic nomenclature shall begin with the year 1872 (Cohn's first -important paper). - -Species nomenclature shall begin with the year 1880 (Koch's discovery -of the pour plate method for the separation of organisms). - -(8) Chromogenesis shall be recorded in standard color terms. - - -TABLE I. - -A NUMERICAL SYSTEM OF RECORDING THE SALIENT CHARACTERS OF AN ORGANISM. -(GROUP NUMBER.) - - 100 Endospores produced - 200 Endospores not produced - 10 Aërobic (strict) - 20 Facultative anaërobic - 30 Anaërobic (strict) - 1 Gelatin liquefied - 2 Gelatin not liquefied - 0.1 Acid and gas from dextrose - 0.2 Acid without gas from dextrose - 0.3 No acid from dextrose - 0.4 No growth with dextrose - 0.01 Acid and gas from lactose - 0.02 Acid without gas from lactose - 0.03 No acid from lactose - 0.04 No growth with lactose - 0.001 Acid and gas from saccharose - 0.002 Acid without gas from saccharose - 0.003 No acid from saccharose - 0.004 No growth with saccharose - 0.0001 Nitrates reduced with evolution of gas - 0.0002 Nitrates not reduced - 0.0003 Nitrates reduced without gas formation - 0.00001 Fluorescent - 0.00002 Violet chromogens - 0.00003 Blue chromogens - 0.00004 Green chromogens - 0.00005 Yellow chromogens - 0.00006 Orange chromogens - 0.00007 Red chromogens - 0.00008 Brown chromogens - 0.00009 Pink chromogens - 0.00000 Non-chromogenics - 0.000001 Diastasic action on potato starch, strong - 0.000002 Diastasic action on potato starch, feeble - 0.000003 Diastasic action on potato starch, absent - 0.0000001 Acid and gas from glycerin - 0.0000002 Acid without gas from glycerin - 0.0000003 No acid from glycerin - 0.0000004 No growth with glycerin - -The genus according to the system of Migula is given its proper symbol -which precedes the number thus:(7) - - BACILLUS COLI (Esch.) Mig. becomes B. 222.111102 - BACILLUS ALCALIGENES Petr. becomes B. 212.333102 - PSEUDOMONAS CAMPESTRIS (Pam.) Sm. becomes Ps. 211.333151 - BACTERIUM SUICIDA Mig. becomes Bact. 222.232103 - -Source............ Date of Isolation.............. Name........ -Group No.(1)............... - - - - -DETAILED FEATURES. - -NOTE--Underscore required terms. Observe notes and glossary of terms on -opposite side of card. - - -I. MORPHOLOGY(2) - - 1. Vegetative Cells, Medium used............................. - temp....................age.................days - - Form, _round_, _short rods_, _long rods_, _short chains_, _long - chains_, _filaments_, _commas_, _short spirals_, _long spirals_, - _clostridium_, _cuneate_, _clavate_, _curved_. - - Limits of Size.......................... - - Size of Majority............................. - - Ends, _rounded_, _truncate_, _concave_. - - {Orientation (grouping)............................ - Agar {Chains (No. of elements)........................ - Hanging-block {_Short chains_, _long chains_ - {Orientation of chains, _parallel_, _irregular_. - - 2. Sporangia, medium - used.....................temp..............age..............days - - Form, _elliptical_, _short rods_, _spindled_, _clavate_, _drumsticks_. - - Limits of Size................ - - Size of Majority.............. - - Agar {Orientation (grouping)........ - Hanging-block {Chains (No. of elements)...... - {Orientation of chains, _parallel_, _irregular_. - - Location of Endospores, _central_, _polar_. - - 3. Endospores. - - Form, _round_, _elliptical_, _elongated_. - - Limits of Size................ - - Size of Majority.............. - - Wall, _thick_, _thin_. - - Sporangium wall, _adherent_, _not adherent_. - - Germination, _equatorial_, _oblique_, _polar_, _bipolar_, _by - stretching_. - - 4. Flagella, No........Attachment _polar_, _bipolar_, - _peritrichiate_. How Stained......... - - 5. Capsules, present on............. - - 6. Zooglea, Pseudozooglea. - - 7. Involution Forms, on........in.....days at....° C. - - 8. Staining Reactions. - - 1:10 watery fuchsin, gentian violet, carbol-fuchsin, Loeffler's - alkaline methylene blue. - - Special Stains. - Gram....................Glycogen............... - Fat.....................Acid-fast................ - Neisser................. - - -II. CULTURAL FEATURES(3) - -1. Agar Stroke. - - Growth, _invisible_, _scanty_, _moderate_, _abundant_. - - Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_, - _plumose_, _arborescent_, _rhizoid_. - - Elevation of growth, _flat_, _effuse_, _raised_, _convex_. - - Lustre, _glistening_, _dull_, _cretaceous_. - - Topography, _smooth_, _contoured_, _rugose_, _verrucose_. - - Optical characters, _opaque_, _translucent_, _opalescent_, - _iridescent_. - - Chromogenesis(3)................ - - Odor, _absent_, _decided_, _resembling_............ - - Consistency, _slimy_, _butyrous_, _viscid_, _membranous_, - _coriaceous_, _brittle_. - - Medium _grayed_, _browned_, _reddened_, _blued_, _greened_. - -2. Potato. - - Growth _scanty_, _moderate_, _abundant_, _transient_, _persistent_. - - Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_, - _plumose_, _arborescent_, _rhizoid_. - - Elevation of growth, _flat_, _effuse_, _raised_, _convex_. - - Lustre, _glistening_, _dull_, _cretaceous_. - - Topography, _smooth_, _contoured_, _rugose_, _verrucose_. - - Chromogenesis(3)...........Pigment in water _insoluble_, _soluble_: - other solvents..................... - - Odor, _absent_, _decided_, _resembling_.................... - - Consistency, _slimy_, _butyrous_, _viscid_, _membranous_, - _coriaceous_, _brittle_. - - Medium, _grayed_, _browned_, _reddened_, _blued_, _greened_. - -3. Loeffler's Blood-serum. - - Stroke _invisible_, _scanty_, _moderate_, _abundant_. - - Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_, - _plumose_, _arborescent_, _rhizoid_. - - Elevation of growth, _flat_, _effuse_, _raised_, _convex_. - - Lustre, _glistening_, _dull_, _cretaceous_. - - Topography, _smooth_, _contoured_, _rugose_, _verrucose_. - - Chromogenesis(3).......................... - - Medium _grayed_, _browned_, _reddened_, _blued_, _greened_. - - Liquefaction begins in.............d, complete in................d, - -4. Agar Stab. - - Growth _uniform_, _best at top_, _best at bottom_: surface growth - _scanty_, _abundant_: _restricted_, _wide-spread_. - - Line of puncture, _filiform_, _beaded_, _papillate_, _villous_, - _plumose_, _arborescent_: _liquefaction_. - -5. Gelatin Stab. - - Growth uniform, _best at top_, _best at bottom_. - - Line of puncture, _filiform_, _beaded_, _papillate_, _villous_, - _plumose_, _arborescent_. - - Liquefaction _crateriform_, _napiform_, _infundibuliform_, - _saccate_, _stratiform_: begins in....................d. complete - in....................d - - Medium _fluorescent_, _browned_............... - -6. Nutrient Broth. - - Surface growth, _ring_, _pellicle_, _flocculent_, _membranous_, - _none_. - - Clouding _slight_, _moderate_, _strong_: _transient_, _persistent_: - _none_: _fluid turbid_. - - Odor, _absent_, _decided_, _resembling_.................. - - Sediment, _compact_, _flocculent_, _granular_, _flaky_, _viscid on - agitation_, _abundant_, _scant_. - -7. Milk. - - Clearing without coagulation. - - Coagulation _prompt_, _delayed_, _absent_. - - Extrusion of whey begins in............days. - - Coagulum _slowly peptonized_, _rapidly peptonized_. - - Peptonization begins on....d, complete on ....d. - - Reaction, 1d...., 2d...., 4d...., 10d...., 20d.... - - Consistency, _slimy_, _viscid_, _unchanged_. - - Medium _browned_, _reddened_, _blued_, _greened_. - - Lab ferment, _present_, _absent_. - -8. Litmus Milk. - - _Acid_, _alkaline_, _acid then alkaline_, _no change_. - - _Prompt reduction_, _no reduction_, _partial slow reduction_. - -9. Gelatin Colonies. - - Growth _slow_, _rapid_. - - Form, _punctiform_, _round_, _irregular_, _ameboid_, _mycelioid_, - _filamentous_, _rhizoid_. - - Elevation, _flat_, _effuse_, _raised_, _convex_, _pulvinate_, - _crateriform_ (_liquefying_). - - Edge, _entire_, _undulate_, _lobate_, _erose_, _lacerate_, - _fimbriate_, _filamentous_, _floccose_, _curled_. - - Liquefaction, _cup_, _saucer_, _spreading_. - -10. Agar Colonies. - - Growth _slow_, _rapid_ (temperature..............) - - Form, _punctiform_, _round_, _irregular_, _ameboid_, _mycelioid_, - _filamentous_, _rhizoid_. - - Surface _smooth_, _rough_, _concentrically ringed_, _radiate_, - _striate_. - - Elevation, _flat_, _effuse_, _raised_, _convex_, _pulvinate_, - _umbonate_. - - Edge, _entire_, _undulate_, _lobate_, _erose_, _lacerate_, - _fimbriate_, _floccose_, _curled_. - - Internal structure, _amorphous_, _finely_, _coarsely granular_, - _grumose_, _filamentous_, _floccose_, _curled_. - -11. Starch Jelly. - - Growth, _scanty_, _copious_. - - Diastatic action, _absent_, _feeble_, _profound_. - - Medium stained................... - -12. Silicate Jelly (Fermi's Solution). - - Growth _copious_, _scanty_, _absent_. - - Medium stained.................. - -13. Cohn's Solution. - - Growth _copious_, _scanty_, _absent_. - - Medium _fluorescent_, _non-fluorescent_. - -14. Uschinsky's Solution. - - Growth _copious_, _scanty_, _absent_. - - Fluid _viscid_, _not viscid_. - -15. Sodium Chloride in Bouillon. - - Per cent. inhibiting growth........................ - -16. Growth in Bouillon over Chloroform, _unrestrained_, - _feeble_, _absent_. - -17. Nitrogen. Obtained from _peptone_, _asparagin_, _glycocoll_, - _urea_, _ammonia salts_, _nitrogen_. - -18. Best media for long-continued growth................... - ..................................................... - -19. Quick tests for differential purposes.................. - ..................................................... - ..................................................... - - -III. PHYSICAL AND BIOCHEMICAL FEATURES. - - +----------------------------------+---+---+---+---+---+---+---+---+ - | | D | S | L | M | G | M | | | - | | e | a | a | a | l | a | | | - | | x | c | c | l | y | n | | | - | | t | c | t | t | c | n | | | - | 1. Fermentation-tubes containing | r | h | o | o | e | i | | | - | peptone-water or | o | a | s | s | r | t | | | - | sugar-tree bouillon and | s | r | e | e | i | | | | - | | e | o | | | n | | | | - | | | s | | | | | | | - | | | e | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Gas production, in per cent. | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | (H/CO{2}) | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Growth in closed arm | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Amount of acid produced 1d. | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Amount of acid produced 2d. | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Amount of acid produced 3d. | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - - 2. Ammonia production, _feeble_, _moderate_, _strong_, _absent_, - _masked by acids_. - - 3. Nitrates in nitrate broth. - - _Reduced_, _not reduced_. - - Presence of nitrites...........ammonia.................. - - Presence of nitrates...........free nitrogen............ - - 4. Indol production, _feeble_, _moderate_, _strong_. - - 5. Toleration of Acids, _great_, _medium_, _slight_. - - _Acids tested_.............. - - 6. Toleration of NaOH, _great_, _medium_, _slight_. - - 7. Optimum reaction for growth in bouillon, stated in terms of - Fuller's scale.......................... - - 8. Vitality on culture media, _brief_, _moderate_, _long_. - - 9. Temperature relations. - - Thermal death-point (10 minutes' exposure in nutrient broth when this - is adapted to growth of organism)............C. - - Optimum temperature for growth......° C.; or best growth at 16° C., - 20° C., 25° C., 30° C., 37° C., 40° C., 50° C., 60° C. - - Maximum temperature for growth.......... ° C. - - Minimum temperature for growth.......... ° C. - - 10. Killed readily by drying: resistant to drying. - - 11. Per cent. killed by freezing (salt and crushed ice or liquid - air)................ - - 12. Sunlight: Exposure on ice in thinly sown agar plates; one-half - plate covered (time 15 minutes), _sensitive_, _not sensitive_. - - Per cent. killed................ - - 13. Acids produced................. - - 14. Alkalies produced............... - - 15. Alcohols....................... - - 16. Ferments, _pepsin_, _trypsin_, _diastase_, _invertase_, - _pectase_, _cytase_, _tyrosinase_, _oxidase_, _peroxidase_, - _lipase_, _catalase_, _glucase_, _galactase_, _lab_, - _etc._........................ - - 17. Crystals formed:..... - - 18. Effect of germicides: - - +-----------+-------------+---+---+---+---+---+ - | | | M | T | K | A | r | - | | | i | e | i | m | e | - | | | n | m | l | t | s | - | | | u | p | l | . | t | - | | | t | e | i | | r | - | | | e | r | n | r | a | - | | | s | a | g | e | i | - | Substance | Method used | | t | | q | n | - | | | | u | q | u | | - | | | | r | u | i | g | - | | | | e | a | r | r | - | | | | | n | e | o | - | | | | | t | d | w | - | | | | | i | | t | - | | | | | t | t | h | - | | | | | y | o | | - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - - -IV. PATHOGENICITY. - - 1. Pathogenic to Animals. - - _Insects_, _crustaceans_, _fishes_, _reptiles_, _birds_, _mice_, - _rats_, _guinea-pigs_, _rabbits_, _dogs_, _cats_, _sheep_, _goats_, - _cattle_, _horses_, _monkeys_, _man_.......................... - - 2. Pathogenic to Plants: - ......................................................... - ......................................................... - ......................................................... - - 3. Toxins, _soluble_, _endotoxins_. - - 4. Non-toxin forming. - - 5. Immunity bactericidal. - - 6. Immunity non-bactericidal. - - 7. Loss of virulence on culture-media: _prompt_, _gradual_, _not - observed in_.....................months. - - +-------------------------------------+ - | BRIEF CHARACTERIZATION. | - | | - | Mark + or 0, and when two terms | - | occur on a line erase the one which | - | does not apply unless both apply. | - | | - +--------------------------------+----+ - | M | Diameter over 1µ |----| - | O | Chains, filaments |----| - | R | Endospores |----| - | P | Capsules |----| - | H | Zooglea, Pseudozooglea |----| - | O | Motile |----| - | L | Involution forms |----| - | O | Gram's stain |----| - | G | |----| - | Y | |----| - |(2)| |----| - +---+----------------------------+----+ - | C | B | Cloudy, turbid |----| - | U | r | Ring |----| - | L | o | Pellicle |----| - | T | t | Sediment |----| - | U | h | |----| - | R +-----+----------------------+----+ - | A | A | Shining |----| - | L | g | Dull |----| - | | a | Wrinkled |----| - | F | r | Chromogenic |----| - | E +-----+----------------------+----+ - | A | G | Round |----| - | T | e | Proteus-like |----| - | U | l. | Rhizoid |----| - | R | | Filamentous |----| - | E | P | Curled |----| - | S | l | |----| - |(3)| a | |----| - | | t | |----| - | | e | |----| - | +-----+----------------------+----+ - | | G S | Surface growth |----| - | | e t | Needle growth |----| - | | l a | |----| - | | . b.| |----| - | +-----+----------------------+----+ - | | P | Moderate, absent |----| - | | o | Abundant |----| - | | t | Discolored |----| - | | a | Starch destroyed |----| - | | t | |----| - | | o | |----| - | +-----+---------------------------+ - | | Grows at 37° C. |----| - | | Grows in Cohn's sol. |----| - | | Grows in Uschinsky's sol. |----| - |---+-----+----------------------+----+ - | B | L f | Gelatin(4) |----| - | I | i a | Blood-serum |----| - | O | q c | Casein |----| - | C | u t | |----| - | H | i i | |----| - | E | - o | |----| - | M | n | |----| - | I +-----+----------------------+----+ - | C | M | Acid curd |----| - | A | i | Rennet curd |----| - | L | l | Casein peptonized |----| - | | k | |----| - | F +-----+----------------------+----+ - | E | Indol(3) |----| - | A | Hydrogen sulphide |----| - | T | Ammonia(3) |----| - | U | Nitrates reduced(3) |----| - | R | Fluorescent |----| - | E | Luminous |----| - | S | |----| - +---+----------------------------+----+ - | D | Animal pathogen, epizoon |----| - | I | Plant pathogen, epiphyte |----| - | S | Soil |----| - | T | Milk |----| - | R | Fresh water |----| - | I | Salt water |----| - | B | Sewage |----| - | U | Iron bacterium |----| - | T | Sulphur bacterium |----| - | I | |----| - | O | |----| - | N | |----| - +---+----------------------------+----+ - - - - -FOOTNOTES. - - -[1] Sir H. A. Blake has called attention to the fact that the "mosquito -theory" of malaria is mentioned in a Sanscrit manuscript of about the -6th century A.D. - -[2] Myxomycetes excepted, and they are probably to be regarded as -animals--Mycetozoa. - -[3] Centralblatt f. Bakteriologie, etc. LXIII. 1 Abt. Orig. 1912, 4, -idem LXVI. 1 Abt. Orig. 1912, 323. - -[4] The pronunciation of this word according to English standards -is kok-si; the continental pronunciation is kok-kee; the commonest -American seems to be kok-ki. We prefer the latter since it is easier -and more natural and should like to see it adopted. (Author.) - -[5] With the possible exception of blue green algæ which have been -found with bacteria in the above-mentioned hot springs. Seeds of -many plants have been subjected to as low temperatures as those -above-mentioned without apparent injury. - -[6] It is popularly supposed that in canning fruit, vegetables, meats, -etc., all the air must be removed, since the organisms which cause -"spoiling" cannot grow in a vacuum. The existence of anaërobic and -facultative anaërobic bacteria shows the fallacy of such beliefs. - -[7] "By cellulose is understood a carbohydrate of the general formula -C{6}H{10}O{5} not soluble in water, alcohol, ether, or dilute acids but -soluble in an ammoniacal solution of copper oxide. It gives with iodine -and sulphuric acid a blue color and with iodine zinc chloride a violet -and yields dextrose on hydrolysis."--H. Fischer. - -[8] The sulphur bacteria are partially prototrophic for S; probably the -iron bacteria also for Fe. Some few soil bacteria have been shown to -be capable of utilizing free H, and it seems certain that the bacteria -associated with the spontaneous heating of coal may oxidize free C. So -far as known no elements other than these six are directly available to -bacteria. - -[9] Only a few kinds of bacteria so far as known are proto-autotrophic. -The nitrous and nitric organisms of Winogradsky which are so essential -in the soil, and which might have been the first of all organisms so -far as their food is concerned, and some of the sulphur bacteria are -examples. - -[10] The term _pathogenic_ is also applied to certain non-parasitic -saprophytic bacteria whose products cause disease conditions, as one -of the organisms causing a type of food poisoning in man (_Clostridium -botulinum_), which also probably causes "forage poisoning" in domestic -animals. - -[11] The term "fermentation" was originally used to denote the process -which goes on in fruit juices or grain extracts when alcohol and gas -are formed. Later it was extended to apply to the decomposition of -almost any organic substance. In recent years the attempt has been made -to give a chemical definition to the word by restricting its use to -those changes in which by virtue of a "wandering" or rearrangement of -the carbon atoms "new substances are formed which are not constitutents -of the original molecule." It may be doubted whether this restriction -is justified or necessary. A definition is at present scarcely possible -except when the qualifying adjective is included as "alcoholic -fermentation," "ammoniacal fermentation," "lactic acid fermentation," -etc. - -[12] See "Oil and Gas in Ohio," Bownocker: Geological Survey of Ohio, -Fourth Series, Bull. I, pp. 313-314. - -[13] It is probable that this is the way "Jack o'lanterns" or "Will o' -the wisps" are ignited. Marsh gas is produced as above outlined from -the vegetable and animal matter decomposing in swampy places under -anaërobic conditions and likewise phosphine. These escape into the air -and the "spontaneous combustion" of the phosphine ignites the marsh gas. - -[14] Dr. H. H. Green, of Pretoria, South Africa, has isolated from -"cattle dips" a bacterium that _reduces arsenates_ to _arsenites_. - -[15] Dr. Green (l. c.) has also isolated an organism which causes some -deterioration of cattle dips by _oxidizing arsenites to arsenates_. - -[16] It will be noted that the names of enzymes (except some of -those first discovered) terminate in _ase_ which is usually added to -the _stem of the name of the substance acted on_, though sometimes -to a word which indicates the substance formed by the action, as -_lactacidase_, _alcoholase_. - -[17] Tetanus toxin is about 120 times as poisonous as strychnin, both -of which act on the same kind of nerve cells. - -[18] In the author's laboratory in the past ten years all sterilization -except those few objects in blood and serum work which must be dry, has -been done in autoclaves of the type shown in Fig. 81 which are supplied -with steam from the University central heating plant. A very great -saving of time is thus secured. - -[19] The author has tested an "electric milk purifier" (Fig. 102) -which was as efficient as a first-class pasteurizer and left the milk -in excellent condition both chemically and as far as "cream line" was -concerned. The cost of operation as compared with steam will depend on -the price of electricity. - -[20] The exact laboratory details for preparing various media are -not given in this chapter. It is the object to explain the choice of -different materials and the reasons for the various processes to which -they are subjected. - -[21] For a discussion of this method of standardization consult the -following: - - Clark & Lubs--J. Bact., 1917, II, 1-34, 109-136, 191-236. - Committee Report--Ibid., 1919. IV, 107-132. - Jones--J. Inf. Dis., 1919, 25, 262-268. - Fennel & Fisher--Ibid., 444-451. - -Additional references will be found in these articles. - -[22] Term also applied to the solidification of serum in media: _e.g._, -the Hiss inulin medium for the differentiation of pneumococci (see -diplococcus of pneumonia). - -[23] The term "antigen" is also used to designate substances which may -take the place of what are supposed to be the true antigens in certain -diagnostic reactions (Chapter XXIX, Complement Fixation Test for -Syphilis). - -[24] If the antitoxin is later concentrated (see last paragraph in -this chapter) a serum containing as little as 175 units per cc. may be -commercially profitable. - -[25] Tho term "allergie" was introduced by Von Pirquet to designate the -state of the animal's being sensitized and "allergic" as the adjective -derived therefrom. It does not seem to the author that there is any -advantage gained by the introduction of these terms. - - - - -INDEX - - - A - - ABBÉ, 17 - condenser, 200 - microscope, improvements in, 30, 36 - - ABILGAARD, 26 - - Abrin, 262 - - Absorption of free nitrogen, 117 - tests, 267 - - Accidental carriers, 241 - structures, 43 - - Acetic acid, 99 - bacteria, carbon oxidation, 114 - fermentation, 32 - - _Acetobacter acidi oxalici_, 83 - _xylinum_, 83 - - _Achorion schoenleinii_, 27, 34 - - Acid, acetic, 99 - fermentation, 32 - agglutination, 266 - amino, relation to green plants, 119 - butyric, 99 - fermentation, 32, 99 - carbolic, first used, 29 - disinfectant action of, 159 - fast bacteria, fat content, 84 - staining of, 209 - fermentation, 93 - Bulgarian fermented milk, 98 - ensilage, 98 - industrial uses, 97 - lactic acid, 96 - sauerkraut, 98 - hydrochloric, 246 - production of, 110 - soils, 81 - - Acquired immunity, 251, 252 - - _Actinomyces bovis_, 30, 36 - - Actinomycosis, cause of, 30, 36 - path of entrance of, 244 - - Actions, reducing, 113 - - Activating enzymes, 125 - - Active immunity, definition of, 251, 252 - production of, 252 - - Activities of bacteria, importance of, 31 - overproduction, of cells, 258 - physiological, definition of, 87 - in identification, 216 - - Acute coryza, 244 - disease, 233 - - Adulteration of food, anaphylactic test in, 293 - complement-fixation test in, 279 - immunity reactions in, 255 - precipitin test in, 269 - - Aërobes, facultative, 76 - strict, 76 - - Aërobic, 76, 215 - - Agar, composition of, 179 - gelatinizing temperature, 179 - medium, preparation of, 179 - melting point of, 179 - plating in, 188 - sterilization of, 180 - - Agent, chemical, for disinfection, 156-163 - choice of, for disinfection, 164 - physical, for disinfection, 131 - - Agglutinating group, 266 - - Agglutination, acid, 266 - diagnostic value of, 266 - in identification of bacteria, 266 - macroscopic, 265 - microscopic, 265 - phenomenon, 265 - - Agglutinin, 265 - absorption test for, 267 - action of, 266 - anti-, 270 - antigenic action of, 270 - bacterial, 265 - chief, 267 - co-, 267 - function of, 266 - normal, 266 - partial, 267 - relation to precipitins, 269 - specificity of, 267 - theory of formation, 265 - use of, 266 - - Agglutinogen, 266 - - Agglutinoid, 270 - - Aggressins, 288 - - Air, bacteria in, 71 - filtration of, 153 - "germ-free," 153 - - Albumin in bacteria, 84 - - Alcohol as antiseptic, 160 - as disinfectant, 160 - - Alcoholase, 125 - - Alcoholic fermentation, 31, 100 - - Alexin, 271, 273 - - Algæ, relation to bacteria, 37 - - Alimentary tract as path of entrance, 246 - - Alkalies as disinfectants, 158 - - Allergic, 290 - - Amboceptor, 273 - anti-, 275 - co-, 274 - in cobra, 275 - formation of, 273 - hemolytic, 278 - partial, 274 - in rattle snake, 275 - specificity of, 274 - theory of formation, 273 - - Amboceptorogen, 274 - - Amebic dysentery, 29, 35 - - Ameboid cells, 247 - colonies, 224 - - Amino-acids, relation to green plants, 119 - - Ammonia, structural formula, 103 - - Ammoniacal fermentation, 32 - - _Amoeba coli_, 29, 35 - - Amphitrichic, 46 - - Amylase, 124 - - Anaërobes, 76 - cultivation, methods of, 188 - principles underlying, 188 - facultative, 76 - isolation of, 190 - relation to elements, 86 - strict, 76 - - Anaërobic, 76, 215 - acid, butyric, 99 - acid fermentation, 98 - bacteria, first discovered, 32 - fermentation of polysaccharides, 95 - - Analysis of ash, 82 - chemical, of tubercle bacilli, 85 - - Anaphylactic, anti-, 290 - phenomena, 292 - reaction, uses of, 293 - - Anaphylatoxin, 290 - - Anaphylaxis, 289 - anti-, 292 - antibodies in, 291 - theory of, 290, 291, 292 - - ANAXIMANDER, 18 - - ANDERSON, 289 - - ANDERSON and MCCLINTIC, phenol coefficient, 165 - - ANDRY, 25, 33 - - Anilin dyes, as antiseptic, 162 - as disinfectants, 162 - introduction of, 30 - as stains, 204 - Weigert, 36 - fuchsin, 205 - gentian violet, 205 - water, 205 - - Animal carriers, 239 - inoculation, uses of, 227 - - Animalcules, 19, 33 - - Animals, disinfection of, 170 - experimental, 227 - food relationships of, 39 - - _Ankylostoma duodenale_, discovery of, 27, 34 - Egyptian chlorosis, cause of, 28, 35 - hookworm disease, cause of, 28 - - Anthrax, 17, 28, 35 - bacterium a facultative saprophyte, 238 - isolation of, 29 - due to a bacterium, 29 - in human beings, 238 - path of entrance, 243 - intestine, 246 - stomach, 246 - persistence due to spores, 251 - produced by exhaustion, 251 - protective inoculation in, 30 - spores, 29, 35 - transmission by flies, 242 - vaccine, 254 - - Anti-agglutinins, 270 - aggressins, 288 - amboceptors, 275 - antisera in snake poisoning, 275 - anaphylactic, 290 - anaphylaxis due to intracellular digestion, 292 - protein immunity compared to, 292 - bacterial immunity, 254, 255 - bodies, 259 - place of production, 295 - tabulation of, 294 - body, action, 260 - chemical composition, 260 - formation of, 128, 260 - complement, 274 - complementophil amboceptor, 275 - cytophil amboceptor, 275 - diphtheritic serum, 263 - enzyme, 122, 262 - function of, 262 - - Antigen, 259 - chemical composition of, 260 - in complement-fixation, 277 - syphilitic, 277, 279 - in Wassermann test, 279 - - Antigens, fats and fatty acids as, 260 - in preparation of vaccine, 285 - tabulation of, 294 - - Antipollenin, 263 - - Antiprecipitins, 270 - - Antisepsis, 131 - Lister, introduced, 35 - primitive, 25 - - Antiseptic, 131 - action of anilin dyes, 162 - carbolic acid as, 159 - cold as, 148 - - Antisera in snake poisoning, 275 - - Antisnake venoms, 275 - - Antitetanic serum, 263 - - Antitoxic immunity, 254, 255 - - Antitoxin, 261 - collection of, 263 - diphtheria, 30, 252 - preparation of, 263 - standard, 264 - tetanus, 252 - - Antitoxins, 261-264 - as factors in immunity, 295 - preservative in, 263 - specific, 261 - - Antivenin, 263 - - Apes, 227 - - Apparatus of Barber, 196 - - Appearance of growth on culture media, 217 - - APPERT, 20, 31, 34 - - Aqueous gentian violet, 205 - - Arborescent growth, 221 - - ARISTOTLE, 18 - - Aromatic compounds, production of, 104, 111 - - Arrak, 100 - - Arsenate, reduction of, 114 - - Arsenite, oxidation of, 115 - - ARTHUS, 289 - phenomenon, 289 - - Articles, unwashable, disinfection of, 169 - washable, disinfection of, 169 - - Artificial immunity, 251, 252 - - Ase, termination of name of enzyme, 124 - - Asepsis, 131 - - Aseptic, 131 - - Ash, analysis of, 82 - - Asiatic cholera, 27, 34, 73, 238, 239, 246, 248, 249 - - Attenuated, 253 - - Autoclave, air pressure sterilizer, 138 - pressure sterilizer, 138 - - Autogenous vaccines, 284 - in epidemic, 241 - - Autoinfection, 234 - - Autolysis, 149 - self-digestion, 126 - - Autotrophic, 86 - - Available nitrogen, loss of, 113 - - Azotobacter, 118 - - - B - - BABES-ERNST corpuscles, 45 - - Bacilli, butter, 209 - colon, 248 - grass, 209 - size and shape of, 52 - tubercle, chemical analysis of, 85 - - Bacillus, 52, 60, 62 - _anthracis_, 17, 36 - spore staining, 209 - - Bacillus of blue milk, 31 - Ducrey's, 245 - _subtilis_, 77, 83 - spore staining, 209 - - Bacteria, absorption of N by, 117 - acid fast, 84, 209 - adaptability, range of, 90 - advantage of motility to, 45 - aids in isolation of, 197 - anaërobic, 32 - cause of disease in animals, 30 - of souring of milk, 32 - cell groupings of, 55 - chains of, 38 - chemical composition of, 39, 81 - elements in, 82 - classed as fungi, 37 - as plants, 33, 35 - definition of, 40 - development of, 90 - distribution of, 71 - energy relationships, 39 - environmental conditions for growth, 72 - first classification of, 34 - drawings of, 20 - seen, 19, 33 - food relationships of, 39 - injurious, 72 - isolation of, 194 - measurement of, 40, 203 - metabolism of, 86 - methods of study of, 171 - morphology of, 41 - motile, 45 - nitric, 114 - nitrous, 114 - nucleus of, 42 - occurrence, 71 - pathogenic, outside the body, 237 - phosphorescent, 111, 112 - position of, 37 - rate of division, 43 - of motion, 45 - relation to algæ, 33, 37 - to elements, 86 - to gas and oil, 95 - to phosphate rock, 115 - to protozoa, 40 - to soil fertility, 120 - to sulphur deposits, 116 - to yeasts and torulæ, 37 - reproduction of, 37, 55 - root tubercle, 86, 87 - size of, 37, 40 - soil, chief function of, 119 - source of N, 102 - speed of, 45 - spiral, 53 - staining of, 204-212 - sulphur, 63 - thermophil, 75, 77 - universal distribution of, 90 - in vinegar-making, 99 - - BACTERIACEÆ, 62, 66, 70 - - Bacterial agglutinin, 265 - vaccines, 282 - preparation of, 283, 284 - - Bacterin, 253 - - Bacteriocidin, 272 - - Bacteriological culture tubes, 184 - examination, material for, 228 - microscope, 200 - - Bacteriology, pathogenic, definition of, 231 - reasons for study of, 217 - as a science, 17, 32 - - Bacteriolysin, 272 - - Bacteriopurpurin, 62, 63, 112 - - Bacteriotropin, 281 - - _Bacterium abortus_, agglutinin of, 265 - _coli_ in autoinfection, 234 - gas formation by, 95 - oxygen limits for, 77 - pneumonia through intestinal route, 246 - in preparation of sugar broths, 176 - definition of, 62, 67, 70 - _enteriditis_, cause of food poisoning, 104 - _fluorescens_, oxygen limits, 77 - _typhosum_, 73 - agglutinin, 265 - in phenol coefficient method, 166 - pneumonia through intestinal route, 246 - - Ballon pipette, 193 - - Balsam, mounting in, 207 - - BARBER, 253 - apparatus, 196 - - Barnyards, disinfection of, 167 - - Baskets, wire, 184 - - BASSI, 27 - silkworm disease, 34 - - BASTIAN, 24 - - BAUMGÄRTNER, 256 - - Beaded growth, 221 - - Bed-bugs, 241 - - Beds, contact, 116 - hot, 117 - - Beer, pasteurization of, 141, 144, 145 - - _Beggiatoa_, 63 - - BEGGIATOACEÆ, 63 - - BEHRING, 30 - - BELFANTI, 271 - - BERG, 27, 34 - - Berkefeld filter, 154 - - Bichloride of mercury as disinfectant, 158 - - BILHARZ, 28, 35 - - Bilharzia disease, 28, 35 - - Biochemical reactions, definition of, 87 - - Biological relationships, immunity reactions, 255, 270 - - Bipolar germination of spore, 48 - - Bismarck brown, 209, 212 - - Black-leg, 51, 73, 238, 243, 248, 251 - vaccine, 254 - - Bleaching powder as disinfectant, 158 - - Blood, collection of, 228 - cytolytic power of, 272 - detection of, 269 - serum, liquid, sterilization of, 182 - Loeffler's, 182 - medium, preparation of, 182, 183 - sterilization of, 182 - vessels in dissemination of organisms, 247 - - Blue milk, bacterial cause of, 34 - fermentation of, 31, 34 - - BOEHM, 27, 34 - - Boiling as disinfectant, 133 - - Boils, 237, 240, 243 - - BOLLINGER, 29, 30, 35, 36 - - BONNET, 20, 33 - - BORDET, 271 - - _Botrytis bassiana_, 27, 34 - - Bottles, staining of, 206 - - Bougies, 154 - - Bouillon, 173 - - BOYER, 260 - - Bread, salt rising, 95, 97 - - Bronchopneumonia, 233, 246 - - Broth, appearance of growth in, 218 - extract of, 176 - glycerine, 176 - medium, 173 - nitrate, 177 - sterilization of, 174 - sugar, 176 - - Brownian movement, 47, 203 - - Brushes, disinfection of, 169 - - Bubonic plague, 239 - - BUCHNER, 271 - - Budding of yeasts, 37 - - Bulgarian fermented milk, 98 - - Burning as disinfectant, 132 - - Burying as disinfectant, 154 - - BÜTSCHLI, 41, 43 - - Butter, 97 - bacilli, staining of, 209 - rancidity of, 101 - - Butyric acid fermentation, 32, 99 - - Buzzards, 241 - - - C - - CABBAGE disease due to protozoa, 36 - - Cadaverin, 104 - - CAIGNARD-LATOUR, 31, 34 - - Calcium hypochlorite as disinfectant, 158 - oxide as disinfectant, 158 - - Candles, filter, 153, 154 - - Canned goods, food poisoning by, 104 - spoilage of, 51 - - Canning, introduced, 21, 34 - principles involved, 133 - - Capsule, 44, 45 - of spore, 48 - staining of, 210 - - Carbohydrates in bacterial cell, 84 - fermentation of, 93-101 - - Carbol-fuchsin, 206 - - Carbolic acid as antiseptic, 159 - as disinfectant, 159 - first used, 29 - - Carbol-xylol, 209 - - Carbon cycle, 107 - dioxide, 108 - function of, in bacteria, 88, 101 - oxidation of, 114 - in proteins, liberation of, 105 - source of, 88 - uses of, 88, 101 - - CARBONI, 271 - - CARDANO, 18 - - Carrier problem, solution of, 240 - - Carriers, 239 - accidental, 241 - carrion eating animals as, 241 - control of, 240 - intermediate hosts as, 242 - protective measures against, 242 - universal, 240 - of unknown organisms, 239 - - Cars, stock, disinfection of, 170 - - Catalase, 125 - - Catalytic agents, function of, 123 - - Catalyzer, 123 - - Cattle, 227 - - Causation of disease, 24, 128 - - Cell, constituents of, 84 - contents of, 41, 83 - forms of, 58, 59 - staining for, 212 - typical, 52 - groupings, 55, 58, 59 - staining for, 195 - metabolism, 90 - structures of, 41 - wall, 41, 59 - composition of, 83 - - Cells, chemical stimuli of, 257 - overproduction activity of, 258 - specific chemical stimuli of, 258 - - Cellular theory of immunity, 256, 280 - - Cellulose, definition of, 83 - occurrence of, 83 - - Chain, 56 - - Channels of infection, 243 - alimentary tract, 246 - conjunctive, 244 - external auditory meatus, 244 - genitalia, 245 - intestines, 246 - lungs, 245 - milk glands, 244 - mouth cavity, 244 - mucosæ, 244 - nasal cavity, 244 - pharynx, 245 - skin, 243 - stomach, 246 - tonsils, 245 - - Chaos, 25 - - Characteristic groupings, 58 - - Characteristics of enzymes, 121 - of toxins, 126 - - CHARRIN, 265 - - Chart, descriptive, 217 - - CHAUVEAU, 256 - - Cheese, eyes in, 96 - failures, 110 - Limburger, 101 - odor of, 99 - poisoning, 104 - ripening of, 35 - - Chemical composition of bacteria, 39, 81, 85 - elements in bacteria, 82 - disinfectants, action of, 156-163 - stimuli, 257-260 - theory, fundamentals of, 256 - - Chemotherapy, 249, 255 - - CHEVREUIL, 21, 27, 31, 34 - - Chicken cholera, 30 - pox, 239, 246 - - Chief agglutinin, 267 - cell, 267 - - Chitin, 72 - - CHLAMYDOBACTERIACEÆ, 63 - - _Chlamydothrix_, 63 - - Chloride of lime as disinfectant, 158 - - Chlorine as disinfectant, 157 - - Chloroform as antiseptic, 162 - as disinfectant, 162 - - Chlorophyl, 37, 112 - - Chlorosis, Egyptian, 27, 35 - - Cholera, Asiatic, carriers of, 239 - organisms in, 27, 34 - facultative saprophytes, 238 - path of elimination of, 248 - of entrance of, 246 - relation to moisture, 73 - specific location of, 249 - hog, 242, 248, 252 - - Cholesterins as cell constituents, 84 - - Chromogenesis, 112 - - Chromoparic, 112 - - Chromophoric, 112 - - Chronic disease, 232 - - Chronological table, 33-36 - - Chymosin, 124 - - Circulation of carbon, 107 - of nitrogen, 107 - of phosphorus, 107 - of sulphur, 108 - - Classification, advantage of, 59 - early, 33, 35, 59 - Migula's, 62-63 - S. A. B., 63-70 - - Cleaning of slides, 207 - - Clearing of sections, 209 - - Closed space disinfection, 161 - - _Clostridium_, 49 - _botulinum_, 87, 104, 128, 238, 261 - _pasteurianum_, 118 - _tetani_, 128, 209, 233, 261, 263 - - Clothing, disinfection of, 170 - - Coagglutinins, 267 - - Coagulases, 124 - - Coagulating enzymes, 124 - - Coagulation temperature of proteins, 51 - - Coal, spontaneous heating of, 88 - - Coamboceptors, 274 - - Cobra, 275 - - COCCACEÆ, 62, 66, 68 - - Coccus, appearance of, on dividing, 57 - cell form of, 52 - groupings of, 52, 56, 57 - division of, 52 - - Coenzymes, 122 - - COHN, 28, 33, 35, 59 - - Cold as antiseptic, 148 - incubator, 215 - storage, 148 - - Colds, due to universal carriers, 240 - path of entrance of, 244 - vaccines in, 241 - - Colonies, characteristics of plate, 223-226 - definition of, 173 - - Color production, 112 - - Colorimetric method of standardization, 175 - - Combustion, spontaneous, 116 - - Commensal, 87 - - Commercial preparation of lactic acid, 99 - products, why keep, 131 - vaccines, 285 - - Communicable disease, 232 - - Complement, 273 - deviation test, 277 - effect of temperature on, 274 - fixation test, 276-279 - lecithin as, 274 - relation to toxins and enzymes, 273 - source of, 277 - - Complementoid, 274 - - Complementophil haptophore, 273 - - Complements, nature of, 274 - - Composition, chemical, 81-85 - related to fungi, 39 - relation to food, 81 - - Concentration of antitoxin, 264 - - Condenser, 200 - - Conditions for growth, general, 72 - maximum, 72 - minimum, 72 - optimum, 72 - spore formation, 51 - - Congenital immunity, 251, 252 - - Conjunctiva as path of entrance, 244 - - Constant temperature apparatus, 213 - - Contact beds, 116 - - Contagion, direct and indirect, 34 - - Contagious abortion, agglutination test, 268 - complement-fixation text, 277 - path of elimination, 248 - of entrance, 245 - - Contagium, definition of, 232 - vivum theory, 25, 28, 33 - - Contamination of food by carriers, 241 - - Continuous pasteurization, 141 - - Contrast stains, 205 - - Convalescents, control of, 239-240 - - CORNALIA, 29 - - Corpuscles, Babes-Ernst, 45 - red, in complement-fixation test, 278, 279 - malaria, etc., in, 249 - - Corrosive sublimate as disinfectant, 158 - - _Corynebacterium diphtheriæ_, 64, 69, 128, 233, 234, 261, 263 - - Coryza, acute, 244 - - Cotton plugs, 21, 184 - - Coughing, 248 - - Crateriform liquefaction, 221 - - Cream ripening, 97 - - CREITE, 271 - - _Crenothrix_, 61 - - Creolin as disinfectant, 160 - - Cresols as disinfectants, 159 - - Culture, definition of, 171 - medium, definition of, 171 - essentials of, 172 - inoculation of, 186, 192 - kinds of, 172 - liquid, 172, 173 - methods of using, 184 - optimum moisture for, 73 - plating of, 188 - reaction of, 81, 216 - selective, 198, 199 - solid, 172, 173 - standardization of, 174, 175 - synthetic, 183 - titration of, 174 - use of, 173 - tubes, description of, 184 - - Cultures, anaërobic, 188-192 - from internal organs, 229 - mass, 188 - plate, 188 - potato, 186 - puncture, 185 - pure, definition of, 171 - isolation of, 194-199 - slant, 186 - slope, 186 - stab, 185 - - Curled edge, 225 - - Cutaneous inoculation, 228 - - Cycle, carbon, 107 - nitrogen, 107 - phosphorus, 107 - sulphur, 108 - - Cystitis, 234 - - Cytolysin, 272 - - Cytolysins, 271-279 - - Cytolytic, 272 - power of blood, 272 - serums, failure of, 275 - substances in immunity, 295 - - Cytophil group, 273 - - Cytoplasm, 41 - - Cytotoxic, 272 - - - D - - DALLERA, 289 - - Dark field illumination, 204 - - DAVAINE, 28, 35 - - Death-point, thermal, 75 - determination of, 215 - - Decomposition, how caused, 108 - importance of, 108 - of urea, 106 - - Deep culture tubes, 190-191 - - Degeneration forms, 54 - - Delousing method in typhus, 242 - - DE MARTIN, 35 - - Denitrification, 114 - - Deodorant, 131 - - Descriptive chart, 217 - - Diagnosis, agglutination test in, 265-267 - anaphylaxis in, 292 - complement-fixation test in, 277 - immunity reactions in, 255 - material for bacteriological, 228-229 - precipitin test in, 269 - - Diastase, 124 - - Diffusion of food through cell wall, 41 - - Digestion of proteins, 102 - - Dilution method of isolation, 194 - plates, 194, 195 - - Dimethylamine, structural formula, 103 - - Diphtheria antitoxin, 30, 263, 264 - bacilli, granules in, 45 - involution forms, 54 - carriers, 239 - location of, 245, 249 - path of entrance, 245 - toxin, M. L. D., 264 - - Diplobacillus, 55 - - Diplococcus, 56 - - _Diplococcus_, 66, 69 - - Diplospirillum, 55 - - Discharges, 228 - intestinal, 248 - nasal, 248 - urethral, 248 - vaginal, 248 - - Discontinuous sterilization, 133 - - Disease, acute, 233 - of animals to man, 232 - Bilharzia, 28, 35 - cabbage, 30, 35 - causation of, 24, 128 - communicable, 232 - contagious, 34, 232 - of flies, 28, 35 - germ, 25, 27, 33 - hookworm, 28, 35 - infectious, 232, 240 - Johne's, 246, 248 - non-specific, 233 - protozoal, eradication, 242 - transmission, 242 - silkworm, 27, 29, 34, 35 - skin, 243 - specific, 27, 30, 233 - transmission of, 26, 232 - - Dishes, Petri, 181 - - Disinfectant, 131 - action of anilin dyes, 162 - closed space, 161 - dry heat as, 133 - moist heat as, 132, 133 - standardization of, 165 - steam as, 132, 133 - - Disinfectants, chemical, action of, 156-163 - first experiment in, 21 - factors affecting, 164-165 - - Disinfection, agents in, 131-163 - by boiling, 132, 133 - by burning, 132 - by burying, 154 - definition of, 130 - first chemical, 34 - hot air, 21, 133 - physical agents, 131-155 - practical, 166-170 - precautions in, 170 - puerperal fever, 28, 34 - steam, 134-138 - surgeon's hands, 28 - - Dissemination of organisms, 247 - - DISTASO, 42, 43 - - Distilling sour mash, 98 - - Division, planes of, 55-58 - rate of, 43, 91 - - DOBELL, 43 - - DORSET, 84 - - Dosage of vaccines, 286 - - Dose, minimum lethal, 264 - standard test, 264 - - DOUGLAS, 42, 43, 280 - - Dourine, 245, 248 - - Drumstick spore, 49 - - Dry heat, 21, 133 - - Drying, 131, 132 - - DUBINI, 27, 34 - - Ducrey's bacillus, 245 - - Dunham's peptone, 177 - - DURHAM, 265 - - Dyes, anilin, as antiseptics, 162 - introduction of, 30 - as stains, 204 - - Dysenteries, 242, 246, 248, 249 - - Dysentery, amebic, 29, 35 - tropical, 29 - - - E - - ECTOPLASM, 41 - - Edema, malignant, 237, 243 - - Edge of colony, 225 - - Effuse colony, 224 - - Egg sensitization, 292 - - EHRENBERG, 33, 34 - - EHRLICH, 256, 276 - - Ehrlich's theory, 256-260 - - EICHSTEDT, 28, 34 - - Electric milk purifier, 152 - - Electricity, 79, 150 - - Elements in bacteria, 82, 86, 88, 89 - - Elimination of organisms, 248 - - _Empusa muscæ_, 28, 29, 35 - - Emulsin, 122 - - Endo-enzymes, 126 - - Endogenous infection, 235 - - Endoplasm, 41 - - Endotoxins, 128, 276 - - Energy relationships, 39 - transformations, 86-90 - - Ensilage, 98 - - Enteritis, 233 - - Entire edge, 225 - - Entrance of organisms, 243-246, 247 - - Environmental conditions, 72, 130, 213 - - Enzymes, 84, 121-126 - in anaphylaxis, 291 - in immunity, 295 - - Enzymoid, 262 - - Epidemics, 241 - - Epitheliolysin, 272 - - Eosin, 204 - - Equatorial spore, 49 - germination of, 48, 49 - - Eradication of disease, 236, 242 - - Erysipelas, hog, 248 - - _Erythrobacillus prodigiosus_, 66, 68, 70, 77, 113 - - Essential structures, 41 - - Essentials of a culture medium, 172 - - Esters, 84, 110 - - Ether as disinfectant, 162 - - EUBACTERIA, 62 - - Exanthemata, 248 - - Exhaustion factor in immunity, 251 - theory of immunity 256 - - Existence, conditions for, 72 - - Exo-enzymes, 126 - - Exogenous infection, 235 - - Exotoxins, 128 - - Experiment, Pasteur's, 21 - Schroeder and Dusch's, 22 - Schultze's, 21 - Schwann's, 22 - Tyndall's, 24 - - Experimental animals, 227 - - External auditory meatus, 244 - genitalia, 245 - - Extracellular enzymes, 126 - - Extract broth, 176 - - Eyes in cheese, 96, 97 - - - F - - Factors affecting disinfectants, 164, 165 - immunity, 250, 251 - in immunity to disease, 295 - - Facultative, 215 - aërobes, 76 - anaërobes, 76, 192 - parasites, 87 - saprophytes, 238 - - Failure of cytolytic serums, 275 - of vaccines, 286 - - Fat colors, 112 - splitting enzymes, 124 - - Father of bacteriology, 19 - of microscope, 19 - - Fats as antigens, 260 - occurrence of, 84 - rancidity of, 101 - in sewage disposal, 101 - splitting of, 101 - - Favus, 27, 34, 243 - - Feces, bacteria in, 72 - - Feeding, as inoculating method, 228 - - FEINBERG, 43 - - Ferment, organized, 126 - unorganized, 126 - - Fermentation, 31, 93 - acid, 93, 96 - acetic, 32, 99 - butyric, 32, 99 - alcoholic, 31, 34, 100 - ammoniacal, 32 - bacterial, 32 - blue milk, 31, 34 - of carbohydrates, 93-101 - definition of, 93 - gaseous, 93, 94, 96 - tubes, 184, 190 - yeast, 34, 99 - - Fermented milk, Bulgarian, 98 - - Fever, due to invisible organisms, 25 - Malta, 268 - recurrent, 29, 35 - Rocky Mountain spotted, 242 - scarlet, 246, 248 - Texas, 232, 233, 242 - typhoid, 232, 248 - typhus, 242 - yellow, 242 - - Fibrin ferment, 124 - - Filament, 56 - - Filiform growth, 221 - - Film, fixing of, 207 - preparation of, 207 - - Filter, Berkefeld, 154 - candles, 153-154 - Mandler, 154 - Pasteur-Chamberland, 154 - sprinkling, 115, 116 - - Filterable virus, 234 - - Filtration, 152-154 - - First order, receptors of, 261, 262 - - FISCHER, 42, 45 - - Fixation test, complement, 276 - - Fixed virus, 253 - - Fixing of film, 207 - - Flagella, 45-47 - staining of, 210 - - Flash process of pasteurization, 145 - - Fleas, 241 - - FLEXNER, 276 - - Flies, 28, 35, 241 - - FLÜGGE, 271 - - FODOR, von, 271 - - Food adulteration, complement-fixation test in, 279 - immunity reactions in, 255 - precipitin test in, 269 - - Food contamination by carriers, 241 - poisoning, 87, 104, 238 - requirement compared with man, 92 - uses of, 86 - - Foot-and-mouth disease, 244, 248 - - Forage poisoning, 87 - - Foreign body pneumonia, 245 - - Formaldehyde as disinfectant, 160 - - Formalin, 160 - - Formol, 160 - - Forms, cell, 52-54 - degeneration, 54 - growth, 55 - involution, 54 - study of, 32-34 - - Fox fire, 111 - - Foxes, 241 - - FRACASTORIUS, 25, 33 - - Free acid, 175 - receptors, 259 - spores, 48 - - Fruiting organs, 37 - - FUCHS, 31, 34 - - Fuchsin, 205 - anilin, 205 - carbol, 206 - - Fungi, bacteria as, 37 - - Funnel-shaped liquefaction, 221 - - - G - - GABBET'S blue, 206 - method of staining, 209 - - Gall-bladder, 248 - - Galvanotaxis, 79 - - Gas formation in cheese, 96, 97 - natural, 95 - production of, 110 - - Gaseous fermentation, 93-95 - - GASPARD, 26, 34 - - Gelatin, advantage of, 178 - composition of, 179 - cultures, first used, 30, 36 - liquefaction of, 103 - medium, 177 - plating of, 188 - standardization of, 178 - sterilization of, 178 - - Gemmation, 37 - - General conditions for growth, 72 - infections, vaccines in, 286 - - Generation, spontaneous, 17-24 - - Generic names introduced, 33 - - Genitals, 245 - - Gentian violet, selective action of, 162 - stain, 205 - - Germ, free air, 153 - theory of disease, 25 - - German measles, 233 - - Germination of spore, 48 - - Germs, 33 - in air, 24, 35 - - GESCHEIDEL, 271 - - Giemsa stain, 43 - - Glanders, 26, 233, 238, 244, 248, 249, 268, 277 - - Glands, mammary, 248 - salivary, 248 - - GLEICHEN, 32 - - Globulin in bacteria, 84 - - Glycerine broth, 176 - - Glycerinized potato, 172 - - Glycogen as cell constituent, 84 - - Goats, 227 - - Gonidia, 63 - - Gonococcus, 245 - - Gonorrhea, 248, 249 - - Good health, 296 - - Grain rust, 26, 34 - - Gram positive organisms, 162, 208 - negative organisms, 162, 208 - - Gram's method of staining, 208 - solution, 208 - - Granular edge, 225 - - Granules, metachromatic, 212 - Neisser's, 45 - polar, 45 - - Granulose in bacteria, 84 - - Grape juice, pasteurization of, 141 - - Grass bacilli, 209 - - Green plants, N nutrition of, 118 - - GRIESINGER, 27, 28, 35 - - Group, agglutinating, 266 - haptophore, 261, 262, 266, 270, 273 - precipitating, 270 - toxophore, 261, 262 - zymophore, 262, 270, 273 - - Groupings, cell, 55-58 - - Growth, appearance in media, 217 - - _Gruber_, 265, 268 - - _Gruby_, 28, 34 - - Gum-like substance in bacteria, 83 - - - H - - HAECKEL, 280 - - Hanging drop slide, 203 - - Haptophore, complementophil, 273 - cytophil, 273 - group, 261, 262, 266, 270, 273 - - Harness, disinfection of, 169 - - Hay fever, 263, 292 - - Health, 296 - - Heat as disinfectant, 132-144 - due to oxidation, 112 - production of, 116 - - Heated serum, 271, 277, 278, 279 - - Heating of manure, 116 - - HELLMICH, 84 - - HELMONT, VAN, 18 - - Hemagglutinin, 265 - - Hemicellulose, 83 - - Hemolysin, 272 - - Hemolytic amboceptor, 278 - - Hemorrhagic septicemia, 246 - - HENLE, 27, 34, 233 - - HERICOURT, 289 - - Herpes tonsurans, 28, 34 - - HESSELING, von, 32 - - Heterologous sera, 276 - - Heterotrophic, 86 - - HILL, 33 - - HILTON, 27 - - HOFFMAN, 24 - - Hog cholera, 231, 242, 248, 252, 253 - erysipelas, 248 - - Holders, 143 - - HOLMES, 28, 34 - - Homologous sera, 276 - - Hookworm disease, 28, 34 - - Horses, 227, 263 - - Host, 87 - - Hot beds, 117 - - Hunger in immunity, 251 - - Hydrochloric acid, 246 - - Hydrogen, function of, 98 - ion concentration standardization, 175, 176 - oxidation of, 114 - peroxide, 162 - sulphide, 115 - - Hydrophobia, 249 - - Hydrostatic pressure, 79 - - Hygienic laboratory, 165 - - Hypochlorites, 157, 158 - - - I - - Ice cream poisoning, 104 - - Identification of bacteria, 216, 217 - in blood, 269 - in meat, 269 - in milk, 269 - - Immersion oil, 201 - - Immunity, 236, 250-296 - acquired, 251, 252 - active, 251, 252-255 - antibacterial, 254, 255 - antitoxic, 254, 255 - artificial, 251, 252 - classification of, 251 - congenital, 251 - factors in, 295 - modifying, 250 - inherited, 251, 252 - natural, 295 - passive, 251, 252, 253 - to protein, 290 - reactions, value, 255 - relative, 250 - summary of, 295 - theories of, 256 - - Inactivate, 272 - - Incubation period, 26, 232 - - Incubator, 213 - cold, 215 - room, 213 - - Index, chronological, 31 - opsonic, 281 - phagocytic, 281 - - Indicator, 278 - - Indol, 104 - - Infection, 232 - auto, 234 - channels of, 243 - endogenous, 235 - exogenous, 235 - mixed, 234 - primary, 234 - secondary, 234 - wound, 17, 25, 26, 27, 30, 34, 36, 233, 234, 240, 243, 248 - - Infectious diseases, 232 - control of, 240 - - Infective organisms, specificity of location, 249 - - Infestation, 232 - - Infested, 232 - - Influenza, 239, 241, 246 - - Infusoria, 33 - - Inhalation, 228 - - Inherited immunity, 251, 252 - - Inoculation of animals, 227 - uses of, 227 - of cultures, 186, 188 - definition of, 192 - methods of, 227 - needles, 192 - - Inoculations, first protective, 30 - of smallpox, 24 - - Insects, 241, 242 - - Instruments, sterilization, 136, 167 - - Intracardiac, 228 - - Intracellular enzyme, 166 - - Invasion, 232 - - Invertase, 124 - - Involution forms, 53, 212 - - Iodine, 157 - - Iron bacteria, 86 - function of, 89 - - Irregular forms, 53 - - Isolation of anaërobes, 190 - of pure cultures, aids in, 197-199 - methods, 194-196 - - Itch mite, 27, 34 - - - J - - JABLOT, 32 - - Jack-o-lantern, 105 - - Jar, Novy, 192 - - JENNER, 26, 34, 253 - - Johne's disease, 248 - - - K - - KETTE, 32, 35 - - Kidneys, 248 - - Kinase, 125 - - KIRCHER, 18, 25, 33 - - KLEBS, 29, 35 - - KLENCKE, 28, 34 - - KOCH, 17, 27, 29, 30, 33, 36 - - Koch's postulates, 233 - - KRAUS, 268 - - KRUSE, 254 - - KÜCHENMEISTER, 28, 35 - - - L - - LAB, 124 - - Lachrymal canal, 244 - - Lactacidase, 125 - - Lactic acid bacteria, 97 - fermentation, 96-99 - - LANCISI, 25, 33 - - LANDOIS, 271 - - LATOUR, 31, 34 - - LAVERAN, 25, 30 - - Lecithin as antigen, 279 - as cell constituent, 84 - as complement, 274 - - LEEUWENHOEK, 19, 32, 33 - - Legumes, 118 - - LEIDY, 27, 33, 34, 35 - - LE MOIGNAC, 284 - - Leprosy, 233, 244, 249 - - LESSER, 32 - - Lethal dose, 264 - - Leukocytes, washing of, 281 - - Lice as carriers, 241 - - LIEBERT, 28, 34 - - Light, action on bacteria, 75 - as disinfectant, 148 - production of, 111 - - LINNÆUS, 25 - - Lipase, 124 - - Lipochromes, 113 - - Lipoids as antigen, 274 - - Lipovaccines, 284 - - Liquefaction of gelatin, 221 - of protein, 103 - - Liquid blood serum, 182 - manure, disinfection of, 169 - media, 172 - - Liquids, sterilization of, 153 - - LISTER, 29, 30, 35 - - Litmus milk, 177 - - Living bacteria, examination of, 201 - cause theory, 28, 33 - - Localized infections, vaccines in, 286 - - Location of organisms, specificity of, 249 - - Lockjaw, 231, 233 - - Loeffler's blood serum, 182 - blue, 206 - - Loop needles, 193 - - Lophotrichic, 46 - - LÖSCH, 29, 35 - - Lungs, 245, 249 - - Lye washes as disinfectants, 159 - - Lymph channels in dissemination, 247 - - Lysol as disinfectant, 160 - - - M - - MCCLINTOCK, 165 - - MCCOY, 160 - - Macrococcus, 52 - - Macroscopic agglutination, 265 - - Malaria, 25, 30, 32, 242 - - Malarial parasite, 30, 249 - - Malignant edema, 237, 243 - - Mallease reaction, 269 - - Mallein test, 292 - - Malta fever, 268 - - Mammary glands, 248 - - Mandler filter, 154 - - Manure, liquid, disinfection of, 169 - heating of, 40 - - _Margaropus annulatus_, 242 - - MARTIN, 32 - - Mass cultures, 188 - - MASSART, 42 - - Maximum conditions, 72, 73, 74, 76 - - Measles, 246, 248, 250 - German, 233, 239 - - Measly pork, 28 - - Measurement of bacteria, 203 - special unit of, 40 - - Meat broth, 173 - identification of, 269 - juice, 173 - poisoning, 104 - - Mechanical vibration, 80 - - Medico-legal examination, 269, 279, 293 - - Medium. _See_ Culture medium - - Meningitis, 239, 244 - - Meningococcus, 244 - - Mercuric chloride, 158 - - Merismopedia, 57 - - Metabiosis, 103 - - Metabolism, 86-91 - - Metachromatic granules, 44, 45, 59, 212 - - Metastases, 235 - - Metatrophic, 86 - - METCHNIKOFF, 256, 280 - - Methods of inoculation of animals, 227 - of cultures, 186-188 - of obtaining pure cultures, 194 - - Methylamine, 103 - - Methylene blue, 205, 206 - - Mice, white, 227 - - Microbiology, 231 - - Micrococcus, 52, 60, 62, 66, 68, 69, 245 - - Micrometer, 203 - - Micromillimeter, 40 - - Micron, 40 - - Microörganisms, 32 - - Microscope, improvements in, 30, 36 - invention, 19 - Leeuwenhoek's, 19 - use of, 200 - - Microspira, 61, 63 - - _Microsporon furfur_, 28, 34 - - Middle ear, 241 - - Migula's classification, 62 - - Milk, blue, 31, 34 - Bulgarian, 98 - digestion of, 102 - flavors in, 110 - glands, 244 - identification of, 269 - litmus, 177 - pasteurization of, 141, 144-147 - as path of elimination, 248 - preparation of, 177 - purifier, electric, 152 - souring of, 32 - sterilization of, 177 - tuberculous, 248 - - Minimum conditions, 72, 73, 74, 77 - lethal dose, 264 - - Mirror, use of, 200 - - Mixed infection, 234 - vaccine, 285 - - Mixotrophic, 86 - - M. L. D., 264 - - MOHLER, 167 - - Moist heat, 133 - - Moisture, 73 - - Mold colonies, 226 - - Molds in alcoholic fermentation, 100 - in relation to bacteria, 37, 39 - - Molecular respiration, 88, 89 - - Monas, 33 - - Monkeys, 227 - - Monotrichic, 45 - - MONTAGUE, 24 - - Mordants, 204, 211 - - Morphology, 41-58 - in identification, 171, 212 - - Mosquitoes and malaria, 25, 242 - - Motile bacteria, 45 - - Motion of bacteria, 47 - Brownian, 47, 203 - - Mounting in balsam, 207 - - Mouth cavity, 244 - - Mu, 40 - - Mucosæ as channels of infection, 244 - - MÜLLER, 33, 34, 59 - - Mumps, 239 - - Municipal disinfection, 170 - - MÜNTZ, 32, 35 - - Muscardine, 34 - - Mycelia, 39, 226 - - _Mycobacteriaceæ_; 64 - - _Mycobacterium_, 64, 69 - of Johne's disease, 209 - _lepræ_, 209 - _smegmatis_, 209 - _tuberculosis_, 83, 176, 209 - - Mycoproteid, 83 - - Mycorrhiza, 119 - - Myxomycetes, 38 - - - N - - NÄGELI, 29, 35 - - Nasal cavity, 244 - discharges, 248 - - Natural gas, 95 - immunity, 251, 252, 296 - - NEEDHAM, 20, 33 - - Needles, inoculation, 192 - - Negative complement-fixation test, 278 - phase, 287 - - Neisser's granules, 45 - stain, 212 - - NENCKI, 83 - - Nephrolysin, 272 - - NEUFELD, 281 - - Neurin, 104 - - Neurotoxin, 272 - - NEUVEL, 43 - - Nichrome wire, 193 - - Nitrate broth, 177 - - Nitrates in soil, 115 - - Nitric bacteria, 114 - - Nitrification, 32, 35 - - Nitrite, oxidation of, 114 - - Nitrogen, absorption of, 117 - in bacterial cell, 89 - circulation, 109 - cycle, 107 - fertilizers, 120 - liberation, 104 - nutrition of green plants, 118 - use of, 103 - - Nitrous bacteria, 114 - - Non-pathogenic, 87 - - Non-specific disease, 233 - - Normal agglutinins, 266 - serum, 272 - - _Nosema bombycis_, 29, 35 - - NOVY, 183 - jar, 192 - - Noxious retention theory, 255 - - Nuclein, 42, 43 - - Nucleoprotein, 43 - - Nucleus, 42, 43 - - Nutrition of green plants, 118 - - NUTTAL, 271 - - - O - - OBERMEIER, 29, 35 - - Objective, oil immersion, 200, 201 - - Oblique germination of spore, 48 - - Occurrence of bacteria, 71 - - Official classification, 59 - - _Oidium albicans_, 27, 34 - - Oil bath, 167 - essential for clearing, 209 - immersion objective, 200, 201 - relation of bacteria to, 116 - - OMODEI, 27 - - Opsonic index, 281, 282, 287 - method, 282 - power, 287 - - Opsonin, 281 - - Opsonins, 281, 282, 295 - - Optimum conditions, 72, 73, 74 - - Order, receptors of first, 261-264 - of second, 265-270, 281 - of third, 271-279 - - Organic acids, 84, 110 - catalyzers, 123 - - Organisms, dissemination of, in body, 247 - filterable, 234 - pathogenic, elimination of, 248 - entrance of, 243-246, 247 - specific relation to tissue, 249 - ultramicroscopic, 234 - - Organized ferments, 126 - - Osmotic pressure, 78, 149, 216 - - Otitis media, 244 - - OTTO, 289 - - Overproduction theory, 257, 258 - - OWEN, 27, 34 - - Oxidation, 114, 115 - - Oxidizing enzymes, 125 - - Oxygen, compressed, 77 - as disinfectant, 156 - function of, 88 - nascent, 77 - relationships, 215, 220 - requirement, 88 - source, 76, 77 - - Oyster sensitization, 292 - - OZNAM, 26 - - Ozone, 77, 150, 157 - - - P - - PANCREAS, 248 - - Papillate, 221 - - PAGET, 27, 34 - - Paraffin oil, 190 - - Parasite, 87 - facultative, 87 - strict, 87 - - PARODKO, 77 - - Partial agglutinin, 267 - amboceptor, 274 - - Passive immunity, 251, 252 - - PASTEUR, 17, 21, 29, 30, 31, 32, 35, 253, 256, 283 - flask, 21, 23, 24, 193 - treatment of rabies, 253 - - Pasteur-Chamberland filter, 154 - - Pasteurization, 139-147 - continuous, 141 - flash process, 145 - - Pathogenic, 87 - bacteria, definition of, 231 - outside the body, 237 - bacteriology, scope of, 235 - organisms, destroyed by boiling, 133 - elimination of, 248 - entrance of, 243-247 - - Paths of elimination, 248 - of entrance, 243-247 - - PEACOCK, 26 - - Pebrine, 29, 35 - - Pedesis, 47 - - Peptone solution, Dunham's, 177 - - Period of incubation, 26, 232 - - Peritonitis, 234 - - Peritrichic, 46 - - _Peronospora infestans_, 28, 34 - - PERTY, 33, 35 - - Pet animals, 241 - - Petri dishes, 181, 188 - - Petroleum, 95 - - PFEIFFER, 271 - - Pfeiffer's phenomenon, 271 - - _Pfeifferella mallei_, 65, 69, 265 - - Phagocytes, 247 - - Phagocytic index, 281 - - Phagocytosis, 243, 280-288, 295 - theory, 256 - - Pharynx, 245 - - Phase, negative, 287 - positive, 287 - - Phenol coefficient, 165, 166 - as disinfectant, 159 - production of, 104, 111 - - Phenolphthalein, 174 - - Phenomenon, anaphylactic, 292 - Arthus', 289 - Pfeiffer's, 271 - - Phosphate reduction, 114 - rock, 115 - - Phosphorescence, 111 - - Phosphorus cycle, 108 - in proteins, 105 - uses of, 89 - - Photogenesis, 111 - - Physical agents for disinfection, 131-155 - - Physiological activities, 93-129 - definition of, 87 - in identification, 216 - - Physiology of bacteria, 71-171 - - Phytotoxins, 127, 128 - - Pickling, 98 - - Pigeons, 227 - - Pigments, 84, 112, 113 - - Pimples, 234, 240, 243 - - PINOY, 284 - - Pipettes for inoculation, 193 - - _Piroplasma bigeminum_, 233, 242, 249 - - Piroplasmoses, 242, 249 - - PIRQUET, von, 289 - - Pityriasis versicolor, 28, 34 - - Plague, 246 - - Planes of division, 56, 57 - - _Planococcus_, 62 - - _Planosarcina_, 62 - - Plants and animals, 39 - - _Plasmodiophora brassicæ_, 30, 36 - - Plasmolysis, 41, 42, 78 - - Plasmoptysis, 42, 78 - - Plate colonies, study of, 224-226 - cultures, 180, 188, 191 - - Plates, dilution, 194, 195 - gelatin first used, 30, 36 - - Platinum needles, 193 - - Plectridium, 49 - - PLENCIZ, 26, 31 - - Plugs, cotton, 21, 184 - - Pneumococcus, 240, 245 - - Pneumonia, 240, 245, 246, 248 - vaccination against, 241 - - Poisoning, cheese, 104 - food, 87, 104, 238 - ice cream, 104 - meat, 104 - - Polar germination, 48, 49 - granules, 45 - - Poliomyelitis, 244 - - POLLENDER, 28, 35 - - Polysaccharides, fermentation of, 95 - - Polyvalent vaccine, 285 - - Pork, measly, 28 - - Position of bacteria, 37 - of flagella, 45, 46 - of spore, 49 - - Positive phase, 287 - test, 278 - - Postulates, Koch's, 233 - - Potato, acidity of, 182 - glycerinized, 182 - media, 180-182 - rot, 28, 34 - - Power, opsonic, 287 - - Practical sterilization and disinfection, 166-170 - - _Pragmidiothrix_, 63 - - Precipitinogen, 269 - - Precipitinoid, 270 - - Precipitins, 268-270 - anti-, 270 - - Preparation of antitoxin, 263 - of bacterial vaccines, 283, 284 - of film, 207 - - Preservation of slides, 207, 208 - - Preservative, alcohol as, 160 - in vaccine, 284 - - Pressure, hydrostatic, 79 - osmotic, 78, 149 - oxygen, 76, 77 - steam, 136 - sterilization, 136-139 - - Prevention of disease, 235, 236, 253, 255, 283 - - Preventive vaccination, colds, 241 - pneumonia, 241 - rabies, 253 - smallpox, 26, 34, 253 - vaccines, autogenous, 285 - stock, 285 - - PREVOST, 26 - - Primary infection, 234 - - Process kettle, 137 - - Pro-enzyme, 121 - - Prophylaxis, 289 - - Protamine in bacteria, 84 - - Protease, 124 - - Protective inoculation, first, 30 - - Protein in bacteria, 84 - coagulation temperature, 51 - composition of, 102 - decomposition of, 105 - differentiation of, 255 - foreign, 289 - identification of, 293 - immunity, 290 - putrefaction of, 102-109 - split products of, 291 - splitting of, 106 - structure of, 291 - synthesis of, 113 - - _Proteus vulgaris_, 67, 70, 77 - - Protoautotrophic, 115 - - Protoplasm, 41, 59 - - Prototrophic, 86 - - Protozoa, cause of disease, 30 - cell wall in, 41 - in intermediate hosts, 242 - relation to bacteria, 40 - specificity of localization, 249 - - Protozoal diseases, transmission of, 242 - - PSEUDOMONADACEÆ, 65, 70 - - _Pseudomonas pyocyanea_, 62, 65, 70, 128, 265 - - Ptomaines, 103, 104 - - _Puccinia graminis_, 26, 34 - - Puerperal fever, 28, 34 - - Punctiform colonies, 223 - - Puncture cultures, 185 - - Pure culture, 171, 194-199 - - Purification of streams, 73 - of water, 150 - - Purin bases in bacteria, 84 - - Pus cocci, 73 - infectious, 26 - organisms in, 35 - - Putrefaction, 27, 31, 33 - definition of, 102 - of proteins, 102-109 - end products of, 103 - in soil, 106 - - Putrescin, 104 - - - Q - - QUARANTINE, 239 - disinfection, 170 - - Quicklime as a disinfectant, 155, 158 - - Quinsy, 245 - - - R - - RABBITS, 227 - - Rabies, bacteriological examination in, 229 - Pasteur treatment of, 253 - path of elimination in, 248 - transmission of, 239 - specificity of localization in, 249 - - Räbiger's method of staining, 210 - - Radiations, 79 - - Radium, 79 - - Rancidity of butter, 101 - - Rashes, serum, 289 - urticarial, 292 - - Rate of division, 43, 91 - of movement, 45 - - Rats, 227, 241 - - RAYER, 28, 35 - - Reaction of medium, 81, 174, 175, 216 - - Reactions, biochemical, 87 - immunity, 255, 269, 279, 292, 293 - surface, 91 - - REAUMUR, 33 - - Receptors, 257, 258, 259, 261-280 - as factors in immunity, 295 - of first order, 262 - free, 259, 261, 262 - of second order, 265 - tabulation of, 294 - of third order, 273 - - Recurrent fever, 29, 35 - - Red corpuscles, 249, 278, 279 - - REDI, 19 - - Reducing actions, 112, 113 - enzymes, 125 - - Refrigeration as antiseptic, 148 - - REINKE, 80 - - Relapses, 235 - - Relationships of bacteria, 37-40 - biological, 255, 270 - - Rennet, 124 - - RENUCCI, 27, 34 - - Reproduction, 37, 63, 90 - - Resistance to disease, 241, 250 - of spores, 50 - - Respiratory function, 88 - tract, 246 - - Retarders, 143 - - Rheumatism, 245 - - _Rhizobium leguminosarum_, 65, 68, 69, 118 - - Rhizoid colonies, 222, 223 - - RHIZOPUS NIGRICANS, 226 - - RHODOBACTERIACEÆ, 63 - - _Rhodococcus_, 66, 69 - - RICHET, 289 - - Ricin, 262 - - RIDEAL, 165 - - Rideal-Walker method, 165 - - RIMPAU, 281 - - RINDFLEISCH, 29, 35 - - Ringworm, 28 - - Ripening of cheese, 32 - of cream, 97 - - ROBIN, 262 - - Rock, phosphate, 115 - - Rocky Mountain spotted fever, 242 - - ROGERS, 265 - - Röntgen rays, 79 - - Room temperature, 213 - - Rooms, disinfection of, 167 - incubator, 213 - - Root tubercle bacteria, 86, 87, 108 - tubercles, 117 - - ROSENAU, 289 - - Rot, potato, 28, 34 - - Round worm, 232 - - Roup, 244 - - ROUX, 30 - - Rubbing as inoculation, 195 - - Rust, grain, 26, 34 - - RUZICKA, 42 - - - S - - SACCATE liquefaction, 222 - - Safranin, 205 - - Saliva, 248 - - Salivary glands, 248 - - Sake, 100 - - Salt-rising bread, 95 - - Saprogenic, 102 - - Saprophilic, 103 - - Saprophyte, 87, 238 - - _Sarcina_, 57, 58, 60, 66, 68, 69 - _lutea_, 77 - _ventriculi_, 83 - - _Sarcoptes scabiei_, 27, 34 - - Sauerkraut, 98 - - Scarlet fever, 246, 248, 250 - - Scavengers, bacteria as, 108 - - SCHICK, 289 - - _Schistosomum hematobium_, 28, 35 - - SCHLÖSING, 32, 35 - - SCHÖNLEIN, 27, 34 - - SCHROEDER and DUSCH, 21 - - SCHULTZE, 21, 34 - - SCHWANN, 21, 31, 34 - - Sea, bacteria in, 71, 111 - - Sealing air-tight, 20 - - Secondary infection, 234 - - Sections, staining of, 209 - - Selective media, 198, 199 - - Self-limited, 233 - - SEMMELWEISS, 28, 35 - - Sensitization, 290 - - Sensitized animal, 290 - bacteria, 254 - vaccine, 254 - - Septicemias, hemorrhagic, 246 - - Sero-bacterins, 254 - - Serum, antidiphtheritic, 263 - antitetanic, 263 - heated, 271, 277, 279 - rashes, 289 - sickness, 289, 292 - simultaneous method, 253 - therapy, 253 - - Serums, cytolytic, failure of, 275 - - Sewage disposal, 101, 116 - sulphate, reduction in, 114 - - Shape of spore, 48 - - Sickness, serum, 289, 292 - - Side-chain theory, 256, 258 - - Silkworm disease, 27, 29, 34, 35 - - Size of bacteria, 37, 40 - - Skatol, 104 - - Skin, channel of infection, 243 - diseases, 243 - glanders, 248 - lesions, 228 - pocket, 227 - - Slant cultures, 186 - - Slide, cleaning of, 207 - hanging drop, 203 - staining on, 207 - - Slope cultures, 186 - - Sludge tanks, 116 - - Small intestine, 249 - - Smallpox, 24, 26, 34, 239, 246, 248 - babies, 252 - vaccines, 253 - - SMITH, 289 - tubes, 184 - - Snake poisons, 263, 275 - venoms, 128 - - Sneezing, 248 - - Soap, 160 - medicated, 160 - - Society of American Bacteriologists, classification, 63 - descriptive chart, 217 - key, 68 - - Sodium hypochlorite, 158 - - Soil, acid, 81 - bacteria, 119 - bacteriology, 35 - enrichment, 117 - fertility, 120 - organisms, 74 - - Solid media, 172, 173 - - Solution, Gram's, 208 - stock, 205 - - Sore throat, 240, 241 - - Sound, 80 - - Sour mash, 98 - - Source of complement, 277 - - Souring, 98 - - SPALLANZANI, 20, 31, 34 - - Species determination, 59, 60 - - Specific amboceptor, 274, 278, 279 - antibody, 291 - chemical stimuli, 257, 258, 259 - disease, 27, 30, 233 - - Specificity of agglutinins, 267 - of amboceptor, 274 - of location, 249 - of opsonins, 281 - - Spermotoxin, 272 - - Spherical form, 52 - - _Spherotilus_, 63 - - _Spirillaceæ_, 63, 65 - - Spirilloses, 241, 242 - - _Spirillum_, 53, 54, 55, 61, 63, 66, 68, 69 - _rubrum_, 113 - - _Spirochæta_, 61 - _obermeieri_, 29, 35 - - Spirochetes, 53, 242 - - _Spirosoma_, 63 - - Splenic fever, 28 - - Split products of proteins, 291 - - Splitting enzymes, 124 - of fats, 101 - - Spoilage of canned goods, 51, 78 - - Spoiling of food, 91 - - Spontaneous combustion, 105, 116 - generation, 17-24, 33, 34 - outbreaks of disease, 239 - - Sporangia, 226 - - Spore, 47-51 - anthrax, 29, 35 - capsule, 48 - germination, 48 - - Spores, cause spoiling of canned goods, 51 - destroyed by boiling, 133 - first recognized, 33, 35 - light on, 75 - in pasteurization, 146 - resistance of, 50, 51 - staining of, 209 - two in bacterium, 50 - - Sprinkling filters, 116 - - Stab cultures, 185 - - Stables, disinfection of, 167 - - Stain, anilin fuchsin, 205 - gentian violet, 205 - aqueous gentian violet, 205 - Bismarck brown, 212 - carbol fuchsin, 206 - contrast, 205 - Gabbet's blue, 206 - Loeffler's blue, 206 - Neisser's, 212 - - Staining, 204-212 - acid-fast bacteria, 209 - bottles, 206 - capsules, 210 - cell forms, 212 - groupings, 212 - flagella, 210 - Gabbet's method, 209 - Gram's method, 208 - metachromatic granules, 212 - Neisser's method, 212 - Räbiger's method, 210 - reasons for, 204 - sections, 209 - spores, 209 - Welch's method, 210 - Ziehl-Neelson, 210 - - Standard antitoxin, 264 - methods, 217 - test dose, 264 - toxin, 264 - - Standardization, colorimetric method, 175 - of culture media, 174 - of disinfectants, 165 - H-ion method, 175 - of vaccines, 284 - - Staphylococcus, 57, 58 - - _Staphylococcus_, 66, 68, 69 - - STARIN, 196 - - Steam at air pressure, 134 - sterilizers, 135 - streaming, 135 - under pressure, 136 - - _Stegomyia_, 242 - - Sterile, 131 - - Sterilization, 130 - in canning, 133 - discontinuous, 133 - by filtration, 21, 152 - first experiment by boiling (moist heat), 20 - by chemicals, 21 - by dry heat (hot air), 21 - by filtration, 21 - - Sterilizers, pressure, 137 - steam, 135 - - Stimuli, chemical, 257, 258, 259 - - Stock cars, 170 - solutions, 205 - vaccines, 285 - - Stomach, 246 - - Straight needles, 192 - - Stratiform liquefaction, 222 - - Strawberry poisoning, 292 - - Streak methods of isolation, 196 - plates, 188 - - Streptobacillus, 53, 56 - - Streptococcus, 56, 60, 245 - - _Streptococcus_, 60, 62, 66, 68, 69 - - Streptospirillum, 55 - - Streptothrix, 38 - - _Streptothrix bovis_, 30, 36 - - Strict aërobe, 76 - anaërobe, 76 - parasite, 87 - - Structures, accidental, 43 - cell, 41 - essential, 41 - - Subcutaneous inoculation, 227 - - Subdural inoculation, 228 - - Substrate, 123 - - Successive existence, 103 - - Sugar broth, 176, 177 - - Sulphate reduction, 114 - - Sulphur bacteria, 63, 86, 115 - deposits, 116 - function of, 89 - in proteins, 105 - - Summary in immunity, 295 - Ehrlich's theory, 259 - - Sunning, 148 - - Surface reactions, 91, 92 - - Surgical instruments, 167 - - Susceptibility, 235 - - Swine, 227 - - Symbionts, 87, 103 - - Symbiosis, 87 - - Synthetic media, 172, 183 - - Syphilitic antigen, 277, 279 - - Syphilis, 233, 245, 248, 249 - Wassermann test, 277, 279 - - - T - - TABULATION of antigens and antibodies, 294 - - _Tænia solium_, 28, 35 - - Tapeworm, 28, 35, 232 - - Taxes, 203 - - Temperature conditions, 74 - effect on growth, 213 - factor in immunity, 251 - room, 213 - - Test, complement deviation, 277 - fixation, 276, 279 - dose, 264 - for enzymes, 123 - Gruber-Widal, 268 - mallein, 292 - negative, 278 - positive, 278 - for toxins, 127 - tuberculin, 292 - Wassermann, 277, 279 - Widal, 268 - - Testicle, 249 - - Tetanus, 231, 238, 243, 249, 251, 252 - antitoxin, 252 - toxin, 126 - - Tetracoccus, 57 - - Tetrad, 57 - - Texas fever, 232, 233, 242 - - THAER, 31 - - Theories of immunity, 256 - - Theory, anaphylaxis (author's), 290-292 - cellular, 256 - chemical, 256 - contagious disease, 34 - contagium vivum, 25, 28, 33 - Ehrlich's, 256-260 - exhaustion, 256 - germ, 25 - living cause, 33 - mosquito, 25 - noxious retention, 256 - overproduction, 257, 258 - phagocytosis, 256 - side-chain, 256 - spontaneous generation, 17 - unfavorable environment, 256 - - Thermal death point, 75, 215 - - Thermophil bacteria, 75, 77 - - Thermoregulator, 213 - - Thermostat, 213 - - THIOBACTERIA, 63 - - _Thiothrix_, 63 - - Thread, 56 - - Thrombin, 124 - - Thrush, 27, 34, 244 - - Ticks, 241 - - TIEDEMANN, 26 - - Tinea, 28 - - Tissue contrast stains, 205 - - Titer, 268 - - Titration, 174 - - Tonsil, 245, 249 - - Tonsillitis, 245 - - TOUISSANT, 283 - - Toxin, diphtheria, 264 - effect of temperature, 262 - final test for, 127 - in food poisoning, 104 - molecule, 261, 262 - standard, 264 - tetanus, 264 - - Toxin-antitoxin method, 254 - - Toxins and enzymes compared, 127 - as cell constituents, 84 - production of, 126-128 - of other organisms, 127 - specific localization, 249 - true, 128 - - Toxoid, 262 - - Toxophore group, 261, 262, 273 - - Tract, alimentary, 246 - - Transmission, accidental carriers in, 241 - agency of, 232 - of contagious diseases, 232 - of disease, 26, 28, 35, 239 - of glanders, 26, 34 - of protozoal diseases, 242 - of tuberculosis, 28, 29, 34, 35, 238 - - Transverse division, 54, 56 - - TRAUBE, 271 - - _Treponema pallidum_, 245 - - Trichina, 27 - - _Trichina spiralis_, 27, 34, 35 - - Trichinosis, 28, 35 - - Trichophyton, 243 - - _Trichophyton tonsurans_, 28, 34 - - Trimethylamine, 104 - - Tropical dysentery, 29 - lands, 242 - - Tropisms, 203 - - True toxins, 128 - - Trypanosomes, 242 - - Trypanosomiases, 241, 243 - - Tubercle bacteria, 85, 209 - - Tuberculin reaction, 292, 293 - - Tuberculosis, 73, 233, 238, 245, 246, 248, 249 - due to bacteria, 30 - produced experimentally, 28, 34 - proved infectious, 29, 35 - - Tuberculous milk, 248 - - Tubes, culture, 184 - deep, 190 - fermentation, 184, 190 - Smith, 184 - Vignal, 189 - - Two spores in a bacterium, 50 - - TYNDALL, 24 - - Tyndallization, 133 - - Tyndall's box, 23, 24, 35 - - Typhoid bacilli, 73, 238 - bacillus, 45 - carriers, 239 - fever, 231, 233, 248, 265, 268 - transmission by flies, 242 - vaccine, 254 - - Typhus, 242 - - Typical cell forms, 52 - - - U - - ULTRAMICROSCOPE, 204 - - Ultramicroscopic organisms, 234 - - Ultraviolet rays, 150 - - Unfavorable environment theory, 256 - - Unit of antitoxin, 264 - of measurement, 40 - - Universal carrier, 240 - - Unorganized ferment, 126 - - Unwashable articles, 169 - - Urea, 106 - - Urease, 125 - - Urethral discharges, 248 - - Urine, 72 - - Urticarial rashes, 292 - - - V - - VACCINATION in chicken cholera, 30 - negative phase in, 287 - in pneumonia, 241 - in smallpox, 26, 34, 253 - - Vaccine, 253 - age of, 285 - anthrax, 254 - antigens for, 285 - autogenous, 285 - black-leg, 254 - derivation of, 253 - mixed, 285 - polyvalent, 285 - preservative in, 284 - sensitized, 254 - smallpox, 253 - - Vaccines, bacterial, 283 - in colds, 241 - dosage of, 286 - in epidemics, 241 - failure of, 285-286 - in infections, 286 - preparation of, 283 - standardization of, 284 - stock, 284 - theory of, 286 - use of, 283 - - Vacuoles, 42, 43, 44, 59 - - Vaginal discharges, 248 - - VARO, 25 - - VAUGHAN, 291 - - Vaughan and Novy's mass cultures, 188 - - Vegetable toxins, 127, 128 - - Vegetables, forcing of, 117 - - Vehicles, disinfection of, 169 - - Venoms, antisnake, 275 - - VIBORG, 26, 34 - - Vibration, mechanical, 80 - - Vibrio, 33, 35, 53, 65, 68, 69 - _choleræ_, 66, 73 - - Vignal tubes, 189 - - VILLEMIN, 29, 35 - - Villous growth, 219, 221 - - Vinegar, 99, 114 - - Virulence, 235 - - Virus, 234 - - Vultures, 241 - - - W - - WALKER, 165 - - Wall, cell, 41 - composition of, 82, 83 - - WARDEN, 260 - - Washable articles, disinfection of, 169 - - Washing leukocytes, 281 - - Wassermann test, 277 - - Water, bacteria in, 73 - filtration of, 153 - purification of, 77, 150 - sterilization of, 157 - - WEBB, 253 - - WEIGERT, 17, 30, 36, 42, 257, 258 - - Welch's method of staining, 210 - - Whooping cough, 246, 250 - - WIDAL, 265 - test, 268 - - Will o' the wisp, 105 - - Wine, pasteurization of, 141 - - WINOGRADSKY, 32, 63, 86 - - Wire baskets, 184 - nichrome, 193 - - WOLLSTEIN, 26, 34 - - WORONIN, 30, 36 - - Wound infections, 17, 25, 26, 27, 30, 34, 36, 233, 234, 240, 243, 248 - - WRIGHT, 280 - - - X - - X-RAYS, 79 - - _Xylinum, acetobacter_, 83 - - - Y - - YEAST, fermentation, 31, 34, 99, 100, 114 - relation to bacteria, 37 - reproduction of, 37, 39 - - Yellow fever, 242 - - - Z - - ZANZ, 18 - - ZENKER, 27, 28, 35 - - ZETTNOW, 43 - - ZIEMANN, 43 - - Ziehl-Neelson method of staining, 210 - - Ziehl's solution, 206 - - Zoögloea, 44 - - Zoötoxins, 128 - - Zymase, 125 - - Zymogens, 121, 125 - - Zymophore group, 273 - - - - -Transcriber's Notes. - -Punctuation has been standardised and simple typographical errors have -been repaired. Hyphenation, quotation mark usage, and obsolete/variant -spelling (including variant spellings of proper nouns) have been -preserved as printed. - -In the original book, the page numbering goes xiii, blank, unnumbered, -18. This is a printer's error: no pages are missing. - -The descriptive chart insert has been moved from between pages 216 and -217 to the end of the book. - -The following changes have also been made: - - Page 26: 'this scourge which had devastated' - for 'this scourge which had devasted' - - Page 30: 'to be the cause of a disease in cabbage,' - [added comma] - - Page 32: 'alcoholic, lactic and butyric' - for 'alcoholic, lactic and butryic' - - Page 32: 'however, workers busied themselves' - for 'however, workers, busied themselves' [deleted extra comma] - - Page 56: 'Fig. 43.--Streptobacillus' - for 'Fig. 43.--Steptobacillus' - - Page 57: 'from a genus of algæ' - for 'from a genus of algae' - - Page 58: 'staphylococcus--irregular' - for 'staphylococcus--irrgular' - - Page 59: 'so that it is impossible' - for 'so that is is impossible' - - Page 62: 'Illustrates the genus Spirochæta' - for 'Illustrates the genus Spirochaeta' - - Page 63: 'since it is without a sheath' - for 'since it is without a a sheath' - - Page 64: 'Corynebacterium diphtheriæ' - for 'Corynebacterium diphtheriae' - - Page 67: 'Prazmowski, 1880; anaërobic' - for 'Prazmowski, 1880; anaerobic' - - Page 70: 'growth processes involving oxidation' - for 'growth processes involving oxidadation' - - Page 70: 'EE--Anaërobes, rods swollen at sporulation' - for 'EE--Anaerobes, rods swollen at sporulation' - - Page 73: 'percentage of water is permissible' - for 'percentage of water is permissable' - - Page 95: 'Material taken from the bottom' - for 'Material taken from the botton' - - Page 102: 'large-moleculed and not diffusible' - for 'large-moleculed and not diffusable' - - Page 104: 'various kinds of "meat poisoning,"' - for 'various kinds of "meat posisoning,"' - - Page 106: 'formed under anaërobic conditions' - for 'formed under anaerobic conditions' - - Page 110: 'volatile fatty acids, ethereal' - for 'volatile fatty acids, etheral' - - Page 127: 'but in much larger doses' - for 'but in much large doses' - - Page 131: '"antiseptic" may become a disinfectant' - for '"antiseptic" may become a disfectant' - - Page 141: 'quarantine station barge' - for 'quaratine station barge' - - Page 147: 'A continuous milk pasteurizer.' - for 'A continuous milk pastuerizer.' - - Page 163: 'especially when a large amount of material' - for 'expecially when a large amount of material' - - Page 179: 'these must be sterilized' - for 'these must be steriliized' - - Page 191: 'Deep tubes showing anaërobic' - for 'Deep tubes showing anaerobic' - - Page 193: 'less than one-twentieth of platinum' - for 'less than one-twentieth of platimum' - - Page 207: 'grease-free cloth, handkerchief' - for 'grease-free cloth, handerchief' - - Page 210: 'Stain with Löffler's blue' - for 'Stain with Löffller's blue' - - Page 211: 'slide to cause precipitates' - for 'slide to cause preciptates' - - Page 213: 'grows at body temperature (37°)' - [added closing parenthesis] - - Page 217: 'working on a revision' - for 'working on a revission' - - Page 220: 'inoculation for facultative anaërobes' - for 'inoculation for facultative anërobes' - - Page 231: 'the unicellular microörganisms' - for 'the unicellular micro-organisms' [split across line] - - Page 232: 'unicellular pathogenic microörganisms' - for 'unicellular pathogenic micro-organisms' [split across line] - - Page 233: 'the fact of self-limitation' - for 'the fact of self-limitaion' - - Page 242: 'the cattle tick (_Margaropus annulatus_).' - for 'the cattle tick (_Margaropus annulatus_.)' - - Page 244: 'B. Mucosæ directly continuous' - for 'f. Mucosæ directly continuous' - - Page 245: 'localized infection as in micrococcal, streptococcal' - for 'localized infection as in micrococcal, strepococcal' - - Page 248: 'ELIMINATION OF PATHOGENIC MICROÖRGANISMS.' - for 'ELIMINATION OF PATHOGENIC MICRO-ORGANISMS.' [split across line] - - Page 254: 'sometimes added to attenuate' - for 'sometimes added to attentuate' - - Page 256: 'Metchnikoff has since elaborated' - for 'Metchinkoff has since elaborated' - - Page 266: 'This is analogous to what' - for 'This is analagous to what' - - Page 280: 'other names, but ascribed' - for 'other names, but asscribed' - - Chart: '(10 minutes' exposure in nutrient broth when this is adapted - to growth of organism)' - for '(10) minutes' exposure in nutrient broth when this is adapted - to growth of organism)' - - Page 299: 'Allergic, 290' - [index entry was printed twice] - - Page 308: 'Foreign body pneumonia' - for 'Foreignbody pneumonia' - - Page 309: 'oxidation of' - for 'ox dation of' - - Page 311: 'Microspira' - for 'Miscospira' - - Page 311: 'Microsporon' - for 'Miscrosporon' - - Page 314: 'Plasmodiophora brassicæ' - for 'Plasmodiophora bassicæ' - - Page 317: 'Starin, 196' - [index entry was printed between Standardization and Staphylococcus] - - Page 318: 'Thermostat' - for 'Thermostadt' - - - - - - -End of the Project Gutenberg EBook of The Fundamentals of Bacteriology, by -Charles Bradfield Morrey - -*** END OF THIS PROJECT GUTENBERG EBOOK THE FUNDAMENTALS OF BACTERIOLOGY *** - -***** This file should be named 43227-8.txt or 43227-8.zip ***** -This and all associated files of various formats will be found in: - http://www.gutenberg.org/4/3/2/2/43227/ - -Produced by Jennifer Linklater, Jason Isbell and the Online -Distributed Proofreading Team at http://www.pgdp.net - - -Updated editions will replace the previous one--the old editions -will be renamed. - -Creating the works from public domain print editions means that no -one owns a United States copyright in these works, so the Foundation -(and you!) can copy and distribute it in the United States without -permission and without paying copyright royalties. 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Hart was the originator of the Project Gutenberg-tm -concept of a library of electronic works that could be freely shared -with anyone. For forty years, he produced and distributed Project -Gutenberg-tm eBooks with only a loose network of volunteer support. - -Project Gutenberg-tm eBooks are often created from several printed -editions, all of which are confirmed as Public Domain in the U.S. -unless a copyright notice is included. Thus, we do not necessarily -keep eBooks in compliance with any particular paper edition. - -Most people start at our Web site which has the main PG search facility: - - www.gutenberg.org - -This Web site includes information about Project Gutenberg-tm, -including how to make donations to the Project Gutenberg Literary -Archive Foundation, how to help produce our new eBooks, and how to -subscribe to our email newsletter to hear about new eBooks. - - - -</pre> - +<div>*** END OF THE PROJECT GUTENBERG EBOOK 43227 ***</div> </body> </html> diff --git a/43227.txt b/43227.txt deleted file mode 100644 index 1aef432..0000000 --- a/43227.txt +++ /dev/null @@ -1,14183 +0,0 @@ -The Project Gutenberg EBook of The Fundamentals of Bacteriology, by -Charles Bradfield Morrey - -This eBook is for the use of anyone anywhere at no cost and with -almost no restrictions whatsoever. You may copy it, give it away or -re-use it under the terms of the Project Gutenberg License included -with this eBook or online at www.gutenberg.org - - -Title: The Fundamentals of Bacteriology - -Author: Charles Bradfield Morrey - -Release Date: July 16, 2013 [EBook #43227] - -Language: English - -Character set encoding: ASCII - -*** START OF THIS PROJECT GUTENBERG EBOOK THE FUNDAMENTALS OF BACTERIOLOGY *** - - - - -Produced by Jennifer Linklater, Jason Isbell and the Online -Distributed Proofreading Team at http://www.pgdp.net - - - - - - -Transcriber's Note: - -Formatting and non-latin characters are indicated thus: - - _italic_ - =bold= - ^{superscript} - {subscript} - #Greek transliteration# - - - - -[Illustration: PLATE I - -ANTHONY VON LEEUWENHOEK - -Who first saw bacteria] - - - - - THE FUNDAMENTALS - OF - BACTERIOLOGY - - BY - CHARLES BRADFIELD MORREY, B.A., M.D. - - PROFESSOR OF BACTERIOLOGY AND HEAD OF THE DEPARTMENT - IN THE OHIO STATE UNIVERSITY, - COLUMBUS, OHIO - - - ILLUSTRATED WITH 171 ENGRAVINGS AND 6 PLATES - - _Second Edition, thoroughly Revised_ - - [Illustration: Publisher's logo] - - LEA & FEBIGER - PHILADELPHIA AND NEW YORK - 1921 - - COPYRIGHT - LEA & FEBIGER - 1921 - - - TO - GRACE HAMILTON MORREY - AMERICAN PIANIST - - - - -PREFACE TO SECOND EDITION - - -The first edition seems to have fulfilled a need for a general -text-book on the subject of bacteriology. The original method of -presentation is preserved. The text-book idea is adhered to, so that -the individual instructor may have full liberty to expand on topics in -which he is especially interested. A number of illustrations have been -added, the text has been improved in many instances by the addition -of further explanatory matter and the most recent general advances in -the Science. Examples are the System of Classification of the Society -of American Bacteriologists, which is used throughout the text, their -Key to the Genera of Bacteria, a discussion of the H-ion concentration -method of standardization, the selective action of anilin dyes, the -mechanism of entrance of pathogenic organisms into the body, a more -detailed explanation of the origin of antibodies, the nature of -antigens and a table of antigens and antibodies. - -Professor Vera McCoy Masters has assisted in the revision by aiding -in the preparation of manuscript and the reading of proof and in the -making of the index, for which services the author's thanks are hereby -expressed. - - C. B. M. - Columbus, Ohio, 1921. - - - - -PREFACE TO FIRST EDITION - - -An experience of nearly twenty years in the teaching of Bacteriology -has convinced the author that students of this subject need a -comprehensive grasp of the entire field and special training in -fundamental technic before specializing in any particular line of work. -Courses at the University are arranged on this basis. One semester is -devoted to General Bacteriology. During the second semester the student -has a choice of special work in Pathogenic, Dairy, Soil, Water, or -Chemical Bacteriology. A second year may be devoted to advanced work in -any of the above lines, to Immunity and Serum Therapy, or to Pathogenic -Protozoa. - -This text-book is intended to cover the first or introductory -semester's work, and requires two classroom periods per week. Each -student is compelled to take two laboratory periods of three hours per -week along with the class work. The outline of the laboratory work is -given at the end of the text. Results attained seem to justify this -plan. A text-book is but one of many pedagogical mechanisms and is not -intended to be an encyclopedia of the subject. - -The author makes no claim to originality of content, since the facts -presented are well known to every bacteriologist, though the method -of presentation is somewhat different from texts in general. During -the preparation of this work he has made a thorough review of the -literature of Bacteriology, covering the standard text-books as well -as works of reference and the leading periodicals dealing with the -subject. Thus the latest information has been incorporated. - -No attempt has been made to give detailed references in a work of this -character. - -The photomicrographs are original except where otherwise indicated -and are all of a magnification of one thousand diameters where no -statement to the contrary appears. These photographs were made with -a Bausch & Lomb Projection Microscope fitted with a home-made camera -box. Direct current arc light was used and exposures were five to ten -seconds. Photographs of cultures are also original with a few indicated -exceptions. All temperatures are indicated in degrees centigrade. - -For use of electrotypes or for prints furnished the author is indebted -to the following: A. P. Barber Creamery Supply Company, Chicago, Ill.; -Bausch & Lomb Optical Company, Rochester, N. Y.; Creamery Package -Manufacturing Company, Chicago, Ill.; Davis Milk Machinery Company, -North Chicago, Ill.; Mr. C. B. Hoover, Superintendent of Sewage -Disposal Plant, Columbus, O.; Mr. C. P. Hoover, Superintendent of Water -Filtration Plant, Columbus, O.; The Hydraulic Press Manufacturing -Company, Mt. Gilead, O.; Loew Manufacturing Company, Cleveland, O.; -Metric Metal Works, Erie, Pa.; Sprague Canning Machine Company, -Chicago, Ill.; U. S. Marine Hospital Service; Wallace and Tiernan -Company, New York City, N. Y. - -For the preparation of many cultures and slides, for great assistance -in the reading of proof and in the preparation of the index, Miss Vera -M. McCoy, Instructor in Bacteriology, deserves the author's thanks. - -The author trusts that the book will find a place in College and -University courses in Bacteriology. - - C. B. M. - - - - -CONTENTS - - - Historical Introduction--Spontaneous Generation--Causation - of Disease--Putrefaction and Fermentation--Study of - Forms--Chronological Table 17 - - CHAPTER I. - - Position of Bacteria--Relationships to - Algae--Yeasts--Molds--Protozoa 37 - - - PART I. - - MORPHOLOGY. - - CHAPTER II. - - Cell Structures--Cell Wall--Protoplasm--Plasmolysis - --Plasmoptysis--Nucleus--Vacuoles--Capsules--Metachromatic - Granules--Flagella--Spores 41 - - CHAPTER III. - - Cell Forms--Coccus--Bacillus--Spirillum--Involution Forms 52 - - CHAPTER IV. - - Cell Groupings 55 - - CHAPTER V. - - Classification--Migula's--Society of American - Bacteriologists'--Key to the Latter 59 - - - PART II. - - PHYSIOLOGY. - - CHAPTER VI. - - Occurrence--General Conditions for - Growth--Moisture--Temperature--Light--Oxygen-- - Osmotic Pressure--Electricity--X-rays and Radium - Emanations--Pressure--Mechanical Vibration 71 - - CHAPTER VII. - - Chemical Environment--Reaction of Medium--Chemical - Composition 81 - - CHAPTER VIII. - - Chemical Environment (Continued)--General Food - Relationships--Metabolism of Elements 86 - - CHAPTER IX. - - Physiological Activities--Fermentation of - Carbohydrates--Splitting of Fats 93 - - CHAPTER X. - - Physiological Activities (Continued)--Putrefaction of - Proteins--Cycles of Nitrogen, Carbon, Sulphur, Phosphorus 102 - - CHAPTER XI. - - Physiological Activities (Continued)--Production of - Acids, Gases, Esters, Alcohols, Aldehydes, Aromatic - Compounds--Phosphorescence--Chromogenesis--Reduction-- - Oxidation--Production of Heat--Absorption of Free - Nitrogen--Nitrogen Nutrition of Green Plants 110 - - CHAPTER XII. - - Physiological Activities (Continued)--Production of - Enzymes--Discussion on Enzymes--Toxins--Causation of - Disease 121 - - CHAPTER XIII. - - Disinfection--Sterilization--Disinfectants--Physical - Agents--Pasteurization 130 - - CHAPTER XIV. - - Disinfection and Sterilization (Continued)--Chemical - Agents--Anilin Dyes 156 - - CHAPTER XV. - - Disinfection and Sterilization (Continued)--Choice - of Agent--Standardization of Disinfectants--Phenol - Coefficient--Practical Sterilization and Disinfection 164 - - - PART III. - - THE STUDY OF BACTERIA. - - CHAPTER XVI. - - Culture Media--Broth, Milk, Gelatin, Agar, Potatoes, Blood - Serum--Standardization of Media--H-ion Concentration - Method--Synthetic Media 171 - - CHAPTER XVII. - - Methods of Using Culture Media--Culture Tubes--Plates-- - Anaerobic Cultures--Vignal Tubes--Fermentation Tubes-- - Deep Culture Tubes--Novy Jars--Inoculation of Culture Media 184 - - CHAPTER XVIII. - - Isolation of Bacteria in Pure Culture--Dilution - --Plating--Streaking--Barber Apparatus--Aids in - Isolation--Heat--Selective Antiseptics--Selective - Food---Indicators--Animal Inoculation 194 - - CHAPTER XIX. - - Study of the Morphology of Bacteria--Bacteriological - Microscope--Hanging Drop Slides--Staining--Gram's - Method--Spores--Acid-fast Bacilli--Capsules-- - Flagella--Metachromatic Granules 200 - - CHAPTER XX. - - Study of the Physiology of Bacteria--Temperature - --Incubators--Thermal Death Point--Oxygen Relationships - --Study of Physiological Activities--Appearance of Growth - on Culture Media--Appearance of Molds on Plate Cultures 213 - - CHAPTER XXI. - - Animal Inoculation--Material for Bacteriological - Examination 227 - - - PART IV. - - GENERAL PATHOGENIC BACTERIOLOGY. - - CHAPTER XXII. - - Introduction--Infection--Acute Infection--Chronic - Infection--Specific--Non-specific--Koch's - Postulates--Virulence--Susceptibility 231 - - CHAPTER XXIII. - - Pathogenic Bacteria Outside the Body--As Saprophytes--As - Facultative Saprophytes--Latent--Carriers--Universal - Carriers--Accidental Carriers--Necessary Intermediate Hosts 237 - - CHAPTER XXIV. - - Channels of Infection--Skin--Mucosae--Respiratory Tract - --Alimentary Tract--Mechanism of Entrance of Organisms - --Dissemination in the Body--Paths of Elimination-- - Specificity of Location 243 - - CHAPTER XXV. - - Immunity--Natural--Artificial--Active--Passive-- - Production of Immunity--Vaccine--Antiserum--Practical - Applications of Immunity Reactions 250 - - CHAPTER XXVI. - - Theories of Immunity--Pasteur--Chauveau--Baumgaertner - --Metchnikoff--Ehrlich--Principles of Ehrlich's Theory 256 - - CHAPTER XXVII. - - Ehrlich's Theory (Continued)--Receptors of the First - Order--Antitoxin--Antienzyme--Preparation of Antitoxins - --Units 261 - - CHAPTER XXVIII. - - Ehrlich's Theory (Continued)--Receptors of the Second - Order--Agglutinins--Agglutination Reaction--Precipitins - --Precipitin Test 265 - - CHAPTER XXIX. - - Ehrlich's Theory (Continued)--Receptors of the Third - Order--Cytolysins--Amboceptor--Complement--Anti-amboceptors - --Antisnake Venoms--Failure of Cytolytic Serums in - Practice--Complement-fixation Test 271 - - CHAPTER XXX. - - Phagocytosis--Opsonins--Opsonic Index--Bacterial - Vaccines--Preparation of--Use of--Lipovaccines - --Aggressins 280 - - CHAPTER XXXI. - - Anaphylaxis--Author's Theory--Tuberculin Test--Table of - Antigens and Antibodies--Summary of Immunity as Applied to - Protection from Disease 289 - - - - -BACTERIOLOGY. - - - - -HISTORICAL INTRODUCTION. - - -Bacteriology as a science is a development of the latter half of the -nineteenth century. It may be said to have begun in the decade between -1870 and 1880, due largely to the wide circulation given to Koch's -work in proving that _Bacillus anthracis_ is the cause of Anthrax in -1876, in devising new culture methods and in demonstrating that wound -infections are due to microoerganisms, 1878. Associated with this -work were the great improvements in the microscope by Abbe and the -introduction of anilin dyes for staining bacteria by Weigert. These -results attracted workers throughout the world to the "new science." -Nevertheless, this work of Koch's was preceded by numerous observations -and experiments which led up to it. Certainly the most important -discoveries immediately responsible were those of Pasteur. He must be -considered as the greatest of the pioneer bacteriologists since he -worked in all fields of the subject. Some of the antecedent work was -done in attempting to disprove the old "spontaneous generation" theory -as to the origin of organisms; some in searching for the causes of -disease and some in the study of fermentation and putrefaction. - - -SPONTANEOUS GENERATION. - -Speculation as to the first origin of life is as old as history and -doubtless older. Every people of antiquity had its own legends, as for -example, the account in Genesis. This question never can be definitely -settled, even though living matter should be made in the laboratory. - -The doctrine of the "spontaneous origin" of particular animals or -plants from dead material under man's own observation is a somewhat -different proposition and may be subjected to experimental test. The -old Greek philosophers believed it. Anaximander (B.C. 610-547) taught -that some animals are derived from moisture. Even Aristotle (B.C. -384-322) said that "animals sometimes arise in soil, in plants, or -in other animals," _i.e._, spontaneously. It can be stated that this -belief was general from his day down through the Dark and Middle Ages -and later. Cardano (A.D. 1501-1576) wrote that water gives rise to -fish and animals and is also the cause of fermentation. Van Helmont -(1578-1644) gives directions for making artificial mice. Kircher -(1602-1680) describes and figures animals _produced under his own eyes_ -by water on plant stems. - -However, many thinkers of the seventeenth century doubted the truth -of this long-established belief. Francesco Redi (1626-1698) made a -number of experiments which tended to prove that maggots did not -arise spontaneously in meat, as was generally believed, but developed -only when flies had an opportunity to deposit their eggs on the -meat. It seems that by the latter part of this century the idea that -organisms large enough to be seen with the naked eye could originate -spontaneously was generally abandoned by learned men. - -The work of Leeuwenhoek served to suspend for a time the subject of -spontaneous generation, only to have it revived more vigorously later -on. He is usually called "The Father of the Microscope," though the -compound microscope was invented probably by Hans Zansz or his son -Zacharias, of Holland, about 1590. Leeuwenhoek used a simple lens, -but his instruments were so much more powerful that they opened up an -entirely new and unknown world. (Fig. 1.) - -Anthony van Leeuwenhoek (1632-1723) was apprenticed to a linen draper -and accumulated a comfortable fortune in this business. He became -interested in the grinding of spectacle lenses, then an important -industry in Delft, Holland, where he lived, and did a great deal of -experimental work in this line, mainly for his own enjoyment. Finally -he succeeded in making a lens so powerful that he could see in water -and various infusions very minute living bodies never before observed. -Leeuwenhoek contributed 112 papers to the Royal Society of Great -Britain, the first in 1673, many of them accompanied by such accurate -descriptions and drawings, for example a paper submitted September -12, 1683, that there is no doubt that he really saw bacteria and was -the first to do so (Fig. 2). Rightly may he be styled "The Father -of Bacteriology," if not of the microscope. He says in one paper: -"With the greatest astonishment I observed that everywhere through -the material I was examining were distributed _animalcules_ of the -most microscopic dimness which moved themselves about in a remarkably -energetic way." Thus he considered these living objects to be animals, -from their motion, and this belief held sway for nearly two hundred -years. - -[Illustration: FIG. 1.--Leeuwenhoek's Microscope. A is the simple -bi-convex lens held firmly in place. In front of this is the small -table, B, with the support, C, on the tip of which the object to be -examined was held. This support could be brought nearer to or removed -further away from the lens and held firmly in place by the screw, D. -E is a second screw for raising or lowering the entire table. A concave -mirror that Leeuwenhoek sometimes used to focus more light on the -object under examination, is shown at the right.] - -Leeuwenhoek was a pure observer of facts and made no attempt at -speculation, but his discoveries soon started the theorists to -discussing the origin of these minute organisms. Most observers, as -was probably to be expected, believed that they arose spontaneously. -Needham, in 1749, described the development of microoerganisms -around grains of barley in water. Bonnet, in 1768, suggested that -probably Needham's animalcules came from ova in the liquid. The Abbot -Spallanzani, in 1769, called attention to the crudeness of Needham's -methods and later, in 1776, attempted to disprove spontaneous origin -by heating infusions of organic material in flasks and then _sealing_ -them. His critics raised the objections that heating the liquids -destroyed their ability to support life, and that sealing prevented -the access of fresh air which was also necessary. The first objection -was disproved by the accidental cracking of some of the flasks which -thereafter showed an abundant growth. This accident seemed also to -support the second objection, and Spallanzani did not answer it. Though -Spallanzani's experiments failed to convince his opponents, they led to -important practical results, since Francois Appert, in 1810, applied -them to the preserving of fruits, meats, etc., and in a sense started -the modern canning industry. - -[Illustration: FIG. 2.--The first drawings of bacteria by Leeuwenhoek. -The dotted line _C-D_ indicates the movement of the organism.] - -[Illustration: FIG. 3.--Schultze's experiment. The set of bulbs next to -the face contained KOH and the other set concentrated H{2}SO{4}. Air was -drawn through at frequent intervals from May until August but no growth -developed in the boiled infusion.] - -From Spallanzani to Schultze, there were no further experiments to -prove or disprove spontaneous generation. Schultze, in 1836, attempted -to meet the second objection to Spallanzani's experiment, _i.e._, -the exclusion of air, by drawing air through his boiled infusions, -first causing it to bubble through concentrated sulphuric acid to -kill the "germs" (Fig. 3.). His flasks fortunately showed no growths, -but his critics claimed that the strong acid changed the properties -of the air so that it would not support life. This experiment of -Schultze's, though devised for a different purpose, was really the -first _experiment_ in the use of _chemical disinfectants_, though -Thaer (page 31) had used chemicals in a practical way. Schwann, in -1837, modified this experiment, by drawing the air through a tube -heated to destroy the living germs (Fig. 4). His experiments were -successful but the "spontaneous generation" theorists raised the same -objection, _i.e._, the change in the air by heating. This was the first -_experiment_ in which the principle of "_dry heat_" or "_hot air_" -sterilization was used. Similar arguments were brought forward, also -to the use of _cotton plugs_ as filters by Schroeder and Dusch in 1859 -(Fig. 5). This was the first use of the principle of _sterilization by -filtration_. It remained for Chevreuil and Pasteur to overcome this -objection in 1861 by the use of flasks with long necks drawn out to a -point and bent over. These permitted a full access of air by diffusion -but kept out living germs, since these cannot fly but are carried -mechanically by air currents or fall of their own weight (Fig. 6.). -Hoffman, the year before (1860), had made similar experiments but these -remained unnoticed. The Pasteur flasks convinced most scientists that -"spontaneous generation" has never been observed by man, though some -few, notably Dr. Charlton Bastian, of England, vigorously supported the -theory from the early seventies until his death in November, 1915. - -[Illustration: FIG. 4.--Schwann's experiment. After boiling, as shown -in the diagram, and cooling, air was drawn into the flask by aspiration -while the coiled tube was kept hot with the flame.] - -[Illustration: FIG. 5.--Schroeder and Dusch's experiment. The -aspirating bottle drew the air through the flask after it had been -filtered by the cotton in the tube.] - -[Illustration: FIG. 6.--Pasteur's flask.] - -[Illustration: FIG. 7.--Tyndall's box. One side is removed to show -the construction. The bent tubes at the top are to permit a free -circulation of air into the interior. The window at the back has one -corresponding in the front (removed). Through these the beam of light -sent through from the lamp at the side was observed. The three tubes -received the infusion and were then boiled in an oil bath. The pipette -was for filling the tubes. (Popular Science Monthly, April, 1877.).] - -John Tyndall, in combating Bastian's views showed that boiled infusions -left open to the air in a closed box through which air circulated -did not show any growth of organisms provided the air was so free of -particles that the path of a ray of light sent through it from side to -side could not be seen (Fig. 7). Or if such sterilized infusions were -exposed to dust-free air, as in the high Alps, the majority showed -no growth, while all infusions in dusty air did show an abundance of -organisms. Tyndall's experiments confirmed those of Pasteur and his -predecessors and showed that the organisms developed from "germs" -present in the air falling into the liquids and not spontaneously. - -While Tyndall's experiments were of great value as indicated, they -probably were harmful in another way. These "germs in the air" were -considered by bacteriologists as well as laymen to include necessarily -many _disease germs_ and to indicate the very general, if not -universal, presence of these latter _in the air_. This idea led to many -erroneous practices in sanitation and disinfection which even to this -day are not eliminated. - - -CAUSATION OF DISEASE. - -The transmission of disease from person to person was recognized by the -ancients of European and Asiatic countries. Inoculation of smallpox was -practiced in China and India probably several thousand years ago and -was introduced by Lady Mary Wortley Montague into England in 1721, from -Constantinople. These beliefs and practices do not seem to have been -associated with any speculations or theories as to the cause of the -disease. - -Apparently the first writer on this subject was Varo, about B.C. 70, -who suggested that fevers in swampy places were due to invisible -organisms. The treatment of wounds during the thirteenth and fourteenth -centuries by hot wine fomentations and by the application of plasters -was based on the theory that the _air_ brought about conditions in the -wounds which led to suppuration. These practices were indeed primitive -antisepsis, yet were not based on a _germ theory_ of the conditions -which were partially prevented. Fracastorius (1484-1553), in a work -published in 1546, elaborated a theory of "disease germs" and "direct -and indirect contagion" very similar to modern views, though based -on no direct pathological knowledge. Nevertheless Kircher (mentioned -already) is usually given undeserved credit for the "contagium vivum" -theory. In 1657 by the use of simple lenses he observed "worms" in -decaying substances, in blood and in the pus from bubonic plague -patients (probably rouleaux of corpuscles in the blood, certainly not -bacteria in any case). Based on these observations and possibly also on -reading the work of Fracastorius, his theory of a "living cause" for -various diseases was published in 1671, but received little support. - -The discoveries of Leeuwenhoek which proved the existence of -microscopic organisms soon revived the "contagium vivum" idea of -Kircher. Nicolas Andry in a work published in 1701 upheld this view. -Lancisi in 1718 advanced the idea that "animalcules" were responsible -for malaria, a view not proved until Laveran discovered the malarial -parasite in 1880.[1] Physicians ascribed the plague which visited -Southern France in 1721 to the same cause, and many even went so far as -to attribute all disease to animalcules, which brought the theory into -ridicule. Nevertheless the "contagium vivum" theory survived, and even -Linnaeus in his _Systema Naturae_ (1753-6) recognized it by placing the -organisms of Leeuwenhoek, the contagia of diseases and the causes of -putrefaction and fermentation in one class called "Chaos." - -Plenciz, a prominent physician and professor in the Vienna Medical -School, published in 1762 a work in which he gave strong arguments for -the "living cause" theory for transmissable diseases. He taught that -the agent is evidently transmitted through the air and that there is -a certain period of incubation pointing to a multiplication within -the body. He also believed that there was a specific agent for each -disease. His writings attracted little attention at the time and the -"contagium vivum" theory seems to have been almost lost sight of for -more than fifty years. Indeed, Oznam, in 1820, said it was no use to -waste time in refuting hypotheses as to the animal nature of contagium. - -Isolated observers, were, however, keeping the idea alive, each in -his own locality. In 1787 Wollstein, of Vienna, showed that the pus -from horses with glanders could infect other horses if inoculated -into the skin. Abilgaard, of Copenhagen, made similar experiments at -about the same time. In 1797 Eric Viborg, a pupil of Abilgaard's, -published experiments in which he showed the infectious nature not -only of the pus but also of the nasal discharges, saliva, urine, etc., -of glandered horses. Jenner in 1795-98 introduced vaccination as a -method of preventing smallpox. This epoch-making discovery attracted -world wide attention and led to the overcoming of this scourge which -had devastated Europe for centuries, but contributed little or nothing -to the question of the causation of disease. Prevost's discovery of -the cause of grain rust (_Puccinia graminis_) in 1807 was the _first -instance of an infectious disease of plants_ shown to be _due to a -microscopic plant organism_, though not a bacterium in this case. - -Doubtless one reason why the work on glanders and grain rust attracted -little attention among the practitioners of human medicine was owing to -the prevalent belief in man's complete separation from all lower forms -of life. The evolutionists had not yet paved the way for experimental -medicine. - -In 1822 Gaspard showed the poisonous nature of material from infected -wounds by injecting it into animals and causing their death. Tiedemann -(1822), Peacock (1828) described "little bodies" in the muscles of -human cadavers which Hilton (1832) considered to be parasitic in -nature. Paget (1835) showed that these bodies were round worms and -Owen (1835) described them more accurately and gave the name _Trichina -spiralis_ to them. Leidy (1846) found organisms in the muscles of -hogs which he considered to be the same as Owen's Trichina and paved -the way for the work of Zenker (1860) in showing the pathological -relation between the Trichina of pork and human Trichinosis. Bearing -on the "contagium vivum" theory was the rediscovery of the "itch mite" -(_Sarcoptes scabiei_) by Renucci (1834), an Italian medical student. -This had been declared several hundred years before but had been lost -sight of. Chevreuil and Pasteur, in 1836, showed that putrefaction did -not occur in meat protected from contamination, and suggested that -wound infection probably resulted from entrance of germs from without. -Bassi, investigating a disease of silkworms in Italy, demonstrated -that a certain mold-like fungus (_Botrytis bassiana_) was the cause in -1837. This was the _first instance of a microscopic vegetable organism_ -proved to be capable of _causing disease in an animal_. - -Boehm, in 1838, observed minute organisms in the stools of cholera -patients and conjectured that they might have a causal connection -with the disease. Dubini of Milan in 1838 discovered the _Ankylostoma -duodenale_ which later was further described by Omodei in 1843 and -shown to be the cause of Egyptian chlorosis by Griesinger (1851). -The fungous nature of favus, a scalp disease, was recognized by -Schoenlein in 1839, and the organism was afterward called "_Achorion -schoenleinii_." Berg, in 1839-41, showed that thrush is likewise due to -a fungus, "_Oidium albicans_." - -These discoveries led Henle, in 1840, to publish a work in which -he maintained that all contagious diseases must be due to living -organisms, and to propound certain postulates (afterward restated -by Koch and now known as "Koch's postulates" p. 233) which must be -demonstrated before one can be sure that a given organism is the -specific cause of a given disease. The methods then in vogue and the -instruments of that period did not enable Henle to prove his claims, -but he must be given the credit for establishing the "contagium vivum" -theory on a good basis and pointing the way for men better equipped to -prove its soundness in after years. - -[Illustration: PLATE II - -SIR JOSEPH LISTER] - -In 1842-43 Gruby showed that Herpes tonsurans, a form of ringworm, is -due to the fungus _Trichophyton tonsurans_. Klencke, in 1843, produced -generalized tuberculosis in a rabbit by injecting tuberculous material -into a vein in the ear, but did not carry his researches further. -In 1843, Doctor Oliver Wendell Holmes wrote a paper in which he -contended that puerperal fever was contagious. Liebert identified the -_Peronospora infestans_ as the cause of one type of potato rot in 1845. -The skin disease Pityriasis (tinea) versicolor was shown to be due to -the _Microsporon furfur_ by Eichstedt in 1846. In 1847 Semmelweiss of -Vienna recommended disinfection of the hands with chloride of lime by -obstetricians because he believed with Holmes in the transmissibility -of puerperal fever through poisons carried in this way from the -dissecting room but his theories were ridiculed. - -[Illustration: PLATE III - -ROBERT KOCH] - -Pollender, in 1849, and Davaine and Rayer, in 1850, independently -observed small rod-like bodies in the blood of sheep and cattle -which had died of splenic fever (anthrax). That Egyptian chlorosis, -afterward identified with Old World "hookworm disease," is caused by -the _Ankylostoma duodenale_ was shown by Greisinger in 1851. In the -same year the _Schistosomum hematobium_ was shown to be the cause -of the "Bilharzia disease" by Bilharz. Kuechenmeister discovered the -tapeworm, _Taenia solium_, in 1852, Cohn, an infectious disease of -flies due to a parasitic fungus (_Empusa muscae_) in 1855, and Zenker -showed the connection between trichinosis of pork ("measly pork") -and human trichinosis (1860) as indicated above. The organisms just -mentioned are, of course, not bacteria, but these discoveries proved -conclusively that _living things of one kind or another, some large, -most of them microscopic, could cause disease in other organisms_ and -stimulated the search for other "living contagiums." In 1863 Davaine, -already mentioned, showed that anthrax could be transmitted from -animal to animal by inoculation of blood, but only if the blood -contained the minute rods which he believed to be the cause. Davaine -later abandoned this belief because he transmitted the disease with -old blood in which he could find no rods. It is now known that this -was because the bacilli were in the "spore" form which Davaine did not -recognize. He thus missed the definite proof of the bacterial nature -of anthrax because he was not familiar with the life history of the -organism which was worked out by Koch thirteen years later. In 1865 -Villemin repeatedly caused tuberculosis in rabbits by subcutaneous -injection of tuberculous material and showed that this disease must be -infectious also. In the same year Lord Lister introduced antiseptic -methods in surgery. He believed that wound infections were due to -microoerganisms getting in from the air, the surgeon's fingers, etc., -and without proving this, he used carbolic acid to kill these germs -and prevent the infection. His pioneer experiments made modern surgery -possible. In this year also, Pasteur was sent to investigate a disease, -Pebrine, which was destroying the silkworms in Southern France. He -showed the cause to be a protozoan which had been seen previously by -Cornalia and described by Naegeli under the name _Nosema bombycis_ and -devised preventive measures. This was the _first infectious disease_ -shown to be _due to a protozoan_. In 1866 Rindfleisch observed small -pin-point-like bodies in the heart muscle of persons who had died -of wound infection. Klebs, in 1870-71, published descriptions and -names of organisms he had found in the material from similar wounds, -though he did not establish their causal relation. Bollinger, in -1872, discovered the spores of anthrax and explained the persistence -of the disease in certain districts as due to the resistant spores. -In 1873 Obermeier observed in the blood of patients suffering from -recurrent fever long, flexible spiral organisms which have been named -_Spirochaeta obermeieri_. Loesch ascribed tropical dysentery to an ameba, -named by him _Amoeba coli_, in 1875. Finally, Koch, in 1876, isolated -the anthrax bacillus, worked out the life history of the organism -and reproduced the disease by the injection of pure cultures and -recovered the organism from the inoculated animals, thus establishing -beyond reasonable doubt its causal relationship to the disease. This -was the _first instance of a bacterium_ proved to be the cause of a -_disease in animals_. Pasteur, working on the disease at the same time, -confirmed all of Koch's findings, though his results were published -the next year, 1877. Bollinger determined that the _Actinomyces bovis_ -(_Streptothrix bovis_) is the cause of actinomycosis in cattle in -1877. Woronin in the same year discovered a protozoan (_Plasmodiophora -brassicae_) to be the cause of a disease in cabbage, the _first proved -instance of a unicellular animal causing a disease in a plant_. In 1878 -Koch published his researches on wound infection in which he showed -beyond question that microoerganisms are the cause of this condition, -though Pasteur in 1837, had suggested the same thing and Lister had -acted on the theory in preventing infection. - -These discoveries, especially those of Koch, immediately attracted -world-wide attention and stimulated a host of workers, so that within -the next ten years most of the bacteria which produce disease in -men and animals were isolated and described. It is well to remember -that the first _specific_ disease of man proved to be caused by a -_bacterium_ was _tuberculosis_, by Koch in 1882. - -Progress was greatly assisted by the introduction of anilin dyes as -suitable stains for organisms by Weigert in 1877, by Koch's application -of special technic and gelatin cultures for isolation and study, 1881, -and the great improvements in the microscope by Prof. Abbe, of Jena. - -Laveran's discovery of the malarial parasite in 1880 turned attention -to protozoa as the causes of disease and led to the discovery of the -various piroplasmoses and trypanosomiases in man and the lower animals. - -Pasteur's protective inoculations in chicken cholera and anthrax -directed attention to the possibility of using bacteria or their -products as a specific protective or curative means against particular -diseases. This finally led to the discovery of diphtheria antitoxin by -Behring, and independently by Roux, in 1890, a discovery which opened -up the wide field of immunity which is so persistently cultivated at -the present time. - -[Illustration: PLATE IV - -LOUIS PASTEUR] - -While the causation of disease by bacteria has probably attracted -most attention, especially in the popular mind, it should not be -forgotten that this is but one of the numerous ways in which these -organisms manifest their activities, and in a sense it is one of -their least-important ways, since other kinds are essential in many -industries (dairying, agriculture) and processes (sewage purification) -and are even _indispensable for the very existence of all green plants -and hence of animals, including man himself_. - - -PUTREFACTION AND FERMENTATION. - -The idea that there is a certain resemblance between some infectious -diseases and the processes of putrefaction and fermentation seems to -have originated during the discussion on spontaneous generation and -the "contagium vivum" theory which followed Leeuwenhoek's discoveries. -Plenciz (1762) appears to have first formulated this belief in writing. -He considered putrefaction to be due to the "animalcules" and said that -it occurred only when there was a coat of organisms on the material -and only when they increased and multiplied. Spallanzani's experiments -tended to support this view since his infusions did not "spoil" when -boiled and sealed. Appert's practical application of this idea has been -mentioned. - -Thaer, in his _Principles of Rational Agriculture_, published in the -first quarter of the nineteenth century, expressed the belief that the -"blue milk fermentation" was probably due to a kind of fungus that -gets in from the air, and stated that he had prevented it by treating -the milk cellars and vessels, with sulphur fumes or with "oxygenated -hydrochloric acid" (hypochlorous acid). - -In 1836 Chevreuil and Pasteur showed that putrefaction did not occur -in meat protected from contamination. In 1837 Caignard-Latour, in -France, and Schwann, in Germany, independently showed that alcoholic -fermentation in beer and wine is due to the growth of a microscopic -plant, the yeast, in the fermenting wort. C. J. Fuchs described the -organism which is commonly called the "blue milk bacillus" in 1841 and -conjectured that the souring of milk was probably bacterial in origin. -It remained for Pasteur to prove this in 1857. During the following -six or seven years Pasteur also proved that acetic acid fermentation, -as in vinegar making, butyric acid fermentation (odor of rancid butter -and old cheese) and the ammoniacal fermentation of urea, so noticeable -around stables, were each due to different species of bacteria. Pasteur -also, during the progress of this work, discovered the class of -organisms which can grow in the absence of free oxygen--the anaerobic -bacteria. There is no question that Pasteur from 1857 on did more to -lay the foundations of the science of bacteriology than any other -one man. Influenced by Pasteur's work von Hesseling, in 1866, stated -his belief that the process of cheese ripening, like the souring of -milk, was associated with the growth of fungi, and Martin also, in -1867, stated that cheese ripening was a process which was akin to -alcoholic, lactic and butyric fermentations. Kette, in 1869, asserted -the probability of Pasteur's researches furnishing a scientific basis -for many processes of change in the soil. In 1873 Schloesing and Muentz -showed that nitrification must be due to the action of microoerganisms, -though the discovery of the particular ones remained for Winogradsky -in 1889. Thus the belief that fermentation and putrefaction are due to -microoerganisms was as well established by the early eighties of the -last century as that similar organisms are the causes of infectious -diseases. - - -STUDY OF FORMS. - -An important part of the scientific knowledge of living organisms is -dependent on a study of their forms and relationships. As has been -stated, Leeuwenhoek considered bacteria to be "animalcules" because -they showed independent movement. But little attention was paid to -the natural history of these animalcules for nearly a hundred years -after Leeuwenhoek. During the last quarter of the eighteenth century, -however, workers busied themselves chiefly with the discovery and -description of new forms. Among these students were Baron Gleichen, -Jablot, Lesser, Reaumur, Hill and others. Mueller, of Copenhagen, in -1786 published the first attempt at classification, a most important -step in the study of these organisms. Mueller introduced the terms -Monas, Proteus and Vibrio, which are still in use. Ehrenberg, in his -work on _Infusoria_, or the organisms found in infusions, published -in 1838, introduced many generic names in use at present, but still -classed the bacteria with protozoa. Joseph Leidy, the American -naturalist, considered that the "vibrios" of previous writers were -plants and not "animalcules." He seems to have been the first to have -made this distinction (1849). Perty (1852) recognized the presence of -spores in some of his organisms. Ferdinand Cohn (1854) classed the -bacteria among plants. Naegeli (1857) proposed the name "Schizomycetes" -or "fission fungi," which is still retained for the entire class of -bacteria. Cohn in the years 1872-1875 established classification on -a modern basis and added greatly to the knowledge of morphology and -natural history of bacteria. He described spore formation and the -development of spores into active bacteria, and showed the close -relationships as well as differences between the bacteria and the lower -algae. Robert Koch was a pupil of Cohn. - -An examination of the accompanying chronological table will show how -the investigations and discoveries in connection with "spontaneous -generation," the "contagium vivum" theory and putrefaction and -fermentation must have been mutually suggestive: - - 1546. Fracastorius, disease germs theory and direct and indirect - contagion. - - 1671. Kircher, "contagium vivum" theory. - - 1675. Leeuwenhoek, first saw bacteria, "animalcules." - - 1701. Andry, "animalcules" cause of diseases. - - 1718. Lancisi, "animalcules" cause of malaria. - - 1749. Needham, described development of organisms in water around - barley grains. - - 1762. Plenciz, arguments for "living cause" theory and that - "animalcules" cause putrefaction. - - 1768. Bonnet, suggested that probably Needham's organisms came from - germs in the liquid. - - 1776. Spallanzani, boiled and sealed infusions. - - 1786. Mueller, first classified "animalcules." - - 1787. Wollstein, glanders pus infectious. - - 1795-1798. Jenner, vaccination against smallpox. - - 1797. Viborg, transmitted glanders repeatedly. - - 1807. Prevost, grain rust, _Puccinia graminis_. _The first instance - of a microscopic plant organism shown to be the cause of a disease in - a higher plant._ - - 1810. Appert, directions for "canning." - - 1822. Gaspard, infectiousness of material from wounds. - - 1834. Renucci, itch--itch mite (_Sarcoptes scabiei_). - - 1835. Paget and Owen, _Trichina spiralis_. - - 1836. Schultze, air through acid to kill "germs." - - 1837. Chevreuil and Pasteur, protected meat did not putrefy; - suggested wound infection due to entrance of germs from without. - - 1837. Caignard-Latour, Schwann, alcoholic fermentation--yeast. - - 1837. Schwann, air through heated tubes to kill germs. - - 1837. Bassi, muscardine of silkworms, _Botrytis bassiana_. _The first - instance of a microscopic plant organism shown to be the cause of a - disease in an animal._ - - 1838. Boehm, cholera, saw organisms in stools (not the cause). - - 1838. Dubini discovered _Ankylostoma duodenale_. - - 1838. Ehrenberg, study of forms. - - 1839. Schoenlein, Favus, _Achorion schoenleinii_. - - 1839-41. Berg, Thrush, _Oidium albicans_. - - 1840. Henle, theory of contagious diseases. - - 1841. Fuchs, bacterial cause of blue milk. - - 1842-43. Gruby, Herpes tonsurans, _Trichophyton tonsurans_. - - 1843. Klencke, inoculations of tuberculous material into rabbit. - - 1843. Holmes, puerperal fever contagious. - - 1845. Liebert, a potato rot, _Peronospora infestans_. - - 1846. Leidy, Joseph (American Naturalist), _Trichina spiralis_ in - pork. - - 1846. Eichstedt, Pityriasis versicolor, _Microsporon furfur_. - - 1847. Semmelweiss, recommended disinfection to prevent puerperal - fever. Not followed. - - 1849. Leidy, considered "vibrios" to be plants. - - 1849. Pollender, Anthrax, saw rods in blood. - - 1850. Davaine and Rayer, Anthrax, saw rods in blood. - - 1851. Griesinger, Egyptian chlorosis, _Ankylostoma duodenale_. - - 1851. Bilharz, Bilharzia disease, _Schistosomum hematobium_. - - 1852. Kueckenmeister, tapeworm, _Taenia solium_. - - 1852. Perty, saw spores in bacteria. - - 1854. Cohn, classed bacteria as plants. - - 1855. Cohn, disease of flies, _Empusa muscae_. - - 1857. Naegeli, named bacteria, Schizomycetes. - - 1857. Pasteur, lactic, acetic, butyric acid fermentation. - - 1860. Zenker, Trichinosis, _Trichinella spiralis_. - - 1861. Pasteur, disproof of spontaneous generation. - - 1863. Davaine, transmitted anthrax by blood injections. - - 1865. Pasteur, Pebrine of silkworms, _Nosema bombycis_. _The first - instance of a protozoan shown to be the cause of a disease in a - higher animal._ - - 1865. Villemin, repeatedly transmitted tuberculosis to rabbits. - - 1865. Lister, introduced antisepsis in surgery. - - 1860. Rindfleisch, Pyemia, organisms in the pus. - - 1866. Von Hesseling, cheese ripening. - - 1867. De Martin, cheese ripening akin to alcoholic fermentation. - - 1869. Kette, Pasteur's researches scientific basis for many processes - in the soil. - - 1871. Klebs, Pyemia, organisms in the pus. - - 1872. Bollinger, spores in anthrax. - - 1872-75. Cohn, definite classification. - - 1873. Obermeier, recurrent fever, _Spirochaeta obermeieri_. - - 1873. Schloesing and Muenz, nitrification due to organisms. - - 1875. Loesch, amebic dysentery, _Amoeba coli_. - - 1875-76. Tyndall, germs in the air. - - 1876. Robert Koch, anthrax, _Bacillus anthracis_. _The first instance - of a bacterium shown to be the cause of disease in an animal._ - - 1877. Bollinger, actinomycosis, _Actinomyces bovis_ (_Streptothrix - bovis_). - - 1877. Weigert, used anilin dyes for staining. - - 1877. Woronin, cabbage disease, _Plasmodiophora brassicae_. _The first - instance of a protozoan shown to be the cause of a disease in a - plant._ - - 1878. Koch, wound infections, bacterial in origin. - - 1881. Koch, gelatin plate cultures, Abbe, improvements in the - microscope. - - - - -CHAPTER I. - -POSITION--RELATIONSHIPS. - - -Bacteria are considered to belong to the plant kingdom not because of -any one character they possess, but because they most nearly resemble -organisms which are generally recognized as plants. While it is not -difficult to distinguish between the higher plants and higher animals, -it becomes almost, if not quite, impossible to separate the lowest, -forms of life. It is only by the method of resemblances above mentioned -that a decision is finally reached. It has even been proposed to make a -third class of organisms neither plants nor animals but midway between -in which the bacteria are included, but such a classification has not -as yet been adopted. - -In many respects the bacteria are most nearly related to the lowest -_algae_, since both are unicellular organisms, both reproduce by -transverse division and the forms of the cell are strikingly similar. -The bacteria differ in one important respect, that is, they do not -contain _chlorophyl_, the green coloring matter which enables all -plants possessing it to absorb and break up carbon dioxide in the -light, and hence belong among the fungi. Bacteria average much smaller -than even the smallest algae. - -Bacteria are closely connected with the _fission yeasts_ and the -_yeasts_ and _torulae_. All are unicellular and without chlorophyl. The -bacteria, as has been stated, reproduce by division but the others -characteristically by budding or gemmation, though the fission yeasts -also by division. - -There is a certain resemblance to the _molds_ in their absence -of chlorophyl. But the molds grow as branching threads and also -have special fruiting organs for producing spores as a means of -reproduction, neither of which characteristics is found among the -_true_ bacteria. The higher thread bacteria do show true branching -and rudimentary fruiting bodies (Streptothrix) and appear to be a link -connecting the true bacteria and the molds. - -[Illustration: FIG. 8.--A thread of blue-green algae.] - -[Illustration: FIG. 9.--A thread of small blue-green algae.] - -[Illustration: FIG. 10.--A thread of bacteria. Compare with Figs. 8 -and 9.] - -[Illustration: FIG. 11.--A chain of spherical blue-green algae.] - -[Illustration: FIG. 12.--A chain of spherical bacteria.] - -[Illustration: FIG. 13.--A pair of spherical blue-green algae.] - -Further the _chemical composition_ of bacteria is more like that of -other fungous plants than of any of the forms classed as animals. - -[Illustration: FIG. 14.--Spherical bacteria. Several pairs are shown.] - -[Illustration: FIG. 15.--Yeast cells. Some show typical budding.] - -The food of bacteria is always taken up in solution by diffusion -through the outer covering of the cell as it is in all plants. Plant -cells never surround and engulf particles of solid food and digest them -within the cell as many single-celled animals do, and as the leukocytes -and similar ameboid cells in practically all multicelled animals do.[2] - -[Illustration: FIG. 16.--A portion of the mycelium of a mold. Note the -large size and the branching.] - -One of the most marked differences between animals and plants is with -respect to their energy relationships. Plants are characteristically -storers of energy while animals are liberators of it. Some bacteria -which have the power of swimming in a liquid certainly liberate -relatively large amounts of energy, and in the changes which bacteria -bring about in the material which they use as food considerable heat is -evolved ("heating" of manure, etc.). Nevertheless the evidence is good -that the bacteria as a class store much more of the energy contained -in the substances actually taken into the body cell as food than is -liberated in any form. - -Bacteria do show some resemblance to the protozoa, or single-celled -animal forms, in that the individuals of each group consist of one cell -only and some bacteria have the power of independent motion from place -to place in a liquid as most "infusoria" do, but here the resemblance -ceases. - -Bacteria are among the smallest of organisms, so small that it requires -the highest powers of the microscope for their successful study, and -the use of a special unit for their measurement. This unit is the -one-thousandth part of a millimeter and is called the micro-millimeter -or micron. Its symbol is the Greek letter _mu_ ( mu). - -The size varies widely among different kinds but is fairly constant in -the same kind. The smallest described form is said to be only 0.18 mu -long by 0.06 mu thick and is just visible with the highest power of the -microscope, though it is possible and even probable that there are -forms still smaller which cannot be seen. Some large rare forms may -measure 40 mu in length, but the vast majority are from 1 mu to 4 mu or 5 mu -long, and from one-third to one-half as wide. - -From the above description a bacterium might be said to be a -_microscopic, unicellular plant, without chlorophyl, which reproduces -by dividing transversely_. - - - - -PART I. - -MORPHOLOGY - - - - -CHAPTER II. - -CELL STRUCTURES. - - -The _essential_ structures which may by appropriate means be -distinguished in the bacterial cell are _cell wall_ and _cell -contents_, technically termed _protoplasm_, cytoplasm. The cell wall is -not so dense, relatively, as that of green plants, but is thicker than -the outer covering of protozoa. It is very similar to the cell wall -of other lower fungi. Diffusion takes place readily through it with -very little selective action on substances absorbed as judged by the -comparative composition of bacteria and their surrounding medium. - -=Cytoplasm.=--The cytoplasm according to Buetschli and others is -somewhat different and slightly denser in its outer portion next to the -cell wall. This layer is designated the _ectoplasm_, as distinguished -from the remainder of the cell contents, the _endoplasm_. When bacteria -are suddenly transferred from a given medium into one of decidedly -_greater_ density, there sometimes results a contraction of the -_endoplasm_, due to the rapid diffusion of water. This phenomenon is -designated _plasmolysis_ (Fig. 17), and is similar to what occurs in -the cells of higher plants when subjected to the same treatment. This -is one of the methods which may be used to show the different parts of -the cell just described. - -If bacteria are suddenly transferred from a relatively dense medium -to one which is of decidedly _less_ density, it occasionally happens -that water diffuses into the cell and swells up the endoplasm so much -more rapidly than the cell wall that the latter ruptures and some of -the endoplasm exudes in the form of droplets on the surface of the cell -wall. This phenomenon is called _plasmoptysis_. Students will seldom -observe the distinction between cell wall and cell contents, except -that in examining living bacteria the outer portion appears more highly -refractive. This is chiefly due to the presence of a cell wall, but is -not a proof of its existence. - -[Illustration: FIG. 17.--Cells of bacteria showing plasmolysis. The -cell substance of three of the cells in the middle of the chain has -shrunk until it appears as a round black mass. The cell wall shows as -the lighter area.] - -[Illustration: FIG. 18.--Vacuoles in the bacterial cell. The lighter -areas are vacuoles.] - -=Nucleus.=--Douglas and Distaso[3] summarize the various opinions with -regard to the nucleus in bacteria as follows: - -1. Those who do not admit, the presence of a nucleus or of anything -equivalent to it. (Fischer, Migula, Massart). - -2. Those who consider that the entire bacterial cell is the equivalent -of a nucleus and contains no protoplasm. (Ruzicka). - -3. Those who admit the presence of nuclein but say that this is not -morphologically differentiated from the protoplasm as a nucleus. -(Weigert). - -4. Those who consider the bacterial protoplasm to consist of a central -endoplasm throughout which the nuclein is diffused and an external -layer of ectoplasm next to the cell wall. (Buetschli, Zettnow). - -5. Those who say that the bacterial cell contains a distinct nucleus, -at least in most instances. These authors base their claims on staining -with a Giemsa stain. (Feinberg, Ziemann, Neuvel, Dobell, Douglass and -Distaso). - -That nucleoproteins are present in the bacterial cell in relatively -large amounts is well established. Also that there are other proteins -and that the protoplasm is not all nuclein. - -Some workers as noted above have been able to demonstrate collections -of nuclein by staining, especially in very young cells. In older cells -this material is in most instances diffused throughout the protoplasm -and can not be so differentiated. - -The following statement probably represents the generally accepted view -at the present time: - -A nucleus _as such_ is not present in bacterial cells, except in a few -large rare forms and in very young cells. _Nuclein_, the characteristic -chemical substance in nuclei, which when aggregated forms the nucleus, -is scattered throughout the cell contents and thus intimately mingled -with the protoplasm, and cannot be differentiated by staining as in -most cells. - -The close association of nuclein and protoplasm may explain the rapid -rate of division of bacteria (Chapter VIII, p. 91). - -The chemical composition of the bacterial cell is discussed in Chapter -VII. - -In addition to the _essential_ parts just described the bacterial cell -may show some of the following _accidental_ structures: _vacuoles_, -_capsules_, _metachromatic granules_, _flagella_, _spores_. - -=Vacuoles.=--_Vacuoles_ appear as clear spaces in the protoplasm when -the organism is examined in the living condition or when stained very -slightly (Fig. 18). During life these are filled with liquid or gaseous -material which is sometimes waste, sometimes reserve food, sometimes -digestive fluids. Students are apt to confuse vacuoles with spores (p. -47). Staining is the surest way to differentiate (Chapter XIX, p. 209). -If vacuoles have any special function, it is an unimportant one. - -[Illustration: FIG. 19.--Bacteria seen within capsules.] - -[Illustration: FIG. 20.--Metachromatic granules in bacteria. The dark -round spots are the granules. The cells of the bacteria are scarcely -visible.] - -=Capsule.=--The _capsule_ is a second covering outside the cell wall -and probably developed from it (Fig. 19). It is usually gelatinous, -so that bacteria which form capsules frequently stick together -when growing in a fluid, so that the whole mass has a jelly-like -consistency. The term _zooegloea_ was formerly applied to such masses, -but it is a poor term and misleading (zooen = an animal) and should -be dropped. The masses of jelly-like material frequently found on -decaying wood, especially in rainy weather, are in some cases masses -of capsule-forming bacteria, though a part of the jelly is a product -of bacterial activity, a gum-like substance which lies among the -capsulated organisms. When these masses dry out, they become tough -and leathery, but it is not to be presumed that capsules are of this -consistency. On the contrary, they are soft and delicate, though they -certainly serve as an additional protection to the organism, doubtless -more by selective absorption than mechanically. Certain bacteria -which cause disease form capsules in the blood of those animals which -they kill and not in the blood of those in which they have no effect -(_Bacterium anthracis_ in guinea pig's blood and in rat's blood). The -presence of capsules around an organism can be proved only by staining -the capsule. Many bacteria when stained in albuminous fluids show a -clear space around them which appears like a capsule. It is due to the -contraction of the fluid away from the organism during drying. - -=Metachromatic Granules.=--The term "_metachromatic_" is applied to -granules which in stained preparations take a color different from -the protoplasm as a whole (Fig. 20). They vary widely in chemical -composition. Some of them are glycogen, some fat droplets. Others are -so-called "granulose" closely related to starch but probably not true -starch. Others are probably nuclein. Of many the chemical composition -is unknown. They are called "Babes-Ernst corpuscles" in certain -bacteria (typhoid bacillus). Since they frequently occur in the ends -of cells the term "polar granules" is also applied. Their presence is -of value in the recognition of but few bacteria ("Neisser granules" in -diphtheria). - -=Flagellum.=--A _flagellum_ is a very minute thread-like process -growing out from the cell wall, probably filled with a strand of -protoplasm. The vibrations of the flagella move the organism through -the liquid medium. Bacteria which are thus capable of independent -movement are spoken of as "motile bacteria." The actual rate of -movement is very slight, though in proportion to the size of the -organism it may be considered rapid. Thus Alfred Fischer determined -that some organisms have a speed for short periods of about 40 cm. per -hour. This is equivalent to a man moving more than 200 miles in the -same time. - -It is obvious that bacteria which can move about in a liquid have an -advantage in obtaining food, since they do not need to wait for it to -be brought to them. This advantage is probably slight. - -[Illustration: FIG. 21.--A bacterium showing a single flagellum at the -end--monotrichic.] - -[Illustration: FIG. 22.--A bacterium showing a bundle of four flagella -at the end--lophotrichic.] - -An organism may have only one flagellum at the end. It is then said -to be monotrichic (Fig. 21) (#monos# = alone, single; #trichos# = hair). -This is most commonly at the front end, so that the bacterium is drawn -through the liquid by its motion. Rarely it is at the rear end. Other -bacteria may possess a bundle of flagella at one end and are called -_lophotrichic_ (Fig. 22) (#lophos# = tuft). Sometimes at approaching -division the flagella may be at both ends and are then _amphitrichic_ -(Fig. 23) (#amphi# = both). It is probable that this condition does not -persist long, but represents the development of flagella at one end -of each of a pair resulting from division of an organism which has -flagella at one end only. In many bacteria the flagella arise from -all parts of the surface of the cell. Such bacteria are _peritrichic_ -(Fig. 24) (#peri# = around). The position and even the number of the -flagella are very constant for each kind and are of decided value in -identification. - -[Illustration: FIG. 23.--A bacterium showing flagella at each -end--amphitrichic.] - -[Illustration: FIG. 24.--A bacterium showing flagella all -around--peritrichic.] - -Flagella are too fine and delicate to be seen on the living organism, -or even on bacteria which have been colored by the ordinary stains. -They are rendered visible only by certain methods which cause a -precipitate on both bacteria and flagella which are thereby made thick -enough to be seen (Chapter XIX, p. 210). The movement of liquid around -a bacterium caused by vibrations of flagella can sometimes be observed -with large forms and the use of "dark-field" illumination. - -Flagella are very delicate and easily broken off from the cell body. -Slight changes in the density or reaction of the medium frequently -cause this breaking off, so that preparations made from actively motile -bacteria frequently show no flagella. For this reason and also on -account of their fineness the demonstration of flagella is not easy, -and it is not safe to say that a non-motile bacterium has no flagella -except after very careful study. - -The motion of bacteria is characteristic and a little practice in -observing will enable the student to recognize it and distinguish -between motility and "Brownian" or molecular motion. Dead and -non-motile bacteria show the latter. In fact, any finely divided -particles suspended in a liquid which is not too viscous and in which -the particles are not soluble show Brownian motion or "pedesis." This -latter is a dancing motion of the particle within a very small area -and without change of place, while motile bacteria move from place to -place or even out of the field of the microscope with greater or less -speed. There is a marked difference in the character of the motion of -different kinds of bacteria. Some rotate around the long axis when -moving, others vibrate from side to side. - -Among the higher thread bacteria there are some which show motility -without possessing flagella. Just how they move is little understood. - -=Spores.=--Under certain conditions some bacterial cells undergo -transformations which result in the formation of so-called _spores_. -If the process is followed under the microscope, the changes observed -are approximately these: A very minute point appears in the protoplasm -which seems to act somewhat like the centrosome of higher cells as a -"center of attraction" so that the protoplasm gradually collects around -it. The spot disappears or is enclosed in the collected protoplasm. -This has evidently become denser as it is more highly refractive than -before. In time all or nearly all of the protoplasm is collected. A new -cell wall is developed around it which is thicker than the cell wall of -the bacterium. This thickened cell wall is called the "spore capsule." -Gradually the remnants of the former cell contents and the old cell -wall disappear or dissolve and the spore becomes "free" (Fig. 25). - -[Illustration: FIG. 25.--The smaller oval bodies in the middle of the -field are free spores.] - -If the spore is placed in favorable conditions the protoplasm absorbs -water, swells, the capsule bursts at some point, a cell wall is formed -and the bacterium grows to normal size and divides, that is, it is an -active growing cell again. This process is called "germination" of the -spore. The point at which the spore capsule bursts to permit the new -cell to emerge is characteristic for each kind of bacterium. It may be -at the end when the germination is said to be _polar_ (Fig. 26). It may -be from the middle of one side which gives _equatorial_ germination -(Fig. 27). Rarely it is diagonally from a point between the equator and -the pole, which type may be styled _oblique_ germination. In one or -two instances the entire spore swells up, lengthens and becomes a rod -without any special germination unless this type might be designated -_bi-polar_. - -[Illustration: FIG. 26.--Spores showing polar germination. The lighter -part of the two organisms just below A and B is the developing -bacterium. In the original slide the spore was stained red and the -developing bacterium a faint blue.] - -[Illustration: FIG. 27.--A spore showing equatorial germination. -The spore in the center of the field shows a rod growing out of it -laterally. In the original slide the spore was stained red and the -developing bacterium blue.] - -[Illustration: FIG. 28.--Spores in the middle of the rod without -enlargement of the rod. The lighter areas in the rods are spores.] - -[Illustration: FIG. 29.--Spores in the middle of the rod with -enlargement of the rod around them. The lighter areas in the rods are -spores.] - -Spores are most commonly oval or elliptical in shape, though sometimes -spherical. A spore may be formed in the middle of the organism without -(Fig. 28) or with (Fig. 29) a change in size of the cell around it. -If the diameter through the cell is increased, then the cell with -the contained spore becomes spindle-shaped. Such a cell is termed a -"_clostridium_." Sometimes the spore develops in the end of the cell -either without (Fig. 30) or with enlarging it (Fig. 31). In a few -forms the spore is placed at the end of the rod and shows a marked -enlargement. This is spoken of as the "_plectridium_" or more commonly -the "drumstick spore" (Fig. 32). The position and shape of the spore -are constant for each kind of bacteria. In one or two instances only, -two spores have been observed in a single organism. - -[Illustration: FIG. 30.--Spores in the end of the rod with no -enlargement of the rod around them. The lighter areas in the rods are -spores.] - -[Illustration: FIG. 31.--Spores in the end of the rod with enlargement -of the rod, _A_, _A_, _A_, _A_.] - -[Illustration: FIG. 32.--Drumstick spores at the end of the rod.] - -The fact that the protoplasm is denser and the spore capsule thicker -(the percentage of water in each is decidedly less than in the growing -cell) gives the spore the property of much greater resistance to all -destructive agencies than the active bacterium has. For example, all -actively growing cells are destroyed by boiling in a very few minutes, -while some spores require several hours' boiling. The same relation -holds with regard to drying, the action of chemicals, light, etc. That -the coagulation temperature of a protein varies inversely with the -amount of water, it contains, is shown by the following table from -Frost and McCampbell, "General Bacteriology": - - Egg albumin plus 50 per cent. water coagulates at 56 deg. - " " " 25 per cent. " " " 74-80 deg. - " " " 18 per cent. " " " 88-90 deg. - " " " 6 per cent. " " " 145 deg. - " " dry " " " 160-170 deg. - -This resistance explains why it happens that food materials boiled -and sealed in cans to prevent the entrance of organisms sometimes -spoil. The spores have not been killed by the boiling. It explains -also in part the persistence of some diseases like anthrax and black -leg in pastures for years. From the above description it follows that -the spore is to be considered as _a condensation of the bacterial -protoplasm surrounded by an especially thick cell wall_. _Its function -is the preservation of the organism under adverse conditions._ It -corresponds most closely to the encystment of certain protozoa--the -ameba for example. Possibly the spore represents a very rudimentary -beginning of a reproductive function such as is gradually evolved in -the higher thread bacteria, the fission yeasts, the yeasts, the molds, -etc. Its characteristics are so markedly different, however, that the -function of preservation is certainly the main one. - -It must not be supposed that spores are formed under adverse conditions -only, because bacteria showing vigorous growth frequently form spores -rapidly. Special conditions are necessary for their formation just as -they are for the growth and other functions of bacteria (Chapters VI -and VII). - - - - -CHAPTER III. - -CELL FORMS. - - -Though there is apparently a wide variation in the shapes of different -bacterial cells, these may all be reduced to _three_ typical _cell -forms_. These are: first and simplest, the round or _spherical_, -typified by a ball and called the _coccus_ form, or _coccus_, plural -cocci[4] (Fig. 33). The coccus may be large, that is, from 1.5 mu to 2 mu -in diameter. The term _macrococcus_ is sometimes applied to these large -cocci. If the _coccus_ is less than 1 mu in diameter, it is sometimes -spoken of as a _micrococcus_; in fact, this term is very commonly -applied to any coccus. When cocci are growing together, many of the -cells do not appear as true spheres but are more or less distorted -from pressure of their neighbors or from failure to grow to full size -after recent division. Most cocci divide into hemispheres and then each -half grows to full size. A few cocci elongate before division and then -appear oval or elliptical. - -The second cell form is that of a _cylinder_ or rod typified by a -section of a lead-pencil. The name _bacillus_, plural _bacilli_, is -applied to this type (Fig. 34). The bacillus may be short (Fig. 35), -1 mu or less in length, or long, up to 40 mu in rare cases. Most bacilli -are from 2 mu to 5 mu or 6 mu long. The ends of the rod are usually rounded, -occasionally square and very rarely pointed. It is evident that a very -short rod with rounded ends approaches a coccus in form and it is not -always easy to differentiate in such cases. Most bacilli are straight, -but some are slightly curved (Fig. 36). - -The third cell form is the _spiral_, typified by a section of a -cork-screw and named _spirillum_, plural _spirilla_ (Fig. 37). A very -short spiral consisting of only a portion of a turn is sometimes called -_vibrio_ (Fig. 38). Vibrios when seen under the microscope look like -short curved rods. The distinction between the two can be made only by -examining the organism alive and moving in a liquid. The vibrio shows a -characteristic spiral twisting motion. Very long, flexible spirals are -usually named _spirochetes_ (Fig. 39). The spirochetes are motile but -flagella have not been shown to be present. - -[Illustration: FIG. 33.--Cocci.] - -[Illustration: FIG. 34.--Bacilli.] - -[Illustration: FIG. 35.--Short bacilli.] - -[Illustration: FIG. 36.--Curved bacilli. Only the one in the center of -the field is in focus. The others curve out of focus.] - -Besides the three typical cell forms bacteria frequently show -very great irregularities in shape. They may be pointed, bulged, -club-shaped or even slightly branched. These peculiar and bizarre -forms practically always occur when some of the necessary conditions -for normal growth, discussed in Chapters VI and VII, are not fulfilled. -They are best regarded as _involution_ or _degeneration_ forms for this -reason (Fig. 40). In a very few cases it is not possible to obtain the -organism without these forms (the diphtheria group). It is probable -that these cell forms are normal in such cases, or else conditions -suitable for the normal growth have not been obtained. - -[Illustration: FIG. 37.--Spirilla.] - -[Illustration: FIG. 38.--Vibrio forms of spirilla. Compare with Fig. -36.] - -[Illustration: FIG. 39.--Spirochetes.] - -[Illustration: FIG. 40.--Involution forms. The organisms are tapering -and branched at one end.] - - - - -CHAPTER IV. - -CELL GROUPINGS. - - -It has been stated that bacteria reproduce by transverse division, that -is, division across the long axis. Following repeated divisions the new -cells may or may not remain attached. In the latter case the bacteria -occur as separate isolated individuals. In the former, arrangements -characteristic of the particular organism almost invariably result. -These arrangements are best described as _cell groupings_ or _growth -forms_. - -[Illustration: FIG. 41.--Streptospirillum grouping.] - -[Illustration: FIG. 42.--Diplobacillus grouping.] - -In the case of spiral forms it is obvious that there is only one -possible grouping, that is, in chains of two or more individuals -adherent end to end. A chain of two spirilla might be called -a _diplospirillum_ (#diplos# = double); of three or more, a -_streptospirillum_ (#streptos# = necklace, chain) (Fig. 41). These terms -are rarely used, since spirilla do not ordinarily remain attached. -Likewise the bacillus can grow only in chains of two or more, and -the terms _diplobacillus_ (Fig. 42), bacilli in groups of two, and -_streptobacillus_ (Fig. 43), bacilli in chains are frequently used. -Still the terms _thread_, _filament_, or _chain_ are more common for -_streptobacillus_. - -[Illustration: FIG. 43.--Streptobacillus grouping.] - -[Illustration: FIG. 44.--Typical diplococcus grouping. Note that the -individual cocci are flattened on the apposing sides.] - -[Illustration: FIG. 45.--Long streptococcus grouping.] - -[Illustration: FIG. 46.--Short streptococcus grouping.] - -Since the coccus is spherical, _transverse_ division may occur in any -direction, though in three planes only at right angles to each other. -Division might occur in _one plane only_ as in spirilla and bacilli, -or in _two planes only_ or in _all three planes_. As a matter of fact -these three methods of division are found among the cocci, but only one -method for each particular kind of coccus. As a result there may be a -variety of cell groupings among the cocci. When division occurs in one -plane only, the possible groupings are the same as among the spirilla -or bacilli. The cocci may occur in groups of two--_diplococcus_ -grouping (Fig. 44), or in chains--_streptococcus_ grouping (Figs. 45 -and 46). When the grouping is in _diplococci_, the individual cocci -most commonly appear as hemispheres with the plane surfaces apposed -(Fig. 44). Sometimes they appear as spheres and occasionally are even -somewhat elongated. The individuals in a streptococcus grouping are -most commonly elongated, either in the same direction as the length of -the chain, or at right angles to it. The latter appearance is probably -due to failure to enlarge completely after division. Streptococci -frequently appear as chains of diplococci, that is, the pair resulting -from the division of a single coccus remain a little closer to each -other than to neighboring cells, as a close inspection of Fig. 45 will -show. - -If division occurs in _two planes only_, there may result the above -groupings and several others in addition. The four cocci which result -from a single division may remain together, giving the _tetracoccus_ -or _tetrad_ grouping. Very rarely all the cocci divide evenly and the -result is a regular _rectangular flat mass_ of cells, the total number -of which is a multiple of four. The term merismopedia (from a genus of -algae which grows the same way) is applied to such a grouping. If the -cells within a group after a few divisions do not reproduce so rapidly -(lack of food), as usually happens, the number of cells becomes uneven -or at least not necessarily a multiple of four and the resultant _flat -mass_ has an _irregular_, _uneven outline_. This grouping is termed -_staphylococcus_ (#staphylos# = a bunch of grapes) (Fig. 47). It is the -most common grouping among the cocci. - -When division occurs in all three planes, there is in addition to all -the groupings possible to one- and two-plane division a third grouping -in which the cells are in _solid packets_, _multiples of eight_. The -name _sarcina_ is applied to this growth form (Fig. 48). The individual -cells in a sarcina packet never show the typical coccus form so long as -they remain together, but are always flattened on two or more sides. - -The above descriptions indicate how the method of division may be -determined. If in examining a preparation the _sarcina_ grouping -appears, that shows _three-plane division_. If there are no sarcina, -but _tetrads_ or _staphylococci_ (rarely merismopedia), then the -division is in _two planes_. If none of the foregoing is observed but -only _diplo-_ or _streptococci_, these indicate _one-plane division_ -only. Cocci show their _characteristic_ groupings only when grown in a -liquid medium, and such should always be used before deciding on the -plane of division. - -[Illustration: FIG. 47.--Staphylococcus grouping. The large flat masses -are staphylococcus grouping. Diplococcus grouping, tetrads and short -streptococci are also evident.] - -[Illustration: FIG. 48.--Sarcina grouping.] - -As the above description shows, these terms which are properly -adjectives describing the cell grouping, are quite generally used as -nouns. Thus the terms a diplococcus, a tetrad, a streptococcus, etc., -are common, meaning a bacterium of the cell form and cell grouping -indicated. - - CELL FORM. CELL GROUPING. - - coccus-- {diplococcus--in 2's. - round or spherical. {streptoccus--in chains. - {tetracoccus, tetrads--in 4's. - {staphylococcus--irregular flat masses. - {sarcina--regular, solid packets, multiples of 8. - - bacillus-- {diplobacillus--in 2's. - rod-shaped {streptobacillus--in chains. - or cylindrical. - - spirillum-- {diplospirillum--in 2's, little used. - spiral-shaped. {streptospirillum--in chains, little used. - - - - -CHAPTER V. - -CLASSIFICATION. - - -The arrangement of living organisms in groups according to their -resemblances and the adoption of _fixed names_ is of the greatest -advantage in their scientific study. For animal forms and for the -higher plants this classification is gradually becoming standardized -through the International Congress of Zooelogists and of Botanists -respectively. Unfortunately, the naming of the bacteria has not as -yet been taken up by the latter body, though announced as one of the -subjects for the Congress of 1916 (postponed on account of the war). -Hence there is at present no system which can be regarded as either -fixed or official. - -[Illustration: FIG. 49.--Illustrates the genus Streptococcus. Typical -chains, no staphylococcus grouping, no sarcina grouping, no flagella.] - -[Illustration: FIG. 50.--Illustrates the genus Micrococcus. -Diplococcus, tetrads short chains and staphylococcus; no sarcina, no -flagella.] - -[Illustration: FIG. 51.--Illustrates the genus Sarcina. Sarcina -grouping, no flagella.] - -[Illustration: FIG. 52.--Illustrates the genus Bacillus. A bacillus -with peritrichic flagella. (Student preparation.)] - -Since Mueller's first classification of "animalcules" in 1786 numerous -attempts have been made to solve the problem. Only those beginning with -Ferdinand Cohn (1872-75) are of any real value. As long as bacteria -are regarded as plants it appears that the logical method is to follow -the well-established botanical principles in any system for naming -them. Botanists depend on morphological features almost entirely in -making their distinctions. The preceding chapters have shown that -the minute plants which are discussed have very few such features. -They are, to recapitulate, _cell wall_, _protoplasm_, _vacuoles_, -_metachromatic granules_, _capsules_, _flagella_, _spores_, _cell -forms_ and _cell groupings_. Most bacteria show not more than three -or four of these features, so that it is impossible by the aid of -morphology alone to distinguish from each other the large number of -different kinds which certainly exist. In the various systems which -are conceded to be the best these characteristics do serve to classify -them down to genera, leaving the "species" to be determined from their -_physiological_ activities. One of these systems was adopted by the -laboratory section of the American Public Health Association and by the -Society of American Bacteriologists and was practically the standard -in this country until superseded by the Society's own classification. -It is that of the German Bacteriologist Migula and is given below for -comparison. Since practically the entire discussion in this book is -concerned with the first three families the generic characteristics -in these only will be given. The full classification as well as a -thorough discussion of this subject is given in Lafar's _Handbuch_, -whence the following is adopted: - -[Illustration: FIG. 53.--Illustrates the genus Pseudomonas. A bacillus -with flagella at the end only.] - -[Illustration: FIG. 54.--Illustrates the genus Microspira. It is -(though the photograph does not prove it) a short spiral with one -flagellum at the end.] - -[Illustration: FIG. 55.--Illustrates the genus Spirillum. Spiral -bacteria with more than three, in this case four, flagella at the end.] - -[Illustration: FIG. 56.--Illustrates the genus Spirochaeta.] - -[Illustration: FIG. 57.--Illustrates the genus Chlamydothrix. Fine -threads with a delicate sheath.] - -[Illustration: FIG. 58.--Illustrates the genus Crenothrix. The -thickness of the cell walls is due to deposits of iron hydroxide. -(After Lafar.)] - -[Illustration: FIG. 59.--Illustrates the genus Beggiatoa. The filament -_A_ is so full of sulphur granules that the individual cells are not -visible. _B_ has fewer sulphur granules. In _C_ the granules are -nearly absent and the separate cells of the filament are seen. (After -Winogradsky, from Lafar.)] - - -ORDER I. Eubacteria. - -Cells without nuclei, free from sulphur granules and from -bacteriopurpurin (p. 112); colorless, or slightly colored. - -1. Family: COCCACEAE (Zopf) Migula, all cocci. - - {Genus 1. _Streptococcus_ Billroth: - { division in one plane only (Fig. 49). - Non-flagellated, { " 2. _Micrococcus_ (Hallier) Cohn: - Non-motile { division in two planes only (Fig. 50). - { " 3. _Sarcina_ Goodsir: - { division in three planes only (Fig. 51). - - { " 4. _Planococcus_ Migula: - Flagellated, { division in two planes only. - motile { " 5. _Planosarcina_ Migula: - { division in three planes only. - -2. Family: BACTERIACEAE Migula, all bacilli. - - Genus 1. _Bacterium_ (Ehrenberg) Migula: no flagella; non-motile. - " 2. _Bacillus_ (Cohn) Migula: flagella peritrichic (Fig. 52). - " 3. _Pseudomonas_ Migula: flagella at the end: - monotrichic, lophotrichic, amphitrichic (Fig. 53). - -3. Family: SPIRILLACEAE Migula, all spirilla. - - {Genus 1. _Spirosoma_ Migula: - { non flagellated; non-motile. - { " 2. _Microspira_ (Schroeter) Migula: - Cells stiff { flagella one to three at the end (Fig. 54). - { " 3. _Spirillum_ (Ehrenberg) Migula: - { flagella more than three - { at the end (Fig. 55). - - Cell flexible { " 4. _Spirochaeta_ Ehrenberg: - { motile; no flagella (Fig. 56). - -4. Family: CHLAMYDOBACTERIACEAE. - -Cells cylindrical in long threads and surrounded by a sheath. -Reproduction also by gonidia formed from an entire cell. - - Genus 1. _Chlamydothrix_ Migula (Fig. 57). - " 2. _Crenothrix_ Colin (Fig. 58). - " 3. _Pragmidiothrix_ Engler. - " 4. _Spherotilus_ (including Cladothrix). - - -ORDER II. THIOBACTERIA: SULPHUR BACTERIA. - -Cells without a nucleus, but containing sulphur granules, may be -colorless or contain bacteriopurpurin and be colored reddish or violet. - -1. Family BEGGIATOACEAE. - - Genus 1. _Thiothrix_ Winogradsky. - - " 2. _Beggiatoa_ Trevisan. Of interest since it is without a - sheath, is motile, but without flagella (Fig. 59). - -2. Family RHODOBACTERIACEAE. - -This has five subfamilies and twelve genera, most of which are due to -the Russian bacteriologist Winogradsky who did more work than anyone -else with the sulphur bacteria. - - -THE CLASSIFICATION OF THE SOCIETY OF AMERICAN BACTERIOLOGISTS. - -The Committee on Classification of the Society of American -Bacteriologists at the meeting held in December, 1919, submitted its -final report. This report has not been formally adopted as a whole, -but in all probability will be substantially as outlined below. This -outline does not attempt to give the detailed characterizations of the -different groups as defined by the committee, but does show the names -to be applied to the commoner organisms. These organisms are included -in the 4th and 5th orders. Details of the first three orders have not -been worked out. They are listed merely for completeness. - -CLASS SCHIZOMYCETES. - -Unicellular, chlorophyl-free plants, reproducing by transverse division -(some forms by gonidia also). - -ORDERS: - - A. Myxobacteriales--Cells united during vegetative stage into - a pseudo-plasmodium which passes over into a highly developed - cyst-producing resting stage. - - B. Thiobacteriales--Sulphur bacteria. - - C. Chlamydobacteriales--Iron bacteria and other sheathed bacteria. - - D. Actinomycetales--Actinomyces, tubercle and diphtheria bacilli. - - E. Eubacteriales--All the other common bacteria. - -GENERA OF ORDERS D AND E. - - D. ACTINOMYCETALES-- - FAMILY I. ACTINOMYCETACEAE Buchanan, 1918. - Genus 1. _Actinobacillus_, Brampt, 1900. - Type species, _Actinobacillus lignieresi_ Brampt, 1900. - Genus 2. _Leptotrichia_ Trevisan, 1879. - Type species, _Leptotrichia buccalis_ (Robin, 1847) Trevisan. - Genus 3. _Actinomyces_ Harz, 1877. - Type species, _Actinomyces bovis_ Harz. - Genus 4. _Erysipelothrix_ Rosenbach, 1909. - Type species, _Erysipelothrix rhusiopathiae_ (Kitt, 1893) - Rosenbach, swine erysipelas. - FAMILY II. MYCOBACTERIACEAE Chester, 1897. - Genus 1. _Mycobacterium_ Lehmann and Neumann, 1896. - Type species, _Mycobacterium tuberculosis_ (Koch, 1882) L. - and N. - Genus 2. _Corynebacterium_ Lehmann and Neumann, 1896. - Type species, _Corynebacterium diphtheriae_ (Loeffler, 1882) - L. and N. - Genus 3. _Fusiformis_ Hoelling, 1910. - Type species, _Fusiformis termitidis_ Hoelling. Vincent's - angina. - Genus 4. _Pfeifferella_ Buchanan, 1918. - Type species, _Pfeifferella mallei_ (Loeffler, 1896) Buchanan. - Glanders bacillus. - - E. EUBACTERIALES - FAMILY I--NITROBACTERIACEAE--Proto- or autotrophic for N - or C and sometimes for both (except Acetobacter). - TRIBE I--NITROBACTEREAE--autotrophic for C. - Genus 1. _Hydrogenomonas_ Jensen, 1909. - Type species, _Hydrogenomonas pantotropha_ (Kaserer, 1906) - Jensen; oxidizes free H. - Genus 2. _Methanomonas_ Jensen, 1909. - Type species, _Methanomonas methanica_ (Soehngen) Jensen; - oxidizes CH{4}. - Genus 3. _Carboxydomonas_ Jensen, 1909. - Type species, _Carboxydomonas oligocarbophila_ (Beijerinck - and Van Delden, 1903) Jensen; oxidizes CO. - Genus 4. _Acetobacter_ Fuhrman, 1905. - Type species, _Acetobacter aceti_ (Thompson, 1852) Fuhrman; - oxidizes alcohol to acetic acid. - Genus 5. _Nitrosomonas_ Winogradsky, 1892. - Type species, _Nitrosomonas europoea_ Winogradsky; oxidizes - ammonia or ammonium salts to nitrous acid, - hence nitrites. - Genus 6. _Nitrobacter_ Winogradsky, 1892. - Type species, _Nitrobacter Winogradskyi_ Committee of 1917; - oxidizes nitrous acid (nitrites) - to nitric acid (nitrates). - TRIBE II--AZOTOBACTEREAE--prototrophic for N. - Genus 7. _Azotobacter_ Beijerinck, 1901; large, free-living, - aerobic N absorbers. - Type species, _Azotobacter chroococcum_ Beijerinck. - Genus 8. _Rhizobium_ Frank, 1889. - Type species, _Rhizobium leguminosarum_ Frank; root tubercle - bacteria of legumes. - FAMILY II--PSEUDOMONADACEAE, Committee of 1917. - Genus 1. _Pseudomonas_ Migula, 1894. - Type species, _Pseudomonas violacea_ (Schroeter, 1872) Migula. - FAMILY III--SPIRILLACEAE Migula, 1894--all spiral bacteria. - Genus 1. _Vibrio_ Mueller, 1786, emended by E. F. Smith, 1905. - Type species, _Vibrio cholerae_ (Koch, 1884) Schroeter, 1886. - Genus 2. _Spirillum_ Ehrenberg, 1830, emended Migula, 1894. - Type species, _Spirillum undula_ (Mueller, 1786) Ehrenberg. - FAMILY IV--COCCACEAE Zopf, 1884, emended Migula, 1894--all cocci. - Tribe I--NEISSEREAE. - Genus 1. _Neisseria_ Trevisan, 1885. - Type species, _Neisseria gonorrhoeae_ Trevisan. - Tribe II--STREPTOCOCCEAE Trevisan, 1889. - Genus 2. _Diplococcus_ Weichselbaum, 1886. - Type species, _Diplococcus pneumoniae_ Weichselbaum. - Genus 3. _Leuconostoc_ Van Tieghem, 1878. - Type species, _Leuconostoc mesenterioides_ (Cienkowski) Van - Tieghem. - Genus 4. _Streptococcus_ Rosenbach, 1884; emended Winslow - and Rogers, 1905. - Type species, _Streptococcus pyogenes_ Rosenbach. - Tribe III--MICROCOCCEAE Trevisan, 1889. - Genus 5. _Staphylococcus_ Rosenbach, 1884; animal parasites. - Type species, _Staphylococcus aureus_ Rosenbach. - Genus 6. _Micrococcus_ Cohn, 1872, emended Winslow and Rogers, - 1905. Facultative parasites or saprophytes. - Type species, _Micrococcus luteus_ (Schroeter, 1872) Cohn. - Genus 7. _Sarcina_ Goodsir, 1842, emended Winslow and - Rogers, 1905. - Type species, _Sarcina ventriculi_ Goodsir. - Genus 8. _Rhodococcus_ Zopf, 1891, emended Winslow and - Rogers, 1905; cocci with red pigment. - Type species, _Rhodococcus rhodochrous_ Zopf. - FAMILY V--BACTERIACEAE Cohn, 1872, emended by Committee of 1917; - bacilli without spores not above included. - Tribe I--CHROMOBACTEREAE Committee of 1919; producing red or - violet pigment, mainly water forms. - Genus 1. _Erythrobacillus_ Fortineau, 1905. - Type species, _Erythrobacillus prodigiosus_ - (Ehrenberg, 1848) Fortineau. - Genus 2. _Chromobacterium_ Bergonzini, 1881. - Type species, _Chromobacterium violaceum_ Bergonzini. - Tribe II--ERWINEAE Committee 1919; plant pathogens. - Genus 3. _Erwinia_ Committee 1917. - Type species, _Erwinia amylovora_ (Burrill, 1883) Committee - 1917. - Tribe III--ZOPFEAE Committee of 1919; Gram +, no pigment, - non-carbohydrate-fermenting. - Genus 4. _Zopfius_ Wenner and Rettger, 1919. - Type species, _Zopfius zopfii_ (Kurth) Wenner and Rettger. - Tribe IV--BACTEREAE Committee of 1919; Gram -, carbohydrate - fermenters. - Genus 5. _Proteus_ Hauser, 1885; liquefy gelatin. - Type species, _Proteus vulgaris_ Hauser. - Genus 6. _Bacterium_ Ehrenberg, 1828, emended Jensen, 1909; - liquefy gelatin rarely. - Type species, _Bacterium coli_. - Tribe VI--LACTOBACILLEAE Committee of 1919; Gram +, high acid, - thermophils. - Genus 7. _Lactobacillus_ Beijerinck, 1901. - Type species, _Lactobacillus caucasicus_ (Kern?) Beijerinck; - Bulgarian bacillus. - Tribe VI--PASTEURELLEAE Committee of 1919; organisms of - hemorrhagic septicemia. - Genus 8. _Pasteurella_ Trevisan, 1888. - Type species, _Pasteurella cholerae-gallinarum_ - (Fluegge, 1886); Trevisan. - Tribe VII--HEMOPHILEAE Committee of 1917; require hemoglobin for - growth. - Genus 9. _Hemophilus_ Committee of 1917. - Type species, _Hemophilus influenzae_ (Pfeiffer, 1893) - Committee of 1917. - FAMILY VI--BACILLACEAE Fischer, 1895. Spore forming rods. - Genus 1. _Bacillus_ Cohn, 1872; aerobic, no change of form - around the spore. - Type species, _Bacillus subtilis_ Cohn. - Genus 2. _Clostridium_ Prazmowski, 1880; anaerobic, frequently - enlarged around spore. - Type species, _Clostridium butyricum_ Prazmowski. - -As compared with Migula's classification it is to be noted that there -are 38 genera listed by the Committee instead of 13 in the same general -groups. - -The following list of _Genera conservanda_ submitted by the Committee -was formally adopted by the Society and these are therefore its -official names for the organisms included in these genera. - - _Acetobacter_ Fuhrman - _Actinomyces_ Harz - _Bacillus_ Cohn - _Bacterium_ Ehrenberg - _Chromobacterium_ Bergonzini - _Clostridium_ Prazmowski - _Erythrobacillus_ Fortineau - _Leptotrichia_ Trevisan - _Leuconostoc_ Van Tieghem - _Micrococcus_ Cohn - _Rhizobium_ Frank - _Sarcina_ Goodsir - _Spirillum_ Ehrenberg - _Staphylococcus_ Rosenbach - _Streptococcus_ Rosenbach - _Vibrio_ Mueller - -_It is greatly to be desired that the Society's Classification when -finally completed shall become the standard in the United States at -least._ - -_Such names as have been adopted by the Society are used throughout -this work._ - -The Committee also submitted the following artificial key for -determining the genera in the two orders _ACTINOMYCETALES AND -EUBACTERIALES_: - - A--Typically filamentous forms _Actinomycetacae_ - B--Mycelium and conidia formed _Actinomyces_ - BB--No true mycelium - C--Cells show branching - D--Gram negative _Actinobacillus_ - DD--Gram positive _Erysipelothrix_ - CC--Cells never branch. Gram positive threads later fragmenting - into rods _Leptotrichia_ - AA--Typically unicellular forms (though chains of cells may occur) - B--Cells spherical--_COCCACEAE_ - C--Parasitic forms (except Leuconostoc), cells generally grouped - in pairs or chains, never in packets, generally active - fermenters. - D--Cells in flattened coffee-bean-like pairs, gram -. - _Neisseria_ - DD--Not as D - E--Saprophytes in zooegloea masses in sugar solutions. - _Leuconostoc_ - EE--Not as E. Gram +. - F--Cells in lanceolate pairs or in chains. Growth on - media not abundant. - G--Cells in lanceolate pairs. Inulin generally fermented. - _Diplococcus_ - GG--Cells in chains. Inulin not generally fermented. - _Streptococcus_ - FF--Cells in irregular groups. Growth in media fairly - vigorous. White or orange pigment. - _Staphylococcus_ - CC--Saprophytic forms. Cells in irregular groups or packets, - not in chains. Fermentative powers low. - D--Packets _Sarcina_ - DD--No packets. - E--Yellow pigment _Micrococcus_ - EE--Red pigment _Rhodococcus_ - BB--Rods: - C--Spiral rods - D--Short, comma-like rods. One to three flagella. - _Vibrio_ - DD--Long spirals. Five to twenty flagella. _Spirillum_ - CC--Straight rods. - D--No endospores. - E--Rods of irregular shape or showing branched or filamentous - involution forms. - F--Cells irregular in shape. Staining unevenly. Animal - parasites. - G--Acid fast _Mycobacterium_ - GG--Not acid fast. - H--Cells elongated, fusiform _Fusiformis_ - HH--Cells not elongated, sometimes branching. - I--Gram positive. Slender, sometimes club-shaped. - _Corynebacterium_ - II--Gram negative. Rods sometimes form threads. - Characteristic honey-like growth on potato. - _Pfeifferella_ - FF--Cells staining unevenly but with branched or filamentous - forms at certain stages. Never acid fast. - Not animal parasites. - G--Metabolism simple, growth processes involving oxidation - of alcohol or fixation of free N (latter in symbiosis - with green plants). - H--Cells minute. Symbiotic in roots of legumes. - _Rhizobium_ - HH--Oxidizing alcohol. Branching forms common. - _Acetobacter_ - GG--Not as G. Proteus-like colonies. - H--Not attacking carbohydrates _Zopfius_ - HH--Fermenting glucose and sucrose at least. - _Proteus_ - EE--Regularly formed rods. - F--Metabolism simple, growth processes involving oxidation - of C, H, or their simple compounds or the fixation - of free N.--_NITROBACTERIACEAE._ - G--Fixing N or oxidizing its simple compounds. - H--Fixing N, cells large, free in soil _Azotobacter_ - HH--Oxidizing N compounds. - I--Oxidizing NH{4} compounds _Nitrosomonas_ - II--Oxidizing nitrites _Nitrobacter_ - GG--Not as G. - H--Oxidizing free H _Hydrogenomonas_ - HH--Oxidizing simple C compounds, not free H. - I--Oxidizing CO _Carboxydomonas_ - II--Oxidizing CH{4} _Methanomonas_ - FF--Not as F. - G--Flagella usually present, polar--_PSEUDOMONADACEAE_ - _Pseudomonas_ - GG--Flagella when present peritrichic--_BACTERIACEAE_ - H--Parasitic forms showing bi-polar staining. - _Pasteurella_ - HH--Not as H. - I--Strict parasites growing only in presence - of hemoglobin - _Hemophilus_ - II--Not as I. - J--Water forms producing red or violet pigment. - K--Pigment red _Erythrobacillus_ - KK--Pigment violet _Chromobacterium_ - JJ--Not as J. - K--Plant pathogens _Erwinia_ - KK--Not plant pathogens. - L--Gram +, forming large amount of acid - from carbohydrates, sometimes CO{2}, - never H _Lactobacillus_ - LL--Gram -, forming H as well as CO{2} if - gas is produced _Bacterium_ - DD--Endospores present--_BACILLACEAE_ - E--Aerobes, rods not swollen at sporulation. _Bacillus_ - EE--Anaerobes, rods swollen at sporulation. _Clostridium_ - - - - -PART II. - -PHYSIOLOGY. - - - - -CHAPTER VI. - -GENERAL CONDITIONS FOR GROWTH. - - -OCCURRENCE. - -Bacteria are probably the most widely distributed of living organisms. -They are found practically everywhere on the surface of the earth. -Likewise in all surface waters, in streams, lakes and the sea. They -occur in the air immediately above the surface, since they are carried -up mechanically by air currents. They cannot fly of themselves. There -is no reason to believe that any increase in numbers occurs to an -appreciable extent in the air. The upper air, for example, on high -mountains, is nearly free from them. So also is the air over midocean, -and in high latitudes. As a rule, the greater the amount of dust in -the air, the more numerous are the bacteria. Hence they are found more -abundantly in the air in cities and towns than in the open country. -The soil is especially rich in numbers in the upper few feet, but they -diminish rapidly below and almost disappear at depths of about six -feet unless the soil is very porous and open, when they may be carried -farther down. Hence the waters from deep wells and springs are usually -devoid of these organisms. In the sea they occur at all levels and have -been found in bottom ooze dredged from depths of several miles. It is -perhaps needless to add that they are found on the bodies and in the -alimentary tract of human beings and animals; on clothing, utensils; -in dwellings, stables, outhouses, etc. From one-fourth to one-half of -the dry weight of the feces of animals and men is due to the bacteria -present. The urine is practically free from them in health. - -While bacteria are thus found nearly everywhere, it is an entirely -mistaken idea to suppose that all are injurious to man. As a matter -of fact, those which are dangerous are relatively few and are for the -most part found only in close association with man. Most bacteria are -harmless and the vast majority are beneficial or even essential to -man's existence on the earth. These facts must be constantly borne in -mind, and it is hoped that the pages which follow will make them clear. - -In order that any organism may thrive there are a number of general -environmental conditions which must be fulfilled. These conditions -vary more or less for each kind of organism. Bacteria are no exception -to this general rule. These conditions may be conveniently considered -under the general heads of _moisture_; _temperature_; _light_; _oxygen -supply_; _osmotic pressure_; _action of electricity_; of _Roentgen_ -and _radium rays_; _pressure_; _mechanical vibration_; and _chemical -environment_, including the _reaction of the medium_, _the effect -of injurious chemicals_, and especially the _food requirements of -bacteria_. For each of these conditions there is a _maximum_, meaning -the greatest amount of the given condition which the organism can -withstand, a _minimum_, or the least amount, and an _optimum_ or that -amount which is most favorable for development. Further, there might be -distinguished a maximum for _mere existence_ and a lower maximum for -_development_; also a minimum for _mere existence_ and a higher minimum -for _development_. These maxima, minima, and optima for bacteria have -been determined with exactness for only a very few of the general -conditions and for comparatively few kinds. - - -MOISTURE. - -The _maximum_ moisture is absolutely pure water, and no organism can -thrive in this alone owing to the factor of too low osmotic pressure -and to the further factor of absence of food material. There are many -bacteria which thrive in water containing only traces of mineral salts -and a large class whose natural habitat is surface water. These "water -bacteria" are of great benefit in the purification of streams. They are -as a class harmless to men and animals. Some of the disease-producing -bacteria like _Bacterium typhosum_ (of typhoid fever) and _Vibrio -cholerae_ (of Asiatic cholera) were undoubtedly originally water -bacteria, and it is rather striking that in these diseases conditions -are induced in the intestine (diarrheas) which simulate the original -watery environment. The _minimum_ moisture condition is absolute -dryness, and no organism can even exist, not to say develop, in such -a condition since water is an essential constituent of living matter. -Some bacteria and especially most spores may live when dried in the -air or by artificial means for months and even years, while some are -destroyed in a few hours or days when dried (typhoid, cholera, etc.). -The optimum amount of moisture has not been determined with any great -accuracy and certainly a rather wide range in percentage of water is -permissible with many, though a liquid medium is usually most favorable -for artificial growth. The "water bacteria" have been mentioned. In the -soil a water content of 5 to 15 per cent. seems to be most suitable for -many of the organisms which aid in plant growth. In animals and man the -organisms infecting the intestinal tract prefer a high percentage of -moisture as a rule, especially those causing disease here. Those found -on the surface of the body (pus cocci) need a less amount of water, -while those invading the tissues (tuberculosis, black-leg, etc.) seem -to be intermediate in this respect. In artificial culture media a water -content of less than 30 per cent. inhibits the growth of most bacteria. - -As a general rule those bacteria which require the largest percentage -of water are most susceptible to its loss and are most readily killed -by drying. The typhoid and cholera organisms die in a few hours when -dried, while pus cocci and tubercle bacilli live much longer. - - -TEMPERATURE. - -The temperature conditions for bacterial existence and growth have been -determined more accurately than any of the other general conditions. -The maximum for existence must be placed at or near 100 deg. since it is -known that all bacteria including spores may be killed by boiling in -time. Nevertheless, certain forms have been reported as thriving in hot -springs where the water temperature was 93 deg.. This is the highest known -temperature for development. The minimum for existence lies at or near -the absolute zero (-273 deg.) since certain organisms have been subjected -to the temperature produced by the sudden evaporation of liquid -hydrogen (-256 deg. to -265 deg.) and have remained alive. Whether they could -withstand such temperatures indefinitely is not known. The minimum for -development is near the freezing-point of water, since reproduction -by division has been observed in the water from melting sea-ice at -a temperature of -1.5 deg.. Thus bacteria as a class have a range for -existence of about 373 deg. (-273 deg. to +100 deg.) and for development of 94.5 deg. -(-1.5 deg. to +93 deg.) certainly much wider ranges than any other group of -organisms.[5] - -The optimum temperature for development varies within rather wide -limits for different organisms. In general it may be stated that the -optimum temperature is approximately that of the natural habitat of -the organism, though there are exceptions. The optimum of the "hot -spring" bacteria just mentioned is apparently that of the springs (93 deg. -in this case). Many soil organisms are known whose optimum is near -70 deg. (a temperature rarely, if ever, attained in the soil), _but only -when grown in air or oxygen_; but is very much lower when grown in the -_absence of oxygen_. Many other soil organisms exhibit very little -difference in rate or amount of growth when grown at temperatures which -may vary as much as 10 deg. or 15 deg., apparently an adaptation to their -normal environment. The disease-producing organisms show much narrower -limits for growth, especially those which are difficult to cultivate -outside the body. For example, the bacterium of tuberculosis in man -scarcely develops beyond the limits of 2 deg. or 3 deg. from the normal body -temperature of man (37 deg.), while the bacterium of tuberculosis in birds -grows best at 41 deg. to 45 deg., the normal for birds, and the bacterium of -so-called tuberculosis of cold-blooded animals at 14 deg. to 18 deg.. - -Those bacteria whose optimum temperature is above 40 deg. are sometimes -spoken of as the "_thermophil_" bacteria. The fixing of the "thermal -death-point" that is, the minimum temperature at which the bacteria -are killed is a matter of great practical importance in many ways -and numerous determinations of this have been made with a great many -organisms and by different observers. The factors which enter into such -determinations are so many and so varied that unless all the conditions -of the experiment are given together with the time of application, -the mere statements are worthless. It may be stated that all _young, -actively growing_ (non-spore-containing) _disease-producing bacteria, -when exposed in watery liquids and in small quantities are killed at -a temperature of 60 deg. within half an hour_. It is evident, that this -fact has very little practical application, since the conditions stated -are rarely, if ever, fulfilled except in laboratory experiments. (See -Sterilization and Pasteurization, Chapter XIII.) - - -LIGHT. - -Speaking generally, it can be said that light is destructive to -bacteria. Many growing forms are killed in a few hours when properly -exposed to direct sunlight and die out in several days in the diffuse -daylight of a well-lighted room. Even spores are destroyed in a -similar manner, though the exposure must be considerably longer. -Certain bacteria which produce colors may grow in the light, since -the pigments protect them. Some few kinds, like the sulphur bacteria, -which contain a purplish-red pigment that serves them to break up -H{2}S, need light for their growth. Since disease-producing bacteria -are all injuriously affected by light, the advantage of well-lighted -habitations both for men and animals is obvious. - - -OXYGEN SUPPLY. - -Oxygen is one of the constituents of protoplasm and is therefore -necessary for all organisms. This does not mean that all organisms -must obtain their supply from _free oxygen_, however, as animals and -plants generally do. This fact is well illustrated by the differences -among bacteria in this respect. Some bacteria _require free oxygen_ for -their growth and are therefore called _aerobic_ bacteria or _aerobes_ -(sometimes _strict aerobes_, though the adjective is unnecessary). -Others _cannot grow in the presence of free oxygen_ and are therefore -named _anaerobic bacteria_ or _anaerobes_ (strict is unnecessary). -There are still other kinds which may grow either in the presence of -free oxygen or in its absence, hence the term _facultative anaerobes_ -(usually) is applied to them. The distinction between _facultative -aerobe_ and _facultative anaerobe_ might be made. The former means -those which grow best in the absence of free oxygen, though capable of -growing in its presence, while the latter term means those which grow -best in the presence of free oxygen, but are capable of growing in its -absence. The amount of oxygen in the atmosphere in which an organism -grows may be conveniently expressed in terms of the oxygen pressure, -_i.e._, in millimeters of mercury. It is evident that the maximum, -minimum and optimum oxygen pressures for anaerobic bacteria are the -same, namely, 0 mm. Hg. This is true only for natural conditions, -since a number of anaerobic organisms have been gradually accustomed -to increasing amounts of O, so that by this process of training they -finally grew in ordinary air, that is, at an oxygen pressure of about -150 mm. Hg. (Normal air pressure is 760 mm. Hg. and oxygen makes up -one-fifth of the air.) The minimum O pressure for facultative anaerobes -is also 0 mm. Hg. Some experiments have been made to determine the -limits for aerobes, but on a few organisms only, so that no general -conclusions can be drawn from them. To illustrate: _Bacillus subtilis_ -(a common "hay bacillus") will grow at 10 mm. Hg. pressure but not at 5 -mm. Hg. It will also grow in compressed oxygen at a pressure of three -atmospheres (2280 mm. Hg.), but not at four atmospheres (3040 mm. Hg.), -though it is not destroyed. - -Parodko has determined the oxygen limits for five common organisms as -follows: - - Minimum - Maximum. Vol. Mm. - In atmospheres. Mm. Hg. per cent. Hg. - - _Bacterium 1.94 to 2.51 1474 to 1908 0.00016 = 0.0012 - fluorescens_ - _Sarcina lutea_ 2.51 to 3.18 1908 to 2417 0.00015 = 0.0011 - _Proteus vulgaris_ 3.63 to 4.35 2749 to 3306 0 0 - _Bacterium coli_ 4.09 to 4.84 3108 to 3478 0 0 - _Erythrobacillus 5.45 to 6.32 3152 to 4800 0 0 - prodigiosus_ - -These few instances do not disclose any general principles which may -be applied either for the growth or for the distinction of aerobes or -facultative anaerobes. - -It has been shown that compressed oxygen will kill some bacteria but -this method of destroying them has little or no practical value. Oxygen -in the form of ozone, O{3}, is rapidly destructive to bacteria, and this -fact is applied practically in the purification of water supplies for -certain cities where the ozone is generated by electricity obtained -cheaply from water power. The same is true of oxygen in the "nascent -state" as illustrated by the use of hypochlorites for the same purpose. - -It was stated (p. 74) that certain thermophil bacteria in the soil have -an optimum temperature for growth _in the air_ which is much higher -than is ever reached in their natural habitat and that they grow at a -moderate temperature under _anaerobic_ conditions. It has been shown -that if these organisms are grown with aerobes or facultative anaerobes -they thrive at ordinary room temperature. These latter organisms by -using up the oxygen apparently keep the tension low, and this explains -how such organisms grow in the soil.[6] - - -OSMOTIC PRESSURE. - -Like all living cells bacteria are very susceptible to changes in -the density of the surrounding medium. If placed in a medium less -concentrated than their own protoplasm water is absorbed and they -"swell up"; while if placed in a denser medium, water is given off and -they shrink (plasmoptysis or plasmolysis). Should these differences -be marked or the transition be sudden, the cell walls may even burst -and the organisms be destroyed. If the differences are not too great -or if the transition is made gradually, the organisms may not be -destroyed, but will either cease to grow and slowly die out, or will -show very much retarded growth, or will produce abnormal cell forms. -This is illustrated in the laboratory in attempting to grow bacteria on -food material which has dried out. A practical application of osmotic -effects is in the use of a high percentage of sugar in preserving -fruits, etc., and in the salting of meats. Neither the cane-sugar nor -the common salt themselves injure the bacteria chemically, but by the -high concentration prevent their development. In drying material in -order to preserve it there are two factors involved: first, the loss of -water necessary for growth and second, the increased osmotic pressure. - -In a medium of greater density diffusion of water is outward from the -cell and this will continue until an equilibrium is established between -cell contents and medium. Food for the organism _must be in solution -and enter the cell by diffusion_. Therefore, growth ceases in a medium -too dense, since water to carry food in solution does not enter the -cell. - - -ELECTRICITY. - -Careful experimenters have shown that the electric current, either -direct or alternating, has no direct destructive effect on bacteria. -In a liquid medium the organisms may be attracted to or repelled -from one or the other pole or may arrange themselves in definite -ways between the poles (galvanotaxis), but are not injured. However, -electricity through the _secondary_ effects produced, may be used to -destroy bacteria. If the passage of the electric current _increases the -temperature_ of the medium sufficiently, the bacteria will be killed, -or if _injurious chemical substances_ are formed (ozone, chlorine, -acids, bases, etc.), the same result will follow (see Ozone, pp. 77 and -157). - - -RADIATIONS. - -Roentgen or _x_-rays and radium emanations when properly applied to -bacteria will destroy them. The practical use of these agents for -the direct destruction of bacteria in diseases of man or animals is -restricted to those cases where they may be applied directly to the -diseased area, since they are just as injurious to the animal cell -as they are to the bacteria, and even more so. Their skilful use as -_stimuli to the body cells_ to enable them to resist and overcome -bacteria and other injurious organisms or cell growths is an entirely -different function and will not be considered here. - - -PRESSURE. - -Hydrostatic pressure up to about 10,000 pounds per square inch is -without appreciable effect on bacteria as has been shown by several -experimenters and also by finding living bacteria in the ooze dredged -from the bottom of the ocean at depths of several miles. - -Pressures from 10,000 to 100,000 pounds show variable effects. Some -bacteria are readily killed and others, even non-spore formers, -are only slightly affected. The time factor is important in this -connection. The presence of acids, even CO{2}, or organic acids, results -in the destruction of most non-spore formers. - - -MECHANICAL VIBRATION. - -Vibrations transmitted to bacteria in a liquid may be injurious to them -under certain circumstances. Some of the larger forms like _Bacillus -subtilis_ may be completely destroyed by shaking in a rapidly moving -shaking machine in a few hours. Bacteria in liquids placed on portions -of machinery where only a slight trembling is felt, have been found -to be killed after several days. Reinke has shown that the passing of -strong sound waves through bacterial growths markedly inhibits their -development. - - - - -CHAPTER VII. - -CHEMICAL ENVIRONMENT. - - -REACTION OF MEDIUM. - -Most bacteria are very susceptible to changes in the degree of acidity -or alkalinity of the medium in which they grow. Some kinds prefer a -slightly acid reaction, some a slightly alkaline, and some a neutral -(with reference to litmus as indicator). The organism which is the -commonest cause of the souring of milk thrives so well in the acid -medium it produces that it crowds out practically all other kinds, -though its own growth is eventually stopped by too much acid. Acid -soils are usually low in numbers of bacteria and as a consequence -produce poor crops. The disease-producing bacteria as a class grow best -in a medium which is slightly alkaline. - -Accurate determination of limits have been made on but few organisms. -The reaction is a most important factor in growing bacteria on -artificial media (see Making of Media, Chapter XVI). - - -INJURIOUS CHEMICAL SUBSTANCES. - -(SEE DISINFECTION AND DISINFECTANTS, Chapter XIII.) - - -CHEMICAL COMPOSITION. - -The chemical composition is subject to wide variation chiefly for -two reasons: First, the cell wall in most instances seems to exert -only a slight selective action in the absorption of mineral salts -so that their concentration within the cell is very nearly that of -the surrounding medium. Second, the chief organic constitutents vary -remarkably with the kind and amount of food material available--a -rich protein pabulum increases the protein, a plentiful supply of -carbohydrates or of fat results in the storing of more fat, especially -and _vice versa_. These facts must be borne in mind in considering the -chemistry of bacteria. - -Of the chemical elements known, only the following seem to be essential -in the structure of bacteria: carbon, hydrogen, oxygen, nitrogen, -sulphur, phosphorus, chlorine, potassium, calcium, magnesium, iron, -manganese. Other elements, as sodium, iodine, silicon, aluminum, -lithium, copper, etc., have been reported by different analysts, but -none of them can be regarded as essential, except possibly in isolated -instances. - -These elements exist in the bacterial cell in a great variety of -combinations of which the most abundant is _water_. The amount of water -varies in different species from 75 to 90 per cent. of the total weight -in growing cells, and is less in spores. The amount of _ash_ has been -shown by different observers to vary from less than 2 per cent. to as -much as 30 per cent. of the _dry weight_. The following table compiled -from various sources will give an idea of the relative abundance of the -different elements in the ash. - - S as SO{3} 7.64 per cent. (much more in sulphur bacteria) - P as P{2}O{5} 18.14 " to 73.94 per cent. - Cl 2.29 " - K as K{2}O 11.1 " to 25.59 " - Ca as CaO 12.64 " to 14.0 " - Mg as MgO 0.7 " to 11.55 " - Fe as Fe{2}O{3} 1.0 " to 8.15 " (iron bacteria) - Mn traces - -As to the form in which the last six elements in the table exist in -the cell, little is known. The sulphur and phosphorus are essential -constituents of various proteins. The high percentage of phosphorus -points to nuclein compounds as its probable source. - -The carbon and nitrogen, together with most of the hydrogen and oxygen -not united as water, make up the great variety of organic compounds -which compose the main substances in the bacterial cell. - -It has already been stated that the essential structures in the -bacterial cell are cell wall and protoplasm, including the nuclein. -These differ markedly in chemical composition. It is well known that -the cell walls of green plants consist largely of cellulose and closely -related substances.[7] _True cellulose_ has been recognized in but -very few bacteria. (_Sarcina ventriculi_, Migula; _Mycobacterium -tuberculosis_, Hammerschlag, Dreyfuss, Nishimura; _Bacillus -subtilis_, Dreyfuss; _Acetobacter xylinum_, Brown; _Acetobacter -acidi oxalici_, Banning; and a few others.) It is certainly not an -important constituent of the cell wall in many. On the other hand, -_hemicellulose_ and _gum-like_ substances have been identified in -numerous organisms of this class as important constituents of the cell -wall and of the capsule which is probably an outgrowth from the latter. -Practically always associated with these substances are compounds -containing nitrogen. One of these has been certainly identified as -_chitin_ or a closely similar substance. Chitin is the nitrogenous -substance which enters largely into the composition of the hard parts -of insects, spiders and crustaceans. It is an interesting fact to find -this substance characteristic of these animals in bacteria, as well as -other fungi. - -Though it is extremely difficult to separate the cell wall of bacteria -from the cell contents, in the light of our present knowledge it can be -stated that the cell walls are composed of a carbohydrate body closely -related to cellulose, though not true cellulose, probably in close -combination with chitin. - -Of the organic constituents of the cell contents the most abundant are -various proteins which ordinarily make up about one-half of the dry -weight of the entire cell. The "Mycoproteid" of Nencki, 1879, and other -earlier workers is deserving of little more than historical interest, -since these substances were certainly very impure and probably -consisted of mixtures of several "proteins" in the more recent sense. - -From later studies it seems probable that substances resembling -the albumin of higher forms do not occur in bacteria, at least in -appreciable quantities. Globulin has been reported by Hellmich in an -undetermined bacterium, but is certainly not commonly found. The larger -portion of the protein is of a comparatively simple type, in fact, -consists of protamins most of which are in combination with nucleic -acid as nucleoprotamins. Practically all recent workers find a high -percentage of nuclein, both actually isolated and as indicated by the -amounts of purin bases--xanthin, guanin, adenin--obtained, as well as -by the abundance of phosphorus in the ash, already mentioned. Some of -these nucleins have been shown to have poisonous properties. - -Closely related to but not identical with the proteins are the enzymes -and toxins which are formed in the cell and exist there as endo-enzymes -or endo-toxins respectively. These substances will be discussed later -under the heading "Physiological Activities of Bacteria" (Chapter XII). - -Carbohydrates are not commonly present in the cell contents, though -glycogen has been observed in a few and a substance staining blue -with iodine in one or two others. This latter substance was at first -considered to be starch "granulose," but is probably more closely -related to glycogen. - -Fats seem to be very generally present. The commoner fats--tri-olein, -tri-palmitin, tri-stearin have been found by many analysts. The -"acid-fast bacteria" are particularly rich in fatty substances, -especially the higher wax-like fats. Lecithins (phosphorized fats) and -cholesterins (not fats but alcohols) have been repeatedly observed and -probably occur in all bacteria as products of katabolism. - -Organic acids and esters occur as cell constituents but will be -discussed in connection with their more characteristic occurrences -as products of bacterial activity, as will also pigments which may -likewise be intracellular in some instances. - -The following analysis of tubercle bacilli, from de Schweinitz and -Dorset, while not intended as typical for all bacteria, still -illustrates the high percentage of protein compounds which undoubtedly -occurs in most, as well as showing the large amount of fatty substance -in a typical "acid-fast" organism: - - { 8.5 per cent. tuberculinic acid - { 24.5 " nucleoprotamin } - In the dried { 23.0 " neucleoprotein } 55.8 per cent. - organisms { 8.3 " proteinoid } protein. - { 26.5 " fat and wax - { 9.2 " ash - - - - -CHAPTER VIII. - -CHEMICAL ENVIRONMENT (CONTINUED). - - -GENERAL FOOD RELATIONSHIPS. METABOLISM. - -The foregoing brief review of the chemical composition of the bacterial -cell illustrates the variety of compounds which necessarily occurs, -but affords no definite clue as to the source of the elements which -enter into these compounds. These elements come from the material -which the organism uses as food. Under this term are included elements -or compounds which serve as building material, either for new cell -substance or to repair waste, or as sources of energy. - -An organism which is capable of making use of an element in the free -state is said to be _prototrophic_ for that particular element. -Thus aerobes and facultative anaerobes are prototrophic for O. The -"root-tubercle bacteria" of leguminous and other plants and certain -free living soil organisms are prototrophic for N.[8] - -On the other hand, if the element must be secured from compounds, then -the organism is _metatrophic_ in respect to the element in question. -Should the compound be inorganic, the term _autotrophic_ is applied -to the organism and _heterotrophic_ if the compound is organic. It is -very probable that anaerobes, exclusive of a few nitrogen absorbers, -are metatrophic for all the elements they utilize. With the exception -of the anaerobes it seems that all bacteria are _mixotrophic_, that is, -prototrophic for one or two elements and auto- or heterotrophic for the -others.[9] - -Those bacteria whose food consists of dead material are spoken of as -_saprophytes_, while those whose natural habitat, without reference to -their food, is in or on other living organisms are called _parasites_. -The _host_ is the organism in or on which the parasite lives. -Parasites may be of several kinds. Those which neither do injury nor -are of benefit to the host are called _non-pathogenic_ parasites or -_commensals_; many of the bacteria in the intestines of man and other -animals are of this class. Those which do injury to the host are -called _pathogenic_ or disease-producing, as the organisms causing the -transmissible diseases of animals and plants.[10] Finally, we have -those parasites which are of benefit to and receive benefit from the -host. These are called _symbionts_ or _symbiotic parasites_ and the -mutual relationship _symbiosis_. Certain of the intestinal bacteria in -man and especially in herbivorous animals are undoubted _symbionts_, as -are also the "root-tubercle bacteria" already mentioned. - -It is evident that all parasites that may be cultivated outside -the body are for the time _saprophytic_, hence the terms _strict_ -parasites and _facultative_ parasites, which should require no further -explanation. - -The changes which the above-mentioned types of food material undergo -in the various anabolic and katabolic processes _within the cell_ are -as yet but very slightly known. Nevertheless there are a number of -reactions brought about by bacteria acting on various food materials, -partly within _but largely without the cell_ which are usually -described as "physiological activities" or "biochemical reactions." -Some of these changes are to be ascribed to the utilization of certain -of the elements and compounds in these materials as tissue builders, -some as energy-yielding reactions and still others as giving rise to -substances that are of direct benefit to the organism concerned in its -competition with other organisms. - -Though all of the twelve elements already mentioned are essential for -the growth of every bacterium, two of them are of especial importance -for the reason that most of the "physiological activities" to be -described in the next chapters are centered around their acquisition -and utilization. These elements are _carbon_ and _nitrogen_. Some few -of the special activities of certain groups have to do with one or the -other of the remaining nine, as will be shown later. But generally -speaking _when a bacterium under natural conditions secures an adequate -supply of carbon and nitrogen, the other elements are readily available -in sufficient amount_. - -Carbon is necessary not only because it is an essential constituent -of protoplasm but because its oxidation is the chief source of the -energy necessary for the internal life of the cell, though nitrogen -and sulphur replace it in this function with a few forms. This -latter use of carbon constitutes what may be called its _respiratory -function_. Bacteria like other organisms in their respiration utilize -oxygen and give off carbon dioxide. The amount of the latter given off -from the cell in this way is very small as compared with that which -is frequently produced as an accompaniment of other reactions (see -Fermentation, next chapter). But there is no doubt of its formation and -it has been determined by a few investigators. On account of this use -of carbon, bacteria require relatively large amounts of this element. -One group of bacteria concerned in the spontaneous heating of coal -seems to be able to use free carbon from this material. Another group -is said to be able to oxidize marsh gas, CH{4}, and use this as its -source of carbon. The nitrite, nitrate and sulphur bacteria mentioned -later utilize carbon dioxide and carbonates as their carbon supply, -and one kind has been described which uses carbon monoxide. With these -few exceptions bacteria are dependent on _organic compounds_ for their -carbon and cannot use CO{2} as green plants do. - -The oxygen requirement is high partly for the same reason that -carbon is, _i.e._, respiration. Oxygen is one of the constituents of -protoplasm, and combined with hydrogen forms water which makes up such -a large part of the living cell. Anaerobic bacteria are dependent on -so-called "molecular respiration" for their energy. That is, through -a shifting or rearrangement of the atoms in the compounds used as -food the oxidation of carbon is brought about. Enzymes are probably -responsible for this action. Carbon dioxide is produced by anaerobes -as well as by aerobes, and frequently in amounts readily collected. A -carbohydrate is usually though not always essential for the growth of -anaerobes and serves them as the best source of energy. - -Nitrogen is the characteristic element of living material. Protoplasm -is a chemical substance in unstable equilibrium and nitrogen is -responsible for this instability. No other of the commoner elements -is brought into combination with such difficulty, nor is so readily -liberated when combined (all commercial explosives are nitrogen -compounds). Bacteria, like other forms of protoplasm, require nitrogen. -More marked peculiarities are shown by bacteria with reference to the -sources from which they derive their nitrogen than for carbon. Some can -even combine the free nitrogen of the air and furnish the only natural -means of any importance for this reaction. Some few forms (the nitrite -and nitrate formers, Chapter XI) obtain their energy from the oxidation -of inorganic nitrogen compounds, ammonia and nitrites respectively, and -not from carbon. These latter bacteria use carbon from carbon dioxide -and carbonates. A great many bacteria can secure their nitrogen from -nitrates but some are restricted to organic nitrogen. Many bacteria -obtain their carbon from the same organic compounds from which their -nitrogen is derived. - -Sulphur serves mainly as a constituent of protein compounds in the -protoplasmic structure. In some of the _sulphur_ bacteria it is a -source of energy, since either free sulphur or H{2}S is oxidized by -them. Some of these bacteria can obtain their carbon from CO{2} or -carbonates, and their nitrogen from nitrates or ammonium salts. - -Whether the _iron_ bacteria, belonging to the genus _Crenothrix_ of the -higher, thread bacteria, use this element or its compounds as sources -of energy is still a disputed question. The evidence is largely in -favor of this view. - -Free hydrogen has been shown to be oxidized by some forms which obtain -their energy in this way. - -Whether there is a special class of _phosphorus_ bacteria remains to be -discovered. That phosphorus is oxidized during the activity of many -bacteria is undoubted, but whether this represents a source of energy -or is the accidental by-product of other activities is undetermined. - -Practically nothing is known about the metabolism of the other elements -as such. - -From the preceding brief review of the relation of certain bacteria -to some of the elements in the free state and from the further fact -that there is scarcely a known natural organic compound which cannot -be utilized by some kind of bacterium, it is evident that this class -of organisms has a far wider range of adaptability than any other -class, and this adaptability helps to explain their seemingly universal -distribution. - -As to the metabolism _within the cell_, no more is known than is the -case with other cells, nor even as much. The materials used for growth -and as sources of energy are taken into the cell, built up into various -compounds some of which have been enumerated and in part broken down -again. Carbon dioxide and water are formed in the latter process. What -other katabolic products occur it is not easy to determine. Certainly -some of the substances mentioned in the next chapters are such products -but it is not always possible to separate those formed _inside_ the -cell from those formed _outside_. Perhaps most of the latter should be -considered true metabolic products. It would seem that on account of -the simplicity of structure of the bacterial cell and of the compounds -which they may use as food they would serve as excellent objects -for the study of the fundamental problems of cell metabolism. Their -minuteness and the nearly impossible task of separating them completely -from the medium in or on which they are grown makes the solution of -these problems one of great difficulty. - -When all of the environmental conditions necessary for the best -development of a given bacterium are fulfilled, it will then develop -to the limit of its capacity. This development is characterized -essentially by its reproduction, which occurs by transverse division. -The rate of this division varies much with the kind even under good -conditions. The most rapid rate so far observed is a division in -eighteen minutes. A great many reproduce every half-hour and this may -be taken as a good average rate. If such division could proceed without -interruption, a little calculation will show that in about sixty-five -hours a mass as large as the earth would be produced. - - Starting with 1 coccus, 1 mu in diameter, - its volume = 0.0000000000005 cc. - - 1/2 hour = 2 - 1 hour = 4 - 2 hours = 16 - 4 hours = 256 - 5 hours = 1024 = 10^{3}+ - 15 hours = 1,000,000,000 = 10^{9} = 0.5 cc. - 35 hours = 10^{21}+ = 500.0 cu.m. - About 65 hours = 2 x 10^{42}+ = 5 x 10^{20} cu.m. = a mass as large - as the earth. - -Such a rate of increase evidently cannot be kept up long on account of -many limiting factors, chief of which is the food supply. - -The foregoing calculation is based on the assumption that the organism -divides in one plane only. If it divides in 2 or 3 planes, the rate is -much faster, as is shown by the following formulae, which indicate the -theoretical rate of division: - - S = number of bacteria after a given number of divisions. - a = number at the beginning, and n = number of divisions. - 1 plane division S = 2^{_n_}a - 2 " " S = 2^{2_n_}a - 3 " " S = 2^{3_n_}a - -With two-plane or three-plane division, assuming that each organism -attains full size, as was assumed in the first calculation, the "mass -as large as the earth" would be attained in about thirty-two and -twenty-two hours respectively. - -This extraordinary rate of increase explains in large measure why -bacteria are able to bring about such great chemical changes in so -short a time as is seen in the rapid "spoiling" of food materials, -especially liquids. The reactions brought about by bacteria on -substances which are soluble and diffusible are essentially "surface -reactions." The material diffuses into the cell over its entire surface -with little hindrance. The bacteria are usually distributed throughout -the medium, so that there is very intimate contact in all parts of -the mass which favors rapid chemical action. The following calculation -illustrates this: - - The volume of a coccus 1 mu in diameter is 0.5236 x 10^{-13} cc. - The surface of a coccus 1 mu in diameter is [pi] x 10^{-8} sq. cm. - -It is not uncommon to find in milk on the point of souring -1,000,000,000 bacteria per cc. - -Assuming these to be cocci of 1 mu diameter the volume of these bacteria -in a liter is only 0.05 cc. or in the liter there would be 19999 parts -of milk and only 1 part bacteria. The surface area of these bacteria is -3141.6 sq. cm. With this large surface exposed, it is not strange that -the change from "on the point of souring" to "sour" occurs within an -hour or less. - -Although large numbers of bacteria can and do cause great chemical -changes the amount of material actually utilized for maintenance of -the cell is very slight, infinitesimal almost, and yet is fairly -comparable to that required for man, as is illustrated by the following -computations: - -E. Kohn has shown that certain water bacteria grew well in water to -which there was added per liter 0.000002 mg. dextrose, 0.00000007 mg. -(NH{4}){2}SO{4} and 0.0000000007 mg. (NH{4}){2}HPO{4}. The bacteria -numbered about 1000 per cc. Taking the specific gravity at 1 (a little -too low) the mass of the bacteria in the liter was about 0.001 mg. -Hence the bacteria used 0.002 of their weight of carbohydrate and -0.00007 of ammonium sulphate. A 150-pound (75-kilo) man can live on 375 -g. of sugar (0.005 of his weight) and 52.5 g. of protein (0.0007 of his -weight). From these figures it can be calculated that the man utilizes -about two and a half times as much carbohydrate and about seven times -as much nitrogen as the bacterium, relatively speaking. - - - - -CHAPTER IX. - -PHYSIOLOGICAL ACTIVITIES. - - -The physiological activities of motion, reproduction and metabolism -within the cell have been discussed in previous chapters. The -objects in view in the discussion of the "physiological activities" -(sometimes spoken of as "biochemical" activities) of bacteria in -this and subsequent chapters are to familiarize the student to some -extent with the great range of chemical changes brought about by these -minute organisms, to show their usefulness, even their necessity, and -to impress the fact that it is chiefly by a careful study of these -"activities" that individual kinds of bacteria are identified. It -should always be borne in mind that the bacteria, in bringing about -these changes which are so characteristic in many instances, are simply -engaged in their own life struggle, in securing the elements which -they need for growth, in liberating energy for vital processes, or -occasionally in providing conditions which favor their own development -and hinder that of their competitors. - - -FERMENTATION OF CARBOHYDRATES. - -By this is meant the changes which different carbohydrates undergo when -subjected to bacterial action.[11] - -These changes are marked chiefly by the production of gas or acid. The -former is called "gaseous fermentation" the latter "acid fermentation." -The gases commonly produced are carbon dioxide (CO{2}) hydrogen and -marsh gas (CH{4}). Other gases of the paraffin series may also be -formed as ethane (C{2}H{6}), acetylene (C{2}H{2}), etc. CO{2} and -H are the ones usually formed from sugars by the few gas-forming -bacteria which produce disease, though even here some CH{4} is present. -The common _Bacterium coli_ forms all three, though the CH{4} is in -smallest quantity. - -[Illustration: FIG. 60.--Cylinder to show the formation of gas by -bacteria. The gauge shows 265 pounds. It went beyond 500 pounds.] - -[Illustration: FIG. 61.--A burning natural gas well at night. From a -photograph colored.] - -In the fermentation of the polysaccharids--starch and especially -cellulose and woody material--large amounts of CH{4} occur, particularly -when the changes are due to anaerobic bacteria. This phenomenon may be -readily observed in sluggish streams, ponds and swamps where vegetable -matter accumulates on the bottom. The bubbles of gas which arise when -the mass is disturbed explode if a lighted match is applied to them. - -The author has conducted a number of experiments to demonstrate this -action as follows: Material taken from the bottom of a pond in the -fall after vegetation had died out was packed into a cylinder five -feet long and six inches in diameter, water was added to within about -2 inches of the top. After leaving them open for a few days to permit -all the dissolved oxygen to be used up by the aerobes, the cylinders -were tightly capped and allowed to stand undisturbed. Pressure gauges -reading to 500 lbs. were attached (Fig. 60). At the end of six months -the gauge showed a pressure beyond the limits of the readings on it. -Most of the gas was collected and measured 146 liters. An analysis -of portions collected when about one-half had been allowed to escape -showed the following composition, according to Prof. D. J. Demorest of -the Department of Metallurgy: - - CO{2} 18.6 per cent. - CH{4} 76.1 " - H 1.0 " - N 4.3 " - -In the author's opinion natural gas and petroleum have been formed in -this way[12] (Figs. 61 and 62). - -[Illustration: FIG. 62.--A "flowing" oil well.] - -One of the very few practical uses of the gaseous fermentation of -carbohydrates is in making "salt rising" bread. The "rising" of the -material is due not to yeasts but to the formation of gas by certain -bacteria which are present on the corn meal or flour used in the -process (Fig. 63). - -[Illustration: FIG. 63.--A loaf of "salt rising" bread. The porous -structure is due to the gas formed by bacilli and not by yeasts.] - -Another is in the formation of the "holes" or "eyes" so characteristic -of Swiss and other types of cheese (Fig. 64). - -[Illustration: FIG. 64.--Ohio Swiss cheese. The "eyes" are due to gas -formed by bacteria during the ripening of the cheese.] - -A great many organic acids are formed during the "acid fermentation" -of carbohydrates by bacteria. Each kind of bacterium, as a rule, forms -several different acids as well as other substances, though usually one -is produced in much larger amounts, and the kind of fermentation is -named from this acid. One of the commonest of these acids is lactic. -The "lactic acid bacteria" form a very large and important group and -are indispensable in many commercial processes. In the making of butter -the cream is first "ripened," as is the milk from which many kinds of -cheese are made (Fig. 65). The chief feature of this "ripening" is the -formation of lactic acid from the milk-sugar by the action of bacteria. -A similar change occurs in the popular "Bulgarian fermented milk." The -reaction is usually represented by the equation: - - Milk-sugar. Lactic acid. - C{12}H{22}O{11} + H{2}O + (bacteria) = 4C{3}H{6}O{3} - -It is not probable that the change occurs quantitatively as indicated, -because a number of other substances are also formed. Some of these -are acetic and succinic acids and alcohol. Another industrial use of -this acid fermentation is in the preparation of "sauer kraut." These -bacteria are chiefly anaerobic and grow best in a relatively high salt -concentration. They occur naturally on the cabbage leaves. - -[Illustration: FIG. 65.--A cream ripener. In this apparatus cream is -"ripened," _i.e._, undergoes lactic acid fermentation, preparatory to -making it into butter.] - -In the formation of ensilage (Fig. 66) the lactic acid bacteria play -a very important part, as they do also in "sour mash" distilling, and -in many kinds of natural "pickling." In fact, whenever green vegetable -material "sours" spontaneously, lactic acid bacteria are always present -and account for a large part of the acid. This property of lactic acid -formation is also taken advantage of in the preparation of lactic acid -on a commercial scale in at least one plant in this country. - -[Illustration: FIG. 66.--Filling a silo on the University farm.] - -Acetic acid is another common product of acid fermentation. However, in -vinegar making the acetic acid is not formed directly from the sugar in -the fruit juice by bacteria. The sugar is first converted into alcohol -by yeasts, then the alcohol is _oxidized_ to acid by the bacteria (Fig. -67). The reaction may be represented as follows: - - Dextrose. Ethyl alcohol. Acetic acid. - C{6}H{12}O{6} = 2C{2}H{5}OH + 2CO{2} - - C{2}H{5}OH + O{2} + (bacteria) = CH{3}COOH + H{2}O. - -Butyric acid is generally produced where fermentation of carbohydrates -occurs under _anaerobic_ conditions. Some of the "strong" odor of -certain kinds of cheese is due to this acid which is formed partly -from the milk-sugar remaining in the cheese. Most of it under these -conditions comes from the proteins of the cheese and especially from -the fat (see page 101). - -As has been indicated alcohol is a common accompaniment of most acid -fermentations, as are the esters of acids other than the chief product. -Bacteria are not used in a commercial way to produce alcohol, however, -as the yield is too small. There are some few bacteria in which the -amount of alcohol is prominent enough to call the process an "alcoholic -fermentation" rather than an acid one. In brewing and distilling -industries, _yeasts_ are used to make the alcohol, though molds replace -them in some countries ("sake" and "arrak" from rice). - -[Illustration: FIG. 67.--A vinegar ripener. The tank shown opened at -the side is filled with a special type of beech shavings which thus -provide a very large surface. The apple juice which has been previously -fermented with yeast, which converts the sugar into alcohol, is allowed -to trickle through the openings at the top over the shavings. The -acetic acid bacteria on the shavings rapidly oxidize the alcohol to -acetic acid. The vinegar is drawn off below.] - -Under ordinary conditions the carbohydrate is never completely -fermented, since the accumulation of the product--acid--stops -the reaction. If the acid is neutralized by the addition of an -alkali--calcium or magnesium carbonate is best--then the sugar -may all be split up. Where such fermentation occurs under natural -conditions, the products are further split up, partly by molds and -partly by acid-destroying bacteria into simpler acids and eventually -to carbon dioxide and water, so that the end-products of the complete -fermentation of carbohydrate material in nature are carbon dioxide, -hydrogen, marsh gas, and water. - -In all of these fermentations the bacteria are utilizing the _carbon_ -both as building material and for oxidation and the fermentations -are incidental to this use. As a rule, the acid-forming bacteria can -withstand a higher concentration of acid than the other bacteria that -would utilize the same material, and in a short time crowd out their -competitors or inhibit their growth, and thus have better conditions -for their own existence, though finally their growth is also checked by -the acid. - - -SPLITTING OF FATS. - -The _splitting of fats_ into glycerin and the particular acid or -acids involved may be brought about by bacteria. An illustration -is the development of rancidity in butter at times and the -"strong" odor of animal fats on long keeping and of many kinds of -cheese--"limburger"--in this country. Generally speaking, however, fats -are not vigorously attacked, as is illustrated by the difficulties due -to accumulation of fats in certain types of sewage-disposal works. The -chemical change is represented by the equation: - - Fat. Glycerin. - C{3}H{5}(C{_n_}H{2}{_n_-1}O{2}){3} + 3 H{2}O = C{3}H{5}(OH){3} - Fatty acid. - + 3 (C{_n_}H{2}{_n_}O{2}). - - - - -CHAPTER X. - -PHYSIOLOGICAL ACTIVITIES (CONTINUED). - - -PUTREFACTION OF PROTEINS. - -The word "_putrefaction_" is now restricted to the action of bacteria -on the _complex nitrogen-containing substances_, proteins, and their -immediate derivatives. The process is usually accompanied by the -development of foul odors. - -Bacteria make use of proteins chiefly as a source of nitrogen, but also -as a source of carbon and other elements. Proteins contain nitrogen, -carbon, hydrogen, oxygen, sulphur and frequently phosphorus. Some of -the metals--potassium, sodium, calcium, magnesium, iron and manganese -and the non-metal chlorine--are nearly always associated with them more -or less intimately. Since these bodies are the most complex of natural -chemical substances it follows that the breaking up of the molecule to -secure a part of the nitrogen gives rise to a great variety of products. - -There are marked differences among bacteria in their ability to -attack this class of compounds. Some can break up the most complex -natural proteins such as albumins, globulins, glyco-, chromo-, and -nucleoproteins, nucleins and albuminoid derivatives like gelatin. The -term _saprogenic_ (#sapros# = rotten) is sometimes applied to bacteria -which have this power. These proteins are large-moleculed and not -diffusible, so that the first splitting up that they undergo must occur -outside the bacterial cell. The products of this first splitting may -diffuse into the cell and be utilized there. The bacteria of this class -attack not only these proteins in the natural state or in solution, -but also in the coagulated state. The coagulum becomes softened and -finally changed into a liquid condition. The process when applied to -the casein of milk is usually called "digestion," also when coagulated -blood serum is acted on. In the latter case the serum is more commonly -said to be "liquefied" as is the case when gelatin is the substance -changed. Most of these bacteria have also the property of coagulating -or curdling milk in an alkaline medium, and then digesting the curd. -A second class of bacteria has no effect on the complex proteins just -mentioned but readily attacks the products of their first splitting, -_i.e._, the proteoses, peptones, polypeptids and amino-acids. They are -sometimes called _saprophilic_ bacteria. - -Other bacteria derive their nitrogen from some of the products of the -first two groups, and still further break down the complex protein -molecule. Under normal conditions these various kinds of bacteria -all occur together and thus mutually assist one another in what is -equivalent to a symbiosis or rather a metabiosis, a "successive -existence," one set living on the products of the other. The result -is the complete splitting up of the complete protein molecule. A part -of the nitrogen is built up into the bodies of the bacteria which are -using it as food. A part is finally liberated as _free nitrogen_ or as -_ammonia_ after having undergone a series of transformations many of -which are still undetermined. - -One class of compounds formed received at one time much attention -because they were supposed to be responsible for a great deal of -illness. These are the "ptomaines," basic nitrogen compounds of -definite composition--amines--some few of which are poisonous, most of -them not. The basic character of ptomaines may be understood if they be -regarded as made up of one or more molecules of ammonia in which the -hydrogen has been replaced by alkyl or other radicals. Thus ammonia -(NH{3}) may be represented as - - /H - / - N--H - \ - \H. - -The simplest ptomaine is - - /CH{3} - / - N--H - \ - \H, - -in which one H is replaced by methyl, methylamine, a gaseous ptomaine. -With two hydrogens replaced by methyl, - - /CH{3} - / - N--CH{3} - \ - \H, - -dimethylamine, also a gas at ordinary temperature, is formed. -Trimethylamine, - - /CH{3} - / - N--CH{3} - \ - \CH{3}, - -a liquid, results when three hydrogens are similarly replaced. -All three of these occur in herring brine and are responsible -for the characteristic odor of this material. Putrescin and -cadaverin--tetramethylene--diamine, and pentamethylenediamine -respectively--occur generally in decomposing flesh, hence the names. -They are only slightly poisonous. One of the highly poisonous ptomaines -is neurin C{5}H{13}NO or C{2}H{3}N(CH{3}){3}OH = trimethyl-vinyl -ammonium hydroxide. This is a stronger base than ammonia, liberating -it from its salts. Numerous other ptomaines have been isolated and -described. These bodies were considered for a long time to be the -cause of various kinds of "meat poisoning," "ice cream poisoning," -"cheese poisoning," etc. It is true that they may sometimes cause these -conditions, but they are very much rarer than the laity generally -believe. Most of the "meat poisonings" in America are due, not to -ptomaines, but to infections with certain bacilli of the _Bacterium -enteritidis_ group. Occasionally a case of poisoning by the true toxin -(see Chapter XII) of _Clostridium botulinum_ occurs, and in recent -years has become entirely too common due to insufficient heating of -canned goods. _The boiling of such material will destroy this toxin. -The safest rule to follow is not to eat any canned material that shows -any departure from the normal in flavor, taste or consistency._ - -As ptomaines result from the putrefaction of proteins, so they are -still further decomposed by bacteria and eventually the nitrogen is -liberated either as free nitrogen or as ammonia. - -Another series of products are the so-called aromatic compounds--phenol -(carbolic acid), various cresols, also indol and skatol or methyl indol -(these two are largely responsible for the characteristic odor of human -feces). All of these nitrogen compounds are attacked by bacteria and -the nitrogen is eventually liberated, so far as it is not locked up in -the bodies of the bacteria, as free nitrogen or as ammonia. - -The carbon which occurs in proteins accompanies the nitrogen in many of -the above products, but also appears in nitrogen-free organic acids, -aldehydes and alcohols which are all eventually split up, so that the -carbon is changed to carbon dioxide or in the absence of oxygen partly -to marsh gas. - -The intermediate changes which the sulphur in proteins undergoes are -not known, but it is liberated as sulphuretted hydrogen (H{2}S) or as -various mercaptans (all foul-smelling), or is partially oxidized to -sulphuric acid. Some of the H{2}S and the sulphur of the mercaptans -are oxidized by the sulphur bacteria to free sulphur and finally to -sulphuric acid. - -Phosphorus is present especially in the nucleoproteins and nucleins. -Just what the intermediate stages are, on whether there are any, so -far as the phosphorus is concerned, in the splitting up of nucleic -acid by bacterial action is not determined. The phosphorus may occur -as phosphoric acid in such decompositions, or when the conditions are -anaerobic, as phosphine (PH{3}), which burns spontaneously in the air to -phosphorus pentoxide (P{2}O{5}), and water.[13] - -The hydrogen in proteins appears in the forms above indicated: H{4}C, -H{3}N, H{3}P, H{2}S, H{2}O and as free H. The oxygen as CO{2} and H{2}O. - -In the breaking down of the complex protein molecule even by a single -kind of bacterium there is not a perfect descending scale of complexity -as might be supposed from the statement that there result proteoses, -peptones, polypeptids, amino-acids. These substances do result, but at -the time of their formation simpler ones are formed also, even CO{2}, -NH{3} and H{2}S. It appears that the entire molecule is shattered in -such a way that less complex proteins are formed from the major part, -while a minor portion breaks up completely to the simplest combinations -possible. A more complete knowledge of these decompositions will aid -in the further unravelling of the structure of proteins. The presence -or absence of free oxygen makes a difference in the end-products, as -has been indicated. There are bacteria which oxidize the ammonia to -nitric acid and the H{2}S to sulphuric acid. (See Oxidation, Chapter -XI.) Bacteria which directly oxidize phosphorus compounds to phosphoric -acid have not been described. It does not seem that such are necessary -since this is either split off from nucleic acid or results from the -spontaneous oxidation of phosphine when this is formed under anaerobic -conditions. - -Not only are proteins decomposed as above outlined, but also their -waste products, that is, the form in which their nitrogen leaves the -animal body. This is largely urea in mammals, with much hippuric acid -in herbivorous animals and uric acid in birds and reptiles. These -substances yield NH{3}, CO{2} and H{2}O with a variety of organic acids -as intermediate products in some cases. The strong odor of ammonia -in stables and about manure piles is the everyday evidence of this -decomposition. - -Where the putrefaction of proteins occurs in the soil with moderate -amounts of moisture and free access of air a large part of the products -is retained in the soil. Thus the ammonia and carbon dioxide in the -presence of water form ammonium carbonate; the nitric, sulphuric and -phosphoric acids unite with some of the metals which are always present -to form salts. Some of the gases do escape and most where the oxygen -supply is least, since they are not oxidized. - -The protein-splitting reactions afford valuable tests in aiding in -the recognition of bacteria. In the study of pathogenic bacteria the -coagulation and digestion of milk, the digestion or liquefaction -of blood serum, the liquefaction of gelatin and the production of -indol and H{2}S are those usually tested for. In dairy bacteriology -the coagulation of milk and the digestion of the casein are common -phenomena. Most bacteria which liquefy gelatin also digest blood serum -and coagulate and digest milk, though there are exceptions. In soil -bacteriology the whole range of protein changes is of the greatest -importance. - -[Illustration: - - +----<----------------------+ - | | - | | - v | - _Nuclein of | - animal cells_ | - | | - | | - v | - | | - _Decomposition | - bacteria_ | - | \ | - | \ ^ - v \ | - _Unknown _PH{3} | - P oxidizes | - compounds_ spontaneously | - | to_ | - | / | - v / | - _Phosphoric | - acid_ | - | | - | | - v | - _Phosphates | - in the | - soil_ | - | ^ - | | - v | - _Green | - plants_ | - | | - | | - v | - _Nuclein of | - plant | - cells_ | - | | - | | - v | - _Animals_----->----------------+ - -FIG. 68.--Diagram to illustrate the circulation of phosphorus through -the agency of bacteria.] - -[Illustration: - - _Fats and - various C <---------------------------+ - compounds_ | - | | - | | - v | - _Decomposition | - bacteria_ | - | | - | | - v | - _Various | - C | - compounds | - eventually ^ - to_ | - | | - | | - v | - _CO{2} <---------------+ | - in | | - the air_<-+ | | - | | | | - | | _Plant respiration_ | - v | | | - _Green | | | - plants_---+ | | - | | | - | | | - v | | - _Carbohydrates, | | - fats and | | - other C compounds_ | | - | | | - | | | - v | | - _Animals_---->_Animal respiration_ | - | | - | | - +---->----------------------------+ - -FIG. 69.--Diagram to illustrate the circulation of carbon through the -agency of bacteria.] - -[Illustration: - - +---------------<-----------------+ - | | - _Dead animal | - protein_ | - | ^ - | | - v | - _Free N taken <-----_Decomposition <-------------+ | - up by free living bacteria_ <----------+ | | - N absorbers and | | | | - root tubercle | | | | - bacteria_ v | | | - | _NH{3} compounds | | | - | in soil_ | | | - | | | | | - | | | | | - | v | | | - | _Nitrite bacteria_ | | | - | | | | | - | | | | | - | v | | | - | _Nitrites_ | | | - | | | | | - | | | | | - | v | | | - | _Nitrate bacteria_ | | | - | | ^ ^ ^ - | | | | | - | v | | | - | _Nitrates in soil_ | | | - | | | | | - | | | | | - | v | | | - | _Green plants_ | | | - | | | | | - | | | | | - | v _Dead plant | | - +------->_Plant protein_----> protein_ | | - | | ^ - | | | - v _Animal waste, | - _Animals_---------->urea, etc._ | - | | - | | - +-------->------------------------+ - -FIG. 70.--Diagram to illustrate the circulation of nitrogen through the -agency of bacteria.] - -[Illustration: - - +--------------<---------------+ - | | - | | - v | - _Dead animal | - protein_ | - | | - | | - v | - _Decomposition bacteria_ | - | | | - | | | - v ^ | - _H{2}S_ | | - | | | - | | | - v | | - _Sulphur bacteria_ | | - | | | - | | | - v | | - _Free S_ | ^ - | | | - | | | - v | | - _Sulphur bacteria_ | | - | | | - | | | - v | | - _Sulphates | | - in the | | - soil_ | | - | | | - | | | - v | | - _Green plants_ ^ | - | | | - | | | - v | | - _Plant protein_----->_Dead plant | - | protein_ | - | | - v | - _Animals_ | - | | - | | - +-------------->---------------+ - -FIG. 71.--Diagram to illustrate the circulation of sulphur through the -agency of bacteria.] - -The three physiological activities already discussed explain how -bacteria break down the chief complex, energy-rich substances ---carbohydrates, fats and proteins which constitute the bulk of the -organic material in the bodies of plants and animals, as well as -the waste products of the latter--into energy-free compounds like -carbon dioxide, water, ammonia, nitric, sulphuric and phosphoric -acids--mineralize them, as is frequently said. By so doing the bacteria -act as the great scavengers of nature removing the dead animal -and vegetable matter of all kinds which but for this action would -accumulate to such an extent that all life, both on land and in the -water, must cease. It is further to be noted that not only is all this -dead organic matter removed; but it is converted into forms which are -again available for plant growth. Carbon dioxide forms the source of -the carbon in all green plants, hence in all animals; the sulphates and -phosphates are likewise taken up by green plants and built up again -into protein compounds; the ammonia is not directly available to green -plants to any large extent but is converted by the nitrifying bacteria -(Chapter XI) into nitrates which is the form in which nitrogen is -assimilated by these higher types. Even the free nitrogen of the air -is taken up by several kinds of bacteria, the symbiotic "root-tubercle -bacteria" of leguminous and other plants, and some free-living forms, -and made available. Hence bacteria are indispensable in nature, -especially in keeping up the circulation of nitrogen. They are also of -great service in the circulation of carbon, sulphur and phosphorus. -Though some few kinds cause disease in man and animals, if it were not -for the saprophytic bacteria above outlined, there could be no animals -and higher plants to acquire these diseases. - - - - -CHAPTER XI. - -PHYSIOLOGICAL ACTIVITIES (CONTINUED). - - -PRODUCTION OF ACIDS. - -The production of organic acids has been sufficiently discussed in -preceding chapters. It should be noted that not only these in great -variety are produced by bacteria but that under certain conditions -mineral acids, such as nitric, sulphuric and phosphoric may be formed -(see Oxidation, p. 114). Acid production is of great value in the -identification of bacteria in dairy and soil work and in connection -with certain types of pathogenic bacteria. - - -GAS PRODUCTION. - -It will be sufficient merely to enumerate collectively the various -gases mentioned in preceding paragraphs and to state that those -commonly observed in the study of pathogenic bacteria are the first six -mentioned. Most of them come in in dairy work either in the study of -bacteria causing milk and cheese "failures" or as affecting the flavors -of butter or cheese. In the study of soil organisms, any or all of -them are liable to be of importance. The gases are: CO{2}, H, CH{4}, -N, NH{3}, H{2}S, gaseous mercaptans, gaseous ptomaines, volatile fatty -acids, ethereal salts or esters and others, both of pleasant and of -foul odor, but of unknown composition. - - -PRODUCTION OF ESTERS. - -The production of esters, as mentioned in Chapters IX and X, of various -alcohols and aldehydes are activities which are sometimes of value in -the study of bacteria, but need not be further discussed. - - -PRODUCTION OF "AROMATIC" COMPOUNDS. - -These have been mentioned in discussing the putrefaction of proteins, -as indol, skatol, phenol and various cresols. Of these only the first -is ordinarily tested for in the study of bacteria, though others of the -group become of value in certain special cases. - -[Illustration: FIG. 72.--Culture of phosphorescent bacteria in an -Ehrlenmeyer flask photographed by their own light. Time of exposure -twelve hours. (Molisch, from Lafar.)] - - -PHOSPHORESCENCE OR PHOTOGENESIS. - -This is a most interesting phenomenon associated with the growth of -some bacteria. The "fox fire" frequently seen on decaying wood which -is covered with a slimy deposit is most commonly due to bacteria, -though also to other fungi. Phosphorescent bacteria are very common -in sea water, hence they are frequently found on various sea foods, -especially when these are allowed to decompose, such as fish, oysters, -clams, etc. The light is due to the conversion of the energy of -unknown easily oxidizable compounds directly into _visible_ radiant -energy through oxidation without appreciable quantities of heat. -The light produced may be sufficient to tell the time on a watch in -absolute darkness, and also to photograph the growths with their own -light, but only after several hours' exposure (Fig. 72). None of the -phosphorescent bacteria so far discovered produce disease in the higher -animals or man. - - -PRODUCTION OF PIGMENT OR CHROMOGENESIS. - -One of the most striking results of bacterial activity is this -phenomenon. The particular color which results may be almost any one -throughout the range of the spectrum, though shades of yellow and of -red are of more frequent occurrence. - -In the red sulphur bacteria the "bacteriopurpurin" which they contain -appears to serve as a true respiratory pigment in a manner similar to -the chlorophyl in green plants, except that these bacteria oxidize -H{2}S in the light as a source of energy instead of splitting up CO{2}. -The red pigment produced by certain bacteria has been shown to have -a capacity for combining with O resembling that of hemoglobin, and -some investigators have believed that such bacteria do store O in this -way for use when the supply is diminished. With these few exceptions -the pigments seem to be merely by-products of cell activity which are -colored and have no known function. - -The red sulphur bacteria above mentioned and one or two other kinds -retain the pigments formed within the cell. Such bacteria are called -_chromophoric_ as distinguished from the _chromoparic_ bacteria whose -pigment lies outside the cell. - -The chemical composition of no bacterial pigment has been determined -up to the present. Some are soluble in water, as shown by the -discoloration of the substances on which they grow. Others are not -soluble in water but are in alcohol, or in some of the fat solvents -as ether, chloroform, benzol, etc. These latter are probably closely -related to the _lipochromes_ or "fat colors" of higher plants and -animals. Attempts have been made to render the production of pigments -a still more reliable means of identification of species of bacteria -through a careful examination of the spectra of their solutions, but -such study has not as yet led to any valuable practical results. - -The production of pigment depends on the same general factors which -determine the growth of the organism but does _not necessarily run -parallel_ with these. It is especially influenced by the oxygen -supply (only a very few organisms are known which produce pigment -anaerobically--_Spirillum rubrum_ is one); by the presence of -certain food substances (starch, as in potato, for many bacteria -producing yellow and red colors; certain mineral salts, as -phosphates and sulphates, for others); by the temperature (many -bacteria cease to produce color at all if grown at body temperature, -37 deg.--_Erythrobacillus prodigiosus_--or if grown for a longer time at -temperatures a few degrees higher). - - -REDUCING ACTIONS. - -Reduction of nitrates to nitrites or to ammonia or even to free -nitrogen is brought about by a great many different kinds of bacteria. -In many instances this phenomenon is due to a lack of free oxygen, -which is obtained by the bacteria from these easily reducible salts. -In other cases a portion of the nitrogen is removed to be used as food -material in the building up of new protein in the bacterial cell. This -latter use of the nitrogen of nitrates by bacteria might theoretically -result in considerable loss of "available nitrogen" in the soil as has -actually been shown in a few experiments. The reduction of nitrates as -above mentioned would also diminish this supply, but probably neither -of these results has any very great practical effect on soil fertility. -The building up of protein from these mineral salts by bacteria in the -intestines of herbivorous animals has been suggested by Armsby as a -considerable source of nitrogenous food, and this suggestion appears -possible. - -The liberation of nitrogen from nitrates or nitrites, either as free -nitrogen or as ammonia, is spoken of as "dentrification," though this -term was formerly applied to such liberations, from compounds of -nitrogen generally even from proteins. - -Certain bacteria may also reduce sulphates and other sulphur compounds -to H{2}S, a phenomenon frequently observed in sewage and likewise of -importance in the soil. It is possible that phosphates may be similarly -reduced.[14] Further and more careful study of the reducing actions of -bacteria is needed. - - -OXIDATION. - -As has been stated in discussing the respiration of bacteria (Chapter -VIII) most of these organisms gain their energy through the oxidation -of carbon in various forms, chiefly organic, so that CO{2} is a product -of the activity of nearly all bacteria. Some few oxidize CO to CO{2}, -others CH{4} and other paraffins to CO{2} for this purpose. One class of -bacteria even oxidizes H in small amounts for its energy and uses the -carbon dioxide of the air or traces of organic carbon in the air as a -source of carbon for "building" purposes. - -One of the familiar oxidations of organic carbon is that of the acetic -acid bacteria in the making of vinegar. These oxidize the alcohol -which results from the action of yeast to acetic acid according to the -formula CH{3}CH{2}OH + O{2} = CH{3}COOH + H{2}O (see Fig. 67). - -Of the various phenomena of oxidation due to bacteria, the formation of -nitrites and nitrates has the greatest practical importance, since it -is by this means that the ammonia which results from the decomposition -of animal and vegetable tissue and waste products is again rendered -available to green plants as food in the form of nitrates. Practically -all the nitrates found in nature, sometimes in large quantities, are -formed in this way. There are two distinct kinds of bacteria involved. -One, the nitrous bacteria, oxidizes the ammonia to nitrous acid -which forms nitrites with bases, and the other, the nitric bacteria, -oxidizes the nitrous to nitric acid, giving nitrates with bases. A -striking peculiarity of these two classes of organisms is that they -may live entirely on inorganic food materials, are proto-autotrophic, -prototrophic for oxygen (aerobic) and autotrophic for the other -elements. Their carbon is derived from CO{2} or carbonates. The -importance of such organisms in keeping up the supply of nitrates in -the soil can scarcely be overestimated. - -[Illustration: FIG. 73.--Sprinkling filters of the Columbus -sewage-disposal plant--devices which provide a good supply of oxygen -for the bacteria that oxidize the organic matter in the sewage.] - -The oxidation of the H{2}S, which is formed in the putrefaction -of proteins, to free S by the sulphur bacteria and the further -oxidation of this free S to sulphuric acid, and of the phosphorus, so -characteristic of the nucleins, to phosphoric acid have been referred -to. These activities of bacteria are of great value in the soil. -Doubtless the commercial "phosphate rock" owes its origin to similar -bacterial action in ages past. - -The oxidation of H{2}S to free S may be an explanation of the origin of -the great deposits of sulphur which are found in Louisiana and along -the Gulf coast. These deposits occur in the same general regions as -natural gas and oil. The sulphur might have been derived from the same -organic material carried down by the Mississippi which yielded the oil -and gas.[15] - -A purposeful utilization of the oxidizing power of bacteria is in -"contact beds," "sprinkling filters" and "aerated sludge tanks" in -sewage disposal works. In these instances the sewage is thoroughly -mixed with air and brought in contact with large amounts of porous -material so as to expose an extensive surface for oxidation (Fig. 73). - -[Illustration: FIG. 74.--One of the University hot beds.] - - -PRODUCTION OF HEAT. - -A direct result of the oxidizing action of bacteria is the production -of heat. Under most conditions of bacterial growth this heat is not -appreciable. It may become well marked. The "heating" of manure is one -of the commonest illustrations. The temperature in such cases may reach -70 deg.. The heating of hay and other green materials is due chiefly to -bacterial action. This heating may lead to "spontaneous combustion." -The high temperatures (60 deg. to 70 deg.) favor the growth of thermophil -bacteria which cause a still further rise. The heat dries out the -material, portions of which are in a state of very fine division due -to the disintegrating action of the organisms. The hot, dry, finely -divided material oxidizes so rapidly on contact with the air that it -ignites. - -A practical use of heat production by bacteria is in the making of "hot -beds" for forcing vegetables (Fig. 74). - - -ABSORPTION OF FREE NITROGEN. - -[Illustration: FIG. 75.--Root tubercles on soy bean. x 3/7.] - -This is likewise one of the most important practical activities of -certain types of bacteria present in the soil. The ability of plants -of the legume family to enrich the soil has been known and taken -advantage of for centuries, but it is only about thirty years since -it was demonstrated that this property is due to bacteria. These -plants, and several other kinds as well, have on their roots larger or -smaller nodules (Fig. 75) spoken of as "root tubercles" which are at -certain stages filled with bacteria. When conditions are favorable, -these bacteria live in symbiotic relationship with the plant tissues, -receiving carbonaceous and other food material from them and in return -furnishing nitrogenous compounds to the plant. This nitrogenous -material is built up from free nitrogen absorbed from the air by the -bacteria. The utilization of this peculiar property through the proper -cultivation of clover, alfalfa, soy beans and other legumes is one of -the best ways of building up and maintaining soil fertility in so far -as the nitrogen is concerned. The technical name of these bacteria is -_Rhizobium leguminosarum_. - -[Illustration: FIG. 76.--Free-living nitrogen absorbing bacteria -"Azotobacter." Note their large size as compared with other bacteria -shown in this book.] - -There are also types of "free-living," as distinguished from these -symbiotic, bacteria which absorb the free nitrogen of the air and aid -materially in keeping up this supply under natural conditions. One -of the most important of these types is the aerobic "Azotobacter" -(Fig. 76), while another is the anaerobic _Clostridium pasteurianum_. -The nitrogen which is absorbed is built up into the protein material -of the cell body and this latter must in all probability be "worked -over" by various types of decomposition bacteria and by the nitrous -and nitric organisms and be converted into utilizable nitrates just -as other protein material is, as has been discussed in Chapter X. At -any rate there is as yet no definite knowledge of any other method of -transformation. Up to the present no intentional practical utilization -of this valuable property of these free-living forms has been made. - -=Nitrogen Nutrition of Green Plants.=--It is the belief of botanists -that green plants obtain their nitrogen chiefly in the form of -nitrates, though ammonium salts may be utilized to some extent by -certain plants at least. Exceptions to this general rule are those -plants provided with root tubercles (and the bog plants and others -which have mycorrhiza?). These plants obtain their nitrogen in the -form of organic compounds made for them by the bacteria growing in -the tubercles. That nitrogen circulates throughout the structure of -plants in organic combination is certain. There does not appear to -be any reason why similar compounds which are soluble and diffusible -(amino-acids?) should not be taken up through the roots of plants and -utilized as such. _It seems to the author that this is very probably -the case._ Arguments in favor of this view are: (1) The nitrogen -nutrition of leguminous and other plants with root nodules. (2) The -close symbiosis between "Azotobacter" and similar nitrogen-absorbing -bacteria and many species of algae in sea water at least. (3) The -vigorous growth of plants in soils very rich in organic matter, which -inhibits the production of nitrates by the nitrous-nitric bacteria -when grown in culture, and possibly (?) in the soil, so that nitrates -may not account for the vigorous growth. (4) The effect of nitrate -fertilizers is to add an amount of nitrogen to the crop much in excess -of the amount added as nitrate. (5) The most fertile soils contain -the largest numbers of bacteria. The doctrine that nitrates furnish -the only nitrogen to plants was established before the activities of -bacteria in the soil were suspected, and, so far as the author is -aware, has not been supported by experiments under conditions rigidly -controlled as to sterility. - -It would seem that one of the chief functions of soil bacteria is to -prepare soluble organic compounds of nitrogen for the use of green -plants and thus to make a "short cut" in the nitrogen cycle (p. 107), -as now believed in, direct from the "decomposition bacteria" to green -plants. - -Experiments have been made by different observers in growing seedling -plants of various kinds in water culture with one or in some cases -several of the amino-acids as sources of nitrogen. Most of these -experiments were disappointing. Plant proteins are not so different -from animal proteins, or plant protoplasm (apart from the chlorophyl -portions of plants) from animal protoplasm as to lead one to suppose -that it could be built up from one or two amino-acids any more than -animal protoplasm can. The author is strongly convinced that this -subject should be thoroughly investigated. It will require careful -experimentation and perhaps rather large funds to provide the amounts -of amino-acids that would probably be needed, but might result in a -decided change in our ideas of soil fertility, and especially in the -use of nitrogen fertilizers. - - - - -CHAPTER XII. - -PHYSIOLOGICAL ACTIVITIES (CONTINUED). - - -PRODUCTION OF ENZYMES. - -Most of the physiological activities of bacteria which have been -discussed are due to the action of these peculiar substances, so that -a knowledge of their properties is essential. This knowledge cannot as -yet be exact because no enzyme has, up to the present, been obtained in -a "pure state," though it must be admitted that there are no certain -criteria which will enable this "pure state" to be recognized. It was -formerly thought that they were protein in nature, but very "pure" and -active enzymes have been prepared which did not give the characteristic -protein reactions, so this idea must be abandoned. That they are large -moleculed colloidal substances closely related to the proteins in many -respects must still be maintained. There are certain characteristics -which belong to enzymes, though no one of them exclusively. These may -be enumerated as follows: - -1. Enzymes are _dead_ organic chemical substances. - -_Dead_ is used in the sense of non-living, never having lived, not in -the sense of "ceased to be alive." - -2. They are always produced by _living_ cells: - -Sometimes as active enzymes, sometimes as _pro-enzymes_ or _zymogens_ -which are converted into enzymes outside the cell by acids, other -inorganic substances or other enzymes. - -3. They produce very great chemical changes without themselves being -appreciably affected. - -Enzymes will not continue to act indefinitely, but are used up in the -process (combination with products?). The amount of change is so great -in proportion to the amount of enzyme that the above statement is -justified in the relative sense. Thus a milk-curdling enzyme has been -prepared that would precipitate 100,000,000 times its own weight of -caseinogen. - -4. Their action is specific in that each enzyme acts on one kind of -chemical substance only, and the products are always the same. - -The substance may be combined with a variety of other chemical -substances so that the action appears to be on several, but in reality -it is on a definite group of molecules in each instance. For example, -emulsin attacks several different glucosides but always sets free -dextrose from them. - -5. The action is inhibited and eventually stopped, and in some cases -the enzyme is destroyed by an accumulation of the products of the -action. If the products are removed, the action will continue, if the -enzyme is not destroyed. This effect is explained partly because the -enzyme probably combines with some of the products, since it does -not act indefinitely, and partly because of the reversibility of the -reaction. - -6. Like many chemical reactions those of enzymes are reversible, that -is, the substance broken up may be reformed by it from the products -produced in many instances. Thus: - - maltose + maltase <-- glucose + glucose + maltase. - --> - fat + lipase <-- glycerin + fatty acid + lipase. - --> - -7. The presence of certain mineral salts seems to be essential for -their action. These and other substances which are necessary are -sometimes called _co-enzymes_. A salt of calcium is most favorable for -a great many. - -8. They may be adsorbed like other colloids by "shaking out" with -finely divided suspensions like charcoal or kaolin, or by other -colloids like aluminum hydroxide or proteins. - -9. When properly introduced into the tissues or blood of an animal, -they cause the body cells to form _anti-enzymes_ which will prevent the -action of the enzyme (see Chapter XXVII). - -10. Though inert, they show many of the characteristics of living -organisms, that is - -(_a_) Each enzyme has an optimum, a maximum and a minimum temperature -for its action. - -All chemical reactions have such temperature limits, the distinction -is that for enzymes as for living substance the _range_ is relatively -narrow. - -(_b_) High temperatures destroy enzymes. All in water are destroyed -by boiling in time and most at temperatures considerably below the -boiling-point. When dry, many will withstand a higher degree of heat -than 100 deg. before they are destroyed. - -(_c_) Temperatures below the minimum stop their action, though they are -not destroyed by cold. - -(_d_) Many poisons and chemical disinfectants (Chapter XIV) which kill -living organisms will also stop the action of enzymes, though generally -more of the substance is required, so that it is possible to destroy -the living cells by such means and yet the action of the enzyme will -continue. - -(_e_) Most enzymes have an optimum reaction of medium either acid, -alkaline or neutral, depending on the particular enzyme, though some -few seem to act equally well within a considerable range on either side -of the neutral point. - -_The final test for an enzyme is the chemical change it brings about in -the specific substance acted on._ - -The most prominent characteristic of enzymes is that they bring about -very great chemical changes without themselves being appreciably -affected. This property is also shown by many inorganic substances -which are spoken of as "catalytic agents" or "catalyzers" so that -enzymes are sometimes called "organic catalyzers." The function of -catalytic agents seems to be to hasten the rate of a reaction which -would occur spontaneously, though in a great many cases with extreme -slowness. - -Just how enzymes act is not certain and probably will not be until -their composition and constitution are known. Most probably they form a -combination with the substance acted on (_the substrate_) as a result -of which there is a rearrangement of the atoms in such a way that new -compounds are formed, nearly always at least two, and the enzyme is at -the same time set free. It is rather remarkable that chiefly optically -active substances are split up by enzymes and where two modifications -exist it is usually the dextro-rotatory one which is attacked. No -single enzyme attacks both. This probably means that the structure of -the enzyme corresponds to that of the substrate, "fits it as a key fits -a lock," as Emil Fischer says. - -The production of enzymes is by no means restricted to bacteria since -all kinds of living cells that have been investigated have been shown -to produce them and presumably _all_ living cells do. Hence the -number of different kinds of enzymes and of substances acted upon -is practically unlimited. Nevertheless they may be grouped into a -comparatively few classes based on the general character of the change -brought about by them. - -I. Class I is the so-called _"splitting" enzymes_ whose action is -for the most part hydrolytic, that is, the substance takes up water -and then splits into compounds that were apparently constituents of -the original molecule. As examples may be mentioned _diastase_, the -enzyme first discovered, which changes starch into a malt-sugar, hence -is more commonly called _amylase_[16] (starch-splitting enzyme); -_invertase_,[16] which splits cane-sugar into dextrose and levulose: -C{12}H{22}O{11} + H{2}O = C{6}H{12}O{6} + C{6}H{12}O{6}. _Lipase_[16] -or a fat-splitting enzyme, which decomposes fat into glycerin and fatty -acid: - - C{3}H{5}(OC{n}H{2}{n-1}O){3} + 3H{2}O = C{3}H{5}(OH){3} - Fat Glycerin - - + 3C{n}H{2}{n}O{2}. - Fatty acid - -_Proteases_, which split up proteins into proteoses and peptones. - -Other classes of "splitting enzymes" break up the products of complex -protein decomposition, such as proteoses, peptones and amino-acids. A -variety of the "splitting enzymes" is the group of - -_"Coagulases" or coagulating enzymes_ as the rennet (lab, chymosin) -which curdles milk; fibrin ferment (thrombin, thrombase) which causes -the coagulation of blood. These apparently act by splitting up a -substance in the fluids mentioned, after which splitting one of the -new products formed combines with other compounds present (usually a -mineral salt, and in the cases mentioned a calcium salt) to form an -insoluble compound, the curd or coagulum. - -_Another variety is the "activating" enzymes or "kinases"_ such as the -enterokinase of the intestine. The action here is a splitting of the -_zymogen_ or mother substance or form in which the enzyme is built up -by the cell so as to liberate the active enzyme. - -Of a character quite distinct, from the splitting enzymes are - -II. The _zymases_. Their action seems to be to cause a "shifting on -rearrangement of the carbon atoms" so that new compounds are formed -which are not assumed to have been constituents of the original -molecule. Most commonly there is a closer combination of the carbon -and oxygen atoms, frequently even the formation of CO{2} so that -considerable energy is thus liberated. Examples are the _zymase_ or -_alcoholase_ of yeast which converts sugar into alcohol and carbon -dioxide; C{6}H{12}O{6} = 2C{2}H{6}O + 2CO{2}: also _urease_, which -causes the change of urea into ammonia and carbon dioxide. Another -common zymase is the _lactacidase_ in lactic acid fermentation. - -III. _Oxidizing enzymes_ also play an important part in many of the -activities of higher plants and animals. Among the bacteria this action -is illustrated by the formation of nitrites, nitrates and sulphates and -the oxidation of alcohol to acetic acid as already described. - -IV. _Reducing enzymes_ occur in many of the dentrifying bacteria and -in those which liberate H{2}S from sulphates. A very widely distributed -reducing enzyme is "catalase" which decomposes hydrogen peroxide. - -As previously stated, most of the physiological activities of -bacteria are due to the enzymes that they produce. It is evident -that for action to occur on substances which do not diffuse into the -bacterial cell--starches, cellulose, complex proteins, gelatin--the -enzymes must _pass out_ of the bacterium and consequently may be -found in the surrounding medium. Substances like sugars, peptones, -alcohol, which are readily diffusible, may be acted on by enzymes -_retained within_ the cell body. In the former case the enzymes are -spoken of as extra-cellular or "_exo-enzymes_," and in the latter as -intra-cellular or "_endo-enzymes_." The endo-enzymes and doubtless also -the exo-enzymes may after the death of the cell digest the contents -to a greater or less extent and thus furnish substances that are -not otherwise obtainable. This process of "self-digestion" is known -technically as "_autolysis_." - -A distinction was formerly made between "organized" and "unorganized -ferments." The former term was applied to the minute living organisms, -bacteria, yeasts, molds, etc., which bring about characteristic -fermentative changes, while the latter term was restricted to enzymes -as just described. Since investigation has shown that the changes -ascribed to the "organized ferments" are really due to their enzymes, -and that enzymes are probably formed by all living cells, the -distinction is scarcely necessary at present. - - -PRODUCTION OF TOXINS. - -The injurious effects of pathogenic bacteria are due in large part to -the action of these substances, which in many respects bear a close -relationship to enzymes. The chemical composition is unknown since -no toxin has been prepared "pure" as yet. It was formerly thought -that they were protein in character, but very pure toxins have been -prepared which failed to show the characteristic protein reactions. It -is well established that they are complex substances, of rather large -molecule and are precipitated by many of the reagents which precipitate -proteins. Toxins will be further discussed in Chapter XXVII. It will -be sufficient at this point to enumerate their chief peculiarities in -order to show their marked resemblance to enzymes. - -1. Toxins are _dead_ organic chemical substances. - -2. They are always produced by _living_ cells. - -3. They are active poisons in _very small quantities_.[17] - -4. Their action is specific in that each toxin acts on a particular -kind of cell. The fact that a so-called toxin acts on several -different kinds of cells, possibly indicates a mixture of several -toxins, or action on the _same substance_ in the cells. - -5. Toxins are very sensitive to the action of injurious agencies such -as heat, light, etc., and in about the same measure that enzymes are, -though as a rule they are somewhat more sensitive or "labile." - -6. Toxins apparently have maxima, optima, and minima of temperature for -their action, as shown by the destructive effect of heat and by the -fact that a frog injected with tetanus toxin and kept at 20 deg. shows no -indication of poison, but if the temperature is raised to 37 deg., symptoms -of poisoning are soon apparent. Cold, however, does not destroy a toxin. - -7. When properly introduced into the tissues of animals they cause the -body cells to form antitoxins (Chapter XXVII) which are capable of -preventing the action of the toxin in question. - -8. _The determining test for a toxin is its action on a living cell._ - -It is true that enzymes are toxic, as are also various foreign -proteins, when injected into an animal, but in much larger doses than -are toxins. - -A marked difference between enzymes and toxins is that the former may -bring about a very great chemical change and still may be recovered -from the mixture of substances acted on and produced, while the toxin -seems to be permanently used up in its toxic action and cannot be -so recovered. _Toxins seem very much like enzymes whose action is -restricted to living cells._ - -Just as enzymes are probably produced by all kinds of cells and not by -bacteria alone, so toxins are produced by other organisms. Among toxins -which have been carefully studied are _ricin_, the poison of the castor -oil plant (_Ricinus communis_); _abrin_ of the jequirity bean (_Abrus -precatorius_); _robin_ of the common locust (_Robinia pseudacacia_); -poisons of spiders, scorpions, bees, fish, snakes and salamanders. - -It has been stated that some enzymes are thrown out from the cell and -others are retained within the cell. The same is true of toxins, hence -we speak of _exo-toxins_ or toxins excreted from, and _endo-toxins_ -or toxins retained within the cell. Among the pathogenic bacteria -there are very few which secrete toxins when growing outside the body. -_Clostridium tetani_ or lockjaw bacillus, _Corynebacterium diphtheriae_ -or the diphtheria bacillus, _Clostridium botulinum_ or a bacillus -causing a type of food poisoning, _Pseudomonas pyocyanea_ or the blue -pus bacillus are the most important. Other pathogenic bacteria do not -secrete their toxins under the above conditions, but only give them up -when the cell is disintegrated either within or outside the body. For -the reason that endotoxins are therefore difficult to obtain, their -characteristics have not been much studied. The description of toxins -as above given is intended to apply to the _exo-toxins_ of bacteria, -sometimes spoken of as _true toxins_, and to the vegetable toxins -(phytotoxins) which resemble them. - -The snake venoms and probably most of the animal toxins (zooetoxins) are -very different substances. (See Chapter XXIX.) - - -CAUSATION OF DISEASE. - -This subject belongs properly in special pathogenic bacteriology. It -will be sufficient to indicate that bacteria may cause disease in one -or more of the following ways: (_a_) blocking circulatory vessels, -either blood or lymph, directly or indirectly; (_b_) destruction of -tissue; (_c_) production of non-specific poisons (ptomaines, bases, -nitrites, acids, gases, etc.); (_d_) production of specific poisons -(toxins). - - -ANTIBODY FORMATION. - -Bacteria cause the formation of specific "antibodies" when properly -introduced into animals. This must be considered as a physiological -activity since it is by means of substances produced within the -bacterial cell that the body cells of animals are stimulated to form -antibodies. (See Chapters XXVI-XXIX.) - - -STAINING. - -The reaction of bacteria to various stains is dependent on their -physico-chemical structure and hence is a result of physiological -processes, but is best discussed separately (Chapter XIX). - - -CULTURAL CHARACTERISTICS. - -The same is true of the appearance and growth on different culture -media. (Chapter XX.) - - - - -CHAPTER XIII. - -DISINFECTION--STERILIZATION--DISINFECTANTS. - - -The discussion of the physiology of bacteria in the preceding chapters -has shown that a number of environmental factors must be properly -correlated in order that a given organism may thrive. Conversely, it -can be stated that any one of these environmental factors may be so -varied that the organism will be more or less injured, may even be -destroyed by such variation. It has been the thorough study of the -above-mentioned relationships which has led to practical methods for -destroying bacteria, for removing them or preventing their growth when -such procedures become necessary. - -The process of killing all the living organisms or of removing them -completely is spoken of as _disinfection_ or as _sterilization_, -according to circumstances. Thus the latter term is applied largely in -the laboratory, while the former more generally in practice outside the -laboratory. So also disinfection is most commonly done with chemical -agents and sterilization by physical means, though exceptions are -numerous. The original idea of disinfection was the destruction of -"infective" organisms, that is, organisms producing disease in man or -animals. A wider knowledge of bacteriology has led to the application -of the term to the destruction of other organisms as well. Thus the -cheese-maker "disinfects" his curing rooms to prevent abnormal ripening -of cheese, and the dairy-worker "disinfects" his premises to avoid bad -flavors, abnormal changes in the butter or milk. _Sterilization_ is -more commonly applied to relatively small objects and _disinfection_ to -larger ones. Thus in the laboratory, instruments, glassware, apparatus, -etc., are "sterilized" while desks, walls and floors are "disinfected." -The surgeon "sterilizes" his instruments, but "disinfects" his -operating table and room. The dairy-workers mentioned above sterilize -their apparatus, pails, milk bottles, etc. Evidently the object of the -two processes is the same, removing or destroying living organisms, the -name to be applied is largely a question of usage and circumstances. -Any agent which is used to destroy microoerganisms is called a -"disinfectant." Material freed from _living_ organisms is "sterile." - -The process of _preventing the growth_ of organisms without reference -to whether they are killed or removed is spoken of as "_antisepsis_," -and the agent as an _antiseptic_. Hence a mildly applied "disinfectant" -becomes an "antiseptic," though it does not necessarily follow that -an "antiseptic" may become a disinfectant when used abundantly. Thus -strong sugar solutions prevent the development of many organisms, -though they do not necessarily kill them. - -_Asepsis_ is a term which is restricted almost entirely to surgical -operations and implies the taking of such precautions that foreign -organisms are _kept out_ of the field of operation. Such an operation -is an _aseptic_ one, or performed _aseptically_. - -A "deodorant or deodorizer" is used to destroy or remove an odor and -does not necessarily have either antiseptic or disinfectant properties. - -The agents which are used for the above-described processes may be -conveniently divided into _physical agents_ and _chemical agents_. - - -PHYSICAL AGENTS. - -=1. Drying.=--This is doubtless the oldest method for _preventing the -growth_ of organisms, and the one which is used on the greatest amount -of material at the present time. A very large percentage of commercial -products is preserved and transported intact because the substances -are kept free from moisture. In the laboratory many materials which -are used as food for bacteria (see Chapter XVI) "keep" because they -are dry. Nevertheless, drying should be considered as an _antiseptic_ -rather than as a _disinfectant_ process. While it is true that the -_complete_ removal of water would result in the death of all organisms -this necessitates a high temperature, in itself destructive, and does -not occur in practice. Further, though many pathogenic bacteria are -killed by drying, many more, including the spore formers, are not. -Hence drying alone is not a practical method of _disinfecting_. - -[Illustration: FIG. 77.--A small laboratory hot-air sterilizer.] - -=2. Heat.=--The use of heat in some form is one of the very best -means for destroying bacteria. It may be made use of by combustion, -or burning, as direct exposure to the open flame, as dry heat (hot -air), or as moist heat (boiling water or steam). Very frequently in -veterinary practice, especially in the country, occasionally under -other conditions, the infected material is best burned. This method is -thoroughly effective and frequently the cheapest in the end. Wherever -there are no valid objections it should be used. Exposure to the open -flame is largely a laboratory procedure to sterilize small metallic -instruments and even small pieces of glassware. It is an excellent -procedure in postmortem examinations to burn off the surface of the -body or of an organ when it is desired to obtain bacteria from the -interior free from contamination with surface organisms. - -_Dry Heat._--Dry heat is not nearly so effective as moist heat as a -sterilizing agent. The temperature must be higher and continued longer -to accomplish the same result. Thus a dry heat of 150 deg. for thirty -minutes is no more efficient than steam under pressure at 115 deg. for -fifteen minutes. Various forms of hot-air sterilizers are made for -laboratory purposes (Fig. 77). On account of the greater length of -time required for sterilization their use is more and more restricted -to objects which must be used dry, as in blood and serum work, for -example. In practice the use of hot air in disinfecting plants is now -largely restricted to objects which might be injured by steam, as -leather goods, furs, and certain articles of furniture, but even here -chemical agents are more frequently used. - -_Moist Heat._--Moist heat may be applied either by boiling in water -or by the use of steam at air pressure, or, for rapid work and on -substances that would not be injured, by steam under pressure. -Boiling is perhaps the best household method for disinfecting all -material which can be so treated. The method is simple, can always -be made use of, and is universally understood. It must be remembered -that all pathogenic organisms, even their spores, are destroyed by a -few minutes' boiling. The process may be applied to more resistant -organisms, such as are met with in canning vegetables, though the -boiling must be continued for several hours, or what is better, -repeated on several different days. This latter process, known as -"_discontinuous sterilization_," or "_tyndallization_," must also be -applied to substances which would be injured or changed in composition -by too long-continued heating, such as gelatin, milk, and certain -sugars. In the laboratory such materials are boiled or subjected to -steaming steam for half an hour on each of three successive days. In -canning vegetables the boiling should be from one to two hours each -day. The principle involved is that the first boiling destroys the -growing cells, but not all spores. Some of the latter germinate by the -next day and are then killed by the second boiling and the remainder -develop and are killed on the third day. Occasionally a fourth boiling -is necessary. It is also true that repeated heating and cooling is more -destructive to bacteria than continuous heating for the same length of -time, but the development of the spores is the more important factor. -Discontinuous heating may also be used at temperatures below the -boiling-point for the sterilization of fluids like blood serum which -would be coagulated by boiling. In this case the material is heated at -55 deg. to 56 deg. for one hour, but on each of seven to ten successive days. -The intermittent heating and cooling is of the same importance as the -development of the spores in this case. (Better results are secured -with such substances by collecting them aseptically in the first place.) - -[Illustration: FIG. 78.--The Arnold steam sterilizer for laboratory -use.] - -[Illustration: FIG. 79.--Vertical gas-heated laboratory autoclave.] - -[Illustration: FIG. 80.--Horizontal gas-heated laboratory autoclave.] - -_Steam._--Steam is one of the most commonly employed agents for -sterilization and disinfection. It is used either as "streaming steam" -at air pressure or confined under pressure so that the temperature is -raised. For almost all purposes where boiling is applicable streaming -steam may be substituted. It is just as efficient and frequently -more easily applied. The principle of the numerous forms of "steam -sterilizers" (Fig. 78) is essentially the same. There is a receptacle -for a relatively small quantity of water and means for conducting the -steam generated by boiling this water to the objects to be treated, -which are usually placed immediately above the water. Surgical -instruments may be most conveniently sterilized by boiling or by -steaming in especially constructed instrument sterilizers. If boiled, -the addition of carbonate of soda, about 1 per cent., usually prevents -injury. - -[Illustration: FIG. 81.--A battery of two horizontal autoclaves in one -of the author's student laboratories. Steam is furnished direct from -the University central heating plant.] - -_Steam under pressure_ affords a much more rapid and certain method of -destroying organisms. Fifteen to twenty pounds pressure corresponding -to temperatures of 121 deg. to 125 deg. is commonly used. Variations depend on -the bulk and nature of the material. Apparatus for this purpose may -now be obtained from sizes as small as one or two gallons up to huge -structures which will take one or two truckloads of material (Figs. -79-91). The latter type is in common use in canning factories, dairy -plants, hospitals, public institutions, municipal and governmental -disinfecting stations. Very frequently there is an apparatus attached -for producing a vacuum, both to exhaust the air before sterilizing, -so that the steam penetrates much more quickly and thoroughly and for -removing the vapor after sterilizing, thus hastening the drying out of -the material disinfected. - -[Illustration: FIG. 82.--A "process kettle" (steam-pressure sterilizer) -used in canning. Diameter, 40 inches; height, 72 inches.] - -The smaller types of pressure sterilizers are called "autoclaves" -and have become indispensable in laboratory work. Fifteen pounds -pressure maintained for fifteen minutes is commonly sufficient for a -few small objects. For larger masses much longer time is needed. The -author found that in an autoclave of the type shown in Fig. 81 it -required ten minutes for 500 cc. of water at 15 pounds pressure to -reach a temperature of 100 deg., starting at room temperatures, 20 deg. to -25 deg.. Autoclaves may be used as simple steam sterilizers by leaving the -escape valves open so that the steam is not confined, hence they have -largely replaced the latter.[18] - -[Illustration: FIG. 83.--Horizontal steam chest used in canning. -Height, 32 inches; width, 28 inches; length, 10 feet.] - -[Illustration: FIG. 84.--A battery of horizontal rectangular steam -chests in actual use in a canning factory.] - -[Illustration: FIG. 85.--A battery of cylindrical process kettles in -actual use in a canning factory.] - -A process closely akin to sterilization by heat is _pasteurization_. -This means the heating of material at a temperature and for a time -which will destroy the actively growing bacteria but not the spores. -The methods for doing this vary but are essentially two in principle. -1. The material in small quantities in suitable containers (bottles) is -placed in the apparatus; the temperature is raised to 60 deg. to 65 deg. and -maintained for twenty to thirty minutes and then the whole is cooled -(beer, wine, grape juice, bottled milk) (Figs. 92, 93 and 94). - -[Illustration: FIG. 86.--A steam chamber used in government -disinfection work. Size, 4 feet 4 inches x 5 feet 4 inches x 9 feet.] - -[Illustration: FIG. 87.--Circular steam chamber used in government -disinfection work, 54 inches in diameter.] - -[Illustration: FIG. 88.--Portable steam chamber used in government -disinfection work.] - -[Illustration: FIG. 89.--Steam chambers on deck of the U. S. quarantine -station barge "Defender."] - -[Illustration: FIG. 90.--Steam chambers in hold of U. S. quarantine -station barge "Protector." Disinfected space.] - -[Illustration: FIG. 91.--Municipal disinfecting station, Washington, -D. C.] - -[Illustration: FIG. 92.--A pasteurizer for milk in bottles.] - -[Illustration: FIG. 93.--A pasteurizer for grape juice, cider, etc., in -bottles.] - -2. Pasteurizing machines are used and the fluid flows through -continuously. In one type the temperature is raised to 60 deg. and by -"retarders" is kept at this temperature for twenty to thirty minutes -(Figs. 95 to 98). In another type the temperature is raised to as -high as 85 deg. for a few seconds only, "flash process" (Fig. 99), and then -the material is rapidly cooled. It is certain that all pathogenic -microoerganisms, except the very few spore formers in that stage, are -killed by proper pasteurization. The process is largely employed in the -fermentation and dairy industries. - -[Illustration: FIG. 94.--A pasteurizer for beer in bottles.] - -[Illustration: FIG. 95.--A continuous milk pasteurizer.] - -[Illustration: FIG. 96.--A pasteurizer for cream to be used in making -ice-cream.] - -[Illustration: FIG. 97.--A continuous milk pasteurizer with holder; -capacity 1500 pounds per hour. _A_, pasteurizer--the milk flows in -tubes inside of a jacket of water heated to the proper temperature; -_B_, holder; _C_, water cooler; _D_, brine cooler.] - -[Illustration: FIG. 98.--A continuous pasteurizing plant in operation. -Similar to Fig. 97 but larger. Capacity, 12,000 pounds per hour. _A_, -pasteurizer; _B_, seven compartment holder; _C_, _D_, coolers.] - -=3. Cold.=--That _cold_ is an excellent _antiseptic_ is illustrated -by the general use of refrigerators and "cold storage." Numerous -experiments have shown that although many pathogenic organisms of a -given kind are killed by temperatures below freezing, not all of the -same kind are, and many kinds are only slightly affected. Hence cold -cannot be considered a practical means for _disinfection_. - -[Illustration: FIG. 99.--A "flash process" pasteurizing outfit, with -holder. _A_, flash pasteurizer; _B_, holder; _C_, cooler.] - -=4. Light.=--It has been stated (p. 75) that light is destructive to -bacteria, and the advisability of having well-lighted habitations -for men and animals has been mentioned. The practice of "sunning" -bedclothing, hangings and other large articles which can scarcely be -disinfected in a more convenient way is the usual method of employing -this agent. Drying and the action of the oxygen of the air assist -the process to some extent. Undoubtedly large numbers of pathogenic -organisms are destroyed under natural conditions by the combined -effects of drying, direct sunlight and oxidation, but it should not -be forgotten that a very slight protection will prevent the action of -light (Figs. 100 and 101). - -[Illustration: FIG. 100.--Effect of light on bacteria. x 7/10. The -plate was inoculated in the usual way. A letter _H_ of black paper was -pasted on the bottom. The plate was then exposed for four hours to the -sun in January outside the window and then incubated. The black paper -protected the bacteria. Outside of it they were killed except where -they happened to be in large masses. Hence the letter shows distinctly. -(Student preparation.)] - -=5. Osmotic Pressure.=--Increase in the concentration of substances -in solution is in practical use as an _antiseptic_ procedure. -Various kinds of "sugar preserves," salt meats and condensed milk -are illustrations. It must be remembered that a similar increase in -concentration occurs when many substances are dried, and is probably -as valuable in the preservative action as the loss of water. That the -process cannot be depended on to _kill_ even pathogenic organisms is -shown by finding living tubercle bacilli in condensed milk. The placing -of bacteria in water or in salt solution in order to have them die and -disintegrate (greatly aided by vigorous shaking in a shaking machine) -("autolysis," p. 126) is a laboratory procedure to obtain cell -constituents. It is not a practical method of disinfection, however. - -[Illustration: FIG. 101.--Effect of light on bacteria. x 7/10. This -plate was treated exactly as the plate in Fig. 100, except that the -letter is _L_, and that it was exposed inside the window and wire -screen. The window was plate glass. It is evident that few of the -bacteria were killed, since the letter _L_ is barely outlined. The -exposure was at the same time as the plate in Fig. 100. (Student -preparation.)] - -=6. Electricity.=--Electricity, though not in itself injurious to -bacteria, is used as an indirect means for destroying bacteria in a -practical way. This is done by electrical production of some substance -which is destructive to bacteria as in ozone water purification -(Petrograd, Florence, and elsewhere), or the use of ultra-violet rays -for the same purpose (Marseilles, Paris) and for treatment of certain -disease conditions. Electricity might be used as a source of heat for -disinfecting purposes should its cheapness justify it. It has also -been used in the preservation of meats to hasten the _penetration of -the salt_ and thus reduce the time of pickling. Electrolyzed sea water -has been tried as a means of flushing and disinfecting streets, but -it is very doubtful if the added expense is justified by any increased -benefit. A number of electric devices have been put forth for various -sterilizing and disinfecting purposes and doubtless will continue to -be, but everyone should be carefully tested before money is invested in -it.[19] - -[Illustration: FIG. 102.--An electric milk purifier (pasteurizer). The -milk flowing from cup to cup completes the circuit when the current is -on. The effect is certainly a heat effect. Sparking occurs at the lips -of the cups.] - -[Illustration: FIG. 103.--One of the ten filter beds of the Columbus -water filtration plant with the filtering material removed. Sand is -the filtering material. All of the beds together have a capacity of -30,000,000 gallons daily.] - -[Illustration: FIG. 104.--Suction filtration. _A_, Berkefeld filter in -glass cylinder containing the liquid to be filtered; _B_, sterile flask -to receive the filtrate as it is drawn through; _C_, water pump; _D_, -manometer, convenient for detecting leaks as well as showing pressure; -_E_, bottle for reflux water.] - -[Illustration: FIG. 105.--Pressure filtration. _A_, cylinder which -contains the filter candle; _B_, cylinder for the liquid to be -filtered; _C_, sterile flask to receive the filtrate; _D_, air pump to -furnish pressure.] - -=7. Filtration.=--Filtration is a process for rendering fluids sterile -by passing them through some material which will hold back the -bacteria. It is used on a large scale in the purification of water for -sanitary or manufacturing reasons (Fig. 103). Air is also rendered -"germ free" in some surgical operating rooms, "serum laboratories" and -breweries by filtration. In the laboratory it is a very common method -of sterilizing liquids which would be injured by any other process. -The apparatus consists of a porous cylinder with proper devices for -causing the liquid to pass through either by suction (Fig. 104) where -the pressure will be only one atmosphere (approximately 15 pounds per -square inch), or by the use of compressed air at any desired pressure -(Fig. 105). The two main types of porous cylinders ("filter candles," -"bougies") are the Pasteur-Chamberland (Fig. 106) and the Berkefeld. -The former are made of unglazed porcelain of different degrees of -fineness, the latter of diatomaceous earth (Fig. 107) The Mandler -filter of this same material is now manufactured in the United States -and is equal if not superior to the Berkefeld. The designs of complete -apparatus are numerous. - -[Illustration: FIG. 106.--Pasteur-Chamberland filter candles about -one-half natural size.] - -=8. Burying.=--This is a time-honored method of disposing of infected -material of all kinds and at first thought might not be considered -a means of _disinfection_. As a matter of fact, under favorable -conditions it is an excellent method. The infected material is -removed. Pathogenic organisms tend to die out in the soil owing to an -unfavorable environment as to temperature and food supply, competition -with natural soil organisms for what food there is, and the injurious -effects of the products of these organisms. Care must be taken that -the burial is done in such a way that the _surface_ soil is not -contaminated either directly or by material brought up from below by -digging or burrowing animals, insects, worms, or movement of ground -water to the surface. Also that the underground water supply which is -drawn upon for use by men or animals is not contaminated. Frequently -infected material, carcasses of animals, etc., are treated in some -way so as to aid the natural process of destruction of the organisms -present, especially by the use of certain chemical agents, as quicklime -(see p. 158). - -[Illustration: FIG. 107.--Berkefeld filter candles about one-half -natural size.] - - - - -CHAPTER XIV. - -DISINFECTION AND STERILIZATION (CONTINUED). - - -CHEMICAL AGENTS. - -A very large number of chemical substances might be used for destroying -bacteria or preventing their growth either through direct injurious -action or by the effect of concentration. Those which are practically -useful are relatively few, though this is one of the commonest methods -of disinfecting and the word "disinfectant" is frequently wrongly -restricted to chemical agents. - -Chemical agents act on bacteria in a variety of ways. Most commonly -there is direct union of the chemical with the protoplasm of the cell -and consequent injury. Some times the chemical is first precipitated -on the surface of the cell without penetrating at once. If removed -soon enough, the organism is not destroyed. This is true of bichloride -of mercury and formaldehyde. If bacteria treated with these agents in -injurious strength be washed with ammonia or ammonium sulphate, even -after a time which would otherwise result in their failure to grow, -they will develop. Some chemicals change the reaction of the material -in a direction unfavorable to growth, and if the change is enough, -may even kill the bacteria. Some agents remove a chemical substance -necessary to the growth of the organism and hence inhibit it. Such -actions are mainly preventive (antiseptic) and become disinfectant only -after a long time. - - -ELEMENTS. - -=Oxygen.=--Oxygen as it occurs in the air is probably not injurious to -living bacteria but aids them with the exception of the anaerobes. In -the nascent state especially as liberated from ozone (O{3}) hydrogen -peroxide (H{2}O{2}) and hypochlorites (Ca(ClO){2}) it is strongly -bactericidal. - -=Chlorine.=--Chlorine is actively disinfectant and is coming into use -for sterilizing water on a large scale in municipal plants (Fig. 108). - -[Illustration: FIG. 108.--Apparatus for sterilizing water with liquid -chlorine.] - -_Iodine_ finds extended use in aseptic surgical operations and -antiseptic dressings. Bromine, mercury, silver, gold, nickel, zinc -and copper are markedly germicidal in the elemental state but are not -practical. - - -COMPOUNDS. - -=Calcium Oxide.=--Calcium oxide (CaO), _quick lime_, is an excellent -disinfectant for stables, yards, outhouses, etc., where it is used -in the freshly slaked condition as "white wash;" also to disinfect -carcasses to be buried. It is very efficient against the typhoid -bacillus in water, where it is much used to assist in the softening. - -=Chloride of Lime.=--Chloride of lime, _bleaching powder_, which -consists of calcium hypochlorite, the active agent, and chloride and -some unchanged quicklime is one of the most useful disinfectants. It -is employed to sterilize water for drinking purposes on a large scale -and to disinfect sewage plant effluents. A 5 per cent. solution is the -proper strength for ordinary disinfection. Only a supply which is fresh -or has been kept in air-tight containers should be used, as it rapidly -loses strength on exposure to the air. The active agent is nascent -oxygen liberated from the decomposition of the hypochlorite. - -=Sodium Hypochlorite.=--Sodium hypochlorite prepared by the -electrolysis of common salt has been used to some extent. - -=Bichloride of Mercury.=--Bichloride of mercury, _mercuric chloride, -corrosive sublimate_ (HgCl{2}), is the strongest of all disinfectants -under proper conditions. It is also extremely poisonous to men and -animals and great care is necessary in its use. It is precipitated by -albuminous substances and attacks metallic objects, hence should not be -used in the presence of these classes of substances. - -It is used in a strength of one part HgCl{2} to 1000 of water for -general disinfection. Ammonium chloride or sodium chloride, common -salt, in quantities equal to the bichloride, or citric acid in one-half -of the amount should be added in making large quantities of solution or -for use with albuminous fluids to prevent precipitation of the mercury -(Fig. 109). - -None of the other metallic salts are of value as practical -disinfectants aside from their use in surgical practice. In this -latter class come boric acid, silver nitrate, potassium permanganate. -The strong mineral acids and alkalies are, of course, destructive to -bacteria, but their corrosive effect excludes them from practical use, -except that "lye washes" are of value in cleaning floors and rough -wood-work, but even here better _disinfection_ can be done more easily -and safely. - -[Illustration: FIG. 109.--Tanks for bichloride of mercury, government -quarantine disinfecting plant.] - - -ORGANIC COMPOUNDS. - -=Carbolic Acid or Phenol.=--Carbolic acid or phenol (C{6}H{5} OH) is one -of the commonest agents in this class. It is used mostly in 5 per -cent. solution as a disinfectant and in 0.5 per cent. solution as an -antiseptic. For use in large quantities the crude is much cheaper and, -according to some experimenters, even more active than the pure acid, -owing to the cresols it contains. The crude acid is commonly mixed with -an equal volume of commercial sulphuric acid and the mixture is added -to enough water to make a 5 per cent. dilution, which is stronger than -either of the ingredients alone in 5 per cent. solution. - -=Cresols.=--The cresols (C{6}H{4}CH{3}OH, ortho, meta and para), -coal-tar derivatives, as phenol, are apparently more powerful -disinfectants. A great number of preparations containing them have -been put on the market. _Creolin_ is one which is very much used in -veterinary practice and forms a milky fluid with water, while _lysol_ -forms a clear frothy liquid owing to the presence of soap. Both -of these appear to be more active than carbolic acid and are less -poisonous and more agreeable to use. They are used in 2 to 5 per cent. -solution. - -=Alcohol.=--Ordinary (ethyl) alcohol (C{2}H{5}OH) is largely used as a -_preservative_, also as a disinfectant for the body surface, hands, and -arms. Experiments show that alcohol of 70 per cent. strength is most -strongly bactericidal and that absolute alcohol is very slightly so. - -=Soap.=--Experimenters have obtained many conflicting results with -soaps when tested on different organisms, as is to be expected from -the great variations in this article. Miss Vera McCoy in the author's -laboratory carried out experiments with nine commercial soaps--Ivory, -Naphtha, Packer's Tar, Grandpa's Tar, Balsam Peru, A. D. S. Carbolic, -German Green, Dutch Cleanser, Sapolio--and obtained abundant growth -from spores of _Bacillus anthracis_, from _Bacterium coli_ and from -_Staphylococcus pyogenes aureus_ in all cases even when the organisms -had been exposed twenty-four hours in 5 per cent. solutions. From -these results and from the wide variations reported in the literature -it is clear that _soap solutions alone cannot be depended on_ as -disinfectants. Medicated soaps do not appear to offer any advantages in -this respect. The amount of the disinfectant which goes into solution -when the soap is dissolved is too small to have any effect. - -=Formaldehyde.=--Formaldehyde (HCHO) is perhaps the most largely used -chemical disinfectant at the present time. The substance is a gas -but occurs most commonly in commerce as a watery solution containing -approximately 40 per cent. of the gas. This solution is variously known -as formalin, formol, and formaldehyde solution. The first two names -are patented and the substance under these names usually costs more. -It is used in the gaseous form for disinfecting closed spaces of all -kinds to the exclusion of most other means today. A great many types -of formalin generators have been devised. The gas has little power of -penetration and all material to be reached should be exposed as much as -possible. The dry gas is almost ineffective, so that the objects must -be moistened or vapor generated along with the gas. A common method -in use is to avoid expensive generators by pouring the formaldehyde -solution on permanganate of potash crystals placed in a vessel removed -from inflammable objects on account of the heat developed which -occasionally sets the gas on fire. The formalin is used in amounts -varying from 20 to 32 ounces to 8-1/2 to 13 ounces of permanganate to -each 1000 cubic feet of space. This method is expensive since one pint -(16 ounces) of formalin is sufficient for each 1000 cubic feet, and -since the permanganate is an added expense. Dr. Dixon, Commissioner of -Health of Pennsylvania, recommends the following mixture to replace the -permanganate, claiming that it works more rapidly and is less expensive -and just as efficient: - - 1. Sodium bichromate, ten ounces. - 2. Saturated solution of formaldehyde, sixteen ounces. - 3. Common sulphuric acid, one and a half ounces. - -Two and three are mixed together and when cool are poured on the -bichromate which is placed in an earthenware jar of a volume about ten -times the quantity of fluid used. The quantities given are for each -1000 cubic feet of space. - -A very simple method is to cause the formalin, diluted about twice -with water to furnish moisture enough, to drop by means of a regulated -"separator funnel" on a heated iron plate. The dropping should be so -regulated that each drop is vaporized as it falls. The plate must -have raised edges, pan-shaped, to prevent the drops rolling off when -they first strike the plate. Formaldehyde has no corrosive (except on -iron) or bleaching action, and is the most nearly ideal closed space -disinfectant today. In disinfecting stations it is made use of in -closed sterilizers such as were described under steam disinfection -particularly in connection with vacuum apparatus. It is also used -in solution as a preservative and as a disinfectant. The commonest -strength is 2 or 3 per cent. of formalin or 0.8 to 1.2 per cent. of -the formaldehyde gas. As an _antiseptic_ it is efficient in dilutions -as high as 1 to 2000 of the gas. It is very irritant to mucous -membranes of most individuals. - -=Anilin Dyes.=--Some of the anilin dyes show remarkable selective -disinfectant and antiseptic action on certain kinds of bacteria with -little effect on others. This has been well shown by Churchman in his -work on Gentian Violet. This dye inhibits the growth of _Gram positive_ -organisms up to a dilution of one part in 300,000 while for _Gram -negative_ organisms it is without effect even in saturated solution. -This is nicely shown in the accompanying illustration. This inhibiting -effect of anilin dyes is taken advantage of in several methods of -isolating bacteria (Chapter XVIII). - -[Illustration: FIG. 110.--The lower half of the plate is plain agar -medium, the upper half the same medium plus gentian violet to make one -part in 300,000. The Gram positive organism is on the right and the -Gram negative on the left. Streak inoculations were made across both -media.] - -In addition to the above-discussed disinfectants a large number of -substances, particularly organic, are used in medicine, surgery, -dentistry, etc., as more or less strong antiseptics, and the list is a -constantly lengthening one. - -In the laboratory chloroform, H{2}O{2}, ether and other volatile or -easily decomposable substances have been used to sterilize liquids -which could not be treated by heat or by filtration. The agent is -removed either by slow evaporation or by exhausting the fluid with -an air pump. The method is not very satisfactory, nor is absolute -sterilization easily accomplished. It is much better to secure such -liquids aseptically where possible. - - - - -CHAPTER XV. - -DISINFECTION AND STERILIZATION (CONTINUED). - - -CHOICE OF AGENT. - -The choice of the above-described agents depends on the conditions. -Evidently a barn is not to be disinfected in the same way that a -test-tube in the laboratory is sterilized. Among the factors to be -considered in making a choice are the thing to be disinfected or -sterilized, its size and nature, that is, whether it will be injured by -the process proposed, cost of the agent, especially when a large amount -of material is to be treated. Among the conditions which affect the -action of all agents the following should be borne in mind particularly -when testing the disinfecting power of chemical agents: - -1. _The kind of bacterium_ to be destroyed, since some are more readily -killed by a given disinfectant than others, even though no spores are -present. - -2. _The age of the culture._ Young bacteria less than twenty-four hours -old are usually more readily killed than older ones since the cell wall -is more delicate and more easily penetrated, though old growths may -be weakened by the accumulation of their products and be more easily -destroyed. - -3. _Presence of spores_, since they are much more resistant than the -growing cells. - -4. Whether the organism is a _"good" or "bad" growth_, _i.e._, whether -it has grown in a favorable environment and hence is vigorous, or under -unfavorable conditions and hence is weak. - -5. _The number of bacteria present_, since with chemical agents the -action is one of relative masses. - -6. _Nature of the substance in which the bacteria are._ Metallic salts, -especially bichloride of mercury, are precipitated by albuminous -substances and if employed at all must be used in several times the -ordinary strength. Solids require relatively more of a given solution -than liquids. - -7. _State of the disinfectant_, whether solid, liquid or gas, and -whether it is ionized or not. Solutions penetrate best and are -therefore more quickly active and more efficient. - -8. _The solvent._ Water is the best solvent to use. Strong alcohol (90 -per cent. +) diminishes the effect of carbolic acid, formaldehyde and -bichloride of mercury. Oil has a similar effect. The action is probably -to prevent the penetration of the disinfectant. - -9. _Strength of solution._ The stronger the solution, the more rapid -and more certain the action, for the same reason as mentioned under 5. -In fact, every disinfectant has a strength below the lethal at which it -stimulates bacterial growth. - -10. _Addition of salts._ Common salt favors the action of bichloride -of mercury and also of carbolic acid. Other salts may hinder by -precipitating the disinfectant. - -11. _Temperature._ Chemical disinfectants, as a rule, follow the -general law that chemical action increases with the temperature, up to -the point where the heat of itself is sufficient to kill. - -12. _Time of action._ It is scarcely necessary to point out that a -certain length of time is necessary for any disinfectant to act. One -may touch a red hot stove and not be burned. All the above-mentioned -conditions are influenced by the time of action. - - -STANDARDIZATION OF DISINFECTANTS--"PHENOL COEFFICIENT." - -Many attempts have been made to devise standard methods for testing -the relative strengths of disinfectants. The one most widely used in -the United States is the so-called "Hygienic Laboratory" method of -determining the "phenol coefficient" of the given substance and is a -modification of the method originally proposed by Rideal and Walker -in England. In this method as proposed by Anderson and McClintic, -formerly of the above laboratory, the strengths of the dilution of the -disinfectant to be tested which kills a culture of _Bacterium typhosum_ -in 2-1/2 minutes is divided by the strength of the dilution of carbolic -acid which does the same; and the dilution which kills in 15 minutes is -likewise divided by the corresponding dilution of carbolic acid. The -two ratios thus obtained are averaged and the result is the "phenol -coefficient." For example - - Phenol 1:80 killed in 2-1/2 minutes - Disinfectant "A" 1:375 " " " " - Phenol 1:110 " " 15 " - Disinfectant "A" 1:650 " " " " - 375 / 80 = 4.69 - 650 / 110 = 5.91 - ----- - 2)10.60 - ----- - Average = 5.30 = "phenol coefficient." - -Standard conditions of temperature, age of culture, medium, reaction, -etc., and of making the dilutions and transfers are insisted on. -Details may be found in the Journal of Infectious Diseases, 1911, 8, p. -1. - -This is probably as good a method as any for arriving at the relative -strengths of disinfectants and in the hands of any given worker -concordant results in comparative tests can usually be attained. -Experience has shown that the results obtained by different workers -with the same disinfectant may be decidedly at variance. This is to -be expected from a knowledge of the factors affecting the action of -disinfectants above stated and from the known specific action of -certain disinfectants on certain organisms (compare anilin dyes, p. -162). - -It seems that the only sure way to test the action of such a substance -is to try it out in the way it is to be used. It is scarcely wise to -adopt the "phenol coefficient" method as a legal standard method as -some states have done. - - -PRACTICAL STERILIZATION AND DISINFECTION. - -The methods for sterilizing in the laboratory have been discussed and -will be referred to again in the next chapter. - -In practical disinfection it is a good plan always to _proceed as -though spores were present_ even if the organism is known. Hence use an -_abundance of the agent_ and _apply it as long as practicable_. Also -it is best to secure the _chemical substances used as such_ and _not -depend on patented mixtures purporting to contain them_. As a rule the -latter are _more expensive_ in _proportion to the results secured_. - -_Surgical instruments_ may be sterilized by boiling in water for -fifteen minutes, provided they are clean, as they should be. If dried -blood, pus, mucus, etc., are adherent, which should never be the case, -they should be boiled one-half hour. The addition of sodium carbonate -(0.5 to 1 per cent.) prevents rusting. Surgeons' sterilizers are to -be had at reasonable prices and are very convenient. Whether the -instruments are boiled or subjected to streaming steam depends on -whether the supporting tray is covered with water or not. The author -finds it a good plan to keep the needles of hypodermic syringes in a -small wire basket in an _oil bath_. The oil may be heated to 150 deg. to -200 deg. and the needles sterilized in a very few minutes. The oil also -prevents rusting. - -_Rooms_, _offices_ and all spaces which may be readily made practically -gas-tight are best disinfected by means of formaldehyde by any of the -methods above described (Figs. 111 and 112). - -_Stables_ and _Barnyards_ (Mohler): "A preliminary cleaning up of all -litter is advisable together with the scraping of the floor, mangers -and walls of the stable with hoes and the removal of all dust and -filth. All this material should be burned since it probably contains -the infective agent. Heat may be applied to the surfaces, including -barnyard, by means of a 'cyclone oil burner.' When such burning is -impracticable, the walls may be disinfected with one of the following: - - 1. Whitewash 1 gallon + chloride of lime 6 ounces. - - 2. Whitewash 1 gallon + crude carbolic acid 7 ounces. - - 3. Whitewash 1 gallon + formalin 4 ounces. - -The same may be applied with brushes or, more rapidly, sprayed on with -a pump; the surface soil of the yard and surroundings should be removed -to a depth of 5 or 6 inches, placed in a heap and thoroughly mixed -with quicklime. The fresh surface of soil thus exposed may be sprinkled -with a solution of a chemical disinfectant as above described. - -[Illustration: FIG. 111.--Formaldehyde generator used in city work for -room disinfection.] - -[Illustration: FIG. 112.--Government formaldehyde generator.] - -"Portions of walls and ceiling not readily accessible may be -disinfected by chlorine gas liberated from chloride of lime by crude -carbolic acid. This is accomplished by making a cone of 5 or 6 pounds -of chloride of lime in the top of which a deep crater is made for the -placement of from 1 to 2 pints of crude carbolic acid. The edge of -the crater is thereupon pushed into the fluid, when a lively reaction -follows. Owing to the heat generated, it is advisable to place the -chloride of lime in an iron crucible (pot), and to have nothing -inflammable within a radius of two feet. The number and location of -these cones of chloride of lime depend on the size and structure -of the building to be disinfected. As a rule it may be stated that -chlorine gas liberated from the above sized cone will be sufficient for -disinfecting 5200 cubic feet of air space." - -_Liquid manure_, _leachings_, etc., where collected are thoroughly -disinfected by chloride of lime applied in the proportion of 2 parts to -1000 of fluid. - -[Illustration: FIG. 113.--Chamber used in government work for -formaldehyde disinfection. The small cylinder at the side is the -generator.] - -_Vehicles_ may be thoroughly washed with 2 per cent. formalin solution, -or if closed space is available, subjected to formaldehyde gas -disinfection, after cushions, hangings, etc., have been removed and -washed with the disinfectant. - -_Harness_, _brushes_, _combs_ should be washed with a solution of -formalin, carbolic acid, or creolin as given under these topics. - -_Washable articles_ should be boiled, dropped into disinfectant, -solutions as soon as soiled, and then boiled or steamed. - -_Unwashable articles_--burn all possible. Use formaldehyde gas method -in a closed receptacle (Fig. 113). - -_Stock cars_--the method described for stables is applicable here. - -_Animals, large and small_, may have the coat and surface of the body -disinfected by washing with 1 to 1000 bichloride or strong hot soapsuds -to which carbolic acid has been added to make a 5 per cent. solution; -they should then be given a good warm bath. - -Frequently time and money are saved by a combination of steam and -formaldehyde disinfection. This is a regular practice in municipal and -quarantine disinfection (Fig. 114). - -[Illustration: FIG. 114.--Chamber in actual use at government -quarantine station for disinfecting baggage and dunnage with steam -or formaldehyde or both. The small cylinder at the side is the steam -formaldehyde generator.] - -Persons engaged in disinfection work should wear rubber boots, coats -and caps which should be washed in a disinfectant solution and the -change to ordinary clothing made in a special room so that no infective -material will be taken away. - - - - -PART III. - -THE STUDY OF BACTERIA. - - - - -CHAPTER XVI. - -CULTURE MEDIA. - - -The study of bacteria may be taken up for the disciplinary and -pedagogic value of the study of a science; with the idea of extending -the limits of knowledge; or for the purpose of learning their -beneficial or injurious actions with the object of taking advantage of -the former and combating or preventing the latter. - -Since bacteria are classed as plants, their successful study implies -their cultivation on a suitable soil. A growth of bacteria is called -a "_culture_" and the "soil" or material on which they are grown is -called a "_culture medium_." In so far as the culture medium is made up -in the laboratory it is an "artificial culture medium" as distinguished -from a natural medium. A culture consisting of one kind of bacteria -only is spoken of as a "pure culture," and accurate knowledge of -bacteria depends on obtaining them in "pure culture." After getting -a "pure culture" the special characteristics of the organism must be -ascertained in order to distinguish it from others. The discussion of -the _morphology_ of bacteria in Chapters II, III, and IV shows that -the morphological structures are too few to separate individual kinds. -They serve at best to enable groups of similarly appearing forms to be -arranged. Hence any further differentiation must be based on a study -of the _physiology_ of the organism as discussed in the chapters on -Physiological Activities of Bacteria. - -The thorough study of a bacterium involves, therefore: - -1. Its isolation in pure culture. - -2. Its study with the microscope to determine morphological features -and staining reactions. - -3. Growth on culture media for determining its physiological activities -as well as morphological characteristics of the growths themselves. - -4. Animal inoculations in certain instances. - -5. Special serum reactions in some cases. - -Since isolation in pure culture requires material for growing the -organism, the first subject to be considered is culture media. - -A culture medium for a given bacterium should show the following -essentials: - -1. It must contain all the elements necessary for the growth of the -organism except those that may be obtained from the surrounding -atmosphere. - -2. These elements must be in a form available to the organism. - -3. The medium must not be too dry, in order to furnish sufficient -moisture for growth and to prevent too great a concentration of the -different ingredients. - -4. The reaction must be adjusted to suit the particular organism dealt -with. - -5. There must be no injurious substances present in concentration -sufficient to inhibit the growth of the organism or to kill it. - -Ordinarily, more attention must be paid to the sources of the two -elements N and C than to the others, for in general the substances -used to furnish these two and the water contain the other elements in -sufficient amount. For very exact work on the products of bacteria, -_synthetic media_ containing definite amounts of chemicals of known -composition have been prepared, but for most of the work with bacteria -pathogenic to animals such media are not needed. - -Culture media may be either _liquid_ or _solid_, or for certain -purposes may be liquid at higher temperatures and solid at lower, as -indicated later. Liquid media are of value for obtaining bacteria for -the study of morphology and cell groupings and for ascertaining many of -the physiological activities of the organisms. Solid media are useful -for studying some few of the physiological activities and especially -for determining characteristic appearances of the isolated growths -of bacteria. These isolated growths of bacteria on solid media are -technically spoken of as "_colonies_," whether they are microscopic in -size or visible to the unaided eye. - -It is clear that the kinds of culture media used for the study of -bacteria may be unlimited but the undergraduate student will need to -use a relatively small number, which will be discussed in this section. - -=Meat Broth (Bouillon).=[20]--This itself is used as a medium and as -the basis for the preparation of other solid and liquid media. - -Finely ground _lean_ beef is selected because it contains the necessary -food materials. Fat is not desired since it is a poor food for most -bacteria and in the further processes of preparation would be melted -and form an undesirable film on the surface of the medium. The meat is -placed in a suitable container and mixed with about twice its weight of -_cold_ water (not distilled) and allowed to soak overnight or longer. -The cold water extracts from the meat water-soluble proteins, blood, -carbohydrates in the form of dextrose (occasionally some glycogen), -nitrogenous extractives and some of the mineral salts. The fluid is -strained or pressed free from the meat. This "meat juice" should now -be thoroughly boiled, which results in a coagulation of a large part -of the proteins and a precipitation of some of the mineral salts, -particularly phosphates of calcium and magnesium, both of which must be -filtered off and the water loss restored by adding the proper amount of -distilled water. The boiling is done at this point because the medium -must later be heated to sterilize it and it is best to get rid of the -coagulable proteins at once. The proteins thus thrown out deprive -the medium of valuable nitrogenous food material which is replaced -by adding about 1 per cent. by weight of commercial peptone. It is -usual also (though not always necessary) to add about 0.5 per cent. by -weight of common salt which helps to restore the proper concentration -of mineral ingredients lost by the boiling. The chlorine is also an -essential element. The reaction is now determined and adjusted to the -desired end point, "standardized," as it is called. The medium is -again _thoroughly_ boiled and filtered boiling hot. The adjusting of -the reaction and the boiling ordinarily cause a precipitate to form -which is largely phosphates of the alkaline earths with some protein. -The filtered medium is collected in suitable containers, flasks or -tubes, which are plugged with well-fitting non-absorbent cotton plugs -and sterilized, best in the autoclave for twenty minutes at 15 pounds -pressure, or discontinuously in streaming steam at 100 deg.. If careful -attention is paid to _titration_ and to _sufficient boiling_ where -indicated, the meat broth prepared as above should be clear, only -faintly yellowish in color and show no precipitate on cooling. - -The conventional method for standardizing an acid medium is as follows: -Take 5 cc of the medium, add 45 cc of distilled water and 1 cc of -_phenolphthalein_ as indicator. Boil the solution and while still hot -run in from a burette N/20 NaOH solution until a faint pink color -appears. From the number of cc of N/20 NaOH used to "neutralize" the -5 cc of medium it is calculated how many cc of N/1 NaOH are necessary -to give the desired end reaction to the volume of medium which is to -be standardized. The resulting reaction is expressed as % _acid or -alkaline to phenolphthalein_. If it is necessary to add to each 100 cc -of the medium 1 cc of N/1 NaOH to make it neutral to phenolphthalein, -the reaction is called 1% acid: if to each 100 cc of medium there is -added 1 cc of N/1 alkali in addition to the quantity necessary to -neutralize, the reaction is called 1% alkaline. - -In order to obtain a pink color when titrating with this indicator -not only must the "free acid" be neutralized by the alkali but also -loosely combined acid and any other substances present which will -combine with the alkali rather than with the indicator so that in many -media _more alkali_ is added than is necessary to neutralize the "free -acid," _i.e._, the free H ions present. - -It is well established that the controlling factor in the growth of -bacteria in so far as "reaction" is concerned is not the _titratable -substances_ present but only the "free acid," _i.e._, the _number of -free H ions_, consequently it is better to determine the concentration -of H ions and to _standardize to a definite H ion concentration_. -Phenolphthalein as shown above is not a good indicator for this purpose. - -The H ions present can be determined accurately in all cases only by -electrolytic methods. The apparatus necessary is usually relatively -expensive and scarcely adapted to the use of large classes of students. -There are a number of indicators each of which will show color changes -within rather narrow ranges of H ion concentration. Standardization by -the use of these indicators, the "colorimetric method," is recommended -by the Society of American Bacteriologists and is coming into general -use. - -The H ion concentration is ordinarily _indicated_ by the conventional -symbol P{H}, _e.g._, the concentration in pure water which is regarded -as neutral is expressed as P{H} 7; of normal HCl, P{H} 0; of normal -NaOH, P{H} 14. The figure after P{H} does not in reality represent the -concentration of H ions in the solution. This, like the concentration -of acids, is expressed on the basis of normality, _i.e._, as compared -with the concentration of a normal solution (1 g. equivalent) of H -ions. Concentration of H ions in pure water is N/10,000,000, _i.e._, -is 1/10,000,000 of the concentration in a normal solution of H ions. -Expressed in other words, it is the concentration in a normal solution -of H ions diluted ten million times. 10,000,000 = 10 to the 7th power -= 10^{7}. Hence the figure after the P{H} indicates the _logarithm of -the number of times the solution is diluted_. Therefore this number -_increases with the dilution_, and the larger the figure after the -P{H}, the _less acid the solution is_. - -Most saprophytic organisms and many parasitic ones grow within a wide -range of H ion concentration so that titration with phenolphthalein -gives sufficient accuracy for media for such organisms. On the -other hand, many organisms grow within a very narrow range of H ion -concentration, hence accurate standardization to a definite H ion -concentration is necessary. It is also evident that for comparative -work, such standardization is essential because this reaction can be -reproduced in other media and by other workers.[21] - -Broth may be prepared from Liebig's or Armour's meat extract by adding -5 grams of either, 10 grams peptone and 5 grams NaCl to 1000 cc of -water, boiling to dissolve, then titrating and filtering as above. - -The author after much experience finds _meat juice_ preferable to meat -extract for broth and other media for pathogenic bacteria, and has -abandoned the use of meat extracts for these organisms. - -=Glycerin Broth.=--Glycerin broth is made by adding 4 to 6 per cent. of -glycerin to the broth just previous to the sterilization. The glycerin -serves as a source of carbon to certain bacteria which will not grow on -the ordinary broth--as _Mycobacterium tuberculosis_. - -=Sugar Broths.=--Sugar broths are used for determining the action of -bacteria on these carbohydrates, since this is a valuable means of -differentiating certain forms, especially those from the intestinal -tract. Broth _free from sugar_ must first be made. This is done by -adding to broth prepared as already described, _just previous to final -filtering and sterilization_, a culture of some sugar-destroying -organism (_Bacterium coli_ is ordinarily used), and then allowing the -organism to grow in the raw broth at body temperature for twenty-four -hours. Any carbohydrate in the broth is destroyed by the _Bacterium -coli_. This mixture is then boiled to kill the _Bacterium coli_, -restandardized and then 1 per cent. by weight of required sugar is -added. Dextrose, saccharose and lactose are the most used, though -many others are used for special purposes. After the sugar is added -the medium must be sterilized by _discontinuous heating_ at 100 deg. for -three or four successive days, because long boiling or heating in the -autoclave splits up the di- and polysaccharids into simpler sugars and -may even convert the simple sugars (dextrose) into acid. - -Various other _modified broths_ are frequently used for special -purposes but need not be discussed here. - -=Dunham's peptone solution=, frequently used to determine indol -production, is a solution of 1 per cent. of peptone and 0.5 per cent. -of salt in tap water. It does not need to be titrated, but should be -boiled and filtered into tubes or flasks and sterilized. - -=Nitrate Broth.=--Nitrate broth for determining nitrate reduction -is 1 per cent. of peptone, 0.2 per cent. of C. P. potassium nitrate -dissolved in distilled water and sterilized. - -=Milk.=--Milk is a natural culture medium much used. It should be -fresh and thoroughly skimmed, best by a separator or centrifuge to -get rid of the _fat_. If the milk is not fresh, it should be titrated -as for broth and the reaction adjusted. The milk should be sterilized -discontinuously to avoid splitting up the lactose as well as action on -the casein and calcium phosphate. - -_Litmus Milk._--Litmus milk is milk as above to which litmus has been -added as an acid production indicator. The milk should show blue when -the litmus is added or be made to by the addition of normal NaOH -solution. It should be sterilized discontinuously. Frequently on -heating litmus milk the blue color disappears due to a reduction of -the litmus. This blue color will reappear on shaking with air or on -standing several days, due to absorption of O and oxidation of the -reduced litmus, provided the heating has produced no other change in -the milk, as proper heating will not. - -=Gelatin Culture Medium.=--Gelatin to the extent of 10 to 15 per -cent. is frequently added to broth and gives a culture medium of -many advantages. It is solid at temperatures up to about 25 deg. and -fluid above this temperature, a property which is of great advantage -in the isolation of bacteria. (See Chapter XVIII.) Further gelatin -is liquefied (that is digested, converted into gelatin proteose and -gelatin peptone, which are soluble in water and do not gelatinize) -by many bacteria and not by others, a valuable diagnostic feature. -The gelatin colonies of many bacteria are very characteristic in -appearance, as is the growth of many on gelatin in culture tubes. - -Gelatin medium may be prepared by adding the proper amount of gelatin -(10 to 15 per cent. by weight) broken into small pieces (powdered -gelatin in the same proportion may be used) to broth, gently warming -until the gelatin is dissolved, standardizing as for broth, filtering -and sterilizing. It is usually cleared before filtering by stirring -into the gelatin solution, cooled to below 60 deg., the white of an egg -for each 1000 cc., and then thoroughly boiling before filtering. The -coagulation of the egg albumen entangles the suspended matter so that -the gelatin filters perfectly clear, though with a slight yellowish -color. The filtering may be done through filter paper if the gelatin -is well boiled and filtered boiling hot, but is more conveniently done -through absorbent cotton, wet with boiling water. - -Or, the gelatin may be added to _meat juice before it is boiled_, then -this is heated to about body temperature (not too hot, or the proteins -will be coagulated too soon) until the gelatin is dissolved. Then -the material is standardized and thoroughly boiled and filtered. The -proteins of the meat juice coagulate and thus clear the medium without -the addition of egg white. Commercial gelatin is markedly acid from the -method of manufacture, hence the medium requires careful titration, -even when made from a standardized broth. - -Gelatin should be sterilized by discontinuous heating at 100 deg. on three -successive days, because long boiling or heating above 100 deg. tends to -hydrolyze the gelatin into gelatin proteose and peptone and it will -not gelatinize on cooling. It may be heated in the autoclave for -ten to fifteen minutes at 10 pounds' pressure and sometimes not be -hydrolyzed, but the procedure is uncertain and very resistant spores -may not be killed. The medium should be put into the culture tubes in -which it is to be used as soon as filtered, and sterilized in these, -since, if put into flasks these must be sterilized, and then when -transferred to tubes for use, it must be again sterilized unless great -care is taken to have the tubes plugged and sterilized first, and in -transferring aseptically to these tubes. These repeated heatings are -very apt to decompose the gelatin, so it will not "set" on cooling. The -prepared and sterilized tubes of gelatin should be kept in an ice-box -or cool room, as they will melt in overheated laboratories in summer or -winter. - -=Agar Medium.=--Agar agar, usually called agar, is a complex -carbohydrate substance of unknown composition obtained from certain -seaweeds along the coast of Japan and Southeastern Asia. It occurs -in commerce as thin translucent strips or as a powder. It resembles -gelatin only in the property its solutions have of gelatinizing when -cooled. Gelatin is an albuminoid closely related to the proteins, -agar a carbohydrate. Agar is much less soluble in water, 1 or 1.5 -per cent. of agar giving a jelly as dense as 10 to 15 per cent. of -gelatin. It dissolves only in water heated to near the boiling-point -(98 deg. to 99 deg.) and only after much longer heating. This hot solution -"jells," "sets" or gelatinizes at about 38 deg. and remains solid until -again heated to near boiling. Hence bacteria may be grown on agar at -the body temperature (37 deg.) and above, and the agar will remain solid, -while gelatin media are fluid above about 25 deg.. No pathogenic bacteria -and none of the saprophytes liable to be met with in the laboratory are -able to "liquefy" agar. - -An agar medium is conveniently prepared from broth by adding 1 or -1.5 per cent. of the finely divided agar to the broth and boiling -until dissolved, standardizing, clearing, filtering, and sterilizing. -The agar must be thoroughly boiled, usually for ten to fifteen -minutes, and the water loss made up by the addition of distilled -water before titration. Agar is practically neutral so that there is -little difference between the titration of the dissolved agar and -the original broth. The agar solution should be kept hot from the -beginning to the end except the cooling down to below 60 deg., when the -egg white for clearing is added. Though filtration through paper is -possible as with gelatin, if the agar solution is thoroughly boiled -and filtered boiling hot, it is more satisfactory for beginners to -use absorbent cotton wet with boiling water and to pour the hot agar -through the same filter if not clear the first time. The solidified -agar medium is never perfectly clear, but always more or less -opalescent. The agar medium may be sterilized in the autoclave for -fifteen minutes at 15 pounds pressure as the high temperature does not -injure the agar. - -=Potato Media.=--Potatoes furnish a natural culture medium which is -very useful for the study of many bacteria. The simplest, and for most -purposes the best, way to use potatoes is in culture tubes as "potato -tube cultures" (No. 8, Fig. 119). These are prepared as follows: -Large tubes are used. Large healthy potatoes are selected. Each end -of the potato is sliced off so as to have parallel surfaces. With a -cork-borer of a size to fit the tubes used, cylinders about one and -one-half inches long are made. Each cylinder is cut diagonally from -base to base. This furnishes two pieces each with a circular base and -an oval, sloping surface. The pieces are then washed clean and dropped -for a minute into boiling water to destroy the oxidizing enzyme on the -surface which would otherwise cause a darkening of the potato. (The -darkening may also be prevented by keeping the freshly cut potatoes -covered with clean water until ready to sterilize.) A bit of cotton -one-fourth to one-half inch in depth is put into each of the test-tubes -to retain moisture and a piece of potato dropped in, circular base -down. The tubes are then plugged with cotton and sterilized in the -autoclave at 15 pounds pressure for not less than twenty-five minutes, -since potatoes usually harbor very resistant spores, and it is not -unusual for a few tubes to spoil even after this thorough heating. - -Potatoes are sometimes used in "potato plate cultures." The term "plate -culture" is a relic of the time when flat glass plates were used for -this and other "plate cultures." Now glass dishes of the general -form shown in Fig. 115, called "Petri dishes," or plates are used for -practically all plate culture work. For "potato plates" slices from -potatoes are cut as large and as thick as the relative sizes of potato -and dish permit (Fig. 116). The slices should be thin enough not to -touch the lid and thick enough to be firm. - -[Illustration: FIG. 115.--Petri dish with the lid partly raised. x 1/2] - -[Illustration: FIG. 116.--A potato plate. x 1/2] - -It is a good plan to wrap each dish separately in paper to retain the -lid securely, then sterilize as for potato tubes, and leave plates -wrapped until wanted. - -It sometimes happens that the natural acidity of potatoes is too great -for the growth of many organisms. The acidity is sufficiently corrected -by soaking the pieces of potato in a 1 per cent. solution of sodium -carbonate for an hour before they are put into the tubes or plates. - -_Glycerinized potato tubes_ are conveniently prepared by covering the -potato in the tube with glycerin broth, sterilizing and pouring off the -excess broth immediately after sterilizing, taking care that the tubes -do not become contaminated which is not very probable if the work is -quickly done while the tubes are still hot. - -=Blood Serum Media.=--Blood serum, usually from the larger, domestic -animals on account of convenience in securing it in quantity, is used -in the study of the bacteria causing disease in man and animals. Most -commonly the serum is collected from the clotted blood after it has -well separated (usually about forty-eight hours is required for this). -It is then run into tubes which are plugged with cotton and placed in -an apparatus for coagulating the serum by heat. A copper water bath -with a tightly closed air compartment or the horizontal autoclave (Fig. -81) is sufficient for this purpose, though special forms of apparatus -are to be had. It is important that the temperature be raised slowly so -that the blood gases escape gradually. Three to five hours or longer -should be allowed for the temperature to reach the boiling-point. If -the tubes are heated too rapidly, the serum is filled with bubbles and -badly torn since the gases are driven off suddenly. _Loeffler's serum_ -is made by adding one part of dextrose broth to three parts of serum -and then coagulating as above. The solidified serum in either case is -best sterilized discontinuously, though with care the autoclave at 15 -pounds pressure may be used for a single sterilization. This is very -apt to cause a greater darkening of the serum and frequently also a -laceration of the solid mass by escaping gases. - -Blood serum is also used in the liquid state. For this purpose it is -best to collect it aseptically; or it may be sterilized discontinuously -at a temperature of 55 deg. or 56 deg. on seven to ten consecutive days. Novy -has recently suggested dialyzing the serum to free it from salts and -thus prevent its coagulation when heated. Whether the removal of the -various "extractives" which diffuse out with the salts deprives the -serum of any of its advantageous properties remains to be ascertained. - -From the discussion of the physiological activities of bacteria in -Chapters IX-XII it is apparent that a very great variety of culture -media other than those described is necessary for the study of special -types of bacteria, but such media are beyond the scope of the present -work. - -The ideal culture media are without a doubt the _synthetic media_, that -is media of definite known chemical composition, so that the various -changes due to the growth of bacteria can be accurately determined -and thus a means of sharply differentiating closely related organisms -be secured. Such media have been prepared and every bacteriologist -believes strongly in their future usefulness when media of wider -application shall have been devised. An example of this type of culture -media is Uschinsky's synthetic medium, of which the following is one of -the modifications: - - Distilled-water 1000 parts - Asparagin 4 " - Ammonium lactate 6 " - Disodium phosphate 2 " - Sodium chloride 5 " - -A criticism of this medium is that the elements K, Ca, Mg, Fe, Mn, and -S which have been shown to be essential are not present if chemically -pure salts are used in the preparation. - - - - -CHAPTER XVII. - -METHODS OF USING CULTURE MEDIA. - - -The way in which culture media shall be used depends on the purpose -in view. By far the larger part of bacteriological work is done with -cultures in "bacteriological culture tubes." Various laboratories have -their own special types but all are more or less after the "Board of -Health" form. They differ from ordinary chemical test-tubes in that -they are usually longer, have no "lip" and have much thicker walls to -prevent breakage and consequent loss of the culture as well as danger -from pathogenic organisms. The author finds two sets of tubes most -serviceable for student use--one size 15 cm. long by 19 mm. outside -diameter (No. 9, Fig. 119), the other 15 cm. long by 13 mm. (Nos. 1 to -7, Fig. 119). Culture tubes are conveniently used in "wire baskets" -circular or square in section and of a size to correspond with the -length and number of tubes used. These baskets are light, do not break, -and if made of good galvanized wire netting do not readily rust (Figs. -117 and 118). - -Liquid media such as broth, milk, litmus milk, indol and nitrate broths -are used in the above-mentioned tubes when small quantities only are -to be worked with. The tubes are filled approximately one-third full, -then plugged with _non-absorbent_ cotton and sterilized. _Cotton plugs_ -are used so much in bacteriological work because they permit a free -circulation of air and gases and at the same time act as filters to -keep out the bacteria of the air. - -Sugar broths or other media in which gas may be produced are used in -fermentation tubes (Smith tubes) of the type shown in Fig. 120 so that -the gas may be collected in the closed arm of the tube, measured (Fig. -121) and tested if desired. - -[Illustration: FIG. 117.--Round wire basket.] - -[Illustration: FIG. 118.--Square wire basket.] - -[Illustration: FIG. 119.--Culture tubes with media in them. x 2/3. _1_ -to _7_ are the smaller tubes mentioned in the text; _9_ the larger -tube; _8_ is extra large for potato tubes; _1_, plain broth; _2_, plain -milk; _3_, litmus milk; _4_, gelatin for "stab" or "puncture" culture; -_5_, agar for "stab" or "puncture" culture; _6_, agar for "slope" or -"slant" culture; _7_, blood serum; _8_, potato tube; _9_, agar for -plating. Note the transparency of the broth and gelatin and the slight -opalescence of the agar.] - -One method of using gelatin and also agar is as "puncture" or "stab" -cultures. The tubes (the narrower tubes are to be preferred for most -"stab" cultures) are filled one-third full of the medium while it is -still fluid, plugged, sterilized and allowed to cool in the vertical -position. The medium is then "inoculated" with a _straight_ platinum -needle by plunging this into the center of the surface down to the -bottom of the tube (Fig. 119, Nos. 4 and 5). - -[Illustration: FIG. 120.--Fermentation tubes. _1_, filled ready for -use; _2_, shows a cloudy growth and the development of gas in the -closed arm.] - -Agar and blood serum are frequently used in the form of "slope" or -"slant" cultures. That is, the medium solidifies with the tubes lying -on their sides which gives a long, sloping _surface_ on which the -bacteria are inoculated (Fig. 119, Nos. 6 and 7). - -[Illustration: FIG. 121.--Method of estimating percentage of gas in a -fermentation tube by means of the "gasometer", the reading is 45 per -cent.] - -[Illustration: FIG. 122.--A toxin flask showing a large surface -growth.] - -Potato tubes are likewise used for "slant" or "slope" cultures (Fig. -119, No. 8). Potatoes as "plate cultures" have been referred to. Agar -and gelatin are very largely used in the form of "plate cultures" -also. For this purpose Petri dishes are first sterilized, then the -melted agar or gelatin poured into them and allowed to "set" while the -plates are kept horizontal. The melted media may be "inoculated" before -they are poured, or a portion of the material to be "plated" may be -placed in the dish, then the melted medium poured in and distributed -over the dish by tilting in various directions, or the medium after -solidifying may be inoculated by "strokes" or "streaks" over its -surface, according to the purpose in view in using the plate. The -larger sized tubes should be used for making plates in order to have -sufficient medium in the plate (No. 9, Fig. 119). - -For using large quantities of medium, Florence flasks, Ehrlenmeyer -flasks, special toxin flasks (Fig. 122) or various other devices -(Vaughan and Novy's "mass cultures," Figs. 123 and 124) have been -employed. - -For growing _anaerobic organisms_ it is evident that some method for -removing and excluding the oxygen of the air must be used. A very great -variety of appliances have been devised for these purposes. Some are -based on the principle of the vacuum, exhausting the air with an air -pump; some on replacing the air with a stream of hydrogen; others on -absorbing the oxygen by chemical means, as with an alkaline solution -of pyrogallic acid, or even by growing a vigorous aerobe in the -same culture or in the same container with the anaerobe, the aerobe -exhausting the oxygen so that the anaerobe then develops, or finally -by excluding the air through the use of deep culture tubes well filled -with the medium, or in the closed arm of fermentation tubes. For many -purposes a combination of two or more of the above methods gives good -results. - -In any event the culture medium should have been _freshly sterilized_ -just before use, or _should be boiled_ in order to drive out the -dissolved oxygen. For most, anaerobes the presence in the medium of -about 1 per cent. of a carbohydrate, as dextrose, is advisable. - -A description of all the various devices is unnecessary in this work, -but the following have answered most of the purposes of general work in -the author's laboratories. - -[Illustration: FIG. 123.--Tank with raised lids. (Vaughan.)] - -[Illustration: FIG. 124.--Tank with lids lowered. (Vaughan.) - -FIGS. 123 and 124.--Vaughan and Novy's mass culture apparatus.] - -_A._ "_Vignal tubes_" of the style shown (Fig. 125) are made from -glass tubes of about 6 to 8 mm. outside diameter, sealed at the small -end, plugged with cotton above the constriction and sterilized. The -medium, agar or gelatin, which has been previously inoculated with the -anaerobic culture, is then drawn up into the tube, after breaking off -the tip, as far as the constriction. The tube is then sealed in the -flame at the small end and also at the constriction. Since it is full -of the medium and sealed, access of air is prevented. This forms an -excellent means for "isolation" (Chapter XVIII); the tube needs merely -to be cut with a file at the point where colonies appear, then these -may be readily transferred. - -[Illustration: FIG. 125.--Vignal tubes. x 1/3 _1_, the sterile tube -ready for inoculation; _2_, fourth dilution tube showing a few isolated -colonies, one near the figure; _3_, third dilution showing colonies -isolated but numerous; _4_, second dilution tube showing colonies still -more numerous; _5_, first dilution tube showing colonies so numerous -and small as to give a cloudy appearance to the tube. In use tube _2_ -would be filed in two at the colony and inoculations made from it.] - -_B._ "_Fermentation tubes_" form a simple means for growing liquid -cultures of anaerobes, the growth occurring in the closed arm only, -while with facultative anaerobes, growth occurs both in the closed arm -and in the open bulb. A little "paraffin oil" (a clear, heavy petroleum -derivative) may be poured on the fluid in the open bulb as a very -efficient seal, though it is not usually necessary. - -_C._ "_Deep culture tubes._"--The medium, agar, gelatin or a liquid is -poured into tubes until they are approximately one-half full, a little -paraffin oil is poured on the surface (not essential always), then the -tubes are plugged and sterilized. Inoculation is made to the bottom -and anaerobes grow well (Fig. 126). - -[Illustration: FIG. 126.--Deep tubes showing anaerobic growth. _1_, -shows a few small gas bubbles; _2_, shows the medium broken up by the -excessive development of gas.] - -_D._ For slope or plate, or any type of surface cultures the Novy jar -(Fig. 127) is the most practical device. It is good practice to combine -the vacuum method, the hydrogen replacement method and the oxygen -absorption method in using these jars. In operation a solution of 20 -per cent. NaOH is poured on the bottom of the jar to a depth of 1 or 2 -cm., the cultures are placed on glass supports above the alkali and a -short wide tube of strong pyrogallol is set in on the bottom in such a -way that it may be easily upset and mixed with the alkali when it is -desired to do so. The cover is now clamped in position with all joints -well vaselined. Then the outlet tube is connected with a suction pump -and the air drawn out. Meanwhile the inlet tube has been connected -with a hydrogen generator, and after the jar is exhausted hydrogen is -allowed to flow in, and this process is repeated until one is satisfied -that the air is replaced. The suction exhausts the air from the tubes -or plates so that much less time is required to replace the air with -hydrogen. Finally the stop-cock is closed, and the pyrogallol solution -is gently shaken down and mixed with the alkali so that any remaining -oxygen will be absorbed. - -[Illustration: FIG. 127.--Novy jars.] - -It must be remembered that facultative anaerobes as well as anaerobes -will grow under any of the above conditions, so that cultures of -organisms so obtained must be further tested aerobically in order to -determine to which group the organisms belong. - -Reference has been made above to the "inoculation" of culture media, -which means introducing into the medium used the desired material in -the proper way. For small quantities this is most conveniently done -with platinum "needles," that is, pieces of platinum wire inserted -into the ends of glass rods. The "straight" needle is a piece of -heavy platinum wire of about 0.022 inch in diameter (Fig. 128). It is -used most frequently to inoculate all forms of _solid media_. The -platinum loop is of lighter wire, 0.018 inch. The loop in the end is -conveniently made by twisting the wire around the lead of an ordinary -lead-pencil. The "loop needle" (Fig. 129) is most used in transferring -liquid media. On account of the high price of platinum, the author -has substituted "nichrome" wire for student use. This is stiffer, not -so easily made into loops and breaks out of the rods more easily. -The latter defect is remedied to some extent, by imbedding the wire -only slightly for about one-fourth of an inch on the side of the end -portion of the rod. The low cost, less than one-twentieth of platinum, -justifies its use. - -[Illustration: FIG. 128.--Straight needle.] - -[Illustration: FIG. 129.--Straight and loop needles.] - -[Illustration: FIG. 130.--Pasteur flask--"ballon pipette."] - -Sterile graduated pipettes varying in capacity from 1 cc graduated in -hundredths, upward, permit the transfer of definite amounts of liquids. -Large quantities are conveniently transferred by means of Pasteur -flasks (Fig. 130). The details of inoculation are best derived from -laboratory practice. - - - - -CHAPTER XVIII. - -ISOLATION OF BACTERIA IN PURE CULTURE. - - -As has been stated, the thorough study of a bacterium depends on -first getting it in pure culture. In the early days of bacteriology -supposedly pure cultures were obtained by (1) _dilution in liquid -media_. A series of tubes or flasks containing sterile liquid media was -prepared. Number one was inoculated with the material to be examined -and thoroughly mixed. A small portion of the mixture was transferred -to number two, and mixed; from this to number three, and so on until a -sufficient number were inoculated, the last three or four in the series -receiving the same amounts of a very high dilution of the original -material. If one or two of these latter showed a growth and the others -not, it was assumed that the dilution had been carried so far that only -a single organism was transferred and therefore the culture obtained -was "pure." The method in this crude form is too uncertain to be of -value today and recourse is had to more exact means. The procedure most -widely used is that of (2) "_plating out_" by means of gelatin or agar -plates. The material to be plated out is diluted by transferring to -three or more tubes of melted gelatin or agar as in the first method -and then all the tubes are poured into Petri dishes and grown under -suitable conditions. By proper mixing in the tubes the bacteria are -well scattered through the medium which holds the individual organisms -separate when it solidifies. On some of the plates a sufficient -dilution will be reached so that the colonies developing from the -bacteria will be so few that they are separate and pure cultures may -be obtained by inoculating from one of these a tube of the appropriate -medium (Figs. 131 to 134). The chief uncertainty with this method is -that occasionally two kinds of bacteria stick together so closely that -even the separate colonies contain both organisms. This is not common, -however. The plate colonies frequently develop from groups of bacteria -which were not separated, but as these are of the same kind the culture -is essentially pure. - -[Illustration: FIG. 131.--Dilution plates. x 3/10. _1_, shows the -first dilution, the colonies are so numerous and small that they -are invisible (compare Fig. 132); _2_, shows fewer and hence larger -colonies, but too crowded to isolate (compare Fig. 133); _3_, shows the -colonies larger and well separated, so that it is easy to isolate from -them (compare Fig. 134).] - -[Illustration: FIG. 132.--A portion of plate _1_ in Fig. 131 as seen -under the low-power objective. x 100. Very small, closely crowded -colonies.] - -Another method which is frequently applicable with material from human -or animal sources is to (3) _rub the material over the surface_ of a -slope tube or of medium solidified in a Petri dish with a sterile heavy -platinum needle, glass rod, or cotton swab. If the bacteria are not -too numerous, pure cultures may frequently be obtained. A modification -of this method is to make a series of (4) _parallel streaks on a -slope tube or plate of medium_ with a needle inserted _but once_ into -the material to be plated. On the first streak most of the bacteria -are rubbed off and a continuous growth results, but usually on the -last of a series only isolated colonies appear, which are presumably -pure. The ideal method for securing pure cultures is to be absolutely -certain that the culture starts from a single organism. This may be -accomplished by means of the (5) _apparatus and pipettes devised by -Professor Barber_ of the University of Kansas (Figs. 135 and 136). With -this instrument a single organism is picked out under the microscope -and isolated in a drop of culture medium and observed until it is -seen to divide, thus proving its viability. Transfers are then made -to the proper media. The method requires much practice to develop the -necessary skill in the making of pipettes, determining the proper -condition of the large cover-glasses used over the isolating box, and -in manipulation, but the results fully compensate. - -[Illustration: FIG. 133.--From the thinnest part of plate _2_, Fig. 131 -as seen under the low-power objective. x 100. Colonies much larger than -on plate _1_, but still crowded.] - -[Illustration: FIG. 134.--The smallest colony on plate _3_, Fig. 131, -as seen under the low-power objective. x 100. Large, single, isolated -colony.] - -Professor W. A. Starin of the author's department, a former student of -Professor Barber, has done some excellent work with this apparatus. - -[Illustration: FIG. 135.--Diagram of Barber's isolation apparatus. _b_, -moist chamber; _ms_, large cover-glass over moist chamber; _p_, small -pipette drawn out to a fine point; _k_, _r_, _g_, pipette holder; _f_, -screw for raising and lowering _k_, _r_, _g_; _s_, screw for lateral -motion of _k_, _r_, _g_; _n_, screw for clamp on pipette which allows -it to be moved in or out; _m_, mechanical stage of microscope; _t_, -rubber tube held in the mouth and used to move the liquid culture -medium in the pipette. (Journal of Infectious Diseases, October 20, -1908, vol. 5, No. 4, p. 381.)] - -[Illustration: FIG. 136.--Photograph of microscope with Barber's -isolation apparatus set up to use.] - -A number of procedures may be used to greatly facilitate the above -methods of isolation by taking advantage of the different physiological -properties of different organisms in a mixture such as ability -to form spores, different resistance to antiseptics, special food -requirements, and pathogenic properties. (_a_) If material contains -resistant spores, it may be _heated to temperatures high_ enough to -kill all of the organisms except the spores (80 deg. for half an hour, for -example) and then plated out. Or (_b_) _an antiseptic which restrains -the growth_ of some organisms and not others may be placed in the -culture media (carbolic acid, various anilin dyes, (p. 162), excess -acid, or alkali, ox bile, etc.), when the more resistant organisms grow -on the final plates, the others not. (_c_) _Special food substances_ -(various carbohydrates) from which the organism desired forms special -products (acids, aldehydes) that may be shown on the plates by various -indicators, is one of the commonest means. Or media in which certain -organisms thrive and others not, so that the former soon "crowd out" -the latter (unsterilized milk for lactic acid bacteria, inorganic -media in soil bacteriology) may be used. A combination of the general -methods (_b_) and (_c_) is much used in the separation of the organisms -of the "intestinal group" in human practice. (_d_) _The inoculation -of a susceptible animal_ with a mixture suspected to contain a given -pathogenic bacterium frequently results in the development of the -latter in pure culture in the body of an animal, from which it may be -readily recovered. In all of the above methods (except Barber's) the -first "pure culture" obtained should be "purified" by replating in a -series of dilution plates to make sure that it is pure. - - - - -CHAPTER XIX. - -STUDY OF INDIVIDUAL BACTERIA--STAINING. - - -When an organism has been obtained in pure culture by any of the -methods described in the preceding chapter the next step is the study -of its morphology as discussed in Chapters II--IV. This involves the -use of the microscope, and since bacteria are so small, objectives -of higher power than the student has presumably used will be needed. -Doubtless only the two-thirds inch or 16 mm. and the one-sixth inch or -4 mm. objectives are all that have been used in previous microscopic -work, while for examining bacteria a one-twelfth inch or 2 mm. is -necessary. It will have been observed that the higher the power of -the objective the smaller is the front lens or object glass and -consequently the less is the amount of light which enters. With the use -of the one-twelfth inch or 2 mm. objective it is necessary to employ -two devices for increasing the amount of light entering it, with which -the student is probably not familiar. One of these is to place a drop -of cedar oil between the front lens and the object and to immerse the -lens in this oil--hence the term "oil-immersion objective;" the other -is the substage or Abbe condenser. The latter is a system of lenses -placed below the stage and so constructed as to bring parallel rays of -light--daylight--from an area much larger than the face of the front -lens of the objective to a focus on the object to be examined, thus -adding very greatly to the amount of light entering the objective. -Since the condenser brings _parallel_ rays to a focus on the object, -the _flat-mirror_ is always used with the condenser when working with -daylight. With _artificial light close_ to the microscope, the concave -mirror may be used to make the divergent rays more nearly parallel and -thus give better illumination. - -The function of immersion oil is to prevent the dispersion of -considerable light that would otherwise occur owing to refraction -as the light passes up through the slide and into the air. The -accompanying diagram will help to make this clearer (Fig. 137). A ray -of light (_A B_) coming through the slide will be refracted in the -direction _B C_ if the medium has a lower refractive index than the -slide, as air has, and hence will not enter the objective _O_. If, -however, there is interposed between the objective and the slide a -medium which has the same refractive index as the slide, as immersion -oil has, then the ray will continue in the same direction (_B D_) at -the point _B_ and hence enter the objective. Evidently the immersion -oil causes much more light to enter the front lens and makes the field -brighter and at the same time prevents considerable refraction and -dispersion of light from the object seen and hence this appears more -distinct and sharply defined. The Abbe condenser and the oil-immersion -objective are practically always used in the microscopic study of -bacteria (Fig. 138). - -[Illustration: FIG. 137.--Diagram of use of immersion oil.] - -[Illustration: FIG. 138.--Diagram of paths of rays of microscope.] - - -HANGING DROP SLIDE. - -It is sometimes necessary to examine living bacteria and for this -purpose the device known as the "hanging drop slide" is used (Fig. -139). The slide has a slight concave depression ground in the middle -of one face. A ring of vaseline is placed around this depression with -the loop needle. On a clean cover-glass, large enough to fit over the -ring of vaseline, several drops of a broth culture, or of material -from a solid culture suspended in broth or physiological normal salt -solution are placed. The slide is inverted on the cover-glass in such -a way that the ring of vaseline seals the latter to the slide. When -the whole preparation is quickly turned cover side up, the drops are -seen "hanging" to the under side of the cover over the depression in -the slide. In examining such a preparation with the microscope great -care is necessary in order to focus on the bacteria, without breaking -the cover. To see the organisms distinctly the _lower iris diaphragm of -the condenser must be nearly closed_, so that the light coming through -consists mainly of parallel vertical rays, otherwise the transparent -bacteria themselves refract and diffract the light and appear blurred -and indistinct. By studying living bacteria with this device it can -be determined whether they are motile or not. The motility should not -be confounded with the familiar "Brownian movement" of all minute -insoluble inert particles which non-motile living bacteria and -also dead bacteria show. The hanging drop slide is of value in the -measurement of bacteria, since this is properly done on the living -organism. Measurement is done with a calibrated ocular micrometer as in -other kinds of measurement with the microscope with which the student -is presumably familiar. The direct effect of various agents on living -bacteria as light, electricity, heat, etc., in the study of "tropisms" -and "taxes" has been investigated on various modifications of the -above-described hanging drop slide. - -[Illustration: FIG. 139.--Hanging drop slide.] - -Cell forms and cell groupings may be studied in the same way but -these features are best determined on _stained_ preparations in many -instances. - -"Dark field" illumination and the ultramicroscope are of great value in -the study of living bacteria and other minute objects, but apparatus -of this type would scarcely be used by the student in an introductory -course, so that they will not be discussed in the present volume. - - -STAINING. - -The main use of the microscope in bacteriology is in the study of -_stained preparations_ of the organisms. Staining makes bacteria opaque -and hence more easily seen than the transparent unstained forms. Some -methods of staining also show morphological structures which are either -imperfectly recognized in the unstained cell, spores, or are not -visible at all--capsules, metachromatic granules, flagella. Finally -certain bacteria are colored by special methods of staining which do -not affect others, so that under proper conditions these bacteria may -be recognized by staining methods alone--tubercle bacilli in the organs -of animals. - -The phenomena of staining are essentially chemical, though sometimes -the chemical union is a very weak one, even resembling an absorption of -the dye rather than true chemical union--most watery stains. In other -cases the chemical compounds formed are decidedly stable and are not -decomposed even by strong mineral acids--staining of tubercle bacilli -and other "acid-fast" organisms. In still other cases the principal -action is a precipitation on the surface of the object stained--methods -for staining flagella. - -In many methods of staining in addition to the dyes used other -substances are added to the solution which assist in fixing the dye in -or on the organism stained. Such substances are called _mordants_. The -principal mordants used are alkalies, anilin, carbolic acid, iodine, -metallic salts, tannic acid. - -While it is true that some bacteria may be stained by that standard -histological nuclear dye, hematoxylin, it is of little value for this -purpose. Practically all bacteriological stains are solutions of the -_anilin dyes_. These dyes, as is well known, are of nearly every -conceivable color and shade but relatively very few are used in -bacteriological work. The beginning student will rarely use solutions -of other than the three dyes _fuchsin_ (red), _methylene blue_ and -_gentian violet_ for staining bacteria, with occasionally Bismarck -brown, or eosin, or safranin as tissue contrast stains. - -The bacteriological dyes are kept "in stock" as saturated solutions in -95 per cent. alcohol which are _never used as stains_, but merely for -convenience in making the various staining solutions. - -The approximate percentages of the three common dyes in such solutions -are indicated in the following table adapted from Woods _Chemical and -Microscopical Diagnosis_, Third Edition, 1917, Appendix: - - Fuchsin 3.0% - Gentian Violet 4.8% - Methylene Blue 2.0% - -The stains made from these dyes which are in most common use are the -following: - - 1. Aqueous (watery) gentian violet solution. - - Saturated alcoholic solution of gentian violet 1 part - Distilled water 20 parts - Mix well and filter. - - 2. Anilin gentian violet. - - Saturated alcoholic solution of gentian violet 1 part - Anilin water (see below) 10 parts - Mix well and filter. - - 3. Anilin Fuchsin. - - Saturated alcoholic solution of fuchsin 1 part - Anilin water (see below) 10 parts - Mix and filter. - -These stains rarely keep longer than ten days in the laboratory (unless -kept in the ice-box) and must be made fresh on the first sign of a -deposit on the glass of the container. - -=Anilin Water.=--Anilin water is made by putting 3 or 4 cc of anilin -"oil" in a 120 cc. flask, adding 100 cc of distilled water, shaking -vigorously for a minute or so and filtering through a wet filter, in -other words, a saturated solution of anilin in water. - - 4. Loeffler's (methylene) blue. - - Saturated alcoholic solution of methylene blue 3 parts - Aqueous solution of NaOH (or KOH), 1 to 10,000 10 " - Mix and filter. - - 5. Carbol-fuchsin (Ziehl's solution). - - Saturated alcoholic solution of fuchsin 1 part - 5 per cent. aqueous solution of carbolic acid 10 parts - Mix and filter. - - 6. Gabbet's (methylene) blue (solution). - - Dry methylene blue 4 parts - Concentrated H{2}SO{4} 25 " - Distilled water 75 " - Dissolve the dry dye in the acid and add the solution to the - distilled water and filter. - -[Illustration: FIG. 140.--Author's staining set. Square bottles are set -in square holes in the block. The capacity of each bottle is 30 cc.] - -Staining solutions are conveniently kept in square dropping bottles -inserted in a block as shown in Fig. 140. This form of holder -necessitates the use of _one hand only_ in securing the stain and -dropping it on the preparation. - -The actual staining of bacteriological preparations can be learned only -by repeated laboratory practice, yet the following methods have given -such uniform results in class work that it is felt they are not out of -place in a text-book. - -=Preparation of the "Film."=--The author learned to stain bacteria, -on the "cover-glass" but does not recall having used this method in -fifteen years and does not teach it to his students. All staining is -done on the slide. To prepare a film from a solid culture medium the -procedure is as follows: - -First, be sure the slide is clean and _free from grease_. This is -accomplished most readily by scouring a few minutes with finely -ground pumice stone and a little water, then washing and drying with -a grease-free cloth, handkerchief, or piece of cheese-cloth. With the -"loop" needle place in the middle of the slide a small loop of water. -This is best done by filling the loop by dipping in water, then tapping -it gently so that all that remains is the water that just fills the -loop level full, and this amount is placed on the slide by touching -the flat side of the loop to the glass. Then the _straight needle_ is -sterilized, dipped into the culture and just touched once into the -small drop of water on the slide. The remainder of the culture on the -straight needle is then burned off and the needle is used to spread the -drop of water containing the bacteria into a thin even film, which will -result, provided the slide is free from grease. This is dried and then -"fixed" by passing three times through the Bunsen flame at intervals of -about one second, passing through slowly for thick slides and a little -more rapidly for thin ones. If the culture is in a liquid medium, the -use of the loop of water is unnecessary; a loop of the fluid from the -surface, middle or bottom as the culture indicates is spread out to a -thin film, dried and fixed. - -After the film is fixed the stain desired is dropped on, allowed -to act for the proper time, which will depend on the stain and the -preparation, washed in water, dried thoroughly and examined with the -oil-immersion lens, without a cover. If it is desired to preserve the -preparation it may then be mounted in balsam. This is not necessary, as -they keep just as well, provided the immersion oil is removed. To do -this, fold a piece of filter paper so that at least three thicknesses -result. Lay this on the slide and press firmly several times, when the -surplus oil will be taken up by the paper. Slides not mounted in balsam -are more apt to become dusty than those that are. This is the only -disadvantage. - -=Gram's Method of Staining.=--It has been ascertained that some -bacteria contain a substance, possibly a protein, which forms a -compound with gentian violet and iodine, which compound is insoluble in -alcohol, and other bacteria do not contain this substance. Consequently -when bacteria are stained by Gram's method (given below), those that -contain this chemical remain colored, while if it is not present the -dye is washed out by the alcohol and the bacteria are colorless and may -be stained by a contrast stain. The bacteria which stain by this method -are said to "take Gram's" or to be "Gram-positive," while those that -decolorize are called "Gram-negative." The method is: - -1. Prepare the film as above given. - -2. Stain with fresh anilin gentian violet 1 minute. - -3. Wash in tap water. - -4. Cover with Gram's solution 1 minute. - -5. Wash in tap water. - -6. Wash with 95 per cent. alcohol three times or until no more color -comes out. - -7. Dry and examine. - -Gram's solution is: - - I 1 part - KI 2 parts - H{2}O 300 " - -This method is excellent for differentiating Gram-positive and -Gram-negative organisms on the same slide. First stain by this -method and after washing with alcohol stain with a counter-stain, -carbol-fuchsin diluted ten to fifteen times with water is excellent. -The Gram-positive bacteria are violet and the Gram-negative are red. - -It is also of great value in staining Gram-positive bacteria in -tissues, but the sections should be stained about five minutes in -the anilin gentian violet and be left about two minutes in the Gram's -solution. Sections are to be counter-stained in Bismarck brown, dilute -eosin or safranin solutions and cleared in oil of bergamot, lavender or -origanum and not in clove oil or carbol-xylol, as these latter dissolve -out the dye from the bacteria. - -=Staining of Spores in the Rod.=--Prepare the films as usual. Cover -with carbol-fuchsin, using plenty of stain so that it will not dry on -the slide; heat until vapor arises, not to boiling; cool until the -stain becomes cloudy and heat again until the stain clears, and repeat -once more; wash in tap water and then wash in 1 per cent. H{2}SO{4} -three times, dropping on plenty of acid, tilting and running this -over the slide three times and then pour off and use fresh acid and -repeat this once. Wash thoroughly in _distilled_ water, then stain with -Loeffler's blue one to three minutes. Wash, dry and examine. The spores -should be bright red in a blue rod. - -This method will give good results if care is taken to secure cultures -of the right age. If the culture is too old the spores will all be free -outside the rods, while if too young they will decolorize with the -acid. For _Bacillus subtilis_ and _Bacillus anthracis_, cultures on -agar slants forty-eight hours in the 37 deg. incubator are just right. For -the spores of _Clostridium tetani_, the culture should be three days -old, but may be as old as a week. - -=Staining of "Acid-fast" Bacilli.=--_Mycobacterium tuberculosis, -Mycobacterium of Johne's disease, "grass" and "butter bacilli," -Mycobacterium leprae, Mycobacterium smegmatis._ - -_Gabbet's method_: - - 1. Prepare the film as usual. - - 2. Stain with carbol-fuchsin as given above for spores. - - 3. Wash with tap water. - - 4. Decolorize and stain at the same time with Gabbet's blue, two or - three minutes. - - 5. Wash, dry and examine. - -The sulphuric acid in Gabbet's blue removes the carbol-fuchsin from -everything except the "acid-fast" bacteria, which remain red, and the -blue stains the decolorized bacteria and nuclei of any tissue cells -present. - -_Ziehl-Neelson method_: - - 1, 2, 3, as in Gabbet's method. - - 4. Decolorize with 10 per cent. HCl until washing with water shows - only a faint pink color left on slide. - - 5. Wash thoroughly. - - 6. Stain with Loeffler's blue one or two minutes. - - 7. Wash, dry and examine. - -The results are the same as with Gabbet's method. - -=Staining of Capsules.=--_Raebiger's Method._--Films of the organism -to show capsules should be _freshly prepared, dried but not fixed_. -Material is usually obtained from milk or blood. A drop of the fluid -is placed on the middle of a slide about one-fourth of the distance -from one end. The narrow edge of another clean slide is placed in this -drop and then drawn lengthwise across the slide with firm pressure. -This gives a _thin layer_ which is necessary if good results are to -be expected. The preparation is covered with a _freshly prepared_ -saturated solution of gentian violet in formalin and this allowed -to stain for 30 seconds. Then wash _lightly_, dry and examine. The -organisms appear deeply violet and much larger than with ordinary -stains and capsules are well stained and show well. - -_Welch's Method._--Prepare films as in the above method. Cover with -glacial acetic acid for 10 to 20 seconds. Wash off the acid with -carbol-fuchsin. Wash the stain off with physiological normal salt -solution (0.85 per cent.) until all surplus stain is removed. Dry and -examine. Capsules and bacteria are red. - -=Staining of Flagella.=--The rendering of flagella visible is -considered one of the most difficult processes in staining. Experience -of a number of years during which whole classes numbering from one -hundred to three hundred students accomplish this result shows that it -is no more difficult than many other staining processes. The essentials -are: (1) clean slides, (2) young cultures on agar slopes, (3) freshly -prepared mordant and stain which are kept free from precipitate, -(4) gentle heating. The author's students are furnished only stock -materials and make their own cultures, mordants and stains. - -The slides are cleaned with pumice in the usual way. An agar slope -culture of the organism to be stained from six to twenty-four hours -old is selected. A bit of the culture is removed and placed in a -watch-glass of water. The bacteria are allowed to diffuse of themselves -without stirring. After several minutes a loop of this water is removed -and three streaks are made across the slide, one in the middle and one -on each side of this about one-quarter of an inch from it. This gives -well scattered bacteria in one of the three streaks at least and very -little other material on the slide to cause precipitates. The slide is -carefully dried and fixed and then covered with an abundance of the -mordant by filtering through a small filter onto the slide so that the -mordant shows transparent on the slide. The preparation is then gently -warmed and cooled three times, adding mordant if necessary. _Do not -heat to steaming._ After mordanting for about five minutes the excess -is washed off under the tap. It is a good plan to hold the slide level -and allow the water to run into the center of the mordant and flow it -off. Inclining the slide is apt to cause the film on the surface of the -mordant to settle down on the slide and spoil the preparation. After -the mordant is washed off and all traces of it removed with a clean -cloth if necessary the stain is applied and gently heated and cooled -the same way for from three to five minutes. The preparation is then -washed, dried and examined. - -The mordant used is a modification of Loeffler's which is somewhat -simpler in preparation since the stock solution of FeCl{3} is more -permanent than FeSO{4} solution. - -Mordant sufficient for one student: - - 5 per cent. solution of FeCl{3} 20.0 cc - 25 per cent. solution of tannic acid 20.0 cc - Anilin fuchsin 4.0 cc - Normal NaOH 1.5 cc - -The solution of FeCl{3} is made up in the cold and must be perfectly -clear. The tannic acid solution must be thoroughly boiled and filtered -until clear. The iron and the acid are carefully mixed, boiled and -filtered clear. The anilin fuchsin must be added slowly with constant -stirring and the mixture boiled and filtered. The NaOH is added in the -same way and this mixture boiled and filtered. The final mordant should -not leave a film on a clean slide when poured on and allowed to run -off. Unless the mordant is in this condition and perfectly clear, it -should not be used, but a new one must be made up. Time and care in the -preparation of the mordant are essential. - -The stain to follow this mordant is anilin fuchsin. - -=Staining of Metachromatic Granules.=--_Neisser's Method._ Prepare the -film in the usual way. Stain with Neisser's stain a few seconds only. -Wash and stain with Bismarck brown a few seconds only. - - _Neisser's Stain_: - - Sat. alcoholic solution of methylene blue 1.0 part - Glacial acetic acid 2.5 parts - Distilled water 50.0 parts - - _Bismarck Brown_: - - Bismarck brown (dry dye) 2 parts - Distilled Water 1000 parts - -By the use of the hanging drop slide and the methods of staining just -described all the various morphological features of the bacterial cell -may be ascertained. - -It is necessary when _cell groupings_ as characteristic of definite -modes of division are to be determined to make slides from a liquid -culture, as broth. Place a drop of the material, preferably from the -bottom of the tube in most instances, from the top in case a pellicle -or scum is formed on the surface, on the slide and allow this to dry -_without spreading it out_, fix, wash gently with water, then stain -lightly with Loeffler's blue. Such slides also show characteristic -_cell forms_ as well. Slides should be made from solid media to show -variations in form and size and involution forms. These latter are -especially apt to occur on potato media. - - - - -CHAPTER XX. - -STUDY OF THE PHYSIOLOGY OF BACTERIA. - - -Of the environmental conditions influencing the growth of bacteria the -following are the chief ones ordinarily determined: - -_A._ Temperature.--The optimum temperature for growth is usually -about the temperature of the natural environment and ordinarily one -determines merely whether the organism grows at body temperature (37 deg.) -and at room temperature (20 deg.) or not. For exact work the maximum, -minimum and optimum temperature must be ascertained by growing in -"incubators" with varying temperatures. - -A bacteriological incubator is an apparatus for growing bacteria at a -constant temperature. This may be any temperature within the limits for -bacterial growth. If temperatures above that of an ordinary room are -desired, some source of artificial heat is needed. Electricity, gas or -oil may be used. A necessary adjunct is some device for maintaining -the temperature constant, a "thermoregulator" or "thermostat." For -lower temperatures a cooling arrangement must be installed. For the -great part of bacteriological work only two temperatures are used, 20 deg. -so-called "room temperature" (this applies to European "rooms" not to -American) and 37 deg. or body temperature. Incubators for 37 deg. of almost any -size and style desired may be secured from supply houses and need not -be further described. Figs. 141 and 142 illustrate some of the types. - -For use with large classes "incubator rooms" are to be preferred. The -author has one such room for 37 deg. work with 200 compartments for student -use which did not cost over $60 to install. - -[Illustration: FIG. 141.--Small laboratory incubator, gas heated.] - -[Illustration: FIG. 142.--Electric incubator.] - -The styles of incubators for lower temperatures, 20 deg. and below, are not -so numerous nor so satisfactory. The author has constructed a device -which answers every purpose for a small class. The diagram, Fig. 143, -explains it. - -[Illustration: FIG. 143.--Diagram of fittings for a cold incubator. -_1._ small tank for constant head, about 1 foot in each dimension. _a_, -inflow; _b_, overflow; _c_, lead pipe. _2_, refrigerator. _a'_, ice; -_b'_, flat coil under ice; _c'_, outflow to incubator. _3_, incubator. -_a"_, cold water inflow; _b"_, overflow; thermometer and burner -omitted. The diagram explains the construction. The water cooled to -about 14 deg. with artificial ice by flowing through the lead coil under -the ice, flows into the incubator which may be heated and regulated in -the usual way.] - -The thermal death-point is determined by exposing the organisms in -thin tubes of broth at varying temperatures for ten-minute periods and -then plating out to determine growth. The effect of heat may also be -determined by exposing at a given temperature, _e.g._, 60 deg., for varying -lengths of time and plating out. - -_B._ Oxygen relations--whether the organism is aerobic, anaerobic, or -facultative is determined by inoculation in gelatin or agar puncture or -stab cultures and noting whether the most abundant growth is at the -top, the bottom or all along the line of inoculation. - -_C._ Reaction of the medium--acid, alkaline or neutral as influencing -the rate and amount of growth. - -_D._ The kind of medium on which the organism grows best. - -_E._ The effect of injurious chemicals, as various disinfectants, on -the growth. - -_F._ Osmotic pressure conditions, though modifying decidedly the growth -of bacteria, are not usually studied as aids in their recognition, nor -are the effects of various forms of energy, such as light, electricity, -_x_-rays, etc. - -Among the "Physiological Activities" discussed in Chapters IX-XII those -which, in addition to the staining reactions described, are of most -use in the identification of non-pathogenic bacteria are the first ten -listed below. For pathogenic bacteria the entire thirteen are needed. - -1. Liquefaction of gelatin. - -2. Digestion of blood serum. - -3. Coagulation and digestion of milk. - -4. Acid or gaseous fermentation in milk, or both. - -5. Acid or gaseous fermentation of various carbohydrates in -carbohydrate broth, or both. - -6. Production of indol in "indol solution." - -7. Production of pigments on various media. - -8. Reduction of nitrates to nitrites, ammonia, or free nitrogen. - -9. Production of enzymes as illustrated in the above activities. - -10. Appearance of growth on different culture media. - -11. Production of free toxins as determined by injection of animals -with broth cultures filtered free from bacteria. - -12. Causation of disease as ascertained by the injection of animals -with the bacteria themselves, and recovery of the organism from the -animals. - -13. Formation of specific antibodies as determined by the -proper injection of animals with the organism or its products -and the subsequent testing of the blood serum of the inoculated -animals. - -For special kinds of bacteria other activities must be determined -(oxidation, nitrate and nitrite formation, action of sulphur and iron -bacteria, etc.). - -The first nine activities are determined by inoculating the different -culture media already described and observing the phenomena indicated, -making chemical tests where necessary. - - -APPEARANCE OF GROWTH ON DIFFERENT CULTURE MEDIA. - -In addition to those changes that are associated with the manifestation -of different physiological activities, many bacteria, show -characteristic appearances on the various culture media which are of -value in their identification. - -Too much stress should not be laid on these appearances alone, however, -since slight variations, particularly in solid media due especially to -the age of the medium, may change decidedly the appearance of a colony. -This is true of variations in the amount of moisture on agar plates. -Colonies which are ordinarily round and regular may assume very diverse -shapes, if there chance to be an excess of moisture on the surface. - -Also in slope and puncture cultures on the various solid media much -variation results from the amount of material on the inoculation needle -and just how the puncture is made, or the needle drawn over the slope. -These variations are largely prevented by the use of standard media and -by inoculating by standard methods. The Laboratory Committee of the -American Public Health Association has proposed standard methods for -all culture media and tests and for methods of inoculation, and these -have been generally adopted in this country for comparative work. - -Likewise the Society of American Bacteriologists has at different times -(1904, 1914, 1917) adopted "descriptive charts" for detailing all the -characteristics of a given organism. A committee is at present working -on a revision of the 1917 chart to be presented at the 1920 meeting. -One of the earlier charts which includes a glossary of descriptive -terms is inserted in this chapter. - -Among the cultural appearances the following are of most importance: - -[Illustration: FIG. 144.--Broth cultures x 2/3. _1_ uninoculated -transparent broth; _2_, broth cloudy from growth of organisms; _3_, -broth slightly cloudy with a deposit in bottom; _4_, broth slightly -cloudy with a heavy membrane at the surface.] - -[Illustration: FIG. 145.--A filiform stab or puncture culture. x 3/5.] - -[Illustration: FIG. 146.--A beaded stab or puncture culture. x 1/2.] - -[Illustration: FIG. 147.--A villous stab or puncture culture. x 1/2.] - -In broth cultures the presence or absence of growth on the surface -and the amount of the same. Whether the broth is rendered cloudy or -remains clear, and whether there is a deposit at the bottom or not -(Fig. 144). An abundant surface growth with little or nothing below -indicates a strict aerobe, while a growth or deposit at bottom and a -clear or nearly clear medium above, an anaerobe. These appearances are -for the first few days only of growth. If the broth is disturbed, or -after the culture stands for several days many surface growths tend to -sink to the bottom. So an actively motile organism causes in general -a cloudiness, especially if the organism is a facultative anaerobe, -which tends to clear up by precipitation after several days when the -organisms lose their motility. Non-motile facultative anaerobes -usually cloud the broth also, but settle out more rapidly than the -motile ones. - -In gelatin and agar punctures the oxygen relationship is shown by -surface growth for aerobes, growth near the bottom of the puncture -for anaerobes, and a fairly uniform growth all along the line of -inoculation for facultative anaerobes. In the case of these last -organisms, a preference for more or less oxygen is indicated by the -approach to the aerobic or anaerobic type of growth. - -[Illustration: FIG. 148 FIG. 149 FIG. 150 FIG. 151 - -FIG. 148.--Crateriform liquefaction of gelatin. x 1/2. - -FIG. 149.--Funnelform liquefaction of gelatin. x 1/2. - -FIG. 150.--Saccate liquefaction of gelatin. x 1/2. - -FIG. 151.--Stratiform liquefaction of gelatin. x 1/2.] - -Along the line of puncture the commonest types are _filiform_ (Fig. -145), which indicates a uniform growth; _beaded_ (Fig. 146), or small -separate colonies; _villous_ (Fig. 147), delicate lateral outgrowths -which do not branch; _arborescent_, tree-like growths branching -laterally from the line. In agar these branchings are usually short and -stubby, or technically, _papillate_. - -[Illustration: FIG. 152.--Filiform slope culture. x 1/2.] - -[Illustration: FIG. 153.--Filiform, slightly spreading, slope culture. -x 1/2.] - -[Illustration: FIG. 154.--Beaded slope culture. x 1/2.] - -Further, in the gelatin puncture the liquefaction which occurs is -frequently characteristic. It may be _crateriform_ (Fig. 148), a -shallow saucer at the surface; or _funnel-shaped_ (Fig. 149); or it -may be of uniform width all along the puncture, _i.e._, _saccate_ (Fig. -150); or it may be _stratiform_, (Fig. 151), _i.e._, the liquefaction -extends to the sides of the tube and proceeds uniformly downward. - -[Illustration: FIG. 155 FIG. 156 FIG. 157 FIG. 158 - -FIG. 155.--Effuse slope culture. x 1/2. - -FIG. 156.--Rhizoid slope culture. x 1/2. - -FIG. 157.--Rugose slope culture. x 1/2. - -FIG. 158.--Verrucose slope culture. x 1/2.] - -On agar, potato and blood serum slope tubes the amount of growth, its -form and elevation, the character of the surface, and the consistency -should be carefully noted, and in some few cases the character of the -edge. Figures 152 to 158 show some of the commoner types. - -[Illustration: FIG. 159.--Punctiform colonies on a plate. x 1/2.] - -[Illustration: FIG. 160.--A rhizoid colony on a plate. Natural size.] - -[Illustration: FIG. 161.--Ameboid colonies on a plate. x 1/2.] - -[Illustration: FIG. 162.--Large effuse colony on a plate. The edge is -lacerated. Incidentally the colony shows the rate of growth for six -successive days. x 2/3.] - -[Illustration: FIG. 163.--Colony with edge entire as seen under the -low-power objective. x 100.] - -[Illustration: FIG. 164.--Colony with edge coarsely granular as seen -under the low-power objective. x 100.] - -[Illustration: FIG. 165.--Colony with edge curled as seen under the -low-power objective. x 100.] - -[Illustration: FIG. 166.--Colony with edge rhizoid as seen under the -low-power objective. x 100.] - -[Illustration: FIG. 167.--A small deep rhizoid colony as seen under the -low-power objective. x 100.] - -On agar and gelatin plates made so that the colonies are well isolated, -the form of the latter, the rate of their growth, the character of the -edge and of the surface, the elevation and the internal structure -as determined by a low-power lens are often of almost diagnostic -value. Also in the case of the gelatin plates, the character of the -liquefaction is important. Figs. 159 to 167 show some of the commoner -characteristics to be noted. - -[Illustration: FIG. 168.--A small mold colony natural size as viewed by -transmitted light.] - -[Illustration: FIG. 169.--The same colony as viewed by reflected light.] - -[Illustration: FIG. 170.--A portion of the thin edge of the same colony -as seen with the lower-power objective. x 100.] - -[Illustration: FIG. 171.--A single fruiting body (sporangium) from the -same colony as seen under the lower-power objective. x 100.] - -Colonies of mold frequently appear on plates. These are readily -differentiated from bacterial colonies after a little experience. With -the naked eye usually the fine radiations of the edge of the colony -are apparent. The surface appears duller and by reflected light more -or less "fuzzy." With the low-power objective the relatively large, -branching threads of the mold (mycelia) show distinctly. Also the large -fruiting bodies (sporangia) are easily distinguished. Figs. 168 to 171 -illustrate a common black mold (_Rhizopus nigricans_). - - - - -CHAPTER XXI. - -ANIMAL INOCULATION. - - -Animal inoculation has been referred to (1) as a method of assisting -in the preparation of pure cultures of pathogenic organisms; (2) as a -means of testing the poisonous properties of substances produced in -bacterial cultures; (3) in order to test the ability of an organism to -cause a disease; (4) for the production of various antibodies; it may -be added (5) that some bacteria produce in the smaller experimental -animals lesions which do not occur in animals naturally infected, but -which nevertheless are characteristic for the given organism. The best -illustration is the testicular reaction of young male guinea-pigs to -intraperitoneal injections of glanders bacilli. Experimental animals -are also inoculated (6) to test the potency of various bacterial and -other biological products, as toxins, antitoxins, etc. - -Guinea-pigs are the most widely used experimental animals because they -are easily kept and are susceptible to so many diseases on artificial -inoculation. Rabbits are used very largely also, as are white mice. For -special purposes white rats, pigeons, goats and swine are necessary. -For commercial products horses (antitoxins) and cattle (smallpox -vaccine) are employed. In the study of many human diseases the higher -monkeys and even the anthropoid apes are necessary, since none of the -lower animals are susceptible. - -The commonest method of animal inoculation is undoubtedly the -_subcutaneous_. This is accomplished most readily with the hypodermic -needle. The skin at the point selected (usually in guinea-pigs the -lateral posterior half of the abdominal surface, in mice the back near -the root of the tail) is pinched up to avoid entering the muscles and -the needle quickly inserted. Clipping the hairs and washing with an -antiseptic solution should precede the inoculation as routine practice. -Frequently a small "skin pocket" is all that is needed. The hair is -clipped off, the skin pinched up with small forceps and a slight snip -with sharp scissors is made. The material may be inserted into this -pocket with a heavy platinum needle. _Cutaneous_ inoculation is made -by shaving the skin and rubbing the material onto the shaved surface -or scratching with a scalpel or special scarifier, but without drawing -blood, and then rubbing in the material to be inoculated. - -_Intravenous_ injections are made with larger animals. In rabbits the -posterior external auricular is a convenient vein. In larger animals -the external jugular is used. - -_Intraperitoneal_, _-thoracic_, _-cardiac_, _-ocular_, _-muscular_ -injections, and injections into the parenchyma of internal organs are -accomplished with the hypodermic needle. In the case of the first two, -injury to contained organs should be carefully avoided. Intracardiac -injection, or aspiration of the heart to secure blood, requires -considerable practice to be successful without causing the death of the -animal at once through internal hemorrhage. In _subdural_ injections -into the cranial cavity it is necessary to trephine the skull first, -while such injections into the spinal canal may be accomplished between -the vertebra with needles longer and stronger than the usual hypodermic -needle. Occasionally animals are caused to _inhale_ the organisms, or -are _fed_ cultures mixed with the feed. - - -SECURING AND TRANSPORTING MATERIAL FROM ANIMALS FOR BACTERIOLOGICAL -EXAMINATION. - -If the site of the lesion is readily accessible from the exterior, -material from the _living animal_ should be collected with sterile -instruments and kept in sterile utensils until the necessary tests can -be made. Testing should be done on material as soon after collection -as possible, in all cases, to avoid the effects of "decomposition" -bacteria. - -If the blood is to be investigated it may be aspirated from a -peripheral vein with a sterile hypodermic syringe of appropriate size -or allowed to flow through a sterile canula into sterile receptacles. -The site of the puncture should be shaved and disinfected before the -instrument is introduced. - -Discharges of whatever kind should likewise be collected in sterile -receptacles and examined as soon as may be. - -If internal organs are to be examined it is best to kill a moribund -animal than to wait for death, since after death, and in severe -infections even sometimes before, the tissues are rapidly invaded by -saprophytic bacteria from the alimentary and respiratory tracts which -complicate greatly the isolation of the specific organism. Hence the -search for specific bacteria in carcasses or organs several hours after -death is frequently negative. Animal inoculation with such material is -very often followed by sepsis or septicemia in a few hours, so that the -specific organism has no opportunity to manifest itself. - -In securing material for cultures from internal organs it is a good -plan to burn the surface of the organ with a gas or alcohol flame, or -to sear it with a hot instrument to kill surface organisms, then make -the incision or puncture through the burned area and secure material -from the interior of the organ. Such punctures made with a stiff -platinum needle frequently give pure cultures of the organism sought. -Slides may be made from such material and culture media inoculated at -once. - -Since a bacteriological diagnosis depends most commonly on growing the -organisms, it is evident that material sent for examination must _never -be treated with an antiseptic or preservative_. If decomposition is to -be feared the only safe procedure is to _pack the material in ice_ and -forward in this way. - -_Tuberculous material from the parenchyma_ of internal organs may be -forwarded in a preservative (not _formalin_, since this makes it very -difficult to stain the bacteria) as _in this special_ case a very -positive diagnosis may be made by staining alone. Even here it is -better to _pack in ice_ in order that the diagnosis by staining may be -confirmed by inoculating the living organisms into guinea-pigs. - -In the case of material _from a rabid animal_ and many protozoal -diseases the rule against preservatives is not absolute, since staining -is a reliable diagnostic means. Even in these cases it is often -desirable to inoculate animals, hence, as before stated, it is best to -make it a uniform practice to _pack material for examination in ice and -use no preservatives_. - - - - -PART IV. - -GENERAL PATHOGENIC BACTERIOLOGY. - - - - -CHAPTER XXII. - -INTRODUCTION. - - -Pathogenic Bacteriology treats of the unicellular microoerganisms which -are responsible for disease conditions, _i.e._, pathological changes -in other organisms. Hence not only are bacteria considered, but also -other low vegetable forms, as yeasts and molds, likewise protozoa in -so far as they may be pathogenic. For this reason the term pathogenic -"Microbiology" has been introduced to include all these organisms. It -is largely for the reason that the methods devised for the study of -bacteria have been applied to the investigation of other microoerganisms -that the term "bacteriology" was extended to cover the entire field. -The general discussion in this chapter is intended to include, -therefore, microoerganisms of whatever kind pathogenic to animals. - -The term pathogenic as applied to an organism must be understood in a -purely _relative_ sense, since there is no single organism that can -cause disease in all of a certain class, but each is limited to a more -or less narrow range. Some form of tuberculosis attacks nearly all -vertebrates, but no other classes of animals and no plants. Lockjaw or -tetanus attacks most mammals, but not any other vertebrates naturally. -Typhoid fever affects human beings; hog cholera, swine, etc. This point -is more fully discussed in Chapter XXIII but can not be too greatly -insisted upon. - - "The greatest enemy to mankind is man." - -Exceptions to this statement do occur and are important and must be -considered in efforts to protect completely human beings from disease -(tuberculosis from cattle, glanders from horses, poisoning from spoiled -canned goods, anthrax from hair, hides, wool, of animals dead of the -disease), but the most common human diseases are derived from other -human beings directly or indirectly. - -Diseases which are due to unicellular pathogenic microoerganisms are -called _infectious_ diseases, while if such diseases are transmitted -under natural conditions from organism to organism they are spoken of -as _contagious_ diseases. Most infectious diseases are contagious but -not all. Tetanus is a good illustration of a non-contagious infectious -disease. There are very few such diseases. - -When a unicellular microoerganism gains entrance into the body and -brings about any pathological changes there, the result is an -_infection_. Undoubtedly many pathogenic organisms get into the body -but never manifest their presence by causing disease conditions, hence -do not cause an infection. It is the pathological conditions which -result that constitute the infection, and not the mere _invasion_. - -The time that elapses between the entrance of the organism and the -appearance of symptoms is called the _period of incubation_ and varies -greatly in different diseases. - -The term _infestation_ is used to denote pathological conditions due to -_multicellular_ parasites. Thus an animal is _infested_ (not infected) -with tapeworms, roundworms, lice, mites, etc. Many of these conditions, -probably all, are contagious, _i.e._, transmissable naturally from -animal to animal. The word _contagious_ has been used in a variety -of ways to mean _communicated by direct contact_, communicated by a -living something (_contagium_) that might be carried to a distance -and finally _communicable_ in any manner, transmissable. The agency -of transmission may be very roundabout--as through a _special tick_ -in Texas fever, a _mosquito_ in malaria, etc.,--or by direct personal -contact, as generally in venereal diseases. After all, though exactness -is necessary, it is better to learn all possible about the _means of -transmission of diseases_, than quibble as to the terms to be used. - -An infectious disease may be _acute_ or _chronic_. An acute infection -is one which runs for a relatively short time and is "self-limited," -so-called, _i.e._, the organisms cease to manifest their presence after -a time. In some acute infections the time is very short--German measles -usually runs five or six days. Typhoid fever may continue eight to -ten weeks, sometimes longer, yet it is an acute infectious disease. -It is not so much the time as the fact of _self-limitation_ that -characterizes acute infections. - -In chronic infections there is little or no evidence of limitation of -the progress of the disease which may continue for years. Tuberculosis -is usually chronic. Leprosy in man is practically always so. Glanders -in horses is most commonly chronic; in mules and in man it is more apt -to be acute. - -Many infections begin acutely and later change to the chronic type. -Syphilis in man is a good illustration. - -The differences between acute and chronic infections are partly due -to the nature of the organism, partly to the number of organisms -introduced and the point of their introduction and partly to the -resistance of the animal infected. - -An infectious disease is said to be _specific_ when one kind of -organism is responsible for its manifestations--as diphtheria due -to the _Corynebacterium diphtheriae_, lockjaw due to _Clostridium -tetani_, Texas fever due to the _Piroplasma bigeminum_, etc. It is -_non-specific_ when it may be due to a variety of organisms, as -_enteritis_ (generally), _bronchopneumonia_, _wound infections_. - -Henle, as early as 1840, stated certain principles that must be -established before a given organism can be accepted as the cause of a -specific disease. These were afterward restated by Koch, and have come -to be known as "Koch's postulates." They may be stated as follows: - -1. The given organism must be found in all cases of the disease in -question. - -2. No other organism must be found in all cases. - -3. The organism must, when obtained in pure culture, reproduce the -disease in susceptible animals. - -4. It must be recovered from such animals in pure culture and this -culture likewise reproduce the disease. - -These postulates have not been fully met with reference to any disease, -but the principles embodied have been applied as far as possible -in all those infections which we recognize as specific, and whose -causative agent is accepted. In many diseases recognized as infectious -and contagious no organism has been found which is regarded as the -specific cause. In some of these the organism appears to be too small -to be seen with the highest powers of the microscope, hence they are -called "_ultramicroscopic_" organisms. Because these agents pass -through the finest bacterial filters, they are also frequently called -"_filterable_." The term "_virus_" or "_filterable virus_" is likewise -applied to these "ultramicroscopic" and "filterable" agents. - -The term _primary infection_ is sometimes applied to the first -manifestation of a disease, either specific or non-specific, while -_secondary_ refers to later developments. For example, a _secondary_ -general infection may follow a _primary_ wound infection, or _primary_ -lung tuberculosis be followed by _secondary_ generalized tuberculosis, -or _primary_ typhoid fever by a _secondary_ typhoid pneumonia. The -terms _primary_ and _secondary_ are also used where the body is -invaded by one kind of an organism and later on by another kind; thus -a _primary_ measles may be followed by _secondary_ infection of the -middle ear, or a _primary_ influenza may be followed by a _secondary_ -pneumonia, or a _primary_ scarlet fever by a _secondary_ nephritis -(inflammation of the kidney). Where several organisms seem to be -associated simultaneously in causing the condition then the term _mixed -infection_ is used--in severe diphtheria, streptococci are commonly -associated with the _Corynebacterium diphtheriae_. In many cases of -hog-cholera, mixed infections in the lungs and in the intestines are -common. Wound infections are usually _mixed_. _Auto-infection_ refers -to those conditions in which an organism commonly present in or on the -body in a latent or harmless condition gives rise to an infectious -process. If the _Bacterium coli_ normal to the intestine escapes into -the peritoneal cavity, or passes into the bladder, a severe peritonitis -or cystitis, respectively, is apt to result. "Boils" and "pimples" -are frequently autoinfections. Such infections are also spoken of as -_endogenous_ to distinguish them from those due to the entrance of -organisms from without--_exogenous_ infections. _Relapses_ are usually -instances of autoinfection. - -Those types of _secondary infection_ where the infecting agent is -transferred from one disease focus to another or several other points -and sets up the infection there are sometimes called _metastases_. Such -are the transfer of tubercle bacilli from lung to intestine, spleen, -etc., the formation of abscesses in internal organs following a primary -surface abscess, the appearance of glanders nodules throughout various -organs following pulmonary glanders, etc. - -The characteristic of a pathogenic microoerganism which indicates -its ability to cause disease is called its _virulence_. If slightly -virulent, the effect is slight; if highly virulent, the effect is -severe, and may be fatal. - -On the other hand, the characteristic of the host which indicates -its capacity for infection is called _susceptibility_. If slightly -susceptible, infection is slight, if highly susceptible, the infection -is severe. - -Evidently the degree of infection is dependent in large measure on -the relation between the _virulence_ of the invading organism and the -_susceptibility_ of the host. High virulence and great susceptibility -mean a severe infection; low virulence and little susceptibility a -slight infection; while high virulence and little susceptibility or -low virulence and great susceptibility might mean a moderate infection -varying in either direction. Other factors influencing the degree of -infection are the number of organisms introduced, the point where they -are introduced and various conditions. These will be discussed in -another connection (Chapter XXV). - -The study of pathogenic bacteriology includes the thorough study -of the individual organisms according to the methods already given -(Chapters XVIII-XXI) as an aid to diagnosis and subsequent treatment, -bacteriological or other, in a given disease. Of far greater -importance than the _treatment_, which in most infectious diseases -is not specific, is the _prevention_ and _ultimate eradication_ of -all infectious diseases. To accomplish these objects involves further -a study of the _conditions under which pathogenic organisms exist -outside the body_, _the paths of entrance into and elimination from -the body_ and those _agencies within the body itself_ which make it -_less susceptible to infection or overcome the infective agent after -its introduction_. That condition of the body itself which prevents any -manifestation of a virulent pathogenic organism after it has been once -introduced is spoken of as _immunity_ in the modern sense. Immunity is -thus the opposite of susceptibility and may exist in varying degrees. - -That scientists are and have been for some years in possession of -sufficient knowledge to permit of the prevention and eradication -of most, if not all, of our infectious diseases can scarcely be -questioned. The practical application of this knowledge presents -many difficulties, the chief of which is the absence of a public -sufficiently enlightened to permit the expenditure of the necessary -funds. Time and educative effort alone can surmount this difficulty. It -will probably be years yet, but it will certainly be accomplished. - - - - -CHAPTER XXIII. - -PATHOGENIC BACTERIA OUTSIDE THE BODY. - - -Pathogenic bacteria may exist outside the body of the host under a -variety of conditions as follows: - - I. In or on inanimate objects or material. - (_a_) As true saprophytes. - (_b_) As facultative saprophytes. - (_c_) Though obligate parasites, they exist in a latent - state. - II. In or on other animals, or products from them: - A. Susceptible to the disease. - (_a_) Sick themselves. - (As far as human beings are concerned these are - mainly: - 1. Other human beings for most diseases. - 2. Rats for plague. - 3. Dogs for rabies. - 4. Horses for glanders. - 5. Cattle, swine, parrots for tuberculosis). - (_b_) Recovered from illness. - (_c_) Never sick but "carriers." - B. Not susceptible. - (_d_) Accidental carriers. - (_e_) Serving as necessary intermediate hosts for certain - stages of the parasite--this applies to _protozoal_ - diseases only, as yet. - - -I. - -(_a_) The bacilli of tetanus, malignant edema and the organisms of -"gas gangrene" are widely distributed. There is no evidence that their -entrance into the body is at all necessary for the continuation of -their life processes, or that one case of either of these diseases -ever has any connection with any other case; they are true saprophytes. -Manifestly it would be futile to attempt to prevent or eradicate such -diseases by attacking the organism in its natural habitat. _Clostridium -botulinum_, which causes a type of food poisoning in man, does not even -multiply in the body, but the disease symptoms are due to a soluble -toxin which is produced during its growth outside the body. - -(_b_) Organisms like the bacterium of anthrax and the bacillus of -black-leg from their local occurrence seem to be distributed from -animals infected, though capable of a saprophytic existence outside the -body for years. These can no more be attacked during their saprophytic -existence than those just mentioned. Doubtless in warm seasons of the -year and in the tropics other organisms pathogenic to animals may live -and multiply in water or in damp soil where conditions are favorable, -just as the cholera organism in India, and occasionally the typhoid -bacillus in temperate climates do. - -(_c_) Most pathogenic organisms, however, when they are thrown off -from the bodies of animals, remain quiescent, do not multiply, in fact -always tend to die out from lack of all that is implied in a "favorable -environment," food, moisture, temperature, light, etc. Disinfection is -sometimes effective in this class of diseases in preventing new cases. - - -II. A. - -(_a_) The most common infectious diseases of animals are transmitted -more or less directly from other animals of the same species. Human -beings get nearly all their diseases from other human beings who are -sick; horses, from other horses; cattle, from other cattle; swine, -from swine, etc. Occasionally transmission from one species to another -occurs. Tuberculosis of swine most frequently results from feeding -them milk of tuberculous cattle or from their eating the droppings of -such cattle. Human beings occasionally contract anthrax from wool, -hair and hides of animals dead of the disease or from postmortems -on such animals; glanders from horses; tuberculosis (in children) -from tuberculous milk; bubonic plague from rats; rabies practically -always from the bites of dogs and other rabid animals, etc. The -mode of limiting this class of diseases is evidently to isolate the -sick, disinfect their discharges and their _immediate_ surroundings, -sterilize such products as must be handled or used, kill lower animals -that are dangerous, and disinfect, bury properly, or destroy their -carcasses. - -Classes of the sick that are especially dangerous for the spread of -disease are the mild cases and the undetected cases. These individuals -do not come under observation and hence not under control. - -(_b_) This class of carriers offers a difficult problem in the -prevention of infectious diseases since they may continue to give off -the organisms indefinitely and thus infect others. Typhoid carriers -have been known to do so for fifty-five years. Cholera, diphtheria, -meningitis and other carriers are well known in human practice. -Carriers among animals have not been so frequently demonstrated, -but there is every reason for thinking that hog-cholera, distemper, -roup, influenza and other carriers are common. Carriers furnish the -explanation for many of the so-called "spontaneous" outbreaks of -disease among men and animals. - -It is the general rule that those who are sick cease to carry the -organisms on recovery and it is the occasional ones who do not that are -the exceptions. In those diseases in which the organism is known it -can be determined by examination of the patient or his discharges how -long he continues to give off the causative agent. In those in which -the cause is unknown (in human beings, the commonest and most easily -transmitted diseases, scarlet fever, measles, German measles, mumps, -chicken-pox, small-pox, influenza), no such check is possible. It is -not known how long such individuals remain carriers. Hence isolation -and quarantine of such convalescents is based partly on experience -and partly on theory. It is highly probable that in the diseases just -mentioned transmission occurs in the _early stages only_, except -in small-pox and chicken-pox where the organism seems to be in the -pustules and transmission by means of material from these is possible, -though only by direct contact with it. - -The fact that such individuals are _known to have had the disease_ is a -guide for control. The methods to be used are essentially the same as -for the sick, (_a_), though obviously such human carriers are much more -difficult to deal with since they are well. - -(_c_) Another class of carriers is those who have never had the -disease. Such individuals are common and are very dangerous sources -of infection. Many of them have _associated with the sick or with -convalescents_ and these should always be suspected of harboring the -organisms. Their control differs in no way from that of class (_b_). -Unfortunately a history of such association is too often not available. -Modern transportation and modern social habits are largely responsible -for the nearly universal distribution of this type of carrier. Their -detection is probably the largest single problem in the prevention -of infectious diseases. A partial solution would be universal -bacteriological examination. In our present stage of progress this is -impossible and would not detect carriers of diseases of unknown cause. - -The various classes of carriers just discussed are in a large part -responsible for the continued presence of the commoner diseases -throughout the country. The difficulties in control have been -mentioned. A complete solution of the problem is not yet obtained. The -army experience of the past few years in the control of infectious -diseases shows what may be done. - -There is another class of carriers which might be called the "universal -carrier," _i.e._, there are certain organisms which seem to be -constantly or almost constantly present in or on the human body. These -are _micrococci_, _streptococci_ and _pneumococci_, all _Gram positive_ -organisms. They are ordinarily harmless parasites, but on occasion may -give rise to serious, even fatal, infection. Infected wounds, pimples, -boils, "common colds," most "sore throats," bronchitis, pneumonia are -pathological conditions that come in this class. Such infections are -usually autogenous. There is a constant interchange of these organisms -among individuals closely associated, so that all of a group usually -harbor the same type though no one individual can be called _the_ -carrier. Whenever, for any reason, the resistance of an individual (see -Chaps. XXV et seq.) is lowered either locally or generally some of -these organisms are liable to gain a foothold and cause infection. It -sometimes happens that a strain of dangerous organisms may be developed -in an individual in this way which is passed around to others with its -virulence increased and thus cause an epidemic. Or, since all of the -group are living under the same conditions the resistance of all or -many of them may be lowered from the same general cause and an epidemic -result from the organism common to all (pneumonia after measles, -scarlet fever and influenza in camps). Protection of the individual is -chiefly a personal question, _i.e._, by keeping up the "normal healthy -tone" in all possible ways: The use of protective vaccines (Chap. XXX) -appears to be advisable in such instances (colds, pneumonia after -measles and influenza, inflammation of throat and middle ear following -scarlet fever and measles). Results obtained in this country during -the recent influenza epidemic have been conflicting but on the whole -appear to show that preventive vaccination against _pneumonia liable to -follow_ should be practiced. - -It would seem that among groups of individuals where infection may be -expected the proper procedure would be to prepare autogenous vaccines -(Chapter XXX) from members of the group and vaccinate all with the -object of protecting them. - - -II. B. - -(_d_) In this class come the "accidental carriers" like flies, fleas, -lice, bed-bugs, ticks, and other biting and blood-sucking insects, -vultures, buzzards, foxes, rats, and carrion-eating animals generally; -pet animals in the household, etc. Here the animals are not susceptible -to the given disease but become contaminated with the organisms -and then through defilement of the food or drink or contact with -individuals or with utensils pass the organisms on to the susceptible. -Some biting and blood-sucking insects transmit the organisms through -biting infected and non-infected animals successively. The spirilloses -and trypanosomiases seem to be transmitted in this way, though there -is evidence accumulating which may place these diseases in the next -class. Anthrax is considered in some instances to be transmitted by -flies and by vultures in the southern United States. Transmission -of typhoid, dysentery, cholera and other diseases by flies is well -established in man. Why not hog-cholera from farm to farm by flies, -English sparrows, pigeons feeding, or by turkey buzzards? Though this -would not be easy to prove, it seems reasonable. - -Preventing contact of such animals with the discharges or with the -carcasses of those dead of the disease, destruction of insect carriers, -screening and prevention of fly breeding are obvious protective -measures. - -(_e_) In this class come certain diseases for which particular -insects are necessary for the parasite in question, so that certain -stages in its life history may be passed therein. The surest means -for eradicating such diseases is the destruction of the insects -concerned. Up to the present no _bacterial_ disease is known in which -this condition exists, unless Rocky Mountain spotted fever and typhus -fever shall prove to be due to bacteria. Such diseases are all due to -protozoa. Among them are Texas fever, due to _Piroplasma bigeminum_ -in this country which has been eradicated in entire districts by -destruction of the cattle tick (_Margaropus annulatus_). - -Piroplasmoses in South Africa among cattle and horses, and in other -countries are transmitted in similar ways. Probably many of the -diseases due to spirochetes and trypanosomes are likewise transmitted -by _necessary_ insect intermediaries. In human medicine the eradication -of yellow fever from Panama and Cuba is due to successful warfare -against, a certain mosquito (_Stegomyia_). So the freeing of large -areas in different parts of the world from _malaria_ follows the -destruction of the mosquitoes. The prevention of typhus fever and -of trench fever by "delousing" methods is familiar from recent army -experience though for typhus this method has been practiced in Russia -for more than ten years to the author's personal knowledge. The -campaign against disease in animals and man from insect sources must be -considered as still in its infancy. The full utilization of tropical -lands depends largely on the solution of this problem. - - - - -CHAPTER XXIV. - -PATHS OF ENTRANCE OF PATHOGENIC ORGANISMS, - -OR - -CHANNELS OF INFECTION. - - -_A._ =The Skin.=--If the skin is healthy there is no opportunity for -bacteria to penetrate it. It is protected not only by the stratified -epithelium, but also in various animals, by coats of hair, wool, -feathers, etc. The secretion pressure of the healthy sweat and oil -glands acts as an effective bar even to motile bacteria. Nevertheless a -very slight injury only is sufficient to give normal surface parasites -and other pathogenics, accidentally or purposely brought in contact -with it, an opportunity for more rapid growth and even entrance -for general infection. Certain diseases due to higher fungi are -characteristically "skin diseases" and rarely become general--various -forms of favus, trichophyton infections, etc. A few disease organisms, -tetanus, malignant edema, usually get in through the skin; others, -black-leg, anthrax, quite commonly; and those diseases transmitted -by biting and blood-sucking insects, piroplasmoses, trypanosomiases, -spirilloses, scarcely in any other way. Defective secretion in the skin -glands from other causes, may permit lodgment and growth of bacteria -in them or in the hair follicles. "Pimples" and boils in man and local -abscesses occasionally in animals are illustrations. Sharp-edged and -freely bleeding wounds are less liable to be infected than contusions, -ragged wounds, burns, etc. The flowing blood washes out the wound and -the clotting seals it, while there is less material to be repaired -by the leukocytes and they are free to care for invading organisms -(phagocytosis). Pathogenic organisms, especially pus cocci, frequently -gain lodgment in the _milk glands_ and cause local (mastitis) or -general infection. - -_B._ =Mucosae directly continuous with the skin and lined with -stratified epithelium= are commonly well protected thereby and by the -secretions. - -(_a_) The external auditory meatus is rarely the seat even of local -infection. The tympanic cavity is normally sterile, though it may -become infected by extension through the Eustachian tube from the -pharynx (_otitis media_). - -(_b_) The conjunctiva is frequently the seat of localized, very rarely -the point of entrance for a generalized infection, except after severe -injury. Those diseases whose path of entrance is generally assumed to -be the respiratory tract (see "Lungs" below) might also be admitted -through the eye. Material containing such organisms might get on the -conjunctiva and be washed down through the lachrymal canal into the -nose. Experiment has shown that bacteria may pass in this way in a -few minutes. In case masks are worn to avoid infection from patients -suffering with these diseases, the eyes should therefore be protected -as well as the nose and mouth. - -(_c_) The nasal cavity on account of its anatomical structure retains -pathogenic organisms which give rise to local infections more -frequently than other mucosae of its character. These may extend from -here to middle ear, neighboring sinuses, or along the lymph spaces of -the olfactory nerve into the cranial cavity (meningitis). Acute coryza -("colds" in man) is characteristic. Glanders, occasionally, is primary -in the nose, as is probably roup in chickens, leprosy in man. The -meningococcus and the virus of poliomyelitis pass from the nose into -the cranial cavity without local lesions in the former. - -(_d_) The mouth cavity is ordinarily protected by its epithelium -and secretions, though the injured mucosa is a common source of -_actinomycosis_ infection, as well as thrush. In foot-and-mouth disease -no visible lesions seem necessary to permit the localization of the -unknown infective agent. - -(_e_) The tonsils afford a ready point of entrance for ever-present -_micrococci_ and _streptococci_ whenever occasion offers (follicular -tonsillitis, "quinsy"), and articular rheumatism is not an uncommon -sequel. The diphtheria bacillus characteristically seeks these -structures for its development. Tubercle and anthrax organisms -occasionally enter here. - -(_f_) The pharynx is the seat of localized infection as in -_micrococcal_, _streptococcal_ and diphtherial "sore throat" in human -beings, but both it and the esophagus are rarely infected in animals -except as the result of injury. - -(_g_) The external genitalia are the usual points of entrance for -the venereal organisms in man (gonococcus, _Treponema pallidum_, and -Ducrey's bacillus). The bacillus of contagious abortion and probably -the trypanosome of dourine are commonly introduced through these -channels in animals. - -_C._ =Lungs.=--The varied types of pneumonia due to many different -organisms (tubercle, glanders, influenza, plague bacilli, pneumococcus, -streptococcus, micrococcus and many others) show how frequently these -organs are the seat of a localized infection, which may or may not be -general. Whether the lungs are the actual point of entrance in these -cases is a question which is much discussed at the present time, -particularly with reference to tuberculosis. The mucous secretion -of the respiratory tract tends to catch incoming bacteria and other -small particles and the ciliary movement along bronchial tubes and -trachea tends to carry such material out. "Foreign body pneumonia" -shows clinically, and many observers have shown experimentally that -microoerganisms may reach the alveoli even though the exchange of -air between them and the bronchioles and larger bronchi takes place -ordinarily only by diffusion. The presence of carbon particles in the -walls of the alveoli in older animals and human beings and in those -that breathe dusty air for long periods indicates strongly, though it -does not prove absolutely, that these came in with inspired air. On the -other hand, experiment has shown that tubercle bacilli introduced into -the intestine may appear in the lungs and cause disease there and not -in the intestine. It is probably safe to assume that in those diseases -which are transmitted most readily through close association though -not necessarily actual contact, the commonest path is through the -respiratory tract, which may or may not show lesions (smallpox, scarlet -fever, measles, chicken-pox, whooping-cough, pneumonic plague in man, -lobar and bronchopneumonias and influenza in man and animals, some -cases of glanders and tuberculosis). On the other hand, the fact that -the _Bacterium typhosum_ and _Bacterium coli_ may cause pneumonia when -they evidently have reached the lung from the intestinal tract, and the -experimental evidence of lung tuberculosis above mentioned show that -this route cannot be excluded in inflammations of the lung. - -_D._ =Alimentary Tract.=--The alimentary tract affords the ordinary -path of entrance for the causal microbes of many of the diseases of -animals and man, since they are carried into the body most commonly and -most abundantly in the food and drink. - -(_a_) The stomach is rarely the seat of local infection, even in -ruminants, except as the result of trauma. The character of the -epithelium in the rumen, reticulum and omasum in ruminants, the -hydrochloric acid in the abomasum and in the stomachs of animals -generally are usually sufficient protection. Occasionally anthrax -"pustules" develop in the gastric mucosa. (The author saw nine such -pustules in a case of anthrax in a man.) - -(_b_) The intestines are frequently the seat of localized infections, -as various "choleras" and "dysenteries" in men and many animals, -anthrax, tuberculosis, Johne's disease. Here doubtless enter the -organisms causing "hemorrhagic septicemias" in many classes of animals, -and numerous others. These various organisms must have passed through -the stomach and the question at once arises, why did the HCl not -destroy them? It must be remembered that the acid is present only -during stomach digestion, and that liquids taken on an "empty stomach" -pass through rapidly and any organisms present are not subjected to -the action of the acid. Also spores generally resist the acid. Other -organisms may pass through the stomach within masses of undigested -food. The fact that digestion is going on in the stomach of ruminants -practically all the time may explain the relative freedom of _adult_ -animals of this class from "choleras" and "dysenteries." - - -MECHANISM OF ENTRANCE OF ORGANISMS. - -In the preceding chapters statements have been made that "bacteria -enter" at various places or they "pass through" different mucous -membranes, skin, etc. Strictly speaking such statements are -incorrect--bacteria do not "enter" or "pass through" of themselves. -It is true that some of the intestinal organisms are motile, but most -of the bacteria which are pathogenic are non-motile. Even the motile -ones can not make their way against fluids secreted or excreted on free -surfaces. Bacteria cannot pass by diffusion through membranes since -they are finite particles and not in solution. - -In the case of penetrating wounds bacteria may be carried mechanically -into the tissues, but this is exceptional in most infections. Also -after gaining lodgment they may gradually grow through by destroying -tissue as they grow, but this is a minor factor. Evidently, there -must be some mechanism by which they _are carried_ through. The known -mechanisms for this in the body are ameboid cells, especially the -phagocytes. It is most probable that these are the chief agents in -getting bacteria into the tissues through various free surfaces. The -phagocytes engulf bacteria, carry them into the tissues and either -destroy them, are destroyed by them, or may disgorge or excrete them -free in the tissues or in the blood. - - -DISSEMINATION OF ORGANISMS. - -Dissemination of organisms within the tissues occurs either through -the lymph channels or the bloodvessels or both. If through the lymph -vessels only it is usually much more restricted in extent, or much more -slowly disseminated, while blood dissemination is characterized by the -number of organs involved simultaneously. - - -PATHS OF ELIMINATION OF PATHOGENIC MICROeORGANISMS. - -I. Directly from the point, of injury. This is true in infected -wounds open to the surface, skin glanders (farcy), black-leg, -surface anthrax, exanthemata in man and animals (scarlet fever (?), -measles (?), smallpox; hog erysipelas, foot-and-mouth disease): -also in case of disease of mucous membranes continuous with the -skin--from nasal discharges (glanders), saliva (foot-and-mouth -disease), material coughed or sneezed out (tuberculosis, influenza, -pneumonias), urethral and vaginal discharges (gonorrhea and syphilis -in man, contagious abortion and dourine in animals), intestinal -discharges (typhoid fever, "choleras," "dysenteries," anthrax, -tuberculosis, Johne's disease). Material from nose, mouth and lungs -may be swallowed and the organisms passed out through the intestines. - -II. Indirectly through the secretions and the excretions where the -internal organs are involved. The _saliva_ of rabid animals contains -the ultramicroscopic virus of rabies (the sympathetic ganglia -within the salivary glands, and pancreas also, are affected in this -disease as well as the cells of the central nervous system). The -_gall-bladder_ in man is known to harbor colon and typhoid bacilli, -as that of hog-cholera hogs does the virus of this disease. It may -harbor analogous organisms in other animals, though such knowledge -is scanty. The _kidneys_ have been shown experimentally to excrete -certain organisms introduced into the circulation within a few minutes -(micrococci, colon and typhoid bacilli, anthrax). Typhoid bacilli occur -in the urine of typhoid-fever patients in about 25 per cent. of all -cases and the urine of hogs with hog cholera is highly virulent. Most -observers are of the opinion, however, that under natural conditions -the kidneys do not excrete bacteria unless they themselves are infected. - -The _milk_ both of tuberculous cattle and tuberculous women has been -shown to contain tubercle bacilli _even when the mammary glands are not -involved_. Doubtless such bacteria are carried through the walls of the -secreting tubules or of the smaller ducts by phagocytes and are then -set free in the milk. - - -SPECIFICITY OF LOCATION OF INFECTIVE ORGANISMS. - -It is readily apparent that certain disease organisms tend to locate -themselves in definite regions and the question arises, Is this due -to any specific relationship between organism and tissue or not? -Diphtheria in man usually attacks the tonsils first, gonorrhea and -syphilis the external genitals, tuberculosis the lung, "choleras" the -small intestine, "dysenteries" the large intestine, influenza the -lungs. In these cases the explanation is probably that the points -attacked are the places where the organism is most commonly carried, -with no specific relationship, since all of these organisms (Asiatic -cholera excepted) also produce lesions in other parts of the body _when -they reach them_. On the other hand, the virus of hydrophobia attacks -nerve cells, leprosy frequently singles out nerves, glanders bacilli -introduced into the abdominal cavity of a young male guinea-pig cause -an inflammation of the testicle, malarial parasites and piroplasms -attack the red blood corpuscles, etc. In fact, most _pathogenic -protozoa_ are specific in their localization either in certain tissue -cells or in the blood or lymph. In these cases there is apparently a -real chemical relationship, as there is also between the _toxins_ of -bacteria and certain tissue cells (tetanus toxin and nerve cells). -Whether "chemotherapy" will ever profit from a knowledge of such -chemical relationships remains to be developed. It appears that a -search for these specific chemical substances with the object of -combining poisons with them so that the organisms might in this way be -destroyed, would be a profitable line of research. - - - - -CHAPTER XXV. - -IMMUNITY. - - -Immunity, as has already been stated, implies such a condition of the -body that pathogenic organisms after they have been introduced are -incapable of manifesting themselves, and are unable to cause disease. -The word has come to have a more specific meaning than resistance in -many instances, in other cases the terms are used synonymously. It is -the opposite of susceptibility. The term must be understood always in a -relative sense, since no animal is immune to all pathogenic organisms, -and conceivably not entirely so to anyone, because there is no question -that a sufficient number of bacteria of any kind might be injected -into the circulation to kill an animal, even though it did it purely -mechanically. - -Immunity may be considered with reference to a single individual or to -entire divisions of the organic world, with all grades between. Thus -plants are immune to the diseases affecting animals; invertebrates to -vertebrate diseases; cold-blooded animals to those of warm blood; man -is immune to most of the diseases affecting other mammals; the rat to -anthrax, which affects other rodents and most mammals; the well-known -race of Algerian sheep is likewise immune to anthrax while other sheep -are susceptible; the negro appears more resistant to yellow fever than -the white; some few individuals in a herd of hogs always escape an -epizooetic of hog cholera, etc. - -Immunity within a given species is modified by a number of -factors--age, state of nutrition, extremes of heat or cold, fatigue, -excesses of any kind, in fact, anything which tends to lower the -"normal healthy tone" of an animal also tends to lower its resistance. -Children appear more susceptible to scarlet fever, measles, -whooping-cough, etc., than adults; young cattle more frequently have -black-leg than older ones (these apparently greater susceptibilities -may be due in part to the fact that most of the older individuals -have had the diseases when young and are immune for this reason). -Animals weakened by hunger or thirst succumb to infection more readily. -Frogs and chickens are immune to tetanus, but if the former be put -in water and warmed up to and kept, at about 37 deg., and the latter be -chilled for several hours in ice-water, then each may be infected. -Pneumonia frequently follows exposure to cold. The immune rat may -be given anthrax if first he is made to run in a "squirrel cage" -until exhausted. Alcoholics are far less resistant to infection than -temperate individuals. "Worry," mental anguish, tend to predispose to -infection. - -The following outlines summarize the different, classifications of -immunity so far as mammals are concerned for the purposes of discussion. - -Immunity. - - { {1. Inherited through - { { the germ cell or cells. - { { {(_a_) By having the - {A. Congenital {2. Acquired { disease _in utero_. - I. Natural { { _in utero_. {(_b_) By absorption - { { { of immune - { { substances - {B. Acquired by { from the mother. - { having the disease. - - II. Artificial--acquired through human agency by: - 1. Introduction of the organism or its products. - 2. Introduction of the blood serum of an immune animal. - -Immunity. - - I. Active--due to the introduction of the organism or due to the - introduction of the products of the organism. - A. Naturally by having the disease. - B. Artificially. - 1. By introducing the organism: - {1. Passage through another animal. - {2. Drying. - (_a_) Alive and virulent. {3. Growing at a higher temperature. - (_b_) Alive and virulence {4. Heating the cultures. - reduced by {5. Treating with chemicals. - (_c_) Dead. {6. Sensitizing. - {7. Cultivation on artificial media. - 2. By introducing the products of the organism. - - II. Passive--due to the introduction of the blood serum of an actively - immunized animal. - -Immunity present in an animal and not due to human interference is to -be regarded as _natural_ immunity, while if brought about by man's -effort it is considered _artificial_. Those cases of natural immunity -mentioned above which are common to divisions, classes, orders, -families, species or races of organisms and to those few individuals -where no special cause is discoverable, must be regarded as instances -of true _inheritance_ through the germ cell as other characteristics -are. All other kinds of immunity are _acquired_. Occasionally young are -born with every evidence that they have had a disease _in utero_ and -are thereafter as immune as though the attack had occurred after birth -("small-pox babies," "hog-cholera pigs"). Experiment has shown that -immune substances may pass from the blood of the mother to the fetus -_in utero_ and the young be immune for a time after birth (tetanus). -This is of no practical value as yet. It is a familiar fact that with -most infectious diseases recovery from one attack confers a more or -less lasting immunity, though there are marked exceptions. - -=Active Immunity.=--By active immunity is meant that which is due -to the actual introduction of the organism, or in some cases of its -products. The term active is used because the body cells of the animal -immunized perform the real work of bringing about the immunity as -will be discussed later. In _passive_ immunity the blood serum of an -actively immunized animal is introduced into a second animal, which -thereupon becomes immune, though its cells are not concerned in the -process. The animal is _passive_, just as a test-tube, in which a -reaction takes place, plays no other part than that of a passive -container for the reagents. - -In _active_ immunity the organism may be introduced in what is to -be considered a natural manner, as when an animal becomes infected, -has a disease, without human interference. Or the organism may be -purposely introduced to bring about the immunity. For certain purposes -the introduction of the products of the organism (toxins) is used to -bring about active immunity (preparation of diphtheria and tetanus -antitoxin from the horse). The method of producing active immunity by -the artificial introduction of the organism is called _vaccination_, -and a _vaccine_ must therefore contain the organism. _Vaccines_ for -_bacterial_ diseases are frequently called _bacterins_. The use of -the blood serum of an immunized animal to confer passive immunity on -a second animal is properly called _serum therapy_, and the serum so -used is spoken of as an _antiserum_, though the latter word is also -used to denote any serum containing any kind of an antibody (Chapters -XXVII-XXXI). In a few instances both the organism and an antiserum are -used to cause both active and passive immunity (_serum-simultaneous -method_ in immunizing against hog cholera). - -In producing active immunity the organism may be introduced (_a_) -_alive and virulent_, but in very small doses, or in combination with -an immune serum, as just mentioned for hog cholera. The introduction -of the live virulent organism alone is done only experimentally as -yet, as it is obviously too dangerous to do in practice, except under -the strictest control (introduction of a _single tubercle_ bacillus, -followed by gradually increasing numbers--Barber and Webb). More -commonly the organisms are introduced (_b_) alive but with their -_virulence reduced_ ("attenuated") in one of several ways: (1) By -passing the organism through another animal as is the case with -_smallpox vaccine_ derived from a calf or heifer. This method was -first introduced by Jenner in 1795 and was the first practical means -of preventing disease by _vaccination_. This word was used because -material was derived from a cow--Latin _vacca_. (2) By drying the -organism, as is done in the preparation of the vaccine for the _Pasteur -treatment of rabies_, where the spinal cords of rabbits are dried for -varying lengths of time--one to four days, Russian method, one to three -days, German method, longer in this country. (It is probable that the -passage of the "fixed virus" through the rabbit is as important in this -procedure as the drying, since it is doubtful if the "fixed virus" is -pathogenic for man.) It would be more correct to speak of this as a -_preventive vaccination against rabies_, since the latter is one of the -few diseases which is not amenable to _treatment_. The patient always -dies if the disease develops. (3) The organism may be attenuated by -growing at a temperature above the normal. This is the method used in -preparing _anthrax vaccine_ as done by Pasteur originally. (4) Instead -of growing at a higher temperature the culture may be heated in such -a way that it is not killed but merely weakened. _Black-leg_ vaccines -are made by this method. (5) Chemicals are sometimes added to attenuate -the organisms, as was formerly done in the preparation of black-leg -vaccine by Kruse's method in Germany. The use of toxin-antitoxin -mixtures in immunizing against diphtheria and in the preparation -of diphtheria antitoxin from horses is an application of the same -principle, though here it is the _product_ of the organism and not the -organism whose action is weakened. (6) Within the past few years the -workers in the Pasteur Institute in Paris have been experimenting with -vaccines prepared by treating living virulent bacteria with antisera -("sensitizing them") so that they are no longer capable of causing -the disease when introduced, but do cause the production of an active -immunity. The method has been used with typhoid fever bacilli in man -and seems to be successful. It remains to be tried out further before -its worth is demonstrated (the procedure is more complicated and the -chance for infection apparently much greater than by the use of killed -cultures). The term _sero-bacterins_ is used by manufacturers in this -country to designate such bacterial vaccines. (7) Growing on artificial -culture media reduces the virulence of most organisms after a longer -or shorter time. This method has been tried with many organisms in the -laboratory, but is not now used in practice. The difficulties are that -the attenuation is very uncertain and that the organisms tend to regain -their virulence when introduced into the body. - -In producing active immunity against many bacterial diseases the -organisms are introduced (_c_) dead. They are killed by heat or by -chemicals, or by using both methods (Chapter XXX). - -When the products of an organism are introduced the resulting immunity -is against the products only and not against the organism. If the -organism itself is introduced there results an immunity against it -and in some cases also against the products, though the latter does -not necessarily follow. Hence the immunity may be _antibacterial_ or -_antitoxic_ or both. - -Investigation as to the causes of immunity and the various methods by -which it is produced has not resulted in the discovery of specific -methods of treatment for as many diseases as was hoped for at one -time. Just at present progress in serum therapy appears to be at a -standstill, though vaccines are giving good results in many instances -not believed possible a few years ago. As a consequence workers in all -parts of the world are giving more and more attention to the search for -_specific chemical substances_, which will destroy invading parasites -and not injure the host (_chemotherapy_). Nevertheless, in the study -of immunity very much of value in the treatment and prevention of -disease has been learned. Also much knowledge which is of the greatest -use in other lines has been accumulated. Methods of _diagnosis_ of -great exactness have resulted, applicable in numerous diseases. Ways -of _detecting adulteration_ in foods, particularly foods from animal -sources, and of _differentiating proteins_ of varied origin, as well -as means of establishing _biological relationships_ and differences -among groups of animals through "immunity reactions" of blood serums -have followed from knowledge gained by application of the facts or the -methods of immunity research. Hence the study of "immunity problems" -has come to include much more than merely the study of those factors -which prevent the development of disease in an animal or result in -its spontaneous recovery. A proper understanding of the principles of -immunity necessitates a study of these various features and they will -be considered in the discussion to follow. - - - - -CHAPTER XXVI. - -THEORIES OF IMMUNITY. - - -Pasteur and the bacteriologists of his time discovered that bacteria -cease to grow in artificial culture media after a time, because of -the exhaustion of the food material in some cases and because of the -injurious action of their own products in other instances. These facts -were brought forward to explain immunity shortly after bacteria were -shown to be the cause of certain diseases. Theories based on these -observations were called (1) "_Exhaustion Theory_" of _Pasteur_, and -(2) "_Noxious Retention Theory_" of _Chauveau_ respectively. The fact, -soon discovered, that virulent pathogenic bacteria are not uncommonly -present in perfectly healthy animals, and the later discovery that -immunity may be conferred by the injection of dead bacteria have led -to the abandonment of both these older ideas. The (3) "_Unfavorable -Environment_" theory of _Baumgartner_, _i.e._, bacteria do not grow -in the body and produce disease because their surroundings are not -suitable, in a sense covers the whole ground, though it is not true as -to the first part, as was pointed out above, and is of no value as a -working basis, since it offers no explanation as to _what the factors -are_ that constitute the "_unfavorable environment_." Metchnikoff -brought forward a rational explanation of immunity with his (4) -"_Cellular or Phagocytosis Theory_." As first propounded it based -immunity on the observed fact that certain white blood corpuscles, -_phagocytes_, engulf and destroy bacteria. Metchnikoff has since -elaborated the original theory to explain facts of later discovery. -Ehrlich soon after published his (5) "_Chemical or Side-chain Theory_" -which seeks to explain immunity on the basis of _chemical substances_ -in the body which may in part destroy pathogenic organisms or in -part neutralize their products; or in some instances there may be -an absence of certain chemical substances in the body cells so that -bacteria or their products _cannot unite_ with the cells and hence can -do no damage. - -[Illustration: PLATE VI - -PAUL EHRLICH] - -At the present time it is generally accepted, in this country at -least, that Ehrlich's theory explains immunity in many diseases as -well as many of the phenomena related to immunity, and in other -diseases the phagocytes, frequently assisted by chemical substances, -are the chief factors. Specific instances are discussed in _Pathogenic -Bacteriologies_ which should be consulted. It is essential that the -student should be familiar with the basic ideas of the chemical -theory, not only from the standpoint of immunity, but also in order to -understand the principles of a number of valuable methods of diagnosis. - -The chemical theory rests on three fundamental physiological -principles: (1) the response of cells to stimuli, in this connection -_specific chemical stimuli_, (2) the presence within cells of _specific -chemical groups_ which combine with chemical stimuli and thus enable -them to act on the cell, which groups Ehrlich has named _receptors_, -and (3) the "_over-production_" activity of cells as announced by -Weigert. - -1. That cells respond to stimuli is fundamental in physiology. These -stimuli may be of many kinds as mechanical, electrical, light, thermal, -chemical, etc. The body possesses groups of cells specially developed -to _receive_ some of these stimuli--touch cells for mechanical stimuli, -retinal cells for light, temperature nerve endings for thermal, -olfactory and gustatory cells for certain chemical stimuli. _Response_ -to chemical stimuli is well illustrated along the digestive tract. That -the chemical stimuli in digestion may be more or less specific is shown -by the observed differences in the enzymes of the pancreatic juice -dependent on the relative amounts of carbohydrates, fats, or proteins -in the food, the specific enzyme in each case being increased in the -juice with the increase of its corresponding foodstuff. The cells of -the body, or certain of them at least, seem to respond in a specific -way when substances are brought into direct contact with them, that is, -without having been subjected to digestion in the alimentary tract, -but injected directly into the blood or lymph stream. Cells may be -affected by stimuli in one of three ways: if the stimulus is too weak, -there is no effect (in reality there is no "stimulus" acting); if the -stimulus is too strong, the cell is injured, or may be destroyed; if -the stimulus is of proper amount then it excites the cell to increased -activity, and in the case of _specific chemical stimuli_ the increased -activity, as mentioned for the pancreas, shows itself in an _increased -production of whatever is called forth by the chemical stimulus_. In -the case of many organic chemicals, the substances produced by the -cells under their direct stimulation are markedly specific for the -particular substance introduced. - -2. Since chemical action always implies at least two bodies to react, -Ehrlich assumes that in every cell which is affected by a chemical -stimulus there must therefore be a chemical group to unite with this -stimulus. He further states that there must be as many different -kinds of these groups as there are different kinds of chemicals which -stimulate the cell. Since these groups are present in the body cells to -_take up_ different kinds of chemical substances, Ehrlich calls them -_receptors_. Since these groups must be small as compared with the cell -as a whole, and must be more or less on the surface and unite readily -with chemical substances he further speaks of them as "side-chains" -after the analogy of compounds of the aromatic series especially. The -term _receptors_ is now generally used. As was stated above, the effect -of _specific chemical stimuli_ is to cause the production of _more of -the particular substance_ for which it is specific and in the class -of bodies under discussion, the _particular product is these cell -receptors_ with which the chemical may unite. - -3. Weigert first called attention to the practically constant -phenomenon that cells ordinarily respond by doing more of a particular -response than is actually called for by the stimulus, that there is -always an "overproduction" of activity. In the case of chemical stimuli -this means an _increased production of the specific substance_ over and -above the amount actually needed. - -The student will better understand this theory if he recalls his -fundamental physiology. Living substance is characterized, among other -things, by irritability which is instability. It is in a constant, -state of unstable equilibrium. Whenever the equilibrium becomes -permanently stable the substance is dead. It is also continually -attempting to restore disturbances in its equilibrium. Whenever a -chemical substance unites with a chemical substance in the cell, a -receptor, the latter is, so far as the cell is concerned, _thrown out -of function_ for that cell. The chemical equilibrium of the latter -is upset. It attempts to restore this and does so by making a _new_ -receptor to take the place of the one thrown out of function. If this -process is continued, _i.e._, if the new receptor is similarly "used -up" and others similarly formed are also, then the cell will prepare -a supply of these and even an excess, according to Weigert's theory. -Whenever a cell accumulates an excess of products the normal result -is that it excretes them from its own substance into the surrounding -lymph, whence they reach the blood stream to be either carried to -the true excretory organs, utilized by other cells or remain for a -longer or shorter time in the blood. Hence the excess of receptors -is _excreted from the cell that forms them_ and they become _free_ -in the blood. These free receptors are termed _antibodies_. _They -are receptors_ but instead of being retained in the cell are _free -in solution in the blood_. One function of the free receptor, the -antibody, is _always to unite with the chemical substance which caused -it to be formed_. _It may have additional functions._ The chemical -substance which caused the excess formation of receptors, antibodies, -is termed an _antigen_ for that particular kind of antibody. - -To recapitulate, Ehrlich's theory postulates _specific chemical -stimuli_, which react with _specific chemical substances in the body -cells, named receptors_, and that these _receptors_, according to -Weigert, are _produced in excess_ and hence are excreted from the -cell and become _free receptors_ in the blood and lymph. These _free -receptors_ are the various kinds of _antibodies_, the kind depending -on the nature of the stimulus, antigen, the substance introduced. Any -substance which when introduced into the body causes the formation of -an antibody of any kind whatsoever is called an _antigen_,[23] _i.e._, -anti (body) former. - -The foregoing discussion explains Ehrlich's theory of immunity. -According to this theory the _manner of formation of all antibodies_ is -the same. The _kind of antibody_ and the _manner of its action_ will -differ with the _different kinds of antigens_ used. - -The succeeding chapters discuss some of the kinds of antibodies, the -theory of their action and some practical applications. It must be -borne in mind throughout the study of these, as has been stated, that -_every antibody has the property of uniting with its antigen whether it -has any property in addition or not_. - -Just what antibodies are chemically has not been determined because -no one has as yet succeeded in isolating them chemically pure. To the -author they appear to be enzymes. - -Antigens were considered by Ehrlich to be proteins or to be related to -proteins. Most workers since Ehrlich have held similar views. Dr. Carl -Warden of the University of Michigan has been doing much work in recent -years in which he is attempting to show that the antigens are not -proteins but are fats or fatty acids. Mr. E. E. H. Boyer, in his work -(not yet published) in the author's laboratory for the degree of Ph.D., -received in June, 1920, succeeded in producing various antibodies from -_Bacterium coli_ antigens. In these antigens he could detect only fatty -acids or salts of fatty acids. If the work of these men is confirmed, -it will open up a most interesting and extremely important field in -immunity and in preventive medicine. It is not apparent that the nature -of the antigen would affect Ehrlich's theory of the formation of -antibodies. - -The author has no doubt that eventually the formation of antibodies and -the reactions between them and their antigens will be explained on the -basis of physical-chemical laws, but this probably awaits the discovery -of their nature. - - - - -CHAPTER XXVII. - -RECEPTORS OF THE FIRST ORDER. - - -ANTITOXINS--ANTIENZYMES. - -The general characteristics of toxins have been described (Chapter -XII). It has been stated that they are more or less specific in -their action on cells. In order to affect a cell it is evident that -a toxin must enter into chemical combination with it. This implies -that the toxin molecule possesses a chemical group which can combine -with a receptor of the cell. This group is called the _haptophore_ or -combining group. The toxic or injurious portion of the toxin molecule -is likewise spoken of as the _toxophore_ group. When a toxin is -introduced into the body its _haptophore_ group combines with suitable -_receptors_ in different cells of the body. If not too much of the -toxin is given, instead of injuring, it acts as a chemical stimulus -to the cell in the manner already described. The cell in response -produces more of the specific thing, which in this instance is more -receptors which can combine with the toxin, _i.e._, with its haptophore -group. If the stimulus is kept up, more and more of these receptors -are produced until an excess for the cell accumulates, which excess is -excreted from the individual cell and becomes free in the blood. These -free receptors have, of course, the capacity to combine with toxin -through its haptophore group. When the toxin is combined with these -free receptors, it cannot combine with any other receptors, _e.g._, -those in another cell and hence cannot injure another cell. These free -receptors constitute, in this case, _antitoxin_, so-called because -they can combine with toxin and hence neutralize it. Antitoxins are -specific--that is, an antitoxin which will combine with the toxin of -_Clostridium tetani_ will not combine with that of _Corynebacterium -diphtheriae_ or of _Clostridium botulinum_, or of any other toxin, -vegetable or animal. - -When a toxin is kept in solution for some time or when it is heated -above a certain temperature (different for each toxin) it loses its -poisonous character. It may be shown, however, that it is still capable -of uniting with antitoxin, and preventing the latter from uniting with -a fresh toxin. This confirms the hypothesis that a toxin molecule has -at least two groups: a combining or _haptophore_, and a poisoning or -_toxophore_ group. A toxin which has lost its poisonous property, its -toxophore group, is spoken of as a _toxoid_. The theory of antitoxin -formation is further supported by the fact that the proper introduction -of _toxoid_, the _haptophore_ group, and hence the real stimulus, can -cause the production of _antitoxin_ to a certain extent at least. - -The close relationship between toxins and enzymes has already been -pointed out. This is still further illustrated by the fact that when -enzymes are properly introduced into the tissues of an animal there -is formed in the animal an _antienzyme_ specific for the enzyme in -question which can prevent its action. The structure of enzymes, -as composed of a _haptophore_, or uniting, and a _zymophore_ or -_digesting_ (or other activity) group, is similar to that of toxins, -and _enzymoids_ or enzymes which can combine with the substance acted -on but not affect it further, have been demonstrated. - -These free cell receptors, antitoxins or antienzymes, which are -produced in the body by the proper introduction of toxins or enzymes, -respectively, have the function of _combining_ with these bodies -_but no other action_. As was pointed out above, this is sufficient -to neutralize the toxin or enzyme and prevent any injurious effect -since they can unite with nothing else. Since these receptors are the -simplest type which has been studied as yet, they are spoken of by -Ehrlich as _receptors of the first order_. Other antibodies which are -likewise free receptors of the first, order and have the function of -combining only have been prepared and will be referred to in their -proper connection. They are mainly of theoretical interest. - -Ehrlich did a large part of his work on toxins and antitoxins -with _ricin_, the toxin of the castor-oil bean, _abrin_, from the -jequirity bean, _robin_ from the locust tree, and with the toxins and -antitoxins for diphtheria and tetanus. Antitoxins have been prepared -experimentally for a large number of both animal and vegetable poisons, -including a number for bacterial toxins. The only ones which, as yet, -are of much practical importance are _antivenin_ for snake poison, (not -a true toxin, however, see p. 275), _antipollenin_ (supposed to be -for the toxin of hay fever) and the antitoxins for the true bacterial -toxins of _Corynebacterium diphtheriae_ and _Clostridium tetani_. - -The method of preparing antitoxins is essentially the same in all -cases, though differing in minor details. For commercial purposes -large animals are selected, usually horses, so that the yield of -serum may be large. The animals must, of course, be vigorous, free -from all infectious disease. The first injection given is either a -relatively small amount of a solution of toxin or of a mixture of -toxin and antitoxin. The animal shows more or less reaction, increased -temperature, pulse and respiration and frequently an edema at the -point of injection, unless this is made intravenously. After several -days to a week or more, when the animal has recovered from the first -injection, a second stronger dose is given, usually with less reaction. -Increasingly large doses are given at proper intervals until the animal -may take several hundred times the amount which would have been fatal -if given at first. The process of immunizing a horse for diphtheria or -tetanus toxin usually takes several months. Variations in time and in -yield of antitoxin are individual and not predictable in any given case. - -After several injections a few hundred cubic centimeters of blood -are withdrawn from the jugular vein and serum from this is tested -for the amount of antitoxin it contains. When the amount is found -sufficiently large (250 "units" at least for diphtheria per cc.)[24] -then the maximum amount of blood is collected from the jugular with -sterile trocar and cannula. The serum from this blood with the addition -of an antiseptic (0.5 per cent. phenol, tricresol, etc.) constitutes -"antidiphtheritic serum" or "antitetanic serum," etc. All sera which -are put on the market must conform to definite standards of strength -expressed in "units" as determined by the U. S. Hygienic Laboratory. -In reality a "unit" of diphtheria antitoxin in the United States is -an amount equivalent to 1 cc. of a given solution of a _standard_ -diphtheria _antitoxin_ which is kept at the above-mentioned laboratory. -This statement, of course, gives no definite idea as to the amount -of antitoxin actually in a "unit." Specifically stated, a "unit" of -antitoxin contains approximately the amount which would protect a 250 -gram guinea-pig from 100 minimum lethal doses of diphtheria toxin, or -protect 100 guinea-pigs weighing 250 grams each from one minimum lethal -dose each. The minimum lethal dose (M. L. D.) of diphtheria toxin is -the least amount that will kill a guinea-pig of the size mentioned -within four days. Since toxins on standing change into toxoids to a -great extent, the amount, of antitoxin in a "unit," though protecting -against 100 M. L. D., in reality would protect against about 200 M. L. -D. of toxin containing no toxoid. - -The official unit for tetanus antitoxin is somewhat different, since it -is standardized against a _standard toxin_ which is likewise kept at -the Hygienic Laboratory. The unit is defined as "ten times the amount -of antitoxin necessary to protect a 350 g. guinea-pig for 96 hours -against the _standard test dose_" of the standard toxin. The standard -test dose is 100 M. L. D. of toxin for a 350 g. guinea-pig. To express -it another way, one could say that a "unit" of tetanus antitoxin -would protect one thousand 350 g. guinea-pigs from 1 M. L. D. each of -standard tetanus toxin. - -Various methods have been devised for increasing the amount of -antitoxin in 1 cc. of solution by precipitating out portions of the -blood-serum proteins and at the same time concentrating the antitoxin -in smaller volume. It is not considered necessary in a work of this -character to enter into these details nor to discuss the process of -standardizing antitoxin so that the exact amount of "units" per cc. may -be known. - - - - -CHAPTER XXVIII. - -RECEPTORS OF THE SECOND ORDER. - - -AGGLUTININS. - -Charrin and Rogers appear to have been the first (1889) to observe the -clumping together of bacteria (_Pseudomonas pyocyanea_) when mixed -with the blood serum of an animal immunized against them. Gruber and -Durham (1896) first used the term "agglutination" in this connection -and called the substance in the blood-serum "agglutinin." Widal (1896) -showed the importance of the reaction for diagnosis by testing the -blood serum of an infected person against a known culture (typhoid -fever). - -It is now a well-known phenomenon that the proper injection of cells -of any kind foreign to a given animal will lead to the accumulation in -the animal's blood of substances which will cause a clumping together -of the cells used when suspended in a suitable liquid. The cells settle -out of such suspension much more rapidly than they would otherwise -do. This clumping is spoken of as "agglutination" and the substances -produced in the animal are called "agglutinins." If blood cells are -injected then "hemagglutinins" result: if bacterial cells "bacterial -agglutinins" for the particular organism used as "glanders agglutinin" -for _Pfeifferella mallei_, "abortion agglutinin" for _Bacterium -abortus_, "typhoid agglutinin" for _Bacterium typhosum_, etc. - -The phenomenon may be observed either under the microscope or in small -test-tubes, that is, either _microscopically_ or _macroscopically_. - -In this case the cells introduced, or more properly, some substances -within the cells, act as stimuli to the body cells of the animal -injected to cause them to produce more of the specific cell receptors -which respond to the stimulus. The substance within the introduced -cell which acts as a stimulus (_antigen_) to the body cells is called -an "_agglutinogen_." That "agglutinogen" is present in the cell has -been shown by injecting animals experimentally with extracts of cells -(bacterial and other cells) and the blood serum of the animal injected -showed the presence of agglutinin for the given cell. It will be -noticed that the receptors which become the free agglutinins have at -least _two functions_, hence at least _two chemical groups_. They must -combine with the foreign cells and also bring about their clumping -together, their agglutination. Hence it can be stated technically that -an agglutinin possesses a _haptophore group_ and an _agglutinating -group_. - -It is probable that the agglutination, the clumping, is a secondary -phenomenon depending on the presence of certain salts and that the -agglutinin acts on its antigen as an enzyme, possibly a "splitting" -enzyme. This is analogous to what occurs in the curdling of milk -by rennet and in the coagulation of blood. This probability is -substantiated by the fact that suspensions of bacteria may be -"agglutinated" by appropriate strengths of various acids. - -The formation of agglutinin in the body for different bacteria does -not as yet appear to be of any special significance in protecting the -animal from the organism, since the bacteria are not killed, even -though they are rendered non-motile, if of the class provided with -flagella, and are clumped together. The fact that such bodies are -formed, however, is of decided value in the diagnosis of disease, and -also in the identification of unknown bacteria. - -In many bacterial diseases, agglutinins for the particular organism -are present in the blood serum of the affected animal. Consequently -if the blood serum of the animal be mixed with a suspension of the -organism supposed to be the cause of the disease and the latter be -agglutinated, one is justified in considering it the causative agent, -provided certain necessary conditions are fulfilled. In the first place -it must be remembered that the blood of normal animals frequently -contains agglutinins ("normal agglutinins") for many different -bacteria when mixed with them in full strength. Hence the serum must -always be diluted with physiological salt solution (0.85 per cent.). -Further, closely related bacteria may be agglutinated to some extent by -the same serum. It is evident that if they are closely related, their -protoplasm must contain some substances of the same kind to account for -this relationship. Since some of these substances may be agglutinogens, -their introduction into the animal body will give rise to agglutinins -for the related cells, as well as for the cell introduced. The -agglutinins for the cell introduced "chief agglutinins," will be -formed in larger quantity, since a given bacterial cell must contain -more of its own agglutinogen than that of any other cell. By _diluting -the blood serum_ from the animal to be tested the agglutinins for the -related organisms (so-called "coagglutinins" or "partial agglutinins") -will become so much diminished as to show no action, while the -agglutinin for the specific organism is still present in an amount -sufficient to cause its clumping. _Agglutinins are specific for their -particular agglutinogens_, but since a given blood serum may contain -many agglutinins, the _serum's specificity for a given bacterium_ -can be determined only by diluting it until this bacterium alone -is agglutinated. Hence the necessity of diluting the unknown serum -in varying amounts when testing against several known bacteria to -determine for which it is specific, _i.e._, which is the cause of the -disease in the animal. - -The agglutinins in the serum may be removed from it by treating it with -a suspension of the cells for which agglutinins are present. If the -"chief" cell is used all the agglutinins will be absorbed. If related -cells are used, only the agglutinins for this particular kind are -removed. These "absorption tests" furnish another means of determining -specificity of serum, or rather of determining the "chief agglutinin" -present. - -Just as an unidentified _disease_ in an animal may be determined by -testing its serum as above described against _known_ kinds of bacteria, -so _unknown bacteria_ isolated from an animal, from water, etc., may -be identified by testing them against the _blood sera_ of different -animals, each of which has been properly inoculated with a different -kind of _known bacteria_. If the unknown organism is agglutinated -by the blood of one of the animals in high dilution, and not by the -others, evidently the bacterium is the same as that with which the -animal has been inoculated, or _immunized_, as is usually stated. This -method of identifying cultures of bacteria is of wide application, -but is used practically only in those cases where other methods of -identification are not readily applied, and especially where other -methods are _not sufficient_ as in the "intestinal group" of organisms -in human practice. - -The diagnosis of disease in an animal by testing its serum is also a -valuable and much used procedure. This is the method of the "Widal" or -"Gruber-Widal" test for typhoid fever in man and is used in veterinary -practice in testing for glanders, contagious abortion, etc. In some -cases a dilution of the serum of from 20 to 50 times is sufficient for -diagnosis (Malta fever), in most cases, however, 50 times is the lowest -limit. Evidently the greater the dilution, that is, the higher the -"titer," the more specific is the reaction. - - -PRECIPITINS. - -Since agglutinins act on bacteria, probably through the presence of -substances within the bacterial cell, it is reasonable to expect that -if these substances be dissolved out of the cell, there would be some -reaction between their (colloidal) solution and the same serum. As -a matter of fact Kraus (1897) showed that broth cultures freed from -bacteria by porcelain filters do show a precipitate when mixed with -the serum of an animal immunized against the particular bacterium and -that the reaction is specific under proper conditions of dilution. -It was not long after Kraus's work until the experiments were tried -of "immunizing" an animal not against a bacterium or its filtered -culture, but against (colloidal) solutions of proteins, such as white -of egg, casein of milk, proteins of meat and of blood serum, vegetable -proteins, etc. It was ascertained that in all these cases the animal's -serum contains a substance which causes a _precipitate_ with solutions -of the protein used for immunization. The number of such precipitating -serums that have been made experimentally is very large and it appears -that protein from any source when properly introduced into the blood -or tissues of an animal will cause the formation of a precipitating -substance for its solutions. This substance is known, technically as a -"_precipitin_." The protein used as antigen to stimulate its formation, -or some part of the protein molecule (haptophore group), which acts -as stimulus to the cell is spoken of as a "precipitinogen," both -terms after the analogy of "agglutinin" and "agglutinogen." In fact -the specific precipitation and agglutination are strictly analogous -phenomena. Precipitins act on proteins in (colloidal) _solution_ and -cause them to settle out, agglutinins act on substances within cells -which cells are in _suspension_ in a fluid and cause the cells to -settle out. Ehrlich's theory of the formation of precipitins is similar -to that of agglutinins, and need not be repeated. Substitute the -corresponding words in the theory of formation of agglutinins as above -given and the theory applies. - -The precipitin reaction has not found much practical use in -bacteriology largely because the "agglutination test" takes its place -as simpler of performance and just as accurate. The reaction is, -however, generally applicable to filtrates of bacterial cultures and -could be used if needed. The so-called "mallease" reaction in glanders -is an instance. - -Precipitins find their greatest usefulness in legal medicine and -in food adulteration work. As was noted above, if animals, rabbits -for example, are immunized with the blood of another animal (human -beings) precipitins are developed which are specific for the injected -blood with proper dilution. This forms an extremely valuable means -of determining the _kind of blood_ present in a given spot shown by -chemical and spectroscopic tests to be blood and has been adopted as -a legal test in countries where such rules of procedure are applied. -Similarly the test has been used to identify the different kinds of -meat in sausage, and different kinds of milk in a mixture. An extract -of the sausage is made and tested against the serum of an animal -previously treated with extract of horse meat, or hog meat, or beef, -etc., the specific precipitate occurring with the specific serum. Such -reactions have been obtained where the protein to be tested was diluted -100,000 times and more. Biological relationships and differences have -been detected by the reaction. Human immune serum shows no reaction -with the blood of any animals except to a slight extent with that of -various monkeys, most with the higher, very slight with the lower Old -World and scarcely any with New World monkeys. - -It is a fact of theoretical interest mainly that if agglutinins -and precipitins themselves be injected into an animal they will -act as _antigens_ and cause the formation of _antiagglutinins_ or -_antiprecipitins_, which are therefore receptors of the first order -since they simply combine with these immune bodies to neutralize their -action, have only a combining or haptophore group. Also if agglutinins -or precipitins be heated to the proper temperature they may retain -their combining power but cause no agglutination or precipitation, -_i.e._, they are converted into agglutinoid or precipitinoid -respectively after the analogy of toxin and toxoid. - -Precipitins like agglutinins possess at least two groups--a combining -or _haptophore_ group and a _precipitating_ (sometimes called -zymophore) group. Hence they are somewhat more complex than antitoxins -or antienzymes which have a combining group only. For this reason -Ehrlich classes agglutinins and precipitins as _receptors of the second -order_. - - - - -CHAPTER XXIX. - -RECEPTORS OF THE THIRD ORDER. - - -CYTOLYSINS. - -Before Koch definitely proved bacteria capable of causing disease -several physiologists had noted that the red corpuscles of certain -animals were destroyed by the blood of other animals (Creite, 1869, -Landois, 1875), and Traube and Gescheidel had shown that freshly drawn -blood destroys bacteria (1874). It was not until about ten years -afterward that this action of the blood began to be investigated in -connection with the subject of immunity. Von Fodor (1885) showed that -saprophytic bacteria injected into the blood are rapidly destroyed. -Fluegge and his pupils, especially Nuttall in combating Metchnikoff's -theory of phagocytosis, announced in 1883, studied the action of the -blood on bacteria and showed its destructive effect (1885-57). Nuttall -also showed that the blood lost this power if heated to 56 deg.. Buchner -(1889) gave the name "alexin" (from the Greek "to ward off") to the -destroying substance and showed that the substance was present in -the _blood serum_ as well as in the whole blood, and that when the -serum lost its power to dissolve, this could be restored by adding -fresh blood. Pfeiffer (1894) showed that the destructive power of the -blood of animals immunized against bacteria (cholera and typhoid) was -markedly specific for the bacteria used. He introduced a mixture of -the blood and the bacteria into the abdominal cavity of the immunized -animal or of a normal one of the same species and noted the rapid -solution of the bacteria by withdrawing portions of the peritoneal -fluid and examining them ("Pfeiffer's phenomenon"). Belfanti and -Carbone and especially Bordet (1898) showed the specific dissolving -action of the serum of one animal on the blood corpuscles of another -animal with which it had been injected. Since this time the phenomenon -has been observed with a great variety of cells other than red blood -corpuscles and bacteria--leukocytes, spermatozoa, cells from liver, -kidney, brain, epithelia, etc., protozoa, and many vegetable cells. - -It is therefore a well-established fact that the proper injection of -an animal with almost any cell foreign to it will lead to the blood of -the animal injected acquiring the power to injure or destroy cells of -the same kind as those introduced. The destroying power of the blood -has been variously called its "cytotoxic" or "cytolytic" power, though -the terms are not strictly synonymous since "cytotoxic" means "cell -poisoning" or "injuring," while "cytolytic" means "cell dissolving." -The latter term is the one generally used and there is said to be -present in the blood a specific "cytolysin." The term is a general one -and a given cytolysin is named from the cell which is dissolved, as a -_bacteriolysin_, a _hemolysin_ (red-corpuscle-lysin), _epitheliolysin_, -_nephrolysin_ (for kidney cells), etc. If the cell is _killed_ but -not _dissolved_ the suffix "cidin" or "toxin" is frequently used as -"bacteriocidin," "spermotoxin," "neurotoxin," etc. - -The use of the term "cytolysin" is also not strictly correct, though -convenient, for the process is more complex than if _one substance -only_ were employed. As was stated above, the immune serum loses its -power to dissolve the cell if it is heated to 55 deg. to 56 deg. for half an -hour, it is _inactivated_. But if there be added to the heated or -inactivated serum a small amount of _normal serum_ (which contains only -a very little cytolytic substance, so that it has no dissolving power -when so diluted) the mixture again becomes cytolytic. It is evident -then that in cytolysis there are _two distinct substances_ involved, -one which is _present in all serum, normal or immune_, and the other -_present only in the immune cytolytic_ serum. This may be more apparent -if the facts are arranged in the following form: - - I. Immune serum dissolves cells in high dilution. - - II. Heated immune serum does not dissolve cells. - - III. Normal serum in high dilution does not dissolve cells. - - II. + III., _i.e._, Heated immune serum plus diluted normal serum - dissolves cells. - -Therefore, there is something in heated immune serum necessary for -cell dissolving and something different in diluted normal serum which -is necessary. This latter something is present in unheated immune -serum also, and is destroyed by heat. Experiment has shown that it is -the substance present in all serum both normal and immune that is the -true dissolving body, while the immune substance serves to unite this -body to the cell to be destroyed, _i.e._, to the antigen. Since the -immune body has therefore _two uniting groups_, one for the dissolving -substance and one for the cell to be dissolved, Ehrlich calls it the -"_amboceptor_." He also uses the word "_complement_" to denote the -dissolving substance, giving the idea that it completes the action of -dissolving after it has been united to the cell by the amboceptor, thus -replacing Buchner's older term "alexin" for the same dissolving body. - - -AMBOCEPTORS. - -The theory of formation of amboceptors is similar to that for the -formation of the other types of antibodies. The cell introduced -contains some substance, which acts as a chemical stimulus to some of -the body cells provided with proper receptors so that more of these -special receptors are produced, and eventually in excess so that they -become free in the blood and constitute the free amboceptors. It will -be noticed that these free receptors differ from either of the two -kinds already described in that they have _two uniting groups_, one for -the antigen (cell introduced) named _cytophil-haptophore_, the other -for the complement, _complementophil haptophore_. Hence amboceptors -are spoken of as _receptors of the third order_. They have no other -function than that of this double combining power. The action which -results is due to the third body--the complement. It will be readily -seen that complement must possess at least two groups, a combining or -_haptophore group_ which unites with the amboceptor, and an active -group which is usually called the _zymophore_ or _toxophore_ group. -Complements thus resemble either toxins, where the specific cell -(antigen) is injured or killed, or enzymes, in case the cell is -likewise dissolved. This action again shows the close relation between -toxins and enzymes. Complement may lose its active group in the same -way that toxin does and becomes then _complementoid_. Complement is -readily destroyed in blood or serum by heating it to 55 deg. to 56 deg. for -half an hour, and is also destroyed spontaneously when serum stands for -a day or two, less rapidly at low temperature than at room temperature. - -Amboceptors appear to be _specific_ in the same sense that agglutinins -are. That is, if a given cell is used to immunize an animal, the -animal's blood will contain amboceptors for the cell used and also for -others closely related to it. Immunization with spermatozoa or with -epithelial or liver cells gives rise to amboceptors for these cells -and also for red blood corpuscles and other body cells. A typhoid -bactericidal serum has also some dissolving effect on colon bacilli, -etc. Hence a given serum may contain a chief amboceptor and a variety -of "coamboceptors," or one amboceptor made up of a number of "partial -amboceptors" each specific for its own antigen ("amboceptorogen"). -Amboceptors may combine with other substances than antigen and -complement, as is shown by their union with lecithin and other -"lipoids," though these substances seem capable of acting as complement -in causing solution of blood corpuscles. - - -COMPLEMENTS. - -As to whether complements are numerous, as Ehrlich claims, or there -is only one complement, according to Buchner and others, need not be -discussed here. In the practical applications given later as means of -diagnosis it is apparent that all the complement or complements are -capable of uniting with at least two kinds of amboceptors. - -If complement be injected into an animal it may act as an antigen and -give rise to the formation of _anticomplement_ which may combine with -it and prevent its action and is consequently analogous to antitoxin. -If amboceptors as antigen are injected into an animal there will be -formed by the animal's cells _antiamboceptors_. As one would expect, -there are two kinds of antiamboceptors, one for each of its combining -groups, since it has been stated that it is always the combining group -of any given antigen that acts as the cell stimulus. Hence we may have -an "anticytophil amboceptor" or an "anticomplementophil amboceptor." -These antiamboceptors and the anticomplements are analogous to -antitoxin, antiagglutinin, etc., and hence are receptors of the first -order. - - -ANTISNAKE VENOMS. - -A practical use of antiamboceptors is in antisnake venoms. Snake -poisons appear to contain only _amboceptors_ for different cells of the -body. In the most deadly the amboceptor is specific for nerve cells -(cobra), in others for red corpuscles, or for endothelial cells of the -bloodvessels (rattlesnake). The complement is furnished by the blood -of the individual bitten, that is, in a sense the individual poisons -himself, since he furnishes the destroying element. The antisera -contain antiamboceptors which unite with the amboceptor introduced and -prevent it joining to cells and thus binding the complement to the -cells and destroying them. With this exception these antibodies are -chiefly of theoretical interest. - - -FAILURE OF CYTOLYTIC SERUMS. - -The discovery of the possibility of producing a strongly bactericidal -serum in the manner above described aroused the hope that such sera -would prove of great value in passive immunization and serum treatment -of bacterial diseases. Unfortunately such expectations have not been -realized and no serum of this character of much practical importance -has been developed as yet (with the possible exception of Flexner's -antimeningococcus serum in human practice. What hog cholera serum is -remains to be discovered). - -The reasons for the failure of such sera are not entirely clear. -The following are some that have been offered: (1) Amboceptors do -not appear to be present in very large amount so that relatively -large injections of blood are necessary, which is not without risk -in itself. (2) Since the complement is furnished by the blood of the -animal to be treated, there may not be enough of this to unite with a -sufficient quantity of amboceptor to destroy all the bacteria present, -hence the disease is continued by those that escape. (3) Or the -complement may not be of the right kind to unite with the amboceptor -introduced, since this is derived from the blood of a _heterologous_ -("other kind") species. In hog-cholera serum, if it is bactericidal, -this difficulty is removed by using blood of a _homologous_ ("same -kind") animal. Hence Ehrlich suggested the use of apes for preparing -bactericidal sera for human beings. The good results which have been -reported in the treatment of human beings with the serum of persons -convalescing from the same disease indicate that this lack of proper -complement for the given amboceptor is probably a chief factor in the -failure of sera from lower animals. (4) The bacteria may be localized -in tissues (lymph glands), within cavities (cranial, peritoneal), in -hollow organs (alimentary tract), etc., so that it is not possible to -get at them with sufficient serum to destroy all. This difficulty is -obviated by injecting directly into the spinal canal when Flexner's -antimeningococcus serum is used. (5) Even if the bacteria are dissolved -it does not necessarily follow that their _endotoxins_ are destroyed. -These may be merely liberated and add to the danger of the patient, -though this does not appear to be a valid objection. (6) Complement -which is present in such a large excess of amboceptor may just as -well unite with amboceptor which is not united to the bacteria to be -destroyed as with that which is, and hence be actually prevented from -dissolving the bacteria. Therefore it is difficult to standardize the -serum to get a proper amount of amboceptor for the complement present. - - -COMPLEMENT-FIXATION TEST. - -Although little practical use has been made of bactericidal sera, -the discovery of receptors of this class and the peculiar relations -between the antigen, amboceptor and complement have resulted in -developing one of the most delicate and accurate methods for the -diagnosis of disease and for the recognition of small amounts of -specific protein that is in use today. - -This method is usually spoken of as the "complement-fixation" or the -"complement-deviation test" ("Wassermann test" in syphilis) and is -applicable in a great variety of microbial diseases, but it is of -practical importance in those diseases only where other methods are -uncertain--syphilis in man, concealed glanders in horses, contagious -abortion in cattle, etc. A better name would be the "Unknown Amboceptor -Test" since it is the amboceptor that is searched for in the test by -making use of its power to "fix" complement. - -The principle is the same in all cases. The method depends, as -indicated above, on the ability of complement to combine with at least -two amboceptor-antigen systems, and on the further fact that if the -combination with one amboceptor-antigen system is once formed, it -does not dissociate so as to liberate the complement for union with -the second amboceptor-antigen system. If an animal is infected with a -microoerganism and a part of its defensive action consists in destroying -the organisms in its blood or lymph, then it follows from the above -discussion of cytolysins that there will be developed in the blood of -the animal amboceptor specific for the organism in question. If the -presence of this _specific amboceptor_ can be detected, the conclusion -is warranted that the organism for which it is specific is the cause of -the disease. Consequently what is sought in the "complement-fixation -test" is a _specific amboceptor_. In carrying out the test, blood -serum from the suspected animal is collected, heated at 56 deg. for half -an hour to destroy any complement it contains and mixed in definite -proportions with the specific antigen and with complement. The -antigen is an extract of a diseased organ (syphilitic fetal liver, -in syphilis), a suspension of the known bacteria, or an extract of -these bacteria. Complement is usually derived from a guinea-pig, -since the serum of this animal is higher in complement than that -of most animals. The blood of the gray rat contains practically as -much. If the specific amboceptor is present, that is, if the animal -is infected with the suspected disease, the complement will unite -with the antigen-amboceptor system and be "fixed," that is, be no -longer capable of uniting with any other amboceptor-antigen system. No -chemical or physical means of telling whether this union has occurred -or not, except as given below, has been discovered as yet, though -doubtless will be by physico-chemical tests, nor can the combination -be seen. Hence an "indicator," as is so frequently used in chemistry, -is put into the mixture of antigen-amboceptor-complement after it has -been allowed to stand in the incubator for one-half to one hour to -permit the union to become complete. The "indicator" used is a mixture -of sheep's corpuscles and the heated ("inactivated") blood serum of -a rabbit which has been injected with sheep's blood corpuscles and -therefore contains a _hemolytic amboceptor specific_ for the corpuscles -which is capable also of uniting with complement. The indicator is -put into the first mixture and the whole is again incubated and -examined. If the mixture is _clear_ and _colorless_ with a _deposit -of red corpuscles_ at the bottom, that would mean that the complement -had been bound to the first complex, since it was not free to unite -with the second sheep's corpuscles (antigen)--rabbit serum (hemolytic -amboceptor) complex--and destroy the corpuscles. Hence if the -complement is bound in the first instance, the _specific amboceptor_ -for the first antigen must have been present in the blood, that is, the -animal was infected with the organism in question. Such a reaction is -called a "positive" test. - -On the other hand, if the final solution is _clear_ but of a _red_ -color, that would mean that complement must have united with the -corpuscles--hemolytic amboceptor system--and destroyed the corpuscles -in order to cause the _clear red_ solution of hemoglobin. If complement -united with this system it could not have united with the first system, -hence there was no _specific amboceptor_ there to bind it; no specific -amboceptor in the animal's blood, means no infection. Hence a _red -solution_ is a "negative test." - -The scheme for the test may be outlined as follows: - - Antigen + Patient's Serum, heated + Complement - (specific for (unknown amboceptor) (derived from - the amboceptor guinea pig's serum) - sought) - -Incubate one-half hour in a water bath or one hour in an incubator. - -Then add the indicator which is - - Antigen + Amboceptor - (red blood corpuscles) (for corpuscles, serum of - a rabbit immunized against - the red corpuscles) - -Incubate as above. - -In practice all the different ingredients must be accurately tested, -standardized and used in exact quantities, and tests must also be run -as controls with a known normal blood of an animal of the same species -as the one examined and with a known positive blood. - - It should be stated that in one variety of the Complement-Fixation - Test, namely, the "Wassermann Test for Syphilis" in human beings, - an antigen is used which is not derived from the specific organism - (_Treponema pallidum_) which causes the disease nor even from - syphilitic tissue. It has been determined that alcohol will extract - from certain tissues, _human or animal_, substances which _act - specifically_ in combining with the syphilitic amboceptor present - in the blood. Alcoholic extracts of beef heart are most commonly - used. Details of this test may be learned in the advanced course in - Immunity and Serum Therapy. - -The complement-fixation test might be applied to the determination -of unknown bacteria, using the unknown culture as antigen and trying -it with the sera of different animals immunized against a variety -of organisms, some one of which might prove to furnish _specific -amboceptor_ for the unknown organism and hence indicate what it is. The -test used in this way has not been shown to be a practical necessity -and hence is rarely employed. It has been used, however, to detect -traces of unknown proteins, particularly blood-serum proteins, in -medico-legal cases in exactly the above outlined manner and is very -delicate and accurate. - - - - -CHAPTER XXX. - -PHAGOCYTOSIS--OPSONINS. - - -It has been mentioned that Metchnikoff, in a publication in 1883, -attempted to explain immunity on a purely cellular basis. It has -been known since Haeckel's first observation in 1858 that certain of -the white corpuscles do engulf solid particles that may get into the -body, and among them bacteria. Metchnikoff at first thought that this -engulfing and subsequent intracellular digestion of the microoerganisms -were sufficient to protect the body from infection. The later -discoveries (discussed in considering Ehrlich's theory of immunity) -of substances present in the blood serum and even in the blood plasma -which either destroy the bacteria or neutralize their action have -caused Metchnikoff to modify his theory to a great extent. He admitted -the presence of these substances, though giving them other names, -but ascribed their formation to the phagocytes or to the same organs -which form the leukocytes--lymphoid tissue generally, bone marrow. It -is not within the province of this work to attempt to reconcile these -theories, but it may be well to point out that Ehrlich's theory is one -of _chemical substances_ and that the _origin_ of these substances is -not an _essential_ part of the theory, so that the two theories, except -in some minor details, are not necessarily mutually exclusive. - -[Illustration: PLATE V - -ELIE METCHNIKOFF] - -Sir A. E. Wright and Douglas, in 1903, showed that even in those -instances where immunity depends on phagocytosis, as it certainly does -in many cases, the phagocytes are more or less inactive unless they are -aided by chemical substances present in the blood. These substances -_act on the bacteria, not on the leukocytes_, and change them in such -a way that they are more readily taken up by the phagocytes. Wright -proposed for these bodies the name _opsonin_, derived from a Greek -word signifying "to prepare a meal for." Neufeld and Rimpau at about -the same time (1904), in studying immune sera, observed substances of -similar action in these sera and proposed the name _bacteriotropins_, -or bacteriotropic substances. There is scarcely a doubt that the two -names are applied to identical substances and that Wright's name -_opsonin_ should have preference. - -The chemical nature of opsonins is not certainly determined, but they -appear to be a distinct class of antibodies and to possess two groups, -a combining or haptophore and a preparing or opsonic group and hence -are similar to antibodies of Ehrlich's second order--agglutinins and -precipitins. Wright also showed that opsonins are just as specific as -agglutinins are--that is, a micrococcus opsonin prepares micrococci -only for phagocytosis and not streptococci or any other bacteria. - -Wright showed that opsonins for many bacteria are present in normal -serum and that in the serum of an animal which has been immunized -against such bacteria the opsonins are _increased_ in amount. Also -that in a person infected with certain bacteria the opsonins are -either increased or diminished, depending on whether the progress of -the infection is favorable or unfavorable. The _opsonic power_ of a -serum normal or otherwise is determined by mixing an emulsion of fresh -leukocytes in normal saline solution with a suspension of the bacteria -and with the serum to be tested. The leukocytes must first be washed in -several changes of normal salt solution to free them from any adherent -plasma or serum. The mixture is incubated for about fifteen minutes -and then slides are made, stained with a good differential blood -stain, Wright's or other, and the average number of bacteria taken up -by at least fifty phagocytes taken in order in a field is determined -by counting under the microscope. The number so obtained Wright calls -the _phagocytic index_ of the serum tested. The phagocytic index of a -given serum divided by the phagocytic index of a normal serum gives -the _opsonic index_ of the serum tested. Assuming the normal opsonic -index to be 1, Wright asserts that in healthy individuals the range -should be not more than from 0.8 to 1.2, and that an index below 0.8 -may show a great susceptibility for the organism tested, infection with -the given organism if derived from the individual, or improper dosage -in case attempts have been made to immunize by using killed cultures, -vaccines, of the organism. - -On the occasion of the author's visit to Wright's clinic (1911) he -stated that he used the determination of the _opsonic index_ chiefly as -a _guide to the dosage_ in the use of vaccines. - -Most workers outside the Wright school have failed to recognize any -essential value of determinations of the opsonic index in the use -of vaccines. Some of the reasons for this are as follows: The limit -of error in phagocytic counts may be as great as 50 per cent. in -different series of fifty, hence several hundred must be counted, which -adds greatly to the tediousness and time involved; the variation in -apparently healthy individuals is frequently great, hence the "normal" -is too uncertain; finally the opsonic index and the clinical course of -the disease do not by any means run parallel. Undoubtedly the method -has decided value in the hands of an individual who makes opsonic -determinations his chief work, as Wright's assistants do, but it can -scarcely be maintained at the present time that such determinations -are necessary in vaccine therapy. Nevertheless that opsonins actually -exist and that they play an essential part in phagocytosis, and hence -in immunity, is now generally recognized. - - -BACTERIAL VACCINES. - -Whether determinations of opsonic index are useful or not is largely -a matter of individual opinion, but there is scarcely room to doubt -that Wright has conferred a lasting benefit by his revival of the -use of _dead cultures of bacteria_, _bacterial vaccines_, both for -protective inoculation and for treatment. It is perhaps better to use -the older terms "vaccination" and "vaccine" (though the cow, _vacca_, -is not concerned) than to use Wright's term "opsonic method" in this -connection, bearing in mind that the idea of a vaccine is that it -contains the _causative organism_ of the infection as indicated on p. -253. - -As early as 1880 Touissant proposed the use of dead cultures of -bacteria to produce immunity. But because injections of such cultures -were so frequently followed by abscess formation, doubtless due to -the _high temperatures_ used to kill the bacteria, the method was -abandoned. Further, Pasteur and the French school persistently denied -the possibility of success with such a procedure, and some of them -even maintain this attitude at the present time. The successes of -Wright and the English school which are being repeated so generally -wherever properly attempted, leave no doubt in the unprejudiced of the -very great value of the method and have unquestionably opened a most -promising field both for preventive inoculation and for treatment in -many infectious diseases. That the practice is no more universally -applicable than are immune serums and that it has been and is still -being grossly overexploited is undoubted. - -The use of a vaccine is based on two fundamental principles. The first -of these is that the cell introduced must not be in a condition to -cause serious injury to the animal by its multiplication and consequent -elaboration of injurious substances. The second is that, on the other -hand, it must contain antigens in such condition that they will act as -stimuli to the body cells to produce the necessary antibodies, whether -these be opsonins, bactericidal substances, or anti-endotoxins. In the -introduction of living organisms there is always more or less risk -of the organism not being sufficiently attenuated and hence of the -possibility of its producing too severe an infection. In using killed -cultures, great care must be exercised in destroying the organisms, -_so that the antigens are not at the same time rendered inactive_. -Hence in the preparation of bacterial vaccines by Wright's method the -_temperature and the length of time used to kill the bacteria are most -important factors_. This method is in general to grow the organisms -on an agar medium, rub off the culture and emulsify in sterile normal -salt solution (0.85 per cent. NaCl). The number of bacteria per cc. -is determined by staining a slide made from a small volume of the -emulsion mixed with an equal volume of human blood drawn from the -finger and counting the relative number of bacteria and of red blood -corpuscles. Since the corpuscles are normally 5,000,000 per c.mm., -a simple calculation gives the number of bacteria. The emulsion of -bacteria is then diluted so that a certain number of millions shall -be contained in each cc., "standardized" as it is called, then heated -to the proper temperature for the necessary time and it is ready for -use. A preservative, as 0.5 per cent. phenol, tricresol, etc., is added -unless the vaccine is to be used up at once. The amounts of culture, -salt solution, etc., vary with the purpose for which the vaccine is to -be used, from one or two agar slant cultures and a few cc. of solution, -when a single animal is to be treated, to bulk agar cultures and liters -of solution as in preparing antityphoid vaccine on a large scale. - -Agar surface cultures are used so that there will be as little -admixture of foreign protein as possible (see Anaphylaxis, p. 289 _et -seq._). Normal saline solution is isotonic with the body cells and -hence is employed as the vehicle. - -=Lipovaccines.=--The suspension of bacteria in neutral oil was first -used by Le Moignac and Pinoy who gave the name "lipovaccines" (#lipos# -= fat) to them. It was claimed that the reaction following injection -of these vaccines was less severe than with saline vaccines in many -instances; also, that the bacteria were much more slowly absorbed. For -these two reasons it was hoped that much larger numbers of bacteria -could be injected at one dose and one injection would suffice instead -of three or more as ordinarily used. The technique of preparation, -standardization and killing of the organisms has not as yet been -sufficiently well established to warrant the general substitution of -lipovaccines for ordinary saline suspensions. - -Vaccines are either "_autogenous_" or "_stock_." An "autogenous" -vaccine is a vaccine that is made from bacteria derived from the -individual or animal which it is desired to vaccinate and contains -not only the particular organism but the particular strain of that -organism which is responsible for the lesion. Stock vaccines are -made up from organisms like the infective agent in a given case but -derived from some other person or animal or from laboratory cultures. -Commercial vaccines are "stock" vaccines and are usually "polyvalent" -or even "mixed." A "polyvalent" vaccine contains several strains of the -infective agent and a "mixed" contains several different organisms. - -Stock vaccines have shown their value when used as preventive -inoculations, notably so in typhoid fever in man, anthrax and black-leg -in cattle. The author is strongly of the opinion, not only from the -extended literature on the subject, but also from his own experience -in animal, and especially in human cases, that stock vaccines are -much inferior and much more uncertain in their action when used in -the _treatment_ of an infection, than are autogenous vaccines. This -applies particularly to those instances in which _pneumococci_, -_streptococci_, _micrococci_, and _colon bacilli_ are the causative -agents but to others as well. The following are some of the reasons for -this opinion: The above organisms are notoriously extremely variable in -their virulence. While there is no necessarily close connection between -virulence and antigenic property, yet since virulence is so variable, -it is rational to assume that antigenic property is also extremely -variable. Individuals vary just as much in susceptibility and hence in -reactive power, and generally speaking, an individual will react better -in the production of antibodies to a stimulus to which he has been more -or less subjected, _i.e._, to organisms derived from his own body. - -In the preparation of a vaccine great care must be used in heating so -that the organisms are killed, but the _antigens_ are not destroyed. -Many of the enzymes present in bacteria, especially the proteolytic -ones, are not any more sensitive to heat than are the antigens, hence -are not destroyed entirely. Therefore a vaccine kept in stock for a -long time gradually has some of its antigens destroyed by the uninjured -enzymes present with them, and so loses in potency. Therefore in -treating a given infection it is well to make up a vaccine from the -lesion, use three or four doses and if more are necessary make up a new -vaccine. - -If the above statements are borne in mind and vaccines are made and -administered accordingly, the author is well satisfied that much better -results will be secured. - -In accordance with the theory on which the use of vaccines is based, -_i.e._, that they stimulate the body cells to produce immunizing -antibodies, it is clear that they are especially suitable in those -infections in which the process is _localized_ and should not be of -much value in _general_ infections. In the latter case the cells of -the body are stimulated to produce antibodies by the circulating -organisms, probably nearly to their limit, hence the introduction of -more of the same organisms, capable of stimulating though dead, is apt -to overtax the cells and do more harm than good. It is not possible to -tell accurately when this limit is reached, but the clinical symptoms -are a guide. If vaccines are used at all in general infections they -should be given in the early stages and in small doses at first with -close watch as to the effect. In localized infections only the cells in -the immediate neighborhood are much stimulated, hence the introduction -of a vaccine calls to their aid cells in the body generally, and much -more of the resulting antibodies are carried to the lesion in question. -Manifestly surgical procedures such as incision, drainage, washing -away of dead and necrotic tissue with normal saline solution, not -necessarily antiseptics, will aid the antibodies in their action and -are to be recommended where indicated. - -In the practical application of any remedy the _dosage_ is most -important. Unfortunately there is no accurate method of determining -this with a vaccine. Wright recommended determining the number of -the organisms per cc. as before mentioned, and his method or some -modification of it is still in general use. From what was said with -regard to variation, both in organisms and in individuals, it can -be seen that the number of organisms is at least only a very rough -guide. This is further illustrated by the doses of micrococcus -(staphylococcus) vaccines recommended by different writers, which -vary from 50,000,000 to 2,000,000,000 per cc. The author is decidedly -of the opinion that _there is no way of determining the dosage of -a vaccine in the treatment of any given case except by the result -of the first dose_. Hence it is his practice to make vaccines of a -particular organism of the same approximate strength, and to give a -dose of a measured portion of a cubic centimeter, judging the amount -by what the individual or animal can apparently withstand, without too -violent a reaction. If there is no local or general reaction or if it -is very slight and there is no effect on the lesion, the dose is too -small. If there is a violent local reaction with severe constitutional -symptoms clinically, and the lesion appears worse, the dose is too -large. There should be some local reaction and some general, but not -enough to cause more than a slight disturbance, easy to judge in human -subjects, more difficult in animals. In cases suitable for vaccine -treatment no _serious_ results should follow from a properly prepared -vaccine, though the process of healing may be delayed temporarily. -Wright claimed, and many have substantiated him, that always following -a vaccination there is a period when the resistance of the animal is -diminished. This is called the "negative phase," and Wright considered -this to last as long as the opsonic index remained low, and when this -latter began to increase the stage of the "positive" or favorable phase -was reached. As has been stated the opsonic index is pretty generally -regarded as of doubtful value, though the existence of a period of -lowered resistance is theoretically probable from the fact that -antibodies already present in the blood will be partially used up in -uniting with the vaccine introduced and that the body cells are called -upon suddenly to do an extra amount of work and it takes them some time -to adapt themselves. This time, the "negative phase," is much better -determined by the clinical symptoms, general and especially local. It -is good practice to begin with a dose relatively small. The result -of this is an indication of the proper dosage and also prepares the -patient for a larger one. The second dose should follow the first not -sooner than three or four days, and should be five to seven days if the -first reaction is severe. These directions are not very definite, but -clinical experience to date justifies them. It is worth the time and -money to one who wishes to use vaccines to learn from one who has had -experience both in making and administering them, and then to remember -that each patient is an individual case, for the use of vaccines as -well as for any other kind of treatment. - - -AGGRESSIN. - -Opsonins have been shown to be specific substances which act on -bacteria in such a way as to render them more readily taken up by the -leukocytes. By analogy one might expect to find bacteria secreting -specific substances which would tend to counteract the destructive -action of the phagocytes and bactericidal substances. Bail and his -co-workers claim to have demonstrated such substances in exudates in -certain diseases and have given the distinctive name "aggressins" to -them. By injecting an animal with "aggressins," antiaggressins are -produced which counteract their effects and thus enable the bacteria to -be destroyed. The existence of such specific bodies is not generally -accepted as proved. The prevailing idea is that bacteria protect -themselves in any given case by the various toxic substances that they -produce, and that "aggressins" as a special class of substances are not -formed. - - - - -CHAPTER XXXI. - -ANAPHYLAXIS. - - -Dallera, in 1874, and a number of physiologists of that period, -observed peculiar skin eruptions following the transfusion of blood, -that is, the introduction of foreign proteins. In the years subsequent -to the introduction of diphtheria antitoxin (1890) characteristic -"serum rashes" were not infrequently reported, sometimes accompanied by -more or less severe general symptoms and occasionally death--a train of -phenomena to which the name "serum sickness" was later applied, since -it was shown that it was the horse serum (foreign protein) that was -the cause, and not the antitoxin itself. In 1898 Richet and Hericourt -noticed that some of the dogs which they were attempting to immunize -against toxic eel serum not only were not immunized but suffered even -more severely after the second injection. They obtained similar results -with an extract of mussels which contain a toxin. Richet gave the name -"anaphylaxis" ("no protection") to this phenomenon to distinguish it -from immunity or prophylaxis (protection). - -All the above-mentioned observations led to no special investigations -as to their cause. In 1903, Arthus noticed abscess formation, -necrosis and sloughing following several injections of horse serum -in immediately adjacent parts of the skin in rabbits ("Arthus' -phenomenon"). Theobald Smith, in 1904, observed the death of -guinea-pigs following properly spaced injections of horse serum. This -subject was investigated by Otto and by Rosenau and Anderson in this -country and about the same time von Pirquet and Schick were making a -study of serum rashes mentioned above. The publications of these men -led to a widespread study of the subject of injections of foreign -proteins. It is now a well-established fact that the injection into an -animal of a foreign protein--vegetable, animal or bacterial, simple or -complex--followed by a second injection after a proper length of time -leads to a series of symptoms indicating poisoning, which may be so -severe as to cause the death of the animal. Richet's term "anaphylaxis" -has been applied to the condition of the animal following the first -injection and indicates that it is in a condition of supersensitiveness -for the protein in question. The animal is said to be "sensitized" -for that protein.[25] The sensitization is specific since an animal -injected with white of chicken's egg reacts to a second injection of -chicken's egg only and not pigeon's egg or blood serum or any other -protein. The specific poisonous substance causing the symptoms has -been called "anaphylotoxin" though what it is, is still a matter of -investigation. It is evident that some sort of an antibody results from -the first protein injected and that it is specific for its own antigen. - -A period of ten days is usually the minimum time that must elapse -between the first and second injections in guinea-pigs in order that a -reaction may result, though a large primary dose requires much longer. -If the second injection is made within less time no effect follows, -and after three or more injections at intervals of about one week the -animal fails to react at all, it has become "immune" to the protein. -Furthermore, after an animal has been sensitized by one injection and -has reacted to a second, then, if it does not die from the reaction, it -fails to react to subsequent injections. In this latter case it is said -to be "antianaphylactic." - -It must be remembered that proteins do not normally get into the -circulation except by way of the alimentary tract. Here all proteins -that are absorbed are first broken down to their constituent -amino-acids, absorbed as such and these are built up into the proteins -characteristic of the animal's blood. Hence when protein as such gets -into the blood it is a foreign substance to be disposed of. The blood -contains proteolytic enzymes for certain proteins normally. It is -also true that the body cells possess the property of digesting the -proteins of the blood and building them up again into those which are -characteristic of the cell. Hence it appears rational to assume that -the foreign proteins act as stimuli to certain cells to produce more -of the enzymes necessary to decompose them, so that they may be either -built up into cell structure or eliminated as waste. If in this process -of splitting up of protein a poison were produced, then the phenomena -of "anaphylaxis" could be better understood. As a matter of fact -Vaughan and his co-workers have shown that by artificially splitting -up proteins from many different sources--animal, vegetable, pathogenic -and saprophytic bacteria--a poison _is produced_ which appears to be -the same in all cases and which causes the symptoms characteristic of -anaphylaxis. On the basis of these facts it is seen that anaphylaxis -is simply another variety of immunity. The _specific antibody_ in -this case is an _enzyme_ which decomposes the protein instead of -precipitating it. The enzyme must be specific for the protein since -these differ in constitution. Vaughan even goes so far as to say that -the poison is really the central ring common to all proteins and that -they differ only in the lateral groups or side chains attached to this -central nucleus. The action of the enzyme in this connection would be -to split off the side chains, and since these are the specific parts of -the protein, the enzyme must be specific for each protein. The pepsin -of the gastric juice and the trypsin of the pancreas split the native -proteins only to peptones. As is well known, these when injected in -sufficient quantity give rise to poisonous symptoms, and will also -give rise to anaphylaxis under properly spaced injections. They do not -poison normally because they are split by the intestinal erepsin to -amino-acids and absorbed as such. Whether Vaughan's theory of protein -structure is the true one or not remains to be demonstrated. It is -not essential to the theory of anaphylaxis above outlined, _i.e._, -a phenomenon due to the action of specific _antibodies_ which are -enzymes. On physiological grounds this appears the most rational of the -few explanations of anaphylaxis that have been offered and was taught -by the author before he had read Vaughan's theory along the same lines. - -On the basis of the author's theory the phenomena of protein immunity -and antianaphylaxis may be explained in the following way which the -author has not seen presented. The enzymes necessary to decompose the -injected protein are present in certain cells and are formed in larger -amount by those cells to meet the increased demand due to injection -of an excess of protein. They are retained in the cell for a time at -least. If a second dose of protein is given before the enzymes are -excreted from the cells as waste, this is digested within the cells in -the normal manner. If a third dose is given, the cells adapt themselves -to this increased intracellular digestion and it thus becomes normal -to them. Hence the _immunity_ is due to this increased intracellular -digestion. - -On the other hand, if the second injection is delayed long enough, then -the _excess_ enzyme, but not all, is excreted from the cells and meets -the second dose of protein in the blood stream and rapidly decomposes -it there, so that more or less intoxication from the split products -results. This uses up _excess_ enzyme, hence subsequent injections are -not digested in the blood stream but within the cells as before. So -that "antianaphylaxis" is dependent on the exhaustion of the excess -enzyme in the blood, and the condition is _fundamentally_ the same as -protein immunity, _i.e._, due to _intracellular_ digestion in each case. - -As has been indicated "serum sickness" and sudden death following -serum injections are probably due to a sensitization of the individual -to the proteins of the horse in some unknown way. Probably hay fever -urticarial rashes and idiosyncrasies following the ingestion of certain -foods--strawberries, eggs, oysters, etc., are anaphylactic phenomena. - -In medical practice the reaction is used as a means of diagnosis in -certain diseases, such as the tuberculin test in tuberculosis, the -mallein test in glanders. The individual or animal with tuberculosis -becomes sensitized to certain proteins of the tubercle bacillus and -when these proteins in the form of tuberculin are introduced into the -body a reaction results, local or general, according to the method of -introduction. The practical facts in connection with the tuberculin -test are also in harmony with the author's theory of anaphylaxis -as above outlined. Milder cases of tuberculosis give more vigorous -reactions because the intracellular enzymes are not used up rapidly -enough since the products of the bacillus are secreted slowly in such -cases. Hence excess of enzyme is free in the blood and the injection of -the tuberculin meets it there and a vigorous reaction results. In old, -far-advanced cases, no reaction occurs, because the enzymes are all -used in decomposing the large amount of tuberculous protein constantly -present in the blood. The fact that an animal which has once reacted -fails to do so until several months afterward likewise depends on the -fact that the _excess_ enzyme is used in the reaction and time must -elapse for a further excess to accumulate. - -The anaphylactic reaction has been made use of in the identification of -various types of proteins and is of very great value since the reaction -is so delicate, particularly when guinea-pigs are used as test animals. -Wells has detected the 0.000,001 g. of protein by this test. It is -evident that the test is applicable in medico-legal cases and in food -examination and has been so used. - - -A TABULATION OF ANTIGENS AND ANTIBODIES AS AT PRESENT RECOGNIZED. - - CLASS OF - ANTIGEN ANTIBODY ACTION OF ANTIBODY RECEPTOR - - Toxin Antitoxin Combines with toxin and I. - hence prevents toxin - from uniting with a - cell and injuring it, - _i.e._, neutralizes toxin. - - Enzyme Antienzyme Combines with enzyme I. - and thus prevents enzyme - from uniting - with anything else and - showing its action, _i.e._, - neutralizes enzyme. - - Solution of Precipitin Unites with its antigen II. - protein and causes its precipitation - from solution. - - Solution of ? Causes phenomenon of (?) - protein anaphylaxis(?) - - Suspension of Agglutinin Unites with its antigen II. - cells causes its clumping together - and settling out - of suspension. - - Suspension of Opsonin Unites with its antigen II. - cells and makes the cells (?) - more easily taken up - by phagocytes. - - Suspension of Amboceptor Unites with its antigen III. - cells and also with complement - which latter then - dissolves the antigen. - - Precipitin Antiprecipitin Neutralizes precipitin. I. - - Agglutinin Antiagglutinin Neutralizes agglutinin. I. - - Opsonin Antiopsonin Neutralizes opsonin. I. - - Amboceptor Antiamboceptor Neutralizes amboceptor. I. - (two kinds) - - Complement Anticomplement Neutralizes complement. I. - - -SUMMARY OF IMMUNITY AS APPLIED TO PROTECTION FROM DISEASE. - -The discussion of "immunity problems" in the preceding chapters serves -to show that protection from disease either as a condition natural to -the animal or as an acquired state is dependent on certain properties -of its body cells or fluids, or both. The actual factors so far as at -present known may be summarized as follows: - -1. _Antitoxins_ which neutralize true toxins; shown to exist for very -few diseases. - -2. _Cytolytic substances_ which destroy the invading organism: in -reality two substances; amboceptor, which is specific, and complement, -the real dissolving enzyme. - -3. _Phagocytosis_ or the destruction of the invading organisms within -the leukocytes. - -4. _Opsonins_ which render the bacteria more readily taken up by the -phagocytes. - -5. _Enzymes_ other than complement possibly play a part in the -destruction of some pathogenic organisms or their products. This -remains to be more definitely established. - -6. It is possible that in natural immunity there might be no receptors -in the body cells to take up the organisms or their products, or the -receptors might be present in certain cells but of a very low chemical -affinity, so that combination does not occur. It is even highly -probable that many substances formed by invading organisms which might -injure specialized cells, such as those of glandular, nervous or muscle -tissue, have a more rapid rate of reaction with, or a stronger affinity -for, lower unspecialized cells, such as connective and lymphoid tissue, -and unite with these so that their effects are not noticed. - -The importance of these different, factors varies in different diseases -and need not be considered in this connection. - -The question "which of the body cells are engaged in the production -of antibodies" is not uncommonly asked. On physiological grounds it -would not seem reasonable that the highly specialized tissues above -mentioned could take up this work, even though they are the ones which -suffer the greatest injury in disease. Hence it is to be expected that -the lower or unspecialized cells are the source, and it has been shown -that the antibodies are produced by the phagocytes (though not entirely -as Metchnikoff maintained), by lymphoid tissue generally, by the bone -marrow and also by connective-tissue cells, though in varying degrees. - -Since immunity depends on the activity of the body cells it is evident -that one of the very best methods for avoiding infectious diseases is -to keep these cells up to their highest state of efficiency, to keep in -"good health." Hence good health means not only _freedom from disease_ -but also _protection against disease_. - - - - -LIST OF LABORATORY EXERCISES GIVEN IN CONNECTION WITH THE CLASS WORK -INCLUDED IN THIS TEXT-BOOK. - - Exercise 1. Cleaning glassware. - - Exercise 2. Preparation of broth medium from meat juice. - - Exercise 3. Preparation of gelatin medium from broth. - - Exercise 4. Preparation of agar medium from broth. - - Exercise 5. Potato tubes. - - Exercise 6. Potato plates. - - Exercise 7. Plain milk tubes. - - Exercise 8. Litmus milk tubes. - - Exercise 9. Sugar broth media. - - Exercise 10. Blood-serum tubes. - - Exercise 11. Inoculation of tubes. Action on complex proteins. - - Exercise 12. Production of gas from carbohydrates. - - Exercise 13. Production of indol. - - Exercise 14. Reduction of nitrates. - - Exercise 15. Chromogenesis: Illustrates nicely the variation with - environment. - - Exercise 16. Enzyme production. - - Exercise 17. Making of plate cultures; isolation in pure culture. - - Exercise 18. Stain making and staining. - - Exercise 19. Cell forms and cell groupings. - - Exercise 20. Hanging drop slides. - - Exercise 21. Staining of spores. - - Exercise 22. Staining of acid-fast bacteria. - - Exercise 23. Staining of capsules. - - Exercise 24. Staining of metachromatic granules. - - Exercise 25. Staining of flagella. - - Exercise 26. Study of individual species. - - Exercise 27. Determination of thermal death-point. - - Exercise 28. Action of disinfectants on bacteria. - - Exercise 29. Action of sunlight on bacteria. - - - - -DESCRIPTIVE CHART--SOCIETY OF AMERICAN BACTERIOLOGISTS. - -_Prepared by Committee on Methods of Identification of Bacterial -Species.--F. D. Chester, F. P. Gorham, Erwin F. Smith._ - -_Endorsed by the Society for general use at the Annual Meeting, -December, 1907._ - - -GLOSSARY OF TERMS. - -AGAR HANGING BLOCK, a small block of nutrient agar cut from a pour -plate, and placed on a cover-glass, the surface next the glass having -been first touched with a loop from a young fluid culture or with a -dilution from the same. It is examined upside down, the same as a -hanging drop. - -AMEBOID, assuming various shapes like an ameba. - -AMORPHOUS, without visible differentiation in structure. - -ARBORESCENT, a branched, tree-like growth. - -BEADED, in stab or stroke, disjointed or semiconfluent colonies along -the lines of inoculation. - -BRIEF, a few days, a week. - -BRITTLE, growth dry, friable under the platinum needle. - -BULLATE, growth rising in convex prominences, like a blistered surface. - -BUTYROUS, growth of a butter-like consistency. - -CHAINS, - Short chains, composed of 2 to 8 elements. - Long chains, composed of more than 8 elements. - -CILIATE, having fine, hair-like extensions, like cilia. - -CLOUDY, said of fluid cultures which do not contain pseudozoogleae. - -COAGULATION,[22] the separation of casein from whey in milk. This may -take place quickly or slowly, and as the result either of the formation -of an acid or of a lab ferment. - -CONTOURED, an irregular, smoothly undulating surface, like that of a -relief map. - -CONVEX surface, the segment of a circle, but flattened. - -COPROPHYL, dung bacteria. - -CORIACEOUS, growth tough, leathery, not yielding to the platinum needle. - -CRATERIFORM, round, depressed, due to the liquefaction of the medium. - -CRETACEOUS, growth opaque and white, chalky. - -CURLED, composed of parallel chains in wavy strands, as in anthrax -colonies. - -DIASTASIC ACTION, same as DIASTATIC, conversion of starch into -water-soluble substances by diastase. - -ECHINULATE, in agar stroke a growth along line of inoculation, with -toothed or pointed margins; in stab cultures growth beset with pointed -outgrowths. - -EFFUSE, growth thin, veily, unusually spreading. - -ENTIRE, smooth, having a margin destitute of teeth or notches. - -EROSE, border irregularly toothed. - -FILAMENTOUS, growth composed of long, irregularly placed or interwoven -filaments. - -FILIFORM, in stroke or stab cultures a uniform growth along line of -inoculation. - -FIMBRIATE, border fringed with slender processes, larger than filaments. - -FLOCCOSE, growth composed of short curved chains, variously oriented. - -FLOCCULENT, said of fluids which contain pseudozoogleae, _i.e._, small -adherent masses of bacteria of various shapes and floating in the -culture fluid. - -FLUORESCENT, having one color by transmitted light and another by -reflected light. - -GRAM'S STAIN, a method of differential bleaching after gentian violet, -methyl violet, etc. The + mark is to be given only when the bacteria -are deep blue or remain blue after counter-staining with Bismarck brown. - -GRUMOSE, clotted. - -INFUNDIBULIFORM, form of a funnel or inverted cone. - -IRIDESCENT, like mother-of-pearl. The effect of very thin films. - -LACERATE, having the margin cut into irregular segments as if torn. - -LOBATE, border deeply undulate, producing lobes (see _Undulate_). - -LONG, many weeks, or months. - -MAXIMUM TEMPERATURE, temperature above which growth does not take place. - -MEDIUM, nutrient substance upon which bacteria are grown. - -MEMBRANOUS, growth thin, coherent, like a membrane. - -MINIMUM TEMPERATURE, temperature below which growth does not take place. - -MYCELIOID, colonies having the radiately filamentous appearance of mold -colonies. - -NAPIFORM, liquefaction with the form of a turnip. - -NITROGEN REQUIREMENTS, the necessary nitrogenous food. This is -determined by adding to _nitrogen-free_ media the nitrogen compound to -be tested. - -OPALESCENT, resembling the color of an opal. - -OPTIMUM TEMPERATURE, temperature at which growth is most rapid. - -PELLICLE, in fluid bacterial growth forming either a continuous or an -interrupted sheet over the fluid. - -PEPTONIZED, said of curds dissolved by trypsin. - -PERSISTENT, many weeks, or months. - -PLUMOSE, a fleecy or feathery growth. - -PSEUDOZOOGLEAE, clumps of bacteria, not dissolving readily in water, -arising from imperfect separation, or more or less fusion of the -components, but not having the degree of compactness and gelatinization -seen in zoogleae. - -PULVINATE, in the form of a cushion, decidedly convex. - -PUNCTIFORM, very minute colonies, at the limit of natural vision. - -RAPID, developing in twenty-four to forty-eight hours. - -RAISED, growth thick, with abrupt or terraced edges. - -RHIZOID, growth of an irregular branched or root-like character, as in -_B. mycoides_. - -RING, same as RIM, growth at the upper margin of a liquid culture, -adhering more or less closely to the glass. - -REPAND, wrinkled. - -SACCATE, liquefaction the shape of an elongated sac, tubular, -cylindrical. - -SCUM, floating islands of bacteria, an interrupted pellicle or bacteria -membrane. - -SLOW, requiring five or six days or more for development. - -SHORT, applied to time, a few days, a week. - -SPORANGIA, cells containing endospores. - -SPREADING, growth extending much beyond the line of inoculation, -_i.e._, several millimetres or more. - -STRATIFORM, liquefying to the walls of the tube at the top and then -proceeding downward horizontally. - -THERMAL DEATH-POINT, the degree of heat required to kill young fluid -cultures of an organism exposed for ten minutes (in thin-walled -test-tubes of a diameter not exceeding 20 mm.) in the thermal -water-bath. The water must be kept agitated so that the temperature -shall be uniform during the exposure. - -TRANSIENT, a few days. - -TURBID, cloudy with flocculent particles; cloudy plus flocculence. - -UMBONATE, having a button-like, raised centre. - -UNDULATE, border wavy, with shallow sinuses. - -VERRUCOSE, growth wart-like, with wart-like prominences. - -VERMIFORM-CONTOURED, growth like a mass of worms or intestinal coils. - -VILLOUS, growth beset with hair-like extensions. - -VISCID, growth follows the needle when touched and withdrawn, sediment -on shaking rises as a coherent swirl. - -ZOOGLEAE, firm gelatinous masses of bacteria, one of the most typical -examples of which is the _Streptococcus mesenterioides_ of sugar vats. -(_Leuconostoc mesenterioides_), the bacterial chains being surrounded -by an enormously thickened, firm covering inside of which there may be -one or many groups of the bacteria. - - -NOTES. - -(1) For decimal system of group numbers see Table I. This will be found -useful as a quick method of showing close relationships inside the -genus, but is not a sufficient characterization of any organism. - -(2) The morphological characters shall be determined and described -from growths obtained upon at least one solid medium (nutrient agar) -and in at least one liquid medium (nutrient broth). Growths at 37 deg. C. -shall be in general not older than twenty-four to forty-eight hours, -and growths at 20 deg. C. not older than forty-eight to seventy-two hours. -To secure uniformity in cultures, in all cases preliminary cultivation -shall be practised as described in the revised Report of the Committee -on Standard Methods of the Laboratory Section of the American Public -Health Association, 1905. - -(3) The observation of cultural and biochemical features shall cover -a period of at least fifteen days and frequently longer, and shall be -made according to the revised Standard Methods above referred to. All -media shall be made according to the same Standard Methods. - -(4) Gelatin stab cultures shall be held for six weeks to determine -liquefaction. - -(5) Ammonia and indol tests shall be made at end of tenth day, nitrite -tests at end of fifth day. - -(6) Titrate with N/20 NaOH, using phenolphthalein as an indicator; make -titrations at same time from blank. The difference gives the amount of -acid produced. - -The titration should be done after boiling to drive off any CO{2} -present in the culture. - -(7) Generic nomenclature shall begin with the year 1872 (Cohn's first -important paper). - -Species nomenclature shall begin with the year 1880 (Koch's discovery -of the pour plate method for the separation of organisms). - -(8) Chromogenesis shall be recorded in standard color terms. - - -TABLE I. - -A NUMERICAL SYSTEM OF RECORDING THE SALIENT CHARACTERS OF AN ORGANISM. -(GROUP NUMBER.) - - 100 Endospores produced - 200 Endospores not produced - 10 Aerobic (strict) - 20 Facultative anaerobic - 30 Anaerobic (strict) - 1 Gelatin liquefied - 2 Gelatin not liquefied - 0.1 Acid and gas from dextrose - 0.2 Acid without gas from dextrose - 0.3 No acid from dextrose - 0.4 No growth with dextrose - 0.01 Acid and gas from lactose - 0.02 Acid without gas from lactose - 0.03 No acid from lactose - 0.04 No growth with lactose - 0.001 Acid and gas from saccharose - 0.002 Acid without gas from saccharose - 0.003 No acid from saccharose - 0.004 No growth with saccharose - 0.0001 Nitrates reduced with evolution of gas - 0.0002 Nitrates not reduced - 0.0003 Nitrates reduced without gas formation - 0.00001 Fluorescent - 0.00002 Violet chromogens - 0.00003 Blue chromogens - 0.00004 Green chromogens - 0.00005 Yellow chromogens - 0.00006 Orange chromogens - 0.00007 Red chromogens - 0.00008 Brown chromogens - 0.00009 Pink chromogens - 0.00000 Non-chromogenics - 0.000001 Diastasic action on potato starch, strong - 0.000002 Diastasic action on potato starch, feeble - 0.000003 Diastasic action on potato starch, absent - 0.0000001 Acid and gas from glycerin - 0.0000002 Acid without gas from glycerin - 0.0000003 No acid from glycerin - 0.0000004 No growth with glycerin - -The genus according to the system of Migula is given its proper symbol -which precedes the number thus:(7) - - BACILLUS COLI (Esch.) Mig. becomes B. 222.111102 - BACILLUS ALCALIGENES Petr. becomes B. 212.333102 - PSEUDOMONAS CAMPESTRIS (Pam.) Sm. becomes Ps. 211.333151 - BACTERIUM SUICIDA Mig. becomes Bact. 222.232103 - -Source............ Date of Isolation.............. Name........ -Group No.(1)............... - - - - -DETAILED FEATURES. - -NOTE--Underscore required terms. Observe notes and glossary of terms on -opposite side of card. - - -I. MORPHOLOGY(2) - - 1. Vegetative Cells, Medium used............................. - temp....................age.................days - - Form, _round_, _short rods_, _long rods_, _short chains_, _long - chains_, _filaments_, _commas_, _short spirals_, _long spirals_, - _clostridium_, _cuneate_, _clavate_, _curved_. - - Limits of Size.......................... - - Size of Majority............................. - - Ends, _rounded_, _truncate_, _concave_. - - {Orientation (grouping)............................ - Agar {Chains (No. of elements)........................ - Hanging-block {_Short chains_, _long chains_ - {Orientation of chains, _parallel_, _irregular_. - - 2. Sporangia, medium - used.....................temp..............age..............days - - Form, _elliptical_, _short rods_, _spindled_, _clavate_, _drumsticks_. - - Limits of Size................ - - Size of Majority.............. - - Agar {Orientation (grouping)........ - Hanging-block {Chains (No. of elements)...... - {Orientation of chains, _parallel_, _irregular_. - - Location of Endospores, _central_, _polar_. - - 3. Endospores. - - Form, _round_, _elliptical_, _elongated_. - - Limits of Size................ - - Size of Majority.............. - - Wall, _thick_, _thin_. - - Sporangium wall, _adherent_, _not adherent_. - - Germination, _equatorial_, _oblique_, _polar_, _bipolar_, _by - stretching_. - - 4. Flagella, No........Attachment _polar_, _bipolar_, - _peritrichiate_. How Stained......... - - 5. Capsules, present on............. - - 6. Zooglea, Pseudozooglea. - - 7. Involution Forms, on........in.....days at.... deg. C. - - 8. Staining Reactions. - - 1:10 watery fuchsin, gentian violet, carbol-fuchsin, Loeffler's - alkaline methylene blue. - - Special Stains. - Gram....................Glycogen............... - Fat.....................Acid-fast................ - Neisser................. - - -II. CULTURAL FEATURES(3) - -1. Agar Stroke. - - Growth, _invisible_, _scanty_, _moderate_, _abundant_. - - Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_, - _plumose_, _arborescent_, _rhizoid_. - - Elevation of growth, _flat_, _effuse_, _raised_, _convex_. - - Lustre, _glistening_, _dull_, _cretaceous_. - - Topography, _smooth_, _contoured_, _rugose_, _verrucose_. - - Optical characters, _opaque_, _translucent_, _opalescent_, - _iridescent_. - - Chromogenesis(3)................ - - Odor, _absent_, _decided_, _resembling_............ - - Consistency, _slimy_, _butyrous_, _viscid_, _membranous_, - _coriaceous_, _brittle_. - - Medium _grayed_, _browned_, _reddened_, _blued_, _greened_. - -2. Potato. - - Growth _scanty_, _moderate_, _abundant_, _transient_, _persistent_. - - Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_, - _plumose_, _arborescent_, _rhizoid_. - - Elevation of growth, _flat_, _effuse_, _raised_, _convex_. - - Lustre, _glistening_, _dull_, _cretaceous_. - - Topography, _smooth_, _contoured_, _rugose_, _verrucose_. - - Chromogenesis(3)...........Pigment in water _insoluble_, _soluble_: - other solvents..................... - - Odor, _absent_, _decided_, _resembling_.................... - - Consistency, _slimy_, _butyrous_, _viscid_, _membranous_, - _coriaceous_, _brittle_. - - Medium, _grayed_, _browned_, _reddened_, _blued_, _greened_. - -3. Loeffler's Blood-serum. - - Stroke _invisible_, _scanty_, _moderate_, _abundant_. - - Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_, - _plumose_, _arborescent_, _rhizoid_. - - Elevation of growth, _flat_, _effuse_, _raised_, _convex_. - - Lustre, _glistening_, _dull_, _cretaceous_. - - Topography, _smooth_, _contoured_, _rugose_, _verrucose_. - - Chromogenesis(3).......................... - - Medium _grayed_, _browned_, _reddened_, _blued_, _greened_. - - Liquefaction begins in.............d, complete in................d, - -4. Agar Stab. - - Growth _uniform_, _best at top_, _best at bottom_: surface growth - _scanty_, _abundant_: _restricted_, _wide-spread_. - - Line of puncture, _filiform_, _beaded_, _papillate_, _villous_, - _plumose_, _arborescent_: _liquefaction_. - -5. Gelatin Stab. - - Growth uniform, _best at top_, _best at bottom_. - - Line of puncture, _filiform_, _beaded_, _papillate_, _villous_, - _plumose_, _arborescent_. - - Liquefaction _crateriform_, _napiform_, _infundibuliform_, - _saccate_, _stratiform_: begins in....................d. complete - in....................d - - Medium _fluorescent_, _browned_............... - -6. Nutrient Broth. - - Surface growth, _ring_, _pellicle_, _flocculent_, _membranous_, - _none_. - - Clouding _slight_, _moderate_, _strong_: _transient_, _persistent_: - _none_: _fluid turbid_. - - Odor, _absent_, _decided_, _resembling_.................. - - Sediment, _compact_, _flocculent_, _granular_, _flaky_, _viscid on - agitation_, _abundant_, _scant_. - -7. Milk. - - Clearing without coagulation. - - Coagulation _prompt_, _delayed_, _absent_. - - Extrusion of whey begins in............days. - - Coagulum _slowly peptonized_, _rapidly peptonized_. - - Peptonization begins on....d, complete on ....d. - - Reaction, 1d...., 2d...., 4d...., 10d...., 20d.... - - Consistency, _slimy_, _viscid_, _unchanged_. - - Medium _browned_, _reddened_, _blued_, _greened_. - - Lab ferment, _present_, _absent_. - -8. Litmus Milk. - - _Acid_, _alkaline_, _acid then alkaline_, _no change_. - - _Prompt reduction_, _no reduction_, _partial slow reduction_. - -9. Gelatin Colonies. - - Growth _slow_, _rapid_. - - Form, _punctiform_, _round_, _irregular_, _ameboid_, _mycelioid_, - _filamentous_, _rhizoid_. - - Elevation, _flat_, _effuse_, _raised_, _convex_, _pulvinate_, - _crateriform_ (_liquefying_). - - Edge, _entire_, _undulate_, _lobate_, _erose_, _lacerate_, - _fimbriate_, _filamentous_, _floccose_, _curled_. - - Liquefaction, _cup_, _saucer_, _spreading_. - -10. Agar Colonies. - - Growth _slow_, _rapid_ (temperature..............) - - Form, _punctiform_, _round_, _irregular_, _ameboid_, _mycelioid_, - _filamentous_, _rhizoid_. - - Surface _smooth_, _rough_, _concentrically ringed_, _radiate_, - _striate_. - - Elevation, _flat_, _effuse_, _raised_, _convex_, _pulvinate_, - _umbonate_. - - Edge, _entire_, _undulate_, _lobate_, _erose_, _lacerate_, - _fimbriate_, _floccose_, _curled_. - - Internal structure, _amorphous_, _finely_, _coarsely granular_, - _grumose_, _filamentous_, _floccose_, _curled_. - -11. Starch Jelly. - - Growth, _scanty_, _copious_. - - Diastatic action, _absent_, _feeble_, _profound_. - - Medium stained................... - -12. Silicate Jelly (Fermi's Solution). - - Growth _copious_, _scanty_, _absent_. - - Medium stained.................. - -13. Cohn's Solution. - - Growth _copious_, _scanty_, _absent_. - - Medium _fluorescent_, _non-fluorescent_. - -14. Uschinsky's Solution. - - Growth _copious_, _scanty_, _absent_. - - Fluid _viscid_, _not viscid_. - -15. Sodium Chloride in Bouillon. - - Per cent. inhibiting growth........................ - -16. Growth in Bouillon over Chloroform, _unrestrained_, - _feeble_, _absent_. - -17. Nitrogen. Obtained from _peptone_, _asparagin_, _glycocoll_, - _urea_, _ammonia salts_, _nitrogen_. - -18. Best media for long-continued growth................... - ..................................................... - -19. Quick tests for differential purposes.................. - ..................................................... - ..................................................... - - -III. PHYSICAL AND BIOCHEMICAL FEATURES. - - +----------------------------------+---+---+---+---+---+---+---+---+ - | | D | S | L | M | G | M | | | - | | e | a | a | a | l | a | | | - | | x | c | c | l | y | n | | | - | | t | c | t | t | c | n | | | - | 1. Fermentation-tubes containing | r | h | o | o | e | i | | | - | peptone-water or | o | a | s | s | r | t | | | - | sugar-tree bouillon and | s | r | e | e | i | | | | - | | e | o | | | n | | | | - | | | s | | | | | | | - | | | e | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Gas production, in per cent. | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | (H/CO{2}) | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Growth in closed arm | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Amount of acid produced 1d. | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Amount of acid produced 2d. | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - | Amount of acid produced 3d. | | | | | | | | | - +----------------------------------+---+---+---+---+---+---+---+---+ - - 2. Ammonia production, _feeble_, _moderate_, _strong_, _absent_, - _masked by acids_. - - 3. Nitrates in nitrate broth. - - _Reduced_, _not reduced_. - - Presence of nitrites...........ammonia.................. - - Presence of nitrates...........free nitrogen............ - - 4. Indol production, _feeble_, _moderate_, _strong_. - - 5. Toleration of Acids, _great_, _medium_, _slight_. - - _Acids tested_.............. - - 6. Toleration of NaOH, _great_, _medium_, _slight_. - - 7. Optimum reaction for growth in bouillon, stated in terms of - Fuller's scale.......................... - - 8. Vitality on culture media, _brief_, _moderate_, _long_. - - 9. Temperature relations. - - Thermal death-point (10 minutes' exposure in nutrient broth when this - is adapted to growth of organism)............C. - - Optimum temperature for growth...... deg. C.; or best growth at 16 deg. C., - 20 deg. C., 25 deg. C., 30 deg. C., 37 deg. C., 40 deg. C., 50 deg. C., 60 deg. C. - - Maximum temperature for growth.......... deg. C. - - Minimum temperature for growth.......... deg. C. - - 10. Killed readily by drying: resistant to drying. - - 11. Per cent. killed by freezing (salt and crushed ice or liquid - air)................ - - 12. Sunlight: Exposure on ice in thinly sown agar plates; one-half - plate covered (time 15 minutes), _sensitive_, _not sensitive_. - - Per cent. killed................ - - 13. Acids produced................. - - 14. Alkalies produced............... - - 15. Alcohols....................... - - 16. Ferments, _pepsin_, _trypsin_, _diastase_, _invertase_, - _pectase_, _cytase_, _tyrosinase_, _oxidase_, _peroxidase_, - _lipase_, _catalase_, _glucase_, _galactase_, _lab_, - _etc._........................ - - 17. Crystals formed:..... - - 18. Effect of germicides: - - +-----------+-------------+---+---+---+---+---+ - | | | M | T | K | A | r | - | | | i | e | i | m | e | - | | | n | m | l | t | s | - | | | u | p | l | . | t | - | | | t | e | i | | r | - | | | e | r | n | r | a | - | | | s | a | g | e | i | - | Substance | Method used | | t | | q | n | - | | | | u | q | u | | - | | | | r | u | i | g | - | | | | e | a | r | r | - | | | | | n | e | o | - | | | | | t | d | w | - | | | | | i | | t | - | | | | | t | t | h | - | | | | | y | o | | - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - | | | | | | | | - +-----------+-------------+---+---+---+---+---+ - - -IV. PATHOGENICITY. - - 1. Pathogenic to Animals. - - _Insects_, _crustaceans_, _fishes_, _reptiles_, _birds_, _mice_, - _rats_, _guinea-pigs_, _rabbits_, _dogs_, _cats_, _sheep_, _goats_, - _cattle_, _horses_, _monkeys_, _man_.......................... - - 2. Pathogenic to Plants: - ......................................................... - ......................................................... - ......................................................... - - 3. Toxins, _soluble_, _endotoxins_. - - 4. Non-toxin forming. - - 5. Immunity bactericidal. - - 6. Immunity non-bactericidal. - - 7. Loss of virulence on culture-media: _prompt_, _gradual_, _not - observed in_.....................months. - - +-------------------------------------+ - | BRIEF CHARACTERIZATION. | - | | - | Mark + or 0, and when two terms | - | occur on a line erase the one which | - | does not apply unless both apply. | - | | - +--------------------------------+----+ - | M | Diameter over 1 mu |----| - | O | Chains, filaments |----| - | R | Endospores |----| - | P | Capsules |----| - | H | Zooglea, Pseudozooglea |----| - | O | Motile |----| - | L | Involution forms |----| - | O | Gram's stain |----| - | G | |----| - | Y | |----| - |(2)| |----| - +---+----------------------------+----+ - | C | B | Cloudy, turbid |----| - | U | r | Ring |----| - | L | o | Pellicle |----| - | T | t | Sediment |----| - | U | h | |----| - | R +-----+----------------------+----+ - | A | A | Shining |----| - | L | g | Dull |----| - | | a | Wrinkled |----| - | F | r | Chromogenic |----| - | E +-----+----------------------+----+ - | A | G | Round |----| - | T | e | Proteus-like |----| - | U | l. | Rhizoid |----| - | R | | Filamentous |----| - | E | P | Curled |----| - | S | l | |----| - |(3)| a | |----| - | | t | |----| - | | e | |----| - | +-----+----------------------+----+ - | | G S | Surface growth |----| - | | e t | Needle growth |----| - | | l a | |----| - | | . b.| |----| - | +-----+----------------------+----+ - | | P | Moderate, absent |----| - | | o | Abundant |----| - | | t | Discolored |----| - | | a | Starch destroyed |----| - | | t | |----| - | | o | |----| - | +-----+---------------------------+ - | | Grows at 37 deg. C. |----| - | | Grows in Cohn's sol. |----| - | | Grows in Uschinsky's sol. |----| - |---+-----+----------------------+----+ - | B | L f | Gelatin(4) |----| - | I | i a | Blood-serum |----| - | O | q c | Casein |----| - | C | u t | |----| - | H | i i | |----| - | E | - o | |----| - | M | n | |----| - | I +-----+----------------------+----+ - | C | M | Acid curd |----| - | A | i | Rennet curd |----| - | L | l | Casein peptonized |----| - | | k | |----| - | F +-----+----------------------+----+ - | E | Indol(3) |----| - | A | Hydrogen sulphide |----| - | T | Ammonia(3) |----| - | U | Nitrates reduced(3) |----| - | R | Fluorescent |----| - | E | Luminous |----| - | S | |----| - +---+----------------------------+----+ - | D | Animal pathogen, epizoon |----| - | I | Plant pathogen, epiphyte |----| - | S | Soil |----| - | T | Milk |----| - | R | Fresh water |----| - | I | Salt water |----| - | B | Sewage |----| - | U | Iron bacterium |----| - | T | Sulphur bacterium |----| - | I | |----| - | O | |----| - | N | |----| - +---+----------------------------+----+ - - - - -FOOTNOTES. - - -[1] Sir H. A. Blake has called attention to the fact that the "mosquito -theory" of malaria is mentioned in a Sanscrit manuscript of about the -6th century A.D. - -[2] Myxomycetes excepted, and they are probably to be regarded as -animals--Mycetozoa. - -[3] Centralblatt f. Bakteriologie, etc. LXIII. 1 Abt. Orig. 1912, 4, -idem LXVI. 1 Abt. Orig. 1912, 323. - -[4] The pronunciation of this word according to English standards -is kok-si; the continental pronunciation is kok-kee; the commonest -American seems to be kok-ki. We prefer the latter since it is easier -and more natural and should like to see it adopted. (Author.) - -[5] With the possible exception of blue green algae which have been -found with bacteria in the above-mentioned hot springs. Seeds of -many plants have been subjected to as low temperatures as those -above-mentioned without apparent injury. - -[6] It is popularly supposed that in canning fruit, vegetables, meats, -etc., all the air must be removed, since the organisms which cause -"spoiling" cannot grow in a vacuum. The existence of anaerobic and -facultative anaerobic bacteria shows the fallacy of such beliefs. - -[7] "By cellulose is understood a carbohydrate of the general formula -C{6}H{10}O{5} not soluble in water, alcohol, ether, or dilute acids but -soluble in an ammoniacal solution of copper oxide. It gives with iodine -and sulphuric acid a blue color and with iodine zinc chloride a violet -and yields dextrose on hydrolysis."--H. Fischer. - -[8] The sulphur bacteria are partially prototrophic for S; probably the -iron bacteria also for Fe. Some few soil bacteria have been shown to -be capable of utilizing free H, and it seems certain that the bacteria -associated with the spontaneous heating of coal may oxidize free C. So -far as known no elements other than these six are directly available to -bacteria. - -[9] Only a few kinds of bacteria so far as known are proto-autotrophic. -The nitrous and nitric organisms of Winogradsky which are so essential -in the soil, and which might have been the first of all organisms so -far as their food is concerned, and some of the sulphur bacteria are -examples. - -[10] The term _pathogenic_ is also applied to certain non-parasitic -saprophytic bacteria whose products cause disease conditions, as one -of the organisms causing a type of food poisoning in man (_Clostridium -botulinum_), which also probably causes "forage poisoning" in domestic -animals. - -[11] The term "fermentation" was originally used to denote the process -which goes on in fruit juices or grain extracts when alcohol and gas -are formed. Later it was extended to apply to the decomposition of -almost any organic substance. In recent years the attempt has been made -to give a chemical definition to the word by restricting its use to -those changes in which by virtue of a "wandering" or rearrangement of -the carbon atoms "new substances are formed which are not constitutents -of the original molecule." It may be doubted whether this restriction -is justified or necessary. A definition is at present scarcely possible -except when the qualifying adjective is included as "alcoholic -fermentation," "ammoniacal fermentation," "lactic acid fermentation," -etc. - -[12] See "Oil and Gas in Ohio," Bownocker: Geological Survey of Ohio, -Fourth Series, Bull. I, pp. 313-314. - -[13] It is probable that this is the way "Jack o'lanterns" or "Will o' -the wisps" are ignited. Marsh gas is produced as above outlined from -the vegetable and animal matter decomposing in swampy places under -anaerobic conditions and likewise phosphine. These escape into the air -and the "spontaneous combustion" of the phosphine ignites the marsh gas. - -[14] Dr. H. H. Green, of Pretoria, South Africa, has isolated from -"cattle dips" a bacterium that _reduces arsenates_ to _arsenites_. - -[15] Dr. Green (l. c.) has also isolated an organism which causes some -deterioration of cattle dips by _oxidizing arsenites to arsenates_. - -[16] It will be noted that the names of enzymes (except some of -those first discovered) terminate in _ase_ which is usually added to -the _stem of the name of the substance acted on_, though sometimes -to a word which indicates the substance formed by the action, as -_lactacidase_, _alcoholase_. - -[17] Tetanus toxin is about 120 times as poisonous as strychnin, both -of which act on the same kind of nerve cells. - -[18] In the author's laboratory in the past ten years all sterilization -except those few objects in blood and serum work which must be dry, has -been done in autoclaves of the type shown in Fig. 81 which are supplied -with steam from the University central heating plant. A very great -saving of time is thus secured. - -[19] The author has tested an "electric milk purifier" (Fig. 102) -which was as efficient as a first-class pasteurizer and left the milk -in excellent condition both chemically and as far as "cream line" was -concerned. The cost of operation as compared with steam will depend on -the price of electricity. - -[20] The exact laboratory details for preparing various media are -not given in this chapter. It is the object to explain the choice of -different materials and the reasons for the various processes to which -they are subjected. - -[21] For a discussion of this method of standardization consult the -following: - - Clark & Lubs--J. Bact., 1917, II, 1-34, 109-136, 191-236. - Committee Report--Ibid., 1919. IV, 107-132. - Jones--J. Inf. Dis., 1919, 25, 262-268. - Fennel & Fisher--Ibid., 444-451. - -Additional references will be found in these articles. - -[22] Term also applied to the solidification of serum in media: _e.g._, -the Hiss inulin medium for the differentiation of pneumococci (see -diplococcus of pneumonia). - -[23] The term "antigen" is also used to designate substances which may -take the place of what are supposed to be the true antigens in certain -diagnostic reactions (Chapter XXIX, Complement Fixation Test for -Syphilis). - -[24] If the antitoxin is later concentrated (see last paragraph in -this chapter) a serum containing as little as 175 units per cc. may be -commercially profitable. - -[25] Tho term "allergie" was introduced by Von Pirquet to designate the -state of the animal's being sensitized and "allergic" as the adjective -derived therefrom. It does not seem to the author that there is any -advantage gained by the introduction of these terms. - - - - -INDEX - - - A - - ABBE, 17 - condenser, 200 - microscope, improvements in, 30, 36 - - ABILGAARD, 26 - - Abrin, 262 - - Absorption of free nitrogen, 117 - tests, 267 - - Accidental carriers, 241 - structures, 43 - - Acetic acid, 99 - bacteria, carbon oxidation, 114 - fermentation, 32 - - _Acetobacter acidi oxalici_, 83 - _xylinum_, 83 - - _Achorion schoenleinii_, 27, 34 - - Acid, acetic, 99 - fermentation, 32 - agglutination, 266 - amino, relation to green plants, 119 - butyric, 99 - fermentation, 32, 99 - carbolic, first used, 29 - disinfectant action of, 159 - fast bacteria, fat content, 84 - staining of, 209 - fermentation, 93 - Bulgarian fermented milk, 98 - ensilage, 98 - industrial uses, 97 - lactic acid, 96 - sauerkraut, 98 - hydrochloric, 246 - production of, 110 - soils, 81 - - Acquired immunity, 251, 252 - - _Actinomyces bovis_, 30, 36 - - Actinomycosis, cause of, 30, 36 - path of entrance of, 244 - - Actions, reducing, 113 - - Activating enzymes, 125 - - Active immunity, definition of, 251, 252 - production of, 252 - - Activities of bacteria, importance of, 31 - overproduction, of cells, 258 - physiological, definition of, 87 - in identification, 216 - - Acute coryza, 244 - disease, 233 - - Adulteration of food, anaphylactic test in, 293 - complement-fixation test in, 279 - immunity reactions in, 255 - precipitin test in, 269 - - Aerobes, facultative, 76 - strict, 76 - - Aerobic, 76, 215 - - Agar, composition of, 179 - gelatinizing temperature, 179 - medium, preparation of, 179 - melting point of, 179 - plating in, 188 - sterilization of, 180 - - Agent, chemical, for disinfection, 156-163 - choice of, for disinfection, 164 - physical, for disinfection, 131 - - Agglutinating group, 266 - - Agglutination, acid, 266 - diagnostic value of, 266 - in identification of bacteria, 266 - macroscopic, 265 - microscopic, 265 - phenomenon, 265 - - Agglutinin, 265 - absorption test for, 267 - action of, 266 - anti-, 270 - antigenic action of, 270 - bacterial, 265 - chief, 267 - co-, 267 - function of, 266 - normal, 266 - partial, 267 - relation to precipitins, 269 - specificity of, 267 - theory of formation, 265 - use of, 266 - - Agglutinogen, 266 - - Agglutinoid, 270 - - Aggressins, 288 - - Air, bacteria in, 71 - filtration of, 153 - "germ-free," 153 - - Albumin in bacteria, 84 - - Alcohol as antiseptic, 160 - as disinfectant, 160 - - Alcoholase, 125 - - Alcoholic fermentation, 31, 100 - - Alexin, 271, 273 - - Algae, relation to bacteria, 37 - - Alimentary tract as path of entrance, 246 - - Alkalies as disinfectants, 158 - - Allergic, 290 - - Amboceptor, 273 - anti-, 275 - co-, 274 - in cobra, 275 - formation of, 273 - hemolytic, 278 - partial, 274 - in rattle snake, 275 - specificity of, 274 - theory of formation, 273 - - Amboceptorogen, 274 - - Amebic dysentery, 29, 35 - - Ameboid cells, 247 - colonies, 224 - - Amino-acids, relation to green plants, 119 - - Ammonia, structural formula, 103 - - Ammoniacal fermentation, 32 - - _Amoeba coli_, 29, 35 - - Amphitrichic, 46 - - Amylase, 124 - - Anaerobes, 76 - cultivation, methods of, 188 - principles underlying, 188 - facultative, 76 - isolation of, 190 - relation to elements, 86 - strict, 76 - - Anaerobic, 76, 215 - acid, butyric, 99 - acid fermentation, 98 - bacteria, first discovered, 32 - fermentation of polysaccharides, 95 - - Analysis of ash, 82 - chemical, of tubercle bacilli, 85 - - Anaphylactic, anti-, 290 - phenomena, 292 - reaction, uses of, 293 - - Anaphylatoxin, 290 - - Anaphylaxis, 289 - anti-, 292 - antibodies in, 291 - theory of, 290, 291, 292 - - ANAXIMANDER, 18 - - ANDERSON, 289 - - ANDERSON and MCCLINTIC, phenol coefficient, 165 - - ANDRY, 25, 33 - - Anilin dyes, as antiseptic, 162 - as disinfectants, 162 - introduction of, 30 - as stains, 204 - Weigert, 36 - fuchsin, 205 - gentian violet, 205 - water, 205 - - Animal carriers, 239 - inoculation, uses of, 227 - - Animalcules, 19, 33 - - Animals, disinfection of, 170 - experimental, 227 - food relationships of, 39 - - _Ankylostoma duodenale_, discovery of, 27, 34 - Egyptian chlorosis, cause of, 28, 35 - hookworm disease, cause of, 28 - - Anthrax, 17, 28, 35 - bacterium a facultative saprophyte, 238 - isolation of, 29 - due to a bacterium, 29 - in human beings, 238 - path of entrance, 243 - intestine, 246 - stomach, 246 - persistence due to spores, 251 - produced by exhaustion, 251 - protective inoculation in, 30 - spores, 29, 35 - transmission by flies, 242 - vaccine, 254 - - Anti-agglutinins, 270 - aggressins, 288 - amboceptors, 275 - antisera in snake poisoning, 275 - anaphylactic, 290 - anaphylaxis due to intracellular digestion, 292 - protein immunity compared to, 292 - bacterial immunity, 254, 255 - bodies, 259 - place of production, 295 - tabulation of, 294 - body, action, 260 - chemical composition, 260 - formation of, 128, 260 - complement, 274 - complementophil amboceptor, 275 - cytophil amboceptor, 275 - diphtheritic serum, 263 - enzyme, 122, 262 - function of, 262 - - Antigen, 259 - chemical composition of, 260 - in complement-fixation, 277 - syphilitic, 277, 279 - in Wassermann test, 279 - - Antigens, fats and fatty acids as, 260 - in preparation of vaccine, 285 - tabulation of, 294 - - Antipollenin, 263 - - Antiprecipitins, 270 - - Antisepsis, 131 - Lister, introduced, 35 - primitive, 25 - - Antiseptic, 131 - action of anilin dyes, 162 - carbolic acid as, 159 - cold as, 148 - - Antisera in snake poisoning, 275 - - Antisnake venoms, 275 - - Antitetanic serum, 263 - - Antitoxic immunity, 254, 255 - - Antitoxin, 261 - collection of, 263 - diphtheria, 30, 252 - preparation of, 263 - standard, 264 - tetanus, 252 - - Antitoxins, 261-264 - as factors in immunity, 295 - preservative in, 263 - specific, 261 - - Antivenin, 263 - - Apes, 227 - - Apparatus of Barber, 196 - - Appearance of growth on culture media, 217 - - APPERT, 20, 31, 34 - - Aqueous gentian violet, 205 - - Arborescent growth, 221 - - ARISTOTLE, 18 - - Aromatic compounds, production of, 104, 111 - - Arrak, 100 - - Arsenate, reduction of, 114 - - Arsenite, oxidation of, 115 - - ARTHUS, 289 - phenomenon, 289 - - Articles, unwashable, disinfection of, 169 - washable, disinfection of, 169 - - Artificial immunity, 251, 252 - - Ase, termination of name of enzyme, 124 - - Asepsis, 131 - - Aseptic, 131 - - Ash, analysis of, 82 - - Asiatic cholera, 27, 34, 73, 238, 239, 246, 248, 249 - - Attenuated, 253 - - Autoclave, air pressure sterilizer, 138 - pressure sterilizer, 138 - - Autogenous vaccines, 284 - in epidemic, 241 - - Autoinfection, 234 - - Autolysis, 149 - self-digestion, 126 - - Autotrophic, 86 - - Available nitrogen, loss of, 113 - - Azotobacter, 118 - - - B - - BABES-ERNST corpuscles, 45 - - Bacilli, butter, 209 - colon, 248 - grass, 209 - size and shape of, 52 - tubercle, chemical analysis of, 85 - - Bacillus, 52, 60, 62 - _anthracis_, 17, 36 - spore staining, 209 - - Bacillus of blue milk, 31 - Ducrey's, 245 - _subtilis_, 77, 83 - spore staining, 209 - - Bacteria, absorption of N by, 117 - acid fast, 84, 209 - adaptability, range of, 90 - advantage of motility to, 45 - aids in isolation of, 197 - anaerobic, 32 - cause of disease in animals, 30 - of souring of milk, 32 - cell groupings of, 55 - chains of, 38 - chemical composition of, 39, 81 - elements in, 82 - classed as fungi, 37 - as plants, 33, 35 - definition of, 40 - development of, 90 - distribution of, 71 - energy relationships, 39 - environmental conditions for growth, 72 - first classification of, 34 - drawings of, 20 - seen, 19, 33 - food relationships of, 39 - injurious, 72 - isolation of, 194 - measurement of, 40, 203 - metabolism of, 86 - methods of study of, 171 - morphology of, 41 - motile, 45 - nitric, 114 - nitrous, 114 - nucleus of, 42 - occurrence, 71 - pathogenic, outside the body, 237 - phosphorescent, 111, 112 - position of, 37 - rate of division, 43 - of motion, 45 - relation to algae, 33, 37 - to elements, 86 - to gas and oil, 95 - to phosphate rock, 115 - to protozoa, 40 - to soil fertility, 120 - to sulphur deposits, 116 - to yeasts and torulae, 37 - reproduction of, 37, 55 - root tubercle, 86, 87 - size of, 37, 40 - soil, chief function of, 119 - source of N, 102 - speed of, 45 - spiral, 53 - staining of, 204-212 - sulphur, 63 - thermophil, 75, 77 - universal distribution of, 90 - in vinegar-making, 99 - - BACTERIACEAE, 62, 66, 70 - - Bacterial agglutinin, 265 - vaccines, 282 - preparation of, 283, 284 - - Bacterin, 253 - - Bacteriocidin, 272 - - Bacteriological culture tubes, 184 - examination, material for, 228 - microscope, 200 - - Bacteriology, pathogenic, definition of, 231 - reasons for study of, 217 - as a science, 17, 32 - - Bacteriolysin, 272 - - Bacteriopurpurin, 62, 63, 112 - - Bacteriotropin, 281 - - _Bacterium abortus_, agglutinin of, 265 - _coli_ in autoinfection, 234 - gas formation by, 95 - oxygen limits for, 77 - pneumonia through intestinal route, 246 - in preparation of sugar broths, 176 - definition of, 62, 67, 70 - _enteriditis_, cause of food poisoning, 104 - _fluorescens_, oxygen limits, 77 - _typhosum_, 73 - agglutinin, 265 - in phenol coefficient method, 166 - pneumonia through intestinal route, 246 - - Ballon pipette, 193 - - Balsam, mounting in, 207 - - BARBER, 253 - apparatus, 196 - - Barnyards, disinfection of, 167 - - Baskets, wire, 184 - - BASSI, 27 - silkworm disease, 34 - - BASTIAN, 24 - - BAUMGAeRTNER, 256 - - Beaded growth, 221 - - Bed-bugs, 241 - - Beds, contact, 116 - hot, 117 - - Beer, pasteurization of, 141, 144, 145 - - _Beggiatoa_, 63 - - BEGGIATOACEAE, 63 - - BEHRING, 30 - - BELFANTI, 271 - - BERG, 27, 34 - - Berkefeld filter, 154 - - Bichloride of mercury as disinfectant, 158 - - BILHARZ, 28, 35 - - Bilharzia disease, 28, 35 - - Biochemical reactions, definition of, 87 - - Biological relationships, immunity reactions, 255, 270 - - Bipolar germination of spore, 48 - - Bismarck brown, 209, 212 - - Black-leg, 51, 73, 238, 243, 248, 251 - vaccine, 254 - - Bleaching powder as disinfectant, 158 - - Blood, collection of, 228 - cytolytic power of, 272 - detection of, 269 - serum, liquid, sterilization of, 182 - Loeffler's, 182 - medium, preparation of, 182, 183 - sterilization of, 182 - vessels in dissemination of organisms, 247 - - Blue milk, bacterial cause of, 34 - fermentation of, 31, 34 - - BOEHM, 27, 34 - - Boiling as disinfectant, 133 - - Boils, 237, 240, 243 - - BOLLINGER, 29, 30, 35, 36 - - BONNET, 20, 33 - - BORDET, 271 - - _Botrytis bassiana_, 27, 34 - - Bottles, staining of, 206 - - Bougies, 154 - - Bouillon, 173 - - BOYER, 260 - - Bread, salt rising, 95, 97 - - Bronchopneumonia, 233, 246 - - Broth, appearance of growth in, 218 - extract of, 176 - glycerine, 176 - medium, 173 - nitrate, 177 - sterilization of, 174 - sugar, 176 - - Brownian movement, 47, 203 - - Brushes, disinfection of, 169 - - Bubonic plague, 239 - - BUCHNER, 271 - - Budding of yeasts, 37 - - Bulgarian fermented milk, 98 - - Burning as disinfectant, 132 - - Burying as disinfectant, 154 - - BUeTSCHLI, 41, 43 - - Butter, 97 - bacilli, staining of, 209 - rancidity of, 101 - - Butyric acid fermentation, 32, 99 - - Buzzards, 241 - - - C - - CABBAGE disease due to protozoa, 36 - - Cadaverin, 104 - - CAIGNARD-LATOUR, 31, 34 - - Calcium hypochlorite as disinfectant, 158 - oxide as disinfectant, 158 - - Candles, filter, 153, 154 - - Canned goods, food poisoning by, 104 - spoilage of, 51 - - Canning, introduced, 21, 34 - principles involved, 133 - - Capsule, 44, 45 - of spore, 48 - staining of, 210 - - Carbohydrates in bacterial cell, 84 - fermentation of, 93-101 - - Carbol-fuchsin, 206 - - Carbolic acid as antiseptic, 159 - as disinfectant, 159 - first used, 29 - - Carbol-xylol, 209 - - Carbon cycle, 107 - dioxide, 108 - function of, in bacteria, 88, 101 - oxidation of, 114 - in proteins, liberation of, 105 - source of, 88 - uses of, 88, 101 - - CARBONI, 271 - - CARDANO, 18 - - Carrier problem, solution of, 240 - - Carriers, 239 - accidental, 241 - carrion eating animals as, 241 - control of, 240 - intermediate hosts as, 242 - protective measures against, 242 - universal, 240 - of unknown organisms, 239 - - Cars, stock, disinfection of, 170 - - Catalase, 125 - - Catalytic agents, function of, 123 - - Catalyzer, 123 - - Cattle, 227 - - Causation of disease, 24, 128 - - Cell, constituents of, 84 - contents of, 41, 83 - forms of, 58, 59 - staining for, 212 - typical, 52 - groupings, 55, 58, 59 - staining for, 195 - metabolism, 90 - structures of, 41 - wall, 41, 59 - composition of, 83 - - Cells, chemical stimuli of, 257 - overproduction activity of, 258 - specific chemical stimuli of, 258 - - Cellular theory of immunity, 256, 280 - - Cellulose, definition of, 83 - occurrence of, 83 - - Chain, 56 - - Channels of infection, 243 - alimentary tract, 246 - conjunctive, 244 - external auditory meatus, 244 - genitalia, 245 - intestines, 246 - lungs, 245 - milk glands, 244 - mouth cavity, 244 - mucosae, 244 - nasal cavity, 244 - pharynx, 245 - skin, 243 - stomach, 246 - tonsils, 245 - - Chaos, 25 - - Characteristic groupings, 58 - - Characteristics of enzymes, 121 - of toxins, 126 - - CHARRIN, 265 - - Chart, descriptive, 217 - - CHAUVEAU, 256 - - Cheese, eyes in, 96 - failures, 110 - Limburger, 101 - odor of, 99 - poisoning, 104 - ripening of, 35 - - Chemical composition of bacteria, 39, 81, 85 - elements in bacteria, 82 - disinfectants, action of, 156-163 - stimuli, 257-260 - theory, fundamentals of, 256 - - Chemotherapy, 249, 255 - - CHEVREUIL, 21, 27, 31, 34 - - Chicken cholera, 30 - pox, 239, 246 - - Chief agglutinin, 267 - cell, 267 - - Chitin, 72 - - CHLAMYDOBACTERIACEAE, 63 - - _Chlamydothrix_, 63 - - Chloride of lime as disinfectant, 158 - - Chlorine as disinfectant, 157 - - Chloroform as antiseptic, 162 - as disinfectant, 162 - - Chlorophyl, 37, 112 - - Chlorosis, Egyptian, 27, 35 - - Cholera, Asiatic, carriers of, 239 - organisms in, 27, 34 - facultative saprophytes, 238 - path of elimination of, 248 - of entrance of, 246 - relation to moisture, 73 - specific location of, 249 - hog, 242, 248, 252 - - Cholesterins as cell constituents, 84 - - Chromogenesis, 112 - - Chromoparic, 112 - - Chromophoric, 112 - - Chronic disease, 232 - - Chronological table, 33-36 - - Chymosin, 124 - - Circulation of carbon, 107 - of nitrogen, 107 - of phosphorus, 107 - of sulphur, 108 - - Classification, advantage of, 59 - early, 33, 35, 59 - Migula's, 62-63 - S. A. B., 63-70 - - Cleaning of slides, 207 - - Clearing of sections, 209 - - Closed space disinfection, 161 - - _Clostridium_, 49 - _botulinum_, 87, 104, 128, 238, 261 - _pasteurianum_, 118 - _tetani_, 128, 209, 233, 261, 263 - - Clothing, disinfection of, 170 - - Coagglutinins, 267 - - Coagulases, 124 - - Coagulating enzymes, 124 - - Coagulation temperature of proteins, 51 - - Coal, spontaneous heating of, 88 - - Coamboceptors, 274 - - Cobra, 275 - - COCCACEAE, 62, 66, 68 - - Coccus, appearance of, on dividing, 57 - cell form of, 52 - groupings of, 52, 56, 57 - division of, 52 - - Coenzymes, 122 - - COHN, 28, 33, 35, 59 - - Cold as antiseptic, 148 - incubator, 215 - storage, 148 - - Colds, due to universal carriers, 240 - path of entrance of, 244 - vaccines in, 241 - - Colonies, characteristics of plate, 223-226 - definition of, 173 - - Color production, 112 - - Colorimetric method of standardization, 175 - - Combustion, spontaneous, 116 - - Commensal, 87 - - Commercial preparation of lactic acid, 99 - products, why keep, 131 - vaccines, 285 - - Communicable disease, 232 - - Complement, 273 - deviation test, 277 - effect of temperature on, 274 - fixation test, 276-279 - lecithin as, 274 - relation to toxins and enzymes, 273 - source of, 277 - - Complementoid, 274 - - Complementophil haptophore, 273 - - Complements, nature of, 274 - - Composition, chemical, 81-85 - related to fungi, 39 - relation to food, 81 - - Concentration of antitoxin, 264 - - Condenser, 200 - - Conditions for growth, general, 72 - maximum, 72 - minimum, 72 - optimum, 72 - spore formation, 51 - - Congenital immunity, 251, 252 - - Conjunctiva as path of entrance, 244 - - Constant temperature apparatus, 213 - - Contact beds, 116 - - Contagion, direct and indirect, 34 - - Contagious abortion, agglutination test, 268 - complement-fixation text, 277 - path of elimination, 248 - of entrance, 245 - - Contagium, definition of, 232 - vivum theory, 25, 28, 33 - - Contamination of food by carriers, 241 - - Continuous pasteurization, 141 - - Contrast stains, 205 - - Convalescents, control of, 239-240 - - CORNALIA, 29 - - Corpuscles, Babes-Ernst, 45 - red, in complement-fixation test, 278, 279 - malaria, etc., in, 249 - - Corrosive sublimate as disinfectant, 158 - - _Corynebacterium diphtheriae_, 64, 69, 128, 233, 234, 261, 263 - - Coryza, acute, 244 - - Cotton plugs, 21, 184 - - Coughing, 248 - - Crateriform liquefaction, 221 - - Cream ripening, 97 - - CREITE, 271 - - _Crenothrix_, 61 - - Creolin as disinfectant, 160 - - Cresols as disinfectants, 159 - - Culture, definition of, 171 - medium, definition of, 171 - essentials of, 172 - inoculation of, 186, 192 - kinds of, 172 - liquid, 172, 173 - methods of using, 184 - optimum moisture for, 73 - plating of, 188 - reaction of, 81, 216 - selective, 198, 199 - solid, 172, 173 - standardization of, 174, 175 - synthetic, 183 - titration of, 174 - use of, 173 - tubes, description of, 184 - - Cultures, anaerobic, 188-192 - from internal organs, 229 - mass, 188 - plate, 188 - potato, 186 - puncture, 185 - pure, definition of, 171 - isolation of, 194-199 - slant, 186 - slope, 186 - stab, 185 - - Curled edge, 225 - - Cutaneous inoculation, 228 - - Cycle, carbon, 107 - nitrogen, 107 - phosphorus, 107 - sulphur, 108 - - Cystitis, 234 - - Cytolysin, 272 - - Cytolysins, 271-279 - - Cytolytic, 272 - power of blood, 272 - serums, failure of, 275 - substances in immunity, 295 - - Cytophil group, 273 - - Cytoplasm, 41 - - Cytotoxic, 272 - - - D - - DALLERA, 289 - - Dark field illumination, 204 - - DAVAINE, 28, 35 - - Death-point, thermal, 75 - determination of, 215 - - Decomposition, how caused, 108 - importance of, 108 - of urea, 106 - - Deep culture tubes, 190-191 - - Degeneration forms, 54 - - Delousing method in typhus, 242 - - DE MARTIN, 35 - - Denitrification, 114 - - Deodorant, 131 - - Descriptive chart, 217 - - Diagnosis, agglutination test in, 265-267 - anaphylaxis in, 292 - complement-fixation test in, 277 - immunity reactions in, 255 - material for bacteriological, 228-229 - precipitin test in, 269 - - Diastase, 124 - - Diffusion of food through cell wall, 41 - - Digestion of proteins, 102 - - Dilution method of isolation, 194 - plates, 194, 195 - - Dimethylamine, structural formula, 103 - - Diphtheria antitoxin, 30, 263, 264 - bacilli, granules in, 45 - involution forms, 54 - carriers, 239 - location of, 245, 249 - path of entrance, 245 - toxin, M. L. D., 264 - - Diplobacillus, 55 - - Diplococcus, 56 - - _Diplococcus_, 66, 69 - - Diplospirillum, 55 - - Discharges, 228 - intestinal, 248 - nasal, 248 - urethral, 248 - vaginal, 248 - - Discontinuous sterilization, 133 - - Disease, acute, 233 - of animals to man, 232 - Bilharzia, 28, 35 - cabbage, 30, 35 - causation of, 24, 128 - communicable, 232 - contagious, 34, 232 - of flies, 28, 35 - germ, 25, 27, 33 - hookworm, 28, 35 - infectious, 232, 240 - Johne's, 246, 248 - non-specific, 233 - protozoal, eradication, 242 - transmission, 242 - silkworm, 27, 29, 34, 35 - skin, 243 - specific, 27, 30, 233 - transmission of, 26, 232 - - Dishes, Petri, 181 - - Disinfectant, 131 - action of anilin dyes, 162 - closed space, 161 - dry heat as, 133 - moist heat as, 132, 133 - standardization of, 165 - steam as, 132, 133 - - Disinfectants, chemical, action of, 156-163 - first experiment in, 21 - factors affecting, 164-165 - - Disinfection, agents in, 131-163 - by boiling, 132, 133 - by burning, 132 - by burying, 154 - definition of, 130 - first chemical, 34 - hot air, 21, 133 - physical agents, 131-155 - practical, 166-170 - precautions in, 170 - puerperal fever, 28, 34 - steam, 134-138 - surgeon's hands, 28 - - Dissemination of organisms, 247 - - DISTASO, 42, 43 - - Distilling sour mash, 98 - - Division, planes of, 55-58 - rate of, 43, 91 - - DOBELL, 43 - - DORSET, 84 - - Dosage of vaccines, 286 - - Dose, minimum lethal, 264 - standard test, 264 - - DOUGLAS, 42, 43, 280 - - Dourine, 245, 248 - - Drumstick spore, 49 - - Dry heat, 21, 133 - - Drying, 131, 132 - - DUBINI, 27, 34 - - Ducrey's bacillus, 245 - - Dunham's peptone, 177 - - DURHAM, 265 - - Dyes, anilin, as antiseptics, 162 - introduction of, 30 - as stains, 204 - - Dysenteries, 242, 246, 248, 249 - - Dysentery, amebic, 29, 35 - tropical, 29 - - - E - - ECTOPLASM, 41 - - Edema, malignant, 237, 243 - - Edge of colony, 225 - - Effuse colony, 224 - - Egg sensitization, 292 - - EHRENBERG, 33, 34 - - EHRLICH, 256, 276 - - Ehrlich's theory, 256-260 - - EICHSTEDT, 28, 34 - - Electric milk purifier, 152 - - Electricity, 79, 150 - - Elements in bacteria, 82, 86, 88, 89 - - Elimination of organisms, 248 - - _Empusa muscae_, 28, 29, 35 - - Emulsin, 122 - - Endo-enzymes, 126 - - Endogenous infection, 235 - - Endoplasm, 41 - - Endotoxins, 128, 276 - - Energy relationships, 39 - transformations, 86-90 - - Ensilage, 98 - - Enteritis, 233 - - Entire edge, 225 - - Entrance of organisms, 243-246, 247 - - Environmental conditions, 72, 130, 213 - - Enzymes, 84, 121-126 - in anaphylaxis, 291 - in immunity, 295 - - Enzymoid, 262 - - Epidemics, 241 - - Epitheliolysin, 272 - - Eosin, 204 - - Equatorial spore, 49 - germination of, 48, 49 - - Eradication of disease, 236, 242 - - Erysipelas, hog, 248 - - _Erythrobacillus prodigiosus_, 66, 68, 70, 77, 113 - - Essential structures, 41 - - Essentials of a culture medium, 172 - - Esters, 84, 110 - - Ether as disinfectant, 162 - - EUBACTERIA, 62 - - Exanthemata, 248 - - Exhaustion factor in immunity, 251 - theory of immunity 256 - - Existence, conditions for, 72 - - Exo-enzymes, 126 - - Exogenous infection, 235 - - Exotoxins, 128 - - Experiment, Pasteur's, 21 - Schroeder and Dusch's, 22 - Schultze's, 21 - Schwann's, 22 - Tyndall's, 24 - - Experimental animals, 227 - - External auditory meatus, 244 - genitalia, 245 - - Extracellular enzymes, 126 - - Extract broth, 176 - - Eyes in cheese, 96, 97 - - - F - - Factors affecting disinfectants, 164, 165 - immunity, 250, 251 - in immunity to disease, 295 - - Facultative, 215 - aerobes, 76 - anaerobes, 76, 192 - parasites, 87 - saprophytes, 238 - - Failure of cytolytic serums, 275 - of vaccines, 286 - - Fat colors, 112 - splitting enzymes, 124 - - Father of bacteriology, 19 - of microscope, 19 - - Fats as antigens, 260 - occurrence of, 84 - rancidity of, 101 - in sewage disposal, 101 - splitting of, 101 - - Favus, 27, 34, 243 - - Feces, bacteria in, 72 - - Feeding, as inoculating method, 228 - - FEINBERG, 43 - - Ferment, organized, 126 - unorganized, 126 - - Fermentation, 31, 93 - acid, 93, 96 - acetic, 32, 99 - butyric, 32, 99 - alcoholic, 31, 34, 100 - ammoniacal, 32 - bacterial, 32 - blue milk, 31, 34 - of carbohydrates, 93-101 - definition of, 93 - gaseous, 93, 94, 96 - tubes, 184, 190 - yeast, 34, 99 - - Fermented milk, Bulgarian, 98 - - Fever, due to invisible organisms, 25 - Malta, 268 - recurrent, 29, 35 - Rocky Mountain spotted, 242 - scarlet, 246, 248 - Texas, 232, 233, 242 - typhoid, 232, 248 - typhus, 242 - yellow, 242 - - Fibrin ferment, 124 - - Filament, 56 - - Filiform growth, 221 - - Film, fixing of, 207 - preparation of, 207 - - Filter, Berkefeld, 154 - candles, 153-154 - Mandler, 154 - Pasteur-Chamberland, 154 - sprinkling, 115, 116 - - Filterable virus, 234 - - Filtration, 152-154 - - First order, receptors of, 261, 262 - - FISCHER, 42, 45 - - Fixation test, complement, 276 - - Fixed virus, 253 - - Fixing of film, 207 - - Flagella, 45-47 - staining of, 210 - - Flash process of pasteurization, 145 - - Fleas, 241 - - FLEXNER, 276 - - Flies, 28, 35, 241 - - FLUeGGE, 271 - - FODOR, von, 271 - - Food adulteration, complement-fixation test in, 279 - immunity reactions in, 255 - precipitin test in, 269 - - Food contamination by carriers, 241 - poisoning, 87, 104, 238 - requirement compared with man, 92 - uses of, 86 - - Foot-and-mouth disease, 244, 248 - - Forage poisoning, 87 - - Foreign body pneumonia, 245 - - Formaldehyde as disinfectant, 160 - - Formalin, 160 - - Formol, 160 - - Forms, cell, 52-54 - degeneration, 54 - growth, 55 - involution, 54 - study of, 32-34 - - Fox fire, 111 - - Foxes, 241 - - FRACASTORIUS, 25, 33 - - Free acid, 175 - receptors, 259 - spores, 48 - - Fruiting organs, 37 - - FUCHS, 31, 34 - - Fuchsin, 205 - anilin, 205 - carbol, 206 - - Fungi, bacteria as, 37 - - Funnel-shaped liquefaction, 221 - - - G - - GABBET'S blue, 206 - method of staining, 209 - - Gall-bladder, 248 - - Galvanotaxis, 79 - - Gas formation in cheese, 96, 97 - natural, 95 - production of, 110 - - Gaseous fermentation, 93-95 - - GASPARD, 26, 34 - - Gelatin, advantage of, 178 - composition of, 179 - cultures, first used, 30, 36 - liquefaction of, 103 - medium, 177 - plating of, 188 - standardization of, 178 - sterilization of, 178 - - Gemmation, 37 - - General conditions for growth, 72 - infections, vaccines in, 286 - - Generation, spontaneous, 17-24 - - Generic names introduced, 33 - - Genitals, 245 - - Gentian violet, selective action of, 162 - stain, 205 - - Germ, free air, 153 - theory of disease, 25 - - German measles, 233 - - Germination of spore, 48 - - Germs, 33 - in air, 24, 35 - - GESCHEIDEL, 271 - - Giemsa stain, 43 - - Glanders, 26, 233, 238, 244, 248, 249, 268, 277 - - Glands, mammary, 248 - salivary, 248 - - GLEICHEN, 32 - - Globulin in bacteria, 84 - - Glycerine broth, 176 - - Glycerinized potato, 172 - - Glycogen as cell constituent, 84 - - Goats, 227 - - Gonidia, 63 - - Gonococcus, 245 - - Gonorrhea, 248, 249 - - Good health, 296 - - Grain rust, 26, 34 - - Gram positive organisms, 162, 208 - negative organisms, 162, 208 - - Gram's method of staining, 208 - solution, 208 - - Granular edge, 225 - - Granules, metachromatic, 212 - Neisser's, 45 - polar, 45 - - Granulose in bacteria, 84 - - Grape juice, pasteurization of, 141 - - Grass bacilli, 209 - - Green plants, N nutrition of, 118 - - GRIESINGER, 27, 28, 35 - - Group, agglutinating, 266 - haptophore, 261, 262, 266, 270, 273 - precipitating, 270 - toxophore, 261, 262 - zymophore, 262, 270, 273 - - Groupings, cell, 55-58 - - Growth, appearance in media, 217 - - _Gruber_, 265, 268 - - _Gruby_, 28, 34 - - Gum-like substance in bacteria, 83 - - - H - - HAECKEL, 280 - - Hanging drop slide, 203 - - Haptophore, complementophil, 273 - cytophil, 273 - group, 261, 262, 266, 270, 273 - - Harness, disinfection of, 169 - - Hay fever, 263, 292 - - Health, 296 - - Heat as disinfectant, 132-144 - due to oxidation, 112 - production of, 116 - - Heated serum, 271, 277, 278, 279 - - Heating of manure, 116 - - HELLMICH, 84 - - HELMONT, VAN, 18 - - Hemagglutinin, 265 - - Hemicellulose, 83 - - Hemolysin, 272 - - Hemolytic amboceptor, 278 - - Hemorrhagic septicemia, 246 - - HENLE, 27, 34, 233 - - HERICOURT, 289 - - Herpes tonsurans, 28, 34 - - HESSELING, von, 32 - - Heterologous sera, 276 - - Heterotrophic, 86 - - HILL, 33 - - HILTON, 27 - - HOFFMAN, 24 - - Hog cholera, 231, 242, 248, 252, 253 - erysipelas, 248 - - Holders, 143 - - HOLMES, 28, 34 - - Homologous sera, 276 - - Hookworm disease, 28, 34 - - Horses, 227, 263 - - Host, 87 - - Hot beds, 117 - - Hunger in immunity, 251 - - Hydrochloric acid, 246 - - Hydrogen, function of, 98 - ion concentration standardization, 175, 176 - oxidation of, 114 - peroxide, 162 - sulphide, 115 - - Hydrophobia, 249 - - Hydrostatic pressure, 79 - - Hygienic laboratory, 165 - - Hypochlorites, 157, 158 - - - I - - Ice cream poisoning, 104 - - Identification of bacteria, 216, 217 - in blood, 269 - in meat, 269 - in milk, 269 - - Immersion oil, 201 - - Immunity, 236, 250-296 - acquired, 251, 252 - active, 251, 252-255 - antibacterial, 254, 255 - antitoxic, 254, 255 - artificial, 251, 252 - classification of, 251 - congenital, 251 - factors in, 295 - modifying, 250 - inherited, 251, 252 - natural, 295 - passive, 251, 252, 253 - to protein, 290 - reactions, value, 255 - relative, 250 - summary of, 295 - theories of, 256 - - Inactivate, 272 - - Incubation period, 26, 232 - - Incubator, 213 - cold, 215 - room, 213 - - Index, chronological, 31 - opsonic, 281 - phagocytic, 281 - - Indicator, 278 - - Indol, 104 - - Infection, 232 - auto, 234 - channels of, 243 - endogenous, 235 - exogenous, 235 - mixed, 234 - primary, 234 - secondary, 234 - wound, 17, 25, 26, 27, 30, 34, 36, 233, 234, 240, 243, 248 - - Infectious diseases, 232 - control of, 240 - - Infective organisms, specificity of location, 249 - - Infestation, 232 - - Infested, 232 - - Influenza, 239, 241, 246 - - Infusoria, 33 - - Inhalation, 228 - - Inherited immunity, 251, 252 - - Inoculation of animals, 227 - uses of, 227 - of cultures, 186, 188 - definition of, 192 - methods of, 227 - needles, 192 - - Inoculations, first protective, 30 - of smallpox, 24 - - Insects, 241, 242 - - Instruments, sterilization, 136, 167 - - Intracardiac, 228 - - Intracellular enzyme, 166 - - Invasion, 232 - - Invertase, 124 - - Involution forms, 53, 212 - - Iodine, 157 - - Iron bacteria, 86 - function of, 89 - - Irregular forms, 53 - - Isolation of anaerobes, 190 - of pure cultures, aids in, 197-199 - methods, 194-196 - - Itch mite, 27, 34 - - - J - - JABLOT, 32 - - Jack-o-lantern, 105 - - Jar, Novy, 192 - - JENNER, 26, 34, 253 - - Johne's disease, 248 - - - K - - KETTE, 32, 35 - - Kidneys, 248 - - Kinase, 125 - - KIRCHER, 18, 25, 33 - - KLEBS, 29, 35 - - KLENCKE, 28, 34 - - KOCH, 17, 27, 29, 30, 33, 36 - - Koch's postulates, 233 - - KRAUS, 268 - - KRUSE, 254 - - KUeCHENMEISTER, 28, 35 - - - L - - LAB, 124 - - Lachrymal canal, 244 - - Lactacidase, 125 - - Lactic acid bacteria, 97 - fermentation, 96-99 - - LANCISI, 25, 33 - - LANDOIS, 271 - - LATOUR, 31, 34 - - LAVERAN, 25, 30 - - Lecithin as antigen, 279 - as cell constituent, 84 - as complement, 274 - - LEEUWENHOEK, 19, 32, 33 - - Legumes, 118 - - LEIDY, 27, 33, 34, 35 - - LE MOIGNAC, 284 - - Leprosy, 233, 244, 249 - - LESSER, 32 - - Lethal dose, 264 - - Leukocytes, washing of, 281 - - Lice as carriers, 241 - - LIEBERT, 28, 34 - - Light, action on bacteria, 75 - as disinfectant, 148 - production of, 111 - - LINNAEUS, 25 - - Lipase, 124 - - Lipochromes, 113 - - Lipoids as antigen, 274 - - Lipovaccines, 284 - - Liquefaction of gelatin, 221 - of protein, 103 - - Liquid blood serum, 182 - manure, disinfection of, 169 - media, 172 - - Liquids, sterilization of, 153 - - LISTER, 29, 30, 35 - - Litmus milk, 177 - - Living bacteria, examination of, 201 - cause theory, 28, 33 - - Localized infections, vaccines in, 286 - - Location of organisms, specificity of, 249 - - Lockjaw, 231, 233 - - Loeffler's blood serum, 182 - blue, 206 - - Loop needles, 193 - - Lophotrichic, 46 - - LOeSCH, 29, 35 - - Lungs, 245, 249 - - Lye washes as disinfectants, 159 - - Lymph channels in dissemination, 247 - - Lysol as disinfectant, 160 - - - M - - MCCLINTOCK, 165 - - MCCOY, 160 - - Macrococcus, 52 - - Macroscopic agglutination, 265 - - Malaria, 25, 30, 32, 242 - - Malarial parasite, 30, 249 - - Malignant edema, 237, 243 - - Mallease reaction, 269 - - Mallein test, 292 - - Malta fever, 268 - - Mammary glands, 248 - - Mandler filter, 154 - - Manure, liquid, disinfection of, 169 - heating of, 40 - - _Margaropus annulatus_, 242 - - MARTIN, 32 - - Mass cultures, 188 - - MASSART, 42 - - Maximum conditions, 72, 73, 74, 76 - - Measles, 246, 248, 250 - German, 233, 239 - - Measly pork, 28 - - Measurement of bacteria, 203 - special unit of, 40 - - Meat broth, 173 - identification of, 269 - juice, 173 - poisoning, 104 - - Mechanical vibration, 80 - - Medico-legal examination, 269, 279, 293 - - Medium. _See_ Culture medium - - Meningitis, 239, 244 - - Meningococcus, 244 - - Mercuric chloride, 158 - - Merismopedia, 57 - - Metabiosis, 103 - - Metabolism, 86-91 - - Metachromatic granules, 44, 45, 59, 212 - - Metastases, 235 - - Metatrophic, 86 - - METCHNIKOFF, 256, 280 - - Methods of inoculation of animals, 227 - of cultures, 186-188 - of obtaining pure cultures, 194 - - Methylamine, 103 - - Methylene blue, 205, 206 - - Mice, white, 227 - - Microbiology, 231 - - Micrococcus, 52, 60, 62, 66, 68, 69, 245 - - Micrometer, 203 - - Micromillimeter, 40 - - Micron, 40 - - Microoerganisms, 32 - - Microscope, improvements in, 30, 36 - invention, 19 - Leeuwenhoek's, 19 - use of, 200 - - Microspira, 61, 63 - - _Microsporon furfur_, 28, 34 - - Middle ear, 241 - - Migula's classification, 62 - - Milk, blue, 31, 34 - Bulgarian, 98 - digestion of, 102 - flavors in, 110 - glands, 244 - identification of, 269 - litmus, 177 - pasteurization of, 141, 144-147 - as path of elimination, 248 - preparation of, 177 - purifier, electric, 152 - souring of, 32 - sterilization of, 177 - tuberculous, 248 - - Minimum conditions, 72, 73, 74, 77 - lethal dose, 264 - - Mirror, use of, 200 - - Mixed infection, 234 - vaccine, 285 - - Mixotrophic, 86 - - M. L. D., 264 - - MOHLER, 167 - - Moist heat, 133 - - Moisture, 73 - - Mold colonies, 226 - - Molds in alcoholic fermentation, 100 - in relation to bacteria, 37, 39 - - Molecular respiration, 88, 89 - - Monas, 33 - - Monkeys, 227 - - Monotrichic, 45 - - MONTAGUE, 24 - - Mordants, 204, 211 - - Morphology, 41-58 - in identification, 171, 212 - - Mosquitoes and malaria, 25, 242 - - Motile bacteria, 45 - - Motion of bacteria, 47 - Brownian, 47, 203 - - Mounting in balsam, 207 - - Mouth cavity, 244 - - Mu, 40 - - Mucosae as channels of infection, 244 - - MUeLLER, 33, 34, 59 - - Mumps, 239 - - Municipal disinfection, 170 - - MUeNTZ, 32, 35 - - Muscardine, 34 - - Mycelia, 39, 226 - - _Mycobacteriaceae_; 64 - - _Mycobacterium_, 64, 69 - of Johne's disease, 209 - _leprae_, 209 - _smegmatis_, 209 - _tuberculosis_, 83, 176, 209 - - Mycoproteid, 83 - - Mycorrhiza, 119 - - Myxomycetes, 38 - - - N - - NAeGELI, 29, 35 - - Nasal cavity, 244 - discharges, 248 - - Natural gas, 95 - immunity, 251, 252, 296 - - NEEDHAM, 20, 33 - - Needles, inoculation, 192 - - Negative complement-fixation test, 278 - phase, 287 - - Neisser's granules, 45 - stain, 212 - - NENCKI, 83 - - Nephrolysin, 272 - - NEUFELD, 281 - - Neurin, 104 - - Neurotoxin, 272 - - NEUVEL, 43 - - Nichrome wire, 193 - - Nitrate broth, 177 - - Nitrates in soil, 115 - - Nitric bacteria, 114 - - Nitrification, 32, 35 - - Nitrite, oxidation of, 114 - - Nitrogen, absorption of, 117 - in bacterial cell, 89 - circulation, 109 - cycle, 107 - fertilizers, 120 - liberation, 104 - nutrition of green plants, 118 - use of, 103 - - Nitrous bacteria, 114 - - Non-pathogenic, 87 - - Non-specific disease, 233 - - Normal agglutinins, 266 - serum, 272 - - _Nosema bombycis_, 29, 35 - - NOVY, 183 - jar, 192 - - Noxious retention theory, 255 - - Nuclein, 42, 43 - - Nucleoprotein, 43 - - Nucleus, 42, 43 - - Nutrition of green plants, 118 - - NUTTAL, 271 - - - O - - OBERMEIER, 29, 35 - - Objective, oil immersion, 200, 201 - - Oblique germination of spore, 48 - - Occurrence of bacteria, 71 - - Official classification, 59 - - _Oidium albicans_, 27, 34 - - Oil bath, 167 - essential for clearing, 209 - immersion objective, 200, 201 - relation of bacteria to, 116 - - OMODEI, 27 - - Opsonic index, 281, 282, 287 - method, 282 - power, 287 - - Opsonin, 281 - - Opsonins, 281, 282, 295 - - Optimum conditions, 72, 73, 74 - - Order, receptors of first, 261-264 - of second, 265-270, 281 - of third, 271-279 - - Organic acids, 84, 110 - catalyzers, 123 - - Organisms, dissemination of, in body, 247 - filterable, 234 - pathogenic, elimination of, 248 - entrance of, 243-246, 247 - specific relation to tissue, 249 - ultramicroscopic, 234 - - Organized ferments, 126 - - Osmotic pressure, 78, 149, 216 - - Otitis media, 244 - - OTTO, 289 - - Overproduction theory, 257, 258 - - OWEN, 27, 34 - - Oxidation, 114, 115 - - Oxidizing enzymes, 125 - - Oxygen, compressed, 77 - as disinfectant, 156 - function of, 88 - nascent, 77 - relationships, 215, 220 - requirement, 88 - source, 76, 77 - - Oyster sensitization, 292 - - OZNAM, 26 - - Ozone, 77, 150, 157 - - - P - - PANCREAS, 248 - - Papillate, 221 - - PAGET, 27, 34 - - Paraffin oil, 190 - - Parasite, 87 - facultative, 87 - strict, 87 - - PARODKO, 77 - - Partial agglutinin, 267 - amboceptor, 274 - - Passive immunity, 251, 252 - - PASTEUR, 17, 21, 29, 30, 31, 32, 35, 253, 256, 283 - flask, 21, 23, 24, 193 - treatment of rabies, 253 - - Pasteur-Chamberland filter, 154 - - Pasteurization, 139-147 - continuous, 141 - flash process, 145 - - Pathogenic, 87 - bacteria, definition of, 231 - outside the body, 237 - bacteriology, scope of, 235 - organisms, destroyed by boiling, 133 - elimination of, 248 - entrance of, 243-247 - - Paths of elimination, 248 - of entrance, 243-247 - - PEACOCK, 26 - - Pebrine, 29, 35 - - Pedesis, 47 - - Peptone solution, Dunham's, 177 - - Period of incubation, 26, 232 - - Peritonitis, 234 - - Peritrichic, 46 - - _Peronospora infestans_, 28, 34 - - PERTY, 33, 35 - - Pet animals, 241 - - Petri dishes, 181, 188 - - Petroleum, 95 - - PFEIFFER, 271 - - Pfeiffer's phenomenon, 271 - - _Pfeifferella mallei_, 65, 69, 265 - - Phagocytes, 247 - - Phagocytic index, 281 - - Phagocytosis, 243, 280-288, 295 - theory, 256 - - Pharynx, 245 - - Phase, negative, 287 - positive, 287 - - Phenol coefficient, 165, 166 - as disinfectant, 159 - production of, 104, 111 - - Phenolphthalein, 174 - - Phenomenon, anaphylactic, 292 - Arthus', 289 - Pfeiffer's, 271 - - Phosphate reduction, 114 - rock, 115 - - Phosphorescence, 111 - - Phosphorus cycle, 108 - in proteins, 105 - uses of, 89 - - Photogenesis, 111 - - Physical agents for disinfection, 131-155 - - Physiological activities, 93-129 - definition of, 87 - in identification, 216 - - Physiology of bacteria, 71-171 - - Phytotoxins, 127, 128 - - Pickling, 98 - - Pigeons, 227 - - Pigments, 84, 112, 113 - - Pimples, 234, 240, 243 - - PINOY, 284 - - Pipettes for inoculation, 193 - - _Piroplasma bigeminum_, 233, 242, 249 - - Piroplasmoses, 242, 249 - - PIRQUET, von, 289 - - Pityriasis versicolor, 28, 34 - - Plague, 246 - - Planes of division, 56, 57 - - _Planococcus_, 62 - - _Planosarcina_, 62 - - Plants and animals, 39 - - _Plasmodiophora brassicae_, 30, 36 - - Plasmolysis, 41, 42, 78 - - Plasmoptysis, 42, 78 - - Plate colonies, study of, 224-226 - cultures, 180, 188, 191 - - Plates, dilution, 194, 195 - gelatin first used, 30, 36 - - Platinum needles, 193 - - Plectridium, 49 - - PLENCIZ, 26, 31 - - Plugs, cotton, 21, 184 - - Pneumococcus, 240, 245 - - Pneumonia, 240, 245, 246, 248 - vaccination against, 241 - - Poisoning, cheese, 104 - food, 87, 104, 238 - ice cream, 104 - meat, 104 - - Polar germination, 48, 49 - granules, 45 - - Poliomyelitis, 244 - - POLLENDER, 28, 35 - - Polysaccharides, fermentation of, 95 - - Polyvalent vaccine, 285 - - Pork, measly, 28 - - Position of bacteria, 37 - of flagella, 45, 46 - of spore, 49 - - Positive phase, 287 - test, 278 - - Postulates, Koch's, 233 - - Potato, acidity of, 182 - glycerinized, 182 - media, 180-182 - rot, 28, 34 - - Power, opsonic, 287 - - Practical sterilization and disinfection, 166-170 - - _Pragmidiothrix_, 63 - - Precipitinogen, 269 - - Precipitinoid, 270 - - Precipitins, 268-270 - anti-, 270 - - Preparation of antitoxin, 263 - of bacterial vaccines, 283, 284 - of film, 207 - - Preservation of slides, 207, 208 - - Preservative, alcohol as, 160 - in vaccine, 284 - - Pressure, hydrostatic, 79 - osmotic, 78, 149 - oxygen, 76, 77 - steam, 136 - sterilization, 136-139 - - Prevention of disease, 235, 236, 253, 255, 283 - - Preventive vaccination, colds, 241 - pneumonia, 241 - rabies, 253 - smallpox, 26, 34, 253 - vaccines, autogenous, 285 - stock, 285 - - PREVOST, 26 - - Primary infection, 234 - - Process kettle, 137 - - Pro-enzyme, 121 - - Prophylaxis, 289 - - Protamine in bacteria, 84 - - Protease, 124 - - Protective inoculation, first, 30 - - Protein in bacteria, 84 - coagulation temperature, 51 - composition of, 102 - decomposition of, 105 - differentiation of, 255 - foreign, 289 - identification of, 293 - immunity, 290 - putrefaction of, 102-109 - split products of, 291 - splitting of, 106 - structure of, 291 - synthesis of, 113 - - _Proteus vulgaris_, 67, 70, 77 - - Protoautotrophic, 115 - - Protoplasm, 41, 59 - - Prototrophic, 86 - - Protozoa, cause of disease, 30 - cell wall in, 41 - in intermediate hosts, 242 - relation to bacteria, 40 - specificity of localization, 249 - - Protozoal diseases, transmission of, 242 - - PSEUDOMONADACEAE, 65, 70 - - _Pseudomonas pyocyanea_, 62, 65, 70, 128, 265 - - Ptomaines, 103, 104 - - _Puccinia graminis_, 26, 34 - - Puerperal fever, 28, 34 - - Punctiform colonies, 223 - - Puncture cultures, 185 - - Pure culture, 171, 194-199 - - Purification of streams, 73 - of water, 150 - - Purin bases in bacteria, 84 - - Pus cocci, 73 - infectious, 26 - organisms in, 35 - - Putrefaction, 27, 31, 33 - definition of, 102 - of proteins, 102-109 - end products of, 103 - in soil, 106 - - Putrescin, 104 - - - Q - - QUARANTINE, 239 - disinfection, 170 - - Quicklime as a disinfectant, 155, 158 - - Quinsy, 245 - - - R - - RABBITS, 227 - - Rabies, bacteriological examination in, 229 - Pasteur treatment of, 253 - path of elimination in, 248 - transmission of, 239 - specificity of localization in, 249 - - Raebiger's method of staining, 210 - - Radiations, 79 - - Radium, 79 - - Rancidity of butter, 101 - - Rashes, serum, 289 - urticarial, 292 - - Rate of division, 43, 91 - of movement, 45 - - Rats, 227, 241 - - RAYER, 28, 35 - - Reaction of medium, 81, 174, 175, 216 - - Reactions, biochemical, 87 - immunity, 255, 269, 279, 292, 293 - surface, 91 - - REAUMUR, 33 - - Receptors, 257, 258, 259, 261-280 - as factors in immunity, 295 - of first order, 262 - free, 259, 261, 262 - of second order, 265 - tabulation of, 294 - of third order, 273 - - Recurrent fever, 29, 35 - - Red corpuscles, 249, 278, 279 - - REDI, 19 - - Reducing actions, 112, 113 - enzymes, 125 - - Refrigeration as antiseptic, 148 - - REINKE, 80 - - Relapses, 235 - - Relationships of bacteria, 37-40 - biological, 255, 270 - - Rennet, 124 - - RENUCCI, 27, 34 - - Reproduction, 37, 63, 90 - - Resistance to disease, 241, 250 - of spores, 50 - - Respiratory function, 88 - tract, 246 - - Retarders, 143 - - Rheumatism, 245 - - _Rhizobium leguminosarum_, 65, 68, 69, 118 - - Rhizoid colonies, 222, 223 - - RHIZOPUS NIGRICANS, 226 - - RHODOBACTERIACEAE, 63 - - _Rhodococcus_, 66, 69 - - RICHET, 289 - - Ricin, 262 - - RIDEAL, 165 - - Rideal-Walker method, 165 - - RIMPAU, 281 - - RINDFLEISCH, 29, 35 - - Ringworm, 28 - - Ripening of cheese, 32 - of cream, 97 - - ROBIN, 262 - - Rock, phosphate, 115 - - Rocky Mountain spotted fever, 242 - - ROGERS, 265 - - Roentgen rays, 79 - - Room temperature, 213 - - Rooms, disinfection of, 167 - incubator, 213 - - Root tubercle bacteria, 86, 87, 108 - tubercles, 117 - - ROSENAU, 289 - - Rot, potato, 28, 34 - - Round worm, 232 - - Roup, 244 - - ROUX, 30 - - Rubbing as inoculation, 195 - - Rust, grain, 26, 34 - - RUZICKA, 42 - - - S - - SACCATE liquefaction, 222 - - Safranin, 205 - - Saliva, 248 - - Salivary glands, 248 - - Sake, 100 - - Salt-rising bread, 95 - - Saprogenic, 102 - - Saprophilic, 103 - - Saprophyte, 87, 238 - - _Sarcina_, 57, 58, 60, 66, 68, 69 - _lutea_, 77 - _ventriculi_, 83 - - _Sarcoptes scabiei_, 27, 34 - - Sauerkraut, 98 - - Scarlet fever, 246, 248, 250 - - Scavengers, bacteria as, 108 - - SCHICK, 289 - - _Schistosomum hematobium_, 28, 35 - - SCHLOeSING, 32, 35 - - SCHOeNLEIN, 27, 34 - - SCHROEDER and DUSCH, 21 - - SCHULTZE, 21, 34 - - SCHWANN, 21, 31, 34 - - Sea, bacteria in, 71, 111 - - Sealing air-tight, 20 - - Secondary infection, 234 - - Sections, staining of, 209 - - Selective media, 198, 199 - - Self-limited, 233 - - SEMMELWEISS, 28, 35 - - Sensitization, 290 - - Sensitized animal, 290 - bacteria, 254 - vaccine, 254 - - Septicemias, hemorrhagic, 246 - - Sero-bacterins, 254 - - Serum, antidiphtheritic, 263 - antitetanic, 263 - heated, 271, 277, 279 - rashes, 289 - sickness, 289, 292 - simultaneous method, 253 - therapy, 253 - - Serums, cytolytic, failure of, 275 - - Sewage disposal, 101, 116 - sulphate, reduction in, 114 - - Shape of spore, 48 - - Sickness, serum, 289, 292 - - Side-chain theory, 256, 258 - - Silkworm disease, 27, 29, 34, 35 - - Size of bacteria, 37, 40 - - Skatol, 104 - - Skin, channel of infection, 243 - diseases, 243 - glanders, 248 - lesions, 228 - pocket, 227 - - Slant cultures, 186 - - Slide, cleaning of, 207 - hanging drop, 203 - staining on, 207 - - Slope cultures, 186 - - Sludge tanks, 116 - - Small intestine, 249 - - Smallpox, 24, 26, 34, 239, 246, 248 - babies, 252 - vaccines, 253 - - SMITH, 289 - tubes, 184 - - Snake poisons, 263, 275 - venoms, 128 - - Sneezing, 248 - - Soap, 160 - medicated, 160 - - Society of American Bacteriologists, classification, 63 - descriptive chart, 217 - key, 68 - - Sodium hypochlorite, 158 - - Soil, acid, 81 - bacteria, 119 - bacteriology, 35 - enrichment, 117 - fertility, 120 - organisms, 74 - - Solid media, 172, 173 - - Solution, Gram's, 208 - stock, 205 - - Sore throat, 240, 241 - - Sound, 80 - - Sour mash, 98 - - Source of complement, 277 - - Souring, 98 - - SPALLANZANI, 20, 31, 34 - - Species determination, 59, 60 - - Specific amboceptor, 274, 278, 279 - antibody, 291 - chemical stimuli, 257, 258, 259 - disease, 27, 30, 233 - - Specificity of agglutinins, 267 - of amboceptor, 274 - of location, 249 - of opsonins, 281 - - Spermotoxin, 272 - - Spherical form, 52 - - _Spherotilus_, 63 - - _Spirillaceae_, 63, 65 - - Spirilloses, 241, 242 - - _Spirillum_, 53, 54, 55, 61, 63, 66, 68, 69 - _rubrum_, 113 - - _Spirochaeta_, 61 - _obermeieri_, 29, 35 - - Spirochetes, 53, 242 - - _Spirosoma_, 63 - - Splenic fever, 28 - - Split products of proteins, 291 - - Splitting enzymes, 124 - of fats, 101 - - Spoilage of canned goods, 51, 78 - - Spoiling of food, 91 - - Spontaneous combustion, 105, 116 - generation, 17-24, 33, 34 - outbreaks of disease, 239 - - Sporangia, 226 - - Spore, 47-51 - anthrax, 29, 35 - capsule, 48 - germination, 48 - - Spores, cause spoiling of canned goods, 51 - destroyed by boiling, 133 - first recognized, 33, 35 - light on, 75 - in pasteurization, 146 - resistance of, 50, 51 - staining of, 209 - two in bacterium, 50 - - Sprinkling filters, 116 - - Stab cultures, 185 - - Stables, disinfection of, 167 - - Stain, anilin fuchsin, 205 - gentian violet, 205 - aqueous gentian violet, 205 - Bismarck brown, 212 - carbol fuchsin, 206 - contrast, 205 - Gabbet's blue, 206 - Loeffler's blue, 206 - Neisser's, 212 - - Staining, 204-212 - acid-fast bacteria, 209 - bottles, 206 - capsules, 210 - cell forms, 212 - groupings, 212 - flagella, 210 - Gabbet's method, 209 - Gram's method, 208 - metachromatic granules, 212 - Neisser's method, 212 - Raebiger's method, 210 - reasons for, 204 - sections, 209 - spores, 209 - Welch's method, 210 - Ziehl-Neelson, 210 - - Standard antitoxin, 264 - methods, 217 - test dose, 264 - toxin, 264 - - Standardization, colorimetric method, 175 - of culture media, 174 - of disinfectants, 165 - H-ion method, 175 - of vaccines, 284 - - Staphylococcus, 57, 58 - - _Staphylococcus_, 66, 68, 69 - - STARIN, 196 - - Steam at air pressure, 134 - sterilizers, 135 - streaming, 135 - under pressure, 136 - - _Stegomyia_, 242 - - Sterile, 131 - - Sterilization, 130 - in canning, 133 - discontinuous, 133 - by filtration, 21, 152 - first experiment by boiling (moist heat), 20 - by chemicals, 21 - by dry heat (hot air), 21 - by filtration, 21 - - Sterilizers, pressure, 137 - steam, 135 - - Stimuli, chemical, 257, 258, 259 - - Stock cars, 170 - solutions, 205 - vaccines, 285 - - Stomach, 246 - - Straight needles, 192 - - Stratiform liquefaction, 222 - - Strawberry poisoning, 292 - - Streak methods of isolation, 196 - plates, 188 - - Streptobacillus, 53, 56 - - Streptococcus, 56, 60, 245 - - _Streptococcus_, 60, 62, 66, 68, 69 - - Streptospirillum, 55 - - Streptothrix, 38 - - _Streptothrix bovis_, 30, 36 - - Strict aerobe, 76 - anaerobe, 76 - parasite, 87 - - Structures, accidental, 43 - cell, 41 - essential, 41 - - Subcutaneous inoculation, 227 - - Subdural inoculation, 228 - - Substrate, 123 - - Successive existence, 103 - - Sugar broth, 176, 177 - - Sulphate reduction, 114 - - Sulphur bacteria, 63, 86, 115 - deposits, 116 - function of, 89 - in proteins, 105 - - Summary in immunity, 295 - Ehrlich's theory, 259 - - Sunning, 148 - - Surface reactions, 91, 92 - - Surgical instruments, 167 - - Susceptibility, 235 - - Swine, 227 - - Symbionts, 87, 103 - - Symbiosis, 87 - - Synthetic media, 172, 183 - - Syphilitic antigen, 277, 279 - - Syphilis, 233, 245, 248, 249 - Wassermann test, 277, 279 - - - T - - TABULATION of antigens and antibodies, 294 - - _Taenia solium_, 28, 35 - - Tapeworm, 28, 35, 232 - - Taxes, 203 - - Temperature conditions, 74 - effect on growth, 213 - factor in immunity, 251 - room, 213 - - Test, complement deviation, 277 - fixation, 276, 279 - dose, 264 - for enzymes, 123 - Gruber-Widal, 268 - mallein, 292 - negative, 278 - positive, 278 - for toxins, 127 - tuberculin, 292 - Wassermann, 277, 279 - Widal, 268 - - Testicle, 249 - - Tetanus, 231, 238, 243, 249, 251, 252 - antitoxin, 252 - toxin, 126 - - Tetracoccus, 57 - - Tetrad, 57 - - Texas fever, 232, 233, 242 - - THAER, 31 - - Theories of immunity, 256 - - Theory, anaphylaxis (author's), 290-292 - cellular, 256 - chemical, 256 - contagious disease, 34 - contagium vivum, 25, 28, 33 - Ehrlich's, 256-260 - exhaustion, 256 - germ, 25 - living cause, 33 - mosquito, 25 - noxious retention, 256 - overproduction, 257, 258 - phagocytosis, 256 - side-chain, 256 - spontaneous generation, 17 - unfavorable environment, 256 - - Thermal death point, 75, 215 - - Thermophil bacteria, 75, 77 - - Thermoregulator, 213 - - Thermostat, 213 - - THIOBACTERIA, 63 - - _Thiothrix_, 63 - - Thread, 56 - - Thrombin, 124 - - Thrush, 27, 34, 244 - - Ticks, 241 - - TIEDEMANN, 26 - - Tinea, 28 - - Tissue contrast stains, 205 - - Titer, 268 - - Titration, 174 - - Tonsil, 245, 249 - - Tonsillitis, 245 - - TOUISSANT, 283 - - Toxin, diphtheria, 264 - effect of temperature, 262 - final test for, 127 - in food poisoning, 104 - molecule, 261, 262 - standard, 264 - tetanus, 264 - - Toxin-antitoxin method, 254 - - Toxins and enzymes compared, 127 - as cell constituents, 84 - production of, 126-128 - of other organisms, 127 - specific localization, 249 - true, 128 - - Toxoid, 262 - - Toxophore group, 261, 262, 273 - - Tract, alimentary, 246 - - Transmission, accidental carriers in, 241 - agency of, 232 - of contagious diseases, 232 - of disease, 26, 28, 35, 239 - of glanders, 26, 34 - of protozoal diseases, 242 - of tuberculosis, 28, 29, 34, 35, 238 - - Transverse division, 54, 56 - - TRAUBE, 271 - - _Treponema pallidum_, 245 - - Trichina, 27 - - _Trichina spiralis_, 27, 34, 35 - - Trichinosis, 28, 35 - - Trichophyton, 243 - - _Trichophyton tonsurans_, 28, 34 - - Trimethylamine, 104 - - Tropical dysentery, 29 - lands, 242 - - Tropisms, 203 - - True toxins, 128 - - Trypanosomes, 242 - - Trypanosomiases, 241, 243 - - Tubercle bacteria, 85, 209 - - Tuberculin reaction, 292, 293 - - Tuberculosis, 73, 233, 238, 245, 246, 248, 249 - due to bacteria, 30 - produced experimentally, 28, 34 - proved infectious, 29, 35 - - Tuberculous milk, 248 - - Tubes, culture, 184 - deep, 190 - fermentation, 184, 190 - Smith, 184 - Vignal, 189 - - Two spores in a bacterium, 50 - - TYNDALL, 24 - - Tyndallization, 133 - - Tyndall's box, 23, 24, 35 - - Typhoid bacilli, 73, 238 - bacillus, 45 - carriers, 239 - fever, 231, 233, 248, 265, 268 - transmission by flies, 242 - vaccine, 254 - - Typhus, 242 - - Typical cell forms, 52 - - - U - - ULTRAMICROSCOPE, 204 - - Ultramicroscopic organisms, 234 - - Ultraviolet rays, 150 - - Unfavorable environment theory, 256 - - Unit of antitoxin, 264 - of measurement, 40 - - Universal carrier, 240 - - Unorganized ferment, 126 - - Unwashable articles, 169 - - Urea, 106 - - Urease, 125 - - Urethral discharges, 248 - - Urine, 72 - - Urticarial rashes, 292 - - - V - - VACCINATION in chicken cholera, 30 - negative phase in, 287 - in pneumonia, 241 - in smallpox, 26, 34, 253 - - Vaccine, 253 - age of, 285 - anthrax, 254 - antigens for, 285 - autogenous, 285 - black-leg, 254 - derivation of, 253 - mixed, 285 - polyvalent, 285 - preservative in, 284 - sensitized, 254 - smallpox, 253 - - Vaccines, bacterial, 283 - in colds, 241 - dosage of, 286 - in epidemics, 241 - failure of, 285-286 - in infections, 286 - preparation of, 283 - standardization of, 284 - stock, 284 - theory of, 286 - use of, 283 - - Vacuoles, 42, 43, 44, 59 - - Vaginal discharges, 248 - - VARO, 25 - - VAUGHAN, 291 - - Vaughan and Novy's mass cultures, 188 - - Vegetable toxins, 127, 128 - - Vegetables, forcing of, 117 - - Vehicles, disinfection of, 169 - - Venoms, antisnake, 275 - - VIBORG, 26, 34 - - Vibration, mechanical, 80 - - Vibrio, 33, 35, 53, 65, 68, 69 - _cholerae_, 66, 73 - - Vignal tubes, 189 - - VILLEMIN, 29, 35 - - Villous growth, 219, 221 - - Vinegar, 99, 114 - - Virulence, 235 - - Virus, 234 - - Vultures, 241 - - - W - - WALKER, 165 - - Wall, cell, 41 - composition of, 82, 83 - - WARDEN, 260 - - Washable articles, disinfection of, 169 - - Washing leukocytes, 281 - - Wassermann test, 277 - - Water, bacteria in, 73 - filtration of, 153 - purification of, 77, 150 - sterilization of, 157 - - WEBB, 253 - - WEIGERT, 17, 30, 36, 42, 257, 258 - - Welch's method of staining, 210 - - Whooping cough, 246, 250 - - WIDAL, 265 - test, 268 - - Will o' the wisp, 105 - - Wine, pasteurization of, 141 - - WINOGRADSKY, 32, 63, 86 - - Wire baskets, 184 - nichrome, 193 - - WOLLSTEIN, 26, 34 - - WORONIN, 30, 36 - - Wound infections, 17, 25, 26, 27, 30, 34, 36, 233, 234, 240, 243, 248 - - WRIGHT, 280 - - - X - - X-RAYS, 79 - - _Xylinum, acetobacter_, 83 - - - Y - - YEAST, fermentation, 31, 34, 99, 100, 114 - relation to bacteria, 37 - reproduction of, 37, 39 - - Yellow fever, 242 - - - Z - - ZANZ, 18 - - ZENKER, 27, 28, 35 - - ZETTNOW, 43 - - ZIEMANN, 43 - - Ziehl-Neelson method of staining, 210 - - Ziehl's solution, 206 - - Zooegloea, 44 - - Zooetoxins, 128 - - Zymase, 125 - - Zymogens, 121, 125 - - Zymophore group, 273 - - - - -Transcriber's Notes. - -Punctuation has been standardised and simple typographical errors have -been repaired. Hyphenation, quotation mark usage, and obsolete/variant -spelling (including variant spellings of proper nouns) have been -preserved as printed. - -In the original book, the page numbering goes xiii, blank, unnumbered, -18. This is a printer's error: no pages are missing. - -The descriptive chart insert has been moved from between pages 216 and -217 to the end of the book. - -The following changes have also been made: - - Page 26: 'this scourge which had devastated' - for 'this scourge which had devasted' - - Page 30: 'to be the cause of a disease in cabbage,' - [added comma] - - Page 32: 'alcoholic, lactic and butyric' - for 'alcoholic, lactic and butryic' - - Page 32: 'however, workers busied themselves' - for 'however, workers, busied themselves' [deleted extra comma] - - Page 56: 'Fig. 43.--Streptobacillus' - for 'Fig. 43.--Steptobacillus' - - Page 57: 'from a genus of algae' - for 'from a genus of algae' - - Page 58: 'staphylococcus--irregular' - for 'staphylococcus--irrgular' - - Page 59: 'so that it is impossible' - for 'so that is is impossible' - - Page 62: 'Illustrates the genus Spirochaeta' - for 'Illustrates the genus Spirochaeta' - - Page 63: 'since it is without a sheath' - for 'since it is without a a sheath' - - Page 64: 'Corynebacterium diphtheriae' - for 'Corynebacterium diphtheriae' - - Page 67: 'Prazmowski, 1880; anaerobic' - for 'Prazmowski, 1880; anaerobic' - - Page 70: 'growth processes involving oxidation' - for 'growth processes involving oxidadation' - - Page 70: 'EE--Anaerobes, rods swollen at sporulation' - for 'EE--Anaerobes, rods swollen at sporulation' - - Page 73: 'percentage of water is permissible' - for 'percentage of water is permissable' - - Page 95: 'Material taken from the bottom' - for 'Material taken from the botton' - - Page 102: 'large-moleculed and not diffusible' - for 'large-moleculed and not diffusable' - - Page 104: 'various kinds of "meat poisoning,"' - for 'various kinds of "meat posisoning,"' - - Page 106: 'formed under anaerobic conditions' - for 'formed under anaerobic conditions' - - Page 110: 'volatile fatty acids, ethereal' - for 'volatile fatty acids, etheral' - - Page 127: 'but in much larger doses' - for 'but in much large doses' - - Page 131: '"antiseptic" may become a disinfectant' - for '"antiseptic" may become a disfectant' - - Page 141: 'quarantine station barge' - for 'quaratine station barge' - - Page 147: 'A continuous milk pasteurizer.' - for 'A continuous milk pastuerizer.' - - Page 163: 'especially when a large amount of material' - for 'expecially when a large amount of material' - - Page 179: 'these must be sterilized' - for 'these must be steriliized' - - Page 191: 'Deep tubes showing anaerobic' - for 'Deep tubes showing anaerobic' - - Page 193: 'less than one-twentieth of platinum' - for 'less than one-twentieth of platimum' - - Page 207: 'grease-free cloth, handkerchief' - for 'grease-free cloth, handerchief' - - Page 210: 'Stain with Loeffler's blue' - for 'Stain with Loeffller's blue' - - Page 211: 'slide to cause precipitates' - for 'slide to cause preciptates' - - Page 213: 'grows at body temperature (37 deg.)' - [added closing parenthesis] - - Page 217: 'working on a revision' - for 'working on a revission' - - Page 220: 'inoculation for facultative anaerobes' - for 'inoculation for facultative anerobes' - - Page 231: 'the unicellular microoerganisms' - for 'the unicellular micro-organisms' [split across line] - - Page 232: 'unicellular pathogenic microoerganisms' - for 'unicellular pathogenic micro-organisms' [split across line] - - Page 233: 'the fact of self-limitation' - for 'the fact of self-limitaion' - - Page 242: 'the cattle tick (_Margaropus annulatus_).' - for 'the cattle tick (_Margaropus annulatus_.)' - - Page 244: 'B. Mucosae directly continuous' - for 'f. Mucosae directly continuous' - - Page 245: 'localized infection as in micrococcal, streptococcal' - for 'localized infection as in micrococcal, strepococcal' - - Page 248: 'ELIMINATION OF PATHOGENIC MICROOeRGANISMS.' - for 'ELIMINATION OF PATHOGENIC MICRO-ORGANISMS.' [split across line] - - Page 254: 'sometimes added to attenuate' - for 'sometimes added to attentuate' - - Page 256: 'Metchnikoff has since elaborated' - for 'Metchinkoff has since elaborated' - - Page 266: 'This is analogous to what' - for 'This is analagous to what' - - Page 280: 'other names, but ascribed' - for 'other names, but asscribed' - - Chart: '(10 minutes' exposure in nutrient broth when this is adapted - to growth of organism)' - for '(10) minutes' exposure in nutrient broth when this is adapted - to growth of organism)' - - Page 299: 'Allergic, 290' - [index entry was printed twice] - - Page 308: 'Foreign body pneumonia' - for 'Foreignbody pneumonia' - - Page 309: 'oxidation of' - for 'ox dation of' - - Page 311: 'Microspira' - for 'Miscospira' - - Page 311: 'Microsporon' - for 'Miscrosporon' - - Page 314: 'Plasmodiophora brassicae' - for 'Plasmodiophora bassicae' - - Page 317: 'Starin, 196' - [index entry was printed between Standardization and Staphylococcus] - - Page 318: 'Thermostat' - for 'Thermostadt' - - - - - - -End of the Project Gutenberg EBook of The Fundamentals of Bacteriology, by -Charles Bradfield Morrey - -*** END OF THIS PROJECT GUTENBERG EBOOK THE FUNDAMENTALS OF BACTERIOLOGY *** - -***** This file should be named 43227.txt or 43227.zip ***** -This and all associated files of various formats will be found in: - http://www.gutenberg.org/4/3/2/2/43227/ - -Produced by Jennifer Linklater, Jason Isbell and the Online -Distributed Proofreading Team at http://www.pgdp.net - - -Updated editions will replace the previous one--the old editions -will be renamed. - -Creating the works from public domain print editions means that no -one owns a United States copyright in these works, so the Foundation -(and you!) can copy and distribute it in the United States without -permission and without paying copyright royalties. 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