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<body>
<div>*** START OF THE PROJECT GUTENBERG EBOOK 51169 ***</div>

<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 />
&emsp;xolol → xylol<br />
&emsp;side → slide<br />
&emsp;overstraining → overstaining</p>

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</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.&nbsp;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.&nbsp;K. LEWIS, 136 GOWER STREET, W.C.<br />
1896</div>


<div class="tp3">PRINTED BY<br />
H.&nbsp;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, &amp;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.&nbsp;S. COLMAN.</p>
<p class="ml2em">
Wimpole Street, W.<br />
&emsp;&emsp;<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>&emsp;</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, &amp;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:&mdash;</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, &amp;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&nbsp;oz. and 2&nbsp;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>&mdash;At least a dozen watch-glasses,
in which to perform the operations of
staining, clarifying, &amp;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>&nbsp;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>&mdash;This instrument is required
for transferring sections from one reagent to
another, or from oil of cloves, &amp;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>&nbsp;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, &amp;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&nbsp;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>&nbsp;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>&nbsp;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>&nbsp;in.
diameter.</p>

<p><b>Microscope.</b>&mdash;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>&mdash;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&nbsp;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>&nbsp;in. or <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">6</span></span></span>&nbsp;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>&nbsp;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.&nbsp;1.</span>&mdash;Double or Triple Nose-​piece.</p></div>
</div>

<p><b>Double or triple nose-piece</b> (fig.&nbsp;1).&mdash;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>&mdash;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.&nbsp;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.&nbsp;2.</span>&mdash;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&nbsp;in. and <span class="nowrap"> <span class="fraction"><span class="fnum">1</span><span class="bar">/</span><span class="fden">4</span></span></span>&nbsp;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, &amp;c., for
£16, meet all requirements.</p>

<p>Leitz of Jena, supplies two good and cheap
microscopes for £3&nbsp;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>&nbsp;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>&nbsp;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, &amp;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, &amp;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:&mdash;<br /><b>Müller’s
fluid</b><span class="nowrap">:&mdash;</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">&emsp;<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">&emsp;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:&mdash;</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:&mdash;</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.&nbsp;<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:&mdash;</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, &amp;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>&mdash;This is a useful
combination for many purposes. It is made thus:&mdash;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>&mdash;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>&mdash;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.&nbsp;<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>&mdash;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:&mdash;</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.&nbsp;<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.&nbsp;<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.).&mdash;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>&mdash;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>&mdash;This consists <span class="nowrap">of:&mdash;</span></p>



<div class="center">
<table border="0" cellpadding="1" cellspacing="0" summary="">
<tr><td class="tal">Müller’s fluid</td><td class="tar">&emsp;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.&nbsp;<a href="#Page_97">97</a>), and also to complete the hardening
of sections of spinal cord, &amp;c., before employing
Schäfer’s modification of the Weigert Pal hæmatoxyline
method (p.&nbsp;<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>&nbsp;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, &amp;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">:&mdash;</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">&emsp;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>&mdash;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, &amp;c. The two best fluids for general use
<span class="nowrap">are:&mdash;</span></p>

<p><b>Chromic and nitric fluid.</b>&mdash;This is made
as <span class="nowrap">follows:&mdash;</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">&emsp;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">&emsp;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">:&mdash;</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">&emsp;<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">&emsp;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">&emsp;100</td><td class="tar">&emsp;<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">&emsp;100</td><td class="tar">&emsp;<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>&mdash;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:&mdash;</span></p>

<p>(1) gum, (2) celloidin, (3) paraffin or wax.</p>

<p><b>Gum.</b>&mdash;<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:&mdash;</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, &amp;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.&nbsp;<a href="#Page_59">59</a>), or in Canada balsam
(p.&nbsp;<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.&nbsp;<a href="#Page_90">90</a>).</p>

<p><i>Paraffin.</i>&mdash;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.&nbsp;<a href="#Page_75">75</a>), alum carmine (p.&nbsp;<a href="#Page_76">76</a>), and
Kleinenberg’s hæmatoxyline (p.&nbsp;<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°&nbsp;F., and another “hard,” melting at 140°&nbsp;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.&nbsp;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.&nbsp;<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.&nbsp;<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>&mdash;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, &amp;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.&nbsp;4.</span>&mdash;Cathcart’s Ether Spray Microtome.</p>

<p>A, B. Wooden frame and supports. C. Glass runners.
G. Screw for raising the zinc plate H.&nbsp;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.&nbsp;4).&mdash;<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>&nbsp;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.&nbsp;<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, &amp;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.&nbsp;5.</span>&mdash;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, &amp;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.&nbsp;<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.&nbsp;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.&nbsp;6.</span>&mdash;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.&nbsp;<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.&nbsp;7.</span>&mdash;Schanze Microtome (see text).</p></div>
</div>

<p><b>Schanze microtome</b> (fig.&nbsp;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>&mdash;This
instrument, as made by the Cambridge
Scientific Instrument Company, or the slightly
modified form made by Messrs. Swift (fig.&nbsp;<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.&nbsp;8.</span>&mdash;Swift’s Modification of the Cambridge Rocking
Microtome.</p></div>
</div>

<p><b>Fresh sections.</b>&mdash;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, &amp;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.&nbsp;<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.&nbsp;<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>&mdash;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.&nbsp;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.&nbsp;9.</span>&mdash;Balsam bottle.</div>

<p><b>Treatment of folded sections.</b>&mdash;The folding
may be <span class="nowrap">due:&mdash;</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">:&mdash;</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">&emsp;<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, &amp;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.&nbsp;<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>:&mdash;The medium is
made <span class="nowrap">thus:&mdash;</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">&emsp;<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.&nbsp;<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>&mdash;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:&mdash;(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:&mdash;</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">&emsp;<img src="images/27x6br.png" width="6" height="27" alt="" /></td><td class="tal" rowspan="2"><span class="ilb">half&nbsp;an&nbsp;ounce</span><br />&emsp;of each.</td></tr>
<tr><td class="tal">Gum dammar</td></tr>
</table></div>

<p><b>Preservation of sections.</b>&mdash;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:&mdash;</span></p>

<p><b>Logwood.</b>&mdash;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:&mdash;<br /><b>Hæmatoxyline.
Schuchardt’s formula.</b>&mdash;</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">&emsp;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">&emsp;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>&mdash;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">&emsp;<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">&emsp;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">&emsp;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">&emsp;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>&mdash;This very useful
nuclear stain is made as <span class="nowrap">follows:&mdash;</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">&emsp;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">&emsp;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>&mdash;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.&nbsp;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">&emsp;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">&emsp;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">&emsp;240</td><td class="tal">c.c.</td><td class="tar">&emsp;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, &amp;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">&emsp;<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">&emsp;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.&nbsp;<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>&mdash;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>&mdash;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>&mdash;It is made as <span class="nowrap">follows:&mdash;</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">&emsp;100</td><td class="tar">&emsp;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">&emsp;<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">&emsp;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>&mdash;</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">&emsp;<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">&emsp;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">&emsp;<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">&emsp;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">:&mdash;</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">&emsp;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">&emsp;<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.&nbsp;<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:&mdash;</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">&emsp;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">&emsp;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>&mdash;Prepared as <span class="nowrap">follows:&mdash;</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">&emsp;10</td><td class="tal">grs.</td></tr>
<tr><td class="tal">Saturated solution lithium carbonate</td><td class="tar">&emsp;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">&emsp;250</td><td class="tal">c.c.</td><td class="tar">&emsp;<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, &amp;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.&nbsp;<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, &amp;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.&nbsp;<a href="#Page_92">92</a>).</p>

<p><b>Methyl violet.</b>&mdash;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>&mdash;Employed as a freshly made
saturated solution in aniline oil water warmed to
60°&nbsp;C. (140°&nbsp;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>&mdash;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:&mdash;</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">&emsp;100</td><td class="tal">parts</td></tr>
<tr><td class="tal">&nbsp;&emsp;&emsp;"&nbsp;&emsp;&emsp; Rubin S</td><td class="tar">20</td><td class="tal">&emsp;"</td></tr>
<tr><td class="tal">&nbsp;&emsp;&emsp;"&nbsp;&emsp;&emsp; Methyl Green OO</td><td class="tar">50</td><td class="tal">&emsp;"</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">&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;70</td><td class="tal">&emsp;"</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.&mdash;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>&mdash;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.&nbsp;<a href="#Page_30">30</a>). When sections are cut they are
transferred at once to Weigert’s hæmatoxyline
<span class="nowrap">solution:&mdash;</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">&emsp;45</td><td class="tal">minims.</td></tr>
<tr><td class="tal">Distilled water</td><td class="tar">&emsp;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°&nbsp;F. in the incubator.
After staining they are transferred to Weigert’s
differentiating <span class="nowrap">solution:&mdash;</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">&emsp;200</td><td class="tal">grs.</td></tr>
<tr><td class="tal">Distilled water</td><td class="tar">&emsp;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.&mdash;By
this method quicker and more complete decolouration
of the neuroglia, nerve cells, &amp;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:&mdash;</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">&emsp;40</td><td class="tal">gr.</td></tr>
<tr><td class="tal">Distilled water</td><td class="tar">&emsp;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:&mdash;</span></p>

<p><b>Schäfer’s modification</b> of Pal’s method.&mdash;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.&nbsp;<a href="#Page_24">24</a>) for six hours.
They are washed and stained all night in the <span class="nowrap">following:&mdash;</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">&emsp;45</td><td class="tal">min.</td></tr>
<tr><td class="tal">Acetic acid (2 per cent. aqueous solution)</td><td class="tar">&emsp;100</td><td class="tar">1</td><td class="tal">oz.</td></tr>
</table></div>

<p>The subsequent processes of differentiation,
bleaching, &amp;c., are exactly the same as in Pal’s
method.</p>

<p><b>Osmic acid.</b>&mdash;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">:&mdash;</span></p>

<p><b>Sihler’s chloral hæmatoxyline method.</b>&mdash;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:&mdash;</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">&nbsp;&ensp;"</td></tr>
<tr><td class="tal">One per cent. aqueous solution of chloral hydrate</td><td class="tar">&emsp;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:&mdash;</span></p>


<div class="center">
<table border="0" cellpadding="1" cellspacing="0" summary="">
<tr><td class="tal">Ehrlich’s hæmatoxyline (p.&nbsp;<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">&nbsp;&ensp;"</td></tr>
<tr><td class="tal">One per cent. aqueous solution of chloral hydrate</td><td class="tar">&emsp;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>&mdash;These
are best stained by the chloride of gold
method (p.&nbsp;<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>:&mdash;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>&nbsp;inch
apart, and perpendicular to the surface of the
convolution, placed on the plate of the freezing
microtome and just frozen&mdash;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>&nbsp;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>&#8201;&ndash;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>&mdash;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.&nbsp;<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>&mdash;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">&emsp;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:&mdash;</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">&emsp;1</td><td class="tal">ounce</td><td class="tar">&emsp;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.&mdash;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>&mdash;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:&mdash;</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:&mdash;</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:&mdash;</span></p>

<p><b>Löffler’s methylene blue.</b>&mdash;In this solution
a weak solution of caustic potash is employed as a
<span class="nowrap">mordant:&mdash;</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">&emsp;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,
&amp;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">&emsp;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>&mdash;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:&mdash;</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">&emsp;<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">&emsp;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>&mdash;The sections must be
placed in a freshly made one per cent. aqueous
solution of methyl violet, gentian violet, fuchsine,
&amp;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>&mdash;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,&mdash;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.&nbsp;<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:&mdash;</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">&emsp;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">&emsp;&emsp;<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>&mdash;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>&mdash;Sections
are placed in Ziehl’s carbol-fuchsine
solution (p.&nbsp;<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>&mdash;</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">&emsp;2</td><td class="tal">grms.</td><td class="tar">&emsp;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">&emsp;37·5</td><td class="tal">grs.</td></tr>
<tr><td class="tal"></td><td class="tal">Rectified spirit</td><td class="tar">&emsp;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">&emsp;15</td><td class="tal">c.c.</td><td class="tar">&emsp;<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&nbsp;<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.&nbsp;<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, &amp;c. (p.&nbsp;<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°&nbsp;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°&nbsp;F.
The coverslips are placed an inch or two further
than this point, and kept there at a temperature
of about 200°&nbsp;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:&mdash;</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">&emsp;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">&emsp;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:&mdash;</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">&emsp;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">&emsp;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.&nbsp;<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.&nbsp;<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°&nbsp;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:&mdash;</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">&emsp;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">&emsp;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>&mdash;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">&emsp;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">&emsp;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">&ensp;"</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">&ensp;"</td></tr>
<tr><td class="tal"></td><td class="tal">Glycerine</td><td class="tar">50</td><td class="tal">&ensp;"</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>&mdash;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&nbsp;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&nbsp;<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.&nbsp;10.</span>&mdash;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.&nbsp;11.</span>&mdash;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°&nbsp;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>&mdash;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, &amp;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,
&amp;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>&mdash;For special methods of examination
see Chapter <a href="#Page_103">VII</a>.</p>

<p><b>Blood crystals&mdash;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>&mdash;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>&mdash;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>&mdash;(<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.&nbsp;<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>&mdash;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>&mdash;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>&mdash;Occurs in
many parts. It may be studied in the salivary
ducts, the intestine, kidney, &amp;c., of any mammal.</p>

<p><b>Goblet-cells.</b>&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;May also be studied in most
sections of skin. If the ligamentum nuchæ of a
large quadruped (horse, bullock), &amp;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>&mdash;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>.&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;Obtained from intervertebral
disc. Prepare and stain as for hyaline
cartilage.</p>

<p><b>Bone</b><span class="nowrap">:&mdash;</span></p>

<p><b>Unsoftened Bone.</b>&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;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&nbsp;<a href="#Page_92">92</a>.</p>

<p><b>Heart muscle.</b>&mdash;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>&mdash;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">:&mdash;</span></p>

<p><b>Meissner’s corpuscles.</b>&mdash;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>&mdash;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.&nbsp;<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>&mdash;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>&mdash;Remove, harden, and stain in the
same way.</p>

<p><b>Capillaries.</b>&mdash;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>&nbsp;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, &amp;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>&mdash;Commencement of lymphatics
in serous membrane. Stain a piece of cat’s omentum
in nitrate of silver (p.&nbsp;<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>&mdash;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>&mdash;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>&mdash;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.&nbsp;<a href="#Page_27">27</a>).</p>

<p><b>Brain and spinal cord.</b>&mdash;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, &amp;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>&mdash;Harden the eye of a recently killed
bullock, cat, or other animal in formal (p.&nbsp;<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>&mdash;Decalcifying the temporal
bone of a cat, dog, guinea pig, &amp;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>&mdash;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, &amp;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>&mdash;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, &amp;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>&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;Make an injection of one specimen
with carmine and gelatin (p.&nbsp;<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>&mdash;Same
preparation as for liver.</p>

<p><b>Spleen.</b>&mdash;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>&mdash;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>.&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;Prepare from patients
who have died of exhausting diseases, phosphorus
poisoning, &amp;c.</p>

<p>Stain in osmic acid. Mount in Farrant’s medium
and keep in the dark.</p>

<p><b>Mucoid degeneration.</b>&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;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>&mdash;May be
studied in brown atrophy of heart, nutmeg liver,
&amp;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.&nbsp;<a href="#Page_23">23</a>).</p>

<p><b>Tumours.</b>&mdash;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, &amp;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&mdash;<i>Whitman</i>.<br />
Practical Pathology&mdash;<i>Woodhead</i>.<br />
Textbook of Bacteriology&mdash;<i>Crookshank</i>.<br />
Manual for Physiological Laboratory&mdash;<i>Harris</i> and <i>Power</i>.<br />
Practical Histology&mdash;<i>Fearnley</i>.<br />
Practical Pathology and Histology&mdash;<i>Gibbes</i>.<br />
Journal of Microscopical Society.<br />
Methods and Formulæ&mdash;<i>Squire</i>.<br />
The Human Brain&mdash;<i>Goodall</i>.<br />
Practical Bacteriology&mdash;<i>Kanthack</i> and <i>Drysdale</i>.<br />
Methods of Microscopical Research&mdash;<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 />
&emsp;&emsp;&ensp;&nbsp;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 />
&emsp;&emsp;&emsp;&ensp;oil, <a href="#Page_102">102</a>, <a href="#Page_108">108</a><br />
&emsp;&emsp;&emsp;&ensp;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 />
&emsp;&emsp;&emsp;&emsp;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 />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&nbsp;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 />
&emsp;&emsp;&emsp;&emsp;method of fixing blood-films, <a href="#Page_116">116</a><br />
&emsp;&emsp;&emsp;&emsp;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 />
&emsp;&emsp;&emsp;&nbsp;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 />
&emsp;&emsp;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 />
&emsp;&emsp;&emsp;&nbsp;sublimate method, <a href="#Page_99">99</a><br />
Gram’s iodide solution, <a href="#Page_105">105</a><br />
&emsp;&emsp;&emsp;&ensp;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 />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;Kleinenberg’s, <a href="#Page_70">70</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;Schuchardt’s, <a href="#Page_68">68</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;Sihler’s, <a href="#Page_92">92</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;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 />
&emsp;&emsp;&emsp;&ensp;&nbsp;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 />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;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 />
&emsp;&emsp;&emsp;&ensp;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 />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;Cambridge rocking, <a href="#Page_49">49</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;Cathcart, <a href="#Page_39">39</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;Cathcart-Frazer, <a href="#Page_42">42</a><span class="pagenum" title="158"><a name="Page_158" id="Page_158"></a></span><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;Jung, <a href="#Page_45">45</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;Schanze, <a href="#Page_47">47</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;Swift’s, <a href="#Page_49">49</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;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 />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;and formal, <a href="#Page_20">20</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;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 />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;endings, <a href="#Page_139">139</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&ensp;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 />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;decalcifying agent, <a href="#Page_26">26</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;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 />
&emsp;&emsp;&ensp;&nbsp;cedar, <a href="#Page_8">8</a>, <a href="#Page_63">63</a><br />
&emsp;&emsp;&ensp;&nbsp;cloves, <a href="#Page_63">63</a><span class="pagenum" title="159"><a name="Page_159" id="Page_159"></a></span><br />
&emsp;&emsp;&ensp;&nbsp;origanum, <a href="#Page_63">63</a><br />
Osmic acid as hardening agent, <a href="#Page_21">21</a><br />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&nbsp;staining reagent, <a href="#Page_81">81</a><br />
<br />
Pal’s method, <a href="#Page_86">86</a><br />
&emsp;&emsp;&nbsp;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 />
&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&nbsp;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 />
&emsp;&emsp;&emsp;&emsp;&ensp;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 &amp; Co.).</p></div></div>

<div>*** END OF THE PROJECT GUTENBERG EBOOK 51169 ***</div>
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