diff options
| author | nfenwick <nfenwick@pglaf.org> | 2025-02-05 08:13:22 -0800 |
|---|---|---|
| committer | nfenwick <nfenwick@pglaf.org> | 2025-02-05 08:13:22 -0800 |
| commit | 683335dde9e708055692b736c42f4c9d86b6ebf9 (patch) | |
| tree | e5ac7abe2236e055dfe7d22617fb7002b9a49b64 /old/51169-h/51169-h.htm | |
| parent | 81851ab07f33b6c7717add3800e50efb671e4222 (diff) | |
Diffstat (limited to 'old/51169-h/51169-h.htm')
| -rw-r--r-- | old/51169-h/51169-h.htm | 5505 |
1 files changed, 0 insertions, 5505 deletions
diff --git a/old/51169-h/51169-h.htm b/old/51169-h/51169-h.htm deleted file mode 100644 index 64406cd..0000000 --- a/old/51169-h/51169-h.htm +++ /dev/null @@ -1,5505 +0,0 @@ -<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Strict//EN" - "http://www.w3.org/TR/xhtml1/DTD/xhtml1-strict.dtd"> -<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"> - <head> - <meta http-equiv="Content-Type" content="text/html;charset=utf-8" /> - <meta http-equiv="Content-Style-Type" content="text/css" /> - <title> - The Project Gutenberg eBook of Section Cutting and Staining, by W. S. Colman. - </title> - <link rel="coverpage" href="images/cover.jpg" /> - <style type="text/css"> - -body { - margin-left: 10%; - margin-right: 10%; -} - -h1, h2, h3 { - text-align: center; - clear: both; -} - -h1 span.t1 { - display: block; - letter-spacing: .2em; - line-height: 1.5em; -} - -h1 span.t2 { - display: block; - font-size: 50%; - margin-top: 1.5em; - margin-bottom: 1.5em; -} - -h1 span.t3 { - display: block; - font-size: 60%; - margin-top: 3em; - margin-bottom: 4em; - letter-spacing: -0.01em; - word-spacing: 0.2em; -} - -h2 {page-break-before: avoid; - font-size: 140%; - -} - -h3 {font-weight: normal; - margin-top: 1.6em; -} - -div.tp1 { - display: block; - text-align: center; - font-size: 85%; - margin-top: 5em; - margin-bottom: 1em; -} - -div.tp2 { - display: block; - text-align: center; - font-size: 150%; - margin-bottom: 0.4em; - letter-spacing: 0.04em; -} - -div.tp3 { - text-align: center; - display: block; - font-size: 70%; -} - -div.tp4 { - text-align: center; - display: block; - font-size: 100%; - margin-bottom: 4em; -} - -p { - text-indent: 1.5em; - text-align: justify; - margin-top: 0em; - margin-bottom: 0em; - line-height: 1.3em; -} - -p.sig { - text-align: right; - margin-right: 5%; -} - -div.caption { - margin-left: auto; - margin-right: auto; - text-align: center; - margin-bottom: 1em; -} - -.epubonly { - display: none; visibility: hidden; -} - -@media handheld { - .epubonly {display: inline; visibility: visible;} - .htmlonly {display: none; visibility: hidden;} -} - -/* Horizontal rules */ -hr { - width: 33%; - margin-top: 2em; - margin-bottom: 2em; - margin-left: 33.5%; - margin-right: 33.5%; - clear: both; -} - -hr.chap { - width: 65%; - margin-left: 17.5%; - margin-right: 17.5%; - margin-top: 2em; - margin-bottom: 2em; -} - -hr.r33 { - width: 33%; - margin-left: 33.5%; - margin-right: 33.5%; -} - -hr.r10 { - width: 10%; - margin-left: 45%; - margin-right: 45%; - margin-top: 1em; - margin-bottom: 1em; -} - -/* Tables */ -table { - margin-left: 3em; - margin-right: auto; - font-size: 95%; - line-height: 1.2em; - border-collapse: collapse; -} - -table#toc { - margin-left: auto; -} - -span.ilb { /*to prevent inappropriate text wrapping in epub version*/ - display: inline-block; - white-space: nowrap; -} - -.tal { - text-align: left; -} - -.tar { - text-align: right; -} - -.tac { - text-align: center; -} - -.vab { - vertical-align: bottom; -} - -.plhi1 { - padding-left: 1em; - text-indent: -1em; -} - -.pt03 { - padding-top: 0.3em; -} - -.pt1 { - padding-top: 1em; -} - -.mb08em { - margin-bottom: 0.8em; -} - -.mb2em { - margin-bottom: 2em; -} - -.ml2em { - margin-left: 2em; -} - -.ml20pc { - margin-left: 20%; -} - -.ti0em { - text-indent: 0em; -} - -.fs70 {font-size: 70%;} - -.fs110 {font-size: 110%;} - -.hide { - visibility: hidden; -} - -.nowrap { - white-space: nowrap; -} - -.center { - text-align: center; -} - -.smcap { - font-variant: small-caps; -} - -sup { - vertical-align: baseline; - font-size: 80%; - position: relative; - top: -0.4em; -} - -sub { - vertical-align: baseline; - font-size: 80%; - position: relative; - top: 0.3em; -} - -@media handheld { -sup { - vertical-align: baseline; - font-size: 80%; - position: relative; - top: -0.4em;} - -sub { - vertical-align: baseline; - font-size: 80%; - position: relative; - top: 0.3em;} -} - -/* page numbering and hyperlinks */ -.pagenum { - position: absolute; - left: 92%; - font-size: 11px; - font-weight: normal; - font-style: normal; - font-variant: normal; - text-align: right; - text-indent: 0em; - color: #585858; -} - -span[title].pagenum:after { - content: attr(title); -} - -a[name] { - position:absolute; /* Fix Opera bug */ -} - -a:link { - text-decoration: none; - color: black; - border-bottom: 1px dotted #000000; -} - -a:link:hover { - background: aqua; -} - -/*fractions*/ -.fraction { - display: inline-block; - vertical-align: middle; - position: relative; - top: 0.1em; - text-align: center; - text-indent: 0em; - font-size: 65%; - line-height: 0.4em; -} - -.fraction>span { - display: block; - padding: 0.1em; -} - -.fnum { - position: relative; - top: -0.25em; -} - -.fraction span.bar { - display: none; -} - -.fraction span.fden { - border-top: thin solid black; - line-height: 0.6em; - padding-top: 0.2em; -} - -.prime { - font-size: 105%; - position: relative; - top: -0.15em -} - -/* Illustrations */ -.figcenter { - margin-left: auto; - margin-right: auto; - margin-top: 1.5em; - text-align: center; - font-size: 90%; -} - -.figcenter div { - text-align: justify; -} - -/* Footnotes */ -.footnotes { - border: dashed 1px; - padding-bottom: 2em; -} - -.footnote p { - margin-left: 10%; - margin-right: 10%; - font-size: 0.9em; - text-indent: 0em; -} - -.footnote .label { - position: absolute; - right: 84%; - text-align: right; - text-indent: 0em; -} - -.label:hover { - background: aqua; -} - -.fnanchor { - vertical-align: baseline; - position: relative; - top: -0.4em; - margin-left: 0.05em; - font-size: 11px; - font-weight: normal; - font-style: normal; - text-decoration: none; - white-space: nowrap; -} - -/* Transcriber's notes */ -.transnote { - background-color: #F5F5F5; - color: black; - font-size: 85%; - padding: 0.5em; - margin-bottom: 5em; - font-family: sans-serif, serif; -} - -ins { - text-decoration: none; - border-bottom: 1px dashed red; -} - - </style> - </head> -<body> - - -<pre> - -Project Gutenberg's Section Cutting and Staining, by Walter S. Colman - -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/license - - -Title: Section Cutting and Staining - A practical introduction to histological methods for - students and practitioners - -Author: Walter S. Colman - -Release Date: February 10, 2016 [EBook #51169] - -Language: English - -Character set encoding: UTF-8 - -*** START OF THIS PROJECT GUTENBERG EBOOK SECTION CUTTING AND STAINING *** - - - - -Produced by Thiers Halliwell and The Online -Distributed Proofreading Team at http://www.pgdp.net (This -file was produced from images generously made available -by The Internet Archive) - - - - - - -</pre> - - -<div class="transnote"> -<p class="ti0em"><b><a id="Transcribers_notes"></a>Transcriber’s -notes</b>:</p> - -<p class="ti0em mb08em">Apart from the following corrected -misspellings the text of this book has been preserved as in the -original:<br /> - xolol → xylol<br /> - side → slide<br /> - overstraining → overstaining</p> - -<p class="ti0em mb08em">In this e-text version a black dotted underline -indicates a link to a page, illustration or footnote – links are also -highlighted when the mouse pointer hovers over them. Page numbers are -shown in the right margin. Footnotes are located at the end of the -book.</p> - -<p class="ti0em mb08em">Where appropriate, illustrations and footnotes -have been positioned adjacent to the relevant text.</p> - -<p class="ti0em mb08em epubonly">The text contains numerous tables -that might not display correctly on handheld devices and also archaic -symbols that might not display at all if suitable fonts are not -available.</p> - -</div> - -<h1><span class="t1">SECTION CUTTING</span> -<span class="t2">AND</span> -<span class="t1">STAINING</span> - -<span class="t3">A PRACTICAL INTRODUCTION TO HISTOLOGICAL METHODS FOR<br /> -STUDENTS AND PRACTITIONERS</span></h1> - - -<div class="tp1">BY</div> -<div class="tp2">W. S. COLMAN, M.D., M.R.C.P.</div> - -<div class="tp3">ASSISTANT PHYSICIAN (FORMERLY PATHOLOGIST) TO THE NATIONAL<br /> -HOSPITAL FOR THE PARALYSED AND EPILEPTIC; AND TO<br /> -THE HOSPITAL FOR SICK CHILDREN, GREAT<br /> -ORMOND STREET, ETC.</div> - - -<div class="tp1">SECOND EDITION</div> - -<div class="tp4"><i>ENLARGED AND IN MOST PART RE-WRITTEN</i></div> - - -<div class="tp4">LONDON<br /> -H. K. LEWIS, 136 GOWER STREET, W.C.<br /> -1896</div> - - -<div class="tp3">PRINTED BY<br /> -H. K. LEWIS, 136 GOWER STREET,<br /> -LONDON, W.C.</div> - - -<hr class="chap" /> - - -<h2>PREFACE TO THE SECOND EDITION.</h2> - -<hr class="r10" /> - -<p>In preparing this edition I have endeavoured to -meet the requirements of students, and of practitioners -who desire to keep up their histological -work. Those methods are selected which have -been found to work well in practice, and it has -been thought better to describe a few in detail -rather than give a short account of many similar -methods.</p> - -<p>I have again to express my obligation to the -various instrument makers for the illustrations of -microtomes, &c.; to Dr. Fearnley, of Bradford, -for the description of his method for injecting -blood vessels, and to Messrs. Macmillan and Co. -for permission to copy figures 10 and 11.</p> - -<p class="sig"> -W. S. COLMAN.</p> -<p class="ml2em"> -Wimpole Street, W.<br /> -  <i>Sept., 1896.</i> -</p> - - -<hr class="chap" /> - - -<h2>CONTENTS.</h2> - -<hr class="r10" /> - - -<div class="center"> -<table id="toc" border="0" cellpadding="2" cellspacing="0" summary="table of contents"> -<tr><td class="tac pt1 fs110" colspan="2">CHAPTER I.</td></tr> -<tr><td class="tal"></td><td class="tar fs70">PAGE</td></tr> -<tr><td class="tal"><span class="smcap">Apparatus Required</span></td><td class="tar vab"><a href="#Page_1">1</a></td></tr> -<tr><td class="tac pt1 fs110" colspan="2">CHAPTER II.</td></tr> -<tr><td class="tal"><span class="smcap">Hardening Processes</span></td><td class="tar vab"><a href="#Page_15">15</a></td></tr> -<tr><td class="tac pt1 fs110" colspan="2">CHAPTER III.</td></tr> -<tr><td class="tal"><span class="smcap">Section Cutting</span></td><td class="tar vab"><a href="#Page_29">29</a></td></tr> -<tr><td class="tac pt1 fs110" colspan="2">CHAPTER IV.</td></tr> -<tr><td class="tal"><span class="smcap">Section Mounting</span></td><td class="tar vab"><a href="#Page_55">55</a></td></tr> -<tr><td class="tac pt1 fs110" colspan="2">CHAPTER V.</td></tr> -<tr><td class="tal"><span class="smcap">General Staining Methods</span></td><td class="tar vab"><a href="#Page_67">67</a></td></tr> -<tr><td class="tac pt1 fs110" colspan="2">CHAPTER VI.</td></tr> -<tr><td class="tal"><span class="smcap">Special Methods for Staining the Nerve Centres</span></td><td class="tar vab"><a href="#Page_87">87</a></td></tr> -<tr><td class="tac pt1 fs110" colspan="2">CHAPTER VII.</td></tr> -<tr><td class="tal plhi1"><span class="smcap">Special Methods for Staining Micro-Organisms and Blood</span> </td><td class="tar vab"><a href="#Page_103">103</a></td></tr> -<tr><td class="tac pt1 fs110" colspan="2">CHAPTER VIII.</td></tr> -<tr><td class="tal"><span class="smcap">Injection of Blood Vessels</span></td><td class="tar vab"><a href="#Page_120">120</a></td></tr> -<tr><td class="tac pt1 fs110" colspan="2">CHAPTER IX.</td></tr> -<tr><td class="tal"><span class="smcap">Directions for Preparing Individual Tissues</span></td><td class="tar vab"><a href="#Page_129">129</a></td></tr> -<tr><td class="tal pt1"><span class="smcap">Index</span></td><td class="tar vab"><a href="#Page_153">153</a></td></tr> -<tr><td class="tal"><span class="pagenum" title="1"><a name="Page_1" id="Page_1"></a></span></td></tr> -</table></div> - - -<hr class="chap" /> - - -<h2>SECTION CUTTING AND STAINING.</h2> - -<hr class="r33" /> - - -<h2>CHAPTER I.</h2> - -<h3><span class="smcap">Apparatus Required.</span></h3> - - -<p>Probably there is nothing more perplexing to a -beginner than to decide what apparatus is required. -If he consult a price list, it is difficult -for him to tell which articles will be necessary, -and which will be either luxuries, or required -only for special investigation.</p> - -<p>In the following account of requisites, those -only will be described which it is useful to have -always at hand. They will be found sufficient -for ordinary work, but for special investigations -a more elaborate equipment will be required.</p> - -<p>All staining and other reagents should be made -as far as possible by the worker himself, according -to the directions given in later chapters. -This should at any rate be done at first, as the -knowledge thus gained will prove invaluable. It<span class="pagenum" title="2"><a name="Page_2" id="Page_2"></a></span> -will also effect a great saving if articles that are -used in any quantity, such as methylated spirit, -distilled water, &c., are bought by the gallon, and -not in small quantities.</p> - -<p>Almost all the processes described here can be -carried out without the use of a fully equipped -laboratory, in fact, in an ordinary room. The -only furniture required is a firm table, and a -cupboard and shelves for storing reagents.</p> - -<p>The following should also be <span class="nowrap">procured:—</span></p> - -<p><b>Jars</b> or <b>bottles</b>, with well fitting stoppers or -corks, to contain the tissues while being hardened. -They should not hold less than two ounces. -Empty drug bottles which can usually be obtained -from druggists for a few pence, serve very -well.</p> - -<p>Smaller bottles should also be procured for -keeping specimens in spirit after they have been -hardened until one is ready to cut sections. After -sections have been cut from a portion of the -specimen, the rest should be preserved, in case -it is wanted for further investigation. Each specimen -must be labelled, with a name or a number -corresponding to a reference in the note-book,<span class="pagenum" title="3"><a name="Page_3" id="Page_3"></a></span> -and a large number of specimens may then be -kept in the same jar. The best way to label them -is to write the name or number on a piece of -vegetable parchment in ordinary “marking ink,” -and warm it until the writing is black. The little -label should then be fixed to a corner of the piece -of tissue with a stitch or by a fine pin, and it may -be identified years afterwards. The importance -of keeping tissues, sections, slides, &c., <b>distinctly -labelled</b> cannot be too strongly impressed -on the beginner. The name, date, and -other particulars should be invariably written on -the label at the time. At first the student will be -inclined to neglect this, as he will recognize his -pieces of tissue and sections so readily merely by -their shape and general appearance. But as time -elapses and similar specimens accumulate, he will -find it most difficult or even impossible to identify -one from the other.</p> - -<p>A number of 1 oz. and 2 oz. <b>stoppered -bottles</b> for staining reagents.</p> - -<p>The stopper of these should be fitted with a rod. -This is done by simply heating the lower end of -the stopper and the upper end of a piece of glass<span class="pagenum" title="4"><a name="Page_4" id="Page_4"></a></span> -rod of suitable length in a blow-pipe, until they -are plastic, and then pressing them together.</p> - -<p><b>Watch glasses.</b>—At least a dozen watch-glasses, -in which to perform the operations of -staining, clarifying, &c. Those with a flat bottom -should be employed as they are less easily upset -than the others.</p> - -<p>Plenty of <b>filter papers</b>.</p> - -<p>Both coarse ones, for use in the manufacture of -reagents, and small fine white ones (<span class="nowrap">2 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span> inch) for -filtering the staining fluids immediately before -using them, should be procured. Before using -them a few drops of alcohol or distilled water -should be placed in them to saturate the paper. -This not only allows the fluid to pass through -more rapidly, but prevents a portion of it being -wasted through being absorbed by the pores of -the paper.</p> - -<p>Several <b>needles</b> mounted in handles.</p> - -<p>They must be kept very bright and smooth, and -care must be taken that the point does not get -turned up.</p> - -<p>A large and small <b>funnel</b>.</p> - -<p>Several <b>pipettes</b> consisting of pieces of glass<span class="pagenum" title="5"><a name="Page_5" id="Page_5"></a></span> -tube with an internal diameter of <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">8</span></span><span class="prime">″</span></span> and about -ten inches long, drawn out almost to a point at -one end.</p> - -<p><b>Section lifter.</b>—This instrument is required -for transferring sections from one reagent to -another, or from oil of cloves, &c., to the slide. -The most convenient form is Woodhead’s, made -of thin sheet copper, which allows the blade to -be bent at any angle to the stem. The stem or -handle is about six inches long, and continuous -with, and at an angle to it, a flat blade about <span class="nowrap"> <span class="fraction"><span class="fnum">3</span><span class="bar">/</span><span class="fden">4</span></span></span> in. -square with the corners rounded off. Larger ones -can be obtained for mounting sections of large -size, <i>e.g.</i>, kidney, medulla oblongata, &c. The -surface of the blade should be brightly polished, -and kept scrupulously clean.</p> - -<p>Ordinary dissecting <b>forceps</b>.</p> - -<p>One or two <b>scalpels</b>.</p> - -<p>A pair of fine <b>scissors</b>.</p> - -<p>A <b>razor</b> or other instrument for cutting sections.</p> - -<p>A smooth <b>oil stone</b> for keeping the razors and -knives properly sharpened.</p> - -<p>A <b>spirit lamp</b> for warming the staining fluids.</p> - -<p><span class="pagenum" title="6"><a name="Page_6" id="Page_6"></a></span></p> - -<p>A few <b>test tubes</b>.</p> - -<p>A <b>minim measure</b>.</p> - -<p><b>Scales</b> and small weights.</p> - -<p>A gross of ground glass slides 3 x 1 in.</p> - -<p>Half a gross of ground glass slides 3 x <span class="nowrap">1 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span> in.</p> - -<p>Half an ounce of thinnest coverslips, <span class="nowrap"> <span class="fraction"><span class="fnum">7</span><span class="bar">/</span><span class="fden">8</span></span></span> in. diameter.</p> - -<p>Quarter of an ounce of thinnest coverslips, <span class="nowrap">1 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">4</span></span></span> in. -diameter.</p> - -<p><b>Microscope.</b>—This is not the place for a description -of the microscope as an optical instrument, -but some hints as to the selection of one -may be found useful.</p> - -<p>Showy microscopes with much brass work -should be avoided, simplicity of construction -being a great recommendation. The microscope -should have a large heavy base, either of the -horse-shoe or tripod pattern, large enough to afford -a firm base when the microscope is tilted.</p> - -<p>Mechanical stages are unnecessary and they -add greatly to the expense, and very little to the -utility of the instrument for ordinary histological -work. Binocular arrangements also are of little -use for this purpose.</p> - -<p><span class="pagenum" title="7"><a name="Page_7" id="Page_7"></a></span></p> - -<p>The microscope should be provided with a -<b>coarse and fine adjustment</b>, which should -be most carefully tested before purchasing the instrument. -They should work freely and smoothly, -and the slightest turn in either direction should at -once alter the focus.</p> - -<p>There should be a <b>reversible mirror</b>, one -side being concave and the other plane. The -concave surface is the one usually employed, the -plane surface being chiefly used in conjunction -with the sub-stage condenser for the examination -of micro-organisms. There should be an <b>eye-piece</b> -of moderate magnifying power. Very -powerful eye-pieces do not reveal additional details, -but merely enlarge the image, and with it -any defects that may be produced there by faults -in the objective. Eye-pieces II. and IV. of most -makers will be ample for most requirements.</p> - -<p><b>Objectives.</b>—These are the most important -parts of the microscope, and the student will be -well advised if he spends a little extra money to -secure good lenses.</p> - -<p>Most objectives and stands are now made with -a universal thread, so that any objective will fit<span class="pagenum" title="8"><a name="Page_8" id="Page_8"></a></span> -any make of stand. Many workers provide themselves -with a cheap stand such as that supplied -by Leitz, and then fit it with lenses by Zeiss, or -other first class maker.</p> - -<p>The most useful lenses are the 1 in. low power -lens, and <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">5</span></span></span> in. or <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">6</span></span></span> in. high power, or No. 3 and -No. 7 of Continental makers, or Zeiss’s A and D. -A <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span> in. lens will also be found very useful.</p> - -<p>For minute work, such as bacteriology and -blood investigations, higher powers will be required, -<span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">8</span></span></span> or <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">12</span></span></span> <b>immersion lenses</b>. These -objectives come extremely close to the object, and -very thin cover glasses must be employed. In -order to avoid the refraction caused by the rays -traversing the air between the coverslip and lens -some immersion fluid is placed between the two. -With some lenses water is employed, but usually -an oil having the same refractive index as glass is -used, and the one most generally employed is cedar -oil (Zeiss prefers the oil from the species Juniperus -virginiana). A spot of oil is placed with a rod -just over the object to be examined and the objective -carefully lowered by the coarse adjustment -till it comes in contact with the droplet of oil.<span class="pagenum" title="9"><a name="Page_9" id="Page_9"></a></span> -The focussing should then be managed with the -fine adjustment only.</p> - -<p>When the section has been examined the oil -must be removed from the lens. For this purpose -a soft silk handkerchief or a special piece of -chamois leather may be employed, and used very -gently. If all the oil cannot be removed, the -handkerchief may be moistened with a little absolute -alcohol, and the lens hastily wiped. The -alcohol must not be allowed to remain in contact -with the lens as it is a solvent of Canada balsam -with which the lenses are often cemented in -position.</p> - -<div class="figcenter" style="width: 240px;"> -<img src="images/i017.jpg" width="190" height="197" alt="" /> -<div class="caption"><p class="tac"><span class="smcap">Fig. 1.</span>—Double or Triple Nose-piece.</p></div> -</div> - -<p><b>Double or triple nose-piece</b> (fig. 1).—This -mechanical arrangement is placed on the lower<span class="pagenum" title="10"><a name="Page_10" id="Page_10"></a></span> -end of the tube. Two or three objectives of different -magnifying power are attached to it. The -nose-piece rotates round a central pivot in such -a way that the objectives can successively be -brought accurately into position above the object -on the stage. It is, therefore, a moment’s work to -replace a high power objective by a low power -one and <i>vice versa</i>. It is an extremely convenient -time-saving appliance, and by its use the risk of -dropping and injuring the objectives when screwing -them on and off frequently is avoided. Those -whose microscopes are not already fitted with this -appliance can easily have one fitted on at a cost of -about a sovereign.</p> - -<p><b>Substage condenser.</b>—This mechanism for -concentrating light on the object is a necessity for -bacteriological work. The most convenient form -is <b>Abbe’s illuminating apparatus</b> (fig. 2).</p> - -<p>This consists of a system of short focus lenses -which collects the light received by the mirror, -and throws it on the object. The amount of -light received from the mirror is controlled by -an “iris diaphragm,” the aperture of which can -be dilated or contracted by moving a small lever<span class="pagenum" title="11"><a name="Page_11" id="Page_11"></a></span> -at the side. It can be fitted on to most microscope -stands, but it is better to get a stand in the -first instance which is constructed to carry one.</p> - -<p>The cost of a microscope varies from two -guineas to two hundred. There are many excellent -microscopes in the market, and of these -several may be mentioned which the writer has -found to work satisfactorily.</p> - -<div class="figcenter" style="width: 255px;"> -<img src="images/i019.jpg" width="255" height="233" alt="" /> -<div class="caption"><p class="tac"><span class="smcap">Fig. 2.</span>—Abbe’s Illuminating Apparatus.</p></div> -</div> - -<p>Of the cheaper student’s microscopes the “Star” -microscope made by Messrs. R. and J. Beck, of -Cornhill, E.C., will be found a safe investment. -It may be obtained with coarse and fine adjustment, -nose-piece, and 1 in. and <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">4</span></span></span> in. objectives,<span class="pagenum" title="12"><a name="Page_12" id="Page_12"></a></span> -for about £5. Those who require a better instrument -will find Beck’s “Pathological” microscope -fitted with nose-piece, Abbe’s illuminator, &c., for -£16, meet all requirements.</p> - -<p>Leitz of Jena, supplies two good and cheap -microscopes for £3 10<i>s.</i> and £5. They are not, -however, of uniform excellence, and they should -be carefully tested by some competent judge before -the purchase is completed. Leitz immersion -lenses are cheap, and often extremely good, but -should be carefully tested beforehand, as their -quality is not quite uniform. The microscopes -can be obtained from Mr. A. Frazer, Teviot Place, -Edinburgh.</p> - -<p>The “Bacteriological” microscope, made by -Messrs. Swift, of Tottenham Court Road, is one -with which no one can be disappointed. It is sold -with Abbe’s condenser, triple nose-piece, <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">6</span></span></span> in., -and a <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">12</span></span></span> in. immersion objective, for just under -£20. Both stand and lenses are turned out in -Swift’s first-class style, and those who can afford -the initial outlay will not regret it. Or the stand -may be purchased, and the objectives and accessories -added singly from time to time.</p> - -<p><span class="pagenum" title="13"><a name="Page_13" id="Page_13"></a></span></p> - -<p>Among Continental makers, excellent microscopes -for histological work are turned out by -many makers. Zeiss’s lenses stand deservedly -high in reputation, as no faulty Zeiss lens ever -leaves the works, and their optical properties are -nearly perfect. For this guarantee, however, the -purchaser has to pay somewhat higher prices, but -the money is well invested. Zeiss’s agency is at -29 Margaret Street, Regent Street, W.</p> - -<p>Reichert, of Vienna, sells microscopes and lenses -which are modelled on the lines of those of Zeiss, -and though cheaper are often equal to them in -excellence, but the quality is not quite uniform. -His instruments can be obtained through any -optician, but his agent in this country is Mr. A. -Frazer, Teviot Place, Edinburgh.</p> - -<p>Before buying a microscope the student should -obtain an illustrated price list from any of the -firms mentioned above, and, having selected an -instrument, he should test it very carefully, or -better get some experienced friend to test it for -him, before deciding to purchase it. Delicate test -objects such as diatoms, scales of butterfly’s wing, -or a stained specimen of micro-organisms should<span class="pagenum" title="14"><a name="Page_14" id="Page_14"></a></span> -be employed. The coarse and fine adjustments -should be tried. They should work freely and -smoothly and without any delay. The definition -of the lens must be tested with the fine objects -mentioned. The field should be quite flat, <i>i.e.</i>, -every part should be in focus at the same time, -and the definition should be perfectly sharp and -accurate, and the test objects without double contour. -The field should be totally free from prismatic -colours. If there is a halo of colour around -the objects it indicates a defect in the optical -properties of the objective, and another should be -selected.</p> - -<p>A microscope must always be treated with the -greatest care. Jars and falls tend to slightly -loosen and shift the lenses, and to permanently -impair its optical properties. Dust must be most -carefully excluded. This is best effected by keeping -the instrument under a glass bell jar when not -in use. The lenses should be wiped as little as -possible, and when it is necessary, very soft -chamois leather should be employed. The microscope -must be kept in a dry room, or the brass -work will soon tarnish and the steel parts will -tend to rust.</p> - -<hr class="chap" /> - -<p><span class="pagenum" title="15"><a name="Page_15" id="Page_15"></a></span></p> - - - - -<h2>CHAPTER II.</h2> - -<h3><span class="smcap">Hardening Processes.</span></h3> - - -<p>For the satisfactory examination of tissues it is -necessary that they should be “hardened” in -certain fluids. The object of this is to give the -specimens greater consistence, so that thin sections -may be more readily obtained and more -safely manipulated, and also to “fix” the tissue -element as far as possible in the same relative -position as in the living body. The hardening -process also acts on the protoplasm of the cells, -and prevents their swelling up when placed in -water, and in the various staining fluids.</p> - -<p>The fluid used must be one which will not itself -injure the specimen, and which can be thoroughly -removed by washing, so that it may not interfere -with staining operations. The specimens should -be kept while hardening in wide mouthed bottles, -on the bottom of which a little cotton wool or tow -has been laid. This allows the hardening fluid to<span class="pagenum" title="16"><a name="Page_16" id="Page_16"></a></span> -come freely in contact with the under surface of -the pieces of tissue, and prevents their being flattened -against the hard glass bottom.</p> - -<p>The hardening fluid requires changing occasionally. -This should always be done at the end of -twenty-four hours, in order to get rid of any deposit -of blood, &c., that may have accumulated. -Besides this, the tissue when placed in the fluid -contained a good deal of water which will have -diluted it and consequently an early change is -desirable. Afterwards the fluid requires to be -changed only as often as it becomes turbid, or any -deposit occurs, usually about once a week.</p> - -<p>While hardening, specimens should be kept in a -cool place, as warmth favours changes in the -cells, &c.</p> - -<p>In manipulating the portions of organs, forceps -should always be used and these with great gentleness. -The specimens should never be impaled -with needles, or unsightly holes, which may even -be mistaken for pathological appearances, will -appear when a section is examined under the -microscope.</p> - -<p>It requires some practice to know when the<span class="pagenum" title="17"><a name="Page_17" id="Page_17"></a></span> -tissue is sufficiently hardened. The object aimed -at is to make them not really hard but tough. It -is almost unnecessary to add that in testing this -with the fingers the utmost gentleness must be -observed, or serious damage may be done to the -tissue.</p> - -<p>When the tissue is sufficiently hardened the -hardening fluid must be thoroughly dissolved out. -This is most quickly effected by placing the -specimen in a basin into which cold water from a -tap is constantly running. The tissue may then -be removed (forceps always being used and never -the needle) and placed in an imbedding medium -as subsequently directed; or, if it is not to be cut -at once, into equal parts of methylated spirit and -water, in which it may be kept indefinitely, the -fluid being changed if it becomes at all cloudy.</p> - -<p>It is unnecessary for ordinary work to have -more than the following hardening fluids:—<br /><b>Müller’s -fluid</b><span class="nowrap">:—</span></p> - - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Potassium Bichromate</td><td class="tar"><span class="nowrap">2 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">4</span></span></span></td><td class="tal">grms.</td><td class="tar"> <span class="nowrap">3 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">drachms.</span></td></tr> -<tr><td class="tal">Sodium Sulphate</td><td class="tar">1</td><td class="tal">grm.</td><td class="tar"><span class="nowrap">1 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">drachms.</span></td></tr> -<tr><td class="tal">Water to</td><td class="tar"> 100</td><td class="tal">c.c.</td><td class="tar">1</td><td class="tal">pint.</td></tr> -</table></div> - - -<p>Two drachms of carbolic acid are sometimes<span class="pagenum" title="18"><a name="Page_18" id="Page_18"></a></span> -added to each pint of the fluid but as a rule it is -not necessary.</p> - -<p>Müller’s fluid is the most generally useful of the -various fluids employed, for the following <span class="nowrap">reasons:—</span></p> - -<p>1. It causes very little shrinking of the elements -of the tissue, and hence may be employed for most -delicate objects, <i>e.g.</i>, the retina and embryos.</p> - -<p>2. In consequence of its not making the tissues -shrink, it does not squeeze the blood out of -the vessels and where the organ has been congested -before death, we may, by using Müller’s -fluid, preserve a natural injection of the capillaries.</p> - -<p>3. There is comparatively little danger of over-hardening -the tissue and rendering it brittle.</p> - -<p>4. Sections of organs hardened in Müller’s fluid -are usually firm and easy to manipulate. They do -not tend to curl up or adhere to one another as -much as those hardened in spirit.</p> - -<p>5. It readily permeates the tissues, and hence -large portions of organs, or even the entire organ -may be satisfactorily hardened in it.</p> - -<p>6. It is very cheap. A gallon can be made up -for about eightpence.</p> - -<p><span class="pagenum" title="19"><a name="Page_19" id="Page_19"></a></span></p> - -<p>The fluid has however certain slight <span class="nowrap">drawbacks:—</span></p> - -<p>1. The hardening process is a slow one occupying -four to eight weeks.</p> - -<p>2. The fluid gives a permanent dingy colour to -the tissue. This does not cause any inconvenience -for microscopic purposes, but it is a disadvantage -when it is intended to preserve the rest of -the specimen, as a naked eye preparation. In -such cases the organ should be hardened in spirit, -carbolic acid, or formal.</p> - -<p>Müller’s fluid can be used for almost any tissue. -It is especially useful for those which contain a -large quantity of fluid, or of blood, and is essential -for nerve tissues which it is intended to stain by -Pal’s method (p. <a href="#Page_89">89</a>).</p> - -<p>To harden a specimen in it at least twenty times -the bulk of fluid must be employed.</p> - -<p>The fluid must be changed on the third day, and -afterwards about every week as may be required.</p> - -<p><b>Methylated spirit</b> is a very useful hardening -agent. It hardens in one to three weeks according -to the size of the tissue and the quantity of -spirit used. Its disadvantages <span class="nowrap">are:—</span></p> - -<p><span class="pagenum" title="20"><a name="Page_20" id="Page_20"></a></span></p> - -<p>1. It is more apt to overharden than Müller’s -fluid.</p> - -<p>2. It causes a great deal of shrinking of the -tissue and thus squeezes much of the blood out of -the vessels.</p> - -<p>It is most useful in hardening tissues containing -much epithelium, <i>e.g.</i>, kidney, epithelioma, &c.</p> - -<p>Spirit is also frequently employed to complete -the hardening by Müller’s fluid and to preserve -tissues after they have been hardened.</p> - -<p>About ten or fifteen times the bulk of spirit -should be used for one of the tissues. The fluid -should be changed on the third day and afterwards -as required.</p> - -<p><b>Müller’s fluid and spirit.</b>—This is a useful -combination for many purposes. It is made thus:—Müller’s -fluid, three parts; methylated spirit, one -part.</p> - -<p>The fluid must be allowed to cool after mixing -before being used, and if necessary filtered. It -will harden specimens satisfactorily in three -weeks.</p> - -<p><b>Müller’s fluid and formal.</b>—Is an extremely -useful mixture made by adding one part<span class="pagenum" title="21"><a name="Page_21" id="Page_21"></a></span> -of formal to nine of Müller’s fluid. It hardens in -a much shorter time than Müller’s fluid and -causes very little shrinkage.</p> - -<p><b>Absolute alcohol.</b>—Used as a hardening -agent where the tissues are to be examined for -micro-organisms, and for specimens to be stained -by Nissl’s method (p. <a href="#Page_101">101</a>). A cheaper and equally -effective hardening medium is made by dehydrating -methylated spirit by adding one ounce of -fused carbonate of potassium to each pint of -methylated spirit, and decanting.</p> - -<p>Small pieces must be used. The depths of the -block should not exceed <span class="nowrap"> <span class="fraction"><span class="fnum">3</span><span class="bar">/</span><span class="fden">8</span></span></span> inch. The fluid should -be changed on the third day. Hardening will be -completed in about ten days or even earlier.</p> - -<p><b>Osmic acid.</b>—For rapidity of action, and for -rapid fixing of all the tissue elements in their -natural position osmic acid is one of the best -hardening reagents we possess.</p> - -<p>Its disadvantages as a hardening agent <span class="nowrap">are:—</span></p> - -<p>1. Its expense.</p> - -<p>2. Its irritating and corrosive vapour.</p> - -<p>3. The fact that only small pieces of tissue can -be hardened in it, since the external surface is<span class="pagenum" title="22"><a name="Page_22" id="Page_22"></a></span> -very rapidly hardened and thus the fluid is prevented -from penetrating into the centre of the -lump.</p> - -<p>It is most frequently used as a hardening agent -for very delicate structures, such as the retina, or -embryos, or for fresh sections of brain (p. <a href="#Page_95">95</a>).</p> - -<p>The acid itself may be procured in sealed -tubes, each containing one gramme. These should -be broken in a bottle under sufficient distilled -water to make a one per cent. solution. The -bottle containing it should be covered with brown -paper to exclude the light. For hardening purposes -small pieces of the tissue, not much larger -than a pea, should be placed in the acid, the one -per cent. solution being diluted with five to ten -volumes of distilled water. The tissues may be -left in this for from three to five days. They -must then be <b>thoroughly</b> washed in distilled -water and may afterwards be preserved in methylated -spirit.</p> - -<p>Both the hardening and the subsequent washing -must be carried on <b>in the dark</b>.</p> - -<p>Osmic acid is also a most valuable staining -reagent (see p. <a href="#Page_81">81</a>).</p> - -<p><span class="pagenum" title="23"><a name="Page_23" id="Page_23"></a></span></p> - -<p><b>Carbolic acid</b> (5 per cent.).—May be used -to harden almost any tissue, but is particularly -useful for hardening nervous tissues such as brain -or spinal cord which are afterwards to be preserved -as museum specimens. It does not discharge -the colour of a specimen so rapidly as -spirit.</p> - -<p>Three or four times the bulk of fluid should be -used. It requires changing at the end of twenty-four -hours, and again at the end of the first -week.</p> - -<p>Saturated aqueous solution of <b>corrosive sublimate</b> -is one of the most convenient hardening -reagents for small pieces of delicate tissue, <i>e.g.</i>, -embryos. It hardens them in a few days. When -they are sufficiently hardened the mercurial salt -should be removed by washing first in methylated -spirit for a few hours and then in running -water.</p> - -<p><b>Formal.</b>—An aqueous solution containing -about 35 per cent. of formaldehyde. It is a -rapid hardening agent, causes very little shrinkage -of the tissues, and does not discharge the -colour of the specimens as much as alcohol. For<span class="pagenum" title="24"><a name="Page_24" id="Page_24"></a></span> -hardening formal should be used as a two to five -per cent. solution in distilled water. It may also -be used as a ten per cent. solution for mounting -museum preparations, but there is some tendency -for a cloudy deposit to form on the glass after a -time. It is the most suitable hardening agent at -our disposal for eyes. It rapidly fixes the tissue -elements, but does not cause much contraction. -It may also be used for hardening the brain and -spinal cord. Large quantities of fluid must be -used for the latter purpose and it must be frequently -changed. As soon as they are sufficiently -hardened they should be transferred to methylated -spirit.</p> - -<p><b>Marchi’s fluid.</b>—This consists <span class="nowrap">of:—</span></p> - - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Müller’s fluid</td><td class="tar"> 2</td><td class="tal">parts.</td></tr> -<tr><td class="tal">Osmic acid solution (one per cent.)</td><td class="tar">1</td><td class="tal">part.</td></tr> -</table></div> - - -<p>It is used for hardening specimens as a preliminary -to Golgi’s method for staining nerve -cells (p. <a href="#Page_97">97</a>), and also to complete the hardening -of sections of spinal cord, &c., before employing -Schäfer’s modification of the Weigert Pal hæmatoxyline -method (p. <a href="#Page_91">91</a>).</p> - -<p>It is also used as a stain for recently degener<span class="pagenum" title="25"><a name="Page_25" id="Page_25"></a></span>ated -nerve tracts and fibres, especially after experimental -lesions.</p> - -<p>The fluid has little penetrating power, and -therefore tissues must be cut into small pieces, -about <span class="nowrap"> <span class="fraction"><span class="fnum">3</span><span class="bar">/</span><span class="fden">8</span></span></span> inch cube. It is not necessary to place -them in this fluid at once on removal from the -body, but the preliminary hardening must be in -Müller’s fluid and not in alcohol, &c.</p> - - -<h3><span class="smcap">Special Hardening Reagents for Rapid Fixation -in Order to Study Cell Structure.</span></h3> - -<p>1. <b>Alcohol.</b></p> - -<p>2. <b>Flemming’s solution</b> (modified by Friedmann)<span class="nowrap">:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Osmic acid (one per cent.)</td><td class="tar">3</td><td class="tal">c.c.</td><td class="tal">(♏xxx.).</td></tr> -<tr><td class="tal">Glacial acetic acid</td><td class="tar">2</td><td class="tal">c.c.</td><td class="tal">(♏xx.).</td></tr> -<tr><td class="tal">Chromic acid (one per cent.)</td><td class="tar"> 42</td><td class="tal">c.c.</td><td class="tal">(℥j.)</td></tr> -</table></div> - -<p>Small pieces should be hardened in this fluid -for twelve to twenty-four hours, and then washed -and transferred to alcohol for some days before -staining.</p> - -<p>3. <b>Nitric acid.</b>—A ten per cent. solution in -distilled water. It hardens the tissue in three to<span class="pagenum" title="26"><a name="Page_26" id="Page_26"></a></span> -four hours, and should be followed by 70 per cent. -alcohol, the hardening being completed in absolute -alcohol.</p> - -<p>In using any of these methods it is necessary -that the tissue be removed from the body during -life or immediately after death. They are employed -for revealing the changes in the cells and -their nuclei in rapidly growing or inflamed tissue, -for studying karyokinesis in cancer cells, and investigating -the appearance of nerve cells and -gland cells at rest, when actively employed and -when fatigued; and they are also most useful in -preparing specimens of the “parasitic bodies” -which have been described in many cancer cells.</p> - - -<h3><span class="smcap">Decalcifying Fluids.</span></h3> - -<p>Used in the preparation of bone, tooth, osseous -tumours, &c. The two best fluids for general use -<span class="nowrap">are:—</span></p> - -<p><b>Chromic and nitric fluid.</b>—This is made -as <span class="nowrap">follows:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Chromic acid</td><td class="tar">1</td><td class="tal">gramme</td><td class="tar"> 45</td><td class="tal">grains.</td></tr> -<tr><td class="tal">Nitric acid</td><td class="tar">2</td><td class="tal">grammes</td><td class="tar"><span class="nowrap">1 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">drachms.</span></td></tr> -<tr><td class="tal">Water</td><td class="tar"> 200</td><td class="tal">c.c.</td><td class="tar">1</td><td class="tal">pint.</td></tr> -</table></div> - -<p><span class="pagenum" title="27"><a name="Page_27" id="Page_27"></a></span></p> - -<p>If the bone is not very compact the fluid may -be used diluted with an equal quantity of water. -A large quantity of fluid should be used, and -like all decalcifying fluids, it should be frequently -changed.</p> - -<p>As soon as the specimen is sufficiently flexible, -it should be thoroughly washed in running water -for some hours, and then transferred to spirit until -it is convenient to cut sections.</p> - -<p><b>Von Ebner’s solution</b><span class="nowrap">:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Hydrochloric acid</td><td class="tar">1</td><td class="tal">gramme</td><td class="tar"> <span class="nowrap">1 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">drachms.</span></td></tr> -<tr><td class="tal">Common salt</td><td class="tar">10</td><td class="tal">grammes</td><td class="tar">2</td><td class="tal">ounces.</td></tr> -<tr><td class="tal">Water to</td><td class="tar"> 100</td><td class="tal">c.c.</td><td class="tar">1</td><td class="tal">pint.</td></tr> -</table></div> - -<p>It is a very useful decalcifying agent, but causes -the fibrous elements to swell up rather more than -chromic and nitric fluid. A large quantity of the -fluid must be used, and it should be changed daily. -It must be very thoroughly washed out in running -water when the decalcification is completed.</p> - -<p><b>Bleaching solution</b> (eau de Javelle).</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(1)</td><td class="tal">“Chloride of lime” (bleaching powder)</td><td class="tar">20</td><td class="tar"><span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">oz.</span></td></tr> -<tr><td class="tal"></td><td class="tal">Water</td><td class="tar"> 100</td><td class="tar"> <span class="nowrap">2 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">oz.</span></td></tr> -</table></div> - -<p>Shake up well.</p> - -<p><span class="pagenum" title="28"><a name="Page_28" id="Page_28"></a></span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(2)</td><td class="tal">Carbonate of potassium</td><td class="tar">20</td><td class="tar"><span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tar"><span class="ilb">oz.</span></td></tr> -<tr><td class="tal"></td><td class="tal">Water</td><td class="tar"> 100</td><td class="tar"> <span class="nowrap">2 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">oz.</span></td></tr> -</table></div> - -<p>Mix the two solutions. Allow them to stand for -an hour and filter.</p> - -<p>It is used chiefly for clearing vegetable sections -but may also be used for sections containing a -large quantity of pigment. It is particularly useful -in decolourizing sections of “madura foot” due to -the presence of a black fungus.</p> - -<hr class="chap" /> - -<p><span class="pagenum" title="29"><a name="Page_29" id="Page_29"></a></span></p> - - - - -<h2>CHAPTER III.</h2> - -<h3><span class="smcap">Section Cutting.</span></h3> - - -<p><b>Embedding of sections.</b>—Before sections are -made the tissues require to be embedded in some -fluid, which will permeate their interstices, and is -capable of being rendered firm so as to support -the most delicate parts when the knife passes -through the tissue.</p> - -<p>The most generally useful substances <span class="nowrap">are:—</span></p> - -<p>(1) gum, (2) celloidin, (3) paraffin or wax.</p> - -<p><b>Gum.</b>—<i>Picked</i> colourless gum arabic 2 parts, -cold water 3 parts.</p> - -<p>Leave with frequent stirring until dissolved. -Add ten drops of carbolic acid to each ounce -of the mucilage.</p> - -<p>Specimens are thoroughly freed from all trace -of the hardening fluid by washing in water, and -the tissue is then placed in the gum solution for at -least twelve hours, or if enough carbolic acid be -added, it may be left there for an indefinite time.</p> - -<p><span class="pagenum" title="30"><a name="Page_30" id="Page_30"></a></span></p> - -<p>When frozen, gum forms a firm non-crystalline -mass, which supports the tissue on all sides. It -must not be frozen too deeply, or it becomes hard -and rather brittle and is apt to injure the razor. -If this have occurred the surface can be softened -sufficiently by breathing gently on it.</p> - -<p>After cutting in gum, the sections are gently -removed from the knife into distilled water by a -soft camel’s hair brush, and left there for an hour -or two, until the medium is entirely dissolved out. -They may then be stained and mounted, or they -may be put away in spirit for an indefinite time, -and then stained and mounted.</p> - -<p><b>Celloidin</b> is for many purposes almost an -ideal embedding medium. (1) It has great penetrating -power; (2) it can be made of an admirable -consistence for cutting purposes; (3) after sections -are made it allows them to be very freely manipulated -without fear of injuring them: (4) and -being perfectly transparent and homogeneous in -thin sections, it does not require to be removed -from a section before mounting. It is insoluble -in water, and in weak spirit; slightly soluble in -alcohol of more than 90 per cent. strength, and<span class="pagenum" title="31"><a name="Page_31" id="Page_31"></a></span> -very readily soluble in ether, or in a mixture of -alcohol and ether. The last solvent is the one -commonly employed.</p> - -<p>The embedding solution is made <span class="nowrap">thus:—</span></p> - -<p>Take some pure celloidin (“Schering’s,” sold in -boxes containing an ounce of shavings, is very -good) and pour on it about eight times its volume -of a mixture of equal parts of absolute alcohol and -ether. Allow this to stand all night until the celloidin -is dissolved. The solution should be made -about the consistence of ordinary mucilage.</p> - -<p>It is also convenient to have a thinner solution -made by using double the proportion of alcohol -and ether. Both solutions should be kept in wide -mouthed stoppered bottles, and the stopper should -be well greased with vaseline as an additional -obstacle to the evaporation of the volatile ether.</p> - -<p>Before embedding a specimen it is necessary to -dehydrate it thoroughly for twelve to twenty-four -hours in absolute alcohol. It should then be placed -in a mixture containing alcohol and ether for an -hour or two, and afterwards transferred to the -thin solution of celloidin for twenty-four hours, -and then to the thick solution for the same period.<span class="pagenum" title="32"><a name="Page_32" id="Page_32"></a></span> -The celloidin penetrates slowly and in the case -of nerve tissues and other delicate structures it -is wise to give the full allowance of time for -the different steps. When the tissue has been -thoroughly permeated by the celloidin, it is gently -removed from the celloidin and placed in position -on a piece of cork of suitable size for clamping -in the holder of the microtome. Celloidin is -painted round the object so that it is supported -on every side. It is then left exposed to the air -until the surface has become firm, when the cork -is placed, with the tissue downwards, in methylated -spirit. The cork floats but the tissue and -celloidin remain submerged. At the end of -twenty-four hours the celloidin will have become -semi-opaque and opalescent, and of the same -consistence as hard boiled white of egg. When -it is impossible to wait so long, rapid hardening of -the celloidin may be secured by immersing it in -methylated chloroform in place of spirit, but the -slower method gives more uniformly satisfactory -results.</p> - -<p>Pieces of tissue embedded in celloidin may -also be cut on a freezing microtome. After<span class="pagenum" title="33"><a name="Page_33" id="Page_33"></a></span> -the celloidin has become firm by immersion in -methylated spirit, the tissue with the celloidin -round it may be cut off the cork, washed in water -to remove the alcohol, and then soaked for an -hour or two in gum, placed on the plate of an -ether spray microtome, frozen and cut in the -usual way.</p> - -<p>Subsequent staining operations are conducted -in the same way as for sections cut by hand or -in gum. As celloidin is only slightly stained by -hæmatoxylin, alum carmine, borax carmine, &c., -it is not necessary to remove it from the sections, -but it exhibits so intense a staining reaction with -aniline dyes that it is necessary to remove it by -treatment with alcohol and ether either before or -after the staining operation.</p> - -<p>The sections after staining may be mounted in -Farrant’s solution (p. <a href="#Page_59">59</a>), or in Canada balsam -(p. <a href="#Page_61">61</a>). If the latter medium is employed, the -section should be clarified, after dehydration in -alcohol, by means of oil of bergamot, or oil of -origanum, instead of oil of cloves, as the latter -dissolves out the celloidin and causes the section -to break up.</p> - -<p><span class="pagenum" title="34"><a name="Page_34" id="Page_34"></a></span></p> - -<p>Celloidin is most useful for cutting sections of -the coats of the eye, of the internal ear, and of -bone marrow. It should always be used for the -Weigert-Pal hæmatoxyline method of staining the -nervous centres, as it protects the section from -being injured by the transference from one fluid -to another which is repeatedly required during the -process. The stain is completely discharged from -the celloidin by the decolourising solution used -(p. <a href="#Page_90">90</a>).</p> - -<p><i>Paraffin.</i>—Paraffin is a very convenient embedding -medium for delicate structures, as very -thin sections can be obtained and the paraffin -need not be removed from the section until the -latter is safely on the slide. It is unsuitable for -large sections. Staining operations are not easily -carried out after cutting in paraffin, and it is better -to stain the blocks of tissue in bulk before embedding. -The best stains for penetrating are -borax carmine (p. <a href="#Page_75">75</a>), alum carmine (p. <a href="#Page_76">76</a>), and -Kleinenberg’s hæmatoxyline (p. <a href="#Page_70">70</a>). The tissue -must be left in them for four to ten days.</p> - -<p>Various kinds of paraffin are employed. It is -usual to keep two kinds, one “soft,” melting at<span class="pagenum" title="35"><a name="Page_35" id="Page_35"></a></span> -110° F., and another “hard,” melting at 140° F. -A mixture of two parts of the hard and one of the -soft will be found most generally useful. In -winter a large proportion of the soft variety and -in hot weather a larger proportion of the hard may -be required. A paraffin mass which is always -available has been suggested recently by Dr. -F. E. Batten, who employs an ordinary white -candle, composed of paraffin and wax. If the -mass is found to be too hard, it can easily be made -of a suitable consistence by adding a little paraffin -with a low melting point.</p> - -<p>To prepare a piece of tissue for embedding in -paraffin, it should be stained, washed in distilled -water, and as much moisture as possible removed -by blotting paper. The block is then dehydrated, -first in methylated spirit for several hours, finally -in absolute alcohol. It is taken carefully by -means of forceps from the alcohol and placed in -xylol for an hour or two according to size. Superfluous -xylol is removed from the surface, and the -tissue placed in the melted paraffin. This will set -round the cold tissue at once, but soon melts -again and must be kept at a temperature just<span class="pagenum" title="36"><a name="Page_36" id="Page_36"></a></span> -above melting point for one to four hours, according -to size. The tissue is then transferred to a -mould (which can be easily made of paper), about -half an inch cube, and melted paraffin poured -round it until the mould is full. The mould may -be made by folding a piece of paper to form a box -about half an inch cube, or a small pill box may -be used. Another convenient method is to place -two L-shaped pieces of lead in contact with each -other so as to enclose a space of suitable size as in -the diagram (fig. <a href="#Fig_3">3</a>). The tissue is now hermetically -sealed, and can be kept indefinitely if it is -not convenient to cut it at the time. To prepare -it for cutting, all superfluous paraffin is trimmed -away with a warm knife, and the block is fixed on -a piece of wood, cut so as to suit the clamp of -the microtome, by melting the lower end of the<span class="pagenum" title="37"><a name="Page_37" id="Page_37"></a></span> -paraffin block with a hot needle or wire and pressing -it down on the wood.</p> - -<div class="figcenter" style="width: 390px;"> -<a id="Fig_3"></a><img src="images/i044.jpg" width="390" height="135" alt="" /> -<div class="caption"><p class="tac"><span class="smcap">Fig. 3.</span></p></div> -</div> - -<p>When sections are cut they may be transferred -singly to the slide (which should be lightly -smeared beforehand with a saturated solution of -celloidin in oil of cloves), or they may be cut so -that the back of one section of the paraffin block -adheres to the front of the next, and in this way -a continuous delicate ribbon of serial sections is -obtained. The ribbon is broken up into lengths -of about two and a half inches and transferred to -the slide, on which several ribbons may be placed -side by side, and so a large number of sections -kept in the order in which they are cut. A mark -should be made on the slide to indicate where the -series begins, and each slide should be numbered, -so that the exact position of each section in the -series can be recognised at once.</p> - -<p>Before mounting, the paraffin must be removed -from the sections. This is easily done on the -slide in the case of single sections and of ribbons. -If the sections are curled, a little warmth will -make them unbend and lie flat. The slide is -warmed over a spirit lamp until the paraffin just<span class="pagenum" title="38"><a name="Page_38" id="Page_38"></a></span> -melts. The sections will keep their places owing -to the celloidin beneath. Xylol is then allowed -to flow over the slide from a pipette, until the -paraffin has been completely dissolved, which can -be ascertained by glancing at the sections under -the low power of the microscope. The slide is -placed in an almost vertical position to let the -xylol drain off, excess is wiped off from the edge -of the slide with blotting paper, a drop of Canada -balsam solution (p. <a href="#Page_61">61</a>) is run on the slide, and a -cover-glass of suitable size is applied.</p> - -<p><b>Microtomes.</b>—After a large amount of practice, -persons with a fair amount of manual dexterity -may acquire sufficient skill to be able to cut -very satisfactory sections of specimens embedded -in paraffin, &c., by hand. In the Pathological -Laboratory of a large German University, until -quite recently the use of a microtome was prohibited -by the Professor, who is himself a most -distinguished histologist. The amount of time -expended before one acquires the necessary skill, -and the cheapness and great convenience of the -modern microtome have combined to throw hand -cutting into the background, and some form of -microtome is now almost universally adopted.</p> - -<p><span class="pagenum" title="39"><a name="Page_39" id="Page_39"></a></span></p> - -<div class="figcenter" style="width: 355px;"> -<img src="images/i047.jpg" width="355" height="313" alt="" /> -<div class="caption"><p class="tac"><span class="smcap">Fig. 4.</span>—Cathcart’s Ether Spray Microtome.</p> - -<p>A, B. Wooden frame and supports. C. Glass runners. -G. Screw for raising the zinc plate H. J. Ether bottle. -L. Tube from air bellows.</p></div> -</div> - -<p>Of these there are a very large number in the -market, each having special advantages, and often -special drawbacks. A few of the more generally -useful only will be described. We have microtomes -for cutting in gum frozen by ether spray or -ice, and those intended for cutting in paraffin or -celloidin.</p> - -<p>Cathcart’s <b>ether spray microtome</b> (fig. 4).—<span class="pagenum" title="40"><a name="Page_40" id="Page_40"></a></span>This, -or its more recent modifications (<i>see</i> later), -is perhaps the most useful and economical microtome -for the purposes of the student. Its prime -cost is low, it is small and portable as well as -being clean and inexpensive to work with.</p> - -<p>It consists of an oak frame which can be firmly -clamped on to a table. On this frame are two -narrow parallel supports about two inches high, -which are covered by strips of plate glass, and -serve as smooth rests along which the razor may -glide in making sections. Between them is a -brass well and in this a zinc plate firmly fixed -in the horizontal position, which is almost at the -level of the glass runners. It is capable of being -raised or lowered through about <span class="nowrap"> <span class="fraction"><span class="fnum">3</span><span class="bar">/</span><span class="fden">8</span></span></span> inch by means -of a screw with a very fine and accurate thread. -This screw is turned by a large milled wheel -beneath the microtome. Just beneath the zinc -plate are two small tubes, one connected with an -india-rubber bellows, the other with a bottle at -the side which contains ether. As the air issues -from the first tube, it passes over the open end -of the second, and thus draws the ether out and -makes it play on the zinc plate, and at the same<span class="pagenum" title="41"><a name="Page_41" id="Page_41"></a></span> -time causes it rapidly to evaporate, and so reduces -the temperature of the zinc plate.</p> - -<p>In cutting sections with this microtome the -tissue is taken out of the gum and placed on the -zinc plate. The bellows are then worked until -the gum on the zinc plate is completely frozen. -The plate should be lowered by means of the -screw until the surface of the piece of tissue is on -a level with the glass runners. These and the -razor should then be wetted with water. The -razor being held firmly in the hand is pushed -along the glass runners in a rather oblique direction. -The plate should then be raised by turning -the screw below through a very small arc, another -section taken off and so on. Sections are carefully -removed from the razor to a vessel of water -by means of a soft wet camel’s hair brush. The -needle should never be used for this purpose.</p> - -<p>If the specimen is very delicate, and likely to be -spoiled by being curled up on the knife, the latter -should be kept cold by frequently dipping it in a -vessel containing lumps of ice in water. The gum -will then remain frozen after cutting, and support -the tissue better. Each section should be at once<span class="pagenum" title="42"><a name="Page_42" id="Page_42"></a></span> -transferred to a glass slide from the knife, washing -it off with a stream of ice water from a pipette.</p> - -<p>The knife that is used may be an ordinary -razor, with the edge ground straight. It requires -to be held steadily with both hands. As this is -rather inconvenient, Dr. Sheridan Délépine suggested -the employment of an ordinary plane iron -such as is used in a carpenter’s plane. This only -requires one hand, and the other can be kept on -the head of the screw beneath to raise the plate -at once after each stroke of the knife. Its disadvantages -are that it is rather heavy for prolonged -working, and that it is less easy to “set” -than a razor.</p> - -<p>A. Frazer has recently introduced a valuable -improvement in the Cathcart microtome (fig. <a href="#Fig_5">5</a>).</p> - -<p>In this the brass frame carrying the zinc plate -and ether spray tubes is surrounded by a brass -cylinder, in which it fits accurately, and is pushed -up as desired by turning the screw beneath the -instrument. This brass frame and with it the -zinc plate, &c., can be easily drawn altogether out -of the outer tube, and replaced by a second brass -well, which exactly fits its place and can be raised<span class="pagenum" title="43"><a name="Page_43" id="Page_43"></a></span><span class="pagenum hide" title="44"><a name="Page_44" id="Page_44"></a></span> -by the screw as desired. In this is a small toothed -clamp which can be screwed up so as to hold a -piece of wood carrying a piece of tissue embedded -in paraffin. Sections can also be cut in celloidin -with this instrument, but as oblique strokes with -the knife cannot be made, it is impossible to get -very thin sections. The combined microtome can -be obtained for a guinea from Frazer, 22 Teviot -Row, Edinburgh.</p> - -<div class="figcenter" style="width: 370px;"> -<a id="Fig_5"></a><img src="images/i051.jpg" width="370" height="558" alt="" /> -<div class="caption"><p class="tac"><span class="smcap">Fig. 5.</span>—Frazer’s Modification of Cathcart’s Microtome.</p> - -<p>A. Microtome arranged for ether spray. B. Cylinder with -clamp for holding object embedded in celloidin, &c. to replace -ether spray apparatus.</p></div> -</div> - -<p>There is another modification which is more -generally useful, and at the same time more expensive -than the original model. In this, instead -of glass runners to support the knife, there is a -flat glass plate about eight inches square sufficiently -large to allow of “Swift’s plough” (fig. <a href="#Fig_6">6</a>) -being used for the purpose of cutting sections. -This instrument consists of a triangular brass -frame, supported on three legs, each of which is -a screw, tipped with ivory. There is one screw -in front and two behind. Beneath the plate, and -held in position by the posterior screws in front, -and a little clamp behind, is a razor with the edge -directed forwards. The edge can be raised or -depressed by turning the anterior screw, on which<span class="pagenum" title="45"><a name="Page_45" id="Page_45"></a></span> -the frame is supported. Before sections are cut -the edge of the razor should be brought down to -the level of the tissue, taking care that all the legs -are equal in length. The plough should then be -firmly grasped with both hands, (the index finger -of one hand being left free to turn the anterior -screw) and pushed rather obliquely through the -tissue. The edge of the razor is then slightly -lowered by turning the screw through a very -small angle, and another section made, and so -on. With a little practice very thin uniform -sections may be made with great rapidity.</p> - -<p>Another useful ether spray microtome is that -made by Jung of Heidelburg. The knife swings -round a pivot, and there is an ingenious -ratchet arrangement which works synchronously -with each swing of the knife, to raise the tissue -automatically the requisite distance for the next -section to be made. The exact thickness of the -sections can be graduated with great nicety by a -simple contrivance. The instrument can be obtained -in this country for about £2. It works satisfactorily, -but, with practice, the student will get -equally good results with the cheaper <span class="pagenum" title="46"><a name="Page_46" id="Page_46"></a></span>“Cathcart.”</p> - -<p><b>Williams’ ice freezing microtome</b> (fig. 6).</p> - -<p>This consists of a round mahogany water-tight -box provided with an exit tube below, and covered -with a strong plate glass lid. Firmly fixed in -the centre of the floor of the box is a stout brass -pillar surmounted by a brass disc which fits into -a hole in the centre of the glass lid, so that its -surface is on a level with that of the lid.</p> - -<div class="figcenter" style="width: 390px;"> -<a id="Fig_6"></a><img src="images/i054.jpg" width="390" height="347" alt="" /> -<div class="caption"><p class="tac"><span class="smcap">Fig. 6.</span>—Williams’ Ice Freezing Microtome, with Swift’s -plough.</p></div> -</div> - -<p>To use it, the box is filled with alternate layers<span class="pagenum" title="47"><a name="Page_47" id="Page_47"></a></span> -of pounded ice and salt; the lid is then put on and -fixed by means of a lateral screw. The tissue to -be frozen is gently removed from the gum and -placed on the brass disc and plenty of gum -painted round it. It should then be covered with -a tin cap for a few minutes until frozen. Sections -are made with a Swift’s Plough (p. <a href="#Page_44">44</a>).</p> - -<div class="figcenter" style="width: 390px;"> -<img src="images/i055.jpg" width="390" height="334" alt="" /> -<div class="caption"><p class="tac"><span class="smcap">Fig. 7.</span>—Schanze Microtome (see text).</p></div> -</div> - -<p><b>Schanze microtome</b> (fig. 7) is the pattern -used in the Leipsic laboratories. It consists of a<span class="pagenum" title="48"><a name="Page_48" id="Page_48"></a></span> -heavy iron frame with a large base. The knife is -carried in a clamp which slides along the full -length of the instrument, gliding upon two smooth -plates of iron which are arranged at an angle to -one another. The knife must be moved very -steadily and gently, as when using a long blade -vibrations are easily set up which prevent good -sections being obtained. The surfaces of contact -must be kept scrupulously free from dust, -and lubricated with equal parts of olive oil and -castor oil. There are several object holders, -which can be removed and interchanged, one -connected with an ether spray apparatus, another -suitable for holding an object embedded in paraffin, -and a third for grasping an object embedded -in celloidin. When celloidin is employed, a -specially long knife must be used, and it must -be fixed very obliquely in the clamp. The -object holder is raised by a fine screw worked -by a large brass toothed wheel. There is a -ratchet arrangement, by which the object may -be raised automatically any desired distance, -after each stroke of the knife. It gives most -satisfactory results with celloidin and paraffin.<span class="pagenum" title="49"><a name="Page_49" id="Page_49"></a></span> -(Messrs. R. and J. Beck are the agents). Its cost -is about £5.</p> - -<p><b>Becker’s microtome</b> is made on exactly the -same principles as the Schanze. The modifications -are that the carrier glides on glass plates -instead of iron ones, and that instead of the whole -surface of the carrier being in contact with the -plates, there are a few smooth ivory buttons only. -Friction is thus reduced to a minimum, and very -uniform sections can be obtained. The price is -the same as that of the Schanze.</p> - -<p>Frazer has introduced a “student’s sliding -microtome” on the same principle as the Schanze -which costs about £3.</p> - -<p><b>The Cambridge Rocking microtome.</b>—This -instrument, as made by the Cambridge -Scientific Instrument Company, or the slightly -modified form made by Messrs. Swift (fig. <a href="#Fig_8">8</a>), is -the best instrument for cutting sections of small -objects embedded in paraffin. Ribbons of serial -sections can be obtained from it with greater ease -and certainty than with other microtomes. This -microtome differs from those which have been -previously described in that the knife is fixed,<span class="pagenum" title="50"><a name="Page_50" id="Page_50"></a></span><span class="pagenum hide" title="51"><a name="Page_51" id="Page_51"></a></span> -while the object is moveable. The microtome -consists of an oblong heavy metal stand. A long -bar is arranged so that it rides in see-saw fashion -on two strong vertical pillars arising from the -frame. One end of this bar is hollow, and receives -the piece of wood carrying the tissue embedded -in paraffin, which is firmly clamped in -position. This end is depressed by means of a -strong spiral spring. In order to raise it there -is an arrangement by which the other end of -the bar is depressed by a cord which revolves -round a pulley. When the handle is turned, -the tissue is raised, and when the cord is relaxed, -the spring pulls the tissue firmly and steadily -down. The razor, which must have a straight -edge, is fixed firmly by screws, with its edge -upwards at the end of the microtome. The object -is then adjusted so that in its descent a thin slice -is taken off by the razor. There is an ingenious -arrangement by which the depression of the bar -to raise the section pushes it a little further in the -direction of the razor. The distance can be -graduated from <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">500</span></span></span> to <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">3000</span></span></span> inch. The actual -working of the machine is therefore very simple.<span class="pagenum" title="52"><a name="Page_52" id="Page_52"></a></span> -The position of the block containing the tissue to -be cut having been adjusted so that the razor just -cuts it, the free end is depressed by means of the -pulley. This also pushes the section a little beyond -the razor. The strong spring then draws -the tissue steadily past the edge of the razor, and -a thin section is left on the blade. This may be -at once transferred to a slide, or if the paraffin -be of the proper consistence, another cut may be -made, when the two sections should adhere by -their edges, and so by repeating the movement a -continuous ribbon may be obtained. If there is -difficulty in obtaining a good ribbon, it will usually -be got over by taking a little soft paraffin and -attaching it by means of a hot needle to the lower -end of the paraffin block. The cost of the instrument -is about £5.</p> - -<div class="figcenter" style="width: 540px;"> -<a id="Fig_8"></a><img src="images/i058.jpg" width="540" height="350" alt="" /> -<div class="caption"><p class="tac"><span class="smcap">Fig. 8.</span>—Swift’s Modification of the Cambridge Rocking -Microtome.</p></div> -</div> - -<p><b>Fresh sections.</b>—Although these are not so -satisfactory as hardened specimens for accurate -histological work, it is often very useful to make -them both in the post-mortem room where an -immediate opinion of the nature of the tumour -or diseased organ is desired, and also in the -operating theatre. With a little practice sections<span class="pagenum" title="53"><a name="Page_53" id="Page_53"></a></span> -may be cut, stained, and mounted, within ten -minutes of the removal of the specimen from the -body. In this way important information may be -afforded to the operating surgeon, and in not a -few cases it has caused the proposed treatment to -be entirely altered. Thus, in one case, a supposed -chronic periostitis was shown to be a sarcoma, -and the limb was amputated. In another, a supposed -sarcoma of the thigh was found to be a -gumma, when a portion was removed and microscopically -examined.</p> - -<p>A portion of the specimen should be placed -without any preparation on the zinc plate of the -freezing microtome, and some gum painted round -it. It is then frozen. The serum in the tissues -is not in sufficient mass to injure the knife when it -is frozen. The knife should be wetted with, and -sections transferred to, either pericardial serum, -or <span class="nowrap"> <span class="fraction"><span class="fnum">3</span><span class="bar">/</span><span class="fden">4</span></span></span> per cent. solution (70 grains to the pint), of -common salt, neither of which causes the cells to -swell up as plain water does. They should be -carefully floated out on a glass slide, an operation -which requires much more patience than in the -case of hardened sections, as fresh sections are<span class="pagenum" title="54"><a name="Page_54" id="Page_54"></a></span> -less coherent and also more sticky, so that the -edges tend to curl up on the knife, &c. They -should then be examined, one unstained, simply -mounted in salt solution; another stained with -picrocarmine and examined in the saline solution; -and a third stained in picrocarmine, mounted in -Farrant’s solution, and preserved. The last usually -gives the best results, the picrocarmine staining -becoming quite brilliant after a week. The -glycerine, however, is apt to make the sections -shrink a good deal, and the weight of the cover-glass -tends to break up the unhardened section.</p> - -<hr class="chap" /> - -<p><span class="pagenum" title="55"><a name="Page_55" id="Page_55"></a></span></p> - - - - -<h2>CHAPTER IV.</h2> - -<h3><span class="smcap">Section Mounting.</span></h3> - - -<p>1. By <b>flotation</b>.</p> - -<p>In this method the section whether stained or -unstained is placed in a bowl of water, or normal -salt solution (p. <a href="#Page_53">53</a>). A clean slide is then introduced -into the water at an angle of about 60°, a -little more than half of its length being submerged. -The section is then brought up by the needle and -floated as far as possible into position on the slide. -One corner is then fixed by the needle, and on -gently withdrawing the slide the section should lie -flat. If any folds are left no attempt should be -made to smoothen them out with a needle, but the -slide should be re-immersed until the folded part -of the section is under water. It should then be -gently withdrawn, when the fold will disappear. -This manœuvre must be repeated in different -directions until the section lies quite smoothly<span class="pagenum" title="56"><a name="Page_56" id="Page_56"></a></span> -on the slide. Stained and unstained sections are -floated out in this way before being mounted in -Farrant’s medium, and unstained sections previous -to staining in picrocarmine.</p> - -<p>2. By <b>transference</b> with a <b>section lifter</b>.</p> - -<p>This method is employed in mounting in Canada -balsam in order to transfer the section from the -clarifying agent (p. <a href="#Page_63">63</a>) to the slide. The lifter is -polished, and insinuated under the section. The -section being held in position by the needle is now -raised from the fluid, excess of which is removed -by holding the section in position with a mounted -needle, and tilting the lifter so as to allow it to -drain off.</p> - -<p><b>Removal of air bubbles from sections.</b>—When -sections contain many air bubbles, the best -plan is to leave them in methylated spirit for a -time. The bubbles then coalesce and escape from -the section.</p> - -<p>For delicate structures and for fresh sections -the transference to spirit, and the subsequent flying -out of the section when returned to water are -risky, and the best method of treating these is to -put the vessel containing them under the receiver<span class="pagenum" title="57"><a name="Page_57" id="Page_57"></a></span> -of an air pump, if one is available, and slightly -exhausting the air.</p> - -<p>The most frequent cause of air bubbles in -mounted specimens, however, is the employment -of cover-glasses which have not been thoroughly -cleansed. Proper cleansing is best effected by -placing the covers when bought in a shallow wide -mouthed stoppered bottle containing strong nitric -acid, and leaving them in this fluid for twenty-four -hours. The acid should then be drained off and -water run through the vessel from a tap, until the -washings no longer give an acid reaction with -litmus paper. The water should then be drained -off, and the glasses covered with absolute alcohol. -They can be removed one by one and rapidly -dried as required. With cover-glasses properly -cleansed in this manner, not only will air bubbles -be avoided, but the covers will be dried much -more easily with the cloth, and fewer will be -broken in the process.</p> - -<p>Another very frequent cause is the transference -of air bubbles with the mounting medium on the -glass rod. This occurs especially if the rod be -fused to the stopper. The proper bottles to use,<span class="pagenum" title="58"><a name="Page_58" id="Page_58"></a></span> -both for Farrant’s medium and balsam are “balsam -bottles” which have no stopper, but the -mouth is closed by a glass cap which fits accurately -(fig. 9). A short glass rod is attached to -the cap, and is used to transfer the medium to the -slide.</p> - -<div class="figcenter" style="width: 105px;"> -<img src="images/i066.jpg" width="105" height="219" alt="" /></div> -<div class="caption" style="width: 150px;"><span class="smcap">Fig. 9.</span>—Balsam bottle.</div> - -<p><b>Treatment of folded sections.</b>—The folding -may be <span class="nowrap">due:—</span></p> - -<p>(1.) To the section having creased through -being cut with a knife whose surface was not -perfectly smooth. This is best remedied by -placing the section in methylated spirit for a -minute, and then transferring it to a bowl of -clean water, when the section will rapidly rise to<span class="pagenum" title="59"><a name="Page_59" id="Page_59"></a></span> -the top, and spread itself out flat on the surface of -the water, in consequence of the alcohol rapidly -diffusing out at the edges into the surrounding -water.</p> - -<p>(2) To the section containing a large amount of -<b>fat</b>, as in those of the skin and subcutaneous -tissue. The fat may be removed from the fat cells -without materially altering the appearance of the -section. This is done by dehydrating the section -in alcohol, and then transferring to a watch glass -containing ether or chloroform to extract the fat. -The tissue should be washed free from ether in the -alcohol and then transferred to the bowl of water, -and allowed to float out. This process does not -interfere with subsequent staining operations.</p> - - -<h3><span class="smcap">Mounting Media.</span></h3> - -<p><b>Farrant’s solution</b><span class="nowrap">:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Gum Arabic (picked, colourless)</td><td class="tar pt03" rowspan="3"> <img src="images/41x6br.png" width="6" height="41" alt="" /></td><td class="tal" rowspan="3">equal parts.</td></tr> -<tr><td class="tal">Glycerine</td></tr> -<tr><td class="tal">Water</td></tr> -</table></div> - -<p>In making this solution the best gum arabic -must be used, and only the clearest pieces of this.<span class="pagenum" title="60"><a name="Page_60" id="Page_60"></a></span> -“Powdered gum acacia” should be avoided, as -though it looks white it often yields a brown -mucilage, and besides is frequently adulterated -with starch, &c.</p> - -<p>The glycerine and water should be mixed and -the gum arabic added. The mixture should be -allowed to stand for some weeks, with frequent -stirring until the whole of the gum is dissolved. -Then allow it to stand for a week or two longer -in order that the dirt may subside, and the bubbles -rise to the top. The scum should be removed and -the clear fluid decanted from the sediment into a -“Balsam bottle” (p. <a href="#Page_58">58</a>) containing a few drops -of a saturated solution of arseniate of sodium and -a small lump of camphor.</p> - -<p>If properly made it is an extremely useful -mounting reagent. It does not clarify the tissues -too much, and in consequence of its containing -gum it dries at the edges and cements the cover-glass -more or less firmly in a week or two. If -this is not the case the medium contains too much -glycerine and more gum must be added to compensate -for this. This drying at the edge prevents -any further evaporation while the glycerine -keeps the section permanently moist.</p> - -<p><span class="pagenum" title="61"><a name="Page_61" id="Page_61"></a></span></p> - -<p>The camphor and arseniate of sodium prevent -the formation of fungi. Sections preserve their -original appearance in this medium for many -years. After a long time they are apt to become -a little cloudy and granular.</p> - -<p>Unstained sections should always be mounted -in Farrant’s medium, as the Canada balsam process -renders them quite transparent. It is suitable -for almost any tissue stained or unstained, but -sections of the nervous centres require to be -mounted in Canada balsam, owing to the opacity -of myelin when mounted in glycerine.</p> - -<p><b>Canada balsam solution</b>:—The medium is -made <span class="nowrap">thus:—</span></p> - -<p>The ordinary Canada balsam which is of a -treacly consistence is heated gently in a water -bath for some hours, to drive off turpentine and -other volatile oils. It is then allowed to cool to a -yellow vitreous mass. Take of</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Dried Canada balsam</td><td class="tar vab" rowspan="2"> <img src="images/27x6br.png" width="6" height="27" alt="" /></td><td class="tal" rowspan="2">equal parts.</td></tr> -<tr><td class="tal">Xylol</td></tr> -</table></div> - -<p>Leave till dissolved, stirring occasionally.</p> - -<p>Unless the solution be perfectly clear, it must -be filtered through a very thin paper, previously<span class="pagenum" title="62"><a name="Page_62" id="Page_62"></a></span> -wetted with xylol. If the medium be too thick -more xylol should be added, if too thin, the xylol -should be allowed to evaporate until the medium -is of the consistence of a thin syrup.</p> - -<p>If the medium is made too thin much annoyance -will be caused by its evaporating at the edge -of the cover-glass, leaving an air-space, which -will increase daily until the section is left quite -dry. This should be remedied by putting another -drop of balsam at the edge of the coverslip -and allowing it to run in and displace the air. A -ring of cement should be put on as early as possible -afterwards.</p> - -<p>The bottle in which the balsam is preserved -must be very carefully dried before being filled -and then rinsed out with absolute alcohol, and -afterwards with xylol. Turpentine or benzol are -often used instead of xylol in the preparation of -the medium, and in the same proportion, but the -latter is less apt to dissolve out the aniline -colours from the sections.</p> - -<p>To mount sections in Canada balsam they must -be transferred first to a watch glass containing -absolute alcohol or an alcoholic solution of some<span class="pagenum" title="63"><a name="Page_63" id="Page_63"></a></span> -staining reagent, <i>e.g.</i>, eosine (p. <a href="#Page_72">72</a>) and left in it, -no attempt being made to spread it out, until it is -perfectly dehydrated, <i>i.e.</i>, in about two minutes. -It should then be transferred to the clarifying oil -on a mounted needle, or on a section lifter, which -must be perfectly dry as any spot of moisture that -gets on to the section will resist the clarifying -action of the oil, and will cause unsightly opaque -areas when the section is mounted. Even breathing -on the section on its way to the clarifying -agent will prevent uniformity of clearing. Should -white spots appear in the section while in the oil -it must be taken out with as little oil as possible, -and again dehydrated in absolute alcohol.</p> - -<p>The process of <b>clarifying</b> must be performed -in some medium in which Canada balsam is readily -soluble, and which is also readily miscible with -alcohol. Those most frequently employed are oil -of cloves, xylol, oil of bergamot, oil of cedar, and -origanum oil. The first named has always been -much used because of its agreeable odour, its -cheapness, and the ease with which it can be -obtained. But it has the disadvantage of dissolving -out many important staining reagents,<span class="pagenum" title="64"><a name="Page_64" id="Page_64"></a></span> -especially eosine and the various aniline colours. -In addition as it dissolves celloidin, sections cut in -this medium tend to fall to pieces when transferred -to oil of cloves, and one of the other oils -(which have no solvent action on celloidin) should -always be employed with celloidin sections. Oil -of bergamot is the most generally useful, but -rather expensive. Where there are special reasons -for employing other dehydrating agents, they will -be indicated in the special directions for particular -staining methods in Chapters <a href="#Page_87">VI</a>. and <a href="#Page_103">VII</a>.</p> - -<p>As soon as the section is plunged into the oil, -the alcohol rapidly diffuses out, so that the edges -of the section fly out with it, and the section floats -quite flat on the surface of the oil. When it is -completely clarified (in about a minute), as shown -by its sinking in the oil, it should be transferred -to the slide by the section lifter, and the oil -drained off. Excess of oil may be removed by -pressing gently on the section with a flat piece of -filter paper folded several times. If carefully performed -this manœuvre will not injure the section, -but it requires practice.</p> - -<p>If the tissue is very delicate, and likely to be<span class="pagenum" title="65"><a name="Page_65" id="Page_65"></a></span> -injured by changing from one vessel to another, -or if it is larger than the section lifter will conveniently -carry, it should be floated out on a glass -slide, and, as much water as possible having been -removed by blotting paper, should be dehydrated -by adding a little alcohol from a pipette once or -twice. Most of the alcohol should then be removed -by tilting the slide, and before the remainder -has evaporated, some oil of cloves or -bergamot should be added from another pipette. -The section will float on the oil at first, but the -latter will gradually come through and appear on -the top of the section. When this occurs the -clarification is complete, and the oil may be run -off by tilting the slide and the section mounted in -Canada balsam.</p> - -<p><b>Cementing of cover-glasses.</b>—The cover-glasses -may be cemented down to prevent their -shifting and spoiling the specimen. If the cover-glass -be circular, a Shadbolt’s turntable should be -used. It consists simply of a horizontal heavy -brass disc, rotating easily on a pivot. There are a -number of circles traced on the disc concentrically. -The slide is then fixed on the disc by<span class="pagenum" title="66"><a name="Page_66" id="Page_66"></a></span> -means of the clips, so that the circumference of -the cover-glass corresponds to one of the circles. -The disc is then rotated and the cement applied -to the edge of the cover-glass with a brush.</p> - -<p>Many materials are employed. The most suitable -are:—(1) Canada balsam, which is almost -colourless and transparent and looks very neat. -(2) Gold size. (3) Marine glue.</p> - -<p>When these are dry a finished appearance may -be given to the slide by laying on a ring of zinc -white. This is made as <span class="nowrap">follows:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal" colspan="2">Oxide of zinc</td><td class="tal"><span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span> drachm.</td></tr> -<tr><td class="tal">Benzole</td><td class="tar vab" rowspan="2"> <img src="images/27x6br.png" width="6" height="27" alt="" /></td><td class="tal" rowspan="2"><span class="ilb">half an ounce</span><br /> of each.</td></tr> -<tr><td class="tal">Gum dammar</td></tr> -</table></div> - -<p><b>Preservation of sections.</b>—They should be -kept <i>flat</i>, and preserved from both light and dust. -Very useful cardboard trays are now sold by -almost all dealers in boxes made to contain -twenty-four dozen slides for about eight shillings, -or suitable cabinets may be constructed by a -carpenter.</p> - -<hr class="chap" /> - -<p><span class="pagenum" title="67"><a name="Page_67" id="Page_67"></a></span></p> - - - - -<h2>CHAPTER V.</h2> - -<h3><span class="smcap">General Staining Methods.</span></h3> - - -<p>Much information may be obtained from unstained -sections, and in most cases one section -should be examined unstained, but the specimens -mounted in this way are so transparent -that it is difficult to study the details of the -tissue. They are therefore usually prepared by -treating them with some staining reagent, not -merely to render them less transparent, but also -to “differentiate” the elements of the section, by -staining one part more deeply than another, or of -a different colour. Thus hæmatoxyline stains the -nuclei and rapidly growing parts of the tissue, -leaving the formed material, as a rule, much more -lightly tinted. Methyl violet again stains healthy -tissues blue, and parts affected with waxy degeneration -a red-violet colour. By combining -stains also much differentiation of the tissue elements -may be obtained. Sections should be<span class="pagenum" title="68"><a name="Page_68" id="Page_68"></a></span> -stained with several reagents, as their effect on -individual specimens varies a good deal.</p> - -<p>The following are the most useful stains for -general <span class="nowrap">purposes:—</span></p> - -<p><b>Logwood.</b>—This or its purified principle -hæmatoxyline is the most useful general stain. -The hæmatoxyline itself is preferable, giving more -constant results, and less diffuse staining.</p> - -<p>For general staining purposes the following -formula will be found to give excellent results:—<br /><b>Hæmatoxyline. -Schuchardt’s formula.</b>—</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(<i>a</i>)</td><td class="tal">Hæmatoxyline</td><td class="tar">3</td><td class="tal">grms.</td><td class="tar"> 30</td><td class="tal">grs.</td></tr> -<tr><td class="tal"></td><td class="tal">Absolute alcohol</td><td class="tar">16</td><td class="tal">c.c.</td><td class="tar"><span class="nowrap">2 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">drms.</td></tr> -<tr><td class="tal">(<i>b</i>)</td><td class="tal">Pure alum</td><td class="tar">3</td><td class="tal">grms.</td><td class="tar">30</td><td class="tal">grs.</td></tr> -<tr><td class="tal"></td><td class="tal">Distilled water</td><td class="tar"> 100</td><td class="tal">c.c.</td><td class="tar">2</td><td class="tal">ozs.</td></tr> -</table></div> - -<p>Add (<i>a</i>) to (<i>b</i>) <i>drop by drop and with constant agitation</i>. -Keep for some days exposed to diffuse daylight -until its colour is so deep that it will not -transmit the light. It should then be filtered, and -a crystal of thymol added. It will not give very -satisfactory staining reactions at first, and should -be allowed to ripen at least a month or six weeks -before using. It improves as a dye with every -month that it is kept. Whenever hæmatoxyline<span class="pagenum" title="69"><a name="Page_69" id="Page_69"></a></span> -has been made up with alum as in the above -formula, an abundant reddish-brown precipitate -forms after some time. This in no way interferes -with the activity of the solution, but it must -always be filtered before being used.</p> - -<p><b>Barrett’s formula.</b>—Introduced by Dr. W. -H. Barrett, of Belfast. It gives almost as good -results as the above. It is made from ordinary -English extract of logwood, and is considerably -cheaper.</p> - -<p>The extract should be dried, and finely powdered, -and then extracted with absolute alcohol -for several days.</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Powdered extract of logwood</td><td class="tar">2</td><td class="tal">grms.</td><td class="tar"> <span class="nowrap">1 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">drms.</td></tr> -<tr><td class="tal">Absolute alcohol</td><td class="tar"> 10</td><td class="tal">c.c.</td><td class="tar">1</td><td class="tal">oz.</td></tr> -</table></div> - -<p>Filter and add slowly to</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Benzoate of sodium</td><td class="tar">1</td><td class="tal">grm.</td><td class="tar"> 36</td><td class="tal">grs.</td></tr> -<tr><td class="tal">Alum</td><td class="tar">1</td><td class="tal">grm.</td><td class="tar">36</td><td class="tal">grs.</td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 100</td><td class="tal">c.c.</td><td class="tar">10</td><td class="tal"><span class="ilb">ozs.</span></td></tr> -</table></div> - -<p>The strength of the solution will vary with -different samples of logwood and must be estimated -by trial. This solution is comparatively -cheap and is useful for class purposes.</p> - -<p><span class="pagenum" title="70"><a name="Page_70" id="Page_70"></a></span></p> - -<p><b>Ehrlich’s hæmatoxyline.</b>—This very useful -nuclear stain is made as <span class="nowrap">follows:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(<i>a</i>)</td><td class="tal">Hæmatoxyline</td><td class="tar">2</td><td class="tal">grms.</td><td class="tar">9</td><td class="tal">grs.</td></tr> -<tr><td class="tal"></td><td class="tal">Absolute alcohol</td><td class="tar">100</td><td class="tal">c.c.</td><td class="tar">2</td><td class="tal">ozs.</td></tr> -<tr><td class="tal">(<i>b</i>)</td><td class="tal">Glycerine</td><td class="tar">100</td><td class="tal">c.c.</td><td class="tar">2</td><td class="tal">ozs.</td></tr> -<tr><td class="tal"></td><td class="tal">Distilled water</td><td class="tar">100</td><td class="tal">c.c.</td><td class="tar">2</td><td class="tal">ozs.</td></tr> -<tr><td class="tal"></td><td class="tal">Alum</td><td class="tar"> 120</td><td class="tal">grms.</td><td class="tar"><span class="nowrap">2 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">ozs.</td></tr> -<tr><td class="tal"></td><td class="tal">Glacial acetic acid</td><td class="tar">5</td><td class="tal">c.c.</td><td class="tar"> 24</td><td class="tal"><span class="ilb">mins.</span></td></tr> -</table></div> - -<p>Add (<i>a</i>) slowly to (<i>b</i>) with constant agitation.</p> - -<p>Allow to ripen in sunlight for two months before -using. It may be employed as a rapid stain -undiluted but far better results are obtained by -using a weak solution, a few drops to a watch-glass -full of distilled water, and staining slowly -for from half an hour to two hours. The solution -improves by keeping. If after a time the staining -becomes diffuse it is an indication that the acetic -acid has evaporated, and a few drops more should -be added.</p> - -<p><b>Kleinenberg’s hæmatoxyline.</b>—This formula -differs from the previous one in being an -alcoholic solution. The calcium chloride is added -because it “sets up diffusion currents between the -alcohol in the material to be stained and the alcoholic<span class="pagenum" title="71"><a name="Page_71" id="Page_71"></a></span> -staining solution, so enabling the latter to -penetrate more rapidly” (Squire). It is much -used in staining embryonic specimens in bulk -before embedding in paraffin, and was strongly -recommended for that purpose by Foster and -Maitland Balfour.</p> - -<p>Various formulæ have been given from time to -time. That advised by Squire (<i>Methods and -Formulæ</i>, p. 25) can be accurately made up without -much difficulty.</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(<i>a</i>)</td><td class="tal"><i>Crystallised</i> calcium chloride</td><td class="tar"> 20</td><td class="tal">grs.</td><td class="tar"><span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">oz.</td></tr> -<tr><td class="tal"></td><td class="tal">Distilled water</td><td class="tar">10</td><td class="tal">c.c.</td><td class="tar">2</td><td class="tal">drms.</td></tr> -<tr><td class="tal">(<i>b</i>)</td><td class="tal">Alum</td><td class="tar">3</td><td class="tal">grms.</td><td class="tar">32</td><td class="tal">grs.</td></tr> -<tr><td class="tal"></td><td class="tal">Distilled water</td><td class="tar">16</td><td class="tal">c.c.</td><td class="tar"> 170</td><td class="tal">mins.</td></tr> -</table></div> - -<p>Mix and add</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Rectified spirit</td><td class="tar"> 240</td><td class="tal">c.c.</td><td class="tar"> 8</td><td class="tal"><span class="ilb">ozs.</span></td></tr> -</table></div> - -<p>Allow it to stand and any excess of calcium -sulphate, &c., to separate. Filter and add</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Hæmatoxyline</td><td class="tar"> <span class="nowrap">2 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">grms.</td><td class="tar"> 25</td><td class="tal">grs.</td></tr> -</table></div> - -<p>A little thymol should be added as a preservative.</p> - -<p>In making up these solutions care must be -taken that <i>only distilled water</i> is used, and that all<span class="pagenum" title="72"><a name="Page_72" id="Page_72"></a></span> -the vessels employed have been previously rinsed -out with it, otherwise precipitation of the hæmatoxyline -will occur.</p> - -<p>Should sections be overstained in hæmatoxyline, -this may be remedied by washing it in a half per -cent. solution of acetic acid, until sufficient of the -stain is discharged, but the staining is more diffuse -than if the happy mean had been hit in the first -instance.</p> - -<p>Hæmatoxyline stains the nuclei of the cells a -beautiful violet colour, and also tints, more or less -lightly, the cell protoplasm and the fibrous elements. -It also stains the axis cylinders of nerves, -and is much used in special staining of the nerve -centres as will be described later, (pp. <a href="#Page_88">88</a>–91).</p> - -<p>The stain is permanent. Sections may be -mounted either in Farrant’s solution, or in Canada -balsam, the latter being preferable.</p> - -<p><b>Eosine.</b>—Much more satisfactory results are -obtained from the commercial eosine (an amorphous -orange powder used in dyeing and in the -manufacture of red ink), than from the pure -crystalline form.</p> - -<p>It may be used as an aqueous solution (<span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">30</span></span></span> per<span class="pagenum" title="73"><a name="Page_73" id="Page_73"></a></span> -cent.) or as a solution in absolute alcohol (<span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">15</span></span></span> per -cent.). Sections stained in the former should be -rapidly passed through a one per cent. solution of -acetic acid in order to “fix” the stain, and then -washed in distilled water.</p> - -<p>It is a very transparent stain, and the most -delicate details of a section stained with it are -perfectly visible.</p> - -<p>It stains the nucleus but slightly, while it stains -the cell protoplasm and fibrous tissues and especially -muscular tissues a beautiful rose colour.</p> - -<p>It will be seen, therefore, that it stains those -parts which are left unstained by hæmatoxyline, -and <i>vice versâ</i>. This complementary action is -applied in the following method.</p> - -<p><b>Double staining with eosine and hæmatoxyline.</b>—Sections -having been stained in -hæmatoxyline in the ordinary way, are washed in -distilled water, and dehydrated in a solution -(about 1 in 1500) of eosine in absolute alcohol. -They should remain in this for about two minutes, -and then be passed through oil of cloves and -mounted in Canada balsam in the ordinary way.</p> - -<p>This method gives extremely useful and beauti<span class="pagenum" title="74"><a name="Page_74" id="Page_74"></a></span>ful -results with almost all tissues, and is superior -to picrocarmine for differentiating the tissue elements. -Thus, the nuclei are stained violet, the -cell protoplasm a much paler and warmer violet, -the fibrous tissues pink, and red blood corpuscles -orange or brick red.</p> - -<p>The alcoholic solution of eosine is also used as -a contrast stain after staining for micro-organisms -with blue or violet dyes.</p> - -<p><b>Carmine.</b>—It is made as <span class="nowrap">follows:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Carmine (best)</td><td class="tar">2</td><td class="tar">1</td><td class="tal"><span class="ilb">drachm.</span></td></tr> -<tr><td class="tal">Strong ammonia</td><td class="tar">2</td><td class="tar">1</td><td class="tal"><span class="ilb">drachm.</span></td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 100</td><td class="tar"> 6</td><td class="tal">ounces.</td></tr> -</table></div> - -<p>Rub the carmine with a little water in a mortar, -add the ammonia, when the liquid will turn black. -Gradually add the rest of the water, rubbing it up -all the time. It should be bottled, allowed to -stand for a few days, and then filtered, and a piece -of camphor put in the bottle.</p> - -<p><b>Lithium carmine</b> resembles closely ammonia -carmine in its staining effects. It is usually -a matter of individual preference which is employed.</p> - -<p><span class="pagenum" title="75"><a name="Page_75" id="Page_75"></a></span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Carmine</td><td class="tar"><span class="nowrap">2 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">grms.</td><td class="tar"> <span class="nowrap">10 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">grs.</td></tr> -<tr><td class="tal">Saturated aqueous solution of lithium carbonate</td><td class="tar"> 100</td><td class="tal">c.c.</td><td class="tar">2</td><td class="tal"><span class="ilb">ozs.</span></td></tr> -</table></div> - -<p>Dissolve and filter.</p> - -<p>Sections may be sufficiently stained in either of -these fluids in from three to five minutes, but -more satisfactory results are to be obtained by -diluting with twenty times the bulk of distilled -water, and leaving sections to stain for twenty-four hours.</p> - -<p>After staining in carmine the sections must be -passed through a half per cent. solution of acetic -acid, in order to fix the carmine in the tissues, as -otherwise the water will dissolve the stain out.</p> - -<p><b>Borax carmine</b>—</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(<i>a</i>)</td><td class="tal">Borax</td><td class="tar">4</td><td class="tal">grms.</td><td class="tar">3</td><td class="tal"><span class="ilb">drachms.</span></td></tr> -<tr><td class="tal"></td><td class="tal">Carmine</td><td class="tar">2</td><td class="tal">grms.</td><td class="tar"> <span class="nowrap">1 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">drachms.</span></td></tr> -<tr><td class="tal"></td><td class="tal">Distilled water</td><td class="tar"> 100</td><td class="tal">c.c.</td><td class="tar">5</td><td class="tal">ounces.</td></tr> -</table></div> - -<p>Dissolve with the aid of heat and add slowly -to (<i>b</i>).</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(<i>b</i>)</td><td class="tal">Alcohol</td><td class="tar">70</td><td class="tal">c.c.</td><td class="tar"> <span class="nowrap">3 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">ounces.</span></td></tr> -<tr><td class="tal"></td><td class="tal">Distilled water</td><td class="tar"> 30</td><td class="tal">c.c.</td><td class="tar"><span class="nowrap">1 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">ounce.</td></tr> -</table></div> - -<p>Allow to stand for a fortnight. Filter, and add -a lump of camphor.</p> - -<p><span class="pagenum" title="76"><a name="Page_76" id="Page_76"></a></span></p> - -<p>To use it, place sections, or the tissue in bulk, -in it for from four to twenty-four hours, according -to size, and then transfer to alcohol (seventy per -cent.) containing a drop to the ounce of hydrochloric -acid, for twenty-four hours, and then wash -thoroughly in water. The tissue may then be -placed in gum if it is to be frozen, dehydrated in -alcohol if paraffin or celloidin is to be employed.</p> - -<p>Its advantage is that it is very diffusible, and so -can be used to stain tissues in bulk. It takes a -considerable time to stain sufficiently deeply, but -there is little fear of overstaining.</p> - -<p>It stains nerve-cells and axis cylinders brightly, -and also the connective tissue, bringing a sclerosed -patch out very prominently.</p> - -<p><b>Alum carmine</b><span class="nowrap">:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Alum five per cent. solution in distilled water</td><td class="tar"> 100</td><td class="tal">c.c.</td><td class="tar">1</td><td class="tal">oz.</td></tr> -<tr><td class="tal">Pure carmine</td><td class="tar">1</td><td class="tal">grm.</td><td class="tar"> <span class="nowrap">4 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">grs.</td></tr> -</table></div> - -<p>Boil for twenty minutes. Filter. Add a few -drops of carbolic acid.</p> - -<p>In using this reagent it should be filtered into a -watch glass, and the sections placed in it for at -least an hour. There is no fear of overstaining,<span class="pagenum" title="77"><a name="Page_77" id="Page_77"></a></span> -and they may be left all night. After they have -been stained they must be <i>thoroughly</i> washed in -water to remove the alum, otherwise numerous -crystals of it will be seen in the field when the -section is mounted. Sections may be mounted in -Farrant’s solution or in Canada balsam. The -staining effect improves very much after the section -has been kept a few days.</p> - -<p>If desired its staining action may be complemented -by dehydrating it in an alcoholic solution, -either of eosine (1 in 1500) or of picric acid, and -then clearing up in oil of cloves, and mounting in -Canada balsam.</p> - -<p>By itself it gives a stain very like that of hæmatoxyline, -only warmer. It picks out the nuclei -and axis cylinders of nerves, stains cell protoplasm -slightly, and the fibrous elements scarcely -at all.</p> - -<p>It may be used for the same purposes as hæmatoxyline. -The colour is less attractive, and not so -deep as that of the latter, but as it does not overstain -sections, even when left in it for a week, it is -a very convenient stain for general purposes.</p> - -<p>It is particularly useful as a contrast stain<span class="pagenum" title="78"><a name="Page_78" id="Page_78"></a></span> -for sections of brain and spinal cord, after the -Weigert-Pal hæmatoxylin process (p. <a href="#Page_88">88</a>).</p> - -<p><b>Ammonia-picrocarmine</b> was formerly very -largely used as a staining reagent. Its place has -now to a large extent been taken by lithio-picrocarmine.</p> - -<p>In its preparation the best carmine must be -used.</p> - -<p>It is made as <span class="nowrap">follows:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Carmine</td><td class="tar">1</td><td class="tal">part.</td></tr> -<tr><td class="tal">Liq. ammon. fort.</td><td class="tar">3</td><td class="tal">parts.</td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 3</td><td class="tal">parts.</td></tr> -</table></div> - -<p>Dissolve with gentle heat, and add</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Cold saturated aqueous solution of picric acid</td><td class="tar"> 200</td><td class="tal">parts.</td></tr> -</table></div> - -<p>Bring the mixture to the boiling point, and then -place in a shallow vessel, covered with a glass -plate, and leave it in full sunlight for a month or -more. Filter, bottle, and add six drops of carbolic -acid to each ounce of the mixture. It will -keep indefinitely and improves with age. It requires -filtering from time to time, as a gelatinous -crimson mud tends to deposit from the solution.</p> - -<p><span class="pagenum" title="79"><a name="Page_79" id="Page_79"></a></span></p> - -<p><b>Lithio-picrocarmine.</b>—Prepared as <span class="nowrap">follows:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Carmine</td><td class="tar">2·5</td><td class="tal">grms.</td><td class="tar"> 10</td><td class="tal">grs.</td></tr> -<tr><td class="tal">Saturated solution lithium carbonate</td><td class="tar"> 100</td><td class="tal">c.c.</td><td class="tar">1</td><td class="tal">oz.</td></tr> -</table></div> - -<p>Dissolve, and add</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Saturated solution picric acid</td><td class="tar"> 250</td><td class="tal">c.c.</td><td class="tar"> <span class="nowrap">2 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal"><span class="ilb">oz.</span></td></tr> -</table></div> - -<p>Add a few drops of carbolic acid to each ounce.</p> - -<p>It should stand a day or two in sunlight and -then be filtered. It improves by keeping.</p> - -<p>It should be kept in a stoppered bottle with a -glass rod fused into the stopper.</p> - -<p>When sections are to be stained they are to be -floated out on a clean glass slide as described on -page <a href="#Page_55">55</a>. The slide should then be tilted to allow -the water to drain off, and superfluous moisture -round the section removed by a soft rag, or blotting -paper. A drop or two of the stain should -then be transferred to the slide, which should be -left lying quite flat for about ten minutes. Unless -the room is very warm it is advisable to heat the -slide very gently over a spirit lamp, as this causes<span class="pagenum" title="80"><a name="Page_80" id="Page_80"></a></span> -the tissues to stain more brightly and more -rapidly.</p> - -<p>The excess of the picrocarmine should be -allowed to run off the slide, and the latter wiped. -Some of the stain should, however, be left on the -section, as its effects go on increasing, and are -often not fully seen until a few weeks have -elapsed. They should be mounted in Farrant’s -medium. As a rule those mounted in Canada -balsam do not give such good results. Should -there be special reasons for using this medium, -as in mounting spinal cord sections, &c., they -should be dehydrated after staining in picrocarmine -in an alcoholic solution of picric acid (one -part of a saturated alcoholic solution to five of -alcohol), before clarifying in oil of cloves, as otherwise -the alcohol will dissolve out the picric acid, -and much of the differential staining effect will be -lost. The nuclei should be stained a bright crimson, -the protoplasm of the cells yellow, or a dull pink, -the fibrous elements a bright pink, red corpuscles -green, and all dead material, <i>e.g.</i>, caseous matter, -bright yellow. It also stains nerve-cells, and the -axis cylinders of nerve fibres very brightly. It is,<span class="pagenum" title="81"><a name="Page_81" id="Page_81"></a></span> -however, a rather uncertain dye. The results are -most brilliant in the case of fresh sections.</p> - -<p><b>Osmic acid</b> is invaluable for staining fatty -particles in the cells.</p> - -<p>For ordinary use the one per cent. stock -solution (p. <a href="#Page_21">21</a>) should be diluted with ten -times its bulk of distilled water, and sections -stained in it all night in a dark cupboard, or -the watch glass containing them may be placed -inside a small box.</p> - -<p>The sections must be washed <i>thoroughly</i> in -plenty of water. If desired they may be stained -subsequently in picrocarmine or methyl violet if -waxy degeneration also be present. Sections -should be mounted in Farrant’s solution, as -Canada balsam usually gives disappointing results.</p> - -<p>It demonstrates the most minute fatty particles -in degenerating cells, &c., staining them black. -It may be employed to demonstrate the globules -of fat blocking up the vessels in fat embolism.</p> - -<p>It stains the myelin sheaths of nerves black, -and will be again referred to when speaking of -methods of staining the spinal cord.</p> - -<p><span class="pagenum" title="82"><a name="Page_82" id="Page_82"></a></span></p> - -<p><b>Nitrate of silver</b> is employed for staining the -intercellular cement of epithelial cells. It stains -this substance a deep black, while the rest of the -tissue takes on a brown colour. It is used as a -half per cent. solution in <b>distilled</b> water, and -kept in a stoppered bottle carefully covered up -with brown paper. To use it take some epithelial -tissue, <i>e.g.</i>, the omentum from a recently killed -animal, or a section of some epithelial tumour, -immediately after excision. Wash thoroughly in -distilled water to remove all chlorides, and then -place in a watch glass containing the silver solution. -Keep this in the dark for half an hour and -then wash thoroughly in plenty of water. The -section should be mounted in glycerine or Farrant’s -medium and kept from the light or it will -become too darkly stained.</p> - -<p><b>Chloride of gold</b> is employed to demonstrate -the peripheral terminations of nerves. It can only -be employed within the first half hour after the -tissue has been removed from the living body. -The pieces of tissue must be small and may be -stained in bulk, sections being subsequently made.</p> - -<p>A half per cent. solution in distilled water is<span class="pagenum" title="83"><a name="Page_83" id="Page_83"></a></span> -employed. The tissue is transferred to this on its -removal from the body, until it becomes lemon -coloured. It is then exposed in a one per cent. -solution of acetic acid to a strong light until it -assumes a purplish tinge, which takes from two -hours to two days. Sections should be mounted -in Farrant’s medium. It stains the cells of the -tissue, and nerve cells reddish purple, and nerve -fibrils, especially the terminal ones, rather more -violet. This is very well seen in the cornea.</p> - -<p>It is useful sometimes for clinical purposes to -excise a portion of muscular tissue and examine -the nerve endings by this method. Unfortunately -the stain is somewhat uncertain in its action. -Better results are obtained by Sihler’s chloral -hæmatoxyline method (p. <a href="#Page_92">92</a>).</p> - -<p><b>Methyl violet.</b>—A very satisfactory solution -may be obtained ready made in the “telegraphen -tinte,” prepared by Leonhardi, of Dresden, as recommended -by Woodhead. It may also be used -as a one per cent. solution in distilled water, a -few drops of carbolic acid being added to prevent -the growth of fungi.</p> - -<p>It is a very useful selective stain. It gives<span class="pagenum" title="84"><a name="Page_84" id="Page_84"></a></span> -two reactions, red violet, and blue violet. Thus -it stains the matrix of hyaline cartilage blue -violet, but the cells red violet. It has also a -most important pathological application, as it -picks out any parts which have undergone -“waxy” or “lardaceous” degeneration, staining -them red violet, but the rest of the section -blue violet.</p> - -<p>About ten drops of a one per cent. solution -should be filtered into a watchglassful of water, -and the sections stained for about five minutes. -They must then be passed through a half per cent. -solution of acetic acid and washed <b>thoroughly</b> -for some time in a large quantity of water till no -more colour comes away.</p> - -<p>If these steps are not taken with care, the dye -will diffuse out after the section has been mounted, -blurring all details and spoiling the appearance of -the section.</p> - -<p>Sections may be mounted in Farrant’s solution -(to which a spot of formic acid may be added): if -mounted in Canada balsam the sections must be -overstained as both the alcohol and oil of cloves -rapidly dissolve out the dye.</p> - -<p><span class="pagenum" title="85"><a name="Page_85" id="Page_85"></a></span></p> - -<p><b>Safranine.</b>—Employed as a freshly made -saturated solution in aniline oil water warmed to -60° C. (140° F.). Filter into a watch glass. Stain -for not more than a minute. Dehydrate in alcohol -which will remove much of the stain, clarify in oil -of cloves or origanum oil. Mount in balsam.</p> - -<p>Another method is to stain for about ten minutes, -and then leave for a minute in Gram’s iodide -solution. The sections are then washed in alcohol, -dehydrated, clarified in oil of cloves, and -mounted in balsam. By these methods the stain is -withdrawn except from certain elements, <i>e.g.</i>, those -undergoing colloid or calcareous degeneration.</p> - -<p>A quarter per cent. watery solution is sometimes -employed as it stains nucleoli and actively -dividing nuclei very brightly, while the rest of -the cell is stained faintly. It may be employed to -study karyokinesis in the cells of a rapidly growing -cancer.</p> - -<p><b>Ehrlich-Biondi stain.</b>—This stain has been -much employed for staining specimens of blood, -for studying karyokinesis, and for investigations -on the supposed parasitic bodies found in cancer -cells.</p> - -<p><span class="pagenum" title="86"><a name="Page_86" id="Page_86"></a></span></p> - -<p>It is prepared by mixing saturated aqueous -solutions of the following aniline dyes, slowly and -with constant <span class="nowrap">agitation:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Solution of Orange G</td><td class="tar"> 100</td><td class="tal">parts</td></tr> -<tr><td class="tal">   "    Rubin S</td><td class="tar">20</td><td class="tal"> "</td></tr> -<tr><td class="tal">   "    Methyl Green OO</td><td class="tar">50</td><td class="tal"> "</td></tr> -</table></div> - -<p>finally add</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Distilled water</td><td class="tar">        70</td><td class="tal"> "</td></tr> -</table></div> - -<p>Filter from the copious precipitate which forms. -The solution must be made up frequently as it -does not keep well.</p> - -<p>Sections may be stained rapidly for half an hour -or an hour, but better results are obtained by -diluting the fluid with twenty volumes of water, -and staining all night. Sections should be washed -in water and then passed rapidly through absolute -alcohol and xylol and mounted in Canada balsam.</p> - -<hr class="chap" /> - -<p><span class="pagenum" title="87"><a name="Page_87" id="Page_87"></a></span></p> - - - - -<h2>CHAPTER VI.</h2> - -<h3><span class="smcap">Special Staining Methods.—Special Methods -for Staining the Nerve Centres.</span></h3> - - -<p>1. For staining <b>nerve fibres</b>.</p> - -<p>Three methods (two of which are modifications -of the first) are employed far more often than any -others. By these methods the myelin coating is -stained. Tissues must have been hardened previously -for many weeks in Müller’s fluid, or some -other bichromate solution. They are then overstained -in a solution of hæmatoxyline, and the -section treated with a suitable bleaching reagent, -when the colour is discharged from all the tissue -elements except the nerve fibres. This method -displays not merely the nerve fibres in the white -matter, but also the fine network in the grey -matter of the brain and spinal cord. Degenerated -fibres are left unstained and so degenerated tracts -shew up as unstained spots on a dark background. -The sections may be subsequently<span class="pagenum" title="88"><a name="Page_88" id="Page_88"></a></span> -stained with alum carmine or eosine to shew -the cells and neuroglia.</p> - -<p><b>Weigert’s method.</b>—The piece of cord to be -cut after prolonged hardening in Müller’s fluid is -transferred without washing to absolute alcohol -and dehydrated preparatory to embedding in -celloidin (p. <a href="#Page_30">30</a>). When sections are cut they are -transferred at once to Weigert’s hæmatoxyline -<span class="nowrap">solution:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Hæmatoxyline</td><td class="tar">1</td><td class="tar">4</td><td class="tal">grains.</td></tr> -<tr><td class="tal">Alcohol</td><td class="tar">10</td><td class="tar"> 45</td><td class="tal">minims.</td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 100</td><td class="tar">1</td><td class="tal">ounce.</td></tr> -</table></div> - -<p>They are stained in this for twenty-four hours -or longer, until they are quite black.</p> - -<p>The staining will take place much more rapidly -if the fluid be kept at 100° F. in the incubator. -After staining they are transferred to Weigert’s -differentiating <span class="nowrap">solution:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Borax</td><td class="tar">2</td><td class="tar">160</td><td class="tal">grs.</td></tr> -<tr><td class="tal">Potassium ferricyanide</td><td class="tar">2·5</td><td class="tar"> 200</td><td class="tal">grs.</td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 100</td><td class="tar">1</td><td class="tal"><span class="ilb">pint.</span></td></tr> -</table></div> - -<p>They are left in this solution for several hours, -until the ground work becomes nearly decolourised.</p> - -<p><span class="pagenum" title="89"><a name="Page_89" id="Page_89"></a></span></p> - -<p>Sections will sometimes stain more satisfactorily -if they are treated, according to Weigert’s original -directions, for a few hours with a half saturated -solution of acetate of copper. If the hardening in -Müller’s fluid has been sufficiently prolonged, this -step is usually superfluous.</p> - -<p><b>Pal’s modification</b> of Weigert’s method.—By -this method quicker and more complete decolouration -of the neuroglia, nerve cells, &c., is -obtained. Sections are prepared in exactly the -same way as in Weigert’s method and then transferred -to Weigert’s hæmatoxyline. Pal recommends -that this solution be diluted to half the -strength and a few drops of a saturated solution of -lithium carbonate added. The writer finds the -results equally good if the ordinary Weigert solution -be employed.</p> - -<p>When the sections have been thoroughly stained -they are washed in distilled water and placed in -a three-quarter per cent. solution of permanganate -of potassium. The time required in this solution -depends on the time the specimen has been in -Müller’s fluid. It should not be less than half a -minute, and in very thoroughly hardened speci<span class="pagenum" title="90"><a name="Page_90" id="Page_90"></a></span>mens, -five minutes may be allowed with advantage. -In this solution the sections will become of -an opaque brown colour. They are washed in -distilled water, and transferred <span class="nowrap">to:—</span></p> - -<p><b>Pal’s differentiating solution.</b></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Potassium sulphite</td><td class="tar">1</td><td class="tal">grm.</td><td class="tar">40</td><td class="tal">gr.</td></tr> -<tr><td class="tal">Oxalic acid</td><td class="tar">1</td><td class="tal">grm.</td><td class="tar"> 40</td><td class="tal">gr.</td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 200</td><td class="tal">cc.</td><td class="tar">1</td><td class="tal"><span class="ilb">pint.</span></td></tr> -</table></div> - -<p>They are kept in this for one to five minutes, -according to the depth of staining, until the white -and grey matter are clearly defined and the brown -colour is completely discharged. If the brown -stain does not readily clear up, the section should -be returned to the permanganate solution for -about half a minute, and again treated with -“Pal’s solution.” This manœuvre may be repeated -several times. As soon as the sections -are thoroughly differentiated they are transferred -one by one to a large vessel of water and -thoroughly washed. The blue stain of the hæmatoxyline -becomes brighter during the washing -process. The sections may be mounted at once, -but more beautiful results will be obtained if they -are stained in alum carmine for 24 hours. They<span class="pagenum" title="91"><a name="Page_91" id="Page_91"></a></span> -should then be washed, dehydrated in alcohol, -clarified in oil of bergamot, and mounted in -Canada balsam.</p> - -<p>The very prolonged hardening in Müller’s fluid -which is a necessary preliminary for this method -led to the introduction <span class="nowrap">of:—</span></p> - -<p><b>Schäfer’s modification</b> of Pal’s method.—In -this method hardening in Müller’s fluid for -three or four weeks is sufficient. The sections -are made exactly as in the previous method, and -transferred to Marchi’s fluid (p. <a href="#Page_24">24</a>) for six hours. -They are washed and stained all night in the <span class="nowrap">following:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Hæmatoxylin</td><td class="tar">1</td><td class="tar">4</td><td class="tal">grs.</td></tr> -<tr><td class="tal">Alcohol</td><td class="tar">10</td><td class="tar"> 45</td><td class="tal">min.</td></tr> -<tr><td class="tal">Acetic acid (2 per cent. aqueous solution)</td><td class="tar"> 100</td><td class="tar">1</td><td class="tal">oz.</td></tr> -</table></div> - -<p>The subsequent processes of differentiation, -bleaching, &c., are exactly the same as in Pal’s -method.</p> - -<p><b>Osmic acid.</b>—Employed with fresh and also -with hardened specimens to demonstrate the medullary -sheath. Much the best results are obtained -with the former. The nerves, or small<span class="pagenum" title="92"><a name="Page_92" id="Page_92"></a></span> -pieces of the central nervous system are placed -in half to one per cent. solution of osmic acid as -soon as possible after death and kept in the dark -for about a week. The tissue must be very -thoroughly washed in running water to remove -all traces of osmic acid, and then stained for a -couple of days in borax carmine to demonstrate -the nuclei and axis cylinders. Sections may be -made in gum, or the tissue may be teased with -needles and then mounted in Farrant. Embedding -in celloidin, and mounting in balsam are -inadvisable, because the ether tends to dissolve -out myelin, and the clarifying oil to render it too -transparent.</p> - -<p>2. <b>Intra-muscular ramifications of -nerves</b><span class="nowrap">:—</span></p> - -<p><b>Sihler’s chloral hæmatoxyline method.</b>—This -method reveals the intra-muscular nerve-endings, -and also brings into prominence the curious -“muscle spindles” which Sherrington has -shown to be connected with the posterior nerve -roots, and which are believed by some to be the -end organs subserving muscular sense.</p> - -<p>A piece of muscle is taken as soon as possible<span class="pagenum" title="93"><a name="Page_93" id="Page_93"></a></span> -after death, or from an amputated limb, and slices -cut about one-tenth of an inch thick with the -freezing microtome. Transfer for twenty-four -hours to the following <span class="nowrap">solution:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Acetic acid</td><td class="tar">1</td><td class="tal">part.</td></tr> -<tr><td class="tal">Glycerine</td><td class="tar">1</td><td class="tal">  "</td></tr> -<tr><td class="tal">One per cent. aqueous solution of chloral hydrate</td><td class="tar"> 6</td><td class="tal">parts.</td></tr> -</table></div> - -<p>The tissues swell up in this fluid and become -translucent and gelatinous in appearance. They -are now placed in pure glycerine until saturated -as shown by their sinking to the bottom of the -dish. This usually takes several days. They -may now be stained in the following <span class="nowrap">solution:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Ehrlich’s hæmatoxyline (p. <a href="#Page_70">70</a>)</td><td class="tar">1</td><td class="tal">part.</td></tr> -<tr><td class="tal">Glycerine</td><td class="tar">1</td><td class="tal">  "</td></tr> -<tr><td class="tal">One per cent. aqueous solution of chloral hydrate</td><td class="tar"> 6</td><td class="tal">parts.</td></tr> -</table></div> - -<p>They may be left in this from three days to a -week with little fear of overstaining. Portions -may then be teased with needles, and mounted in -glycerine, or the stained tissue may be pressed out -into a sufficiently thin layer by squeezing it forcibly -between two glass slides.</p> - -<p><span class="pagenum" title="94"><a name="Page_94" id="Page_94"></a></span></p> - -<p><b>Motor and sensory nerve endings.</b>—These -are best stained by the chloride of gold -method (p. <a href="#Page_82">82</a>).</p> - -<p>Specimens must be taken from the body immediately -after death. The method is therefore -useless for the post-mortem room, but may be -used for tissues removed by operation. Small -pieces of tissue must be employed and must be -stained in bulk, sections being made subsequently.</p> - -<p>For motor nerve endings the muscle of a frog or -human muscle from a limb just amputated may -be taken. Specimens should be prepared after -staining by teasing in preference to making sections. -Mount in Farrant.</p> - -<p>Sensory nerve endings may be conveniently -studied in the cornea of a recently killed frog or -rabbit, or in a freshly extirpated human eye. -Tactile end organs may be studied in the lip or -finger tips, taste buds in the papilla foliata of the -rabbit’s tongue, and Pacini’s corpuscles are well -seen in the mesentery of a thin cat.</p> - -<p>3. <b>Staining nerve cells.</b></p> - -<p><b>Bevan Lewis’s aniline blue-black method</b>:—This -method is the best for demonstrating<span class="pagenum" title="95"><a name="Page_95" id="Page_95"></a></span> -the wealth of nerve cells in the fresh cerebral -cortex. The solution of aniline blue-black should -be of the strength of 1 in 400, about a grain to the -ounce. A piece of the cerebral cortex with pia -mater attached, should be removed as soon as -possible after death by parallel cuts about <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">8</span></span></span> inch -apart, and perpendicular to the surface of the -convolution, placed on the plate of the freezing -microtome and just frozen—not too hard or the -tissue will be brittle and will also injure the edge -of the razor. As soon as a good section is obtained -the razor should be plunged into a large -bowl of cold water to detach the section, which is -at once floated on a glass slide, and osmic acid -solution, <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">4</span></span></span> per cent. allowed to flow over it from a -pipette. This will fix the tissue elements in about -two minutes. The section is again floated off into -the bowl of water and thoroughly washed to free -it from the osmic acid. It is then stained either -on the slide, or in a watch-glass, with the aniline -blue-black solution for an hour in the cold, or half-an-hour -if the solution is slightly warmed. The -dye is thoroughly washed away with distilled -water, excess of moisture wiped off the slide with<span class="pagenum" title="96"><a name="Page_96" id="Page_96"></a></span> -blotting paper, and the section allowed to dry -under a glass bell jar. It is not practicable to dehydrate -by means of alcohol as it would cause -sudden shrinking of the tissues. When the section -is dry a drop of Canada balsam is applied -and it is covered with alcohol in the usual way. -The nerve cells and their processes are stained a -deep slate colour, as are the nuclei of the connective -tissue cells, while the ground work of the -neuroglia is faintly stained and of a neutral grey -tint. This method gives beautiful results both -with normal and morbid specimens.</p> - -<p>Various other aniline dyes, indulin, methylene -blue, gentian violet, have been employed in the -same way, but none of them give such good or -uniform results as aniline blue-black.</p> - -<p>Hardened specimens may also be stained with -aniline blue-black, but the results are not to be -compared with those obtained by the fresh method. -The stain is usually diffuse, but this can be improved -by placing the sections for <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span> –2</span> minutes in -a 2 per cent. solution of chloral hydrate in distilled -water after staining.</p> - -<p><b>Golgi’s metallic stains</b> for nerve cells.</p> - -<p><span class="pagenum" title="97"><a name="Page_97" id="Page_97"></a></span></p> - -<p>Golgi introduced the methods of producing a -metallic deposit of mercury or silver in the nerve -cell, revealing both the cell and its processes. -This method has been very fruitful in discoveries, -especially in the hands of Ramon y Cajal, -Köllicher, Van Gehucten and others. It gives -best results with embryonic tissues. To ensure -good results it is important that the tissue be removed -immediately after death. Sections of -brains removed some hours after death usually -give disappointing results.</p> - -<p>There are several methods now in vogue, all -slight modifications of Golgi’s original methods.</p> - -<p><b>Silver nitrate method.</b>—Small pieces not -more than a quarter of an inch cube, are transferred -straight from the body to a large quantity -of Marchi’s fluid (p. <a href="#Page_24">24</a>) and kept in it for about a -week, or longer in the case of adult specimens. -On removal from Marchi’s solution the tissue -should be washed for a few seconds in distilled -water, and then placed in a large quantity of a <span class="nowrap"> <span class="fraction"><span class="fnum">3</span><span class="bar">/</span><span class="fden">4</span></span></span> -per cent. solution of nitrate of silver solution in -distilled water for at least a week. The lump of -tissue becomes of a brick red colour owing to a<span class="pagenum" title="98"><a name="Page_98" id="Page_98"></a></span> -coating of silver chromate. On removal from the -silver solution the tissue should be washed in -methylated spirit for a few minutes and the -incrustation of silver chromate brushed off. Sections -may be cut in gum and celloidin; or they -may be fixed on a cork with celloidin or spirit -varnish and cut without embedding: very thin -sections are not required. Dehydrate in alcohol, -clear in xylol, and mount in balsam. Goodall -advises a mixture of pyridine and xylol for clearing, -and mounts in strong xylol-dammar solution, -without a cover-glass.</p> - -<p>Very careful attention to details and much -practice are required before uniformly good results -can be obtained. The results are extremely -beautiful and well repay the labour expended on -them. The cells and their processes appear black -on a yellowish ground.</p> - -<p>A method has been employed for deepening the -colour of the stain, but the writer has no experience -of it. Kallus (<i>Zeitsch. f. Wiss. Mikr.</i>, 1893, -477) dilutes an ordinary hydrokinone developing -solution (prepared as for developing an ordinary -photographic plate) with about ten times its<span class="pagenum" title="99"><a name="Page_99" id="Page_99"></a></span> -volume of distilled water. Just before using a third -part of absolute alcohol is added. Sections which -have been through the silver process when placed -in it become grey or black in a few minutes, and, -after washing in methylated spirit, are transferred -to a 20 per cent. aqueous solution of hyposulphite -of soda for a couple of minutes and then washed -very thoroughly in distilled water for twenty-four -hours. They are then dehydrated and mounted -in balsam.</p> - -<p><b>Buckley’s modification of the silver -method.</b>—Described in <i>Brain</i>, Winter number, -1895.</p> - -<p>The method is applicable to specimens that -have been hardened in Müller’s fluid. Thin slices -are cut in the usual way, and then immersed in</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Bichromate of potassium, 3 per cent. solution</td><td class="tar"> 5</td><td class="tal">parts</td></tr> -<tr><td class="tal">Osmic acid, 1 per cent. solution</td><td class="tar">1</td><td class="tal">part</td></tr> -</table></div> - -<p class="ti0em">for three to five days. Excess of bichromate is -removed from the sections by blotting paper, and -they are transferred to the <i>freshly prepared</i> staining -m<span class="nowrap">ixture:—</span></p> - -<p><span class="pagenum" title="100"><a name="Page_100" id="Page_100"></a></span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Phospho-molybdic acid (10 per cent.)</td><td class="tar">1</td><td class="tal">minim</td><td class="tar">2</td><td class="tal">drops.</td></tr> -<tr><td class="tal">Nitrate of silver (1 per cent.)</td><td class="tar"> 1</td><td class="tal">ounce</td><td class="tar"> 60</td><td class="tal">c.c.</td></tr> -</table></div> - -<p class="ti0em">which must not be filtered. Stain for several -days.</p> - -<p>The sections should be cut at once after removal -from the staining solution. It is claimed that the -minute details of structure of the cell processes -are better shewn by this method.</p> - -<p><b>Corrosive sublimate</b> method.—This method -is similar in its mode of action to the last, -mercury being deposited in the cell instead of -silver. It is rather less certain and requires more -practice. It seldom stains uniformly. One cell -will be found exquisitely stained while those in its -vicinity are unaffected.</p> - -<p>Small pieces of cortex are hardened for several -weeks in Müller’s fluid, or other bichromate solution, -and are then transferred direct to a one-half -per cent. aqueous solution of corrosive sublimate, -in which they should be left from three to six -weeks. Shorter periods will only give disappointing -and inconstant results. Sections should be<span class="pagenum" title="101"><a name="Page_101" id="Page_101"></a></span> -cut, if possible, in gum. They may be mounted -in Farrant, or dehydrated and mounted in balsam. -Tal has proposed to render the effect sharper by -transforming the deposit of mercury into mercuric -sulphide, by treating the sections with a solution -of sulphide of sodium, which he prepares by saturating -a ten per cent. solution of caustic soda with -sulphuretted hydrogen and then adding an equal -quantity of fresh soda solution. They are stained -in this for a few minutes and then thoroughly -washed.</p> - -<p>By this method the pyramidal cells and their -delicate processes appear as black opaque objects -on a light ground. The neuroglia cells with their -fine delicate processes are often also beautifully -stained.</p> - -<p><b>Nissl’s aniline method.</b>—This method is -complementary to Golgi’s method. The latter impregnates -the cell rendering it opaque and shewing -its form with great definiteness.</p> - -<p>Nissl’s method stains the protoplasm without -greatly reducing its transparency and allows us to -study details of cell structure. Small portions of -tissue, <i>removed as soon as possible after death</i>, are<span class="pagenum" title="102"><a name="Page_102" id="Page_102"></a></span> -hardened in alcohol. Sections are then cut, preferably -in gum, as celloidin is inconvenient owing -to its staining so deeply with aniline dyes.</p> - -<p>Sections are transferred from alcohol to a one-half -per cent. aqueous solution of methylene blue, -which is heated in a watch glass till it steams -freely, but short of the boiling point. Stain for -about a quarter of an hour and allow to cool. -Transfer the sections to a mixture containing one -part of aniline oil and ten of absolute alcohol, and -move them about till no more colour comes away. -Transfer the section to a slide with a section lifter, -drain, and dry well by pressing folded filter paper -carefully on the section. Allow some origanum -oil to flow over the section and remove excess of -this by pressure with blotting paper. Moisten -with benzine<span class="nowrap">,<a id="FNanchor_1" href="#Footnote_1" class="fnanchor">1</a></span> and add a drop of colophonium -resin dissolved in benzine. The slide is warmed -cautiously till the benzine is driven off and the -colophonium liquefied by heat alone, and then the -cover-glass is applied.</p> - -<p>Magenta and other aniline dyes may also be -employed in a similar manner.</p> -<hr class="chap" /> - -<p><span class="pagenum" title="103"><a name="Page_103" id="Page_103"></a></span></p> - - - -<h2>CHAPTER VII.</h2> - -<h3><span class="smcap">Special Methods for Staining Micro-organisms -and Blood.</span></h3> - - -<p>It is impossible, within the limits of this work, to -attempt any adequate description of the modern -methods of bacteriological investigation. Some of -these are very lengthy and complicated, and require -much skill and practice before good results -can be relied on. But those who do not desire to -make a special study of bacteriology may often -require to examine for the presence of organisms -in sections, or in various excretions, and it is -hoped that they may find the following short description -of special methods sufficient for their -purpose. For more elaborate work they must -consult one of the many excellent textbooks on -the subject.</p> - -<p>The student should provide himself with the -following dyes in <span class="nowrap">powder:—</span></p> - -<p><b>Methylene blue.</b></p> - -<p><span class="pagenum" title="104"><a name="Page_104" id="Page_104"></a></span></p> - -<p><b>Gentian violet.</b></p> - -<p><b>Methyl violet.</b></p> - -<p><b>Fuchsine.</b></p> - -<p><b>Bismarck brown.</b></p> - -<p>The following solutions of these dyes are <span class="nowrap">used:—</span></p> - -<p>1. Saturated alcoholic solutions which may be -kept in stoppered bottles.</p> - -<p>2. One per cent. aqueous solutions. These -must be freshly made each time of using.</p> - -<p>In filtering either alcoholic or aqueous solutions -it is well to moisten the filter paper beforehand -with alcohol or water as the case may be.</p> - -<p>The following special solutions will also be -<span class="nowrap">wanted:—</span></p> - -<p><b>Löffler’s methylene blue.</b>—In this solution -a weak solution of caustic potash is employed as a -<span class="nowrap">mordant:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Saturated alcoholic solution of methylene blue</td><td class="tar">3</td><td class="tal"><span class="ilb">volumes.</span></td></tr> -<tr><td class="tal">Caustic potash, aqueous solution 1 : 10,000</td><td class="tar"> 10</td><td class="tal"><span class="ilb">volumes.</span></td></tr> -</table></div> - -<p>Filter.</p> - -<p>This solution is perhaps the most generally useful -stain. It colours most bacilli and micrococci,<span class="pagenum" title="105"><a name="Page_105" id="Page_105"></a></span> -and while rapid in its action rarely overstains. It -must be made up fresh on each occasion. It is -the best counterstain after staining tubercle bacilli, -&c., with fuchsine.</p> - -<p><b>Ziehl’s carbol-fuchsine.</b></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal"><i>Carbolic acid</i> (5 per cent. aqueous solution)</td><td class="tar"> 100</td><td class="tal"><span class="ilb">volumes.</span></td></tr> -<tr><td class="tal"><i>Fuchsine</i> (saturated alcoholic solution)</td><td class="tar">11</td><td class="tal"><span class="ilb">volumes.</span></td></tr> -</table></div> - -<p>The solution must be filtered immediately before -being used.</p> - -<p><b>Gram’s iodine solution.</b>—Sections are -placed in this solution after being stained with -aniline dyes. The iodine in some way fixes the -dye in the organisms, so that they are not decolourised -along with the rest of the tissues.</p> - -<p>It is made <span class="nowrap">thus:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Iodine</td><td class="tar">1</td><td class="tal">grm.</td><td class="tar"> <span class="nowrap">1 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">grains.</td></tr> -<tr><td class="tal">Iodide of potassium</td><td class="tar">2</td><td class="tal">grms.</td><td class="tar">3</td><td class="tal">grains.</td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 300</td><td class="tal">c.c.</td><td class="tar">1</td><td class="tal">ounce.</td></tr> -</table></div> - -<p>Ten per cent. aqueous solutions of <b>nitric</b> and -<b>sulphuric</b> acids should be prepared and may be -kept indefinitely.</p> - -<p><span class="pagenum" title="106"><a name="Page_106" id="Page_106"></a></span></p> - -<p>The following are the general methods of employing -these reagents for the purpose of staining -organisms in sections. Special methods are required -for special organisms, but one or two only -can be given.</p> - -<p><b>Weigert’s method.</b>—The sections must be -placed in a freshly made one per cent. aqueous -solution of methyl violet, gentian violet, fuchsine, -&c. The solution may be kept at the temperature -of the body in an incubator. The organisms will -often stain more readily if the section be passed -through a 1 in 2000 solution of corrosive sublimate -before putting it into the staining fluid. After -staining the section is washed in distilled water -and then in methylated spirit until it appears -almost decolourised. Some prefer to decolourise -the tissues by washing in a half per cent. solution -of acetic acid instead of methylated spirit. Practice -is required before the correct time for decolourising -is accurately estimated. The beginner -should float a section rapidly on the slide now -and then, put on a cover-glass and examine it -under a low power to see if the decoloration has -been carried far enough. A contrast stain may<span class="pagenum" title="107"><a name="Page_107" id="Page_107"></a></span> -then be used, such as picrocarmine, after which -the section may be mounted in Farrant’s medium: -or a weak solution of another aniline colour may -be used as a counter stain, after which the section -is clarified in xylol, and mounted in balsam dissolved -in xylol.</p> - -<p><b>Gram’s method.</b>—Place some aniline oil in a -test tube and add ten times its volume of distilled -water. Close the end with the thumb and shake -very thoroughly. Filter ninety drops into another -clean test tube, and add ten drops of a saturated -solution of gentian violet or some similar dye. -Filter the mixture into a watch glass. Stain -sections in it for from three minutes to half-an-hour -according to the temperature,—the shorter -time for the incubator at 100°, the longer when the -sections are stained at the ordinary temperature -of the room. Wash in distilled water, and -transfer to Gram’s iodine solution until they become -black, usually in a few minutes. They are -then decolourised in absolute alcohol. This often -takes some time. It may be hastened, as -Crookshank suggests, by placing the section in -clove oil, returning to alcohol, and so on.</p> - -<p><span class="pagenum" title="108"><a name="Page_108" id="Page_108"></a></span></p> - -<p><b>Ehrlich’s modification</b> of Gram’s method. -The contrast stain is here used first.</p> - -<p>Stain the section (<i>e.g.</i>, that of a mitral valve in -a case of ulcerative endocarditis), in an alcoholic -solution of eosine (1 in 1500). Transfer to a -solution of some aniline dye, such as gentian -violet, dissolved in aniline oil water, exactly as in -Gram’s method. The section floats on the surface -and spreads out, owing to the alcohol diffusing -out. Stain for about twenty minutes. Wash the -section in water, and float out (p. <a href="#Page_55">55</a>) on a glass -slide. Allow the water to drain off and add -Gram’s iodine slowly from a pipette so as not to -disarrange the section. When the section has -become quite black pour off the Gram’s solution. -Remove all superfluous fluid from the slide with -blotting paper, and dry the section by carefully -and firmly pressing on it a folded piece of blotting -paper. If this is done with care the section -need not be injured in the least. Decoloration -is effected on the slide with aniline oil, instead -of alcohol as in the preceding method. The slide -is rocked about so that the colour may be evenly -discharged by the aniline. When no more colour<span class="pagenum" title="109"><a name="Page_109" id="Page_109"></a></span> -comes away, the aniline oil is poured off, the section -clarified in xylol, and mounted in Canada -balsam.</p> - -<p>As soon as the section is decolourised it may be -treated with a contrast stain, the most suitable -being alcoholic solutions of eosine or Bismarck -brown if a blue stain has been employed, or -methylene blue if fuchsine has been the first -stain used.</p> - -<p>The following will be found the most useful -stains and contrast <span class="nowrap">stains:—</span></p> - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tac" colspan="2"><span class="smcap">Stains.</span></td><td class="tac" colspan="2"><span class="smcap"> Contrast Stains.</span></td></tr> -<tr><td class="tal">Gentian violet.<br />Methyl violet.<br />Methylene blue.</td><td class="tar pt03"><img src="images/41x6br.png" width="6" height="41" alt="" /></td><td class="tal pt03">  <img src="images/59x6bl.png" width="6" height="59" alt="" /></td><td class="tal">Picrocarmine.<br />Eosine.<br />Bismarck brown.<br />Safranine.</td></tr> -<tr><td class="tal">Magenta.<br />Fuchsine.</td><td class="tar pt03"><img src="images/27x6br.png" width="6" height="27" alt="" /></td><td class="tal"></td><td class="tal">Methylene blue.</td></tr> -<tr><td class="tac" colspan="4">And <i>vice versâ</i>.</td></tr> -</table></div> - -<p>Much practice is required in using either of -the methods before one can judge accurately -how long to leave sections in the staining reagents -or decolourising agents, and the beginner must -not be discouraged if at first he is unable to obtain<span class="pagenum" title="110"><a name="Page_110" id="Page_110"></a></span> -good results although he follows the book directions -most minutely.</p> - -<p><b>Ehrlich method for tubercle bacilli.</b>—Sections -are stained for six to twenty-four hours -in a one per cent. solution of gentian violet, -methyl violet, methyl blue or fuchsine. They will -stain more rapidly if the staining fluid be kept in -an incubator at the body temperature. They -should be removed from the staining fluid, and -washed in distilled water, and then transferred -(preferably on a glass section lifter) to a ten per -cent. solution of nitric acid in distilled water until -they are nearly decolourised. They should then -be very thoroughly washed in distilled water. -They may then be treated with some suitable -contrast stain and mounted in Canada balsam.</p> - -<p><b>Neelsen’s stain for tubercle bacilli.</b>—Sections -are placed in Ziehl’s carbol-fuchsine -solution (p. <a href="#Page_103">103</a>) which should be warmed for ten -minutes to half-an-hour. They are then decolourised -in a solution of sulphuric acid. Twenty-five -per cent. is the strength originally recommended, -but a ten per cent. solution does equally well and -injures the section less. They are then very<span class="pagenum" title="111"><a name="Page_111" id="Page_111"></a></span> -thoroughly washed in a large quantity of water, -and afterwards may be treated with a contrast -stain.</p> - -<p><b>Gibbes’ double stain for tubercle bacilli.</b>—</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(1)</td><td class="tal">Rosaniline hydrochlorate</td><td class="tar"> 2</td><td class="tal">grms.</td><td class="tar"> 25</td><td class="tal">grs.</td></tr> -<tr><td class="tal"></td><td class="tal">Methyl blue</td><td class="tar">1</td><td class="tal">grm.</td><td class="tar">12·5</td><td class="tal">grs.</td></tr> -</table></div> - -<p>Triturate in a glass mortar,</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(2)</td><td class="tal">Aniline oil</td><td class="tar">3</td><td class="tal">c.c.</td><td class="tar"> 37·5</td><td class="tal">grs.</td></tr> -<tr><td class="tal"></td><td class="tal">Rectified spirit</td><td class="tar"> 15</td><td class="tal">c.c.</td><td class="tar"><span class="nowrap">3 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">drms.</td></tr> -</table></div> - -<p>Dissolve and add slowly to (1).</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(3)</td><td class="tal" colspan="5">Lastly add slowly to the mixture</td></tr> -<tr><td class="tal"></td><td class="tal">Distilled water</td><td class="tar"> 15</td><td class="tal">c.c.</td><td class="tar"> <span class="nowrap">3 <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span></td><td class="tal">drms.</td></tr> -</table></div> - -<p>Some of the solution is filtered into a watch -glass and warmed. The sections are placed in it -and left for some hours. They are then washed -in methylated spirit till they are sufficiently decolourised, -and then rapidly passed through absolute -alcohol and oil of cloves and mounted in -balsam and xylol. It is a very useful stain for -examining the sputum for tubercle bacilli.</p> - -<p>In order to stain fluids, such as blood, pus, or -sputum, for organisms, a very thin layer should -be obtained by placing a little of the fluid between<span class="pagenum" title="112"><a name="Page_112" id="Page_112"></a></span> -two clean cover-glasses and pressing them together. -They are then separated and allowed to -dry. The film is fixed by holding the cover-glass -in a pair of forceps, and passing it slowly through -the flame of a spirit lamp two or three times. -Films of pus should be ‘cleared’ after fixing by -placing them in a twenty per cent. solution of -acetic acid for three minutes.</p> - -<p>For clinical purposes it is often necessary to -examine urine, fæces or vomited matter for bacilli. -Films are prepared in the usual way and allowed -to evaporate slowly, and then fixed by passing -through the flame, and then washed in distilled -water before staining. In the case of vomited -matter and fæces this is usually done without -difficulty. In the case of urine however it is often -difficult to get the urine to evaporate completely. -A syrupy layer remains, and if more heat be applied -it decomposes and chars, and the products -cause precipitation of aniline during subsequent -staining processes. This may be partly avoided -by gently washing the film in distilled water -before staining.</p> - -<p>Another plan is to mix the urinary deposit with<span class="pagenum" title="113"><a name="Page_113" id="Page_113"></a></span> -a little gelatine free from organisms, such as that -in unused culture tubes. The gelatin is liquefied -by heat, and mixed with the deposit. Films are -made from this mixture, and allowed to set, and -then thoroughly washed in distilled water. The -film is then dried thoroughly, and the cover-glass -laid flat with the film uppermost, and a few drops -of the staining fluid filtered on to it. After it has -been stained sufficiently the stain is drained off, -and the slip gently washed. The film may then -be stained with some contrast stain in exactly the -same way as sections, again washed, dried between -folds of blotting paper, and mounted in -balsam.</p> - -<p>It is sometimes difficult to tell which is the side -of the cover-glass which bears the film. This is -readily done by holding the glass obliquely so that -light from a window is reflected from its surface. -The side which is coated appears dull; while the -other is smooth and bright.</p> - - -<h3><span class="smcap">Methods of Examining Blood.</span></h3> - -<p>In all these methods blood is obtained by pricking -the skin of one of the fingers, or the lobule of<span class="pagenum" title="114"><a name="Page_114" id="Page_114"></a></span> -the ear, preferably the latter. The skin must previously -be washed with soap and water or ether, -to remove any grease or epithelial scales. The -puncture should be made firmly so that blood may -escape freely. The finger or ear must not be -squeezed. Specimens must be made rapidly before -red corpuscles have run into rouleaux. The -slides and coverslips employed must be scrupulously -clean, or it is impossible to get really good -films. They should be cleaned with nitric acid -and alcohol according to the directions on page <a href="#Page_57">57</a>.</p> - -<p>Fresh specimens should be examined. The -coverslip is made just to touch the drop of blood -at one edge, so as to transfer a small quantity -only, and is at once lowered on to the slide with -the aid of a mounted needle. If slide and coverslip -be perfectly clean the blood will spread out -into a thin film, the corpuscles lying quite flat. If -there be any delay, or if the cover-glass be not -quite clean the red corpuscles will run into masses -and the specimen will be useless for minute examination. -Another specimen may be mixed with -a little of Ferrier’s solution (p. <a href="#Page_129">129</a>) before mount<span class="pagenum" title="115"><a name="Page_115" id="Page_115"></a></span>ing. -Permanent coverslip films may also be prepared.</p> - -<p>Here again the use of absolutely clean coverslips -is essential, and the blood must be taken -immediately it escapes from the puncture. A -little blood is taken on a cover-glass which is held -horizontally. Another cover-glass is lowered on to -this and by its weight and by capillary attraction, -the drop of blood quickly becomes transformed -into a thin film. The two covers are separated -as soon as the film is formed by rapidly sliding -them off one another. This manœuvre requires a -little practice and dexterity. The movement of -the slips must be in an exactly parallel direction -otherwise the coating left will be uneven, just as -when two pieces of bread and butter are pulled -apart. Even with practice it is difficult to get -more than one good film, the lower being usually -best. There are four ways of fixing the film.</p> - -<p>1. Exposure to <b>osmic acid vapour</b>.</p> - -<p>The film while still moist is held over the mouth -of a bottle containing at least one per cent. solution -of osmic acid. In a minute or two the fixation -will be complete, and the film becomes of a<span class="pagenum" title="116"><a name="Page_116" id="Page_116"></a></span> -dirty brown colour. It is then left exposed to the -air to get rid of all traces of osmic acid, and may -afterwards be stained as described below.</p> - -<p>2. Treatment with <b>saturated aqueous solution -of corrosive sublimate</b> (Muir’s method).</p> - -<p>The cover-glass on which the film has been -spread, is floated before the latter has time to dry, -film downwards on a saturated solution of corrosive -sublimate in a watch glass for half an hour. -The cover-glass is placed in distilled water and -then in alcohol to remove excess of corrosive -sublimate, and then stained. A little care is required -when washing the film to prevent it sliding -bodily off the cover-glass.</p> - -<p>3. By drying and passing rapidly through the -flame of a Bunsen burner, exactly as in preparing -specimens of sputum, &c. (p. <a href="#Page_111">111</a>). This method -is handy for ordinary clinical purposes.</p> - -<p>4. By keeping the coverslips at a temperature -of about 200° F. (Ehrlich’s method).</p> - -<p>Ehrlich uses for this purpose a strip of copper -about two inches wide and a foot long which is -supported on a retort stand in a horizontal position. -One end is heated by a Bunsen’s burner<span class="pagenum" title="117"><a name="Page_117" id="Page_117"></a></span> -beneath. The point in the copper strip at which -the temperature is at boiling point is readily ascertained -by dropping a little water on. The point -at which a drop of water assumes the spherical -state indicates a temperature there of 212° F. -The coverslips are placed an inch or two further -than this point, and kept there at a temperature -of about 200° F. for some hours.</p> - - -<h3><span class="smcap">Staining Methods.</span></h3> - -<p>Fresh blood may be stained by mixing with -Ferrier’s fuchsine <span class="nowrap">solution:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Fuchsine</td><td class="tar">1</td><td class="tal"><span class="ilb">grm.</span></td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 150</td><td class="tal">c.c.</td></tr> -</table></div> - -<p>Dissolve and add</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Alcohol (80 per cent.)</td><td class="tar">50</td><td class="tal">c.c.</td></tr> -<tr><td class="tal">Neutral glycerine</td><td class="tar"> 200</td><td class="tal">c.c.</td></tr> -</table></div> - -<p>A spot of this solution is mixed with the blood -on a slide by means of a mounted needle, and -covered with a clean cover-glass. The red corpuscles -are slightly stained, while the nuclei of the -white corpuscles are stained a bright crimson, and -the “blood plates” a deep pink colour.</p> - -<p><span class="pagenum" title="118"><a name="Page_118" id="Page_118"></a></span></p> - -<p>Stained preparations may also be obtained by -using <b>Toison’s fluid</b>, which serves also for -diluting the blood in order to determine the exact -number of red and white corpuscles present by -means of Gowers’ or the Thoma-Zeiss hæmocytometer. -It is prepared <span class="nowrap">thus:—</span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Glycerine</td><td class="tar">30</td><td class="tal">c.c.</td><td class="tar">1</td><td class="tal">oz.</td></tr> -<tr><td class="tal">Sodium sulphate</td><td class="tar">8</td><td class="tal">grms.</td><td class="tar">2</td><td class="tal">drms.</td></tr> -<tr><td class="tal">Sodium chloride</td><td class="tar">1</td><td class="tal">grm.</td><td class="tar"> 15</td><td class="tal">grs.</td></tr> -<tr><td class="tal">Methyl violet</td><td class="tar">·25</td><td class="tal">grm.</td><td class="tar">4</td><td class="tal">grs.</td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 160</td><td class="tal">c.c.</td><td class="tar">5</td><td class="tal">oz.</td></tr> -</table></div> - -<p>It stains the nuclei and blood plates, but does -not alter the shape of the red cells. It requires to -be made up fresh occasionally as torulæ are apt -to form and multiply in it.</p> - -<p>Dried films may be stained with hæmatoxyline, -picrocarmine, or any of the general stains. The -nuclei of the leucocytes may be stained rapidly in -a couple of minutes in a one per cent. solution of -methyl violet, washing in water, drying between -blotting paper and mounting in balsam. The best -method for general purposes is to stain with a -saturated aqueous solution of methyl blue for half -an hour or longer. Wash in water, and then<span class="pagenum" title="119"><a name="Page_119" id="Page_119"></a></span> -stain for ten minutes in a half saturated aqueous -solution of eosine. In this way the eosinophile -granules of the leucocytes and the red corpuscles, -are stained by the eosine, while the nuclei of the -leucocytes are stained by the methyl blue.</p> - -<p>Kanthack and Drysdale recommend that the -film should first be stained with a half per cent. -solution of eosine in 50 per cent. alcohol, then -washed, dried and fixed in the flame, and stained -for a short time in Löffler’s solution of methylene -blue (p. <a href="#Page_104">104</a>).</p> - -<p>These films may be stained for micro-organisms -in the way described for cover-glass preparations -(p. <a href="#Page_112">112</a>).</p> - -<hr class="chap" /> - -<p><span class="pagenum" title="120"><a name="Page_120" id="Page_120"></a></span></p> - - - - -<h2>CHAPTER VIII.</h2> - -<h3><span class="smcap">Injection of Blood Vessels.</span></h3> - - -<p>Injection of blood vessels may be performed on -small animals, or on individual human organs -after removal from the body. The object is to -fill the vessels with a coloured fluid which will -solidify afterwards. It is possible in the same -organ to inject the arteries with a red medium, -the veins blue, and secretory ducts, such as bile -ducts, yellow or blue.</p> - -<p>The most convenient basis for an injection mass -is gelatine, as its solutions liquefy at a temperature -of about 100° F., and solidify a little below -that point, and when solidified cut readily, and do -not tend to become brittle. The various masses -are prepared as <span class="nowrap">follows:—</span></p> - -<p><b>Red injection mass</b> (Woodhead’s formula) -consists of gelatine softened by mixture with water -and coloured by carmine.</p> - -<p><span class="pagenum" title="121"><a name="Page_121" id="Page_121"></a></span></p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(1)</td><td class="tal">Carmine</td><td class="tar">4</td><td class="tal">grms.</td></tr> -<tr><td class="tal"></td><td class="tal">Liq. ammoniæ B.P.</td><td class="tar">8</td><td class="tal">grms.</td></tr> -<tr><td class="tal"></td><td class="tal">Distilled water</td><td class="tar"> 150</td><td class="tal">c.c.</td></tr> -</table></div> - -<p>Dissolve the carmine in the ammonia in a mortar. -Pour on the water. Mix thoroughly and -filter.</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(2)</td><td class="tal">Gelatine</td><td class="tar">10</td><td class="tal">grms.</td></tr> -<tr><td class="tal"></td><td class="tal">Distilled water</td><td class="tar"> 50</td><td class="tal">c.c.</td></tr> -</table></div> - -<p>Allow it to stand in the cold water until the -water is absorbed and the gelatine has become -soft.</p> - -<p>Warm (1) almost to boiling point over a Bunsen -burner, and add the gelatine slowly. Stir thoroughly -and add a ten per cent. solution of acetic -acid until the solution becomes slightly acid. -This will be shewn by the mass assuming a -darker and duller colour. A little salicylic acid -may be added to preserve it.</p> - -<p><b>Blue injection mass.</b>—To the gelatine mass -(2) prepared as above, and liquefied by heat, add -instead of carmine</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">Soluble Prussian blue</td><td class="tar">5</td><td class="tal">grms.</td></tr> -<tr><td class="tal">Distilled water</td><td class="tar"> 60</td><td class="tal">c.c.</td></tr> -</table></div> - -<p>Every trace of alkali must be kept away from<span class="pagenum" title="122"><a name="Page_122" id="Page_122"></a></span> -the mass during and after the preparation. Sections -of injected organs should be mounted in -Farrant’s solution slightly acidulated with formic -or acetic acid. With every care, however, the -blue colour is apt to fade in the course of time.</p> - -<p><b>Green injection mass.</b> Robin’s formula -(modified).</p> - - -<div class="center"> -<table border="0" cellpadding="1" cellspacing="0" summary=""> -<tr><td class="tal">(1)</td><td class="tal">Arseniate of soda (sat. sol.)</td><td class="tar"> 80</td><td class="tal">c.c.</td></tr> -<tr><td class="tal"></td><td class="tal">Glycerine</td><td class="tar">50</td><td class="tal"> "</td></tr> -<tr><td class="tal">(2)</td><td class="tal">Sulphate of copper (sat. sol.)</td><td class="tar">40</td><td class="tal"> "</td></tr> -<tr><td class="tal"></td><td class="tal">Glycerine</td><td class="tar">50</td><td class="tal"> "</td></tr> -</table></div> - -<p>Mix and add one part to three parts of the -gelatine mass made as for the red and blue injections.</p> - -<p><b>Method of injection.</b>—In injecting the vessels -of tissues it is necessary that the organ or the -entire animal, as the case may be, shall be kept -during injection at a temperature well above that -at which the gelatine mass will melt, otherwise -the gelatine will “set” in the arteries and will -never reach the capillaries. This warming is -effected by immersing the animal in a water bath. -The liquefied gelatine is forced into the artery by a -syringe or by air pressure. It is essential that the<span class="pagenum" title="123"><a name="Page_123" id="Page_123"></a></span> -pressure be uniform and steady. This is so much -more easily managed with air pressure that this -method is strongly recommended to the beginner. -But, whatever method be adopted, perfect results -can only be obtained with certainty after long -practice. Sometimes too high pressure will be -employed and the vessels give way, at others the -injection may not reach the capillaries at all. The -most scrupulous attention to details is essential.</p> - -<p>By far the most effective apparatus for injecting -is the modification of Ludwig’s constant pressure -apparatus devised by Fearnley<span class="nowrap">.<a id="FNanchor_2" href="#Footnote_2" class="fnanchor">2</a></span> Although the -apparatus appears complicated, the various parts -are easily obtained and it would be easy to improvise -a substitute for the water bath.</p> - -<p>The apparatus which is shewn in figures 10 and -11 consists of a bath deep enough to contain the -animal, and a vessel containing the injection fluid. -The bath is kept at a temperature of about 110° -by an ordinary Bunsen burner. A large Wolff’s -bottle (20–40 oz.) with three necks, is fitted with -three india-rubber stoppers perforated by glass -tubes. Through the central stopper a glass tube<span class="pagenum" title="124"><a name="Page_124" id="Page_124"></a></span> -connected by a rubber tube, with an ordinary -Higginson’s syringe, passes almost to the bottom of -the bottle. From one of the other necks a rubber -tube passes to an ordinary mercurial manometer, -while from the third a tube passes to the flask -containing the liquefied injection mass, which is<span class="pagenum" title="125"><a name="Page_125" id="Page_125"></a></span> -immersed in the water bath. This flask is also -firmly stoppered, and should be about half filled -with injection material. The delivery tube from -the large Wolff’s bottle should only just come -through the cork. Another glass tube passes<span class="pagenum" title="126"><a name="Page_126" id="Page_126"></a></span> -down almost to the bottom of the flask, and is connected -by a rubber tube with the cannula inserted -into the artery. It will be evident from figure <a href="#Fig_11">11</a> -that when water is pumped by the Higginson’s -syringe into the Wolff’s bottle the pressure there -will be raised (as indicated by the manometer). -This increase of pressure will equally affect the -air inside the bottle containing the injection fluid, -and the fluid will be forced out along the tube and -through the cannula into the artery.</p> - -<div class="figcenter" style="width: 450px;"> -<img src="images/i132.jpg" width="450" height="389" alt="" /> -<div class="caption"><span class="smcap">Fig. 10.</span>—Fearnley’s arrangement for injecting blood -vessels. (Reproduced by permission of Messrs. Macmillan, -from Fearnley’s <i>Practical Histology</i>).</div> -</div> - -<div class="figcenter" style="width: 440px;"> -<a id="Fig_11"></a><img src="images/i133.jpg" width="440" height="243" alt="" /> -<div class="caption"><span class="smcap">Fig. 11.</span>—Scheme shewing distribution of pressure in Fearnley’s Injection -Apparatus (from Fearnley’s <i>Practical Histology</i>).</div> -</div> - -<p>Before using the apparatus a clamp should be -placed on the exit tube of the vessel containing -the injection fluid, and the pressure should be -raised to see that the apparatus is everywhere air-tight. -Any leaks should be sealed before the -actual injection is commenced.</p> - -<p>If an isolated organ is to be injected, a cannula -of glass or brass should first be inserted into the -artery and securely tied in position. The organs, -if cold, must be soaked in water at 120° F. for -about half an hour and then transferred to a water -bath.</p> - -<p>In the case of injecting an entire animal, such -as a rabbit, rat, or guinea pig, the injection is best<span class="pagenum" title="127"><a name="Page_127" id="Page_127"></a></span> -made a few minutes after death. The animal -may be chloroformed, and then bled to death by -opening a large vein. As soon as death has occurred -incise the skin over the thorax in the -middle line. Cut through the costal cartilages -to the right of the sternum, and through the -junction of the manubrium and body of the -sternum. These incisions being for most part -through non-vascular parts will not lead to escape -of fluid during injection. The sternum being -forcibly raised towards the left, the pericardium -will be exposed and must be carefully divided. -An incision must be made into the left ventricle, -and a cannula passed up into the aorta and firmly -secured by a ligature passed round the aorta with -the assistance of forceps or an aneurism needle. -Any blood is cleaned away and the animal is then -placed in the water bath for about ten minutes. -The tube from the bottle containing the injection -fluid is then filled by gentle pressure on the -syringe, and clamped when full. Its end is then -placed on the cannula and secured there by a -ligature. The pressure should be raised by -squeezing the syringe until the manometer regis<span class="pagenum" title="128"><a name="Page_128" id="Page_128"></a></span>ters -one inch. The clamp should then be removed -and the injection commenced. The pressure -should be raised very gently and constantly by -working the syringe, and the condition of the -gums, lips, and eyes of the animal observed. The -gums will soon shew a pink tinge. The best -indications are obtained by watching the effect on -the small vessels of the sclerotic. When these -are completely filled, which will be in about five -to ten minutes according to the rate at which the -air pressure has been increased, the injection may -be stopped. This result will be obtained, under -good conditions, before the manometer indicates a -pressure of five inches. The aorta should now be -ligatured, and the animal placed in cold water -frequently renewed until it is thoroughly cooled. -The organs may then be removed and placed in -methylated spirit and hardened. Sections are -afterwards cut and mounted in the usual way.</p> - -<hr class="chap" /> - -<p><span class="pagenum" title="129"><a name="Page_129" id="Page_129"></a></span></p> - - - - -<h2>CHAPTER IX.</h2> - -<h3><span class="smcap">Directions for Preparing Individual Tissues.</span></h3> - - -<p><b>Normal histology.</b>—It cannot be too strongly -impressed on the beginner that a thorough mastery -of the normal appearances of tissues and -organs is absolutely necessary before attempting -to make an accurate study of morbid changes in -them. He should not be satisfied with examining -one specimen of an organ but as many as he conveniently -can, in order to be fully acquainted with -the many deviations from normal which may exist -without actual disease. He should therefore obtain -several animals, such as small dogs, cats, -rabbits, frogs, &c., and remove their organs with -all care, and harden them in the various appropriate -fluids. He should also obtain specimens of -normal human organs from the post-mortem room. -Many normal tissues (skin, muscle, tendon, bone, -&c.), can also be prepared from a limb amputated -for an accident to a healthy patient. By prepar<span class="pagenum" title="130"><a name="Page_130" id="Page_130"></a></span>ing -specimens in this way he will not only become -the possessor of a set of slides illustrating normal -histology, but will find also that he has acquired -that proficiency in hardening and staining the -specimens which practice alone can give.</p> - -<p>The following account of the method of preparing -different tissues is merely intended to indicate -the lines on which the beginner should proceed. -After some practice he will be quite able to -select the modes of hardening and staining which -special circumstances or cases may seem to demand.</p> - -<p>The first part of these directions will refer to -the preparation of normal tissues, the second part -to morbid histology.</p> - -<p><b>Blood.</b>—For special methods of examination -see Chapter <a href="#Page_103">VII</a>.</p> - -<p><b>Blood crystals—Hæmoglobin crystals</b>, -obtained from the blood of an animal, or enough -may be collected at any operation. A little water -or a little ether is added to the blood which is -allowed to stand for half-an-hour after which a -drop is allowed to evaporate slowly on a clean -slide.</p> - -<p><span class="pagenum" title="131"><a name="Page_131" id="Page_131"></a></span></p> - -<p><b>Hæmatin crystals.</b>—The student should -make himself thoroughly familiar with these, as -their presence affords positive proof of the existence -of blood colouring matter in a stain.</p> - -<p>To obtain them a drop of blood should be allowed -to dry on a slide. The dried blood is -scraped into a little heap with a small piece of -clean glass, and a drop of glacial acetic acid -added. As it evaporates minute reddish-brown -acicular crystals will appear.</p> - -<p><b>Hæmatoidin crystals.</b>—Obtained from the -site of a bruise, or an old hæmorrhage, <i>e.g.</i>, a -cerebral apoplexy or a hæmatocele.</p> - -<p><b>Simple squamous epithelium.</b>—(<i>Endothelium</i>). -Carefully strip off the lining of the -parietal pericardium or parietal pleura, of a recently -killed animal, or spread out its omentum on -a piece of cork, and (1) stain the intercellular -cement with nitrate of silver (p. <a href="#Page_82">82</a>) so as to reveal -the outlines of the cells. (2) Stain other -specimens with hæmatoxyline or alum carmine to -reveal the nuclei.</p> - -<p><b>Stratified squamous epithelium.</b>—Specimens -from skin of various parts, finger, groin, lip,<span class="pagenum" title="132"><a name="Page_132" id="Page_132"></a></span> -tongue should be prepared. Harden in Müller’s -fluid.</p> - -<p><b>Transitional epithelium.</b>—Occurs in the -pelvis of the kidney, ureter and bladder. It is -very readily detached, especially if not hardened -immediately after death. Remove as early as -possible. If the bladder is taken it should be cut -open and pinned out as flat as possible. Harden -in osmic acid, or Müller’s fluid and spirit. Embed -preferably in celloidin.</p> - -<p><b>Simple columnar epithelium.</b>—Occurs in -many parts. It may be studied in the salivary -ducts, the intestine, kidney, &c., of any mammal.</p> - -<p><b>Goblet-cells.</b>—Seen abundantly among the -columnar cells of the intestinal glands, and in the -mucous glands of the mouth and of the cervix -uteri.</p> - -<p><b>Stratified columnar epithelium.</b>—Occurs -only in the urethra. Harden the penis of a cat in -Müller’s fluid, and cut transverse sections.</p> - -<p><b>Ciliated epithelium.</b>—Harden the trachea -of a recently killed cat in osmic acid or Müller’s -fluid. Beautiful specimens may also be obtained -from an ordinary nasal polypus, which should be<span class="pagenum" title="133"><a name="Page_133" id="Page_133"></a></span> -put into hardening fluid immediately after removal.</p> - -<p>Stain all sections of epithelium in picrocarmine, -and in eosine and hæmatoxyline.</p> - -<p><b>Ordinary areolar tissue.</b>—Difficult to obtain -free from fat. It may be studied in the subcutaneous -tissue of the section of the cat’s penis -already made. A fragment of the tissue should -also be removed and carefully teased in a drop of -picrocarmine. Areolar tissue may also be studied -in sections of skin, and in the capsules of the -different internal organs.</p> - -<p><b>Elastic tissue.</b>—May also be studied in most -sections of skin. If the ligamentum nuchæ of a -large quadruped (horse, bullock), &c., is available -it yields the best specimens, or the human ligamenta -subflava may be examined. Pin a piece -out on a piece of wood or wax. Harden in -Müller’s fluid. Stain in picrocarmine. Both -sections and teased specimens should be prepared.</p> - -<p><b>Tendon.</b>—Readily obtained from an amputated -limb. Harden in Müller’s fluid. Make -transverse and longitudinal sections. Stain with -eosine and hæmatoxyline.</p> - -<p><span class="pagenum" title="134"><a name="Page_134" id="Page_134"></a></span></p> - -<p>A preparation should also be made by teasing -a little of the fresh tendon in normal salt solution, -and staining with picrocarmine.</p> - -<p><b>Retiform</b> or <b>lymphadenoid tissue</b>.—Seen -in lymphatic glands and in the lymphoid follicles -scattered along the sub-mucous coat of the alimentary -canal.</p> - -<p>Prepare sections in the ordinary way. Stain in -eosine and hæmatoxyline or in picrocarmine.</p> - -<p>Some sections should also be prepared by pencilling -(<i>i.e.</i>, dabbing with a camel’s hair brush) or -by shaking sections up in a test tube with water -or normal salt solution. By this means the leucocytes -are removed, and the structure of the -adenoid tissue itself becomes more evident.</p> - -<p><b>Fat.</b>—Best studied in sections of skin and -subcutaneous tissue, or in the mesentery of the -cat. One specimen should be stained with osmic -acid and picrocarmine and mounted in Farrant’s -medium, and another in eosine and hæmatoxyline -and mounted in Canada balsam.</p> - -<p><b>Pigment cells.</b>—Branched cells are best -studied in the living foot of the frog, where amœboid -movements may be seen in them when the<span class="pagenum" title="135"><a name="Page_135" id="Page_135"></a></span> -light falling on the retina is made to vary in intensity. -Permanent preparations are most conveniently -made from the pallium of the common snail. -The shell is removed, and the pallium snipped out -with the scissors. It is then pinned out flat, -hardened for a day in methylated spirit, and -mounted unstained in Farrant’s medium. They -are also well seen in sections of the choroid coat -of the eye.</p> - -<p><b>Hyaline cartilage.</b>—Specimens may be obtained -from any joint, from the costal cartilages -of young animals, or from the thyroid cartilage -and tracheal rings. It may be hardened in spirit. -Stain with picrocarmine, eosine and hæmatoxyline, -and with methyl violet.</p> - -<p><b>Elastic cartilage.</b>—Prepared from the epiglottis, -or from the cartilages of the ear, <i>e.g.</i>, of a -cat. Harden in spirit. Stain in picrocarmine or -in dilute fuchsin.</p> - -<p><b>White fibro-cartilage.</b>—Obtained from intervertebral -disc. Prepare and stain as for hyaline -cartilage.</p> - -<p><b>Bone</b><span class="nowrap">:—</span></p> - -<p><b>Unsoftened Bone.</b>—Cut as thin a section as<span class="pagenum" title="136"><a name="Page_136" id="Page_136"></a></span> -possible with a fine saw. Rub the section with -the hand on a dry oil stone until it is as thin as -possible. Then cement it by Canada balsam -(liquefied by warming) to a piece of plate glass -and continue the rubbing process with this, examining -it now and then with the low power to see -if it is thin enough. As soon as it is thin enough -it is washed off the slide with methylated spirit, -and washed to get rid of the fine bone dust. It -should then be transferred to turpentine and may -be mounted in balsam.</p> - -<p><b>Softened bone.</b>—Specimens may be obtained -from an amputated limb or from the femur of a cat.</p> - -<p>Specimens should be decalcified in chromic and -nitric fluid, and the hardening completed in spirit. -In studying the process of ossification, <i>e.g.</i>, in the -head of the humerus of a kitten, it is best to -embed the specimen in celloidin before cutting -sections, as the trabeculæ of bone are very delicate, -and easily detached.</p> - -<p>Very beautiful double staining effects may be -obtained with either picrocarmine, or eosine and -hæmatoxyline, and with eosine and methyl violet.</p> - -<p><b>Bone marrow.</b>—To obtain good sections of<span class="pagenum" title="137"><a name="Page_137" id="Page_137"></a></span> -red bone marrow, take a piece of the clavicle or a -rib, or of one of the carpal or tarsal bones. Decalcify -in chromic and nitric fluid. Embed in -celloidin. Stain with eosine and logwood, eosine -and alum carmine, or alum carmine and picric -acid. Mount in Canada balsam. The various -cells present in bone marrow may also be studied -by squeezing some fresh marrow from a rib, and -making a cover-glass film, and preparing in -exactly the same way as is directed in the case -of blood films on page <a href="#Page_116">116</a>.</p> - -<p><b>Tooth.</b>—Best cut <i>in situ</i> from the jaw of a cat. -Decalcify in chromic and nitric fluid, and cut both -vertical and transverse sections. Stain in picrocarmine, -or eosine and hæmatoxyline.</p> - -<p><b>Developing tooth.</b>—Extremely good specimens -may be obtained from the jaw of a newly-born -kitten or puppy. Sections can easily be -made shewing a milk tooth and a developing permanent -tooth by its side.</p> - -<p>The enamel is dissolved by decalcifying fluids. -To study it a specimen of unsoftened tooth should -be made, according to the directions given for -bone.</p> - -<p><span class="pagenum" title="138"><a name="Page_138" id="Page_138"></a></span></p> - -<p><b>Striped muscle.</b>—Should be studied in various -animals.</p> - -<p>The leg of an insect such as a cockroach may -be hardened in osmic acid. One leg should be -hardened in a straight position so as to fix the -fibrils in the fully extended position, another -should be bent up so as to get specimens of relaxed -fibrils.</p> - -<p>Portions of muscle should be removed, and -teased on a glass slide in some staining fluid -such as picrocarmine, a tenth per cent. solution -of eosine or quarter per cent. of safranine.</p> - -<p>Sections of amphibian and mammalian muscle -should be prepared to show their differences in -structure. The most convenient part to select is -the tongue, as a view of the fibres is obtained both -in longitudinal and transverse sections. Sections -should be stained in eosine and hæmatoxyline -which gives a beautiful effect. For special stains -for intra-muscular nerve endings see page <a href="#Page_92">92</a>.</p> - -<p><b>Heart muscle.</b>—A portion should be teased -fresh in picrocarmine or eosine, another portion -hardened in Müller’s fluid, and sections made and -stained with eosine and hæmatoxyline.</p> - -<p><span class="pagenum" title="139"><a name="Page_139" id="Page_139"></a></span></p> - -<p><b>Unstriped muscle</b> may be obtained by teasing -a fresh portion of the muscular coat of the small -intestine of an animal, or by sections of the hardened -intestine, bladder or uterus. Stain in picrocarmine -or preferably eosine and hæmatoxyline.</p> - -<p><b>Nerves.</b>—The special methods for staining -nerve tissues are detailed in Chapter <a href="#Page_87">VI</a>. The -student must remember that the ordinary staining -methods are also applicable to nervous tissues.</p> - -<p><b>Nerve terminations</b><span class="nowrap">:—</span></p> - -<p><b>Meissner’s corpuscles.</b>—Take the tip of an -index finger immediately after amputation. Place -part of it at once in chloride of gold solution, and -the rest in Müller’s fluid until it is hardened.</p> - -<p>Sections stained with chloride of gold should be -mounted in Farrant’s medium. The other sections -may be stained in picrocarmine or eosine -and hæmatoxyline.</p> - -<p><b>Pacini’s corpuscles.</b>—May be dissected out -on the smaller branches of the digital nerves, or -may be found in the mesentery of the cat. The -latter should be spread out on wood, hardened in -Müller’s fluid, stained in hæmatoxyline, and -mounted in balsam.</p> - -<p><span class="pagenum" title="140"><a name="Page_140" id="Page_140"></a></span></p> - -<p>Other forms of tactile corpuscles may be studied -in the tongues of frogs, ducks, or geese. A network -of nervous fibrils should be studied in the -cornea. Take the cornea of a newly killed frog -or cat and stain with chloride of gold (p. <a href="#Page_82">82</a>).</p> - -<p>The end plates in which the nerves terminate -in muscle may be studied by placing specimens of -living muscle of some cold blooded animal into -chloride of gold solution, and staining rather -deeply.</p> - -<p><b>Arteries.</b>—Take a piece of the aorta, a piece -of some medium artery, as the renal or radial, and -harden in Müller’s fluid. Stain in picrocarmine -and always in eosine and hæmatoxyline. Arterioles -are best studied in sections of the various -organs. Thus they are seen in each Malpighian -body of the spleen, in the boundary zone of the -kidney, and so on. A longitudinal surface view -can also be obtained by staining and examining -the pia mater.</p> - -<p><b>Veins.</b>—Remove, harden, and stain in the -same way.</p> - -<p><b>Capillaries.</b>—May be very well seen in the -foot of the frog.</p> - -<p><span class="pagenum" title="141"><a name="Page_141" id="Page_141"></a></span></p> - -<p>Stun a frog by striking its head, or by chloroforming -it. Fix it on a piece of card with a V -shaped notch at one end. Tie one of the hind -feet by means of threads attached to its toes so -that the web of the foot is gently stretched over -the V. The foot can then be readily examined -under a <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span> inch objective. The foot must be -brushed from time to time with normal salt solution -to keep it moist. The movement of blood in -the capillaries, &c., can then be studied for an -hour or two. After death the mesentery should -be spread out on a piece of wood, and hardened -for a few days in Müller’s fluid.</p> - -<p>Stain with eosine and hæmatoxyline.</p> - -<p><b>Lymphatics.</b>—Commencement of lymphatics -in serous membrane. Stain a piece of cat’s omentum -in nitrate of silver (p. <a href="#Page_82">82</a>) for some minutes. -After washing keep in glycerine for about a week -and then stain in hæmatoxyline and mount in -Farrant’s medium.</p> - -<p><b>Lymphatic glands.</b>—The lymphatic glands -of the neck of the cat may be used. Harden in -Müller’s fluid. Stain in picrocarmine, eosine and -hæmatoxyline.</p> - -<p><span class="pagenum" title="142"><a name="Page_142" id="Page_142"></a></span></p> - -<p><b>Skin and sweat glands.</b>—Sections should -be made from pieces taken (<i>a</i>) from the sole, (<i>b</i>) -from the skin of the body, (<i>c</i>) from the axilla of an -adult to study the pigment. Harden in Müller’s -fluid. Stain in picrocarmine or eosine and hæmatoxyline.</p> - -<p><b>Hairs and sebaceous glands.</b>—Take a -portion of the scalp, or of the skin of a puppy. -Harden in Müller’s fluid. Stain in eosine and -hæmatoxyline, and mount others unstained.</p> - -<p>Hairs from various parts of the body should -also be soaked for some hours in liq. potassæ and -mounted unstained in Farrant’s medium. They -may be bleached subsequently by treatment with -eau de Javelle (p. <a href="#Page_27">27</a>).</p> - -<p><b>Brain and spinal cord.</b>—Must be removed -from the body with extreme care, all stretching or -squeezing being avoided. Harden slowly in -Müller’s fluid to which a fourth of its bulk of -water may be added.</p> - -<p>The best staining reagents to employ are eosine -and hæmatoxyline, alum carmine or borax carmine, -aniline blue-black, &c. Staining methods, -see Chapter <a href="#Page_87">VI</a>.</p> - -<p><span class="pagenum" title="143"><a name="Page_143" id="Page_143"></a></span></p> - -<p><b>Eye.</b>—Harden the eye of a recently killed -bullock, cat, or other animal in formal (p. <a href="#Page_23">23</a>), -puncturing the sclerotic in places to allow the -hardening fluid to penetrate. In about a week -make a horizontal section through the eye. The -anterior half (the lens having been removed) may -be satisfactorily cut in gum. Sections of the -crystalline lens are not very satisfactory. The -best way to get specimens of the fibres is to tease -a piece of the fresh lens of a fish (<i>e.g.</i>, a cod) in -a <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">40</span></span></span> per cent. aqueous solution of eosine. Wash -the eosine off the slide with <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">2</span></span></span> per cent. acetic -acid, and mount in Farrant’s solution.</p> - -<p>The posterior half of the eye should be embedded -in celloidin, as otherwise it is extremely -difficult to get sections of the retina in its proper -relation to the other coats.</p> - -<p>Mount some specimens unstained. Stain others -with the ordinary stains.</p> - -<p><b>Internal ear.</b>—Decalcifying the temporal -bone of a cat, dog, guinea pig, &c., in chromic -and nitric fluid. As soon as the bone is decalcified -complete the hardening of the soft parts in -methylated spirit, embed in celloidin, and cut<span class="pagenum" title="144"><a name="Page_144" id="Page_144"></a></span> -sections in the longitudinal axis of the cochlea. -Owing to the extreme hardness of the bone in -adults it will be found best to use the petrous -bone of newly born animals.</p> - -<p>The semi-circular canals will be most readily -studied in the temporal bone of fishes, or of birds, -<i>e.g.</i>, the common fowl. They also must be cut in -celloidin, and stained in the ordinary way.</p> - -<p><b>Nose and olfactory epithelium.</b>—It is -difficult to obtain specimens from the human -subject, but very satisfactory preparations may be -made from the dog, or more conveniently in a new -born puppy where the bones are still cartilaginous. -Harden the latter in Müller’s fluid, decalcify adult -specimens in chromic and nitric fluid. Specimens -of ciliated epithelium, &c., will be obtained from -the lower part, and of the special olfactory epithelium -from the upper part. Stain in eosine and -hæmatoxyline.</p> - -<p><b>Lungs.</b>—Carefully remove the lungs of a cat -without injuring the bronchi or trachea. Introduce -a cannula into the trachea and gently inflate -the trachea with air. Ligature the trachea and -place the lung in Müller’s fluid, a weight being<span class="pagenum" title="145"><a name="Page_145" id="Page_145"></a></span> -attached to keep the organ submerged. Harden -for about six weeks, and then make sections of the -various parts.</p> - -<p>To demonstrate the endothelium of the alveoli, -inject instead of air, nitrate of silver. Allow it to -remain in for half an hour, then remove it by -washing, and harden in Müller’s fluid.</p> - -<p>Beautiful casts of the alveoli, &c., may be obtained -by placing a cat’s or human lung under the -receiver of an air-pump, and when the air is completely -exhausted, injecting fusible metal into the -bronchus. The lung tissue is then removed by corrosion -or by maceration. Portions of the casts should -be removed, fixed in a glass cell with a spot of -Canada balsam, and examined by reflected light.</p> - -<p><b>Thyroid gland.</b>—Best obtained from a young -subject either human or an animal.</p> - -<p>Harden in Müller’s fluid. Stain in picrocarmine -or eosine and hæmatoxyline. Also stain sections -in safranine, which stains the colloid material, and -also picks out any colloid formation in the cells -themselves.</p> - -<p><b>Thymus.</b>—Remove from a fœtus or a very -young animal, and prepare in the usual way.</p> - -<p><span class="pagenum" title="146"><a name="Page_146" id="Page_146"></a></span></p> - -<p><b>Tongue.</b>—That of the cat or rabbit serves -very well.</p> - -<p>Ordinary transverse sections should be made, -and also sections through the circumvallate -papillæ in order to study the “taste buds.”</p> - -<p><b>Salivary glands.</b>—Those of a cat or dog do -very well.</p> - -<p>Sections should be made from each of the three -glands.</p> - -<p><b>Stomach.</b>—That of the cat or dog should be -studied. The organ must be removed immediately -after death before any post-mortem digestion of -the coats has occurred. The stomach should be -opened, washed gently and pinned out flat, with -as little stretching as possible on a piece of wood, -and hardened in Müller’s fluid.</p> - -<p>Sections should be made (<i>a</i>) longitudinally -through the cardiac end to show the transition -from the œsophageal to the gastric mucous membrane, -(<i>b</i>) from a portion of the greater curvature, -(<i>c</i>) longitudinally through the pyloric valve.</p> - -<p>Eosine and hæmatoxyline form the best stain -for the alimentary canal.</p> - -<p><b>Intestine.</b>—Prepare in the same way as the<span class="pagenum" title="147"><a name="Page_147" id="Page_147"></a></span> -stomach. Make sections from (<i>a</i>) the upper part -of the duodenum to show Brunner’s glands, (<i>b</i>) the -ileum, (<i>c</i>) a Peyer’s patch, (<i>d</i>) the vermiform appendix, -(<i>e</i>) the colon.</p> - -<p><b>Liver.</b>—Make an injection of one specimen -with carmine and gelatin (p. <a href="#Page_120">120</a>). Harden in -methylated spirit. Others should be hardened in -Müller’s fluid and stained in the usual way.</p> - -<p><b>Kidney, supra-renal, and pancreas.</b>—Same -preparation as for liver.</p> - -<p><b>Spleen.</b>—Harden in Müller’s fluid.</p> - -<p>Mount one section unstained. Shake another -up with water in a test tube to shew the structure -of the pulp. Stain others in eosine and hæmatoxyline.</p> - -<p><b>Bladder.</b>—Must be removed and pinned out -immediately after death, as otherwise the epithelium -will be macerated off. Consequently it must -be taken from an animal, as a cat. Harden in -osmic acid. Cut in celloidin as the coats are very -apt to become detached.</p> - -<p><b>Penis</b> and <b>testis</b>.—Readily obtained from -dog, cat, or rat.</p> - -<p>Stain with eosine and hæmatoxyline.</p> - -<p><span class="pagenum" title="148"><a name="Page_148" id="Page_148"></a></span></p> - -<p><b>Uterus, ovaries, and Fallopian tubes.</b>—May -be obtained from the post-mortem room or -from the lower animals. Harden in Müller’s fluid, -and make sections from the cervix, the body of -the uterus, the Fallopian tube, and the ovary.</p> - -<p>Stain with eosine and hæmatoxyline.</p> - -<p><b>Embryological specimens.</b>—For systematic -work special manuals should be consulted.</p> - -<p>Specimens should be hardened in osmic acid or -in Müller’s fluid, and cut in celloidin, or paraffin.</p> - -<p><b>Cloudy swelling.</b>—Specimens are obtained -from organs of subjects who have died in the early -stage of some fever. They should be always hardened -in Müller’s fluid, as the appearances alter -if the tissue is kept in spirit for any length of time.</p> - -<p><b>Fatty degeneration.</b>—Prepare from patients -who have died of exhausting diseases, phosphorus -poisoning, &c.</p> - -<p>Stain in osmic acid. Mount in Farrant’s medium -and keep in the dark.</p> - -<p><b>Mucoid degeneration.</b>—Study in goblet -cells of normal intestine or of ovarian cysts. -There are no satisfactory selective stains for -mucin.</p> - -<p><span class="pagenum" title="149"><a name="Page_149" id="Page_149"></a></span></p> - -<p><b>Colloid degeneration.</b>—Occurs in the thyroid -gland, in the tubules of the kidney in many -diseases, and the prostate of the old.</p> - -<p>Stain in safranine.</p> - -<p><b>Waxy or lardaceous degeneration.</b>—Best -studied in liver, spleen, or kidneys. It should -be searched for in persons who have died from -a long illness, accompanied by suppuration, <i>e.g.</i>, -phthisis or bone disease. Mount one section unstained, -stain another in methyl violet, a third in -a weak solution of iodine, and examine the latter -at once both by transmitted and reflected light. -The iodine stain is not permanent. Another section -should be stained in osmic acid, followed by -methyl violet, as waxy and fatty degeneration -frequently co-exist.</p> - -<p><b>Hyaline degeneration.</b>—Seen in the arterioles -of the spleen in some cases of typhoid and -diphtheria. The ordinary staining methods must -be used.</p> - -<p><b>Calcareous degeneration.</b>—Occurs after -fatty degeneration in gummata and in atheromatous -arteries. It also occurs in the matrix of the -costal cartilages after middle life. Mount one<span class="pagenum" title="150"><a name="Page_150" id="Page_150"></a></span> -section unstained and examine if possible with the -polariscope. Stain others in safranine.</p> - -<p><b>Pigmentary degeneration.</b>—May be -studied in brown atrophy of heart, nutmeg liver, -&c. It is also seen well in spinal and cerebral -nerve cells of the aged. Harden in Müller’s fluid -and mount sections unstained.</p> - -<p>It will be unnecessary to recapitulate the -methods for hardening the various diseased organs -as the directions for the normal organs hold good. -If the presence of micro-organisms be suspected, -harden in methylated spirit or absolute alcohol, -but as a rule both for diseased organs and tumours -Müller’s fluid will be found the most satisfactory -reagent for general use.</p> - -<p>It sometimes happens, however, that it is inconvenient -to wait several weeks, until the Müller’s -fluid has hardened the specimen sufficiently, before -making sections. In this case the best plan is -to make fresh sections, or else to cut a slice about -one-eighth of an inch thick, and harden for about -three days in plenty of methylated spirit, or in -formal (p. <a href="#Page_23">23</a>).</p> - -<p><b>Tumours.</b>—Müller’s fluid should be employed, -unless a more rapid agent is required.</p> - - - -<p><span class="pagenum" title="151"><a name="Page_151" id="Page_151"></a></span></p> - -<p>Methylated spirit may be used in the case -of epithelioma, adenoma, &c., but for sarcoma, -myxoma, tumours containing cysts or much blood, -Müller’s fluid yields by far the best results.</p> - - -<hr class="chap" /> - -<p><span class="pagenum hide" title="152"><a name="Page_152" id="Page_152"></a></span></p> -<h2>BOOKS OF REFERENCE.</h2> - - -<p class="ti0em ml20pc"> -Methods in Microscopical Anatomy—<i>Whitman</i>.<br /> -Practical Pathology—<i>Woodhead</i>.<br /> -Textbook of Bacteriology—<i>Crookshank</i>.<br /> -Manual for Physiological Laboratory—<i>Harris</i> and <i>Power</i>.<br /> -Practical Histology—<i>Fearnley</i>.<br /> -Practical Pathology and Histology—<i>Gibbes</i>.<br /> -Journal of Microscopical Society.<br /> -Methods and Formulæ—<i>Squire</i>.<br /> -The Human Brain—<i>Goodall</i>.<br /> -Practical Bacteriology—<i>Kanthack</i> and <i>Drysdale</i>.<br /> -Methods of Microscopical Research—<i>Cole</i>. -</p> - -<hr class="chap" /> - - -<p><span class="pagenum hide" title="153"><a name="Page_153" id="Page_153"></a></span></p> -<h2>INDEX.</h2> - - -<p class="mb2em ti0em">Abbe’s condenser, <a href="#Page_11">11</a><br /> -Absolute alcohol, <a href="#Page_21">21</a><br /> -Acetate of copper, <a href="#Page_89">89</a><br /> -Air bubbles, removal of, <a href="#Page_56">56</a><br /> -Alum carmine, <a href="#Page_76">76</a><br /> -    hæmatoxyline, <a href="#Page_68">68</a>, <a href="#Page_70">70</a><br /> -Amyloid degeneration, <a href="#Page_149">149</a><br /> -Aniline blue-black, <a href="#Page_94">94</a><br /> -    oil, <a href="#Page_102">102</a>, <a href="#Page_108">108</a><br /> -    oil water, <a href="#Page_107">107</a><br /> -Apparatus required, <a href="#Page_1">1</a><br /> -Areolar tissue, <a href="#Page_133">133</a><br /> -<br /> -Bacteria, stains for, <a href="#Page_103">103</a><br /> -Balsam bottle, <a href="#Page_58">58</a><br /> -Barrett’s logwood solution, <a href="#Page_69">69</a><br /> -Bevan Lewis’s method, <a href="#Page_94">94</a><br /> -Bichromate of potassium, <a href="#Page_17">17</a><br /> -Bismarck brown, <a href="#Page_104">104</a><br /> -Bleaching solution, <a href="#Page_27">27</a><br /> -Blood crystals, <a href="#Page_130">130</a><br /> -Blood, methods of examining, <a href="#Page_113">113</a><br /> -Blood-vessels, injection of, <a href="#Page_120">120</a><span class="pagenum" title="154"><a name="Page_154" id="Page_154"></a></span><br /> -Blue injection mass, <a href="#Page_121">121</a><br /> -Bone marrow, <a href="#Page_136">136</a><br /> -Bone, sections of, <a href="#Page_136">136</a><br /> -Borax carmine, <a href="#Page_75">75</a><br /> -Brain, methods of staining, <a href="#Page_94">94</a><br /> -Buckley’s modification of Golgi’s method, <a href="#Page_99">99</a><br /> -<br /> -Calcareous degeneration, <a href="#Page_149">149</a><br /> -Canada balsam solution, <a href="#Page_61">61</a><br /> -Carbolic acid, <a href="#Page_23">23</a><br /> -Carmine, <a href="#Page_74">74</a><br /> -    injection mass, <a href="#Page_120">120</a><br /> -Cathcart microtome, <a href="#Page_39">39</a><br /> -Cathcart-Frazer microtome, <a href="#Page_42">42</a><br /> -Cedar oil, <a href="#Page_8">8</a>, <a href="#Page_63">63</a><br /> -Celloidin, <a href="#Page_30">30</a><br /> -Cementing cover-glasses, <a href="#Page_65">65</a><br /> -Chloral hæmatoxyline, <a href="#Page_92">92</a><br /> -Chloride of gold, <a href="#Page_82">82</a>, <a href="#Page_94">94</a><br /> -Chromic and nitric decalcifying fluid, <a href="#Page_26">26</a><br /> -Ciliated epithelium, <a href="#Page_132">132</a><br /> -Circulation in frog’s foot, <a href="#Page_141">141</a><br /> -Clarifying sections, <a href="#Page_63">63</a><br /> -Clearing agents, <a href="#Page_63">63</a><br /> -Cloudy swelling, <a href="#Page_148">148</a><br /> -Clove oil, <a href="#Page_63">63</a><br /> -Colloid degeneration, <a href="#Page_149">149</a><br /> -Columnar epithelium, <a href="#Page_132">132</a><span class="pagenum" title="155"><a name="Page_155" id="Page_155"></a></span><br /> -Corrosive sublimate hardening, <a href="#Page_23">23</a><br /> -          staining, <a href="#Page_100">100</a><br /> -Cover-glasses, cleansing of, <a href="#Page_57">57</a><br /> -Cover-glass preparations, <a href="#Page_111">111</a><br /> -<br /> -Decalcifying solutions, <a href="#Page_26">26</a><br /> -Dehydration, <a href="#Page_63">63</a><br /> -<br /> -Eau de Javelle, <a href="#Page_27">27</a><br /> -Ebner’s solution, <a href="#Page_27">27</a><br /> -Ehrlich-Biondi fluid, <a href="#Page_85">85</a><br /> -Ehrlich’s hæmatoxyline, <a href="#Page_70">70</a><br /> -    method of fixing blood-films, <a href="#Page_116">116</a><br /> -    method for tubercle bacilli, <a href="#Page_110">110</a><br /> -Ehrlich-Gram method for staining bacteria, <a href="#Page_108">108</a><br /> -Elastic cartilage, <a href="#Page_135">135</a><br /> -    tissue, <a href="#Page_133">133</a><br /> -Embedding methods, <a href="#Page_29">29</a><br /> -Endothelium, <a href="#Page_131">131</a><br /> -Eosine, <a href="#Page_72">72</a><br /> -Eosine and hæmatoxyline, <a href="#Page_73">73</a><br /> -Epithelial cement, <a href="#Page_131">131</a><br /> -Ether spray microtome, <a href="#Page_39">39</a><br /> -<br /> -Fæces, staining for bacilli, <a href="#Page_112">112</a><br /> -Farrant’s solution, <a href="#Page_59">59</a><br /> -Fat, removal from sections, <a href="#Page_59">59</a><br /> -  staining of, <a href="#Page_134">134</a><br /> -Fatty degeneration, <a href="#Page_148">148</a><br /> -Fearnley’s injection apparatus, <a href="#Page_123">123</a><span class="pagenum" title="156"><a name="Page_156" id="Page_156"></a></span><br /> -Ferrier’s fuchsine solution, <a href="#Page_117">117</a><br /> -Flemming’s solution, <a href="#Page_25">25</a><br /> -Flotation of sections, <a href="#Page_55">55</a><br /> -Folded sections, treatment of, <a href="#Page_58">58</a><br /> -Formal, <a href="#Page_23">23</a><br /> -Fresh sections, <a href="#Page_52">52</a><br /> -Fuchsine, <a href="#Page_104">104</a><br /> -<br /> -Gentian violet, <a href="#Page_104">104</a><br /> -Gibbes’ stain for tubercle bacilli, <a href="#Page_111">111</a><br /> -Gold chloride, <a href="#Page_82">82</a>, <a href="#Page_94">94</a><br /> -Golgi’s silver method, <a href="#Page_96">96</a><br /> -    sublimate method, <a href="#Page_99">99</a><br /> -Gram’s iodide solution, <a href="#Page_105">105</a><br /> -    method for staining bacteria, <a href="#Page_107">107</a><br /> -Green injection mass, <a href="#Page_122">122</a><br /> -Gum, <a href="#Page_29">29</a><br /> -<br /> -Hæmatin crystals, <a href="#Page_131">131</a><br /> -Hæmatoidin, <a href="#Page_131">131</a><br /> -Hæmatoxyline, Ehrlich’s, <a href="#Page_70">70</a><br /> -       Kleinenberg’s, <a href="#Page_70">70</a><br /> -       Schuchardt’s, <a href="#Page_68">68</a><br /> -       Sihler’s, <a href="#Page_92">92</a><br /> -       Weigert’s, <a href="#Page_88">88</a><br /> -Hæmoglobin crystals, <a href="#Page_130">130</a><br /> -Hardening processes, <a href="#Page_15">15</a><br /> -Hyaline cartilage, <a href="#Page_135">135</a><br /> -     degeneration, <a href="#Page_149">149</a><span class="pagenum" title="157"><a name="Page_157" id="Page_157"></a></span><br /> -<br /> -Ice freezing microtome, <a href="#Page_46">46</a><br /> -Immersion lenses, <a href="#Page_8">8</a><br /> -Injection of blood-vessels, <a href="#Page_120">120</a><br /> -      pulmonary alveoli, <a href="#Page_145">145</a><br /> -Internal ear, <a href="#Page_143">143</a><br /> -Intestines, <a href="#Page_146">146</a><br /> -Iodine solution, <a href="#Page_105">105</a><br /> -<br /> -Jung’s ether spray microtome, <a href="#Page_45">45</a><br /> -<br /> -Kleinenberg’s hæmatoxyline, <a href="#Page_70">70</a><br /> -<br /> -Lardaceous degeneration, <a href="#Page_149">149</a><br /> -Lithio-picrocarmine, <a href="#Page_79">79</a><br /> -Lithium carmine, <a href="#Page_74">74</a><br /> -Liver, <a href="#Page_147">147</a><br /> -Löffler’s methyl blue, <a href="#Page_104">104</a><br /> -Logwood, <a href="#Page_68">68</a><br /> -Lymphoid tissue, <a href="#Page_134">134</a><br /> -<br /> -Marchi’s fluid, <a href="#Page_24">24</a><br /> -Marrow, <a href="#Page_136">136</a><br /> -Methyl blue, <a href="#Page_101">101</a>, <a href="#Page_104">104</a><br /> -    violet, <a href="#Page_83">83</a><br /> -Methylated spirit, <a href="#Page_19">19</a><br /> -Micro-organisms, stains for, <a href="#Page_103">103</a><br /> -Microscope, <a href="#Page_6">6</a><br /> -Microtome, Becker, <a href="#Page_49">49</a><br /> -      Cambridge rocking, <a href="#Page_49">49</a><br /> -      Cathcart, <a href="#Page_39">39</a><br /> -      Cathcart-Frazer, <a href="#Page_42">42</a><span class="pagenum" title="158"><a name="Page_158" id="Page_158"></a></span><br /> -      Jung, <a href="#Page_45">45</a><br /> -      Schanze, <a href="#Page_47">47</a><br /> -      Swift’s, <a href="#Page_49">49</a><br /> -      Williams’, <a href="#Page_46">46</a><br /> -Mould for paraffin embedding, <a href="#Page_36">36</a><br /> -Mounting methods, <a href="#Page_55">55</a><br /> -Mucoid degeneration, <a href="#Page_148">148</a><br /> -Muscle, <a href="#Page_138">138</a><br /> -Muir’s method of hardening films, <a href="#Page_116">116</a><br /> -Müller’s fluid, <a href="#Page_17">17</a><br /> -      and formal, <a href="#Page_20">20</a><br /> -      and spirit, <a href="#Page_20">20</a><br /> -<br /> -Neelsen’s stain for tubercle bacilli, <a href="#Page_110">110</a><br /> -Nerve cells, stains for, <a href="#Page_94">94</a><br /> -      endings, <a href="#Page_139">139</a><br /> -      fibres, stains for, <a href="#Page_87">87</a><br /> -Nissl’s aniline method, <a href="#Page_101">101</a><br /> -Nitrate of silver, <a href="#Page_82">82</a><br /> -Nitric acid as hardening agent, <a href="#Page_25">25</a><br /> -      decalcifying agent, <a href="#Page_26">26</a><br /> -      decolourising agent, <a href="#Page_110">110</a><br /> -Normal salt solution, <a href="#Page_53">53</a><br /> -Nose piece, <a href="#Page_9">9</a><br /> -<br /> -Objectives, <a href="#Page_7">7</a><br /> -Oil of bergamot, <a href="#Page_63">63</a><br /> -    cedar, <a href="#Page_8">8</a>, <a href="#Page_63">63</a><br /> -    cloves, <a href="#Page_63">63</a><span class="pagenum" title="159"><a name="Page_159" id="Page_159"></a></span><br /> -    origanum, <a href="#Page_63">63</a><br /> -Osmic acid as hardening agent, <a href="#Page_21">21</a><br /> -       staining reagent, <a href="#Page_81">81</a><br /> -<br /> -Pal’s method, <a href="#Page_86">86</a><br /> -   solution, <a href="#Page_90">90</a><br /> -Paraffin, <a href="#Page_34">34</a><br /> -Picrocarmine, <a href="#Page_78">78</a><br /> -Pigment cells, <a href="#Page_134">134</a><br /> -Pigmentary degeneration, <a href="#Page_150">150</a><br /> -Plane iron microtome knife, <a href="#Page_42">42</a><br /> -<br /> -Rapid hardening, <a href="#Page_150">150</a><br /> -Retina, <a href="#Page_143">143</a><br /> -<br /> -Safranine, <a href="#Page_85">85</a><br /> -Salivary glands, <a href="#Page_146">146</a><br /> -Schäfer-Pal method, <a href="#Page_91">91</a><br /> -Schanze microtome, <a href="#Page_47">47</a><br /> -Schuchardt’s hæmatoxyline, <a href="#Page_68">68</a><br /> -Sihler’s chloral hæmatoxyline, <a href="#Page_92">92</a><br /> -Silver nitrate, stain for nerve cells, <a href="#Page_96">96</a><br /> -           epithelial cement, <a href="#Page_131">131</a><br /> -Skin, <a href="#Page_142">142</a><br /> -Spinal cord, <a href="#Page_86">86</a>, <a href="#Page_142">142</a><br /> -Spleen, <a href="#Page_147">147</a><br /> -Sputum, staining of, <a href="#Page_111">111</a><br /> -Squamous epithelium, <a href="#Page_131">131</a><br /> -Staining methods, <a href="#Page_67">67</a> <i>seq.</i><br /> -Stomach, <a href="#Page_146">146</a><span class="pagenum" title="160"><a name="Page_160" id="Page_160"></a></span><br /> -Striped muscle, <a href="#Page_138">138</a><br /> -Sulphuric acid, <a href="#Page_105">105</a><br /> -Sweat glands, <a href="#Page_142">142</a><br /> -<br /> -Tendon, <a href="#Page_133">133</a><br /> -Testing a microscope, <a href="#Page_13">13</a><br /> -Thymus gland, <a href="#Page_145">145</a><br /> -Thyroid gland, <a href="#Page_145">145</a><br /> -Toison’s fluid, <a href="#Page_118">118</a><br /> -Tooth, sections of, <a href="#Page_137">137</a><br /> -Transitional epithelium, <a href="#Page_132">132</a><br /> -Tubercle bacillus, stains for, <a href="#Page_110">110</a><br /> -Tumours, hardening of, <a href="#Page_150">150</a><br /> -<br /> -Unstriped muscle, <a href="#Page_139">139</a><br /> -Urine, examination for bacilli, <a href="#Page_112">112</a><br /> -Uterus, <a href="#Page_148">148</a><br /> -<br /> -Von Ebner’s decalcifying solution, <a href="#Page_27">27</a><br /> -<br /> -Waxy degeneration, <a href="#Page_149">149</a><br /> -Weigert’s hæmatoxyline method, <a href="#Page_88">88</a><br /> -     method for staining bacteria, <a href="#Page_106">106</a><br /> -Williams’ ice freezing microtome, <a href="#Page_46">46</a><br /> -Woodhead’s injection mass, <a href="#Page_120">120</a><br /> -<br /> -Xylol, <a href="#Page_61">61</a><br /> -<br /> -Ziehl’s carbol-fuchsine, <a href="#Page_105">105</a></p> - - -<div class="footnotes"><h3>FOOTNOTES:</h3> - -<div class="footnote"> - -<p><a id="Footnote_1" href="#FNanchor_1" class="label">1</a> -The student will bear in mind the danger of working -with benzine near a naked light.</p></div> - -<div class="footnote"> - -<p><a id="Footnote_2" href="#FNanchor_2" class="label">2</a> -“Practical Histology,” (Macmillan & Co.).</p></div></div> - - - - - - - - - -<pre> - - - - - -End of Project Gutenberg's Section Cutting and Staining, by Walter S. Colman - -*** END OF THIS PROJECT GUTENBERG EBOOK SECTION CUTTING AND STAINING *** - -***** This file should be named 51169-h.htm or 51169-h.zip ***** -This and all associated files of various formats will be found in: - http://www.gutenberg.org/5/1/1/6/51169/ - -Produced by Thiers Halliwell and The Online -Distributed Proofreading Team at http://www.pgdp.net (This -file was produced from images generously made available -by The Internet Archive) - - -Updated editions will replace the previous one--the old editions -will be renamed. - -Creating the works from public domain print editions means that no -one owns a United States copyright in these works, so the Foundation -(and you!) can copy and distribute it in the United States without -permission and without paying copyright royalties. Special rules, -set forth in the General Terms of Use part of this license, apply to -copying and distributing Project Gutenberg-tm electronic works to -protect the PROJECT GUTENBERG-tm concept and trademark. Project -Gutenberg is a registered trademark, and may not be used if you -charge for the eBooks, unless you receive specific permission. If you -do not charge anything for copies of this eBook, complying with the -rules is very easy. You may use this eBook for nearly any purpose -such as creation of derivative works, reports, performances and -research. They may be modified and printed and given away--you may do -practically ANYTHING with public domain eBooks. Redistribution is -subject to the trademark license, especially commercial -redistribution. - - - -*** START: FULL LICENSE *** - -THE FULL PROJECT GUTENBERG LICENSE -PLEASE READ THIS BEFORE YOU DISTRIBUTE OR USE THIS WORK - -To protect the Project Gutenberg-tm mission of promoting the free -distribution of electronic works, by using or distributing this work -(or any other work associated in any way with the phrase "Project -Gutenberg"), you agree to comply with all the terms of the Full Project -Gutenberg-tm License (available with this file or online at -http://gutenberg.org/license). - - -Section 1. General Terms of Use and Redistributing Project Gutenberg-tm -electronic works - -1.A. By reading or using any part of this Project Gutenberg-tm -electronic work, you indicate that you have read, understand, agree to -and accept all the terms of this license and intellectual property -(trademark/copyright) agreement. If you do not agree to abide by all -the terms of this agreement, you must cease using and return or destroy -all copies of Project Gutenberg-tm electronic works in your possession. -If you paid a fee for obtaining a copy of or access to a Project -Gutenberg-tm electronic work and you do not agree to be bound by the -terms of this agreement, you may obtain a refund from the person or -entity to whom you paid the fee as set forth in paragraph 1.E.8. - -1.B. "Project Gutenberg" is a registered trademark. It may only be -used on or associated in any way with an electronic work by people who -agree to be bound by the terms of this agreement. There are a few -things that you can do with most Project Gutenberg-tm electronic works -even without complying with the full terms of this agreement. See -paragraph 1.C below. There are a lot of things you can do with Project -Gutenberg-tm electronic works if you follow the terms of this agreement -and help preserve free future access to Project Gutenberg-tm electronic -works. See paragraph 1.E below. - -1.C. The Project Gutenberg Literary Archive Foundation ("the Foundation" -or PGLAF), owns a compilation copyright in the collection of Project -Gutenberg-tm electronic works. Nearly all the individual works in the -collection are in the public domain in the United States. If an -individual work is in the public domain in the United States and you are -located in the United States, we do not claim a right to prevent you from -copying, distributing, performing, displaying or creating derivative -works based on the work as long as all references to Project Gutenberg -are removed. Of course, we hope that you will support the Project -Gutenberg-tm mission of promoting free access to electronic works by -freely sharing Project Gutenberg-tm works in compliance with the terms of -this agreement for keeping the Project Gutenberg-tm name associated with -the work. You can easily comply with the terms of this agreement by -keeping this work in the same format with its attached full Project -Gutenberg-tm License when you share it without charge with others. - -1.D. The copyright laws of the place where you are located also govern -what you can do with this work. Copyright laws in most countries are in -a constant state of change. If you are outside the United States, check -the laws of your country in addition to the terms of this agreement -before downloading, copying, displaying, performing, distributing or -creating derivative works based on this work or any other Project -Gutenberg-tm work. The Foundation makes no representations concerning -the copyright status of any work in any country outside the United -States. - -1.E. Unless you have removed all references to Project Gutenberg: - -1.E.1. The following sentence, with active links to, or other immediate -access to, the full Project Gutenberg-tm License must appear prominently -whenever any copy of a Project Gutenberg-tm work (any work on which the -phrase "Project Gutenberg" appears, or with which the phrase "Project -Gutenberg" is associated) is accessed, displayed, performed, viewed, -copied or distributed: - -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/license - -1.E.2. If an individual Project Gutenberg-tm electronic work is derived -from the public domain (does not contain a notice indicating that it is -posted with permission of the copyright holder), the work can be copied -and distributed to anyone in the United States without paying any fees -or charges. If you are redistributing or providing access to a work -with the phrase "Project Gutenberg" associated with or appearing on the -work, you must comply either with the requirements of paragraphs 1.E.1 -through 1.E.7 or obtain permission for the use of the work and the -Project Gutenberg-tm trademark as set forth in paragraphs 1.E.8 or -1.E.9. - -1.E.3. If an individual Project Gutenberg-tm electronic work is posted -with the permission of the copyright holder, your use and distribution -must comply with both paragraphs 1.E.1 through 1.E.7 and any additional -terms imposed by the copyright holder. Additional terms will be linked -to the Project Gutenberg-tm License for all works posted with the -permission of the copyright holder found at the beginning of this work. - -1.E.4. Do not unlink or detach or remove the full Project Gutenberg-tm -License terms from this work, or any files containing a part of this -work or any other work associated with Project Gutenberg-tm. - -1.E.5. Do not copy, display, perform, distribute or redistribute this -electronic work, or any part of this electronic work, without -prominently displaying the sentence set forth in paragraph 1.E.1 with -active links or immediate access to the full terms of the Project -Gutenberg-tm License. - -1.E.6. You may convert to and distribute this work in any binary, -compressed, marked up, nonproprietary or proprietary form, including any -word processing or hypertext form. However, if you provide access to or -distribute copies of a Project Gutenberg-tm work in a format other than -"Plain Vanilla ASCII" or other format used in the official version -posted on the official Project Gutenberg-tm web site (www.gutenberg.org), -you must, at no additional cost, fee or expense to the user, provide a -copy, a means of exporting a copy, or a means of obtaining a copy upon -request, of the work in its original "Plain Vanilla ASCII" or other -form. Any alternate format must include the full Project Gutenberg-tm -License as specified in paragraph 1.E.1. - -1.E.7. Do not charge a fee for access to, viewing, displaying, -performing, copying or distributing any Project Gutenberg-tm works -unless you comply with paragraph 1.E.8 or 1.E.9. - -1.E.8. You may charge a reasonable fee for copies of or providing -access to or distributing Project Gutenberg-tm electronic works provided -that - -- You pay a royalty fee of 20% of the gross profits you derive from - the use of Project Gutenberg-tm works calculated using the method - you already use to calculate your applicable taxes. The fee is - owed to the owner of the Project Gutenberg-tm trademark, but he - has agreed to donate royalties under this paragraph to the - Project Gutenberg Literary Archive Foundation. Royalty payments - must be paid within 60 days following each date on which you - prepare (or are legally required to prepare) your periodic tax - returns. Royalty payments should be clearly marked as such and - sent to the Project Gutenberg Literary Archive Foundation at the - address specified in Section 4, "Information about donations to - the Project Gutenberg Literary Archive Foundation." - -- You provide a full refund of any money paid by a user who notifies - you in writing (or by e-mail) within 30 days of receipt that s/he - does not agree to the terms of the full Project Gutenberg-tm - License. You must require such a user to return or - destroy all copies of the works possessed in a physical medium - and discontinue all use of and all access to other copies of - Project Gutenberg-tm works. - -- You provide, in accordance with paragraph 1.F.3, a full refund of any - money paid for a work or a replacement copy, if a defect in the - electronic work is discovered and reported to you within 90 days - of receipt of the work. - -- You comply with all other terms of this agreement for free - distribution of Project Gutenberg-tm works. - -1.E.9. If you wish to charge a fee or distribute a Project Gutenberg-tm -electronic work or group of works on different terms than are set -forth in this agreement, you must obtain permission in writing from -both the Project Gutenberg Literary Archive Foundation and Michael -Hart, the owner of the Project Gutenberg-tm trademark. Contact the -Foundation as set forth in Section 3 below. - -1.F. - -1.F.1. Project Gutenberg volunteers and employees expend considerable -effort to identify, do copyright research on, transcribe and proofread -public domain works in creating the Project Gutenberg-tm -collection. Despite these efforts, Project Gutenberg-tm electronic -works, and the medium on which they may be stored, may contain -"Defects," such as, but not limited to, incomplete, inaccurate or -corrupt data, transcription errors, a copyright or other intellectual -property infringement, a defective or damaged disk or other medium, a -computer virus, or computer codes that damage or cannot be read by -your equipment. - -1.F.2. LIMITED WARRANTY, DISCLAIMER OF DAMAGES - Except for the "Right -of Replacement or Refund" described in paragraph 1.F.3, the Project -Gutenberg Literary Archive Foundation, the owner of the Project -Gutenberg-tm trademark, and any other party distributing a Project -Gutenberg-tm electronic work under this agreement, disclaim all -liability to you for damages, costs and expenses, including legal -fees. YOU AGREE THAT YOU HAVE NO REMEDIES FOR NEGLIGENCE, STRICT -LIABILITY, BREACH OF WARRANTY OR BREACH OF CONTRACT EXCEPT THOSE -PROVIDED IN PARAGRAPH 1.F.3. YOU AGREE THAT THE FOUNDATION, THE -TRADEMARK OWNER, AND ANY DISTRIBUTOR UNDER THIS AGREEMENT WILL NOT BE -LIABLE TO YOU FOR ACTUAL, DIRECT, INDIRECT, CONSEQUENTIAL, PUNITIVE OR -INCIDENTAL DAMAGES EVEN IF YOU GIVE NOTICE OF THE POSSIBILITY OF SUCH -DAMAGE. - -1.F.3. LIMITED RIGHT OF REPLACEMENT OR REFUND - If you discover a -defect in this electronic work within 90 days of receiving it, you can -receive a refund of the money (if any) you paid for it by sending a -written explanation to the person you received the work from. If you -received the work on a physical medium, you must return the medium with -your written explanation. The person or entity that provided you with -the defective work may elect to provide a replacement copy in lieu of a -refund. If you received the work electronically, the person or entity -providing it to you may choose to give you a second opportunity to -receive the work electronically in lieu of a refund. If the second copy -is also defective, you may demand a refund in writing without further -opportunities to fix the problem. - -1.F.4. Except for the limited right of replacement or refund set forth -in paragraph 1.F.3, this work is provided to you 'AS-IS' WITH NO OTHER -WARRANTIES OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO -WARRANTIES OF MERCHANTABILITY OR FITNESS FOR ANY PURPOSE. - -1.F.5. Some states do not allow disclaimers of certain implied -warranties or the exclusion or limitation of certain types of damages. -If any disclaimer or limitation set forth in this agreement violates the -law of the state applicable to this agreement, the agreement shall be -interpreted to make the maximum disclaimer or limitation permitted by -the applicable state law. The invalidity or unenforceability of any -provision of this agreement shall not void the remaining provisions. - -1.F.6. INDEMNITY - You agree to indemnify and hold the Foundation, the -trademark owner, any agent or employee of the Foundation, anyone -providing copies of Project Gutenberg-tm electronic works in accordance -with this agreement, and any volunteers associated with the production, -promotion and distribution of Project Gutenberg-tm electronic works, -harmless from all liability, costs and expenses, including legal fees, -that arise directly or indirectly from any of the following which you do -or cause to occur: (a) distribution of this or any Project Gutenberg-tm -work, (b) alteration, modification, or additions or deletions to any -Project Gutenberg-tm work, and (c) any Defect you cause. - - -Section 2. Information about the Mission of Project Gutenberg-tm - -Project Gutenberg-tm is synonymous with the free distribution of -electronic works in formats readable by the widest variety of computers -including obsolete, old, middle-aged and new computers. It exists -because of the efforts of hundreds of volunteers and donations from -people in all walks of life. - -Volunteers and financial support to provide volunteers with the -assistance they need, are critical to reaching Project Gutenberg-tm's -goals and ensuring that the Project Gutenberg-tm collection will -remain freely available for generations to come. In 2001, the Project -Gutenberg Literary Archive Foundation was created to provide a secure -and permanent future for Project Gutenberg-tm and future generations. -To learn more about the Project Gutenberg Literary Archive Foundation -and how your efforts and donations can help, see Sections 3 and 4 -and the Foundation web page at http://www.pglaf.org. - - -Section 3. Information about the Project Gutenberg Literary Archive -Foundation - -The Project Gutenberg Literary Archive Foundation is a non profit -501(c)(3) educational corporation organized under the laws of the -state of Mississippi and granted tax exempt status by the Internal -Revenue Service. The Foundation's EIN or federal tax identification -number is 64-6221541. Its 501(c)(3) letter is posted at -http://pglaf.org/fundraising. Contributions to the Project Gutenberg -Literary Archive Foundation are tax deductible to the full extent -permitted by U.S. federal laws and your state's laws. - -The Foundation's principal office is located at 4557 Melan Dr. S. -Fairbanks, AK, 99712., but its volunteers and employees are scattered -throughout numerous locations. Its business office is located at -809 North 1500 West, Salt Lake City, UT 84116, (801) 596-1887, email -business@pglaf.org. Email contact links and up to date contact -information can be found at the Foundation's web site and official -page at http://pglaf.org - -For additional contact information: - Dr. Gregory B. Newby - Chief Executive and Director - gbnewby@pglaf.org - - -Section 4. Information about Donations to the Project Gutenberg -Literary Archive Foundation - -Project Gutenberg-tm depends upon and cannot survive without wide -spread public support and donations to carry out its mission of -increasing the number of public domain and licensed works that can be -freely distributed in machine readable form accessible by the widest -array of equipment including outdated equipment. Many small donations -($1 to $5,000) are particularly important to maintaining tax exempt -status with the IRS. - -The Foundation is committed to complying with the laws regulating -charities and charitable donations in all 50 states of the United -States. Compliance requirements are not uniform and it takes a -considerable effort, much paperwork and many fees to meet and keep up -with these requirements. We do not solicit donations in locations -where we have not received written confirmation of compliance. To -SEND DONATIONS or determine the status of compliance for any -particular state visit http://pglaf.org - -While we cannot and do not solicit contributions from states where we -have not met the solicitation requirements, we know of no prohibition -against accepting unsolicited donations from donors in such states who -approach us with offers to donate. - -International donations are gratefully accepted, but we cannot make -any statements concerning tax treatment of donations received from -outside the United States. U.S. laws alone swamp our small staff. - -Please check the Project Gutenberg Web pages for current donation -methods and addresses. Donations are accepted in a number of other -ways including checks, online payments and credit card donations. -To donate, please visit: http://pglaf.org/donate - - -Section 5. General Information About Project Gutenberg-tm electronic -works. - -Professor Michael S. Hart is the originator of the Project Gutenberg-tm -concept of a library of electronic works that could be freely shared -with anyone. For thirty years, he produced and distributed Project -Gutenberg-tm eBooks with only a loose network of volunteer support. - - -Project Gutenberg-tm eBooks are often created from several printed -editions, all of which are confirmed as Public Domain in the U.S. -unless a copyright notice is included. Thus, we do not necessarily -keep eBooks in compliance with any particular paper edition. - - -Most people start at our Web site which has the main PG search facility: - - http://www.gutenberg.org - -This Web site includes information about Project Gutenberg-tm, -including how to make donations to the Project Gutenberg Literary -Archive Foundation, how to help produce our new eBooks, and how to -subscribe to our email newsletter to hear about new eBooks. - - -</pre> - -</body> -</html> |