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+The Project Gutenberg eBook of The Elements of Bacteriological Technique,
+by John William Henry Eyre
+
+
+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 Elements of Bacteriological Technique
+       A Laboratory Guide for Medical, Dental, and Technical Students. Second Edition Rewritten and Enlarged.
+
+
+Author: John William Henry Eyre
+
+
+
+Release Date: January 5, 2009  [eBook #27713]
+
+Language: English
+
+Character set encoding: ISO-646-US (US-ASCII)
+
+
+***START OF THE PROJECT GUTENBERG EBOOK THE ELEMENTS OF BACTERIOLOGICAL
+TECHNIQUE***
+
+
+E-text prepared by Suzanne Lybarger, Brian Janes, Josephine Paolucci, and
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+Transcriber's note:
+
+      Text enclosed by tilde marks was in bold face in the original
+      (~bold~).
+
+      Text enclosed by underscore marks is in italics (_italics_).
+      The italic designation for single italized letters (such as
+      variables in equations) and "foreign" abbreviations has been
+      omitted for ease of reading.
+
+      In numbers, equations, and chemical formulas, an underscore
+      indicates that the following term enclosed within curly
+      brackets is a subscript. Examples: CO_{2}, H_{2}SO_{4}.
+      A carat character indicates that the following term enclosed
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+      is 11.1 to the third power.
+
+      Minor typographical errors have been corrected.
+
+
+
+
+
+THE ELEMENTS OF BACTERIOLOGICAL TECHNIQUE
+
+A Laboratory Guide for Medical, Dental, and Technical Students
+
+by
+
+J. W. H. EYRE, M.D., M.S., F.R.S. (EDIN.)
+
+Director of the Bacteriological Department of Guy's Hospital, London,
+and Lecturer on Bacteriology in the Medical and Dental Schools; formerly
+Lecturer on Bacteriology at Charing Cross Hospital Medical School, and
+Bacteriologist to Charing Cross Hospital; sometime Hunterian Professor,
+Royal College of Surgeons, England
+
+Second Edition Rewritten and Enlarged
+
+
+
+
+
+
+
+Philadelphia and London
+W. B. Saunders Company
+1913
+
+Copyright, 1902, by W. B. Saunders and Company Revised, entirely
+reset, reprinted, and recopyrighted July, 1913
+
+Copyright, 1913, by W. B. Saunders Company
+
+Registered at Stationers' Hall, London, England
+
+Printed in America
+Press of
+W. B. Saunders Company
+Philadelphia
+
+
+
+
+TO THE MEMORY OF
+
+JOHN WICHENFORD WASHBOURN, C.M.G., M.D., F.R.C.P.
+
+Physician to Guy's Hospital and Lecturer on Bacteriology in the
+Medical School, and Physician to the London Fever Hospital
+
+MY TEACHER, FRIEND, AND CO-WORKER
+
+
+
+
+PREFACE TO THE SECOND EDITION
+
+
+Bacteriology is essentially a practical study, and even the elements of
+its technique can only be taught by personal instruction in the
+laboratory. This is a self-evident proposition that needs no emphasis,
+yet I venture to believe that the former collection of tried and proved
+methods has already been of some utility, not only to the student in the
+absence of his teacher, but also to isolated workers in laboratories far
+removed from centres of instruction, reminding them of forgotten details
+in methods already acquired. If this assumption is based on fact no
+further apology is needed for the present revised edition in which the
+changes are chiefly in the nature of additions--rendered necessary by
+the introduction of new methods during recent years.
+
+I take this opportunity of expressing my deep sense of obligation to my
+confrere in the Physiological Department of our medical school--Mr. J.
+H. Ryffel, B. C., B. Sc.--who has revised those pages dealing with the
+analysis of the metabolic products of bacterial life; to successive
+colleagues in the Bacteriological Department of Guy's Hospital, for
+their ready co-operation in working out or in testing new methods; and
+finally to my Chief Laboratory Assistant, Mr. J. C. Turner whose
+assistance and experience have been of the utmost value to me in the
+preparation of this volume. I have also to thank Mrs. Constant Ponder
+for many of the new line drawings and for redrawing a number of the
+original cuts.
+
+    JOHN W. H. EYRE.
+
+    GUY'S HOSPITAL, S. E.
+    _July, 1913._
+
+
+
+
+PREFACE TO THE FIRST EDITION
+
+
+In the following pages I have endeavoured to arrange briefly and
+concisely the various methods at present in use for the study of
+bacteria, and the elucidation of such points in their life-histories as
+are debatable or still undetermined.
+
+Of these methods, some are new, others are not; but all are reliable,
+only such having been included as are capable of giving satisfactory
+results even in the hands of beginners. In fact, the bulk of the matter
+is simply an elaboration of the typewritten notes distributed to some of
+my laboratory classes in practical and applied bacteriology;
+consequently an attempt has been made to present the elements of
+bacteriological technique in their logical sequence.
+
+I make no apology for the space devoted to illustrations, nearly all of
+which have been prepared especially for this volume; for a picture, if
+good, possesses a higher educational value and conveys a more accurate
+impression than a page of print; and even sketches of apparatus serve a
+distinct purpose in suggesting to the student those alterations and
+modifications which may be rendered necessary or advisable by the
+character of his laboratory equipment.
+
+The excellent and appropriate terminology introduced by Chester in his
+recent work on "Determinative Bacteriology" I have adopted in its
+entirety, for I consider it only needs to be used to convince one of its
+extreme utility, whilst its inclusion in an elementary manual is
+calculated to induce in the student habits of accurate observation and
+concise description.
+
+With the exception of Section XVII--"Outlines for the Study of
+Pathogenic Bacteria"--introduced with the idea of completing the volume
+from the point of view of the medical and dental student, the work has
+been arranged to allow of its use as a laboratory guide by the technical
+student generally, whether of brewing, dairying, or agriculture.
+
+So alive am I to its many inperfections that it appears almost
+superfluous to state that the book is in no sense intended as a rival to
+the many and excellent manuals of bacteriology at present in use, but
+aims only at supplementing the usually scanty details of technique, and
+at instructing the student how to fit up and adapt apparatus for his
+daily work, and how to carry out thoroughly and systematically the
+various bacterioscopical analyses that are daily demanded of the
+bacteriologist by the hygienist.
+
+Finally, it is with much pleasure that I acknowledge the valuable
+assistance received from my late assistant, Mr. J. B. Gall, A. I. C., in
+the preparation of the section dealing with the chemical products of
+bacterial life, and which has been based upon the work of Lehmann.
+
+    JOHN W. H. EYRE.
+
+    GUY'S HOSPITAL, S. E.
+
+
+
+
+CONTENTS
+
+
+                                                                 PAGE
+
+I. LABORATORY REGULATIONS                                           1
+
+
+II. GLASS APPARATUS IN COMMON USE                                   3
+
+    The Selection, Preparation, and Care of
+    Glassware, 8--Cleaning of Glass
+    Apparatus, 18--Plugging Test-tubes and
+    Flasks, 24.
+
+
+III. METHODS OF STERILISATION                                      26
+
+    Sterilising Agents, 26--Methods of
+    Application, 27--Electric Signal Timing
+    Clock, 38.
+
+
+IV. THE MICROSCOPE                                                 49
+
+    Essentials, 49--Accessories, 57--Methods
+    of Micrometry, 61.
+
+
+V. MICROSCOPICAL EXAMINATION OF BACTERIA AND OTHER
+MICRO-FUNGI                                                        69
+
+    Apparatus and Reagents used in Ordinary
+    Microscopical Examination, 69--Methods of
+    Examination, 74.
+
+
+VI. STAINING METHODS                                               90
+
+    Bacteria Stains, 90--Contrast Stains,
+    93--Tissue Stains, 95--Blood Stains,
+    97--Methods of Demonstrating Structure of
+    Bacteria, 99--Differential Methods of
+    Staining, 108.
+
+
+VII. METHODS OF DEMONSTRATING BACTERIA IN TISSUES                 114
+
+    Freezing Method, 115--Paraffin Method,
+    117--Special Staining Methods for
+    Sections, 121.
+
+
+VIII. CLASSIFICATION OF FUNGI                                     126
+
+    Morphology of the Hyphomycetes,
+    126--Morphology of the Blastomycetes,
+    129.
+
+
+IX. SCHIZOMYCETES                                                 131
+
+    Anatomy, 134--Physiology,
+    136--Biochemistry, 144.
+
+
+X. NUTRIENT MEDIA                                                 146
+
+    Meat Extract, 148--Standardisation of
+    Media, 154--The Filtration of Media,
+    156--Storing Media in Bulk, 159--Tubing
+    Nutrient Media, 160.
+
+
+XI. ORDINARY OR STOCK CULTURE MEDIA                               163
+
+
+XII. SPECIAL MEDIA                                                182
+
+
+XIII. INCUBATORS                                                  216
+
+
+XIV. METHODS OF CULTIVATION                                       221
+
+    Aerobic, 222--Anaerobic, 236.
+
+
+XV. METHODS OF ISOLATION                                          248
+
+
+XVI. METHODS OF IDENTIFICATION AND STUDY                          259
+
+    Scheme of Study, 259--Macroscopical
+    Examination of Cultivations,
+    261--Microscopical Methods,
+    272--Biochemical Methods, 276--Physical
+    Methods, 295--Inoculation Methods,
+    315--Immunisation, 321--Active
+    Immunisation, 322--The Preparation of
+    Haemolytic Serum, 327--The Titration of
+    Haemolytic Serum, 328--Storage of
+    Haemolysin, 331.
+
+
+XVII. EXPERIMENTAL INOCULATION OF ANIMALS                         332
+
+    Selection and Care of Animals,
+    335--Methods of Inoculation, 352.
+
+
+XVIII. THE STUDY OF EXPERIMENTAL INFECTIONS DURING LIFE           370
+
+    General Observations, 371--Blood
+    Examinations, 373--Serological
+    Investigations, 378--Agglutinin,
+    381--Opsonin, 387--Immune Body, 393.
+
+
+XIX. POST-MORTEM EXAMINATION OF EXPERIMENTAL ANIMALS              396
+
+
+XX. THE STUDY OF THE PATHOGENIC BACTERIA                          408
+
+
+XXI. BACTERIOLOGICAL ANALYSES                                     415
+
+    Bacteriological Examination of Water,
+    416--Examination of Milk, 441--Ice Cream,
+    457--Examination of Cream and Butter,
+    457--Examination of Unsound Meats,
+    460--Examination of Oysters and Other
+    Shellfish, 463--Examination of Sewage and
+    Sewage Effluents, 466--Examination of
+    Air, 468--Examination of Soil,
+    470--Testing Filters, 478--Testing of
+    Disinfectants, 480.
+
+
+APPENDIX                                                          492
+
+
+INDEX                                                             505
+
+[Illustration]
+
+
+
+
+BACTERIOLOGICAL TECHNIQUE.
+
+
+
+
+I. LABORATORY REGULATIONS.
+
+
+The following regulations are laid down for observance in the
+Bacteriological Laboratories under the direction of the author. Similar
+regulations should be enforced in all laboratories where pathogenic
+bacteria are studied.
+
+     _Guy's Hospital._
+
+
+     ~BACTERIOLOGICAL DEPARTMENT.~
+
+     HANDLING OF INFECTIVE MATERIALS.
+
+     The following Regulations have been drawn up in the interest
+     of those working in the Laboratory as well as the public at
+     large, and will be strictly enforced.
+
+     Their object is to avoid the dangers of infection which may
+     arise from neglect of necessary precautions or from
+     carelessness.
+
+     Everyone must note that by neglecting the general rules laid
+     down he not only runs grave risk himself, but is a danger to
+     others.
+
+     REGULATIONS.
+
+     1. Each worker must wear a gown or overall, provided at his
+     own expense, which must be kept in the Laboratory.
+
+     2. The hands must be disinfected with lysol 2 per cent.
+     solution, carbolic acid 5 per cent. solution, or corrosive
+     sublimate 1 per mille solution, after dealing with
+     infectious material, and ~before using towels~.
+
+     3. On no account must Laboratory towels or dusters be used
+     for wiping up infectious material, and if such towels or
+     dusters do become soiled, they must be immediately
+     sterilised by boiling.
+
+     4. Special pails containing disinfectant are provided to
+     receive any waste material, and nothing must be thrown on
+     the floor.
+
+     5. All instruments must be flamed, boiled, or otherwise
+     disinfected immediately after use.
+
+     6. Labels must be moistened with water, and not by the
+     mouth.
+
+     7. All disused cover-glasses, slides, and pipettes after use
+     in handling infectious material, etc., must be placed in 2
+     per cent. lysol solution. A vessel is supplied on each bench
+     for this purpose.
+
+     8. All plate and tube cultures of pathogenic organisms when
+     done with, must be placed for immediate disinfection in the
+     boxes provided for the purpose.
+
+     9. No fluids are to be discharged into sinks or drains
+     unless previously disinfected.
+
+     10. Animals are to be dissected only after being nailed out
+     on the wooden boards, and their skin thoroughly washed with
+     disinfectant solution.
+
+     11. Immediately after the post-mortem examination is
+     completed each cadaver must be placed in the zinc
+     animal-box--_without removing the carcase from the
+     post-mortem board_--and the cover of the box replaced, ready
+     for carriage to the destructor.
+
+     12. Dead animals, when done with, are cremated in the
+     destructor, and the laboratory attendant must be notified
+     when the bodies are ready for cremation.
+
+     13. None of the workers in the laboratory are allowed to
+     enter the animal houses unless accompanied by the special
+     attendant in charge, who must scrupulously observe the same
+     directions regarding personal disinfection as the workers in
+     the laboratories.
+
+     14. No cultures are to be taken out of the laboratory
+     without the permission of the head of the Department.
+
+     15. All accidents, such as spilling infected material,
+     cutting or pricking the fingers, must be at once reported to
+     the bacteriologist in charge.
+
+
+
+
+II. GLASS APPARATUS IN COMMON USE.
+
+
+The equipment of the bacteriological laboratory, so far as the glass
+apparatus is concerned, differs but little from that of a chemical
+laboratory, and the cleanliness of the apparatus is equally important.
+The glassware comprised in the following list, in addition to being
+clean, must be stored in a sterile or germ-free condition.
+
+~Test-tubes.~--It is convenient to keep several sizes of test-tubes in
+stock, to meet special requirements, viz.:
+
+1. ~18 x 1.5~ cm., to contain media for ordinary tube cultivations.
+
+2. ~18 x 1.3~ cm., to contain media used for pouring plate cultivations,
+and also for holding sterile "swabs."
+
+3. ~18 x 2~ cm., to contain wedges of potato, beetroot, or other vegetable
+media.
+
+4. ~13 x 1.5~ cm., to contain inspissated blood-serum.
+
+The tubes should be made from the best German potash glass,
+"blue-lined," stout and heavy, with the edge of the mouth of the tube
+_slightly_ turned over, but not to such an extent as to form a definite
+rim. (Cost about $1.50, or 6 shillings per gross.) Such tubes are
+expensive it is true, but they are sufficiently stout to resist rough
+handling, do not usually break if accidentally allowed to drop (a point
+of some moment when dealing with cultures of pathogenic bacteria), can
+be cleaned, sterilised, and used over and over again, and by their
+length of life fully justify their initial expense.
+
+A point be noted is that the manufacturers rarely turn out such tubes as
+these absolutely uniform in calibre, and a batch of 18 by 1.5 cm. tubes
+usually contains such extreme sizes as 18 by 2 cm. and 18 by 1.3 cm.
+Consequently, if a set of standard tubes is kept for comparison or
+callipers are used each new supply of so-called 18 by 1.5 cm. tubes may
+be easily sorted out into these three sizes, and so simplify ordering.
+
+5. ~5 x 0.7~ cm., for use in the inverted position inside the tubes
+containing carbohydrate media, as gas-collecting tubes.
+
+These tubes, "unrimmed," may be of common thin glass as less than two
+per cent. are fit for use a second time.
+
+[Illustration: FIG. 1.--Bohemian flask.]
+
+[Illustration: FIG. 2.--Pear-shaped flask.]
+
+[Illustration: FIG. 3.--Erlenmeyer flask (narrow neck).]
+
+~Bohemian Flasks~ (Fig. 1).--These are the ordinary flasks of the chemical
+laboratory. A good variety, ranging in capacity from 250 to 3000 c.c.,
+should be kept on hand. A modified form, known as the "pear-shaped"
+(Fig. 2), is preferable for the smaller sizes--i. e., 250 and 500 c.c.
+
+~Erlenmeyer's Flasks~ (Fig. 3).--Erlenmeyer's flasks of 75, 100, and 250
+c.c. capacity are extremely useful. For use as culture flasks care
+should be taken to select only such as have a narrow neck of about 2 cm.
+in length.
+
+~Kolle's Culture Flasks~ (Fig. 4).--These thin, flat flasks (to contain
+agar or gelatine, which is allowed to solidify in a layer on one side)
+are extremely useful on account of the large nutrient surface available
+for growth. A surface cultivation in one of these will yield as much
+growth as ten or twelve "oblique" tube cultures. The wide mouth,
+however, is a disadvantage, and for many purposes thin, flat culture
+bottles known as ~Roux's bottles~ (Fig. 5) are to be preferred.
+
+[Illustration: FIG. 4.--Kolle's culture flask.]
+
+[Illustration: FIG. 5.--Roux's culture bottle.]
+
+[Illustration: FIG. 6.--Guy's culture bottle.]
+
+[Illustration: FIG. 7.--Filter flask.]
+
+An even more convenient pattern is that used in the author's laboratory
+(Fig. 6), as owing to the greater depth of medium which it is possible
+to obtain in these flasks an exceedingly luxuriant growth is possible;
+the narrow neck reduces the chance of accidental contamination to a
+minimum and the general shape permits the flasks to be stacked one upon
+the other.
+
+~Filter Flasks or Kitasato's Serum Flasks~ (Fig. 7).--Various sizes, from
+250 to 2000 c.c. capacity. These must be of stout glass, to resist the
+pressure to which they are subjected, but at the same time must be
+thoroughly well annealed, in order to withstand the temperature
+necessary for sterilisation.
+
+All flasks should be either of Jena glass or the almost equally
+well-known Resistance or R glass, the extra initial expense being
+justified by the comparative immunity of the glass from breakage.
+
+~Petri's Dishes or "Plates"~ (Fig. 8, a).--These have now completely
+replaced the rectangular sheets of glass introduced by Koch for the
+plate method of cultivation. Each "plate" consists of a pair of circular
+discs of glass with sharply upturned edges, thus forming shallow dishes,
+one of slightly greater diameter than the other, and so, when inverted,
+forming a cover or cap for the smaller. Plates having an outside
+diameter of 10 cm. and a height of 1.5 cm. are the most generally
+useful. A batch of eighteen such plates is sterilised and stored in a
+cylindrical copper box (30 cm. high by 12 cm. diameter) provided with a
+"pull-off" lid. Inside each box is a copper stirrup with a circular
+bottom, upon which the plates rest, and by means of which each can be
+raised in turn to the mouth of the box (Fig. 9) for removal.
+
+~Capsules~ (Fig. 8, b and c).--These are Petri's dishes of smaller
+diameter but greater depth than those termed plates. Two sizes will be
+found especially useful--viz., 4 cm. diameter by 2 cm. high, capacity
+about 14 c.c.; and 5 cm. diameter by 2 cm. high, capacity about 25 c.c.
+These are stored in copper cylinders of similar construction to those
+used for plates, but measuring 20 by 6 cm. and 20 by 7 cm.,
+respectively.
+
+[Illustration: FIG. 8.--Petri dish (a), and capsules (b, c).]
+
+[Illustration: FIG. 9.--Plate box with stirrup.]
+
+~Graduated Pipettes.~--Several varieties of these are required, viz.:
+
+1. Pipettes of 1 c.c. capacity graduated in 0.1 c.c.
+
+2. Pipettes of 1 c.c. capacity graduated in 0.01 c.c. (Fig. 10, a).
+
+3. Pipettes of 10 c.c. capacity graduated in 0.1 c.c. (Fig. 10, b).
+
+These should be about 30 cm. in length (1 and 2 of fairly narrow bore),
+graduated to the extreme point, and having at least a 10 cm. length of
+clear space between the first graduation and the upper end; the open
+mouth should be plugged with cotton-wool. Each variety should be
+sterilised and stored in a separate cylindrical copper case some 36 by 6
+cm., with "pull-off" lid, upon which is stamped, in plain figures, the
+capacity of the contained pipettes.
+
+[Illustration: FIG. 10.--Measuring pipettes, a and b.]
+
+The laboratory should also be provided with a complete set of "Standard"
+graduated pipettes, each pipette in the set being stamped and
+authenticated by a certificate from one of the recognised Physical
+Measurement Laboratories, such as Charlottenburg. These instruments are
+expensive and should be reserved solely for standardising the pipettes
+in ordinary use, and for calibrating small pipettes manufactured in the
+laboratory. Such a set should comprise, at least, pipettes delivering 10
+c.c., 5 c.c., 2.5 c.c., 2 c.c., 1 c.c., 0.5 c.c., 0.25 c.c., 0.2 c.c.,
+0.1 c.c., 0.05 c.c., and 0.01 c.c., respectively.
+
+In the immediately following sections are described small pieces of
+glass apparatus which should be prepared in the laboratory from glass
+tubing of various sizes. In their preparation three articles are
+essential; first a three-square hard-steel file or preferably a
+glass-worker's knife of hard Thuringian steel for cutting glass tubes
+etc.; next a blowpipe flame, for although much can be done with the
+ordinary Bunsen burner, a blowpipe flame makes for rapid work; and
+lastly a bat's-wing burner.
+
+[Illustration: FIG. 11.--Glass-cutting knife. a. handle. b. double
+edged blade. c. shaft. d. locking nut. e. spanner for nut.]
+
+1. The glass-cutting knife. This article is sold in two forms, a bench
+knife (Fig. 11) and a pocket knife. The former is provided with a blade
+some 8 cm. in length and having two cutting edges. The cutting edge when
+examined in a strong light is seen to be composed of small closely set
+teeth, similar to those in a saw. The knife should be kept sharp by
+frequent stroppings on a sandstone hone. The pocket form, about 6-cm.
+long over all, consists of a small spring blade with one cutting edge
+mounted in scales like an ordinary pocket knife.
+
+2. For real convenience of work the blowpipe should be mounted on a
+special table connected up with cylindrical bellows operated by a pedal.
+That figured (Fig. 12) is made by mounting a teak top 60 cm. square upon
+the uprights of an enclosed double-action concertina bellows (Enfer's)
+and provided with a Fletcher's Universal gas blowpipe.
+
+3. An ordinary bat's-wing gas-burner mounted at the far corner of the
+table top is invaluable in the preparation of tubular apparatus with
+sharp curves, and for coating newly-made glass apparatus with a layer of
+soot to prevent too rapid cooling, and its usually associated
+result--cracking.
+
+[Illustration: FIG. 12.--Glass blower's table with Enfer's foot
+bellows.]
+
+6. ~Sedimentation tubes 5x0.5~ cm., for sedimentation reactions, etc., and
+for containing small quantities of fluid to be centrifugalised in the
+haematocrit. These are made by taking 14-cm. lengths of stout glass
+tubing of the requisite diameter and heating the centre in the Bunsen or
+blowpipe flame. When the central portion is quite soft draw the ends
+quickly apart and then round off the pointed ends of the two test-tubes
+thus formed. With the glass-cutting knife cut off whatever may be
+necessary from the open ends to make the tubes the required length.
+
+A rectangular block of "plasticine" (modelling clay) into which the
+conical ends can be thrust makes a very convenient stand for these small
+tubes.
+
+~Capillary Pipettes or Pasteur's Pipettes~ (Fig. 13 a).--These little
+instruments are invaluable, and a goodly supply should be kept on hand.
+They are prepared from soft-glass tubing of various-sized calibre (the
+most generally useful size being 8 mm. diameter) in the following
+manner: Hold a 10 cm. length of glass tube by each end, and whilst
+rotating it heat the central portion in the Bunsen flame or the blowpipe
+blast-flame until the glass is red hot and soft. Now remove it from the
+flame and steadily pull the ends apart, so drawing the heated portion
+out into a roomy capillary tube; break the capillary portion at its
+centre, seal the broken ends in the flame, and round off the edges of
+the open end of each pipette. A loose plug of cotton-wool in the open
+mouth completes the capillary pipette. After a number have been
+prepared, they are sterilised and stored in batches, either in metal
+cases similar to those used for the graduated pipettes or in large-sized
+test-tubes--sealed ends downward and plugged ends toward the mouth of
+the case.
+
+[Illustration: FIG. 13.--Capillary pipettes. a, b, c.]
+
+The filling and emptying of the capillary pipette is most satisfactorily
+accomplished by slipping a small rubber teat (similar to that on a
+baby's feeding bottle but _not perforated_) on the upper end, after
+cutting or snapping off the sealed point of the capillary portion. If
+pressure is now exerted upon the elastic bulb by a finger and thumb
+whilst the capillary end is below the surface of the fluid to be taken
+up, some of the contained air will be driven out, and subsequent
+relaxation of that pressure (resulting in the formation of a partial
+vacuum) will cause the fluid to ascend the capillary tube. Subsequent
+compression of the bulb will naturally result in the complete expulsion
+of the fluid from the pipette (Fig. 14).
+
+[Illustration: FIG. 14.--Filling the capillary teat-pipette.]
+
+A modification of this pipette, in which a constriction or short length
+of capillary tube is introduced just below the plugged mouth (Fig. 13,
+b), will also be found extremely useful in the collection and storage
+of morbid exudations.
+
+A third form, where the capillary portion is about 4 or 5 cm. long and
+only forms a small fraction of the entire length of the pipette (Fig.
+13, c), will also be found useful.
+
+~"Blood" Pipettes~ (Fig 15).--Special pipettes for the collection of
+fairly large quantities of blood (as suggested by Pakes) should also be
+prepared. These are made from _soft_ glass tubing of 1 cm. bore, in a
+similar manner to the Pasteur pipettes, except that the point of the
+blowpipe flame must be used in order to obtain the sharp shoulder at
+either end of the central bulb. The terminal tubes must retain a
+diameter of at least 1 mm., in order to avoid capillary action during
+the collection of the fluid.
+
+[Illustration: FIG. 15.--Blood pipettes and hair-lip pin in a
+test-tube.]
+
+[Illustration: FIG. 16.--Blood-pipette in metal thermometer case.]
+
+For sterilisation and storage each pipette is placed inside a test-tube,
+resting on a wad of cotton-wool, and the tube plugged in the ordinary
+manner. As these tubes are used almost exclusively for blood work, it is
+usual to place a lance-headed hare-lip pin or a No. 9 flat Hagedorn
+needle inside the tube so that the entire outfit may be sterilised at
+one time.
+
+For the collection of small quantities of blood for agglutination
+reactions and the like, many prefer a short straight piece of narrow
+glass tubing drawn out at either extremity to almost capillary
+dimensions. Such pipettes, about 8 cm. in length over all, are most
+conveniently sterilized in ordinary metal thermometer cases (Fig. 16).
+
+~Graduated Capillary Pipettes~ (Fig. 17).--These should also be made in
+the laboratory--from manometer tubing--of simple, convenient shape, and
+graduated by the aid of "standard" pipettes (in hundredths) to contain
+such quantities as 10, 50, and 90 c. mm., and carefully marked with a
+writing diamond. These, previously sterilised in large test-tubes, will
+be found extremely useful in preparing accurate percentage solutions,
+when only minute quantities of fluid are available.
+
+[Illustration: FIG. 17.--Capillary graduated pipettes.]
+
+~Automatic ("Throttle") Pipettes.~--These ingenious pipettes, introduced
+by Wright, can easily be calibrated in the laboratory and are
+exceedingly useful for graduating small pipettes, for measuring small
+quantities of fluids, in preparing dilutions of serum for agglutination
+reactions, etc. They are usually made from the Capillary Pasteur
+pipettes (Fig. 13, a). The following description of the manufacture of
+a 5 c. mm. pipette will serve to show how the small automatic pipettes
+are calibrated.
+
+1. Select a pipette the capillary portion of which is fairly roomy in
+bore and possesses regular even walls, and remove the cotton-wool plug
+from the open end.
+
+2. Heat the capillary portion near the free extremity in the by-pass
+flame of the bunsen burner and draw it out into a very fine hair-like
+tube and break this across. This hair-like extremity will permit the
+passage of air but is too fine for metallic mercury to pass.
+
+3. From a standard graduated pipette deliver 5 c. mm. clean mercury into
+the upper wide portion of the pipette.
+
+4. Adjust a rubber teat to the pipette and by pressure on the bulb
+gradually drive the mercury in an unbroken column down the capillary
+tube until it is stopped by the filiform extremity.
+
+5. Cut off the capillary tube exactly at the upper level of the column
+of mercury, invert it and allow the mercury to run out.
+
+6. Snap off the remainder of the capillary tube from the broad upper
+portion of the pipette which is now destined to form the covering tube
+or air chamber, or what we may term the "barrel." This barrel now has
+the lower end in the form of a truncated cone, the upper end being cut
+square. Remove the teat.
+
+7. Introduce the capillary tube into this barrel with the filiform
+extremity uppermost, and the square cut end projecting about 0.5 cm.
+beyond the tapering end of the barrel.
+
+[Illustration: FIG. 18.--Throttle pipette--small capacity.]
+
+8. Drop a small pellet of sealing wax into the barrel by the side of the
+capillary tube and then warm the tube at the gas flame until the wax
+becomes softened and makes an air-tight joint between the capillary tube
+and the end of the barrel.
+
+9. Fit a rubber teat to the open end of the barrel, and so complete a
+pipette which can be depended upon to always aspirate and deliver
+exactly 5 cm. of fluid.
+
+Slight modification of this procedure is necessary in making tubes to
+measure larger volumes than say 75 c. mm. Thus to make a throttle
+pipette to measure 100 c. mm.:
+
+1. Take a short length of quill tubing and draw out one end into a roomy
+capillary stem, and again draw out the extremity into a fine hair point,
+thus forming a small Pasteur pipette with a hair-like capillary
+extremity.
+
+2. With a standard pipette fill 100 c. mm. into the neck of this
+pipette, and make a scratch with a writing diamond at the upper level
+(a) of the mercury meniscus (Fig. 19, A).
+
+[Illustration: FIG. 19.--Making throttle pipettes--large capacity]
+
+Now force the mercury down into the capillary stem as far as it will go,
+so as to leave the upper part of the tube in the region of the diamond
+scratch empty (Fig. 19, B).
+
+3. Heat the tube in the region of the diamond scratch in the blowpipe
+flame, and removing the tube from the flame draw it out so that the
+diamond scratch now occupies a position somewhere near the centre of
+this new capillary portion (Fig. 19, C).
+
+4. Heat the tube in this position in the peep flame of the Bunsen
+burner, and draw it out into a hair-like extremity. Snap off the glass
+tube, leaving about 5 mm. of hair-like extremity attached to the upper
+capillary portion (Fig. 19, D). Allow the glass to cool.
+
+5. Lift up the bulb by the long capillary stem and allow the mercury to
+return to its original position--an operation which will be facilitated
+by snapping off the hair-like extremity from the long piece of capillary
+tubing.
+
+6. Mark on the capillary stem with a grease pencil the position of the
+end of the column of mercury (Fig. 19, E.)
+
+7. Warm the capillary tubing at this spot in the peep flame of the
+Bunsen burner, and draw it out very slightly so that when cut at this
+position a pointed extremity will be obtained.
+
+8. With a glass-cutting knife cut the capillary tube through at the
+point "b," and allow the mercury to run out.
+
+9. Now apply a thick layer of sealing wax to the neck of the bulb.
+
+10. Take a piece of 5 mm. bore glass tubing and draw it out as if making
+an ordinary Pasteur pipette.
+
+11. Break the capillary portion off so as to leave a covering tube
+similar to that already used for the smaller graduated pipettes. Into
+this covering tube drop the graduated bulb and draw the capillary stem
+down through the conical extremity until further progress is stopped by
+the layer of sealing wax.
+
+12. Warm the pipette in the gas flame so as to melt the sealing wax and
+make an air-tight joint.
+
+13. Fit an india-rubber teat over the open end of the covering tube, and
+the automatic pipette is ready for use (Fig. 19, F).
+
+~Sedimentation Pipettes~ (Fig. 20).--These are prepared from 10 cm.
+lengths of narrow glass tubing by sealing one extremity, blowing a
+small bulb at the centre, and plugging the open end with cotton-wool;
+after sterilisation the open end is provided with a short piece of
+rubber tubing and a glass mouthpiece. When it is necessary to observe
+sedimentation reactions in very small quantities of fluid, these tubes
+will be found much more convenient than the 5 by 0.5 cm. test-tubes
+previously mentioned.
+
+[Illustration: FIG. 20.--Sedimentation pipette.]
+
+Pasteur pipettes fitted with india-rubber teats will also be found
+useful for sedimentation tests when dealing with minute quantities of
+serum, etc.
+
+[Illustration: FIG. 21.--Fermentation tubes.]
+
+~Fermentation Tubes~ (Fig. 21).--These are used for the collection and
+analysis of the gases liberated from the media during the growth of some
+varieties of bacteria and may be either plain (a) or graduated (b).
+A simple form (Fig. 21, c) may be made from 14 cm. lengths of soft
+glass tubing of 1.5 cm. diameter. The Bunsen flame is applied to a spot
+some 5 cm. from one end of such a piece of tubing and the tube slightly
+drawn out to form a constriction, the constricted part is bent in the
+bat's-wing flame, to an acute angle, and the open extremity of the long
+arm sealed off in the blowpipe flame. The open end of the short arm is
+rounded off and then plugged with cotton-wool, and the tube is ready for
+sterilisation.
+
+
+CLEANING OF GLASS APPARATUS.
+
+All glassware used in the bacteriological laboratory must be thoroughly
+cleaned before use, and this rule applies as forcibly to new as to old
+apparatus, although the methods employed may vary slightly.
+
+~To Clean New Test-tubes.~--
+
+1. Place the tubes in a bucket or other convenient receptacle, fill with
+water and add a handful of "Sapon" or other soap powder. See that the
+tubes are full and submerged.
+
+2. Fix the bucket over a large Bunsen flame and boil for thirty
+minutes--or boil in the autoclave for a similar period.
+
+3. Cleanse the interior of the tubes with the aid of test-tube brushes,
+and rinse thoroughly in cold water.
+
+4. Invert the tubes and allow them to drain completely.
+
+5. Dry the tubes and polish the glass inside and out with a soft cloth,
+such as selvyt.
+
+~New flasks, plates, and capsules~ must be cleaned in a similar manner.
+
+~To Clean New Graduated Pipettes.~--
+
+1. Place the pipettes in a convenient receptacle, filled with water to
+which soap powder has been added.
+
+2. Boil the water vigorously for twenty minutes over a Bunsen flame.
+
+3. Rinse the pipettes in running water and drain.
+
+4. Run distilled water through the pipettes and drain.
+
+5. Run rectified spirits through the pipette and drain as completely as
+possible.
+
+6. Place the pipettes in the hot-air oven (_vide_ page 31), close the
+door, open the ventilating slide, and run the temperature slowly up to
+about 80 deg. C. Turn off the gas and allow the oven to cool.
+
+Or 6a. Attach each pipette in turn to the rubber tube of the foot
+bellows, or blowpipe air-blast, and blow air through the pipette until
+the interior is dry.
+
+Glassware that has already been used is regarded as _infected_, and is
+treated in a slightly different manner.
+
+~Infected Test-tubes.~--
+
+1. Pack the tubes in the wire basket of the autoclave (having previously
+removed the cotton-wool plugs, caps, etc.), in the vertical position,
+and before replacing the basket see that there is a sufficiency of water
+in the bottom of the boiler. Now attach a piece of rubber tubing to the
+nearest water tap, and by means of this fill each tube with water.
+
+2. Disinfect completely by exposing the tubes, etc., to a temperature of
+120 deg. C. for twenty minutes (_vide_ page 37).
+
+(If an autoclave is not available, the tubes must be placed in a
+digester, or even a large pan or pail with a tightly fitting cover, and
+boiled vigorously for some thirty to forty-five minutes to ensure
+disinfection.)
+
+3. Whilst still hot, empty each tube in turn and roughly clean its
+interior with a stiff test-tube brush.
+
+4. Place the tubes in a bucket or other convenient receptacle, fill with
+water and add a handful of Sapon or other soap powder. See that the
+tubes are full and submerged.
+
+5. Fix the bucket over a large Bunsen flame and boil for thirty minutes.
+
+6. Cleanse the interior of the tubes with the aid of test-tube brushes,
+and rinse thoroughly in cold water.
+
+7. Drain off the water and immerse tubes in a large jar containing water
+acidulated with 2 to 5 per cent. hydrochloric acid. Allow them to remain
+there for about fifteen minutes.
+
+8. Remove from the acid jar, drain, rinse thoroughly in running water,
+then with distilled water.
+
+9. Invert the tubes and allow them to drain completely.
+
+Dry the tubes and polish the glass inside and out with a soft cloth,
+such as selvyt.
+
+~Infected flasks, plates, and capsules~ must be treated in a similar
+manner.
+
+~Flasks~ which have been used only in the preparation of media must be
+cleaned immediately they are finished with. Fill each flask with water
+to which some soap powder and a few crystals of potassium permanganate
+have been added, and let boil over the naked flame. The interior of the
+flask can then usually be perfectly cleaned with the aid of a flask
+brush, but in some cases water acidulated with 5 per cent. nitric acid,
+or a large wad of wet cotton-wool previously rolled in silver sand, must
+be shaken around the interior of the flask, after which rinse thoroughly
+with clean water, dry, and polish.
+
+
+~Infected Pipettes.~--
+
+1. Plunge infected pipettes immediately after use into tall glass
+cylinders containing a 2 per cent. solution of lysol, and allow them to
+remain therein for some days.
+
+2. Remove from the jar and drain. Boil in water to which a little soap
+has been added, for thirty minutes.
+
+3. Rinse thoroughly in cold water.
+
+4. Immerse in 5 per cent. nitric acid for an hour or two.
+
+5. Rinse again in running water to remove all traces of acid.
+
+6. Complete the cleaning as described under "new pipettes."
+
+When dealing with graduated capillary pipettes employed for blood or
+serum work (whether new or infected), much time is consumed in the
+various steps from 5 onward, and the cleansing process can be materially
+hastened if the following device is adopted.
+
+Fit up a large-sized Kitasato's filter flask to a Sprengel's suction
+pump or a Geryk air pump (see page 43). To the side tubulure of the
+filter flask attach a 20 cm. length of rubber pressure tubing having a
+calibre sufficiently large to admit the ends of the pipettes.
+
+Next fill a small beaker with distilled water. Attach the first pipette
+to the free end of the rubber tubing, place the pipette point downward
+in the beaker of water and start the pump (Fig. 22).
+
+[Illustration: FIG. 22.--Cleaning blood pipettes.]
+
+When all the water has been aspirated through the pipette into the
+filter flask, fill the beaker with rectified spirit and when this is
+exhausted refill with ether. Detach the pipette and dry in the hot-air
+oven.
+
+~Slides and cover-slips~ (Fig. 23), when first purchased, have "greasy"
+surfaces, upon which water gathers in minute drops and effectually
+prevents the spreading of thin, even films.
+
+~Microscopical Slides.~--The slides in general use are those known as
+"three by one" slips (measuring 3 inches by 1 inch, or 76 by 26 mm.),
+and should be of good white crown glass, with ground edges.
+
+~New slides~ should be allowed to remain in alcohol acidulated with 5 per
+cent. hydrochloric acid for some hours, rinsed in running water, roughly
+drained on a towel, dried, and finally polished with a selvyt cloth.
+
+[Illustration: FIG. 23.--Slides and cover-slips, actual size.]
+
+If only a few slides are required for immediate use a good plan is to
+rub the surface with jeweler's emery paper (Hubert's 00). A piece of
+hard wood 76x26x26 mm. with a piece of this emery paper gummed tightly
+around it is an exceedingly useful article on the microscope bench.
+
+~Cover-slips.~--The most useful sizes are the 19 mm. squares for ordinary
+cover-glass film preparations, and 38 by 19 mm. rectangles for blood
+films and serial sections; both varieties must be of "No. 1" thickness,
+which varies between 0.15 and 0.22 mm., that they may be available for
+use with the high-power immersion lenses.
+
+Cover-slips should be cleaned in the following manner:
+
+1. Drop the cover-slips one by one into an enamelled iron pot or tall
+glass beaker, containing a 10 per cent. solution of chromic acid.
+
+2. Heat over a Bunsen flame and allow the acid to boil gently for twenty
+minutes.
+
+     NOTE.--A few pieces of pipe-clay or pumice may be placed in
+     the beaker to prevent the "spurting" of the chromic acid.
+
+3. Turn the cover-slips out into a flat glass dish and wash in running
+water under the tap until all trace of yellow colour has disappeared.
+During the washing keep the cover-slips in motion by imparting a
+rotatory movement to the dish.
+
+4. Wash in distilled water in a similar manner.
+
+5. Wash in rectified spirit.
+
+6. Transfer the cover-slips, by means of a pair of clean forceps,
+previously heated in the Bunsen flame to destroy any trace of grease, to
+a small beaker of absolute alcohol.
+
+Drain off the alcohol and transfer the cover-slips, by means of the
+forceps, to a wide-mouthed glass pot, containing absolute alcohol, in
+which they are to be stored, and stopper tightly.
+
+     NOTE.--After once being placed in the chromic acid, the
+     cover-slips must on no account be touched by the fingers.
+
+~Used Slides and Cover-slips.~--Used slides with the mounted cover-slip
+preparations, and cover-slips used for hanging-drop mounts, should, when
+discarded, be thrown into a pot containing a 2 per cent. solution of
+lysol.
+
+After immersion therein for a week or so, even the cover-slips mounted
+with Canada balsam can be readily detached from their slides.
+
+
+_Slides._--
+
+1. Wash the slides thoroughly in running water.
+
+2. Boil the slides in water to which "sapon" has been added, for half an
+hour.
+
+3. Rinse thoroughly in cold water.
+
+4. Dry and polish with a dry cloth.
+
+
+_Cover-slips._--
+
+1. Wash the cover-slips thoroughly in running water.
+
+2. Boil the cover-slips in 10 per cent. solution of chromic acid, as for
+new cover-slips.
+
+3. Wash thoroughly in running water.
+
+4. Pick out those cover-slips which show much adherent dirty matter, and
+rub them between thumb and forefinger under the water tap. The dirt
+usually rubs off easily, as it has become friable from contact with the
+chromic acid.
+
+5. Return all the cover-slips to the beaker, fill in _fresh_ chromic
+acid solution, and treat as new cover-slips.
+
+     NOTE.--_Test-tubes, plates, capsules_, etc., which, from
+     long use, have become scratched and hazy, or which cannot be
+     cleaned in any other way, may be dealt with by immersing
+     them in an enamelled iron bath, containing water acidulated
+     to 1 per cent. with hydrofluoric acid, for ten minutes,
+     rinsing thoroughly in water, drying, and polishing.
+
+
+PLUGGING TEST-TUBES AND FLASKS.
+
+Before sterilisation all test-tubes and flasks must be carefully plugged
+with cotton-wool, and for this purpose best absorbent cotton-wool
+(preferably that put up in cylindrical one-pound packets and interleaved
+with tissue paper--known as surgeons' wool) should be employed.
+
+1. For a test-tube or a small flask, tear a strip of cotton-wool some 10
+cm. long by 2 cm. wide from the roll.
+
+2. Turn in the ends neatly and roll the strip of wool lightly between
+the thumb and fingers of both hands to form a long cylinder.
+
+3. Double this at the centre and introduce the now rounded end into the
+open mouth of the tube or flask.
+
+4. Now, whilst supporting the wool between the thumb and fingers of the
+right hand, rotate the test-tube between those of the left, and
+gradually screw the plug of wool into its mouth for a distance of about
+2.5 cm., leaving about the same length of wool projecting.
+
+[Illustration: FIG 24..--Plugging test-tubes: a, cylinder of wool
+being rolled; b, cylinder of wool being doubled; c, cylinder of wool
+being inserted in tube.]
+
+The plug must be firm and fit the tube or flask fairly tightly,
+sufficiently tightly in fact to bear the weight of the glass plus the
+amount of medium the vessel is intended to contain, but not so tightly
+as to prevent it from being easily removed by a screwing motion when
+grasped between the fourth, or third and fourth, fingers, and the palm
+of the hand.
+
+For a large flask a similar but larger strip of wool must be taken; the
+method of making and inserting the plug is identical.
+
+
+
+
+III. METHODS OF STERILISATION.
+
+
+STERILISING AGENTS.
+
+Sterilisation--i. e., the removal or the destruction of germ life--may
+be effected by the use of various agents. As applied to the practical
+requirements of the bacteriological laboratory, many of these agents,
+such as electricity, sunlight, etc., are of little value, others are
+limited in their applications; others again are so well suited to
+particular purposes that their use is almost entirely restricted to
+such.
+
+The sterilising agents in common use are:
+
+~Chemical Reagents.~--_Disinfectants_ (for the disinfection of glass and
+metal apparatus and of morbid tissues).
+
+~Physical Agents.~ HEAT.--(a) _Dry Heat:_
+
+1. Naked flame (for the sterilisation of platinum needles, etc.).
+
+2. Muffle furnace (for the sterilisation of filter candles, and for the
+destruction of morbid tissues).
+
+3. Hot air (for the sterilisation of all glassware and of metal
+apparatus).
+
+(b) _Moist Heat:_
+
+1. Water at 56 deg. C. (for the sterilisation of certain albuminous
+fluids).
+
+2. Water at 100 deg. C. (for the sterilisation of surgical instruments,
+rubber tubing, and stoppers, etc.).
+
+3. Streaming steam at 100 deg. C. (for the sterilisation of media).
+
+4. Superheated steam at 115 deg. C. or 120 deg. C. (for the disinfection
+of contaminated articles and the destruction of old cultivations of
+bacteria).
+
+FILTRATION.--
+
+1. Cotton-wool filters (for the sterilisation of air and gases).
+
+2. Porcelain filters (for the sterilisation of various liquids).
+
+
+METHODS OF APPLICATION.
+
+~Chemical Reagents~, such as belong to the class known as antiseptics (_i.
+e._, substances which inhibit the growth of, but do not destroy,
+bacterial life), are obviously useless. Disinfectants or germicides (_i.
+e._, substances which destroy bacterial life), on the other hand, are of
+value in the disinfection of morbid material, and also of various pieces
+of apparatus, such as pipettes, pending their cleansing and complete
+sterilisation by other processes. To this class (in order of general
+utility) belong:
+
+    Lysol, 2 per cent. solution;
+    Perchloride of mercury, 0.1 per cent. solution;
+    Carbolic acid, 5 per cent. solution;
+    Absolute alcohol;
+    Ether;
+    Chloroform;
+    Camphor;
+    Thymol;
+    Toluol;
+    Volatile oils, such as oil of mustard, oil of garlic.
+
+Formaldehyde is a powerful germicide, but its penetrating vapor
+restricts its use. These disinfectants are but little used in the final
+sterilisation of apparatus, chiefly on account of the difficulty of
+effecting their complete removal, for the presence of even traces of
+these chemicals is sufficient to so inhibit or alter the growth of
+bacteria as to vitiate subsequent experiments conducted by the aid of
+apparatus sterilised in this manner.
+
+     NOTE.--Tubes, flasks, filter flasks, pipettes, glass tubing,
+     etc., may be rapidly sterilised, in case of emergency, by
+     washing, in turn, with distilled water, perchloride of
+     mercury solution, alcohol, and ether, draining, and finally
+     gently heating over a gas flame to completely drive off the
+     ether vapor. Chloroform or other volatile disinfectants may
+     be added to various fluids in order to effect the
+     destruction of contained bacteria, and when this has been
+     done, may be completely driven off from the fluid by the
+     application of gentle heat.
+
+~Dry Heat.~--The _naked flame_ of the Bunsen burner is invariably used for
+sterilising the platinum needles (which are heated to redness) and may
+be employed for sterilising the points of forceps, or other small
+instruments, cover-glasses, pipettes, etc., a very short exposure to
+this heat being sufficient.
+
+_Ether Flame._--In an emergency small instruments, needles, etc., may be
+sterilised by dipping them in ether and after removal lighting the
+adherent fluid and allowing it to burn off the surface of the
+instruments. Repeat the process twice. It may then be safely assumed
+that the apparatus so treated is sterile.
+
+[Illustration: FIG. 25.--Muffle furnace.]
+
+_Muffle Furnace_ (Fig. 25).--Although this form of heat is chiefly used
+for the destruction of the dead bodies of small infected animals, morbid
+tissues, etc., it is also employed for the sterilisation of porcelain
+filter candles (_vide_ p. 42).
+
+Filter candles are disinfected immediately after use by boiling in a
+beaker of water for some fifteen or twenty minutes. This treatment,
+however, leaves the dead bodies of the bacteria upon the surface and
+blocking the interstices of the filter.
+
+To destroy the organic matter and prepare the filter candle for further
+use proceed as follows:
+
+1. Roll each bougie up in a piece of asbestos cloth, secure the ends of
+the cloth with a few turns of copper wire, and place inside the muffle
+(a small muffle 76x88x163 mm. will hold perhaps four small filter
+candles).
+
+2. Light the gas and raise the contents of the muffle to a white heat;
+maintain this temperature for five minutes.
+
+3. Extinguish the gas, and when the muffle has become quite cold remove
+the filter candles, and store them (without removing the asbestos
+wrappings) in sterile metal boxes.
+
+     NOTE.--The too rapid cooling of the candles, such as takes
+     place if they are removed from the muffle before it has
+     cooled down to the room temperature, may give rise to
+     microscopic cracks and flaws which will effectually destroy
+     their efficiency.
+
+_Hot Air._--Hot air at 150 deg. C. destroys all bacteria, spores, etc:, in
+about thirty minutes; a momentary exposure to a temperature of 175 deg. to
+180 deg. C. will effect the same result and offers the more convenient
+method of sterilisation. This method is only applicable to glass and
+metallic substances, and the small bulk of cotton-wool comprised in the
+test-tube plugs, etc. Large masses of fabric are not effectually
+sterilised by dry heat--short of charring--as its power of penetration
+is not great.
+
+Sterilisation by hot air is effected in the hot-air oven (Fig. 18). This
+is a rectangular, double-walled metal box, mounted on a stand and heated
+from below by a large Bunsen burner. The interior of the oven is
+provided with loose shelves upon which the articles to be sterilised are
+arranged, either singly or packed in square wire baskets or crates, kept
+specially for this purpose. One of the sides is hinged to form a door.
+The central portion of the metal bottom, on which the Bunsen flame would
+play, is cut away, and replaced by firebrick plates, which slide in
+metal grooves and are easily replaced when broken or worn out. The top
+of the oven is provided with a perforated ventilator slide and two
+tubulures, the one for the reception of a centigrade thermometer
+graduated to 200 deg. or 250 deg. C., the other for a thermo-regulator. An
+ordinary mercurial thermo-regulator may be used but it is preferable to
+employ a regulating capsule of the Hearson type (see p. 219) with a
+spring arm adjusted to the lever so that when the boiling-point of the
+capsule (e. g., 175 deg. C.) is reached the gas supply is absolutely cut
+off and the jet cannot again be lighted until the spring-arm has been
+readjusted by hand. The thermo-regulator is by no means a necessity, and
+may be replaced by a large bore thermometer with a sliding platinum
+point, connected with an electric bell, which can be easily adjusted to
+ring at any given temperature. Even if the steriliser is provided with
+the capsule regulator above described the contact thermometer should
+also be fitted.
+
+[Illustration: FIG. 26.--Hot-air oven.]
+
+
+TO USE THE HOT-AIR OVEN.--
+
+1. Place the crates of test-tubes, metal cases containing plates and
+pipettes, loose apparatus, etc., inside the oven, taking particular care
+that none of the cotton-wool plugs are in contact with the walls,
+otherwise the heat transmitted by the metal will char or even flame
+them.
+
+     To prepare a wire crate for the reception of test-tubes,
+     etc., cover the bottom with a layer of thick asbestos cloth;
+     or take some asbestos fibre, moisten it with a little water
+     and knead it into a paste; plaster the paste over the bottom
+     of the crate, working it into the meshes and smoothing the
+     surface by means of a pestle. When several crates have been
+     thus treated, place them inside the hot-air oven, close the
+     door, open the ventilating slide, light the gas, and run the
+     temperature of the interior up to about 160 deg. C. After an
+     interval of ten minutes extinguish the gas, open the oven
+     door, and allow the contents to cool. The asbestos now forms
+     a smooth, dry, spongy layer over the bottom, which will last
+     many months before needing renewal, and will considerably
+     diminish the loss of tubes from breakage.
+
+     Copper cylinders and large test-tubes intended for the
+     reception of pipettes are prepared in a similar manner, in
+     order to protect the points of these articles from injury.
+
+2. Close the oven door, and open the ventilating slide, in order that
+any moisture left in the tubes, etc., may escape; light the gas below;
+set the electric alarm to ring at 100 deg. C.
+
+3. When the temperature of the oven has reached 100 deg. C., close the
+ventilating slide; reset the alarm to ring at 175 deg. C.
+
+4. Run the temperature up to 175 deg. C.
+
+5. Extinguish the gas at once, and allow the apparatus to cool.
+
+6. When the temperature of the interior, as recorded by the thermometer,
+has fallen to 60 deg. C.--_but not before_--the door may be opened and
+the sterile articles removed and stored away.
+
+     NOTE.--Neglect of this precautionary cooling of the oven to
+     60 deg. C. will result in numerous cracked and broken tubes.
+
+On removal from the oven, the cotton-wool plugs will probably be
+slightly brown in colour.
+
+Metal instruments, such as knives, scissors, and forceps, may be
+sterilised in the hot-air oven as described above, but exposure to 175 deg.
+C. is likely to seriously affect the temper of the steel and certainly
+blunts the cutting edges. If, however, it is desired to sterilise
+surgical instruments by hot air, they should be packed in a metal box,
+or boxes, and heated to 130 deg. C. and retained at that temperature for
+about thirty minutes.
+
+~Moist Heat.~--_Water at 56 deg. C._--This temperature, if maintained for
+thirty minutes, is sufficient to destroy the vegetative forms of
+bacteria, but has practically no effect on spores. Its use is limited to
+the sterilisation of such albuminous "fluid" media as would coagulate at
+a higher temperature.
+
+METHOD.--
+
+1. Fit up a water-bath, heated by a Bunsen flame which is controlled by
+a thermo-regulator, so that the temperature of the water remains at 56
+deg. C.
+
+2. Immerse the tubes or flasks containing the albuminous fluid in the
+water-bath so that the upper level of such fluid is at least 2 cm. below
+the level of the water. (The temperature of the bath will now fall
+somewhat, but after a few minutes will again rise to 56 deg. C).
+
+3. After thirty minutes' exposure to 56 deg. C, extinguish the gas, remove
+the tubes or flasks from the bath, and subject them to the action of
+running water so that their contents are rapidly cooled.
+
+4. The vegetative forms of bacteria present in the liquid being killed,
+stand it for twenty-four hours in a cool, dark place; at the end of that
+time some at least of such spores as may be present will have germinated
+and assumed the vegetative form.
+
+5. Destroy these new vegetative forms by a similar exposure to 56 deg. C.
+on the second day, whilst others, of slower germination, may be caught on
+the third day, and so on.
+
+6. In order to ensure thorough sterilisation, repeat the process on each
+of six successive days.
+
+This method of exposing liquids to a temperature of 56 deg. C. in a
+water-bath for half an hour on each of six successive days is termed
+_fractional sterilisation_.
+
+_Water at 100 deg. C._ destroys the vegetative forms of bacteria almost
+instantaneously, and spores in from five to fifteen minutes. This method
+of sterilisation is applicable to the metal instruments, such as knives,
+forceps, etc., used in animal experiments; syringes, rubber corks,
+rubber and glass tubing, and other small apparatus, and is effected in
+what is usually spoken of as the "water steriliser" (Fig. 27).
+
+[Illustration: FIG. 27.--Water sterilizer.]
+
+This is a rectangular copper box, 26 cm. long, 18 cm. wide, and 12 cm.
+deep, mounted on legs, heated from below by a Bunsen or radial gas
+burner, and containing a movable copper wire tray, 2 cm. smaller in
+every dimension than the steriliser itself, and provided with handles.
+The top of the steriliser is hinged to form a lid.
+
+METHOD.--
+
+1. Place the instruments, etc., to be sterilised inside the copper
+basket, and replace the basket in the steriliser.
+
+2. Pour a sufficient quantity of water into the steriliser, shut down
+the lid, and light the gas below.
+
+[Illustration: FIG. 28.--Koch's steriliser.]
+
+[Illustration: FIG. 29.--Arnold's steriliser.]
+
+3. After the water has boiled and steam has been issuing from beneath
+the lid for at least ten minutes, extinguish the gas, open the lid, and
+lift out the wire basket by its handles and rest it diagonally on the
+walls of the steriliser; the contained instruments, etc., are now
+sterile and ready for use.
+
+4. After use, or when accidentally contaminated, replace the instruments
+in the basket and return that to the steriliser; completely disinfect by
+a further boiling for fifteen minutes.
+
+5. After disinfection, and whilst still hot, take out the instruments,
+dry carefully and at once, and return them to their store cases.
+
+_Streaming steam_--i. e., steam at 100 deg. C.--destroys the vegetative
+forms of bacteria in from fifteen to twenty minutes, and the sporing
+forms in from one to two hours. This method is chiefly used for the
+sterilisation of the various nutrient media intended for the cultivation
+of bacteria, and is carried out in a steam kettle of special
+construction, known as Koch's steam steriliser (Fig. 28) or in one of
+its many modifications, the most efficient of which is Arnold's (Fig.
+29).
+
+The steam steriliser in its simplest form consists of a tall tinned-iron
+or copper cylindrical vessel, divided into two unequal parts by a
+movable perforated metal diaphragm, the lower, smaller portion serving
+for a water reservoir, and the upper part for the reception of wire
+baskets containing the articles to be sterilised. The vessel is closed
+by a loose conical lid, provided with handles, and perforated at its
+apex by a tubulure; it is mounted on a tripod stand and heated from
+below by a Bunsen burner. The more elaborate steriliser is cased with
+felt or asbestos board, and provided with a water gauge, also a tap for
+emptying the water compartment.
+
+
+TO USE THE STEAM STERILISER.--
+
+1. Fill the water compartment to the level of the perforated diaphragm,
+place the lid in position, and light the Bunsen burner.
+
+2. After the water has boiled, allow sufficient time to elapse for steam
+to replace the air in the sterilising compartment, as shown by the steam
+issuing in a steady, continuous stream from the tubulure in the lid.
+
+3. Remove the lid, quickly lower the wire basket containing media tubes,
+etc., into the sterilising compartment until it rests on the diaphragm,
+and replace the lid.
+
+4. After an interval of twenty minutes in the case of fluid media, or
+thirty minutes in the case of solid media, take off the lid and remove
+the basket with its contents.
+
+5. Now, but not before, extinguish the gas.
+
+     NOTE.--After removing tubes, flasks, etc., from the steam
+     steriliser, they should be at once separated freely in order
+     to prevent moisture condensing upon the cotton-wool plugs
+     and soaking through into the interior of the tubes.
+
+This treatment will destroy any vegetative forms of bacteria; during the
+hours of cooling any spores present will germinate, and the young
+organisms will be destroyed by repeating the process twenty-four hours
+later; a third sterilisation after a similar interval makes assurance
+doubly sure.
+
+The method of sterilising by exposure to streaming steam at 100 deg. C.
+for twenty minutes on each of three consecutive days is termed
+_discontinuous_ or _intermittent sterilisation_.
+
+Exposure to steam at 100 deg. C. for a period of one or two hours, or
+_continuous sterilisation_, cannot always be depended upon and is
+therefore not to be recommended.
+
+_Superheated steam_--i. e., steam under pressure (see
+Pressure-temperature table, Appendix, page 500) in sealed vessels at a
+temperature of 115 deg. C.--will destroy both the vegetative and the
+sporing forms of bacteria within fifteen minutes; if the pressure is
+increased, and the temperature raised to 120 deg. C., the same end is
+attained in ten minutes. This method was formerly employed for the
+sterilisation of media (and indeed is so used in some laboratories
+still), but most workers now realise that media subjected to this high
+temperature undergo hydrolytic changes which render them unsuitable for
+the cultivation of the more delicate micro-organisms. The use of
+superheated steam should be restricted almost entirely to the
+disinfection of such contaminated articles, old cultivations, etc., as
+cannot be dealt with by dry heat or the actual furnace. Sterilisation
+by means of superheated steam is carried out in a special
+boiler--Chamberland's autoclave (Fig. 30). The autoclave consists of a
+stout copper cylinder, provided with a copper or gun-metal lid, which
+is secured in place by means of bolts and thumbscrews, the joint between
+the cylinder and its lid being hermetically sealed by the interposition
+of a rubber washer. The cover is perforated for a branched tube carrying
+a vent cock, a manometer, and a safety valve. The copper boiler is
+mounted in the upper half of a cylindrical sheet-iron case--two
+concentric circular rows of Bunsen burners, each circle having an
+independent gas-supply, occupying the lower half. In the interior of the
+boiler is a large movable wire basket, mounted on legs, for the reception
+of the articles to be sterilised.
+
+
+TO USE THE AUTOCLAVE.--
+
+1. Pack the articles to be sterilised in the wire basket.
+
+2. Run water into the boiler to the level of the bottom of the basket;
+also fill the contained flasks and tubes with water.
+
+3. See that the rubber washer is in position, then replace the cover and
+fasten it tightly on to the autoclave by means of the thumbscrews.
+
+4. Open the vent cock and light both rings of burners.
+
+5. When steam is issuing in a steady, continuous stream from the vent
+tube, shut off the vent cock and extinguish the outer ring of gas
+burners.
+
+6. Wait until the index of the manometer records a temperature of 120 deg.
+C., then regulate the gas and the spring safety valve in such a manner
+that this temperature is just maintained, and leave it thus for twenty
+minutes. In the more expensive patterns of autoclave this regulation of
+the safety valve is carried out automatically, the manometer being
+fitted with an adjustable pointer which can be set to any required
+pressure-temperature and so arranged that when the index of the
+manometer coincides with the adjustable hand the safety valve is opened.
+
+7. Extinguish the gas and allow the manometer index to fall to zero.
+
+[Illustration: FIG. 30.--Chamberland's Autoclave.]
+
+8. Now open the vent cock slowly, and allow the internal pressure to
+adjust itself to that of the atmosphere.
+
+9. Remove the cover and take out the sterilised contents.
+
+~Sterilisation Periods.~--An exceedingly useful device for the timing of
+sterilisation periods (and indeed for many other operations in the
+laboratory) is the
+
+
+ELECTRIC SIGNAL TIMING CLOCK.
+
+This is a clock of American type in which the face is surrounded by a
+metal plate having a series of 60 holes at equal distances apart,
+corresponding to the minutes on the dial. This plate is connected with
+one of the poles of a dry battery, the other pole of which is connected
+to the metal case of the clock for the purpose of actuating an ordinary
+magnet alarm bell. In the centre of each of the holes in the plate a
+metal rod is fixed, which then passes through an insulating ring and
+projects inside the clock face, where it makes contact with the hour
+hand. The clock is mounted on a heavy base, with a key-board containing
+20 numbered plugs. If one of the plugs is inserted in a hole in the
+plate it makes contact with the rod, and when the hour hand of the clock
+touches the other end the circuit is completed and the bell starts
+ringing. The period of this friction contact is approximately 20
+seconds. The clock can therefore be used for electrically noting the
+periods of time from one minute by multiples of one minute up to one
+hour.
+
+[Illustration: FIG. 31.--Electric signal timing clock.]
+
+~Filtration.~--(a) _Cotton-wool Filter._--Practically the only method in
+use in the laboratory for the sterilisation of air or of a gas is by
+filtration through dry cotton-wool or glass-wool, the fibres of which
+entangle the micro-organisms and prevent their passage.
+
+Perhaps the best example of such a filter is the cotton-wool plug which
+closes the mouth of a culture tube. Not only does ordinary diffusion
+take place through it, but if a tube plugged in the usual manner with
+cotton-wool is removed from the hot incubator, the temperature of the
+contained air rapidly falls to that of the laboratory, and a partial
+vacuum is formed; air passes into the tube, through the cotton-wool
+plug, to restore the equilibrium, and, so long as the plug remains dry,
+in a germ-free condition. If, however, the plug becomes moist, either by
+absorption from the atmosphere, or from liquids coming into contact with
+it, micro-organisms (especially the mould fungi) commence to multiply,
+and the long thread forms rapidly penetrate the substance of the plug,
+and gain access to and contaminate the interior of the tube.
+
+[Illustration: FIG. 32.--Cotton-wool air filter.]
+
+
+METHOD.--
+
+If it is desired to sterilise gases before admission to a vessel
+containing a pure cultivation of a micro-organism, as, for instance,
+when forcing a current of oxygen over or through a broth cultivation of
+the diphtheria bacillus, this can be readily effected as follows:
+
+1. Take a length of glass tubing of, say, 1.5 cm. diameter, in the
+centre of which a bulb has been blown, fill the bulb with dry
+cotton-wool (Fig. 32), wrap a layer of cotton-wool around each end of
+the tube, and secure in position with a turn of thin copper wire or
+string; then sterilise the piece of apparatus in the hot-air oven.
+
+2. Prepare the cultivation in a Ruffer or Woodhead flask (Fig. 33) the
+inlet tube of which has its free extremity enveloped in a layer of
+cotton-wool, secured by thread or wire, whilst the exit tube is plugged
+in the usual manner.
+
+[Illustration: FIG. 33.--Ruffer's flask.]
+
+3. Sterilise a short length of rubber tubing by boiling. Transfer it
+from the boiling water to a beaker of absolute alcohol.
+
+4. When all is ready remove the rubber tube from the alcohol by means of
+a pair of forceps, drain it thoroughly, and pass through the flame of a
+Bunsen burner to burn off the last traces of alcohol.
+
+5. Remove the cotton-wool wraps from the entry tube of the flask and
+from one end of the filter tube and rapidly couple them up by means of
+the sterile rubber tubing.
+
+6. Connect the other end of the bulb tube with the delivery tube from
+the gas reservoir.
+
+The gas in its passage through the dry sterile cotton-wool in the bulb
+of the filter tube will be freed from any contained micro-organisms and
+will enter the flask in a sterile condition.
+
+(b) _Porcelain Filter._--The sterilisation of liquids by filtration is
+effected by passing them through a cylindrical vessel, closed at one end
+like a test-tube, and made either of porous "biscuit" porcelain,
+hard-burnt and unglazed (Chamberland system), or of Kieselguhr, a fine
+diatomaceous earth (Berkefeld system), and termed a "bougie" or "candle"
+(Fig. 34).
+
+     NOTE.--In selecting candles for use in the laboratory avoid
+     those with metal fittings, since during sterilisation cracks
+     develop at the junction of the metal and the siliceous
+     material owing to the unequal expansion.
+
+In this method the bacteria are retained in the pores of the filter
+while the liquid passes through in a germ-free condition.
+
+It is obvious that to be effective the pores of the filter must be
+extremely minute, and therefore the rate of filtration will usually be
+slow. Chamberland filter candles possess finer channels than Berkefeld
+candles and consequently filter much more slowly. To overcome this
+disadvantage, either aspiration or pressure, or a combination of these
+two forces, may be employed to hasten the process.
+
+Doultons white porcelain filters it may be noted are as efficient as the
+Chamberland candles and filter rather more rapidly.
+
+_Apparatus Required._--
+
+1. Separatory funnel containing the unfiltered fluid.
+
+2. Sterile filter candle (Fig. 34), the open end fitted with a rubber
+stopper (Fig. 34, a) perforated to receive the delivery tube of the
+separatory funnel, and its neck passed through a large rubber washer
+(Fig. 34, b) which fits the mouth of the filter flask.
+
+3. Sterile filter flask of suitable size, for the reception of the
+filtered fluid, its mouth closed by a cotton-wool plug.
+
+4. Water injector Sprengel (see Fig. 38, c) pump, or Geryk's pump (an
+air pump on the hydraulic principle, sealed by means of low
+vapor-tension oil, Fig. 35).
+
+If this latter is employed, a Wulff's bottle, fitted as a wash-bottle
+and containing sulphuric acid, must be interposed between the filter
+flask and the pump, in order to prevent moist air reaching the oil in
+the pump.
+
+5. Air filter (_vide_ page 40) sterilised.
+
+6. Pressure tubing.
+
+7. Screw clamps (Fig. 36).
+
+METHOD.--
+
+1. Couple the exhaust pipe of the suction pump with the lateral tube of
+the filter flask (first removing the cotton-wool plug from this latter),
+by means of pressure tubing, interposing, if necessary, the wash-bottle
+of sulphuric acid.
+
+[Illustration: FIG. 34.--Porcelain filter candle.]
+
+[Illustration: FIG. 35.--Geryk air pump.]
+
+2. Remove the cotton-wool plug from the neck of the filter flask and
+adjust the porcelain candle in its place.
+
+[Illustration: FIG. 36.--Screw clamps.]
+
+3. Attach the nozzle of the separatory funnel to the filter candle by
+means of the perforated rubber stopper (Fig. 37).
+
+[Illustration: FIG. 37.--Apparatus arranged for filtering--aspiration.]
+
+4. Open the tap of the funnel, and exhaust the air from the filter flask
+and wash-bottle; maintain the vacuum until the filtration is complete.
+
+5. When the filtration is completed close the tap of the funnel; adjust
+a screw clamp to the pressure tubing attached to the lateral branch of
+the filter flask; screw it up tightly, and disconnect the acid
+wash-bottle.
+
+6. Attach the air filter to the open end of the pressure tubing; open
+the screw clamp gradually, and allow filtered air to enter the flask, to
+abolish the negative pressure.
+
+7. Detach the rubber tubing from the lateral branch of the flask, flame
+the end of the branch in the Bunsen, and plug its orifice with sterile
+cotton-wool.
+
+8. Remove the filter candle from the mouth of the flask, flame the
+mouth, and plug the neck with sterile cotton-wool.
+
+9. Disinfect the filter candle and separatory funnel by boiling.
+
+If it is found necessary to employ pressure in addition to or in place
+of suction, insert a perforated rubber stopper into the mouth of the
+separatory funnel and secure in position with copper wire; next fit a
+piece of glass tubing through the stopper, and connect the external
+orifice with an air-pressure pump of some kind (an ordinary foot pump
+such as is employed for inflating bicycle tyres is one of the most
+generally useful, for this purpose) or with a cylinder of compressed air
+or other gas.
+
+In order to filter a large bulk of fluid very rapidly it is necessary to
+use a higher pressure than glass would stand, and in these cases the
+metal receptacle designed by Pakes (Fig. 38, a), to hold the filter
+candle itself as well as the fluid to be filtered, should be employed.
+(A vacuum must also be maintained in the filter flask, by means of an
+exhaust pump, during the entire process.)
+
+This piece of apparatus consists of a brass cylinder, capacity 2500
+c.c., with two shoulders; and an opening in the neck at each end,
+provided with screw threads.
+
+A nut carrying a pressure gauge fits into the top screw; and into the
+bottom is fitted a brass cylinder carrying the filter candle and
+prolonged downwards into a delivery tube. Leakage is prevented by means
+of rubber washers.
+
+Into the top shoulder a tube is inserted, bent at right angles and
+provided with a tap. All the brass-work is tinned inside (Fig. 38, a).
+In use the reservoir is generally mounted on a tripod stand.
+
+~To Sterilise.~--
+
+1. Insert the filter candle into its cylinder and screw this loosely on.
+
+[Illustration: FIG. 38.--Pakes' filtering reservoir--pressure and
+aspiration.]
+
+2. Wrap a layer of cotton-wool around the delivery tube and fasten in
+position.
+
+3. Remove the nut carrying the pressure gauge and plug the neck with
+cotton-wool.
+
+4. Heat the whole apparatus in the autoclave at 120 deg. C. for twenty
+minutes.
+
+METHOD.--
+
+1. Remove the apparatus from the autoclave, and allow it to cool.
+
+2. Screw home the box carrying the bougie.
+
+3. Set the apparatus up in position, with its delivery tube (from which
+the cotton-wool wrapping has been removed) passing through a perforated
+rubber stopper in the neck of a filter flask.
+
+[Illustration: FIG. 39.--Closed candle arranged for filtering.]
+
+4. Fill the fluid to be filtered into the cylinder and screw on the nut
+carrying the pressure gauge. (This nut should be immersed in boiling
+water for a few minutes previous to screwing on, in order to sterilise
+it.)
+
+5. Connect the horizontal arm of the entry tube with a cylinder of
+compressed oxygen (or carbon dioxide, Fig. 38, b), by means of
+pressure tubing.
+
+6. Connect the lateral arm of the filter flask with the exhaust pump
+(Fig. 38, c) and start the latter working.
+
+7. Open the tap of the gas cylinder; then open the tap on the entry tube
+of the filter cylinder and raise the pressure in its interior until the
+desired point is recorded on the manometer. Maintain this pressure,
+usually one or one and a half atmospheres, until filtration is
+completed, by regulating the tap on the entry tube.
+
+Some forms of filter candle are made with the open end contracted into a
+delivery nozzle, which is glazed. In this case the apparatus is fitted
+up in a slightly different manner; the fluid to be filtered is contained
+in an open cylinder into which the candle is plunged, while its delivery
+nozzle is connected with the filter flask by means of a piece of
+flexible pressure tubing (previously sterilised by boiling), as in
+figure 39.
+
+
+
+
+IV. THE MICROSCOPE.
+
+
+The essentials of a microscope for bacteriological work may be briefly
+summed up as follows:
+
+[Illustration: FIG. 40.--Microscope stand.]
+
+The instrument, of the monocular type, must be of good workmanship and
+well finished, rigid, firm, and free from vibration, not only when
+upright, but also when inclined to an angle or in the horizontal
+position. The various joints and movements must work smoothly and
+precisely, equally free from the defects of "loss of time" and
+"slipping." All screws, etc., should conform to the Royal Microscopical
+Society's standard. It must also be provided with good lenses and a
+sufficiently large stage. The details of its component parts, to which
+attention must be specially directed, are as follows:
+
+[Illustration: FIG. 41.--Foot, three types.]
+
+~1. The Base or Foot~ (Fig. 40, a).--Two elementary forms--the tripod
+(Fig. 41, a) and the vertical column set into a plate known as the
+"horse-shoe" (Fig. 41, b)--serve as the patterns for countless
+modifications in shape and size of this portion of the stand. The chief
+desiderata--stability and ease of manipulation--are attained in the
+first by means of the "spread" of the three feet, which are usually shod
+with cork; in the second, by the dead weight of the foot-plate. The
+tripod is mechanically the more correct form, and for practical use is
+much to be preferred. Its chief rival, the Jackson foot (Fig. 41, c),
+is based upon the same principle, and on the score of appearance has
+much to recommend it.
+
+~2.~ The ~body tube~ (Fig. 40, b) may be either that known as the "long"
+or "English" (length 250 mm.), or the "short" or "Continental" (length
+160 mm.). Neither length appears to possess any material advantage over
+the other, but it is absolutely necessary to secure objectives which
+have been manufactured for the particular tube length chosen. In the
+high-class microscope of the present day the body tube is usually
+shorter than the Continental, but is provided with a draw tube which,
+when fully extended, gives a tube length greater than the English, thus
+permitting the use of either form of objective.
+
+[Illustration: FIG. 42.--Coarse adjustment.]
+
+[Illustration: FIG. 43.--Fine adjustment.]
+
+
+     For practical purposes the tube length = distance from the
+     end of the nosepiece to the eyeglass of the ocular. This is
+     the measurement referred to in speaking of "long" or "short"
+     tube.
+
+~3.~ The ~coarse adjustment~ (Fig. 40, c) should be a rack-and-pinion
+movement, steadiness and smoothness of action being secured by means of
+accurately fitting dovetailed bearings and perfect correspondence
+between the teeth of the rack and the leaves of the pinion (Fig. 42).
+Also provision should be made for taking up the "slack" (as by the
+screws _AA_, Fig. 42).
+
+~4.~ The ~fine adjustment~ (Fig. 40, d) should on no account depend upon
+the direct action of springs, but should be of the lever pattern,
+preferably the Nelson (Fig. 43). In this form the unequal length of the
+arms of the lever secures very delicate movement, and, moreover, only a
+small portion of the weight of the body tube is transmitted to the
+thread of the vertical screw actuating the movement.
+
+[Illustration: FIG. 44.--Spindle head to fine adjustment.]
+
+A spindle milled head (Fig. 44) will be found a very useful device to
+have fitted in place of the ordinary milled head controlling the fine
+adjustment. In this contrivance the axis of the milled head is prolonged
+upward in a short column, the diameter of which is one-sixth of that of
+the head. The spindle can be rapidly rotated between the fingers for
+medium power adjustments while the larger milled head can be slowly
+moved when focussing high powers.
+
+~5.~ The ~stage~ (Fig. 40, e) should be square in shape and large in
+area--at least 12 cm.--flat and rigid, in order to afford a safe support
+for the Petri dish used for plate cultivations; and should be supplied
+with spring clips (removable at will) to secure the 3 by 1 glass slides.
+
+A mechanical stage must be classed as a necessity rather than a luxury
+so far as the bacteriologist is concerned, as when working with high
+powers, and especially when examining hanging-drop specimens, it is
+almost impossible to execute sufficiently delicate movements with the
+fingers. In selecting a mechanical stage, preference should be given to
+one which forms an integral part of the instrument (Fig. 45) rather than
+one which needs to be clamped on to an ordinary plain stage every time
+it is required, and its traversing movements should be controlled by
+stationary milled heads (Fig. 45, _AA'_). The shape of the aperture is a
+not unimportant point; it should be square to allow of free movement
+over the substage condenser. The mechanical stage should be tapped for
+three (removable) screw studs to be used in place of the sliding bar, so
+that if desired the Vernier finder (Fig. 45, _BB'_), such as is usually
+fitted to this class of stage, or a Maltwood finder, may be employed.
+
+[Illustration: FIG. 45.--Mechanical stage.]
+
+[Illustration: FIG. 46.--Iris diaphragm.]
+
+~6. Diaphragm.~--Separate single diaphragms must be avoided; a revolving
+plate pierced with different sized apertures and secured below the stage
+is preferable, but undoubtedly the best form is the "iris" diaphragm
+(Fig. 46) which enters into the construction of the substage condenser.
+
+~7.~ The ~substage condenser~ is a necessary part of the optical outfit.
+Its purpose is to collect the beam of parallel rays of light reflected by
+the plane mirror, by virtue of a short focus system of lenses, into a
+cone of large aperture (reducible at will by means of an iris diaphragm
+mounted as a part of the condenser), which can be accurately focussed on
+the plane of the object. This focussing must be performed anew for each
+object, on account of the variation in the thickness of the slides.
+
+The form in most general use is that known as the Abbe (Fig. 47) and
+consists of a plano-convex lens mounted above a biconvex lens. This
+combination is carried in a screw-centering holder known as the substage
+below the stage of the microscope (Fig. 40 f), and must be accurately
+adjusted so that its optical axis coincides with that of the objective.
+Vertical movement of the entire substage apparatus effected by means of
+a rack and pinion is a decided advantage, and some means should be
+provided for temporarily removing the condenser from the optical axis of
+the microscope.
+
+[Illustration: FIG. 47--Optical part of Abbe illuminator.]
+
+With the oil immersion objective, however, an ~achromatic condenser~,
+giving an illuminating cone of about 0.9, should be used if the full
+value of the lens is to be obtained. It is generally assumed that a good
+objective requires an illuminating cone equivalent to two-thirds of its
+numerical aperture. The best Abbe condenser transmits a cone of about
+.45 whilst the aperture of the 1/12 inch immersion lenses of different
+makers varies from 1.0 to 1.4, hence, the efficiency of these lenses is
+much curtailed if the condenser is merely the Abbe. These improved
+condensers must be absolutely centered to the objective and capable of
+very accurate focussing otherwise much of their value is lost.
+
+~8. Mirrors.~--Below the substage condenser is attached a gymbal carrying
+a reversible circular frame with a plane mirror on one side and a
+concave mirror on the other (Fig. 40, g). The plane mirror is that
+usually employed, but occasionally, as for example when using low powers
+and with the condenser racked down and thrown out of the optical axis,
+the concave mirror is used.
+
+~9. Oculars, or Eyepieces.~--Those known as the Huyghenian oculars (Fig.
+48) will be sufficient for all ordinary work without resorting to the
+more expensive "compensation" oculars. Two or three, magnifying the
+"real" image (formed by the objective) four, six, or eight times
+respectively, form a useful equipment.
+
+As an accessory ~Ehrlich's Eyepiece~ is a very useful piece of apparatus
+when the enumeration of cells or bacteria has to be carried out. This is
+an ordinary eyepiece fitted with an adjustable square diaphragm operated
+by a lever projecting from the side of the mount. Three notches are made
+in one of the sides of the square and by moving the lever square
+aperture can be reduced to three-quarters, one-half or one-quarter of
+the original size.
+
+~10. Objectives.~--Three objectives are necessary: one for low-power
+work--e. g., 1 inch, 2/3 inch, or 1/2 inch; one for high-power
+work--e. g., 1/12 inch oil immersion lens; and an intermediate
+"medium-power" lens--e. g., 1/6 inch or 1/8 inch (dry). These lenses
+must be carefully selected, especial attention being paid to the
+following points:
+
+(a) _Correction of Spherical Aberration._--Spherical aberration gives
+rise to an ill-defined image, due to the central and peripheral rays
+focussing at different points.
+
+(b) _Correction of Chromatic Aberration._--Chromatic aberration gives
+rise to a coloured fringe around the edges of objects due to the fact
+that the different-coloured rays of the spectrum possess varying
+refrangibilities and that a simple lens acts toward them as a prism.
+
+(c) _Flatness of Field._--The ideal visual field would be large and,
+above all, _flat_; in other words, objects at the periphery of the field
+would be as distinctly "in focus" as those in the centre. Unfortunately,
+however, this is an optical impossibility and the field is always
+spherical in shape. Some makers succeed in giving a larger central area
+that is in focus at one time than others, and although this may
+theoretically cause an infinitesimal sacrifice of other qualities, it
+should always be sought for. Successive zones and the entire peripheral
+ring should come into focus with the alteration of the fine adjustment.
+This simultaneous sharpness of the entire circle is an indication of the
+perfect centering of the whole of the lenses in the objective.
+
+[Illustration: FIG. 48.--Huyghenian eyepiece.]
+
+(d) _Good Definition._--Actual magnification is, within limits, of
+course, of less value than clear definition and high resolving power,
+for it is upon these properties we depend for our knowledge of the
+detailed structure of the objects examined.
+
+(e) _Numerical Aperture_ (_N. A._).--The numerical aperture may be
+defined, in general terms, as the ratio of the _effective_ diameter of
+the back lens of the objective to its equivalent focal length. The
+determination of this point is a process requiring considerable
+technical skill and mathematical ability, and is completely beyond the
+powers of the average microscopist.[1]
+
+Although with the increase in power it is correspondingly difficult to
+combine all these corrections in one objective, they are brought to a
+high pitch of excellence in the present-day "achromatic" objectives, and
+so remove the necessity for the use of the higher priced and less
+durable apochromatic lenses.
+
+In selecting objectives the best "test" objects to employ are:
+
+1. A thin (one cell layer), even    }     { 1", 2/3", 1/2":
+"blood film," stained with Jenner's } for { 1/6", 1/8"
+or Romanowsky's stain.              }     { 1/12" oil
+
+2. A thin cover-slip preparation    }
+of a young cultivation of           }     { 1/8" dry
+_B. diphtheriae_ (showing           } for {
+segmentation) stained with          }     { 1/12" oil
+methylene-blue.                     }
+
+~Accessories.~--_Eye Shade_ (Fig. 49).--This piece of apparatus consists
+of a pear-shaped piece of blackened metal or ebonite, hinged to a collar
+which rotates on the upper part of the body tube of the microscope. It
+can be used to shut out the image of surrounding objects from the
+unoccupied eye, and when carrying out prolonged observations will be
+found of real service.
+
+_Nosepiece._--Perhaps the most useful accessory is a nosepiece to carry
+two of the objectives (Fig. 50), or, better still, all three (Fig. 51).
+This nosepiece, preferably constructed of aluminium, must be of the
+covered-in type, consisting of a curved plate attached to the lower end
+of the body tube--a circular aperture being cut to correspond to the
+lumen of that tube. To the under surface of this plate is pivoted a
+similarly curved plate, fitted with three tubulures, each of which
+carries an objective. By rotating the lower plate each of the objectives
+can be brought successively in to the optical axis of the microscope.
+
+[Illustration: FIG. 49.--Eye shade.]
+
+For critical work and particularly for photo-micrography, however, the
+interchangeable nosepiece is by no means perfect as it is next to
+impossible to secure accurate centreing of each lens in the optical
+axis. For special purposes, therefore, it is necessary to employ a
+special nosepiece such as that made by Zeiss or Leitz into which each
+objective slides on its own carrier and upon which it is accurately
+centred.
+
+[Illustration: FIG. 50.--Double nosepiece.]
+
+[Illustration: FIG. 51.--Triple nosepiece.]
+
+_Warm Stage_ (Fig. 52).--This is a flat metal case containing a system
+of tubes through the interior of which water of any required temperature
+can be circulated. It is made to clamp on to the stage of the
+microscope by the screws _A A'_, and is perforated with a large hole
+coinciding with the optical axis of the microscope; a short tube B,
+projecting from one end of the warm stage permits water of the desired
+temperature to be conducted from a reservoir through a length of rubber
+tubing to the interior of the stage and a similar tube at the other end
+_B'_ of the stage allows exit to the waste water. By raising the
+temperature of hanging-drop preparations, etc., placed upon it, above
+that of the surrounding atmosphere, the warm stage renders possible
+exact observations on spore germination, hanging-drop cultivations, etc.
+
+[Illustration: FIG. 52.--Warm stage.]
+
+A better form is the electrical hot stage designed by Lorrain Smith;[2]
+it requires the addition of a lamp resistance and sliding rheostat, also
+a delicate ammeter reading to .01 of an ampere. It consists of a wooden
+frame supporting a flat glass bulb with a long neck bent upward at an
+obtuse angle (Fig. 53). The bulb is filled with liquid paraffin, which
+rises in the open neck when expanded by heat. The neck also accommodates
+the thermometer. Two coils of manganin wire run in the paraffin at
+opposite sides of the bulb (outside the field of vision), coupled to
+brass terminals on the wooden frame by platinum wire fused into the
+glass. The resistance of the two coils in series is about 10 ohms. A
+current of 2-1/2 amperes is needed, and is conducted to the coils in the
+stage through the rheostat. With the help of the ammeter any desired
+temperature can be obtained and maintained, up to about 200 deg. C. If
+immersion oil contact is made between the top lens of the condenser and
+the lower surface of the bulb, this stage works very well indeed with
+the 1/12-inch oil immersion lens.
+
+[Illustration: FIG. 53.--Lorrain Smith's warm stage.]
+
+_Dark Ground or Paraboloid Condenser._--This is an immersion substage
+condenser of high aperture by means of which unstained objects such as
+bacteria can be shown as bright white particles upon a dense black
+background. The central rays of light are blocked out by means of an
+opaque stop while the peripheral rays are reflected from the
+paraboloidal sides of the condenser and refracted by the object viewed.
+To obtain the best results with this type of condenser a powerful
+illuminant--such as a small arc lamp or an incandescent gas lamp--is
+needed, together with picked slides of a certain thickness (specified
+for the particular make of condenser but generally 1 mm.) and specially
+thin cover-glasses (not more than 0.17 mm.) The objective must not have
+a higher NA than 1.0, consequently immersion lenses must be fitted with
+an internal stop to cut down the aperture.
+
+_Micrometer._--Some form of micrometer for the purpose of measuring
+bacteria and other objects is also essential. Details of those in
+general use will be found in the following pages.
+
+[Illustration: FIG. 54--Diamond Object marker.]
+
+_Object Marker_ (Fig. 54).--This is an exceedingly useful piece of
+apparatus. Made in the form of an objective, the lenses are replaced by
+a diamond point, set slightly out of the centre, which can be rotated by
+means of a milled plate. Screwed on to the nosepiece in place of the
+objective, rotation of the diamond point will rule a small circle on the
+object slide to permanently record the position of an interesting
+portion of the specimen. The diamond is mounted on a spring which
+regulates the pressure, and the size of the circle can be adjusted by
+means of a lateral screw.
+
+
+METHODS OF MICROMETRY.
+
+The unit of length as applied to the measurement of microscopical
+objects is the one-thousandth part of a millimetre (0.001 mm.),
+denominated a _micron_ (sometimes, and erroneously, referred to as a
+micro-millimetre), and indicated in writing by the Greek letter mu. Of
+the many methods in use for the measurement of bacteria, three only will
+be here described, viz.:
+
+(a) By means of the Camera Lucida.
+
+(b) By means of the ocular or Eyepiece Micrometer.
+
+(c) By means of the Filar Micrometer (Ramsden's micrometer eyepiece).
+
+For each of these methods a ~stage micrometer~ is necessary. This is a 3
+by 1 inch glass slip having engraved on it a scale divided to hundredths
+of a millimetre (0.01 mm.), every tenth line being made longer than the
+intervening ones, to facilitate counting; and from these engraved lines
+the measurement in every case is evaluated. A cover-glass is cemented
+over the scale to protect it from injury.
+
+[Illustration: FIG. 55.--Camera lucida, Abbe pattern.]
+
+(a) By means of the Camera Lucida.
+
+1. Attach a camera lucida (of the Wollaston, Beale, or Abbe pattern)
+(Fig. 55) to the eyepiece of the microscope.
+
+2. Adjust the micrometer on the stage of the microscope and accurately
+focus the divisions.
+
+3. Project the scale of the stage micrometer on to a piece of paper and
+with pen or pencil sketch in the magnified image, each division of which
+corresponds to 10 mu. Mark on the paper the optical combination (ocular
+objective and tube length) employed to produce this particular
+magnification.
+
+4. Repeat this procedure for each of the possible combinations of
+oculars and objectives fitted to the microscope supplied, and carefully
+preserve the scales thus obtained.
+
+To measure an object by this method simply project the image on to the
+scale corresponding to the particular optical combination in use at the
+moment. Read off the number of divisions it occupies and express them as
+_micra_.
+
+In place of preserving a scale for each optical combination, the object
+to be measured and the micrometer scale may be projected and sketched,
+in turn, on the same piece of paper, taking particular care that the
+centre of the eyepiece is 25 cm. from the paper on which the divisions
+are drawn.
+
+[Illustration: FIG. 56.--Eyepiece micrometer, ordinary.]
+
+[Illustration: FIG. 57.--Eyepiece micrometer, net.]
+
+(b) By means of the Eyepiece Micrometer.
+
+The ~eyepiece micrometer~ is a circular glass disc having engraved on it a
+scale divided to tenths of a millimetre (0.1 mm.) (Fig. 56), or the
+entire surface ruled in 0.1 mm. squares (the net micrometer) (Fig. 57).
+It can be fitted inside the mount of any ocular just above the aperture
+of the diaphragm and must be adjusted exactly in the focus of the eye
+lens.
+
+Some makers mount the glass disc together with a circular cover-glass in
+such a way that when placed in position in any Huyghenian eyepiece of
+their own manufacture, the scale is exactly in focus for normal vision.
+Special eyepieces are also obtainable having a sledging adjustment to
+the eye lens for focussing the micrometer.
+
+The value of one division of the micrometer scale must first be
+ascertained for each optical combination by the aid of the stage
+micrometer, thus:
+
+1. Insert the eyepiece micrometer inside the ocular and adjust the stage
+micrometer on the stage of the microscope.
+
+2. Focus the scale of the stage micrometer accurately; the lines will
+appear to be immediately below those of the eyepiece micrometer. Make
+the lines on the two micrometers parallel by rotating the ocular.
+
+3. Make two of the lines on the ocular micrometer coincide with those
+bounding one division of the stage micrometer; this is effected by
+increasing or diminishing the tube length; and note the number of
+included divisions.
+
+4. Calculate the value of each division of the eyepiece micrometer in
+terms of  mu, by means of the following formula:
+
+    x = 10 y.
+
+    Where x = the number of included divisions of the
+                        eyepiece micrometer.
+
+          y = the number of included divisions of the
+                       stage micrometer.
+
+5. Note the optical combination employed in this experiment and record
+it with the calculated micrometer value.
+
+Repeat this process for each of the other combinations. Carefully record
+the results.
+
+To measure an object by this method read off the number of divisions of
+the eyepiece micrometer it occupies and express the result in _micra_ by
+a reference to the standard value for the particular optical combination
+employed.
+
+Zeiss prepares a compensating eyepiece micrometer for use with his
+apochromatic objectives, the divisions of which are so computed that
+(with a tube length of 160 mm.) the value of each is equivalent to as
+many _micra_ as there are millimetres in the focal length of the
+objective employed.
+
+_Wright's Eikonometer_ is really a modification of the eyepiece
+micrometer for rapidly measuring microscopical objects by direct
+inspection, having previously determined the magnifying power of the
+particular optical combination employed. It is a small piece of
+apparatus resembling an eyepiece, with a sliding eye lens, which can be
+accurately focussed on a micrometer scale fixed within the instrument.
+When placed over the microscope ocular the divisions of this scale
+measure the actual size of the virtual image in millimetres.
+
+In order to use this instrument for direct measurement, it is first
+necessary to determine the magnifying power of each combination of
+ocular, tube length and objective.
+
+Place a stage micrometer divided into hundredths of a millimetre on the
+microscope stage and focus accurately.
+
+Rest the eikonometer on the eyepiece. Observation through the
+eikonometer shows its micrometer scale superposed on the image of the
+stage micrometer.
+
+Rotate the eikonometer until the lines on the two scales are parallel,
+and make the various adjustments to ensure that two lines on the
+eikonometer scale coincide with two lines on the stage micrometer.
+
+For the sake of illustration it may be assumed that five of the
+divisions on the stage micrometer accurately fill one of the divisions
+of the eikonometer scale; this indicates a magnifying power of 500 as
+the constant for that particular optical combination, and a record
+should be made of the fact.
+
+The magnification constants of the various other optical combinations
+should be similarly made and recorded.
+
+To measure any object subsequently it should be first focussed carefully
+in the ordinary way.
+
+The eikonometer should then be applied to the eyepiece and the size of
+the object read off on the eikonometer scale as millimetres, and the
+actual size calculated by dividing the observed size by the
+magnification constant for the particular optical combination employed
+in the observation.
+
+(c) By means of the filar micrometer.
+
+[Illustration: FIG. 58.--Ramsden's Filar micrometer.]
+
+[Illustration: FIG. 59.--Ramsden's micrometer field, a, fixed wire;
+b, reference wire (fixed); c, travelling wire.]
+
+The ~Filar~ or cobweb Micrometer (Ramsden's micrometer) eyepiece (Fig. 58)
+consists of an ocular having a fine "fixed" wire stretching horizontally
+across the field (Fig. 59), a vertical reference wire--fixed--adjusted
+at right angles to the first; and a fine wire, parallel to the reference
+wire, which can be moved across the field by the action of a micrometer
+screw; the drum head is divided into one hundred parts, which
+successively pass a fixed index as the head is turned. In the lower part
+of the field is a comb with the intervals between its teeth
+corresponding to one complete revolution of this screw-head.
+
+As in the previous method, the value of each division of the micrometer
+scale (i. e., the comb) must first be determined for each optical
+combination. This is effected as follows:
+
+1. Place the filar micrometer and the stage micrometer in their
+respective positions.
+
+2. Rotate the screw of the filar micrometer until the movable wire
+coincides with the fixed one, and the index marks zero on the drum head.
+(If when the drum head is at zero the two wires do not exactly coincide
+they must be adjusted by loosening the drum screw and resetting the
+drum.)
+
+3. Focus the scale of each micrometer accurately, and make the lines on
+them parallel.
+
+4. Rotate the head of the micrometer screw until the movable line has
+transversed one division of the stage micrometer. Note the number of
+complete revolutions (by means of the recording comb) and the fractions
+of a revolution (by means of scale on the head of the micrometer screw),
+which are required to measure the 0.01 mm.
+
+5. Make several such estimations and average the results.
+
+6. Note the optical combination employed in this experiment and record
+it carefully, together with the micrometer value in terms of  mu.
+
+7. Repeat this process for each of the different optical combinations
+and record the results.
+
+To measure an object by this method, simply note the number of
+revolutions and fractions of a revolution of the screw-head required to
+traverse such object from edge to edge, and express the result as
+_micra_ by reference to the recorded values for that particular optical
+combination.
+
+_Microscope Illuminant._--In tropical and subtropical regions diffuse
+daylight is the best illuminant. In temperate climes however daylight of
+the desirable quantity is not always available, and recourse must be
+had to oil lamps, gas lamps--preferably those with incandescent
+mantles--and electricity; and of these the last is undoubtedly the best.
+A handy lamp holder which can be manufactured in the laboratory is shown
+in Fig. 60. It consists of a base board weighted with lead to which is
+attached the ordinary domestic lamp holder, and behind this is fastened
+a curved sheet-iron reflector. An obscured metal filament lamp of about
+16 candle power gives the most suitable light, and if monochromatic
+light is needed, the blue grease pencil is streaked over the side of the
+lamp nearest the microscope; the current is switched on and when the
+glass bulb is warm, rubbing with a wad of cotton-wool will readily
+distribute the blue greasy material in an even film over the ground
+glass.
+
+[Illustration: FIG. 60.--Electric microscope lamp.]
+
+FOOTNOTES:
+
+[1] Its importance will be realised, however, when it is stated in the
+words of the late Professor Abbe: "The numerical aperture of a lens
+determines all its essential qualities; the brightness of the image
+increases with a given magnification and other things being equal, as
+the square of the aperture; the resolving and defining powers are
+directly related to it, the focal depth of differentiation of depths
+varies inversely as the aperture, and so forth."
+
+[2] Made by Mr. Otto Baumbach, 10, Lime Grove, Manchester.
+
+
+
+
+V. MICROSCOPICAL EXAMINATION OF BACTERIA AND OTHER MICRO-FUNGI.
+
+
+APPARATUS AND REAGENTS USED IN ORDINARY MICROSCOPICAL EXAMINATION.
+
+The following comprises the essential apparatus and reagents for routine
+work with which each student should be provided.
+
+1. India-rubber "change-mat" upon which cover-glasses may be rested
+during the process of staining.
+
+2. Squares of blotting paper about 10 cm., for drying cover-slips and
+slides.
+
+(The filter paper known as "German lined"--a highly absorbent, closely
+woven paper, having an even surface and no loose "fluff" to adhere to
+the specimens--is the most useful for this purpose.)
+
+[Illustration: FIG. 61.--Disinfectant Jar.]
+
+3. Glass jar filled with 2 per cent. lysol solution for the reception of
+infected cover-glasses and infected pipettes, etc.
+
+4. A square glazed earthenware box with a loose lining containing 2 per
+cent. lysol solution for the reception of infected material and used
+slides. The bottom of the lining is perforated so that when full the
+lining and its contents can be lifted bodily out of the box, when the
+disinfectant solution drains away and the slides, etc., can easily be
+emptied out. The empty lining is then returned to the box with its
+disinfectant solution (Fig. 61).
+
+5. Bunsen burner provided with "peep-flame" by-pass.
+
+6. Porcelain trough holding five or six hanging-drop slides (Fig. 62).
+
+[Illustration: FIG. 62.--Hanging-drop slides: a, Double cell seen from
+above; b, single cell seen from the side.]
+
+The best form of hanging-drop slide is a modification of Boettcher's
+glass ring slide, and is prepared by cementing a circular cell of tin,
+13 to 15 mm. diameter, and 1 to 2 mm. in height, to the centre of a 3 by
+1 slip by means of Canada balsam. It is often extremely convenient to
+have two of these cells cemented close together on one slide (Fig. 62,
+a).
+
+     Another form of hanging-drop slide is made in which a
+     circular or oval concavity or "cell" is ground out of the
+     centre of a 3 by 1 slip. These are more expensive, less
+     convenient to work with, and are more easily contaminated by
+     drops of material under examination, and should be carefully
+     avoided.
+
+7. Three aluminium rods (Fig. 63), each about 25 cm. long and carrying a
+piece of 0.015 gauge platino-iridium wire 7.5 cm. in length. The end of
+one of the wires is bent round to form an oval loop, of about 1 mm. in
+its short diameter, and is termed a loop or an oese; the terminal 3 or 4
+mm. of another wire is flattened out by hammering it on a smooth iron
+surface to form a "spatula"; the third is left untouched or is pointed
+by the aid of a file. These instruments are used for inoculating culture
+tubes and preparing specimens for microscopical examination.
+
+[Illustration: FIG. 63.--Ends of platinum rods. a, loop; b, spatula;
+c, needle.]
+
+The method of mounting these wires may be described as follows:
+
+Take a piece of aluminium wire 25 cm. long and about 0.25 cm. in
+diameter, and drill a fine hole completely through the wire about a
+centimetre from one end. Sink a straight narrow channel along one side
+of the wire, in its long axis, from the hole to the nearest end, shallow
+at first, but gradually becoming deeper.
+
+On the opposite side of the wire make a short cut, 2 mm. in length,
+leading from the hole in the same direction. [The use of a fine dental
+drill and small circular saw, worked by a dental motor facilitates the
+manufacture of these aluminium handled instruments.]
+
+Now pass one end of the platinum wire through the hole, turn up about 2
+mm. at right angles and press the short piece into the short cut. Turn
+the long end of the wire sharply, also at right angles, and sink it into
+the long channel so that it emerges from about the centre of the cut end
+of the aluminium wire (Fig. 63). A few sharp taps with a watch maker's
+hammer will now close in the sides of the two channels over the wire and
+hold it securely.
+
+[Illustration: FIG. 64.--Platinum rod in aluminium handle--method of
+mounting.
+
+The platinum wire may be fused into the end of a piece of glass rod, but
+such a handle is vastly inferior to aluminium and is not to be
+recommended.]
+
+8. Two pairs of sharp-pointed spring forceps (10 cm. long), one of which
+must be kept perfectly clean and reserved for handling clean
+cover-slips, the other being for use during staining operations.
+
+9. A box of clean 3 by 1 glass slips.
+
+10. A glass capsule with tightly fitting (ground on) glass lid,
+containing clean cover-slips in absolute alcohol.
+
+11. One of Faber's "grease pencils" (yellow, red, or blue) for writing
+on glass.
+
+12. A wooden rack (Fig. 65) with twelve drop-bottles (Fig. 66) each 60
+c.c. capacity, containing
+
+    Aniline water.
+
+    Gentian violet, saturated alcoholic solution.
+
+    Lugol's (Gram's) iodine.
+
+    Absolute alcohol.
+
+    Methylene-blue, }
+    Fuchsin, basic, } saturated alcoholic solution.
+
+    Neutral red, 1 per cent. aqueous solution.
+
+    Leishman's modified Romanowsky stain.
+
+    Carbolic acid, 5 per cent. aqueous solution.
+
+    Acetic acid, 1 per cent. solution.
+
+    Sulphuric acid, 25 per cent. solution.
+
+    Xylol.
+
+[Illustration: FIG. 65.--Staining rack, rubber change mat and lysol
+pot.]
+
+[Illustration: FIG. 66.--Drop bottle.]
+
+[Illustration: FIG. 67.--Canada balsam pot.]
+
+And two pots with air-tight glass caps (Fig. 67), each provided with a
+piece of glass rod and filled respectively with Canada balsam dissolved
+in xylol, and sterile vaseline.
+
+
+METHODS OF EXAMINATION.
+
+Bacteria, etc., are examined microscopically.
+
+    1. In the living state, unstained, or stained.
+    2. In the "fixed" condition (i. e., fixed, killed,
+       and stained by suitable methods).
+
+The preparation of a specimen from a tube cultivation for examination by
+these methods may be described as follows:
+
+~1. Living, Unstained.~--(a) _"Fresh" Preparation._--
+
+1. Clean and dry a 3 by 1 glass slip and place it on one of the squares
+of filter paper. Deposit a drop of water (preferably distilled) or a
+drop of 1 per cent. solution of caustic potash, on the centre of the
+slip, by means of the platinum loop.
+
+[Illustration: FIG. 68.--Holding tubes for removing bacterial growth, as
+seen from the front.]
+
+     TECHNIQUE OF OPENING AND CLOSING A CULTURE TUBE.
+
+     2. Remove the tube cultivation from its rack or jar with the
+     left hand and ignite the cotton-wool plug by holding it to
+     the flame of the Bunsen burner. Extinguish the flame by
+     blowing on the plug, whilst rotating the tube on its long
+     axis, its mouth directed vertically upward, between the
+     thumb and fingers. (This operation is termed "flaming the
+     plug," and is intended to destroy any micro-organisms that
+     may have become entangled in the loose fibres of the
+     cotton-wool, and which, if not thus destroyed, might fall
+     into the tube when the plug is removed and so accidentally
+     contaminate the cultivation.)
+
+     3. Hold the tube at or near its centre between the ends of
+     the thumb and first two fingers of the left hand, and allow
+     the sealed end to rest upon the back of the hand between the
+     thumb and forefinger, the plug pointing to the right. Keep
+     the tube as nearly in the horizontal position as is
+     consistent with safety, to diminish the risk of the
+     accidental entry of organisms (Fig. 68).
+
+     4. Take the handle of the loop between the thumb and
+     forefinger of the right hand, holding the instrument in a
+     position similar to that occupied by a pen or a paint-brush,
+     and sterilise the platinum portion by holding it in the
+     flame of a Bunsen burner until it is red hot. Sterilise the
+     adjacent portion of the aluminium handle by passing it
+     rapidly twice or thrice through the flame. After sterilising
+     it, the loop must not be allowed to leave the hand or to
+     touch against anything but the material it is intended to
+     examine, until it is finished with and has been again
+     sterilised.
+
+     5. Grasp the cotton-wool plug of the test-tube between the
+     little finger and the palm of the right hand (whilst still
+     holding the loop as directed in step 4), and remove it from
+     the mouth of the tube by a "screwing" motion of the right
+     hand.
+
+     6. Introduce the platinum loop into the tube and hold it in
+     this position until satisfied that it is quite cool. (The
+     cooling may be hastened by touching the loop on one of the
+     drops of moisture which are usually to be found condensed on
+     the interior of the glass tube, or by dipping it into the
+     condensation water at the bottom; at the same time care must
+     be taken in the case of cultures on solid media to avoid
+     touching either the medium or the growth.)
+
+     7. Remove a small portion of the growth by taking up a drop
+     of liquid, in the case of a fluid culture, in the loop; or
+     by touching the loop on the surface of the growth when the
+     culture is on solid medium; and withdraw the loop from the
+     tube without again touching the medium or the glass sides of
+     the tube.
+
+     8. Replace the cotton-wool plug in the mouth of the tube.
+
+9. Replace the tube cultivation in its rack or jar.
+
+10. Mix the contents of the loop thoroughly with the drop of water on
+the 3 by 1 slide.
+
+11. Again sterilise the loop as directed in step 4, and replace it in
+its stand.
+
+12. Remove a cover-slip from the glass capsule by means of the
+cover-slip forceps, rest it for a moment on its edge, on a piece of
+filter paper to remove the excess of alcohol, then pass it through the
+flame of the Bunsen burner. This burns off the remainder of the alcohol,
+and the cover-slip so "flamed" is now clean, dry, and sterile.
+
+13. Lower the cover-slip, still held in the forceps, on to the surface
+of the drop of fluid on the 3 by 1 slip, carefully and gently, to avoid
+the inclusion of air bubbles.
+
+14. Examine microscopically (_vide infra_).
+
+During the microscopical examination, stains and other reagents may be
+run in under a cover-slip by the simple method of placing a drop of the
+reagent in contact with one edge of the cover-glass and applying the
+torn edge of a piece of blotting paper to the opposite side. The reagent
+may then be observed to flow across the field and come into contact with
+such of the micro-organisms as lie in its path.
+
+The non-toxic basic dyes most generally employed for the intra-vitam
+staining of bacteria are
+
+    Neutral red,    }
+    Quinoleine blue }
+    Methylene green }  in 0.5 per cent. aqueous solutions.
+    Vesuvin,        }
+
+_Negative Stain_ (Burri).--By this method of demonstration the
+appearances presented by dark ground illumination (by means of a
+paraboloid condenser) are closely simulated, since minute particles,
+bacteria, blood or pus cells etc. stand out as brilliantly white or
+colourless bodies on a dark grey-brown background.
+
+_Reagent required:_
+
+Any one of the liquid waterproof black drawing inks (Chin-chin, Pelican,
+etc.). This is prepared for use as follows:
+
+Measure out and mix:
+
+    Liquid black ink,       25 c.c.
+    Tincture of iodine       1 c.c.
+
+Allow the mixture to stand 24 hours, centrifugalise thoroughly, pipette
+off the supernatant liquid to a clean bottle and then add a crystal of
+thymol or one drop of formalin as a preservative.
+
+METHOD.--
+
+1. With the sterilised loop deposit one drop of the liquid ink close to
+one end of a 3 by 1 slide.
+
+2. With the sterilised loop deposit a drop of the fluid culture (or of
+an emulsion from a solid culture) by the side of the drop of ink (Fig.
+69, a); mix the two drops thoroughly by the aid of the loop.
+
+3. Sterilise the loop.
+
+4. Hold the slide firmly on the bench with the thumb and forefinger of
+the left hand applied to the end nearest the drop of fluid.
+
+5. Take another clean 3 by 1 slide in the right hand and lower its short
+end obliquely (at an angle of about 60 deg.) transversely on to the mixed
+ink and culture on the first slide, and allow the fluid to spread across
+the slide and fill the angle of incidence.
+
+6. Maintaining the original angle, draw the second slide firmly and
+evenly along the first toward the end farthest from the left hand (Fig.
+69, b).
+
+7. Throw the second slide into a pot of disinfectant; allow the first
+slide to dry in the air.
+
+[Illustration: FIG. 69.--Spreading negative film.]
+
+8. Place a drop of immersion oil on the centre of the film, lower the
+1/12-inch objective into the oil and examine microscopically without the
+intervention of a cover-slip.
+
+(The film of ink may be covered with a long cover-glass and xylol balsam
+as a permanent preparation.)
+
+(<b) _Hanging-drop Preparation._--
+
+1. Smear a layer of sterile vaseline on the upper surface of the ring
+cell of a hanging-drop slide by means of the glass rod provided with the
+vaseline bottle, and place the slide on a piece of filter paper.
+
+2. "Flame" a cover-slip and place it on the filter paper by the side of
+the hanging-drop slide.
+
+3. Place a drop of water on the centre of the cover-slip by means of the
+platinum loop.
+
+4. Obtain a small quantity of the material it is desired to examine, in
+the manner detailed above (pages 74-76, steps 2 to 11 must be followed
+in their entirety and with the strictest exactitude whenever tube
+contents are being handled), and mix it with the drop of water on the
+cover-slip.
+
+5. Raise the cover-slip in the points of the forceps and rapidly invert
+it on to the ring cell of the hanging-drop slide, so that the drop of
+fluid occupies the centre of the ring. (Carefully avoid contact between
+the drop of fluid and either the ring cell or the layer of vaseline.
+Should this happen, the now _infected_ hanging-drop slide and its
+cover-slip must be dropped into the pot of lysol and a new preparation
+made.)
+
+6. Press the cover-slip firmly down into the vaseline on to the top of
+the ring cell. (This spreads out the vaseline into a thin layer, and
+besides ensuring the adhesion of the cover-slip, seals the cells and so
+retards evaporation.)
+
+7. Examine microscopically.
+
+The examination of a "fresh" specimen or a "hanging-drop" preparation is
+directed to the determination of the following data:
+
+1. The nature of the bacteria present--e. g., cocci, bacilli, etc.
+
+2. The purity of the cultivation; this can only be determined when gross
+morphological differences exist between the organisms present.
+
+3. The presence or absence of spores; when present, spores show their
+typical refrangibility exceedingly well by this method.
+
+4. The presence or absence of mobility. In a hanging-drop specimen some
+form of movement can practically always be observed, and its character
+must be carefully determined by noting the relative positions of
+adjacent micro-organisms.
+
+(a) Brownian or molecular movement. Minute particles of solid matter
+(including bacteria), when suspended in a fluid, will always show a
+vibratory movement affecting the entire field, but never altering the
+relative positions of the bacteria. (Cocci exhibit this movement, but
+with the exception of the Micrococcus agilis, the cocci are non-motile.)
+
+(b) Streaming movement. This is due to currents set up in the hanging
+drop as a result of jarring of the specimen or of evaporation, or to the
+fact that the cover-slip is not perfectly level, and although the
+relative positions of the bacteria may vary, still the flowing movement
+of large numbers of organisms in some one direction will usually be
+sufficient to demonstrate the nature of this motion.
+
+(c) Locomotive movement, or ~true motility~, is determined by observing
+some one particular bacillus changing its position in the field
+independently of, and in a direction contrary to, other organisms
+present.
+
+When the examination is completed and the specimen finished with, the
+"fresh specimen"--i. e., the slide with the cover-slip attached--must
+be dropped into the lysol pot. In the hanging-drop specimen, however,
+the cover-slip only is infected, and this may be raised from the ring
+cell by means of forceps and dropped into the disinfectant.
+
+_Permanent Staining of the Hanging-drop Specimen._--Occasionally it is
+necessary to fix and stain a hanging-drop preparation. This may be done
+as follows:
+
+1. Remove the cover-slip from the cell by the aid of the forceps.
+
+2. If the drop is small, fix it by dropping it face downward, whilst
+still wet, on to the surface of some Gulland's solution or corrosive
+sublimate solution (_vide_ page 82) in a watch-glass. If the drop is
+large, place it face upward on the rubber mat, cover it with an inverted
+watch-glass, and allow it to dry. Then fix it in the alcohol and ether
+solution (_vide_, page 82).
+
+3. Dip the cover-glass into a beaker containing hot water in order to
+remove some of the vaseline adhering to it.
+
+4. Wash successively in alcohol, xylol, ether, and alcohol, to remove
+the last traces of grease.
+
+5. Wash in water.
+
+6. Stain, wash, dry, and mount as for an ordinary cover-slip film
+preparation (_vide_ pages 83-85).
+
+~2. Killed, Stained.~--In this method three distinct processes are
+necessary:
+
+    "Preparing" and "fixing" the film.
+    Staining.
+    Mounting.
+
+_Preparing the Film._--
+
+1. Flame a cover-slip and place it on a piece of filter paper.
+
+2. Place a drop of water on the centre of the cover-slip by means of
+platinum loop.
+
+3. Obtain a small quantity of the material to be examined upon a
+sterilised platinum loop (see pages 74-76, steps 2 to 11) and mix it
+with the drops of water on the cover-slip.
+
+4. Spread the drop of emulsion evenly over the cover-slip in the form of
+a square film to within 1 mm. of each edge of the cover-slip.
+
+5. Allow it to dry completely in the air.
+
+_Fixing._--Fix by passing the cover-slip, held in the fingers, three or
+four times through the flame of a Bunsen burner.
+
+In some instances (e. g., when the films after staining are intended
+for micrometric observations) it is almost essential to fix by exposure
+to a uniform temperature of 115 deg. C., for twenty minutes. This is best
+done in a carefully regulated hot-air oven.
+
+Fixation may also be effected by immersing in some fixative fluid, such
+as one of the following:
+
+1. Absolute alcohol, for five to fifteen minutes.
+
+                         { equal parts, for five to thirty
+    2. Absolute alcohol, { minutes (e. g., for blood or
+    Ether,               { milk).
+
+3. Osmic acid, 1 per cent. aqueous solution, for thirty seconds.
+
+4. Corrosive sublimate, saturated aqueous solution, for five minutes.
+
+5. Corrosive sublimate (Lang), for five minutes. This solution is
+prepared by dissolving:
+
+      Sodium chloride           0.75 gramme
+      Hydrarg. perchloride     12.00 grammes
+      Acetic acid               5.00 grammes
+      In distilled water      100.00 c.c.
+    Filter.
+
+6. Gulland's solution, for five minutes. This solution is prepared by
+mixing:
+
+    Absolute alcohol                                            25.0 c.c.
+    Ether                                                       25.0 c.c.
+    Corrosive sublimate, 20 per cent. alcoholic solution         0.4 c.c.
+
+7. Formalin 10 per cent. aqueous solution (= 4 per cent. aqueous
+solution of formaldehyde since formalin is a 40 per cent. solution of
+the gas in water).
+
+Either of these methods of fixation coagulates the albuminous material
+and ensures perfect adhesion of the film to the cover-slip.
+
+_Clearing._--Wash the cover-slip thoroughly in running water and proceed
+with the staining.
+
+If the film has been prepared from broth, liquefied gelatine, or pus or
+other morbid exudations, saturate the film after fixation with acetic
+acid 2 per cent. and allow it to act for two minutes.
+
+Wash with alcohol, then let the alcohol remain on the cover-slip for two
+minutes. (This will "clear" the groundwork and give a much sharper and
+cleaner film than would otherwise be obtained.)
+
+If the film has been prepared from blood or bloodstained fluid, treat
+with acetic acid 2 per cent. for two minutes after fixation. Wash with
+water, dry, and proceed with the staining. (This will remove the
+haemoglobin and facilitate examination.)
+
+_Staining._--
+
+1. Rest the cover-slip, film side uppermost, on the rubber mat.
+
+2. By means of a drop-bottle, cover the film side of the cover-slip with
+the selected stain, allow it to act for a few minutes, then wash off the
+excess in running water.
+
+The penetrating power of stains is increased by (a) physical
+means--e. g., heating the stain; (b) chemical means--e. g., by the
+addition of carbolic acid, 5 per cent. aqueous solution; caustic
+alkalies, 2 per cent. aqueous solutions; water saturated with aniline
+oil; borax, 0.5 per cent. aqueous solution.
+
+The most commonly used dyes for cover-slip film preparations are the
+aniline dyes.
+
+    (A) Basic:
+      (a) Methylene-blue.
+      (b) Gentian violet.
+      (c) Fuchsin.
+
+These dyes are kept in saturated alcoholic (90 per cent.) solutions so
+that decomposition may be retarded.
+
+Two or three drops of alcoholic solution of these dyes to, say, 4 c.c.
+water, usually makes a sufficiently strong staining fluid for cover-slip
+film preparations.
+
+Carbolic methylene-blue (C.M.B.) and carbol fuchsin (C.F.) are prepared
+by covering the cover-slip with 5 per cent. solution of carbolic acid
+and adding a few drops of the saturated alcoholic solution of
+methylene-blue or fuchsin respectively to it. For aniline gentian violet
+(A.G.V.) the stain is added to a saturated solution of aniline oil in
+water.
+
+      (d) Thionine blue.
+      (e) Bismarck brown.
+      (f) Neutral red.
+    (B) Acid:
+      (a) Eosin, aqueous yellowish.
+      (b) Safranine.
+
+These dyes are kept in 1 per cent. aqueous solution to which is added 5
+per cent. of alcohol, as a preservative. They are generally used in this
+form.
+
+A few nuclear stains (carmine, haematoxylin) are occasionally used more
+especially in "section" work.
+
+_Decolourisation._--After overstaining, films may be decolourised by
+washing for a longer or shorter time in one of the following reagents
+arranged in ascending order of power
+
+1. Water.
+2. Chloroform.
+3. Acetic acid, 1 per cent.
+4. Alcohol.
+5. Alcohol absolute,         }   equal parts.
+   Acetic acid, 1 per cent., }
+
+                             {Hydrochloric, 1 per cent. aqueous solution.
+                             {Hydrochloric, 1 per cent. Alcoholic
+                             {  (90 per cent.) solution.
+6. Mineral acids:            {Sulphuric, 25 per cent. aqueous solution.
+                             {Nitric, 33 per cent. aqueous solution.
+
+_Counterstaining._--Use colours which will contrast with the first
+stain; e. g.,
+
+Vesuvin,                     }
+Neutral red,                 }for films stained by methylene-blue or
+Eosin,                       }Gram's method.
+Fuchsin,                     }
+
+Methylene-blue,              }for films stained by fuchsin.
+Gentian violet,              }
+
+8. _Mounting._--
+
+1. Wash the film carefully in running water.
+
+2. Blot off the superfluous water with the filter paper, or dry more
+completely between two folds of blotting paper.
+
+3. Complete the drying in the air, or by holding the cover-slip in the
+fingers at a safe distance above the flame of the Bunsen burner.
+
+4. Place a drop of xylol balsam on the centre of a clean 3 by 1 glass
+slide and invert the cover-slip over the balsam, and lower it carefully
+to avoid the inclusion of air bubbles.
+
+     NOTE.--Xylol is used in preference to chloroform to dissolve
+     Canada balsam, as it does not decolourise the specimen.
+
+~Impression films~ (_Klatschpraeparat_) are prepared from isolated
+colonies of bacteria in order that their characteristic formation may be
+examined by higher powers than can be brought to bear on the living
+cultivation. They are prepared from plate cultivations (_vide_ page 230)
+in the following manner.
+
+1. Remove a clean cover-slip from the alcohol pot with sterile forceps
+and burn off the spirit.
+
+2. Open the plate and rest one edge of the cover-slip on the surface of
+the medium a little to one side of the selected colony. Lower it
+cautiously over the colony until horizontal. Avoid any lateral movement
+or the inclusion of bubbles of air.
+
+3. Make gentle vertical pressure on the centre of the cover-slip with
+the points of the forceps to ensure perfect contact with the colony.
+
+4. Steady one edge of the cover-slip with the forceps and pass the point
+of a mounted needle just under the opposite edge and raise the
+cover-slip carefully; the colony will be adherent to it. When nearly
+vertical, grasp the cover-slip with the forceps and remove it from the
+plate. Re-cover the plate.
+
+5. Place the cover-slip, film uppermost, on the rubber mat, and cover
+it with an inverted watch-glass until dry.
+
+6. Fix by immersing in one of the fixing fluids previously mentioned
+(_vide_ page 82).
+
+7. Clear with acetic acid and alcohol.
+
+8. Stain and mount as an ordinary cover-slip film preparation, being
+careful to perform all washing operations with extreme gentleness.
+
+~Microscopical Examination of the Unstained Specimens.~--
+
+1. Place the body tube of the microscope in the vertical position.
+
+2. Arrange the hanging-drop slide on the microscope stage so that the
+drop of fluid is in the optical axis of the instrument, and secure it in
+that position by means of the spring clips.
+
+3. Use the 1/6-inch objective, rack down the body tube until the front
+lens of the objective is almost in contact with the cover-slip--that is,
+well within its focal distance. This is best done whilst bending down
+the head to one side of the microscope, so that the eyes are on a level
+with the stage.
+
+4. Apply the eye to the ocular and adjust the plane mirror to the
+position which secures the best illumination.
+
+5. Rack the condenser down slightly and cut down the aperture of the
+iris diaphragm so that the light, although even, is dim.
+
+6. Rack up the body tube by means of the coarse adjustment until the
+bacteria come into view; then focus exactly by means of the fine
+adjustment.
+
+Some difficulty is often experienced at first in finding the hanging
+drop, and if the first attempt is unsuccessful, the student must not on
+any account, whilst still applying his eye to the ocular, rack the body
+tube down (for by so doing there is every likelihood of the front lens
+of the objective being forced through the cover-glass, and not only
+spoiling the specimen, but also contaminating the objective); but, on
+the contrary, withdraw his eye, rack the tube up, and commence again
+from step 2.
+
+
+~Dark Ground Illumination.~--
+
+1. Set up the microscope stand in the vertical position and insert the
+highest eyepiece available.
+
+2. Remove the nosepiece from the microscope tube and fit the 2/3 inch
+objective in place.
+
+3. Remove the substage condenser and replace it by the dark ground
+condenser.
+
+4. Fit up the source of illumination some 30-50 cm. distant from the
+microscope. (This should be the Liliput Arc Lamp (Leitz), Nernst Lamp or
+incandescent gas lamp; if either of the two latter are employed, a
+bull's eye condenser to produce parallel rays must be interposed between
+light and microscope); and adjust illuminant and microscope so that the
+substage plane mirror is completely filled with light.
+
+5. Focus the two concentric rings engraved upon the upper surface of the
+condenser and centre them accurately by means of the centring screws.
+
+6. Prepare a "fresh" specimen (see pages 74-76) of the material it is
+desired to observe, using selected, new, 3 by 1 glass slips of less than
+1 mm. thickness, and No. 1 cover-glasses (0.17 mm. thick), which should
+be cleaned with a piece of soft washleather and not with the emery
+paper, as scratches on the glass produce haziness in the preparation.
+
+7. Deposit a large drop of immersion oil (or pure water) on the upper
+surface of the condenser and rack it down a few millimetres.
+
+8. Adjust the fresh preparation on the microscope stage and fasten it in
+position with the stage clips.
+
+9. Rack up the condenser until the immersion fluid makes contact with
+the under surface of the slide; avoid the formation of air bubbles.
+
+10. Adjust the substage mirror so that the light is reflected upward. A
+bright spot will be seen on the fresh preparation near the centre of the
+field.
+
+11. Replace the 2/3-inch objective by the 1/12-inch oil immersion lens
+which has been fitted with the special stop to reduce its N. A.; place a
+drop of immersion oil upon the centre of the cover-glasses of the fresh
+preparation and lower the microscope tube until the front lens of the
+objective has entered the oil drop.
+
+12. Focus the bright spot referred to in step 10. If it no longer
+occupies the centre of the field, alter the angle of the substage mirror
+until it does.
+
+13. Now focus the lens accurately on the film, cautiously vary the
+height of the dark ground condenser until the best position is found.
+The intensely illuminated bacteria will stand out in vivid contrast to
+the dark background.
+
+[Illustration: FIG. 70.--Immersion oil bottle.]
+
+~Microscopical Examination of the Stained Specimen.~--(The body tube of
+the microscope may be vertical or inclined to an angle.)
+
+1. Secure the slide on the stage of the microscope by means of the
+spring clips.
+
+2. Place a drop of cedarwood oil on the centre of the cover-slip.
+
+     The immersion oil is pure cedarwood oil, and is kept in a
+     small bottle of stout glass (Fig. 70), the cavity of which
+     is shaped like an inverted cone, and is provided with a
+     safety funnel (so that the oil does not escape if the bottle
+     is accidentally overturned) and a dust cap of boxwood fitted
+     with a wooden rod with which the drop of oil is applied to
+     the cover-glass or lens.
+
+3. Use the 1/12-inch oil immersion lens of the microscope. Rack down the
+body tube till the front lens of the objective is in contact with the
+oil and nearly touching the cover-slip.
+
+4. Rack up the condenser until it is in contact with the under surface
+of the slide.
+
+5. Apply the eye to the ocular and arrange the plane mirror so as to
+obtain the greatest possible amount of light.
+
+6. Rack up the body tube until the stained film comes into view.
+
+7. Focus the condenser accurately on the film.
+
+8. Focus the film accurately by means of the fine adjustment.
+
+
+
+
+VI. STAINING METHODS.
+
+
+In the following pages are collected the various "stock" stains in
+everyday use in the bacteriological laboratory, together with a
+selection of the most convenient and generally useful staining methods
+for demonstrating particular structures or differentiating groups of
+bacteria. The stains employed should either be those prepared by
+Gruebler, of Leipzig, or Merck, of Darmstadt. The methods printed in
+ordinary type are those which a long experience has shown to be the most
+reliable, and to give the best results--those relegated to small type
+comprise such as are not so generally useful, but give excellent results
+in the hands of the experienced worker.
+
+
+BACTERIA STAINS.
+
+~Methylene-blue.~--
+
+1. _Saturated Aqueous Solution._
+
+Weigh out
+
+    Methylene-blue         1.5 grammes
+
+Place in a stoppered bottle having a capacity of from 150 to 200 c.c.
+and add
+
+    Distilled water      100.0 c.c.
+
+Allow the water to remain in contact with the dye for two weeks, shaking
+the contents of the bottle vigourously for a few moments every day.
+Filter.
+
+2. _Saturated Alcoholic Solution._
+
+Weigh out
+
+    Methylene-blue          1.5 grammes
+
+Place in a stoppered bottle of 150 c.c. capacity and add
+
+    Alcohol, 90 per cent         100.0 c.c.
+
+Allow the alcohol to remain in contact with the dye for two hours,
+shaking vigourously every few minutes. Filter.
+
+3. _Carbolic Methylene-blue_ (Kuehne).
+
+Weigh out
+
+    Methylene-blue       1.5 grammes
+    Carbolic acid        5.0 grammes
+
+and dissolve in
+
+    Distilled water    100.0 c.c.
+
+and add
+
+    Absolute alcohol    10.0 c.c.
+
+Filter.
+
+4. _Alkaline Methylene-blue_ (Loeffler).
+
+Measure out and mix
+
+    Methylene-blue, saturated alcoholic solution       30.0 c.c.
+    Caustic potash, 0.1 per cent. aqueous solution    100.0 c.c.
+
+Filter.
+
+~Gentian Violet.~--
+
+5. _Saturated Aqueous Solution._
+
+Weigh out
+
+    Gentian violet      2.25 grammes
+
+and proceed as in preparing the corresponding solution of
+methylene-blue.
+
+6. _Saturated Alcoholic Solution._
+
+Weigh out
+
+    Gentian violet              5.0 grammes
+
+and proceed as in preparing the corresponding solution of
+methylene-blue.
+
+7. _Carbolic Gentian Violet_ (Nicolle).
+
+Measure out and mix
+
+    Gentian violet, saturated alcoholic solution   10.0 c.c.
+    Carbolic acid, 1 per cent. aqueous solution   100.0 c.c.
+
+Filter.
+
+8. _Anilin Water Solution_ (Koch-Ehrlich).
+
+Measure out
+
+    Distilled water           100 c.c.
+
+Add anilin oil drop by drop (shaking well after the addition of each
+drop) until the solution is opaque.
+
+Filter until clear.
+
+and add
+
+    Absolute alcohol                             10 c.c.
+    Saturated alcoholic solution gentian violet  11 c.c.
+
+Filter.
+
+     NOTE.--This solution will not keep longer than 14 days.
+
+~Thionine Blue (or Lauth's Violet).~--
+
+9. _Carbolic Thionine Blue_ (Nicolle).
+
+Weigh out
+
+    Thionine blue            1.0 gramme
+    Carbolic acid            2.5 grammes
+
+and dissolve in
+
+    Distilled water        100.0 c.c.
+
+Filter.
+
+Before use dilute with equal quantity of distilled water and again
+filter.
+
+~Fuchsin (Basic).~--
+
+10. _Saturated Aqueous Solution._
+
+Weigh out
+
+    Basic fuchsin             1.5 grammes
+
+and proceed as in preparing the corresponding solution of methylene-blue
+(_q. v._).
+
+11. _Saturated Alcoholic Solution._
+
+Weigh out
+
+    Basic fuchsin   3.5 grammes
+
+and proceed as in preparing the corresponding solution of
+methylene-blue.
+
+12. _Carbolic Fuchsin_ (Ziehl).
+
+Weigh out
+
+    Basic fuchsin     1.0 gramme
+    Carbolic acid     5.0 grammes
+
+dissolve in
+
+    Distilled water   100.0 c.c.
+
+and add
+
+    Absolute alcohol   10.0 c.c.
+
+Filter.
+
+
+CONTRAST STAINS.
+
+~Eosin.~--There are several commercial varieties of eosin, which, from the
+bacteriological point of view, possess very different values. Gruebler
+lists four varieties, of which two only are useful for bacteriological
+work:
+
+    Eosin, aqueous yellowish.
+    Eosin, aqueous bluish.
+
+13. _Eosin Aqueous Solution_ (Yellowish or Bluish Shade), 1 per cent.
+
+Weigh out
+
+    Eosin, aqueous   1.0 gramme
+
+dissolve in
+
+    Distilled water   100.0 c.c.
+
+and add
+
+    Absolute alcohol   5.0 c.c.
+
+Filter.
+
+14. _Eosin Alcoholic Solution_, 0.5 per cent.
+
+Weigh out
+
+    Eosin, alcoholic      0.5 gramme
+
+and dissolve in
+
+    Alcohol (70 per cent.)      100.0 c.c.
+
+Filter.
+
+~Safranine.~--
+
+15. _Aqueous Solution._
+
+Weigh out.
+
+    Safranine                  0.5 gramme
+
+and dissolve in
+
+    Distilled water      100.0 c.c.
+
+Filter.
+
+~Neutral Red.~--
+
+16. _Aqueous Solution._
+
+Weigh out
+
+    Neutral red      1.0 gramme
+
+and dissolve in
+
+    Distilled water      100.0 c.c.
+
+Filter.
+
+~Vesuvin (or Bismarck Brown).~--
+
+17. _Saturated Aqueous Solution._
+
+Weigh out
+
+    Vesuvin      0.5 gramme
+
+and dissolve in
+
+    Distilled water      100.0 c.c.
+
+Filter.
+
+
+TISSUE STAINS.
+
+
+~Aniline Gentian Violet~ (For Weigert's Fibrin Stain).--
+
+Weigh out
+
+    Gentian violet      1.0 gramme
+
+and dissolve in
+
+    Absolute alcohol         15.0 c.c.
+    Distilled water          80.0 c.c.
+
+then add
+
+    Aniline oil               3.0 c.c.
+
+Shake well and filter before use.
+
+
+~Haematoxylin~ (Ehrlich).--
+
+1. Weigh out
+
+    Haematoxylin               2.0 grammes
+
+and dissolve in
+
+    Absolute alcohol        100.0 c.c.
+
+2. Weigh out
+
+    Ammonium alum             2.0 grammes
+
+and dissolve in
+
+    Distilled water         100.0 c.c.
+
+3. Mix 1 and 2, allow the mixture to stand forty-eight hours, then
+filter.
+
+4. Add
+
+    Glycerine                85.0 c.c.
+    Acetic acid, glacial     10.0 c.c.
+
+5. Allow the stain to stand for one month exposed to light; then filter
+again ready for use.
+
+
+~Haematin~ (Mayer's).--
+
+A. Weigh out
+
+    Haematin                   1.0 gramme
+
+and dissolve in
+
+    Alcohol 90 per cent. (warmed to 37 deg. C.)      50 c.c.
+
+B. Weigh out
+
+    Potash alum       50 grammes
+
+and dissolve in
+
+    Distilled water      100 c.c.
+
+Prepare these two solutions in separate flasks. Take a clean flask of
+250 c.c. capacity and insert a large funnel in its neck. Pour the
+solutions A and B simultaneously and slowly into the funnel to mix
+thoroughly. Store for future use.
+
+     NOTE.--If acid haematin is required, introduce glacial acetic
+     acid (3 c.c.) into the mixing flask before adding the
+     solutions A and B.
+
+
+~Alum Carmine~ (Mayer).--
+
+Weigh out
+
+    Alum         2.5 grammes
+    Carmine      1.0 gramme
+
+and place in a glass beaker.
+
+Measure out in a measuring cylinder,
+
+    Distilled water      100.0 c.c.
+
+Place the beaker on a sand-bath, add the water in successive small
+quantities, and keep the mixture boiling for twenty minutes. Measure the
+solution and make up to 100 c.c. by the addition of distilled water.
+Filter.
+
+
+~Lithium Carmine~ (Orth).--
+
+Weigh out
+
+    Carmine      2.5 grammes
+
+and dissolve in
+
+    Lithium carbonate, cold saturated solution      100.0 c.c.
+
+Filter.
+
+
+~Picrocarmine.~--
+
+Weigh out
+
+    Picrocarmine      2.0 grammes
+
+and dissolve in
+
+    Distilled water      100.0 c.c.
+
+
+BLOOD STAINS
+
+When watery solutions of medicinal methylene blue and water soluble
+eosins are mixed a precipitate is formed which is soluble only in
+alcohol, and solutions of this precipitate impart a peculiar
+reddish-purple colour to chromatin. This compound was first used by
+Romanowsky to demonstrate malarial parasites, but various modifications
+are now employed for staining blood films generally, and also for
+bacteria and protozoa. The best modifications of the original Romanowsky
+are those of Jenner and Leishman--Jenner being most suitable for the
+histological study of the blood, and Leishman for the demonstration of
+protozoa.
+
+
+~Jenner's Stain.~--
+
+A. Weigh out:
+
+    Eosin aqueous yellow      6.0 grammes
+
+Dissolve in
+
+    Distilled water (non-alkaline)      250 c.c.
+
+This will make a thick solution.
+
+B. Weigh out:
+
+    Methylene blue (medicinally pure) Hoechst      5.0 grammes
+
+Dissolve in
+
+    Distilled water (non-alkaline)      250 c.c.
+
+1. Add B to A very slowly, stirring all the time. A viscous precipitate
+forms which frequently loses its viscosity when heat is applied. (This
+explains the necessity of mixing slowly).
+
+2. Evaporate slowly in a porcelain basin, stirring occasionally, on a
+water bath at 55 deg. C. When a paste begins to form scrape and break up
+occasionally. (On no account must the paste be allowed to fuse.)
+
+3. Grind the resulting mass into an amorphous powder.
+
+4. Weigh out:
+
+    Amorphous powder      0.5 grammes
+
+Dissolve in
+
+    Methylic alcohol (Merck's puriss, for analysis)      100 c.c.
+
+Allow time for true solution. (About three days is sufficient.)
+
+METHOD.--
+
+1. Prepare film, dry, but _do not fix_.
+
+2. Flood the unfixed film with the stain, allow it to act for 3 minutes
+(the methylic alcohol of the stain fixes the film).
+
+3. Pour off the stain and wash in distilled water until the film
+presents a pink colour.
+
+4. Dry and mount.
+
+
+~Leishman's Stain.~--
+
+_A._ Weigh out:
+
+    Methylene blue (medicinal)      1 gramme
+
+Dissolve in
+
+    Sodium carbonate, 0.5 per cent. aqueous solution      100 c.c.
+
+Keep at 65 deg. C. for 12 hours in either a hot incubator or a water-bath;
+then stand in dark place at room temperature (20 deg. C.) for ten days.
+
+_B._ Weigh out:
+
+    Eosin, extra B. A.      0.1 gramme
+
+Dissolve in
+
+    Distilled water      100 c.c.
+
+1. Mix the two solutions A and B in equal volumes, and allow the
+mixture to stand for 12 hours with occasional stirring.
+
+2. Filter, and collect precipitate on filter paper.
+
+3. Wash precipitate thoroughly with distilled water, and dry.
+
+4. Weigh out 0.15 gramme of the dried precipitate; rub up in a mortar
+with 5 c.c. of methylic alcohol (Merck's puriss, for analysis).
+
+Allow undissolved powder to settle, then decant the supernatant fluid to
+a clean 100 c.c. measuring cylinder.
+
+5. Add further 5 c.c. alcohol to sediment in mortar and repeat the
+process, and so on until all the sediment has been dissolved.
+
+6. Now make up the fluid in the measuring cylinder to 100 c.c. by the
+addition of more methylic alcohol.
+
+METHOD.--
+
+1. Prepare film, dry, but _do not fix_.
+
+2. Flood the unfixed film with stain, allow it to act 30 seconds.
+
+3. Add double the volume of distilled water to the stain on the film,
+and mix with glass rod or platinum loop.
+
+4. Allow this diluted stain to act five minutes.
+
+5. Wash off with distilled water.
+
+6. Leave some water on film for thirty seconds to intensify the colour
+contrasts.
+
+7. Dry and mount.
+
+
+METHODS OF DEMONSTRATING STRUCTURE OF BACTERIA, ETC.
+
+~To Demonstrate Capsules.~
+
+~1. MacConkey.~--
+
+_Stain._--
+
+Weigh out
+
+    Dahlia                          0.5 gramme
+    Methyl green (00 crystals)      1.5 grammes
+
+rub up in a mortar with
+
+    Distilled water      100.0 c.c.
+
+Add
+
+    Fuchsin, saturated alcoholic solution      10.0 c.c.
+
+and make up to 200 c.c. by the addition of
+
+    Distilled water      90.0 c.c.
+
+Filter.
+
+Allow the stain to stand for two weeks before use; keep in a dark place
+or in an amber glass bottle. Owing to the unstable character of the
+methyl green, this stain deteriorates after about six months.
+
+METHOD.--
+
+1. Prepare and fix film in the usual manner.
+
+2. Flood the cover-slip with the stain and allow it to act for five to
+ten minutes.
+
+3. Wash very thoroughly in water; if necessary, direct a powerful stream
+of water on the film from a wash-bottle.
+
+4. Dry and mount.
+
+     ~2. Muir's Method.~--
+
+     1. Prepare, dry and fix film in the ordinary manner.
+
+     2. Flood the film with carbolic fuchsin, warm until steam
+     begins to rise. Allow the stain to act for thirty seconds.
+
+     3. Wash quickly with methylated spirit.
+
+     4. Wash thoroughly with water.
+
+     5. Subject the film to the action of the following mordant
+     for five seconds:
+
+    Corrosive sublimate, saturated aqueous solution      2 c.c.
+    Tannic acid, 20 per cent. aqueous solution           2 c.c.
+    Potash alum saturated aqueous solution               5 c.c.
+
+     6. Wash thoroughly in water.
+
+     7. Treat with methylated spirit for about sixty seconds.
+     (The preparation should now be pale red.)
+
+     8. Wash thoroughly in water.
+
+     9. Counterstain in methylene blue, aqueous solution thirty
+     seconds.
+
+     10. Wash in water.
+
+     11. Dehydrate in alcohol.
+
+     12. Clear in xylol and mount in xylol balsam.
+
+     ~3. Welch's Method.~--
+
+     1. Prepare and fix film in the usual manner.
+
+     2. Flood the slide with acetic acid 2 per cent.; allow the
+     acid to remain in contact with the film for two minutes.
+     This swells up and fixes the capsule and enables it to take
+     the stain.
+
+     3. Blow off the acetic acid by the aid of a pipette.
+
+     4. Immerse in aniline gentian violet, five to thirty
+     seconds.
+
+     5. Wash in water.
+
+     6. Dry and mount.
+
+     ~4. Ribbert's Method.~--
+
+     _Stain._--
+
+     Measure out and mix:
+
+    Acetic acid, glacial      12.5 c.c.
+    Alcohol, absolute         50.0 c.c.
+    Distilled water          100.0 c.c.
+
+     Warm to 36 deg. C. (e. g., in the "hot" incubator) and
+     saturate with dahlia. Filter.
+
+     METHOD.--
+
+     1. Prepare and fix films in the usual manner.
+
+     2. Cover the film with the stain and allow it to act for one
+     or two seconds only.
+
+     3. Wash thoroughly in water.
+
+     4. Dry and mount.
+
+
+~To Demonstrate Flagella.~
+
+~1. Muir's Modified Pitfield.~--This is the best method and gives the most
+reliable results, for not only is the percentage of successful
+preparations higher than with any other, but the bacilli and flagella
+retain their relative proportions.
+
+     (a) ~Mordant.~--
+
+    Tannic acid, 10 per cent. aqueous solution            10 c.c.
+    Corrosive sublimate, saturated aqueous solution        5 c.c.
+    Alum, saturated aqueous solution                       5 c.c.
+    Carbolic fuchsin (Ziehl)                               5 c.c.
+
+
+
+Mix thoroughly.
+
+A precipitate forms which must be allowed to settle for a few hours.
+
+Decant off the clear fluid into tubes and centrifugalise thoroughly.
+
+This solution is at its best some four or five days after manufacture;
+it keeps for about a couple of weeks, but must be re-centrifugalised
+each time, before use.
+
+(b) _Stain._--
+
+    Alum, saturated aqueous solution                 25 c.c.
+    Gentian violet, saturated alcoholic solution      5 c.c.
+
+Filter.
+
+This stain must be freshly prepared.
+
+METHOD.--The cultivations employed should be smear agar cultures, twelve
+to eighteen hours old if incubated at 37 deg. C, twenty-four to thirty
+hours if incubated at 22 deg. C.
+
+1. Remove a very small quantity of the growth by means of the platinum
+spatula.
+
+2. Emulsify it with a few cubic centimetres of distilled water in a
+watch-glass, by gently moving the spatula to and fro in the water. Do
+not rub up the growth on the side of the watch-glass. Some workers
+prefer to use tap water, others employ normal saline solution, but
+distilled water gives the best emulsion.
+
+3. Spread a thin film of the emulsion on a newly flamed cover-slip,
+using no force, but rather _leading_ the drop over the cover-slip with
+the platinum loop.
+
+4. Allow the film to dry in the air, properly protected from falling
+dust.
+
+5. Fix by passing thrice through the Bunsen flame, holding the
+cover-slip whilst doing so by one corner between the finger and thumb.
+
+6. Pour on the film as much of the mordant as the cover-glass will hold.
+Grasp the cover-slip with the forceps and hold it, high above the flame,
+until steam rises. Allow the steaming mordant to remain in contact with
+the film two minutes.
+
+7. Wash well in water and dry carefully.
+
+8. Pour on the film as much of the stain as the cover-glass will hold.
+Steam over the flame as before for two minutes.
+
+9. Wash well in water.
+
+10. Dry and mount.
+
+     ~2. "Pitfield" Original Method.~--
+
+     (a) _Mordant._--
+
+    Tannic acid     1 gramme
+    Water          10 c.c.
+
+     (b) _Stain._--
+
+    Saturated aqueous solution of alum              10 c.c.
+    Saturated alcoholic solution of gentian violet   1 c.c.
+    Distilled water                                  5 c.c.
+
+     Mix equal parts of a and b before using.
+
+     1. Prepare and fix the film in the manner described above.
+
+     2. Boil the mixture and immerse the cover-slip in it, whilst
+     still hot, for one minute.
+
+     3. Wash in water.
+
+     4. Examine in water; if satisfactory, dry and mount in
+     Canada balsam.
+
+     ~3. MacCrorrie's Method.~--
+
+     _Mordant-Stain._--
+
+     Measure out and mix.
+
+    Night blue, saturated alcoholic solution    10 c.c.
+    Potash alum, saturated aqueous solution     10 c.c.
+    Tannin, 10 per cent. aqueous solution       10 c.c.
+
+     NOTE.--The addition of gallic acid, 0.1 to 0.2 gramme, may
+     improve the solution, but is not necessary.
+
+     METHOD.--
+
+     1. Prepare and fix the films as above.
+
+     2. Pour some of the mordant-stain on the film and warm
+     gently, high above the flame, for two minutes (or place in
+     the "hot" incubator for a like period).
+
+     3. Wash thoroughly in water.
+
+     4. Dry and mount.
+
+     ~4. Loeffler's Method.~--
+
+     (a) _Mordant._--
+
+    Tannic acid, 20 per cent. aqueous solution     10 c.c.
+    Ferrous sulphate, saturated aqueous solution    5 c.c.
+    Haematoxylin solution                           3 c.c.
+    Carbolic acid, 1 per cent. aqueous solution     4 c.c.
+
+     This solution must be freshly prepared.
+
+     _Haematoxylin solution_ is prepared by boiling 1 gramme
+     logwood
+
+with 8 c.c. distilled water, filtering and replacing the loss from
+evaporation.
+
+    _Alternative Mordant_ (Bunge's Mordant).--
+
+    Tannic acid, 20 per cent. aqueous solution       10 c.c.
+    Ferrous sulphate, saturated aqueous solution      5 c.c.
+    Fuchsin, saturated alcoholic solution             1 c.c.
+
+    (b) _Stain._--
+
+    Weigh out
+        Methylene-blue        }
+        Or methylene-violet   } 4 grammes
+        Or fuchsin            }
+
+and dissolve in
+
+    Aniline water, freshly saturated and filtered     100 c.c.
+
+METHOD.--
+
+1. Prepare and fix films as above.
+
+2. Pour the mordant on to the film and warm cautiously over the flame
+till steam rises; keep the mordant gently steaming for one minute.
+
+3. Wash well in distilled water till no more colour is discharged; if
+necessary, wash carefully with absolute alcohol.
+
+4. Filter a few drops of the stain on to the film, warm as before, and
+allow the steaming stain to act for one minute.
+
+5. Wash well in distilled water.
+
+6. Dry and mount.
+
+NOTE.--The flagella of some organisms can be demonstrated better by
+means of an alkaline stain or an acid stain--a point to be determined
+for each. Speaking generally, those bacilli which give rise to an acid
+reaction in the culture medium require an alkali; those which form
+alkali in cultivation require an acid. According to requirements,
+therefore, Loeffler recommends the addition of sodium hydrate, 1 per
+cent. aqueous solution, 1 c.c.; or an equal quantity of an exactly
+comparable solution of sulphuric acid.
+
+~5. Van Ermengem's Method.~--This method, being merely a precipitation of
+a silver salt on the micro-organisms and not a true stain, creates a
+false impression as to the relative proportions of bacteria and
+flagella.
+
+
+    (a) _Fixing Fluid._--
+
+    Osmic acid, 2 per cent. aqueous solution        10 c.c.
+    Tannic acid, 20 per cent. aqueous solution      20 c.c.
+    Acetic acid, glacial                             1 c.c.
+
+     The fixing fluid should be prepared some days before use and
+     filtered as required. In colour it should be distinctly
+     violet.
+
+     (b) _Sensitising Solution._--
+
+     Silver nitrate, 0.5 per cent. aqueous solution.
+
+     This solution must be kept in a dark blue glass bottle or in
+     a dark cupboard.
+
+     Filter immediately before use.
+
+     (c) _Reducing Solution._--
+
+     Weigh out
+
+    Gallic acid                    5 grammes
+    Tannic acid                    3 grammes
+    Potassium acetate, fused      10 grammes
+
+     and dissolve in
+
+    Distilled water           350 c.c.
+
+     Filter.
+
+     This solution will keep active for several days, but fresh
+     solution must be used for each preparation.
+
+     METHOD.--
+
+     1. Prepare emulsion, make and fix films as above in the
+     preceding method, steps 1 to 4.
+
+     2. Pour on the film as much of the fixing solution as the
+     cover-glass will hold, heat carefully over the flame till
+     steam rises, and allow the steaming fixing fluid to act for
+     five minutes.
+
+     3. Wash well in water.
+
+     4. Wash in absolute alcohol.
+
+     5. Wash in distilled water.
+
+     6. Pour some of the sensitising solution on the film and
+     allow it to act for from thirty seconds to one minute; blot
+     off the excess of fluid with filter paper.
+
+     7. Without washing, transfer the film to a watch-glass
+     containing the reducing solution and allow it to remain
+     therein for from thirty seconds to one minute; blot off the
+     excess of fluid with filter paper.
+
+     8. Without washing, again treat the film with the
+     sensitising solution, this time until the film commences to
+     turn black.
+
+     9. Wash in distilled water.
+
+     10. Dry and mount.
+
+~To Stain Nuclei of Yeast Cells.~
+
+1. Prepare and fix film in the usual manner.
+
+2. Soak in ferric ammonia sulphate 3 per cent. aqueous solution for two
+hours.
+
+3. Wash thoroughly in water.
+
+4. Stain in haematoxylin solution (see page 95) for thirty minutes.
+
+5. Wash in water.
+
+6. Differentiate in ferric ammonia sulphate solution for 1-1/2-2
+minutes, examining wet under microscope during the process.
+
+
+~To Stain Spores.~
+
+~1. Single Stain.~--
+
+1. Prepare cover-slip film in the usual way.
+
+2. In fixing, pass the cover-slip film fifteen or thirty times through
+the flame instead of only three. This destroys the resisting power of
+the spore membrane and allows the stain to reach the interior.
+
+3. Stain in the usual way with methylene-blue or fuchsin.
+
+4. Wash in water.
+
+5. Dry and mount.
+
+~2. Double Stain.~--
+
+1. Prepare and fix film in the usual way--i. e., pass three times
+through flame to fix.
+
+2. Cover the film with hot carbol-fuchsin and hold in the forceps above
+a small flame until the fluid begins to steam. Set the cover-slip down
+and allow it to cool. Repeat the process when the stain ceases to steam
+and continue to repeat until the stain has been in contact with the film
+for twenty minutes. (This stains both spores and bacteria.)
+
+3. Wash in water.
+
+4. Decolourise in alcohol, 2 parts; acetic acid, 1 per cent., 1 part.
+(This removes the stain from everything but the spores.)
+
+5. Wash in water.
+
+6. Mount the cover-slip in water and examine microscopically with the
+1/6-inch objective. (Spores should be red, and the rest of the film
+colourless or a very light pink.) If satisfactory, pass on to section 7;
+if unsatisfactory, repeat steps 2 to 5.
+
+7. Counterstain in weak methylene-blue. (Now spores red, bacilli blue.)
+
+8. Wash in water.
+
+9. Dry and mount.
+
+The spores of different bacilli differ greatly in their resistance to
+decolourising reagents; even the spores of the same species of organisms
+vary according to their age. Young spores are more easily decolourised
+than those more mature.
+
+Sulphuric acid, 1 per cent. aqueous solution, and hydrochloric acid, 0.5
+per cent. alcoholic (90 per cent.) solution, are useful decolourising
+reagents.
+
+     ~3. Moeller's Method.~--
+
+     1. Prepare and fix films in the usual manner.
+
+     2. Immerse in absolute alcohol for two minutes, then in
+     chloroform for two minutes; wash in water. This dissolves
+     out any fat or crystals that might otherwise retain the
+     "spore" stain.
+
+     3. Immerse in chromic acid, 5 per cent. aqueous solution,
+     for one minute; wash in water.
+
+     4. Pour Ziehl's carbolic fuchsin on the film, warm as in
+     previous methods, and allow it to act for ten minutes.
+
+     5. Wash in water.
+
+     6. Decolourise in sulphuric acid, 5 per cent. aqueous
+     solution, for five seconds.
+
+     7. Wash in water.
+
+     8. Counterstain with Kuehne's carbolic methylene-blue for
+     one or two minutes.
+
+     9. Wash in water.
+
+     10. Dry and mount.
+
+     (Spores red, bacilli blue.)
+
+     ~4. Abbott's Method.~--
+
+     1. Prepare and fix films in the usual manner.
+
+     2. Pour Loeffler's alkaline methylene-blue on the film; warm
+     cautiously over the flame till steam rises and allow the hot
+     steam to act for one to five minutes.
+
+     3. Wash thoroughly in water.
+
+     4. Decolourise in nitric acid, 2 per cent. alcoholic
+     (alcohol 80 per cent.) solution.
+
+     5. Wash thoroughly in water.
+
+     6. Counterstain in eosin, 1 per cent. aqueous solution.
+
+     7. Wash.
+
+     8. Dry and mount.
+
+     (Spores blue, bacilli red.)
+
+
+DIFFERENTIAL METHODS OF STAINING.
+
+~Gram's Method.~--This method depends upon the fact that the protoplasm of
+some bacteria permits aniline gentian violet and Lugol's iodine
+solution, when applied consecutively, to enter into a chemical
+combination which results in the formation of a new blue-black pigment,
+only very sparingly soluble in absolute alcohol. Such organisms are said
+to "stain by Gram," or to be "Gram positive."
+
+1. Prepare a cover-slip film and fix in the usual way.
+
+2. Stain in aniline gentian violet three to five minutes. Filter as much
+aniline water on to the cover-slip as it will hold; then add the
+smallest quantity of alcoholic solution of gentian violet which suffices
+to saturate the aniline water and form a "bronze scum" upon its
+surface--if too much of the alcoholic gentian violet is added the
+alcohol present redissolves this scum.
+
+     To prepare aniline water, pour 4 or 5 c.c. aniline oil into
+     a stoppered bottle and add distilled water, 100 c.c. Shake
+     vigourously and filter immediately before use. The excess of
+     oil sinks to the bottom of the bottle and may be used again.
+
+3. Wash in water.
+
+4. Treat with Lugol's iodine solution until the film is black or dark
+brown.
+
+To do this treat with iodine solution for a few seconds, wash in water,
+and examine the film over a piece of white filter paper. Note the
+colour. Repeat this process until the film ceases to darken with the
+fresh application of iodine solution.
+
+Lugol's solution is prepared by dissolving
+
+    Iodine                  1 gramme
+    Iodide of potassium     3 grammes
+    In distilled water    300 c.c.
+
+5. Wash in water.
+
+6. Wash with alcohol until no more colour is discharged and the alcohol
+runs away clear and colourless.
+
+The following mixture may be substituted for absolute alcohol as a
+decolouriser
+
+    Acetone             10 c.c.
+    Absolute alcohol   100 c.c.
+
+7. Wash in water.
+
+8. Counterstain very lightly with aqueous solution of Neutral Red. Other
+counterstains may be used such as dilute eosin, dilute fuchsin, or
+vesuvin.
+
+     NOTE.--This section may be omitted when dealing with films
+     prepared from pure cultivations.
+
+9. Wash in water.
+
+10. Dry and mount.
+
+
+~Gram-Claudius Method.~--
+
+1. Prepare a cover-slip film and fix in the usual way.
+
+2. Stain in methyl violet, 1 per cent. aqueous solution for three to
+five minutes.
+
+3. Treat with two lots picric acid, saturated aqueous solution.
+
+4. Wash in water and dry.
+
+5. Decolourise with clove oil.
+
+6. Wash off clove oil with xylol.
+
+7. Mount in xylol balsam.
+
+
+~Gram-Weigert Method.~--
+
+1-5. Proceed as for the corresponding sections of Gram's method (_quod
+vide_).
+
+6. Dry in the air.
+
+7. Wash in aniline oil, 1 part, xylol, 2 parts, until no more colour is
+discharged.
+
+8. Wash in xylol.
+
+9. Mount in xylol balsam.
+
+
+~Modified Gram-Weigert Method.~--(To demonstrate trichophyta in hair.)
+
+1. Soak the hairs in ether for ten minutes to remove the fat.
+
+2. Stain thirty minutes in a tar-like solution of aniline gentian violet
+(prepared by adding 15 drops of the alcoholic solution of gentian violet
+to 3 drops of aniline water).
+
+3. Dry the hairs between pieces of blotting paper.
+
+4. Treat with perfectly fresh iodine solution.
+
+5. Again dry between blotting paper.
+
+6. Treat with aniline oil to remove excess of stain. (If necessary, add
+a drop or two of nitric acid to the oil.)
+
+7. Again treat with aniline oil.
+
+8. Treat with aniline oil and xylol, equal parts.
+
+9. Clear with xylol.
+
+10. Mount in xylol balsam.
+
+To obtain the best differentiation the preparation should be repeatedly
+examined microscopically (with a 1/6-inch objective) between steps 5 and
+9, as the actual time involved varies with different specimens.
+
+~Ziehl-Neelsen's Method.~--(To demonstrate tubercle and other acid-fast
+bacilli.)
+
+1. Smear a thin, even film of the specimen on the cover-slip by means of
+the platinum loop. (In the case of sputum, if it is a very watery
+specimen, allow the film to dry, then spread a second and even a third
+layer over the first.)
+
+2. Fix by passing three times through the flame.
+
+3. Stain in hot carbol-fuchsin (as in staining for spores) for five to
+ten minutes. (This stains everything on the film.) Avoid over-heating.
+
+4. Decolourise by dipping in sulphuric acid, 25 per cent. (This removes
+stain from everything but acid-fast bacilli; e. g., tubercle, leprosy,
+and smegma bacilli and the film turns yellow.)
+
+5. Wash in water. (A pale red colour returns to the film).
+
+6. Wash in alcohol till no more colour is discharged. (This often, but
+not invariably, removes the stain from acid-fast bacilli other than
+tubercle; e. g., smegma bacillus.)
+
+7. Wash in water.
+
+8. Counterstain in weak methylene-blue. (Stains non-acid-fast bacilli,
+leucocytes, epithelial cells, etc.)
+
+9. Wash in water, dry, and mount.
+
+~Pappenheim's Method.~--
+
+This method is supposed to differentiate between B. tuberculosis and
+other acid-fast micro-organisms.
+
+1. Prepare and fix film in the usual way.
+
+2. Stain in carbol-fuchsin _without heat_ for three minutes.
+
+3. Without previously washing in water treat the film with three or four
+successive applications of corallin (Rosolic acid) solution.
+
+    Corallin                             1 gramme
+    Methylene-blue
+    (saturated alcoholic solution)     100 c.c.
+    Glycerine                           20 c.c.
+
+4. Wash in water.
+
+5. Dry and mount.
+
+~Neisser's Method--Modified.~--(To demonstrate diphtheroid bacilli.)
+
+_Stain I._--
+
+Measure out and mix
+
+    Methylene-blue, saturated alcoholic solution     4.0 c.c.
+    Acetic acid, 5 per cent. aqueous solution       96.0 c.c.
+
+Filter.
+
+_Stain II._--
+
+Weigh out
+
+    Neutral red                2.5 grammes
+
+and dissolve in
+
+    Distilled water              1000 c.c.
+
+Filter.
+
+METHOD.--
+
+1. Prepare and fix films in the usual way.
+
+2. Pour stain I on the film and allow it to act for two minutes.
+
+3. Wash thoroughly in water.
+
+4. Treat with Lugol's iodine for ten seconds.
+
+5. Wash thoroughly in water.
+
+6. Pour stain II on to the film and allow it to act for thirty seconds.
+
+7. Wash thoroughly in water.
+
+8. Dry and mount.
+
+     NOTE.--The cultivation from which the films are prepared
+     must be upon blood-serum which has been incubated at 37 deg. C.
+     for from nine to eighteen hours.
+
+The bacilli are stained a light red by the neutral red, which contrasts
+well with the two or three black spots, situated at the poles and
+occasionally one in the centre representing protoplasmic aggregations (?
+metachromatic granules) stained by the acid methylene-blue.
+
+     ~Wheal and Chown (Oxford) Method.~--(To demonstrate
+     actinomyces.)
+
+     1. Stain briefly with Ehrlich's haematoxylin (until nuclei
+     are faint blue after washing with tap water).
+
+     2. Wash in tap water.
+
+     3. Stain in hot carbol-fuchsin (as for tubercle bacilli) for
+     five to ten minutes.
+
+     4. Wash in tap water.
+
+     5. Decolourise with Spengler's picric acid alcohol. This is
+     prepared by mixing:
+
+        Alcohol, absolute                          20 c.c.
+        Picric acid, saturated aqueous solution    10 c.c.
+        Distilled water                            10 c.c.
+
+     During the progress of steps 1-5 the preparation must be
+     repeatedly examined microscopically with the 1/6-inch
+     objective.
+
+     When properly differentiated the clubs appear brilliant red
+     on greenish ground.
+
+     6. Dehydrate in alcohol.
+
+     7. Clear in xylol.
+
+     8. Mount in xylol balsam.
+
+     This method serves equally well for films and for sections.
+
+
+
+
+VII. METHODS OF DEMONSTRATING BACTERIA IN TISSUES.
+
+
+For bacteriological purposes, sections of tissue are most conveniently
+prepared by either the ~freezing method~ or the ~paraffin method~.
+
+The latter is decidedly preferable, but as it is of greater importance
+to demonstrate the bacteria, if such are present, than to preserve the
+tissue elements unaltered, the "frozen" sections are often of value.
+
+Whichever method is selected, it is necessary to take small pieces of
+the tissue for sectioning,--2 to 5 mm. cubes when possible, but in any
+case not exceeding half a centimetre in thickness. Post-mortem material
+should be secured as soon after the death of the animal as possible.
+
+The tissue is prepared for cutting by--
+
+(a) Fixation; that is, by causing the death of the cellular elements
+in such a manner that they retain their characteristic shape and form.
+
+The fixing fluids in general use are: Absolute alcohol; corrosive
+sublimate, saturated aqueous solution; corrosive sublimate, Lang's
+solution (_vide_ page 82); formaldehyde, 4 per cent. aqueous solution.
+(Of these, Lang's corrosive sublimate solution is decidedly the best
+all-round "fixative.")
+
+(b) Hardening; that is, by rendering the tissue of sufficient
+consistency to admit of thin slices or "sections" being cut from it.
+This is effected by passing the tissue successively through alcohols of
+gradually increasing strength: 30 per cent. alcohol, 50 per cent.
+alcohol, 75 per cent. alcohol, 90 per cent. alcohol, absolute alcohol.
+
+In both these processes a large excess of fluid should always be used.
+
+
+FREEZING METHOD.
+
+1. ~Fixation.~ Place the pieces of tissue in a wide-mouthed glass bottle
+and fill with absolute alcohol. Allow the tissues to remain therein for
+twenty-four hours.
+
+2. ~Hardening.~ Remove the alcohol (no longer absolute, as it has taken up
+water from the tissues) from the bottle and replace it with fresh
+absolute alcohol. Allow the tissues to remain therein for twenty-four
+hours.
+
+[Illustration: FIG. 71.--Washing tissues.]
+
+     NOTE.--If not needed for cutting immediately, the hardened
+     tissues can be stored in 75 per cent. alcohol.
+
+3. Remove the alcohol from the tissues by soaking in water from one to
+two hours. Remove the stopper from the bottle; rest a glass funnel in
+the open mouth and place under a tap of running water. The water of
+course, overflows, but the tissues remain in the bottle (Fig. 71).
+
+4. Impregnate the tissues with mucilage for twelve to twenty-four hours,
+according to size. Transfer the pieces of tissue to a bottle containing
+sterilised gum mixture.
+
+~Formula.~--
+
+    Gum arabic      5 grammes
+    Saccharose      1 gramme
+    Boric acid      1 gramme
+    Water         100 c.c.
+
+5. Place the tissue on the plate of a freezing microtome (Cathcart's is
+perhaps the best form), cover and surround with fresh gum mixture;
+freeze with ether, or for preference, carbon dioxide, and cut sections.
+
+6. Float the sections off the knife into a glass dish containing tepid
+water and allow them to remain therein for about an hour to dissolve out
+the gum.
+
+(If not required at once, store in 90 per cent. alcohol.)
+
+7. Transfer to a glass capsule containing the selected staining fluid,
+by means of a section lifter.
+
+8. Transfer the sections in turn to a capsule containing absolute
+alcohol (to dehydrate) and to one containing xylol or oil of cloves (to
+clear).
+
+9. Mount in xylol balsam.
+
+_Alternative Rapid Method._--
+
+     1. Cut very small blocks of the tissue.
+
+     2. Fix in formalin 10 per cent. aqueous solution (fixation
+     fluid No. 7, page 82) for 24 hours.
+
+     3. Transfer block to plate of freezing microtome and freeze
+     with carbon dioxide vapour.
+
+     4. Float the sections off the knife into a glass dish of
+     tepid water.
+
+     5. Stain the sections in glass capsules containing selected
+     stains.
+
+     6. Place the stained section in a dish of clean water and
+     introduce a glass slide obliquely beneath the section; with
+     a mounted needle draw the section on to the slide and hold
+     it there; gently remove the slide from the water, taking
+     care that any folds in the section are floated out before
+     the slide is finally removed from the water.
+
+     7. Drain away as much water as possible from the section.
+     Drop absolute alcohol on to the section from a drop bottle,
+     to dehydrate it.
+
+     8. Double a piece of blotting paper and gently press it on
+     the section to dry it.
+
+     9. Drop on xylol to clear the section.
+
+     10. Place a large drop of xylol balsam on the section and
+     carefully lower a cover-glass on to the balsam.
+
+
+PARAFFIN METHOD.
+
+1. ~Fixation.~ Place the pieces of tissue, resting on cotton-wool, in a
+wide-mouthed glass bottle. Pour on a sufficient quantity of the
+corrosive sublimate fixing fluid; allow the tissue to remain therein for
+twelve to twenty-four hours according to size.
+
+2. Pour off the fixing fluid and wash thoroughly in running water for
+twenty minutes to half an hour to remove the excess of corrosive
+sublimate.
+
+[Illustration: FIG. 72.--~L~-shaped brass moulds.]
+
+[Illustration: FIG. 73.--Paraffin kettle.]
+
+3. ~Hardening.~ Place the tissues in each of the following strengths of
+alcohol in turn for from twelve to twenty-four hours: 50 per cent., 75
+per cent., 90 per cent., absolute.
+
+4. ~Dehydration~ is effected by transferring the tissues to fresh absolute
+alcohol.
+
+5. ~Clearing.~ Half fill a wide-mouthed bottle with chloroform. On the
+surface of the chloroform float a layer of absolute alcohol about five
+to ten millimetres in depth. Place the pieces of tissue in the layer of
+alcohol and when they have sunk through this layer, transfer them to
+pure chloroform for from six to twenty-four hours according to the size
+of the pieces. When "cleared," the tissue becomes more or less
+transparent.
+
+6. ~Infiltration.~ Place the cleared tissues in fresh chloroform with
+several pieces of paraffin wax and stand in a warm place, such as on the
+top of the warm incubator. The warmth gradually melts the paraffin and
+the tissues should remain in the mixture about twenty-four hours.
+
+7. Transfer the tissues to a vessel containing pure melted paraffin.
+Place this vessel in a paraffin water-bath regulated for 2 deg. C. above
+the melting-point of the paraffin used, and allow the tissues to soak for
+some four to six hours to ensure complete impregnation. The paraffin
+used should have a melting-point of not more than 58 deg. C. For all
+ordinary purposes 54 deg. C. will be found quite high enough.
+
+8. Imbed in fresh paraffin in a metal (or paper) mould.
+
+(a) Arrange a pair of ~L~-shaped pieces of metal on a plate of glass to
+form a rectangular trough (Fig. 72).
+
+(b) Pour fresh melted paraffin into the mould from a special vessel
+(Fig. 73).
+
+(c) Lift the piece of tissue from the paraffin bath and arrange it in
+the mould.
+
+(d) Blow gently on the surface of the paraffin in the mould, and as
+soon as a film of solid paraffin has formed, carefully lift the glass
+plate on which the mould is set and lower plate and mould together into
+a basin of cold water.
+
+(e) When the block is cold, break off the metal ~L~'s; trim off the
+excess of paraffin from around the tissue with a knife, taking care to
+retain the rectangular shape, and store the block in a pill-box.
+
+When several pieces of tissue have to be imbedded at one time, shapes of
+stout copper, 10 cm., 5 cm., and 2.5 cm. square respectively, and 0.75
+cm. deep (Fig. 74) will be found extremely useful. These placed upon
+plates of glass replace the pair of L's in the above process. When the
+paraffin has set firmly the screw a should be loosened to allow the
+two halves of the flange b to separate slightly--this facilitates
+removal of the paraffin block.
+
+[Illustration: FIG. 74.--Paraffin mould.]
+
+8. Cement the block on the carrier of a "paraffin" microtome (the Minot,
+the Jung, or the Cambridge Rocker) with a little melted paraffin.
+Greater security is obtained if the paraffin around the base of the
+block is melted by means of a hot metal or glass rod.
+
+9. Cut sections--thin, and if possible in ribbands.
+
+
+~Mounting Paraffin Sections.~--
+
+1. Place a large drop of 30 per cent. alcohol on the centre of a slide
+(or cover-slip) and float the section on to the surface of the drop,
+from a section lifter.
+
+2. Hold the slide in the fingers of one hand and warm cautiously over
+the flame of a Bunsen burner, touching the under surface of the glass
+from time to time on the back of the other hand. As soon as the slide
+feels distinctly warm to the skin, the paraffin section will flatten out
+and all wrinkles disappear.
+
+(The slide with the section floating on it may be rested on the top of
+the paraffin bath for two or three minutes, instead of warming over the
+flame as here described.)
+
+3. Cautiously tilt up the slide and blot off the excess of spirit with
+blotting paper, leaving the section attached to the centre of the
+slide.
+
+4. Place the slide in a wire rack (Fig. 75), section downward, in the
+"hot" incubator for twelve to twenty-four hours. At the end of this time
+the section is firmly adherent to the glass, and is treated during the
+subsequent steps as a "fixed" cover-glass film preparation.
+
+     NOTE.--If large, thick sections have to be manipulated, or
+     if time is of importance or acids are used during the
+     staining process, it is often advisable to add a trace of
+     Mayer's albumin to the alcohol before floating out the
+     section. If this substance is employed, a sojourn of twenty
+     minutes to half an hour in the "hot" incubator will be found
+     ample to ensure firm adhesion of the section to the slide.
+     The albuminous fluid is prepared as follows:
+
+[Illustration: FIG. 75.--Section rack.]
+
+
+~Mayer's Albumin.~--
+
+    Weigh out
+          Salicylate of soda    1 gramme
+    and dissolve in
+          Glycerine            50 c.c.
+    Add
+          White of egg         50 c.c.
+
+     Mix thoroughly by means of an egg whisk.
+
+     Filter into a clean bottle.
+
+     As an alternative method paint a thin layer of Schallibaum's
+     solution on the slide with a camel's hair pencil; lay the
+     section carefully on this film and heat gently to fix the
+     section.
+
+
+_Schallibaum's solution_:
+
+    Clove oil      30 c.c.
+    Collodion      10 c.c.
+
+Keep in a dark blue bottle in a cool place.
+
+
+~Staining Paraffin Sections.~--
+
+1. Warm paraffin section over the Bunsen flame to soften (_but not to
+melt_) the paraffin, then dissolve out the wax with xylol poured on from
+a drop bottle.
+
+2. Remove xylol by flushing the section with alcohol.
+
+3. If the tissue was originally "fixed" in a corrosive sublimate
+solution, the section must now be treated with Lugol's iodine solution
+for two minutes and subsequently immersed in 90 per cent. alcohol to
+remove all traces of yellow staining.
+
+4. Wash in water.
+
+5. Stain deeply, if using a single stain, as the subsequent processes
+decolourise.
+
+6. Wash in water, decolourise if necessary.
+
+7. Flood with several changes of absolute alcohol to dehydrate the
+section.
+
+8. Clear in xylol. (Oil of cloves is not usually employed, as it
+decolourises the section.)
+
+9. Mount in xylol balsam.
+
+
+SPECIAL STAINING METHODS FOR SECTIONS.
+
+
+~Double-staining Carmine and Gram-Weigert.~--
+
+1. Prepare the section for staining as above, sections 1 to 3.
+
+2. Stain in lithium carmine (Orth's) or picrocarmine for ten to thirty
+minutes, in a porcelain staining pot (Fig. 76).
+
+3. Wash in picric acid solution until yellow. At this stage cell nuclei
+are red, protoplasm is yellow, and bacteria are colourless.
+
+Picric acid solution is prepared by mixing
+
+    Picric acid, saturated aqueous solution   40 c.c.
+    Hydrochloric acid                          1 c.c.
+    Alcohol (90 per cent.)                   160 c.c.
+
+4. Wash in water.
+
+5. Wash in alcohol.
+
+6. Stain in aniline gentian violet.
+
+7. Wash in iodine solution till dark brown or black.
+
+8. Wash in water.
+
+9. Dip in absolute alcohol for a second.
+
+10. Decolourise with aniline oil till no more colour is discharged.
+
+[Illustration: FIG. 76.--Staining pot.]
+
+11. Wash with aniline oil, 2 parts, xylol, 1 part.
+
+12. Clear with xylol.
+
+13. Mount in xylol balsam.
+
+~Alternative Gram-Weigert Method for Sections.~--
+
+1. Fix paraffin section on slide and prepare for staining in the usual
+manner.
+
+2. Stain in alum carmine for about fifteen minutes.
+
+3. Wash thoroughly in water.
+
+4. Filter aniline gentian violet solution on to the section on the slide
+and allow to stain about twenty-five minutes.
+
+5. Wash thoroughly in water.
+
+6. Treat with Lugol's iodine until section ceases to become any blacker.
+
+7. Wash thoroughly in water.
+
+8. Treat with a mixture of equal parts of aniline oil and xylol until no
+more colour comes away.
+
+9. Wash thoroughly with xylol.
+
+10. Decolourise and dehydrate rapidly with absolute alcohol until there
+remains only a very faint bluish tint.
+
+11. Clear with xylol.
+
+12. Mount in xylol balsam.
+
+(Then fibrin and hyaline tissue are stained deep blue, whilst bacteria
+which "stain Gram" appear of a deep blue-violet colour.)
+
+~Unna-Pappenheim Method.~--
+
+Stain.--
+
+Weigh out and mix
+
+    Methylene green   0.15 gramme
+    Pyronin           0.25 gramme
+
+and dissolve in
+
+    Carbolic acid 0.5 per cent. aqueous solution  78 c.c.
+
+Measure out
+
+    Alcohol       2.5 c.c.   }
+    Glycerine    20.0 c.c.   } and add to the stain.
+
+~Method.~--
+
+1. Place tissue in the above stain for ten minutes.
+
+2. Differentiate and dehydrate with absolute alcohol.
+
+3. Clear in xylol.
+
+4. Mount in xylol balsam.
+
+~To Demonstrate Capsules.~--
+
+1. _MacConkey's Method._--Stain precisely as for cover-slip films
+(_vide_ page 100).
+
+2. _Friedlaender's Method._--
+
+Stain.--
+
+    Gentian violet, saturated alcoholic solution     50 c.c.
+    Acetic acid, glacial                             10 c.c.
+    Distilled water                                 100 c.c.
+
+     METHOD.--
+
+     1. Prepare the sections for staining, _secundum artem_.
+
+     2. Stain sections in the warm (e. g., in the hot
+     incubator) for twenty-four hours.
+
+     3. Wash with water.
+
+     4. Decolourise lightly with acetic acid, 1 per cent.
+
+     5. Dehydrate rapidly with absolute alcohol.
+
+     6. Clear with xylol.
+
+     7. Mount in xylol balsam.
+
+
+~To Demonstrate Acid-fast Bacilli.~--
+
+1. Prepare the sections for staining in the usual way.
+
+2. Stain with haematin solution ten to twenty seconds, to obtain a pure
+nuclear stain; then wash in water.
+
+3. Stain with carbolic fuchsin twenty to thirty minutes at 47 deg. C.;
+then wash in water.
+
+4. Treat with aniline hydrochlorate, 2 per cent. aqueous solution, for
+two to five seconds.
+
+5. Decolourise in 75 per cent. alcohol till section appears free from
+stain--fifteen to thirty minutes.
+
+6. Dehydrate with absolute alcohol.
+
+7. Clear very rapidly with xylol.
+
+8. Mount in xylol balsam.
+
+
+~To Demonstrate Spirochaetes in Tissues.~
+
+~Piridin Method (Levaditi).~--
+
+1. Cut slices of tissue 1 mm. thick.
+
+2. Fix in 10 per cent. formalin solution for twenty-four hours.
+
+3. Wash in water for one hour.
+
+4. Place in 96 per cent. alcohol for twenty-four hours.
+
+5. Measure into a dark green or amber bottle 100 c.c. silver nitrate
+solution 1 per cent., and 10 grammes pyridin puriss. Transfer slices of
+tissue to this. Stopper and keep at room temperature three hours, then
+in thermostat at 50 deg. C. for four to six hours.
+
+6. Wash quickly in 10 per cent. pyridin solution.
+
+7. Reduce silver by transferring slices of tissue to following solution
+for forty-eight hours.
+
+    Pyrogallic acid      4 grammes
+    Acetone             10 c.c.
+    Pyridin puriss      15 grammes
+    Distilled water    100 c.c.
+
+8. Wash well in water.
+
+Take through alcohols of increasing strength up to absolute, keeping in
+each strength for twenty-four hours.
+
+9. Clear, embed, cut very thin sections, mount, remove paraffin, again
+clear and mount in xylol balsam.
+
+The spirochaetes if present are black and show up against the pale yellow
+color of the background.
+
+Weak carbol fuchsin, neutral red or toluidin blue can also be used to
+stain the background if desired, after the removal of the paraffin in
+step 9.
+
+~To Demonstrate Protozoa in Sections (Leishman).~--
+
+Reagents required:
+
+    Leishman's Polychrome stain.
+    Acetic acid 1 in 1500 aqueous solution.
+    Caustic soda 1 in 7000 aqueous solution.
+    Distilled water.
+
+1. Mount section, remove paraffin and take into distilled water as usual
+(_vide_ page 121).
+
+2. Drain off the excess of water.
+
+3. Cover the section with diluted Leishman (1 part stain, 2 parts
+distilled water) and allow to act for five to ten minutes (until tissue
+appears a deep blue).
+
+4. Decolourise with acetic acid solution until only the nuclei appear
+blue (examine the section wet, with low power objective).
+
+5. If the eosin colour is too well marked treat with the caustic soda
+solution until the desired tint is obtained (as seen with the 1/6-inch
+objective).
+
+6. Wash with distilled water.
+
+7. Rapidly dehydrate with alcohol.
+
+8. Clear with xylol.
+
+9. Mount in xylol balsam.
+
+
+
+
+~VIII. CLASSIFICATION OF FUNGI.~
+
+
+For practical purposes FUNGI may be divided into:
+
+    ~1. Hymenomycetes~ (including the mushrooms, etc.).
+    ~2. Hyphomycetes~ (moulds).
+    ~3. Blastomycetes~ (yeasts and torulae).
+    ~4. Schizomycetes~ (bacteria).
+
+     NOTE.--Formerly myxomycetes were included in the fungi; they
+     are now recognized as belonging to the animal kingdom, and
+     are termed "mycetozoa."
+
+
+~MORPHOLOGY OF THE HYPHOMYCETES.~
+
+At the commencement of his studies, the attention of the student is
+directed to the various non-pathogenic moulds and yeasts, not only that
+he may gain the necessary technique whilst handling cultivations of
+harmless organisms, but also because these very species are amongst the
+commonest of those that may accidentally contaminate his future
+preparations.
+
+The hyphomycetes are composed of a mycelium of short jointed rods or
+"hyphae" springing from an axis or germinal tube which develops from the
+spore. Hyphae are--
+
+(a) Nutritive or submerged.
+
+(b) Reproductive or aerial.
+
+The protoplasm of these cells contains granules, pigment, oil globules,
+and sometimes crystals of calcium oxalate.
+
+~Reproduction.~--Apical spore formation--asexual;
+                               zoospores--sexual.
+
+~Mucorinae.~--_Mucor_ (Fig. 77).--Note the branching filaments--"mycelium"
+(a), "hyphae" (b).
+
+Note the asexual reproduction.
+
+1. A filament grows upward. At its apex a septum forms, then a globular
+swelling appears--"sporagium" (d). This possesses a definite membrane.
+
+2. From the septum grows a club-shaped mass of protoplasm--"columella"
+(c).
+
+[Illustration: FIG. 77.--Mucor mucedo.]
+
+[Illustration: FIG. 78.--Aspergillus]
+
+3. The rest of the contained protoplasm breaks up into "swarm spores"
+(e).
+
+Finally the membrane ruptures and spores escape.
+
+~Perisporaceae.~--_Aspergillus_ (Fig. 78).--Note the branching
+filaments--"mycelium" (a).
+
+[Illustration: FIG. 79.--Penicillium.]
+
+Note the asexual reproduction.
+
+1. A filament (b) grows upward, its termination becomes clubbed; on
+the clubbed extremity flask-shaped cells appear--"sterigmata" (c).
+
+2. At free end of each sterigma is formed an oval body--a spore or
+"gonidium" (d), which, when ripe, is thrown off from the sterigma. Two
+or more gonidia may be supported upon each sterigma.
+
+_Penicillium_ (Fig. 79).--Note the branching filaments--"mycelium" (a)
+(frequently containing globules).
+
+Note the asexual reproduction.
+
+1. A filament grows upward--"goniodophore" (b)--and its apex divides
+up into several branches--"basidia" (c).
+
+2. At the apex of each basidium a flask-shaped cell, "sterigma" (d),
+appears.
+
+3. At the apex of each sterigma appears a row of oval cells--"spores" or
+"conidia" (e). These, when ripe, are cast off from the sterigmata.
+
+[Illustration: FIG. 80.--Oidium.]
+
+~Ascomycetae.~--_Oidium_ (Fig. 80).--(This family is perhaps as nearly
+related to the blastomycetes as it is to the hyphomycetes.)
+
+Note the branching filaments--"pseudomycelium" (a). Here and there
+filaments are broken up at their ends into oval or rod-shaped segments,
+"oidia," and behave as spores.
+
+Note the asexual reproduction. From the pseudomycelium arise true hyphae
+(b), each of which in turn ends in a chain of spores (c).
+
+
+~MORPHOLOGY OF THE BLASTOMYCETES.~
+
+The blastomycetes are composed of spherical or oval cells (8 to 9.5 mu in
+diameter), which, when rapidly multiplying by budding, may form a
+spurious mycelium. A thin cell-wall encloses the granular protoplasm, in
+which vacuoles and sometimes a nucleus may be noted. This latter is best
+seen when stained with haematoxylin (see page 105).
+
+During their growth and multiplication the blastomycetes split up
+solutions containing sugar into alcohol and CO_{2}.
+
+~Saccharomyces~ (Fig. 81).--Note the round or oval cells of granular
+protoplasm (a) containing solid particles and vacuoles (c), and
+surrounded by a definite envelope.
+
+~Reproduction.~--Budding; ascospores--asexual.
+
+Note the asexual _reproduction_.
+
+1. "Gemmation"--that is, the budding out of daughter cells (b) from
+various parts of the gradually enlarging mother cell. These are
+eventually cast off and in turn become mother cells and form fresh
+groups of buds.
+
+[Illustration: FIG. 81.--Saccharomyces with ascospores.]
+
+[Illustration: FIG. 82.--Torula.]
+
+2. Spore formation--"ascospores" (e). These are formed at definite
+temperatures and within well-defined periods; e. g., Saccharomyces
+cerevisiae, thirty hours at 25 deg. to 37 deg. C., or ten days at
+12 deg. C.
+
+~Torulae~ (Fig. 82).--Torulae, whilst resembling yeasts in almost
+every other respect, never form endo-spores. Note the elongated,
+sausage-shaped cells (a) the larger oval cells (b) and the globular
+cells (c) the former two often interlacing and growing as a film.
+
+Note the absence of ascospore formation.
+
+
+
+
+IX. SCHIZOMYCETES.
+
+
+~Classification and Morphology.~--Bacteria are often classified, in
+general terms, according to their life functions, into--
+
+    _Saprogenic_, or putrefactive bacteria;
+    _Zymogenic_, or fermentative bacteria;
+    _Pathogenic_, or disease-producing bacteria;
+
+or according to their food requirements into--
+
+    _Prototrophic_, requiring no organic food (e. g., nitrifying bacteria);
+    _Metatrophic_, requiring organic food (e. g., saprophytes
+         and facultative parasites);
+    _Paratrophic_, requiring living food (obligate parasites);
+
+or according to their metabolic products into--
+
+    _Chromogenic_, or pigment-producing bacteria;
+    _Photogenic_, or light-producing bacteria;
+    _Aerogenic_, or gas-producing bacteria;
+
+and so on.
+
+Such broad groupings as these have, however, but little practical value
+when applied to the systematic study of the fission fungi.
+
+On the other hand, no really scientific classification of the
+schizomycetes has yet been drawn up, and the varying morphological
+appearances of the members of the family are still utilised as a basis
+for classification, as under--
+
+~1. Cocci.~ (Fig. 83).--Rounded or oval cells, subdivided according to the
+arrangement of the individuals after fission, into--
+
+_Diplococci_ and _Streptococci_, where division takes place in one plane
+only, and the individuals remain attached (a) in pairs or (b) in
+chains.
+
+_Tetrads_, _Merismopedia_, or _Pediococci_, where division takes place
+alternately in two planes at right angles to each other, and the
+individuals remain attached in flat tablets of four, or its multiples.
+
+[Illustration: FIG. 83.--Types of bacteria--cocci: 1, Diagram of sphere
+indicating planes of fission; 2, diplococci; 3, streptococci; 4,
+tetrads; 5, sarcinae; 6, staphylococci.]
+
+_Sarcinae_, where division takes place in three planes successively,
+and the individuals remain attached in cubical packets of eight and its
+multiples.
+
+[Illustration: FIG. 84.--Types of bacteria--bacilli, etc.: 1, Bacilli;
+2, diplobacilli; 3 streptobacilli; 4, spirilla; 5, vibrios; 6,
+spirochaetae.]
+
+_Micrococci_ or _Staphylococci_, where division takes place in three
+planes, but with no definite sequence; consequently the individuals
+remain attached in pairs, short chains, plates of four, cubical packets
+of eight, and irregular masses containing numerous cocci.
+
+~2. Bacilli~ (Fig. 84, 1 to 3).--Rod-shaped cells. A bacillus, however
+short, can usually be distinguished from a coccus in that two sides are
+parallel. Some bacilli after fission retain a characteristic arrangement
+and may be spoken of as _Diplobacilli_ or _Streptobacilli_.
+
+Leptothrix is a term that in the past has been loosely used to signify a
+long thread, but is now restricted to such forms as belong to the
+leptothriciae (_vide infra_).
+
+~3. Spirilla~ (Fig. 84, 4 to 6).--Curved and twisted filaments.
+Classified, according to shape, into--
+
+    Spirillum.
+    Vibrio (comma).
+    Spirochaeta.
+
+Many Spirochaetes appear to belong to the animal kingdom and are grouped
+under protozoa; other organisms to which this name has been given are
+undoubtedly bacteria.
+
+Higher forms of bacteria are also met with, which possess the following
+characteristics: They are attached, unbranched, filamentous forms,
+showing--
+
+(a) Differentiation between base and apex;
+
+(b) Growth apparently apical;
+
+(c) Exaggerated pleomorphism;
+
+(d) "Pseudo-branching" from apposition of cells; and are classified
+into--
+
+    1. Beggiotoa.    }   Free swimming forms, which
+    2. Thiothrix.    }   contain sulphur granules.
+
+    3. Crenothrix.   }
+    4. Cladothrix.   }  These forms do not contain
+    5. Leptothrix.   }  sulphur granules.
+
+    6. Streptothrix. A group which exhibits true but
+    not dichotomous branching, and contains some pathogenic
+    species.
+
+The morphology of the same bacterium may vary greatly under different
+conditions.
+
+For example, under one set of conditions the examination of a pure
+cultivation of a bacillus may show a short oval rod as the predominant
+form, whilst another culture of the same bacillus, but grown under
+different conditions, may consist almost entirely of long filaments or
+threads. This variation in morphology is known as "pleomorphism."
+
+Some of the factors influencing pleomorphism are:
+
+1. The composition, reaction, etc., of the _nutrient medium_ in which
+the organism is growing.
+
+2. _The atmosphere_ in which it is cultivated.
+
+3. _The temperature_ at which it is incubated.
+
+4. Exposure to or protection from _light_.
+
+The various points in the anatomy morphology and physiology of bacteria
+upon which stress is laid in the following pages should be studied as
+closely as is possible in preparations of the micro-organisms named in
+connection with each.
+
+
+~ANATOMY.~
+
+1. _Capsule_ (Fig. 85, b).--A gelatinous envelope (probably akin to
+mucin in composition) surrounding each individual organism, and
+preventing absolute contact between any two. In some species the capsule
+(e. g., B. pneumoniae) is well marked, but it cannot be demonstrated in
+all. In very well marked cases of gelatinisation of the cell wall, the
+individual cells are cemented together in a coherent mass, to which the
+term "zoogloea" is applied (e. g., Streptococcus mesenteroides). In
+some species colouring matter or ferric oxide is stored in the capsule.
+
+2. _Cell Wall_ (Fig. 85, c).--A protective differentiation of the
+outer layer of the cell protoplasm; difficult to demonstrate, but
+treatment with iodine or salt solution sometimes causes shrinkage of the
+cell contents--"plasmolysis"--and so renders the cell wall apparent (_e.
+g._, B. megatherium) in the manner shown in figure 85. Stained bacilli,
+when examined with the polarising microscope, often show a doubly
+refractile cell wall (e. g., B. tuberculosis and B. anthracis).
+
+In some of the higher bacteria the cell wall exhibits this
+differentiation to a marked degree and forms a hard sheath within which
+the cell protoplasm is freely movable; and during the process of
+reproduction the cell protoplasm may be extruded, leaving the empty tube
+unaltered in shape.
+
+[Illustration: FIG. 85.--Dragrammatic sketch of composite bacterium to
+illustrate details of anatomical structure.]
+
+[Illustration: FIG. 86.--Plasmolysis.]
+
+3. _Cell Contents._--Protoplasm (mycoprotein) contains a high percentage
+of nitrogen, but is said to differ from proteid in that it is not
+precipitated by C_{2}H_{6}O. It is usually homogeneous in
+appearance--sometimes granular--and may contain oil globules or sap
+vacuoles (Fig. 85, d), chromatin granules, and even sulphur granules.
+Sap vacuoles must be distinguished from spores, on the one hand, and the
+vacuolated appearance due to plasmolysis, on the other.
+
+The cell contents may sometimes be differentiated into a parietal layer,
+and a central body (e. g., beggiotoa) when stained by haematoxylin.
+
+4. _Nucleus._--This structure has not been conclusively proved to
+exist, but in some bacteria chromatin particles have been observed near
+the centre of the bacterial cell and denser masses of protoplasm
+situated at the poles which exhibit a more marked affinity than the rest
+of the cell protoplasm for aniline dyes. These latter are termed polar
+granules or _Polkoerner_ (Fig. 85, e). Occasionally these aggregations
+of protoplasm alter the colour of the dye they take up. They are then
+known as metachromatic bodies or _Ernstschen Koerner_ (e. g., B.
+diphtheriae).
+
+5. _Flagella_ (Organs of Locomotion, Fig. 85, a).--These are
+gelatinous elongations of the cell protoplasm (or more probably of the
+capsule), occurring either at one pole, at both poles, or scattered
+around the entire periphery. Flagella are not pseudopodia. The
+possession of flagella was at one time suggested as a basis for a system
+of classification, when the following types of ciliation were
+differentiated (Fig. 87):
+
+[Illustration: FIG. 87.--Types of ciliation.]
+
+1. Polar: (a) _Monotrichous_ (a single flagellum situated at one pole;
+e. g., B. pyocyaneus).
+
+(b) _Amphitrichous_ (a single flagellum at each pole; e. g.,
+Spirillum volutans).
+
+(c) _Lophotrichous_ (a tuft or bunch of flagella situated at each
+pole; e. g., B. cyanogenus).
+
+2. Diffuse: _Peritrichous_ (flagella scattered around the entire
+periphery e. g., B. typhosus).
+
+
+~PHYSIOLOGY.~
+
+~Reproduction.~--_Active Stage._--Vegetative, i. e., by the division of
+cells, or "fission."
+
+1. The cell becomes elongated and the protoplasm aggregated at opposite
+poles.
+
+2. A circular constriction of the organism takes place midway between
+these aggregations, and a septum is formed in the interior of the cell
+at right angles to its length.
+
+3. The division deepens, the septum divides into two lamellae, and
+finally two cells are formed.
+
+[Illustration: FIG. 88.--Fission of cocci.]
+
+[Illustration: FIG. 89.--Fission of bacteria.]
+
+4. The daughter cells may remain united by the gelatinous envelope for a
+variable time. Eventually they separate and themselves subdivide.
+
+Cultures on artificial media, after growing in the same medium for some
+time--i. e., when the pabulum is exhausted--show "involution forms"
+(Fig. 90), well exemplified in cultures of B. pestis on agar two days
+old, B. diphtheriae on potato four to six days old.
+
+[Illustration: FIG. 90.--Involution forms.]
+
+They are of two classes, viz.:
+
+(a) Involution forms characterised by alterations of shape (Fig. 90).
+(Not necessarily dead.)
+
+(b) Involution forms characterised by loss of staining power. (Always
+dead.)
+
+_Resting Stage._--Spore Formation.--Conditions influencing spore
+formation: In an old culture nothing may be left but spores. It used to
+be supposed that spores were _always_ formed, so that the species might
+not become extinct, when
+
+(a) The supply of nutrient was exhausted.
+
+(b) The medium became toxic from the accumulation of metabolic
+products.
+
+(c) The environment became unfavourable; e. g., change of
+temperature.
+
+This is not altogether correct; e. g., the temperature at which spores
+are best formed is constant for each bacterium, but varies with
+different species; again, aerobes require oxygen for sporulation, but
+anaerobes will not spore in its presence.
+
+(A) Arthrogenous: Noted only in the micrococci. One complete element
+resulting from ordinary fission becomes differentiated for the purpose,
+enlarges, and develops a dense cell wall. One or more of the cells in a
+series may undergo this alteration.
+
+This process is probably not real spore formation, but merely relative
+increase of resistance. These so-called arthrospores have never been
+observed to "germinate," nor is their resistance very marked, as they
+fail to initiate new cultures, after having been exposed to a
+temperature of 80 deg. C. for ten minutes.
+
+(B) Endogenous: The cell protoplasm becomes differentiated and condensed
+into a spherical or oval mass (very rarely cylindrical). After further
+contraction the outer layers of the mass become still more highly
+differentiated and form a distinct spore membrane, and the spore itself
+is now highly refractile. It has been suggested, and apparently on good
+grounds, that the spore membrane consists of two layers, the exosporium
+and the endosporium. Each cell forms one spore only, usually in the
+middle, occasionally at one end (some exceptions, however, are recorded;
+e. g., B. inflatus). The shape of the parent cell may be unaltered, as
+in the anthrax bacillus, or altered, as in the tetanus bacillus, and
+these points serve as the basis for a classification of spore-bearing
+bacilli, as follows:
+
+(A) Cell body of the parent bacillus unaltered in shape (Fig. 91, a).
+
+(B) Cell of the parent bacillus altered in shape.
+
+1. _Clostridium_ (Fig. 91, b): Rod swollen at the centre and
+attenuated at the poles; spindle shape; e. g., B. butyricus.
+
+2. _Cuneate_ (Fig. 91, c): Rods swollen slightly at one pole and more
+or less pointed at the other; wedge-shaped.
+
+[Illustration: FIG. 91--Types of spore-bearing bacilli.]
+
+3. _Clavate_ (Fig. 91, d): Rods swollen at one pole and cylindrical
+(unaltered) at the other; keyhole-shaped; e. g., B. chauvei.
+
+4. _Capitate_ (Fig. 91, e): Rods with a spherical enlargement at one
+pole; drumstick-shaped; e. g., B. tetani.
+
+The endo-spores remain within the parent cell for a variable time (in
+one case it is stated that germination of the spore occurs within the
+interior of the parent cell--"endo-germination"), but are eventually set
+free, as a result of the swelling up and solution of the cell membrane
+of the parent bacillus in the surrounding liquid, or of the rupture of
+that membrane. They then present the following characteristics:
+
+1. Well-formed, dense cell membranes, which renders them extremely
+difficult to stain, but when once stained equally difficult to
+decolourise.
+
+2. High refractility, which distinguished them from vacuoles.
+
+3. Higher resistance than the parent organism to such lethal agents as
+heat, desiccation, starvation, time, etc., this resistance being due to
+
+(a) Low water contents of plasma of the spore.
+
+    (b) Low heat-conducting power  } of the spore
+    (c) Low permeability           } membrane.
+
+This resistance varies somewhat with the particular species--e. g.,
+some spores may resist boiling for a few minutes--but practically all
+are killed if the boiling is continued for ten minutes.
+
+~Germination.~--When transplanted to suitable media and placed under
+favourable conditions, the spores germinate, usually within twenty-four
+to thirty-six hours, and successively undergo the following changes
+which may be followed in hanging-drop cultures on a warm stage:
+
+1. Swell up slowly and enlarge, through the absorption of water.
+
+2. Lose their refrangibility.
+
+3. At this stage one of three processes (but the particular process is
+always constant for the same species) may be observed:
+
+(a) The spore grows out into the new bacillus without discarding the
+spore membrane (which in this case now becomes the cell membrane); _e.
+g._, B. leptosporus.
+
+(b) It loses its spore membrane by solution; e. g., B. anthracis.
+
+(c) It loses its spore membrane by rupture.
+
+In this process the rupture may be either polar (at one pole only _e.
+g._, B. butyricus), or bipolar (e. g., B. sessile), or equatorial;
+(e. g., B. subtilis).
+
+In those cases where the spore membrane is discarded the cell membrane
+of the new bacillus may either be formed from--
+
+(a) The inner layer of the spore membrane, which has undergone a
+preliminary splitting into parietal and visceral layers; e. g., B.
+butyricus.
+
+(b) The outer layers of the cell protoplasm, which become
+differentiated for that purpose; e. g., B. megatherium.
+
+The new bacillus now increases in size, elongates, and takes on a
+vegetative growth--i. e., undergoes fission--the bacilli resulting
+from which may in their turn give rise to spores.
+
+[Illustration: FIG. 92. Simple.]
+
+[Illustration: FIG. 93. Solution.]
+
+[Illustration: FIG. 94. Polar.]
+
+[Illustration: FIG. 95. Bipolar.]
+
+[Illustration: FIG. 96. Equatorial.]
+
+
+~Food Stuffs.~--1. _Organic Foods._--
+
+(a) The pure parasites (e. g., B. leprae) will not live outside the
+living body.
+
+(b) Both saprophytic and facultative parasitic bacteria agree in
+requiring non-concentrated food.
+
+(c) The facultative parasites need highly organised foods; e. g.,
+proteids or other sources of nitrogen and carbon, and salts.
+
+(d) The saprophytic bacteria are more easily cultivated; e. g.,
+
+1. Some bacteria will grow in almost pure distilled water.
+
+2. Some bacteria will grow in pure solutions of the carbohydrates.
+
+3. _Water_ is absolutely essential to the _growth_ of bacteria.
+
+Food of a definite reaction is needed for the growth of bacteria. As a
+general rule growth is most active in media which react slightly acid to
+phenolphthalein--that is, neutral or faintly alkaline to litmus. Mould
+growth, on the other hand, is most vigourous in media that are strongly
+acid to phenolphthalein.
+
+~Environment.~--The influence of physical agents upon bacterial life and
+growth is strongly marked.
+
+1. _Atmosphere._--The presence of _oxygen_ is necessary for the growth
+of some bacteria, and death follows when the supply is cut off. Such
+organisms are termed _obligate aerobes_.
+
+Some bacteria appear to thrive equally well whether supplied with or
+deprived of oxygen. These are termed _facultative anaerobes_.
+
+A third class will only live and multiply when the access of free oxygen
+is completely excluded. These are termed _obligate anaerobes_.
+
+2. _Temperature._--Practically no bacterial growth occurs below 5 deg. C,
+and very little above 40 deg. C. 30 deg. C. to 37 deg. C is the most
+favorable for the large majority of micro-organisms.
+
+The maximum and minimum temperatures at which growth takes place, as
+well as the optimum, are fairly constant for each bacterium.
+
+Bacteria have been classified, according to their optimum temperature,
+into--
+
+                                       MIN.       OPT.        MAX.
+
+1. Psychrophilic bacteria
+   (chiefly water organisms)         0 deg. C.  15 deg. C.  30 deg. C.
+2. Mesophilic bacteria
+   (includes pathogenic bacteria)   15 deg. C.  37 deg. C.  45 deg. C.
+3. Thermophilic bacteria            45 deg. C.  55 deg. C.  70 deg. C.
+
+The thermal death-point of an organism is another biological constant;
+and is that temperature which causes the death of the vegetative forms
+when the exposure is continued for a period of ten minutes (see pages
+298-301).
+
+3. _Light._--Many organisms are indifferent to the presence of light. On
+the other hand, light frequently impedes growth, and alters to a greater
+or lesser extent the biochemical characters of the organisms--e. g.,
+chromogenicity or power of liquefaction. Pathogenic bacteria undergo a
+progressive loss of virulence when cultivated in the presence of light.
+
+4. _Movements._--Movements, if slight and simply of a flowing character,
+do not appear to injuriously affect the growth of bacteria; but violent
+agitation, such as shaking, absolutely kills them.
+
+A condition of perfect rest would seem to be that most conducive to
+bacterial growth.
+
+~The Metabolic Products of Bacteria.~--_Pigment Production._--Many
+micro-organisms produce one or more vivid pigments--yellow, orange, red,
+violet, fluorescent, etc.--during the course of their life and growth.
+The colouring matter usually exists as an intercellular excrementitious
+substance. Occasionally, however, it appears to be stored actually
+within the bodies of the bacteria. The chromogenic bacteria are
+therefore classified, in accordance with the final destination of the
+colouring matter they elaborate, into--
+
+_Chromoparous_ Bacteria: in which the pigment is diffused out upon and
+into the surrounding medium.
+
+_Chromophorous_ Bacteria: in which the pigment is stored in the cell
+protoplasm of the organism.
+
+_Parachromophorous_ Bacteria: in which the pigment is stored in the cell
+wall of the organism.
+
+Different species of chromogenic bacteria differ in their requirements
+as to environment, for the production of their characteristic pigments;
+e. g., some need oxygen, light, or high temperature; others again
+favor the converse of these conditions.
+
+_Light Production._--Some bacteria, and usually those originally derived
+from water, whether fresh or salt, exhibit marked phosphorescence when
+cultivated under suitable conditions. These are classed as "photogenic."
+
+_Enzyme Production._--Many bacteria produce soluble ferments or enzymes
+during the course of their growth, as evidenced by the liquefaction of
+gelatine, the clotting of milk, etc. These ferments may belong to either
+of the following well-recognised classes: proteolytic, diastatic,
+invertin, rennet.
+
+_Toxin Production._--A large number, especially of the pathogenic
+bacteria, elaborate or secrete poisonous substances concerning which but
+little exact knowledge is available, although many would appear to be
+enzymic in their action.
+
+These toxins are usually differentiated into--
+
+_Extracellular_ (or Soluble) Toxins: those which are diffused into, and
+held in solution by, the surrounding medium.
+
+_Intracellular_ (or Inseparate) Toxins: those which are so closely bound
+up with the cell protoplasm of the bacteria elaborating them that up to
+the present time no means has been devised for their separation or
+extraction.
+
+_End-products of Metabolism._--Under this heading are included--
+
+Organic Acids (e. g., lactic, butyric, etc.).
+
+Alkalies (e. g., ammonia).
+
+Aromatic Compounds (e. g., indol, phenol).
+
+Reducing Substances (e. g., those reducing nitrates to nitrites).
+
+Gases (e. g., sulphuretted hydrogen, carbon dioxide, etc.).
+
+And while the discussion of their formation, etc., is beyond the scope
+of a laboratory handbook, the methods in use for their detection and
+separation come into the ordinary routine work and will therefore be
+described (_vide_ page 276 _et seq._).
+
+
+
+
+X. NUTRIENT MEDIA.
+
+
+In order that the life and growth of bacteria may be accurately observed
+in the laboratory, it is necessary--
+
+1. To _isolate_ individual members of the different varieties of
+micro-organisms.
+
+2. To _cultivate_ organisms, thus isolated, apart from other associated
+or contaminating bacteria--i. e., in _pure culture_.
+
+For the successful achievement of these objects it is necessary to
+provide nutriment in a form suited to the needs of the particular
+bacterium or bacteria under observation, and in a general way it may be
+said that the nutrient materials should approximate as closely as
+possible, in composition and character, to the natural pabulum of the
+organism.
+
+The general requirements of bacteria as to their food-supply have
+already been indicated (page 142) and many combinations of proteid and
+of carbohydrate have been devised, from time to time, on those lines.
+These, together with various vegetable tissues, physiological or
+pathological fluid secretions, etc., are collectively spoken of as
+_nutrient media_ or _culture media_.
+
+The greater number of these media are primarily _fluid_, but, on account
+of the rapidity with which bacterial growth diffuses itself through a
+liquid, it is impossible to study therein the characteristics of
+individual organisms. Many such media are, therefore, subsequently
+rendered solid by the addition of substances like gelatine or agar, in
+varying proportions, the proportions of such added material being
+generally mentioned when referring to the media; e. g., 10 per cent.
+gelatine, 2 per cent. agar. Gelatine is employed for the solidification
+of those media it is intended to use in the cultivation of bacteria at
+the room temperature or in the "cold" incubator. In the percentages
+usually employed, gelatine media become fluid at 25 deg. C.; higher
+percentages remain solid at somewhat higher temperatures, but the
+difficulty of filtering strong solutions of gelatine militates against
+their general use.
+
+Media, on the other hand which have been solidified by the addition of
+agar, only become liquid when exposed to 90 deg. C. for about ten
+minutes, and again solidify when the temperature falls to 40 deg. C.
+
+When it becomes necessary to render these media fluid, heat is applied,
+upon the withdrawal of which they again assume their solid condition.
+Such media should be referred to as _liquefiable media_; in point of
+fact, however, they are usually grouped together with the solid media.
+
+     NOTE.--It must here be stated that the designation 10 per
+     cent. gelatine or 2 per cent. agar refers only to the
+     quantity of those substances actually added in the process
+     of manufacture, and _not_ to the percentage of gelatine or
+     agar, as the case may be, present in the finished medium;
+     the explanation being that the commercial products employed
+     contain a large proportion of insoluble material which is
+     separated off by filtration during the preparation of the
+     liquefiable media.
+
+Other media, again--e. g., potato, coagulated blood-serum,
+etc.--cannot be again liquefied by physical means, and these are spoken
+of as _solid_ media.
+
+The following pages detail the method of preparing the various nutrient
+media, in ordinary use (see also Chapter XI), those which are only
+occasionally required for more highly specialised work are grouped
+together in Chapter XII. It must be premised that scrupulous cleanliness
+is to be observed with regard to all apparatus, vessels, funnels, etc.,
+employed in the preparation of media; although in the preliminary stages
+of the preparation of most media absolute sterility of the apparatus
+used is not essential.
+
+
+MEAT EXTRACT.
+
+A watery solution of the extractives, etc., of lean meat (usually beef)
+forms the basis of several nutrient media. This solution is termed "meat
+extract" and it has been determined empirically that its preparation
+shall be carried out by extracting half a kilo of moist meat with one
+litre of water. For many purposes, however, it is more convenient to
+have a more concentrated extract; one kilo of meat should therefore be
+extracted with one litre of water, to form "Double Strength" meat
+extract.
+
+It was customary at one time, and is even now in some laboratories to
+use either "shin of beef" or "beef-steak"--both contain muscle sugar
+which often needs to be removed before the nutrient medium can be
+completed. Heart muscle (bullock's heart or sheep's heart) is much to be
+preferred and from the point of economy, ease and cleanliness of
+manipulation, and extractive value, the imported frozen bullock's hearts
+provide the best extract.
+
+Meat extract (Fleischwasser) is prepared as follows:
+
+1. Measure 1000 c.c. of distilled water into a large flask (or glass
+beaker, or enamelled iron pot) and add 1000 grammes (roughly, 2-1/2
+pounds) of fresh lean meat--e. g., bullock's heart--finely minced in a
+mincing machine.
+
+2. Heat the mixture gently in a water-bath, taking care that the
+temperature of the contents of the flask does not exceed 40 deg. C. for
+the first twenty minutes. (This dissolves out the soluble proteids,
+extractives, salts, etc.)
+
+3. Now raise the temperature of the mixture to the boiling-point, and
+maintain at this temperature for ten minutes. (This precipitates some
+of the albumins, the haemoglobin, etc., from the solution.)
+
+4. Strain the mixture through sterile butter muslin or a perforated
+porcelain funnel, then filter the liquid through Swedish filter paper
+into a sterile "normal" litre flask, and when cold make up to 1000 c.c.
+by the addition of distilled water--to replace the loss from
+evaporation.
+
+5. If not needed at once, sterilise the meat extract in bulk in the
+steam steriliser for twenty minutes on each of three consecutive days.
+
+Calf, sheep, or chicken flesh is occasionally substituted for the beef;
+or the meat extract may be prepared from animal viscera, such as brain,
+spleen, liver, or kidneys.
+
+     NOTE.--As an alternative method, 5 c.c. of Brand's meat
+     juice or 3 grammes of Wyeth's beef juice, or 10 grammes
+     Liebig's extract of meat (Lemco) may be dissolved in 1000
+     c.c. distilled water, and heated and filtered as above to
+     form ordinary or single strength meat extract.
+
+     Media, prepared from such meat extracts are, however,
+     eminently unsatisfactory when used for the cultivation of
+     the more highly parasitic bacteria; although when working in
+     tropical and subtropical regions their use is well-nigh
+     compulsory.
+
+~Reaction of Meat Extract.~--Meat extract thus prepared is acid in its
+reaction, owing to the presence of acid phosphates of potassium and
+sodium, weak acids of the glycolic series, and organic compounds in
+which the acid character predominates. Owing to the nature of the
+substances from which it derives its reaction, the total acidity of meat
+extract can only be estimated accurately when the solution is at the
+boiling-point.
+
+Moreover, it has been observed that prolonged boiling (such as is
+involved in the preparation of nutrient media) causes it to undergo
+hydrolytic changes which increase its acidity, and ~the meat extract only
+becomes stable in this respect after it has been maintained at the
+boiling-point for forty-five minutes~.
+
+Although meat extract always reacts acid to phenolphthalein, it
+occasionally reacts neutral or even alkaline to litmus; and again, meat
+extract that has been rendered exactly neutral to litmus still reacts
+acid to phenolphthalein. This peculiar behaviour depends upon two
+factors:
+
+1. Litmus is insensitive to many weak organic acids the presence of
+which is readily indicated by phenolphthalein.
+
+2. Dibasic sodium phosphate which is formed during the process of
+neutralisation is a salt which reacts alkaline to litmus, but neutral to
+phenolphthalein. In order, therefore, to obtain an accurate estimation
+of the reaction of any given sample of meat extract, it is essential
+that--
+
+1. The meat extract be previously exposed to a temperature of 100 deg. C.
+for forty-five minutes.
+
+2. The estimation be performed at the boiling-point.
+
+3. Phenolphthalein be used as the indicator.
+
+The estimation is carried out by means of titration experiments against
+standard solutions of caustic soda, in the following manner:
+
+_Method of Estimating the Reaction._--
+
+_Apparatus Required_:                  _Solutions Required_:
+
+1. 25 c.c. burette graduated           1. 10N NaOH, accurately
+in tenths of a centimetre.             standardised.
+
+2. 1 c.c. pipette graduated in         2. n/1 NaOH, accurately
+hundredths, and provided               standardised
+with rubber tube, pinch-cock,
+and delivery nozzle.
+
+3. 25 c.c. measure (cylinder or        3. n/10 NaOH, accurately
+pipette, calibrated for                standardised.
+98 deg. C.--_not_ 15 deg. C).
+
+4. Several 60 c.c. conical             4. 0.5 per cent. solution of
+beakers or Erlenmeyer                  phenolphthalein in 50 per
+flasks.                                cent. alcohol.
+
+5. White porcelain evaporating basin, filled with boiling water and
+arranged over a gas flame as a water-bath.
+
+6. Bohemian glass flask, fitted as a wash-bottle, and filled
+with distilled water, which is kept boiling on a tripod stand.
+
+METHOD.--Arrange the apparatus as indicated in figure 97.
+
+(A) 1. Fill the burette with n/10 NaOH.
+
+2. Fill the pipette with n/1 NaOH.
+
+[Illustration: FIG. 97.--Arrangement of apparatus for titrating media.]
+
+3. Measure 25 c.c. of the meat extract (previously heated in the steamer
+at 100 deg. C. for forty-five minutes) into one of the beakers by means of
+the measure; rinse out the measure with a very small quantity of boiling
+distilled water from the wash-bottle, and then add this rinse water to
+the meat extract already in the beaker.
+
+4. Run in about 0.5 c.c. of the phenolphthalein solution and immerse the
+beaker in the water-bath, and raise to the boil.
+
+5. To the medium in the beaker run in n/10 NaOH cautiously from the
+burette until the end-point is reached, as indicated by the development
+of a pinkish tinge, shown in figure 98 (b). Note the amount of
+decinormal soda solution used in the process.
+
+     NOTE.--Just before the end-point is reached, a very slight
+     opalescence may be noted in the fluid, due to the
+     precipitation of dibasic phosphates. After the true
+     end-point is reached, the further addition of about 0.5 c.c.
+     of the decinormal soda solution will produce a deep magenta
+     colour (Fig. 98, c), which is the so-called "end-point" of
+     the American Committee of Bacteriologists.
+
+[Illustration: FIG. 98.--a, Sample of filtered meat extract or
+nutrient gelatine to which phenolphthalein has been added. The medium is
+acid, as evidenced by the unaltered colour of the sample. b, The same
+neutralised by the addition of n/10 NaOH. The production of this faint
+rose-pink colour indicates that the "end-point," or neutral point to
+phenolphthalein, has been reached. If such a sample is cooled down to
+say 30 deg. or 20 deg. C., the colour will be found to become more distinct
+and decidedly deeper and brighter, resembling that shown in c. c, Also
+if, after the end-point is reached, a further 0.5 c.c. or 1.0 c.c. n/10
+NaOH be added to the sample, the marked alkalinity is evidenced by the
+deep colour here shown.]
+
+(B) Perform a "control" titration (occasionally two controls may be
+necessary), as follows:
+
+1. Measure 25 c.c. of the meat extract into one of the beakers, wash out
+the measure with boiling water, and add the phenolphthalein as in the
+first estimation.
+
+2. Run in n/1 NaOH from the pipette, just short of the equivalent of the
+amount of _deci_-normal soda solution required to neutralise the 25 c.c.
+of medium. (For example, if in the first estimation 5 c.c. of n/10 NaOH
+were required to render 25 c.c. of medium neutral to phenolphthalein,
+only add 0.48 c.c. of n/1 NaOH.) Immerse the beaker in the water-bath.
+
+3. Complete the titration by the aid of the n/10 NaOH.
+
+4. Note the amount of n/10 NaOH solution required to complete the
+titration, and add it to the equivalent of the n/1 NaOH solution
+previously run in. Take the total as the correct estimation.
+
+
+_Method of Expressing the Reaction._--
+
+The reaction or _titre_ of meat extract, medium, or any solution
+estimated in the foregoing manner, is most conveniently expressed by
+indicating the number of cubic centimetres of normal alkali (or normal
+acid) that would be required to render _one litre_ of the solution
+exactly neutral to phenolphthalein.
+
+[Illustration: FIG. 99.--Stock bottle for dekanormal soda solution.]
+
+The sign + (plus) is prefixed to this number if the original solution
+reacts acid, and the sign - (minus) if it reacts alkaline.
+
+For example, "meat extract + 10," indicates a sample of meat extract
+which reacts acid to phenolphthalein, and would require the addition of
+10 c.c. of _normal_ NaOH per litre, to neutralise it.
+
+     NOTE.--Such a solution would probably react alkaline to
+     litmus.
+
+Conversely, if as the result of our titration experiments we find that
+25 c.c. of meat extract require the addition of 5 c.c. n/10 NaOH to
+neutralise, then 1000 c.c. of meat extract will require the addition of
+200 c.c. n/10 NaOH = 20 c.c. n/1 NaOH.
+
+And this last figure, 20, preceded by the sign + (i. e., +20), to
+signify that it is acid, indicates the reaction of the meat extract.
+
+     NOTE.--The standard soda solutions should be prepared by
+     accurate measuring operations, controlled by titrations,
+     from a stock solution of 10N NaOH, which should be very
+     carefully standardised. If a large supply is made or the
+     consumption is small this stock solution must be kept in an
+     aspirator bottle to which air can only gain access after it
+     has been dried and rendered free from CO_{2}. This may be
+     done by first leading it over H_{2}SO_{4} and soda lime, or
+     soda lime alone, by some such arrangement as is shown in
+     figure 99, which also shows a constant burette arrangement
+     for the delivery of small measured quantities of the
+     dekanormal soda solution.
+
+
+STANDARDISATION OF MEDIA.
+
+Differences in the reaction of the medium in which it is grown will
+provoke not only differences in the rate of growth of any given
+bacterium, but also well-marked differences in its cultural and
+morphological characters; and nearly every organism will be found to
+affect a definite "optimum reaction"--a point to be carefully determined
+for each. For most bacteria, however, the "optimum" usually approximates
+fairly closely to +10; and as experiment has shown that this reaction is
+the most generally useful for routine laboratory work, it is the one
+which may be adopted as the standard for all nutrient media derived from
+meat extract.
+
+Briefly, the method of standardising a litre of media to +10 consists in
+subtracting 10 from the initial _titre_ of the medium mass; the
+remainder indicates the number of cubic centimetres of normal soda
+solution that must be added to the medium, per litre, to render the
+reaction +10.
+
+~Standardising Nutrient Bouillon.~--For example, 1000 c.c. bouillon are
+prepared; at the first titration it is found
+
+1. 25 c.c. require the addition of 5.50 c.c. n/10 NaOH to neutralise.
+
+Two controls give the following results:
+
+2. 25 c.c. require the addition of 5.70 c.c. n/10 NaOH to neutralise.
+
+3. 25 c.c. require the addition of 5.60 c.c. n/10 NaOH to neutralise.
+
+Averaging these two controls, 25 c.c. require the addition of 5.65 c.c.
+n/10 NaOH to neutralise, and therefore 1000 c.c. require the addition of
+226 c.c. n/10 NaOH, or 22.60 c.c. n/1 NaOH, or 2.26 c.c. n/10 NaOH.
+
+Initial _titre_ of the bouillon = +22.6, and as such requires the
+addition of (22.6 c.c. - 10 c.c.) = 12.6 c.c. of n/1 NaOH per litre to
+leave its finished reaction +10.
+
+But the three titrations, each on 25 c.c. of medium, have reduced the
+original bulk of bouillon to (1000 - 75 c.c.) = 925 c.c. The amount of
+n/1 NaOH required to render the reaction of this quantity of medium +10
+may be deduced thus:
+
+    1000 c.c.:925 c.c.::12.6 c.c.:x.
+
+Then x = 11.65 c.c. n/1 NaOH.
+
+Whenever possible, however, the required reaction is produced by the
+addition of dekanormal soda solution, on account of the minute increase
+it causes in the bulk, and the consequent insignificant disturbance of
+the percentage composition of the medium. By means of a pipette
+graduated to 0.01 c.c. it is possible to deliver very small quantities;
+but if the calculated amount runs into thousandth parts of a cubic
+centimetre, these are replaced by corresponding quantities of normal or
+even decinormal soda.
+
+In the above example it is necessary to add 11.65 c.c. normal NaOH or
+its equivalent, 1.165 c.c. dekanormal NaOH. The first being too bulky a
+quantity, and the second inconveniently small for exact measurement, the
+total weight of soda is obtained by substituting 1.16 c.c. dekanormal
+soda solution, and either 0.05 c.c. of normal soda solution or 0.5 c.c.
+of decinormal soda solution.
+
+~Standardising Nutrient Agar and Gelatine.~--The method of standardising
+agar and gelatine is precisely similar to that described under bouillon.
+
+
+THE FILTRATION OF MEDIA.
+
+~Fluid media~ are usually filtered through stout Swedish filter paper
+(occasionally through a porcelain filter candle), and in order to
+accelerate the rate of filtration the filter paper should be folded in
+that form which is known as the "physiological filter," not in the
+ordinary "quadrant" shape, as by this means a large surface is available
+for filtration and a smaller area in contact with the glass funnel
+supporting it.
+
+To fold the filter proceed thus:
+
+1. Take a circular piece of filter paper and fold it exactly through its
+centre to form a semicircle (Fig. 100, a).
+
+2. Fold the semicircle exactly in half to form a quadrant; make the
+crease 2, distinct by running the thumbnail along it, then open the
+filter out to a semicircle again.
+
+3. Fold each end of the semicircle in to the centre and so form another
+quadrant; smooth down the two new creases 3 and 3a, thus formed and
+again open out to a semicircle.
+
+4. The semicircle now appears as in figure 100, a, the dark lines
+indicating the creases already formed.
+
+5. Fold the point 1 over to the point 3, and 1a to 3a, to form the
+creases 4 and 4a, indicated in the diagram by the light lines. Fold
+point 1 over to 3a, and 1a to 3, to form the creases 5 and 5a.
+
+[Illustration: FIG. 100.--Filter folding: a, Filter folded in half,
+showing creases; b, appearance of filter on completion of folding;
+c, filter opened out ready for use.]
+
+6. Thus far the creases have all been made on the same side of the
+paper. Now subdivide each of the eight sectors by a crease through its
+centre on the opposite side of the paper, indicated by the faint broken
+lines in the diagram. Fold up the filter gradually as each crease is
+made, and when finished the filter has assumed the shape of a wedge, as
+in figure 100, b.
+
+When opened out the filter assumes the shape represented in figure 100,
+c.
+
+The folded filter is next placed inside a glass funnel supported on a
+retort stand, and moistened with hot distilled water before the
+filtration of the medium is commenced.
+
+~Liquefiable solid media~ are filtered through a specially made filter
+paper--"papier Chardin"--which is sold in boxes of twenty-five
+ready-folded filters.
+
+[Illustration: FIG. 101.--Hot-water filter funnel and ring burner.]
+
+Gelatine, when properly made, filters through this paper as quickly as
+bouillon does through the Swedish filter paper, and does _not_ require
+the use of the hot-water funnel.
+
+Agar, likewise, if properly made, filters readily, although not at so
+rapid a rate as gelatine. If badly "egged," and also during the winter
+months, it is necessary to surround the glass funnel, in which the
+filtration of the agar is carried on, by a hot-water jacket. This is
+done by placing the glass funnel inside a double-walled copper
+funnel--the space between the walls being filled with water at about
+90 deg. C.--and supporting the latter on a ring gas burner fixed to a
+retort stand (Fig. 101). The gas is lighted and the water jacket
+maintained at a high temperature until filtration is completed. If the
+steam steriliser of the laboratory is sufficiently large, it is sometimes
+more convenient to place the flask and filtering funnel bodily inside,
+close the steriliser and allow filtration to proceed in an atmosphere of
+live steam, than to use the gas ring and hot-water funnel.
+
+
+STORING MEDIA IN BULK.
+
+After filtration fill the medium into sterile litre flasks with
+cotton-wool plugs and sterilise in the steamer for twenty minutes on
+each of three consecutive days. After the third sterilisation, and when
+the flasks and contents are cool, cut off the top of the cotton-wool
+plug square with the mouth of the flask; push the plug a short distance
+down into the neck of the flask and fill in with melted paraffin wax to
+the level of the mouth. When the wax has set the flasks are stored in a
+cool dark cupboard for future use.
+
+[Illustration: FIG. 102.--Rubber cap closing store bottle. a, before,
+and b, after sterilizing.]
+
+This plan is not absolutely satisfactory, although very generally
+employed on occasion, and it is preferable to fill the medium into
+long-necked flint glass bottles (the quart size, holding nearly 1000
+c.c., such as those in which Pasteurised milk is retailed) and to close
+the neck of the bottle by a special rubber cap.[3] This cap is made of
+soft rubber, the lower part, dome-shaped with thin walls, being slipped
+over the neck of the bottle (Fig. 102, a). The upper part is solid,
+but with a sharp clean-cut (made with a cataract or tenotomy knife)
+running completely through its axis from the centre of the disc to the
+top of the dome. During sterilisation the air in the neck of the bottle,
+expanded by the heat, is driven out through the valvular aperture in the
+solid portion of the stopper. On removing the bottle from the steam
+chamber, the liquid contracts as it cools, and the pressure of the
+external air drives the solid piece of rubber down into the neck of the
+bottle, and forces together the lips of the slit (Fig. 102, b). Thus
+sealed, the bottle will preserve its contents sterile for an indefinite
+period without loss from evaporation.
+
+
+TUBING NUTRIENT MEDIA.
+
+After the final filtration, the nutrient medium is usually "tubed"--_i.
+e._, filled into sterile tubes in definite measured quantities, usually
+10 c.c. This process is sometimes carried out by means of a large
+separator funnel fitted with a "three-way" tap which communicates with a
+small graduated tube (capacity 20 c.c. and graduated in cubic
+centimetres) attached to the side. The shape of this piece of apparatus,
+known as Treskow's funnel, renders it particularly liable to damage. It
+is better, therefore, to arrange a less expensive piece of apparatus
+which will serve the purpose equally well (Fig. 103).
+
+A Geissler's three-way stop-cock has the tube on one side of the tap
+ground obliquely at its extremity, and the tube on the opposite side cut
+off within 3 cm. of the tap. The short tube is connected by means of a
+perforated rubber cork with a 10 cm. length of stout glass tubing (1.5
+cm. bore). The third channel of the three-way tap is connected, by means
+of rubber tubing, with the nozzle of an ordinary separator funnel.
+Finally, the receiving cylinder above the three-way tap is graduated in
+cubic centimetres up to 20, by pouring into it measured quantities of
+water and marking the various levels on the outside with a writing
+diamond.
+
+Fluid media containing carbohydrates are filled into fermentation tubes
+(_vide_ Fig. 21); or into ordinary media tubes which already have
+smaller tubes, inverted, inside them (Fig. 104), to collect the products
+of growth of gas-forming bacteria. When first filled, the small tubes
+float on the surface of the medium after the first sterilisation nearly
+all the air is replaced by the medium, and after the final sterilisation
+the gas tubes will be submerged and completely filled with the medium.
+
+[Illustration: FIG. 103.--Separatory funnel and three-way tap arranged
+for tubing media.]
+
+[Illustration: FIG. 104.--Gas tube (Durham).]
+
+~Storing "Tubed" Media.~--Media after being tubed are best stored by
+packing, in the vertical position, in oblong boxes having an internal
+measurement of 37 cm. long by 12 cm. wide by 10 cm. deep. Each box (Fig.
+105) has a movable partition formed by the vertical face of a weighted
+triangular block of wood, sliding free on the bottom (Fig. 105, A); or
+by a flat piece of wood sliding in a metal groove in the bottom of the
+box, which can be fixed at any spot by tightening the thumbscrew of a
+brass guide rod which transfixes the partition (Fig. 105, B). The front
+of the box is provided with a handle and a celluloid label for the name
+of the contained medium. These boxes are arranged upon shelves in a dark
+cupboard--or preferably an iron safe--which should be rendered as nearly
+air-tight as possible, and should have the words "media stores" painted
+on its doors.
+
+[Illustration: FIG. 105.--Medium box, showing alternative partitions A
+and B.]
+
+FOOTNOTES:
+
+[3] This rubber cap has been made for me by the Holborn Surgical
+Instrument Co., Thavies Inn, London, W. C.
+
+
+
+
+XI. CULTURE MEDIA.
+
+ORDINARY OR STOCK MEDIA.
+
+
+~Nutrient Bouillon.~--
+
+1. Measure out double strength meat extract, 500 c.c., into a litre
+flask and add 300 c.c. distilled water.
+
+2. Weigh out Witte's peptone, 10 grammes (= 1 per cent.), salt, 5
+grammes (= 0.5 per cent.), and mix into a smooth paste with 200 c.c. of
+distilled water previously heated to 60 deg. C. (Be careful to leave no
+unbroken globular masses of peptone.)
+
+3. Add the peptone emulsion to the meat extract in the flask and heat in
+the steamer for forty-five minutes (to completely dissolve the peptone,
+and to render the acidity of the meat extract stable).
+
+4. Estimate the reaction of the medium; control the result; render the
+reaction of the finished medium +10 (_vide_ page 155).
+
+5. Heat for half an hour in the steamer at 100 deg. C. (to complete the
+precipitation of the phosphates, etc.).
+
+6. Filter through Swedish filter paper into a sterile flask.
+
+7. Fill into sterile tubes (10 c.c. in each tube).
+
+8. Sterilise in the steamer for twenty minutes on each of three
+consecutive days--i. e., by the discontinuous method (_vide_ page 35).
+
+     NOTE.--As an alternative method when neither fresh nor
+     frozen meat is available nutrient bouillon may be prepared
+     from a commercial meat extract, as follows:
+
+     ~Lemco Broth.~--
+
+     1. Measure out 250 c.c. distilled water into a litre flask.
+
+     2. Weigh out 10 grammes Liebig's Lemco Meat Extract on a
+     piece of clean filter paper and add to the water in the
+     flask. Shake the flask well to make an even emulsion of the
+     meat extract.
+
+     3. Weigh out Witte's peptone (10 grammes), salt (5 grammes).
+     Mix into smooth paste with 100 c.c. distilled water
+     previously heated to 60 deg. C.
+
+     4. Add the peptone salt emulsion to the meat extract
+     emulsion in the flask and add 650 c.c. distilled water. Heat
+     in the steamer for forty-five minutes.
+
+     5. Standardise the medium and complete as for nutrient
+     bouillon.
+
+~Nutrient Gelatine.~--
+
+1. Weigh a 2-litre flask on a trip balance (Fig. 106) and note the
+weight, or counterpoise carefully.
+
+[Illustration: FIG. 106.--Trip balance.]
+
+An extremely useful counterpoise is a small sheet-brass cylinder about
+38 mm. high and 38 mm. in diameter, with a funnel-shaped top and
+provided with a side tube by which its contents, fine "dust" shot, may
+be emptied out (Fig. 107).
+
+[Illustration: FIG. 107.--Counterpoise; weight when empty, 35 grammes;
+when full of dust shot, 200 grammes.]
+
+2. Measure out double strength meat extract, 500 c.c., into the "tared"
+flask.
+
+3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and make
+into a thick paste with 150 c.c. distilled water; then add the emulsion
+to the meat extract in the flask; also add 100 grammes sheet gelatine
+cut into small pieces; place the flask in the water-bath and raise to
+the boil.
+
+[Illustration: FIG. 108.--Arrangement of steam can and water-bath for
+the preparation of media.]
+
+4. Arrange a 5-litre tin can (with copper bottom, such as is used in the
+preparation of distilled water) by the side of the water bath, fill the
+can with boiling water and place a lighted Bunsen burner under it. Fit a
+long safety tube to the neck of the can and also a delivery tube, bent
+twice at right angles; adjust the tube to reach to the bottom of the
+interior of the flask containing the gelatine, etc. (Fig. 108).
+
+5. Keep the water in the steam can vigourously boiling, and so steam at
+100 deg. C, bubbling through the medium mass, for ten minutes, by
+which time complete solution of the gelatine is effected. A certain
+amount of steam will condense as water in the medium flask during this
+process--hence the necessity for the use of double strength meat
+extract--but if the water bath is kept boiling this condensation will
+not exceed 100 c.c.
+
+6. Weigh the flask and its contents; then (1115[4] grammes + weight of
+the flask) minus (weight of the flask and its contents) equals the
+weight of water required to make up the bulk to 1 litre. The addition of
+the requisite quantity of water is carried out as follows:
+
+In one pan of the trip balance place the counterpoise of the tared flask
+(or its equivalent in weights) together with the weights making up the
+_calculated medium weight_. In the opposite pan place the flask
+containing the medium mass. Now add boiling distilled water from a wash
+bottle until the two pans are exactly balanced.
+
+7. Titrate and estimate the reaction of the medium mass; control the
+result. Calculate the amount of soda solution required to make the
+reaction of the medium mass +10 (i. e., calculate for 1000 c.c., less
+the quantity used for the titrations).
+
+8. Add the necessary amount of soda solution and heat in the steamer at
+100 deg. C. for twenty minutes, to precipitate the phosphates, etc.
+
+9. Allow the medium mass to cool to 60 deg. C. Well whip the whites of
+two eggs, add to the contents of the flask and replace in the steamer at
+100 deg. C. for about half an hour (until the egg-albumen has coagulated
+and formed large, firm masses floating on and in clear gelatine).
+
+10. Filter through papier Chardin into a sterile flask.
+
+11. Tube in quantities of 10 c.c.
+
+12. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
+three consecutive days--i. e., by the discontinuous method.
+
+
+~Nutrient Agar-agar.~--
+
+1. Weigh a 2-litre flask and note the weight--or counterpoise exactly.
+
+2. Measure out double strength meat extract, 500 c.c., into the "tared"
+flask.
+
+3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and 20
+grammes of powdered agar, and make into a thick paste with 150 c.c.
+distilled water, and add to the meat extract in the flask; place the
+flask in a water-bath.
+
+4. Arrange the steam can and water-bath as already directed (for the
+preparation of gelatine) and figured.
+
+5. Bubble live steam (at 100 deg. C.) through the medium mass, for
+twenty-five minutes, by which time complete solution of the agar is
+effected.
+
+6. Now weigh the flask and its contents; then (1035[5] grammes + weight
+of flask) minus (weight of flask and its contents) equals the weight of
+water required to make up the bulk of the medium to 1 litre. Add the
+requisite amount (see preparation of gelatine, page 166, step 6).
+
+7. Titrate, and estimate the reaction of the medium mass; control the
+result. Calculate the amount of soda solution required to make the
+reaction of the medium mass + 10 (i. e., calculated for 1000 c.c.,
+less the quantity used for the titrations).
+
+8. Add the necessary amount of soda solution and replace in the steamer
+for twenty minutes (to complete the precipitation of the phosphates,
+etc.).
+
+9. Allow the medium mass to cool to 60 deg. C. Well whip the whites of
+two eggs, add to the contents of the flask, and replace in the steamer at
+100 deg. C. for about _one hour_ (until the egg-albumen has coagulated
+and formed large, firm masses floating on and in clear agar.)
+
+10. Filter through papier Chardin, by the aid of a hot-water funnel, if
+necessary (Fig. 101), into a sterile flask.
+
+11. Tube in quantities of 10 c.c. or 15 c.c.
+
+12. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
+three consecutive days--i. e., by the discontinuous method.
+
+
+~Blood-serum (Inspissated).~--
+
+1. Sterilise cylindrical glass jar (Fig. 109) and its cover by dry heat,
+or by washing first with ether and then with alcohol and drying.
+
+2. Collect blood at the slaughter house from ox or sheep in the sterile
+cylinder.
+
+3. Allow the vessel to stand for fifteen minutes for the blood to
+coagulate. (This must be done before leaving the slaughterhouse,
+otherwise the serum will be stained with haemoglobin.)
+
+4. Separate the clot from the sides of the vessel by means of a sterile
+glass rod (the yield of serum is much smaller when this is not done),
+and place the cylinder in the ice-chest for twenty-four hours.
+
+5. Remove the serum with sterile pipettes, or syphon it off, and fill
+into sterile tubes (5 c.c. in each) or flasks.
+
+6. Heat tubes containing serum to 56 deg. C. in a water-bath for half an
+hour on each of two successive days.
+
+7. On the third day, heat the tubes, in a sloping position, in a serum
+inspissator to about 72 deg. C. (A coagulum is formed at this temperature
+which is fairly transparent; above 72 deg. C., a thick turbid coagulum is
+formed.)
+
+[Illustration: FIG. 109.--Blood-serum jar with wicker basket for
+transport.]
+
+The serum inspissator (Fig. 110) in its simplest form is a double-walled
+rectangular copper box, closed in by a loose glass lid, and cased in
+felt or asbestos--the space between the walls is filled with water. The
+inspissator is supported on adjustable legs so that the serum may be
+solidified at any desired "slant," and is heated from below by a Bunsen
+burner controlled by a thermo-regulator. The more elaborate forms
+resemble the hot-air oven (Fig. 26) in shape and are provided with
+adjustable shelves so that any desired obliquity of the serum slope can
+be obtained.
+
+8. Place the tubes in the incubator at 37 deg. C. for forty-eight hours
+in order to eliminate those that have been contaminated. Store the
+remainder in a cool place for future use.
+
+_Alternative Method._
+
+_Steps 1-5 as above._
+
+6. Sterilise the serum by the fractional method--that is, by exposure in
+a water-bath to a temperature of 56 deg. C. for half an hour on each of
+six consecutive days; store in the fluid condition.
+
+7. Coagulate in the inspissator when needed.
+
+[Illustration: FIG. 110.--Serum inspissator.]
+
+     ~Serum Water.~--
+
+     This forms the basis of many useful media, and is prepared
+     as follows:
+
+     1. Collect blood in the slaughterhouse (see page 168) and
+     when firmly clotted collect all the expressed serum and
+     measure in a graduated cylinder.
+
+     2. For every 100 c.c. of serum add 300 c.c. distilled water
+     and mix in a flask.
+
+     3. Heat the mixture in the steamer at 100 deg. C. for thirty
+     minutes. (This destroys any diastatic ferment present in the
+     serum and partially sterilises the fluid.)
+
+     4. Filter if turbid.
+
+     5. If not needed at once complete the sterilisation of the
+     serum water by two subsequent steamings at 100 deg. C. for
+     twenty minutes at twenty-four hour intervals.
+
+
+~Citrated Blood Agar. Guy's.~--
+
+1. Kill a small rabbit with chloroform vapour, and nail it out on a
+board (as for a necropsy); moisten the hair thoroughly with 2 per cent.
+solution of lysol.
+
+2. Sterilise several pairs of forceps, scissors, etc. by boiling.
+
+3. Reflect the skin over the thorax with sterile instruments.
+
+4. Open the thoracic cavity by the aid of a fresh set of sterile
+instruments.
+
+5. Open the pericardium with another set of sterile instruments.
+
+6. Sear the surface of the left ventricle with a red-hot iron.
+
+7. Take a sterile capillary pipette (Fig. 13, c); break off the sealed
+extremity with a pair of sterile forceps.
+
+8. Steady the heart in a pair of forceps and thrust the point of the
+pipette through the wall of the ventricle and through the seared area,
+apply suction to the plugged end of the pipette and fill it with blood.
+
+9. Transfer the entire quantity of blood collected from the rabbit's
+heart to a small Erlenmeyer flask containing a number of sterile glass
+beads and 5 c.c. concentrated sod. citrate solution. (See page 378.)
+
+10. Agitate thoroughly and set aside for a couple of hours.
+
+11. Melt up several tubes of nutrient agar (see page 167) and cool to
+42 deg. C.
+
+12. With a sterile 10 c.c. graduated pipette transfer 1 c.c. citrated
+blood from the Erlenmeyer flask to each tube of liquefied agar. Rotate
+the tube between the hands in order to diffuse the citrated blood evenly
+throughout the agar.
+
+13. Place the tubes in a sloping position and allow the medium to set.
+
+14. Place tubes of blood agar for forty-eight hours in the incubator at
+37 deg. C. and at the end of that time eliminate any contaminated tubes.
+
+15. Store such tubes as remain sterile for future use.
+
+
+~Milk.~--
+
+1. Pour 1 litre of fresh cow's or goat's milk into a large separating
+funnel, and heat in the steamer at 100 deg. C. for one hour.
+
+2. Remove from the steamer and estimate the reaction of the milk (normal
+cows' milk averages +17). If of higher acidity than +20, or lower than
++10, reject this sample of milk and proceed with another supply of milk
+from a different source.
+
+Reject milk to which antiseptics have been added as preservatives.
+
+3. Allow the milk to cool, when the fat or cream will rise to the
+surface and form a thick layer.
+
+4. Draw off the subnatant fat-free milk into sterile tubes (10 c.c. in
+each).
+
+5. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
+five successive days.
+
+6. Incubate at 37 deg. C. for forty-eight hours and eliminate any
+contaminated tubes. Store the remainder for future use.
+
+
+~Litmus Milk.~--
+
+1. Prepare milk as described above, sections 1 to 3.
+
+2. Draw off the subnatant fat-free milk into a flask.
+
+3. Add sterile litmus solution, sufficient to colour the milk a deep
+lavender.
+
+4. Tube, sterilise, etc., as for milk.
+
+
+~Nutrose Agar (Eyre).~--
+
+(This is a modification of the well known Drigalski-Conradi medium
+originally introduced for the isolation of B. typhosus).
+
+1. Collect 250 c.c. perfectly fresh ox serum (_vide_ Blood Serum, page
+168, steps 1 to 5) and add to it 450 c.c. sterile distilled water.
+
+2. Weigh out agar powder, 20 grammes, and emulsify it with 250 c.c. of
+the cold serum water.
+
+3. Weigh out
+
+    Witte's peptone        10 grammes
+    Sodium chloride         5 grammes
+    Nutrose                10 grammes
+
+and dissolve in 200 c.c. of serum water heated to 80 deg. C.
+
+4. Mix the agar emulsion and the peptone-nutrose solution in a "tared"
+flask of 2-litre capacity and add a further 100 c.c. serum water.
+
+5. Complete the solution of the various ingredients by bubbling live
+steam through the flask as in making nutrient agar.
+
+6. Add further 250 c.c. serum water.
+
+7. Weigh the flask and its contents: then (1045 grammes + weight of
+flask) minus (weight of flask and its present contents) = weight of
+fluid required to make up the bulk of the medium to 1 litre. Add the
+requisite amount of sterile distilled water.
+
+8. Titrate and estimate the reaction of the medium mass. Then
+standardise to reaction of +2.5.
+
+9. Clarify with egg, and filter as for nutrient agar. (In clarifying,
+after the addition of the egg white the mixture should be in the steamer
+for full two hours.)
+
+10. After filtration is complete measure the filtrate, and to every 150
+c.c. of the medium add:
+
+Litmus solution (Kahlbaum)                              20   c.c.
+Krystal violet aqueous solution (1:1000) (B. Hoechst)    1.5 c.c.
+Lactose                                                  1.5 grammes
+
+11. Tube in quantities of 15 c.c.
+
+12. Sterilise in the steamer at 100 deg. C. for thirty minutes on each
+of three successive days--i. e., by the discontinuous method for three
+days.
+
+
+~Egg Medium (Dorset).~--
+
+1. Prepare 1000 c.c. of a 0.85 per cent. solution of sodium chloride in
+a stout 2-litre flask.
+
+2. Sterilise in the autoclave at 120 deg. C. for twenty minutes. Cool
+to 20 deg. C.
+
+3. Take 12 fresh eggs; wash the shells first with water then with
+undiluted formalin: allow the shells to dry.
+
+4. Break the eggs into a sterile graduated cylinder and measure the
+total volume of the mixed whites and yolks. Add one part sterile saline
+solution to three parts mixed eggs.
+
+5. Transfer this mixture to a large wide-mouthed stoppered bottle
+previously sterilised. Add sterile glass beads and shake thoroughly in a
+mechanical shaker for about thirty minutes, or whip with an egg-whisk.
+
+6. Filter through coarse butter muslin into a sterile flask.
+
+     NOTE.--A few drops of alcoholic solution of basic fuchsin
+     (sufficient to give a definite pink colour), or a few drops
+     of waterproof Chinese ink added to the medium at this stage
+     facilitates the subsequent "fishing" of colonies.
+
+7. Tube in quantities of 10 c.c.
+
+8. Solidify in the sloping position in the inspissator at 75 deg. C.
+for one hour.
+
+9. Place the tubes for forty-eight hours in the incubator at 37 deg. C.,
+and eliminate any contaminated tubes.
+
+To prevent drying, 0.5 c.c. glycerine bouillon (see page 209) may be
+added to each tube between steps 8 and 9.
+
+10. Cap those tubes of media which remain sterile with india-rubber caps
+and store for future use.
+
+
+~Potato.~--
+
+1. Choose fairly large potatoes, wash them well, and scrub the peel with
+a stiff nail-brush.
+
+2. Peel and take out the eyes.
+
+3. Remove cylinders from the longest diameter of each potato by means of
+an apple-corer or a large cork-borer (i. e., one of about 1.4 cm.
+diameter).
+
+The reaction of the fresh potato is strongly acid to phenolphthalein.
+If, therefore, the potatoes are required to approximate +10, as for the
+cultivation of some of the vibrios, the cylinders should be soaked in a
+1 per cent. solution of sodium carbonate for thirty minutes.
+
+4. Cut each cylinder obliquely from end to end, forming two wedge-shaped
+portions.
+
+5. Place a small piece of sterilised cotton-wool, moistened with sterile
+water, at the bottom of a sterile test-tube; insert the potato wedge
+into the tube so that its base rests upon the cotton-wool. Now plug the
+tube with cotton-wool (Fig. 111).
+
+6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
+_five_ consecutive days.
+
+[Illustration: FIG. 111.--Potato tube.]
+
+     NOTE.--The cork borer reserved for cutting the potato
+     cylinders should be silver electro-plated both inside and
+     out, and the knife used for dividing the cylinders should be
+     of silver or silver plated. When these precautions are
+     adopted the potato wedges will retain their white color and
+     will not show the discoloration so often observed when steel
+     instruments are employed.
+
+~Beer Wort.~--Wort is chiefly used as a medium for the cultivation of
+yeasts, moulds, etc., both in its fluid form and also when made solid by
+the addition of gelatine or agar. The wort is prepared as follows:
+
+1. Weigh out 250 grammes crushed malt and place in a 2-litre flask.
+
+2. Add 1000 c.c. distilled water, heated to 70 deg. C., and close the
+flask with a rubber stopper.
+
+3. Place the flask in a water-bath regulated to 60 deg. C. and allow
+the maceration to continue for one hour.
+
+4. Strain through butter muslin into a clean flask and heat in the
+steamer for thirty minutes.
+
+5. Filter through Swedish filter paper.
+
+6. Tube in quantities of 10 c.c. or store in flasks.
+
+7. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
+three consecutive days.
+
+The natural reaction of the wort should _not_ be interfered with.
+
+     NOTE.--It is sometimes more convenient to obtain
+     "_unhopped_"[6] beer wort direct from the brewery. In this
+     case it is diluted with an equal quantity of distilled
+     water, steamed for an hour, filtered, filled into sterile
+     flasks or tubes, and sterilised by the discontinuous method.
+
+
+~Wort Gelatine.~--
+
+1. Measure out wort (prepared as above), 900 c.c., into a sterile flask.
+
+2. Weigh out gelatine, 100 grammes (= 10 per cent.), and add it to the
+wort in the flask.
+
+3. Bubble live steam through the mixture for ten minutes, to dissolve
+the gelatine.
+
+4. Cool to 60 deg. C.; clarify with egg as for nutrient gelatine
+(_vide_ page 164).
+
+5. Filter through papier Chardin.
+
+6. Tube, and sterilise as for nutrient gelatine.
+
+
+~Wort Agar.~--
+
+1. Measure out wort (as above), 700 c.c., into a sterile flask.
+
+2. Weigh out powdered agar, 20 grammes; mix into a smooth paste with 200
+c.c. of cold wort and add to the wort in the flask.
+
+3. Bubble live steam through the mixture for twenty minutes, to dissolve
+the agar.
+
+4. Cool to 60 deg. C.; clarify with egg as for nutrient agar (_vide_ page
+167).
+
+5. Filter through papier Chardin, using the hot-water funnel.
+
+6. Tube, and sterilise as for nutrient agar.
+
+
+~Peptone Water (Dunham).~--
+
+1. Weigh out Witte's peptone, 10 grammes, and salt, 5 grammes, and
+emulsify with about 250 c.c. of distilled water previously heated to
+60 deg. C.
+
+2. Pour the emulsion into a litre flask and make up to 1000 c.c. by the
+addition of distilled water.
+
+3. Heat in the steamer at 100 deg. C. for thirty minutes.
+
+4. Filter through Swedish filter paper.
+
+5. Tube in quantities of 10 c.c. each.
+
+6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
+three consecutive days.
+
+~"Sugar" or "Carbohydrate" Media.~--
+
+Formerly the ability of bacteria to induce hydrolytic changes in
+carbohydrate substances was observed only in connection with a few
+well-defined sugars, but of recent years it has been shown that when
+using litmus as an indicator these so-called "fermentation reactions"
+facilitate the differentiation of closely allied species, and the list
+of substances employed in this connection has been considerably
+extended. The media prepared with them are now no longer regarded as
+special, but are comprised in the "stock media" of the laboratory. The
+chief of these substances are the following, arranged in accordance with
+their chemical constitution:
+
+    _Monosaccharides_       Dextrose (glucose), laevulose, galactose,
+                              mannose, arabinose, xylose.
+    _Disaccharides_         Maltose, lactose, saccharose.
+    _Trisaccharides_        Raffinose (mellitose).
+    _Polysaccharides_       Dextrin, inulin, starch, glycogen, amidon.
+    _Glucosides_            Amygdalin, coniferin, salicin,
+                              helicin, phlorrhizin.
+    _Polyatomic alcohols_   _Trihydric_, Glycerin.
+                            _Tetrahydric_, Erythrite.
+                            _Pentahydric_, Adonite.
+                            _Hexahydric_, Dulcite, (dulcitol or
+                              melampirite), isodulcite (rhamnose),
+                              mannite (mannitol), sorbite (sorbitol),
+                              inosite.
+
+These substances should be obtained from Kahlbaum (of Berlin); in the
+pure form, and when possible as large crystals, and the method of
+preparing a medium containing either of them may be exemplified by
+describing Dextrose Solution.
+
+
+~Dextrose Solution.~--
+
+1. Weigh out
+
+    Peptone    20 grammes
+    Glucose    10 grammes
+
+and grind together in a mortar; then emulsify in 100 c.c. of distilled
+water heated to 60 deg. C.
+
+2. Place in a flask and add
+
+    Distilled water   850 c.c.
+
+3. Steam in the steamer at 100 deg. C. for twenty minutes to dissolve
+the peptone and glucose.
+
+4. Add
+
+    Kubel-Tiemann litmus solution (Kahlbaum)    50 c.c.
+
+(The substances enumerated above react acid to phenolphthalein, but
+variously toward the neutral litmus solution. To such as react acid, add
+very cautiously n/1 sodium hydrate solution to the medium in bulk until
+the neutral tint has returned).
+
+5. Fill into tubes in which have previously been placed the inverted
+Durham's gas tubes.
+
+6. Sterilise in the steamer at 100 deg. C. for _twenty minutes_ on
+each of three successive days.
+
+     NOTE.--On no account should these media be sterilised in the
+     autoclave, as temperatures above 100 deg. C. themselves induce
+     hydrolytic changes in the substances in question. It is
+     equally important that the twenty minutes should not be
+     exceeded in sterilisation, as neglect of this precaution may
+     discolour the litmus or lead to the production of yellowish
+     tints when the tubes are subsequently inoculated with
+     acid-forming bacteria.
+
+
+~Neutral Litmus Solution.~
+
+The most satisfactory is the Kubel-Tiemann, prepared by Kahlbaum. It can
+however be made in the laboratory as follows:
+
+1. Weigh out
+
+    Commercial litmus      50 grammes,
+
+and place in a well stoppered 500 c.c. bottle; measure out and add 300
+c.c. alcohol 95 per cent.
+
+2. Shake well at least once a day for seven days--the alcohol acquires a
+green colour.
+
+3. Decant off the green alcohol and fill a further 300 c.c. 95 per cent.
+alcohol into the bottle and repeat the shaking.
+
+4. Repeat this process until on adding fresh alcohol the fluid only
+becomes tinged with violet.
+
+5. Pour off the alcohol, leaving the litmus as dry as possible. Connect
+up the bottle to an air pump and evaporate off the last traces of
+alcohol.
+
+6. Transfer the dry litmus to a litre flask, measure in 600 c.c.
+distilled water and allow to remain in contact 24 hours with frequent
+shakings.
+
+7. Filter the solution into a clean flask and add one or two drops of
+pure concentrated sulphuric acid until the litmus solution is distinctly
+wine-red in colour.
+
+8. Add excess of pure solid baryta and allow to stand until the reaction
+is again alkaline.
+
+9. Filter.
+
+10. Bubble CO_{2} through the solution until reaction is definitely
+acid.
+
+11. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
+three consecutive days. This sterilises the solution and also drives off
+the carbon dioxide, leaving the solution neutral.
+
+~Media for anaerobic cultures.~ In addition to the foregoing media, all of
+which can be, and are employed in the cultivation of anaerobic bacteria,
+certain special media containing readily oxidised substances are
+commonly used for this purpose. The principal of these are as follows:
+
+     ~Bile Salt Broth (MacConkey).~--
+
+     1. Weigh out Witte's peptone, 20 grammes (= 2 per cent.),
+     and emulsify with 200 c.c. distilled water previously warmed
+     to 60 deg. C.
+
+     2. Weigh out sodium taurocholate (commercial), 5 grammes (=
+     0.5 per cent.), and glucose, 5 grammes (= 0.5 per cent.),
+     and dissolve in the peptone emulsion.
+
+     3. Wash the peptone emulsion into a flask with 800 c.c.
+     distilled water, and heat in the steamer at 100 deg. C. for
+     twenty minutes.
+
+     4. Filter through Swedish filter paper into a sterile flask.
+
+     5. Add sterile litmus solution sufficient to colour the
+     medium to a deep purple, usually 13 per cent. required.
+
+     6. Fill, in quantities of 10 c.c., into tubes containing
+     small gas tubes (_vide_ Fig. 104, page 161). Sterilise in
+     the steamer at 100 deg. C. for twenty minutes on each of three
+     consecutive days.
+
+     ~Glucose Formate Bouillon (Kitasato).~--
+
+     1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page
+     163, sections 1 to 6).
+
+     2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
+     formate, 4 grammes (= 0.4 per cent.), and dissolve in the
+     fluid.
+
+     3. Tube, and sterilise as for bouillon.
+
+     ~Glucose Formate Gelatine (Kitasato).~--
+
+     1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to
+     7) and measure out 1000 c.c.
+
+     2. Weigh out glucose, 20 grammes (= 2 per cent.), and sodium
+     formate, 4 grammes (= 0.4 per cent.), and dissolve in the
+     hot gelatine.
+
+     3. Filter through papier Chardin.
+
+     4. Tube, and sterilise as for nutrient gelatine.
+
+     ~Glucose Formate Agar (Kitasato).~--
+
+     1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8).
+     Measure out 1000 c.c.
+
+     2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
+     formate, 4 grammes (= 0.4 per cent.), and dissolve in the
+     agar.
+
+     3. Tube, and sterilise as for nutrient agar.
+
+     ~Sulphindigotate Bouillon (Weyl).~--
+
+     1. Measure out nutrient bouillon (_vide_ page 163, sections
+     1 to 6 1000 c.c.).
+
+     2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
+     sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in
+     the fluid.
+
+     3. Tube, and sterilise as for bouillon.
+
+     ~Sulphindigotate Gelatine (Weyl).~--
+
+     1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to
+     7). Measure out 1000 c.c.
+
+     2. Weigh out glucose, 20 grammes (= 2 per cent.), and sodium
+     sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in
+     the hot gelatine.
+
+     3. Filter through papier Chardin.
+
+     4. Tube, and sterilise as for nutrient gelatine.
+
+     ~Sulphindigotate Agar.~--
+
+     1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8).
+     Measure out 1000 c.c.
+
+     2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
+     sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in
+     the hot agar.
+
+     3. Tube, and sterilise as for nutrient agar.
+
+     NOTE.--The Sulphindigotate media are of a blue colour, which
+     during the growth of anaerobic bacteria is oxidised and
+     decolourised to a light yellow.
+
+FOOTNOTES:
+
+[4] This figure is obtained by adding together 1 litre water, 1000
+grammes; 10 per cent. gelatine, 100 grammes; 1 per cent. peptone, 10
+grammes; 0.5 per cent. salt, 5 grammes; total, 1115 grammes.
+Modifications of the above process, as to quantities and percentages,
+will require corresponding alterations of the figures. The average
+weight of a measured litre of 10 per cent. nutrient gelatine when
+prepared in this way _after filtration_ is 1080 grammes.
+
+[5] This figure is obtained by adding together 1 litre of water (meat
+extract), 1000 grammes; 2 per cent. agar, 20 grammes; 1 per cent.
+peptone, 10 grammes; 0.5 per cent. salt, 5 grammes--total 1035 grammes.
+Modifications of the process as to quantities or percentages will
+necessitate corresponding alterations in the calculated medium figure.
+The average weight of a measured litre of 2 per cent. agar when prepared
+in this way, _after filtration_, is 1010.5 grammes.
+
+[6] "Hopped" wort exerts a toxic effect upon many bacteria, including
+the lactic acid bacteria.
+
+
+
+
+XII. SPECIAL MEDIA.
+
+
+In this chapter are collected a number of media which have been
+elaborated by various workers for special purposes, grouped together
+under headings which indicate their chief utility. In many instances the
+name of the originator of the medium is given, but without reference to
+his original instructions, since these are in many cases inadequate to
+the requirements of the isolated worker, who would probably fail to
+reproduce the medium in a form giving the results attributed to it by
+its author. Such modifications have therefore been introduced as make
+for uniformity between the different batches of media.
+
+A considerable number of coloured media, chiefly intended for work with
+intestinal bacteria, have been included; but beyond the fact that the
+author's modification of the Drigalski-Conradi medium has been included
+amongst the routine media of the laboratory, no comment has been made
+upon their relative values, since only by observation and practice can
+the skill necessary to utilise their full value be acquired.
+
+The instructions as to sterilisation are rarely given in full; the
+routine method of exposure in the steam steriliser at 100 deg. C. (without
+pressure) for twenty minutes on each of three successive days for all
+fluid media, and thirty minutes on each of three successive days for all
+liquefiable or solid media must be carried out; and only when these
+general rules are to be departed from are further details given.
+
+_Media for the Study of the Chemical Composition of Bacteria._
+
+
+~Asparagin Medium (Uschinsky).~--
+
+1. Weigh out and mix
+    Asparagin                                 3.4 grammes
+    Ammonium lactate                         10.0 grammes
+    Sodium chloride                           5.0 grammes
+    Magnesium sulphate                        0.2 gramme
+    Calcium chloride                          0.1 gramme
+    Acid potassium phosphate (KH_{2}PO_{4})   1.0 gramme
+
+2. Dissolve the mixture in distilled water 1000 c.c.
+
+3. Add glycerine, 40 c.c.
+
+4. Tube, and sterilise as for nutrient bouillon.
+
+~Asparagin Medium (Frankel and Voges).~--
+
+1. Weigh out and mix
+    Asparagin                                4 grammes
+    Sodium phosphate, (Na_{2}HPO_{4}) 12OH   2 grammes
+    Ammonium lactate                         6 grammes
+    Sodium chloride                          5 grammes
+and dissolve in
+    Distilled water                          1000 c.c.
+
+2. Tube, and sterilise as for nutrient bouillon.
+
+     NOTE.--Either of the above asparagin media, after the
+     addition of 10 per cent. gelatine or 1.5 per cent. agar, may
+     be advantageously employed in the solid condition.
+
+
+~Proteid Free Broth (Uschinsky).~--
+
+1. Weigh out and mix
+    Calcium chloride                           0.1 gramme
+    Magnesium sulphate                         0.2 gramme
+    Acid potassium phosphate (KH_{2}PO_{4})    2.0 grammes
+    Potassium aspartate                        3.0 grammes
+    Sodium chloride                            5.0 grammes
+    Ammonium lactate                           6.0 grammes
+
+2. Dissolve the mixture in distilled water 1000 c.c.
+
+3. Add glycerine 30 c.c.
+
+4. Tube and sterilise as for nutrient broth.
+
+
+_Media for the Study of Biochemical Reaction._
+
+
+~Inosite-free Media--Bouillon (Durham).~--
+
+1. Prepare meat extract, 1000 c.c. (_vide_ page 148), from bullock's
+heart which has been "hung" for a couple of days.
+
+2. Prepare nutrient bouillon (+10), 1000 c.c. (_vide_, page 161), from
+the meat extract, and store in 1-litre flask.
+
+3. Inoculate the bouillon from a pure cultivation of the B. lactis
+aerogenes, and incubate at 37 deg. C. for forty-eight hours.
+
+4. Heat in the steamer at 100 deg. C. for twenty minutes to destroy the
+bacilli and some of their products.
+
+5. Estimate the reaction of the medium and if necessary restore to +10.
+
+6. Inoculate the bouillon from a pure cultivation of the B. coli
+communis and incubate at 37 deg. C. for forty-eight hours.
+
+7. Heat in the steamer at 100 deg. C. for twenty minutes.
+
+Now fill two fermentation tubes with the bouillon, tint with litmus
+solution, and sterilise; inoculate with B. lactis aerogenes. If no acid
+or gas is formed, the bouillon is in a sugar-free condition; but if acid
+or gas is present, again make the bouillon in the flask +10, reinoculate
+with one or other of the above-mentioned bacteria, and incubate; then
+test again. Repeat this till neither acid nor gas appears in the medium
+when used for the cultivation of either of the bacilli referred to
+above.
+
+8. After the final heating, stand the flask in a cool place and allow
+the growth to sediment. Filter the supernatant broth through Swedish
+filter paper. If the filtrate is cloudy, filter through a porcelain
+filter candle.
+
+9. Tube, and sterilise as for bouillon.
+
+Bouillon prepared in the above-described manner will prove to be
+absolutely sugar-free; and from it may be prepared nutrient sugar-free
+gelatine or agar, by dissolving in it the required percentage of
+gelatine or agar respectively and completing the medium according to
+directions given on pages 166 and 167. The most important application of
+inosite-free bouillon is its use in the preparation of sugar bouillons,
+whether glucose, maltose, lactose, or saccharose, of exact percentage
+composition.
+
+
+~Sugar (Dextrose) Bouillon.~--
+
+1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
+to 6) or sugar-free bouillon (_vide supra_).
+
+2. Weigh out glucose (anhydrous), 20 grammes (= 2 per cent.), and
+dissolve in the fluid.
+
+3. Tube, and sterilise as for bouillon.
+
+Ordinary commercial glucose serves the purpose equally well, but is not
+recommended, as during the process of sterilisation it causes the medium
+to gradually deepen in colour.
+
+     NOTE.--In certain cases a corresponding percentage of
+     lactose, maltose, or saccharose is substituted for glucose.
+
+~Sugar Gelatine.~--
+
+1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). Measure
+out 1000 c.c.
+
+2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the
+hot gelatine.
+
+3. Filter through papier Chardin.
+
+4. Tube, and sterilise as for nutrient gelatine.
+
+
+~Sugar Agar.~--
+
+1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out
+1000 c.c.
+
+2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the
+clear agar.
+
+3. Tube, and sterilise as for nutrient agar.
+
+     NOTE.--Other "sugar" media are prepared by substituting a
+     corresponding percentage of lactose, maltose (or any other
+     of the substances referred to under "Sugar Media," page 177)
+     for the glucose.
+
+
+~Iron Bouillon.~--
+
+1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 141, sections 1
+to 6).
+
+2. Weigh out ferric tartrate, 1 gramme (= 0.1 per cent.), and dissolve
+it in the bouillon.
+
+3. Tube, and sterilise as for bouillon.
+
+     NOTE.--The lactate of iron may be substituted for the
+     tartrate.
+
+
+~Lead Bouillon.~--
+
+1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
+to 6).
+
+2. Weigh out lead acetate, 1 gramme (= 0.1 per cent.), and dissolve it
+in the bouillon.
+
+3. Tube, and sterilise as for bouillon.
+
+
+~Nitrate Bouillon.~--
+
+1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
+to 6).
+
+2. Weigh out potassium nitrate, 5 grammes (= 0.5 per cent.), and
+dissolve it in the bouillon.
+
+3. Tube, and sterilise as for bouillon.
+
+     NOTE.--The nitrate of sodium or ammonium may be substituted
+     for that of potassium, or the salt may be added in the
+     proportion of from 0.1 to 1 per cent. to meet special
+     requirements.
+
+
+~Iron Peptone Solution (Pakes).~--
+
+1. Weigh out peptone, 30 grammes, and emulsify it with 200 c.c. tap
+water, previously heated to about 60 deg. C.
+
+2. Wash the emulsion into a litre flask with 800 c.c. tap water.
+
+3. Weigh out salt, 5 grammes, and sodium phosphate, 3 grammes, and
+dissolve in the mixture in the flask.
+
+4. Heat the mixture in the steamer at 100 deg. C. for thirty minutes,
+to complete the solution of the peptone, and filter into a clean flask.
+
+5. Fill into tubes in quantities of 10 c.c. each.
+
+6. Add to each tube 0.1 c.c. of a 2 per cent. neutral solution of ferric
+tartrate. (A yellowish-white precipitate forms.)
+
+7. Sterilise as for nutrient bouillon.
+
+
+~Lead Peptone Solution.~--
+
+Prepare as for iron peptone solution but in step 6 substitute 0.1 c.c.
+of a 1 per cent. neutral aqueous solution of lead acetate.
+
+
+~Nitrate Peptone Solution (Pakes).~--
+
+1. Weigh out Witte's peptone, 10 grammes, and emulsify it with 200 c.c.
+ammonia-free distilled water previously heated to 60 deg. C.
+
+2. Wash the emulsion into a flask and make up to 1000 c.c., with
+ammonia-free distilled water.
+
+3. Heat in the steamer at 100 deg. C. for twenty minutes.
+
+4. Weigh out sodium nitrate, 1 gramme, and dissolve in the contents of
+the flask.
+
+5. Filter through Swedish filter paper.
+
+6. Tube, and sterilise as for nutrient bouillon.
+
+
+~Litmus Bouillon.~--
+
+1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
+to 6).
+
+2. Add sufficient sterile litmus solution to tint the medium a dark
+lavender colour. (Media rendered +10 will usually react very faintly
+alkaline or occasionally neutral to litmus.)
+
+3. Tube, and sterilise as for bouillon.
+
+
+~Rosolic Acid Peptone Solution.~--
+
+1. Weigh out rosolic acid (corallin), 0.5 gramme, and dissolve it in 80
+per cent. alcohol, 100 c.c. Keep this as a stock solution.
+
+2. Measure out peptone water (Dunham), 100 c.c., and rosolic acid
+solution, 2 c.c., and mix.
+
+3. Heat in the steamer at 100 deg. C. for thirty minutes.
+
+4. Filter through Swedish filter paper.
+
+5. Tube, and sterilise as for nutrient bouillon.
+
+
+~Capaldi-Proskauer Medium, No. I.~--
+
+1. Weigh out and mix
+
+    Sodium chloride            2.0 grammes
+    Magnesium sulphate         0.1 gramme
+    Calcium chloride           0.2 gramme
+    Monopotassium phosphate    2.0 grammes
+
+2. Dissolve in water 1000 c.c. in a 2-litre flask
+
+3. Weigh out and mix
+
+    Asparagin    2 grammes
+    Mannite      2 grammes
+
+and add to contents of flask.
+
+4. Measure out 25 c.c. of the solution and titrate it against decinormal
+sodic hydrate, using litmus as the indicator. Control the result and
+estimate the amount of sodic hydrate necessary to be added to render the
+remainder of the solution neutral to litmus. Add this quantity of sodic
+hydrate.
+
+5. Filter.
+
+6. Add litmus solution 47.5 c.c. (= 5 per cent.).
+
+7. Tube, and sterilise as for nutrient bouillon.
+
+
+~Capaldi-Proskauer Medium No. II.~--
+
+1. Weigh out and mix
+
+    Peptone   20 grammes
+    Mannite    1 gramme
+
+2. Dissolve in water 1000 c.c. in a 2-litre flask.
+
+3. Neutralise to litmus as in No. I (_vide supra_, Step 4).
+
+4. Filter.
+
+5. Add litmus solution 47.5 c.c. (= 5 per cent.).
+
+6. Tube, and sterilise as for nutrient bouillon.
+
+
+~Urine Media. Bouillon.~--
+
+1. Collect freshly passed urine in sterile flask.
+
+2. Place the flask in the steamer at 100 deg. C. for thirty minutes.
+
+3. Filter through two thicknesses of Swedish filter paper.
+
+4. Tube, and sterilise as for nutrient bouillon. (Leave the reaction
+unaltered.)
+
+
+~Urine Gelatine.~--
+
+1. Collect freshly passed urine in sterile flask.
+
+2. Take the specific gravity, and, if above 1010, dilute with sterile
+water until that gravity is reached.
+
+3. Estimate (with control) at the boiling-point, and note the reaction
+of the urine.
+
+4. Weigh out gelatine, 10 per cent., and add to the urine in the flask.
+
+5. Heat in the steamer at 100 deg. C. for one hour to dissolve the
+gelatine.
+
+6. Estimate the reaction and add sufficient caustic soda solution to
+restore the reaction of the medium mass to the equivalent of the
+original urine.
+
+7. Cool to 60 deg. C. and clarify with egg as for nutrient gelatine
+(_vide_ page 166).
+
+8. Filter through papier Chardin.
+
+9. Tube, and sterilise as for nutrient gelatine.
+
+
+~Urine Gelatine (Heller).~--
+
+1. Collect freshly passed urine in sterile flask.
+
+2. Filter through animal charcoal to remove part of the colouring
+matter.
+
+3. Take the specific gravity, and if above 1010, dilute with sterile
+water till this gravity is reached.
+
+4. Add Witte's peptone, 1 per cent.; salt, 0.5 per cent.; gelatine, 10
+per cent.
+
+5. Heat in the steamer at 100 deg. C. for one hour, to dissolve the
+gelatine, etc.
+
+6. Add normal caustic soda solution in successive small quantities, and
+test the reaction from time to time with litmus paper, until the fluid
+reacts faintly alkaline.
+
+7. Cool to 60 deg. C. and clarify with egg as for nutrient gelatine
+(_vide_ page 166).
+
+8. Filter through papier Chardin.
+
+9. Tube, and sterilise as for nutrient gelatine.
+
+
+~Urine Agar.~--
+
+1. Collect freshly passed urine in sterile flask.
+
+2. Take the specific gravity and if above 1010, dilute with sterile
+water till this gravity is reached.
+
+3. Weigh out 1.5 per cent. or 2 per cent. powdered agar, and add it to
+the urine.
+
+4. Heat in the steamer at 100 deg. C. for ninety minutes to dissolve the
+agar.
+
+5. Cool to 60 deg. C. and clarify with egg as for nutrient agar (_vide_
+page 168).
+
+6. Filter through papier Chardin, using the hot-water funnel.
+
+7. Tube, and sterilise as for nutrient agar.
+
+(Leave the reaction unaltered.)
+
+
+~Serum Sugar Media (Hiss).~--
+
+In these media the fermentation of carbohydrate substance by bacterial
+action is indicated by the coagulation of the serum proteids in addition
+to the production of an acid reaction.
+
+
+~Serum Dextrose Water (Hiss).~--
+
+1. Measure out into a litre flask
+
+    Serum water (See page 170)    1000 c.c.
+
+2. Weigh out
+
+    Dextrose                     10 grammes
+
+and dissolve in the serum water.
+
+3. Filter through Swedish filter paper.
+
+4. Measure out and add to the medium
+
+    Litmus solution (Kahlbaum)      50 c.c.
+
+5. Tube in quantities of 10 c.c. and sterilise in the steamer at 100
+deg. C. for twenty minutes on each of three successive days.
+
+Laevulose, galactose, maltose, lactose, etc., can be substituted in
+similar amounts for dextrose and the medium completed as above.
+
+
+~Omeliansky's Nutrient Fluid~ (_For Cellulose Fermenters_).--
+
+1. Weigh out and mix
+
+    Potassium phosphate   4.0  grammes
+    Magnesium sulphate    2.0  grammes
+    Ammonium sulphate     4.0  grammes
+    Sodium chloride       0.25 gramme
+
+2. Dissolve in distilled water 4000 c.c.
+
+3. Flask in quantities of 250 c.c.
+
+4. Weigh out and add 5 grammes precipitated chalk to each flask.
+
+5. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
+three successive days.
+
+
+_Media for the Study of Chromogenic Bacteria._
+
+
+~Milk Rice (Eisenberg).~--
+
+1. Measure out nutrient bouillon, 70 c.c., and milk, 210 c.c., and mix
+thoroughly.
+
+2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with
+the milk and broth mixture.
+
+3. Fill the paste into sterile capsules, spreading it out so as to form
+a layer about 0.5 cm. thick, over the bottom of each.
+
+4. Heat over a water-bath at 100 deg. C. until the mixture solidifies.
+
+5. Replace the lids of the capsules. Sterilise in the steamer at 100
+deg. C. for thirty minutes on each of three consecutive days.
+
+(A solid medium of the colour of _cafe au lait_ is thus produced.)
+
+
+~Milk Rice (Soyka).~--
+
+1. Measure out nutrient bouillon, 50 c.c., and milk, 150 c.c., and mix
+thoroughly.
+
+2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with
+the milk and broth mixture.
+
+3. Fill the paste into sterile capsules, to form a layer over the bottom
+of each.
+
+4. Replace the lids of the capsules.
+
+5. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
+three consecutive days.
+
+(A pure white, opaque medium is thus formed.)
+
+
+_Media for the Study of Phosphorescent and Photogenic Bacteria._
+
+
+~Fish Bouillon.~--
+
+1. Weigh out herring, mackerel, or cod, 500 grammes, and place in a
+large porcelain beaker (or enamelled iron pot).
+
+2. Weigh out sodium chloride, 26.5 grammes; potassium chloride, 0.75
+gramme; magnesium chloride, 3.25 grammes; and dissolve in 500 c.c.
+distilled water. Add the solution to the fish in the beaker.
+
+3. Place the beaker in a water-bath and proceed as in preparing meat
+extract--i. e., heat gently at 40 deg. C. for twenty minutes, then rapidly
+raise the temperature to, and maintain at, the boiling-point for ten
+minutes.
+
+4. Strain the mixture through butter muslin into a clean flask.
+
+5. Weigh out peptone, 5 grammes, and emulsify with about 200 c.c. of the
+hot fish water; incorporate thoroughly with the remainder of the fish
+water in the flask.
+
+6. Heat in the steamer at 100 deg. C. for twenty minutes to complete the
+solution of the peptone.
+
+7. Filter through Swedish filter paper.
+
+8. When the fish bouillon is cold, if it is to be used as fluid medium,
+make up to 1000 c.c. by the addition of distilled water. If, however, it
+is to be used as the basis for agar or gelatine media store it in the
+"Double Strength" condition.
+
+9. Tube and sterilise as for nutrient bouillon.
+
+As an alternative method "Marvis" fish food (16 grammes) may be
+substituted for the 500 grammes of fresh fish.
+
+
+~Fish Gelatine.~--
+
+1. Measure out double strength fish bouillon, 500 c.c., into a "tared"
+2-litre flask.
+
+2. Add sheet gelatine, 100 grammes, cut into small pieces.
+
+3. Bubble live steam through the mixture for fifteen minutes to dissolve
+the gelatine.
+
+4. Weigh the flask and its contents; adjust the weight to the calculated
+figure for one litre of medium (1135.5 grammes) by the addition of
+distilled water at 100 deg. C. (_vide_ page 166).
+
+5. Cool to below 60 deg. C., and clarify with egg.
+
+6. Filter through papier Chardin.
+
+7. Tube, and sterilise as for nutrient gelatine.
+
+Shake well after the final sterilisation, to aerate the medium.
+
+
+~Fish Gelatine-Agar.~--
+
+1. Weigh out powdered agar, 5 grammes, and emulsify it with 200 c.c.
+double strength fish bouillon.
+
+2. Wash the emulsion into a "tared" 2-litre flask with 300 c.c. fish
+bouillon.
+
+3. Weigh out sheet gelatine, 70 grammes, cut it into small pieces and
+add it to the contents of the flask.
+
+4. Bubble live steam through the mixture to dissolve the gelatine and
+agar.
+
+5. Weigh the flask and contents. Adjust the weight to the calculated
+figure for one litre of medium (1110.5 grammes) by the addition of
+distilled water at 100 deg. C. (_vide_ page 166).
+
+6. Cool to below 60 deg. C. and clarify with egg.
+
+7. Filter through papier Chardin.
+
+8. Tube, and sterilise as for nutrient gelatine.
+
+Shake well after the final sterilisation, to aerate the medium.
+
+
+_Media for the Study of Yeasts and Moulds._
+
+
+~Pasteur's Solution.~--
+
+(Reaction alkaline).
+
+1. Weigh out and mix the ash from 10 grammes of yeast; ammonium
+tartrate, 10 grammes; cane sugar, 100 grammes.
+
+2. Dissolve the mixture in distilled water, 1000 c.c.
+
+3. Tube or flask, and sterilise as for nutrient bouillon.
+
+
+~Yeast Water (Pasteur).~--
+
+1. Weigh out pressed yeast, 75 grammes; place in a 2-litre flask and add
+1000 c.c. distilled water.
+
+2. Heat in the steamer at 100 deg. C. for thirty minutes.
+
+3. Filter through papier Chardin.
+
+4. Tube or flask, and sterilise as for nutrient bouillon.
+
+
+~Cohn's Solution.~--
+
+1. Weigh out and mix
+
+    Acid potassium phosphate (KH_{2}PO_{4})  5.0 grammes
+    Calcium phosphate                        0.5 gramme
+    Magnesium sulphate                       5.0 grammes
+    Ammonium tartrate                       10.0 grammes
+
+and dissolve in
+
+    Distilled water                       1000 c.c.
+
+2. Tube, or flask and sterilise as for nutrient bouillon.
+
+
+~Naegeli's Solution.~--
+
+1. Weigh out and mix
+
+    Dibasic potassium phosphate (K_{2}HPO_{4})  1.0 gramme
+    Magnesium sulphate                          0.2 gramme
+    Calcium chloride                            0.1 gramme
+    Ammonium tartrate                          10.0 grammes
+
+and dissolve in
+
+    Distilled water                        1000 c.c.
+
+2. Tube or flask; sterilise as for nutrient bouillon.
+
+
+~Plaster-of-Paris Discs.~--
+
+1. Take large corks, 2.5 cm. diameter, and roll a piece of stiff
+note-paper round each, so that about a centimetre projects as a ridge
+above the upper surface of the cork, and secure in position with a pin
+(Fig. 112).
+
+2. Mix plaster-of-Paris into a stiff paste with distilled water, and
+fill each of the cork moulds with the paste.
+
+3. When the plaster has set, remove the paper from the corks, and raise
+the plaster discs.
+
+4. Place the plaster discs on a piece of asbestos board and sterilise by
+exposing in the hot-air oven to 150 deg. C. for half an hour.
+
+[Illustration: Fig. 112.--Cork and paper mould for plaster-of-Paris
+disc.]
+
+5. Remove the sterile discs from the oven by means of sterile forceps,
+place each inside a sterile capsule, and moisten with a little sterile
+water.
+
+6. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
+three consecutive days.
+
+
+~Gypsum Blocks (Engel and Hansen).~--
+
+These are in the form of truncated cones and for their preparation small
+tin moulds are required, each having a diameter of 5.5 cm. at the base
+and 4 cm. at the truncated apex. The height (or depth) of a mould is 4.5
+to 5 cm.
+
+1. Mix powdered calcined gypsum into a stiff paste with distilled water.
+
+2. Fill the paste into the moulds and allow it to set and dry by
+exposure to air.
+
+3. Remove the block from the mould and transfer it to a double glass
+dish of adequate size (7 cm. diameter x 7 cm. high).
+
+4. Sterilise block in its dish for one hour in the hot-air oven at
+115 deg. C.
+
+5. Carefully open the dish and add sterile distilled water to moisten
+the block and form a layer in the bottom of the dish 1 cm. deep.
+
+
+~Wine Must.~--(Wine must is obtained from Sicily, in hermetically sealed
+tins, in a highly concentrated form--as a thick syrup--but not
+sterilised.)
+
+1. Weigh out "wine must," 200 grammes, place in a 2-litre flask and add
+distilled water, 800 c.c.
+
+2. Weigh out ammonium tartrate, 5 grammes, and add to the dilute must.
+
+3. Place the flask in a water-bath regulated to 60 deg. C. for one hour
+and incorporate the mixture thoroughly by frequent shaking.
+
+4. Filter through papier Chardin.
+
+5. Tube, and sterilise as for nutrient bouillon.
+
+
+~Wheat Bouillon (Gasperini).~--
+
+1. Weigh out and mix wheat flour, 150 grammes; magnesium sulphate, 0.5
+gramme; potassium nitrate, 1 gramme; glucose, 15 grammes.
+
+2. Dissolve the mixture in 1000 c.c. of water heated to 100 deg. C.
+
+3. Filter through papier Chardin.
+
+4. Tube, and sterilise as for nutrient bouillon.
+
+
+~Bread Paste.~--
+
+1. Grate stale bread finely on a bread-grater.
+
+2. Distribute the crumbs in sterile Erlenmeyer flasks, sufficient to
+form a layer about one centimetre thick over the bottom of each.
+
+3. Add as much distilled water as the crumbs will soak up, but not
+enough to cover the bread.
+
+4. Plug the flasks and sterilise in the steamer at 100 deg. C. for
+thirty minutes on each of _four_ consecutive days.
+
+
+_Media for the Study of Parasitic Moulds._
+
+
+~French Proof Agar (Sabouraud).~--
+
+1. Weigh out Chassaing's peptone, 10 grammes, and emulsify it with 200
+c.c. distilled water previously heated to 60 deg. C.
+
+2. Weigh out powdered agar, 13 grammes, and emulsify with 200 c.c. cold
+distilled water.
+
+3. Mix the two emulsions and wash into a tared 2-litre flask with 600
+c.c. distilled water.
+
+4. Bubble live steam through the mixture for twenty minutes, to dissolve
+the agar.
+
+5. Cool to 60 deg. C. and clarify with egg as for nutrient agar (_vide_
+page 168).
+
+6. Filter through Papier Chardin, using the hot-water funnel.
+
+7. Weigh out _French_ maltose, 40 grammes, and dissolve in the agar.
+
+8. Tube, and sterilise as for nutrient agar.
+
+~English Proof Agar (Blaxall).~--Substitute Witte's peptone for that of
+Chassaing, and proceed as for French proof agar.
+
+~French Mannite Agar, Sabouraud.~--(_For cultivation of Favus._)
+
+Proceed exactly as in preparing French Proof agar _vide supra_
+substituting Mannite (38 grammes) for maltose.
+
+
+_Media for the Study of Milk Bacteria._
+
+
+~Gelatine Agar.~--This medium is prepared by adding to nutrient gelatine
+sufficient agar to ensure the solidity of the medium when incubated at
+temperatures above 22 deg. C. If it is intended to employ an incubating
+temperature of 30 deg. C., 10 per cent. gelatine and 0.5 per cent. agar
+must be dissolved in the meat extract before the addition of the peptone
+and salt; while for incubating at 37 deg. C., 12 per cent. gelatine and
+0.75 per cent. agar must be used. Avoid the addition of more agar than is
+absolutely necessary, otherwise the action upon the medium of such
+organisms as elaborate a liquefying ferment may be retarded or
+completely absent.
+
+1. Measure out 400 c.c. double strength meat extract into a "tared"
+2-litre flask, and add to it gelatine, 100 grammes.
+
+2. Weigh out powdered agar, 5 grammes, emulsify with 100 c.c., cold
+distilled water and add to the contents of the flask.
+
+3. Dissolve the agar and gelatine by bubbling live steam through the
+flask for twenty minutes.
+
+4. Weigh out peptone, 10 grammes; salt, 5 grammes; emulsify with 100
+c.c. double strength meat extract previously heated to 60 deg. C., and
+add to the contents of the flask.
+
+5. Replace in the steamer for fifteen minutes. Then adjust the weight to
+the calculated figure for one litre (in this instance 1120 grammes) by
+the addition of distilled water at 100 deg. C.
+
+6. Estimate the reaction; control the result. Then add sufficient
+caustic soda solution to render the reaction +10.
+
+7. Replace in the steamer at 100 deg. C. for twenty minutes.
+
+8. Cool to 60 deg. C. Clarify with egg as for nutrient agar.
+
+9. Filter through papier Chardin, using the hot-water funnel.
+
+10. Tube, and sterilise as for nutrient agar.
+
+
+~Agar Gelatine (Guarniari).~--
+
+1. Measure out double strength meat extract, 400 c.c., into a "tared"
+2-litre flask, and add to it gelatine, 50 grammes.
+
+2. Weigh out powdered agar, 3 grammes; emulsify with cold distilled
+water, 50 c.c., and add to the contents of the flask.
+
+3. Dissolve the agar and gelatine by bubbling live steam through the
+flask for twenty minutes.
+
+4. Weigh out Witte's peptone, 25 grammes; salt, 5 grammes, and emulsify
+with 100 c.c. double strength meat extract previously heated to 60 deg.
+C., and add to the contents of the flask.
+
+5. Replace in the steamer for fifteen minutes.
+
+6. Weigh the flask and make up the medium mass to the calculated figure
+for one litre (1083 grammes) by the addition of distilled water at
+100 deg. C.
+
+7. Neutralise carefully to litmus paper by the successive additions of
+small quantities of normal soda solution.
+
+8. Replace in the steamer at 100 deg. C. for twenty minutes.
+
+9. Cool to 60 deg. C. Clarify with egg as for nutrient agar.
+
+10. Filter through papier Chardin, using the hot-water funnel.
+
+11. Tube, and sterilise as for nutrient agar.
+
+
+~Whey Gelatine.~--
+
+1. Curdle fresh milk by warming to 60 deg. C., and adding rennet; filter
+off the whey into a sterile "tared" flask.
+
+2. Estimate and note the reaction of the whey.
+
+3. Weigh out gelatine, 10 per cent., and add it to the whey in the
+flask.
+
+4. Bubble live steam through the mixture fifteen minutes to dissolve the
+gelatine; and weigh.
+
+5. Estimate the reaction of the medium mass; then add sufficient caustic
+soda solution to restore the reaction of the medium mass (i. e., total
+weight minus weight of flask) to the equivalent of the original whey.
+
+6. Cool to 60 deg. C. and clarify with egg as for nutrient gelatine
+(_vide_ page 166).
+
+7. Filter through papier Chardin.
+
+8. Tube, and sterilise as for nutrient gelatine.
+
+
+~Whey Agar.~--
+
+1. Curdle fresh milk by warming to 60 deg. C., and adding rennet; filter
+off the whey into a sterile flask.
+
+2. Weigh out agar, 1.5 or 2 per cent., and add it to the whey in the
+flask.
+
+3. Bubble live steam through the mixture for twenty minutes, to dissolve
+the agar.
+
+4. Cool to 60 deg. C.; clarify with egg as for nutrient agar (_vide_ page
+168).
+
+5. Filter through papier Chardin, using the hot-water funnel.
+
+6. Tube, and sterilise as for nutrient agar.
+
+
+~Litmus Whey.~--
+
+1. Curdle fresh milk by warming to 60 deg. C. and adding rennet.
+
+2. Filter off the whey through butter muslin into a sterile flask.
+
+3. Neutralise to litmus by the cautious addition of citric acid solution
+4 per cent. (Do not neutralise with _mineral_ acid.)
+
+4. Heat in the steamer at 100 deg. C. for one hour to coagulate all the
+proteid.
+
+(If the whey is cloudy when removed from the steamer allow it to stand
+for forty-eight hours in the ice chest and then decant off the clear
+fluid--or filter through a Berkefeld filter candle.)
+
+5. Filter into a sterile flask.
+
+6. Tint the whey with litmus solution to a deep purple red.
+
+7. Tube, and sterilise as for milk.
+
+
+~Litmus Whey (Petruschky).~--
+
+1. Measure out into a flask
+
+    Fresh milk                                    1000 c.c.
+
+2. Add
+
+    Hydrochloric acid (or glacial acetic acid)     1.5 c.c.
+
+and boil.
+
+3. Filter off coagulated casein.
+
+4. Neutralise to litmus by the addition of n/1 caustic soda solution and
+boil. Whey now cloudy and acid again.
+
+5. Again neutralise to litmus by addition of n/10 caustic soda solution.
+
+6. Filter.
+
+7. Tint the whey with neutral litmus solution to a deep purple colour.
+
+8. Tube and sterilise as for milk.
+
+
+~Litmus Whey Gelatine.~--
+
+1. Measure out milk 1000 c.c. into a tared 2-litre flask.
+
+2. Add hydrochloric acid (or glacial acetic acid) 1.5 c.c. and boil for
+five minutes.
+
+3. Filter off the casein, and make the whey faintly alkaline to litmus.
+
+4. Weigh out
+
+    Peptone                 10 grammes
+
+and emulsify in a few cubic centimeters of the whey and return to the
+flask.
+
+5. Weigh out
+
+    Gelatine                50 grammes
+
+add it to the whey in the flask and incorporate the mixture by bubbling
+through live steam.
+
+6. Clear with egg and filter.
+
+7. Make the weight of the medium mass to the calculated figure for one
+litre (1060 grammes) by the addition of distilled water.
+
+8. Weigh out
+
+    Dextrose                15 grammes
+
+and dissolve in the fluid whey gelatine.
+
+9. Add sterile litmus solution to the required tint.
+
+10. Tube and sterilise for twenty minutes in steamer at 100 deg. C. on
+each of five successive days.
+
+This medium will remain semi-fluid at the room temperature, and may be
+used for cultures in the cool or hot incubator.
+
+
+~Litmus Whey Agar~ is prepared in a similar manner to Whey Gelatine, with
+the substitution of 15 grammes of agar for the gelatine.
+
+
+~Malt Extract Solution (Herschell).~--
+
+1. Measure into a flask distilled water 1000 c.c.
+
+2. Weigh out
+
+    Extractum malti (malt extract)        25 grammes
+
+and add to distilled water in flask.
+
+3. Boil for five minutes, allow to stand, and decant off clear fluid
+from sediment.
+
+4. Tube and sterilise as for nutrient bouillon.
+
+
+_Media for the Study of Earth Bacteria, Nitrogen Fixers._
+
+
+~Earthy Salts Agar (Lipman and Brown).~--(_For the enumeration of soil
+organisms._)
+
+1. Measure out
+
+    Agar      20 grammes.
+
+Emulsify in 200 c.c. distilled water.
+
+2. Wash the agar emulsion into a tared 2-litre flask with 400 c.c.
+distilled water.
+
+3. Weigh out
+
+    Peptone      0.5 gramme.
+
+Emulsify in 50 c.c. distilled water and add to the contents of the
+flask.
+
+4. Bubble live steam through the mixture for twenty minutes to dissolve
+the agar.
+
+5. Weigh out and mix
+
+    Dextrose             10.0 grammes.
+    Potassium phosphate   0.5 gramme.
+    Magnesium sulphate    0.2 gramme.
+    Potassium nitrate    0.06 gramme.
+
+and add to the contents of the flask.
+
+6. Adjust the weight of the medium mass to the calculated figure for one
+litre (1025 grammes) by the addition of distilled water at 100 deg. C.
+
+7. Titrate the medium mass and adjust the reaction to +5.
+
+8. Cool to 60 deg. C. Clarify with egg and filter.
+
+9. Tube in quantities of 10 c.c. and sterilise as for nutrient agar.
+
+
+~Beyrinck's Solution. I.~--(_For the cultivation of nitrogen fixing
+organisms._)
+
+1. Weigh out and mix 1 gramme potassium hydrogen phosphate, 0.2 gramme
+magnesium sulphate, and 0.02 gramme sodium chloride.
+
+2. Dissolve in water 1000 c.c., in a 2-litre flask.
+
+3. Add 1 c.c. of a one per thousand aqueous solution of ferrous
+sulphate.
+
+4. Add 1 c.c. of a one per thousand solution manganese sulphate.
+
+5. Weigh out 20 grammes dextrose and add to the contents of the flask
+(dextrose up to 40 grammes may be used for the different organisms).
+
+6. Steam for twenty minutes, filter.
+
+7. Tube, and sterilise as for nutrient bouillon.
+
+
+~Beyrinck's Solution. II.~--(_For growth of Azobacter._)
+
+Proceed as in preparing solution No. I, substituting mannite for
+dextrose in step 5.
+
+
+~Winogradsky's Solution (for Nitric Organisms).~--
+
+1. Weigh out and mix.
+
+    Potassium phosphate      1.0 gramme
+    Magnesium sulphate       0.5 gramme
+    Calcium chloride         0.01 gramme
+    Sodium chloride          2.0 grammes
+
+and dissolve in
+
+    Distilled water       1000 c.c.
+
+2. Fill into flasks, in quantities of 20 c.c. and add to each a small
+quantity of freshly washed magnesium carbonate.
+
+3. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
+three consecutive days.
+
+4. Add to each flask containing 20 c.c. solution, 2 c.c. of a sterile 2
+per cent. solution of ammonium sulphate.
+
+5. Incubate at 37 deg. C. for forty-eight hours and eliminate any
+contaminated culture flasks. Store the remainder for future use.
+
+~Winogradsky's Solution (for Nitrous Organisms).~--
+
+1. Weigh out and mix
+
+    Ammonium sulphate        1 gramme
+    Potassium sulphate       1 gramme
+
+and dissolve in
+
+    Distilled water       1000 c.c.
+
+2. Add 5 to 10 grammes basic magnesium carbonate, previously sterilised
+by boiling.
+
+3. Fill into flasks and sterilise, etc., as for previous solution.
+
+
+~Silicate Jelly (Winogradsky).~--
+
+1. Weigh out and mix
+
+    Ammonium sulphate        0.40 gramme
+    Magnesium sulphate       0.05 gramme
+    Calcium chloride         0.01 gramme
+
+and dissolve in
+
+    Distilled water         50 c.c.
+
+Label--Solution A.
+
+2. Weigh out and mix
+
+    Potassium phosphate      0.10 gramme
+    Sodium carbonate         0.60 gramme
+
+and dissolve in
+
+    Distilled water         50 c.c.
+
+Label--Solution B.
+
+3. Weigh out
+
+    Silicic acid            3.4 grammes
+
+and dissolve in
+
+    Distilled water         100 c.c.
+
+4. Pour the silicic acid solution into a large porcelain basin.
+
+5. Mix equal quantities of the solutions A and B; then add successive
+small quantities of the mixed salts to the silicic acid solution,
+stirring continuously with a glass rod, until a jelly of sufficiently
+firm consistence has been formed.
+
+6. Spread a layer of this jelly over the bottom of each of several large
+capsules or "plates."
+
+7. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
+three consecutive days.
+
+
+_Media for the Study of Water Bacteria._
+
+
+~Naehrstoff Agar (Hesse and Niedner).~--(_For enumeration of water
+organisms._)
+
+1. Weigh out: agar, 12.5 grammes and emulsify in 250 c.c. distilled
+water.
+
+2. Wash the agar emulsion into a tared 2-litre flask with a further 250
+c.c. distilled water.
+
+3. Dissolve by bubbling live steam through the mixture.
+
+4. Emulsify Naehrstoff-Heyden (albumose) 7.5 grammes in 200 c.c. cold
+distilled water and add to melted agar.
+
+5. Adjust weight of medium mass to the calculated figure for one litre
+(1020 grammes) by addition of distilled water at 100 deg. C.
+
+6. Clarify with white of egg and filter.
+
+7. Tube in quantities of 10 c.c. and sterilise in the steamer at 100
+deg. C. for twenty minutes on each of three successive days.
+
+
+~Bile Salt Broth--Double Strength.~--
+
+1. Weigh out Witte's peptone, 40 grammes, and emulsify with 300 c.c.
+distilled water previously warmed to 60 deg. C.
+
+2. Wash the peptone emulsion into a litre flask with 600 c.c. distilled
+water.
+
+3. Weigh out sodium taurocholate, 10 grammes, and glucose, 10 grammes;
+dissolve in 100 c.c. distilled water and add to the peptone emulsion in
+the flask.
+
+4. Heat in the steamer at 100 deg. C. for twenty minutes.
+
+5. Filter through Swedish filter paper into a sterile flask.
+
+6. Add sterile neutral litmus solution sufficient to colour the medium
+to a deep purple.
+
+7. Fill into small Erlenmeyer flasks in quantities of 25 c.c.
+
+8. Sterilise as for nutrient bouillon.
+
+
+_Media for the Study of Plant Bacteria._
+
+    ~Beetroot.~--  }
+    ~Carrot.~--    } are prepared tubes and sterilised in a manner
+    ~Turnip.~--    } precisely similar to that described for potato.
+    ~Parsnip.~--   }
+
+
+~Hay Infusion.~--
+
+1. Weigh out dried hay, 10 grammes, chop it up into fine particles and
+place in a flask.
+
+2. Add 1000 c.c. distilled water, heated to 70 deg. C.; close the flask
+with a solid rubber stopper.
+
+3. Macerate in a water-bath at 60 deg. C. for three hours.
+
+4. Replace the stopper by a cotton-wool plug, and heat in the steamer at
+100 deg. C. for one hour.
+
+5. Filter through Swedish filter paper.
+
+6. Tube, and sterilise as for nutrient bouillon.
+
+
+~Haricot Bouillon.~--(_For cultivation of bacteria from tubercles of
+Legumes._)
+
+1. Measure out 1000 c.c. distilled water into a 2-litre flask.
+
+2. Weigh out 250 grammes haricot beans and add to the water in the
+flask.
+
+3. Weigh out 10 grammes sodium chloride and add to the contents of the
+flask.
+
+4. Add 1 c.c. of a 1 per cent. solution of sodium bicarbonate.
+
+5. Place in the steamer at 100 deg. C. for thirty minutes.
+
+6. Filter.
+
+7. Weigh out 20 grammes saccharose and add to the filtrate.
+
+8. Tube, and sterilise as for nutrient bouillon.
+
+
+~Haricot Agar.~--
+
+1. Measure out 400 c.c. distilled water into a "tared" 2-litre flask.
+
+2. Weigh out 15 grammes agar and mix into a thick paste with 100 c.c.
+cold distilled water, and add to the flask.
+
+3. Dissolve the agar by bubbling live steam through the mixture as in
+making nutrient agar.
+
+4. Weigh out 250 grammes haricot beans, place in the flask with the agar
+mixture.
+
+5. Add 1 c.c. of 1 per cent. aqueous solution sodium bicarbonate.
+
+6. Weigh out 10 grammes sodium chloride and add to the contents of the
+flask.
+
+7. Place in the steamer at 100 deg. C. for thirty minutes.
+
+8. Adjust the weight of the medium mass to 1030 grammes (the figure per
+litre obtained experimentally) by the addition of distilled water at
+100 deg. C.
+
+9. Cool to 60 deg. C., clarify with egg and filter.
+
+10. Weigh out 20 grammes saccharose and add to the contents of the
+flask.
+
+11. Tube, and sterilise as for nutrient agar.
+
+
+~Wood Ash Agar.~--
+
+1. Measure 400 c.c. distilled water into a tared 2-litre flask.
+
+2. Weigh out 10 grammes agar and make into a thick paste with 100 c.c.
+cold distilled water.
+
+3. Add this agar paste to the distilled water in the flask.
+
+4. Dissolve the agar by passing live steam through it, as in preparing
+nutrient agar.
+
+5. Weigh out 5 grammes clean wood ash and place in a second flask
+containing 200 c.c. distilled water with some sterile glass beads: shake
+thoroughly in a mechanical shaker for ten minutes.
+
+6. Heat in steamer at 100 deg. C., for thirty minutes.
+
+7. After removal from the steamer dry the outside of the flask
+thoroughly, place it over a Bunsen flame and boil for one minute.
+
+8. Filter directly into the flask containing the melted agar mixture.
+
+9. Weigh out 4 grammes maltose. Add to the contents of the flask.
+
+10. Adjust the weight of the medium mass to the calculated figure for
+one litre (1019 grammes) by the addition of distilled water at 100
+deg. C.
+
+11. Replace the flask in the steamer for twenty minutes, cool to 60
+deg. C., and clarify with egg and filter.
+
+12. Tube, and sterilise as for nutrient agar.
+
+
+_Media for the Study of Special Bacilli._
+
+_B. Acnes._
+
+
+~Oleic Acid Agar (Fleming).~--
+
+1. Measure out into a sterile stout glass bottle which already contains
+about 10 sterile glass beads
+
+    Ascitic fluid               250 c.c.
+
+2. Weigh out
+
+    Oleic acid                  25 grammes
+
+and add it to the ascitic fluid in the bottle.
+
+3. Emulsify evenly by shaking (either by hand or in a shaking machine)
+for ten minutes.
+
+4. Liquefy and measure out into a flask
+
+    Nutrient agar               750 c.c.
+
+then cool to 55 deg. C.
+
+5. Mix the oleic acid emulsion with the agar.
+
+6. Add 10 c.c. sterile neutral red, 1 per cent. aqueous solution.
+
+7. Tube in quantities of 10 c.c., slant, and allow to set.
+
+8. Incubate for forty-eight hours at 37 deg. C. and reject any
+contaminated tubes. Store the sterile tubes for future use.
+
+
+_Coli-typhoid Group._
+
+~Parietti's Bouillon.~--
+
+1. Measure out pure hydrochloric acid, 4 c.c., and add to it carbolic
+acid solution (5 per cent.), 100 c.c. Allow the solution to stand at
+least a few days before use.
+
+2. This solution is added in quantities of 0.1, 0.2. and 0.3 c.c.
+(delivered by means of a sterile graduated pipette) to tubes each
+containing 10 c.c. of previously sterilised nutrient bouillon (_vide_
+page 163).
+
+3. Incubate at 37 deg. C. for forty-eight hours to eliminate contaminated
+tubes. Store the remainder for future use.
+
+~Carbolised Bouillon.~--
+
+1. Prepare nutrient bouillon (_vide_ page 163, sections 1 to 6). Measure
+out 1000 c.c.
+
+2. Weigh out carbolic acid, 1 gramme (2.5 or 5 grammes may be needed for
+special purposes), and dissolve it in the medium.
+
+3. Tube, and sterilise as for bouillon.
+
+~Carbolised Gelatine.~--
+
+1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). Measure
+out 1000 c.c.
+
+2. Weigh out carbolic acid, 5 grammes (= 0.5 per cent.), and dissolve it
+in the gelatine.
+
+3. Filter if necessary through papier Chardin.
+
+4. Tube, and sterilise as for nutrient gelatine.
+
+One or 2.5 grammes of carbolic acid (= 0.1 per cent. or 0.25 per cent.)
+are occasionally used in place of the 5 grammes to meet special
+requirements.
+
+
+~Carbolised Agar.~--
+
+1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out
+1000 c.c.
+
+2. Weigh out 1 gramme pure phenol and dissolve in the medium.
+
+3. Filter if necessary through papier Chardin.
+
+4. Tube, and sterilise as for nutrient agar.
+
+~Litmus Gelatine.~--
+
+1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 8).
+
+2. Add sterile litmus solution, sufficient to tint the medium a deep
+lavender colour.
+
+3. Tube, and sterilise as for nutrient gelatine.
+
+
+~Lactose Litmus Bouillon (Lakmus Molke).~--
+
+1. Weigh out peptone, 4 grammes, and emulsify it with 200 c.c. meat
+extract (_vide_ page 148), previously heated to 60 deg. C.
+
+2. Weigh out salt, 2 grammes, and lactose, 20 grammes, and mix with the
+emulsion.
+
+3. Wash the mixture into a sterile litre flask with 200 c.c. meat
+extract and add 600 c.c. distilled water.
+
+4. Heat in the steamer at 100 deg. C. for thirty minutes, to completely
+dissolve the peptone, etc.
+
+5. _Neutralise carefully to litmus paper_ by the successive additions of
+small quantities of decinormal soda solution.
+
+6. Replace in the steamer for twenty minutes to precipitate phosphates,
+etc.
+
+7. Filter through two thicknesses of Swedish filter paper.
+
+8. Add sterile litmus solution, sufficient to colour the medium a deep
+purple.
+
+9. Tube, and sterilise as for bouillon.
+
+
+~Lactose Litmus Gelatine (Wurtz).~--
+
+1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 4).
+
+2. Render the reaction of the medium mass -5.
+
+3. Replace in the steamer at 100 deg. C. for twenty minutes.
+
+4. Clarify with egg as for gelatine.
+
+5. Weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the
+medium.
+
+6. Filter through papier Chardin.
+
+7. Add sufficient sterile litmus solution to colour the medium pale
+lavender.
+
+8. Tube, and sterilise as for nutrient gelatine.
+
+
+~Lactose Litmus Agar (Wurtz).~--
+
+1. Prepare nutrient agar (_vide_ page 167, sections 1 to 4).
+
+2. Render the reaction of the medium mass -5.
+
+3. Replace in the steamer at 100 deg. C. for twenty minutes.
+
+4. Cool to 60 deg. C. and clarify with egg as for nutrient agar.
+
+5. Weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the
+medium.
+
+6. Filter through papier Chardin, using the hot-water funnel.
+
+7. Add sterile litmus solution, sufficient to colour the medium a pale
+lavender.
+
+8. Tube, and sterilise as for nutrient agar.
+
+
+~Glycerine Potato Bouillon.~--
+
+1. Take 1 kilo of potatoes, wash thoroughly in water, peel, and grate
+finely on a bread-grater.
+
+2. Weigh the potato gratings, place them in a 2-litre flask, and add
+distilled water in the proportion of 1 c.c. for every gramme weight of
+potato. Allow the flask to stand in the ice-chest for twelve hours.
+
+3. Strain the mixture through butter muslin and filter through Swedish
+filter paper into a graduated cylinder. Note the amount of the filtrate.
+
+4. Place the filtrate in a flask, add an equal quantity of distilled
+water, and heat in the steam steriliser for sixty minutes.
+
+5. Add glycerine, 4 per cent., mix thoroughly, and again filter.
+
+6. Tube and sterilise as for nutrient bouillon.
+
+~Potato Gelatine (Elsner).~--
+
+1. Take 1 kilo of potatoes, wash thoroughly in water, peel, and finally
+grate finely on a bread-grater.
+
+2. Weigh the potato gratings, place them in a 2-litre flask, and add
+distilled water in the proportion of 1 c.c. for every gramme weight of
+potato. Allow the flask to stand in the ice-chest for twelve hours.
+
+3. Strain the mixture through butter muslin, and filter through Swedish
+filter paper into a graduated cylinder.
+
+4. Add 15 per cent. gelatine to the potato decoction and bubble live
+steam through the mixture for ten minutes.
+
+5. Estimate the reaction; adjust the reaction of the medium mass to +25.
+
+6. Cool the medium to below 60 deg. C.; clarify with egg as for nutrient
+gelatine (_vide_ page 166).
+
+7. Add 1 per cent. potassium iodide (powdered) to the medium.
+
+8. Filter through papier Chardin.
+
+9. Tube and sterilise as for nutrient gelatine.
+
+~Aesculin Agar.~--(B. coli and allied organisms give black colonies
+surrounded by black halo.)
+
+1. Measure out 400 c.c. distilled water into a tared 2-litre flask.
+
+2. Weigh out
+
+    Agar                       15 grammes
+    Peptone                    10 grammes
+    Sodium taurocholate         5 grammes
+
+and make into a thick paste with 150 c.c. distilled water.
+
+3. Add this paste to the distilled water in the flask.
+
+4. Dissolve the ingredients by bubbling live steam through the mixture.
+
+5. Weigh out
+
+    Aesculin               1.0 gramme
+    Ferric citrate         0.5 gramme
+
+and dissolve in a second flask containing 100 c.c. distilled water.
+
+6. Mix the contents of the two flasks--adjust the weight to the
+calculated medium figure (in this case 1031.5 grammes) by the addition
+of distilled water at 100 deg. C.
+
+7. Clarify with egg and filter.
+
+8. Tube and sterilise as for nutrient agar.
+
+~Bile Salt Agar (MacConkey).~--
+
+1. Weigh out powdered agar, 15 grammes (= 1.5. per cent.), and emulsify
+with 200 c.c. _cold tap_ water.
+
+2. Weigh out peptone, 20 grammes (= 2 per cent.), and emulsify with 200
+c.c. _tap_ water previously warmed to 60 deg. C.
+
+3. Mix the peptone and agar emulsions thoroughly.
+
+4. Weigh out sodium taurocholate, 5 grammes (= 0.5 per cent.), dissolve
+it in 300 c.c. _tap_ water, and use the solution to wash the
+agar-peptone emulsion into a tared 2-litre flask.
+
+5. Bubble live steam through the mixture for twenty minutes.
+
+6. Adjust the weight of the medium mass to the calculated figure for one
+litre (1040 grammes).
+
+7. Cool to 60 deg. C. and clarify with egg as for nutrient agar (_vide_
+page 168).
+
+8. Filter through papier Chardin, using the hot-water funnel.
+
+9. Weigh out lactose, 10 grammes (= 1 per cent.), and dissolve it in the
+agar.
+
+If desired, add 5 c.c. of a 1 per cent. (= 0.5 per cent.) aqueous
+solution of neutral red.
+
+10. Tube, and sterilise as for nutrient agar.
+
+
+~Litmus Nutrose Agar (Drigalski-Conradi).~--
+
+This medium should be prepared in precisely the same manner as the
+Nutrose agar described on page 172 substituting meat extract for serum
+water, and increasing the percentage of agar added per litre to 3 per
+cent.
+
+
+~Fuchsin Agar (Braun).~--
+
+1. Liquefy and measure out into a sterile flask:
+
+    Nutrient agar          1000 c.c.
+
+2. Weigh out: lactose 10 grammes and dissolve in the fluid agar.
+
+3. Adjust the reaction to -5 and filter.
+
+4. Measure out and mix thoroughly with agar:
+
+    Fuchsin, alcoholic solution           5 c.c.
+
+The fuchsin solution is prepared by mixing:
+
+    Fuchsin (basic)           3 grammes.
+    Absolute alcohol          60 c.c.
+
+Allow to stand twenty-four hours, then centrifugalise thoroughly and
+decant the supernatant fluid into a well-stoppered bottle.
+
+5. Measure out and add to the nutrient agar, sodium sulphite, 10 per
+cent. aqueous solution, freshly prepared 25 c.c.
+
+6. Tube and sterilise as for nutrient agar.
+
+7. Store in a dark cupboard.
+
+
+~Fuchsin Sulphite Agar (Endo).~--
+
+1. Liquefy and measure out into a sterile flask:
+
+    Nutrient agar     1000 c.c.
+
+2. Weigh out
+
+    Lactose     10 grammes.
+
+and dissolve in the fluid agar.
+
+3. Adjust the reaction to +3 and filter.
+
+4. Measure out and mix thoroughly with the fluid agar.
+
+    Fuchsin, alcoholic solution (_vide supra_)     5 c.c.
+
+5. Measure out and add to the medium
+
+    Sodium sulphite, 10 per cent. aqueous solution     25 c.c.
+
+6. Tube and sterilise as for nutrient agar.
+
+
+~Brilliant Green Agar (Conradi).~--
+
+1. Liquefy and measure out into a sterile flask
+
+    Nutrient agar     1000 c.c.
+
+2. Adjust reaction to +30 by the addition of normal phosphoric acid; and
+filter.
+
+3. Measure out and mix thoroughly with the fluid medium
+
+    Brilliant green (Hoechst) 1 per thousand aqueous solution     6.5 c.c.
+
+4. Measure out and add to the medium
+
+    Picric acid (Gruebler), 1 per cent. aqueous solution      6.5 c.c.
+
+5. Tube and sterilise as for nutrient agar.
+
+
+~Brilliant Green Bile Salt Agar (Fawcus).~--
+
+1. Weigh out agar 20 grammes and emulsify in 100 c.c. cold distilled
+water.
+
+2. Wash the emulsion into a "tared" 2-litre flask with 500 c.c.
+distilled water.
+
+3. Dissolve the agar by bubbling live steam through the flask.
+
+4. Cool, clarify with egg and filter.
+
+5. Weigh out
+
+    Sodium taurocholate     5 grammes
+    Peptone                20 grammes
+
+and add to the medium in the flask.
+
+6. Weigh out
+
+    Lactose     5 grammes
+
+and add to the medium in the flask.
+
+7. Adjust reaction to +15 and filter if necessary.
+
+8. Measure out
+
+    Brilliant green, 1 per thousand aqueous solution     20 c.c.
+
+and mix thoroughly with the fluid agar.
+
+9. Measure out and add to the medium
+
+    Picric acid, 1 per cent. aqueous solution     20 c.c.
+
+10. Tube and sterilise as for nutrient agar.
+
+
+~China Green Agar (Werbitski).~--
+
+1. Liquefy and measure out into a sterile flask
+
+    Nutrient agar     1000 c.c.
+
+2. Adjust the reaction accurately to +13 and filter.
+
+3. Measure out and mix thoroughly with the fluid agar
+
+    China green 0.2 per cent. aqueous solution     15 c.c.
+
+4. Tube and sterilise as for nutrient agar.
+
+
+~Malachite Green Agar (Loeffler).~--
+
+1. Liquefy and measure out into a sterile flask
+
+    Nutrient agar     1000 c.c.
+
+2. Weigh out
+
+    Dextrose     10 grammes.
+
+and dissolve in nutrient agar.
+
+3. Adjust the reaction to +3, and filter.
+
+4. Measure out and mix thoroughly in the fluid agar
+
+    Malachite green, 0.1 per cent. aqueous solution     16 c.c.
+        for ~"weak"~ medium.
+
+_4a._ To the filtered agar add
+
+    Malachite green, 2 per cent. aqueous solution     25 c.c.
+        for ~"strong"~ medium.
+
+5. Tube and sterilise as for nutrient agar.
+
+~Double Sugar Agar (Russell).~--
+
+1. Liquefy and measure out into a sterile flask
+
+    Nutrient agar     1000 c.c.
+
+2. Add 100 c.c. litmus solution to the fluid agar.
+
+3. Weigh out and dissolve in the fluid agar.
+
+    Lactose      10 grammes
+    Dextrose     10 grammes.
+
+4. Render the reaction of the medium neutral to litmus paper by the
+cautious addition of normal caustic soda.
+
+5. Tube in quantities of 10 c.c. and sterilise in the steamer at 100
+deg. C. for twenty minutes on each of three successive days.
+
+6. Store for use in a cool dark place.
+
+
+_B. Diphtheriae._
+
+~Glycerine Blood-serum.~--
+
+1. Prepare blood-serum as described, page 168, sections 1 to 4.
+
+2. Add 5 per cent. pure glycerine.
+
+3. Complete as described above for ordinary blood-serum, sections 5 to
+7.
+
+     NOTE.--Different percentages of glycerine--from 4 per cent.
+     to 8 per cent.--are used for special purposes. Five per
+     cent. is that usually employed.
+
+
+~Blood-serum (Loeffler).~--
+
+1. Prepare nutrient bouillon (_vide_ page 163), using meat extract made
+from veal instead of beef.
+
+2. Add 1 per cent. glucose to the bouillon, and allow it to dissolve
+completely.
+
+3. Now add 300 c.c. clear blood-serum (_vide_ page 168, sections 1 to 4)
+to every 100 c.c. of this bouillon.
+
+4. Fill into sterile tubes and complete as for ordinary blood-serum.
+
+
+~Blood-serum (Lorrain Smith).~--
+
+1. Collect blood-serum (_vide_ page 168, sections 1 to 4), as free from
+haemoglobin as possible.
+
+2. Weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid
+(or add 0.375 c.c. of dekanormal soda solution for every 100 c.c. of
+serum).
+
+3. Tube, and stiffen at 100 deg. C. in the serum inspissator.
+
+4. Incubate at 37 deg. C. for forty-eight hours to eliminate any
+contaminated tubes. Store the remainder for future use.
+
+
+~Blood Serum (Councilman and Mallory).~--
+
+1. Collect blood serum in slaughterhouse, coagulate, remove serum and
+tube (_vide_ page 168).
+
+Great care must be taken to avoid the inclusion of air bubbles--indeed
+if only a few tubes are filled at one time, it is a good plan to stand
+them upright in the receiver of an air pump and to exhaust as completely
+as possible before transferring to the serum inspissator.
+
+2. Heat the tubes in a slanting position in hot-air steriliser at 90 deg.
+C. till firmly coagulated, say half an hour.
+
+3. Sterilise in steam steriliser at 100 deg. C. for 20 minutes on each of
+three successive days.
+
+Resulting medium not translucent, but opaque and firm.
+
+
+_B. Tuberculosis._
+
+~Egg Medium (Lubenau).~--
+
+This modification of Dorset's egg medium (_quod vide_ page 174) is
+preferred by some for the growth of the tubercle bacillus of the human
+type. It consists in the addition of one part of 6 per cent. glycerine
+in normal saline solution, to the egg mixture between steps 4 and 5.
+
+
+~Glycerine Bouillon.~--
+
+1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
+to 6).
+
+2. Measure out glycerine, 60 c.c. (= 6 per cent.), and add to the
+bouillon.
+
+3. Tube, and sterilise as for bouillon.
+
+
+~Glycerine Agar.~--
+
+1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out
+1000 c.c.
+
+2. Measure out pure glycerine, 60 c.c. (= 6 per cent.), and add to the
+agar.
+
+3. Tube, and sterilise as for nutrient agar.
+
+
+~Glycerine Blood-serum.~--
+
+1. Prepare blood-serum as described, page 168, sections 1 to 4.
+
+2. Add 5 per cent. pure glycerine.
+
+3. Complete as described above for ordinary blood-serum, sections 5 to
+7.
+
+     NOTE.--Different percentages of glycerine--from 4 per cent.
+     to 8 per cent.--are used for special purposes. Five per
+     cent. is that usually employed.
+
+
+~Glycerinated Potato.~--
+
+1. Prepare ordinary potato wedges (_vide_ page 174, sections 1 to 4).
+
+2. Soak the wedges in 25 per cent. solution of glycerine for fifteen
+minutes.
+
+3. Moisten the cotton-wool pads at the bottom of the potato tubes with a
+25 per cent. solution of glycerine.
+
+4. Insert a wedge of potato in each tube and replug the tubes.
+
+5. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
+_five_ consecutive days.
+
+
+~Animal Tissue Media (Frugoni).~--
+
+1. Take a number of sterile test-tubes 16 x 3 or 4 cm., plugged with
+cotton wool, and into each insert a 2 cm. length of stout glass tubing
+(about 1 cm. diameter); fill in glycerine (6 per cent.) bouillon to the
+upper level of the piece of glass tubing. Sterilise in the steamer at
+100 deg. C. for twenty minutes on each of three successive days.
+
+2. Kill a small rabbit by means of chloroform vapour.
+
+3. Under strictly aseptic precautions remove the lungs, liver and other
+solid organs and transfer them to a sterile double glass dish.
+
+4. With the help of sterile scissors and forceps divide the organs into
+roughly rectangular blocks 3 x 1.5 x 1 cm.
+
+5. Pour into the dish a sufficient quantity of sterile glycerine
+solution (6 per cent. in normal saline), cover, and allow to stand for
+one hour.
+
+6. Introduce a block of tissue into each tube so that it rests upon the
+upper end of the piece of glass tubing. (The surface of the tissue will
+now be kept moist by capillary attraction and condensation).
+
+7. Sterilise in the autoclave at 120 deg. C. for thirty minutes.
+
+8. Cap the tubes and store them in the ice chest for future use.
+
+Tissues obtained at postmortems can also be used after preliminary
+sterilisation by boiling or autoclaving.
+
+
+_Media for the Study of Special Cocci._
+
+_Diplococcus Gonorrhoeae._
+
+
+~Ascitic Bouillon (Serum Bouillon).~--
+
+1. Collect ascitic fluid (pleuritic fluid, hydrocele fluid, etc.), by
+aspiration directly into sterile flasks, under strictly aseptic
+precautions.
+
+2. Mix the serum with twice its bulk of sterile nutrient bouillon
+(_vide_ page 163).
+
+3. If considered necessary (on account of the presence of blood,
+crystals, etc.), filter the serum bouillon through porcelain filter
+candle.
+
+4. Tube, and sterilise in the water bath at 56 deg. C. for half an hour
+on each of five consecutive days.
+
+5. Incubate at 37 deg. C. for forty-eight hours and eliminate
+contaminated tubes. Store the remainder for future use.
+
+
+~Serum Agar (Heiman).~--
+
+1. Prepare nutrient agar (_vide_ page 167), to following formula:
+
+    Agar               2.0 per cent.
+    Peptone            1.5 per cent.
+    Salt               0.5 per cent.
+    Meat extract       _quantum sufficit._
+
+2. Make reaction of medium + 10.
+
+3. Filter; tube in quantities of 6 c.c.
+
+4. Sterilise as for nutrient agar.
+
+5. After the third sterilisation cool the tubes to 42 deg. C., and add
+to each 3 c.c. of sterile hydrocele fluid, ascitic fluid, or pleuritic
+effusion (previously sterilised, if necessary, by the fractional
+method); allow the tubes to solidify in a sloping position.
+
+6. When solid, incubate at 37 deg. C. for forty-eight hours, and eliminate
+any contaminated tubes. Store the remainder for future use.
+
+
+~Serum Agar (Wertheimer).~--
+
+1. Prepare nutrient agar (_vide_ page 167), to the following formula:
+
+    Agar              2.0 per cent.
+    Peptone           2.0 per cent.
+    Salt              0.5 per cent.
+    Meat extract      _quantum sufficit._
+
+2. Make reaction of medium +10.
+
+3. Filter; tube in quantities of 5 c.c.
+
+4. Sterilise as for nutrient agar.
+
+5. After the last sterilisation cool to 42 deg. C., then add 5 c.c.
+sterile blood-serum from human placenta (sterilised, if necessary, by
+the fractional method) to each tube; slope the tubes.
+
+6. When solid, incubate at 37 deg. C. for forty-eight hours, and eliminate
+any contaminated tubes. Store the remainder for future use.
+
+
+~Serum Agar (Kanthack and Stevens).~--
+
+1. Collect ascitic, pleuritic, or hydrocele fluid in sterile flasks and
+allow to stand in the ice-chest for twelve hours to sediment.
+
+2. Decant 1000 c.c. of the clear fluid into a measuring cylinder and
+transfer to sterile litre flask.
+
+3. Add 0.5 c.c. dekanormal NaOH solution for every 100 c.c. serum (_i.
+e._, 5.0 c.c.), and mix thoroughly.
+
+4. Heat in the steamer for twenty minutes.
+
+5. Weigh out 15 grammes agar, emulsify in a separate vessel with 200
+c.c. of the alkaline fluid previously cooled to about 20 deg. C., and
+then add to the remainder of the fluid in the flask.
+
+6. Bubble live steam through the mixture for twenty minutes to dissolve
+the agar.
+
+7. Filter through papier Chardin, using a hot-water funnel.
+
+8. Weigh out glucose 10 grammes (= 1 per cent.), and dissolve it in the
+clear agar.
+
+8a. If desired, add glycerine, 5 per cent., to the clear agar.
+
+9. Tube, and sterilise as for nutrient agar.
+
+
+~Serum Agar (Libman).~--
+
+1. Prepare nutrient agar (_vide_, page 167) using, however, 1.5 per
+cent. peptone (that is 15 grammes per litre instead of 10 grammes).
+
+2. Adjust the reaction to 0 (i. e., neutral to phenolphthalein).
+
+3. Filter and transfer 1000 c.c. liquefied medium to a sterile flask.
+
+4. Weigh out dextrose 20 grammes and dissolve in the fluid agar.
+
+5. Tube in quantities of 6 c.c.; and sterilise in the steamer at 100
+deg. C. for thirty minutes on each of three consecutive days.
+
+6. After the third sterilisation cool to 42 deg. C. and add to each tube
+3 c.c. of sterile hydrocele fluid, ascitic fluid or pleuritic effusion
+(previously sterilised, if necessary, by the fractional method); allow
+the tubes to solidify in a sloping position.
+
+7. When solid, incubate at 37 deg. C. for forty-eight hours, and eliminate
+any contaminated tubes. Store the remainder for future use.
+
+
+~Egg-albumen, Inspissated.~--
+
+1. Break several fresh eggs (hens', ducks', or turkeys' eggs), and
+collect the "whites" in a graduated cylinder, taking care to avoid
+admixture with the yolks.
+
+2. Add 40 per cent. distilled water, and incorporate the mixture
+thoroughly by the aid of an egg-whisk.
+
+3. Weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid
+(or add the amount of dekanormal caustic soda solution calculated to
+yield the required percentage of soda in the total bulk of the
+fluid--i. e., 0.375 c.c. of dekanormal NaOH solution per 100 c.c. of
+the mixture).
+
+_3a._ Glucose to the extent of 1 to 2 per cent. may now be added, if
+desired.
+
+4. Strain the mixture through butter muslin and filter through a
+porcelain filter candle into a sterile filter flask.
+
+5. Tube, and stiffen at 100 deg. C. in the serum inspissator.
+
+6. Incubate at 37 deg. C. for forty-eight hours and eliminate any
+contaminated tubes; store the remainder for future use.
+
+
+~Egg-albumen (Tarchanoff and Kolesnikoff).~--
+
+1. Place unbroken hens' eggs in dekanormal caustic soda solution for ten
+days. (After this time the white becomes firm like gelatine.)
+
+2. Carefully remove the shell and cut the egg into fine slices.
+
+3. Wash for two hours in running water.
+
+4. Place the egg slices in a large beaker and sterilise in the steamer
+at 100 deg. C. for one hour.
+
+5. Transfer each slice of egg by means of a pair of sterilised forceps
+to a Petri dish or large capsule.
+
+6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
+three consecutive days.
+
+
+~Egg Albumin Broth (Lipschuetz).~--
+
+1. Weigh out
+
+    Egg albumin (extra fine powder, Merck).    4 grammes
+
+and place in a 2-litre flask with a number of sterile glass beads.
+
+2. Measure out distilled water 200 c.c. into a half-litre flask and warm
+to 37 deg. C. in the incubator.
+
+3. Add the water to the flask containing the albumin and beads and
+dissolve by shaking.
+
+4. Add n/10-NaOH, 40 c.c. Allow the mixture to stand for thirty minutes
+with frequent shaking.
+
+5. Filter through Swedish filter paper.
+
+6. Sterilise by boiling two or three times at intervals of two hours.
+
+7. Add ordinary nutrient bouillon 600 c.c.
+
+8. Fill into small Erlenmeyer flasks in quantities of 50 c.c.
+
+9. Incubate for forty-eight hours at 37 deg. C.--discard any contaminated
+flasks and store the remainder for future use.
+
+
+~Egg Albumin Agar.~--
+
+1. Prepare egg albumin solution as above 1-6.
+
+2. Liquefy and measure out ordinary nutrient agar 600 c.c. and add to
+the egg albumin solution (in place of the nutrient broth).
+
+3. Complete as above 8-9.
+
+
+_Diplococcus Meningitidis Intracellularis._
+
+~Ascitic Fluid Agar (Wassermann)~ _Synonym_ ~N-as-gar (Mervyn Gordon).~
+
+1. Liquefy and measure out into a sterile flask:
+
+    Nutrient agar     600 c.c.
+
+2. Measure out into a half litre flask
+
+    Distilled water     210 c.c.
+
+and add to it
+
+    Ascitic fluid     90 c.c.
+    Nutrose     6 grammes
+
+3. Heat over a bunsen flame, shaking constantly until the fluid boils,
+and the nutrose is dissolved.
+
+4. Add the nutrose ascitic solution to the fluid agar.
+
+5. Heat in the steamer for thirty minutes, then filter.
+
+6. Tube and sterilise as for nutrient agar.
+
+     NOTE.--The finished medium in this case measures 900 c.c.
+     only since inconvenient fractions would be introduced in
+     making up to one litre exactly.
+
+
+_Diplococcus Pneumoniae._
+
+~Blood Agar (Washbourn).~--
+
+1. Melt up several tubes of nutrient agar (_vide_ page 167) and allow
+them to solidify in the oblique position.
+
+2. Place the tubes, in the horizontal position, in the "hot" incubator
+for forty-eight hours, to evaporate off some of the condensation water.
+
+3. Kill a small rabbit with chloroform and nail it out on a board (as
+for a necropsy). Moisten the hair thoroughly with 2 per cent. solution
+of lysol.
+
+4. Sterilise several pairs of forceps, scissors, etc., by boiling.
+
+5. Reflect the skin over the thorax with sterile instruments.
+
+6. Open the thoracic cavity by the aid of a fresh set of sterile
+instruments.
+
+7. Open the pericardium with another set of sterile instruments.
+
+8. Sear the surface of the left ventricle with a red-hot iron and remove
+fluid blood from the heart by means of sterile pipettes (e. g., those
+shown in Fig. 13, c).
+
+9. Deliver a small quantity of the blood on the slanted surface of the
+agar in each of the tubes, and allow it to run over the entire surface
+of the medium.
+
+10. Place the tubes in the slanting position and allow the blood to
+coagulate.
+
+11. Return the "blood agar" to the hot incubator for forty-eight hours
+and eliminate any contaminated tubes. Store the remainder for future
+use.
+
+
+_Media for the Study of Mouth Bacteria Generally._
+
+~Potato Gelatine (Goadby).~--
+
+1. Prepare glycerine potato broth (see page 203, sections 1 to 5).
+
+2. Add 10 per cent. gelatine to the potato decoction and bubble live
+steam through the mixture for ten minutes.
+
+3. Estimate the reaction; adjust the reaction of the medium to +5.
+
+4. Cool the medium to below 60 deg. C., clarify with egg as for nutrient
+gelatine.
+
+5. Filter through papier Chardin.
+
+6. Tube, and sterilise as for nutrient gelatine.
+
+
+_Media for the Study of Protozoa._
+
+~Tissue Medium (Noguchi).~--_For spirochaetes (cultivations must be grown
+anaerobically)._
+
+1. Plug and sterilise test-tubes 20 x 2 cm.
+
+2. Kill a small rabbit with chloroform vapour. Open the abdomen with
+all aseptic precautions, remove kidneys and testicles and transfer to a
+sterile glass dish. Cut up the organs with sterile scissors into small
+pieces--say 4 millimetre cubes. The four organs should yield from 25 to
+30 pieces of tissue.
+
+3. Drop a small piece of sterile tissue into the bottom of each
+sterilised tube.
+
+4. Take a flask containing about 400 c.c. nutrient agar (+10 reaction),
+liquefy the medium by heat and cool in a water bath to 50 deg. C.
+
+5. Add 200 c.c. ascitic or hydrocele fluid (horse or sheep serum may be
+employed, but is not so good) to the liquid agar and mix carefully to
+avoid formation of air bubbles.
+
+6. Fill about 20 c.c. of the ascitic agar into each of the sterilised
+tubes which already contains a piece of sterile rabbit's tissue, stand
+all the tubes upright in racks or a jar, and allow agar to set.
+
+7. After solidification pour sterile paraffin oil on the surface of the
+medium in each tube to the depth of 3 centimetres.
+
+8. Incubate tubes at 37 deg. C. for several days and discard any which
+prove to be contaminated.
+
+9. Store such tubes as are sterile for future use.
+
+
+
+
+XIII. INCUBATORS.
+
+
+[Illustration: FIG. 113.--Incubator.]
+
+An incubator (Fig. 113) consists essentially of a chamber for the
+reception of cultivations, etc., surrounded by a water jacket, the walls
+of which are of metal, usually copper, and outside all an asbestos or
+felt jacket, or wooden casing. The water in the jacket is heated by gas
+or electricity and maintained at some constant temperature by a
+thermo-regulator. The cellular incubator (Fig. 114) which was made for
+me[7] some years ago is of the greatest practical utility. Here the
+central cavity is subdivided by five double-walled partitions (in which
+water circulates in connection with the water tanks at the top and base
+of the incubator) and again by iron shelves to form twenty-four pigeon
+holes. Into each of these slides an iron drawer 35 cm. long x 12 cm.
+wide x 22 cm. high forming a self-contained incubator. The drawer is
+fitted with a wooden form to which is fixed a handle and a numbered
+label. The thermo-regulating apparatus is the well-known Hearson
+capsule.
+
+[Illustration: FIG. 114.--Cellular incubator.]
+
+Two incubators at least are required in the laboratory, for the
+cultivation of bacteria the one regulated to maintain a temperature of
+37 deg. C., and known as the "hot" incubator; the other, 20 deg. C. to
+22 deg. C., and known as the "cool" or "cold" incubator.
+
+Two other incubators, regulated to 42 deg. C. and 60 deg. C. respectively,
+whilst not absolutely, necessary very soon justify their purchase.
+
+~Thermo-regulators.~--The thermo-regulator is the most essential portion
+of the incubator, as upon its efficient working depends the maintenance
+of a constant temperature in the cultivation chamber. It is also used in
+the fitting up of water and paraffin baths, and for many other purposes.
+
+[Illustration: FIG. 115.--Reichert's thermo-regulator.]
+
+Of the many forms and varieties of thermo-regulator (other than
+electrical), two only are of sufficiently general use to need mention.
+In one of these the flow of gas to the gas-jet is controlled by the
+expansion or contraction of mercury within a glass bulb; in the other,
+by alterations in the position of the walls of a metallic capsule
+containing a fluid, the boiling-point of which corresponds to the
+temperature at which the incubator is intended to act. They are:
+
+(a) _Reichert's_ (Fig. 115), consists of a bulb containing mercury
+which is to be suspended in the medium, whether air or water, the
+temperature of which it is desired to regulate. Gas enters at A, and
+passes out to the jet by B. As the temperature rises the mercury expands
+and cuts off the main gas supply. As the temperature falls the mercury
+contracts and reopens the narrow tube C. By means of a thumbscrew D
+(which mechanically raises or lowers the column of mercury irrespective
+of the temperature) and the aid of a thermometer the apparatus can be
+set to keep the incubator at any desired temperature. With this form a
+special gas burner is required, with separate supply of gas to a pilot
+jet at the side.
+
+(b) _Hearson's capsule regulator_ consists of a metal capsule
+hermetically sealed and filled with a liquid which boils at the required
+temperature, this is adjusted in the interior of the incubator. Soldered
+to the upper side of the capsule is a thick piece of metal having a
+central cup to receive the lower end of a rigid rod, through which the
+movements of the walls of the capsule are transmitted to the gas valve
+fixed outside the incubator.
+
+The gas valve or governor is shown in figure 116. A is the inlet for
+gas, C the outlet to burner heating the water jacket, B D a lever
+pivoted to standards at G, and acted upon by the capsule, through the
+rigid rod which enters the socket below the screw P.
+
+[Illustration: FIG. 116.--Capsule thermo-regulator.]
+
+The construction of the valve is such that, whenever the short arm of
+the lever B D presses on the disc below the end B, the main supply of
+gas is entirely cut off. At such times, however, a very small portion of
+gas passes from A to C, through an aperture inside the valve, the size
+of which aperture can be adjusted by the screw needle S, hence the gas
+flame below the incubator is never extinguished.
+
+The expansion of the metal walls of the capsule, which takes place upon
+the boiling of its contents, provides the motive force, transmitted
+through the rigid rod to raise the long arm of the lever B D, and as
+this expansion only takes place at a predetermined temperature, the
+lever will only be acted upon when the critical temperature is reached,
+no sensible effect being produced at even 1 deg. C. below that at which
+the capsule is destined to act.
+
+W is a weight sliding on the lever rod D; by increasing the distance
+between the weight and the fulcrum of the lower increased pressure is
+brought to bear upon the walls of the capsule with the result that the
+boiling-point of the liquid in the capsule is slightly raised, and a
+range of about two degrees can thus be obtained with any particular
+capsule.
+
+FOOTNOTES:
+
+[7] Made by the firm of Chas. Hearson & Co., 235 Regent St., London, W.
+
+
+
+
+XIV. METHODS OF CULTIVATION.
+
+
+Cultivations of micro-organisms are usually prepared in the laboratory
+in one of three ways:
+
+    ~Tube cultures.~
+    ~Plate cultures.~
+    ~Hanging-drop cultures.~
+
+These may be incubated either ~aerobically~ (i. e., in the presence of
+oxygen) or ~anaerobically~ (i. e., in the absence of oxygen, or in the
+presence of an indifferent gas, such as hydrogen, nitrogen, or carbon
+dioxide).
+
+With regard to the temperature at which the cultivations are grown, it
+may be stated as a general rule that all media rendered solid by the
+addition of gelatine are incubated at 20 deg. C., or at any rate at a
+temperature not exceeding 22 deg. C. (that is, in the "cold" incubator);
+whilst fluid media and all other solid media are incubated at 37 deg. C.
+(that is, in the "hot" incubator). Exceptions to this rule are numerous.
+For instance, in studying the growth of the psychrophylic bacteria, the
+yeasts and the moulds, the cold incubator is employed for all media.
+
+Tube cultivations are usually packed in the incubator in small tin
+cylinders, such as those in which American cigarettes are sold, or in
+square tin boxes. Beakers or tumblers may be used for the same purpose,
+but being fragile are not so convenient. Metal test-tube racks, long
+enough to just fit into the interior of the incubator and each
+accommodating two rows of tubes, are also exceedingly useful.
+
+
+~AEROBIC.~
+
+~The Preparation of Tube Cultivations.~
+
+
+The preparation of a tube cultivation consists in:
+
+(a) Inoculating a tube of sterile nutrient medium with a portion of
+the material to be examined.
+
+(b) Incubating the inoculated tube at a suitable temperature.
+
+The details of the first of these processes must be varied somewhat
+according to whether the tubes of nutrient media are inoculated or
+"planted" from--
+
+1. Pre-existing cultivations.
+
+2. Morbid material previously collected (_vide_ page 373).
+
+3. Fluids, tissues, etc., or from the animal body direct.
+
+The method of preparing tube cultivations from pre-existing cultivations
+is as follows:
+
+[Illustration: FIG. 117.--Inoculating tubes, seen from the front.]
+
+~1. Fluid Media~ (e. g., Nutrient Bouillon).--
+
+1. Flame the cotton-wool plug of the tube containing the cultivation and
+also that of the tube of sterile bouillon.
+
+2. Hold the two tubes, side by side, between the left thumb and the
+first and third fingers, allowing the sealed ends to rest on the dorsum
+of the hand, and separating the mouths of the tubes (which are pointed
+to the right) by the tip of the second finger. Keep the tubes as nearly
+horizontal as is possible without allowing the fluid in the bouillon
+tube to reach the cotton-wool plug (Fig. 117).
+
+3. Sterilise the platinum loop and allow it to cool.[8]
+
+4. Grasp the plug of the tube containing the cultivation between the
+little finger and palm of the hand and remove it from the tube.
+
+5. Grasp the plug of the bouillon tube between the fourth finger and the
+ball of the thumb and remove it from the tube.
+
+6. Pass the platinum loop into the tube containing the culture--do not
+allow the loop to touch the sides of the tube, or the handle to touch
+the medium--and remove a small portion of the growth; withdraw the loop
+from the tube, keeping the infected side of the loop downward.
+
+7. Pass the loop into the bouillon tube almost down to the level of the
+fluid, reverse the loop so that the infected side faces upward, emulsify
+the portion of the growth in the moisture adhering to the side of the
+tube which is uppermost. Withdraw the loop.
+
+8. Replug both tubes.
+
+9. Sterilise the platinum loop.
+
+10. Label the bouillon tube with (a) the name of the organism and
+(b) the date of inoculation.
+
+11. Incubate.
+
+~2. Solid Media.~--Solid media are stored in tubes in one of two ways:
+
+1. Oblique tube or slanted tube (Fig. 118), in which the medium has been
+allowed to solidify whilst the tube was retained in an inclined
+position, so forming an extensive surface of medium extending from the
+bottom of the tube almost to its mouth.
+
+This is employed for "streak" or "smear" cultivations (_Strichcultur_).
+
+2. Straight tube (Fig. 119), in which the medium forms a cylindrical
+mass in the lower portion of the tube and presents an upper surface
+which is at right angles to the long axis of the tube.
+
+This is employed for "stab" or "stick" cultivations (_Stichcultur_), or
+by inoculating the medium whilst fluid, and allowing to solidify in this
+position, for "shake" cultivations.
+
+
+_Streak Culture._--
+
+1. Flame the plugs, sterilise the platinum loop (or spatula). Open the
+tubes and charge the loop as in previous inoculation.
+
+2. Pass the infected loop to the bottom of the tube to be inoculated and
+draw it, as lightly as possible, along the centre of the surface of the
+medium, terminating the "streak" over the thin layer of medium near the
+mouth of the tube.
+
+3. Replug the tubes, sterilise the platinum loop.
+
+4. Label the newly inoculated tube and incubate.
+
+_Smear Culture._--Proceed generally as in streak culture, but rub the
+infected loop all over the surface of the medium, instead of restricting
+the inoculation to a narrow line.
+
+     NOTE.--Gelatine and agar oblique tubes should be freshly
+     "slanted" before use.
+
+
+_Stab Culture._--
+
+1. Flame the plugs, open the tubes, sterilise the platinum needle and
+charge it with the inoculum as in the previous cultivations.
+
+2. Pass the platinum needle into the tube to be inoculated until it
+touches the centre of the surface of the medium. Now thrust it deeply
+into the substance of the medium, keeping the needle as nearly as
+possible in the axis of the cylinder of medium. Then withdraw the
+needle.
+
+3. Replug the tubes. Sterilise the platinum needle.
+
+4. Label the newly planted tube and incubate.
+
+     NOTE.--When gelatine is stored for some time the upper
+     surface of the cylinder becomes concave owing to
+     evaporation. Tubes showing this appearance should be
+     liquefied and again allowed to set before use for stab
+     culture, otherwise when the needle enters the medium, the
+     surface tension will cause the gelatine cylinder to split.
+
+[Illustration: FIG. 118.--Sloped or slanted medium for streak or smear
+culture.]
+
+[Illustration: FIG. 119.--Straight tube.]
+
+_Shake Culture._--
+
+1. Liquefy a tube of nutrient gelatine (or agar, or other similar
+medium), by heating in a water-bath (Fig. 121).
+
+2. Inoculate the liquefied medium and label it, etc., precisely as if
+dealing with a tube of bouillon.
+
+3. Place the newly planted tube in the upright position (e. g., in a
+test-tube rack) and allow it to solidify.
+
+4. Label the tube; when solid, incubate.
+
+     _Esmarch's Roll Cultivation._--
+
+     1. Liquefy three tubes of gelatine by heat.
+
+     2. Prepare three dilutions of the inoculum (as described for
+     plate cultivations, page 228, steps 4 to 7).
+
+     3. Roll the tubes, held almost horizontally, in a groove
+     made in a block of ice, until the gelatine has set in a thin
+     film on the inner surface of tube (Fig. 120); or under the
+     cold-water tap.
+
+     [Illustration: FIG. 120. Esmarch's roll culture on block of
+     ice.]
+
+     In order that the medium may adhere firmly to the glass, the
+     agar used for roll cultivation should have 1 per cent.
+     gelatine or 1 per cent. gum arabic added to it before
+     sterilisation.
+
+     Roll cultivations, which served a most important purpose in
+     the days before the introduction of Petri dishes for plate
+     cultivations, are now obsolete in modern laboratories and
+     are merely mentioned for the benefit of students, since
+     examiners who are interested in the academic and historical
+     aspects of bacteriology sometimes expect candidates to be
+     acquainted with the method of preparing them.
+
+
+The Preparation of Plate Cultures.
+
+If a small number of bacteria are suspended in liquefied gelatine, agar,
+or other similar medium, and the infected medium spread out in an even
+layer over a flat surface and allowed to solidify, each individual
+micro-organism becomes fixed to a certain spot and its further
+development is restricted to the vicinity of this spot. After a variable
+interval the growth of this organism becomes visible to the naked eye
+as a "colony." This is the principle upon which the method of plate
+cultivation is based and its practice enables the bacteriologist to
+study the particular manner of development affected by each species of
+microbe when growing (a) unrestricted upon the surface of the medium,
+(b) in the depths of the medium. The method itself is as follows:
+
+     ~Apparatus Required.~--
+
+     1. Tripod levelling stand.
+
+     2. Large shallow glass dish, with a square sheet of plate
+     glass to cover it.
+
+     3. Spirit level.
+
+     4. Case of sterile Petri dishes.
+
+     5. Tubes of sterile nutrient media, gelatine (or agar)
+     previously liquefied by heating in the water-bath and cooled
+     to 42 deg. C., otherwise the heat of the medium would destroy
+     many, if not all, of the bacteria introduced.
+
+     6. Tube of cultivation to be planted from.
+
+     7. Platinum loop.
+
+     8. Bunsen burner.
+
+     9. Grease pencil.
+
+[Illustration: FIG. 121.--Handy form of water-bath for melting tubes of
+agar and gelatine previous to slanting them; or to making shake cultures
+or pouring plates.]
+
+
+Method of "Pouring" Plates.--
+
+1. Place the glass dish on the levelling tripod (Figs. 122, 123); if
+gelatine plates are to be poured fill the dish with ice water--gelatine
+solidifies so slowly that it is necessary to hasten the process; if agar
+is to be used fill with water at 50 deg. C.--agar sets almost immediately
+at the room temperature and by slightly retarding the process lumpiness
+is avoided; cover the dish with the square sheet of glass.
+
+2. Place the spirit level on the sheet of glass and by means of the
+levelling screws adjust the surface of the glass to the horizontal.
+
+This leveling is an important matter since the development of a colony
+is to some extent proportionate to the supply of medium available for
+its nutrition. Thus in a "smear" on sloped tube culture, the colonies at
+the upper part of the medium are stunted and small but increase in size
+and luxuriance of growth the nearer they approach to the bottom of the
+tube, where there is the greatest depth of medium.
+
+[Illustration: FIG. 122.--Plate-levelling stand.]
+
+3. Place three sterile Petri dishes in a row on the surface of the glass
+plate and number them 1, 2, and 3, from left to right.
+
+[Illustration: FIG. 123.--Plate-levelling stand, side view.]
+
+4. Number the previously liquefied tubes of nutrient media 1, 2, and 3.
+Flame the plugs and see that each plug can be readily removed from the
+mouth of its tube.
+
+5. Add one loopful of the inoculum to tube No. 1, treating the
+liquefied medium as bouillon. After replugging, grasp the tube near its
+mouth by the thumb and first finger of the right hand, and with an even
+circular movement of the whole arm, diffuse the inoculum throughout the
+medium; avoid jerky movements, as these cause bubbles of air to form in
+the medium.
+
+[Illustration: FIG. 124.--Mixing emulsion for plates.]
+
+The knack of mixing evenly without producing air bubbles, is not always
+easily acquired, by this method. An alternative plan is to hold the
+inoculated tube vertically upright between the opposed palms and to
+rotate it between them by rapid backward and forward movements of the
+two hands (Fig. 124).
+
+[Illustration: FIG. 125.--Pouring plates.]
+
+6. Sterilise the platinum loop, and add two loopfuls of diluted inoculum
+to tube No. 2, and mix as before.
+
+7. In a similar manner transfer three loopfuls of liquefied medium from
+tube No. 2 to tube No. 3, and mix thoroughly.
+
+8. Flame the plug of tube No. 1, remove it, then flame the lips of the
+tube; slightly raise the cover of Petri dish No. 1, introduce the mouth
+of the tube; then, elevating the bottom of the tube, pour the liquefied
+medium into the Petri dish, to form a thin layer. Remove the mouth of
+the tube and close the "plate." If the medium has failed to flow evenly
+over the bottom of the plate, raise the plate from the levelling
+platform and by tilting in different directions rectify the fault.
+
+9. Pour plates No. 2 and No. 3, in a similar manner, from tubes Nos. 2
+and 3.
+
+10. Label the plates with the distinctive name or number of the
+inoculum, also the date; the number of the dilution having been
+previously indicated (step 3).
+
+11. Place in the cool incubator for three or more days, as may be
+necessary.
+
+In this way colonies may be obtained quite pure and separate from each
+other.
+
+In plate No. 1, probably, the colonies will be so numerous and crowded,
+and therefore so small, as to render it useless. In plate No. 2 they
+will be more widely separated, but usually No. 3 is the plate reserved
+for careful examination, as in this the colonies are usually widely
+separated, few in number, and large in size.
+
+_Agar plates_ are poured in a similar manner, but the agar must be
+melted in boiling water and then allowed to cool to 45 deg. C. or 42 deg.
+C. in a carefully regulated water-bath before being inoculated, and the
+entire process must be carried out very rapidly, otherwise the agar will
+have solidified before the operation is completed.
+
+     NOTE.--In pouring plates, since tube No. 1 (for the first
+     dilution) rarely gives a plate that is of any practical
+     value it is frequently replaced by a tube of bouillon or
+     sterile salt solution, and in such case plate No. 1 is not
+     poured.
+
+
+~Surface Plates.~--
+
+This method of pouring what may be termed "whole" plates (since colonies
+may appear both on the surface and in the depths of the medium) is
+essential to the accurate study of the formation of colonies under
+various conditions, but when the main object of the separation of the
+bacteria is to obtain subcultivations from a number of individual
+bacteria, "surface" plates must be prepared, since here colony formation
+is restricted to the surface of the medium. The method adopted varies
+slightly according to whether the medium employed is gelatine or agar,
+or one of the derivatives or variants of the latter.
+
+
+(a) ~Gelatine Surface Plates.~--
+
+1. Liquefy three tubes of nutrient gelatine.
+
+2. Pour each tube into a separate Petri dish and allow it to solidify.
+Then turn each plate and its cover upside down.
+
+[Illustration: FIG. 126.--Surface plate spreader.]
+
+3. When quite cold raise the bottom of plate 1, revert it and deposit a
+drop of the inoculum (whether a fluid culture or an emulsion from solid
+culture) upon the surface of the gelatine with a platinum loop--close to
+one side of the plate; replace the bottom half of the Petri dish in its
+cover.
+
+4. Take a piece of thin glass rod, stout platinum wire or best of all a
+piece of aluminium wire (say 2 mm. diameter) about 28 cm. long. Bend the
+terminal 4 cm. at right angles to the remainder, making an L-shaped rod
+(Fig. 126). Sterilise the short arm and adjacent portion of the long
+arm, in the Bunsen flame, and allow it to cool.
+
+5. Now raise the bottom of the Petri dish in the left hand, leaving the
+cover on the laboratory bench, and holding it vertically, smear the drop
+of inoculum all over the surface of the gelatine with the short arm of
+the spreader by a rotatory motion, (Fig. 127). Replace the dish in its
+cover.
+
+6. Raise the bottom of plate 2 and rub the infected spreader all over
+the surface of the gelatine--then go on in like manner to the third
+plate in the series.
+
+7. Sterilise the spreader.
+
+8. Label and incubate the plates.
+
+[Illustration: FIG. 127.--Spreading surface plate.]
+
+After incubation, plate No. 1 will probably yield an enormous number of
+colonies; plate 2 will show fewer colonies, since only those bacteria
+adhering to the rod after rubbing over plate 1 would be deposited on its
+surface, and by the time the rod reached plate 3 but very few organisms
+should remain upon it. So that the third plate as a rule will only show
+a very few scattered colonies, eminently suitable for detailed study.
+
+
+(b) ~Agar Surface Plates.~--
+
+1. Liquefy three tubes of nutrient agar--nutrose agar or the like.
+
+2. Pour each tube into a separate Petri dish and allow it to solidify.
+
+3. When quite solid invert each dish, raise the bottom half and rest it
+obliquely on its inverted cover (Fig. 128) and place it in this position
+in an incubator at 60 deg. C. for forty-five minutes (or in an incubator
+at 42 deg. C. for two hours). This evaporates the water of condensation
+and gives the medium a firm, dry surface.
+
+4. On removing the plates from the incubator close each dish and place
+it--still upside down--on the laboratory bench.
+
+[Illustration: FIG. 128.--Drying surface plate of agar.]
+
+5. Inoculate the plates in series of three, as described for gelatine
+surface plates 3-8.
+
+
+Hanging-drop Cultivation.
+
+    ~Apparatus Required.~--
+
+    Hanging-drop slides.
+    Cover-slips.
+    Section rack (Fig. 75).
+    Blotting paper.
+    Bell glass to cover slides.
+    Original culture.
+    Tubes of broth, or liquefied gelatine or agar.
+    Forceps.
+    Platinum loop.
+    Bunsen burner.
+    Grease pencil.
+    Sterile vaseline.
+    Lysol.
+
+
+(a) ~Fluid Media.~--
+
+1. Prepare first and second dilutions of the inoculum as directed for
+plate cultivations (_vide_ pages 228-229, sections 4 to 6), substituting
+tubes of nutrient broth for the liquefied gelatine.
+
+2. Sterilise a hanging-drop slide by washing thoroughly in water and
+drying, then plunging it into a beaker of absolute alcohol, draining off
+the greater part of the spirit, grasping the slide in a pair of forceps,
+and burning off the remainder of the alcohol in the flame.
+
+3. Place the hanging-drop slide on a piece of blotting paper moistened
+with 2 per cent. lysol solution and cover it with a small bell glass
+that has been rinsed out with the same solution and _not dried_.
+
+4. Raise the bell glass slightly and smear sterile vaseline around the
+rim of the metal cell by means of a sterile spatula of stout platinum
+wire.
+
+5. Remove a clean cover-slip from the alcohol pot with sterile forceps
+and burn off the alcohol; again raise the bell glass and place the
+sterile cover-slip on the blotting paper by the side of the hanging-drop
+slide.
+
+6. Remove a drop of the broth from the second dilution tube with a large
+platinum loop; raise the bell glass and deposit the drop on the centre
+of the cover-slip. Sterilise the loop.
+
+7. Raise the bell glass sufficiently to allow of the cover-slip being
+grasped with forceps, inverted, and adjusted over the cell of the
+hanging-drop slide. Remove the bell glass altogether and press the
+cover-slip firmly on to the cell.
+
+8. Either incubate and examine at definite intervals, or observe
+continuously with the microscope, using a warm stage if necessary (Fig.
+53).
+
+(b) ~Solid Media.~--Observing precisely similar technique, a few drops of
+liquefied gelatine or agar from the second dilution tube may be run over
+the surface of the sterile cover-slip and a hanging-drop plate
+cultivation thereby prepared.
+
+This method is extremely useful in connection with the study of yeasts,
+when the circular cell on the hanging-drop slide should be replaced by a
+rectangular cell some 38 by 19 mm., and the gelatine spread over a
+cover-slip of similar size. After sealing down the preparation, the
+upper surface of the cover-slip may be ruled into squares by the aid of
+the grease pencil or a writing diamond and numbered to facilitate the
+subsequent identification of the colonies which are observed to develop
+from solitary germs.
+
+
+~Hanging-block Culture~ (Hill).--
+
+_Apparatus required_: As for hanging-drop cultivation with the addition
+of a scalpel.
+
+Carry out the method as far as possible under cover of a bell glass, to
+avoid aerial contamination.
+
+1. Liquefy a tube of nutrient agar (or gelatine) and pour into a Petri
+dish to the depth of about 4 mm. and allow to set.
+
+2. With a sharp scalpel cut out a block some 8 mm. square, from the
+entire thickness of the agar layer.
+
+3. Raise the agar block on the blade of the scalpel and transfer it,
+under side down, to the centre of a sterile slide.
+
+4. Spread a drop of fluid cultivation (or an emulsion of growth from a
+solid medium) over the upper surface of the agar block as if making a
+cover-slip film.
+
+5. Place the slide and block covered by the bell glass in the incubator
+at 37 deg. C. for ten minutes to dry slightly.
+
+6. Take a clean dry sterile cover-slip in a pair of forceps, and with
+the help of a second pair of forceps lower it carefully on the
+inoculated surface of the agar (avoiding air bubbles), so as to leave a
+clear margin of cover-slip overlapping the agar block.
+
+7. Invert the preparation and with the blade of the scalpel remove the
+slide from the agar block.
+
+8. With a platinum loop run a drop or two of melted agar around the
+edges of the block. This solidifies at once and seals the block to the
+cover-slip.
+
+9. Prepare a sterile hanging-drop slide, and smear hard vaseline or
+melted white wax on the rim of the metal cell.
+
+10. Invert the cover-slip with the block attached on to the hanging-drop
+slide, and seal the cover-slip firmly in place.
+
+11. Observe as for hanging-drop cultivations.
+
+
+ANAEROBIC CULTIVATIONS.
+
+Numerous methods have been devised for the cultivation of anaerobic
+bacteria, the majority requiring the employment of special apparatus.
+The principle upon which any method is based and upon which it depends
+for its success falls under one or another of the following headings:
+
+(a) ~Exclusion of air~ from the cultivation.
+
+(b) ~Exhaustion of air~ from the vessel containing the cultivation by
+means of an air pump--i. e., cultivation _in vacuo_.
+
+(c) ~Absorption of oxygen~ from the air in contact with the cultivation
+by means of pyrogallic acid rendered alkaline with caustic soda--i. e.,
+cultivation in an atmosphere of nitrogen.
+
+(d) ~Displacement of air~ by an indifferent gas, such as hydrogen or coal
+gas--i. e., cultivation in an atmosphere of hydrogen.
+
+(e) A combination of two or more of the above methods.
+
+A selection of the simplest and most generally useful methods is given
+here.
+
+Whenever possible, the nutrient media that are employed in any of the
+processes should contain some easily oxidisable substance, such as
+sodium formate (0.4 per cent.) or sodium sulphindigotate (0.1 per
+cent.), which will absorb all the available oxygen held in solution by
+the medium. The further addition of glucose, 2 per cent., favors the
+growth of anaerobic bacteria (_vide_, pages 189-190).
+
+Further, it is advisable to seal all joints between india-rubber
+stoppers and tubulures or the mouths of the tubes with melted paraffin;
+glass stoppers and taps should be lubricated with resin ointment or a
+mixture of beeswax 1 part, olive oil 4 parts.
+
+
+(A) ~Method I~ (Hesse's Method).--
+
+1. Make a stab culture in gelatine or agar, choosing for the purpose a
+straight tube containing a deep column of medium, and thrusting the
+inoculating needle to the bottom of the tube.
+
+2. Pour a layer of sterilised oil (olive oil, vaseline, or petroleum), 1
+or 2 cm. deep, upon the surface of the medium.
+
+3. Incubate.
+
+
+~Method II.~--This method is only available when dealing with pure
+cultivations.
+
+1. Liquefy a tube of gelatine (or agar) by heat, pour it into a Petri
+dish, and allow it to solidify.
+
+2. Inoculate the surface of the medium in one spot only.
+
+3. Remove a cover-slip from the pot of absolute alcohol with sterile
+forceps; burn off the alcohol in the gas flame.
+
+4. Lower the now sterile cover-slip carefully on to the inoculated
+surface of the medium, carefully excluding air bubbles, and press it
+down firmly with the points of the forceps. (A sterile disc of mica may
+be substituted for the cover-slip.)
+
+5. Incubate.
+
+
+~Method III~ (Roux's Physical Method).--
+
+1. Prepare tube cultures of fluid media (or solid media rendered fluid
+by heat) in the usual way.
+
+2. Aspirate some of the inoculated media into capillary pipettes.
+
+3. Seal both ends of each pipette in the blowpipe flame.
+
+4. Incubate.
+
+
+~Method IV~ (Roux's Biological Method).--
+
+1. Plant a deep stab, as in method I.
+
+2. Pour a layer, 1 or 2 cm. deep, of broth cultivation of a vigourous
+aerobe--e. g., B. aquatilis sulcatus or B. prodigiosus--upon the
+surface of the medium; or an equal depth of liquefied gelatine, which is
+then inoculated with the aerobic organism.
+
+3. Incubate.
+
+The growth of the aerobe will use up all the oxygen that reaches it and
+will not allow any to pass through to the medium below, which will
+consequently remain in an anaerobic condition.
+
+
+(B) ~Method V.~--
+
+1. Prepare tube or flask cultivations in the usual way.
+
+2. Replace the cotton-wool plug by an india-rubber stopper perforated
+with one hole and fitted with a length of glass tubing which has a
+constriction about 3 cm. above the stopper and is then bent at right
+angles (Fig. 129). The stopper and glass tubing are sterilised by being
+boiled in a beaker of water for five minutes.
+
+[Illustration: FIG. 129.--Vacuum culture.]
+
+3. Connect the tube leading from the culture vessel with a water or air
+pump, interposing a Wulff's bottle fitted as a wash-bottle and
+containing sulphuric acid.
+
+4. Exhaust the air from the culture vessel.
+
+5. Before disconnecting the apparatus, seal the glass tube from the
+culture vessel at the constriction, using the blowpipe flame.
+
+6. Incubate.
+
+
+(C) ~Method VI~ (Buchner's Method).
+
+~Apparatus and Solutions Required.~--
+
+     Buchner's tube (a stout glass test-tube 23 cm. long and 4
+     cm. in diameter, fitted with india-rubber stopper, Fig.
+     130).
+
+     Pyrogallic acid in compressed tablets each containing 1
+     gram.
+
+     Dekanormal solution of caustic soda.
+
+METHOD.--
+
+1. Prepare the tube cultivation in the usual way.
+
+2. Moisten the india-rubber stopper of the Buchner's tube with water and
+see that it fits the mouth of the tube accurately.
+
+3. Remove the stopper from the caustic soda bottle.
+
+4. Drop one of the pyrogallic acid tablets[9] into the Buchner's tube
+(roughly, use 1 gramme pyrogallic acid for every 100 c.c. air capacity
+of the receiving vessel).
+
+5. Add about 1 c.c. of the soda solution.
+
+6. Place the inoculated tube inside the Buchner's tube. The pyrogallic
+tablet acts as a buffer and prevents damage to either the inoculated
+tube or the Buchner's tube even should it be slipped in hurriedly.
+
+7. Fit the india-rubber stopper tightly into the mouth of the Buchner's
+tube.
+
+[Illustration: FIG. 130.--Buchner's tube.]
+
+The pyrogallic acid tablet dissolves slowly in the soda solution and its
+oxidation proceeds very slowly at first so that ample time is available
+when this method is adopted.
+
+8. Restopper the caustic soda bottle.
+
+9. Place Buchner's tube in a wire support, and incubate.
+
+
+~Method VII~ (Wright's Method).--
+
+1. Prepare tube cultivation in the usual way.
+
+2. Cut off that portion of the cotton-wool plug projecting above the
+mouth of the tube with scissors, then push the plug into the tube for a
+distance of 2 or 3 cm.
+
+3. By means of a pipette drop about 1 c.c. of pyrogallic acid 10 per
+cent. aqueous solution on to the plug. It will immediately be absorbed
+by the cotton-wool.
+
+4. With another pipette run in an equal quantity of the caustic soda
+solution.
+
+5. Quickly close the mouth of the tube with a tightly fitting
+india-rubber stopper.
+
+6. Incubate.
+
+[Illustration: FIG. 131.--McLeod's anaerobic plate base with half petri
+dish inverted _in situ_]
+
+
+~Method VIII~ (McLeod's Method).--
+
+~Apparatus and Solutions Required.~--
+
+     McLeod's plate base (a hollow glazed earthenware disc 9 cm.
+     in diameter and 2 cm. deep: the upper surface is pierced by
+     a central hole, 2 cm. in diameter, giving access to the
+     interior, the lower part of which is divided into two by a
+     low partition. A shallow groove encircles the upper surface
+     near to the edge).
+
+    Plasticine.
+    Pyrogallic acid (1 gramme) compressed tablets.
+    Sodic hydroxide (0.4 gramme) compressed tablets.
+    Wash bottle of distilled water.
+    Surface plates of one or other agar medium (in petri dishes
+      of 8 cm. diameter).
+    Surface plate spreader.
+
+METHOD.--
+
+1. Roll out a long cylinder of plasticine and fit it into the groove on
+the upper surface of the earthenware base.
+
+2. Place a tablet of pyrogallic acid in one division of the interior of
+the plate base, and two tablets of sodic hydroxide in the other.
+
+3. Prepare surface plate culture of the organism to be cultivated.
+
+4. Run a few cubic centimetres of distilled water into that division of
+the plate base containing the sodic hydroxide.
+
+5. Invert the bottom half of the surface plate over the plate base and
+press its edges firmly down into the plasticine filling the groove.
+
+6. Label and incubate.
+
+
+(D) ~Method IX.~--
+
+~Apparatus Required.~--
+
+    Small Ruffer's or Woodhead's flask (Fig. 33).
+    Sterile india-rubber stopper.
+    India-rubber tubing.
+    Glass tubing.
+    Metal screw clips.
+    Cylinder of compressed hydrogen; or hydrogen gas apparatus
+
+METHOD.--
+
+1. Sterilise a glass vessel, shaped as in a Ruffer's or Woodhead's
+flask, in the hot-air oven. (The tubulure and the side tubes are plugged
+with cotton-wool.) After sterilisation, fix a short piece of rubber
+tubing occluded by a metal clip to each side tube.
+
+2. Inoculate a large quantity (e. g., 200 c.c.) of the medium. Where
+solid media are employed they must first be liquefied by heat.
+
+3. Remove the cotton-wool plug from the tubulure and pour the inoculated
+medium into the glass vessel.
+
+4. Close the tubulure by means of an india-rubber stopper previously
+sterilised by boiling in a beaker of water.
+
+[Illustration: FIG. 132.--Kipp's hydrogen apparatus, (a) connected up
+to two washing bottles containing (b) lead acetate 10 per cent.
+solution, to remove H_{2}S and (c) silver nitrate solution to remove
+AsH_{3}. A third washing bottle containing pyrogallic acid 10 per cent.
+solution, rendered alkaline, to remove any trace of oxygen, is sometimes
+introduced.]
+
+[Illustration: FIG. 133.--Improved gas apparatus; the metal is contained
+in a perforated glass tube which is submerged in acid when the
+triangular bottle is upright (a), but is above the level of the liquid
+when the bottle is turned on its side (b).]
+
+5. Connect up the india-rubber tubing on one of the side tubes with a
+cylinder of compressed hydrogen (or the delivery tube of a Kipp's Fig.
+132 or other hydrogen apparatus, Fig. 133), interposing a short piece of
+glass tubing; and in like manner connect a long piece of rubber tubing
+which should be led into a basin of water, to the opposite side tube.
+
+6. Open both metal clips and pass hydrogen through the vessel until the
+atmospheric air is replaced by hydrogen. This is determined by
+collecting some of the gas which bubbles through the water in the basin
+in a test-tube and testing it by means of a lighted taper.
+
+7. Close the metal clip on the tube through which the gas is entering;
+close the clip on the exit tube.
+
+8. Disconnect the gas apparatus.
+
+9. Incubate.
+
+
+~Method X~ (Botkin's Method).--
+
+~Apparatus Required.~--
+
+    Large glass dish 20 cm. diameter and 8 cm. deep. Flat leaden
+    cross slightly shorter than the internal diameter of the glass dish.
+    Bell glass about 15 cm. diameter and 20 to 25 cm. high.
+    Metal frame for plate cultivations.
+    _Or_, glass battery jar for tube cultivations.
+    Cylinder of compressed hydrogen.
+    Rubber tubing.
+    Two pieces of ~U~-shaped glass tubing (each arm 8 cm. in length).
+    Half a litre of glycerine (or metallic mercury).
+
+METHOD.--
+
+1. Place the leaden cross inside the glass dish, resting on the bottom.
+
+2. Prepare the cultivations in the usual way.
+
+3. Place the tube cultivations in a glass battery jar (or the plate
+cultivations on a metal frame), resting on the centre of the leaden
+cross.
+
+4. Cover the cultivations with the bell jar.
+
+5. Adjust the U-shaped pieces of glass tubing in a vertical position on
+opposite sides of the bell jar, one arm of each inside the jar, the
+other outside. These tubes are best held in position by embedding the
+U-shaped bends in two lumps of plasterine stuck on the bottom of the
+glass dish. Fix a short length of rubber tubing clamped with a metal
+clip to each of the outside arms (Fig. 134).
+
+6. Fill the glass dish with glycerine or metallic mercury to a depth of
+about 5 cm.
+
+[Illustration: FIG. 134.--Botkin's apparatus.]
+
+7. Connect up one U-shaped tube with the hydrogen cylinder (or gas
+apparatus) by means of rubber tubing. Replace the atmospheric air by
+hydrogen, as in method IX.
+
+8. Clamp the tubes and disconnect the gas apparatus.
+
+9. Incubate.
+
+
+~Method XI~ (Novy's Method).--
+
+~Apparatus Required.~--
+
+    Jar for plate cultivations (Fig. 135).
+    _Or_, jar for tube cultivations (Fig. 136).
+    Lubricant for stopper of jar.
+    Rubber tubing.
+    Cylinder of compressed hydrogen.
+
+METHOD.--
+
+1. Prepare cultivations in the usual way.
+
+2. Place these inside the jar.
+
+3. Lubricate the stopper and insert it in the mouth of the jar, with the
+handle in a line with the two side tubes.
+
+4. Connect up the delivery tube a with the hydrogen gas supply by
+means of rubber tubing.
+
+[Illustration: FIG. 135.--Novy's jar for plate cultivations.]
+
+[Illustration: FIG. 136.--Novy's jar for tube cultivations.]
+
+5. Attach a piece of rubber tubing to the exit tube b and collect
+samples of the issuing gas (over water) and test from time to time.
+
+6. When the air is completely displaced by hydrogen, turn the handle of
+the stopper at right angles to the line of entry and exit tubes; this
+seals the orifice of both tubes.
+
+7. Disconnect the gas apparatus and incubate.
+
+
+(E) ~Method XII~ (Bulloch's Method).--
+
+~Apparatus Required.~--
+
+    Bulloch's jar.
+    Pot of resin ointment.
+    Small glass dish 14 cm. diameter by 5 cm. deep.
+    Vessel for tube cultures or metal rack for plate cultures.
+    Pyrogallic acid tablets.
+    Cylinder of compressed hydrogen.
+    Geryk or other air pump.
+    Rubber pressure tubing.
+    10 c.c. pipette.
+    Glass tubing.
+    Dry granulated caustic soda or compressed tablets each, containing
+      0.4 grammes sodic hydroxide.
+    Small beaker of water.
+
+METHOD.--
+
+1. Prepare the cultivations in the usual way.
+
+2. Place the glass dish in the centre of the glass slab, and stand the
+cultivations inside this.
+
+3. Place a sufficient number of pyrogallic acid tablets at one side of
+the glass dish (i. e., 1 tablet for each 100 cubic centimeters air
+capacity of the bell jar). Place a small heap of dry granulated soda (or
+half a dozen tablets of sodic hydroxide) by the side of the pyro
+tablets.
+
+4. Smear the flange of the bell jar with resin ointment and apply the
+jar firmly to the glass slab, covering the cultivations--so arranged
+that the long tube passes with its lower end into the glass dish at a
+point directly opposite to the pyrogallic acid tablets. Lubricate the
+two stop-cocks with resin ointment (Fig. 137).
+
+5. Connect up the short tube a with the gas-supply by means of rubber
+pressure tubing and open both stop-cocks.
+
+6. Connect a long, straight piece of glass tubing to the long tube b
+by means of a piece of rubber tubing interposing a screw clamp: and
+collect samples of the issuing gas from time to time and test.
+
+7. When the air is displaced, shut off the stop-cock of the entry tube,
+then that of the exit tube b. Screw down the clamp and remove the
+glass tube from the rubber connection and connect up the short tube a
+to the air pump by means of pressure tubing.
+
+8. Open the stop-cock of tube a and with two or three strokes of the
+air pump, aspirate a small quantity of gas, so creating a slight vacuum.
+Then shut off the stop-cock and disconnect the air pump.
+
+9. Fill the 10 c.c. bulb pipette with water; insert its point into the
+rubber tubing on the long tube b as far as the screw clamp. Open the
+screw clamp and run in water until stopped by the internal pressure.
+Shut off stop-cock. (The water dissolves the soda and pyrogallic acid
+converting the latter into alkaline pyro. and so bringing its latent
+capacity for oxygen into action).
+
+[Illustration: FIG. 137.--Bulloch's jar.]
+
+10. Reverse the tubes from the tubulures so that they meet, out of
+harm's way, over the top of the bell glass; again see that all joints
+are tight and transfer the apparatus to the incubator.
+
+This last method is the most satisfactory for anaerobic cultivations, as
+by its means complete anaerobiosis can be obtained with the least
+expenditure of time and trouble.
+
+FOOTNOTES:
+
+[8] See also method of opening and closing culture tubes, pages 74-76.
+
+[9] If compressed tablets of pyrogallic acid cannot be obtained make up
+a stock "acid pyro" solution
+
+     Pyrogallic acid,          10 grammes
+     Hydrochloric acid,       1.5 c.c.
+     Distilled water,         100 c.c.
+
+and at step 4, run in 10 c.c. of the solution.
+
+
+
+
+XV. METHODS OF ISOLATION.
+
+
+The work in the preceding sections, arranged to demonstrate the chief
+biological characters of bacteria in general, is intended to be carried
+out by means of cultivations of various organisms previously isolated
+and identified and supplied to the student in a state of purity. A
+cultivation which comprises the progeny of a single cell is termed a
+"pure culture"; one which contains representatives of two or more
+species of bacteria is spoken of as an "impure," or "mixed"
+"cultivation," and it now becomes necessary to indicate the chief
+methods by which one or more organisms may be isolated in a state of
+purity from a mixture; whether that mixture exists as an impure
+laboratory cultivation, or is contained in pus and other morbid
+exudations, infected tissues, or water or food-stuffs.
+
+[Illustration: FIG. 138.--Haematocytometer cell, showing, a, section
+through the centre of the cell, and b, a magnified image of the cell
+rulings.]
+
+Before the introduction of solid media the only method of obtaining pure
+cultivations was by "dilution"--by no means a reliable method.
+"Dilution" consisted in estimating approximately the number of bacteria
+present in a given volume of fluid (by means of a graduated-celled slide
+resembling a haematocytometer, Fig. 138), and diluting the fluid by the
+addition of sterile water or bouillon until a given volume (usually 1
+c.c.) of the dilution contained but one organism. By planting this
+volume of the fluid into several tubes or flasks of nutrient media, it
+occasionally happened that the resulting growth was the product of one
+individual microbe. A method so uncertain is now fortunately replaced by
+many others, more reliable and convenient, and in those methods selected
+for description here, the segregation and isolation of the required
+bacteria may be effected--
+
+A. ~By Mechanical Separation.~
+
+1. By surface plate cultivation:
+
+    (a) Gelatine.
+    (b) Agar.
+    (c) Serum agar.
+    (d) Blood agar.
+    (e) Hanging-drop or block.
+
+[2. By Esmarch's roll cultivation:
+
+This archaic method (see page 226) is no longer employed for the
+isolation of bacteria.]
+
+3. By serial cultivation.
+
+B. ~By Biological Differentiation.~
+
+4. By differential media.
+
+    (a) Selective.
+    (b) Deterrent.
+
+5. By differential incubation.
+
+6. By differential sterilisation.
+
+7. By differential atmosphere cultivation.
+
+8. By animal inoculation.
+
+The selection of the method to be employed in any specific instance will
+depend upon a variety of circumstances, and often a combination of two
+or more will ensure a quicker and more reliable result than a rigid
+adherence to any one method. Experience is the only reliable guide, but
+as a general rule the use of either the first or the third method will
+be found most convenient, affording as each of them does an opportunity
+for the simultaneous isolation of several or all of the varieties of
+bacteria present in a mixture.
+
+~1. Surface Plate Cultivations.~--
+
+(a) _Gelatine_ (_vide_ page 164).
+
+(b) _Agar_ (_vide_ page 167).
+
+(c) _Alkaline serum agar_ (_vide_ page 211).
+
+These plates are prepared in a manner precisely similar to that adopted
+for nutrient gelatine and agar surface plates (_vide_ pages 231-233).
+
+(d) _Serum Agar._--
+
+1. Melt three tubes of nutrient agar, label them 1, 2, and 3, and place
+them, with three tubes of sterile fluid serum, also labelled 1a, 2a,
+and 3a, in a water-bath regulated at 45 deg. C.; allow sufficient time to
+elapse for the temperature of the contents of each tube to reach that of
+the water-bath.
+
+2. Take serum tube No. 1a and agar tube No. 1. Flame the plugs and
+remove them from the tubes (retaining the plug of the agar tube in the
+hand); flame the mouths of the tubes, pour the serum into the tube of
+liquefied agar and replace the plug of the agar tube.
+
+3. Mix thoroughly and pour plate No. 1 _secundum artem_.
+
+4. Treat the remaining tube of agar and serum in a similar fashion, and
+pour plates Nos. 2 and 3.
+
+5. Dry the serum agar plates in the incubator running at 60 deg. C. for
+one hour (see page 232).
+
+6. Inoculate the plates in series as described for gelatine surface
+plates (page 231).
+
+(e) _Blood Agar, Human._--
+
+1. Melt a tube of sterile agar and pour it into a sterile plate; let it
+set.
+
+2. Collect a few drops of human blood, under all aseptic conditions, in
+a sterile capillary teat pipette.
+
+3. Raise the cover of the Petri dish very slightly, insert the extremity
+of the capillary pipette, and deposit the blood on the centre of the
+agar surface. Close the dish.
+
+4. Charge a platinum loop with a small quantity of the inoculum. Raise
+the cover of the plate, introduce the loop, mix its contents with the
+drop of blood, remove the loop, close the dish and sterilise the loop.
+
+5. Finally smear the mixture over the surface of the agar with a
+sterilised L-shaped rod.
+
+6. Label and incubate.
+
+(If considered necessary, two, three, or more similar plates may be
+inoculated in series.)
+
+(f) _Blood Agar, Animal._--
+
+When preparing citrated blood agar (page 171) it is always advisable to
+pour several blood agar tubes into plates, which can be stored in the
+ice chest ready for use at any moment for surface plate cultures.
+
+(g) Hanging-drop or block culture, (_vide_ page 233).
+
+~3. Serial Cultivations.~--These are usually made upon agar or
+blood-serum, although gelatine may also be used.
+
+The method is as follows:
+
+1. Take at least four "slanted" tubes of media and number them
+consecutively.
+
+2. Flame all the plugs and see that each can be readily removed.
+
+3. Charge the platinum loop with a small quantity of the inoculum,
+observing the usual routine, and plant tube No. 1, smearing thoroughly
+all over the surface. If any water of condensation has collected at the
+bottom of the tube, use this as a diluent before smearing the contents
+of the loop over the surface of the medium.
+
+4. Without sterilising or recharging the loop, inoculate tube No. 2, by
+making three parallel streaks from end to end of the slanted surface.
+
+5. Plant the remainder of the tubes in the series as "smears" like tube
+No. 1.
+
+6. Label with distinctive name or number, and date; incubate.
+
+The growth that ensues in the first two or three tubes of the series
+will probably be so crowded as to be useless. Toward the end of the
+series, however, discrete colonies will be found, each of which can be
+transferred to a fresh tube of nutrient medium without risk of
+contamination from the neighbouring colonies.
+
+
+~"Working" up Plates.~--
+
+Having succeeded in obtaining a plate (or tube cultivation) in which the
+colonies are well grown and sufficiently separated from each other, the
+process of "working up," "pricking out," or "fishing" the colonies in
+order to obtain subcultures in a state of purity from each of the
+different bacteria present must now be proceeded with.
+
+Occasionally it happens that this is quite a simple matter. For example,
+the original mixed cultivation when examined microscopically was found
+to contain a Gram positive micrococcus, a Gram positive straight
+bacillus and a Gram negative short bacillus. The third gelatine plate
+prepared from this mixture, on inspection after four day's incubation,
+showed twenty-five colonies--seven moist yellow colonies, each sinking
+into a shallow pit of liquefied gelatine, fourteen flat irridescent
+filmy colonies, and four raised white slimy colonies. A film preparation
+(stained Gram) from each variety examined microscopically showed that
+the yellow liquefying colony was composed of Gram positive micrococci;
+the flat colony of Gram positive bacilli and the white colony of gram
+negative bacilli. One of each of these varieties of colonies would be
+transferred by means of the sterilised loop to a fresh gelatine culture
+tube, and after incubation the growth in each subculture would
+correspond culturally and microscopically with that of the plate colony
+from which it was derived,--the object aimed at would therefore be
+achieved.
+
+Usually, however, the colonies cannot be thus readily differentiated,
+and unless they are "worked up" in an orderly and systematic manner much
+labour will be vainly expended and valuable time wasted. The following
+method minimises the difficulties involved.
+
+
+(A) Inspection.
+
+a. Without opening the plate carefully study the various colonies with
+the naked eye, with the assistance of a watchmaker's lens or by
+inverting the plate on the stage of the microscope and viewing with the
+1-inch objective through the bottom of the plate and the layer of
+medium.
+
+b. If gross differences can be detected mark a small circle on the
+bottom of the plate around the site of each of the selected colonies,
+with the grease pencil.
+
+c. If no obvious differences can be made out choose nine colonies
+haphazard and indicate their positions by pencil marks on the bottom of
+the plate.
+
+
+(B) Fishing Colonies.--
+
+a. Take a sterile Petri dish and invert it upon the laboratory bench.
+Rule two parallel lines on the bottom of the dish with a grease pencil,
+and two more parallel lines at right angles to the first pair--so
+dividing the area of the dish into nine portions. Number the top
+right-hand portion 1, and the central bottom portion 8 (Fig. 139).
+Revert the dish. The numbers 1 and 8 can be readily recognised through
+the glass and by their positions enable any of the other divisions to be
+localised by number. This is the stock dish.
+
+b. Slightly raise the cover of the dish, and with a sterile
+teat-pipette deposit a small drop of sterile water in the centre of each
+of the nine divisions.
+
+c. With the sterilised platinum spatula raise one of the marked
+colonies from the "plate 3" and transfer it to the first division in the
+ruled plate and emulsify it in the drop of water awaiting it. Repeat
+this process with the remaining colonies, emulsifying a separate colony
+in each drop of water.
+
+
+(C) Preliminary Differentiation of Bacteria.--
+
+a. Prepare a cover-slip film preparation from each drop of emulsion in
+the "stock dish" and number to correspond to the division from which it
+was taken. Stain by Gram's method.
+
+b. Examine microscopically, using the oil immersion lens and note the
+numbers of those cover-slips which morphologically and by Gram results
+appear to be composed of different species of bacteria.
+
+[Illustration: FIG. 139.--Diagram for stock plate.]
+
+
+(D) Preparing Isolation Subcultures.--
+
+a. Inoculate an agar slope and a broth tube from the emulsion in the
+stock dish corresponding to each of these specially selected numbers.
+
+b. Ascertain whether the cover-slips from the nine emulsions in the
+stock dish include all the varieties represented in the cover-slip film
+preparation made from the original mixture before plating.
+
+c. If some varieties are missing prepare a second stock dish from
+other colonies on plate 3, and repeat the process until each
+morphological form or tinctorial variety has been secured in subculture.
+
+_d._ Place the stock dishes in the ice chest to await the results of
+incubation. (If any of the subcultures fail, further material can be
+obtained from the corresponding emulsion; or if it has dried, by
+moistening it with a further drop of sterile distilled water.)
+
+_e._ Incubate all the subcultures and identify the organisms picked out.
+
+
+4. Differential Media.--
+
+(a) _Selective._--Some varieties of media are specially suitable for
+certain species of bacteria and enable them to overgrow and finally
+choke out other varieties; e. g., wort is the most suitable
+medium-base for the growth of torulae and yeasts and should be employed
+when pouring plates for the isolation of these organisms. To obtain a
+pure cultivation of yeast from a mixture containing bacteria as well, it
+is often sufficient to inoculate wort from the mixture and incubate at
+37 deg. C. for twenty-four hours. Plant a fresh tube of wort from the
+resulting growth and incubate. Repeat the process once more, and from
+the growth in this third tube plant a streak on wort gelatine, and
+incubate at 20 deg. C. The resulting growth will almost certainly be
+a pure culture of the yeast.
+
+(b) _Deterrent._--The converse of the above also obtains. Certain
+media possess the power of inhibiting the growth of a greater or less
+number of species. For instance, media containing carbolic acid to the
+amount of 1 per cent. will inhibit the growth of practically everything
+but the Bacillus coli communis.
+
+
+~5. Differential Incubation.~--
+
+In isolating certain bacteria, advantage is taken of the fact that
+different species vary in their optimum temperature. A mixture
+containing the Bacillus typhosus and the Bacillus aquatilis sulcatus,
+for example, may be planted on two slanted agar tubes, the one incubated
+at 40 deg. C., and the other at 12 deg. C. After twenty-four hours'
+incubation the first will show a pure cultivation of the Bacillus
+typhosus, whilst the second will be an almost pure culture of the
+Bacillus aquatilis.
+
+
+6. Differential Sterilisation.--
+
+(a) _Non-sporing Bacteria._--Similarly, advantage may be taken of the
+varying thermal death-points of bacteria. From a mixture of two
+organisms whose thermal death-points differ by, say, 4 deg. C.--e. g.,
+Bacillus pyocyaneus, thermal death-point 55 deg. C., and Bacillus
+mesentericus vulgatus, thermal death-point 60 deg. C.--a pure cultivation
+of the latter may be obtained by heating the mixture in a water-bath to
+58 deg. C. and keeping it at that point for ten minutes. The mixture is
+then planted on to fresh media and incubated, when the resulting growth
+will be found to consist entirely of the B. mesentericus.
+
+(b) _Sporing Bacteria._--This method finds its chief practical
+application in the differentiation of a spore-bearing organism from one
+which does not form spores. In this case the mixture is heated in a
+water-bath at 80 deg. C. for fifteen to twenty minutes. At the end of this
+time the non-sporing bacteria are dead, and cultivations made from the
+mixture will yield a growth resulting from the germination of the spores
+only.
+
+Differential sterilisation at 80 deg. C. is most conveniently carried out
+in a water-bath of special construction, designed by Balfour Stewart (Fig.
+140). It consists of a double-walled copper vessel mounted on legs, and
+provided with a tubulure communicating with the space between the walls.
+This space is nearly filled with benzole (boiling-point 80 deg. C.; pure
+benzole, free from thiophene must be employed for the purpose, otherwise
+the boiling-point gradually and perceptibly rises in the course of
+time), and to the tubulure is fitted a long glass tube, some 2 metres
+long and about 0.75 cm. diameter, serving as a condensing tube (a tube
+half this length if provided with a condensing bulb at the centre will
+be equally efficient). The interior of the vessel is partly filled with
+water and covered with a lid which is perforated for a thermometer. This
+latter dips into the water and records its temperature. A very small
+Bunsen flame under the apparatus suffices to keep the benzole boiling
+and the water within at a constant temperature of 80 deg. C. The bath is
+thus always ready for use.
+
+METHOD.--To use the apparatus.
+
+1. Place some of the mixture itself, if fluid, containing the spores, or
+an emulsion of the same if derived from solid material, in a test-tube.
+
+2. Immerse the test-tube in the water contained in the benzole bath,
+taking care that the upper level of the liquid in the tube is at least 2
+cm. beneath the surface of the water in the copper vessel.
+
+3. The temperature of the water, of course, falls a few degrees after
+opening the bath and introducing a tube of colder liquid, but after a
+few minutes the temperature will have again reached 80 deg. C.
+
+4. When the thermometer again records 80 deg. C., note the time, and
+fifteen minutes later remove the tube containing the mixture from the
+bath.
+
+5. Make cultures upon suitable media; incubate.
+
+[Illustration: FIG. 140.--Benzole bath.]
+
+
+7. Differential Atmosphere Cultivation.--
+
+(a) By adapting the atmospheric conditions to the particular organism
+it is desired to isolate, it is comparatively easy to separate a strict
+aerobe from a strict anaerobe, and _vice versa_. In the first case,
+however, it is important that the cultivations should be made upon
+solid media, for if carried out in fluid media the aerobes multiplying
+in the upper layers of fluid render the depths completely anaerobic, and
+under these conditions the growth of the anaerobes will continue
+unchecked.
+
+(b) When it is desired to separate a facultative anaerobe from a
+strict anaerobe, it is generally sufficient to plant the mixture upon
+the sloped surface agar, incubate aerobically at 37 deg. C., and examine
+carefully at frequent intervals. At the first sign of growth,
+subcultivations must be prepared and treated in a similar manner. As a
+result of these rapid subcultures, the facultative anaerobe will be
+secured in pure culture at about the third or fourth generation.
+
+(c) If, on the other hand, the strict anaerobe is the organism
+required from a mixture of facultative and strict anaerobes, pour plates
+of glucose formate agar (or gelatine) in the usual manner, place them in
+a Bulloch's or Novy's jar, and incubate at a suitable temperature. Pick
+off the colonies of the required organism when the growth appears, and
+transfer to tubes of the various media.
+
+Incubate under suitable conditions as to temperature and atmosphere.
+
+
+~8. Animal Inoculation.~--
+
+Finally, when dealing with pathogenic organisms, it is often advisable
+to inoculate some of the impure culture (or even some of the original
+_materies morbi_) into an animal specially chosen on account of its
+susceptibility to the particular pathogenic organism it is desired to
+inoculate. Indeed, with some of the more sensitive and strictly
+parasitic bacteria this method of animal inoculation is practically the
+only method that will yield a satisfactory result.
+
+
+
+
+XVI. METHODS OF IDENTIFICATION AND STUDY.
+
+
+In order to identify an organism after isolation, tube, plate, and other
+cultivations must be prepared, incubated under suitable conditions as to
+temperature and environment, and examined from time to time (a)
+~macroscopically~, (b) by ~microscopical methods~, (c) by ~chemical
+methods~, (d) by ~physical methods~, (e) by ~inoculation methods~, and
+the results of these examinations duly recorded.
+
+It must be stated definitely that no micro-organism can be identified by
+any _one_ character or property, whether microscopical, biological or
+chemical, but that on the contrary its entire life history must be
+carefully studied and then its identity established from a consideration
+of the sum total of these observations.
+
+In order to give to the recorded results their maximum value it is
+essential that they should be exact and systematic, therefore some such
+scheme as the following should be adhered to; and especially is this
+necessary in describing an organism not previously isolated and studied.
+
+
+SCHEME OF STUDY.
+
+Designation:
+
+Originally isolated by (_observer's name_) in (_date_), from (_source of
+organism_).
+
+     ~1. Cultural Characters.~--(_Vide_ Macroscopical Examination
+     of Cultivation, page 261.)
+
+    Gelatine plates, }
+    Gelatine streak, } at 20 deg. C.
+    Gelatine stab,   }
+    Gelatine shake,  }
+
+    Agar plates,             }
+    Agar streak or smear,    }
+    Agar stab,               }
+    Inspissated blood-serum, } at 20 deg. C. and 37 deg. C.
+    Bouillon,                }
+    Litmus milk,             }
+    Potato,                  }
+
+     Special media for the purpose of demonstrating
+     characteristic appearances.
+
+     ~2. Morphology~.--(_Vide_ Microscopical Examination of
+     Cultivations, page 272.)
+
+    Vegetative forms:
+      Shape.
+      Size.
+      Motility.
+      Flagella (if present).
+      Capsule (if present).
+      Involution forms.
+      Pleomorphism (if observed).
+    Sporing forms (if observed). Of which class?
+    Staining reactions.
+
+     ~3. Chemical Products of Growth.~--(_Vide_ Chemical
+     Examination of Cultivations, page 276.)
+
+    Chromogenesis.
+    Photogenesis.
+    Enzyme formation.
+      Fermentation of carbohydrates:
+    Acid formation.
+    Alkali formation.
+    Indol formation.
+    Phenol formation.
+    Reducing and oxidising substances.
+    Gas formation.
+
+     ~4. Biology.~--(_Vide_ Physical Examination of Cultures, page
+     295.)
+
+    Atmosphere.
+    Temperature.
+
+    Reaction of nutrient media.
+    Resistance to lethal agents:
+      Physical:
+        Desiccation.
+        Light.
+        Colours.
+      Chemical germicides.
+    Vitality.
+
+     ~5. Pathogenicity:~
+
+    Susceptible animals, subsequently arranged in order of susceptibility.
+    Immune animals.
+    Experimental inoculation, symptoms of disease.
+    Post-mortem appearances.
+    Virulence:
+      Length of time maintained.
+      Optimum medium?
+      Minimal lethal dose.
+      Exaltation and attenuation of virulence?
+    Toxin formation.
+
+
+MACROSCOPICAL EXAMINATION OF CULTIVATIONS.
+
+In describing the naked-eye and low-power appearances of the bacterial
+growth the descriptive terms introduced by Chester (and included in the
+following scheme) should be employed.
+
+SOLID MEDIA.
+
+~Plate Cultures.~--
+
+_Gelatine._--Note the presence or absence of liquefaction of the
+surrounding medium. If liquefaction is present, note shape and character
+(_vide_ page 269, "stab" cultures).
+
+_Agar._--No liquefaction takes place in this medium. The liquid found on
+the surface of the agar (or at the bottom of the tube in agar tube
+cultures) is merely water which has been expressed during the rapid
+solidification of the medium and has subsequently condensed.
+
+_Gelatine and Agar._--Examine the colonies at intervals of twenty-four
+hours.
+
+(a) With the naked eye.
+
+(b) With a hand lens or watchmaker's glass.
+
+(c) Under a low power (1 inch) of the microscope, or by means of a small
+dissecting microscope.
+
+Distinguish superficial from deep colonies and note the characters of
+the individual colonies.
+
+(A) ~Size.~--The diameter in millimetres, at the various ages.
+
+(B) ~Shape.~--
+
+Punctiform: Dimensions too slight for defining form by naked eye;
+minute, raised, hemispherical.
+
+Round: Of a more or less circular outline.
+
+Elliptical: Of a more or less oval outline.
+
+Irregular: Outlines not conforming to any recognised shape.
+
+Fusiform: Spindle-shaped, tapering at each end.
+
+Cochleate: Spiral or twisted like a snail shell (Fig. 141, a).
+
+[Illustration: FIG. 141.--Types of colonies: a, Cochleate; b,
+amoeboid; c, mycelioid.]
+
+Amoeboid: Very irregular, streaming (Fig. 141, b).
+
+Mycelioid: A filamentous colony, with the radiate character of a mould
+(Fig. 141, c).
+
+Filamentous: An irregular mass of loosely woven filaments (Fig. 142,
+a).
+
+Floccose: Of a dense woolly structure.
+
+Rhizoid: Of an irregular, branched, root-like character (Fig. 142, b).
+
+Conglomerate: An aggregate of colonies of similar size and form (Fig.
+142, c).
+
+Toruloid: An aggregate of colonies, like the budding of the yeast plant
+(Fig. 142, d).
+
+Rosulate: Shaped like a rosette.
+
+[Illustration: FIG. 142.--Types of colonies: a, Filamentous; b,
+rhizoid; c, conglomerate; d, toruloid.]
+
+(C) ~Surface Elevation.~--
+
+1. _General Character of Surface as a Whole_:
+
+Flat: Thin, leafy, spreading over the surface (Fig. 143, a).
+
+Effused: Spread over the surface as a thin, veily layer, more delicate
+than the preceding.
+
+Raised: Growth thick, with abrupt terraced edges (Fig. 143, b).
+
+Convex: Surface the segment of a circle, but very flatly convex (Fig.
+143, c).
+
+Pulvinate: Surface the segment of a circle, but decidedly convex (Fig.
+143, d).
+
+Capitate: Surface hemispherical (Fig. 143, e).
+
+Umbilicate: Having a central pit or depression (Fig. 143, f).
+
+Conical: Cone with rounded apex (Fig. 143, g).
+
+Umbonate: Having a central convex nipple-like elevation (Fig. 143, h).
+
+2. _Detailed Characters of Surface_:
+
+Smooth: Surface even, without any of the following distinctive
+characters.
+
+Alveolate: Marked by depressions separated by thin walls so as to
+resemble a honeycomb (Fig. 144).
+
+Punctate: Dotted with punctures like pin-pricks.
+
+Bullate: Like a blistered surface, rising in convex prominences, rather
+coarse.
+
+Vesicular: More or less covered with minute vesicles due to gas
+formation; more minute than bullate.
+
+[Illustration: FIG. 143.--Surface elevation of colonies: a, Flat; b,
+raised; c, convex; d, pulvinate; e, capitate; f, umbilicate;
+g, conical; h, umbonate.]
+
+[Illustration: FIG. 144.--Types of colonies--alveolate.]
+
+Verrucose: Wart-like, bearing wart-like prominences.
+
+Squamose: Scaly, covered with scales.
+
+Echinate: Beset with pointed prominences.
+
+Papillate: Beset with nipple or mamma-like processes.
+
+Rugose: Short irregular folds, due to shrinkage of surface growth.
+
+Corrugated: In long folds, due to shrinkage.
+
+Contoured: An irregular but smoothly undulating surface, resembling the
+surface of a relief map.
+
+Rimose: Abounding in chinks, clefts, or cracks.
+
+(D) ~Internal Structure of Colony~ (_Microscopical_).--
+
+Refraction Weak: Outline and surface of relief not strongly defined.
+
+Refraction Strong: Outline and surface of relief strongly defined;
+dense, not filamentous colonies.
+
+[Illustration: FIG. 145.--Types of colonies: a, Grumose; b,
+moruloid; c, clouded.]
+
+1. _General_:
+
+Amorphous: Without any definite structure, such as is specified below.
+
+Hyaline: Clear and colourless.
+
+Homogeneous: Structure uniform throughout all parts of the colony.
+
+Homochromous: Colour uniform throughout.
+
+2. _Granulations or Blotchings_:
+
+Finely granular.
+
+Coarsely granular.
+
+Grumose: Coarser than the preceding, with a clotted appearance, and
+particles in clustered grains (Fig. 145, a).
+
+Moruloid: Having the character of a mulberry, segmented, by which the
+colony is divided in more or less regular segments (Fig. 145, b).
+
+Clouded: Having a pale ground, with ill-defined patches of a deeper tint
+(Fig. 145, c).
+
+[Illustration: FIG. 146.--Types of colonies: a, Reticulate; b,
+gyrose; c, marmorated.]
+
+3. _Colony Marking or Striping_:
+
+Reticulate: In the form of a network, like the veins of a leaf (Fig.
+146, a).
+
+Areolate: Divided into rather irregular, or angular, spaces by more or
+less definite boundaries.
+
+Gyrose: Marked by wavy lines, indefinitely placed (Fig. 146, b).
+
+Marmorated: Showing faint, irregular stripes, or traversed by vein-like
+markings, as in marble (Fig. 146, c).
+
+Rivulose: Marked by lines like the rivers of a map.
+
+Rimose: Showing chinks, cracks, or clefts.
+
+[Illustration: FIG. 147.--Types of colonies--curled.]
+
+4. _Filamentous Colonies:_
+
+Filamentous: As already defined.
+
+Floccose: Composed of filaments, densely placed.
+
+Curled: Filaments in parallel strands, like locks or ringlets (Fig.
+147).
+
+(E) ~Edges of Colonies.~--
+
+Entire: Without toothing or division (Fig. 148, a).
+
+Undulate: Wavy (Fig. 148, b).
+
+Repand: Like the border of an open umbrella (Fig. 148, c).
+
+Erose: As if gnawed, irregularly toothed (Fig. 148, d).
+
+[Illustration: FIG. 148.--Edges of colonies: a, Entire; b, undulate;
+c, repand; d, erose.]
+
+Lobate.
+
+Lobulate: Minutely lobate (Fig. 149, e).
+
+Auriculate: With ear-like lobes (Fig. 149, f).
+
+Lacerate: Irregularly cleft, as if torn (Fig. 149, g).
+
+Fimbriate: Fringed (Fig. 149, h).
+
+Ciliate: Hair-like extensions, radiately placed (Fig. 149, j).
+
+Tufted.
+
+Filamentous: As already defined.
+
+Curled: As already defined.
+
+[Illustration: FIG. 149.--Edges of colonies: e, Lobar-lobulate; f,
+auriculate; g, lacerate; h, fimbriate; i, ciliate.]
+
+(F) ~Optical Characters~ (after Shuttleworth).--
+
+1. _General Characters_:
+
+Transparent: Transmitting light.
+
+Vitreous: Transparent and colourless.
+
+Oleaginous: Transparent and yellow; olive to linseed-oil coloured.
+
+Resinous: Transparent and brown, varnish or resin-coloured.
+
+Translucent: Faintly transparent.
+
+Porcelaneous: Translucent and white.
+
+Opalescent: Translucent; greyish-white by reflected light.
+
+Nacreous: Translucent, greyish-white, with pearly lustre.
+
+Sebaceous: Translucent, yellowish or greyish-white.
+
+Butyrous: Translucent and yellow.
+
+Ceraceous: Translucent and wax-coloured.
+
+Opaque.
+
+Cretaceous: Opaque and white, chalky.
+
+Dull: Without lustre.
+
+Glistening: Shining.
+
+Fluorescent.
+
+Iridescent.
+
+2. _Chromogenicity_:
+
+Colour of pigment.
+
+Pigment restricted to colonies.
+
+Pigment restricted to medium surrounding colonies.
+
+Pigment present in colonies and in medium.
+
+
+~Streak or Smear Cultures.~--
+
+_Gelatine and Agar._--Note general points as indicated under plate
+cultivations.
+
+_Inspissated Blood-serum._--Note the presence or absence of liquefaction
+of the medium. (The presence of condensation water at the bottom of the
+tube must not be confounded with liquefaction of the medium.)
+
+_All Oblique Tube Cultures._--
+
+1. Colonies Discrete: Size, shape, etc., as for plate cultivations
+(_vide_ page 261).
+
+2. Colonies Confluent: Surface elevation and character of edge, as for
+plate cultivations (_vide_ page 263).
+
+Chromogenicity: As for plate cultures.
+
+
+~Gelatine Stab Cultures.~--
+
+(A) _Surface Growth._--As for individual colonies in plate cultures
+(_vide_ page 261).
+
+[Illustration: FIG. 150.--Stab cultivations--types of growth: a,
+Filiform; b, beaded; c, echinate; d, villous; e, arborescent.]
+
+(B) _Line of Puncture._--
+
+Filiform: Uniform growth, without special characters (Fig. 150, a).
+
+Nodose: Consisting of closely aggregated colonies.
+
+Beaded: Consisting of loosely placed or disjointed colonies (Fig. 150,
+b).
+
+Papillate: Beset with papillate extensions.
+
+Echinate: Beset with acicular extensions (Fig. 150, c).
+
+Villous: Beset with short, undivided, hair-like extensions (Fig. 150,
+d).
+
+Plumose: A delicate feathery growth.
+
+[Illustration: FIG. 151.--Stab cultivations--types of growth: f,
+Crateriform; g, saccate; h, infundibuliform; j, napiform; k,
+fusiform; l, stratiform.]
+
+Arborescent: Branched or tree-like, beset with branched hair-like
+extensions (Fig. 150, e).
+
+(C) _Area of Liquefaction_ (if present).--
+
+Crateriform: A saucer-shaped liquefaction of the gelatine (Fig. 151,
+f).
+
+Saccate: Shape of an elongated sack, tubular cylindrical (Fig. 151,
+g).
+
+Infundibuliform: Shape of a funnel, conical (Fig. 151, h).
+
+Napiform: Shape of a turnip (Fig. 151, j).
+
+Fusiform: Outline of a parsnip, narrow at either end, broadest below the
+surface (Fig. 151, k).
+
+Stratiform: Liquefaction extending to the walls of the tube and downward
+horizontally (Fig. 151, l).
+
+(D) _Character of the Liquefied Gelatine._--
+
+1. Pellicle on surface.
+
+2. Uniformly turbid.
+
+3. Granular.
+
+4. Mainly clear, but containing flocculi.
+
+5. Deposit at apex of liquefied portion.
+
+
+(E) _Production of Gas Bubbles._
+
+
+~Shake Cultures.~--
+
+1. Presence or absence of liquefaction.
+
+2. Production of gas bubbles.
+
+3. Bulk of growth at the surface--aerobic.
+
+4. Bulk of growth in depths--anaerobic.
+
+
+~Fluid Media.~
+
+
+~1. Surface of the Liquid.~--
+
+Presence or absence of froth due to gas bubbles.
+
+Presence or absence of pellicle formation.
+
+Character of pellicle.
+
+
+~2. Body of the Liquid.~--
+
+Uniformly turbid.
+
+Flocculi in suspension.
+
+Granules in suspension.
+
+Clear, with precipitate at bottom of tube.
+
+Colouration of fluid, presence or absence of.
+
+
+~3. Precipitate.~--
+
+Character.
+
+Amount.
+
+Colour.
+
+
+~Carbohydrate Media.~--
+
+Growth.
+
+Reaction.
+
+Gas formation.
+
+Coagulation or not of serum albumen (when serum water media are
+employed).
+
+
+~Litmus Milk Cultivations.~--
+
+
+                    {Unaltered.
+    1. Reaction:    {Acid.
+                    {Alkaline.
+    2. Odour.
+
+    3. Formation of gas.
+
+                      {Unaltered.
+    4. Consistency:   {Peptonised (character of solution).
+                      {Coagulated.
+
+                         {hard: solid.
+    5. Clot: Character   {soft: floculent.
+                         {ragged and broken up by gas
+                         {bubbles.
+
+(a) Coagulum undissolved.
+
+(b) Coagulum finally peptonised, completely: incompletely.
+
+Resulting solution, clear: turbid.
+
+               {Abundant.
+               {Scanty.
+    6. Whey:   {Clear.
+               {Turbid.
+               {Coagulated by boiling, or not.
+
+
+~BY MICROSCOPICAL METHODS.~
+
+As a council of perfection preparations must be made from pure
+cultivations 4, 6, 8, 12, 18, and 24 hours; and subsequently at
+intervals of, say, twenty-four hours, during the entire period they are
+under observation, and examined--
+
+(A) ~Living.--1.~ In ~hanging drop~, to determine _motility_ or
+_non-motility_.
+
+In this connection it must be remembered that under certain conditions
+as to environment (e. g., when examined in an unsuitable medium,
+atmosphere, temperature, etc.) motile bacilli may fail to exhibit
+activity. No organism, therefore, should be recorded as non-motile from
+one observation only; a series of observations at different ages and
+under varying conditions should form the basis of an opinion as to the
+absence of true locomotion.
+
+_Size._--In the case of non-motile or sluggishly motile organisms,
+endeavour to measure several individuals in each hanging drop by means
+of the eyepiece micrometer or the eikonometer (_vide_ page 63), and
+average the results.
+
+If the organism is one which forms spores, observe--
+
+(a) _Spore Formation._--Prepare hanging-drop cultivations (_vide_ page
+78) from vegetative forms of the organism, adding a trace of magenta
+solution (0.5 per cent.) or other intra vitam stain (see page 77) to the
+drop, on the point of the platinum needle, to facilitate the observation
+of the phenomenon by rendering the bacilli more distinct.
+
+Place the preparation on the stage of the microscope; if necessary,
+using a warm stage.
+
+Arrange illumination, etc., and select a solitary bacillus for
+observation, by the help of the 1/6-inch lens.
+
+Substitute the 1/12-inch oil-immersion lens for the sixth, and observe
+the formation of the spore; if possible, measure any alteration in size
+which may occur by means of the Ramsden micrometer.
+
+(b) _Spore Germination._--Prepare hanging-drop cultivations from old
+cultivations in which no living vegetative forms are present, and
+observe the process of germination in a similar manner.
+
+The comfort of the microscopist is largely enhanced in those cases where
+the period of observation is at all lengthy, by use of some form of eye
+screen before the unemployed eye, such as is figured on page 58 (Fig.
+49).
+
+If it is impossible to carry out the method suggested above, proceed as
+follows:
+
+(a) _Spore Formation._--Plant the organism in broth and incubate under
+optimum conditions.
+
+At regular intervals, say every thirty minutes, remove a loopful of the
+cultivation and prepare a cover-slip film preparation.
+
+Fix, while still wet, in the corrosive sublimate fixing solution.
+
+Stain with aniline gentian violet, and partially decolourise with 2 per
+cent. acetic acid.
+
+Mount and number consecutively; then examine.
+
+(b) _Spore Germination._--Expose a thick emulsion of the spores to a
+temperature of 80 deg. C. for ten minutes in the differential steriliser
+(_vide_ page 257).
+
+Transfer the emulsion to a tube of sterile nutrient broth and incubate.
+
+Remove specimens from the tube culture at intervals of, say, five
+minutes.
+
+Fix, stain, etc., wet, as under (a), and examine.
+
+(B) ~Fixed.--2.~ In ~stained preparations~.
+
+(a) To determine points in _morphology_:
+
+_Shape_ (_vide_ classification, page 131).
+
+_Size_:
+
+(a) Prepare cover-slip film preparations at the various ages, and fix
+by exposure to a temperature of 115 deg. C. for twenty minutes in hot-air
+oven.
+
+(b) Stain the preparations by Gram's method (if applicable) or with
+dilute carbol-fuchsin, and mount in the usual way.
+
+(c) Measure (_vide_ page 66) some twenty-five individuals in each film
+by means of the Ramsden's or the stage micrometer and average the
+result.
+
+_Pleomorphism_; If noted, record--
+
+    The predominant character of the variant forms.
+    On what medium or media they are observed.
+    At what period of development.
+
+(b) To demonstrate details of _structure_:
+
+_Flagella_: If noted, record--
+
+    Method of staining (_vide_ page 101).
+    Position and arrangement (_vide_ page 136).
+    Number.
+
+_Spores_: If noted, record--
+
+    Method of staining.
+    Shape.
+    Size.
+    Position within the parent cell.
+    Condition, as to shape, of the parent cell (_vide_
+    page 139).
+    Optimum medium and temperature.
+    Age of cultivation.
+    Conditions of environment as to temperature,
+    atmosphere.
+    Method of germination (_vide_ page 140).
+
+_Involution Forms_: If noted, record--
+
+    Method of staining.
+    Character (e. g., if living or dead).
+    Shape.
+    On what medium they are observed.
+    Age of medium.
+    Environment.
+
+_Metachromatic Granules_: If noted, record--
+
+    Method of staining.
+    Character of granules.
+    Number of granules.
+    Colour of granules.
+
+~3. Staining Reactions.~--
+
+1. _Gram's Method._--Positive or negative.
+
+2. _Neisser's Method._--If granules are noted, record--
+
+    1. Position.
+    2. Number.
+
+3. _Ziehl-Neelsen's Method._--Acid-fast or decolourised.
+
+4. _Simple Aniline Dyes._--(Noting those giving the best results, with
+details of staining processes.)
+
+    Methylene-blue }
+    Fuchsin        } and their modifications.
+    Gentian violet }
+    Thionine blue  }
+
+
+BY BIOCHEMICAL METHODS.
+
+Test cultivations of the organism for the presence of--
+
+Soluble enzymes--proteolytic, diastatic, invertase.
+
+Organic acids--(a) quantitatively--i. e., estimate the total acid
+production; (b) qualitatively for formic, acetic, propionic, butyric,
+lactic.
+
+Ammonia.
+
+Neutral volatile substances--ethyl alcohol, aldehyde, acetone.
+
+Aromatic products--indol, phenol.
+
+Soluble pigments.
+
+Test the power of reducing (a) colouring matters, (b) nitrates to
+nitrites.
+
+Investigate the gas production--H_{2}S, CO_{2}, H_{2}. Estimate the
+ratio between the last two gases.
+
+Prepare all cultivations for these methods of examination under
+_optimum_ conditions, previously determined for each of the organisms it
+is intended to investigate, as to
+
+    (a) Reaction of medium;
+    (b) Incubation temperature;
+    (c) Atmospheric environment;
+
+and keep careful records of these points, and also of the age of the
+cultivation used in the final examination.
+
+Examine the cultivations for the various products of bacterial
+metabolism after forty-eight hours' growth, and ~never omit to examine
+"control" (uninoculated) tube or flask of medium from the same batch,
+kept for a similar period under identical conditions~.
+
+If the results are negative, test further cultivations at three days,
+five days, and ten days.
+
+
+~1. Enzyme Production.~--
+
+(A) _Proteolytic Enzymes._--(Convert proteins into proteose, peptone
+and further products of hydrolysis; e. g., B. pyocyaneus.)
+
+     _Media Required_:
+
+     Blood-serum and milk-serum which have been carefully
+     filtered through a porcelain candle.
+
+     _Reagents Required_:
+
+    Ammonium sulphate.
+    Thirty per cent. caustic soda solution.
+    Copper sulphate, 0.5 per cent. aqueous solution.
+    One per cent. acetic acid solution.
+    Millon's reagent.
+    Glyoxylic acid solution.
+    Concentrated sulphuric acid.
+
+METHOD.--
+
+1. Prepare cultivations in bulk (50 c.c.) in a flask and incubate.
+
+2. Make the liquid faintly acid with acetic acid, then boil. (This
+precipitates the unaltered proteins.)
+
+3. Filter.
+
+4. Take 10 c.c. of the filtrate in a test-tube and add 1 c.c. of the
+caustic soda, then add the copper sulphate drop by drop.
+
+     Pink colour which becomes violet with more copper sulphate =
+     proteose and peptone.
+
+5. Saturate the rest of the filtrate with ammonium sulphate.
+
+Precipitate = proteose.
+
+6. Filter and divide the filtrate into three parts a, b and c.
+
+a. Repeat the copper sulphate test, using excess of caustic soda to
+displace the ammonia from the ammonium sulphate.
+
+Pink colour = peptone.
+
+b. Boil with Millon's reagent.
+
+Red colour = tyrosine.
+
+c. Add glyoxylic acid solution and run in concentrated sulphuric acid.
+
+Violet ring at upper level of acid = tryptophane.
+
+Both the tyrosine and tryptophane may be either in the free state or in
+combination as polypeptid or peptone.
+
+(B) _Diastase._--(Converts starch into sugar; e. g., B. subtilis.)
+
+     _Medium Required_:
+
+     Inosite-free bouillon.
+
+     _Reagents Required_:
+
+    Starch.
+    Thymol.
+    Fehling's solution.
+
+METHOD.--
+
+1. Prepare tube cultivation and incubate.
+
+2. Prepare a thin starch paste and add 2 per cent. thymol to it.
+
+3. Mix equal parts of the cultivation to be tested and the starch paste,
+and place in the incubator at 37 deg. C. for six to eight hours.
+
+4. Filter.
+
+Test the filtrate for sugar.
+
+Boil some of the Fehling's solution in a test-tube.
+
+Add the filtrate drop by drop until, if necessary, a quantity has been
+added equal in amount to the Fehling's solution employed, keeping the
+mixture at the boiling-point during the process.
+
+Yellow or orange precipitate = sugar.
+
+(C) _Invertase._--(Convert saccharose into a mixture of dextrose and
+laevulose e. g., B. fluorescens liquefaciens.)
+
+    _Medium Required_:
+    Inosite-free bouillon.
+
+    _Reagents Required_:
+    Cane sugar, 2 per cent. aqueous solution.
+    Carbolic acid.
+
+METHOD.--
+
+1. Prepare tube cultivations and incubate.
+
+2. Add 2 per cent. of carbolic acid to the sugar solution.
+
+3. Mix equal quantities of the carbolised sugar solution and the
+cultivation in a test-tube; allow the mixture to stand for several
+hours.
+
+4. Filter.
+
+Test the filtrate for reducing sugar as in the preceding section.
+
+(D) _Rennin and "Lab" Enzymes._--(Coagulate milk independently of the
+action of acids; e. g., B. prodigiosus.)
+
+    _Media Required_:
+    Inosite-free bouillon.
+    Litmus milk.
+
+METHOD.--
+
+1. Prepare tube cultivations and incubate.
+
+2. After incubation heat the cultivation to 55 deg. C. for half an hour,
+to sterilise.
+
+3. By means of a sterile pipette run 5 c.c. of the cultivation into each
+of three tubes of litmus milk.
+
+4. Place in the cold incubator at 22 deg. C. and examine each day for
+ten days.
+
+Absence of coagulation at the end of that period will indicate absence
+of rennin ferment formation.
+
+
+Fermentation Reactions.
+
+As tested upon carbohydrate substances and organic salts.
+
+_Media Required_:
+
+Peptone water containing various percentages (generally 2 per cent.) of
+each of the substances referred to under "sugar" media (page 177), also
+tubes of peptone water containing 1 per cent. respectively of each of
+the following:
+
+     Organic salts: Sodium citrate, formate, lactate, malate,
+     tartrate.
+
+METHOD.--
+
+1. Prepare tube cultivations in each of the above media.
+
+2. Observe from day to day up to the expiration of ten days if
+necessary.
+
+3. Note growth, reaction, gas production.
+
+
+2. Acid Production.
+
+    (a) _Quantitative._--
+
+    _Medium Required_:
+    Sugar (glucose) bouillon of known "optimum" reaction.
+
+    _Apparatus and Reagents Required_:
+    As for estimating reaction of media (_vide_ page 150).
+
+METHOD.--
+
+1. Prepare cultivation in bulk (100 c.c.) in a flask; also "control"
+flask of medium from same batch.
+
+2. After suitable incubation, heat both flasks in the steamer at 100
+deg. C. for thirty minutes to sterilise.
+
+3. Determine the _titre_ of the medium in "inoculated" and "control"
+flasks as described in the preparation of nutrient media (_vide_ page
+151).
+
+4. The difference between the titre of the medium in the two flasks
+gives the total acid production of the bacterium under observation in
+terms of normal NaOH.
+
+     NOTE.--If the growth is very heavy it may be a difficult
+     matter to determine the end-point. The cultivation should
+     then be filtered through a Berkefeld filter candle previous
+     to step 2, and the filtrate employed in the titration.
+
+    (b) _Qualitative_ (of all the organic acids present).--
+
+    _Medium Required_:
+    Sugar (glucose or lactose) bouillon as in quantitative examination.
+
+    _Reagents Required_:
+    Hydrochloric acid, concentrated.
+    Hydrochloric acid, 25 per cent.
+    Sulphuric acid, concentrated (pure).
+    Phosphoric acid, concentrated solution.
+    Ammonia.
+    Ammonium sulphate.
+    Baryta water.
+    Sodium carbonate, saturated aqueous solution.
+    Absolute alcohol.
+    Ether.
+    Calcium chloride.
+    Calcium chloride solution.
+    Zinc carbonate.
+    Copper sulphate saturated aqueous solution.
+    Alcoholic thiophene solution (0.15 c.c. in 100 c.c.).
+    Animal charcoal.
+    Five per cent. sodium nitroprusside solution.
+    Potassium bichromate.
+    Schiff's reagent.
+    Arsenious oxide.
+    Ferric chloride, 4 per cent. aqueous solution.
+    Silver nitrate, 1 per cent. aqueous solution.
+    Lugol's iodine.
+    Ten per cent. caustic soda solution.
+    Hard paraffin wax (melting-point about 52 deg. C.).
+
+METHOD.--
+
+1. Prepare cultivation in bulk (500 c.c.) in a litre flask and add
+sterilised precipitated chalk, 10 grammes. Incubate at the optimum
+temperature.
+
+2. After incubation throw a piece of paraffin wax (about a centimetre
+cube) into the cultivation and connect up the flask with a condenser.
+
+The paraffin, which liquefies and forms a thin layer on the surface of
+the fluid, is necessary to prevent the cultivation frothing up and
+running unaltered through the condenser during the subsequent process of
+distillation.
+
+3. Distill over 200 to 300 c.c.
+
+Use a rose-top burner to minimise the danger of cracking the flask; and
+to the same end, well agitate the contents of the flask to prevent the
+chalk settling.
+
+The distillate "A" will contain alcohol, etc. (_vide_ page 285); the
+residue "a" will contain the volatile and fixed acids.
+
+4. Disconnect the flask and filter. The residue "a" then = filtrate B
+and residue b.
+
+[Illustration: FIG. 152.--Arrangement of distillation apparatus for
+acids, etc.]
+
+5. Residue b. Wash the residue from the filter paper, dissolve by
+heating with dilute hydrochloric acid, and add calcium chloride solution
+and ammonia until alkaline.
+
+White precipitate insoluble in acetic acid = oxalic acid.
+
+6. Make up filtrate B to 500 c.c. with distilled water and divide into
+two parts.
+
+7. Acidify 250 c.c. with 20 c.c. concentrated phosphoric acid (this
+liberates the volatile acids) and distil to small bulk.
+
+The distillate "B" may contain formic, acetic, propionic, butyric and
+benzoic acids.
+
+                              DISTILLATE "B."
+                              (Volatile Acids.)
+                                      |
+                                      |
+                   1. Add baryta water till alkaline,
+                      and evaporate to dryness.
+
+                   2. Add 50 c.c. absolute alcohol and allow
+                      to stand, with frequent stirring, for
+                      two to three hours.
+
+                   3. Filter and wash with alcohol.
+                                      |
+                                      |
+                  |---------------------------------------|
+                  |                                       |
+                  |                                       |
+               FILTRATE                                RESIDUE
+                  |                                       |
+                  |                                       |
+   may contain barium propionate,         may contain barium acetate,
+   barium butyrate.                       barium formate, barium benzoate.
+                  |                                       |
+                  |                                       |
+   1. Evaporate to dryness.               1. Evaporate off alcohol and
+                                          dissolve up the residue on
+   2. Dissolve residue in 150             the filter in hot water and
+   c.c. water.                            neutralise.
+
+   3. Acidify with phosphoric             2. Divide the solution into
+   acid and distil.                       four portions:
+
+   4. Saturate distillate with            (a) Add ferric chloride solution.
+   calcium chloride and distill
+   over a few c.c.                            ~Brown~ colour = _acetic_ or
+                                              _formic_ acids.
+   5. Test distillate for butyric
+   acid:                                      ~Buff ppt.~ = _benzoic_ acid
+                                              (see ether soluble acids).
+   Add 3 c.c. alcohol and 4 drops
+   concentrated sulphuric acid.           (b) Add silver nitrate
+                                          solution; then add one drop
+      ~Smell of pineapple~ = _butyric_    ammonia water, and boil.
+      acid.
+                                            ~Black~ precipitate of metallic
+      Propionic acid in small               silver = _formic_ acid.
+      quantities cannot be
+      distinguished from butyric         (c) Evaporate to dryness; mix
+      acid by tests within the           with equal quantity of
+      scope of the bacteriological       arsenious oxide and heat
+      laboratory.                        on platinum foil.
+
+                                             Unpleasant ~smell of cacodyl~
+                                             = _acetic_ acid.
+
+                                         (d) Add a few drops of
+                                         mercuric chloride solution
+                                         in test-tube, and heat to
+                                         70 deg. C.
+
+                                             ~Precipitate~ of mercurous
+                                             chloride which is slowly
+                                             reduced to mercury =
+                                             _formic_ acid.
+
+8. If the distillation of "B" is continued as long as acid comes over
+(distilled water being occasionally added to the distilling flask) the
+distillate can be measured and 50 c.c. used for titration. This will
+give the amount of volatile acid formation.
+
+9. The second part of the filtrate "B" (see page 282) should be examined
+for lactic, oxalic, succinic, benzoic, salicylic, gallic and tannic
+acids, as follows:
+
+
+~Ether Soluble Acids.~--
+
+1. Evaporate to a thin syrup, acidify strongly with phosphoric acid.
+
+2. Extract with five times its volume of ether by agitation in a
+separatory funnel.
+
+3. Evaporate the ethereal extract to a thin syrup.
+
+4. Add 100 c.c. water and mix thoroughly.
+
+5. To a small portion of this solution add slight excess of sodium
+carbonate, evaporate to dryness on the water-bath, dissolve in 5-10 c.c.
+pure sulphuric acid, add 2 drops saturated copper sulphate solution,
+place in a test-tube and heat in a boiling water-bath for 2 minutes,
+cool, add 2 or 3 drops of the alcoholic thiophene and warm gently.
+
+Cherry red colour = lactic acid.
+
+If a brown colour is produced on the addition of sulphuric acid, another
+sample should be taken and boiled with animal charcoal before
+evaporating.
+
+6. If lactic acid is definitely present, prepare zinc lactate by boiling
+part of the solution of the ether extract with excess of zinc carbonate,
+filtering and evaporating to crystallise. The crystals so obtained have
+a characteristic form, and if dried at 110 deg. C, should contain 26.87
+per cent. of zinc.
+
+7. Test a portion of the rest of the solution of the ether extract for
+oxalic acid (page 282, step 5). Carefully neutralise the remainder and
+add ferric chloride solution.
+
+Red brown gelatinous precipitate = succinic acid.
+
+Buff precipitate = benzoic acid, and other acids related to benzoic
+acid.
+
+Violet colour = salicylic acid.
+
+Inky black colour or precipitate = gallic acid or tannic acid.
+
+For further identification the melting-points of the crystalline acids,
+and the percentage of silver in their silver salts should be determined.
+
+
+~3. Ammonia Production.~--
+
+    _Medium Required_:
+    Nutrient bouillon.
+
+    _Reagent Required_:
+    Nessler reagent.
+
+METHOD.--
+
+1. Prepare cultivation in bulk (100 c.c.) in a 250 c.c. flask and
+incubate together with a control flask.
+
+Test the cultivation and the control for ammonia in the following
+manner:
+
+2. To each flask add 2 grammes of calcined magnesia, then connect up
+with condensers and distil.
+
+3. Collect 50 c.c. distillate, from each, in a Nessler glass.
+
+4. Add 1 c.c. Nessler reagent to each glass by means of a clean pipette.
+
+Yellow colour = ammonia.
+
+The depth of colour is proportionate to the amount present.
+
+
+~4. Alcohol, etc., Production.~--Divide the distillate "A" obtained in the
+course of a previous experiment (_vide_ page 282, step 3) into four
+portions and test for the production of alcohol, acetaldehyde, acetone.
+
+1. Add Lugol's iodine, then a little NaOH solution, and stir with a
+glass rod till the colour of the iodine disappears.
+
+Pale-yellow crystalline precipitate of iodoform, with its characteristic
+smell, appearing in the cold, indicates acetaldehyde, or acetone;
+appearing only on warming indicates alcohol.
+
+The precipitate may be absent even when the odour is pronounced.
+
+2. Add Schiff's reagent.
+
+Violet or red colour = aldehyde.
+
+3. To 10 c.c. of solution add 2.5 c.c., 25 per cent. sulphuric acid, and
+a crystal or two of potassium bichromate and distil. Reduction of the
+bichromate to a green colour and a distillate, which smells of
+acetaldehyde and reacts with Schiff's reagent, shows the presence of
+alcohol in the original liquid.
+
+4. Add a few drops of sodium nitroprusside solution, make alkaline with
+ammonia, then saturate with ammonium sulphate crystals. Acetone gives
+little colour on the addition of ammonia, but after the addition of
+ammonium sulphate a deep permanganate colour, which takes ten minutes to
+reach its full intensity. Aldehyde gives a carmine red unaltered by
+ammonium sulphate.
+
+
+~5. Indol Production.~--
+
+    _Media Required_:
+
+    Inosite-free bouillon (_vide_ page 183).
+    Or peptone water (_vide_ page 177).
+
+    _Reagents Required_:
+
+    Potassium persulphate, saturated aqueous solution.
+    Paradimethylamino-benzaldehyde solution. This is prepared by mixing:
+
+      Paradimethylamino-benzaldehyde      4 grammes
+      Absolute alcohol                  380 c.c.
+      Hydrochloric acid, concentrated    80 c.c.
+
+METHOD.--
+
+Prepare several test-tube cultivations of the organism to be tested, and
+incubate.
+
+Test for indol by means of the Rosindol reaction in the following
+manner. (If the culture has been incubated at 37 deg. C., it must be
+allowed to cool to the room temperature before applying the test.)
+
+1. Remove 2 c.c. of the cultivation by means of a sterile pipette and
+transfer to a clean tube, then,
+
+2. Add 2 c.c. paradimethylamino-benzaldehyde solution.
+
+3. Add 2 c.c. potassium persulphate solution.
+
+The presence of indol is indicated by the appearance of a delicate
+rose-pink colour throughout the mixture which deepens slightly on
+standing.
+
+     Indol is tested for in many laboratories by the ordinary
+     nitrosoindol reaction which, however, is not so delicate a
+     method as that above described. The test is carried out as
+     follows:
+
+     1. Remove the cotton-wool plug from the tube, and run in 1
+     c.c. pure concentrated sulphuric acid down the side of the
+     tube by means of a sterile pipette. Place the tube upright
+     in a rack, and allow it to stand, if necessary, for ten
+     minutes.
+
+     A rose-pink or red colour at the junction of the two liquids
+     = indol (_plus a nitrite_).
+
+     2. If the colour of the medium remains unaltered, add 2 c.c.
+     of a 0.01 per cent. aqueous solution sodium nitrite, and
+     again allow the culture to stand for ten minutes.
+
+     Red colouration = indol.
+
+     NOTE.--In place of performing the test in two stages as
+     given above, 2 c.c. concentrated _commercial_ sulphuric,
+     hydrochloric, or nitric acid (all of which hold a trace of
+     nitrite in solution), may be run into the cultivation. The
+     development of a red colour within twenty minutes will
+     indicate the presence of indol.
+
+
+~5a. Phenol Production.~--
+
+    _Medium Required_:
+
+    Nutrient bouillon.
+
+    _Reagents Required_:
+
+    Hydrochloric acid, concentrated.
+    Millon's reagent.
+    Ferric chloride, 1 per cent. aqueous solution.
+
+METHOD.--
+
+1. Prepare cultivation in a Bohemian flask containing at least 50 c.c.
+of medium, and incubate.
+
+Test for phenol in the following manner:
+
+2. Add 5 c.c., 25 per cent. sulphuric acid to the cultivation and
+connect up the flask with a condenser.
+
+3. Distil over 15 to 20 c.c. Divide the distillate into three portions
+a, b and c.
+
+4. Add to (a) 0.5 c.c. Millon's reagent and boil.
+
+Red colour = phenol.
+
+5. Add to (b) about 0.5 c.c. ferric chloride solution. Violet colour =
+phenol.
+
+(If the distillate be acid the reaction will be negative.)
+
+6. Add to (c) bromine water. Crystalline white ppt. of tribromo-phenol
+= phenol.
+
+     NOTE.--If both indol and phenol appear to be present in
+     cultivations of the same organism, it is well to separate
+     them before testing. This may be done in the following
+     manner:
+
+1. Prepare inosite-free bouillon cultivation, say 200 or 300 c.c., in a
+flask as before.
+
+2. Render definitely acid by the addition of acetic acid and connect up
+the flask with a condenser.
+
+3. Distil over 50 to 70 c.c.
+
+Distillate will contain both indol and phenol.
+
+4. Render the distillate strongly alkaline with caustic potash and
+redistil.
+
+Distillate will contain indol; residue will contain phenol.
+
+5. Test the distillate for indol (_vide ante_).
+
+6. Saturate the residue, when cold, with carbon dioxide and redistil.
+
+7. Test this distillate for phenol (_vide ante_).
+
+
+~6. Pigment Production.~--
+
+1. Prepare tube cultivations upon the various media and incubate under
+varying conditions as to temperature (at 37 deg. C. and at 20 deg. C.),
+atmosphere (aerobic and anaerobic), and light (exposure to and
+protection from).
+
+Note the conditions most favorable to pigment formation.
+
+2. Note the solubility of the pigment in various solvents, such as water
+(hot and cold), alcohol, ether, chloroform, benzol, carbon bisulphide.
+
+3. Note the effect of acids and alkalies respectively upon the pigmented
+cultivation, or upon solutions of the pigment.
+
+4. Note spectroscopic reactions.
+
+
+~7. Reducing Agent Formation.~--
+
+(a) _Colour Destruction._--
+
+1. Prepare tube cultivations in nutrient bouillon tinted with litmus,
+rosolic acid, neutral red, and incubate.
+
+2. Examine the cultures each day and note whether any colour change
+occurs.
+
+(b) _Nitrates to Nitrites._--
+
+    _Medium Required_:
+
+    Nitrate bouillon (_vide_ page 185).
+    Or nitrate peptone solution (_vide_ page 186).
+
+    _Reagents Required_:
+
+    Sulphuric acid (25 per cent.).
+    Metaphenylene diamine, 5 per cent. aqueous solution.
+
+METHOD.--
+
+1. Prepare tube cultivations and incubate together with control tubes
+(i. e., uninoculated tubes of the same medium, placed under identical
+conditions as to environment).
+
+This precaution is necessary as the medium is liable to take up nitrites
+from the atmosphere, and an opinion as to the absence of nitrites in the
+cultivation is often based upon an equal colouration of the medium in
+the control tube.
+
+Test both the culture tube and the control tube for the presence of
+nitrites.
+
+2. Add a few drops of sulphuric acid to the medium in each of the tubes.
+
+3. Then run in 2 or 3 c.c. metaphenylene diamine into each tube.
+Brownish-red colour = nitrites.
+
+The depth of colour is proportionate to the amount present.
+
+
+~8. Gas Production.~--
+
+(A) _Carbon Dioxide and Hydrogen._--
+
+     _Apparatus Required_:
+
+     Fermentation tubes (_vide_ page 161) containing sugar
+     bouillon (glucose, lactose, etc.). The medium should be
+     prepared from inosite-free bouillon (_vide_ page 183).
+
+     _Reagent Required_:
+
+     n/2 caustic soda.
+
+METHOD.--
+
+1. Inoculate the surface of the medium in the bulb of a fermentation
+tube and incubate.
+
+2. Mark the level of the fluid in the closed branch of the fermentation
+tube, at intervals of twenty-four hours, and when the evolution of gas
+has ceased, measure the length of the column of gas with the millimetre
+scale.
+
+Express this column of gas as a percentage of the entire length of the
+closed branch.
+
+3. To analyse the gas and to determine roughly the relative proportions
+of CO_{2} and H_{2}, proceed as follows:
+
+Fill the bulb of the fermentation tube with caustic soda solution.
+
+Close the mouth of the bulb with a rubber stopper.
+
+Alternately invert and revert the tube six or eight times, to bring the
+soda solution into intimate contact with the gas.
+
+Return the residual gas to the end of the closed branch, and measure.
+
+The loss in volume of gas = carbon dioxide.
+
+The residual gas = hydrogen.
+
+Transfer gas to the bulb of the tube, and explode it by applying a
+lighted taper.
+
+(B) _Sulphuretted Hydrogen._--
+
+    _Media Required_:
+
+    Iron peptone solution (_vide_ page 185).
+    Lead peptone solution.
+
+1. Inoculate tubes of media, and incubate together with control tubes.
+
+2. Examine from day to day, at intervals of twenty-four hours.
+
+The liberation of the H_{2}S will cause the yellowish-white precipitate
+to darken to a brownish-black, or jet black, the depth of the colour
+being proportionate to the amount of sulphuretted hydrogen present.
+
+Quantitative: For exact quantitative analyses of the gases produced by
+bacteria from certain media of definite composition, the methods devised
+by Pakes must be employed, as follows:
+
+[Illustration: FIG. 153.--Gas-collecting apparatus.]
+
+     _Apparatus Required_:
+
+     Bohemian flask (300 to 1500 c.c. capacity) containing from
+     100 to 400 c.c. of the medium. The mouth of the flask is
+     fitted with a perforated rubber stopper, carrying an
+     L-shaped piece of glass tubing (the short arm passing just
+     through the stopper). To the long arm of the tube is
+     attached a piece of pressure tubing some 8 cm. in length,
+     plugged at its free end with a piece of cotton-wool. Measure
+     accurately the total capacity of the flask and exit tube,
+     also the amount of medium contained. Note the difference.
+
+     Gas receiver. This is a bell jar of stout glass, 14 cm. high
+     and 9 cm. in diameter. At its apex a glass tube is fused in.
+     This rises vertically 5 cm., and is then bent at right
+     angles, the horizontal arm being 10 cm. in length. A
+     three-way tap is let horizontally into the vertical tube
+     just above its junction with the bell jar.
+
+     An iron cylinder just large enough to contain the bell jar.
+
+     About 15 kilos of metallic mercury.
+
+     Melted paraffin.
+
+An Orsat-Lunge working with mercury instead of water, provided with two
+gas tubes of extra length (capacity 120 and 60 c.c. respectively and
+graduated throughout, both being water-jacketed) or other gas analysis
+apparatus, capable of dealing with CO_{2}, O_{2}, H_{2}, and N_{2}.
+
+METHOD.--
+
+1. Inoculate the medium in the flask in the usual manner, by means of a
+platinum needle, taking care that the neck of the flask and the rubber
+stopper are thoroughly flamed before and after the operation.
+
+[Illustration: FIG. 154.--Orsat-Lunge gas analysis apparatus.]
+
+2. Fill the iron cylinder with mercury.
+
+3. Place the bell jar mouth downward in the mercury--first seeing that
+there is free communication between the interior of the jar and the
+external air--and suck up the mercury into the tap; then shut off the
+tap.
+
+4. Plug the open end of the three-way tap with melted wax.
+
+5. Connect up the horizontal arm of the culture flask with that of the
+gas receiver by means of the pressure tubing (after removing the
+cotton-wool plug from the rubber tube), as shown in Fig. 153.
+
+6. Give the three-way tap half turn to open communication between flask
+and receiver, and seal _all_ joints by coating with a film of melted
+wax. When the tap is turned, the mercury in the receiver will naturally
+fall.
+
+7. Place the entire apparatus in the incubator. (Two hours later, by
+which time the temperature of the apparatus is that of the incubator,
+mark the height of the mercury on the receiver.)
+
+8. Examine the apparatus from day to day and mark the level of the
+mercury in the receiver at intervals of twenty-four hours.
+
+9. When the evolution of gas has ceased, remove the apparatus from the
+incubator; clear out the wax from the nozzle of the three-way tap (first
+adjusting the tap so that no escape of gas shall take place) and connect
+it with the Orsat.
+
+10. Remove, say, 100 c.c. of gas from the receiver, reverse the tap and
+force it into the culture flask. Remove 100 c.c. of mixed gases from the
+culture flask and replace in the receiver.
+
+Repeat these processes three or four times to ensure thorough admixture
+of the contents of flask and receiver.
+
+11. Now withdraw a sample of the mixed gases into the Orsat and analyse.
+
+In calculating the results be careful to allow for the volume of air
+contained in the flask at the commencement of the experiment.
+
+For the collection of gases formed under anaerobic conditions a slightly
+different procedure is adopted:
+
+1. Fix a culture flask (500 c.c. capacity) with a perforated rubber
+stopper carrying an ~L~-shaped piece of manometer tubing, each arm 5 cm.
+in length.
+
+2. Prepare a second ~L~-shaped piece of tubing, the short arm 5 cm. and
+the long arm 20 cm., and connect its short arm to the horizontal arm of
+the tube in the culture flask by means of a length of pressure tubing,
+provided with a screw clamp.
+
+3. Fill the culture flask completely with boiling medium and pass the
+long piece of tubing through the plug of an Erlenmeyer flask (150 c.c.
+capacity) which contains 100 c.c. of the same medium.
+
+4. Sterilise these coupled flasks by the discontinuous method, in the
+usual manner.
+
+Immediately the last sterilisation is completed, screw up the clamp on
+the pressure tubing which connects them, and allow them to cool.
+
+As the fluid cools and contracts it leaves a vacuum in the neck of the
+flask below the rubber stopper.
+
+5. To inoculate the culture flask, withdraw the long arm of the bent
+tube from the Erlenmeyer flask and pass it to the bottom of a test-tube
+containing a young cultivation (in a fluid medium similar to that
+contained in the culture flask) of the organism it is desired to
+investigate.
+
+6. Slightly release the clamp on the pressure tubing to allow 4 or 5
+c.c. of the culture to enter the flask.
+
+7. Clamp the rubber tube tightly; remove the bent glass tube from the
+culture tube and plunge it into a flask containing recently boiled and
+quickly cooled distilled water.
+
+8. Release the clamp again and wash in the remains of the cultivation
+until the culture flask and tubing are completely filled with water.
+
+9. Clamp the rubber tubing tightly and take away the long-armed glass
+tubing.
+
+10. Prepare the gas receiver as in the previous method (in this case,
+however, the mercury should be warmed slightly) and fill the horizontal
+arm of the receiver with hot water.
+
+11. Connect up the culture flask with the horizontal arm of the gas
+receiver.
+
+12. Remove the screw clamp from the rubber tubing, adjust the three-way
+tap, seal all joints with melted wax, and incubate.
+
+13. Complete the investigation as described for the previous method.
+
+
+BY PHYSICAL METHODS.
+
+Examine cultivations of the organism with reference to its growth and
+development under the following headings:
+
+Atmosphere:
+
+(a) In the presence of oxygen.
+
+(b) In the absence of oxygen.
+
+(c) In the presence of gases other than oxygen.
+
+Temperature:
+
+(a) Range.
+
+(b) Optimum.
+
+(c) Thermal death-point:
+
+    Moist: Vegetative forms.
+
+           Spores.
+
+    Dry: Vegetative forms.
+
+           Spores.
+
+Reaction of medium.
+
+Resistance to lethal agents:
+
+(a) Desiccation.
+
+    (b) Light: Diffuse.
+
+               Direct.
+
+               Primary colours.
+
+(c) Heat.
+
+(d) Chemical antiseptics and disinfectants.
+
+Vitality in artificial cultures.
+
+~I. Atmosphere.~--The question as to whether the organism under
+observation is (a) an obligate aerobe, (b) a facultative anaerobe, or
+(c) an obligate anaerobe is roughly decided by the appearance of
+cultivations in the fermentation tubes. Obvious growth in the closed
+branch as well as in the bulb or in the inverted gas tube as well as in
+the bulk of the medium will indicate that it is a facultative anaerobe;
+whilst growth only occurring in the bulb or in the closed branch shows
+that it is an obligate aerobe or anaerobe respectively. This method,
+however, is not sufficiently accurate for the present purpose, and the
+examination of an organism with respect to its behaviour in the absence
+of oxygen is carried out as follows:
+
+     _Apparatus Required:_
+
+    Buchner's tubes.
+    Bulloch's apparatus.
+    Exhaust pump.
+    Pyrogallic acid.
+    Dekanormal caustic soda.
+
+     _Media Required:_
+
+    Glucose formate agar.
+    Glucose formate gelatine.
+    Glucose formate bouillon.
+
+METHOD.--
+
+1. Prepare four sets of cultivations:
+
+(A) Sloped glucose formate agar, and incubate aerobically at 37 deg. C.
+
+Sloped glucose formate gelatine, and incubate aerobically at 20 deg. C.
+
+(B) Sloped glucose agar to incubate anaerobically at 37 deg. C.
+
+Sloped glucose formate gelatine to incubate anaerobically at 20 deg. C.
+
+(C) Sloped glucose formate agar to incubate anaerobically at 37 deg. C.
+
+Glucose formate bouillon to incubate anaerobically at 37 deg. C.
+
+(D) Sloped glucose formate gelatine to incubate anaerobically at
+20 deg. C.
+
+Glucose formate bouillon to incubate anaerobically at 20 deg. C.
+
+2. Seal the cultures forming set B in Buchner's tubes (_vide_ page 239).
+
+3. Seal the cultures forming set C in Bulloch's apparatus; exhaust the
+air by means of a vacuum pump, and provide for the absorption of any
+residual oxygen by the introduction of pyrogallic acid and caustic soda
+in solution (_vide_ page 245). Treat set D in the same way.
+
+4. Observe the cultivations macroscopically and microscopically at
+intervals of twenty-four hours until the completion, if necessary, of
+seven days' incubation.
+
+5. Control these results.
+
+_Gases Other than Oxygen._--
+
+
+_Apparatus Required:_
+
+    Bulloch's apparatus.
+    Sterile gas filter (_vide_ page 40).
+    Gasometer containing the gas it is desired to test (SO_{2}, N_{2}O, NO,
+      CO_{2}, etc.) or gas generator for its production.
+
+METHOD.--
+
+1. Prepare at least seven tube cultivations upon solid media and deposit
+them in Bulloch's apparatus.
+
+2. Connect up the inlet tube of the Bulloch's jar with the sterile gas
+filter, and this again with the delivery tube of the gasometer or gas
+generator.
+
+3. Open both stop-cocks of the Bulloch's apparatus and pass the gas
+through until it has completely replaced the air in the bell jar as
+shown by the result of analyses of samples collected from the exit tube.
+
+4. Incubate under optimum conditions as to temperature.
+
+5. Examine the cultivations at intervals of twenty-four hours, until the
+completion of seven days.
+
+6. Remove one tube from the interior of the apparatus each day. If no
+growth is visible, incubate the tube under optimum conditions as to
+temperature _and_ atmosphere, and in this way determine the length of
+exposure to the action of the gas necessary to kill the organisms under
+observation.
+
+7. Control these results.
+
+~II. Temperature.~--
+
+(A) _Range._--
+
+1. Prepare a series of ten tube cultivations, in fluid media, of optimum
+reaction.
+
+2. Arrange a series of incubators at fixed temperatures, varying 5 deg. C.
+and including temperatures between 5 deg. C. and 50 deg. C.
+
+(In the absence of a sufficient number of incubators utilise the
+water-bath employed in testing the thermal death-point of vegetative
+forms.)
+
+3. Incubate one tube cultivation of the organism aerobically or
+anaerobically, as may be necessary, in each incubator, and examine at
+half-hour intervals for from five to eighteen hours.
+
+4. Note that temperature at which growth is first observed
+macroscopically (Optimum temperature).
+
+5. Continue the incubation until the completion of seven days. Note the
+extremes of temperature at which growth takes place (Range of
+temperature).
+
+6. Control these results--if considered necessary arranging the series
+of incubators to include each degree centigrade for five degrees beyond
+each of the extremes previously noted.
+
+(B) _Optimum._--
+
+1. Prepare a second series of ten tube cultivations under similar
+conditions as to reaction of medium.
+
+2. Incubate in a series of incubators in which the temperature is
+regulated at intervals of 1 deg. C. for five degrees on either side of
+optimum temperature observed in the previous experiment (A, step 4).
+
+3. Observe again at half-hour intervals and note that temperature at
+which growth is first visible to the naked eye = Optimum temperature.
+
+(C) _Thermal Death-point (t. d. p.)_--
+
+Moist--Vegetative Forms:
+
+The _t. d. p._ here is that ~temperature~ which with certainty kills a
+watery suspension of the organisms in question after an exposure of ~10
+minutes~.
+
+[Illustration: FIG. 155.--Hearson's water-bath.]
+
+     _Apparatus Required:_
+
+     Water-bath. For the purpose of observing the thermal
+     death-point a special water-bath is necessary. The
+     temperature of this piece of apparatus is controlled by
+     means of a capsule regulator that can be adjusted for
+     intervals of half a degree centigrade through a range of
+     30 deg., from 50 deg. C. to 80 deg. C. by means of a spring,
+     actuated by the handle a, which increases the pressure
+     in the interior of the capsule. A hole is provided for the
+     reception of the nozzle of a blast pump, so that a current
+     of air may be blown through the water while the bath is in
+     use, and thus ensure a uniform temperature of its contents.
+     Through a second hole is suspended a certified centigrade
+     thermometer, the bulb of which although completely immersed
+     in the water is raised at least 2 cm. above the floor of
+     the bath.
+
+     Sterile glass capsules.
+
+     Flask containing 250 c.c. sterile normal saline solution.
+
+     Case of sterile pipettes, 10 c.c. (in tenths of a cubic
+     centimetre).
+
+     Special platinum loop.
+
+     Test-tubes, 18 by 1.5 cm., of thin German glass.
+
+     Case of sterile petri dishes.
+
+     Tubes of agar or gelatine.
+
+METHOD.--
+
+1. Prepare tube cultivations on solid media of optimum reaction;
+incubate forty-eight hours under optimum conditions as to temperature
+and atmosphere.
+
+2. Examine preparations from the cultivation microscopically to
+determine the absence of spores.
+
+3. Pipette 5 c.c. salt solution into each of twelve capsules.
+
+4. Suspend three loopfuls of the surface growth (using a special
+platinum loop, _vide_ page 316) in the normal saline solution by
+emulcifying evenly against the moist walls of each capsule.
+
+5. Transfer emulsion from each capsule to sterile 250 c.c. flask, and
+mix.
+
+6. Pipette 5 c.c. emulsion into each of twelve sterile test-tubes
+numbered consecutively.
+
+7. Adjust the first tube in the water-bath, regulated at 40 deg. C, by
+means of two rubber rings around the tube, one above and the other below
+the perforated top of the bath, so that the upper level of the fluid in
+the tube is about 4 cm. below the surface of the water in the bath, and
+the bottom of the tube is a similar distance above the bottom of the bath.
+
+8. Arrange a control test-tube containing 5 c.c. sterile saline solution
+under similar conditions. Plug the tube with cotton-wool and pass a
+thermometer through the plug so that its bulb is immersed in the water.
+
+9. Close the unoccupied perforations in the lid of the water-bath by
+means of glass balls.
+
+10. Watch the thermometer in the test-tube until it records a
+temperature of 40 deg. C. Note the time. Ten minutes later remove the tube
+containing the suspension, and cool rapidly by immersing its lower end
+in a stream of running water.
+
+11. Pour three gelatine (or agar) plates containing respectively 0.2,
+0.3, and 0.5 c.c. of the suspension, and incubate.
+
+12. Pipette the remaining 4 c.c. of the suspension into a culture flask
+containing 250 c.c. of nutrient bouillon, and incubate.
+
+13. Observe these cultivations from day to day. "No growth" must not be
+recorded as final until after the completion of seven days' incubation.
+
+14. Extend these observations to the remaining tubes of the series, but
+varying the conditions so that each tube is exposed to a temperature 2
+deg. C. higher than the immediately preceding one--i. e., 42 deg. C.,
+44 deg. C., 46 deg. C., and so on.
+
+15. Note that temperature, after exposure to which no growth takes place
+up to the end of seven days' incubation, = the thermal death-point.
+
+16. If greater accuracy is desired, a second series of tubes may be
+prepared and exposed for ten minutes to fixed temperatures varying only
+0.5 deg. C., through a range of 5 deg. C. on either side of the previously
+observed death-point.
+
+Moist--Spores: The thermal death-point in the case of spores is that
+~time exposure~ to a ~fixed temperature of 100 deg. C.~ necessary to
+effect the death of all the spores present in a suspension.
+
+     NOTE.--If it is desired to retain the ~time constant 10
+     minutes~ and investigate the temperature necessary to destroy
+     the spores, varying amounts of calcium chloride must be
+     added to the water in the bath, when the boiling-point will
+     be raised above 100 deg. C. according to the percentage of
+     calcium in solution. In such case use the bath figured on
+     page 227; the bath figured on page 299 can only be used if
+     the capsule is first removed.
+
+It is determined in the following manner
+
+     _Apparatus Required:_
+
+     Steam-can fitted with a delivery tube and a large bore
+     safety-valve tube.
+
+     Water-bath at 100 deg. C.
+
+     Erlenmeyer flask, 500 c.c. capacity, containing 140 c.c.
+     sterile normal saline solution and fitted with rubber
+     stopper perforated with four holes.
+
+     The rubber stopper is fitted as follows:
+
+     (a) Thermometer to 120 deg. C., its bulb immersed in the normal
+     saline.
+
+     (b) Straight entry tube, reaching to the bottom of the
+     flask, the upper end plugged with cotton-wool.
+
+     (c) Bent syphon tube, with pipette nozzle attached by means
+     of rubber tubing and fitted with pinch-cock.
+
+     The nozzle is protected from accidental contamination by
+     passing it through the cotton-wool plug of a small
+     test-tube.
+
+     (d) A sickle-shaped piece of glass tubing passing just
+     through the stopper, plugged with cotton-wool, to act as a
+     vent for the steam.
+
+     Sterile plates.
+
+     Sterile pipettes.
+
+     Sterile test-tubes graduated to contain 5 c.c.
+
+     _Media Required:_
+
+     Gelatine or agar.
+
+     Culture flasks containing 200 c.c. nutrient bouillon.
+
+[Illustration: FIG. 156.--Apparatus arranged for the determination of
+the death-point of spores.]
+
+METHOD.--
+
+1. Prepare twelve tube cultivations upon the surface (or two cultures in
+large flat culture bottles--_vide_ page 5) of nutrient agar and
+incubate under the optimum conditions (previously determined), for the
+formation of spores.
+
+Examine preparations from the cultures microscopically to determine the
+presence of spores.
+
+2. Pipette 5 c.c. sterile normal saline into each culture tube or 30
+c.c. into each bottle and by means of a sterile platinum spatula
+emulsify the entire surface growth with the solution.
+
+3. Add the 60 c.c. emulsion to 140 c.c. normal saline contained in the
+fitted Erlenmeyer flask.
+
+4. Place the flask in the water-bath of boiling water.
+
+5. Connect up the straight tube, after removing the cotton-wool plug,
+with the delivery tube of the steam can; remove the plug from the vent
+tube.
+
+6. When the thermometer reaches 100 deg. C., open the spring clip on the
+_syphon_, discard the first cubic centimeter of suspension that syphons
+over (i. e., the contents of the syphon tube); collect the next 5 c.c.
+of the suspension in the sterile graduated test-tube and pour plates and
+prepare flask cultures therefrom as in the previous experiments.
+
+7. Repeat this process at intervals of twenty-five minutes' steaming.
+
+8. Observe the inoculated plates and flasks up to the completion, if
+necessary, of seven days' incubation.
+
+9. Control these experiments, but in this instance syphon off portions
+of the suspension at intervals of one-half to one minute during the five
+or ten minutes preceding the previously determined death-point.
+
+_Thermal Death-point._--
+
+Dry--Vegetative Forms: The thermal death-point in this case is that
+~temperature~ which with certainty kills a thin film of the organism in
+question after a time exposure of ~ten minutes~.
+
+     _Apparatus Required:_
+
+     Hot-air oven, provided with thermo-regulator.
+
+     Sterile cover-slips.
+
+     Flask containing 250 c.c. sterile normal saline solution.
+
+     Case of sterile pipettes, 10 c.c. (in tenths of a cubic
+     centimetre).
+
+     Case of sterile capsules.
+
+     Crucible tongs.
+
+METHOD.--
+
+1. Prepare an emulsion with three loopfuls from an optimum cultivation
+in 5 c.c. normal saline in a sterile capsule and examine microscopically
+to determine the absence of spore forms.
+
+2. Make twelve cover-slip films on sterile cover-slips; place each in a
+sterile capsule to dry.
+
+3. Expose each capsule in turn in the hot-air oven for ten minutes to a
+different fixed temperature, varying 5 deg. C. between 60 deg. C. and
+120 deg. C.
+
+4. Remove each capsule from the oven with crucible tongs immediately
+after the ten minutes are completed; remove the cover-glass from its
+interior with a sterile pair of forceps.
+
+5. Deposit the film in a flask containing 200 c.c. nutrient bouillon.
+
+6. Prepare subcultivations from such flasks as show evidence of growth,
+to determine that no accidental contamination has taken place but that
+the organism originally spread on the film is responsible for the
+growth.
+
+7. Control the result of these experiments.
+
+Dry--Spores: The thermal death-point in this case is that ~temperature~
+which with certainty kills the spores of the organism in question when
+present in a thin film after a time exposure of ~10 minutes~.
+
+     _Apparatus Required:_
+
+     As for vegetative forms.
+
+METHOD.--
+
+1. Prepare a sloped agar tube cultivation and incubate under optimum
+conditions as to spore formations.
+
+2. Pipette 5 c.c. sterile normal saline into the culture tube and
+emulsify the entire surface growth in it. Examine microscopically to
+determine the presence of spores in large numbers.
+
+3. Spread thin even films on twelve sterile cover-slips and place each
+cover-slip in a separate sterile capsule.
+
+4. Expose each capsule in turn for ten minutes to a different fixed
+temperature, varying 5 deg. C, between 100 deg. C. and 160 deg. C.
+
+5. Complete the examination as for vegetative forms.
+
+
+~III. Reaction of Medium.~
+
+(A) _Range._--
+
+1. Prepare a bouillon culture of the organism and incubate, under
+optimum conditions as to temperature and atmosphere, for twenty-four
+hours.
+
+2. Pipette 0.1 c.c. of the cultivation into a sterile capsule; add 9.9
+c.c. sterile bouillon and mix thoroughly.
+
+3. Prepare a series of tubes of nutrient bouillon of varying reactions,
+from +25 to -30 (_vide_ page 155), viz.: +25, +20, +15, +10, +5,
+neutral, -5, -10, -15, -20, -25, -30.
+
+4. Inoculate each of the bouillon tubes with 0.1 c.c. of the diluted
+cultivation by means of a sterile graduated pipette and incubate under
+optimum conditions.
+
+5. Observe the cultures at half-hourly intervals from the third to the
+twelfth hours. Note the reaction of the tube or tubes in which growth is
+first visible macroscopically (probably optimum reaction).
+
+6. Continue the incubation until the completion, if necessary, of seven
+days. Note the extremes of acidity and alkalinity in which macroscopical
+growth has developed (Range of reaction).
+
+7. Control the result of these observations.
+
+(B) _Optimum Reaction._--The optimum reaction has already been
+roughly determined whilst observing the range. It can be fixed within
+narrower limits by inoculating in a similar manner a series of tubes of
+bouillon which represent smaller variations in reaction than those
+previously employed (say, 1 instead of 5) for five points on either side
+of the previously observed optimum. For example, the optimum reaction
+observed in the set of experiments to determine the range was +10. Now
+plant tubes having reactions of +15, +14, +13, +12, +11, +10, +9, +8,
++7, + 6, +5, and observe as before.
+
+
+~IV. Resistance to Lethal Agents.~--
+
+(A) _Desiccation._--
+
+     _Apparatus Required:_
+
+     Mueller's desiccator. This consists of a bell glass fitted
+     with an exhaust tube and stop-cock (d), which can be
+     secured to a plate-glass base (c) by means of wax or
+     grease. It contains a cylindrical vessel of porous clay
+     (a) into the top of which pure sulphuric acid is poured
+     whilst the material to be dried is placed within its walls
+     on a glass shelf (b). The air is exhausted from the
+     interior and the acid rapidly converts the clay vessel into
+     a large absorbing surface (Fig. 157).
+
+     Exhaust pump.
+
+     Pure concentrated sulphuric acid.
+
+     Sterile cover-slips.
+
+     Sterile forceps.
+
+     Culture flask containing 200 c.c. nutrient bouillon.
+
+     Sterile ventilated Petri dish. This is prepared by bending
+     three short pieces of aluminium wire into V shape and
+     hanging these on the edge of the lower dish and resting the
+     lid upon them (Fig. 158).
+
+METHOD.--
+
+1. Prepare a surface cultivation on nutrient agar in a culture bottle
+and incubate under optimum conditions for forty-eight hours.
+
+2. Examine preparations from the cultivation, microscopically, to
+determine the absence of spores.
+
+3. Pipette 5 c.c. sterile normal saline solution into the flask and
+suspend the entire growth in it.
+
+4. Spread the suspension in thin, even films on sterile cover-slips and
+deposit inside sterile "plates" to dry.
+
+5. As soon as dry, transfer the cover-slip films to the ventilated Petri
+dish by means of sterile forceps.
+
+[Illustration: FIG. 157.--Mueller's desiccator.]
+
+6. Place the Petri dish inside the Mueller's desiccator; fill the upper
+chamber with pure sulphuric acid, cover with the bell jar, and exhaust
+the air from its interior. Ten minutes later connect up the desiccator
+to a sulphuric acid wash-bottle interposing an air filter so that only
+dry sterile air enters.
+
+[Illustration: FIG. 158.--Petri dish for drying cultivations.]
+
+7. At intervals of five hours open the apparatus, remove one of the
+cover-slip films from the Petri dish, and transfer it to the interior of
+a culture flask, with every precaution against contamination. Reseal the
+desiccator and again exhaust, and subsequently admit dry sterile air as
+before.
+
+8. Incubate the culture flask under optimum conditions until the
+completion of seven days, if necessary; and determine the time exposure
+at which death occurs.
+
+9. Pour plates from those culture flasks which grow, to determine the
+absence of contamination.
+
+10. Repeat these observations at hourly intervals for the five hours
+preceding and succeeding the death time, as determined in the first set
+of experiments.
+
+(B) _Light._--
+
+(a) Diffuse Daylight:
+
+1. Prepare a tube cultivation in nutrient bouillon, and incubate under
+optimum conditions, for forty-eight hours.
+
+[Illustration: FIG. 159.--Plate with star for testing effect of light.]
+
+2. Pour twenty plate cultivations, ten of nutrient gelatine and ten of
+nutrient agar, each containing 0.1 c.c. of the bouillon culture.
+
+3. Place one agar plate and one gelatine plate into the hot and cold
+incubators, respectively, as _controls_.
+
+4. Fasten a piece of black paper, cut the shape of a cross or star, on
+the centre of the cover of each of the remaining plates (Fig. 159).
+
+5. Expose these plates to the action of diffuse daylight (not direct
+sunlight) in the laboratory for one, two, three, four, five, six, eight,
+ten, twelve hours.
+
+6. After exposure to light, incubate under optimum conditions.
+
+7. Examine the plate cultivations after twenty-four and forty-eight
+hours' incubation, and compare with the two controls. Record results. If
+growth is absent from that portion of the plate unprotected by the black
+paper, continue the incubation and daily observation until the end of
+seven days.
+
+8. Control the results.
+
+(b) Direct Sunlight:
+
+1. Prepare plate cultivations precisely as in the former experiments and
+place the two controls in the incubators.
+
+2. Arrange the remaining plates upon a platform in the direct rays of
+the sun.
+
+3. On the top of each plate stand a small glass dish 14 cm. in diameter
+and 5 cm. deep.
+
+4. Fill a solution of potash alum (2 per cent. in distilled water) into
+each dish to the depth of 2 cm. to absorb the heat of the sun's rays and
+so eliminate possible effects of temperature on the cultivations.
+
+5. After exposures for periods similar to those employed in the
+preceding experiment, incubate and complete the observation as above.
+
+(c) Primary Colours: Each colour--violet, blue, green and red--must be
+tested separately.
+
+1. Prepare plate cultivations, as in the previous "light" experiments,
+and incubate controls.
+
+2. Fasten a strip of black paper, 3 cm. wide, across one diameter of the
+cover of each plate.
+
+3. Coat the remainder of the surface of the cover with a film of pure
+photographic collodion which contains 2 per cent. of either of the
+following aniline dyes, as may be necessary:
+
+    Chrysoidin (for red).
+    Malachite green (for green).
+    Eosin, bluish (for blue).
+    Methyl violet (for violet).
+
+4. Expose the plates, thus prepared, to bright daylight (but not direct
+sunlight) for varying periods, and complete the observations as in the
+preceding experiments. The bactericidal action of light appears to
+depend upon the more refrangible rays of the violet end of the spectrum
+and is noted whether the red yellow rays are transmitted or not.
+
+5. Control the results.
+
+     NOTE.--The ultra-violet rays obtained from a quartz mercury
+     vapour lamp destroy bacterial life with great rapidity under
+     laboratory conditions.
+
+(C) _Heat._--(_Vide_ Thermal Death-point, page 298.)
+
+(D) _Antiseptics and Disinfectants._--The resistance exhibited by any
+given bacterium toward any specified disinfectant or germicide should be
+investigated with reference to the following points:
+
+(A) ~Inhibition coefficient~--i. e., that _percentage of the
+disinfectant_ present in the nutrient medium which is sufficient to
+prevent the growth and multiplication of the bacterium.
+
+(B) ~Inferior lethal coefficient~--i. e., the _time exposure_ necessary
+to kill _vegetative forms_ of the bacterium suspended in water at
+20 deg. to 25 deg. C, in which the disinfectant is present in _medium_
+concentration (concentration insufficient to cause plasmolysis). And if
+the bacterium is one which forms spores,
+
+(C) ~Superior lethal coefficient~--i. e., the _time exposure_ necessary
+to kill the _spores_ of the bacterium under conditions similar to those
+obtaining in B.
+
+The example here detailed only specifically refers to certain of the
+disinfectants:
+
+    viz:--Bichloride of mercury;
+    Formaldehyde;
+    Carbolic acid;
+
+investigated with regard to B. anthracis, but the technique is
+practically similar for all other chemical disinfectants.
+
+~Inhibition Coefficient.~--
+
+     _Apparatus Required:_
+
+     Case of sterile pipettes, 10 c.c. (in tenths).
+
+     Case of sterile pipettes, 1 c.c. (in tenths).
+
+     Sterile tubes or capsules for dilutions.
+
+     Tubes of nutrient bouillon each containing a measured 10
+     c.c. of medium.
+
+     Twenty-four-hour-old agar culture of a recently isolated B.
+     Anthracis.
+
+     _Germicides:_
+
+     1. Five per cent. aqueous solution of carbolic acid.
+
+     2. One per cent. aqueous solution of perchloride of mercury.
+
+     3. One-tenth per cent. aqueous solution of formaldehyde.
+
+METHOD.--
+
+1. Number six bouillon tubes consecutively 1 to 6. Inoculate each from
+the stock cultivation of B. anthracis and at once add varying
+quantities[10] of the carbolic acid solution, viz.:
+
+    To tube 1 add 2.0 c.c. (= 1:100)
+    To tube 2 add 1.0 c.c. (= 1:200)
+    To tube 3 add 0.6 c.c. (= 1:300)
+    To tube 4 add 0.5 c.c. (= 1:400)
+    To tube 5 add 0.4 c.c. (= 1:500)
+    To tube 6 add 0.2 c.c. (= 1:1,000)
+
+2. Prepare a similar series of tube cultivations numbered consecutively
+7 to 12 and add varying quantities of the mercuric perchloride solution,
+viz.:
+
+    To tube  7 add 0.1   (= 1:1,000)
+    To tube  8 add 0.05  (= 1:2,000)
+    To tube  9 add 0.03  (= 1:3,000)
+    To tube 10 add 0.025 (= 1:4,000)
+    To tube 11 add 0.02  (= 1:5,000)
+    To tube 12 add 0.01  (= 1:10,000)
+
+
+3. Prepare a similar series of tube cultivations numbered consecutively
+13 to 18 and add varying quantities of the formaldehyde solution, viz.:
+
+    To tube No. 13 add 1.0   c.c. (= 1:1,000)
+    To tube No. 14 add 0.4   c.c. (= 1:2,500)
+    To tube No. 15 add 0.2   c.c. (= 1:5,000)
+    To tube No. 16 add 0.1   c.c. (= 1:10,000)
+    To tube No. 17 add 0.075 c.c. (= 1:15,000)
+    To tube No. 18 add 0.05  c.c. (= 1:20,000)
+
+4. Incubate all three sets of cultivations under optimum conditions as
+to temperature and atmosphere.
+
+5. Examine each of the culture tubes from day to day, until the
+completion of seven days, and note those tubes, if any, in which growth
+takes place.
+
+6. From such tubes as show growth prepare subcultivations upon suitable
+media, and ascertain that the organism causing the growth is the one
+originally employed in the test and not an accidental contamination.
+
+
+~Inferior Lethal Coefficient.~--
+
+     _Apparatus Required:_
+
+     Highly concentrated solutions of the disinfectants.
+
+     Sterile test-tubes in which to make dilutions from the
+     concentrated solutions of the disinfectants.
+
+     Hanging-drop slides.
+
+     Cover-slips.
+
+     Erlenmeyer flask containing 100 c.c. sterile distilled
+     water.
+
+     Case of sterile pipettes, 10 c.c. (in tenths of a cubic
+     centimetre).
+
+     Case of sterile pipettes, 1 c.c. (in tenths of a cubic
+     centimetre).
+
+METHOD.--
+
+1. Prepare a surface cultivation of the "test" organism B. anthracis
+upon nutrient agar in a culture bottle and incubate under optimum
+conditions for twenty-four hours; then examine the cultivation
+microscopically to determine the absence of spores.
+
+2. Prepare solutions of different percentages of each disinfectant.
+
+3. Make a series of hanging-drop preparations from the agar culture,
+using a loopful of disinfectant solution of the different percentages to
+prepare the emulsion on each cover-slip.
+
+4. Examine microscopically and note the strongest solution which does
+not cause plasmolysis and the weakest solution which does plasmolyse the
+organism.
+
+5. Make control preparations of these two solutions and determine the
+percentage to be tested.
+
+6. Pipette 10 c.c. sterile water into the culture bottle and suspend the
+entire surface growth in it.
+
+7. Transfer the suspension to the Erlenmeyer flask and mix it with the
+90 c.c. of sterile water remaining in the flask.
+
+8. Pipette 10 c.c. of the diluted suspension into each of ten sterile
+test-tubes.
+
+9. Label one of the tubes "Control" and place it in the incubator at
+18 deg. C.
+
+10. Add to each of the remaining tubes a sufficient quantity[11] of a
+concentrated solution of the disinfectant to produce the percentage
+previously determined upon (_vide_ step 5).
+
+11. Incubate the tubes at 18 deg. C. to 20 deg. C.
+
+12. At hourly intervals remove the control tube and one of the tubes
+with added disinfectant from the incubator.
+
+13. Make a subcultivation from both the control and the test suspension,
+upon the surface of nutrient agar; incubate under optimum conditions.
+
+14. Observe these culture tubes from day to day until the completion of
+seven days, and determine the shortest exposure necessary to cause the
+death of vegetative forms.
+
+
+~Superior Lethal Coefficient.~--
+
+1. Prepare surface cultivations of the "test" organisms upon nutrient
+agar in a culture bottle, and incubate under optimum conditions, for
+three days, for the formation of their spores.
+
+2. Transfer the emulsion to a sterile test-tube and heat in the
+differential steriliser for ten minutes at 80 deg. C. to destroy all
+vegetative forms.
+
+3. Employing that percentage solution of the disinfectant determined in
+the previous experiment, and complete the investigations as detailed
+therein, steps 7 to 14, increasing the interval between planting the
+subcultivations to two, three, or five hours if considered advisable.
+
+     NOTE.--Where it is necessary to leave the organisms in
+     contact with a strong solution of the disinfectant for
+     lengthy periods, some means must be adopted to remove every
+     trace of the disinfectant from the bacteria before
+     transferring them to fresh culture media; otherwise,
+     although not actually killed, the presence of the
+     disinfectant may prevent their development, and so give rise
+     to an erroneous conclusion. Consequently it is essential in
+     all germicidal experiments to determine first of all the
+     inhibition coefficient of the germicide employed. Under the
+     circumstances referred to above it is usually sufficient to
+     prepare the subcultures in such a volume of fluid nutrient
+     medium as would suffice to reduce the concentration of the
+     germicide to about one hundredth of the inhibition
+     percentage, assuming that the entire bulk of inoculum was
+     made up of that strength of germicide employed in the test.
+     In some cases it is a simple matter to neutralise the
+     germicide and render it inert by washing the organisms in
+     some non-germicidal solution (such for example as ammonium
+     sulphide when using mercurial salts as the germicide). When,
+     however, it is desired to remove the last traces of
+     germicide proceed as follows:
+
+     1. Transfer the suspension of bacteria to sterile
+     centrifugal tubes; add the required amount of disinfectant,
+     and allow it to remain in contact with the bacteria for the
+     necessary period.
+
+     2. Centrifugalise thoroughly, pipette off the supernatant
+     fluid; fill the tube with sterile water and distribute the
+     deposit evenly throughout the fluid.
+
+     3. Centrifugalise again, pipette off the supernatant fluid;
+     fill the tube with sterile water; distribute the deposit
+     evenly throughout the fluid, and transfer the suspension to
+     a litre flask.
+
+     4. Make up to a litre by the addition of sterile water;
+     filter the suspension through a sterile porcelain candle.
+
+     5. Emulsify the bacterial residue with 5 c.c. sterile
+     bouillon.
+
+     6. Prepare the necessary subcultivations from this emulsion.
+
+
+PATHOGENESIS.
+
+_Living Bacteria._--
+
+(a) Psychrophilic Bacteria: When the organism will only grow at or below
+18 deg. to 20 deg. C.,
+
+1. Prepare cultivations in nutrient broth and incubate under optimum
+conditions.
+
+2. After seven days' incubation inject that amount of the culture
+corresponding to 1 per cent. of the body-weight of a healthy frog, into
+the reptile's dorsal lymph sac.
+
+3. Observe until death takes place, or, in the event of a negative
+result, until the completion of twenty-eight days (_vide_ Chapter
+XVIII).
+
+4. If, and when, death occurs, make a careful post-mortem examination
+(_vide_ Chapter XIX).
+
+(b) Mesophilic Bacteria: When the organism grows at 35 deg. to 37 deg. C.,
+
+1. Prepare cultivations in nutrient broth and incubate under optimum
+conditions for forty-eight hours.
+
+2. Select two white mice, as nearly as possible of the same age, size,
+and weight.
+
+3. Inoculate the first mouse, subcutaneously at the root of the tail,
+with an amount of cultivation equivalent to 1 per cent. of its
+body-weight.
+
+4. Inoculate the second mouse intraperitoneally with a similar dose.
+
+5. Observe carefully until death occurs, or until the lapse of
+twenty-eight days.
+
+6. If the inoculated animals succumb, make complete post-mortem
+examination.
+
+If death follows shortly after the injection of cultivations of
+bacteria, the inoculation experiments should be repeated two or three
+times. Then, if the organism under observation invariably exhibits
+pathogenic effects, steps should be taken to ascertain, if possible, the
+minimal lethal dose (_vide infra_) of the growth upon solid media for
+the frog or white mouse respectively. Other experimental animals--_e.
+g._, the white rat, guinea-pig, and rabbit--should next be tested in a
+similar manner.
+
+7. If the inoculated mice are unaffected, test the action of the
+organism in question upon white rats, guinea-pigs, rabbits, etc.
+
+_Minimal Lethal Dose_ (_m. l. d._); If the purpose of the inoculation is
+to determine the minimal lethal dose, a slightly different procedure
+must be followed. For this and other exact experiments a special
+platinum loop is manufactured, some 2.5 mm. by 0.75 mm., with parallel
+sides, and calibrated by careful weighing, to determine approximately
+the amount of moist bacterial growth, the loop will hold when filled.
+
+1. The cultivation must be prepared on a solid medium of the optimum
+reaction, incubated at the optimum temperature, and injected at the
+period of greatest activity and vigour, of the particular organism it is
+desired to test.
+
+2. Arrange four sterile capsules in a row and label them I, II, III, and
+IV. Into the first deliver 10 c.c. sterile bouillon by means of a
+sterile graduated pipette; and into each of the remaining three, 9.9
+c.c.
+
+3. Remove one loopful of the bacterial growth from the surface of the
+medium in the culture tube, observing the usual precautions against
+contamination, and emulsify it evenly with the bouillon in the first
+capsule. Each cubic centimetre of the emulsion will now contain
+one-tenth of the organisms contained in the original loopful (written
+shortly 0.1 loop).
+
+4. Remove 0.1 c.c. of the emulsion in the first capsule by means of a
+sterile graduated pipette and transfer it to the second capsule and mix
+thoroughly. Drop the infected pipette into a jar of lysol solution. This
+makes up the bulk of the fluid in the second capsule to 10 c.c., and
+therefore every cubic centimetre of bouillon in capsule II contains
+0.001 loop.
+
+5. Similarly, 0.1 c.c. of the mixture is transferred from capsule II to
+capsule III (1 c.c. of bouillon in capsule III contains 0.00001 loop),
+and then from capsule III to capsule IV (1 c.c. of bouillon in capsule
+IV contains 0.0000001 loop).
+
+The dilutions thus prepared may be summarised in a table;
+
+Capsule   I = 1 loopful + 10 c.c. water             [.'.] 1 c.c.=0.1 loop.
+Capsule  II = 0.1 c.c. capsule I + 9.9 c.c. water   [.'.] 1 c.c.=0.001 loop.
+Capsule III = 0.1 c.c. capsule II + 9.9 c.c. water  [.'.] 1 c.c.=0.00001 loop.
+Capsule  IV = 0.1 c.c. capsule III + 9.9 c.c. water
+      [.'.] 1 c.c. = 0.0000001 loop.
+
+6. With sterile graduated pipettes remove the necessary quantity of
+bouillon corresponding to the various divisors of ten of the loop from
+the respective capsules, and transfer each "dose" to a separate sterile
+capsule and label; and to such doses as are small in bulk, add the
+necessary quantity of sterile bouillon to make up to 1 c.c.
+
+7. Multiples of the loop are prepared by emulsifying 1, 2, 5, or 10
+loops each with 1 c.c. sterile bouillon in separate sterile capsules.
+
+8. Inoculate a series of animals with these measured doses, filling the
+syringe first from that capsule containing the smallest dose, then from
+the capsule containing the next smallest, and so on. If care is taken,
+it will not be found necessary to sterilise the syringe during the
+series of inoculations.
+
+9. Plant tubes of gelatine or agar, liquefied by heat, from each of the
+higher dilutions, say from 0.0000001 loop to 0.01 loop; pour plates and
+incubate. When growth is visible enumerate the number of organisms
+present in each, average up and calculate the number of bacteria present
+in one loopful of the inoculum.
+
+10. The smallest dose which causes the infection and death of the
+inoculated animal is noted as the minimal lethal dose.
+
+_Toxins._--
+
+Prepare flask cultivations of the organism under observation in glucose
+formate broth, and incubate for fourteen days under optimum conditions.
+
+(a) Intracellular or Insoluble Toxins:
+
+1. Heat the fluid culture in a water-bath at 60 deg. C. for thirty
+minutes. (The resulting sterile, turbid fluid is often spoken of as
+"killed" culture,)
+
+2. Inoculate a tube of sterile bouillon with a similar quantity, and
+incubate under optimum conditions. This "control" then serves to
+demonstrate the freedom of the toxin from living bacteria.
+
+[Illustration: FIG. 160.--Apparatus arrange for toxin filtration.]
+
+3. Inject intraveneously that amount of the cultivation corresponding to
+1 per cent. of the body-weight of the selected animal, usually one of
+the small rodents.
+
+4. Observe during life or until the completion of twenty-eight days, and
+in the event of death occurring during that period, make a complete
+post-mortem examination.
+
+5. Repeat the experiment at least once. In the event of a positive
+result estimate the minimal lethal dose of "killed" culture for each of
+the species of animals experimented upon.
+
+(b) Extracellular or Soluble Toxins:
+
+1. Filter the cultivation through a porcelain filter candle (Berkefeld)
+into a sterile filter flask, arranging the apparatus as in the
+accompanying figure (Fig. 160).
+
+2. Inoculate mice, rats, guinea-pigs, and rabbits subcutaneously with
+that quantity of toxin corresponding to 1 per cent. of the body-weight
+of each respectively, and observe, if necessary, until the completion of
+one month.
+
+3. Inoculate a "control" tube of bouillon with a similar quantity and
+incubate, to determine the freedom of the filtered toxin from living
+bacteria.
+
+4. In the event of a fatal termination make complete and careful
+post-mortem examinations.
+
+5. Repeat the experiments and, if the results are positive, ascertain
+the minimal lethal dose of toxin for each of the susceptible animals.
+
+The estimation of the _m. l. d._ of a toxin is carried out on lines
+similar to those laid down for living bacteria (_vide_ page 316) merely
+substituting 1 c.c. of toxin as the unit in place of the unit "loopful"
+of living culture.
+
+It frequently happens, during the course of casual investigations that a
+bouillon-tube culture is available for a toxin test whilst a flask
+cultivation is not. In such cases, Martin's small filter candle and tube
+(Fig. 161) specially designed for the filtration of small quantities of
+fluid, is invaluable. This consists of a narrow filter flask just large
+enough to accommodate an ordinary 18 x 2 cm. test-tube. The mouth of the
+tubular Chamberland candle 15 x 1.5 cm. is closed by a perforated rubber
+cork into which fits the end of the stem of a thistle headed funnel,
+whilst immediately below the butt of the funnel is situated a rubber
+cork to close the mouth of the filter flask. When the apparatus is fixed
+in position and connected to an exhaust pump, the cultivation is poured
+into the head of the funnel and owing to the relatively large filtering
+surface the germ free filtrate is rapidly drawn through into the
+test-tube receiver.
+
+~Raising the Virulence of an Organism.~--If it is desired to raise or
+"exalt" the virulence of a feebly pathogenic organism, special methods
+of inoculation are necessary, carefully adjusted to the exigencies of
+each individual case. Among the most important are the following:
+
+1. _Passage of Virus._--The inoculation of pure cultivations of the
+organism into highly susceptible animals, and passing it as rapidly as
+possible from animal to animal, always selecting that method of
+inoculation-e. g., intraperitoneal--which places the organism under
+the most favorable conditions for its growth and multiplication.
+
+[Illustration: FIG. 161--Martin's filtering apparatus for small
+quantities of fluid.]
+
+2. _Virus Plus Virulent Organisms._--The inoculation of pure
+cultivations of the organism together with pure cultivations of some
+other microbe which in itself is sufficiently virulent to ensure the
+death of the experimental animal, either into the same situation or into
+some other part of the body. By this association the organism of low
+virulence will frequently acquire a higher degree of virulence, which
+may be still further raised by means of "passages" (_vide supra_).
+
+3. _Virus Plus Toxins._--The inoculation of pure cultivations of the
+organism into some selected situation, together with the subcutaneous,
+intraperitoneal, or intravenous injection of a toxin--e. g., one of
+those elaborated by the proteus group--either simultaneously with,
+before, or immediately after, the injection of the feeble virus. By
+this means the natural resistance of the animal is lowered, and the
+organism inoculated is enabled to multiply and produce its pathogenic
+effect, its virulence being subsequently exalted by means of "passages."
+
+~Attenuating the Virulence of an Organism.~--Attenuating or lowering the
+virulence of a pathogenic microbe is usually attained with much less
+difficulty than the exaltation of its virulence, and is generally
+effected by varying the environment of the cultivations, as for example:
+
+1. Cultivating in such media as are unsuitable by reason of their (a)
+composition or (b) reaction.
+
+2. Cultivating in suitable media, but at an unsuitable temperature.
+
+3. Cultivating in suitable media, but in an unsuitable atmosphere.
+
+4. Cultivation in suitable media, but under unfavorable conditions as to
+light, motion, etc.
+
+Attenuation of the virus can also be secured by
+
+5. Passage through naturally resistant animals.
+
+6. Exposure to desiccation.
+
+7. Exposure to gaseous disinfectants.
+
+8. By a combination of two or more of the above methods.
+
+
+IMMUNISATION.
+
+The further study of the pathogenetic powers of any particular bacterium
+involves the active immunisation of one or more previously normal
+animals. This end may be attained by various means; but it must be
+remembered that immunisation is not carried out by any hard and fast
+rule or by one method alone, but usually by a combination of methods
+adapted to the exigencies of each particular case. The ordinary methods
+include:
+
+     A. Active Immunisation.
+
+     I. By inoculation with dead bacteria (i. e., bacteria
+     killed by heat; the action of ultra-violet rays, of chemical
+     germicides, or by autolysis).
+
+     II. By the inoculation of attenuated strains of bacteria.
+
+     III. By the inoculation of living virulent bacteria (exalted
+     in virulence if necessary).
+
+     B. Combined Active and Passive Immunisation:
+
+     IV. By the inoculation of toxin-antitoxin mixtures.
+
+
+ACTIVE IMMUNISATION.
+
+The immunisation of the rabbit against the Diplococcus pneumoniae may be
+instanced as an example of the general methods of immunisation of
+laboratory animals.
+
+1. Take a full grown rabbit weighing not less than 1200 to 1500 grammes
+(large rabbits of 2000 grammes and over are the most suitable for
+immunising experiments). Observe weight and temperature carefully during
+the few days occupied in the following steps.
+
+2. Inoculate a small rabbit intraperitoneally with one or two loopfuls
+of a twenty-four-hour-old blood agar cultivation of a _virulent_ strain
+of Diplococcus pneumoniae.
+
+Death should follow within twenty-four hours, and in any case will not
+be delayed beyond forty-eight hours.
+
+3. Under aseptic precautions, at the post-mortem, transfer a loopful of
+heart blood to an Erlenmeyer flask containing 50 c.c. sterile nutrient
+broth. Incubate at 37 deg. C. for twenty-four hours.
+
+4. Prepare also several blood agar cultures from the heart blood of the
+rabbit, label them all O.C. (original culture). After twenty-four hours
+incubation at 37 deg. C. place an india-rubber cap over the plugged mouth
+of the tube of all but one of these cultures and paint the cap with Canada
+balsam or shellac varnish, dry, and replace in the hot incubator.
+
+This will prevent evaporation, and cultures thus sealed will remain
+unaltered in virulence for a considerable time.
+
+5. Make a fresh subcultivation on blood agar from the uncapped O.C.
+cultivation and after twenty-four hours incubation at 37 deg. C.
+determine the minimal lethal dose of this strain upon a series of mice
+(see page 316).
+
+6. Suspend the flask containing the twenty-four-hour-old broth culture
+(step 3) in the water-bath at 60 deg. C. for one hour. Cool the flask
+rapidly under a stream of cold water.
+
+7. Determine the sterility of this (?) killed cultivation by
+transferring one cubic centimetre to each of several tubes of nutrient
+broth, and incubate at 37 deg. C. for twenty-four hours. If growth of
+Diplococcus pneumoniae occurs, again heat culture in water-bath at 60
+deg. C. for one hour and again test for sterility.
+
+8. Inject the selected rabbit intravenously (see page 363) with 2 c.c.
+of the killed cultivation, and inject a further 10 c.c. into the
+peritoneal cavity.
+
+During the next few days the animal will lose some weight and perhaps
+show a certain amount of pyrexia.
+
+9. When the temperature and weight have again returned to
+normal--generally about seven days after the inoculation--again inject
+killed cultivation, this time giving a dose of 5 c.c. intravenously and
+20 c.c. intraperitoneally. A temperature and weight reaction similar to,
+but less marked than that following the first injection will probably be
+observed, but after about a week's interval the animal will be ready for
+the next injection.
+
+10. When ready to give the third injection prepare a fresh blood agar
+subculture from another O.C. tube and after twenty-four hours incubation
+prepare a minimal lethal dose (as determined in 5) and inject it
+subcutaneously into the rabbit's abdominal wall.
+
+A slight local reaction will probably be observed as well as the weight
+and temperature reactions.
+
+11. A week to ten days later inject a similar minimal lethal dose into
+the peritoneal cavity.
+
+12. Observe the weight and temperature of the rabbit very carefully, and
+regulating the dates of inoculation by the animal's general condition,
+continue to inject living cultivations of the pneumococcus into the
+peritoneal cavity, gradually increasing the dose by multiples of ten.
+
+13. At intervals of two months samples of blood may be collected from
+the posterior auricular vein and the serum tested for specific
+antibodies.
+
+14. Under favourable conditions it will be found after some six months
+steady work that the rabbit may be injected intraperitoneally with an
+entire blood agar cultivation without any ill effects being apparent;
+and this characteristic--resistance to the lethal effects of large doses
+of the virus--is the sole criterion of _immunity_. Further, the serum
+separated from blood withdrawn from the animal about a week after an
+injection, if used in doses of .01 c.c., will protect a mouse against
+the lethal effects of at least ten minimal lethal doses of living
+pneumococci.
+
+In the foregoing illustration it has been assumed that complete acquired
+active immunity has been conferred upon the experimental rabbit in
+consequence of the formation of antibody, specific to the diplococcus
+pneumoniac, sufficient in amount to ensure the destruction of enormous
+doses of the living cocci--the _antigen_ (that is the substance injected
+in response to which _antibody_ has been elaborated) in this particular
+case being the bacterial protoplasm of the pneumococcus with its
+endo-toxins.
+
+But provided death does not immediately follow the injection of the
+antigen, specific antibody is always formed in greater or lesser amount;
+and in experimental work a sufficient amount of any required antibody
+can often be obtained without carrying the process of immunisation to
+its logical termination.
+
+For instance, if the immunisation of a rabbit toward Bacillus typhosus
+is commenced on the lines already set out it will often be found, after
+a few injections of "killed" cultivation that the blood serum of the
+animal (even when diluted with several hundred times its volume of
+normal saline) contains specific agglutinin for B. typhosus--and if the
+sole object of the experiment has been the preparation of agglutinin the
+inoculations may well be stopped at this point, although the animal is
+not yet immune in the strict meaning of the word.
+
+Again, antibodies may be formed in response to antigens other than
+infective particles--thus the injection into suitable animals of foreign
+proteins such as egg albumin, heterologous blood sera or red blood discs
+from a different species of animal, will result in the formation of
+specific antibodies possessing definite affinities for their respective
+antigens.
+
+The most important antibody of this latter type is Haemolysin, a
+substance that makes its appearance in the blood serum of an animal
+previously injected with washed blood cells from an animal of a
+different species. The serum from such an animal possesses the power of
+disintegrating red blood discs of the variety employed as antigen and
+causing the discharge of their contained haemoglobin, and is specific in
+its action to the extent of failing to exert any injurious effect upon
+the red blood cells of any other species of animal.
+
+The action of this serum is due to the presence of two distinct bodies,
+complement and haemolysin.
+
+_Complement_ (or alexine) is a thermo-labile readily oxidised body
+present in variable but unalterable amount in the normal serum of every
+animal. It is a substance which exerts a lytic effect upon all foreign
+matter introduced into the blood or tissues; but by itself is a
+comparatively inert body, and is only capable of exerting its maximum
+lytic effect in the presence of and in combination with a specific
+antibody, or immune body.
+
+Complement is obtained (unmixed with antibody) by collecting fresh blood
+serum from any healthy normal (that is uninoculated) animal.
+Guinea-pigs' serum is that most frequently employed for experimental
+work.
+
+_Haemolysin_ (immune body, copula, sensitising body, amboceptor) is a
+_thermostable_ antibody formed in response to the injection of red cells
+which although in itself inert is capable of linking up complement
+present in the normal serum to the red cells of the variety used as
+antigen--a combination resulting in haemolysis.
+
+Haemolysin is obtained by collecting fresh blood serum from a suitably
+inoculated animal and exposing it to a temperature of 56 deg. C. (to
+destroy the thermo-labile complement) for 15 to 30 minutes before use.
+It is then referred to as _inactivated_, and is _reactivated_ by the
+addition of fresh normal serum--that is serum containing complement.
+
+Haemolysin is of importance academically owing to the fact that many of
+the problems of immunity have been elucidated by its aid; but its
+present practical importance lies in the application of the _haemolytic
+system_ (that is haemolysin, corresponding erythrocyte solution and
+complement) to certain laboratory methods having for their object either
+the identification of the infective entity or the diagnosis of the
+existence of infection.
+
+For use in these laboratory methods of diagnosis it is most convenient
+to prepare haemolytic serum specific for human blood--whether the
+laboratory is isolated or attached to a large hospital. Ox blood, sheep
+blood or goat blood if readily obtainable, may however be used instead,
+and although the following method is directed to the preparation of
+human haemolysin the same procedure serves in all cases.
+
+
+THE PREPARATION OF HAEMOLYTIC SERUM.
+
+_Apparatus Required:_
+
+    Small centrifuge, preferably electrically driven, with two
+      receptacles for tubes, and enclosed in a safety shield (Fig. 162).
+    Sterile centrifuge tubes (10 c.c. capacity), Fig. 163.
+    Sterile pipettes (10 c.c. graduated) in case.
+    Sterile glass capsules (in case).
+    Sterile test-tubes.
+    Sterile all glass syringe (5 c.c. or 10 c.c. capacity)
+      and needle.
+
+[Illustration: FIG. 162.--Small electrical centrifuge.]
+
+[Illustration: FIG. 163.--Centrifuge tube.]
+
+_Reagents Required:_
+
+    Normal saline solution.
+    10 per cent. sodium citrate solution in normal saline.
+    Human blood (_vide infra_).
+
+METHOD.--
+
+1. Select a healthy full-grown rabbit of not less than 2500 grammes
+weight in accordance with the directions already given (page 322) and
+prepare it for intraperitoneal inoculation.
+
+2. Measure out 2 c.c. citrated human blood (collected at a surgical
+operation or a venesection, or withdrawn by venipuncture from the median
+basilic or median cephalic vein of a normal adult) into a centrifuge
+tube and centrifugalise thoroughly.
+
+3. Wash with three changes of normal saline (_vide_ also page 388).
+
+4. Transfer the washed cells to a sterile capsule by means of a sterile
+pipette. Add 5 c.c. of normal saline and mix thoroughly.
+
+5. Take up the mixture of cells and saline in the all-glass syringe and
+inject into the peritoneal cavity of the rabbit.
+
+6. Seven days later inject intraperitoneally the washed cells from 5
+c.c. human blood mixed with 5 c.c. normal saline.
+
+7. Seven days later inject the washed cells from 10 c.c. human blood
+mixed with 5 c.c. normal saline.
+
+8. After a further interval of seven days repeat the injection of washed
+cells from 10 c.c. human blood mixed with 5 c.c. normal saline.
+
+     NOTE.--Better results are obtained if the second and
+     subsequent injections are made intravenously, even when
+     smaller quantities of washed red cells are employed. If,
+     however, the intravenous route is selected exceeding great
+     care must be exercised to avoid the introduction of air into
+     the vein--an accident which is followed, within a few
+     minutes, by the death of the rabbit from pulmonary embolism.
+
+9. Allow five days to elapse, then collect a preliminary sample of
+blood, say about 2 c.c., from the rabbit's ear. Allow it to clot,
+separate off the serum and transfer to a sterile test-tube. Place the
+test-tube in a water-bath at 56 deg. C. for fifteen minutes (to
+inactivate) and test the serum quantitatively for haemolytic properties
+in the following manner:
+
+
+THE TITRATION OF HAEMOLYTIC SERUM.
+
+_Apparatus Required:_
+
+    Electrical centrifuge.
+    Sterile centrifuge tubes.
+    Water-bath regulated at 56 deg. C.
+    Sterilised pipettes 10 c.c. graduated in tenths.
+    Sterilised pipettes 1 c.c. graduated in tenths.
+    Sterile test-tubes, 16 x 2 cm.
+    Small sterile test-tubes, 9 x 1 cm.
+    Small test-tube rack, or roll of plasticine.
+    Capillary teat pipettes.
+    Stout rubber band or length of small rubber tubing.
+
+_Reagents Required and Method of Preparation:_
+
+     1. Normal saline solution.
+
+     2. Haemolytic serum inactivated by preliminary heating to 56 deg.
+     C. for 15 minutes (_vide supra_) in test-tube labelled H. S.
+
+     3. Complement. Fresh guinea-pig serum in test-tube labelled
+     C.
+
+     Kill a normal guinea-pig with chloroform vapour.
+
+     Open the thorax with all aseptic precautions, and collect as
+     much blood as possible from the heart with a sterile Pasteur
+     pipette.
+
+     Transfer it to a sterile centrifuge tube and place the tube
+     in the incubator at 37 deg. C. Two hours later separate the clot
+     from the sides of the tube, and centrifugalise thoroughly.
+
+     Pipette off the clear serum to a clean sterilised test-tube.
+
+     4. Erythrocyte solution, in test-tube labelled E.
+
+     Collect and wash human red blood cells (see page 388, 1-8).
+     Measure the volume of red cells available and prepare a 2
+     per cent. suspension in normal saline solution.
+
+METHOD.--
+
+1. Take two test-tubes and number them 1 and 2, and pipette into each 9
+c.c. of normal saline solution.
+
+2. Add 1 c.c. of haemolytic rabbit serum to tube No. 1 and mix
+thoroughly: take up 1 c.c. of the mixture and add it to tube No. 2; mix
+thoroughly.
+
+3. Set up ten small test-tubes in test-tube rack or in roll of
+plasticine, and number 1 to 10.
+
+    4. Pipette into tube No. 1 0.5 c.c. = 0.5 c.c.}
+    haemolytic serum                              }    From tube
+    Pipette into tube No. 2 0.1 c.c. = 0.1 c.c.   }     H. S.
+    haemolytic serum                              }
+
+    Pipette into tube No. 3 0.5 c.c. = 0.05 c.c.  }
+    haemolytic serum                              }
+    Pipette into tube No. 4 0.3 c.c. = 0.03 c.c.  }
+    haemolytic serum                              }    From
+    Pipette into tube No. 5 0.2 c.c. = 0.02 c.c.  }    tube 1.
+    haemolytic serum                              }
+    pipette into tube No. 6 0.1 c.c. = 0.01 c.c.  }
+    haemolytic serum                              }
+
+    Pipette into tube No. 7 0.5 c.c. = 0.005 c.c.  }
+    haemolytic serum                               }
+    Pipette into tube No. 8 0.3 c.c. = 0.003 c.c.  }
+    haemolytic serum                               } From
+    Pipette into tube No. 9 0.2 c.c. = 0.002 c.c.  } tube 2.
+    haemolytic serum                               }
+    Pipette into tube No. 10 0.1 c.c. = 0.001 c.c. }
+    haemolytic serum                               }
+
+5. To each tube add 1 c.c. of erythrocyte solution.
+
+6. When necessary (that is to say in tubes 2, 4, 5, 6, 8, 9 and 10) add
+normal saline solution to the mixture in the test-tubes till the column
+of fluid in each reaches to the same level.
+
+7. Shake each tube in turn, so as to thoroughly mix its contents. Plug
+the mouth of each tube with cotton wool, and place entire set in the
+incubator at 37 deg. C. for one hour.
+
+8. Remove the tubes from the incubator and into each tube pipette 0.1
+c.c. complement (guinea-pig's serum) and replace tubes in incubator at
+37 deg. C. for further period of one hour.
+
+9. Remove the tubes from the incubator, and if complete haemolysis has
+not taken place in every tube, stand on one side, preferably in the ice
+chest, for an hour.
+
+10. Then examine the tubes.
+
+     Complete haemolysis is indicated by a clear red solution,
+     with no deposit of red cells at the bottom of the test-tube.
+
+     Absence of haemolysis is indicated by a clear or turbid
+     colourless fluid, with a deposit of red cells at the bottom
+     of the test-tubes.
+
+The smallest amount of haemolytic serum that has caused complete
+haemolysis is known as the minimal haemolytic dose (_M. H. D._) and if
+haemolysis has occurred in all the tubes down to No. 7--the m. h. d. of
+this particular serum is .005 c.c. = 200 minimal haemolytic doses per
+cubic centimetre. Such a serum is strong enough for experimental work;
+indeed, for many purposes, complete haemolysis down to tube 6 will
+indicate a serum sufficiently strong(= 100 m. h. d. per cubic
+centimetre). If, however, only the first one or two tubes are completely
+haemolysed, this is an indication that the rabbit should receive further
+injections in order to raise the haemolytic power to a sufficiently high
+level.
+
+
+STORAGE OF HAEMOLYSIN.
+
+If, and when the haemolysin content of the rabbit's serum is found to be
+sufficient, destroy the animal by chloroform vapour.
+
+Remove as much of its blood as possible from the heart under aseptic
+precautions into sterilized centrifuge tubes.
+
+Transfer the tubes of blood to the incubator at 37 deg. C. for two
+hours--then centrifugalize thoroughly.
+
+Pipette off the clear serum, and fill in quantities of 1 c.c., into
+small glass ampoules or pipettes, and hermetically seal in the blowpipe
+flame, care being taken to avoid scorching the serum.
+
+Place the ampoules when filled with serum and sealed, in a water-bath at
+56 deg. C. for 30 minutes. This destroys the complement, i. e.,
+inactivates the serum, and at the same time, provided the various
+operations have been carried out under aseptic precautions, ensures its
+sterility. A longer exposure reduces the haemolytic power.
+
+Place the ampoules in a closed metal box and store in the ice chest for
+future use.
+
+FOOTNOTES:
+
+[10] The quantities here given are not absolutely correct. If exactitude
+is essential the student must calculate the amount required by the aid
+of the Percentage Formula, Appendix, page 496.
+
+[11] See Percentage Formula, Appendix, page 496.
+
+
+
+
+XVII. EXPERIMENTAL INOCULATION OF ANIMALS.
+
+
+The use of living animals for inoculation experiments may become a
+necessary procedure in the Bacteriological Laboratory for some one or
+more of the following reasons:
+
+A. ~Determination of Pathogenetic Properties of Bacteria already Isolated
+in Pure Culture~ (see page 315).
+
+The exact study of the conditions influencing the virulence (including
+its maintenance, exaltation and attenuation) of an organism, and precise
+observations upon the pathogenic effects produced by its entrance into,
+and multiplication within the body tissues can obviously only be carried
+out by means of experimental inoculation; whilst many points relating to
+vitality, longevity, etc., can be most readily elucidated by such
+experiments.
+
+B. ~Isolation of Pathogenetic Bacteria.~
+
+Certain highly parasitic bacteria (which grow with difficulty upon the
+artificial media of the laboratory) can only be isolated with
+considerable difficulty from associated saprophytic bacteria when
+cultural methods alone are employed; but if the mixture of parasite and
+saprophytes is injected into an animal susceptible to the action of the
+former, the pathogenic organism can readily be isolated from the tissues
+of the infected animal. The pneumococcus for example occurs in the
+sputum of patients suffering from acute lobar pneumonia, but usually in
+association with various saprophytes derived from the mouth and pharynx.
+The optimum medium for the growth of the pneumococcus, blood agar, is
+also an excellent pabulum for the saprophytes of the mouth, and plate
+cultures are rapidly overgrown by them to the destruction of the more
+delicate pneumococcus. But inoculate some of the sputum under the skin
+of a mouse and three or four days later the pneumococcus will have
+entered the blood stream (leaving the saprophytes at the seat of
+inoculation) and killed the animal. Cultivations made at the post-mortem
+(see page 398) from the mouse's heart blood will yield a pure growth of
+the pneumococcus.
+
+C. ~Identification of Pathogenetic Bacteria.~
+
+The resemblances, morphological and cultural, existing between certain
+pathogenetic bacteria are in some cases so great as to completely
+overwhelm the differences; again the same bacterium may under varying
+conditions assume appearances so different from those regarded as
+typical or normal as to throw doubt on its identity. In each case a
+simple inoculation experiment may decide the point at once. As a
+concrete example may be instanced an autopsy on an animal dead from an
+unknown infection. Cultivations from the heart blood gave a pure growth
+of a typical (capsulated) pneumococcus. Cultivations from the liver gave
+a pure growth of what appeared to be a typical (non-capsulated)
+Streptococcus pyogenes longus. The latter inoculated into a rabbit
+caused the death of the animal from pneumococcic septicaemia, and
+cultures from the rabbit's blood gave a pure growth of a typical
+(capsulated) pneumococcus.
+
+~D. Study of the Problems of Immunity.~
+
+It is only by a careful and elaborate study of the behaviour of the
+animal cell and the body fluids vis-a-vis with the infecting bacterium
+that it becomes possible to throw light upon the complex problem whereby
+the cell opposes successful resistance to the diffusion of the invading
+microbe, or succeeds in driving out the microbe subsequently to the
+occurrence of that diffusion.
+
+At the moment, however, our attention is directed to the first of these
+broad headings, for it is by the application of the knowledge acquired
+in its pursuit that we are able to deal with problems arising under any
+of the remainder.
+
+For whatever purpose the inoculation is performed, it is essential that
+the experiment should be planned to secure the maximum amount of
+information and the minimum of discomfort to the animal used. Every care
+therefore must be taken to ensure that the virus is introduced into the
+exact tissue or organ selected; and the operation itself must be carried
+out with skill and expedition, and under strictly aseptic conditions.
+
+In the course of inoculation studies many instances of natural immunity,
+both racial and individual, will be met with; but it must be recollected
+that natural immunity is relative only and never absolute, and care be
+taken not to label an organism as _non-pathogenic_ until many different
+methods of inoculation have been performed upon different species of
+animals, combined when necessary with various procedures calculated to
+overcome any apparent immunity, and have invariably given negative
+results.
+
+In some countries experiments upon animals are only permitted under
+direct license from the Government, and then only within premises
+specially licensed for the purpose. In England this license is in the
+grant of the Home Secretary, and confers the permission to experiment
+upon animals under general anaesthesia, provided that after the
+experiment is completed the animal must be destroyed before regaining
+consciousness. If it is intended to carry out simple hypodermic
+inoculations and superficial venesections, Certificate A, granting this
+specific permission and dispensing with the necessity for general
+anaesthesia must be obtained _in addition to the license_; whilst if the
+inoculation entails more extensive operative procedures, and it is
+necessary to observe the subsequent course of the infection, should such
+occur, the license must be _coupled with Certificate B_--since this
+certificate removes the compulsion to destroy the animal whilst under
+the anaesthetic. Further special certificates and combinations of
+certificates are required if cats, dogs, horses, asses or cattle are to
+be the subjects of experiment. Under every certificate it is expressly
+stipulated that if the animal shows signs of pain it must be destroyed
+immediately.
+
+The animals generally employed in the study of the pathogenic properties
+of the various micro-organisms are:
+
+    _Cold Blooded._       _Warm Blooded._         _Hot Blooded._
+        Frog.                 Mouse.                  Fowl.
+        Toad.                 Rat.                    Pigeon.
+        Lizard.               Guinea pig.
+                              Rabbit.
+                              Monkey.
+
+~Preparation.~--Before inoculation, the experimental animals should be
+carefully examined, to avoid the risk of employing such as are already
+diseased: since it must be remembered that in a state of nature, as well
+as in captivity, the animals employed for laboratory inoculations are
+subject to infection by various animal and vegetable parasites, and in
+some instances such infection presents no symptoms which are obvious to
+the casual examination; the sex should be noted, the weight recorded,
+and the rectal temperature taken. The remaining items of importance are
+the time of the inoculation, the material that is inoculated, and the
+method of inoculation, and finally under what authority the experiment
+is performed. In the author's laboratory these data are entered upon a
+pink card which forms part of a card index system. The card further
+provides space for notes on the course of the resulting infection, and
+carries on the reverse the weight and temperature chart (Figs. 164 and
+165).
+
+[Illustration: Fig. 164.--Front of inoculation card.]
+
+~Preliminary Inspection and Examination.~--The preliminary examination
+should comprise observation of the animal at rest and in motion; the
+appearance of the fur, feathers or scales, inspection of the eyes, and
+of external orifices of the body; tactile examination of the body and
+limbs, and palpation of the groins and abdomen; and in many cases the
+microscopical examination of fresh and stained blood-films.
+
+Some of the commoner forms of naturally acquired infection may be
+briefly mentioned, without however touching upon the various fleas, lice
+and ticks which at times infect the ordinary laboratory animals.
+
+[Illustration: FIG. 165.--Back of inoculation card.]
+
+~The Rabbit~, particularly in captivity, is subject to attacks of Psoric
+Acari, and the infection is readily transmitted to rabbits in
+neighbouring cages and also to guinea pigs, but not to rats and mice.
+One species (_Sarcoptes minor_ var. _cuniculi_) gives rise to the
+ordinary mange. The infection first shows itself as thick yellowish
+scales and crusts around the nose, mouth and eyes, spreads to the bases
+and outer surfaces of the ears (never to the inside of the concha), to
+the fore and hind legs and into the groins and around the genitals. The
+acari can be readily demonstrated microscopically in scrapings of the
+skin, treated with liquor potassae. Another form of scabies (due to
+Psoroptes _communis cuniculi_) commences at the bottom of the concha,
+which is filled with whitish-yellow masses consisting of dried crusts,
+scales, faeces, and dead acari. The base of the ear is hard and swollen,
+and lifting the animal by the ears--as is usually done--gives rise to
+considerable pain; indeed this symptom may be the one which first
+attracts attention to an infection, which causes progressive wasting and
+terminates in death. A mixed infection--sarcoptic plus psorotic
+acariasis--is sometimes seen.
+
+If it is decided to try and save animals suffering from infection by
+these parasites, they must be segregated, the scabs carefully cleaned
+from the infected areas and the denuded surfaces washed with 5 per cent.
+solution of Potassium persulphate (a few drops being allowed to run into
+the concha), or with a preparation containing equal parts of soft
+paraffin and vaseline with a few drops of lysol. This treatment should
+be repeated daily until the acarus is destroyed and the animal has
+regained its normal condition. The cages should be disinfected and all
+neighbouring animals carefully examined, and any which show signs of
+infection should be treated in a similar manner. Favus also attacks the
+rabbit, and the typical spots are first noted around the base of the
+ear.
+
+Infection by _Coccidium oviforme_ is very common, without however
+presenting any symptoms by which the infection may be recognised.
+Usually the condition is only noted post-mortem, when the liver is found
+to be studded with numerous cascating tubercles, which on examination
+prove to be cystic areas crowded with coccidia. Sometimes too the liver
+of a rabbit dead from some intentional or accidental bacterial infection
+is found at the post-mortem to be marked by fine yellowish streaks and
+small tubercles due to the embryos of _Taenia serrata_, while the cystic
+form (_Cysticercus pisiformis_) is often noted free in the peritoneal
+cavity, or invading the mesentery.
+
+Abscess formation from infection with ordinary pyogenic bacteria occurs
+naturally in the rabbit, and frequently the animal house of a laboratory
+is decimated by an infective septicaemia due to _B. cuniculicida_.
+
+The ~Mouse~ and ~Rat~ suffer from septicaemia, and from the cysticercus
+form of _Taenia murina_; the cystic form (_Cysticercus fasciolaris_) of
+_T. crassicollis_ has its habitat in their livers. These small rodents are
+frequently infected with scabies, but if freely provided with clean
+straw will clean themselves by rubbing through it. The mouse is also
+attacked by favus, and the rat is often infected with _Trypanosoma
+Lewisi_.
+
+The ~Guinea pig~, like the rabbit, suffers from scabies and coccidiosis.
+In addition it is often naturally infected with _B. tuberculosis_, and
+it is a wise precaution to test animals as soon as they reach the
+laboratory by injecting Koch's Old Tuberculin--0.5 c.c. causing death in
+the tuberculous cavy within 48 hours.
+
+The ~Monkey~ is naturally prone to tuberculosis, and should be injected
+with 1 c.c. Old Tuberculin on arrival in the laboratory. The tissues of
+the monkey also serve as the habitat for a Nematode worm parasitic in
+cattle (_Oesophagostoma inflatum_) resembling the Anchylostomum, and
+this parasite frequently bores through the intestinal wall, and
+provokes the formation of small cysts in the immediately adjacent
+mesentery. The presence of these cysts may give rise to considerable
+speculation at the post-mortem.
+
+The ~Pigeon~ may be infected by _Haemosporidia_, and its blood show the
+presence of halteridia. This bird may also be the subject of a bacterial
+infection known as pigeon diphtheria; while the fowl may be subject to
+scabies and ringworm, or suffer from fowl cholera or fowl
+septicaemia--infections due to members of the haemorrhagic septicaemia
+group.
+
+~Weighing.~--The larger animals are most conveniently weighed in a decimal
+scale provided with a metal cage for their reception instead of the
+ordinary pan (Fig. 166). Mice and rats are weighed in a modification of
+the letter balance, weighing to 250 grammes, which has a conical wire
+cage, (carefully counterpoised) substituted for its original pan (Fig.
+167).
+
+[Illustration: FIG. 166.--Rabbit scales.]
+
+~Temperature.~--To take the rectal temperature of any of the laboratory
+animals, the animal should be carefully and firmly held by an assistant.
+Introduce the bulb of an ordinary clinical thermometer, well greased
+with vaseline, just within the sphincter ani. Allow it to remain in this
+position for a few seconds, and then push it on gently and steadily
+until the entire bulb and part of the stem, as far as the constriction,
+have passed into the rectum. Three to five minutes later, the time
+varying of course with the sensibility of the thermometer used, withdraw
+the instrument and take the reading. The thermometers employed for
+recording temperature should be verified from time to time by comparison
+with a standard Kew certified Thermometer kept in the laboratory for
+that purpose.
+
+[Illustration: FIG. 167.--Mouse scales]
+
+~Cages.~--During the period which elapses between inoculation and death,
+or complete recovery, the experimental animals must be kept in suitable
+receptacles which can easily be kept clean and readily disinfected.
+
+The _mouse_ is usually stored in a glass jar (Fig. 168) 11 cm. high and
+11 cm. in diameter, closed by a wire gauze cover which is weighted with
+lead or fastened to the mouth of the jar by a bayonet catch. A small
+oblong label, 5 cm. by 2.5 cm., sand-blasted on the side of the
+cylinder, is a very convenient device as notes made upon this with an
+ordinary lead pencil show up well and only require the use of a damp
+cloth to remove them (Fig. 168).
+
+The _rat_ is kept under observation in a glass jar similar, but larger,
+to that used for the mouse.
+
+[Illustration: FIG. 168.--Mouse jar.]
+
+[Illustration: FIG. 169.--Tripod.]
+
+A layer of sawdust at the bottom of the jar absorbs any moisture and
+cotton-wool or paper shavings should be provided for bedding. The food
+should consist of bran and oats with an occasional feed of
+bread-and-milk sop.
+
+The use of a metal tripod, on the platform of which are soldered two
+small cups for the reception of the food, inside the cage, prevents
+waste of food or its contamination with excreta (Fig. 169).
+
+After use the jars and tripods are sterilised either by chemical
+reagents or by autoclaving.
+
+The _rabbit_ and the _guinea-pig_ are confined in cages of suitable
+size, made entirely of metal (Fig. 170). The sides and top and bottom
+are of woven wire work; beneath the cage is a movable metal tray filled
+with sawdust, for the reception of the excreta. The cage as a whole is
+raised from the ground on short legs. The sides, etc., are generally
+hinged so that the cage packs up flat, for convenience of storing and
+also of sterilising.
+
+The ordinary rat cage, a rectangular wire-work box, 30 cm. from front to
+back, 20 cm. wide, and 14 cm. high, makes an excellent cage for
+guinea-pigs if fitted with a shallow zinc tray, 35 cm. by 24 cm., for it
+to stand upon.
+
+[Illustration: FIG. 170.--Metal rabbit rage.]
+
+A plentiful supply of straw should be provided for bedding and the food
+should consist of fresh vegetables, cabbage leaves, carrot and turnip
+tops and the like for the morning meal and broken animal biscuits for
+the evening meal. Occasionally a little water may be placed in the cage
+in an earthenware dish.
+
+The tray which receives the dejecta should be cleaned out and supplied
+with fresh sawdust each day, and the soiled sawdust, remains of food,
+etc., should be cremated.
+
+These cages are sterilised after use either by autoclaving or spraying
+with formalin.
+
+As ~animal inoculation~ is purely a surgical operation, the necessary
+instruments will be similar to those employed by the surgeon, and, like
+them, must be sterile. In the performance of the inoculation strict
+attention must be paid to asepsis, and suitable precautions adopted to
+guard against accidental contamination of the material to be introduced
+into the animal. In addition, the hands of the operator should be
+carefully disinfected.
+
+The list of apparatus used in animal inoculations given below comprises
+practically everything needed for any inoculation. Needless to remark,
+all the apparatus will never be required for any one inoculation.
+
+[Illustration: FIG. 171.--Hypodermic syringe with finger rests.]
+
+     Apparatus Required for Animal Inoculation:
+
+     1. Water steriliser (_vide_ page 33). It is also convenient
+     to have a second water steriliser, similar but smaller (23
+     by 7 by 5 cm.), for the sterilisation of the syringes.
+
+     2. Injection syringe. The best form is one of the ordinary
+     hypodermic pattern, 1 c.c. capacity graduated in twentieths
+     of a cubic centimeter (0.05 c.c.), fitted with finger rests,
+     but with the leather washers and the packing of the piston
+     replaced by those made of asbestos (Fig. 171). The
+     instrument must be easily taken to pieces, and spare parts
+     should be kept on hand to replace accidental breakage or
+     loss. Other useful syringes are those of 2 c.c., 5 c.c., 10
+     c.c., and 20 c.c. capacity. A good supply of needles must be
+     kept on hand, both sharp-pointed and with blunt ends. To
+     sterilise the syringe, fill it with water, loosen the
+     packing of the piston and all the screw joints, place it in
+     the steriliser and boil for at least five minutes. Disinfect
+     the syringe _after use_, in a similar manner. The needles,
+     which are exceedingly apt to rust after being boiled, should
+     be stored in a pot of absolute alcohol when not in use.
+
+     3. Operating table.
+
+     4. Surgical instruments. Sterilise these before use by
+     boiling, and disinfect them _after use_ by the same means.
+     Wipe perfectly dry immediately after the disinfection is
+     completed.
+
+     Scissors, probe and sharp-pointed.
+
+     Dissecting forceps of various patterns.
+
+     Pressure forceps.
+
+     Retractors (small self retaining Fig. 172).
+
+     Aneurism needles, sharp and blunt.
+
+     Scalpels, } Keratomes, } with metal handles. Trephines, }
+
+     Michel's steel clips and special forceps for applying the
+     same. These small steel clips enable the operator to easily
+     and rapidly close skin incisions and are most satisfactory
+     for animal operations.
+
+     Surgical needles.
+
+     Needle holder.
+
+     Soft rubber catheters, various sizes.
+
+     Gum elastic oesophageal bougies with connection to fit
+     syringe.
+
+[Illustration: FIG. 172. Small self retaining retractors.]
+
+5. Anaesthetic.
+
+(a) General: The safest general anaesthetic for animals is an A. C. E.
+mixture, freshly prepared, containing by volume alcohol 1 part,
+chloroform 2 parts, ether 6 parts, and should be administered on a
+"cone" formed by twisting up one corner of a towel and placing a wad of
+cotton-wool inside it, or from a saturated cotton-wool pad packed into
+the bottom of a small beaker.
+
+(b) Local:
+
+    1. Cocaine hydrochloride, 2 per cent. in adrenalin 1 per mille
+       solution.
+    2. Beta-eucaine, 2 per cent. in adrenalin, 1 per mille solution.
+    3. Ethyl chloride jet.
+
+6. Sterile glass capsules of various sizes.
+
+7. Cases of sterile pipettes { 10 c.c. (in tenths of a cubic centimetre).
+                             {  1 c.c. (in hundredths of a cubic
+                                        centimetre).
+
+8. Flasks (75 c.c.) containing sterilised normal saline solution (or
+sterile bouillon).
+
+9. Sterilised cotton-wool. Cotton-wool (absorbent) is packed loosely in
+a copper cylinder similar to that used for storing capsules, and
+sterilised in the hot-air oven.
+
+10. Sterilised gauze. Gauze is sterilised in the same way as
+cotton-wool.
+
+11. Sterilised silk and catgut for sutures. These are sterilised, as
+required, by boiling for some ten minutes in the water steriliser.
+
+12. Flexible collodion (or compound tincture of benzoin).
+
+13. Grease pencil.
+
+14. Tie-on celluloid labels, to affix to the cages.
+
+15. Razor.
+
+16. Small pot of warm water.
+
+17. Liquid soap. Liquid soap is prepared as follows: Measure out 100
+grammes of soft soap and add to 500 c.c. of 2 per cent. lysol solution
+in a large glass beaker; dissolve by heating in a water-bath at about
+90 deg. C. Bottle and label "Liquid Soap."
+
+18. In place of the liquid soap and razor it is sometimes convenient to
+use a Depilatory powder.
+
+    Barium sulphide     1 part
+    Rice starch         3 parts
+
+Dust the powder thickly over the area to be denuded of hair, sprinkle
+with water and mix into a thin paste _in situ_; allow the paste to act
+for three minutes, then scrape off with a bone spatula--the hair comes
+away with the paste and leaves a perfectly bare patch. This process is
+preferably carried out, the day previous to the operation.
+
+~Material Utilised for Inoculation.~--The material inoculated may be
+either--
+
+1. Cultures of bacteria--grown in fluid media, or on solid media.
+
+2. Metabolic products of bacterial activity--e. g., toxins in
+solution.
+
+3. Pathological products (fluid secretions and excretions, solid
+tissues).
+
+~The Preparation of the Inoculum.~--
+
+(a) _Cultivations in Fluid Media._--
+
+1. Flame the plug of the culture tube.
+
+2. Remove the plug and flame the mouth of the tube.
+
+3. Slightly raise the lid of a sterile capsule, insert the mouth of the
+culture tube into the aperture and pour some of the cultivation into the
+capsule.
+
+4. Remove the mouth of the culture tube from the capsule, replace the
+lid of the latter, flame the mouth of the tube, and replug.
+
+5. Remove the syringe from the steriliser, squirt out the water from its
+interior, and allow to cool.
+
+6. Raise the lid of the capsule sufficiently to admit the needle of the
+syringe and draw the required amount of the cultivation into the barrel
+of the syringe.
+
+(Or, remove a definite measured quantity of the cultivation directly
+from the tube or flask by means of a sterile graduated pipette,
+discharge the measured amount into a sterile capsule, and fill into the
+syringe; or take up the required quantity of the cultivation directly
+into the graduated syringe from the tube or flask.)
+
+[Illustration: FIG. 173.--Conical separatory funnel, fitted for
+injection of fluid cultivations.]
+
+If it is necessary to introduce a large bulk of fluid into the animal,
+the cultivation should be transferred with aseptic precautions, to a
+sterile separatory funnel, preferably of the shape shown in figure 173,
+and graduated if necessary. This is supported on a retort stand and
+raised sufficiently above the level of the animal to be injected, so as
+to secure a good "fall." A piece of sterilised rubber tubing of suitable
+length, fitted with an injection needle and provided with a screw clamp,
+is now attached to the nozzle of the funnel and the operation completed
+according to the requirements of the particular case.
+
+This method is quite satisfactory when the injection is made into the
+pleural or abdominal cavities or directly into a vein but if the
+injection has to be made into the subcutaneous tissue the "fall" may not
+be sufficient to force the fluid in. In this case it will be necessary
+to transfer the culture to a sterile wash-bottle and fasten a rubber
+hand bellows to the air inlet tube (interposing an air filter) and
+attach the tubing with the injection needle to the outlet tube (Fig.
+174). By careful use sufficient force can be obtained to drive the
+injection in.
+
+(b) _Cultivations on Solid Media (e. g., Sloped Agar)._--
+
+1. By means of a sterile graduated pipette introduce a suitable small
+quantity of sterile bouillon (or sterile normal saline solution) into
+the culture tube.
+
+[Illustration: FIG. 174.--Arrangement of pressure injection apparatus.]
+
+2. With a sterile platinum loop or spatula scrape the bacterial growth
+off the surface of the medium, and emulsify it with the bouillon. It
+then becomes to all intents and purposes a fluid inoculum.
+
+3. Pour the emulsion into a sterile capsule and fill the syringe
+therefrom.
+
+(c) _Toxins._--Prepared by previously described methods (_vide_ page
+318), are manipulated in a similar manner to cultivations in fluid
+media.
+
+(d) _Pathological Products._--Fluid secretions, excretions, etc., such
+as serous exudation, pus, blood, etc., are treated as fluid
+cultivations; but if the material is very thick or viscous, a small
+quantity of sterile bouillon or normal saline solution may be used to
+dilute it, and thorough incorporation effected by the help of a sterile
+platinum rod.
+
+Solid tissues, such as spleen, lymph glands, etc., may be divided into
+small pieces by sterile instruments and rubbed up in a sterilised agate
+mortar (using an agate pestle), with a small quantity of sterile
+bouillon, and the syringe filled from the resulting emulsion.
+
+[Illustration: FIG. 175.--Holding rabbit for shaving.]
+
+If it is desired to inoculate tissue _en masse_, remove from the
+material a small cube of 1 or 2 mm. and introduce it into a wound made
+by sterile instruments in a suitable situation, and occlude the wound by
+means of Michel's steel clips and a sealed dressing.
+
+~Method of Securing Animals During Inoculation.~--
+
+For the majority of inoculations, especially when no anaesthetic is
+administered, it is customary to employ an assistant to hold the animal
+(see Fig. 175).
+
+If working single handed Voge's holder for guinea-pigs, is a useful
+piece of apparatus the method of using which is readily seen from the
+accompanying figures (Figs. 176, 177).
+
+The instrument itself consists of a hollow copper cylinder, one end of
+which is turned over a ring of stout copper wire, and from this open end
+a slot is cut extending about half way along one side of the cylinder.
+The opposite end is closed by a "pull-off" cap and is perforated around
+its edge by a row of ventilating holes, which correspond with holes cut
+in the rim of the cap. In the event of the animal resisting attempts to
+remove it from the holder backwards, this cap is taken off and the
+holder placed on the table and the guinea-pig allowed to walk out.
+
+[Illustration: FIG. 176.--Taking guinea-pig's temperature.]
+
+To provide for different-sized animals, two sizes of this holder will be
+found useful:
+
+1. Length, 16 cm.; breadth, 6 cm.; size of slot, 8 cm. by 2.5 cm.
+
+2. Length, 20 cm.; breadth, 8 cm.; size of slot, 10 cm. by 2.5 cm.
+
+A convenient holder for mice and even small rats is shown in figure 178,
+the tail being securely held by the spring clip. Needless to say, the
+holder should be entirely of metal, and the wire cage detachable and
+easily renewed.
+
+[Illustration: FIG. 177.--Voge's holder.]
+
+When the animal is anaesthetised, it is more convenient to secure it
+firmly to some simple form of operating table, such as Tatin's (Fig.
+179), which will accommodate rabbits, guinea-pigs, and rats: or to the
+more elaborate table devised by the author (Fig. 180).
+
+[Illustration: FIG. 178.--Mouse holder.]
+
+[Illustration: FIG. 179.--Tatin's operation table.]
+
+~Operation Table.~--This is a table of the "aseptic" type, composed of
+steel tubing, nickel-plated or enamelled. The table-top frame is
+sufficiently large to accommodate rabbits, dogs and monkeys; and is
+supported upon telescopic uprights, so that it is adjustable as to
+height; in its long axis it can be inclined (at either end) to 45 deg.
+from the horizontal. Further it can be completely rotated about its long
+axis. The table-top itself is composed of a sheet of copper wire gauze
+loosely suspended from the long sides of the tubular frame. The
+slackness of the gauze bed permits of an india rubber hot water bottle,
+or an electrotherm being placed under the animal, and if during the
+course of an experiment it is necessary to reverse the animal, the
+table-top frame is completely rotated, the device adopted for suspending
+the gauze is detached and the gauze reversed also, so that it again
+supports the animal from below.
+
+[Illustration: FIG. 180.--Author's operating table[12].]
+
+
+METHODS OF INOCULATION.
+
+The following methods of inoculation apply more particularly to the
+rabbit, but from them it will readily be seen what modifications in
+technique, if any, are necessary in the case of the other experimental
+animals.
+
+~1. Cutaneous Inoculation.~--(_Anaesthetic, none._)
+
+1. Have the animal firmly held by an assistant (or secured to the
+operating table).
+
+2. Apply the liquid soap to the fur, over the area selected for
+inoculation, with a wad of cotton-wool, and lather freely by the aid of
+warm water; shave carefully and thoroughly; or apply the depilatory
+powder.
+
+3. Wash the denuded area of skin thoroughly with 2 per cent. lysol
+solution.
+
+4. Wash off the lysol with ether and allow the latter to evaporate.
+
+5. Make numerous short, parallel, superficial incisions with the point
+of a sterile scalpel.
+
+6. When the oozing from the incisions has ceased, rub the inoculum into
+the scarifications by means of the flat of a scalpel blade, or a sterile
+platinum spatula.
+
+7. Cover the inoculated area with a pad of sterile gauze secured _in
+situ_ by strips of adhesive plaster or by sealing down the edges of the
+gauze with collodion.
+
+8. Release the animal, place it in its cage, and affix a label upon
+which is written:
+
+    (a) Distinctive name or number of the animal.
+    (b) Its weight.
+    (c) Particulars as to source and dose of inoculum.
+    (d) Date of inoculation.
+
+~2. Subcutaneous Inoculation.~--
+
+(a) _Fluid Inoculum._--(_Anaesthetic, none._)
+
+Steps 1-4. As for cutaneous inoculation.
+
+5. Pinch up a fold of skin between the forefinger and thumb of the left
+hand; take the charged hypodermic syringe in the right hand, enter the
+needle into a ridge of skin raised by the left finger and thumb, and
+push it steadily onward until about 2 cm. of the needle are lying in the
+subcutaneous tissue. Now release the grasp of the left hand and slowly
+inject the fluid contained in the syringe.
+
+6. Withdraw the needle, and at the same moment close the puncture with a
+wad of cotton wool, to prevent the escape of any of the inoculum. The
+injected fluid, unless large in amount, will be absorbed within a very
+short time.
+
+7. Label, etc.
+
+(b) _Solid Inoculum.--(Anaesthetic, none; or Ethyl chloride spray.)_
+
+Steps 1-4. As for cutaneous inoculation.
+
+5. Raise a small fold of skin in a pair of forceps, and make a small
+incision through the skin with a pair of sharp-pointed scissors or with
+the point of a scalpel.
+
+6. Insert a probe through the opening and push it steadily onward in the
+subcutaneous tissue, and by lateral movements separate the skin from the
+underlying muscles to form a funnel-shaped pocket with its apex toward
+the point of entrance.
+
+7. By means of a pair of fine-pointed forceps introduce a small piece of
+the inoculum into this pocket and deposit it as far as possible from the
+point of entrance.
+
+[Illustration: FIG. 181.--Glass tube syringe for subcutaneous "solid"
+inoculation.]
+
+Or, improvise a syringe by sliding a piece of glass rod (to serve as a
+piston) into the lumen of a slightly shorter length of glass tubing and
+secure in position by a band of rubber tubing. Sterilise by boiling.
+Withdraw the rod a few millimetres and deposit the piece of tissue
+within the orifice of the tube, by means of sterile forceps. Now pass
+the tube into the depths of the "pocket," push on the glass rod till it
+projects beyond the end of the tube, and withdraw the apparatus, leaving
+the tissue behind in the wound.
+
+8. Close the wound in the skin with Michel's clips and a dressing of
+gauze sealed with collodion (or Tinct. benzoin).
+
+9. Label, etc.
+
+~3. Intramuscular.~--
+
+(a) _Fluid Inoculum.--(Anaesthetic, none.)_
+
+Steps 1-4. As for cutaneous inoculation.
+
+5. Steady the skin over the selected muscle or muscles with the slightly
+separated left forefinger and thumb.
+
+6. Thrust the needle of the injecting syringe boldly into the muscular
+tissue and inject the inoculum slowly.
+
+7. Label, etc.
+
+(b) _Solid Inoculum.--(Anaesthetic, A. C. E.)_
+
+1. Secure the animal to the operation table and anaesthetise.
+
+2. Shave and disinfect the skin at the seat of operation.
+
+3. Surround the field of operation by strips of gauze wrung out in 2 per
+cent. lysol solution.
+
+4. Incise skin, aponeurosis, and muscle in turn.
+
+5. Deposit the inoculum in the depths of the incision.
+
+6. Close the wound in the muscle with buried sutures and the cutaneous
+wound with either continuous or interrupted sutures or with Michel's
+steel clips.
+
+7. Apply a sealed dressing of gauze and collodion.
+
+8. Remove the animal from the operating table.
+
+9. Label, etc.
+
+
+~4. Intraperitoneal.~--
+
+(a) _Fluid Inoculum.--(Anaesthetic, none.)_
+
+Steps 1-4. As for cutaneous inoculation. Shave a fairly broad transverse
+area, stretching from flank to flank.
+
+5. Place the left forefinger on one flank and the thumb on the opposite,
+and pinch up the entire thickness of the abdominal parietes in a
+triangular fold. Now, by slipping the peritoneal surfaces (which are in
+apposition) one over the other, ascertain that no coils of intestine are
+included in the fold.
+
+6. Take the syringe in the right hand and with the needle transfix the
+fold near its base (Fig. 182).
+
+7. Now release the fold, but hold the syringe steady; as the parietes
+flatten out, the point of the needle is left free in the peritoneal
+cavity (see Fig. 183).
+
+[Illustration: FIG. 182.--Intraperitoneal inoculation--fluid.]
+
+8. Inject the fluid from the syringe.
+
+9. Label, etc.
+
+[Illustration: FIG. 183.--Section of abdominal wall, etc., showing point
+of needle lying free in the peritoneal cavity above the coils of
+intestine.]
+
+Second Method:
+
+Steps 1-4. As in the first method.
+
+5. Anaesthetise a small selected area of skin by spraying it with ethyl
+chloride.
+
+6. Heat platinum searing wire (0.5 mm. wire, twisted to the shape
+indicated in figure 184, mounted in an aluminium handle) to redness, and
+with it burn a hole through the anaesthetic area of skin and abdominal
+muscle down to, but not through, the visceral peritoneum.
+
+7. Fix a blunt-ended needle on to the charged syringe, and by pressing
+the rounded end firmly against the peritoneum it can easily be pushed
+through into the peritoneal cavity.
+
+8. Inject the fluid from the syringe.
+
+9. Label, etc.
+
+This method is especially useful when it is desired to collect samples
+of the peritoneal fluid from time to time during the period of
+observation, as fluid can be removed from the peritoneal cavity, at
+intervals, through this aperture in the abdominal parietes, by means of
+a sterile capillary pipette.
+
+[Illustration: FIG. 184.--Platinum wire for burning hole through
+parietes.]
+
+(b) _Solid Inoculum_ (or the implantation of capsules containing fluid
+cultivations).--(_Anaesthetic, A. C. E._)
+
+1. Anaesthetise the animal and secure it to the operating table.
+
+2. Shave a large area of the abdominal parietes.
+
+3. Make an incision through the skin in the middle line about 2 cm. in
+length, midway between the lower end of the sternum and the pubes.
+
+4. Divide the aponeuroses between the recti upon a director.
+
+5. Divide the peritoneum upon a director.
+
+6. Introduce the inoculum into the peritoneal cavity.
+
+7. Close the peritoneal cavity with Lembert's sutures.
+
+8. Close the skin and aponeurosis incisions together with interrupted
+sutures or Michel's steel clips, and apply a sealed dressing.
+
+9. Release the animal from the operating table.
+
+10. Label, etc.
+
+Suitable sacs may be readily prepared by either of the following
+methods:
+
+A. ~Collodion Sacs.~
+
+1. Dip a small test-tube (5 by 0.5 cm.), bottom downward, into a beaker
+of collodion, and dry in the air; repeat this process three or four
+times.
+
+2. Dip the tube, with its coating of collodion, alternately into a
+beaker of alcohol and one of water. This loosens the collodion and
+allows it to be peeled off in the shape of a small test-tube.
+
+3. Take a 20 cm. length of glass tubing, of about the diameter of the
+test-tube used in forming the sac, and insert one end into the open
+mouth of the sac.
+
+4. Suspend the glass tube with attached sac, inside a larger test-tube,
+by packing cotton-wool in the mouth of the test-tube around the glass
+tubing, and place in the incubator at 37 deg. C. for twenty-four hours.
+When removed from the incubator, the sac will be firmly adherent to the
+extremity of the glass tubing.
+
+5. Plug the open end of the glass tubing with cotton-wool, and sterilise
+the test-tube and its contents in the hot-air oven.
+
+To use the sac, remove the plug from the glass tubing, partly fill the
+sac with cultivation to be inoculated, by means of a sterile capillary
+pipette, and replug the tubing. When the abdominal cavity has been
+opened, remove the tubing and attached sac from the protecting
+test-tube, close the sac by tying a sterilised silk thread tightly
+around it a little below the end of the glass tubing, and separate it
+from the tubing by cutting through the collodion above the ligature, and
+the sac is ready for insertion in the peritoneal cavity.
+
+B. ~Celloidin Sacs~ (_Harris_).
+
+_Materials Required._
+
+     Quill glass tubing.
+
+     Gelatine capsules such as pharmacists prepare for the
+     exhibition of bulky powders.
+
+     Various grades of celloidin, thick and thin, in wide-mouthed
+     bottles.
+
+1. Take a piece of quill glass tubing some 4 cm. long by 5 mm. diameter;
+heat one end in the bunsen flame.
+
+2. Thrust the heated end of the tube just through one end of a gelatine
+capsule and allow it to cool (Fig. 185).
+
+3. Remove any gelatine from the lumen of the tube with a heated platinum
+needle; paint the joint between capsule and tube with moderately thick
+celloidin and allow to dry.
+
+[Illustration: FIG. 185.--Making celloidin capsules.]
+
+4. Dip the capsule into a beaker containing thin celloidin, beyond the
+junction with the glass and after removal rotate it in front of the
+blowpipe air blast to dry it evenly. Repeat these manoeuvres until a
+sufficiently thick coating is obtained.
+
+5. Apply thick celloidin to the tube-capsule joint, the opposite end of
+the capsule, and the line of junction of the capsule with its cap; dry
+thoroughly.
+
+6. With a teat pipette fill the capsule (through the attached tube) with
+hot water, and stand the capsule in a beaker of boiling water for a few
+minutes to melt the gelatine.
+
+7. Remove the solution of gelatine from the interior of the celloidin
+case with a pipette.
+
+8. Fill the sac with nutrient broth and place it, _glass tube downward_,
+in a tube containing sufficient sterile nutrient broth to cover the sac
+to the depth of 1 cm. Plug the tube and sterilise in the steamer in the
+usual manner.
+
+9. To prepare the sac for use, empty it out of the broth tube into a
+sterile glass dish.
+
+10. Grasp the tube near its junction with the sac in the jaws of sterile
+forceps, and with a teat pipette remove sufficient of the contained
+broth to leave a small space in the sac. Introduce the inoculum in the
+form of an emulsion by means of another pipette.
+
+11. Still holding the tube in the forceps, draw it out and seal off near
+the sac in the blowpipe flame.
+
+12. When cool wash the sac in sterile water, then transfer to a tube of
+nutrient broth and incubate over night to determine its impermeability
+to bacteria.
+
+13. If the broth outside the sac remains sterile, insert the sac in the
+peritoneal cavity of the experimental animal.
+
+~5. Intracranial.~--(_Anaesthetic, A. C. E._)
+
+[Illustration: FIG. 186.--Guarded trephine.]
+
+_Trephines and Surgical Engine._--The most useful instrument for
+intracranial operations upon animals is the small nasal trephine
+(Curtis) having a tooth cutting circle of 7 mm. The addition of an
+adjustable collar guard--secured by a screw--prevents accidental
+laceration of the dura mater or brain substance[13] (Fig. 186). This
+size is suitable for monkeys, dogs, cats and large rabbits. Other
+smaller sizes which will be found useful for guinea pigs and other small
+animals cut circles of 6 and 4 mm.; for very small animals--young guinea
+pigs and rats--a small dental drill or screw will make a sufficiently
+large hole to admit the syringe needle. The trephine can be set in
+ordinary metal handles and rotated by hand, but a surgical engine of
+some kind is much preferable on the score of rapidity and safety to the
+animal. The Guy's electrical Dental engine[14] (Fig. 187) which can be
+connected to a lamp socket or wall plug, and is operated by a foot
+switch, although inexpensive is eminently satisfactory.
+
+     NOTE.--A fine dental drill attached to the dental engine
+     renders the manufacture of aluminium handles needles (see
+     page 71) quite an easy matter.
+
+
+
+(a) _Subdural._
+
+1. Anaesthetise the animal and secure it to the operating table, dorsum
+uppermost.
+
+2. Shave a portion of the scalp immediately in front of the ears.
+
+[Illustration: FIG. 187.--Guy's electrical dental engine.]
+
+3. Mark out with a sharp scalpel a crescentic flap of skin muscle, etc.,
+convexity forward, commencing 0.5 cm. in front of the root of one ear
+and terminating at a similar spot in front of the other ear. Reflect the
+marked flap.
+
+4. Make a corresponding incision through the periosteum and raise it
+with a blunt dissector.
+
+5. With a small trephine (diameter 6 mm.) remove a circular piece of
+bone from the parietal segment. The centre of the trephine hole should
+be at the intersection of the median line and a line joining the
+posterior canthi (Fig. 188).
+
+6. Introduce the inoculum by means of a hypodermic syringe, perforating
+the dura mater with the needle and depositing the material immediately
+below this membrane, at the same time taking care to avoid injuring the
+sinuses.
+
+7. Turn back the flap of skin and secure it in position with Michel's
+steel clips.
+
+8. Dress with sterile gauze and wool and seal the dressing with
+collodion.
+
+9. Label, etc.
+
+(b) _Intracerebral._--This inoculation is performed precisely as for
+subdural save in step 6 the needle after perforating the dura mater is
+pushed onward into the substance of one or other cerebral hemispheres
+before the contents are ejected.
+
+[Illustration: FIG. 188.--Intracranial inoculation of rabbit. The circle
+indicates the situation of the trephine hole.]
+
+~6. Intraocular.~--
+
+(a) _Fluid Inoculum._--(_Anaesthetic, cocaine._)
+
+1. Instil a few drops of a sterile solution of cocaine, and repeat the
+instillation in two minutes.
+
+2. Five minutes later have the animal firmly held by an assistant as in
+intravenous injection (see Fig. 189), the head being steadied by the
+assistant's hands.
+
+3. Select two needles to accurately fit the same syringe and sterilise.
+
+4. Attach one needle to the syringe and take up the required dose of
+inoculum and remove the needle.
+
+5. Steady the eye with fixation forceps; then pierce the cornea with the
+other syringe needle and allow the aqueous to escape through the needle.
+
+6. Without removing the needle from the cornea attach the syringe and
+make the injection into the anterior chamber.
+
+7. Irrigate the conjunctival sac with sterile saline solution.
+
+8. Label, etc.
+
+(b) _Solid Inoculum._--(_Anaesthetic, A. C. E._)
+
+1. Anaesthetise the animal and secure it firmly to the operating table.
+
+2. Irrigate the conjunctival sac thoroughly with sterile saline
+solution.
+
+3. Make an incision through the upper quadrant of the cornea into the
+anterior chamber by means of a triangular keratome.
+
+4. Separate the lips of the corneal wound with a flexible silver
+spatula; seize the solid inoculum in a pair of iris forceps, introduce
+it through the corneal wound, and deposit it on the anterior surface of
+the iris; withdraw the forceps.
+
+5. Again irrigate the sac and the surface of the cornea.
+
+6. Release the animal from the operating table.
+
+7. Label, etc.
+
+~7. Intrapulmonary.~--
+
+_Fluid Inoculum._--(_Anaesthetic, none._)
+
+1. Have the animal firmly held by an assistant. (In this case the
+foreleg of the selected side is drawn up by the assistant and held with
+the ear of that side.)
+
+2. Shave carefully in the axillary line and disinfect the denuded skin.
+
+3. Thrust the needle of the syringe boldly through the fifth or sixth
+intercostal space into the lung tissue.
+
+4. Inject the contents of the syringe slowly.
+
+5. Label, etc.
+
+~8. Intravenous.~--
+
+_Fluid Inoculum._--(_Anaesthetic, none._)
+
+The site selected for the injection in the rabbit is the posterior
+auricular vein (see Fig. 192). Although this is smaller than the median
+vein, it is firmly bound down to the cartilage of the ear by dense
+connective tissue, and is therefore more readily accessible. (In the
+guinea-pig the jugular vein must be utilised, and in order to perform
+the inoculation satisfactorily a general anaesthetic must be
+administered to the animal. In the monkey or the dog, the internal
+saphenous vein is the most convenient and before puncturing should be
+distended or rendered prominent by compressing the vein above the
+selected site.)
+
+_Preparation of the Inoculum._--Care must be taken in preparing the
+inoculum, as the injection of even small fragments may cause fatal
+embolism. To obviate this risk the fluid should, if possible, be
+filtered through sterile filter paper before filling into the syringe.
+
+Air bubbles, when injected into a vein, frequently cause immediate
+death. To prevent this, the syringe after being filled should be held in
+the vertical position, needle uppermost. A piece of sterile filter paper
+is then impaled on the needle and the piston of the syringe pressed
+upward until all the air is expelled from the barrel and needle. Should
+any drops of the inoculum be forced out, they will fall on the filter
+paper, which should be immediately burned.
+
+1. Have the animal firmly held by an assistant. The selected ear is
+grasped at its root and stretched forward toward the operator.
+
+2. Shave the posterior border of the dorsum of the ear.
+
+3. Disinfect the skin over the vein, rubbing it vigourously with
+cotton-wool soaked in lysol. The friction will make the vein more
+conspicuous. Wash the lysol off with ether and allow the latter to
+evaporate.
+
+4. Direct the assistant to compress the vein at the root of the ear.
+This will cause its peripheral portion to swell up and increase in
+calibre.
+
+5. Hold the syringe as one would a pen and thrust the point of the
+needle through the skin and the wall of the vein till it enters the
+lumen of the vein (Fig. 189). Now press it onward in the direction of
+the blood stream--i. e., toward the body of the animal.
+
+6. Direct the assistant to cease compressing the root of the ear, and
+_slowly_ inject the inoculum. (If the fluid is being forced into the
+subcutaneous tissue, a condition which is at once indicated by the
+swelling that occurs, the injection must be stopped and another attempt
+made at a spot closer to the root of the ear or at some point on the
+corresponding vein on the opposite ear.)
+
+7. Withdraw the needle and press a pledget of cotton-wool over the
+puncture to ensure closure of the aperture in the vein wall.
+
+8. Label, etc.
+
+[Illustration: FIG. 189.--Intravenous inoculation.]
+
+~9. Inhalation.~--
+
+(a) _Fluid Inoculum._--(_Anaesthetic, none._)
+
+1. Place the animal in a closed metal box.
+
+2. Through a hole in one side introduce the nozzle of some simple
+spraying apparatus, such as is used for nasal medicaments.
+
+3. Fill the reservoir of the instrument (previously sterilised) with the
+fluid inoculum, and having attached the bellows, spray the inoculum into
+the interior of the box.
+
+4. On the completion of the spraying, open the box, spray the animal
+thoroughly with a 10 per cent. solution of formaldehyde (to destroy any
+of the virus that may be adhering to fur or feathers).
+
+5. Transfer the animal to its cage.
+
+6. Label, etc.
+
+7. Thoroughly disinfect the inhalation chamber.
+
+(b) _Fluid or Powdered Inoculum._--_Anaesthetic, A. C. E._
+
+1. Anaesthetise the animal and secure it firmly to the operating table.
+
+[Illustration: FIG. 190.--Gag for rabbits.]
+
+2. Prop open the mouth by means of some form of gag; seize the tongue
+with a pair of forceps and draw it forward.
+
+The most convenient form of gag for the rabbit or cat is that shown in
+Fig. 190. It is simply a strip of hard wood shaped at the middle and
+provided with a square orifice through which a tracheal or oesophageal
+tube can be passed.
+
+3. Pass a previously sterilised glass tube (17 cm. long, 0.5 cm.
+diameter, with its terminal 2 cm. slightly curved) down through the
+larynx into the trachea.
+
+4. Connect the straight portion of a ~Y~-shaped piece of tubing to the
+upper end of the sterilised tube and couple one branch of the ~Y~ to a
+separatory funnel containing the fluid inoculum, or insufflator
+containing the powdered inoculum, and the other to a hand bellows.
+
+5. Allow the fluid inoculum to run into the lungs by gravity, or blow in
+the powdered inoculum by means of a rubber-ball bellows.
+
+6. Remove the intratracheal tube; release the animal from the table.
+
+7. Label, etc.
+
+As an alternative method in the case of fairly large animals, such as
+rabbits, etc., a sterile piece of glass tubing of suitable diameter may
+be passed through the larynx down the trachea almost to its
+bifurcation. Fluid cultivations may then be literally poured into the
+lungs, or cultivations, dried and powdered, may be blown into the lung
+by the aid of a small hand bellows or even a teat pipette.
+
+~10. Intragastric Inoculation.~--_Fluid or semi-fluid inoculum.
+(Anaesthetic none.)_
+
+The method of performing the operation is varied slightly according to
+the size of the experimental animal.
+
+_A. Monkey, Rabbit, Guinea-pig._
+
+1. Secure the animal to the operating table ventral surface uppermost.
+
+2. Prop the mouth open with a gag; draw the tongue forward with forceps.
+
+3. Sterilise a soft rubber catheter (No. 10 or 8 English scale, or No.
+18 or 15 French) and lubricate it with sterile glycerine.
+
+4. Pass it to the back of the pharynx, keeping the end in the middle
+line.
+
+5. Gently assist the progress of the catheter down the oesophagus
+until it passes the cardiac orifice of the stomach. Do not use any
+force.
+
+6. Take up the required dose of inoculum into a sterilised pipette.
+Insert the point of the pipette into the open end of the catheter and
+allow the fluid to run down into the stomach. Remove the pipette and
+drop it into a jar of lysol.
+
+7. With another sterile pipette run one cubic centimetre of sterile
+saline solution through the catheter to wash out the last traces of the
+inoculum.
+
+8. Withdraw the catheter.
+
+9. Label, etc.
+
+_B. Rats and Mice (Mark's Method)._
+
+1. Secure the animal in the vertical position.
+
+(a) _Rat._--Take a pair of catch sinus forceps about 22 cm. in length
+and seize the animal by the loose skin of the head as far forward as
+possible--fix the forceps, and holding the instrument vertically upward,
+transfer to the left hand of an assistant who secures the animal's tail
+between the fingers grasping the handle of the forceps. (See Fig. 191.)
+
+[Illustration: FIG. 191.--Intragastric inoculation of rat.]
+
+(b) _Mouse._--An assistant grasps the loose skin between the ears as far
+forwards as possible between the forefinger and thumb of the left hand.
+He now grasps the tail with the right hand, draws the mouse straight and
+passes the tail between the fourth and little fingers of the left hand
+and secures it there.
+
+2. The assistant takes a closed pair of thin-bladed forceps in his right
+hand, passes the ends into the animal's mouth, then allows the blades to
+separate. This opens the animal's jaw and serves as a gag.
+
+3. Moisten the sterilised oesophageal tube with sterile water. (This
+tube is of silk rubber, 6.5 cm. in length, with the distal end rounded,
+the proximal end mounted in a syringe needle head, which fits the
+nozzles of the two sterile syringes to be used.)
+
+4. Grasp the tube about its middle and pass it into the animal's mouth,
+downwards and a little to one side or the other until its length is lost
+in the digestive tract and mouth. Gentle guidance is alone necessary. Do
+not use any force.
+
+5. Take up the required dose of inoculum into the syringe; insert the
+nozzle of the syringe into the needle-mount, and force the piston down.
+
+6. Steadying the needle-mount with the left hand, detach the syringe.
+
+7. Draw up some sterile water in the second (sterile) syringe, and
+inserting its nozzle into the needle-mount force a few drops of water
+through the tube to wash it out.
+
+8. With one quick upward movement remove the tube from the animal's
+mouth.
+
+9. Label, etc.
+
+One other method of inoculation remains to be described, which does not
+require operative interference.
+
+~11. Feeding.~--
+
+1. _Fluid Inoculum._--Small pieces of sterilised bread or sop
+(sterilised in the steamer at 100 deg. C.) are soaked in the fluid
+inoculum and offered to the animals in a sterile Petri dish or capsule.
+
+2. _Solid Inoculum._--Small pieces of tissue are placed in sterile
+vessels and offered to the animals.
+
+FOOTNOTES:
+
+[12] This table is made by Messrs. Down Bros., St. Thomas's Street,
+London, S. E.
+
+[13] This modification is made for the author by Messrs. Down Bros., St.
+Thomas's Street, London, S. E.
+
+[14] Manufactured by Messrs. Francis Lepper, 56, Great Marlborough
+Street, London, W.
+
+
+
+
+XVIII. THE STUDY OF EXPERIMENTAL INFECTIONS DURING LIFE.
+
+
+The possession of pathogenetic properties by an organism under study is
+indicated by the "infection" of the experimental animal--a term which is
+employed to summarise the condition resulting from the successful
+invasion of the tissues of the experimental animal by the
+micro-organisms inoculated and by their multiplication therein.
+Infection is considered to have taken place:
+
+1. When the death of the animal is produced as a direct consequence of
+the inoculation.
+
+2. When without necessarily producing death the inoculation causes local
+or general changes of a pathological character.
+
+3. When either with or without death, or local or general changes
+occurring, certain substances make their appearance in the body fluids,
+which can be shown (_in vitro_ or _in vivo_) to exert some profound and
+specific effect when brought into contact with subcultivations of the
+organism originally inoculated.
+
+The important factors in the production of infection are:
+
+A. Seed.  Virulence of organism.
+          Dose of organism.
+
+B. Soil.  Resistance offered by the cells of the experimental animal.
+
+The first two factors, although variable, are to a certain extent under
+the control of the experimenter. Thus by suitable means the virulence of
+an organism can be exalted or attenuated, whilst the size of the dose
+may be increased or diminished. The third factor also varies, not only
+amongst different species of animals, but also amongst different
+individuals of the same species. The essential causes of this variation
+are not so obvious, so that beyond selecting the animals intended for
+similar experiments with regard to such points as age, size or sex, but
+little can be done to standardise cell resistance.
+
+Immediately an animal has been inoculated a period of clinical
+observation must be entered upon, which should only terminate with the
+death of the animal. The general observations should at first and if the
+infection is an acute one, be made daily--later, and if the animal
+appears to be unaffected or if the infection is chronic, both general
+and special observations should be carried out at weekly intervals. If
+the animal appears to be still unaffected, it should be killed with
+chloroform vapour at the end of two or three months and a complete
+post-mortem carried out.
+
+A. The ~general observations~ should take cognisance of:
+
+1. _General appearance._ The experimental animal should be inspected
+daily, not only with a view to detecting symptoms due to the
+experimental infection, but also to prevent any intercurrent infection,
+naturally acquired, from escaping notice (_vide_ page 337).
+
+2. _The weight_ of the inoculated animal should be observed and recorded
+each day during the course of an experimental infection at precisely the
+same hour, preferably just before the morning feed.
+
+3. _The temperature_ should similarly be recorded daily, if not more
+frequently, during the whole period the animal is under observation, and
+carefully charted--individual variations will at once become apparent.
+It should be borne in mind that the temperature regarded as normal for
+man (37.5 deg. C.) is not the normal average temperature of any of the
+lower animals save the rat and mouse. The accompanying table of normal
+averages for the animals usually employed in bacteriological research
+may be of use in preventing the erroneous assumption that pyrexia is
+present in an animal, which merely shows its own normal temperature.
+
+                  NORMAL AVERAGES.
+----------------------------------------------------
+                |  Rectal  | Pulse. | Respirations.
+    Animal.     |   Temp.  |------------------------
+                |  deg. C. |    Rate per minute.
+----------------------------------------------------
+                |          |        |
+Frog            | 8.9-17.2 |   80   |      12
+Mouse           |   37.4   |  120   |     ...
+Rat             |   37.5   |  ...   |     210
+Guinea pig      |   38.6   |  150   |      80
+Rabbit          |   38.7   |  135   |      55
+Cat             |   38.7   |  130   |      24
+Dog             |   38.6   |   95   |      15
+Goat            |   40.0   |   75   |      16
+Ox              |   38.8   |   45   |      ..
+Horse           |   37.9   |   38   |      11
+Monkey (Rhesus) |   38.4   |  100   |      19
+Pigeon          |   40.9   |  136   |      30
+Fowl            |   41.6   |  140   |      12
+                |          |        |
+----------------------------------------------------
+
+B. ~Special observations~ comprise some or all of the following, according
+to the method of inoculation and the character of the virus.
+
+1. _The site of inoculation_ should be minutely examined at least at
+weekly intervals, and the neighbouring lymphatic glands palpated.
+
+2. Any _local reaction_ at the site of inoculation and any other readily
+accessible lesion should be carefully investigated. Any suppurative
+process which may occur, whether in the subcutaneous tissues or in
+joints, should be explored and the pus carefully examined both
+microscopically and culturally.
+
+Fluid secretions and excretions, such as pus or serous exudates when
+accessible are collected direct from the body in sterile capillary
+pipettes (_vide_ Fig. 13a,) in the following manner:
+
+1. Open the case containing the pipettes, grasp one by the plugged end,
+remove it from the case, and replace the lid of the latter.
+
+2. Attach a rubber teat (_vide_ page 10) to the plugged end of the
+pipette and use the teat as the handle of the pipette.
+
+3. Pass the entire length of the pipette twice or thrice through the
+flame of the Bunsen burner.
+
+4. Snap off the sealed end of the pipette with a pair of sterile
+forceps.
+
+5. Compress the india-rubber teat, thrust the point of the pipette into
+the secretion; now relax the pressure on the teat and allow the pipette
+to fill.
+
+6. Remove the point of the pipette from the secretion, allow the fluid
+to run a short distance up the capillary stem and seal the point of the
+pipette in the flame. (If using a pipette with a constriction below the
+plugged mouthpiece (Fig 13b), this portion of the pipette may also be
+sealed in the flame.)
+
+When ready to examine the morbid material snap off the sealed end of the
+pipette with sterile forceps and eject the contents of the pipette into
+a sterile capsule. The material can now be utilized for cover-slip
+preparations, cultivations and inoculation experiment.
+
+3. _The peripheral blood_ should be examined from time to time for from
+this tissue is often obtained the fullest information as to the course
+and progress of an infection.
+
+a. The ~histological examination of the blood~ should be directed
+chiefly to observations on the number and kind of white cells; and since
+but few bacteriologists are at the same time expert comparative
+haematologists, some notes on the normal characters of the blood of the
+commoner laboratory animals, contrasted with those of man, are inserted
+for reference. These have been very kindly compiled for me by my friend
+and one time colleague Dr. Cecil Price Jones.
+
+
+COMPARATIVE HAEMOCYTOLOGY OF LABORATORY ANIMALS.
+
+--------------------------------------------------------------------
+       |       Totals    |                Percentages
+       |------------------------------------------------------------
+Animal |          |      | Hb, |Lympho-|Large |Poly- |Eosin-| Mast
+       |Red cells |White | per | cytes,|monos,|morph,| oph, |cells,
+       |          | cells|cent.|  per  | per  | per  | per  | per
+       |          |      |     | cent. | cent.| cent.|cent. |cent.
+--------------------------------------------------------------------
+Frog   |   490,000| 8,000|  58 |   40  | 10.0 | 22.0 |15    | 13
+Mouse  | 8,700,000| 8,000|  78 |   60  | 21.5 | 17.0 | 1.4  |  0.1
+Rat    | 9,000,000| 9,000|  85 |   54  |  7.0 | 37.5 | 1.3  |  0.2
+Guinea-|          |      |     |       |      |      |      |
+  pig  | 5,700,000|10,000|  99 |   55  |  9.0 | 32.8 | 3.0  |  0.2
+Rabbit | 6,000,000| 7,000|  70 |   50  |  2.0 | 46.0 | 0.6  |  1.4
+Rhesus | 4,500,000|13,000|  77 |   43  |  5.0 | 50.0 | 1.3  |  0.7
+Goat   |14,600,000|15,000|  58 |   35  |  6.3 | 56.7 | 1.25 |  0.75
+Fowl   | 3,500,000|30,000| 100 |   49  |  3.0 | 42.0 | 1.0  |  5.0
+Pigeon | 3,500,000|20,000| 101 |   43  |  9.0 | 43.0 | 3.0  |  2.0
+--------------------------------------------------------------------
+Man    |          |      |     |       |      |      |      |
+(adult)| 5,000,000| 7,500| 100 |   25  |  5.5 | 65   | 4.0  |  0.5
+Normal | (4.5-5)  | (7-9)|(95- |(20-30)| (4-8)|(55-  |(3-5) |(0.5-2)
+limits.| millions.| thou-| 101)|       |      |  68) |      |
+       |          |sands.|     |       |      |      |      |
+--------------------------------------------------------------------
+
+The above table represents in each case the average of a large number of
+counts.
+
+
+REMARKS.
+
+_Frog._--The _red cells_ are large oval nucleated (20-25 mu by 12-15 mu)
+discs, the nucleus relatively small and irregularly elongated or oval,
+about 10 mu in length. Many primitive and developing forms are usually
+observed--also free nuclei and many cells in various stages of
+degeneration. Haemoglobin estimation is difficult owing to turbidity of
+the blood after dilution with water. The _polymorphonuclear_ leucocytes
+are large cells, about 20 mu; no definite granules can be observed. The
+_eosinophile_ cells contain large deeply staining coccal-shaped
+granules.
+
+_Mouse._--The granules of the _polymorphonuclear_ leucocytes are usually
+not stained, or only very faintly so. The nucleus of the _eosinophile
+cell_ is ring-shaped or much divided, and the granules are coccal and
+stain oxyphile. The remarkable character of the blood is the high
+percentage of large _mononuclear_ cells.
+
+_Rat._--The fine rod-shaped granules of the _polymorphonuclear_
+leucocytes are usually very faintly stained. The granules of
+_eosinophile_ cells are well stained and coccal-shaped, the nucleus is
+often ring shaped. The _basophile_ granular cells are few--but the
+granules are large, and stain deeply basophile.
+
+_Guinea-pig._--Polychromasia and punctate basophilia of _red cells_ are
+very commonly observed--nucleated red cells are also frequent. The large
+_mononuclear_ cells often contain vacuoles--"Kurlow cells"--possibly of
+a parasitic nature.
+
+_Rabbit._--It is not uncommon to find nucleated _red cells_ in films
+from quite healthy animals. The granules of the _polymorphonuclear_
+leucocytes stain oxyphile. The coarse granules of the _eosinophile_
+cells appear to stain less deeply oxyphile, probably owing to the
+basophile staining of the cytoplasm.
+
+_Rhesus monkey._--The blood cells resemble those met with in human
+blood. The minute neutrophile granules of the _polymorphonuclear_
+leucocytes are often very scanty, and sometimes apparently absent. The
+_eosinophile_ cells are not so densely packed with coarse oxpyhile
+granules as in the human eosinophile, and the nuclei of these cells are
+usually much divided, or polymorphous.
+
+_Goat._--The _red cells_ are small, nonnucleated discs, only about 4.5
+mu diameter, not much more than half that of the human red cell. The
+_polymorphonuclear_ leucocytes have only a few very minute
+coccal-shaped oxyphile granules, the nucleus is polymorphous. The
+_eosinophile_ cells are large cells up to 20 mu, the cytoplasm is
+basophile and contains coarse coccal-shaped oxyphile granules, and the
+nucleus is often much divided.
+
+_Fowl._--The _red cells_ are oval nucleated discs about 12 mu by 6 mu, the
+nucleus being relatively small (about 4 mu long), irregularly elongated or
+oval; round, more deeply stained cells with round or diffuse nuclei,
+also free nuclei and degenerated forms of red cells are often present.
+The granules of the cells corresponding to the _polymorphonuclear_
+leucocytes are rod-shaped, often beaded or with clubbed ends. The
+nucleus is not polymorphous, but usually divided into two, though it may
+be single. The cells probably corresponding to _eosinophile_ leucocytes
+have fine coccal-shaped granules, faintly staining eosinophile or
+neutrophile. The basophile granules of the "mast" cells are
+coccal-shaped, of various size--often quite powdery.
+
+_Pigeon._--_Red cells_ resemble those of the fowl, and similar varieties
+of appearance may be noted. The granules of those cells which correspond
+to _polymorphonuclear_ leucocytes are rod-shaped, but smaller and finer
+than in the fowl, and do not show clubbed appearances. The nucleus is
+not polymorphous, and only occasionally divided. The coccal-shaped
+granules of the _eosinophile_ cells are stained more deeply oxyphile
+than those of the corresponding cells of the fowl.
+
+_The preparation of dried films_ for this histological examination of
+the blood is carried out as follows:
+
+1. Small samples of blood for the preparation of blood films are most
+conveniently obtained from the veins of the ear in most of the ordinary
+laboratory animals, viz., monkey, goat, dog, cat, rabbit, guinea-pig; in
+the pigeon and fowl the axillary vein should be punctured; in the rat
+and mouse either a vein in the ear or preferably by wounding the tip of
+the tail; in the frog, the web of the foot should be selected.
+
+2. Puncture the selected vein with a sharp needle. A flat Hagedorn
+needle (size No. 8) with a cutting edge is the most useful for this
+purpose. If the vein cannot be distended by proximal compression,
+vigourous friction with a piece of dry lint may have the desired
+effect--or a test-tube full of water at about 40 deg. C. may be placed
+close to the vein. Failing these methods, a drop or two of xylol may be
+dropped on the skin just over the vein, left on for a few seconds and
+then wiped off with a piece of dry lint.
+
+3. One of the short ends of a 3 by 1 glass slip is brought into contact
+with the exuding drop of blood, so that it picks up a small drop.
+
+4. The slide is then lowered transversely on to the surface of a second
+3 by 1 slip, which rests on the bench near to one end at an angle of
+about 45 deg., and retained in this position for a few seconds, while the
+drop of blood spreads along the whole of the line of contact (see also
+Fig. 69).
+
+5. Draw the first slide firmly and evenly along the entire length of the
+lower slide, leaving a thin regular film which will probably show the
+blood cells only one layer thick.
+
+6. Allow the film to dry in the air.
+
+7. Stain with one of the polychrome blood stains (see page 97).
+
+8. Examine microscopically.
+
+b. The ~bacteriological examination of the blood~ is directed solely to
+the demonstration of the presence in the circulating blood of the
+organisms previously injected into the animal. For this purpose several
+cubic centimetres of blood should be taken in an all-glass syringe from
+an accessible vein corresponding to one of those suggested as the site
+of intravenous inoculation--and under similar aseptic precautions.
+
+1. Sterilise an all-glass syringe of suitable size, and when cool draw
+into the syringe some sterile sodium citrate solution and moisten the
+whole of the interior of the barrel; then eject all the citrate solution
+if less than 5 c.c. blood are to be withdrawn; if more than 5 c.c. are
+required retain about half a cubic centimetre of the fluid in the
+syringe. This prevents coagulation of the blood.
+
+The sodium citrate solution is prepared by dissolving:
+
+    Sodium citrate           10 gramme.
+    Sodium chloride           0.75 grammes.
+    In distilled water      100 c.c.
+
+Sterilise by boiling.
+
+2. Prepare the animal as for intravenous inoculation (see page 363) and
+introduce the syringe needle into the lumen of the selected vein.
+
+3. Slowly withdraw the piston of the syringe. When sufficient blood has
+been collected direct the assistant to release the proximal compression
+of the vein; and withdraw the needle.
+
+4. Remove the needle from the nozzle of the syringe and deliver the
+citrated blood into a small Ehlenmeyer flask containing about 250 c.c.
+of nutrient broth.
+
+5. Label, incubate and examine daily until growth occurs or until the
+expiration of ten days.
+
+c. The ~serological examination of the blood~ is directed to the
+demonstration of the presence of certain specific antibodies in the sera
+of experimentally infected animals, and within certain limits to an
+estimation of their amounts.
+
+The chief of these bodies are:
+
+    Antitoxin.
+    Agglutinin.
+    Precipitin.
+    Opsonin.
+    Immune body or Bacteriolysin.
+
+None of these substances are capable of isolation in a state of purity
+apart from the blood serum, consequently special methods have been
+elaborated to permit of their recognition. In every instance the
+behaviour of serum from the experimental animal, which may be termed
+"specific" serum, is studied in comparison with that of serum from an
+uninoculated animal of the same species, and which is termed "normal"
+serum. In view of minor differences in constitution exhibited by the
+serum of various individuals of the same series, it is usual to employ a
+mixture of sera obtained from several different normal animals of the
+same species as the inoculated animal, under the term "pooled serum."
+The method of collecting blood (e. g., from the rabbit) for
+serological tests is as follows:
+
+~Collection of Serum.~
+
+_Apparatus required:_
+
+    Razor.
+    Liquid soap.
+    Cotton-wool.
+    Lysol 2 per cent. solution, in drop bottle.
+    Ether in drop bottle.
+    Flat Hagedorn needles.
+    Blood pipettes (Fig. 16, page 12).
+    Centrifugal machine.
+    Centrifuge tubes.
+    Glass cutting knife.
+    Bunsen flame.
+    Writing diamond or grease pencil.
+
+METHOD.
+
+1. Shave the dorsal surface of the ear over the course of the posterior
+auricular vein (see Fig. 192).
+
+2. Sterilise the skin by washing with lysol.
+
+The lysol should be applied with sterile cotton-wool and the ear
+vigourously rubbed, not only to remove superficial scales of epithelium,
+but also to render the ear hyperaemic and the vein prominent.
+
+3. Remove the lysol with ether dropped from a drop bottle, and allow the
+ether to evaporate.
+
+4. Puncture the vein with a sterile Hagedorn needle.
+
+5. Take a small blood-collecting pipette (Fig. 161) and hold it at an
+angle to the ear, one end touching the issuing drop of blood, the other
+depressed.
+
+The blood will now enter the pipette at first by capillarity; afterward
+gravity will also come into play and the pipette can be two-thirds
+filled without difficulty.
+
+6. Hold the tube by the end containing the blood, the clean end pointing
+obliquely upward--warm this end at the bunsen flame to expel some of the
+contained air; then seal the clean point in the flame.
+
+[Illustration: FIG. 192.--Collecting blood from rabbit.]
+
+7. Place the pipette down on a cool surface (e. g., a glass slide).
+The rapid cooling of the air in the clean end of the pipette creates a
+negative pressure, and the blood is sucked back into the pipette,
+leaving the soiled end free from blood. Seal this end in the bunsen
+flame.
+
+8. Mark the distinctive title of the specimen (e. g., animal's number)
+upon the pipette with a writing diamond or grease pencil.
+
+9. When the sealed ends are cold and the blood has clotted, place the
+pipette on the centrifuge, clean end downward; counterpoise and
+centrifugalise thoroughly. On removing the pipette from the centrifuge,
+the red cells will be collected in a firm mass at one end, and above
+them will appear the clear serum.
+
+10. By marking the blood pipette above the level of the serum with the
+glass cutting knife and snapping the tube at that point, the blood-serum
+becomes readily accessible for testing purposes.
+
+If larger quantities of blood are required, the animal, after puncturing
+the vein, should be inverted, an assistant holding it up by the legs.
+Blood to the volume of several cubic centimetres will now drop from the
+punctured vein, and should be caught in a tapering centrifuge tube, the
+tube transferred to the incubator at 37 deg. C. for two hours, then
+placed in the centrifugal machine, counterpoised and centrifugalised
+thoroughly. The three most important of the antibodies referred to which
+can be demonstrated with a certain amount of facility are agglutinin,
+opsonin and bacteriolysin; and the methods of testing for these bodies
+will now be considered.
+
+
+AGGLUTININ.
+
+Agglutinin is the name given to a substance present in the blood-serum
+of an animal that has successfully resisted inoculation with a certain
+micro-organism. This substance possesses the power of collecting
+together in clumps and masses, or agglutinating watery suspensions of
+that particular microbe.
+
+
+~Dilution of the Specific Serum~:
+
+_Apparatus required_:
+
+Sterile graduated capillary pipettes to contain 10 c. mm. (Fig. 17).
+Sterile graduated capillary pipettes to contain 90 c. mm. (Fig. 17).
+Small sterile test-tubes 5 x 0.5 cm.
+Normal saline solution in flask or test-tube.
+Pipette of specific serum.
+Glass cutting knife, or three-square file.
+Glass capsule, nearly full of dry silver sand, or roll of plasticine.
+Grease pencil.
+
+METHOD.--
+
+1. Take three sterile test-tubes and number them 1, 2 and 3.
+
+2. Pipette 0.9 c.c. sterile normal saline solution into each tube, and
+stand tubes upright in the sand in the capsule, or in the plasticine
+block.
+
+3. Make a scratch with the glass cutting knife on the blood pipette
+above the upper level of the clear serum, and snap off and discard the
+empty portion of the tube.
+
+4. Remove 0.1 c.c. of the serum from the blood pipette tube, and mix it
+thoroughly with the fluid in tube No. 1; and label ~s.s.~, (specific
+serum), 10 per cent.
+
+5. Remove 0.1 c.c. of the solution from tube No. 1 by means of a fresh
+pipette, and mix it with the contents of tube No. 2; and label ~s.s.~, 1
+per cent.
+
+6. Remove 0.1 c.c. of the solution from tube No. 2 by means of a fresh
+pipette, and mix it with the contents of tube No. 3; and label ~s.s.~, 0.1
+per cent.
+
+When the yield of serum from the specimen of blood which has been
+collected, or is available, is small, the above method of diluting is
+not practicable, and the dilution should be carried out by Wright's
+method in a capillary teat pipette.
+
+
+~Dilution of Serum by Means of a Teat Pipette.~
+
+_Materials required:_
+
+    Blood pipette containing sample of specific serum after
+      centrifugalisation.
+    Capsule of diluting fluid--normal saline solution.
+    Supply of Pasteur pipettes (Fig. 13a).
+    India-rubber teats.
+    Small test-tubes.
+    A block of plasticine to act as a test-tube stand.
+    Grease pencil.
+
+METHOD:
+
+1. Mark three small test-tubes 10 per cent., 1 per cent. and 0.1 per
+cent. respectively, and stand them upright in the plasticine block.
+
+2. Take a Pasteur pipette, nick the capillary stem just above the sealed
+end with a glass cutting knife, and snap off the sealed end with a quick
+movement so that the fracture is clean cut and at right angles to the
+long axis of the capillary stem--cut "square", in fact. Prepare several,
+say a dozen, in this manner.
+
+3. Fit a rubber teat to the barrel of each of the pipettes.
+
+4. Make a mark with the grease pencil on the stem of one of the pipettes
+about 2 or 3 cm. from the open extremity.
+
+[Illustration: FIG. 193.--Filling the capillary teat pipette.]
+
+5. Compress the teat between the finger and thumb (Fig. 193) to such an
+extent as to drive out the greater part of the contained air.
+
+6. Maintaining the pressure on the teat pass the stem of the pipette
+into the capsule holding the saline solution, until the open end of the
+pipette is below the level of the fluid.
+
+7. Now cautiously relax the pressure on the teat and let the fluid enter
+the pipette and rise in the stem until it reaches the level of the
+grease pencil mark. As soon as this point is reached, check the movement
+of the column of fluid by maintaining the pressure on the teat, neither
+relaxing nor increasing it.
+
+8. Withdraw the point of the pipette clear of the fluid, and again relax
+the pressure on the teat very slightly. The column of saline solution
+rises higher in the stem, and a column of air will now enter the pipette
+and serve as an index to separate the first volume of fluid drawn into
+the stem from the next succeeding one.
+
+9. Again introduce the end of the pipette into the fluid and draw up a
+second volume of saline to the level of the grease pencil mark, and
+follow this with a second air index.
+
+10. In like manner take up seven more equal volumes of saline solution
+and their following air bubbles. There are now nine equal volumes of
+normal saline in the pipette.
+
+11. Now pass the point of the pipette into the blood tube and dip the
+open end below the surface of the serum. Proceeding as before, aspirate
+a volume of serum into the capillary stem up to the level of the pencil
+mark.
+
+12. Eject the contents of the pipette into the small tube marked 10 per
+cent. by compressing the rubber teat between thumb and finger.
+
+13. Mix the one volume of serum with the nine volumes of saline solution
+very thoroughly by repeatedly drawing up the whole of the fluid into the
+pipette and driving it out again into the test-tube.
+
+14. Now take a clean pipette and proceed precisely as before, 4 to 10.
+
+15. Having aspirated nine equal volumes of saline into this second
+pipette, now take up one similar volume of the fluid in the "10 per
+cent. tube."
+
+16. Eject the contents of this pipette into the second tube marked 1 per
+cent. and mix thoroughly as before.
+
+17. In similar fashion make the 0.1 per cent. solution and transfer to
+the third tube.
+
+18. Further dilutions in multiples of ten can be prepared in the same
+way, and by varying the number of volumes of diluting fluid or serum any
+required dilution can be made (see Appendix, Dilution Tables).
+
+     NOTE.--The saline diluting fluid _must always_ be taken into
+     the pipette first, otherwise if the serum contains a very
+     large amount of agglutinin the traces of this serum added to
+     the saline solution may be sufficient to entirely vitiate
+     the subsequent observations--whilst if more than one sample
+     of serum is diluted from the same saline solution serious
+     errors may be introduced into the experiments.
+
+
+~The Microscopical Reaction:~
+
+_Apparatus Required:_
+
+     Five hanging-drop slides (or preferably two slide), with two
+     cells mounted side by side on each (Fig. 62, a), and one
+     slide with one cell only.
+
+     Vaseline.
+
+     Cover-slips.
+
+     Platinum loop.
+
+     Grease pencil.
+
+     Eighteen to twenty-four-hour-old bouillon cultivation of the
+     organism to be tested (e. g., Bacillus typhi abdominalis)
+
+     Pipette end with the remainder of the specific serum
+     labelled ~s.s.~
+
+     Tubes containing the three solutions of the specific serum,
+     10, 1, and 0.1 per cent. respectively.
+
+     Pipette end with pooled normal serum labelled ~p.s.~
+
+METHOD.--
+
+1. Make five hanging-drop preparations, thus:
+
+(a) One loopful of bouillon cultivation + one loopful pooled serum;
+label "Control."
+
+(b) One loopful culture + one loopful undiluted specific serum; label
+50 per cent.
+
+Mount these two cover-slips on a double-celled slide.
+
+(c) One loopful bouillon culture + one loopful 10 per cent. serum;
+label 5 per cent.
+
+Mount this on single-cell slide.
+
+(d) One loopful bouillon culture + one loopful 1 per cent. serum;
+label 0.5 per cent.
+
+(e) One loopful bouillon culture + one loopful 0.1 per cent. serum;
+label 0.05 per cent.
+
+Mount these two cover-slips on a double-celled slide.
+
+2. Note the time: Examine the control to determine that the bacilli are
+motile and uniformly scattered over the field--not collected into
+masses.
+
+3. Next examine the 50 per cent. serum preparation.
+
+If agglutinin is present and the test is giving a positive reaction, the
+bacilli _will_ be collected in large clumps.
+
+If the test is giving a negative reaction, the bacilli _may_ be
+collected in large clumps owing to the viscosity of the concentrated
+serum.
+
+4. Observe the 5 per cent. preparation microscopically.
+
+If the bacilli are aggregated into clumps, positive reaction.
+
+If the bacilli are _not_ aggregated into clumps, observe until thirty
+minutes from the time of preparation before recording a negative
+reaction.
+
+5. Examine the 0.5 and 0.05 per cent. preparations.
+
+These may or may not show agglutination when the result of the
+examination of the 5 per cent. preparation is positive, according to the
+potency of the specific serum; and by the examination of a series of
+dilutions a quantitative comparison of the valency of specific sera from
+different sources, or of serum from the same animal at different periods
+during the course of active immunisation may be obtained.
+
+     NOTE.--The graduated pipettes supplied with Thoma's
+     haematocytometer (intended for the collection of the specimen
+     of blood required for the enumeration of leucocytes), giving
+     a dilution of 1 in 10--i. e., 10 per cent.--may be
+     substituted for the graduated capillary pipettes referred to
+     above, if the vessel in which the serum has been separated
+     is of sufficiently large diameter to permit of their use.
+
+
+~The Macroscopical Reaction:~
+
+     Sterile graduated capillary pipettes to contain 90 c. mm.
+
+     Eighteen to twenty-four-hours-old bouillon cultivation of
+     the organism to be tested.
+
+     Three test-tubes containing the 10, 1, and 0.1 per cent.
+     solutions of specific serum (about 90 c. mm. remaining in
+     each).
+
+     Tube containing 50 per cent. solution of pooled serum.
+
+     Sedimentation pipettes (_vide_ page 17) or teat pipettes.
+
+METHOD.
+
+1. Pipette 90 c. mm. of the bouillon culture into each of the tubes
+containing the diluted serum; and the same quantity into the tube
+containing the pooled serum.
+
+2. Fill a sedimentation tube (by aspirating) or a teat pipette from the
+contents of each tube. Seal off the lower ends of the sedimentation
+tubes in the Bunsen flame.
+
+3. Label each tube with the dilution of serum that it contains--viz., 5,
+0.5, and 0.05 per cent.
+
+4. Place the pipettes in a vertical position, in a beaker, in the
+incubator at 37 deg. C., for one or two hours.
+
+5. Observe the granular precipitate which is thrown down when the
+reaction is positive, and the uniform turbidity of the negative reaction
+as compared with the appearances in the control pooled serum.
+
+
+OPSONIN.
+
+Opsonin is the term applied by Wright to a substance, present in the
+serum of an inoculated animal, which is able to act upon or sensitise
+bacteria of the species originally injected, so as to render them an
+easy prey to the phagocytic activity of polymorphonuclear leucocytes. In
+the method for demonstrating opsonin about to be described, a comparison
+is made between the opsonic "power" of the pooled serum and the specific
+serum.
+
+     _Apparatus:_
+
+     Small centrifuge and tubes for same (made from the barrels
+     of broken capillary pipettes by sealing the conical ends in
+     the bunsen flame).
+
+     Capillary Pasteur pipettes.
+
+     India-rubber teats.
+
+     Grease pencil.
+
+     Bunsen burner with peep flame.
+
+     Electrical signal clock (see page 39) stop watch, or watch.
+
+     Rectangular glass box or tray to hold pipettes.
+
+     Incubator regulated at 37 deg. C.
+
+     3 x 1 slides.
+
+     Piece of light rubber tubing.
+
+     Rectangular block of plasticine.
+
+     Flask of normal saline solution.
+
+     Flask of sodium citrate (1.5 per cent.) in normal saline
+     solution.
+
+     _Materials required_, and their preparation:
+
+     Small tube of "washed cells" (red blood discs and
+     leucocytes); human cells are used in estimating the
+     opsonising power of the serum of experimental animals.
+
+     Small tube of emulsion of bacteria of the species
+     responsible for the infection of the experimental animal.
+
+     Blood pipette containing specific serum.
+
+     Blood pipette containing "pooled" serum.
+
+_Washed Cells._--
+
+1. Take a small centrifuge tube and half fill it with sodium citrate
+solution. Mark with the grease pencil the upper limit of the fluid.
+
+2. Cleanse the skin of the distal phalanx of the second finger of the
+left hand above the root of the nail with lint and ether. Wind the
+rubber tubing tightly round the second phalanx; puncture with a sterile
+Hagedorn needle through the cleansed area of skin.
+
+3. Take up a sufficiency of the issuing blood (more or less according to
+the number of tests to be performed) with a teat pipette, transfer it to
+the tube of citrate solution and mix thoroughly. Make a second mark on
+the tube at the upper level of the mixed citrate solution and blood.
+
+4. Place the tube in the centrifuge, counterpoise accurately and
+centrifugalise until the blood cells are thrown down in a compact mass
+occupying approximately the same volume as is included between the two
+pencil marks.
+
+The column of fluid in the tube now shows clear supernatant fluid
+(citrate solution and blood plasma) separated from the sharp cut upper
+surface of the red deposit of corpuscles by a narrow greyish layer of
+leucocytes.
+
+5. Remove the supernatant column of citrate solution by means of a teat
+pipette, fill normal saline solution into the tube up to the upper
+pencil mark, and distribute the blood cells throughout the saline by
+means of the teat pipette. Centrifugalise as before.
+
+6. Again remove the supernatant fluid and fill in a fresh supply of
+saline solution and centrifugalise once more.
+
+7. Remove the supernatant saline solution as nearly down to the level of
+the leucocytes as can be safely done without removing any of the
+leucocytes.
+
+8. Next distribute the leucocytes evenly throughout the mass of red
+cells by rotating the tube between the palms of the hands--just as is
+done with a tube of liquefied medium prior to pouring a plate.
+
+9. Set the tube upright in the plasticine block near to one end.
+
+_Bacterial Emulsion._--
+
+1. Take an 18- to 24-hour culture of the required bacterium (e. g.,
+Diplococcus pneumoniae) grown upon sloped blood agar at 37 deg. C. Pour
+over the surface of the medium some 5 c.c. of normal saline solution.
+
+2. With a platinum loop emulsify the growth from the surface of the
+medium as evenly as possible in the saline solution.
+
+3. Allow the tube to stand for a few minutes so that the large masses of
+growth may settle down; transfer the upper portion of the saline
+suspension to a centrifuge tube and centrifugalise thoroughly.
+
+4. Examine a drop of the supernatant opalescent emulsion microscopically
+to determine its freedom from clumps and masses. If unsatisfactory
+prepare another emulsion, this time scraping up the surface growth with
+a platinum spatula, transferring it to an agate mortar and grinding it
+up with successive small quantities of normal saline. If satisfactory
+insert the tube in the plasticine block next to that containing the
+washed cells.
+
+
+~Specific Serum.~--
+
+~Pooled Serum.~--
+
+These sera are collected and treated as already described (see page
+379), and the portions of the blood pipettes containing them are
+arranged in the remaining space in plasticine block.
+
+[Illustration: FIG. 194.--Plasticine block with materials arranged for
+opsonin estimations.]
+
+The plasticine block now presents the appearances shown in Fig. 194.
+
+METHOD FOR DETERMINING THE OPSONIC INDEX.--
+
+1. Take a capillary pipette fitted with a teat, cut the distal end
+_square_ and make a pencil mark about 2 cm. from the end.
+
+2. Aspirate into the pipette one volume of washed cells, air index, one
+volume of bacterial emulsion, air index, and one volume of specific
+serum (see Fig. 195).
+
+[Illustration: FIG. 195. Opsonin pipette.]
+
+3. Mix thoroughly on a 3 by 1 slide by compressing the teat and ejecting
+the contents of the pipette on to the surface of the slide, relaxing the
+pressure and so drawing the fluid up into the pipette again. These two
+processes should be repeated several times; finally take up the mixture
+in an unbroken column to the central portion of the capillary stem.
+
+4. Seal the point of the pipette in the peep flame of the bunsen burner
+and remove teat.
+
+5. Mark the pipette (with the grease pencil) with the distinctive number
+of the serum and place it in the glass box or tray.
+
+6. Take another similarly prepared pipette and aspirate into it equal
+volumes of washed cells, bacterial emulsion and pooled serum. Treat
+precisely as in 3 and 4, label it "control" or "N.S." (normal serum) and
+place in the box by the side of the specific serum preparation.
+
+7. Place the box with the pipettes in the incubator and set the signal
+clock to ring at 15 minutes (or start the stop watch).
+
+8. At the expiration of the incubation time remove the pipettes from the
+incubator.
+
+9. Cut off the sealed end of the specific serum preparation. Mix its
+contents thoroughly as in step 3, and then divide the mixture between
+two 3 by 1 slips and carefully spread a blood film (_vide_ page 376) on
+each in such a way that only one-half of the surface of each slide is
+covered with blood--the free edge of the blood film approximating to the
+longitudinal axis of the slide.
+
+Allow films to dry and label the slides with writing diamond.
+
+10. Treat the contents of the control pipette in similar fashion.
+
+11. Select the better film from each pair for fixing and staining.
+
+12. Fixing and staining must be carried out under strictly comparable
+conditions, and to this end the slides are best handled by placing in a
+glass staining rack which can be lowered in turn into each of a series
+of glass troughs containing the various reagents (Fig. 196). Place the
+rack in the first trough which contains the alcoholic solution of
+Leishman's stain for two minutes to fix.
+
+Transfer to the second trough containing the diluted stain for ten
+minutes.
+
+Transfer to the third trough containing distilled water, and holding the
+trough over a sink, run in a stream of distilled water until washing is
+complete. Remove slides from the rack and dry.
+
+Leishman's stain is the best for routine work for all bacteria other
+than B. tuberculosis. Films containing tubercle bacilli must of course
+be stained by the Ziehl Neelsen method.
+
+[Illustration: FIG. 196. Glass staining trough for blood films.]
+
+13. Examine specific serum slide microscopically with 1/12 inch oil
+immersion. Find the edge of the blood film--along this the bulk of the
+leucocytes will be collected. Starting at one end of the film move the
+slide slowly across the microscope stage and as each leucocyte comes
+into view count and record the number of ingested bacteria. The sum of
+the contents of the first 50 consecutive polymorphonuclears that are
+encountered is marked down. (The _average_ number of bacilli ingested
+per leucocyte = the "_phagocytic index_.")
+
+14. In precisely similar manner enumerate the bacteria present in the
+first 50 cells of the control preparation. This number is recorded as
+the denominator of a vulgar fraction of which the numerator is the
+number recorded for the specific serum. This fraction, expressed as a
+percentage of unity = the _opsonic index_.
+
+
+IMMUNE BODY.
+
+Immune body or amboceptor is the name given to a substance present in
+the serum of an infected animal that has successfully resisted
+inoculation with some particular micro-organism, and which possesses the
+power of linking the complement normally present in the serum to
+bacteria of the species used as antigen in such a manner that the
+micro-organisms are rendered innocuous, and ultimately destroyed. The
+presence of the immune body in the serum can be demonstrated _in vitro_
+by the reaction elaborated by Bordet and Gengou, known as the complement
+fixation test, the existence or the absence of the phenomenon of
+complement fixation being rendered obvious macroscopically by the
+absence or presence of haemolysis on the subsequent addition of
+"sensitised" red blood corpuscles, (e. g., a mixture of crythrocyte
+solution and the appropriate haemolysin--two of the three essentials in
+the haemolytic system, _vide_ page 326).
+
+
+     _Apparatus Required:_
+
+     Sterile pipettes 1 c.c., (graduated in tenths).
+
+     16 x 2 cm. test-tubes.
+
+     9 x 1 cm. test-tubes.
+
+     Test-tube racks for each size of test-tube.
+
+
+     _Reagents Required:_
+
+     Normal saline solution.
+
+     Erythrocyte solution (human red cells, page 329) = E.
+
+     Haemolytic serum (for human cells) = H.S.
+
+     Complement (fresh guinea-pig serum) = C.
+
+     Specific serum from inoculated animal, inactivated = S.S.
+
+     Control pooled serum from normal animals of same species,
+     Inactivated = P.S.
+
+     _Antigen_ (cultivation upon solid medium of the organism
+     (e. g., B. typhosus) which has already served as antigen
+     in the inoculation of the experimental animal) = A.
+
+To prepare the antigen for use, emulsify the whole of the bacterial
+growth in 5 c.c. normal saline solution.
+
+Shake the emulsion in a test-tube with some sterilised glass beads to
+ensure a homogenous emulsion, and sterilise by heating to 60 deg. C. in
+a water-bath for one hour.
+
+METHOD.--
+
+1. Take five small test-tubes, and number them 1 to 5 with a grease
+pencil.
+
+2. Into tubes Nos. 1, 3, 4 and 5 pipette 0.1 c.c. of complement.
+
+3. Into tubes Nos. 1 and 2 pipette 0.2 c.c. of the serum to be tested.
+
+4. Into tube No. 4 pipette 0.2 c.c. of control serum.
+
+5. Into tubes Nos. 1, 2, 3 and 4 pipette 1 c.c. of the bacterial
+emulsion which forms the antigen.
+
+6. Place the whole set of tubes in the incubator at 37 deg. C. for a
+period of one hour.
+
+7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte
+solution and 4 minimal haemolytic doses of the corresponding haemolysin
+into each tube.
+
+8. Mix thoroughly and return the tubes to the incubator at 37 deg. C.
+for further period of one hour.
+
+9. At the expiration of that time transfer the tubes to the ice chest,
+and allow them to stand for three hours.
+
+10. Examine the tubes.
+
+Tubes 3, 4 and 5 should show complete haemolysis; tube 2 should give no
+evidence whatever of haemolysis.
+
+These tubes form the controls to the first tube, which contains the
+serum to be tested.
+
+In tube No. 1 the absence of haemolysis would indicate the presence in
+the serum of the inoculated animal of a specific antibody to the
+micro-organism used in the inoculations; since it shows that the
+complement has been bound by the immune body to the bacterial antigen,
+and none has been left free to enter into the haemolytic system; on the
+other hand the presence of haemolysis would show that no appreciable
+amount of antibody has yet been formed in response to the inoculations.
+In other words, there is an absence of infection, since the complement
+remained unfixed at the time of the addition of the erythrocyte solution
+and haemolytic serum, and was ready to combine with those reagents to
+complete the haemolytic system.
+
+The method may be shown diagramatically as under using the symbols
+already indicated
+
+                       Test-tubes.
+
+     1              2               3               4               5
+
+0.1 c.c. C.      ........       0.1 c.c. C.     0.1 c.c. C.    0.1 c.c. C.
+
+0.2 c.c. S.S.   0.2 c.c. S.S.    .........      0.2 c.c. P.S.   ........
+
+    A.               A.              A.              A.         ........
+--------------------------------------------------------------------------
+                 Incubate at 37 deg. C. for one hour.
+--------------------------------------------------------------------------
+
+1 c.c. E.       1. c.c. E.        1 c.c. E.        1 c.c. E.     1 c.c. E.
+
+ H.S.^{4}         H.S.^{4}         H.S.^{4}         H.S.^{4}      H.S.^{4}
+--------------------------------------------------------------------------
+                 Incubate at 37 deg. C. for one hour.
+--------------------------------------------------------------------------
+(?)            No haemolysis.         |__________________________________|
+
+                                                   Haemolysis.
+
+     NOTE.--It is sometimes more convenient to _sensitise_ the
+     erythrocytes just before they are needed. This is done
+     forty-five minutes after the experiment has been started
+     (page 394, step 6), that is to say, before the completion of
+     the first period of incubation, thus:
+
+     1. Measure out into a sterile test-tube (or flask) five c.c.
+     of erythrocyte solution.
+
+     2. Measure out twenty minimal haemolytic doses of haemolysin,
+     add to the erythrocyte solution on the test-tube.
+
+     3. Allow the erythrocyte and haemolysin to remain in contact
+     for fifteen minutes at room temperature. The red cells are
+     then sensitised and ready for use.
+
+     4. When the tubes are removed from the incubator at the end
+     of the first hour (i. e., step 7) add 1 c.c. sensitised
+     red cells to each tube by means of a graduated pipette.
+
+     5. Mix thoroughly, return the tubes to the incubator at
+     37 deg. C. and complete the experiment as previously described
+     (steps 8 onward).
+
+
+
+
+XIX. POST-MORTEM EXAMINATIONS OF EXPERIMENTAL ANIMALS.
+
+
+The post-mortem examination should be carried out as soon as possible
+after the death of the animal, for it must be remembered that even in
+cold weather the tissues are rapidly invaded by numerous bacteria
+derived from the alimentary tract or the cavities of the body, and from
+external sources.
+
+The following outlines refer to a complete and exhaustive necropsy, and
+in routine work the examination will rarely need to be carried out in
+its entirety.
+
+     NOTE.--Throughout the autopsy the searing irons must be
+     freely employed, and it must be recollected that one
+     instrument is only to be employed to seize or cut one
+     structure. This done, it must be regarded as contaminated
+     and a fresh instrument taken for the next step.
+
+~Apparatus Required~:
+
+Water steriliser.
+
+                           { Scalpels.
+    Surgical instruments:  { Scissors.
+                           { Forceps.
+                           { Bone forceps.
+
+Spear-headed platinum spatula (Fig. 199).
+
+Searing irons (Fig. 198).
+
+Tubes of media--bouillon and sloped agar.
+
+Surface plates in petri dishes (of agar or one of its derivatives).
+
+Platinum loop.
+
+Aluminium "spreader."
+
+Grease pencil.
+
+Sterile capillary pipettes (Fig. 13, a).
+
+Sterile glass capsules, large and small.
+
+Cover-slips or slides.
+
+Bottles of fixing fluid (_vide_ page 114) for pieces of tissue intended
+for sectioning.
+
+1. Place the various instruments, forceps, scissors, scalpels, etc.,
+needed for the autopsy inside the steriliser and sterilise by boiling
+for ten minutes; then open the steriliser, raise the tray from the
+interior and rest it crosswise on the edges.
+
+2. Heat the searing irons to redness in a separate gas stove.
+
+[Illustration: FIG. 197.--Apparatus for post-mortem examination, animal
+on board.]
+
+3. Drench the fur (or feathers) with lysol solution, 2 per cent. This
+serves the twofold purpose of preventing the hairs from flying about and
+entering the body cavities during the autopsy, and of rendering
+innocuous any vermin that may be present on the animal.
+
+[Illustration: FIG. 198.--Searing iron.]
+
+4. Examine the cadaver carefully. Recollect that laboratory animals are
+not always hardy; death may be due to exposure to heat or cold, to
+starvation or over- or improper feeding or to the attack of rats--and
+not to the bacterial infection.
+
+5. Fasten the body of the animal, ventral surface upward (unless there
+is some special reason for having the dorsum exposed), out on a board
+by means of copper nails driven through the extremities.
+
+6. With sterile forceps and scalpel incise the skin in the middle line
+from the top of the sternum to the pubes. Make other incisions at right
+angles to the first out to the axillae and groins, and reflect the skin
+in two lateral flaps. (Place the now infected instruments on the board
+by the side of the body or support them on a porcelain knife rest.)
+
+
+~Seat of Inoculation.~--
+
+7. Inspect the seat of inoculation. If any local lesion is visible, sear
+its exposed surface and with the platinum loop, remove material from the
+deeper parts to make tube and surface plate cultivations and cover-slip
+preparations.
+
+Collect specimens of pus or other exudation in capillary pipettes for
+subsequent examination.
+
+8. Inspect the neighbouring lymphatic glands and endeavour to trace the
+path of the virus.
+
+9. Sear the whole of the exposed surface of the thorax with the searing
+irons.
+
+
+~Pleural Cavity.~--
+
+10. Divide the ribs on either side of the sternum and remove a
+rectangular portion of the anterior chest wall with sterile scissors and
+a fresh pair of forceps, exposing the heart. Place the infected
+instruments by the side of the first set.
+
+11. Observe the condition of the anterior mediastinal glands, the thymus
+and the lungs. Collect a quantity of pleuritic effusion, if such is
+present, in a pipette for further examination later.
+
+12. Raise the pericardial sac in a fresh pair of forceps and burn
+through this structure with a searing iron.
+
+Collect a sample of pericardial fluid in a pipette for microscopical and
+cultural examination.
+
+13. Grasp the apex of the heart in the forceps and sear the surface of
+the right ventricle.
+
+14. Plunge the open point of a capillary pipette through the seared area
+into the ventricle and fill with blood.
+
+Make cultivations and cover-slip preparations of the heart blood.
+
+15. Collect a further sample of blood or serum for subsequent
+investigation as to the presence of antibodies.
+
+
+~Peritoneal Cavity.~--
+
+16. Sear a broad track in the middle line of the abdominal wall; open
+the peritoneal cavity by an incision in the centre of the seared line.
+Observe the condition of the omentum, the mesentery, the viscera and the
+peritoneal surface of the intestines.
+
+17. Collect a specimen of the peritoneal fluid (or pus, if present) in a
+capillary pipette. Make cultivations, tube and surface plate, and
+cover-slip preparations from this situation.
+
+18. Collect a specimen of the urine from the distended bladder in a
+large pipette (in the manner indicated for heart blood), for further
+examination, by cultivations, microscopical preparations, and chemical
+analysis.
+
+19. Collect a specimen of bile from the gall bladder in similar manner.
+
+20. Excise the spleen and place it in a sterile capsule. Later, sear the
+surface of this organ; plunge the spear-headed spatula through the
+centre of the seared area, twist it round between the finger and thumb,
+and remove it from the organ. Sufficient material will be brought away
+in the eye in its head to make cultivations. A repetition of the process
+will afford material for cover-slip preparations.
+
+21. Seize one end of the spleen with sterile forceps. Sear a narrow band
+of tissue, right around the organ and divide the spleen in this
+situation with a pair of scissors. Holding the piece of spleen in the
+forceps, dab the cut surface on to a surface plate in a number of
+different spots.
+
+22. In like manner examine the other organs--liver, lungs, kidneys,
+lymphatic glands (mesenteric, hepatic, lumbar, etc), etc. Prepare
+cultivations and cover-slip preparations.
+
+23. Dissect out a long bone from one upper and one lower limb and one of
+the largest ribs. Prepare cultures from the bone marrow in each case.
+Set aside these bones for the subsequent preparation of marrow films.
+
+24. Film preparations of bone marrow are best made by the Price-Jones
+method. Seize the bone in a pair of pliers and squeeze out some of the
+marrow; receive it in a platinum loop, and transfer to a watch glass of
+dissociating fluid and emulsify. The dissociating fluid is a neutral 10
+per cent. solution of glycerine prepared as follows:--
+
+     Measure out 10 c.c. Price's best glycerine and 90 c.c.
+     sterile ammonia-free distilled water. Mix. Titrate against
+     n/10 sodic hydrate solution using phenolphthalein as the
+     indicator. The initial reaction is usually + 0.1 to + 0.5;
+     add the calculated amount of n/10 sodic hydrate solution to
+     neutralise.
+
+25. Place a loopful of fresh desiccating fluid on a 3 x 1 glass slide;
+add a similar loopful of the marrow emulsion, and spread very gently
+over the surface of the slip.
+
+26. Allow film to dry in the air (protected from dust) without heating.
+
+27. Stain with Jenner's polychrome stain (page 97) for two and a half
+minutes.
+
+28. Wash with ammonia-free distilled water, dry thoroughly and mount in
+xylol balsam.
+
+
+~Cranial and Spinal Cavities.~--
+
+29. In some instances it may be necessary (e. g., experimental
+inoculation of rabies) to examine the cranial cavity or to remove the
+spinal cord. Return the viscera to the abdominal cavity; draw the flaps
+of skin together and secure with Michel's steel clips. Draw the copper
+nails securing the limbs to the board, reverse the animal and again nail
+the limbs down--the body now being dorsum uppermost.
+
+30. Make a longitudinal incision in the mesial line from snout to root
+of tail, and four transverse incisions--one joining the roots of the two
+ears, one across the body at the level of the spinis of the scapulae,
+another at the level of the costal margin and the last across the upper
+level of the pelvis. Reflect these flaps of skin.
+
+31. With forceps and scalpel dissect out the muscles lying in the furrow
+on either side of the spinal processes.
+
+32. Cut through the bases of the transverse processes with bone forceps.
+Cut away the vault of the skull, cut through the roots of the nerves and
+remove the brain and spinal cord, place in a large glass dish for
+examination. Prepare cultivations from the cerebro-spinal fluid. The
+removal of the brain and cord is a tedious process and during the
+dissection it is difficult to avoid injury to these structures.
+
+The operation is, however, carried out very expeditiously and neatly
+with the aid of the surgical engine (_vide_ page 361). A small circular
+saw is fitted to the hand piece. The bones of the skull are cut through
+and the whole of the vault removed, exposing the entire vertex of the
+brain. Similarly all the spinous processes can be removed in one string
+by running the saw down first one side of the spinal column and then the
+other. In this way ample space for the removal of the nervous tissues is
+obtained with a minimum of labour.
+
+33. Having completed the preparation of cultures remove small portions
+of various organs at leisure and place each in separate bottles of
+fixing fluid for future sectioning. Affix to each bottle a label bearing
+all necessary details as to its contents.
+
+34. If necessary, remove portions of the organs for preservation and
+display as museum specimens (_vide_ page 404).
+
+35. Gather up all the infected instruments, return them to the
+steriliser, and disinfect by boiling for ten minutes.
+
+[Illustration: FIG. 199.--Spear-headed platinum spatula (actual size.)]
+
+36. Sprinkle dry sawdust into the exposed body cavities to absorb blood
+and fluid. Cover the body with blotting or filter paper, moistened with
+2 per cent. lysol solution. Place in a galvanised iron pail, provided
+with a lid, ready for transport to the crematorium.
+
+37. Cremate the cadaver together with the board upon which it is fixed.
+
+38. Stain the cover-slip preparations by suitable methods and examine
+microscopically.
+
+39. Incubate the cultivations and examine carefully from day to day.
+
+40. Make full notes of the condition of the various body cavities and of
+the viscera immediately the autopsy is completed; and add the result of
+the microscopical and cultural investigation when available.
+
+As part of the card index system in use in the author's laboratory
+already referred to (_vide_ page 335) there is a special yellow card for
+P-M notes. On the face of the card are printed headings for various
+data--some of which are sometimes unintentionally omitted--and on the
+reverse is a schematic figure which can be utilised for indicating the
+position of the chief lesions in the cadaver of any of the laboratory
+animals.
+
+AUTOPSY CARD       Laboratory No. _________
+
+Date ________
+
+Animal ______  No. in Series ______ [Symbols: male female] Weight ________
++------------------------------------------------------------------------+
+Died (or killed) _____ o'clock ____ m.  Autopsy made _____ o'clock ____ m.
++------------------------------------------------------------------------+
+Notes on Post Mortem Examinations.
+
+_General._
+
+A. Seat of Inoculation.
+
+B. Thoracic Cavity.
+
+C. Abdominal Cavity.
+
+D. Cranial Cavity.
+
++-------------------+---- -------------+--------------------------+
+_Bacteriological_   |  _Histological_  |  _Organs Preserved._     |
+    _Examination._  |  _Examination._  |                          |
+A.                  |                  |                          |
+                    |                  |                          |
+B.                  |                  |                          |
+                    |                  |                          |
+C.                  |                  |                          |
+                    |                  |                          |
+D.                  |                  |                          |
+
+[Illustration: FIG. 200.--Front of post-mortem card.]
+
+41. Finally, the results of the action of the organism or organisms
+isolated may be correlated with the symptoms observed during life and
+the observations summarised under the following headings:
+
+Tissue changes:
+
+    1. Local--i. e., produced in the neighbourhood of the bacteria.
+
+       Position: (a) At primary lesion.
+
+                 (b) At secondary foci.
+
+       Character: (a) Vascular changes and tissue    }   Acute
+                      reactions.                     }    or
+                  (b) Degeneration and necrosis.     }   chronic.
+
+    2. General (i. e., produced at a distance from the bacteria, by
+       absorption of toxins):
+
+         (a) In special tissues--e. g., nerve cells and fibres, secreting
+             cells, vessel walls, etc.
+
+         (b) General effects of malnutrition, etc.
+
+    Symptoms:
+
+        (a) Associated with known tissue changes.
+
+        (b) Without known tissue changes.
+
+[Illustration: FIG. 201.--Back of post-mortem card.]
+
+
+~Permanent Preparations--Museum Specimens.~--
+
+_I. Tissues._--The naked-eye appearances of morbid tissues may be
+preserved by the following method:
+
+1. Remove the tissue or organ from the cadaver as soon after death as
+possible, using great care to avoid distortion or injury.
+
+2. Place it in a wide-mouthed stoppered jar, large enough to hold it
+conveniently, resting on a pad of cotton-wool, and arrange it in the
+position it is intended to occupy (but if it is intended to show a
+section of the tissue or organ, do not incise it yet).
+
+3. Cover with the Kaiserling fixing solution, and stopper the jar; allow
+the tissues to remain in this solution for from forty-eight hours to
+seven days (according to size) to fix. Make any necessary sections.
+
+Kaiserling modified solution is prepared as follows:
+
+Weigh out
+
+    Potassium acetate      30 grammes.
+    Potassium nitrate      15 grammes.
+
+and dissolve in
+
+    Distilled water        1000 c.c.
+
+then add
+
+    Formalin               150 c.c.
+
+Filter.
+
+This fixing solution can be used repeatedly so long as it remains clear.
+Even when it has become turbid, if simple filtration is sufficient to
+render it clear, the filtrate may be used again.
+
+4. Transfer the tissue to a bath of methylated spirit (95 per cent.) for
+thirty minutes to one hour.
+
+5. Remove to a fresh bath of spirit and watch carefully. When the
+natural colours show in their original tints, average time three to six
+hours, remove the tissues from the spirit bath, dry off the spirit from
+the cut surfaces by mopping with a soft cloth, then transfer to the
+mounting solution.
+
+Jore's mounting solution (modified) consists of
+
+    Glycerine                  500 c.c.
+    Distilled water            750 c.c.
+    Formalin                     2 c.c.
+
+Equally good but much cheaper is Frost's mounting solution:
+
+    Potassium acetate               160 grammes.
+    Sodium fluoride                  80 grammes.
+    Chloral hydrate                  80 grammes.
+    Cane sugar (Tate's cubes)     3,500 grammes.
+    Saturated thymol water        8,000 c.c.
+
+6. After twenty-four hours in this solution, or as soon as the tissue
+sinks, transfer to a museum jar, fill with fresh mounting solution, and
+seal.
+
+_6a._ Or transfer to museum jar and fill with liquefied gelatine, to
+which has been added 1 per cent. formalin. Cover the jar and allow the
+gelatine to set. When solid, seal the cover of the jar in place.
+
+7. To seal the museum preparation first warm the glass plate which forms
+the cover. This is most conveniently done by placing the cleaned and
+polished cover-plate upon a piece of asbestos millboard over a bunsen
+flame turned low.
+
+8. Smear an even layer of hot cement over the flange of the jar. The
+cement is prepared as follows:
+
+Weigh out and mix in an iron ladle
+
+    Gutta percha (pure)        4 parts.
+    Asphaltum                  5 parts.
+
+and melt together over a bunsen flame, stirring with an iron rod until
+solution is complete.
+
+9. Invert the glass plate over the jar and press down firmly into the
+cement. Place a piece of asbestos board on the top and on that rest a
+suitable weight until the cement is cold and has thoroughly set.
+
+10. Trim off any projecting pieces of cement with an old knife, burr
+over the joint between jar and cover-plate with a hot smooth piece of
+metal (e. g., the searing iron).
+
+11. Paint a narrow band of Japan black to finish off, round the joint,
+overlapping on to the cover-plate.
+
+_II. Tube Cultivations of Bacteria._--When showing typical appearances
+these may be preserved, if not permanently, at least for many years, as
+museum specimens, by the following method:
+
+1. Take a large glass jar 25 cm. high by 18 cm. diameter, with a firm
+base and a broad flange, carefully ground, around the mouth. The jar
+must be fitted with a disc of plate glass ground on one side, to serve
+as a lid.
+
+2. Smear a thick layer of resin ointment (B.P.) on the flange around the
+mouth of the jar.
+
+3. Cover the bottom of the jar with a layer of cotton-wool and saturate
+it with formalin.
+
+4. Remove the cotton-wool plug from the culture tubes and place them,
+mouth upward, inside the jar. (If water of condensation is present in
+any of the culture tubes, it should be removed by means of a capillary
+pipette before placing the tubes in the formalin chamber.)
+
+5. Adjust the glass disc, ground side downward, over the mouth of the
+jar and secure it by pressing it firmly down into the ointment, with a
+rotary movement.
+
+6. Remove the tubes from the formalin chamber after the lapse of a week,
+and dry the exterior of each.
+
+[Illustration: FIG. 202.--Bulloch's tubes.]
+
+7. Seal the open mouth of each tube in the blowpipe flame and label.
+
+If the cultivations are intended for museum purposes when they are first
+planted, it is more convenient to employ Bulloch's tubes. These are
+slightly longer than the ordinary tubes, and are provided with a
+constriction some 2 cm. below the mouth (Fig. 202)--a feature which
+renders sealing in the blowpipe flame an easy matter.
+
+
+
+
+XX. THE STUDY OF THE PATHOGENIC BACTERIA.
+
+
+The student, who has conscientiously worked out the methods, etc.,
+previously dealt with, is in a position to make accurate observations
+and to write precise descriptions of the results of such observations.
+He is, therefore, now entrusted with pure cultivations of the various
+pathogenic bacteria, in order that he may study the life-history of each
+and record the results of his own observations--to be subsequently
+corrected or amplified by the demonstrator. In this way he is rendered
+independent of text-book descriptions, the statements in which he is
+otherwise too liable to take for granted, without personally attempting
+to verify their accuracy.
+
+During the course of this work attention must also be directed, as
+occasion arises, to such other bacteria, pathogenic or saprophytic, as
+are allied to the particular organisms under observation, or so resemble
+them as to become possible sources of error, by working them through on
+parallel lines--in other words the various bacteria should be studied in
+"groups." In the following pages the grouping in use in the author's
+elementary classes for medical and dental students and for candidates
+for the Public Health service is adopted, since a fairly long experience
+has completely vindicated the value and utility of this arrangement, and
+by its means a fund of information is obtained with regard to the
+resemblances and differences, morphological and cultural, of a large
+number of bacteria. The fact that some bacteria appear in more than one
+of these groups, so far from being a disadvantage, is a positive gain to
+the student, since with repetition alone will the necessary familiarity
+with the cultural characters of important bacteria be acquired. The
+study of the various groups will of course vary in detail with
+individual demonstrators, and with the student's requirements--the
+general line it should take is indicated briefly in connection with the
+first group only (pages 410-411). This section should be carefully
+worked through before the student proceeds to the study of
+bacterioscopical analysis.
+
+It is customary to commence the study of the pathogenic bacteria with
+the Organisms of Suppuration. This is a large group, for all the
+pathogenic bacteria possess the power, under certain conditions, of
+initiating purely pyogenic processes in place of or in addition to their
+specific lesions, (e. g., Bacillus tuberculosis, Streptococcus
+lanceolatus, Bacillus typhosus, etc.). There are, however, a certain few
+organisms which commonly express their pathogenicity in the formation of
+pus. These are usually grouped together under the title of "pyogenic
+bacteria," as distinct from those which only occasionally exercise a
+pyogenic role.
+
+The organisms included in this group are:
+
+    1. Staphylococcus pyogenes albus.
+    2. Staphylococcus pyogenes aureus.
+    3. Staphylococcus pyogenes citreus.
+    4. Streptococcus pyogenes longus.
+    5. Micrococcus tetragenus.
+    6. Bacillus pyocyaneus.
+    7. Bacillus pneumoniae.
+
+and in certain special tissues
+
+    8. Micrococcus gonorrhoeae.
+    9. Micrococcus intracellularis meningitidis (Meningococcus).
+    10. Micrococcus catarrhalis.
+    11. Bacillus aegypticus (Koch-Weeks Bacillus).
+
+The group may with advantage be subdivided as indicated in the following
+pages:
+
+I. _Pyogenic cocci._
+
+    Staphylococcus pyogenes albus.
+    Staphylococcus pyogenes aureus.
+    Staphylococcus pyogenes citreus.
+        to contrast with
+    Micrococcus candicans.
+    Micrococcus agilis.
+
+1. Prepare subcultivations from each:
+
+    Bouillon,         }
+    Agar streak,      }
+    Blood serum,      }
+    Litmus milk.      }  and incubate at 37 deg. C.
+    Agar streak,      }
+    Gelatine stab,    }
+    Potato.           }  and incubate at 20 deg. C.
+
+Compare the naked-eye appearances of the cultures from day to day. Note
+M. agilis refuses to grow at 37 deg. C.
+
+2. Make hanging-drop preparations from the bouillon and agar
+cultivations after twenty-four hours' incubation. Examine
+microscopically and compare. Note the locomotive activity of M. agilis
+and the Brownian movement of the remaining micrococci.
+
+3. Prepare cover-slip films from the agar cultures, after twenty-four
+hours' incubation. Stain for flagella by the modified Pitfield's method.
+Note M. agilis is the only micrococcus showing flagella.
+
+4. Make microscopical preparations of each from all the various media
+after twenty-four and forty-eight hours and three days' incubation.
+Stain carbolic methylene-blue, carbolic fuchsin, and Gram's method.
+Examine the films microscopically and compare. Note in the Gram
+preparation, the Gram negative character of certain individual cocci in
+each film prepared from the three days' growth--such cocci are dead.
+
+5. Stain section of kidney tissue provided (showing abscess formation
+by Staphylococcus aureus) by Gram's method, and counterstain with cosin.
+
+6. Stain film preparation of pus from an abscess (containing
+Staphylococcus pyogenes aureus) with carbolic methylene-blue and also by
+Gram's method, counterstained with cosin.
+
+7. Inoculate[15] a white mouse subcutaneously with three loopfuls of a
+forty-eight-hour agar cultivation of the Staphylococcus aureus,
+emulsified with 0.2 c.c. sterile broth.
+
+Observe carefully during life, and when death occurs make a careful
+post-mortem examination.
+
+II. _Pyogenic cocci._
+
+    Micrococcus gonorrhoeae.
+    Micrococcus intracellularis meningitidis (meningococcus).
+    Micrococcus catarrhalis.
+    Micrococcus tetragenus.
+    Micrococcus paratetragenus.
+
+III. _Pyogenic cocci._
+
+    Streptococcus pyogenes longus.
+    Streptococcus of bovine mastitis.
+    Streptococcus lanceolatus (Diplococcus pneumoniae or pneumococcus).
+        to contrast with
+    Streptococcus brevis.
+    Streptococcus lebensis.
+
+IV. _Pyogenic bacilli._
+
+    Bacillus pneumoniae (Friedlaender).
+    Bacillus rhinoscleromatis.
+    Bacillus lactis aerogenes.
+
+V. _Pyogenic bacilli._
+
+     Bacillus pyocyaneus.
+        to contrast with
+    Bacillus fluorescens liquefaciens.
+    Bacillus fluorescens non-liquefaciens.
+
+VI. _Pneumonia group._
+
+    Streptococcus lanceolatus (pneumococcus).
+    Bacillus pneumoniae (Friedlaender).
+    Streptococcus pyogenes longus.
+
+VII. _Diphtheroid group._
+
+    Bacillus diphtheriae (Klebs-Loeffler).
+    Bacillus Hoffmanni.
+    Bacillus xerosis.
+    Bacillus septus.
+
+VIII. _Coli-typhoid group._
+
+    B. typhi abdominalis (B. typhosus).
+    B. coli communis.
+    B. enteritidis (Gaertner).
+      to contrast with
+    B. aquatilis sulcatus.
+
+IX. _Escherich group._
+
+    B. coli communis (Escherich).
+    B. coli communior.
+    B. lactis aerogenes.
+    B. cloacae.
+
+X. _Gaertner group._
+
+    Bacillus enteritidis (Gaertner).
+    B. paratyphosus A.
+    B. paratyphosus B.
+    Bacillus cholerae suum (Hog Cholera).
+    B. psittacosis.
+
+XI. _Eberth group._
+
+    B. typhosus (Eberth).
+    B. dysenteriae (Shiga).
+    B. dysenteriae (Flexner).
+    B. faecalis alcaligines.
+
+XII. _Spirillum group._
+
+    Vibrio cholerae.
+    Vibrio metschnikovi.
+        to contrast with
+    Vibrio proteus (Finkler and Prior).
+    Spirillum rubrum.
+    Spirillum rugula.
+
+XIII. _Anthrax group._
+
+    Bacillus anthracis.
+        to contrast with
+    Bacillus subtilis.
+    Bacillus mycoides.
+    Bacillus mesentericus fuscus.
+
+XIV. _Acid fast group._
+
+    Bacillus tuberculosis (human).
+       "          "       (bovine).
+       "          "       (avian).
+       "          "       (fish).
+      to contrast with
+    Bacillus phlei (Timothy grass bacillus).
+    Butter bacillus of Rabinowitch.
+
+XV. _Plague group._
+
+    Bacillus pestis.
+    B. septicaemiae haemorrhagicae.
+    B. suipestifer.
+
+XVI. _Influenzae group._
+
+    B. influenzae.
+    Bacillus aegypticus (Koch-Weeks).
+    Bacillus pertussis.
+
+XVII. _Miscellaneous._
+
+    Bacillus leprae.
+    Bacillus mallei.
+    Micrococcus melitensis.
+
+XVIII. _Streptothrix group._
+
+    Streptothrix actinomycotica.
+    Streptothrix madurae.
+        to contrast with
+    Cladothrix nivea.
+
+XIX. _Tetanus group._
+
+    Bacillus tetani.
+    Bacillus oedematis maligni.
+    Bacillus chauvei (symptomatic anthrax).
+
+XX. _Enteritidis sporogenes group._
+
+    Bacillus enteritidis sporogenes.
+    B. botulinus.
+    B. butyricus.
+    B. cadaveris.
+
+FOOTNOTES:
+
+[15] See note on Vivisection License, page 334.
+
+
+
+
+XXI. BACTERIOLOGICAL ANALYSES.
+
+
+Each bacteriological or bacterioscopical analysis of air, earth, sewage,
+various food-stuffs, etc., includes, as a general rule, two distinct
+investigations yielding results of very unequal value:
+
+    1. Quantitative.
+    2. Qualitative.
+
+The first is purely quantitative and as such is of minor importance as
+it aims simply at enumerating (approximately) the total number of
+bacteria present in any given unit of volume irrespective of the nature
+and character of individual organisms.
+
+The second and more important is both qualitative and quantitative in
+character since it seeks to accurately identify such pathogenic bacteria
+as may be present while, incidentally, the methods advocated are
+calculated to indicate, with a fair degree of accuracy, the numerical
+frequency of such bacteria, in the sample under examination.
+
+The general principles underlying the bacteriological analyses of water,
+sewage, air and dust, soil, milk, ice cream, meat, and other tinned
+stuffs, as exemplified by the methods used by the author, are indicated
+in the following pages, together with the methods of testing filters and
+chemical germicides; and the technique there set out will be found to be
+capable of expansion and adaptation to any circumstance or set of
+circumstances which may confront the student.
+
+~Controls.~--The necessity for the existence of adequate controls in all
+experimental work cannot be too urgently insisted upon. Every batch of
+plates that is poured should include at least one of the presumably
+"sterile" medium; plate or tube cultures should be made from the various
+diluting fluids; every tube of carbohydrate medium that is inoculated
+should go into the incubator in company with a similar but uninoculated
+tube, and so on.
+
+
+BACTERIOLOGICAL EXAMINATION OF WATER.
+
+The bacteria present in the water may comprise not only varieties which
+have their normal habitat in the water and will consequently develop at
+20 deg. C., but also if the water has been contaminated with excremental
+matter, varieties which have been derived from, or are pathogenic for,
+the animal body, and which will only develop well at a temperature of
+37 deg. C. In order to demonstrate the presence of each of these classes
+it will be necessary to incubate the various cultivations at each of these
+temperatures.
+
+Further, the sample of water may contain moulds, yeasts, or torulae, and
+the development of these will be best secured by plating in wort
+gelatine and incubating at 20 deg. C.
+
+~1. Quantitative.~--
+
+_Collection of the Sample._--The most suitable vessels for the reception
+of the water sample are small glass bottles, 60 c.c. capacity, with
+narrow necks and overhanging glass stoppers (to prevent contamination of
+the bottle necks by falling dust). These must be carefully sterilised in
+the hot-air steriliser (_vide_ page 31).
+
+(a) If the sample is obtained from a ~tap~ or ~pipe~, turn on the water
+and allow it to run for a few minutes. Remove the stopper from the
+bottle and retain it in the hand whilst the water is allowed to run into
+the bottle and three parts fill it. Replace the stopper and tie it down,
+but _do not seal it_.
+
+(b) If the sample is obtained from a ~stream~, ~tank~, or ~reservoir~,
+fasten a piece of stout wire around the neck of the bottle, remove the
+stopper, and retain it in the hand. Then, using the wire as a handle,
+plunge the bottle into the water, mouth downward, until it is well
+beneath the surface; then reverse it, allow it to fill, and withdraw it
+from the water. Pour out a few cubic centimetres of water from the
+bottle, replace the stopper, and tie it down.
+
+[Illustration: FIG. 203.--Esmarch's collecting bottle for water
+samples.]
+
+(c) If the sample is obtained from a ~lake~, ~river~ or the ~sea~; or when
+it is desired to compare samples taken at varying depths, the apparatus
+designed by v. Esmarch (Fig. 203) is employed. In this the sterilised
+bottle is enclosed in a weighted metal cage which can be lowered, by
+means of a graduated line, until the required depth is reached. At this
+point the bottle is opened by a thin wire cord attached to the stopper;
+when the bottle is full (as judged by the air bubbles ceasing to rise)
+the pull on the cord is released and the tension of the spiral spring
+above the stopper again forces it into the neck of the bottle. When the
+apparatus is taken out of the water, the small bottles are filled from
+it, and packed in the ice-box mentioned below.
+
+An inexpensive substitute for Esmarch's bottle can be made in the
+laboratory thus:
+
+Select a wide-mouthed glass stoppered bottle of about 500 c.c. capacity
+(about 20 cm. high and 8 cm. in diameter).
+
+Remove the glass stopper and insert a rubber cork with two perforations
+in its place.
+
+Through one perforation pass a piece of glass tubing about 5 cm. long
+and through the other a piece 22 cm. long, reaching to near the bottom
+of the bottle, each tube projecting about 2.5 cm. above the rubber
+stopper. Plug the open ends of the tubes with cotton wool. Secure the
+stopper in place with thin copper wire.
+
+[Illustration: FIG. 204.--Thresh's deep water sampling bottle.]
+
+Sterilise the fitted bottle in the autoclave. Remove the cotton wool
+plugs and connect the projecting tubes by a piece of loosely fitting
+stout rubber pressure tubing about 5 cm. long, previously sterilised by
+boiling.
+
+Take a piece of stout rubber cord about 33 cm. long, and of 10 mm.
+diameter (such as is used for door springs) thread a steel split ring
+upon it and secure the free ends tightly to the neck of the bottle by
+cord or catgut.
+
+Attach the cord used for lowering the bottle into the water to the split
+ring on the rubber suspender. The best material for this purpose is
+cotton insulated electric wire knotted at every metre.
+
+Connect the split ring also with the short piece of rubber tubing
+uniting the two glass tubes by a piece of catgut (or thin copper wire)
+of such length that when the bottle is suspended there is no pull upon
+the rubber tube, but which, however, will be easily jerked off when a
+sharp pull is given to the suspending cord.
+
+Now wind heavy lead tubing about 1 cm. diameter around the upper part of
+the bottle, starting at the neck just above the shoulder. This ensures
+the sinking of the bottle in the vertical position (Fig. 204).
+
+The apparatus being arranged is lowered to the required depth, a sharp
+jerk is then given to the suspending cord, which detaches the rubber
+tube and so opens the two glass tubes. Water enters through the longer
+tube and the air is expelled through the shorter tube. The bubbles of
+air can be seen or heard rising through the water, until the bottle is
+nearly full, a small volume of compressed air remaining in the neck of
+the bottle.
+
+As the apparatus is raised, the air thus imprisoned expands, and
+prevents the entry of more water from nearer the surface.
+
+[Illustration: FIG. 205.--Ice-box for transmission of water samples,
+etc.]
+
+_Transport of Sample._--If the examination of the sample cannot be
+commenced immediately, steps must be taken to prevent the multiplication
+of the bacteria contained in the water during the interval occupied in
+transit from the place of collection to the laboratory. To this end an
+ice-box such as that shown (in Fig. 205) is essential. It consists of a
+double-walled metal cylinder into which slides a cylindrical chamber of
+sufficient capacity to accommodate four of the 60 c.c. bottles; this in
+turn is covered by a metal disc--the three portions being bolted
+together by thumb screws through the overhanging flanges. When in use,
+place the bottles, rolled in cotton-wool, in the central chamber, pack
+the space between the walls with pounded ice, securely close the metal
+box by screwing down the fly nuts, and place it in a felt-lined wooden
+case. (It has been shown that whilst bacteria will survive exposure to
+the temperature of melting ice, practically none will multiply at this
+temperature.)
+
+On reaching the laboratory, the method of examination consists in adding
+measured quantities of the water sample to several tubes of nutrient
+media previously liquefied by heat, pouring plate cultivations from each
+of these tubes, incubating at a suitable temperature, and finally
+counting the colonies which make their appearance on the plates.
+
+_Apparatus Required_:
+
+    Plate-levelling stand.
+    Case of sterile plates.
+    Case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre).
+    Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).
+    Case of sterile capsules, 25 c.c. capacity.
+    Tubes of nutrient gelatine.
+    Tubes of nutrient agar.
+    Tubes of wort gelatine.
+    One 250 c.c. flask of sterile distilled water.
+    Tall cylinder containing 2 per cent. lysol solution.
+    Bunsen burner.
+    Grease pencil.
+    Water-bath regulated at 42 deg. C.
+
+METHOD.--
+
+1. Arrange the plate-levelling platform with its water compartment
+filled with water, at 45 deg. C.
+
+2. Number the agar tubes, consecutively, 1 to 6; the gelatine tubes,
+consecutively, 1 to 6, and the wort tubes, 1, 2, and 3. Flame the plugs
+and see that they are not adherent to the lips of the tubes.
+
+3. Place the agar tubes in boiling water until the medium is melted,
+then transfer them to the water-bath regulated at 42 deg. C. Liquefy the
+nutrient gelatine and wort gelatine tubes by immersing them in the same
+water-bath.
+
+4. Remove the bottle containing the water sample from the ice-box,
+distribute the bacterial contents evenly throughout the water by
+shaking, cut the string securing the stopper, and loosen the stopper,
+but do not take it out.
+
+[Illustration: FIG. 206.--Withdrawing water from water sample bottle.]
+
+5. Remove one of the 1 c.c. pipettes from the case, holding it by the
+plain portion of the tube. Pass the graduated portion twice through the
+Bunsen flame. Tilt the bottle containing the water sample on the bench
+holding the neck between the middle and ring fingers of the left hand;
+grasp the head of the stopper between the forefinger and thumb, and
+remove it from the bottle.
+
+6. Pass the pipette into the mouth of the bottle, holding its point well
+below the surface of the water (Fig. 206). Suck up rather more than 1
+c.c. into the pipette and allow the pipette to empty; this moistens the
+interior of the pipette and renders accurate measurement possible. Now
+draw up exactly 1 c.c. into the pipette. Withdraw the pipette from the
+bottle, replace the stopper, and stand the bottle upright.
+
+7. Take the first melted agar tube in the left hand, remove the
+cotton-wool plug, and add to its contents 0.5 c.c. of the water sample
+from the pipette; replug the tube and replace it in the water-bath. In a
+similar manner add 0.3 c.c. water to the contents of the second tube,
+and 0.2 c.c. to the contents of the third.
+
+8. In a similar manner add 1 c.c. of the sample to the contents of the
+fourth tube.
+
+9. Similarly, add 0.5 c.c. and 0.1 c.c. respectively to the contents of
+the fifth and sixth tubes.
+
+10. Drop the pipette into the cylinder containing lysol solution.
+
+11. Mix the water sample with the medium in each tube in the manner
+described under plate cultivations; pour a plate from each tube. Label
+each plate with (a) the distinctive number of the sample, (b) the
+quantity of water sample it contains, and (c) the date.
+
+12. Pour the contents of a tube of liquefied agar--not inoculated--into
+a Petri dish to act as a control to demonstrate the sterility of the
+batch of agar employed.
+
+13. Allow the plates to set, and incubate at 37 deg. C.
+
+14. Empty the water chamber of the levelling apparatus and refill it
+with ice-water.
+
+15. By means of the sterile 10 c.c. pipette deliver 9.9 c.c. sterile
+distilled water into a sterile glass capsule.
+
+16. Add 0.1 c.c. of the water sample to the 9.9 c.c. sterile water in
+the capsule. This will give a dilution of 1 in 100.
+
+17. Plant the six tubes of nutrient gelatine in the following manner: To
+the first tube add 0.5 c.c. of the water sample direct from the bottle;
+to the second, 0.3 c.c.; and to the third, 0.2 c.c.; and pour a plate of
+each tube. To the fourth tube add 0.5 c.c. of the diluted water sample
+from the capsule; to the fifth, 0.3 c.c.; and to the sixth, 0.2 c.c.;
+and pour a plate from each.
+
+18. Label each plate with the quantity of the water sample it
+contains--that is, 0.5 c.c., 0.3 c.c., 0.2 c.c., 0.005 c.c., 0.003 c.c.,
+and 0.002 c.c.
+
+19. Pour a control (uninoculated) gelatine plate.
+
+20. Allow the plates to set, and incubate at 20 deg. C.
+
+21. To the first tube of liquefied wort gelatine add 0.5 c.c. water
+sample; to the second, 0.3 c.c.; and to the third, 0.2 c.c.
+
+22. Label the plates, allow them to set, and incubate at 20 deg. C.
+
+23. Count and record the number of colonies that have developed upon the
+agar at 37 deg. C. after forty-eight hours' incubation.
+
+24. Note the number of colonies present on each of the gelatine and wort
+gelatine plates after forty-eight hours' incubation.
+
+25. Replace the gelatine and wort plates in the incubator; observe again
+at three days, four days, and five days.
+
+26. Calculate and record the number of organisms present per cubic
+centimetre of the original water from the average of the six gelatine
+plates at the latest date possible up to seven days--the presence of
+liquefying bacteria may render the calculation necessary at an earlier
+date, hence the importance of daily observations.
+
+_Method of Counting._--The most accurate method of counting the colonies
+on each of the plates is by means of either Jeffery's or Pakes' counting
+disc. Each of these discs consists of a piece of paper, upon which is
+printed a dead black disc, subdivided by concentric circles and radii,
+printed in white. In Jeffery's counter (Fig. 207), each subdivision has
+an area of 1 square centimetre; in Pakes' counter (Fig. 208), radii
+divide the circle into sixteen equal sectors, and counting is
+facilitated by concentric circles equidistant from the centre.
+
+[Illustration: FIG. 207.--Jeffery's disc, reduced.]
+
+[Illustration: FIG. 208.--Pakes' disc, reduced.]
+
+(a) In the final counting of each plate, place the plate over the
+counting disc, and centre it, if possible, making its periphery coincide
+with one or other of the concentric circles.
+
+(b) Remove the cover of the plate, and by means of a hand lens count the
+colonies appearing in each of the sectors in turn. Make a note of the
+number present in each.
+
+(c) If the colonies present are fewer than 500, the entire plate should
+be counted. If, however, they exceed this number, enumerate one-half, or
+one-quarter of the plate, or count a sector here and there, and from
+these figures estimate the number of colonies present on the entire
+plate. In practice it will be found that Pakes' disc is more suitable
+for the former class of plate; Jeffery's disc for the latter. It should
+be recollected however that unless the plates have been carefully
+leveled and the medium is of equal thickness all over it is useless to
+try and average from small areas--since where the medium is thick all
+the bacteria will develop, where the layer is a thin one, only a few
+bacteria will find sufficient pabulum for the production of visible
+colonies.
+
+It will be noted that the quantities of water selected for addition to
+each set of tubes of nutrient media have been carefully chosen in order
+to yield workable results even when dealing with widely differing
+samples. Plates prepared in agar with 0.1 c.c. and in gelatin with 0.02
+c.c. can be counted even when large numbers of bacteria are present in
+the sample; whereas if micro-organisms are relatively few, agar plate 4
+and gelatine plate 1 will give the most reliable counts. Again the
+counts of the plates in a measure control each other; for example, the
+second and third plates of each gelatine series should together contain
+as many colonies as the first, and the second should contain about half
+as many more than the third and so on.
+
+2. Qualitative Examination.--
+
+_Collection of Sample._--The water sample required for the routine
+examination, which it will be convenient to consider first, amounts to
+about 110 c.c. It is collected in the manner previously described
+(_vide_ page 416); similar bottles are used, and if four are filled the
+combined contents, amounting to about 240 c.c., will provide ample
+material for both the qualitative and quantitative examinations. Unless
+the examination is to be commenced at once, the ice-box must be
+employed, otherwise water bacteria and other saprophytes will probably
+multiply at the expense of the microbes indicative of pollution, and so
+increase the difficulties of the investigation.
+
+In the routine examination of water supplies it is customary to limit
+the qualitative examination to a search for
+
+A. B. coli and its near allies.
+
+B. Streptococci,
+
+organisms which are frequently spoken of as microbes of indication, as
+their presence is held to be evidence of pollution of the water by
+material derived from the mammalian alimentary canal, and so to
+constitute a danger signal.
+
+C. Some observers still attach importance to the presence of B.
+enteritidis sporogenes, but as the search for this bacterium,
+(relatively scarce in water) necessitates the collection of a fairly
+large quantity of water it is not usually included in the routine
+examination.
+
+In the case of water samples examined during the progress of an
+epidemic, of new supplies and of unknown waters the search is extended
+to embrace other members of the coli-typhoid group; and on occasion the
+question of the presence or absence of Vibrio cholerae or (more rarely)
+such bacteria as B. anthracis or B. tetani, may need investigation.
+
+When pathogenic or excremental bacteria are present in water, their
+numbers are relatively few, owing to the dilution they have undergone,
+and it is usual in commencing the examination, to adopt one or other of
+the following methods:
+
+A. _Enrichment_, in which the harmless non-pathogenic bacteria may be
+destroyed or their growth inhibited, whilst the growth of the parasitic
+bacteria is encouraged.
+
+This is attained by so arranging the environment, (i. e., Media,
+incubation temperature, and atmosphere) as to favor the growth of the
+pathogenic organisms at the expense of the harmless saprophytes.
+
+B. _Concentration_, whereby all the bacteria present in the sample of
+water, pathogenic or otherwise, are concentrated in a small bulk of
+fluid.
+
+This is usually effected by filtration of the water sample through a
+porcelain filter candle, and the subsequent emulsion of the bacterial
+residue remaining on the walls of the candle with a small measured
+quantity of sterile bouillon.
+
+A. ~Enrichment Method.~
+
+(Dealing with the demonstration of bacteria of intestinal origin.)
+
+_Apparatus Required_ (_Preliminary Stage_):
+
+    Incubator running at 42 deg. C.
+    Case of sterile pipettes, 1 c.c. graduated in tenths.
+    Case of sterile pipettes, 10 c.c. graduated in c.c.
+    Case of sterile pipettes, graduated to deliver 25 c.c.
+    Tubes of bile salt broth (_vide_ page 180).
+    Flask of double strength bile salt broth (_vide_ page 199).
+    Tubes of litmus silk.
+    Sterile flasks, 250 c.c. capacity.
+    Buchner's tubes.
+    Tabloids pyrogallic acid.
+    Tabloids sodium hydrate.
+    Bunsen burner.
+    Grease pencil.
+
+(_Later stage_):
+
+    Incubator running at 37 deg. C.
+    Surface plates of nutrose agar (see page 232).
+    Aluminium spreader.
+    Tubes of various media, including carbohydrate media.
+    Agglutinating sera, etc.
+
+METHOD.--
+
+1. Number a set of bile salt broth, tubes 1-5, and a duplicate set
+1a-5a.
+
+2. Number one flask 7 and another 8.
+
+3. To Tubes No. 1 and 1a add 0.1 c.c. water sample.
+
+To Tubes No. 2 and 2a add 1 c.c. water sample.
+
+To Tubes No. 3 and 3a add 2 c.c. water sample.
+
+To Tubes No. 4 and 4a add 5 c.c. water sample.
+
+To Tubes No. 5 and 5a add 10 c.c. water sample.
+
+4. Put up all the tubes in Buchner's tubes and incubate anaerobically at
+42 deg. C.
+
+     NOTE.--The bile salt medium is particularly suitable for the
+     cultivation of bacteria of intestinal origin, and at the
+     same time inhibits the growth of bacteria derived from other
+     sources.
+
+The anaerobic conditions likewise favor the multiplication of intestinal
+bacteria, and also their fermentative activity. The temperature 42 deg. C.
+destroys ordinary water bacteria and inhibits the growth of many
+ordinary mesophilic bacteria.
+
+5. Pipette 25 c.c. of double strength bile salt broth into flask 6, and
+50 c.c. double strength bile salt broth into flask 7.
+
+6. Pipette 25 c.c. water sample into flask 6, and 50 c.c. water sample
+into flask 7.
+
+7. Incubate the two flasks aerobically at 42 deg. C.
+
+8. After twenty-four hours incubation note in each culture:
+
+a. The presence or absence of visible growth.
+
+b. The reaction of the medium as indicated by the colour change, if
+any, the litmus has undergone.
+
+c. The presence or absence of gas formation, as indicated by a froth
+on the surface of the medium, and the collection of gas in the inner
+"gas" tube.
+
+9. Replace those tubes which show no signs of growth in the incubator.
+Examine after another period of twenty-four hours (total forty-eight
+hours incubation) with reference to the same points.
+
+10. Remove culture tubes which show visible growth from the Buchner's
+tubes, whether acid production and gas formation are present or not.
+
+11. Examine all tubes which show growth by hanging-drop preparations.
+Note such as show the presence of chains of cocci.
+
+12. Prepare surface plate cultivations upon nutrose agar from each tube
+that shows growth either macroscopically or microscopically, and
+incubate for twenty-four hours aerobically at 37 deg. C.
+
+13. Examine the growth on the plates either with the naked eye or with
+the help of a small hand lens. Practice will facilitate the recognition
+of colonies of the coli group, the typhoid group and the paratyphoid
+group; also those due to the growth of streptococci. The investigation
+from this stage proceeds along two divergent lines of enquiry--the first
+being concerned with the identity of the bacilli--typhoid bacilli, the
+second with that of the cocci.
+
+A. _B. Coli and its allies._
+
+14. Pick off coliform or typhiform colonies; make streak or smear
+subcultivations upon nutrient agar; incubate aerobically for twenty-four
+hours at 37 deg. C.
+
+15. Examine the growth in each tube carefully both macroscopically and
+microscopically. If the growth is impure, replate on nutrose agar, pick
+off colonies and subcultivate again. When the growth in a tube is pure,
+add 5 c.c. sterile normal saline solution or sterile broth, and emulsify
+the entire surface growth with it.
+
+16. Utilise the emulsion for the preparation of a series of
+subcultivations upon the media enumerated below, using the ordinary loop
+to make the subcultures upon solid media, but adding one-tenth of a
+cubic centimetre of the emulsion to each of the fluid media by means of
+a sterile pipette.
+
+    Gelatine streak.
+    Agar streak.
+    Potato.
+    Nutrient broth.
+    Litmus milk.
+    Dextrose peptone solution.
+    Laevulose peptone solution.
+    Galactose peptone solution.
+    Maltose peptone solution.
+    Lactose peptone solution.
+    Saccharose peptone solution.
+    Raffinose peptone solution.
+    Dulcite peptone solution.
+    Mannite peptone solution.
+    Glycerin peptone solution.
+    Inulin peptone solution.
+    Dextrin peptone solution.
+
+17. Differentiate the bacilli after isolation by means of their cultural
+reactions and biological characters into members of:
+
+I. The Escherich Group.
+
+    B. coli communis.
+    B. coli communior.
+    B. lactis aerogenes.
+    B. cloacae.
+
+II. The Gaertner Group.
+
+    Bacillus enteritidis (of Gaertner).
+    B. paratyphosus A.
+    B. paratyphosus B.
+    Bacillus cholerae suum.
+
+III. The Eberth Group.
+
+    B. typhosus.
+    B. dysenteriae (Shiga).
+    B. dysenteriae (Flexner).
+    B. faecalis alcaligines.
+
+18. Confirm these results by testing the organisms isolated against
+specific agglutinating sera obtained from experimentally inoculated
+animals.
+
+If a positive result is obtained when using this method, it only needs a
+simple calculation to determine the smallest quantity (down to 0.1 c.c.)
+of the sample that contains at least one of the microbes of indication.
+For instance, if growth occurs in all the tubes from 4 to 10, and that
+growth is subsequently proved to be due to the multiplication of B.
+coli, then it follows that at least one colon bacillus is present in
+every 10 c.c. of the water sample, but not in every 5 c.c. If, on the
+other hand, the presence of the B. coli can only be proved in flask No.
+7, then the average number of colon bacilli present in the sample is at
+least one in every 50 c.c. (i. e., twenty per litre), but not one in
+every 25 c.c. and so on.
+
+The general outline of the method of identifying the members of the
+coli-typhoid group is given in the form of an analytical schema--whilst
+the full differential details are set out in tabular form.
+
+ANALYTICAL SCHEME FOR ISOLATION OF MEMBERS OF THE COLI AND TYPHOID
+GROUPS.
+
+                           Nutrose agar.
+                                  |
+               -----------------------------------
+               |                                  |
+         Red colonies.                       Blue colonies.
+        Escherich group.                   Gaertner and Eberth groups.
+               ||                                 |
+               ====================---------------
+                                  ||
+                        Lactose peptone solution.
+                                  ||
+               ====================---------------
+               ||                                |
+              Gas.                            No gas.
+               ||                                |
+B. coli communis and its allies.                 |
+               ||                   Gaertner and Eberth groups.
+Acid and gas in glucose peptone solution.        |
+Acid and coagulation in milk.                    |
+General turbidity and indol in bouillon.   Glucose peptone solution.
+                                                 |
+               ==================================|
+               ||                                |
+               ||                                |
+              Gas.                            No gas.
+               ||                                |
+          Gaertner group.                  Eberth group.
+               ||                                |
+      ===================                ----------------
+      ||                ||               |               |
+      ||                ||               |               |
+Litmus milk.   Peptone solution.   Litmus milk.     Peptone solution.
+      ||                ||               |               |
+Acid at first.    General turbidity.   Acid.            General turbidity.
+Alkaline later.   No indol.            No coagulation.  No indol.
+No coagulation.   Serum reaction.                       Serum reaction.
+
+_B. Streptococci._
+
+19. Pick off streptococcus colonies and subcultivate upon nutrient agar
+exactly as directed in steps 14, 15 and 16.
+
+20. Differentiate the streptococci isolated into members of the
+saprophytic group of short-chained cocci, or members of the parasitic
+(pathogenic) group of long-chained cocci, by means of their cultural
+characters, and record their numerical frequency in the manner indicated
+for the members of the coli-typhoid group.
+
+DIFFERENTIAL TABLE OF COLI-TYPHOID GROUP
+
+Transcriber's note: Table split to fit 80 spaces.
+
++-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+
+|                         | M | D   | L   | G   | M   | L   | S   | R   | D   |
+|A = acid reaction        | o | e   | a   | a   | a   | a   | a   | a   | e   |
+|G = gas formation        | t | x   | e   | l   | l   | c   | c   | f   | t   |
+|                         | i | t   | v   | a   | t   | t   | c   | f   | r   |
+|                         | l | r   | u   | c   | o   | o   | h   | i   | i   |
+|                         | i | o   | l   | t   | s   | s   | a   | n   | n   |
+|                         | t | s   | o   | o   | e   | e   | r   | o   |     |
+|                         | y | e   | s   | s   |     |     | o   | s   |     |
+|                         |   |     | e   | e   |     |     | s   | e   |     |
+|                         |   |     |     |     |     |     | e   |     |     |
+|                         |   +-----+-----+-----+-----+-----+-----+-----+-----+
+|                         |   | A G | A G | A G | A G | A G | A G | A G | A G |
++-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+
+|_The Escherich Group._   |   |     |     |     |     |     |     |     |     |
+| B. coli communis        | + | + + | + + | + + | + + | + + |  O  | + + | + + |
+| B. coli communior       | + | + + | + + | + + | + + | + + | + + | + + | + + |
+| B. lactis aerogenes     | - | + + | + + | + + | + + | + + |  O  |  O  | + + |
+| B. acidi lactici        | - | + + | + + | + + | + + | + + |  O  |  O  |  O  |
+| B. pneumoniae           | - | + + | + + | + + | + + | + + | + + | + + | + + |
+| B cloaceae(A)           | + | + + | + + | + + | + + | + + | + + | + + | + + |
+|                         |   |     |     |     |     |     |     |     |     |
+|_The Gaertner Group._    |   |     |     |     |     |     |     |     |     |
+| B. enteritidis          | + | + + | + + | + + | + + |  O  |  O  |  O  |  O  |
+| B. paratyphosus A       | + | + + | + + | + + | + + |  O  |  O  |  O  |  O  |
+| B. paratyphosus B       | + | + + | + + | + + | + + |  O  |  O  |  O  |  O  |
+| B. cholerae suum        | + | + + | + + | + + | + + |  O  |  O  |     |  O  |
+| B. suipestifer          | + | + + | + + | + + | + + |  O  |  O  |     |  O  |
+|                         |   |     |     |     |     |     |     |     |     |
+|_The Eberth Group._      |   |     |     |     |     |     |     |     |     |
+| B. typhosus             | + | +   | +   | +   | +   | O   | O   | O   | +   |
+| B. dysenteriae (Shiga)  | - | +   | +   | +   | O   | O   | O   | O   | O   |
+| B. dysenteriae (Flexner)| - | +   | +   | +   | +   | O   | O   | +/- | O   |
+| B. faecalis alkaligines | + | O   | O   | O   | O   | O   | O   | O   | O   |
+|                         |   |     |     |     |     |     |     |     |     |
++-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+
+| Table Notes:            |(B)| (C)                                           |
++-------------------------+---+-----------------------------------------------+
+
++-------------------------+-----+-----+-----+-----+-----+-----+---+-----------+
+|                         | I   | S   | G   | D   | M   | S   | I |Litmus Milk|
+|A=acid reaction          | n   | a   | l   | u   | a   | o   | n |           |
+|G=gas formation          | u   | l   | y   | l   | n   | r   | d |           |
+|                         | l   | i   | c   | c   | n   | b   | o +-----+-----+
+|                         | i   | c   | e   | i   | i   | i   | l |Early|Late |
+|                         | n   | i   | r   | t   | t   | t   |   |     |     |
+|                         |     | n   | i   | e   | e   | e   |   |     |     |
+|                         |     |     | n   |     |     |     |   |     |     |
+|                         |     |     |     |     |     |     |   |     |     |
+|                         |     |     |     |     |     |     |   |     |     |
+|                         |-----+-----+-----+-----+-----+-----+   |     |     |
+|                         | A G | A G | A G | A G | A G | A G |   |     |     |
++-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+
+|_The Escherich Group_    |     |     |     |     |     |     |   |     |     |
+| B. coli communis        |  O  |  O  | + + | + + | + + | + + | + | +   | + C |
+| B. coli communior       |  O  |  O  | + + | + + | + + | + + | + | +   | + C |
+| B. lactis aerogenes     |  O  |  O  |  O  |  O  | + + | + + | - | +   | + C |
+| B. acidi lactici        |  O  |  O  |  O  | + + | + + | + + | + | +   | + C |
+| B. pneumoniae           |  O  |  O  | + + | + + | + + | + + | - | +   | + C |
+| B cloaceae[A]           |  O  |  O  | + + |  O  | + + | - + | + | +   | + C |
+|                         |     |     |     |     |     |     |   |     |     |
+|_The Gaertner Group._    |     |     |     |     |     |     |   |     |     |
+| B. enteritidis          |  O  |  O  |  O  | + + | + + | + + | - | +/- | -   |
+| B. paratyphosus A       |  O  | +/- |  O  | + + | + + | + + | - | +   | O   |
+| B. paratyphosus B       |  O  |  O  |  O  | + + | + + | + + | - | +   | -   |
+| B. cholerae suum        |  O  |  O  |  O  |  O  |  O  | + + |+/-| +   | -   |
+| B. suipestifer          |  O  |  O  |  O  | + + | + + | + + | - | +   | -   |
+|                         |     |     |     |     |     |     |   |     |     |
+|_The Eberth Group._      |     |     |     |     |     |     |   |     |     |
+| B. typhosus             | O   | O   | O   | O   | +   | +   | - | +   | +   |
+| B. dysenteriae (Shiga)  | O   | O   | O   | O   | O   | O   | - | +   | -   |
+| B. dysenteriae (Flexner)| O   | O   | O   | O   | +   | O   |+/-| +   | -   |
+| B. faecalis alkaligines | O   | O   | O   | O   | O   | O   | - | -   | -   |
+|                         |     |     |     |     |     |     |   |     |     |
++-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+
+| Table Notes:            |                                   |(D)| (E)       |
++-------------------------+-----------------------------------+---+-----------+
+
+Table Notes:
+
+(A) * Liquefies gelatine.
+
+(B) + = motile. - = non-motile.
+
+(C) + = acid or gas production. +/- = slight acid production. O = no
+change.
+
+(D) + = indol production. +/- = slight indol production. - = no indol
+formed.
+
+(E) + = acid production. - = alkali production. O = no change in
+reaction. C = clot.
+
+21. Determine the pathogenicity for mice (subcutaneous inoculation) and
+rabbits (intravenous inoculation) of the streptococci isolated.
+
+On the facing insert page is reproduced a blank from the author's
+Laboratory Water Analysis Book, by means of which an exact record can be
+kept, with a minimum of labour, of every sample examined.
+
+
+B. ~Concentration Method.~
+
+The remaining organisms referred to on page 426 are more conveniently
+sought for by the concentration method.
+
+_Collection of the Sample._--The quantity of water required for this
+method of examination is about 2000 c.c., and the vessel usually chosen
+for its reception is an ordinary blue glass Winchester quart bottle,
+sterilised in the hot-air oven, and over this a paper or parchment cap
+fastened with string. The bottle may be packed in a wooden box or in an
+ordinary wicker case. The method of collecting the sample is identical
+with that described under the heading of Quantitative Examination; there
+is, however, not the same imperative necessity to pack the sample in ice
+for transmission to the laboratory.
+
+_Apparatus required_:
+
+     Sterile Chamberland or Doulton "white" porcelain open mouth
+     filter candle, fitted with rubber washer.
+
+     Rubber cork to fit mouth of the filter candle, perforated
+     with one hole.
+
+     Kitasato serum flask, 2500 c.c. capacity.
+
+     Geryk air pump or water force pump.
+
+     Wulff's bottle, fitted as wash-bottle, and containing
+     sulphuric acid (to act as a safety valve between filter and
+     pump).
+
+     Pressure tubing, clamps, pinch-cock.
+
+     Retort stand, with ring and clamp.
+
+     Rubber cork for the neck of Winchester quart, perforated
+     with two holes and fitted with one 6 cm. length of straight
+     glass tubing, and one V-shaped piece of glass tubing, one
+     arm 32 cm. in length, the other 52 cm., the shorter arm
+     being plugged with cotton-wool. The rubber stopper must be
+     sterilised by boiling and the glass tubing by hot air,
+     before use.
+
+     Flask containing 250 c.c. sterile broth.
+
+     Test-tube brush to fit the lumen of the candle, enclosed in
+     a sterile test-tube (and previously sterilised by dry heat
+     or by boiling).
+
+     Case of sterile pipettes, 10 c.c. in tenths.
+
+     Case of sterile pipettes, 1 c.c. in tenths.
+
+     Case of sterile pipettes, 1 c.c. in hundredths.
+
+     Tubes of various nutrient media (according to requirements).
+
+     Twelve Buchner's tubes with rubber stoppers.
+
+     Pyrogallic acid tablets.
+
+     Caustic soda tablets.
+
+[Illustration: Sample form]]
+
+[Illustration: FIG. 209.--Water filtering apparatus. That portion of the
+figure to the left of the vertical line is drawn to a larger scale than
+that on the right, in order to show details of Sprengel's pump.]
+
+METHOD.--
+
+1. Fit up the filtering apparatus as in the accompanying diagram (Fig.
+209), interposing the wash-bottle with sulphuric acid between the
+filter flask and the force-pump (in the position occupied in the diagram
+by the central vertical line), and placing another screw clamp on the
+rubber tubing connecting the lateral arm of the filter flask with the
+wash-bottle.
+
+[Illustration: FIG. 210. Sterile test-tube brush.]
+
+2. Filter the entire 2000 c.c. of water through the filter candle.
+
+3. When the nitration is completed, screw up the clamps and so occlude
+the two pieces of pressure tubing.
+
+4. Reverse the position of the glass tubes in the Wulff's bottle so that
+the one nearest the air pump now dips into the sulphuric acid.
+
+5. Slowly open the metal clamps and allow air to gradually pass through
+the acid, and enter filter flask, and so restore the pressure.
+
+6. Unship the apparatus, remove the cork from the mouth of the candle.
+
+7. Pipette 10 c.c. of sterile broth into the interior of the candle, and
+by means of the sterile test-tube brush (Fig. 210) emulsify the slimy
+residue which lines the candle, with the broth.
+
+Practically all the bacteria contained in the original 2000 c.c. of
+water are now suspended in 10 c.c. of broth, so that 1 c.c. of the
+suspension is equivalent, so far as the contained organisms are
+concerned, to 200 c.c. of the original water. (Some bacteria will of
+course be left behind on the walls of the filter and in its pores.)
+
+Up to this point the method is identical, irrespective of the particular
+organism whose presence it is desired to demonstrate; but from this
+point onward the methods must be specially adapted to the isolation of
+definite groups of organisms or of individual bacteria.
+
+The Coli-Typhoid Group.--
+
+1. Number nine tubes of bile salt broth (_vide_ page 180), consecutively
+from 1 to 9.
+
+2. To No 1 add 1 c.c. } of the original water sample
+         2 add 2 c.c. } before the nitration is commenced.
+         3 add 5 c.c. }
+
+3. To the remaining tubes of bile salt broth add varying quantities of
+the suspension by means of suitably graduated sterile pipettes, as
+follows:
+
+No. 4   0.05  c.c. (equivalent to  10 c.c. of the original water sample).
+No. 5   0.125 c.c. (equivalent to  25 c.c. of the original water sample).
+No. 6   0.25  c.c. (equivalent to  50 c.c. of the original water sample).
+No. 7   0.5   c.c. (equivalent to 100 c.c. of the original water sample).
+No. 8   1.0   c.c. (equivalent to 200 c.c. of the original water sample).
+No. 9   2.5   c.c. (equivalent to 500 c.c. of the original water sample).
+
+4. Put up each tube anaerobically in a Buchner's tube and incubate at
+42 deg. C.
+
+5. The subsequent steps are identical with those described under the
+Enrichment method (see page 428 to 431; Steps 8 to 18).
+
+     _Alternative Methods._--
+
+     A few of the older methods for the isolation of the members
+     of the coli-typhoid groups are referred to but they are
+     distinctly inferior to those already described.
+
+     (A) The Carbolic Method:
+
+     1. Take ten tubes of carbolised bouillon (_vide_ page 202)
+     and number them consecutively from 1 to 10.
+
+     2. Inoculate each tube with a different amount of the water
+     sample or suspension, as in the previous method.
+
+     3. Incubate aerobically at 37 deg. C.
+
+     4. Examine the culture tubes after twenty-four hours'
+     incubation.
+
+     5. From those tubes which shows signs of growth, pour plates
+     in the usual manner, using carbolised gelatine (_vide_ page
+     202) in place of the ordinary gelatine, and incubate at 20 deg.
+     C. for three, four, or five days as may be necessary.
+
+     6. Subcultivate from any colonies that make their
+     appearance, and determine their identity on the lines laid
+     down in the previous method.
+
+     (B) Parietti's Method:
+
+     1. Take nine tubes of Parietti's bouillon (_vide_ page
+     202)--i. e., three each of those containing 0.1 c.c., 0.2
+     c.c., and 0.5 c.c. of Parietti's solution respectively.
+     Mark plainly on the outside of each tube the quantity of
+     Parietti's solution it contains.
+
+     2. To each tube add a different amount of the original
+     water, or of the suspension, and incubate at 37 deg. C.
+
+     3. Examine the culture tubes after twenty-four and
+     forty-eight hours' incubation, and plate in nutrient
+     carbolised or potato gelatine from such as have grown.
+
+     4. Pick off suspicious colonies, if any such appear on the
+     plates, subcultivate them upon the various media, and
+     identify them.
+
+     (C) Elsner's Method: This method simply consists in
+     substituting Elsner's potato gelatine (_vide_ page 204) for
+     ordinary nutrient gelatine in any of the previously
+     mentioned methods.
+
+     (D) Cambier's Candle Method:
+
+     Treat a large volume of the water sample by the
+     concentration method (_vide_ page 434).
+
+     1. Remove the rubber stopper from the mouth of the filter
+     candle, introduce 10 c.c. sterile bouillon into its
+     interior, and emulsify the bacterial sediment; replug the
+     mouth of the candle with a wad of sterile cotton-wool.
+
+     2. Remove the filter candle from the filter flask and insert
+     it into the mouth of a flask or a glass cylinder containing
+     sterile bouillon sufficient to reach nearly up to the rubber
+     washer on the candle.
+
+     3. Incubate for twenty-four to thirty-six hours at 37 deg. C.
+
+     4. From the now turbid bouillon in the glass cylinder pour
+     gelatine plates and incubate at 20 deg. C.
+
+     5. Subcultivate and identify any suspicious colonies that
+     appear.
+
+     (The method depends upon the assumption that members of the
+     typhoid and coli groups find their way through the porcelain
+     filter from the interior to the surrounding bouillon at a
+     quicker rate than the associated bacteria.)
+
+
+B. ~Enteritidis Sporogenes.~--
+
+1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile
+test-tube and plug carefully.
+
+2. Place the test-tube in the interior of the benzole bath employed in
+separating out spore-bearing organisms (_vide_ page 257), and expose to
+a temperature of 80 deg. C. for twenty minutes.
+
+3. Number ten tubes of litmus milk consecutively from 1 to 10.
+
+4. Remove the test-tube from the benzole bath and shake well to
+distribute the spores evenly through the fluid.
+
+5. To each tube of litmus milk add a measured quantity of the suspension
+corresponding to the amounts employed in isolating the coli group
+(_vide_ page 437).
+
+6. Incubate each tube anaerobically at 37 deg. C. Anaerobic conditions
+can be obtained by putting the cultures up in Buchner's tubes or in
+Bulloch's apparatus. If, however, whole milk has been used in making the
+litmus milk the layer of cream that rises to the surface will be
+sufficient to ensure anaerobiosis; whilst if separated milk has been
+employed it will be sufficient to pour a layer of sterile vaseline or
+liquid paraffin on the surface of the fluid.
+
+7. Examine after twenty-four hours' incubation. Note (if B. enteritidis
+sporogenes is present)--
+
+(a) Acid reaction of the medium as indicated by the colour of the
+litmus or its complete decolourisation.
+
+(b) Presence of clotting, and the separation of clear whey.
+
+(c) Presence of gas, as indicated by fissures and bubbles in the
+coagulum, and possibly masses of coagulum driven up the tube almost to
+the plug.
+
+8. Replace the tubes which show no signs of growth in the incubator for
+a further period of twenty-four hours and again examine with reference
+to the same points.
+
+9. Remove those tubes which give evidence of growth from the Buchner's
+tubes and carefully pipette off the whey; examine the whey
+microscopically.
+
+10. Inoculate two guinea-pigs each subcutaneously with 0.5 c.c. of the
+whey and observe the result.
+
+
+~Vibrio Cholerae.~--
+
+1. Number ten tubes of peptone water consecutively from 1 to 10.
+
+2. To each of the tubes of peptone water add a measured quantity of the
+suspension, corresponding to those amounts employed in isolating the
+members of the coli group (_vide_ page 437).
+
+3. Incubate aerobically at 37 deg. C. for twenty-four hours. Examine
+the tubes carefully for visible growth, especially delicate pellicle
+formation, which if present should be examined microscopically for
+vibrios, both by stained preparations or by fresh specimens with dark
+ground illumination.
+
+4. Inoculate fresh tubes of peptone water from such of the tubes as
+exhibit pellicle formation--from the pellicle itself--and incubate at
+37 deg. C. for twenty-four hours.
+
+5. Test the peptone water itself for the presence of indol and nitrite
+by the addition of pure concentrated H_{2}SO_{4}.
+
+5. Prepare gelatine and agar plates in the usual way from such of these
+tubes as show pellicle formation.
+
+6. Pick off from the plates any colonies resembling those of the Vibrio
+cholerae and subcultivate upon all the ordinary laboratory media.
+
+7. Test the vibrio isolated against the serum of an animal immunised to
+the Vibrio cholerae for agglutination.
+
+
+~B. Anthracis.~--
+
+1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile
+test-tube and plug carefully.
+
+2. Place the test-tube in the interior of the benzole bath employed in
+separating out spore-bearing organisms (_vide_ page 257), and expose to
+a temperature of 80 deg. C. for twenty minutes.
+
+3. Inoculate a _young_ white rat subcutaneously (on the inner aspect of
+one of the hind legs) with 1 c.c. of the emulsion. Observe during life,
+and, if the animal succumbs, make a complete post-mortem examination.
+
+4. Melt three tubes of nutrient agar in boiling water and cool to 42
+deg. C.
+
+5. Number the tubes 1, 2, and 3. To No. 1 add 0.2 c.c., to No. 2 add 0.3
+c.c., and to No. 3 add 0.5 c.c. of the suspension, and pour plates
+therefrom.
+
+6. Incubate at 37 deg. C. for twenty-four or forty-eight hours.
+
+7. Pick off any colonies resembling those of anthrax and subcultivate on
+all the ordinary laboratory media.
+
+8. Inoculate another young white rat as in 3, using two loopfuls of the
+agar subcultivation emulsified with 1 c.c. sterile bouillon. Observe
+during life, and if the animal succumbs, make a complete post-mortem
+examination.
+
+
+~B. Tetani.~--
+
+1. Proceed as detailed above in steps 1 and 2 for the isolation of the
+B. anthracis.
+
+2. Add 1 c.c. of the suspension to each of three tubes of glucose
+formate broth, and incubate anaerobically in Buchner's tubes at 37 deg. C.
+
+3. From such of the tubes as show visible growth (with or without the
+production of gas) after twenty-four hours' incubation inoculate
+guinea-pigs, subcutaneously (under the skin of the abdomen), using 0.1
+c.c. of the bouillon cultivation as a dose. Observe carefully during
+life, and, if death occurs, make a complete post-mortem examination.
+
+4. From the same tubes pour agar plates and incubate anaerobically in
+Bulloch's apparatus, at 37 deg. C.
+
+5. Subcultivate suspicious colonies on the various media, incubate
+anaerobically, making control cultivations on glucose formate agar, stab
+and streak, to incubate aerobically and carry out further inoculation
+experiments with the resulting growths.
+
+
+EXAMINATION OF MILK.
+
+"One-cow" or "whole" milk, if taken from the apparently healthy animal
+(that is, an animal without any obvious lesion of the udder or teats)
+with ordinary precautions as to cleanliness, avoidance of dust, etc.,
+contains but few organisms. In dealing with one-cow milk, from a
+suspected, or an obviously diseased animal, a complete analysis should
+include the examination (both qualitative and quantitative) of samples
+of (a) fore-milk, (b) mid-milk, (c) strippings, and, if possible,
+from each quarter of the udder. "Mixed" milk, on the other hand, by the
+time it leaves the retailer's hands, usually contains as many
+micro-organisms as an equal volume of sewage and indeed during the
+examination it is treated as such.
+
+It is possible however to collect and store mixed milk in so cleanly a
+manner that its germ content does not exceed 5000 micro-organisms per
+cubic centimetre. Such comparative freedom from extraneous bacteria is
+usually secured by the purveyor only when he resorts to the process of
+pasteurisation (heating the milk to 65 deg. C. for twenty minutes or to
+77 deg. C. for one minute) or the simpler plan of adding preservatives to
+the milk. Information regarding the employment of these methods for the
+destruction of bacteria should always be sought in the case of mixed
+milk samples, and in this connection the following tests will be found
+useful:
+
+1. _Raw Milk_ (Saul).
+
+To 10 c.c. milk in a test tube, add 1 c.c. of a 1 per cent. aqueous
+solution of ortol (ortho-methyl-amino-phenol sulphate), recently
+prepared and mix. Next add 0.2 c.c. of a 3 per cent. peroxide of
+hydrogen solution. The appearance of a brick red color within 30 seconds
+indicates raw milk. Milk heated to 74 deg. C. for thirty minutes undergoes
+no alteration in color; if heated to 75 deg. C. for ten minutes only, the
+brick red color appears after standing for about two minutes.
+
+2. _Boric Acid._
+
+Evaporate to dryness, 50 c.c. of the milk which has been rendered
+slightly alkaline to litmus, then incinerate.
+
+Dissolve in distilled water, add slight excess of dilute hydrochloric
+acid and again evaporate to dryness.
+
+Dissolve the residue in a small quantity of hot water and moisten a
+piece of turmeric paper with the solution. Dry the turmeric paper.
+_Rose_ or _cherry-red_ color = borax or boric acid.
+
+3. _Formaldehyde_ (Hehner).
+
+To 10 c.c. milk in a test tube add 5 c.c. concentrated _commercial_
+sulphuric acid slowly, so that the two fluids do not mix. Hold the tube
+vertically and agitate very gently. _Violet zone_ at the junction of the
+two liquids = formaldehyde.
+
+4. _Hydrogen Peroxide._
+
+To 10 c.c. milk (diluted with equal quantities of water) in a test tube
+add 0.4 c.c. of a 4 per cent. alcoholic solution of benzidine and 0.2
+c.c. acetic acid. _Blue coloration_ of the mixture = hydrogen peroxide.
+
+5. _Salicylic Acid._
+
+Precipitate the caseinogen by the addition of acetic acid and filter. To
+the filtrate add a few drops of 1 per cent. aqueous solution of ferric
+chloride. _Purple coloration_ = salicylic acid.
+
+6. _Sodium Carbonate or Bicarbonate._
+
+To 10 c.c. of the milk in a test tube add 10 c.c. of alcohol and 0.3
+c.c. of a 1 per cent. alcoholic solution of rosolic acid. _Brownish_
+color = pure milk; _rose_ color = preserved milk.
+
+[Illustration: FIG. 211.--Milk-collecting bottle and dipper in case.]
+
+Quantitative.--
+
+_Collection of Sample._--
+
+The apparatus used for the collection of a retail mixed milk sample
+consists of a cylindrical copper case, 16 cm. high and 9 cm. in
+diameter, provided with a "pull-off" lid, containing a milk dipper, also
+made of copper; and inside this, again, a wide-mouthed, stoppered glass
+bottle of about 250 c.c. capacity (about 14 cm. high by 7 cm. diameter),
+having a tablet for notes, sand-blasted on the side. The copper cylinder
+and its contents, secured from shaking by packing with cotton-wool, are
+sterilised in the hot-air oven (Fig. 26).
+
+When collecting a sample,
+
+1. Remove the cap from the cylinder.
+
+2. Draw out the cotton-wool.
+
+3. Lift out the bottle and dipper together.
+
+4. Receive the milk in the sterile dipper, and pour it directly into the
+sterile bottle.
+
+5. Enter the particulars necessary for the identification of the
+specimen, on the tablet, with a lead pencil, or pen and ink.
+
+6. Pack the apparatus in the ice-box for transmission to the laboratory
+in precisely the same manner as an ordinary water sample.
+
+"Whole" milk may with advantage be collected in the sterile bottle
+directly since the mouth is sufficiently wide for the milker to direct
+the stream of milk into it.
+
+~Condensed milk~ must be diluted with sterile distilled water in
+accordance with the directions printed upon the label, then treated as
+ordinary milk.
+
+_Apparatus Required_:
+
+    Case of sterile capsules (25 c.c. capacity).
+    Case of sterile graduated pipettes, 10 c.c.
+      (in tenths of a cubic centimetre).
+    Case of sterile graduated pipettes, 1 c.c.
+      (in tenths of a cubic centimetre).
+    Flask containing 250 c.c. sterile bouillon.
+    Tall cylinder containing 2 per cent. lysol solution.
+    Plate-levelling stand.
+    Case of sterile plates.
+    Tubes nutrient gelatine or gelatine agar.
+    Tubes of wort gelatine.
+    Tubes of nutrient agar.
+    Water-bath regulated at 42 deg. C.
+    Bunsen burner.
+    Grease pencil.
+
+METHOD.--
+
+1. Arrange four sterile capsules in a row; number them I, II, III, and
+IV.
+
+2. Fill 9 c.c. sterile bouillon into the first, and 9.9 c.c. bouillon
+into each of the three remaining capsules.
+
+3. Remove 1 c.c. milk from one of the bottles by means of a sterile
+pipette and add it to the bouillon in capsule I; mix thoroughly by
+repeatedly filling and emptying the pipette.
+
+4. Remove 0.1 c.c. of the milky bouillon from capsule I, add it to the
+contents of capsule II, and mix as before.
+
+5. In like manner add 0.1 c.c. of the contents of capsule II to capsule
+III; and then 0.1 c.c. of the contents of capsule III to capsule IV.
+
+    Then 1 c.c. of dilution   I contains 0.1       c.c. milk sample.
+         1 c.c. of dilution  II contains 0.001     c.c. milk sample.
+         1 c.c. of dilution III contains 0.00001   c.c. milk sample.
+         1 c.c. of dilution  IV contains 0.0000001 c.c. milk sample.
+
+6. Melt the gelatine and the agar tubes in boiling water; then transfer
+to the water-bath and cool them down to 42 deg. C.
+
+7. Number the gelatine tubes consecutively 1 to 12.
+
+8. Inoculate the tubes with varying quantities of the material as
+follows:
+
+    To tube No.  1 add 1.0 c.c. of the milk sample.
+                 2 add 0.1 c.c. of the milk sample.
+              {  3 add 1.0 c.c. from capsule I.
+              {  4 add 0.1 c.c. from capsule I.
+              {  5 add 1.0 c.c. from capsule II.
+              {  6 add 0.1 c.c. from capsule II.
+              {  7 add 0.5 c.c. from capsule III.
+              {  8 add 0.3 c.c. from capsule III.
+              {  9 add 0.2 c.c. from capsule III.
+              { 10 add 0.5 c.c. from capsule IV.
+              { 11 add 0.3 c.c. from capsule IV.
+              { 12 add 0.2 c.c. from capsule IV.
+
+9. Pour plates from the gelatine tubes; label, and incubate at 20 deg. C.
+
+10. Liquefy five wort gelatine tubes and to them add 1.0 c.c. of the
+milk sample and a similar quantity of the diluted milk from capsules I,
+II, and III and IV respectively.
+
+11. Pour plates from the wort gelatine; label, and incubate at 20 deg. C.
+
+12. Inoculate the liquefied agar tubes as follows:
+
+    To tube No. 1 add 0.1 c.c. of the milk sample.
+                2 add 0.1 c.c. from capsule I.
+                3 add 0.1 c.c. from capsule II.
+                4 add 0.1 c.c. from capsule III.
+                5 add 1.0 c.c. from capsule IV.  }
+                6 add 0.1 c.c. from capsule IV.  }
+
+13. Pour plates from the agar tubes; label, and incubate at 37 deg. C.
+
+14. After twenty-four hours' incubation "inspect," and after forty-eight
+hours' incubation, "count" the agar plates and estimate the number of
+"organisms growing at 37 deg. C." present per cubic centimetre of the
+sample of milk.
+
+15. After three, four, or five days' incubation, "count" the gelatine
+plates and estimate therefrom the number of "organisms growing at 20 deg.
+C." present per cubic centimetre of the sample of milk.
+
+16. After a similar interval "count" the wort gelatine plates and
+estimate the number of moulds and yeasts present per cubic centimetre of
+the sample of milk.
+
+     NOTE.--Many observers prefer to employ gelatine agar (see
+     page 193) for the quantitative examination. In this case
+     gelatine-agar plates should be poured from tubes containing
+     the quantities of material indicated in step 8, incubated at
+     28 deg. C. to 30 deg. C. and after five days the "total number
+     of organisms developing at 28 deg. C." recorded.
+
+~Qualitative.~--The qualitative bacteriological examination of milk is
+chiefly directed to the detection of the presence of one or more of the
+following pathogenic bacteria and when present to the estimation of
+their numerical frequency.
+
+    Members of the Coli-typhoid group.
+    Vibrio cholerae.
+    Streptococcus pyogenes longus.
+    Micrococcus melitensis.
+    Staphylococcus pyogenes aureus.
+    Bacillus enteritidis sporogenes.
+    Bacillus diphtheriae.
+    Bacillus tuberculosis.
+
+Some of these occur as accidental contaminations, either from the water
+supply to the cow farm, or from the farm employees, whilst others are
+derived directly from the cow.
+
+In milk, as in water examinations, two methods are available, viz.:
+Enrichment and Concentration--the former is used for the demonstration
+of bacteria of intestinal origin, the latter for the isolation of the
+micro-organisms of diphtheria and tubercle. The first essential in the
+latter process is the concentration of the bacterial contents of a large
+volume of the sample into a small compass; but in the case of milk,
+thorough centrifugalisation is substituted for filtration.
+
+     _Apparatus Required_:
+
+     A large centrifugal machine. This machine, to be of real
+     service in the bacteriological examination of milk, must
+     conform to the following requirements:
+
+     1. The centrifugal machine must be of such size, and should
+     carry tubes or bottles of such capacity, as to enable from
+     200 to 500 c.c. of milk to be manipulated at one time.
+
+     2. The rate of centrifugalisation should be from 2500 to
+     3000 revolutions per minute.
+
+     3. The portion of the machine destined to carry the tubes
+     should be a metal disc, of sufficient weight to ensure good
+     "flank" movement, continuing over a considerable period of
+     time. In other words, the machine should run down very
+     gradually and slowly after the motive power is removed, thus
+     obviating any disturbance of the relative positions of
+     particulate matter in the solution that is being
+     centrifugalised.
+
+     4. The machine should preferably be driven by electricity,
+     or by power, but in the case of hand-driven machines--
+
+     (a) The gearing should be so arranged that the requisite
+     speed is obtained by not more than forty or fifty
+     revolutions of the crank handle per minute, so that it may
+     be maintained for periods of twenty or thirty minutes
+     without undue exertion.
+
+     (b) The handle employed should be provided with a special
+     fastening (e. g., a clutch similar to that employed for
+     the free wheel of a bicycle), or should be readily
+     detachable so that, on ceasing to turn, the handle should
+     not, by its weight and air resistance, act as a brake and
+     stop the machine too suddenly.
+
+     One of the few satisfactory machines of this class is shown
+     in figure 212.
+
+[Illustration: FIG. 212.--Electrically driven centrifugal machine, with
+flexible (broken) spindle encircled by the field magnets of the motor.]
+
+     Sterile centrifugal tubes, of some 60-70 c.c. capacity,
+     tapering to a point at the closed end, plugged with
+     cotton-wool.
+
+     Small centrifugal machine to run two tubes of 10 c.c.
+     capacity at 2500 to 3000 revolutions per minute preferably
+     driven by electricity, of the type figured on page 327 (Fig.
+     162).
+
+     Sterile centrifugal tubes of 10 c.c. capacity with the
+     distal extremity contracted to a narrow tube and graduated
+     in hundredths of a cubic centimetre (Fig. 213).
+
+     Sterilised cork borer.
+
+     Case of sterile pipettes, 10 c.c. (in tenths of a cubic
+     centimetre).
+
+     Case of sterile pipettes, 1 c.c. (in tenths of a cubic
+     centimetre).
+
+     Sterile teat pipettes.
+
+     Flask of sterile normal saline solution.
+
+METHOD.--
+
+1. Fill 50 c.c. of the milk sample into each of four tubes, and replace
+the cotton-wool plugs by solid rubber stoppers (sterilised by boiling),
+and fit the tubes in the centrifugal machine.
+
+     NOTE.--One or two cubic centimetres of paraffinum liquidum
+     introduced into the buckets of the centrifuge before the
+     glass tubes are inserted will obviate any risk of breakage
+     to the latter.
+
+[Illustration: FIG. 213.--Milk sedimenting tubes.]
+
+[Illustration: FIG. 214.--Milk in centrifuge tube.]
+
+2. Centrifugalise the milk sample for thirty minutes at a speed of 2500
+revolutions per minute.
+
+3. Remove the motive power and allow the machine to slow down gradually.
+
+4. Remove the tubes of milk from the centrifuge. Each tube will now show
+(Fig. 214):
+
+(a) A superficial layer of cream (varying in thickness with different
+samples) condensed into a semi-solid mass, which can be shown to
+contain some organisms and a few leucocytes.
+
+(b) A central layer of separated milk, thin, watery, and opalescent, and
+containing extremely few bacteria.
+
+(c) A sediment or deposit consisting of the great majority of the
+contained bacteria and leucocytes, together with adventitious matter,
+such as dirt, hair, epithelial cells, faecal debris, etc.
+
+5. Withdraw the rubber stopper and remove a central plug of cream from
+each tube by means of a sterile cork borer; place these masses of cream
+in two sterile capsules. Label C^{1} and C^{2}.
+
+6. Remove all but the last one or two c.c. of separated milk from each
+tube, by means of sterile pipettes.
+
+7. Mix the deposits thoroughly with the residual milk, pipette the
+mixture from each pair of tubes into one sterile 10 c.c. tube
+(graduated) by means of sterile teat pipettes, then fill to the 10 c.c.
+mark with sterile normal saline solution and mix together. Label D^{1}
+and D^{2}.
+
+8. Place the two tubes of mixed deposit in the centrifuge, adjust by the
+addition or subtraction of saline solution so that they counterpoise
+exactly, and centrifugalise for ten minutes.
+
+     NOTE.--Each tube now contains the deposit from 100 c.c. of
+     the milk sample and the amount can be read off in hundredths
+     of a centimetre. The multiplication of this figure by 100
+     will give the amount of "Apparent Filth," in "parts per
+     million"--the usual method of recording this quality of
+     milk.
+
+9. Pipette off all the supernatant fluid and invert the tube to drain on
+to a pad of sterilised cotton-wool, contained in a beaker. (This wool is
+subsequently cremated.)
+
+10. Examine both cream (C^{1}) and deposit (D^{1}) microscopically--
+
+(a) In hanging-drop preparations.
+
+(b) In film preparations stained carbolic methylene-blue, by Gram's
+method, by Neisser's method, and by Ziehl-Neelsen's method.
+
+Note the presence or absence of altered and unaltered vegetable fibres;
+pus cells, blood discs; cocci in groups or chains, diphtheroid bacilli,
+Gram negative bacilli or cocci, spores and acid fast bacteria.
+
+11. Adapt the final stages of the investigation to the special
+requirements of each individual sample, thus:
+
+~1. Members of the Coli-typhoid Group.~--
+
+1. Emulsify the deposit from the second centrifugal tube (D^{2}) with 10
+c.c. sterile bouillon and inoculate three tubes of bile salt broth as
+follows:
+
+    To Tube No. 1 add 2.5 c.c. milk deposit emulsion
+      (=25 c.c. original milk.)
+    To Tube No. 2 add 1.0 c.c. milk deposit emulsion
+      (=10 c.c. original milk.)
+    To Tube No. 3 add 0.5 c.c. milk deposit emulsion
+      (= 5 c.c. original milk.)
+
+2. Inoculate tube of bile salt broth No. 4 with 1 c.c. of the original
+milk.
+
+3. Inoculate further tubes of bile salt broth with previously prepared
+dilutions (see page 445) as follows:
+
+    To tube No.  5 add 1.0 c.c. from capsule I.
+    To tube No.  6 add 0.1 c.c. from capsule I.
+    To tube No.  7 add 1.0 c.c. from capsule II.
+    To tube No.  8 add 0.1 c.c. from capsule II.
+    To tube No.  9 add 1.0 c.c. from capsule III.
+    To tube No. 10 add 0.1 c.c. from capsule III.
+    To tube No. 11 add 1.0 c.c. from capsule IV.
+    To tube No. 12 add 0.1 c.c. from capsule IV.
+
+and incubate anaerobically (in Buchner's tubes) at 42 deg. C. for a
+maximum period of forty-eight hours.
+
+4. If growth occurs complete the investigation as detailed under the
+corresponding section of water examination (see pages 428 to 431).
+
+     NOTE.--The B. coli communis, derived from the alvine
+     discharges of the cow, is almost universally present in
+     large or small numbers, in retail milk. Its detection,
+     therefore, unless in enormous numbers, (when it indicates
+     want of cleanliness), is of little value.
+
+~2. Vibrio Cholerae.~--Inoculate tubes of peptone water by using the same
+amounts as in the search for members of the Coli-typhoid groups (_vide
+ante_ 1-3); incubate aerobically at 37 deg. C. and complete the
+examination as detailed under the corresponding section of water
+examination (see page 439).
+
+~3. B. Enteritidis Sporogenes.~--Inoculate tubes of litmus milk with
+similar amounts to those used in the previous searches, omitting tube
+No. 1 (_vide ante_ 1-3) place in the differential steriliser at 80 deg.
+C. for ten minutes and then incubate anaerobically at 37 deg. C. for a
+maximum period of forty-eight hours. Complete the investigation as
+detailed under the corresponding section of water examination (see
+page 438).
+
+~4. B. Diphtheriae.~--
+
+(A) 1. Plant three sets of serial cultivations, twelve tubes in each
+set, from (a) cream C^{2}, (b) deposit D^{1} upon oblique
+inspissated blood-serum, and incubate at 37 deg. C.
+
+2. Pick off any suspicious colonies which may have made their appearance
+twelve hours after incubation, examine microscopically and subcultivate
+upon blood-serum and place in the incubator; return the original tubes
+to the incubator.
+
+3. Repeat this after eighteen hours' incubation.
+
+4. From the resulting growths make cover-slip preparations and stain
+carbolic methylene-blue, Neisser's method, Gram's method. Subcultivate
+such as appear to be composed of diphtheria bacilli in glucose peptone
+solution. Note those in which acid production takes place.
+
+5. Inoculate guinea-pigs subcutaneously with one or two cubic
+centimetres forty-eight-hour-old glucose bouillon cultivation derived
+from the first subcultivation of each glucose fermenter, and observe the
+result.
+
+6. If death, apparently from diphtheritic toxaemia, ensues, inoculate two
+more guinea pigs with a similar quantity of the lethal culture. Reserve
+one animal as a control and into the other inject 1000 units of
+antidiphtheritic serum. If the control dies and the treated animal
+survives, the proof of the identity of the organism isolated with the
+Klebs-Loeffler bacillus becomes absolute.
+
+7. Inoculate guinea-pigs subcutaneously with filtered glucose bouillon
+cultivations (toxins?) and observe the result.
+
+(B) 1. Emulsify the remainder of the deposit with 5 c.c. sterile
+bouillon and inoculate two guinea-pigs, thus: guinea-pig a,
+subcutaneously with 1 c.c. emulsion; guinea-pig b, subcutaneously with
+2 c.c. emulsion; and observe the result.
+
+2. If either or both of the inoculated animals succumb, make complete
+post-mortem examination and endeavour to isolate the pathogenic
+organisms from the local lesion. Confirm their identity as in A5 and 6
+(_vide supra_).
+
+~5. Bacillus Tuberculosis.~--
+
+(A) 1. Inoculate each of three guinea-pigs (previously tested with
+tuberculin, to prove their freedom from spontaneous tuberculosis)
+subcutaneously at the inner aspect of the bend of the left knee, with 1
+c.c. of the deposit emulsion remaining in one or other tube (D^{1} or
+D^{2}).
+
+2. Introduce a small quantity of the cream into a subcutaneous pocket
+prepared at the inner aspect of the bend of the right knee of each of
+these three animals. Place a sealed dressing on the wound.
+
+3. Observe carefully, and weigh accurately each day.
+
+4. Kill one guinea-pig at the end of the second week and make a
+complete post-mortem examination.
+
+5. If the result of the examination is negative or inconclusive, kill a
+second guinea-pig at the end of the third week and examine carefully.
+
+[Illustration: FIG. 215.--Cadaver of guinea-pig experimentally infected
+with B. tuberculosis.]
+
+6. If still negative or inconclusive, kill the third guinea-pig at the
+end of the _sixth_ week. Make a careful post-mortem examination.
+Examine material from any caseous glands microscopically and inoculate
+freely on to Dorset's egg medium.
+
+     NOTE.--Every post-mortem examination of animals infected
+     with tuberculous material should include the naked eye and
+     microscopical examination of the popliteal, superficial and
+     deep inguinal, iliac, lumbar and axillary glands on each
+     side of the body, also the retrohepatic, bronchial and
+     sternal glands, the spleen, liver and lungs (Fig. 215).
+
+(B) 1. Intimately mix all the available cream and deposit from the milk
+sample, and transfer to a sterile Erlenmeyer flask.
+
+2. Treat the mixture by the antiformin method (_vide_ Appendix, page
+502).
+
+3. Inoculate each of two guinea-pigs, intraperitoneally, with half of
+the emulsion thus obtained.
+
+4. Kill one of the guinea-pigs at the end of the first week and examine
+carefully.
+
+5. Kill the second guinea-pig at the end of the second week and examine
+carefully.
+
+6. Utilise the remainder of the deposit for microscopical examination
+and cultivations upon Dorset's egg medium.
+
+     NOTE.--No value whatever attaches to the result of a
+     microscopical examination for the presence of the B.
+     tuberculosis unless confirmed by the result of inoculation
+     experiments.
+
+~6. Streptococcus Pyogenes Longus.~--
+
+(A) 1. Spread serial surface plates upon nutrose agar. Also plant serial
+cultivations upon sloped nutrient agar (six tubes in series).
+
+2. If the resulting growth shows colonies which resemble those of the
+streptococcus, make subcultivations upon agar and in bouillon, in the
+first instance, and study carefully.
+
+(B) 1. Plant a large loopful of the deposit D^{2} into each of three
+tubes of glucose formate bouillon, and incubate anaerobically (in
+Buchner's tubes) for twenty-four hours at 37 deg. C.
+
+2. If the resulting growth resembles that of the streptococcus, make
+subcultivations upon nutrient agar.
+
+3. Prepare subcultivations of any suspicious colonies that appear, upon
+all the ordinary media, and study carefully.
+
+If the streptococcus is successfully isolated, inoculate serum bouillon
+cultivations into the mouse, guinea-pig, and rabbit, to determine its
+pathogenicity and virulence.
+
+~7. Staphylococcus Pyogenes Aureus.~--
+
+1. Examine carefully the growth upon the serial blood serum cultivations
+prepared to isolate B. diphtheriae and the serial agar cultivations to
+isolate streptococci after forty-eight hours' incubation.
+
+2. Pick off any suspicious orange coloured colonies, plant on sloped
+agar, and incubate at 20 deg. C. Observe pigment formation.
+
+3. Prepare subcultivations from any suspicious growths upon all the
+ordinary media, study carefully and investigate their pathogenicity.
+
+~8. Micrococcus Melitensis.~--The milk from an animal infected with M.
+melitensis usually contains the organisms in large numbers and but few
+other bacteria.
+
+1. Spread several sets of surface plates upon nutrose agar, each from
+one loopful of the deposit in tube D^{1} or D^{2}.
+
+2. Spread several sets of surface plates upon nutrose agar, each from
+one drop of the original milk sample.
+
+3. Incubate aerobically at 37 deg. C. and examine daily up to the end
+of ten days.
+
+4. Pick off suspicious colonies, examine them microscopically and
+subcultivate upon nutrose agar in tubes; upon glucose agar and in litmus
+milk.
+
+5. Test the subsequent growth against the serum of an experimental
+animal inoculated against M. melitensis to determine its
+agglutinability.
+
+6. If apparently M. melitensis, inoculate growth from a nutrose agar
+culture after three days incubation intracranially into the guinea-pig.
+
+
+ICE CREAM.
+
+~Collection of the Sample.~--
+
+1. Remove the sample from the drum in the ladle or spoon with which the
+vendor retails the ice cream, and place it at once in a sterile copper
+capsule, similar to that employed for earth samples (_vide_ page 471).
+
+2. Pack for transmission in the ice-box.
+
+3. On arrival at the laboratory place the copper capsules containing the
+ice cream in the incubator at 20 deg. C. for fifteen minutes--that is,
+until at least some of the ice cream has become liquid.
+
+~Qualitative and Quantitative Examination.~--Treat the fluid ice cream as
+milk and conduct the examination in precisely the same manner as
+described for milk (_vide_ page 443).
+
+
+EXAMINATION OF CREAM AND BUTTER.
+
+~Collection of the Sample.~--Collect, store, and transmit samples to the
+laboratory, precisely as is done in the case of ice cream.
+
+~Quantitative.~--
+
+_Apparatus Required_:
+
+    Sterile test-tube.
+    Sterilised spatula.
+    Water-bath regulated at 42 deg. C.
+    Case of sterile plates.
+    Case of sterile graduated pipettes, 1 c.c. (in hundredths).
+    Tubes of gelatine-agar (+10 reaction).
+    Plate-levelling stand, with its water chamber filled with water at
+      42 deg. C.
+
+METHOD.--
+
+1. Transfer a few grammes of the sample to a sterile test-tube by means
+of the sterilised spatula.
+
+2. Place the tube in the water-bath at 42 deg. C. until the contents are
+liquid.
+
+3. Liquefy eight tubes of gelatine-agar and place them in the water-bath
+at 42 deg. C, and cool down to that temperature.
+
+4. Inoculate the gelatine-agar tubes with the following quantities of
+the sample by the help of a sterile pipette graduated to hundredths of a
+cubic centimetre--viz.,
+
+    To tube No. 1 add 1    c.c. liquefied butter.
+                2 add 0.5  c.c. liquefied butter.
+                3 add 0.3  c.c. liquefied butter.
+                4 add 0.2  c.c. liquefied butter.
+                5 add 0.1  c.c. liquefied butter.
+                6 add 0.05 c.c. liquefied butter.
+                7 add 0.03 c.c. liquefied butter.
+                8 add 0.02 c.c. liquefied butter.
+                9 add 0.01 c.c. liquefied butter.
+
+5. Pour a plate cultivation from each of the gelatine-agar tubes and
+incubate at 28 deg. C.
+
+6. "Count" the plates after three days' incubation, and from the figures
+thus obtained estimate the number of organisms present per cubic
+centimetre of the sample.
+
+~Qualitative.~--
+
+_Apparatus Required_:
+
+     Sterile beaker, its mouth plugged with sterile cotton-wool.
+
+     Counterpoise for beaker.
+
+     Scales and weights.
+
+     Sterilised spatula.
+
+     Water-bath regulated at 42 deg. C.
+
+     Separatory funnel, 250 c.c. capacity, its delivery tube
+     protected against contamination by passing it through a
+     cotton-wool plug into the interior of a small Erlenmeyer
+     flask which serves to support the funnel. This piece of
+     apparatus is sterilised _en masse_ in the hot-air oven.
+
+     Large centrifugal machine.
+
+     Sterile tubes (for the centrifuge) closed with solid rubber
+     stoppers.
+
+     Case of sterile pipettes, 10 c.c.
+
+     Case of sterile graduated pipettes, 1 c.c. (in tenths of a
+     cubic centimetre).
+
+METHOD.--
+
+1. Weigh out 100 grammes of the sample in a sterile beaker.
+
+2. Plug the mouth of the beaker with sterile cotton-wool and immerse the
+beaker in a water-bath at 42 deg. C. until the contents are completely
+liquefied.
+
+3. Fill the liquefied butter into the sterile separatory funnel.
+
+4. Transfer the funnel to the incubator at 37 deg. C. and allow it to
+remain there for four days.
+
+At the end of this time the contents of the funnel will have separated
+into two distinct strata.
+
+(a) A superficial oily layer, practically free from bacteria.
+
+(b) A deep watery layer, turbid and cloudy from the growth of bacteria.
+
+5. Draw off the subnatant turbid layer into sterile centrifugal tubes,
+previously warned to about 42 deg. C., and centrifugalise at once.
+
+6. Pipette off the supernatant fluid and fill the tubes with sterile 1
+per cent. sodium carbonate solution previously warmed slightly; stopper
+the tubes and shake vigourously for a few minutes.
+
+7. Centrifugalise again.
+
+8. Pipette off the supernatant fluid; filling the tubes with warm
+sterile bouillon, shake well, and again centrifugalise, to wash the
+deposit.
+
+9. Pipette off the supernatant fluid.
+
+10. Prepare cover-slip preparations, fix and clear as for milk
+preparations, stain carbolic methylene-blue, Gram's method,
+Ziehl-Neelsen's method, and examine microscopically with a 1/12 inch
+oil-immersion lens.
+
+11. Proceed with the examination of the deposit as in the case of milk
+deposit (see pages 450 _et seq._).
+
+
+EXAMINATION OF UNSOUND MEATS.
+
+(INCLUDING TINNED OR POTTED MEATS, FISH, ETC.)
+
+The bacterioscopic examination of unsound food is chiefly directed to
+the detection of those members of the Coli-typhoid group--B. enteritidis
+of Gaertner and its allies--which are usually associated with epidemic
+outbreaks of food poisoning, and such anaerobic bacteria as initiate
+putrefactive changes in the food which result in the formation of
+poisonous ptomaines, consequently the quantitative examination pure and
+simple is frequently omitted.
+
+A. Cultural Examination.
+
+Quantitative.--
+
+_Apparatus Required_:
+
+     Sterilised tin opener, (if necessary.)
+
+     Erlenmeyer flask (500 c.c. capacity) containing 200 c.c.
+     sterile bouillon and fitted with solid rubber stopper.
+
+     Counterpoise.
+
+     Scissors and forceps.
+
+     Scales and weights.
+
+     Water steriliser.
+
+     Hypodermic syringe.
+
+     Syringe with intragastric tube.
+
+     Rat forceps.
+
+     Case of sterile capsules.
+
+     Filtering apparatus as for water analysis.
+
+     Case of sterile plates.
+
+     Case of sterile graduated pipettes, 10 c.c. (in tenths of a
+     cubic centimetre).
+
+     Case of sterile graduated pipettes, 1 c.c. (in tenths of a
+     cubic centimetre).
+
+     Plate-levelling stand.
+
+     Tubes of nutrient gelatine.
+
+     Tubes of nutrient agar.
+
+     Water-bath regulated at 42 deg. C.
+
+     Bulloch's apparatus.
+
+METHOD.--
+
+1. Place the flask containing 200 c.c. sterile broth on one pan of the
+scales and counterpoise accurately.
+
+2. Mince a portion of the sample by the aid of sterile scissors and
+forceps, and add the minced sample to the bouillon in the flask to the
+extent of 20 grammes.
+
+3. Make an extract by standing the flask in the incubator running at 42
+deg. C. (or in a water-bath regulated to that temperature) for half an
+hour, shaking its contents from time to time. Better results are obtained
+if an electrical shaker is fitted inside the incubator and the flask kept
+in motion throughout the entire thirty minutes.
+
+Now every centimetre contains the bacteria washed out from 0.1 gramme of
+the original food.
+
+4. Inoculate tubes of liquefied gelatine as follows:
+
+    To tube No. 1 add 1.0 c.c. of the extract.
+                2 add 0.5 c.c. of the extract.
+                3 add 0.3 c.c. of the extract.
+                4 add 0.2 c.c. of the extract.
+                5 add 0.1 c.c. of the extract.
+
+Pour plates from these tubes and incubate at 20 deg. C.
+
+5. Prepare a precisely similar set of agar plates and incubate at 37
+deg. C.
+
+6. Pipette 5 c.c. of the extract into a sterile tube, heat in the
+differential steriliser at 80 deg. C. for ten minutes.
+
+7. From the heated extract prepare duplicate sets of agar and gelatine
+plates and incubate anaerobically in Bulloch's apparatus at 37 deg. C. and
+20 deg. C. respectively.
+
+8. After three days' incubation examine the agar plates both aerobic and
+anaerobic and enumerate the colonies developed from spores (7), and from
+vegetative forms and spores (5), and calculate and record the numbers of
+each group per gramme of the original food.
+
+9. After seven days' incubation (or earlier if compelled by the growth
+of liquefying colonies) enumerate the gelatine plates in the same way.
+
+10. Subcultivate from the colonies that make their appearance and
+identify the various organisms.
+
+11. Continue the investigations with reference to the detection of
+pathogenic organisms as described under water (page 429 _et seq._).
+
+Qualitative.--
+
+I. _Cultural._
+
+The micro-organisms sought for during the examination of unsound foods
+comprise the following:
+
+Members of the Coli-typhoid groups (chiefly those of the Gaertner
+class).
+
+B. anthracis.
+
+Streptococci
+
+Anaerobic Bacteria:
+
+    B. enteritidis sporogenes.
+    B. botulinus.
+    B. cadaveris.
+
+The methods by which these organisms if present may be identified and
+isolated have already been described under the corresponding section of
+water examination with the exception of those applicable to B.
+botulinus, and B. cadaveris. These can only be isolated satisfactorily
+from the bodies of experimentally inoculated animals.
+
+II _Experimental._
+
+_Tissue._--
+
+1. Feed rats and mice on portions of the sample and observe the result.
+
+2. If any of the animals die, make complete post-mortem examinations and
+endeavour to isolate the pathogenic organisms.
+
+_Extract._--
+
+1. Introduce various quantities of the bouillon extract into the
+stomachs of several rats, mice and guinea-pigs repeatedly over a period
+of two or three days by the intragastric method of inoculation (see page
+367) and observe the result. Guinea-pigs and mice are very susceptible
+to infection by B. botulinus by this method; rabbits less so.
+
+2. Inoculate rats, mice, and guinea-pigs subcutaneously into deep
+pockets, and intraperitoneally with various quantities of the bouillon
+extract, and observe the result.
+
+3. Filter some of the extract through a Chamberland candle and incubate
+the filtrate to determine the presence of soluble toxins.
+
+4. If any of the animals succumb to either of these methods of
+inoculation, make careful post-mortem examinations and endeavour to
+isolate the pathogenic organisms.
+
+
+THE EXAMINATION OF OYSTERS AND OTHER SHELLFISH.
+
+On opening the shell of an oyster a certain amount of fluid termed
+"liquor" is found to be present. This varies in amount from a drop to
+many cubic centimetres (0.1 c.c. to 10 c.c.)--in the latter case the
+bulk of the fluid is probably the last quantum of water ingested by the
+bivalve before closing its shell. In order to obtain a working average
+of the bacteriological flora of a sample, ten oysters should be taken
+and the body, gastric juice and liquor should be thoroughly mixed before
+examination. The examination, as in dealing with other food stuffs, is
+directed to the search for members of the Coli-typhoid group, sewage
+streptococci and perhaps also B. enteritidis sporogenes.
+
+_Apparatus Required_:
+
+     Two hard nail brushes.
+
+     Liquid soap.
+
+     Sterile water in aspirator jar with delivery nozzle
+     controlled by a spring clip.
+
+     Sterile oyster knives.
+
+     Sterile glass dish, with cover, sufficiently large to
+     accommodate ten oysters.
+
+     Sterile forceps.
+
+     Sterile scissors.
+
+     Sterile towels or large gauze pads.
+
+     Sterile graduated cylinders 1000 c.c. capacity, with either
+     the lid or the bottom of a sterile Petri dish inverted over
+     the open mouth as a cover.
+
+     Glass rods.
+
+     Corrosive sublimate solution, 1 per mille.
+
+     Bile salt broth tubes.
+
+     Litmus milk tubes.
+
+     Surface plates of nutrose agar.
+
+     Case of sterile pipettes, 1 c.c. (in tenths of a c.c.)
+
+     Case of sterile pipettes, 10 c.c. (in tenths of a c.c.)
+
+     Case of sterile glass capsules.
+
+     Erlenmeyer flasks, 250 c.c. capacity.
+
+     Double strength bile salt broth.
+
+METHOD.--
+
+1. Thoroughly clean the outside of the oyster shells by scrubbing each
+in turn with liquid soap and nail brush under a tap of running water.
+Then, holding an oyster shell in a pair of sterile forceps wash every
+part of the outside of the shell with a stream of sterile water running
+from an aspirator jar; deposit the oyster inside the sterile glass dish.
+Repeat the process with each of the remaining oysters.
+
+2. Before proceeding further, cleanse the hands thoroughly with clean
+nail brush, soap and water, then plunge them in lysol 2 per cent.
+solution, and finally in sterile water.
+
+3. Spread a sterile towel on the bench.
+
+4. Remove one of the oysters from the sterile glass dish and place it,
+resting on its convex shell, on the towel. Turn a corner of the sterile
+towel over the upper flat shell to give a firmer grip to the left hand,
+which holds the shell in position.
+
+5. With the sterile oyster knife (in the right hand) open the shell and
+separate the body of the oyster from the inner surface of the upper flat
+shell. Bend back and separate the flat shell, leaving the body of the
+oyster in and attached to the concave shell. Avoid spilling any of the
+liquor.
+
+(Some dexterity in opening oysters should be acquired before undertaking
+these experiments).
+
+6. Cut up the body of the oyster with sterile scissors into small pieces
+and allow the liquor freed from the body during the process to mix with
+the liquor previously in the shell.
+
+7. Transfer the comminuted oyster and the liquor to the cylinder.
+
+8. Treat each of the remaining oysters in similar fashion.
+
+9. Mix the contents of the cylinder thoroughly by stirring with a
+sterile glass rod. The total volume will amount to about 100 c.c.
+
+10. Use 0.1 c.c. of the mixed liquor to inseminate each of a series of
+three nutrose surface plates.
+
+11. Inoculate 0.1 c.c. of the mixed liquor into each of three tubes of
+litmus milk.
+
+12. Add sterile distilled water to the contents of the cylinder up to
+1000 c.c. and stir thoroughly with a sterile glass rod and allow to
+settle. The bacterial content of each oyster may be regarded, for all
+practical purposes, as comprised in 100 c.c. of fluid.
+
+13. Arrange four glass capsules in a row and number I, II, III, IV.
+Pipette 9 c.c. sterile distilled water into each.
+
+14. To capsule No. I add 1 c.c. of the diluted liquor, etc. from the
+cylinder, and mix thoroughly. To capsule II add 1 c.c. of dilution in
+capsule I and mix thoroughly. Carry over 1 c.c. of fluid from capsule
+II to capsule III, afterwards adding 1 c.c. of fluid from capsule III to
+capsule IV.
+
+15. Label tubes of bile salt broth and inoculate with the following
+amounts of diluted oysters:
+
+    No. 6 with 10 c.c. cylinder    fluid = 0.1      oyster.
+    No. 5 with  1 c.c. cylinder    fluid = 0.01     oyster.
+    No. 4 with  1 c.c. capsule   I fluid = 0.001    oyster.
+    No. 3 with  1 c.c. capsule  II fluid = 0.0001   oyster.
+    No. 2 with  1 c.c. capsule III fluid = 0.00001  oyster.
+    No. 1 with  1 c.c. capsule  IV fluid = 0.000001 oyster.
+
+16. Transfer 100 c.c. cylinder fluid (= 1 oyster) to an Erlenmeyer flask
+and add 50 c.c. double strength bile salt broth, and label 7.
+
+17. Duplicate all the above indicated cultures.
+
+18. Put up the tube cultures in Buchner's tubes and incubate
+anaerobically at 42 deg. C.
+
+If growth occurs in tube 1 the organism finally isolated, e. g., B.
+coli, must have been present to the extent of one million per oyster.
+
+19. Complete the examination for members of the Coli-typhoid group and
+sewage streptococci, as directed under Water Examination, page 429
+(steps 11-21).
+
+20. Inoculate a series of 6 tubes of litmus milk with quantities of the
+material similar to those indicated in step 15; heat to 80 deg. C. for ten
+minutes, and incubate under anaerobic conditions at 37 deg. C. Examine for
+the presence of B. enteritidis sporogenes as directed under Water
+Examination, page 438 (steps 7-10).
+
+
+EXAMINATION OF SEWAGE AND SEWAGE EFFLUENTS.
+
+Quantitative.--
+
+_Collection of the Sample._--As only small quantities of material are
+needed, the samples should be collected in a manner similar to that
+described under water for quantitative examination and transmitted in
+the ice apparatus used in packing those samples.
+
+_Apparatus Required._--As for water (_vide_ page 420).
+
+METHOD.--
+
+1. Arrange four sterile capsules in a row and number them I, II, III,
+IV.
+
+2. Pipette 9 c.c. sterile bouillon into capsule No. I.
+
+3. Pipette 9.9 c.c. sterile bouillon into capsules II, III, and IV.
+
+4. Add 1 c.c. of the sewage to capsule No. I by means of a sterile
+pipette, and mix thoroughly.
+
+5. Take a fresh sterile pipette and transfer 0.1 c.c. of the mixture
+from No. I to No. II and mix thoroughly.
+
+6. In like manner transfer 0.1 c.c. from No. II to No. III, and then 0.1
+c.c. from No. III to No. IV.
+
+Now 1 c.c. of dilution No.   I contains 0.1       c.c. of the original sewage.
+    1 c.c. of dilution No.  II contains 0.001     c.c. of the original sewage.
+    1 c.c. of dilution No. III contains 0.00001   c.c. of the original sewage.
+    1 c.c. of dilution No.  IV contains 0.0000001 c.c. of the original sewage.
+
+7. Pour a set of gelatine plates from the contents of each capsule,
+three plates in a set, and containing respectively 0.2, 0.3, and 0.5
+c.c. of the dilution. Label carefully; incubate at 20 deg. C. for three,
+four, or five days.
+
+8. Enumerate the organisms present in those sets of plates which have
+not liquefied, probably those from dilution III or IV, and calculate
+therefrom the number present per cubic centimetre of the original sample
+of sewage.
+
+Qualitative.--The qualitative examination of sewage is concerned with
+the identification and enumeration of the same bacteria dealt with under
+the corresponding section of water examination; it is consequently
+conducted on precisely similar lines to those already indicated (_vide_
+pages 426 to 441).
+
+
+EXAMINATION OF AIR.
+
+Quantitative.--
+
+_Apparatus Required_:
+
+     Aspirator bottle, 10 litres capacity, fitted with a delivery
+     tube, and having its mouth closed by a perforated rubber
+     stopper, through which passes a short length of glass
+     tubing.
+
+     Erlenmeyer flask, 250 c.c. capacity (having a wide mouth
+     properly plugged with wool), containing 50 c.c. sterile
+     water.
+
+     Rubber stopper to fit the mouth of the flask, perforated
+     with two holes, and fitted as follows:
+
+     Take a 9 cm. length of glass tubing and bend up 3 cm. at one
+     end at right angles to the main length of tubing. Pass the
+     long arm of the angle through one of the perforations in the
+     stopper; plug the open end of the short arm with
+     cotton-wool.
+
+     Take a glass funnel 5 or 6 cm. in diameter with a stem 12
+     cm. in length and bend the stem close up to the apex of the
+     funnel, in a gentle curve through a quarter of a circle;
+     pass the long stem through the other perforation in the
+     rubber stopper.
+
+     A battery jar or a small water-bath to hold the Erlenmeyer
+     flask when packed round with ice.
+
+     Supply of broken ice.
+
+     Rubber tubing.
+
+     Screw clamps and spring clips, for tubing.
+
+     Water steriliser.
+
+     Retort stand and clamps.
+
+     Apparatus for plating (as for enumeration of water
+     organisms, _vide_ page 420).
+
+METHOD.--
+
+1. Fill 10 litres of water into the aspirating bottle and attach a piece
+of rubber tubing with a screw clamp to the delivery tube. Open the taps
+fully and regulate the screw clamp, by actual experiment, so that the
+tube delivers 1 c.c. of water every second. The screw clamp is not
+touched again during the experiment.
+
+At this rate the aspirator bottle will empty itself in just under three
+hours. Shut off the tap and make up the contents of the aspirator bottle
+to 10 litres again.
+
+2. Sterilise the fitted rubber cork, with its funnel and tubing, by
+boiling in the water steriliser for ten minutes.
+
+3. Remove the cotton-wool plug from the flask, and replace it by the
+rubber stopper with its fittings. Make sure that the end of the stem of
+the funnel is immersed in the bouillon.
+
+4. Place the flask in a glass or metal vessel and pack it round with
+pounded ice. Arrange the flask with its ice casing just above the neck
+of the aspirator bottle.
+
+[Illustration: FIG. 216.--Arrangement of apparatus for air analysis.]
+
+5. Connect up the free end of the glass tube from the flask--after
+removing the cotton-wool plug--with the air-entry tube in the mouth of
+the aspirating bottle (Fig. 216).
+
+6. Open the tap fully, and allow the water to run.
+
+Replenish the ice from time to time if necessary.
+
+(In emptying itself the aspirator bottle will aspirate 10 litres of air
+slowly through the water in the Erlenmeyer flask.)
+
+7. When the aspiration is completed, disconnect the flask and remove it
+from its ice packing.
+
+8. Liquefy three tubes of nutrient gelatine and add to them 0.5 c.c.,
+0.3 c.c., and 0.2 c.c., respectively, of the water from the flask, by
+means of a sterile graduated pipette, as in the quantitative examination
+of water. Pour plates.
+
+9. Pour a second similar set of gelatine plates.
+
+10. Incubate both sets of plates at 20 deg. C.
+
+11. Enumerate the colonies present in the two sets of gelatine plates
+after three, four, or five days and average the results from the numbers
+so obtained; estimate the number of micro-organisms present in 1 c.c.,
+and then in the 50 c.c. of broth in the flask.
+
+12. The result of air examination is usually expressed as the number of
+bacteria present per cubic metre (i. e., kilolitre) of air; and as the
+number of organisms present in the 50 c.c. water only represent those
+contained in 10 litres of air, the resulting figure must be multiplied
+by 100.
+
+Qualitative.--
+
+1. Proceed exactly as in the quantitative examination of air (_vide
+supra_), steps 1 to 10.
+
+2. Pour plates of wort agar with similar quantities of the air-infected
+water, and incubate at 37 deg. C.
+
+3. Pour plates of nutrient agar with similar quantities of the water and
+incubate at 37 deg. C.
+
+4. Pour similar plates of wort gelatine and incubate at 20 deg. C.
+
+5. Pick off the individual colonies that appear in the several plates,
+subcultivate them on the various media, and identify them.
+
+
+EXAMINATION OF SOIL.
+
+The bacteriological examination of soil yields information of value to
+the sanitarian during the progress of the process of homogenisation of
+"made soil" (e. g., a dumping area for the refuse of town) and
+determines the period at which such an area may with propriety and
+safety be utilised for building purposes; or to the agriculturalist in
+informing him of the suitability of any given area for the growth of
+crops.
+
+The surface of the ground, exposed as it is to the bactericidal
+influence of sunlight and to rapid alternations of heat and cold, rain
+and wind, contains but few micro-organisms. Again, owing to the density
+of the molecules of deep soil and lack of aeration on the one hand, and
+the filtering action of the upper layers of soil and bacterial
+antagonism on the other, bacterial life practically ceases at a depth of
+about 2 metres. The intermediate stratum of soil, situated from 25 to 50
+cm. below the surface, invariably yields the most numerous and the most
+varied bacterial flora.
+
+~Collection of Sample.~--A small copper capsule 6 cm. high by 6 cm.
+diameter, with "pull-off" cap secured by a bayonet catch, previously
+sterilised in the hot-air oven, is the most convenient receptacle for
+samples of soil.
+
+[Illustration: FIG. 217.--Soil scoop.]
+
+The instrument used for the actual removal of the soil from its natural
+position will vary according to whether we require surface samples or
+soil from varying depths.
+
+(a) For ~surface~ samples, use an iron scoop, shaped like a shoe horn,
+but provided with a sharp spine (Fig. 217). This is wrapped in asbestos
+cloth and sterilised in the hot-air oven. When removed from the oven,
+wrap a piece of oiled paper, silk, or gutta-percha tissue over the
+asbestos cloth, and secure it with string, as a further protection
+against contamination.
+
+On reaching the spot whence the samples are to be taken, the coverings
+of the scoop are removed, and the asbestos cloth employed to brush away
+loose stones and debris from the selected area. The surface soil is then
+broken up with the point of the scoop, scraped up and collected in the
+body of the scoop, and transferred to the sterile capsule for
+transmission.
+
+[Illustration: FIG. 218.--Fraenkel's borer.]
+
+(b) For ~deep~ samples collected at various distances from the surface,
+an experimental trench may be cut to the required depth and samples
+collected at the required points on the face of the section. It is,
+however, preferable to utilise some form of borer, such as that designed
+by Fraenkel (Fig. 218).
+
+_Fraenkel's Earth Borer._--This instrument consists of a stout
+hard-steel rod, 150 cm. long, marked in centimetres from the
+drill-pointed extremity. It is provided with a cross handle (adjustable
+at any point along the length of the rod by means of a screw nut). The
+terminal centimeters are thicker than the remainder of the rod, and on
+one side a vertical cavity about 0.5 cm. deep is cut. This is covered by
+a flanged sleeve so long as the borer is driven into the soil clockwise,
+and is opened for the reception of the sample of soil, when the required
+depth is reached, by reversing the screwing motion, and again closed
+before withdrawal of the borer from the earth by resuming the original
+direction of twist. It can be sterilised in a manner similar to that
+adopted for the scoop, or by repeatedly filling the cavity with ether
+and burning it off.
+
+~Quantitative.~--Four distinct investigations are included in the complete
+quantitative bacteriological examination of the soil:
+
+1. The enumeration of the aerobic organisms.
+
+2. The enumeration of the spores of aerobes.
+
+3. The enumeration of the anaerobic organisms (including the facultative
+anaerobes).
+
+4. The enumeration of the spores of anaerobes.
+
+Further, by a combination of the results of the first and second, and of
+the third and fourth of these, the ratio of spores to vegetative forms
+is obtained.
+
+_Apparatus Required_:
+
+     Case of sterile capsules (25 c.c. capacity).
+
+     Case of sterile graduated pipettes, 10 c.c. (in tenths of a
+     cubic centimetre).
+
+     Case of sterile graduated pipettes, 1 c.c. (in tenths of a
+     cubic centimetre).
+
+     Flask containing 250 c.c. sterile bouillon.
+
+     Tall cylinder containing 2 per cent. lysol solution.
+
+     Plate-levelling stand.
+
+     12 sterile plates.
+
+     Tubes of nutrient gelatine.
+
+     Tubes of wort gelatine.
+
+     Tubes of nutrient agar.
+
+     Tubes of glucose formate gelatine.
+
+     Tubes of glucose formate agar.
+
+     Water-bath regulated at 42 deg. C.
+
+     Bunsen burner.
+
+     Grease pencil.
+
+     Sterile mortar and pestle (agate).
+
+     Sterile wide-mouthed Erlenmeyer flask (500 c.c. capacity).
+
+     Sterile metal funnel with short wide bore delivery tube to
+     just fit mouth of flask.
+
+     Solid rubber stopper to fit the flask (sterilised by
+     boiling).
+
+     Pair of scales.
+
+     Counterpoise (Fig. 107).
+
+     Sterile metal (nickel) spoon or spatula.
+
+     Fractional steriliser (Fig. 140).
+
+METHOD.--
+
+1. Arrange four sterile capsules numbered I, II, III, and IV; pipette 9
+c.c. sterile bouillon into the first capsule, and 9.9 c.c. into each of
+the remaining three.
+
+2. Pipette 100 c.c. sterile bouillon into the Erlenmeyer flask.
+
+3. Remove the cotton-wool plug from the flask and replace it by the
+sterile funnel.
+
+4. Place flask and funnel on one pan of the scales, and counterpoise
+accurately.
+
+5. Empty the sample of soil into the mortar and triturate thoroughly.
+
+6. By means of the sterile spatula add 10 grammes of the earth sample to
+the bouillon in the flask.
+
+The final results will be more reliable if steps 2, 3, 4, and 5 are
+performed under a hood--to protect from falling dust, etc.
+
+7. Remove the funnel from the mouth of the flask; replace it by the
+rubber stopper and shake vigourously; then allow the solid particles to
+settle for about thirty minutes. One cubic centimetre of the turbid
+broth contains the washings from 0.1 gramme of soil.
+
+8. Pipette off 1 c.c. of the supernatant bouillon, termed the "soil
+water," and add it to the contents of capsule I; mix thoroughly.
+
+9. Remove 0.1 c.c. of the infected bouillon from capsule I and add it to
+capsule II, and mix.
+
+10. In like manner add 0.1 c.c. of the contents of capsule II to capsule
+III, and then 0.1 c.c. of the contents of capsule III to capsule IV.
+
+Then 1 c.c. fluid from capsule   I contains soil water
+  from .01       gm. earth.
+Then 1 c.c. fluid from capsule  II contains soil water
+  from .0001     gm. earth.
+Then 1 c.c. fluid from capsule III contains soil water
+  from .000001   gm. earth.
+Then 1 c.c. fluid from capsule  IV contains soil water
+  from .00000001 gm. earth.
+
+(A) _Aerobes (Vegetative Forms and Spores)._--
+
+11. Pour a set of gelatine plates from the contents of each capsule--two
+plates in a set, and containing respectively 0.1 c.c. and 0.4 c.c. of
+the diluted soil water. Label and incubate.
+
+12. Pour similar sets of wort gelatine plates from the contents of
+capsules II and III, label, and incubate at 20 deg. C.
+
+13. Pour similar sets of agar plates from the contents of capsules II
+and III; label and incubate at 37 deg. C.
+
+14. Weigh out a second sample of soil--10 grammes--dry over a water-bath
+until of constant weight and calculate the ratio
+
+    wet soil weight
+    ---------------
+    dry soil weight
+
+15. "Count" the plates after incubation for three, four, or five days,
+and correcting the figures thus obtained by means of the "wet" to "dry"
+soil ratio estimate--
+
+(a) The number of aerobic micro-organisms present per gramme of the
+soil.
+
+(b) The number of yeasts and moulds present per gramme of the soil.
+
+(c) The number of aerobic organisms "growing at 37 deg. C." present per
+gramme of the soil.
+
+(B) _Anaerobes (Vegetative Forms and Spores)._--
+
+16. Pour similar sets of plates in glucose formate gelatine and agar and
+incubate in Bulloch's anaerobic apparatus.
+
+(C) _Aerobes and Anaerobes (Spores Only)._--
+
+17. Pipette 5 c.c. soil water into a sterile tube.
+
+18. Place in the differential steriliser at 80 deg. C. for ten minutes.
+
+19. Pour two sets of four gelatine plates containing 0.1, 0.2, 0.5, and
+1 c.c. respectively of the soil water; label and incubate at 20 deg. C.,
+one set aerobically, the other anaerobically in Bulloch's apparatus.
+
+20. "Count" the plates (delay the enumeration as long as possible) and
+estimate the number of spores of aerobes and anaerobes respectively
+present per gramme of the soil.
+
+21. Calculate the ratio existing between spores and spores + vegetative
+forms under each of the two groups, aerobic and anaerobic
+micro-organisms.
+
+~Qualitative Examination.~--The qualitative examination of soil is usually
+directed to the detection of one or more of the following:
+
+Members of the Coli-typhoid group.
+
+Streptococci.
+
+Bacillus anthracis.
+
+Bacillus tetani.
+
+Bacillus oedematis maligni.
+
+The nitrous organisms.
+
+The nitric organisms.
+
+1. Transfer the remainder of the soil water (88 c.c.) to a sterile
+Erlenmeyer flask by means of a sterile syphon.
+
+2. Fix up the filtering apparatus as for the qualitative examination of
+water, and filter the soil water.
+
+3. Suspend the bacterial residue in 5 c.c. sterile bouillon (technique
+similar to that described for the water sample, _vide_ pages 434-436).
+
+Every cubic centimetre of suspension now contains the soil water from
+nearly 1 gramme of earth.
+
+The methods up to this point are identical no matter which organism or
+group of organisms it is desired to isolate; but from this stage onward
+the process is varied slightly for each particular bacterium.
+
+~I. The Coli-typhoid Group.~--
+
+~II. Streptococci.~--
+
+~III. Bacillus Anthracis.~--
+
+~IV. Bacillus Tetani.~--
+
+The methods adopted for the isolation of these organisms are identical
+with those already described under water (page 437 _et seq._).
+
+~V. Bacillus Oedematis Maligni.~--Method precisely similar to that
+employed for the B. tetani.
+
+~VI. The Nitrous Organisms.~--
+
+1. Take ten tubes of Winogradsky's solution No I (_vide_ page 198) and
+number them consecutively from 1 to 10.
+
+2. Inoculate each tube with varying quantities of the material as
+follows:
+
+    To tube No.  1 add 1.0 c.c. of the soil water.
+    To tube No.  2 add 0.1 c.c. of the soil water.
+    To tube No.  3 add 1.0 c.c. from Capsule I.
+    To tube No.  4 add 0.1 c.c. from Capsule I.
+    To tube No.  5 add 1.0 c.c. from Capsule II.
+    To tube No.  6 add 0.1 c.c. from Capsule II.
+    To tube No.  7 add 1.0 c.c. from Capsule III.
+    To tube No.  8 add 0.1 c.c. from Capsule III.
+    To tube No.  9 add 1.0 c.c. from Capsule IV.
+    To tube No. 10 add 0.1 c.c. from Capsule IV.
+
+Label and incubate at 30 deg. C.
+
+
+~VII. The Nitric Organisms.~--
+
+3. Take ten tubes of Winogradsky's solution No II, number them
+consecutively from 1 to 10 and inoculate with quantities of soil water
+similar to those enumerated in section VI step 2. Label and incubate at
+30 deg. C.
+
+4. Examine after twenty-four and forty-eight hours' incubation. From
+those tubes that show signs of growth make subcultivations in fresh
+tubes of the same medium and incubate at 30 deg. C.
+
+5. Make further subcultivations from such of those tubes as show growth,
+and again incubate.
+
+6. If growth occurs in these subcultures, make surface smears on plates
+of Winogradsky's silicate jelly (_vide_ page 198).
+
+7. Pick off such colonies as make their appearance and subcultivate in
+each of these two media.
+
+TESTING FILTERS.
+
+Porcelain filter candles are examined with reference to their power of
+holding back _all_ the micro-organisms suspended in the fluids which are
+filtered through them, and permitting only the passage of germ-free
+filtrates. In order to determine the freedom of the filter from flaws
+and cracks which would permit the passage of bacteria no matter how
+perfect the general structure of the candle might be, the candle must
+first be attached by means of a long piece of pressure tubing, to a
+powerful pump, such as a foot bicycle pump, fitted with a manometer. The
+candle is then immersed in a jar of water and held completely submerged
+whilst the internal pressure is gradually raised to two atmospheres by
+the action of the pump. Any crack or flaw will at once become obvious by
+reason of the stream of air bubbles issuing from it.
+
+The examination for permeability is conducted as follows:
+
+_Apparatus Required_:
+
+     Filtering apparatus: The actual filter candle that is used
+     must be the one it is intended to test and must be
+     previously carefully sterilised; the arrangement of the
+     apparatus will naturally vary with each different form of
+     filter, one or other of those already described (_vide_
+     pages 42-48).
+
+     Plate-levelling stand.
+
+     Case of sterile plates.
+
+     Case of sterile pipettes, 10 c.c. (in tenths).
+
+     Case of sterile pipettes, 1 c.c. (in tenths).
+
+     Tubes of nutrient gelatine.
+
+     Flask containing sterile normal saline solution.
+
+     Sterile measuring flask, 1000 c.c. capacity.
+
+METHOD.--
+
+1. Prepare surface cultivations, on nutrient agar in a culture bottle,
+of the Bacillus mycoides, and incubate at 20 deg. C., for forty-eight
+hours.
+
+2. Pipette 5 c.c. sterile normal saline into the culture bottle and
+emulsify the entire surface growth in it.
+
+3. Pipette the emulsion into the sterile measuring flask and dilute up
+to 1000 c.c. by the addition of sterile water.
+
+4. Pour the emulsion into the filter reservoir and start the filtration.
+
+5. When the filtration is completed, pour six agar plates each
+containing 1 c.c. of the filtrate.
+
+6. Incubate at 37 deg. C. until, if necessary, the completion of seven
+days.
+
+7. If the filtrate is not sterile, subcultivate the organism passed and
+determine its identity with the test bacterium before rejecting the
+filter--since the filtrate may have been accidentally contaminated.
+
+8. If the filtrate is sterile, resterilise the candle and repeat the
+test now substituting a cultivation of B. prodigiosus--a bacillus of
+smaller size.
+
+9. If the second test is satisfactory, test the candle against a
+cultivation of a very small coccus, e. g., Micrococcus melitensis, in
+a similar manner; in this instance continuing the incubation of
+cultivations from the filtrate for fourteen days.
+
+
+TESTING OF DISINFECTANTS.
+
+Methods have already been detailed (page 310) for the purpose of
+studying the vital resistance offered by micro-organisms to the lethal
+effect of germicides. But it frequently happens that the bacteriologist
+has to determine the relative efficiency of "disinfectants" from the
+standpoints of the sanitarian and commercial man rather than from the
+research worker's point of view. In pursuing this line of investigation,
+it is convenient to compare the efficiency, under laboratory conditions,
+of the proposed disinfectant with that of some standard germicide, such
+as pure phenol. In so doing, and in order that the work of different
+observers may be compared, conditions as nearly uniform as possible
+should be aimed at. The method described is one that has been in use by
+the writer for many years past, modified recently by the adoption of
+some of the recommendations of the Lancet Commission on the
+Standardisation of Disinfectants--particularly of the calculation for
+determining the phenol coefficient.
+
+This method has many points in common with that modification of the
+"drop" method known as the Rideal-Walker test.
+
+
+~General Considerations.~--
+
+These may be grouped under three headings: Test Germ, Germicide, and
+Environment.
+
+1. _Test Germ._--~B. coli.~
+
+As disinfectants are tested for sanitary purposes, it is obvious that a
+member of the coli-typhoid group should be selected as the test germ. B.
+coli is selected on account of its relative nonpathogenicity, the ease
+with which it can be isolated and identified by different observers in
+various parts of the world, the stability of its fundamental characters,
+and evenness of its resistance when utilised for these tests; finally
+since the colon bacillus is an organism which is slightly more
+resistant to the lethal action of germicides than the more pathogenic
+members of this group, a margin of safety is introduced into the test
+which certainly enhances its value.
+
+B. coli should be recently isolated from a normal stool, and plated at
+least twice to ensure the purity of the strain; and a stock agar culture
+prepared which should be used throughout any particular test. For any
+particular experiment prepare a smear culture on agar and incubate at
+37 deg. C. for 24 hours anaerobically. Then emulsify the whole of the
+surface growth in 10 c.c. of sterile water. Transfer the emulsion to a
+sterile test-tube with some sterile glass beads and shake thoroughly to
+ensure homogenous emulsion. Transfer to a centrifuge tube and
+centrifugalise the emulsion to throw down any masses of bacteria which
+may have escaped the disintegrating action of the beads. Pipette off the
+supernatant emulsion for use in the test.
+
+_2. Germicide._--
+
+_a. Disinfectant to be tested._--
+
+The first essential point is to test the unknown disinfectant, which may
+be referred to as germicide-x, on the lines set out on page 311 to
+determine its inhibition coefficient.
+
+This constant having been fixed, prepare various solutions of
+germicide-x with sterilised distilled water by accurate volumetric
+methods, commencing with a solution somewhat stronger than that
+representing the inhibition coefficient. The solutions must be prepared
+in fairly large bulk, not less than 5 c.c. of the disinfectant being
+utilised for the preparation of any given percentage solution.
+
+
+_b. Standard Control._--~Phenol.~
+
+The standard germicide used for comparison should be one which is not
+subject to variation in its chemical composition, and the one which has
+obtained almost universal use is Phenol.
+
+The following table shows the effect of different percentages of
+carbolic acid upon B. coli for varying contact times, compiled from an
+experiment conducted under the standard conditions referred to under
+Environment. The results closely correspond to those recorded by the
+Lancet Commission on Disinfectants, 1909.
+
+---------------------+-----------------------------------
+                     |        Contact time in minutes.
+Percentage of phenol +------+---+---+---+---+---+---+----
+                     | 2-1/2| 5 |10 | 15| 20| 25| 30| 35
+---------------------+------+---+---+---+---+---+---+----
+1.20                 |   -  | - | - | - | - | - | - | -
+1.10                 |   -  | - | - | - | - | - | - | -
+1.0                  |   +  | - | - | - | - | - | - | -
+0.9                  |   +  | - | - | - | - | - | - | -
+0.85                 |   +  | + | - | - | - | - | - | -
+0.80                 |   +  | + | + | - | - | - | - | -
+0.75                 |   +  | + | + | + | + | - | - | -
+0.7                  |   +  | + | + | + | + | + | - | -
+0.65                 |   +  | + | + | + | + | + | + | -
+---------------------+------+---+---+---+---+---+---+----
+
+- = No growth, i. e., bacteria killed.
++ = Growth, i. e., bacteria still living.
+
+From this it will be seen that the following percentage solutions will
+need to be prepared, namely: 1.1 per cent., 1.0 per cent., 0.9 per
+cent., 0.75 per cent., 0.7 per cent., as controls for each experiment.
+
+Prepare solutions of varying percentages by weighing out the quantity of
+carbolic acid required for each and dissolving in 100 c.c. of pure
+distilled water in an accurately standardised measuring flask. The
+solutions must be prepared freshly as required each day.
+
+
+~Environment.~--
+
+_a. General._--
+
+Close the windows and doors of the laboratory in which the investigation
+is carried out, to avoid draughts. Flush over the work bench and
+adjacent floor with 1:1000 solution of corrosive sublimate. Caution the
+assistant, if one is employed, to avoid unnecessary movement or speech.
+
+_b. Contact Temperature_, ~15-18 deg. C.~--
+
+This is the temperature at which contact between the germicide and the
+test germ takes place, and is of importance, since some germicides (_e.
+g._, Phenol) appear to be more powerful at high temperatures. 18 deg.
+C.--practically the ordinary room temperature--is a temperature at which
+the multiplication of B. coli is a comparatively slow process, but
+variation of a degree above this temperature or of two or three degrees
+below is of no moment. If the room temperature is below 15 deg. C. when
+the experiments are in progress, arrange a water-bath regulated at 18
+deg. C. for the reception of the tubes containing the mixture of germ and
+germicide; if above 19 deg. C. immerse the tubes in cold water, to which
+small pieces of ice are added from time to time to prevent the
+temperature rising above 18 deg. C.
+
+_c. Relative Proportional Bulk of Test Germ and Germicide_, ~50:1.~--
+
+Five cubic centimetres is a convenient amount of germicidal solution to
+employ, and to this 0.1 c.c. of the emulsion of test germ should be
+added.
+
+_d. Bulk of Sample Removed from Germ + Germicide Mixture at Each of the
+Time Periods_, ~0.1 c.c.~--
+
+This is sufficient to afford a fair sample of the germ content of the
+mixture, and at the same time is insufficient to exert any inhibitory
+action when transferred to the subculture medium.
+
+_e. Subculture Medium._ ~Bile Salt Broth.~--
+
+A _fluid_ medium is essential in order to obtain immediate dilution of
+the germicide carried over; at the same time it is advantageous to
+employ a selective medium which favours the growth of the test germ to
+the exclusion of organisms likely to contaminate the preparation, and
+if possible one which affords characteristic cultural appearances.
+
+Bile Salt Broth (page 180) combines these desiderata; it permits only
+the growth of intestinal bacteria, whilst the formation of an acid
+reaction and the production of gas in subcultures prepared from the
+germ-germicide mixture is fairly complete evidence of the presence of
+living B. coli.
+
+The amount of medium present in each test-tube is a matter of
+importance, since the medium not only provides pabulum for the test
+germ, but also acts as a diluent to the germicide, to reduce its
+strength below its inhibition coefficient. For routine work each
+subculture tube contains 10 c.c. of medium, but it is obvious that if
+germicide-x possesses an inhibition coefficient of 0.1 per cent. the
+addition of 0.1 c.c. of a 10 per cent. solution to 10 c.c. of medium
+would effectually prevent the subsequent growth of the test germ after a
+contact period insufficient to destroy its vitality. Hence the
+preliminary tests may in some instances indicate the necessity for the
+presence of 12 c.c., 15 c.c. or more of the fluid medium in the culture
+tubes.
+
+_f. Incubation Temperature_, ~37 deg. C.~--
+
+_g. Observation Period of the Subcultivations_, ~Seven Days.~--
+
+In order to determine whether or no the test germs have been destroyed,
+observations must always be continued--when growth appears to be
+absent--up to the end of seven days before recording "no growth."
+
+_h. Identification of the Organisms Developing in the Subcultivations
+after Contact in the Germ + Germicide Solution._--
+
+This is based on the naked eye characters of the growth in the bile salt
+broth, supplemented where necessary by plating methods, further
+subcultivations upon carbohydrate media and agglutination experiments.
+The sign (+) is used to indicate that growth of the test organism
+occurred in the subcultivations, and the sign (-) to indicate that the
+test germs have been destroyed and no subsequent growth has taken place.
+
+METHOD.--
+
+     _Apparatus Required_:
+
+     Sterile test-tubes (narrow, not exceeding 1.3 cm. diameter).
+
+     Test-tube rack (Fig. 219).
+
+     Sterile graduated pipettes in case, 1 c.c. (in tenths).
+
+     Sterile graduated pipettes in case, 5 c.c. (in c.c.).
+
+     Circular rubber washers, 2.5 cm. diameter with central hole,
+     sterilised by boiling immediately before use, then
+     transferred to sterilised glass double dish.
+
+     Electric signal clock or stop watch.
+
+     Sterile forceps.
+
+     Sterilised glass beads.
+
+     Shaking machine.
+
+     Grease pencil.
+
+     _Material Required_:
+
+     Percentage solutions of germicide-x (_vide_ page 481).
+
+     Percentage solutions of pure phenol (_vide_ page 482).
+
+     Aqueous emulsion of B. coli (_vide_ page 481).
+
+     Tubes of bile salt broth.
+
+
+~Preliminary Tests.~--
+
+_a. Inhibition Coefficient._--
+
+Determine the lowest percentage of germicide-x which inhibits growth of
+B. coli in the bile salt broth, and the highest percentage which fails
+to inhibit (page 311). On the result of this experiment determine the
+bulk of medium required in the subculture tubes and the percentage
+solutions to be employed in the trial trip. Assuming the inhibition
+coefficient to be 1:1000, it will be quite safe to employ the ordinary
+culture tubes containing 10 c.c. medium in the subsequent experiments.
+
+_b. Trial Trip._--
+
+Determine the lethal effect of a series of five solutions of germicide-x
+(say 1:100, 1:250, 1:300, 1:500, 1:600) at contact times of 2-1/2, 5, 25
+and 30 minutes in the following manner:
+
+1. Arrange five test-tubes marked A to E in the lower tier of the
+test-tube rack.
+
+2. Into tube A pipette 5 c.c. germicide-x 1:100 solution.
+
+Into tube B pipette 5 c.c. germicide-x 1:200 solution.
+
+Into tube C pipette 5 c.c. germicide-x 1:300 solution.
+
+Into tube D pipette 5 c.c. germicide-x 1:500 solution.
+
+Into tube E pipette 5 c.c. germicide-x 1:600 solution.
+
+3. Arrange 20 tubes of bile salt broth in the upper tier of the
+test-tube rack in two rows, those in the front row numbered
+consecutively from left to right 1-10, those in the back row 11-20.
+
+4. Place a square wire basket of about 50 tubes capacity close to the
+left of the test-tube rack, for the reception of the inoculated tubes.
+
+5. Take a sterile 1 c.c. pipette from the case, pick up a sterile rubber
+washer with forceps and push the point of the pipette into the central
+hole.
+
+6. Put down the forceps on the bench with the sterile points projecting
+over the edge. Without taking the tube from the rack remove the
+cotton-wool plug from tube A, and lower the pipette, with the rubber
+washer affixed, on to the open mouth of the tube; with the help of the
+forceps to steady the washer, push the pipette on through the hole until
+the point of the pipette has reached to within a few millimetres of the
+bottom of the tube (see fig. 219).
+
+7. Adjust in the same way a pipette and a washer in the mouth of each of
+the other tubes, B, C, D and E.
+
+8. Set the electric signal clock to ring for the commencement of the
+experiment and at subsequent intervals of 2-1/2, 5, 25 and 30 minutes.
+
+9. Take up 0.5 c.c. of B. coli emulsion in sterile pipette graduated in
+tenths of a cubic centimetre and stand by.
+
+10. As soon as the bell rings lift the pipette from tube A with the left
+hand and from the charged pipette held in the right hand deliver 0.1
+c.c. of B. coli emulsion into the 1:100 solution. Then replace the
+pipette and washer.
+
+[Illustration: FIG. 219.--Test-tube rack.]
+
+11. Raise the tube with the left hand and shake it to mix germ and
+germicide, whilst returning the delivery pipette in the right hand.
+
+12. Repeat the process with tubes B, C, D and E; then drop the infected
+delivery pipette in the lysol jar. The inoculation of the five tubes can
+be carried out very expeditiously, but a period of 10 seconds must be
+allowed for each tube.
+
+13. When the bell rings at 2-1/2 minutes blow through the pipette in
+tube A (this agitates the germ + germicide mixture and ensures the
+collection of a fair sample); allow the mixture to enter the pipette,
+and as the column of fluid extends well above the terminal graduation,
+the right forefinger adjusted over the butt-end of the pipette before it
+is lifted will retain more than 0.1 c.c. of the mixture within the bore
+when the point of the pipette is clear of the fluid in the tube. Touch
+the point of the pipette on the inner wall of the tube, and allow any
+excess of fluid to escape, only retaining 0.1 c.c. in the pipette.
+
+14. At the same time, with the left hand remove Bile Salt Tube No. 1
+from the upper tier of the rack, take out the cotton-wool plug with the
+hand already holding the pipette (the relative positions of pipette,
+plug and culture tubes being practically the same as those of platinum
+loop, plug and culture tube shown in Fig. 68, page 74).
+
+15. Insert the point of the pipette into the subculture tube, and blow
+out the mixture into the medium--replug the tube and drop it into the
+wire basket. Replace the washer-pipette in tube A.
+
+As soon as the point of the pipette has entered the mouth of tube A it
+may be released, since it has already been so adjusted that it just
+clears the bottom of the test-tube, and the elastic washer will prevent
+any damage to the tube.
+
+Steps 13, 14 and 15 occupy on an average 10 seconds.
+
+16. Repeat steps 13, 14 and 15 with each of the other tubes B, C, D and
+E.
+
+17. Repeat these various steps 13-16 when the bell rings at 5, 25 and 30
+minutes.
+
+18. Place all the inoculated tubes in the incubator at 37 deg. C.
+
+19. Examine the tubes at intervals of 24 hours, and record the results
+in tabular form as shown in Table page 491 (the figures in the squares
+indicate the number of hours at which the changes in the medium due to
+the growth of B. coli first appeared).
+
+20. If a consideration of the tabulated results indicates strengths of
+Germicide-x lethal at 2-1/2 and 30 minutes the final test can be
+arranged, but if this result has not been attained, sufficient evidence
+will probably be available to enable a second trial test to be planned
+which will give the required information.
+
+
+~Final Test.~--
+
+c. _Determination of Phenol Coefficient._--
+
+_X-Disinfectant._--This comprises two distinct tests, one of the
+Germicide-x, the other of the standard phenol.
+
+1. Arrange five test-tubes clearly marked in the lower tier of the rack.
+
+2. Pipette into each 5 c.c. respectively of the five percentage
+solutions of x-disinfectant which the trial run has already shown will
+include those affording lethal values at 2-1/2 and 30 minutes.
+
+3. Arrange 20 tubes of bile salt broth in the upper tier of the
+test-tube rack in two rows, those in the front row numbered
+consecutively from left to right 1-10, those in the back row 11-20.
+
+4. Arrange further 20 tubes of bile salt broth numbered 21-40 in two
+rows in a second smaller rack which can be stood on the upper tier of
+the rack as soon as the first 20 tubes have been inoculated.
+
+5. Place a square wire basket of about 50 tube capacity close to the
+left of the test-tube rack, for the reception of the inoculated tubes.
+
+6. Adjust a sterile 1 c.c. pipette in the mouth of each of the tubes, A,
+B, C, D and E, by means of a washer, as previously described.
+
+7. Set the electric signal clock to ring for the commencement of the
+experiment and subsequently at 2-1/2, 5, 10, 15, 20, 25, 30 and 35
+minutes.
+
+8. Complete precisely as indicated in Trial Runs, steps 9-19.
+
+_Control Phenol._--
+
+Immediately the subculture tube from the 30-minute contact period have
+been inoculated, carry out a precisely similar experiment, in which
+five percentage strengths of Phenol, (e. g., 1.1, 1.0, 0.9, 0.75, 0.7)
+are arranged in the lower tier of the test-tube rack in place of the
+five strengths of Germicide-x.
+
+Calculate the phenol coefficient by the following method:
+
+(a) Divide the figure representing the percentage strength of the
+weakest lethal dilution of the carbolic acid control at the 2-1/2-minute
+contact period by the figure representing the percentage strength of the
+weakest lethal dilution of the x-disinfectant at the same period. The
+quotient = phenol coefficient at 2-1/2 minutes.
+
+(b) Similarly obtain the phenol coefficient at 30 minutes contact
+period.
+
+(c) Record the mean of the two coefficients obtained in (a) and (b) as
+the _mean phenol coefficient_, or simply as the ~Phenol Coefficient~.
+
+The details of the Final Test of an actual determination are set out in
+the accompanying table.
+
+
+TABLE 27
+
+Organism employed, B. Coli Communis.
+
+Culture Medium, Nutrient Agar (+10). Age, 24 hrs.
+Temp. of Incubation, 37 deg. C.
+
+Quantities used { Culture  } Emulsion 0.1 c.c. + 5 c.c. Germicide.
+                { Emulsion }
+
+Room Temperature during Experiments, 17 deg. C.
+
+  Germicide  Strength          Time of exposure             Incubation
+                     2-1/2  5  10  15  20  25  30  35    Time     Temp.
+1 Germicide-x  4%      --  --  --  --  --  --  --  --   7 days.  37 deg. C.
+2 Germicide-x  3%      48  --  --  --  --  --  --  --   7 days.  37 deg. C.
+3 Germicide-x  2%      24  24  24  24  48  72           7 days.  37 deg. C.
+4 Germicide-x  1%      24  24  24  24  72  24  72       7 days.  37 deg. C.
+5 Germicide-x  0.5%    24  24  24  24  24  24  24  24  24 hours. 37 deg. C.
+
+1 Phenol       1.10%   --  --  --  --  --  --  --  --   7 days.  37 deg. C.
+2 Phenol       1.00%   24                               7 days.  37 deg. C.
+3 Phenol       0.75%   24  24  24  24  48               7 days.  37 deg. C.
+4 Phenol       0.70%   24  24  24  24  24  72           7 days.  37 deg. C.
+5 Phenol       0.65%   24  24  24  24  24  48  24  24   2 days.  37 deg. C.
+
+
+                    ((1.10/4.00) + (0.7/2.0))   0.27 + 0.35   .62
+Phenol Coefficient = ------------------------ = ----------- = --- = 0.31
+                                2                    2         2
+
+
+
+
+APPENDIX.
+
+
+METRIC AND IMPERIAL SYSTEMS OF WEIGHTS AND MEASURES.
+
+The initial unit of the metric system is the Metre (_m._) or unit of
+length, representing one-fourth-millionth part of the circumference of
+the earth round the poles.
+
+The unit of mass is the Gramme (_g._), and represents the weight of one
+cubic centimetre of water at its maximum density (viz. 4 deg. C. and
+760 mm. mercury pressure).
+
+The unit of the measure of capacity is the Litre (_l._), and represents
+the volume of a kilogramme of distilled water at its maximum density.
+
+The decimal subdivisions of each of the units are designated by the
+Latin prefixes _milli_ = 1/1000; _centi_ = 1/100; _deci_ = 1/10; the
+multiples of each unit by the Greek prefixes _deka_ = 10; _hecto_ = 100;
+_kilo_ = 1000; _myria_ = 10,000.
+
+For a comparison of the values of some of the more frequently employed
+expressions of the Metric System and the Imperial System, the following
+may be found convenient for reference:
+
+     ~Length:~
+
+     1 millimetre (= 1 mm.) = 1/25 of an inch.
+
+     1 centimetre (= 1 cm.) = 2/5 of an inch.
+
+     1 inch (1") = 25 millimetres or 2-1/2 centimetres.
+
+
+     ~Mass:~
+
+     1 milligramme (= 1 mg.) = 0.01543 grain (or approximately
+     1/64 grain).
+
+     1 gramme (= 1 g.) = 15.4323 grains.
+
+     1 "kilo" or kilogramme (= 1 kgm.) = 2 pounds, 3-1/4 ounces
+     avoirdupois.
+
+     1 pound avoirdupois (= 1 lb.) = 453.592 grammes.
+
+     1 ounce avoirdupois (= 1 oz.) = 28.35 grammes.
+
+     1 grain = 0.0648 gramme or 64.8 milligrammes.
+
+
+     ~Capacity:~
+
+     1 cubic centimetre (= 1 c.c.) = 16.9 minims imperial
+     measure.
+
+     1 litre (= 1 _l._) = 35.196 fluid ounces imperial measure.
+
+     1 fluid ounce imperial measure (= 1 [Symbol: ounce]) =
+     28.42 cubic centimetres.
+
+     1 pint imperial measure (= 1 O.) = 568.34 cubic centimetres.
+
+     1 gallon imperial measure (= 1 C.) = 4.546 litres, or 10
+     pounds avoirdupois, of pure water at 62 deg. F. and under an
+     atmospheric pressure of 30 inches of mercury.
+
+
+FACTORS FOR CONVERTING FROM ONE SYSTEM TO THE OTHER.
+
+    To convert grammes into grains                            x 15.432.
+    To convert grammes into ounces avoirdupois                x 0.03527.
+    To convert kilogrammes into pounds                        x 2.2046.
+    To convert cubic centimetres into fluid ounces imperial   x 0.0352.
+    To convert litres into fluid ounces imperial              x 35.2.
+    To convert metres into inches                             x 39.37.
+    To convert grains into grammes                            x 0.0648.
+    To convert avoirdupois ounces into grammes                x 28.35.
+    To convert troy ounces into grammes                       x 31.104.
+    To convert fluid ounces into cubic centimetres            x 28.42.
+    To convert pints into litres                              x 0.568.
+    To convert inches into metres                             x 0.0254.
+
+
+TABLE FOR THE CONVERSION OF DEGREES CENTIGRADE INTO DEGREES FAHRENHEIT.
+
+
+_X. deg. C. = ((9x/5) + 32) deg. F._
+
+| Cent. | Faht.  || Cent. | Faht.  || Cent. | Faht.  |
+|   0   |  32.0  ||  34   |  93.2  ||  68   | 154.4  |
+|   1   |  33.8  ||  35   |  95.0  ||  69   | 156.2  |
+|   2   |  35.6  ||  36   |  96.8  ||  70   | 158.0  |
+|   3   |  37.4  ||  37   |  98.6  ||  71   | 159.8  |
+|   4   |  39.2  ||  38   | 100.4  ||  72   | 161.6  |
+|   5   |  41.0  ||  39   | 102.2  ||  73   | 163.4  |
+|   6   |  42.8  ||  40   | 104.0  ||  74   | 165.2  |
+|   7   |  44.6  ||  41   | 105.8  ||  75   | 167.0  |
+|   8   |  46.4  ||  42   | 107.6  ||  76   | 168.8  |
+|   9   |  48.2  ||  43   | 109.4  ||  77   | 170.6  |
+|  10   |  50.0  ||  44   | 111.2  ||  78   | 172.4  |
+|  11   |  51.8  ||  45   | 113.0  ||  79   | 174.2  |
+|  12   |  53.6  ||  46   | 114.8  ||  80   | 176.0  |
+|  13   |  55.4  ||  47   | 116.6  ||  81   | 177.8  |
+|  14   |  57.2  ||  48   | 118.4  ||  82   | 179.6  |
+|  15   |  59.0  ||  49   | 120.2  ||  83   | 181.4  |
+|  16   |  60.8  ||  50   | 122.0  ||  84   | 183.2  |
+|  17   |  62.6  ||  51   | 123.8  ||  85   | 185.0  |
+|  18   |  64.4  ||  52   | 125.6  ||  86   | 186.8  |
+|  19   |  66.2  ||  53   | 127.4  ||  87   | 188.6  |
+|  20   |  68.0  ||  54   | 129.2  ||  88   | 190.4  |
+|  21   |  69.8  ||  55   | 131.0  ||  89   | 192.2  |
+|  22   |  71.6  ||  56   | 132.8  ||  90   | 194.0  |
+|  23   |  73.4  ||  57   | 134.6  ||  91   | 195.8  |
+|  24   |  75.2  ||  58   | 136.4  ||  92   | 197.6  |
+|  25   |  77.0  ||  59   | 138.2  ||  93   | 199.4  |
+|  26   |  78.8  ||  60   | 140.0  ||  94   | 201.2  |
+|  27   |  80.6  ||  61   | 141.8  ||  95   | 203.0  |
+|  28   |  82.4  ||  62   | 143.6  ||  96   | 204.8  |
+|  29   |  84.2  ||  63   | 145.4  ||  97   | 206.6  |
+|  30   |  86.0  ||  64   | 147.2  ||  98   | 208.4  |
+|  31   |  87.8  ||  65   | 149.0  ||  99   | 210.2  |
+|  32   |  89.6  ||  66   | 150.8  ||  100  | 212.0  |
+|  33   |  91.4  ||  67   | 152.6  ||       |        |
+
+
+TABLE FOR THE CONVERSION OF DEGREES FAHRENHEIT INTO DEGREES CENTIGRADE.
+
+
+_X deg. F. = (5(x - 32))/9 deg. C._
+
+ Faht.| Cent.|| Faht.| Cent.|| Faht.|Cent. || Faht.| Cent.|| Faht.| Cent.
+  32  |    0.||  68  | 20.0 ||  104 | 40.0 ||  140 | 60.0 ||  176 | 80.0
+  33  |  0.6 ||  69  | 20.6 ||  105 | 40.6 ||  141 | 60.6 ||  177 | 80.6
+  34  |  1.1 ||  70  | 21.1 ||  106 | 41.1 ||  142 | 61.1 ||  178 | 81.1
+  35  |  1.7 ||  71  | 21.7 ||  107 | 41.7 ||  143 | 61.7 ||  179 | 81.7
+  36  |  2.2 ||  72  | 22.2 ||  108 | 42.2 ||  144 | 62.2 ||  180 | 82.2
+  37  |  2.8 ||  73  | 22.8 ||  109 | 42.8 ||  145 | 62.8 ||  181 | 82.8
+  38  |  3.3 ||  74  | 23.3 ||  110 | 43.3 ||  146 | 63.3 ||  182 | 83.3
+  39  |  3.9 ||  75  | 23.9 ||  111 | 43.9 ||  147 | 63.9 ||  183 | 83.9
+  40  |  4.4 ||  76  | 24.4 ||  112 | 44.4 ||  148 | 64.4 ||  184 | 84.4
+  41  |  5.0 ||  77  | 25.0 ||  113 | 45.0 ||  149 | 65.0 ||  185 | 85.0
+  42  |  5.6 ||  78  | 25.6 ||  114 | 45.6 ||  150 | 65.6 ||  186 | 85.6
+  43  |  6.1 ||  79  | 26.1 ||  115 | 46.1 ||  151 | 66.1 ||  187 | 86.1
+  44  |  6.7 ||  80  | 26.7 ||  116 | 46.7 ||  152 | 66.7 ||  188 | 86.7
+  45  |  7.2 ||  81  | 27.2 ||  117 | 47.2 ||  153 | 67.2 ||  189 | 87.2
+  46  |  7.8 ||  82  | 27.8 ||  118 | 47.8 ||  154 | 67.8 ||  190 | 87.8
+  47  |  8.3 ||  83  | 28.3 ||  119 | 48.3 ||  155 | 68.3 ||  191 | 88.3
+  48  |  8.9 ||  84  | 28.9 ||  120 | 48.9 ||  156 | 68.9 ||  192 | 88.9
+  49  |  9.4 ||  85  | 29.4 ||  121 | 49.4 ||  157 | 69.4 ||  193 | 89.4
+  50  | 10.0 ||  86  | 30.0 ||  122 | 50.0 ||  158 | 70.0 ||  194 | 90.0
+  51  | 10.6 ||  87  | 30.6 ||  123 | 50.6 ||  159 | 70.6 ||  195 | 90.6
+  52  | 11.1 ||  88  | 31.1 ||  124 | 51.1 ||  160 | 71.1 ||  196 | 91.1
+  53  | 11.7 ||  89  | 31.7 ||  125 | 51.7 ||  161 | 71.7 ||  197 | 91.7
+  54  | 12.2 ||  90  | 32.2 ||  126 | 52.2 ||  162 | 72.2 ||  198 | 92.2
+  55  | 12.8 ||  91  | 32.8 ||  127 | 52.8 ||  163 | 72.8 ||  199 | 92.8
+  56  | 13.3 ||  92  | 33.3 ||  128 | 53.3 ||  164 | 73.3 ||  200 | 93.3
+  57  | 13.9 ||  93  | 33.9 ||  129 | 53.9 ||  165 | 73.9 ||  201 | 93.9
+  58  | 14.4 ||  94  | 34.4 ||  130 | 54.4 ||  166 | 74.4 ||  202 | 94.4
+  59  | 15.0 ||  95  | 35.0 ||  131 | 55.0 ||  167 | 75.0 ||  203 | 95.0
+  60  | 15.6 ||  96  | 35.6 ||  132 | 55.6 ||  168 | 75.6 ||  204 | 95.6
+  61  | 16.1 ||  97  | 36.1 ||  133 | 56.1 ||  169 | 76.1 ||  205 | 96.1
+  62  | 16.7 ||  98  | 36.7 ||  134 | 56.7 ||  170 | 76.7 ||  206 | 96.7
+  63  | 17.2 ||  99  | 37.2 ||  135 | 57.2 ||  171 | 77.2 ||  207 | 97.2
+  64  | 17.8 ||  100 | 37.8 ||  136 | 57.8 ||  172 | 77.8 ||  208 | 97.8
+  65  | 18.3 ||  101 | 38.3 ||  137 | 58.3 ||  173 | 78.3 ||  209 | 98.3
+  66  | 18.9 ||  102 | 38.9 ||  138 | 58.9 ||  174 | 78.9 ||  210 | 98.9
+  67  | 19.4 ||  103 | 39.4 ||  139 | 59.4 ||  175 | 79.4 ||  211 | 99.4
+      |      ||      |      ||      |      ||      |      ||  212 |100.0
+
+~Percentage Formula~ for addition of salts, etc., to completed media.
+
+~Formula for preparing any desired percentage~ of a given salt, etc., in
+tubed media; e. g., to make 4 per cent. solution of KNO_{3} in a
+series of tubes of broth each containing 10 c.c. of medium, when there
+is already available a 25 per cent. stock aqueous solution of potassium
+nitrate.
+
+    (_N_ + ~X~) _Y_    _A_ (~X~)
+    --------------- = ----------
+          100            100
+
+_N_ = number of cubic centimetres contained in each tube.
+
+~X~ = amount of stock solution to be added to each tube.
+
+_Y_ = percentage required in the medium.
+
+_A_ = percentage of stock solution.
+
+Then
+
+    (10 + ~X~) 4   25 ~X~
+    ------------ = ------
+         100        100
+
+    Therefore, 40 + 4~X~ = 25~X~.
+
+    Therefore,     21~X~ = 40.
+
+                     ~X~ = 1.9 c.c.
+
+This allows for solution added to the original bulk of medium.
+
+Therefore, 10 c.c. broth + 1.9 c.c. of a 25 per cent. aqueous solution
+KNO_{3} makes 11.9 c.c. medium containing 4 per cent. KNO_{3}.
+
+
+~TABLES FOR PREPARING DILUTIONS~
+
+(of Serum, Disinfectants or other substances.)
+
+In estimating the agglutinin content or _titre_ of a serum, testing
+disinfectants and for many other purposes, it becomes necessary to
+prepare a series of dilutions of the material under examination, and in
+order to avoid unnecessary expenditure of labour it is convenient to
+adhere to some definite scale of increment, such for example as the
+following:
+
+From dilutions of 1:10 to 1:80 rise by increments of 5.
+
+From dilutions of 1:80 to 1:200 rise by increments of 10.
+
+From dilutions of 1:200 to 1:400 rise by increments of 25.
+
+From dilutions of 1:400 to 1:500 rise by increments of 50.
+
+From dilutions of 1:500 to 1:1000 rise by increments of 100.
+
+From dilutions of 1: 1000 to 1:5000 rise by increments of 250.
+
+From dilutions of 1: 5000 to 1:10,000 rise by increments of 1000.
+
+From dilutions of 1:10,000 to 1:100,000 rise by increments of 5000.
+
+From dilutions of 1:100,000 to 1:1,000,000 rise by increments of 100,000.
+
+When dealing with a substance of unknown powers--and this is especially
+true with regard to agglutinating sera--it is customary to run a
+preliminary test, using a few widely separated dilutions such as may be
+obtained in the following manner:
+
+FIRST DILUTION--I.
+
+1 c.c. serum + 9 c.c. normal saline solution = 10 per cent. solution or
+1: 10 dilution (of which 1 c.c. contains 0.1 c.c. of the original
+serum).
+
+When dealing with fluids other than serum the diluent is usually
+distilled water; whilst if the original substance is a solid the
+instructions would read:
+
+1 gram o.s. + 10 c.c. distilled water = 10 per cent. solution, etc.
+
+SECOND DILUTION--II.
+
+1 c.c. first dilution + 9 c.c. normal saline solution = 1 per cent.
+solution or 1: 100 dilution.
+
+THIRD DILUTION--III.
+
+1 c.c. second dilution + 9 c.c. normal saline solution = 1 per mille
+solution or 1: 1000 dilution.
+
+FOURTH DILUTION--IV.
+
+1 c.c. second dilution + 9 c.c. normal saline solution = 0.1 per mille
+solution or 1: 10,000 dilution.
+
+The following tables showing the secondary dilutions that can readily be
+prepared from each of these four primary dilutions for use in the
+subsequent determination of the exact _titre_ will probably be found of
+service by those who are not ready mathematicians.
+
+
+TABLES FOR PREPARING DILUTIONS.
+
+-----------------------------------+----------------------------------
+                                   |
+            TABLE I                |          TABLE II
+    Using 10 % stock solution      |    Using 1% stock solution
+    First       }                  |      Second      }
+    dilution    } + Diluent        |      dilution    } + Diluent
+                                   |
+-----------------------------------+----------------------------------
+                                   |
+     1:  10 = 1 c.c. +  0 c.c.     |    1: 100  = 1 c.c. + 0 c.c.
+     1:  15 = 1 c.c. +  0.5 c.c.   |    1: 110  = 1 c.c. + 0.1 c.c.
+     1:  20 = 1 c.c. +  1.0 c.c.   |    1: 120  = 1 c.c.  + 0.2 c.c.
+     1:  25 = 1 c.c. +  1.5 c.c.   |   [1: 125  = 1 c.c. + 0.25 c.c.]
+     1:  30 = 1 c.c. +  2.0 c.c.   |    1: 130  = 1 c.c. + 0.3 c.c.
+     1:  35 = 1 c.c. +  2.5 c.c.   |    1: 140  = 1 c.c. + 0.4 c.c.
+     1:  40 = 1 c.c. +  3.0 c.c.   |    1: 150  = 1 c.c. + 0.5 c.c.
+     1:  45 = 1 c.c. +  3.5 c.c.   |    1: 160  = 1 c.c. + 0.6 c.c.
+     1:  50 = 1 c.c. +  4.0 c.c.   |    1: 170  = 1 c.c. + 0.7 c.c.
+     1:  55 = 1 c.c. +  4.5 c.c.   |   [1: 175  = 1 c.c. + 0.75 c.c.]
+     1:  60 = 1 c.c. +  5.0 c.c.   |    1: 180  = 1 c.c. + 0.8 c.c.
+     1:  65 = 1 c.c. +  5.5 c.c.   |    1: 190  = 1 c.c. + 0.9 c.c.
+     1:  70 = 1 c.c. +  6.0 c.c.   |    1: 200  = 1 c.c. + 1.0 c.c.
+     1:  75 = 1 c.c. +  6.5 c.c.   +---------------------------------
+     1:  80 = 1 c.c. +  7.0 c.c.   |    1: 200  = 1 c.c. + 1.0 c.c.
+     ------------------------------+    1: 225  = 1 c.c. + 1.25 c.c.
+     1:  80 = 1 c.c. +  7.0 c.c.   |    1: 250  = 1 c.c. + 1.5 c.c.
+     1:  90 = 1 c.c. +  8.0 c.c.   |    1: 275  = 1 c.c. + 1.75 c.c.
+     1: 100 = 1 c.c. +  9.00 c.c.  |    1: 300  = 1 c.c. + 2.0 c.c.
+     1: 110 = 1 c.c. + 10.0 c.c.   |    1: 325  = 1 c.c. + 2.25 c.c.
+     1: 120 = 1 c.c. + 11.0 c.c.   |    1: 350  = 1 c.c. + 2.5 c.c.
+    [1: 125 = 1 c.c. + 11.5 c.c.]  |    1: 375  = 1 c.c. + 2.75 c.c.
+     1: 130 = 1 c.c. + 12.0 c.c.   |    1: 400  = 1 c.c. + 3.0 c.c.
+     1: 140 = 1 c.c. + 13.0 c.c.   +---------------------------------
+     1: 150 = 1 c.c. + 14.0 c.c.   |    1: 400  = 1 c.c. + 3.0 c.c.
+     1: 160 = 1 c.c. + 15.0 c.c.   |    1: 450  = 1 c.c. + 3.5 c.c.
+     1: 170 = 1 c.c. + 16.0 c.c.   |    1: 500  = 1 c.c. + 4.0 c.c.
+    [1: 175 = 1 c.c. +-16.5 c.c.]  +---------------------------------
+     1: 180 = 1 c.c. + 17.0 c.c.   |    1: 500  = 1 c.c. + 4.0 c.c.
+     1: 190 = 1 c.c. + 18.0 c.c.   |    1: 600  = 1 c.c. + 5.0 c.c.
+     1: 200 = 1 c.c. + 19.0 c.c.   |    1: 700  = 1 c.c. + 6.0 c.c.
+     ----------------- ------------+   [1: 750  = 1 c.c. + 6.5 c.c.]
+     1: 200 = 1 c.c. + 19.0 c.c.   |    1: 800  = 1 c.c. + 7.0 c.c.
+     1: 225 = 1 c.c. + 21.5 c.c.   |    1: 900  = 1 c.c. + 8.0 c.c.
+     1: 250 = 1 c.c. + 24.0 c.c.   |    1: 1000 = 1 c.c. + 9.0 c.c.
+     1: 275 = 1 c.c. + 26.5 c.c.   +--------------------------------
+     1: 300 = 1 c.c. + 29.0 c.c.   |    1: 1000 = 1 c.c. + 9.0 c.c.
+     1: 325 = 1 c.c. +-31.5 c.c.   |    1: 2000 = 1 c.c. + 19.0 c.c.
+     1: 350 = 1 c.c. + 34.0 c.c.   |    1: 3000 = 1 c.c. + 29.0 c.c.
+     1: 375 = 1 c.c. + 36.5 c.c.   |    1: 4000 = 1 c.c. + 39.0 c.c.
+     1: 400 = 1 c.c. + 39.0 c.c.   |    1: 5000 = 1 c.c. + 49.0 c.c.
+     ------------------------------+--------------------------------
+     1: 400 = 1 c.c. + 39.0 c.c.   |
+     1: 450 = 1 c.c. + 44.5 c.c.   |
+     1: 500 = 1 c.c. + 49.0 c.c.   |
+
+   ---------------------------------+-------------------------------
+                                    |
+       TABLE III                    |         TABLE IV
+       Using 0.1% stock solution    |    Using 0.01% stock solution
+       Third    }                   |       Fourth   }
+       dilution } + Diluent         |       Dilution } + Diluent
+                                    |
+   ---------------------------------+-------------------------------
+                                    |
+    1:   1000 = 1 c.c. +  0 c.c.    |  1:   10,000 = 1 c.c. + 0 c.c.
+    1:   1250 = 1 c.c. +  0.25 c.c. |  1:   15,000 = 1 c.c. + 0.5 c.c.
+    1:   1500 = 1 c.c. +  0.5 c.c.  |  1:   20,000 = 1 c.c. + 1.0 c.c.
+    1:   1750 = 1 c.c. +  0.75 c.c. |  1:   25,000 = 1 c.c. + 1.5 c.c.
+    1:   2000 = 1 c.c. +  1.0 c.c.  |  1:   30,000 = 1 c.c. + 2.0 c.c.
+    1:   2250 = 1 c.c. +  1.25 c.c. |  1:   35,000 = 1 c.c. + 2.5 c.c.
+    1:   2500 = 1 c.c. +  1.5 c.c.  |  1:   40,000 = 1 c.c. + 3.0 c.c.
+    1:   2750 = 1 c.c. +  1.75 c.c. |  1:   45,000 = 1 c.c. + 3.5 c.c.
+    1:   3000 = 1 c.c. +  2.0 c.c.  |  1:   50,000 = 1 c.c. + 4.0 c.c.
+    1:   3250 = 1 c.c. +  2.25 c.c. |  1:   55,000 = 1 c.c. + 4.5 c.c.
+    1:   3500 = 1 c.c. +  2.5 c.c.  |  1:   60,000 = 1 c.c. + 5.0 c.c.
+    1:   3750 = 1 c.c. +  2.75 c.c. |  1:   65,000 = 1 c.c. + 5.5 c.c.
+    1:   4000 = 1 c.c. +  3.0 c.c.  |  1:   70,000 = 1 c.c. + 6.0 c.c.
+    1:   4250 = 1 c.c. +  3.25 c.c. |  1:   75,000 = 1 c.c. + 6.5 c.c.
+    1:   4500 = 1 c.c. +  3.5 c.c.  |  1:   80,000 = 1 c.c. + 7.0 c.c.
+    1:   4750 = 1 c.c. +  3.75 c.c. |  1:   85,000 = 1 c.c. + 7.5 c.c.
+    1:   5000 = 1 c.c. +  4.0 c.c.  |  1:   90,000 = 1 c.c. + 8.0 c.c.
+    --------------------------------+  1:   95,000 = 1 c.c. + 8.5 c.c.
+    1:   5000 = 1 c.c. +  4.0 c.c.  |  1:  100,000 = 1 c.c. + 9.0 c.c.
+    1:   6000 = 1 c.c. +  5.0 c.c.  +-----------------------------------
+    1:   7000 = 1 c.c. +  6.0 c.c.  |  1:  100,000 = 0.1 c.c. + 0.9 c.c.
+   [1:   7500 = 1 c.c. +  6.5 c.c.] |  1:  200,000 = 0.1 c.c. + 1.9 c.c.
+    1:   8000 = 1 c.c. +  7.0 c.c.  | [1:  250,000 = 0.1 c.c. + 2.4 c.c.]
+    1:   9000 = 1 c.c. +  8.0 c.c.  |  1:  300,000 = 0.1 c.c. + 2.9 c.c.
+    1: 10,000 = 1 c.c. +  9.0 c.c.  |  1:  400,000 = 0.1 c.c. + 3.9 c.c.
+    ------------------------------- +  1:  500,000 = 0.1 c.c. + 4.9 c.c.
+    1: 10,000 = 1 c.c. +  9.0 c.c.  +-----------------------------------
+    1: 15,000 = 1 c.c. + 14.0 c.c.  |  1:  500,000 = 0.1 c.c. + 4.9 c.c.
+    1: 20,000 = 1 c.c. + 19.0 c.c.  |  1:  600,000 = 0.1 c.c. + 5.9 c.c.
+    1: 25,000 = 1 c.c. + 24.0 c.c.  |  1:  700,000 = 0.1 c.c. + 6.9 c.c.
+    1: 30,000 = 1 c.c. + 29.0 c.c.  | [1:  750,000 = 0.1 c.c. + 7.4 c.c.]
+    --------------------------------+  1:  800,000 = 0.1 c.c. + 7.9 c.c.
+                                    |  1:  900,000 = 0.1 c.c. + 8.9 c.c.
+                                    |  1:1,000,000 = 0.1 c.c. + 9.9 c.c.
+                                   -+-------------------------------------
+
+
+TEMPERATURE PRESSURE TABLE.
+
+ ---------------+--------------+---------------------+-------------
+    Temperature |              |  Pounds per sq. in. |
+    Centigrade  |  Mm. of Hg.  |  absolute pressure  | Atmospheres
+                |              |                     |
+ ---------------+--------------+---------------------+-------------
+                |              |                     |
+     98 deg.    |    707.1     |         13.7        |     0.93
+     99 deg.    |    733.1     |         14.2        |     0.96
+    100 deg.    |    760.0     |         14.7        |     1.00
+                |              |                     |
+    101 deg.    |    787.8     |         15.2        |     1.03
+    102 deg.    |    816.0     |         15.8        |     1.07
+    103 deg.    |    845.2     |         16.3        |     1.11
+    104 deg.    |    875.4     |         16.9        |     1.15
+    105 deg.    |    906.4     |         17.5        |     1.19
+                |              |                     |
+    106 deg.    |    938.3     |         18.1        |     1.23
+    107 deg.    |    971.1     |         18.8        |     1.27
+    108 deg.    |   1004.9     |         19.4        |     1.32
+    109 deg.    |   1039.6     |         20.1        |     1.36
+    110 deg.    |   1075.3     |         20.8        |     1.41
+                |              |                     |
+    111 deg.    |   1112.0     |         21.5        |     1.46
+    112 deg.    |   1149.8     |         22.2        |     1.51
+    113 deg.    |   1188.6     |         22.9        |     1.56
+    114 deg.    |   1228.4     |         23.7        |     1.61
+    115 deg.    |   1269.4     |         24.5        |     1.67
+                |              |                     |
+    116 deg.    |   1311.4     |         25.3        |     1.72
+    117 deg.    |   1354.6     |         26.2        |     1.78
+    118 deg.    |   1399.0     |         27.0        |     1.84
+    119 deg.    |   1444.5     |         27.9        |     1.90
+    120 deg.    |   1491.2     |         28.8        |     1.96
+                |              |                     |
+    121 deg.    |   1539.2     |         29.7        |     2.02
+    122 deg.    |   1588.4     |         30.7        |     2.09
+    123 deg.    |   1638.9     |         31.7        |     2.15
+    124 deg.    |   1690.7     |         32.7        |     2.22
+    125 deg.    |   1743.8     |         33.7        |     2.29
+ ---------------+--------------+---------------------+-------------
+
+
+TABLE FOR DESICCATION AT LOW TEMPERATURES IN VACUO.
+
++--------------------------+
+| Temperature |            |
+| Centigrade  | Mm. of Hg. |
++-------------+------------+
+| 21 deg.     | 18.4       |
+| 22 deg.     | 19.6       |
+| 23 deg.     | 20.8       |
+| 24 deg.     | 22.1       |
+| 25 deg.     | 23.5       |
+|             |            |
+| 26 deg.     | 24.9       |
+| 27 deg.     | 26.4       |
+| 28 deg.     | 28.0       |
+| 29 deg.     | 29.7       |
+| 30 deg.     | 31.5       |
+|             |            |
+| 31 deg.     | 33.3       |
+| 32 deg.     | 35.3       |
+| 33 deg.     | 37.3       |
+| 34 deg.     | 39.5       |
+| 35 deg.     | 41.7       |
+|             |            |
+| 36 deg.     | 44.1       |
+| 37 deg.     | 46.6       |
+| 38 deg.     | 49.2       |
+| 39 deg.     | 51.9       |
+| 40 deg.     | 54.8       |
+|             |            |
+| 41 deg.     | 57.8       |
+| 42 deg.     | 61.0       |
+| 43 deg.     | 64.3       |
+| 44 deg.     | 67.7       |
+| 45 deg.     | 71.3       |
+|             |            |
+| 46 deg.     | 75.1       |
+| 47 deg.     | 79.0       |
+| 48 deg.     | 83.1       |
+| 49 deg.     | 87.4       |
+| 50 deg.     | 91.9       |
++-------------+------------+
+
+
+ANTIFORMIN METHOD
+
+For the detection of B. Tuberculosis.
+
+_Antiformin_ was introduced into bacteriological technique by Uhlenhuth
+in 1908 for the purpose of demonstrating tubercle bacilli when present
+in small numbers, in sputum or other material. It is a powerful
+oxidising agent and rapidly destroys most bacteria, but tubercle and
+other acid-fast organisms resist its lethal action for considerable
+periods, and upon this fact the method is based.
+
+_To prepare Antiformin_ measure out and mix:--
+
+Eau de Javelle (Liquor sodae chlorinatae--B.P.)  50 c.c.
+Sodic hydrate 15 per cent. aqueous solution      50 c.c.
+
+METHOD.
+
+1. Introduce the sputum or other material (e. g. milk deposit and cream;
+pus; minced gland or other organ; caseous material; broken down foci,
+etc.) into a sterile tube and then add an equal volume of antiformin.
+
+2. Close the tube with a rubber cork and shake vigorously (a sample of
+antiformin that does not "foam" at this stage is of little use).
+Disintegration of the material at once starts, associated bacteria are
+destroyed and the mixture rapidly becomes a homogenous but turbid
+fluid--a process which may be hastened by:--
+
+3. Placing the tube in the incubator at 37 deg. C. for 30 minutes--shaking
+from time to time.
+
+4. Centrifugalise the fluid thoroughly, at high speed.
+
+5. Pipette off the supernatant fluid, fill up with sterile distilled
+water, cork the tube and shake to distribute the deposit throughout the
+water. Again centrifugalise.
+
+6. Repeat steps 4 and 5 twice more.
+
+7. Employ one portion of the final deposit to inoculate guinea pigs.
+
+8. Plant the remainder of the deposit freely on Dorset's Egg medium; cap
+and incubate at 37 deg. C.
+
+     NOTE.--If only microscopical films are needed, fill up the
+     centrifuge tube with Ligroin (a petroleum ether) in place of
+     sterile distilled water in step 5 and prepare the films from
+     the _surface_ of the fluid, to stain by the Ziehl-Neelsen
+     process.
+
+
+
+
+INDEX
+
+
+Abbe's condenser, 7
+
+Abbott's stain for spores, 107
+
+Aberration, chromatic, 56
+  spherical, 55
+
+Absolute alcohol as a fixative, 82
+  as an antiseptic, 27
+
+Absorbent paper for drying cover-slips, 69
+
+A. C. E. mixture, 345
+
+Acetic acid for clearing films, 82
+
+Achromatic condenser, 54
+
+Acid haematin, 96
+ production, analysis table, 283
+   by bacteria, 145
+   investigation of, 280
+   qualitative examination, 283, 284
+   quantitative examination, 280
+
+Acid-fast bacilli in tissues, to stain, 124
+
+Action of various gases on bacteria, 295
+
+Active immunisation, illustrative example, 322
+
+Adjustable water bath, 299
+
+Aerobic cultures, 221
+
+Aerogenic bacteria, 131
+
+Aesculin agar, 204
+
+Agar gelatine (guarniari), 194
+  methods of preparation, 167
+  surface plates, 232
+
+Agar-agar, preparation of, 167
+
+Agglutination reaction, macroscopical, 386
+  microscopical, 385
+
+Agglutinin, 381
+
+Air, analysis of, 468
+  filter, 40
+  pump, Geryk, 43
+
+Albumin solution, Mayer's, 120
+
+Alcohol production, test for, 285
+
+Alkaline pyro, 239
+
+Alum carmine, 96
+
+Ammonia production test for, 285
+
+Amphitrichous bacteria, 136
+
+Anaerobic cultures, 236
+  Botkin's method, 243
+  Buchner's method, 238
+  Bulloch's method, 245
+  Hesse's method, 237
+  McLeod's method, 240
+  media, 180
+  Novy's method, 244
+
+Anaerobic cultures, Roux's biological method, 237
+  physical method, 237
+  vacuum method, 238
+  Wright's method, 239
+
+Anaesthetics, 345
+
+Analysis of air, apparatus for, 469
+  method of, 468
+  qualitative bacteriological, 470
+  quantitative bacteriological, 468
+  of butter, qualitative bacteriological, 458
+    quantitative bacteriological, 457
+  of cream, qualitative bacteriological, 458
+    quantitative bacteriological, 457
+  of fish, 460
+  of ice cream, qualitative bacteriological, 457
+  of meat, apparatus for, 460
+    method of, 460
+    qualitative bacteriological, 462
+  of milk, apparatus for, 444
+    collection of samples, 441
+    method of, 441
+    qualitative bacteriological, 446.
+    quantitative bacteriological, 444
+  of oysters, 463
+  of sewage, qualitative bacteriological, 467
+    quantitative bacteriological, 466
+  of shellfish, 463
+  of soil, apparatus for, 473
+    collection of samples, 471
+    method of, 470
+    qualitative bacteriological, 476
+    quantitative bacteriological, 473
+  of water, apparatus for, 420, 427
+    collection of samples, 416
+    method of, 416
+    qualitative bacteriological, 426
+
+Analysis of water, quantitative bacteriological, 420
+
+Aniline dyes, 83
+  Gentian violet, 95
+  water, to prepare, 108
+
+Animal tissue media (Frugoni), 210
+
+Animals, natural infections of, 337
+
+Antiformin method for B. tuberculosis, 502
+
+Antigen, definition of, 324
+
+Antiseptics, 27
+  action of, 310
+
+Apparent filth in milk, 450
+
+Arnold's steam steriliser, 34
+
+Arthrogenous spores, 138
+
+Ascitic bouillon, 210
+  fluid agar (Wassermann), 213
+
+Ascomycetae, 128
+
+Ascopores, 129
+
+Asparagin Media (Frankel and Voges), 183
+  (Uschinsky), 183
+
+Aspergillus, 127
+
+Atmospheric conditions, 295
+
+Attenuating the virulence of organisms, 321
+
+Autoclave, 37
+  to use, 37
+
+Automatic pipettes, 13
+
+Autopsies, 396
+
+Autopsy, card index for, 402
+
+
+Bacilli, morphology of, 132
+
+Bacillus anthracis in soil, 477
+  in water, 440
+  coli in water, detection of, 429
+  diphtheriae in milk, 452
+  enteritidis in water, 437
+  sporogenes in milk, 452
+    in water, 438
+  oedematis maligni in soil, 477
+  tetani in soil, 477
+    in water, 441
+  tuberculosis in milk, 453
+  antiformin method, 502
+  typhosus in water, 441
+
+Bacteria, anatomy of, 134
+  classification of, 131
+  grouping of, for study, 410
+  in tissues, demonstration of, 114
+  influence of environment on, 142
+  metabolic products of, 143
+  methods of identification, 259
+  microscopical examination of, stained, 81
+    unstained, 74
+  physiology of, 136
+
+Bacteria, simple stains for, 90
+
+Bacterial emulsion, preparation of, 389
+  enzymes, 144, 277
+  ferments, 144
+  food stuffs, 142
+  toxins, 144
+
+Bacteriological analyses, general considerations, 415
+  examination of blood, 377
+
+Base of microscope, 50
+
+Basidium, 128
+
+Beer wort, preparation of, 175
+
+Beetroot media, 200
+
+Beggiotoa, morphology of, 133
+
+Benzole bath, 256
+
+Berkefeld filter, 42
+
+Beyrinck's solution I, 197
+  II, 198
+
+Bile salt agar (MacConkey), 205
+  broth, double strength, 199
+    (MacConkey), 180
+
+Biochemical examination of cultures, 276
+
+Biochemistry of bacteria, 276
+
+Biological differentiation of bacteria, 249
+
+Bipolar germination, 140
+
+Bismarck brown, 94
+
+Blastomycetes, morphology of, 129
+
+Blood agar, 171, 214
+  plates, animal, 251
+    human, 250
+  (Washbourn), 214
+  bacteriological examination of, 377
+  cells, washing of, 388
+  collection of, for serological examination, 379
+  films, preparations of, 376
+    staining of, 97
+  histological examination of, 373
+  pipettes, 11
+  serological examination of, 378
+  stains, 97
+
+Blood-serum (Councilman and Mallory), 208
+  inspissated, 168
+  (Loeffler), 208
+  (Lorrain Smith), 208
+
+Blowpipe table, 9
+
+Body tube of microscope, 50
+
+Bohemian flask, 4
+
+Boiling water, 33
+
+Bone marrow, films, preparation of, 400
+
+Bordet-Gengou reaction, 393
+
+Boric acid in milk, test for, 442
+
+Botkin's anaerobic method, 243
+
+Bouillon, preparation of, 163
+
+Brain extract, 149
+
+Bread paste, 193
+
+Brilliant green agar (Conradi), 206
+      bile salt agar (Fawcus), 206
+
+Brownian movement, 79
+
+Buchner's anaerobic method, 238
+
+Bulloch's anaerobic method, 245
+  tubes for permanent preparations, 407
+
+Bunge's mordant, 104
+
+Burri's Chinese ink stain, 77
+
+Butter, analysis of, 457
+  qualitative analysis of, 458
+  quantitative analysis of, 457
+
+
+Cadaver, preparation of, for autopsy, 397
+
+Cages for guinea-pigs, 343
+  for laboratory animals, 341
+  for mice, 342
+  for rabbits, 343
+  for rats, 342
+
+Calculated figure for weight of
+  medium mass, 166, 167
+
+Cambier's candle method of isolating
+  coli-typhoid groups, 438
+
+Camera lucida, 62
+
+Capaldi-Proskauer medium, No I, 186
+    No II, 187
+
+Capillary pipettes, 10
+    graduated, 13
+
+Capitate bacilli, 139
+
+Capsule formation, 134
+  of bacteria, 134
+  thermo-regulator, 218
+
+Capsules, collodion, inoculation of, 357
+    preparation of, 357
+  glass, 6
+  to clean infected, 20
+    new, 18
+  to stain, 99
+  to sterilise, 31
+
+Carbohydrate media, preparation of, 177
+
+Carbolic acid as a germicide, 27, 481
+  method of isolating coli-typhoid group, 437
+
+Carbolised agar, 202
+  bouillon, 202
+  gelatine, 202
+
+Carbon dioxide in cultures, test for, 289
+
+Card index, 336, 402
+
+Carrot media, 200
+
+Cedarwood oil for immersion lens, 88
+
+Cell wall of bacteria, 134
+
+Celloidin sacs, manufacture of, 358
+
+Cellular incubator, 216
+
+Centrifugal machine for blood and serum work, 327
+    for milk work, 447
+
+Centrifugalised milk, 449
+
+Centrigade degrees, conversion of, 494
+
+Chemical products of bacteria, 145
+
+China green agar (Werbitski), 207
+
+Chloroform as an antiseptic, 27
+
+Chromatic aberration, 56
+
+Chromogenic bacteria, 131
+
+Chromoparous bacteria, 144
+
+Chromophorous bacteria, 144
+
+Citrated blood agar, 191
+
+Cladothrix, morphology, 193
+
+Classification of bacteria, 131
+  of fungi, 126
+
+Clavate bacilli, 139
+
+Clearing films with acetic acid, 82
+
+Clostridium, 139
+
+Coarse adjustment, 51
+
+Cobweb micrometer, 66
+
+Cocaine, 345
+
+Cocci, morphology of, 131
+
+Coccidium infection, 339
+
+Coefficient, inferior lethal, 312
+  of inhibition, 311
+  phenol, 489
+  superior lethal, 313
+
+Cohn's solution, 191
+
+Cold incubator, 217
+
+Coli-typhoid group, differential table, 433
+    in milk, 451
+    in soil, 477
+    isolation of, 432
+    members of, 430
+
+Collection of blood for bacteriological examination, 378
+    for media making, 168
+  of milk samples, 443
+  of pathological material during life, 373
+  of pus, 373
+  of soil sample, 471
+  of water samples, 416
+
+Collodion capsules, 357
+  sacs, manufacture of, 357
+
+Colonies of bacteria, edges, 267
+
+Coloured light, action of, 309
+
+Columella, 127
+
+Comparative haemocytology, 374
+
+Complement, definition of, 325
+  fixation test, 393
+
+Concentration method in water, analysis, 434
+
+Condenser achromatic, 54
+  dark ground, 60
+  paraboloid, 60
+  substage, 54
+
+Condidium, 128
+
+Continuous sterilisation, 36
+
+Contrast stains, 93
+
+Corrosive sublimate (Lang), 82
+
+Cotton-wool filter, 40
+
+Counterstaining films, 84
+
+Counting plate colonies, 423
+
+Cover-slip films, 81
+  to clean new, 22
+    used, 24
+
+Crates for test-tubes, 31
+
+Cream, analysis of, 457
+  qualitative analysis of, 458
+  quantitative analysis of, 457
+
+Crenothrix morphology, 133
+
+Criteria of infection, 370
+
+Criterion of immunity, 324
+
+Cultural characters, macroscopical examination, 261
+
+Culture flask, Guy's, 5
+    Kolle, 4
+    Roux, 5
+
+Cuneate bacilli, 139
+
+Cutaneous inoculation, 352
+
+
+Dark ground condenser, 60
+    illumination, 87
+
+Daughter cells, 129
+
+Daylight, diffuse, action of, 308
+
+Decimal scales, 340
+
+Decolourising agents, 84
+
+Definition of objective, 56
+
+Depilatory powder, 346
+
+Description of plate culture, 261
+
+Descriptive terms, 261
+
+Desiccation, effects of, 306
+  table, 501
+
+Desiccator, Mueller's, 307
+
+Dextrose solution, preparation of, 178
+
+Diaphragm, iris, 53
+
+Diastatic enzymes, tests for, 278
+
+Differential atmosphere cultivation, 257
+  incubation, 255
+  media, 255
+  staining, 108
+  sterilisation, 256
+
+Diluting chamber, 248
+
+Dilution by teat pipette, 383
+  of serum, 382
+  tables, 498
+
+Dilutions, preparations of, 496
+
+Diphtheria, bacillus of, in milk, 452
+
+Diplobacilli, morphology of, 133
+
+Diplococci, morphology of, 133
+
+Diplococcus pneumoniae, immunisation against, 322
+
+Discontinuous sterilisation, 36
+
+Discs of plaster-of-Paris, 192
+
+Disinfectants, action of, 310
+  chemical, 27
+  testing of, 480
+
+Dissociating fluid, Price Jones; 400
+
+Dosage of inoculum, 316
+
+Double nosepiece, 58
+  stains for spores, 106
+  sugar agar (Russell), 207
+
+Drop-bottle, 73
+
+Dry heat, 28
+
+Dunham's solution, 177
+
+Dyes, aniline, 83
+
+
+Earthenware box for dirty slides, 70
+
+Earthy salts agar (Lipman and Brown), 197
+
+Edge of individual colonies, characters of, 267
+
+Egg albumin agar, 213
+    broth, (Lipschuetz), 213
+  media (Dorset), preparation of, 174
+    inspissated, 212
+    (Lubenau), 209
+    (Tarchanoff and Kolesnikoff), 212
+  to clear nutrient media with, 166
+
+Ehrlich's eyepiece, 55
+
+Eikonometer, 65
+
+Eisenberg's milk-rice medium, 189
+
+Electric dental engine, 360
+  signal clock, 38
+  warm stage, 59
+
+Elevation of colonies, 263
+
+Eisner's gelatine, 204
+  method of isolating coli: typhoid group, 438
+
+Endogenous spores, 138
+  varieties of, 139
+
+Endo-germination, 139
+
+English proof agar, Blaxall, 193
+
+Enumerating colonies on plates, 423
+  discs, Jeffer's, 424
+    Pakes', 424
+
+Enrichment method in water analysis, 427
+
+Enumeration of micro-organisms, 423
+
+Environmental conditions, 142
+
+Enzyme production, investigation of, 277
+
+Eosin, 93
+
+Equatorial germination, 140
+
+Erlenmeyer flask, 4
+
+Ernstschen Koerner, 136
+
+Esmarch's roll culture, 226
+  water collecting bottle, 417
+
+Estimation of reaction of media, 280
+
+Ether flame, 28
+  soluble acids, 284
+
+Eucaine, 345
+
+Exalting virulence of organisms, 320
+
+Examination of milk, 441
+
+Experimental infections, study of, during life, 370
+  inoculation of animals, 332
+
+Extracellular toxins, 144
+
+Eyepiece, Ehrlich, 55
+  _micrometer_, 63
+
+Eyepieces, 55
+
+Eye-shade, 57
+
+
+Fahrenheit degrees, conversion of, 495
+
+Feeding experiments, 369
+
+Fermentation reactions, 279
+  tubes, 17
+
+Field of objective, 56
+
+Filar micrometer, 66
+
+Filling tubes, etc., with medium, 160
+
+Film preparations, 81
+  fixing, 81
+  making, 81
+  mounting, 85
+  staining, 83
+
+Filter candle, closed, 47
+    open, 43
+    testing efficiency of, 478
+    to disinfect, 28
+    to sterilise, 29
+  flask, 6
+  papers, to fold, 156
+
+Filters, cotton-wool, 40
+  porcelain, 42
+  testing of, 478
+
+Filtration, 40
+  by aspiration, 42
+  of media, 156
+  under pressure, 45
+
+Fine adjustment, 51
+  spindle head, 52
+
+Fish, analysis of, 460
+  bouillon, 190
+
+Fish gelatine, 190
+  gelatine-agar, 190
+
+Fishing colonies, 253
+
+Fission, reproduction by, 136
+
+Fixation, 81
+  by heat, 81
+  of tissues, 114
+
+Fixing fluids, for films, 82
+
+Flagella, classification of bacilli by, 136
+  to stain, 101
+
+Flask Bohemian, 4
+  Erlenmeyer, 4
+  filter, 6
+  Kitasato'a serum, 6
+  Kolle's culture, 4
+
+Flasks and test tubes, to plug, 24
+  to clean dirty, 20
+    new, 18
+  to sterilise, 31
+
+Fleischwasser, 148
+
+Fluid cultures, description of, 271
+  media, 146
+
+Foot of microscope, 50
+
+Formaldehyde in milk, Hehner's test for, 442
+
+Formalin method of preserving cultures, 407
+  tissues, 404
+
+Fractional sterilisation, 33
+
+Fraenkel and Voge's solution, 183
+
+Fraenkel's earth borer, 472
+
+Freezing method for sections, 115
+
+French Mannite Agar (Sabouraud), 193
+  proof agar (Sabouraud), 193
+
+Fresh preparations of bacteria, 74
+
+Friedlaender's capsule stain for sections, 123
+
+Frost's mounting fluid, 406
+
+Frozen sections, rapid method, 116
+
+Fuchsin, 92
+  agar (Braun), 205
+  sulphite agar (Endo), 206
+
+
+Gas analysis, qualitative, 290
+    quantitative, 290
+  collecting apparatus, 291
+  generators, 242
+  production by bacteria, 289
+  tubes for media, 161
+
+Gasperini's solution, 193
+
+Gelatin agar, 193
+  preparation of, 164
+  surface plates, 231
+
+General anaesthetics, 345
+
+Gentian violet, 91
+
+German lined paper, 69
+
+Germicides, 27
+  testing power of, 480
+
+Germination, 140
+
+Geryk air-pump, 43
+
+Glass apparatus in common use, 3
+  to clean, 18
+
+Glass-cutting knife, 8
+
+Glucose formate agar (Kitasato), 180
+  bouillon (Kitasato), 180
+  gelatine (Kitasato), 180
+
+Glycerinated potato, 209
+
+Glycerine agar, 209
+  blood-serum, 208
+  bouillon, 209
+  potato bouillon, 203
+    broth, 203
+
+Goadby's gelatine, 214
+
+Gonidium, 128
+
+Goniodophore, 128
+
+Graduated capillary pipettes, 13
+  pipettes, 6
+
+Gram-Claudius' differential stain, 109
+
+Gram's differential stain, 108
+
+Gram-Weigert for sections, 121, 122
+
+Gram-Weigert's differential stain, 109
+  modified, 110
+
+Grease pencils, 72
+
+Grouping of bacteria for study, 410
+
+Guarded trepine, 360
+
+Guarniari's agar gelatine, 194
+
+Guinea-pig cages, 343
+  holder, 350
+
+Gulland's solution, 82
+
+Gum solution, preparation of, 116
+
+Guy's culture bottle, 5
+
+Gypsum blocks (Engel and Hansen), 192
+
+
+Haematin, 95
+
+Haematocytometer, 248
+
+Haematoxilin, 95
+
+Haemolysin, definition of, 326
+  preparation of, 327
+  storage of, 331
+
+Haemolytic serum, titration of, 328
+
+Hanging-block culture (Hill), 235
+
+Hanging-drop cultures, 233
+  examination of, 86, 79
+  preparation of, 78
+    permanent staining of, 80
+  slides, 70
+
+Hardening tissues, 114
+
+Haricot agar, 200
+  bouillon, 200
+
+Hay infusion, 200
+
+Hearson's water bath, 299
+
+Heat effect of, 299
+
+Hehner's test, 442
+
+Heiman's serum agar, 210
+
+Hesse's anaerobic culture method, 237
+
+Histological examination of blood, 373
+
+Holder for guinea-pigs, 350
+
+Hot air, 29
+    steriliser, 30
+      to use, 31
+  incubator, 217
+
+Hot-water funnel, 158
+
+Human blood agar plates, 250
+
+Huyghenian eyepiece, 55
+
+Hydrogen, generating apparatus, 242
+  in culture, test for, 289
+  peroxide in milk, test for, 442
+
+Hyphomycetes, morphology of, 126
+  reproduction of, 126
+
+
+Ice-box, for water samples, 419
+
+Ice cream, analysis of, 457
+
+Illuminant for microscope, 67
+
+Immune body, 393
+
+Immunisation, methods of, 321
+
+Imperial system, 492
+  factors for converting, 493
+
+Impression films, 85
+
+Incubators, 216
+
+Index cards, 336, 403
+
+Indol, test for, 286
+
+Infection, definition of, 370
+  general observations during life, 371
+  results of, 404
+
+Influence of environment on bacterial growth, 142
+
+Inhalation, fluid inoculum, 365
+  powdered inoculum, 366
+
+Inhibition coefficient, 310, 311
+
+Inoculation card index, 336
+  cutaneous, 352
+  intracranial, 360
+  intramuscular, 355
+  intraocular, 362
+  intraperitoneal, 355
+  intrapulmonary, 363
+  intravenous, 363
+  of collodion capsules, 357
+  subcutaneous, 353
+  syringe, 344
+
+Inoculum, character of, 346
+  preparation of, 346
+
+Inosite-free media--bouillon (Durham), 183
+
+Inseparate toxins, 144
+
+Intermittent sterilisation, 36
+
+Intracellular toxins, 144
+
+Intracerebral inoculation, 362
+
+Intracranial inoculation, 360
+
+Intragastric inoculation, large animals, 367
+    Marks method, 367
+
+Intramuscular inoculation, 355
+
+Intraocular inoculation, 362
+
+Intraperitoneal inoculation, 355
+
+Intrapulmonary inoculation, 363
+
+Intravenous inoculation, 363
+
+In vacuo anaerobia cultures, 289
+
+Invertin enzymes, tests for, 279
+
+Involution forms, 137
+
+Iodine solution, 108
+
+Iron bouillon, 185
+  peptone solution (Pakes), 185
+
+Isolation by animal experiments, 258
+  by differential atmosphere, 257
+    incubation, 255
+    media, 255
+    sterilisation, 256
+  by dilution, 248
+  by plate cultures, 250
+  subcultures, preparation of, 254
+
+
+Jeffer's counting disc, 424
+
+Jenner's stain, 97
+
+Jores' mounting fluid, 405
+
+
+Kaiserling fixing solution, 405
+
+Kanthack's serum agar, 211
+
+Killed cultivations, 318
+
+Kipp's hydrogen apparatus, 242
+
+Kitasato's serum flask, 6
+
+Klebs-Loeffler bacillus in milk, 452
+
+Koch's steam steriliser, 34
+
+Kohle's culture flask, 4
+
+
+Lab enzymes, test for, 279
+
+Laboratory animals, 335
+    comparative haematocytology of, 374
+    normal temperature, 372
+  regulations, 1
+
+Lactose litmus agar (Wurtz), 203
+  bouillon, 203
+  gelatine (Wurtz), 203
+
+Lakmus Molke, 203
+
+Lang's solution, 82
+
+Lead bouillon, 185
+  peptone solution, 186
+
+Leishman's stain, 98
+  for sections, 125
+
+Lemco broth, 163
+
+Leptothrix, morphology, 133
+
+Lethal dose, minimal, 316
+
+Leviditi's staining method, 124
+
+Light, action of, 308
+
+Liquefiable media, 147
+
+Liquid soap, 346
+
+Lithium carmine, 96
+
+Litmus bouillon, 186
+  gelatine, 202
+  milk cultures, description of, 272
+    preparation of, 172
+  nutrose agar (Drigalski-Conradi), 205
+  whey, 195
+    agar, 196
+    gelatine, 196
+    (Petruschky), 195
+
+Local anaesthetics, 345
+  reaction to infection, 372
+
+Locomotive movement, 80
+
+Loeffler's capsule stain, 103
+  serum, 208
+
+Lophotrichous bacilli, 136
+
+Lorrain Smith electric warm stage, 59
+  serum, 208
+
+Lugol's solution, to prepare, 108
+
+Lysol, 27
+
+
+MacConkey's capsule stain, 99
+  media, 180, 199, 205
+
+MacCrorrie's capsule stain, 103
+
+Macroscopical examination of cultures, 261
+
+Malachite green agar (Loeffler), 207
+
+Malt extract solution (Herschell), 196
+
+Margin of individual colonies, 267
+
+Martin's filtering apparatus, 320
+
+Material for inoculation, 346
+
+Mayer's albumin, 120
+
+Mean phenol coefficient, 490
+
+Measuring bacteria, 61
+
+Meat, bacteriological analysis of, 460
+  extract preparation of, 148
+    reaction of, 149
+
+Mechanical separation of bacteria, 249
+  stage, 52
+
+Media, filtration of, 156
+  preparation of, 163
+    aerobic culture, 222
+    aesculin agar, 204
+    agar-agar, 167
+    agar gelatine (Guarniari), 194
+
+Media, preparation of anaerobic culture, 180
+  animal tissue (Frugoni), 210
+  ascitic bouillon, 210
+   fluid agar (Wassermann), 213
+  asparagin (Fraenkel and Voge's), 183
+    (Uschinsky), 183
+  beer wort, 175
+  beetroot, 200
+  Beyrinck's solution I, 197
+    II, 198
+  bile salt agar (MacConkey), 205
+    broth (MacConkey), 180
+      double strength, 199
+  blood agar (Washbourn), 214
+  blood-serum, 168
+    (Councilman and Mallory), 208
+    (Loeffler), 208
+    (Lorrain Smith), 208
+  bouillon, 163
+  bread paste, 193
+  brilliant green agar (Conradi), 206
+    bile salt agar (Fawcus), 206
+  Capaldi-Proskauer, No. I, 186
+    No. II, 187
+  carbohydrate, 177
+  carbolised agar, 202
+    bouillon, 202
+    gelatine, 202
+  carrot, 200
+  China green agar (Werbitski), 207
+  citrated blood agar, 171
+  Cohn's solution, 191
+  dextrose solution, 178
+  double sugar agar (Russell), 207
+  earthy salt agar (Lipman and Brown), 197
+  egg Dorset, 174
+    Lubenau, 209
+  egg-albumen, inspissated, 212
+    (Tarchanoff and Kolesnikoff), 212
+  egg-albumin agar, 213
+    broth (Lipschuetz), 213
+  English proof agar (Blaxall), 193
+  fish bouillon, 190
+    gelatine, 190
+      agar, 190
+  fluid, 146
+  French mannite agar (Sabouraud), 193
+
+Media, preparation of French proof agar (Sabouraud), 193
+  Fuchsin agar (Braun), 205
+    sulphite agar (Endo), 206
+  gelatine, 193
+    agar, 193
+  glucose formate agar (Kitasato), 180
+    bouillon (Kitasato), 180
+    gelatine (Kitasato), 180
+  glycerinated broth, 209
+    potato, 209
+  glycerine agar, 209
+    blood-serum, 208, 209
+    bouillon, 209
+    potato bouillon, 203
+  gypsum blocks (Engel and Hansen), 192
+  haricot agar, 200
+    bouillon, 200
+  hay infusion, 200
+  inosite free-bouillon (Durham), 183
+  iron bouillon, 185
+    peptone solution (Pakes), 185
+  lactose litmus agar (Wurtz), 203
+    bouillon, 203
+    gelatine (Wurtz), 203
+  lakmus molke, 203
+  lead bouillon, 185
+    peptone solution, 186
+  lemco broth, 163
+  liquefiable, 147
+  litmus bouillon, 186
+    gelatine, 202
+    milk, 172
+    nutrose agar (Drigalski-Conradi), 205
+    whey, 195
+      agar, 196
+      gelatine, 196
+      (Petruschky), 195
+  malachite green agar (Loeffler), 207
+  malt extract solution (Herschell), 196
+  milk, 172
+    rice (Eisenberg), 189
+      (Soyka), 189
+  Naegeli's solution, 191
+  Naehrstoff agar (Hesse and Niedner), 199
+  neutral litmus solution, 179
+  nitrate bouillon, 185
+    peptone solution (Pakes), 186
+  nutrient, 146
+    agar-agar, 167
+
+Media, preparation of nutrient bouillon, 163
+    gelatine, 164
+  nutrose agar (Eyre), 172
+  oleic acid agar (Fleming), 201
+  Omeliansky's nutrient fluid, 189
+  Parietti's bouillon, 202
+  parsnip, 200
+  Pasteur's solution, 191
+  peptone rosolic acid water, 186
+    water (Dunham), 177
+  plaster-of-Paris discs, 192
+  potato, 174
+    gelatine (Elsner), 204
+      (Goadby), 214
+  proteid free broth (Uschinsky), 183
+  rosolic acid peptone solutions, 186
+  serum, bouillon, 210
+    dextrose water, (Hiss), 188
+    sugar, (Hiss), 188
+    water, 170
+  serum-agar (Heiman), 210
+    (Kanthack and Stevens), 211
+    (Libman), 212
+    (Wertheimer), 211
+  silicate jelly (Winogradsky), 198
+  solid, 147
+  special, 182
+  stock nutrient, 163
+  sugar, 177
+    agar, 185
+    (dextrose) bouillon, 184
+    gelatine, 184
+  sulphindigotate agar, 181
+    bouillon (Weyl), 181
+    gelatine (Weyl), 181
+  tissue (Noguchi), 214
+  turnip, 200
+  urine agar, 188
+    bouillon, 187
+    gelatine, 187
+      (Heller), 188
+  wheat bouillon (Gasperini), 193
+  whey agar, 195
+    gelatine, 195
+  wine must, 192
+  Winogradsky's solution (for nitric organisms), 198
+    (for nitrous organisms), 198
+  wood ash agar, 201
+  wort agar, 176
+    gelatine, 176
+
+Media, preparation of yeast water (Pasteur), 191
+  standardisation of, 154
+  storage of, in bulk, 159
+  storing tubes of, 161
+  sore boxes, 162
+  titration of, 150
+  tubing of nutrient, 160
+
+Merismopedia, morphology of, 132
+
+Mesophilic bacteria, 143
+  pathogenic effects, 315
+
+Metabolic end-products, 145
+
+Metachromatic granules, 136
+
+Metal instruments, to sterilise, 28
+
+Metatrophic bacteria, 131
+
+Methods of cultivation, 221
+  of identification of bacteria, 259
+  of inoculation, 352
+  of isolation, 248
+  of sterilisation, 26
+
+Methylene-blue, 90
+
+Metric system, 492
+  factors for converting, 493
+
+Meyer's carmine, 96
+
+Microbes of indication, 426
+
+Micrococci, morphology, 132
+
+Micrococcus, melitensis in milk, 456
+
+Micrometer, filar, 66
+  net, 63
+  ocular, 63
+  stage, 62
+
+Micrometry, methods of, 61
+
+Micron, 61
+
+Microscope, 49
+
+Microscopical examination of bacteria, 86
+    stained, 88
+    unstained, 86
+  observations of cultures, 272
+
+Milk, analysis of, qualitative, 446
+    quantitative, 444
+  condensed, analysis of, 444
+  media, 193
+  preparation of, 172
+  rice (Eisenberg), 193
+    (Soyka), 189
+  samples, collection of, 443
+  sedimenting tubes, 449
+
+Minimal lethal dose, 316
+
+Mirror for microscope, 55
+
+Moeller's stain for spores, 107
+
+Moist heat, 32
+
+Molecular movement, 79
+
+Monotrichous bacilli, 136
+
+Motility, examination for, 79
+  true, 80
+
+Moulds, examination of, 126
+  for paraffin imbedding, 117, 119
+
+Mounting film preparations, 85
+  paraffin sections, 119
+
+Mouse cages, 342
+  holder, 351
+  scales, 341
+
+Mucor mucedo, 126
+
+Mucorinae, 126
+
+Mueller's desiccator, 307
+
+Muffle furnace, 28
+
+Muirs's capsule stain, 100
+  flagella stain, 101
+
+Museum preparations of bacteria, 407
+    of tissues, 404
+    sealing of, 406
+
+Mycelium, 126
+
+Mycoprotein, 135
+
+
+Naegeli's solution, 191
+
+Naehrstoff agar (Hesse and Niedner), 199
+
+Naked flame, 28
+
+Neisser's stain modified, 111
+
+Net micrometer, 63
+
+Neutral litmus solution, preparation of, 179
+  red, 94
+
+Nitrate bouillon, 185
+  peptone solution (Pakes), 186
+
+Nitric organisms in soil, 478
+
+Nitrosoindol reaction, 287
+
+Nitrous organisms in soil, 477
+
+Normal averages (_t.p.r._), 372
+  serum, 375
+
+Nosepiece, 57
+  double, 58
+  triple, 58
+
+Navy's anaerobic method, 244
+  jars, 245
+
+Nuclei, to stain, 105
+
+Nucleus of bacteria, 135
+
+Numerical aperture, 56
+
+Nutrient media, 146
+
+Nutrose agar (Eyre), preparation of, 172
+
+
+Object marker, 61
+
+Objectives, 55
+
+Oblique tube cultures, 223
+
+Ocular micrometer, 63
+
+Oculars, 55
+
+Oese, platinum, 71
+
+Oidium, 128
+
+Oil of garlic, 27
+  of mustard, 27
+
+Oleic acid agar (Fleming), 201
+
+Omeliansky's nutrient fluid, 189
+
+Operation tables (Eyre's), 352
+    (Tatin's), 351
+
+Opsonic index, 393
+
+Opsonic index, determination of, 390
+
+Opsonin, 387
+
+Optical characters of colonies, 267
+
+Optimum reaction of medium, determination of, 305
+  temperature, determination of, 298
+
+Organisms of suppuration, 409
+
+Orsat-Lunge gas apparatus, 292
+
+Orth's carmine, 96
+
+Oxford stain for Actinomyces, 112
+
+Oysters, analysis of, 463
+
+
+Pakes' counting disc, 424
+  filter reservoir, 45
+
+Papier chardin, 158
+
+Pappenheim's stain, 111
+
+Paraboloid condenser, 60
+
+Parachromophorous bacteria, 144
+
+Paraffin method for sections, 117
+  sections, mounting of, 119
+    to stain, 121
+
+Paratrophic bacteria, 131
+
+Parietti's bouillon, 202
+  method of isolating coli-typhoid group, 437
+
+Parsnip medium, 200
+
+Passages of virus, 320
+
+Pasteur-Chamberland filter, 42
+
+Pasteur's pipettes, 10
+  solution, 191
+
+Pathogenesis, investigation of, 315
+
+Pathogenic bacteria, 131
+    study of, 408
+
+Pediococci, morphology of, 132
+
+Penicillium, 128
+
+Peptone rosolic acid water, 186
+  water (Dunham), preparation of, 177
+
+Percentage formula, 496
+
+Perchloride of mercury, 27
+
+Perisporaceae, 127
+
+Peritrichous bacilli, 136
+
+Permanent preparations of bacteria, 407
+    of tissues, 404
+
+Petri's dishes, 6
+
+Phagocytic index, 392
+
+Phenol coefficient, 489
+  production, test for, 287
+
+Photogenic bacteria, 131, 144
+
+Physiological filter, 156
+
+Picric acid solution, 121
+      (Spengler's), 112
+
+Picrocarmine, 97
+
+Pigment production, observations on, 288
+
+Pipettes, automatic, 13
+  blood, 11
+  capillary, 10
+  cases for, 7
+  graduated, 6
+    capillary, 13
+  Pasteur's, 10
+  sedimentation, 16
+  standard graduated, 7
+  teat, 10
+  throttle, 13
+  to clean infected, 20
+    new, 18
+  to sterilise, 31
+
+Piridin method of staining spirochaetes, 124
+
+Pitfield's flagella stain, 103
+
+Plasmolysis, 135
+
+Plaster-of-Paris discs, 192
+
+Plate box, 7
+  cultures, description of, 261
+    preparation of, 226
+  levelling stand, 228
+
+Plates, Petri's, 6
+  to clean infected, 20
+    new, 18
+  to sterilise, 31
+
+Platinum needles, 71
+    method of mounting, 71
+
+Pleomorphism, 133
+
+Polar germination, 140
+  granules, 136
+
+Polkoerner, 136
+
+Polychrome blood stains, 97
+
+Pooled serum, 379
+
+Porcelain filter, 42
+    Berkefeld, 42
+    Chamberland, 42
+    Doulton, 42
+
+Post-mortem examination of experimental animals, 396
+
+Potato gelatine (Eisner), 204
+    (Goadby), 214
+  medium, preparation of, 174
+
+Potted meat, analysis of, 460
+
+Pouring plates, 227
+
+Preparation of experimental animals, 335
+
+Preservatives in milk, 442
+
+Pressure temperature table, 500
+
+Primary colours, action of, 309
+
+Proteid free broth (Uschinsky), 183
+
+Proteolytic enzymes, tests for, 277
+
+Prototrophic bacteria, 131
+
+Psychrophilic bacteria, 143
+    pathogenic effects, 315
+
+Pus, collection of, 373
+
+Pyrogallic acid solution, 293
+
+
+Qualitative analysis of air, 470
+    of milk, 446
+    of sewage, 467
+    of soil, 476
+    of unsound meat, 462
+    of water, 426
+
+Quantitative analysis of air, 468
+    of milk, 444
+    of sewage, 466
+    of soil, 473
+    of unsound meat, 460
+
+
+Rabbit cages, 343
+  scabies, treatment of, 338
+  scales, 340
+
+Raising virulence of organisms, 320
+
+Ramsden's micrometer, 66
+
+Range of medium reaction, measurement of, 305
+  of temperature, measurement of, 298
+
+Rat cages, 342
+
+Raw milk, Saul's test for, 442
+
+Reaction of medium, 305
+    optimum, 305
+    range of, 305
+  scale, 153
+
+Reduced pressure and temperature table, 501
+
+Reducing agents, production, 389
+    tests for, 289
+
+Reduction of nitrates, 389
+
+Reichert's thermo-regulator, 218
+
+Relation of bacteria to environment, 142
+
+Removal of material from culture tubes, 74
+
+Rennin enzymes, tests for, 279
+
+Reproduction of bacteria, 136
+
+Resistance glass, 6
+  to lethal agents, 306
+
+Resting stage of bacteria, 137
+
+Restrictions upon experimental inoculations, 334
+
+Ribbert's capsule stain, 101
+
+Roll cultures, 226
+
+Rosolic acid peptone solution, 186
+
+Rosindol reaction, 286
+
+Roux's anaerobic culture method, 237
+  culture bottle, 5
+
+
+Sabouraud's medium, 193
+
+Saccharomyces, morphology of, 129
+
+Safranine, 94
+
+Salicylic acid in milk, test for, 443
+
+Saprogenic bacteria, 131
+
+Sarcinae, morphology of, 132
+
+Saul's test, 442
+
+Scales, decimal, 340
+  trip, 164
+
+Scalpels, to sterilise, 32, 33
+
+Schallibaum's solution, 121
+
+Scheme for study of bacteria, 259
+
+Schizomycetes, classification of, 131
+  morphology of, 131
+
+Scissors, to sterilise, 32
+
+Sealing museum jars, 406
+
+Searing iron, 397
+
+Sections, special staining methods for, 121
+
+Sedimentation pipettes, 16
+  tubes, 9
+
+Selecting objectives, 57
+
+Sensitising red blood cells, 395
+
+Serial cultivations, 251
+
+Serological examination of blood, 378
+
+Serum agar (Heiman), 210
+    (Kanthack and Stevens), 211
+    (Libman), 212
+    plates, 250
+    (Wertheimer), 211
+  bouillon, 210
+  collection of, 379
+  dextrose water (Hiss), 188
+  inspissator, 169
+  sugar media (Hiss), 188
+  water, preparation of, 170
+
+Sewage, analysis of, qualitative, 467
+    quantitative, 466
+
+Shake cultivations, 225
+    description of, 271
+
+Shape of colonies, 262
+
+Shaving experimental animals, 349
+
+Shellfish, analysis of, 463
+
+Silicate jelly (Winogradsky), 198
+
+Single stain for spores, 106
+
+Size of colonies, 262
+
+Slanted tube cultures, 223
+
+Slides, to clean new, 22
+    used, 23
+
+Smear culture, 224
+    description of, 268
+
+Soap liquid, 346
+
+Soda solution, storage of stock, 154
+
+Sodium bicarbonate in milk, test for, 443
+
+Soil, analysis of, qualitative, 476
+    quantitative, 473
+    collection of samples, 471
+
+Solid media, 147
+
+Soluble toxins, 144
+
+Soyka's milk rice, 189
+
+Spear-headed spatula, 402
+
+Special media, 182
+
+Specific serum, 379
+    dilution of, 382
+
+Spherical aberration, 55
+
+Spirillum, morphology of, 133
+
+Spirochaeta, morphology of, 133
+
+Spirochaetes in tissues, to stain, 124
+
+Spleen extract, 149
+
+Sporangium, 127
+
+Spore formation, arthrogenous, 138
+    endogenous, 138
+    method of, 138, 273
+  germination, method of, 140, 274
+    observation of, 140, 273
+
+Spores, characters of, 139
+  classification of, 139
+  double stain for, 106
+  to stain, 106
+
+Stab culture, 224
+    description of, 265
+
+Stage micrometer, 62
+  of microscope, 52
+
+Staining methods, 90
+  paraffin sections, 121
+  reactions of bacteria, 274
+
+Stains intra-vitam, 77
+  negative (Burri), 77
+  rack for, 72
+
+Standard graduated pipettes, 7
+  soda solution, 154
+
+Standardisation of media, 154
+
+Standardising bouillon, 155
+
+Staphylococci, morphology, 132
+
+Staphylococcus in milk, 456
+
+Steam steriliser, Arnold, 35
+    Koch, 35
+    to use, 35
+  streaming, 35
+
+Sterigma, 127
+
+Sterilisation by chemicals, 27
+  by dry heat, 28
+  by filters, 40
+  by moist heat, 32
+  by streaming steam, 35
+  by superheated steam, 36
+  of albuminous liquids, 32
+  of gases, 40
+
+Sterilising agents, 26
+
+Stichcultur, 224
+
+Stock dilutions, 497
+  nutrient media, 163
+  plate for isolation work, 253
+
+Storage of media in bulk, 159
+  of tubed media, 161
+
+Store boxes for media, 161
+
+Streak culture, 224
+    description of, 268
+
+Streaming movement, 80
+  steam, 35
+
+Streptobacilli, morphology, 133
+
+Streptococci in soil, 477
+  in water, detection of, 432
+  morphology of, 132
+
+Streptococcus pyogenes longus in milk, 455
+
+Streptothrix, morphology of, 133
+
+Strichcultur, 223
+
+Structure, internal, of colonies, 265
+
+Study of pathogenic bacteria, 408
+
+Subcutaneous inoculation, 353
+
+Subdural inoculation, 361
+
+Substage condenser, 54
+
+Sugar agar, 185
+    dextrose bouillon, 184
+  gelatine, 184
+  media, preparation of, 177
+
+Sulphindigotate agar, 181
+  bouillon (Weyl), 181
+  gelatine (Weyl), 181
+
+Sulphuretted hydrogen in cultures, test for, 290
+
+Sunlight, action of, 309
+
+Superheated steam, 36
+
+Superior lethal coefficient, 310, 313
+
+Suppuration, organisms of, 409
+
+Surface characters of colonies, 264
+  plates, 230
+
+Surgical motor, electric, 360
+
+Swarm spores, 127
+
+Syringe for subcutaneous inoculation of solid material, 354
+  hypodermic, 344
+
+
+Tatin's operating table, 351
+
+Taxonomy, 262
+
+Teat-pipettes, 10
+
+Temperature, action of, 299
+  optimum, 298
+  pressure table, 500
+  range, 298
+  taking, 340
+
+Test objects for objectives, 57
+
+Testing filters, 478
+
+Test-tubes, 3
+  to clean infected, 19
+    new, 18
+  to plug, 24
+  to sterilise, 31
+
+Tetracocci, morphology of, 132
+
+Thermal death-point, 143
+    determination of, 298
+    of spores, 301, 304
+    of vegetative forms, 298, 303
+
+Thermophilic bacteria, 143
+
+Thermo-regulators, Hearson's capsule, 218
+  Reichert's, 218
+
+Thionine blue, 92
+
+Thiothrix, morphology of, 133
+
+Thresh's water collecting bottle, 418
+
+Throttle pipettes, 13
+
+Tinned meat, analysis of, 460
+
+Tissue medium (Noguchi), 214
+  stains, 95
+
+Tissues for sectioning, fixing, 114
+    freezing, 116
+    hardening, 114
+    imbedding, 118
+    preparation of, 114
+    washing, 115
+
+Titration of media, 150
+
+Torulae, differentiation from saccharomyces, 130
+
+Total acidity, 280
+
+Toxins, testing of, 318
+
+Trephines, 360
+
+Triple nosepiece, 58
+
+True motility, 80
+
+Tube cultures, preparation of, 222
+  length, 50
+
+Tubercle bacillus in milk, 453
+    to stain, 110, 124
+
+Tuberculous guinea-pig, cadaver of, 454
+
+Tubing nutrient media, 160
+
+Turnip media, 200
+
+
+Unna-Pappenheim's stain for sections, 123
+
+Unsound meat, analysis of, 460
+
+Urine agar, 188
+  gelatine, 187
+    (Heller), 188
+  media bouillon, 187
+
+Uschinsky's solution, 183
+
+
+Valency of specific sera, 386
+
+Van Ermengem's flagella stain, 104
+
+Vegetative stage of bacteria, 136
+
+Vesuvin, 94
+
+Vibrio cholerae in milk, 452
+    in water, 439
+  morphology of, 133
+
+Virulence, attenuating, 321
+  of organisms, 320
+  raising, 320
+
+Vivisection license, 334
+
+Voges holder, 350
+
+Volatile oils as disinfectants, 27
+
+
+Warm stage, 58
+
+Washing red blood cells, 388
+  tissues, 115
+
+Water, analysis of, qualitative, 426
+    quantitative, 416
+  steriliser, 33
+
+Weighing animals, 340
+
+Welch's capsule stain, 101
+
+Wertheimer's serum agar, 211
+
+Wheat bouillon (Gasperini), 193
+
+Whey agar, 195
+  gelatine, 195
+
+Wine must, 192
+
+Winogradsky's solution I, 198
+    II, 198
+
+Wire crates for test-tubes, 31
+
+Wood ash agar, 201
+
+Working up plates, 252
+
+Wort agar, 176
+  gelatine, 176
+
+Wright's anaerobic method, 239
+
+
+Yeast water (Pasteur), 191
+
+
+Ziehl-Neelsen's stain, 110
+
+Zoogloea, 134
+
+Zymogenic bacteria, 131
+
+
+
+
+SAUNDERS' BOOKS
+
+on
+
+Pathology, Physiology
+
+Histology, Embryology
+
+Bacteriology, Biology
+
+       *       *       *       *       *
+
+    W. B. SAUNDERS COMPANY
+    WEST WASHINGTON SQUARE    PHILADELPHIA
+    9, HENRIETTA STREET    COVENT GARDEN, LONDON
+
+
+
+       *       *       *       *       *
+
+
+
+LITERARY SUPERIORITY
+
+The excellent judgment displayed in the publications of the house at the
+very beginning of its career, and the success of the modern business
+methods employed by it, at once attracted the attention of leading men
+in the profession, and many of the most prominent writers of America
+offered their books for publication. Thus, there were produced in rapid
+succession a number of works that immediately placed the house in the
+front rank of Medical Publishers. One need only cite such instances as
+Musser and Kelly's Treatment, Keen's Surgery, Kelly and Noble's
+Gynecology and Abdominal Surgery, Cabot's Differential Diagnosis, De
+Lee's Obstetrics, Mumford's Surgery, Cotton's Dislocations and Joint
+Fractures, Crandon and Ehrenfried's Surgical After-treatment, Sisson's
+Veterinary Anatomy, Anders and Boston's Medical Diagnosis, Gant's
+Constipation and Obstruction, Jordan's Bacteriology, and Kemp on
+Stomach, Intestines, and Pancreas. These books have made for themselves
+places among the best works on their respective subjects.
+
+     ~A Complete Catalogue of our Publications will be Sent upon
+     Request~
+
+
+Mallory's
+
+Pathologic Histology
+
+~Pathologic Histology.~ By FRANK B. MALLORY, M. D., Associate Professor of
+Pathology, Harvard University Medical School. Octavo of 677 pages, with
+497 figures containing 683 original illustrations, 124 in colors. Cloth,
+$5.50 net; Half Morocco, $7.00 net.
+
+~REPRINTED IN THREE MONTHS~
+
+     Dr. Mallory here presents _pathology_ from the morphologic
+     point of view. He presents his subject biologically, first
+     by ascertaining the cellular elements out of which the
+     various lesions are built up; then he traces the development
+     of the lesions from the simplest to the most complex. He so
+     presents pathology that you are able to trace backward from
+     any given end-result, such as sclerosis of an organ
+     (cirrhosis of the liver, for example), through all the
+     various acute lesions that may terminate in that particular
+     end-result to the primal _cause_ of the lesion. The
+     _illustrations_ are most beautiful.
+
+~Dr. W. G. MacCallum~, _Columbia University_
+
+     "I have looked over the book and think the plan is admirably
+     carried out and that the book supplies a need we have felt
+     very much. I shall be very glad to recommend it."
+
+       *       *       *       *       *
+
+Howell's Physiology
+
+~A Text-Book of Physiology.~ By WILLIAM H. HOWELL, PH.D., M. D., Professor
+of Physiology in the Johns Hopkins University, Baltimore, Md. Octavo of
+1020 pages, 306 illustrations. Cloth, $4.00 net.
+
+~THE NEW (5th) EDITION~
+
+     Dr. Howell has had many years of experience as a teacher of
+     physiology in several of the leading medical schools, and is
+     therefore exceedingly well fitted to write a text-book on
+     this subject. Main emphasis has been laid upon those facts
+     and views which will be directly helpful in the practical
+     branches of medicine. At the same time, however, sufficient
+     consideration has been given to the experimental side of the
+     science. The entire literature of physiology has been
+     thoroughly digested by Dr. Howell, and the important views
+     and conclusions introduced into his work. Illustrations have
+     been most freely used.
+
+~The Lancet, London~
+
+     "This is one of the best recent text-books on physiology,
+     and we warmly commend it to the attention of students who
+     desire to obtain by reading a general, all-round, yet
+     concise survey of the scope, facts, theories, and
+     speculations that make up its subject matter."
+
+
+Mallory _and_ Wright's Pathologic Technique
+
+~Fifth Edition~
+
+~Pathologic Technique.~ A Practical Manual for Workers in Pathologic
+Histology, including Directions for the Performance of Autopsies and for
+Clinical Diagnosis by Laboratory Methods. By FRANK B. MALLORY, M. D.,
+Associate Professor of Pathology, Harvard University; and JAMES H.
+WRIGHT, M. D., Director of the Pathologic Laboratory, Massachusetts
+General Hospital. Octavo of 500 pages, with 152 illustrations. Cloth,
+$3.00 net.
+
+     In revising the book for the new edition the authors have
+     kept in view the needs of the laboratory worker, whether
+     student, practitioner, or pathologist, for a practical
+     manual of histologic and bacteriologic methods in the study
+     of pathologic material. Many parts have been rewritten, many
+     new methods have been added, and the number of illustrations
+     has been considerably increased.
+
+~Boston Medical and Surgical Journal~
+
+     "This manual, since its first appearance, has been
+     recognized as the standard guide in pathological technique,
+     and has become well-nigh indispensable to the laboratory
+     worker."
+
+       *       *       *       *       *
+
+Eyre's Bacteriologic Technic
+
+~Bacteriologic Technic.~ A Laboratory Guide for the Medical, Dental, and
+Technical Student. By J. W. H. EYRE, M. D., F. R. S. Edin., Director of
+the Bacteriologic Department of Guy's Hospital, London. Octavo of 520
+pages, 219 illustrations. Cloth, $3.00 net.
+
+~JUST READY--NEW (2d) EDITION, REWRITTEN~
+
+     Dr. Eyre has subjected his work to a most searching
+     revision. Indeed, so thorough was his revision that the
+     entire book, enlarged by some 150 pages and 50
+     illustrations, had to be reset from cover to cover. He has
+     included all the latest technic in every division of the
+     subject. His thoroughness, his accuracy, his attention to
+     detail make his work an important one. He gives clearly the
+     technic for the bacteriologic examination of water, sewage,
+     air, soil, milk and its products, meats, etc. And he gives
+     you good technic--methods attested by his own large
+     experience. To any one interested in this line of endeavor
+     the new edition of Dr. Eyre's work is indispensable. The
+     illustrations are as practical as the text.
+
+
+McFarland's Pathology
+
+~A Text-Book of Pathology.~ By JOSEPH MCFARLAND, M. D., Professor of
+Pathology and Bacteriology in the Medico-Chirurgical College of
+Philadelphia. Octavo of 856 pages, with 437 illustrations, many in
+colors. Cloth, $5.00 net; Half Morocco, $6.50 net.
+
+~THE NEW (2d) EDITION~
+
+     You cannot successfully treat disease unless you have a
+     practical, _clinical_ knowledge of the pathologic changes
+     produced by disease. For this purpose Dr. McFarland's work
+     is well fitted. It was written with just such an end in
+     view--to furnish a ready means of acquiring a thorough
+     training in the subject, a training such as would be of
+     daily help in your practice. For this edition every page has
+     been gone over most carefully, correcting, omitting the
+     obsolete, and adding the new. Some sections have been
+     entirely rewritten. You will find it a book well worth
+     consulting, for it is the work of an authority.
+
+~St. Paul Medical Journal~
+
+     "It is safe to say that there are few who are better
+     qualified to give a resume of the modern views on this
+     subject than McFarland. The subject-matter is thoroughly up
+     to date."
+
+~Boston Medical and Surgical Journal~
+
+     "It contains a great mass of well-classified facts. One of
+     the best sections is that on the special pathology of the
+     blood."
+
+       *       *       *       *       *
+
+McFarland's Biology: Medical and General
+
+~Biology: Medical and General~--By JOSEPH MCFARLAND, M. D., Professor of
+Pathology and Bacteriology in the Medico-Chirurgical College of Phila.
+12mo, 457 pages, 160 illustrations. Cloth, $1.75 net.
+
+~JUST READY--NEW (2d) EDITION~
+
+     This work is both a _general_ and _medical_ biology. The
+     former because it discusses the peculiar nature and
+     reactions of living substance generally; the latter because
+     particular emphasis is laid on those subjects of special
+     interest and value in the study and practice of medicine.
+     The illustrations will be found of great assistance.
+
+~Frederic P. Gorham, A. M.~, _Brown University_.
+
+     "I am greatly pleased with it. Perhaps the highest praise
+     which I can give the book is to say that it more nearly
+     approaches the course I am now giving in general biology
+     than any other work."
+
+
+McFarland's Pathogenic Bacteria and Protozoa
+
+~Pathogenic Bacteria and Protozoa.~ By JOSEPH MCFARLAND, M. D., Professor
+of Pathology and Bacteriology in the Medico-Chirurgical College of
+Philadelphia. Octavo of 878 pages, finely illustrated. Cloth, $3.50 net.
+
+~NEW (7th) EDITION, ENLARGED~
+
+     Dr. McFarland has subjected his book to a most vigorous
+     revision, bringing this edition right down to the minute.
+     Important new additions have increased it in size some 180
+     pages. By far the most important addition is the inclusion
+     of an entirely new section on _Pathogenic Protozoa_. This
+     section considers every protozoan pathogenic to man; and in
+     that same clean-cut, definite way that won for McFarland's
+     work a place in the very front of medical bacteriologies.
+     The illustrations are the best the world affords, and are
+     beautifully executed.
+
+~H. B. Anderson, M. D.~, _Professor of Pathology and Bacteriology, Trinity
+Medical College, Toronto._
+
+     "The book is a satisfactory one, and I shall take pleasure
+     in recommending it to the students of Trinity College."
+
+~The Lancet, London~
+
+     "It is excellently adapted for the medical students and
+     practitioners for whom it is avowedly written.... The
+     descriptions given are accurate and readable."
+
+       *       *       *       *       *
+
+Hill's Histology and Organography
+
+~A Manual of Histology and Organography.~ By CHARLES HILL, M. D., formerly
+Assistant Professor of Histology and Embryology, Northwestern
+University, Chicago. 12mo of 468 pages, 337 illustrations. Flexible
+leather, $2.00 net.
+
+~THE NEW (2d) EDITION~
+
+     Dr. Hill's work is characterized by a completeness of
+     discussion rarely met in a book of this size. Particular
+     consideration is given the mouth and teeth.
+
+~Pennsylvania Medical Journal~
+
+     "It is arranged in such a manner as to be easy of access and
+     comprehension. To any contemplating the study of histology
+     and organography we would commend this work."
+
+       *       *       *       *       *
+
+      GET                                             THE NEW
+    THE BEST                                         STANDARD
+                           American
+                    Illustrated Dictionary
+
+~New (7th) Edition--5000 Sold in Two Months~
+
+~The American Illustrated Medical Dictionary.~ A new and complete
+dictionary of the terms used in Medicine, Surgery, Dentistry, Pharmacy,
+Chemistry, Veterinary Science, Nursing, and kindred branches; with over
+100 new and elaborate tables and many handsome illustrations. By W. A.
+NEWMAN DORLAND, M.D., Editor of "The American Pocket Medical
+Dictionary." Large octavo, 1107 pages, bound in full flexible leather.
+Price, $4.50 net; with thumb index, $5.00 net.
+
+~IT DEFINES ALL THE NEW WORDS--IT IS UP TO DATE~
+
+     The American Illustrated Medical Dictionary defines hundreds
+     of the newest terms not defined in any other dictionary--bar
+     none. These new terms are live, active words, taken right
+     from modern medical literature.
+
+     It gives the capitalization and pronunciation of all words.
+     It makes a feature of the derivation or etymology of the
+     words. In some dictionaries the etymology occupies only a
+     secondary place, in many cases no derivation being given at
+     all. In the American Illustrated practically every word is
+     given its derivation.
+
+     Every word has a separate paragraph, thus making it easy to
+     find a word quickly.
+
+     The tables of arteries, muscles, nerves, veins, etc., are of
+     the greatest help in assembling anatomic facts. In them are
+     classified for quick study all the necessary information
+     about the various structures.
+
+     Every word is given its definition--a definition that
+     _defines_ in the fewest possible words. In some dictionaries
+     hundreds of words are not defined at all, referring the
+     reader to some other source for the information he wants at
+     once.
+
+~Howard A, Kelly, M. D.~, _Johns Hopkins University, Baltimore._
+
+     "The American Illustrated Dictionary is admirable. It is so
+     well gotten up and of such convenient size. No errors have
+     been found in my use of it."
+
+~J. Collins Warren, M. D., LL.D., F.R.C.S. (Hon.)~, _Harvard Medical
+School_
+
+     "I regard it as a valuable aid to my medical literary work.
+     It is very complete and of convenient size to handle
+     comfortably. I use it in preference to any other."
+
+       *       *       *       *       *
+
+Stengel's Text-Book of Pathology
+
+~Fifth Edition~
+
+~A Text-Book of Pathology.~ By ALFRED STENGEL, M. D., Professor of
+Medicine in the University of Pennsylvania. Octavo volume of 979 pages,
+with 400 text-illustrations, many in colors, and 7 full-page colored
+plates. Cloth, $5.00 net; Sheep or Half Morocco, $6.50 net.
+
+~WITH 400 TEXT-CUTS, MANY IN COLORS, AND 7 COLORED PLATES~
+
+     In this work the practical application of pathologic facts
+     to clinical medicine is considered more fully than is
+     customary in works on pathology. While the subject of
+     pathology is treated in the broadest way consistent with the
+     size of the book, an effort has been made to present the
+     subject from the point of view of the clinician. In the
+     second part of the work the pathology of individual organs
+     and tissues is treated systematically and quite fully under
+     subheadings that clearly indicate the subject-matter to be
+     found on each page. In this edition the section dealing with
+     General Pathology has been most extensively revised, several
+     of the important chapters having been practically rewritten.
+
+~The Lancet, London~
+
+     "This volume is intended to present the subject of pathology
+     in as practical a form as possible, and more especially from
+     the point of view of the 'clinical pathologist.' These
+     objects have been faithfully carried out, and a valuable
+     text-book is the result. We can most favorably recommend it
+     to our readers as a thoroughly practical work on clinical
+     pathology."
+
+       *       *       *       *       *
+
+Stiles' Nutritional Physiology
+
+~Nutritional Physiology.~ By PERCY GOLDTHWAIT STILES, Assistant Professor
+of Physiology at Simmons College, Boston. 12mo of 295 pages,
+illustrated. Cloth, $1.25 net.
+
+~ILLUSTRATED~
+
+     This new work expresses the most advanced views on this
+     important subject. It discusses in a concise way the
+     processes of digestion and metabolism. The key-word of the
+     book throughout is "energy"--its source and its
+     conservation.
+
+     "It is remarkable for the fineness of its diction and for
+     its clear presentation of the subject, relieved here and
+     there by a quaintly humorous turn of phrase that is
+     altogether delightful."--_Colin C. Stewart, Ph. D.,
+     Dartmouth College._
+
+       *       *       *       *       *
+
+Jordan's General Bacteriology
+
+~A Text-Book of General Bacteriology.~ By EDWIN O. JORDAN, PH.D.,
+Professor of Bacteriology in the University of Chicago and in Rush
+Medical College. Octavo of 623 pages, illustrated. Cloth, $3.00 net.
+
+~NEW (3d) EDITION~
+
+     Professor Jordan's work embraces the entire field of
+     bacteriology, the non-pathogenic as well as the pathogenic
+     bacteria being considered, giving greater emphasis, of
+     course, to the latter. There are extensive chapters on
+     methods of studying bacteria, including staining,
+     biochemical tests, cultures, etc.; on the development and
+     composition of bacteria; on enzymes and
+     fermentation-products; on the bacterial production of
+     pigment, acid and alkali; and on ptomaines and toxins.
+     Especially complete is the presentation of the serum
+     treatment of gonorrhea, diphtheria, dysentery, and tetanus.
+     The relation of bovine to human tuberculosis and the ocular
+     tuberculin reaction receive extensive consideration.
+
+     This work will also appeal to academic and scientific
+     students. It contains chapters on the bacteriology of
+     plants, milk and milk-products, air, agriculture, water,
+     food preservatives, the processes of leather tanning,
+     tobacco curing, and vinegar making; the relation of
+     bacteriology to household administration and to sanitary
+     engineering, etc.
+
+~Prof. Severance Burrage~, _Associate Professor of Sanitary Science,
+Purdue University._
+
+     "I am much impressed with the completeness and accuracy of
+     the book. It certainly covers the ground more completely
+     than any other American book that I have seen."
+
+       *       *       *       *       *
+
+Buchanan's Veterinary Bacteriology
+
+~Veterinary Bacteriology.~ By ROBERT E. BUCHANAN, PH.D., Professor of
+Bacteriology in the Iowa State College of Agriculture and Mechanic Arts.
+Octavo, 516 pages, 214 illustrations. Cloth, $3.00 net.
+
+~THE BEST PUBLISHED~
+
+     Professor Buchanan discusses thoroughly all bacteria causing
+     diseases of the domestic animals. He goes minutely into the
+     consideration of immunity, opsonic index, reproduction,
+     sterilization, antiseptics, biochemic tests, culture-media,
+     isolation of cultures, the manufacture of the various
+     toxins, antitoxins, tuberculins, and vaccines that have
+     proved of diagnostic or therapeutic value. Then, in addition
+     to bacteria and protozoa proper, he considers molds,
+     mildews, smuts, rusts, toadstools, puff-balls, and the other
+     fungi pathogenic for animals.
+
+~B. F. Kaupp, D. V. S.~, _State Agricultural College, Fort Collins._
+
+     "It is the best in print on the subject. What pleases me
+     most is that it contains all the late results of research.
+     It fills a long felt want."
+
+
+Heisler's Embryology
+
+~A Text-Book of Embryology.~ By JOHN C. HEISLER, M.D., Professor of
+Anatomy in the Medico-Chirurgical College, Philadelphia. Octavo volume
+of 435 pages, with 212 illustrations, 32 of them in colors. Cloth, $3.00
+net.
+
+~THIRD EDITION--WITH 212 ILLUSTRATIONS, 32 IN COLORS~
+
+This edition represents all the advances recently made in the science of
+embryology. Many portions have been entirely rewritten, and a great deal
+of new and important matter added. A number of new illustrations have
+also been introduced and these will prove very valuable. Heisler's
+Embryology has become a standard work.
+
+~G. Carl Huber, M.D.~, _Professor of Embryology at the Wistar Institute,
+University of Pennsylania._
+
+     "I find this edition of 'A Text-Book of Embryology,' by Dr.
+     Heisler, an improvement on the former one. The figures added
+     increase greatly the value of the work. I am again
+     recommending it to our students."
+
+       *       *       *       *       *
+
+Boehm, Davidoff, _and_ Huber's Histology
+
+~A Text-Book of Human Histology.~ Including Microscopic Technic. By DR. A.
+A. BOEHM and DR. M. VON DAVIDOFF, of Munich, and G. CARL HUBER, M.D.,
+Professor of Embryology at the Wistar Institute, University of
+Pennsylvania. Handsome octavo of 528 pages, with 361 beautiful original
+illustrations. Flexible cloth, $3.50 net.
+
+~SECOND EDITION, ENLARGED~
+
+     The work of Drs. Boehm and Davidoff is well known in the
+     German edition, and has been considered one of the most
+     practically useful books on the subject of Human Histology.
+     This second edition has been in great part rewritten and
+     very much enlarged by Dr. Huber, who has also added over one
+     hundred original illustrations. Dr. Huber's extensive
+     additions have rendered the work the most complete students'
+     text-book on Histology in existence.
+
+~Boston Medical and Surgical Journal~
+
+     "Is unquestionably a text-book of the first rank, having
+     been carefully written by thorough masters of the subject,
+     and in certain directions it is much superior to any other
+     histological manual."
+
+       *       *       *       *       *
+
+Wells' Chemical Pathology
+
+~Chemical Pathology.~--Being a Discussion of General Pathology from the
+Standpoint of the Chemical Processes Involved. By H. GIDEON WELLS,
+PH.D., M.D., Assistant Professor of Pathology in the University of
+Chicago. Octavo of 616 pages. Cloth, $3.25 net.
+
+~JUST READY--NEW (2d) EDITION~
+
+     Dr. Wells' work is written for the physician, for those
+     engaged in research in pathology and physiologic chemistry,
+     and for the medical student. In the introductory chapter are
+     discussed the chemistry and physics of the animal cell,
+     giving the essential facts of ionization, diffusion, osmotic
+     pressure, etc., and the relation of these facts to cellular
+     activities. Special chapters are devoted to _Diabetes_ and
+     to _Uric-acid Metabolism and Gout_.
+
+~Wm. H. Welch, M.D.~ _Professor of Pathology, Johns Hopkins University._
+
+     "The work fills a real need in the English literature of a
+     very important subject, and I shall be glad to recommend it
+     to my students."
+
+       *       *       *       *       *
+
+Lusk's Elements of Nutrition
+
+~Elements of the Science of Nutrition.~ By GRAHAM LUSK, PH.D., Professor
+of Physiology at Cornell Medical School. Octavo volume of 302 pages.
+Cloth, $3.00 net.
+
+~THE NEW (2d) EDITION--TRANSLATED INTO GERMAN~
+
+     Prof. Lusk presents the scientific foundations upon which
+     rests our knowledge of nutrition and metabolism, both in
+     health and in disease. There are special chapters on the
+     metabolism of diabetes and fever, and on purin metabolism.
+     The work will also prove valuable to students of _animal
+     dietetics_ at agricultural stations.
+
+~Lewellys F. Barker, M. D.~ _Professor of the Principles and Practice of
+Medicine, Johns Hopkins University._
+
+     "I shall recommend it highly to my students. It is a comfort
+     to have such a discussion of the subject in English."
+
+
+Daugherty's Economic Zoology
+
+~Economic Zoology.~ By L. S. DAUGHERTY, M. S., PH. D., Professor of
+Zoology, State Normal School, Kirksville, Mo., and M. C. DAUGHERTY,
+author with Jackson of "Agriculture Through the Laboratory and School
+Garden." Part I: _Field and Laboratory Guide_. 12mo of 237 pages,
+interleaved. Cloth, $1.25 net. Part II: _Principles._ 12mo of 406 pages,
+illustrated. Cloth, $2.00 net.
+
+~ILLUSTRATED~
+
+     There is no other book just like this. Not only does it give
+     the salient facts of structural zoology and the development
+     of the various branches of animals, but also the natural
+     history--the _life and habits_--thus showing the
+     interrelations of structure, habit, and environment. In a
+     word, it gives the principles of zoology and _their actual
+     application_. The economic phase is emphasized.
+
+     Part I--the _Field and Laboratory Guide_--is designed for
+     practical instruction in the field and laboratory. To
+     enhance its value for this purpose blank pages are inserted
+     for notes.
+
+       *       *       *       *       *
+
+Drew's Invertebrate Zoology
+
+~A Laboratory Manual of Invertebrate Zoology.~ By GILMAN A. DREW, PH. D.,
+Assistant Director at Marine Biological Laboratory, Woods Hole, Mass.
+With the aid of Former and Present Members of the Zoological Staff of
+Instructors. 12mo of 213 pages. Cloth, $1.25 net.
+
+~JUST READY--NEW (2d) EDITION~
+
+     The subject is presented in a logical way, and the type
+     method of study has been followed, as this method has been
+     the prevailing one for many years.
+
+~Prof. Allison A. Smyth, Jr., Virginia Polytechnic Institute~
+
+     "I think it is the best laboratory manual of zoology I have
+     yet seen. The large number of forms dealt with makes the
+     work applicable to almost any locality."
+
+       *       *       *       *       *
+
+Norris' Cardiac Pathology
+
+~Studies in Cardiac Pathology.~ By GEORGE W. NORRIS, M.D., Associate in
+Medicine at the University of Pennsylvania. Large octavo of 235 pages,
+with 85 superb illustrations. Cloth, $5.00 net.
+
+~SUPERB ILLUSTRATIONS~
+
+     The wide interest being manifested in heart lesions makes
+     this book particularly opportune. The illustrations are
+     superb and are faithful reproductions of the specimens
+     photographed. Each illustration is accompanied by a detailed
+     description; besides, there is ample letter press
+     supplementing the pictures. Considerable matter of a
+     diagnostic and therapeutic nature has been interwoven.
+
+~Boston Medical and Surgical Journal~
+
+     "The illustrations are arranged in such a way as to
+     illustrate all the common and many of the rare cardiac
+     lesions, and the accompanying descriptive text constitutes a
+     fairly continuous didactic treatise."
+
+       *       *       *       *       *
+
+McConnell's Pathology
+
+~A Manual of Pathology.~ By GUTHRIE MCCONNELL, M.D., Professor of
+Bacteriology and Pathology at Temple University, Philadelphia. 12mo of
+523 pages, with 170 illustrations. Flexible leather, $2.50 net.
+
+~NEW (2d) EDITION~
+
+     Dr. McConnell has discussed his subject with a clearness and
+     precision of style that make the work of great assistance to
+     both student and practitioner. The illustrations have been
+     introduced for their practical value.
+
+~New York State Journal of Medicine~
+
+     "The book treats the subject of pathology with a
+     thoroughness lacking in many works of greater pretension.
+     The illustrations--many of them original--are profuse and of
+     exceptional excellence."
+
+       *       *       *       *       *
+
+Hektoen and Riesman's Pathology
+
+AMERICAN TEXT-BOOK OF PATHOLOGY. Edited by LUDVIG HEKTOEN, M.D.,
+Professor of Pathology, Rush Medical College, Chicago; and DAVID
+RIESMAN, M.D., Professor of Clinical Medicine, Philadelphia Polyclinic.
+Octavo of 1245 Pages, 443 illustrations, 66 in colors. Cloth, $7.50 net;
+Half Morocco, $9.00 net.
+
+
+Duerck _and_ Hektoen's Special Pathologic Histology
+
+~Atlas and Epitome of Special Pathologic Histology.~ By DR. H. DUERCK, of
+Munich. Edited, with additions, by LUDVIG HEKTOEN, M. D., Professor of
+Pathology, Rush Medical College, Chicago. In two parts. Part
+I.--Circulatory, Respiratory, and Gastro-intestinal Tracts. 120 colored
+figures on 62 plates, and 158 pages of text. Part II.--Liver, Urinary
+and Sexual Organs, Nervous System, Skin, Muscles, and Bones. 123 colored
+figures on 60 plates, and 192 pages of text. Per part: Cloth, $3.00 net.
+_In Saunders' Hand-Atlas Series._
+
+     The great value of these plates is that they represent in
+     the exact colors the effect of the stains, which is of such
+     great importance for the differentiation of tissue. The text
+     portion of the book is admirable, and, while brief, it is
+     entirely satisfactory in that the leading facts are stated,
+     and so stated that the reader feels he has grasped the
+     subject extensively.
+
+~William H. Welch, M.D.,~ _Professor of Pathology, Johns Hopkins
+University, Baltimore._
+
+     "I consider Duerck's 'Atlas of Special Pathologic Histology,'
+     edited by Hektoen, a very useful book for students and
+     others. The plates are admirable."
+
+       *       *       *       *       *
+
+Sobotta _and_ Huber's Human Histology
+
+~Atlas and Epitome of Human Histology.~ By PRIVATDOCENT DR. J. SOBOTTA, of
+Wuerzburg. Edited, with additions, by G. CARL HUBER, M. D., Professor of
+Histology and Embryology in the University of Michigan, Ann Arbor. With
+214 colored figures on 80 plates, 68 text-illustrations, and 248 pages
+of text. Cloth, $4.50 net. _In Saunders' Hand-Atlas Series._
+
+~INCLUDING MICROSCOPIC ANATOMY~
+
+     The work combines an abundance of well-chosen and most
+     accurate illustrations, with a concise text, and in such a
+     manner as to make it both atlas and text-book. The great
+     majority of the illustrations were made from sections
+     prepared from human tissues, and always from fresh and in
+     every respect normal specimens. The colored lithographic
+     plates have been produced with the aid of over thirty
+     colors.
+
+~Boston Medical and Surgical Journal~
+
+     "In color and proportion they are characterized by
+     gratifying accuracy and lithographic beauty."
+
+
+Bosanquet on Spirochaetes
+
+~Spirochaetes~: A Review of Recent Work, with Some Original Observations.
+By W. CECIL BOSANQUET, M.D., Fellow of the Royal College of Physicians,
+London. Octavo of 152 pages, illustrated. $2.50 net.
+
+~ILLUSTRATED~
+
+     This is a complete and authoritative monograph on the
+     spirochaetes, giving morphology, pathogenesis,
+     classification, staining, etc. Pseudospirochaetes are also
+     considered, and the entire text well illustrated. The high
+     standing of Dr. Bosanquet in this field of study makes this
+     new work particularly valuable.
+
+       *       *       *       *       *
+
+Levy _and_ Klemperer's Clinical Bacteriology
+
+~The Elements of Clinical Bacteriology.~ By DRS. ERNST LEVY and FELIX
+KLEMPERER, of the University of Strasburg. Translated and edited by
+AUGUSTUS A. ESHNER, M. D., Professor of Clinical Medicine, Philadelphia
+Polyclinic. Octavo volume of 440 pages, fully illustrated. Cloth, $2.50
+net.
+
+~S. Solis-Cohen, M.D.~, _Professor of Clinical Medicine, Jefferson Medical
+College_, Philadelphia.
+
+     "I consider it an excellent book. I have recommended it in
+     speaking to my students."
+
+       *       *       *       *       *
+
+Lehmann, Neumann, _and_ Weaver's Bacteriology
+
+~Atlas and Epitome of Bacteriology~: INCLUDING A TEXT-BOOK OF SPECIAL
+BACTERIOLOGIC DIAGNOSIS. By PROF. DR. K. B. LEHMANN and DR. R. O.
+NEUMANN, of Wuerzburg. _From the Second Revised and Enlarged German
+Edition._ Edited, with additions, by G. H. WEAVER, M. D., Assistant
+Professor of Pathology and Bacteriology, Rush Medical College, Chicago.
+In two parts. Part I.--632 colored figures on 69 lithographic plates.
+Part II.--511 pages of text, illustrated. Per part: Cloth, $2.50 net.
+_In Saunders' Hand-Atlas Series._
+
+       *       *       *       *       *
+
+Duerck and Hektoen's General Pathologic Histology
+
+ATLAS AND EPITOME OF GENERAL PATHOLOGIC HISTOLOGY. By PR. DR. H. DUERCK,
+of Munich. Edited, with additions, by LUDVIG HEKTOEN, M. D., Professor
+of Pathology in Rush Medical College, Chicago. 172 colored figures on 77
+lithographic plates, 36 text-cuts, many in colors, and 353 pages. Cloth,
+$5.00 net. _In Saunders' Hand Atlas Series._
+
+
+    American Text-Book of Physiology      Second Edition
+
+AMERICAN TEXT-BOOK OF PHYSIOLOGY. In two volumes. Edited by WILLIAM H.
+HOWELL, PH. D., M.D., Professor of Physiology in the Johns Hopkins
+University, Baltimore, Md. Two royal octavos of about 600 pages each,
+illustrated. Per volume: Cloth, $3.00 net; Half Morocco, $4.25 net.
+
+     "The work will stand as a work of reference on physiology.
+     To him who desires to know the status of modern physiology,
+     who expects to obtain suggestions as to further physiologic
+     inquiry, we know of none in English which so eminently meets
+     such a demand."--_The Medical News._
+
+
+    Warren's Pathology and Therapeutics      Second Edition
+
+SURGICAL PATHOLOGY AND THERAPEUTICS. By JOHN COLLINS WARREN, M. D., LL.
+D., F. R. C. S. (Hon.), Professor of Surgery, Harvard Medical School.
+Octavo, 873 pages, 136 relief and lithographic illustrations, 33 in
+colors. With an Appendix on Scientific Aids to Surgical Diagnosis and a
+series of articles on Regional Bacteriology. Cloth, $5.00 net; Half
+Morocco, $6.50 net.
+
+
+Gorham's Bacteriology
+
+A LABORATORY COURSE IN BACTERIOLOGY. For the Use of Medical,
+Agricultural, and Industrial Students. By FREDERIC P. GORHAM, A. M.,
+Associate Professor of Biology in Brown University, Providence, R. I.,
+etc. 12mo of 192 pages, with 97 illustrations. Cloth, $1.25 net.
+
+     "One of the best students' laboratory guides to the study of
+     bacteriology on the market.... The technic is thoroughly
+     modern and amply sufficient for all practical
+     purposes."--_American Journal of the Medical Sciences._
+
+
+    Raymond's Physiology      New (3d) Edition
+
+HUMAN PHYSIOLOGY. By JOSEPH H. RAYMOND, A. M., M. D., Professor of
+Physiology and Hygiene, Long Island College Hospital, New York. Octavo
+of 685 pages, with 444 illustrations. Cloth, $3.50 net.
+
+     "The book is well gotten up and well printed, and may be
+     regarded as a trustworthy guide for the student and a useful
+     work of reference for the general practitioner. The
+     illustrations are numerous and are well executed."--_The
+     Lancet_, London.
+
+
+    Ball's Bacteriology      Seventh Edition, Revised
+
+ESSENTIALS OF BACTERIOLOGY: being a concise and systematic introduction
+to the Study of Micro-organisms. By M. V. BALL, M. D., Late
+Bacteriologist to St. Agnes' Hospital, Philadelphia. 12mo of 289 pages,
+with 135 illustrations, some in colors. Cloth, $1.00 net. _In Saunders'
+Question-Compend Series._
+
+     "The technic with regard to media, staining, mounting, and
+     the like is culled from the latest authoritative
+     works."--_The Medical Times_, New York.
+
+
+    Budgett's Physiology      New (3d) Edition
+
+ESSENTIALS OF PHYSIOLOGY. Prepared especially for Students of Medicine,
+and arranged with questions following each chapter. By SIDNEY P.
+BUDGETT, M. D., formerly Professor of Physiology, Washington University,
+St. Louis. Revised by HAVAN EMERSON, M. D., Demonstrator of Physiology,
+Columbia University. 12mo volume of 250 pages, illustrated. Cloth, $1.00
+net. _Saunders' Question-Compend Series._
+
+     "He has an excellent conception of his subject.... It is one
+     of the most satisfactory books of this class"--_University
+     of Pennsylvania Medical Bulletin._
+
+
+    Leroy's Histology      New (4th) Edition
+
+ESSENTIALS OF HISTOLOGY. By LOUIS LEROY, M. D., Professor of Histology
+and Pathology, Vanderbilt University, Nashville, Tennessee. 12mo, 263
+pages, with 92 original illustrations. Cloth, $1.00 net. _In Saunders'
+Question-Compend Series._
+
+     "The work in its present form stands as a model of what a
+     student's aid should be; and we unhesitatingly say that the
+     practitioner as well would find a glance through the book of
+     lasting benefit."--_The Medical World_, Philadelphia.
+
+
+Barton and Wells' Medical Thesaurus
+
+A THESAURUS OF MEDICAL WORDS AND PHRASES. By WILFRED M. BARTON, M. D.,
+Assistant Professor of Materia Medica and Therapeutics, and WALTER A.
+WELLS, M. D., Demonstrator of Laryngology, Georgetown University,
+Washington, D.C. 12mo, 534 pages. Flexible leather, $2.50 net; thumb
+indexed, $3.00 net.
+
+
+    American Pocket Dictionary      New (8th) Edition
+
+DORLAND'S POCKET MEDICAL DICTIONARY. Edited by W. A. NEWMAN DORLAND, M.
+D., Editor "American Illustrated Medical Dictionary." Containing the
+pronunciation and definition of the principal words used in medicine and
+kindred sciences, with 64 extensive tables. 677 pages. Flexible leather,
+with gold edges, $1.00 net; with patent thumb index, $1.25 net.
+
+     "I can recommend it to our students without reserve."--J. H.
+     HOLLAND, M.D., _of the Jefferson Medical College_,
+     Philadelphia.
+
+
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