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Title: Section Cutting and Staining
 - A practical introduction to histological methods for students and practitioners
Author: Colman, Walter S.
Language: English
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Copyright Status: Not copyrighted in the United States. If you live elsewhere check the laws of your country before downloading this ebook. See comments about copyright issues at end of book.

*** Start of this Doctrine Publishing Corporation Digital Book "Section Cutting and Staining
 - A practical introduction to histological methods for students and practitioners" ***

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Transcriber’s notes:

Apart from the following corrected misspellings the text of this book
has been preserved as in the original:

  xolol —> xylol
  side —> slide
  overstraining —> overstaining

Where appropriate, illustrations and footnotes have been positioned
adjacent to the relevant text.



 SECTION CUTTING AND STAINING

A PRACTICAL INTRODUCTION TO HISTOLOGICAL METHODS FOR STUDENTS AND
PRACTITIONERS


BY W. S. COLMAN, M.D., M.R.C.P.

ASSISTANT PHYSICIAN (FORMERLY PATHOLOGIST) TO THE NATIONAL HOSPITAL FOR
THE PARALYSED AND EPILEPTIC; AND TO THE HOSPITAL FOR SICK CHILDREN,
GREAT ORMOND STREET, ETC.


SECOND EDITION

_ENLARGED AND IN MOST PART RE-WRITTEN_


LONDON H. K. LEWIS, 136 GOWER STREET, W.C. 1896



                              PRINTED BY
                    H. K. LEWIS, 136 GOWER STREET,
                             LONDON, W.C.



PREFACE TO THE SECOND EDITION.


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.

I have again to express my obligation to the various instrument makers
for the illustrations of microtomes, &c.; to Dr. Fearnley, of Bradford,
for the description of his method for injecting blood vessels, and to
Messrs. Macmillan and Co. for permission to copy figures 10 and 11.

                                                    W. S. COLMAN.

    Wimpole Street, W.
        _Sept., 1896._



  CONTENTS.                                                  PAGE

  CHAPTER I.
  Apparatus Required                                            1

  CHAPTER II.
  Hardening Processes                                          15

  CHAPTER III.
  Section Cutting                                              29

  CHAPTER IV.
  Section Mounting                                             55

  CHAPTER V.
  General Staining Methods                                     67

  CHAPTER VI.
  Special Methods for Staining the Nerve Centres               87

  CHAPTER VII.
  Special Methods for Staining Micro-Organisms and Blood      103

  CHAPTER VIII.
  Injection of Blood Vessels                                  120

  CHAPTER IX.
  Directions for Preparing Individual Tissues                 129

  Index                                                       153



SECTION CUTTING AND STAINING.



CHAPTER I.

APPARATUS REQUIRED.


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.

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.

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 will also effect a great saving
if articles that are used in any quantity, such as methylated spirit,
distilled water, &c., are bought by the gallon, and not in small
quantities.

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.

The following should also be procured:--

*Jars* or *bottles*, 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.

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, and a large number of specimens may then
be kept in the same jar. The best way to label them is to write the
name or number on a piece of vegetable parchment in ordinary “marking
ink,” and warm it until the writing is black. The little label should
then be fixed to a corner of the piece of tissue with a stitch or by a
fine pin, and it may be identified years afterwards. The importance of
keeping tissues, sections, slides, &c., *distinctly labelled* 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.

A number of 1 oz. and 2 oz. *stoppered bottles* for staining reagents.

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 rod of suitable length in a blow-pipe, until they are
plastic, and then pressing them together.

*Watch glasses.*--At least a dozen watch-glasses, in which to perform
the operations of staining, clarifying, &c. Those with a flat bottom
should be employed as they are less easily upset than the others.

Plenty of *filter papers*.

Both coarse ones, for use in the manufacture of reagents, and small
fine white ones (2-1/2 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.

Several *needles* mounted in handles.

They must be kept very bright and smooth, and care must be taken that
the point does not get turned up.

A large and small *funnel*.

Several *pipettes* consisting of pieces of glass tube with an internal
diameter of 1/8″ and about ten inches long, drawn out almost to a point
at one end.

*Section lifter.*--This instrument is required for transferring
sections from one reagent to another, or from oil of cloves, &c., to
the slide. The most convenient form is Woodhead’s, made of thin sheet
copper, which allows the blade to be bent at any angle to the stem. The
stem or handle is about six inches long, and continuous with, and at an
angle to it, a flat blade about 3/4 in. square with the corners rounded
off. Larger ones can be obtained for mounting sections of large size,
_e.g._, kidney, medulla oblongata, &c. The surface of the blade should
be brightly polished, and kept scrupulously clean.

Ordinary dissecting *forceps*.

One or two *scalpels*.

A pair of fine *scissors*.

A *razor* or other instrument for cutting sections.

A smooth *oil stone* for keeping the razors and knives properly
sharpened.

A *spirit lamp* for warming the staining fluids.

A few *test tubes*.

A *minim measure*.

*Scales* and small weights.

A gross of ground glass slides 3 x 1 in.

Half a gross of ground glass slides 3 x 1-1/2 in.

Half an ounce of thinnest coverslips, 7/8 in. diameter.

Quarter of an ounce of thinnest coverslips, 1-1/4 in. diameter.

*Microscope.*--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.

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.

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.

The microscope should be provided with a *coarse and fine adjustment*,
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.

There should be a *reversible mirror*, 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
*eye-piece* 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.

*Objectives.*--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.

Most objectives and stands are now made with a universal thread, so
that any objective will fit 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.

The most useful lenses are the 1 in. low power lens, and 1/5 in. or
1/6 in. high power, or No. 3 and No. 7 of Continental makers, or
Zeiss’s A and D. A 1/2 in. lens will also be found very useful.

For minute work, such as bacteriology and blood investigations,
higher powers will be required, 1/8 or 1/12 *immersion lenses*. 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. The focussing should then be managed
with the fine adjustment only.

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.

[Illustration: Fig. 1.--Double or Triple Nose-piece.]

*Double or triple nose-piece* (fig. 1).--This mechanical arrangement
is placed on the lower 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 _vice versa_. 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.

*Substage condenser.*--This mechanism for concentrating light on the
object is a necessity for bacteriological work. The most convenient
form is *Abbe’s illuminating apparatus* (fig. 2).

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 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.

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.

[Illustration: Fig. 2.--Abbe’s Illuminating Apparatus.]

Of the cheaper student’s microscopes the “Star” microscope made by
Messrs. R. and J. Beck, of Cornhill, E.C., will be found a safe
investment. It may be obtained with coarse and fine adjustment,
nose-piece, and 1 in. and 1/4 in. objectives, for about £5. Those who
require a better instrument will find Beck’s “Pathological” microscope
fitted with nose-piece, Abbe’s illuminator, &c., for £16, meet all
requirements.

Leitz of Jena, supplies two good and cheap microscopes for £3 10_s._
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.

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, 1/6 in., and a 1/12 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.

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.

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.

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 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.e._, 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.

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.



CHAPTER II.

HARDENING PROCESSES.


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.

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
come freely in contact with the under surface of the pieces of tissue,
and prevents their being flattened against the hard glass bottom.

The hardening fluid requires changing occasionally. This should always
be done at the end of twenty-four hours, in order to get rid of any
deposit of blood, &c., that may have accumulated. Besides this, the
tissue when placed in the fluid contained a good deal of water which
will have diluted it and consequently an early change is desirable.
Afterwards the fluid requires to be changed only as often as it becomes
turbid, or any deposit occurs, usually about once a week.

While hardening, specimens should be kept in a cool place, as warmth
favours changes in the cells, &c.

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.

It requires some practice to know when the 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.

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.

It is unnecessary for ordinary work to have more than the following
hardening fluids:--*Müller’s fluid*:--

  Potassium Bichromate    2-1/4 grms.        3-1/2 drachms.
  Sodium Sulphate             1 grm.         1-1/2 drachms.
  Water to                  100 c.c.         1     pint.

Two drachms of carbolic acid are sometimes added to each pint of the
fluid but as a rule it is not necessary.

Müller’s fluid is the most generally useful of the various fluids
employed, for the following reasons:--

1. It causes very little shrinking of the elements of the tissue, and
hence may be employed for most delicate objects, _e.g._, the retina and
embryos.

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.

3. There is comparatively little danger of over-hardening the tissue
and rendering it brittle.

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.

5. It readily permeates the tissues, and hence large portions of
organs, or even the entire organ may be satisfactorily hardened in it.

6. It is very cheap. A gallon can be made up for about eightpence.

The fluid has however certain slight drawbacks:--

1. The hardening process is a slow one occupying four to eight weeks.

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.

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. 89).

To harden a specimen in it at least twenty times the bulk of fluid must
be employed.

The fluid must be changed on the third day, and afterwards about every
week as may be required.

*Methylated spirit* 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 are:--

1. It is more apt to overharden than Müller’s fluid.

2. It causes a great deal of shrinking of the tissue and thus squeezes
much of the blood out of the vessels.

It is most useful in hardening tissues containing much epithelium,
_e.g._, kidney, epithelioma, &c.

Spirit is also frequently employed to complete the hardening by
Müller’s fluid and to preserve tissues after they have been hardened.

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.

*Müller’s fluid and spirit.*--This is a useful combination for many
purposes. It is made thus:--Müller’s fluid, three parts; methylated
spirit, one part.

The fluid must be allowed to cool after mixing before being used, and
if necessary filtered. It will harden specimens satisfactorily in three
weeks.

*Müller’s fluid and formal.*--Is an extremely useful mixture made by
adding one part of formal to nine of Müller’s fluid. It hardens in a
much shorter time than Müller’s fluid and causes very little shrinkage.

*Absolute alcohol.*--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. 101). 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.

Small pieces must be used. The depths of the block should not exceed
3/8 inch. The fluid should be changed on the third day. Hardening will
be completed in about ten days or even earlier.

*Osmic acid.*--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.

Its disadvantages as a hardening agent are:--

1. Its expense.

2. Its irritating and corrosive vapour.

3. The fact that only small pieces of tissue can be hardened in it,
since the external surface is very rapidly hardened and thus the fluid
is prevented from penetrating into the centre of the lump.

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. 95).

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 *thoroughly* washed in
distilled water and may afterwards be preserved in methylated spirit.

Both the hardening and the subsequent washing must be carried on *in
the dark*.

Osmic acid is also a most valuable staining reagent (see p. 81).

*Carbolic acid* (5 per cent.).--May be used to harden almost any
tissue, but is particularly useful for hardening nervous tissues such
as brain or spinal cord which are afterwards to be preserved as museum
specimens. It does not discharge the colour of a specimen so rapidly as
spirit.

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.

Saturated aqueous solution of *corrosive sublimate* is one of the
most convenient hardening reagents for small pieces of delicate
tissue, _e.g._, 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.

*Formal.*--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 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.

*Marchi’s fluid.*--This consists of:--

  Müller’s fluid                          2 parts.
  Osmic acid solution (one per cent.)     1 part.

It is used for hardening specimens as a preliminary to Golgi’s method
for staining nerve cells (p. 97), and also to complete the hardening of
sections of spinal cord, &c., before employing Schäfer’s modification
of the Weigert Pal hæmatoxyline method (p. 91).

It is also used as a stain for recently degenerated nerve tracts and
fibres, especially after experimental lesions.

The fluid has little penetrating power, and therefore tissues must be
cut into small pieces, about 3/8 inch cube. It is not necessary to
place them in this fluid at once on removal from the body, but the
preliminary hardening must be in Müller’s fluid and not in alcohol, &c.


SPECIAL HARDENING REAGENTS FOR RAPID FIXATION IN ORDER TO STUDY CELL
STRUCTURE.

1. *Alcohol.*

2. *Flemming’s solution* (modified by Friedmann):--

  Osmic acid (one per cent.)      3 c.c. (♏xxx.).
  Glacial acetic acid             2 c.c. (♏xx.).
  Chromic acid (one per cent.)   42 c.c. (℥j.)

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.

3. *Nitric acid.*--A ten per cent. solution in distilled water. It
hardens the tissue in three to four hours, and should be followed by
70 per cent. alcohol, the hardening being completed in absolute alcohol.

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.


DECALCIFYING FLUIDS.

Used in the preparation of bone, tooth, osseous tumours, &c. The two
best fluids for general use are:--

*Chromic and nitric fluid.*--This is made as follows:--

  Chromic acid      1 gramme         45 grains.
  Nitric acid       2 grammes     1-1/2 drachms.
  Water           200 c.c.            1 pint.

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.

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.

*Von Ebner’s solution*:--

  Hydrochloric acid     1 gramme  1-1/2 drachms.
  Common salt          10 grammes     2 ounces.
  Water to            100 c.c.        1 pint.

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.

*Bleaching solution* (eau de Javelle).

  (1) “Chloride of lime” (bleaching powder)    20     1/2 oz.
      Water                                   100   2-1/2 oz.

Shake up well.

  (2) Carbonate of potassium                   20     1/2 oz.
      Water                                   100   2-1/2 oz.

Mix the two solutions. Allow them to stand for an hour and filter.

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.



CHAPTER III.

SECTION CUTTING.


*Embedding of sections.*--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.

The most generally useful substances are:--

(1) gum, (2) celloidin, (3) paraffin or wax.

*Gum.*--_Picked_ colourless gum arabic 2 parts, cold water 3 parts.

Leave with frequent stirring until dissolved. Add ten drops of carbolic
acid to each ounce of the mucilage.

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.

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.

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.

*Celloidin* 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 very readily soluble in ether, or
in a mixture of alcohol and ether. The last solvent is the one commonly
employed.

The embedding solution is made thus:--

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.

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.

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.
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.

Pieces of tissue embedded in celloidin may also be cut on a freezing
microtome. After 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.

Subsequent staining operations are conducted in the same way as for
sections cut by hand or in gum. As celloidin is only slightly stained
by hæmatoxylin, alum carmine, borax carmine, &c., it is not necessary
to remove it from the sections, but it exhibits so intense a staining
reaction with aniline dyes that it is necessary to remove it by
treatment with alcohol and ether either before or after the staining
operation.

The sections after staining may be mounted in Farrant’s solution
(p. 59), or in Canada balsam (p. 61). 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.

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. 90).

_Paraffin._--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. 75), alum carmine
(p. 76), and Kleinenberg’s hæmatoxyline (p. 70). The tissue must be
left in them for four to ten days.

Various kinds of paraffin are employed. It is usual to keep two kinds,
one “soft,” melting at 110° F., and another “hard,” melting at 140° F.
A mixture of two parts of the hard and one of the soft will be found
most generally useful. In winter a large proportion of the soft variety
and in hot weather a larger proportion of the hard may be required. A
paraffin mass which is always available has been suggested recently by
Dr. F. E. Batten, who employs an ordinary white candle, composed of
paraffin and wax. If the mass is found to be too hard, it can easily be
made of a suitable consistence by adding a little paraffin with a low
melting point.

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 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. 3). 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 paraffin block with a hot
needle or wire and pressing it down on the wood.

[Illustration: Fig. 3.]

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.

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 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. 61) is run on
the slide, and a cover-glass of suitable size is applied.

*Microtomes.*--After a large amount of practice, persons with a fair
amount of manual dexterity may acquire sufficient skill to be able to
cut very satisfactory sections of specimens embedded in paraffin, &c.,
by hand. In the Pathological Laboratory of a large German University,
until quite recently the use of a microtome was prohibited by the
Professor, who is himself a most distinguished histologist. The amount
of time expended before one acquires the necessary skill, and the
cheapness and great convenience of the modern microtome have combined
to throw hand cutting into the background, and some form of microtome
is now almost universally adopted.

[Illustration: Fig. 4.--Cathcart’s Ether Spray Microtome.

A, B. Wooden frame and supports. C. Glass runners. G. Screw for raising
the zinc plate H. J. Ether bottle. L. Tube from air bellows.]

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.

Cathcart’s *ether spray microtome* (fig. 4).--This, or its more recent
modifications (_see_ 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.

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 3/8 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 time causes it rapidly to evaporate, and so reduces the
temperature of the zinc plate.

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.

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 transferred to a glass slide from the knife,
washing it off with a stream of ice water from a pipette.

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.

A. Frazer has recently introduced a valuable improvement in the
Cathcart microtome (fig. 5).

In this the brass frame carrying the zinc plate and ether spray tubes
is surrounded by a brass cylinder, in which it fits accurately, and
is pushed up as desired by turning the screw beneath the instrument.
This brass frame and with it the zinc plate, &c., can be easily drawn
altogether out of the outer tube, and replaced by a second brass
well, which exactly fits its place and can be raised 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.

[Illustration: Fig. 5.--Frazer’s Modification of Cathcart’s Microtome.

A. Microtome arranged for ether spray. B. Cylinder with clamp for
holding object embedded in celloidin, &c. to replace ether spray
apparatus.]

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. 6) 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 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.

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 “Cathcart.”

*Williams’ ice freezing microtome* (fig. 6).

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.

[Illustration: Fig. 6.--Williams’ Ice Freezing Microtome, with Swift’s
plough.]

To use it, the box is filled with alternate layers 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. 44).

[Illustration: Fig. 7.--Schanze Microtome (see text).]

*Schanze microtome* (fig. 7) is the pattern used in the Leipsic
laboratories. It consists of a 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. (Messrs. R. and J.
Beck are the agents). Its cost is about £5.

*Becker’s microtome* 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.

Frazer has introduced a “student’s sliding microtome” on the same
principle as the Schanze which costs about £3.

*The Cambridge Rocking microtome.*--This instrument, as made by the
Cambridge Scientific Instrument Company, or the slightly modified form
made by Messrs. Swift (fig. 8), 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, 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 1/500 to 1/3000 inch. The actual working of the machine
is therefore very simple. 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.

[Illustration: Fig. 8.--Swift’s Modification of the Cambridge Rocking
Microtome.]

*Fresh sections.*--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 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.

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 3/4
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 less coherent and also more sticky, so that the
edges tend to curl up on the knife, &c. They should then be examined,
one unstained, simply mounted in salt solution; another stained with
picrocarmine and examined in the saline solution; and a third stained
in picrocarmine, mounted in Farrant’s solution, and preserved. The last
usually gives the best results, the picrocarmine staining becoming
quite brilliant after a week. The glycerine, however, is apt to make
the sections shrink a good deal, and the weight of the cover-glass
tends to break up the unhardened section.



CHAPTER IV.

SECTION MOUNTING.


1. By *flotation*.

In this method the section whether stained or unstained is placed in a
bowl of water, or normal salt solution (p. 53). 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 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.

2. By *transference* with a *section lifter*.

This method is employed in mounting in Canada balsam in order to
transfer the section from the clarifying agent (p. 63) 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.

*Removal of air bubbles from sections.*--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.

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 of an air pump, if one is
available, and slightly exhausting the air.

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.

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, both for Farrant’s
medium and balsam are “balsam bottles” which have no stopper, but the
mouth is closed by a glass cap which fits accurately (fig. 9). A short
glass rod is attached to the cap, and is used to transfer the medium to
the slide.

[Illustration: Fig. 9.--Balsam bottle.]

*Treatment of folded sections.*--The folding may be due:--

(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 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.

(2) To the section containing a large amount of *fat*, 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.


MOUNTING MEDIA.

*Farrant’s solution*:--

  Gum Arabic (picked, colourless)  }
  Glycerine                        } equal parts.
  Water                            }

In making this solution the best gum arabic must be used, and only the
clearest pieces of this. “Powdered gum acacia” should be avoided, as
though it looks white it often yields a brown mucilage, and besides is
frequently adulterated with starch, &c.

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. 58)
containing a few drops of a saturated solution of arseniate of sodium
and a small lump of camphor.

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.

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.

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.

*Canada balsam solution*:--The medium is made thus:--

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

  Dried Canada balsam  } equal parts.
  Xylol                }

Leave till dissolved, stirring occasionally.

Unless the solution be perfectly clear, it must be filtered through a
very thin paper, previously 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.

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.

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.

To mount sections in Canada balsam they must be transferred first to
a watch glass containing absolute alcohol or an alcoholic solution
of some staining reagent, _e.g._, eosine (p. 72) and left in it, no
attempt being made to spread it out, until it is perfectly dehydrated,
_i.e._, 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.

The process of *clarifying* 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, 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 VI. and VII.

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.

If the tissue is very delicate, and likely to be 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.

*Cementing of cover-glasses.*--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 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.

Many materials are employed. The most suitable are:--(1) Canada balsam,
which is almost colourless and transparent and looks very neat. (2)
Gold size. (3) Marine glue.

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 follows:--

  Oxide of zinc        1/2 drachm.
  Benzole            } half an ounce
  Gum dammar         }    of each.

*Preservation of sections.*--They should be kept _flat_, 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.



CHAPTER V.

GENERAL STAINING METHODS.


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 stained with several reagents, as
their effect on individual specimens varies a good deal.

The following are the most useful stains for general purposes:--

*Logwood.*--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.

For general staining purposes the following formula will be found to
give excellent results:--*Hæmatoxyline. Schuchardt’s formula.*--

  (_a_) Hæmatoxyline         3 grms.      30 grs.
        Absolute alcohol    16 c.c.    2-1/2 drms.
  (_b_) Pure alum            3 grms.      30 grs.
        Distilled water    100 c.c.        2 ozs.

Add (_a_) to (_b_) _drop by drop and with constant agitation_. 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 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.

*Barrett’s formula.*--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.

The extract should be dried, and finely powdered, and then extracted
with absolute alcohol for several days.

  Powdered extract of logwood    2 grms.  1-1/2 drms.
  Absolute alcohol              10 c.c.       1 oz.

Filter and add slowly to

  Benzoate of sodium             1 grm.      36 grs.
  Alum                           1 grm.      36 grs.
  Distilled water              100 c.c.      10 ozs.

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.

*Ehrlich’s hæmatoxyline.*--This very useful nuclear stain is made as
follows:--

  (_a_) Hæmatoxyline             2 grms.      9 grs.
        Absolute alcohol       100 c.c.       2 ozs.
  (_b_) Glycerine              100 c.c.       2 ozs.
        Distilled water        100 c.c.       2 ozs.
        Alum                   120 grms.  2-1/2 ozs.
        Glacial acetic acid      5 c.c.      24 mins.

Add (_a_) slowly to (_b_) with constant agitation.

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.

*Kleinenberg’s hæmatoxyline.*--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 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.

Various formulæ have been given from time to time. That advised by
Squire (_Methods and Formulæ_, p. 25) can be accurately made up without
much difficulty.

  (_a_) _Crystallised_ calcium chloride  20 grs.     1/2 oz.
        Distilled water                  10 c.c.     2 drms.
  (_b_) Alum                              3 grms.   32 grs.
        Distilled water                  16 c.c.   170 mins.

Mix and add

  Rectified spirit                      240 c.c.     8 ozs.

Allow it to stand and any excess of calcium sulphate, &c., to separate.
Filter and add

  Hæmatoxyline                        2-1/2 grms.   25 grs.

A little thymol should be added as a preservative.

In making up these solutions care must be taken that _only distilled
water_ is used, and that all the vessels employed have been previously
rinsed out with it, otherwise precipitation of the hæmatoxyline will
occur.

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.

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. 88–91).

The stain is permanent. Sections may be mounted either in Farrant’s
solution, or in Canada balsam, the latter being preferable.

*Eosine.*--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.

It may be used as an aqueous solution (1/30 per cent.) or as a
solution in absolute alcohol (1/15 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.

It is a very transparent stain, and the most delicate details of a
section stained with it are perfectly visible.

It stains the nucleus but slightly, while it stains the cell protoplasm
and fibrous tissues and especially muscular tissues a beautiful rose
colour.

It will be seen, therefore, that it stains those parts which are left
unstained by hæmatoxyline, and _vice versâ_. This complementary action
is applied in the following method.

*Double staining with eosine and hæmatoxyline.*--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.

This method gives extremely useful and beautiful 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.

The alcoholic solution of eosine is also used as a contrast stain after
staining for micro-organisms with blue or violet dyes.

*Carmine.*--It is made as follows:--

  Carmine (best)        2    1 drachm.
  Strong ammonia        2    1 drachm.
  Distilled water     100    6 ounces.

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.

*Lithium carmine* resembles closely ammonia carmine in its staining
effects. It is usually a matter of individual preference which is
employed.

  Carmine                      2-1/2 grms. 10-1/2 grs.
  Saturated aqueous solution
    of lithium carbonate         100 c.c.       2 ozs.

Dissolve and filter.

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.

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.

*Borax carmine*--

  (_a_) Borax                      4 grms.      3 drachms.
        Carmine                    2 grms.  1-1/2 drachms.
        Distilled water 100 c.c.                5 ounces.

Dissolve with the aid of heat and add slowly to (_b_).

  (_b_) Alcohol                   70 c.c.   3-1/2 ounces.
        Distilled water           30 c.c.   1-1/2 ounce.

Allow to stand for a fortnight. Filter, and add a lump of camphor.

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.

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.

It stains nerve-cells and axis cylinders brightly, and also the
connective tissue, bringing a sclerosed patch out very prominently.

*Alum carmine*:--

  Alum five per cent. solution
    in distilled water           100 c.c.       1 oz.
  Pure carmine                     1 grm.   4-1/2 grs.

Boil for twenty minutes. Filter. Add a few drops of carbolic acid.

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, and they may be left all night. After they have been
stained they must be _thoroughly_ 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.

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.

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.

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.

It is particularly useful as a contrast stain for sections of brain
and spinal cord, after the Weigert-Pal hæmatoxylin process (p. 88).

*Ammonia-picrocarmine* was formerly very largely used as a staining
reagent. Its place has now to a large extent been taken by
lithio-picrocarmine.

In its preparation the best carmine must be used.

It is made as follows:--

  Carmine                            1 part.
  Liq. ammon. fort.                  3 parts.
  Distilled water                    3 parts.

Dissolve with gentle heat, and add

  Cold saturated aqueous solution
    of picric acid                 200 parts.

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.

*Lithio-picrocarmine.*--Prepared as follows:--

  Carmine                                2·5 grms.    10 grs.
  Saturated solution lithium carbonate   100 c.c.      1 oz.

Dissolve, and add

  Saturated solution picric acid         250 c.c.  2-1/2 oz.

Add a few drops of carbolic acid to each ounce.

It should stand a day or two in sunlight and then be filtered. It
improves by keeping.

It should be kept in a stoppered bottle with a glass rod fused into the
stopper.

When sections are to be stained they are to be floated out on a clean
glass slide as described on page 55. 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 the tissues to stain more brightly and more rapidly.

The excess of the picrocarmine should be allowed to run off the slide,
and the latter wiped. Some of the stain should, however, be left on the
section, as its effects go on increasing, and are often not fully seen
until a few weeks have elapsed. They should be mounted in Farrant’s
medium. As a rule those mounted in Canada balsam do not give such good
results. Should there be special reasons for using this medium, as in
mounting spinal cord sections, &c., they should be dehydrated after
staining in picrocarmine in an alcoholic solution of picric acid (one
part of a saturated alcoholic solution to five of alcohol), before
clarifying in oil of cloves, as otherwise the alcohol will dissolve out
the picric acid, and much of the differential staining effect will be
lost. The nuclei should be stained a bright crimson, the protoplasm of
the cells yellow, or a dull pink, the fibrous elements a bright pink,
red corpuscles green, and all dead material, _e.g._, caseous matter,
bright yellow. It also stains nerve-cells, and the axis cylinders of
nerve fibres very brightly. It is, however, a rather uncertain dye.
The results are most brilliant in the case of fresh sections.

*Osmic acid* is invaluable for staining fatty particles in the cells.

For ordinary use the one per cent. stock solution (p. 21) 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.

The sections must be washed _thoroughly_ 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.

It demonstrates the most minute fatty particles in degenerating cells,
&c., staining them black. It may be employed to demonstrate the
globules of fat blocking up the vessels in fat embolism.

It stains the myelin sheaths of nerves black, and will be again
referred to when speaking of methods of staining the spinal cord.

*Nitrate of silver* 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 *distilled* water, and kept in a stoppered bottle
carefully covered up with brown paper. To use it take some epithelial
tissue, _e.g._, 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.

*Chloride of gold* 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.

A half per cent. solution in distilled water is 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.

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. 92).

*Methyl violet.*--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.

It is a very useful selective stain. It gives 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.

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 *thoroughly* for some time in a large quantity
of water till no more colour comes away.

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.

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.

*Safranine.*--Employed as a freshly made saturated solution in aniline
oil water warmed to 60° C. (140° F.). Filter into a watch glass. Stain
for not more than a minute. Dehydrate in alcohol which will remove much
of the stain, clarify in oil of cloves or origanum oil. Mount in balsam.

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,
_e.g._, those undergoing colloid or calcareous degeneration.

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.

*Ehrlich-Biondi stain.*--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.

It is prepared by mixing saturated aqueous solutions of the following
aniline dyes, slowly and with constant agitation:--

  Solution of Orange G          100 parts
     "        Rubin S            20   "
     "        Methyl Green OO    50   "

finally add

  Distilled water                70   "

Filter from the copious precipitate which forms. The solution must be
made up frequently as it does not keep well.

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.



CHAPTER VI.

SPECIAL STAINING METHODS.--SPECIAL METHODS FOR STAINING THE NERVE
CENTRES.


1. For staining *nerve fibres*.

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
stained with alum carmine or eosine to shew the cells and neuroglia.

*Weigert’s method.*--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. 30).
When sections are cut they are transferred at once to Weigert’s
hæmatoxyline solution:--

  Hæmatoxyline               1       4 grains.
  Alcohol                   10      45 minims.
  Distilled water          100       1 ounce.

They are stained in this for twenty-four hours or longer, until they
are quite black.

The staining will take place much more rapidly if the fluid be kept
at 100° F. in the incubator. After staining they are transferred to
Weigert’s differentiating solution:--

  Borax                      2     160 grs.
  Potassium ferricyanide     2·5   200 grs.
  Distilled water          100       1 pint.

They are left in this solution for several hours, until the ground work
becomes nearly decolourised.

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.

*Pal’s modification* of Weigert’s method.--By this method quicker and
more complete decolouration of the neuroglia, nerve cells, &c., is
obtained. Sections are prepared in exactly the same way as in Weigert’s
method and then transferred to Weigert’s hæmatoxyline. Pal recommends
that this solution be diluted to half the strength and a few drops of
a saturated solution of lithium carbonate added. The writer finds the
results equally good if the ordinary Weigert solution be employed.

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 specimens,
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 to:--

*Pal’s differentiating solution.*

  Potassium sulphite      1 grm.    40 gr.
  Oxalic acid             1 grm.    40 gr.
  Distilled water       200 cc.      1 pint.

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 should then
be washed, dehydrated in alcohol, clarified in oil of bergamot, and
mounted in Canada balsam.

The very prolonged hardening in Müller’s fluid which is a necessary
preliminary for this method led to the introduction of:--

*Schäfer’s modification* of Pal’s method.--In this method hardening in
Müller’s fluid for three or four weeks is sufficient. The sections are
made exactly as in the previous method, and transferred to Marchi’s
fluid (p. 24) for six hours. They are washed and stained all night in
the following:--

  Hæmatoxylin                                    1    4 grs.
  Alcohol                                       10   45 min.
  Acetic acid (2 per cent. aqueous solution)   100    1 oz.

The subsequent processes of differentiation, bleaching, &c., are
exactly the same as in Pal’s method.

*Osmic acid.*--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 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.

2. *Intra-muscular ramifications of nerves*:--

*Sihler’s chloral hæmatoxyline method.*--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.

A piece of muscle is taken as soon as possible 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
solution:--

  Acetic acid                                          1 part.
  Glycerine                                            1  "
  One per cent. aqueous solution of chloral hydrate    6 parts.

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
solution:--

  Ehrlich’s hæmatoxyline (p. 70)                       1 part.
  Glycerine                                            1  "
  One per cent. aqueous solution of chloral hydrate    6 parts.

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.

*Motor and sensory nerve endings.*--These are best stained by the
chloride of gold method (p. 82).

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.

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.

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.

3. *Staining nerve cells.*

*Bevan Lewis’s aniline blue-black method*:--This method is the best for
demonstrating 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 1/8 inch apart, and perpendicular to the surface
of the convolution, placed on the plate of the freezing microtome and
just frozen--not too hard or the tissue will be brittle and will also
injure the edge of the razor. As soon as a good section is obtained
the razor should be plunged into a large bowl of cold water to detach
the section, which is at once floated on a glass slide, and osmic acid
solution, 1/4 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 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.

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.

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 1/2 - 2 minutes in a 2 per cent. solution of chloral
hydrate in distilled water after staining.

*Golgi’s metallic stains* for nerve cells.

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.

There are several methods now in vogue, all slight modifications of
Golgi’s original methods.

*Silver nitrate method.*--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. 24) 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 3/4 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 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.

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.

A method has been employed for deepening the colour of the stain,
but the writer has no experience of it. Kallus (_Zeitsch. f. Wiss.
Mikr._, 1893, 477) dilutes an ordinary hydrokinone developing solution
(prepared as for developing an ordinary photographic plate) with about
ten times its 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.

*Buckley’s modification of the silver method.*--Described in _Brain_,
Winter number, 1895.

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

  Bichromate of potassium, 3 per cent. solution   5 parts
  Osmic acid, 1 per cent. solution                1 part

for three to five days. Excess of bichromate is removed from the
sections by blotting paper, and they are transferred to the _freshly
prepared_ staining mixture:--

  Phospho-molybdic acid (10 per cent.)            1 minim   2 drops.
  Nitrate of silver (1 per cent.)                 1 ounce  60 c.c.

which must not be filtered. Stain for several days.

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.

*Corrosive sublimate* method.--This method is similar in its mode of
action to the last, mercury being deposited in the cell instead of
silver. It is rather less certain and requires more practice. It seldom
stains uniformly. One cell will be found exquisitely stained while
those in its vicinity are unaffected.

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
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.

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.

*Nissl’s aniline method.*--This method is complementary to Golgi’s
method. The latter impregnates the cell rendering it opaque and shewing
its form with great definiteness.

Nissl’s method stains the protoplasm without greatly reducing its
transparency and allows us to study details of cell structure. Small
portions of tissue, _removed as soon as possible after death_, are
hardened in alcohol. Sections are then cut, preferably in gum, as
celloidin is inconvenient owing to its staining so deeply with aniline
dyes.

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,[1] 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.

[1] The student will bear in mind the danger of working with benzine
near a naked light.

Magenta and other aniline dyes may also be employed in a similar
manner.



CHAPTER VII.

SPECIAL METHODS FOR STAINING MICRO-ORGANISMS AND BLOOD.


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.

The student should provide himself with the following dyes in powder:--

*Methylene blue.*

*Gentian violet.*

*Methyl violet.*

*Fuchsine.*

*Bismarck brown.*

The following solutions of these dyes are used:--

1. Saturated alcoholic solutions which may be kept in stoppered bottles.

2. One per cent. aqueous solutions. These must be freshly made each
time of using.

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.

The following special solutions will also be wanted:--

*Löffler’s methylene blue.*--In this solution a weak solution of
caustic potash is employed as a mordant:--

  Saturated alcoholic solution of methylene blue    3 volumes.
  Caustic potash, aqueous solution 1 : 10,000      10 volumes.

Filter.

This solution is perhaps the most generally useful stain. It colours
most bacilli and micrococci, and while rapid in its action rarely
overstains. It must be made up fresh on each occasion. It is the best
counterstain after staining tubercle bacilli, &c., with fuchsine.

*Ziehl’s carbol-fuchsine.*

  _Carbolic acid_ (5 per cent. aqueous solution)  100 volumes.
  _Fuchsine_ (saturated alcoholic solution)        11 volumes.

The solution must be filtered immediately before being used.

*Gram’s iodine solution.*--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.

It is made thus:--

  Iodine                  1 grm.    1-1/2 grains.
  Iodide of potassium     2 grms.       3 grains.
  Distilled water       300 c.c.        1 ounce.

Ten per cent. aqueous solutions of *nitric* and *sulphuric* acids
should be prepared and may be kept indefinitely.

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.

*Weigert’s method.*--The sections must be placed in a freshly made
one per cent. aqueous solution of methyl violet, gentian violet,
fuchsine, &c. The solution may be kept at the temperature of the body
in an incubator. The organisms will often stain more readily if the
section be passed through a 1 in 2000 solution of corrosive sublimate
before putting it into the staining fluid. After staining the section
is washed in distilled water and then in methylated spirit until it
appears almost decolourised. Some prefer to decolourise the tissues
by washing in a half per cent. solution of acetic acid instead of
methylated spirit. Practice is required before the correct time for
decolourising is accurately estimated. The beginner should float a
section rapidly on the slide now and then, put on a cover-glass and
examine it under a low power to see if the decoloration has been
carried far enough. A contrast stain may 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.

*Gram’s method.*--Place some aniline oil in a test tube and add ten
times its volume of distilled water. Close the end with the thumb and
shake very thoroughly. Filter ninety drops into another clean test
tube, and add ten drops of a saturated solution of gentian violet
or some similar dye. Filter the mixture into a watch glass. Stain
sections in it for from three minutes to half-an-hour according to the
temperature,--the shorter time for the incubator at 100°, the longer
when the sections are stained at the ordinary temperature of the room.
Wash in distilled water, and transfer to Gram’s iodine solution until
they become black, usually in a few minutes. They are then decolourised
in absolute alcohol. This often takes some time. It may be hastened, as
Crookshank suggests, by placing the section in clove oil, returning to
alcohol, and so on.

*Ehrlich’s modification* of Gram’s method. The contrast stain is here
used first.

Stain the section (_e.g._, 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. 55) 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 comes away, the
aniline oil is poured off, the section clarified in xylol, and mounted
in Canada balsam.

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.

The following will be found the most useful stains and contrast
stains:--

  STAINS.              CONTRAST STAINS.

                       { Picrocarmine.
  Gentian violet. }    { Eosine.
  Methyl violet.  }    { Bismarck brown.
  Methylene blue. }    { Safranine.

  Magenta.  }            Methylene blue.
  Fuchsine. }

            And _vice versâ_.

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 good results although he follows the book
directions most minutely.

*Ehrlich method for tubercle bacilli.*--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.

*Neelsen’s stain for tubercle bacilli.*--Sections are placed in
Ziehl’s carbol-fuchsine solution (p. 103) 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 thoroughly washed in a large
quantity of water, and afterwards may be treated with a contrast stain.

*Gibbes’ double stain for tubercle bacilli.*--

  (1) Rosaniline hydrochlorate           2 grms.    25 grs.
      Methyl blue                        1 grm.   12·5 grs.

Triturate in a glass mortar,

  (2) Aniline oil                        3 c.c.   37·5 grs.
      Rectified spirit                  15 c.c.  3-1/2 drms.

Dissolve and add slowly to (1).

  (3) Lastly add slowly to the mixture
      Distilled water                   15 c.c.  3-1/2 drms.

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.

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 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.

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.

Another plan is to mix the urinary deposit with 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.

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.


METHODS OF EXAMINING BLOOD.

In all these methods blood is obtained by pricking the skin of one of
the fingers, or the lobule of 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 57.

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. 129) before mounting. Permanent coverslip films may also
be prepared.

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.

1. Exposure to *osmic acid vapour*.

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 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.

2. Treatment with *saturated aqueous solution of corrosive sublimate*
(Muir’s method).

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.

3. By drying and passing rapidly through the flame of a Bunsen burner,
exactly as in preparing specimens of sputum, &c. (p. 111). This method
is handy for ordinary clinical purposes.

4. By keeping the coverslips at a temperature of about 200° F.
(Ehrlich’s method).

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 beneath. The point
in the copper strip at which the temperature is at boiling point is
readily ascertained by dropping a little water on. The point at which a
drop of water assumes the spherical state indicates a temperature there
of 212° F. The coverslips are placed an inch or two further than this
point, and kept there at a temperature of about 200° F. for some hours.


STAINING METHODS.

Fresh blood may be stained by mixing with Ferrier’s fuchsine solution:--

  Fuchsine                   1 grm.
  Distilled water          150 c.c.

Dissolve and add

  Alcohol (80 per cent.)    50 c.c.
  Neutral glycerine        200 c.c.

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.

Stained preparations may also be obtained by using *Toison’s fluid*,
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 thus:--

  Glycerine           30    c.c.    1 oz.
  Sodium sulphate      8    grms.   2 drms.
  Sodium chloride      1    grm.   15 grs.
  Methyl violet         ·25 grm.    4 grs.
  Distilled water    160    c.c.    5 oz.

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.

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 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.

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. 104).

These films may be stained for micro-organisms in the way described for
cover-glass preparations (p. 112).



CHAPTER VIII.

INJECTION OF BLOOD VESSELS.


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.

The most convenient basis for an injection mass is gelatine, as its
solutions liquefy at a temperature of about 100° F., and solidify a
little below that point, and when solidified cut readily, and do not
tend to become brittle. The various masses are prepared as follows:--

*Red injection mass* (Woodhead’s formula) consists of gelatine softened
by mixture with water and coloured by carmine.

  (1) Carmine                4 grms.
      Liq. ammoniæ B.P.      8 grms.
      Distilled water      150 c.c.

Dissolve the carmine in the ammonia in a mortar. Pour on the water. Mix
thoroughly and filter.

  (2) Gelatine              10 grms.
      Distilled water       50 c.c.

Allow it to stand in the cold water until the water is absorbed and the
gelatine has become soft.

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.

*Blue injection mass.*--To the gelatine mass (2) prepared as above, and
liquefied by heat, add instead of carmine

  Soluble Prussian blue                 5 grms.
  Distilled water                      60 c.c.

Every trace of alkali must be kept away from 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.

*Green injection mass.* Robin’s formula (modified).

  (1) Arseniate of soda (sat. sol.)    80 c.c.
      Glycerine                        50  "

  (2) Sulphate of copper (sat. sol.)   40  "
      Glycerine                        50  "

Mix and add one part to three parts of the gelatine mass made as for
the red and blue injections.

*Method of injection.*--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 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.

By far the most effective apparatus for injecting is the modification
of Ludwig’s constant pressure apparatus devised by Fearnley.[2]
Although the apparatus appears complicated, the various parts are
easily obtained and it would be easy to improvise a substitute for the
water bath.

[2] “Practical Histology,” (Macmillan & Co.).

The apparatus which is shewn in figures 10 and 11 consists of a
bath deep enough to contain the animal, and a vessel containing the
injection fluid. The bath is kept at a temperature of about 110° by an
ordinary Bunsen burner. A large Wolff’s bottle (20–40 oz.) with three
necks, is fitted with three india-rubber stoppers perforated by glass
tubes. Through the central stopper a glass tube 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 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 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 11 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.

[Illustration: Fig. 10.--Fearnley’s arrangement for injecting blood
vessels. (Reproduced by permission of Messrs. Macmillan, from
Fearnley’s _Practical Histology_).]

[Illustration: Fig. 11.--Scheme shewing distribution of pressure in
Fearnley’s Injection Apparatus (from Fearnley’s _Practical Histology_).]

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.

If an isolated organ is to be injected, a cannula of glass or brass
should first be inserted into the artery and securely tied in position.
The organs, if cold, must be soaked in water at 120° F. for about half
an hour and then transferred to a water bath.

In the case of injecting an entire animal, such as a rabbit, rat, or
guinea pig, the injection is best 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 registers 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.



CHAPTER IX.

DIRECTIONS FOR PREPARING INDIVIDUAL TISSUES.


*Normal histology.*--It cannot be too strongly impressed on the
beginner that a thorough mastery of the normal appearances of tissues
and organs is absolutely necessary before attempting to make an
accurate study of morbid changes in them. He should not be satisfied
with examining one specimen of an organ but as many as he conveniently
can, in order to be fully acquainted with the many deviations from
normal which may exist without actual disease. He should therefore
obtain several animals, such as small dogs, cats, rabbits, frogs, &c.,
and remove their organs with all care, and harden them in the various
appropriate fluids. He should also obtain specimens of normal human
organs from the post-mortem room. Many normal tissues (skin, muscle,
tendon, bone, &c.), can also be prepared from a limb amputated for an
accident to a healthy patient. By preparing 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.

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.

The first part of these directions will refer to the preparation of
normal tissues, the second part to morbid histology.

*Blood.*--For special methods of examination see Chapter VII.

*Blood crystals--Hæmoglobin crystals*, 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.

*Hæmatin crystals.*--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.

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.

*Hæmatoidin crystals.*--Obtained from the site of a bruise, or an old
hæmorrhage, _e.g._, a cerebral apoplexy or a hæmatocele.

*Simple squamous epithelium.*--(_Endothelium_). 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. 82) so as
to reveal the outlines of the cells. (2) Stain other specimens with
hæmatoxyline or alum carmine to reveal the nuclei.

*Stratified squamous epithelium.*--Specimens from skin of various
parts, finger, groin, lip, tongue should be prepared. Harden in
Müller’s fluid.

*Transitional epithelium.*--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.

*Simple columnar epithelium.*--Occurs in many parts. It may be studied
in the salivary ducts, the intestine, kidney, &c., of any mammal.

*Goblet-cells.*--Seen abundantly among the columnar cells of the
intestinal glands, and in the mucous glands of the mouth and of the
cervix uteri.

*Stratified columnar epithelium.*--Occurs only in the urethra. Harden
the penis of a cat in Müller’s fluid, and cut transverse sections.

*Ciliated epithelium.*--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 put into hardening
fluid immediately after removal.

Stain all sections of epithelium in picrocarmine, and in eosine and
hæmatoxyline.

*Ordinary areolar tissue.*--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.

*Elastic tissue.*--May also be studied in most sections of skin. If
the ligamentum nuchæ of a large quadruped (horse, bullock), &c., is
available it yields the best specimens, or the human ligamenta subflava
may be examined. Pin a piece out on a piece of wood or wax. Harden
in Müller’s fluid. Stain in picrocarmine. Both sections and teased
specimens should be prepared.

*Tendon.*--Readily obtained from an amputated limb. Harden in Müller’s
fluid. Make transverse and longitudinal sections. Stain with eosine and
hæmatoxyline.

A preparation should also be made by teasing a little of the fresh
tendon in normal salt solution, and staining with picrocarmine.

*Retiform* or *lymphadenoid tissue*.--Seen in lymphatic glands and
in the lymphoid follicles scattered along the sub-mucous coat of the
alimentary canal.

Prepare sections in the ordinary way. Stain in eosine and hæmatoxyline
or in picrocarmine.

Some sections should also be prepared by pencilling (_i.e._, 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.

*Fat.*--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.

*Pigment cells.*--Branched cells are best studied in the living foot
of the frog, where amœboid movements may be seen in them when the
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.

*Hyaline cartilage.*--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.

*Elastic cartilage.*--Prepared from the epiglottis, or from the
cartilages of the ear, _e.g._, of a cat. Harden in spirit. Stain in
picrocarmine or in dilute fuchsin.

*White fibro-cartilage.*--Obtained from intervertebral disc. Prepare
and stain as for hyaline cartilage.

*Bone*:--

*Unsoftened Bone.*--Cut as thin a section as 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.

*Softened bone.*--Specimens may be obtained from an amputated limb or
from the femur of a cat.

Specimens should be decalcified in chromic and nitric fluid, and the
hardening completed in spirit. In studying the process of ossification,
_e.g._, 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.

Very beautiful double staining effects may be obtained with either
picrocarmine, or eosine and hæmatoxyline, and with eosine and methyl
violet.

*Bone marrow.*--To obtain good sections of 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 116.

*Tooth.*--Best cut _in situ_ 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.

*Developing tooth.*--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.

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.

*Striped muscle.*--Should be studied in various animals.

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.

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.

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 92.

*Heart muscle.*--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.

*Unstriped muscle* 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.

*Nerves.*--The special methods for staining nerve tissues are detailed
in Chapter VI. The student must remember that the ordinary staining
methods are also applicable to nervous tissues.

*Nerve terminations*:--

*Meissner’s corpuscles.*--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.

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.

*Pacini’s corpuscles.*--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.

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. 82).

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.

*Arteries.*--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.

*Veins.*--Remove, harden, and stain in the same way.

*Capillaries.*--May be very well seen in the foot of the frog.

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 1/2 inch objective. The foot must be brushed from time
to time with normal salt solution to keep it moist. The movement of
blood in the capillaries, &c., can then be studied for an hour or two.
After death the mesentery should be spread out on a piece of wood, and
hardened for a few days in Müller’s fluid.

Stain with eosine and hæmatoxyline.

*Lymphatics.*--Commencement of lymphatics in serous membrane. Stain a
piece of cat’s omentum in nitrate of silver (p. 82) for some minutes.
After washing keep in glycerine for about a week and then stain in
hæmatoxyline and mount in Farrant’s medium.

*Lymphatic glands.*--The lymphatic glands of the neck of the cat may
be used. Harden in Müller’s fluid. Stain in picrocarmine, eosine and
hæmatoxyline.

*Skin and sweat glands.*--Sections should be made from pieces taken
(_a_) from the sole, (_b_) from the skin of the body, (_c_) from the
axilla of an adult to study the pigment. Harden in Müller’s fluid.
Stain in picrocarmine or eosine and hæmatoxyline.

*Hairs and sebaceous glands.*--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.

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. 27).

*Brain and spinal cord.*--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.

The best staining reagents to employ are eosine and hæmatoxyline, alum
carmine or borax carmine, aniline blue-black, &c. Staining methods, see
Chapter VI.

*Eye.*--Harden the eye of a recently killed bullock, cat, or other
animal in formal (p. 23), 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 (_e.g._, a cod)
in a 1/40 per cent. aqueous solution of eosine. Wash the eosine off the
slide with 1/2 per cent. acetic acid, and mount in Farrant’s solution.

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.

Mount some specimens unstained. Stain others with the ordinary stains.

*Internal ear.*--Decalcifying the temporal bone of a cat, dog,
guinea pig, &c., in chromic and nitric fluid. As soon as the bone is
decalcified complete the hardening of the soft parts in methylated
spirit, embed in celloidin, and cut 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.

The semi-circular canals will be most readily studied in the temporal
bone of fishes, or of birds, _e.g._, the common fowl. They also must be
cut in celloidin, and stained in the ordinary way.

*Nose and olfactory epithelium.*--It is difficult to obtain specimens
from the human subject, but very satisfactory preparations may be made
from the dog, or more conveniently in a new born puppy where the bones
are still cartilaginous. Harden the latter in Müller’s fluid, decalcify
adult specimens in chromic and nitric fluid. Specimens of ciliated
epithelium, &c., will be obtained from the lower part, and of the
special olfactory epithelium from the upper part. Stain in eosine and
hæmatoxyline.

*Lungs.*--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 attached to keep the organ
submerged. Harden for about six weeks, and then make sections of the
various parts.

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.

Beautiful casts of the alveoli, &c., may be obtained by placing a cat’s
or human lung under the receiver of an air-pump, and when the air is
completely exhausted, injecting fusible metal into the bronchus. The
lung tissue is then removed by corrosion or by maceration. Portions
of the casts should be removed, fixed in a glass cell with a spot of
Canada balsam, and examined by reflected light.

*Thyroid gland.*--Best obtained from a young subject either human or an
animal.

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.

*Thymus.*--Remove from a fœtus or a very young animal, and prepare in
the usual way.

*Tongue.*--That of the cat or rabbit serves very well.

Ordinary transverse sections should be made, and also sections through
the circumvallate papillæ in order to study the “taste buds.”

*Salivary glands.*--Those of a cat or dog do very well.

Sections should be made from each of the three glands.

*Stomach.*--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.

Sections should be made (_a_) longitudinally through the cardiac end to
show the transition from the œsophageal to the gastric mucous membrane,
(_b_) from a portion of the greater curvature, (_c_) longitudinally
through the pyloric valve.

Eosine and hæmatoxyline form the best stain for the alimentary canal.

*Intestine.*--Prepare in the same way as the stomach. Make sections
from (_a_) the upper part of the duodenum to show Brunner’s glands,
(_b_) the ileum, (_c_) a Peyer’s patch, (_d_) the vermiform appendix,
(_e_) the colon.

*Liver.*--Make an injection of one specimen with carmine and gelatin
(p. 120). Harden in methylated spirit. Others should be hardened in
Müller’s fluid and stained in the usual way.

*Kidney, supra-renal, and pancreas.*--Same preparation as for liver.

*Spleen.*--Harden in Müller’s fluid.

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.

*Bladder.*--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.

*Penis* and *testis*.--Readily obtained from dog, cat, or rat.

Stain with eosine and hæmatoxyline.

*Uterus, ovaries, and Fallopian tubes.*--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.

Stain with eosine and hæmatoxyline.

*Embryological specimens.*--For systematic work special manuals should
be consulted.

Specimens should be hardened in osmic acid or in Müller’s fluid, and
cut in celloidin, or paraffin.

*Cloudy swelling.*--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.

*Fatty degeneration.*--Prepare from patients who have died of
exhausting diseases, phosphorus poisoning, &c.

Stain in osmic acid. Mount in Farrant’s medium and keep in the dark.

*Mucoid degeneration.*--Study in goblet cells of normal intestine or of
ovarian cysts. There are no satisfactory selective stains for mucin.

*Colloid degeneration.*--Occurs in the thyroid gland, in the tubules of
the kidney in many diseases, and the prostate of the old.

Stain in safranine.

*Waxy or lardaceous degeneration.*--Best studied in liver, spleen, or
kidneys. It should be searched for in persons who have died from a long
illness, accompanied by suppuration, _e.g._, 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.

*Hyaline degeneration.*--Seen in the arterioles of the spleen in some
cases of typhoid and diphtheria. The ordinary staining methods must be
used.

*Calcareous degeneration.*--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 section unstained and
examine if possible with the polariscope. Stain others in safranine.

*Pigmentary degeneration.*--May be studied in brown atrophy of
heart, nutmeg liver, &c. It is also seen well in spinal and cerebral
nerve cells of the aged. Harden in Müller’s fluid and mount sections
unstained.

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.

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. 23).

*Tumours.*--Müller’s fluid should be employed, unless a more rapid
agent is required.

Methylated spirit may be used in the case of epithelioma, adenoma,
&c., but for sarcoma, myxoma, tumours containing cysts or much blood,
Müller’s fluid yields by far the best results.



BOOKS OF REFERENCE.


    Methods in Microscopical Anatomy--_Whitman_.

    Practical Pathology--_Woodhead_.

    Textbook of Bacteriology--_Crookshank_.

    Manual for Physiological Laboratory--_Harris_ and _Power_.

    Practical Histology--_Fearnley_.

    Practical Pathology and Histology--_Gibbes_.

    Journal of Microscopical Society.

    Methods and Formulæ--_Squire_.

    The Human Brain--_Goodall_.

    Practical Bacteriology--_Kanthack_ and _Drysdale_.

    Methods of Microscopical Research--_Cole_.



INDEX.


  Abbe’s condenser, 11

  Absolute alcohol, 21

  Acetate of copper, 89

  Air bubbles, removal of, 56

  Alum carmine, 76
    hæmatoxyline, 68, 70

  Amyloid degeneration, 149

  Aniline blue-black, 94
    oil, 102, 108
    oil water, 107

  Apparatus required, 1

  Areolar tissue, 133


  Bacteria, stains for, 103

  Balsam bottle, 58

  Barrett’s logwood solution, 69

  Bevan Lewis’s method, 94

  Bichromate of potassium, 17

  Bismarck brown, 104

  Bleaching solution, 27

  Blood crystals, 130

  Blood, methods of examining, 113

  Blood-vessels, injection of, 120

  Blue injection mass, 121

  Bone marrow, 136

  Bone, sections of, 136

  Borax carmine, 75

  Brain, methods of staining, 94

  Buckley’s modification of Golgi’s method, 99


  Calcareous degeneration, 149

  Canada balsam solution, 61

  Carbolic acid, 23

  Carmine, 74
    injection mass, 120

  Cathcart microtome, 39

  Cathcart-Frazer microtome, 42

  Cedar oil, 8, 63

  Celloidin, 30

  Cementing cover-glasses, 65

  Chloral hæmatoxyline, 92

  Chloride of gold, 82, 94

  Chromic and nitric decalcifying fluid, 26

  Ciliated epithelium, 132

  Circulation in frog’s foot, 141

  Clarifying sections, 63

  Clearing agents, 63

  Cloudy swelling, 148

  Clove oil, 63

  Colloid degeneration, 149

  Columnar epithelium, 132

  Corrosive sublimate hardening, 23
    staining, 100

  Cover-glasses, cleansing of, 57

  Cover-glass preparations, 111


  Decalcifying solutions, 26

  Dehydration, 63


  Eau de Javelle, 27

  Ebner’s solution, 27

  Ehrlich-Biondi fluid, 85

  Ehrlich’s hæmatoxyline, 70
    method of fixing blood-films, 116
    method for tubercle bacilli, 110

  Ehrlich-Gram method for staining bacteria, 108

  Elastic cartilage, 135
    tissue, 133

  Embedding methods, 29

  Endothelium, 131

  Eosine, 72

  Eosine and hæmatoxyline, 73

  Epithelial cement, 131

  Ether spray microtome, 39


  Fæces, staining for bacilli, 112

  Farrant’s solution, 59

  Fat, removal from sections, 59
    staining of, 134

  Fatty degeneration, 148

  Fearnley’s injection apparatus, 123

  Ferrier’s fuchsine solution, 117

  Flemming’s solution, 25

  Flotation of sections, 55

  Folded sections, treatment of, 58

  Formal, 23

  Fresh sections, 52

  Fuchsine, 104


  Gentian violet, 104

  Gibbes’ stain for tubercle bacilli, 111

  Gold chloride, 82, 94

  Golgi’s silver method, 96
    sublimate method, 99

  Gram’s iodide solution, 105
    method for staining bacteria, 107

  Green injection mass, 122

  Gum, 29


  Hæmatin crystals, 131

  Hæmatoidin, 131

  Hæmatoxyline, Ehrlich’s, 70
    Kleinenberg’s, 70
    Schuchardt’s, 68
    Sihler’s, 92
    Weigert’s, 88

  Hæmoglobin crystals, 130

  Hardening processes, 15

  Hyaline cartilage, 135
    degeneration, 149


  Ice freezing microtome, 46

  Immersion lenses, 8

  Injection of blood-vessels, 120
    pulmonary alveoli, 145

  Internal ear, 143

  Intestines, 146

  Iodine solution, 105


  Jung’s ether spray microtome, 45


  Kleinenberg’s hæmatoxyline, 70


  Lardaceous degeneration, 149

  Lithio-picrocarmine, 79

  Lithium carmine, 74

  Liver, 147

  Löffler’s methyl blue, 104

  Logwood, 68

  Lymphoid tissue, 134


  Marchi’s fluid, 24

  Marrow, 136

  Methyl blue, 101, 104
    violet, 83

  Methylated spirit, 19

  Micro-organisms, stains for, 103

  Microscope, 6

  Microtome, Becker, 49
    Cambridge rocking, 49
    Cathcart, 39
    Cathcart-Frazer, 42
    Jung, 45
    Schanze, 47
    Swift’s, 49
    Williams’, 46

  Mould for paraffin embedding, 36

  Mounting methods, 55

  Mucoid degeneration, 148

  Muscle, 138

  Muir’s method of hardening films, 116

  Müller’s fluid, 17
    and formal, 20
    and spirit, 20


  Neelsen’s stain for tubercle bacilli, 110

  Nerve cells, stains for, 94
    endings, 139
    fibres, stains for, 87

  Nissl’s aniline method, 101

  Nitrate of silver, 82

  Nitric acid as hardening agent, 25
    decalcifying agent, 26
    decolourising agent, 110

  Normal salt solution, 53

  Nose piece, 9


  Objectives, 7

  Oil of bergamot, 63
    cedar, 8, 63
    cloves, 63
    origanum, 63

  Osmic acid as hardening agent, 21
    staining reagent, 81


  Pal’s method, 86
    solution, 90

  Paraffin, 34

  Picrocarmine, 78

  Pigment cells, 134

  Pigmentary degeneration, 150

  Plane iron microtome knife, 42


  Rapid hardening, 150

  Retina, 143


  Safranine, 85

  Salivary glands, 146

  Schäfer-Pal method, 91

  Schanze microtome, 47

  Schuchardt’s hæmatoxyline, 68

  Sihler’s chloral hæmatoxyline, 92

  Silver nitrate, stain for nerve cells, 96
    epithelial cement, 131

  Skin, 142

  Spinal cord, 86, 142

  Spleen, 147

  Sputum, staining of, 111

  Squamous epithelium, 131

  Staining methods, 67 _seq._

  Stomach, 146

  Striped muscle, 138

  Sulphuric acid, 105

  Sweat glands, 142


  Tendon, 133

  Testing a microscope, 13

  Thymus gland, 145

  Thyroid gland, 145

  Toison’s fluid, 118

  Tooth, sections of, 137

  Transitional epithelium, 132

  Tubercle bacillus, stains for, 110

  Tumours, hardening of, 150


  Unstriped muscle, 139

  Urine, examination for bacilli, 112

  Uterus, 148


  Von Ebner’s decalcifying solution, 27


  Waxy degeneration, 149

  Weigert’s hæmatoxyline method, 88
    method for staining bacteria, 106

  Williams’ ice freezing microtome, 46

  Woodhead’s injection mass, 120


  Xylol, 61


  Ziehl’s carbol-fuchsine, 105





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