PRACTICAL
HISTOLOGY AND PATHOLOGY
BY
HENEAGE GIBBES, M.B.
LONDON:
fl. K. LEWIS, 136 GOWER STREET, W.C.
1880.
PREFACE.
THE object of this small work is to lay before the
practitioner and student of medicine, a few concise
and simple methods, by which the various tissues of
the body may be prepared for examination with the
microscope.
I do not claim any originality in these methods, but
I recommend them from my own personal experience
as the best, easiest, and cheapest to carry out.
The use of dyes for staining the tissues is a com-
paratively new branch of the science of Histology, and
its value is being proved every day.
I have been engaged in experiments with all the
various colouring agents for a long time, and in trying
their effect in double and treble staining, and I have
given the result of those which have proved most suc-
cessful hitherto. I have also added a list of the aniline
dyes, those soluble in water, and those soluble in spirit,
which will be found very useful by anyone wishing to
make experiments in staining.
iv Preface.
Briefly, but sincerely, I offer my best thanks to
Dr. Klein, F.E.S., for the assistance which he has at
all times freely and generously given me.
H. G.
94 Gower Street.
October 1, 1880.
TABLE OF CONTENTS,
CHAPTEE I.
INTRODUCTION.
PAGE
The Microscope ... . .4
Achromatic Condenser . .
On large stands for high powers . . . .
On the Binocular Microscope . . 9
CHAPTEE II.
ON PREPARING TISSUES FOR EXAMINATION.
Chromic Acid Mixture . . . . . .11
Muller's Fluid .12
Dilute Spirit ... . , 13
Bichromate of Potash ..... 13
Chromate of Ammonia . . . . . .13
Chloride of Gold ....... 14
Picric Acid ...... .15
CHAPTEE III.
ON CUTTING SECTIONS.
Freezing Microtome ...... 18
To prepare material for freezing . . . . .19
To make Mucilage . . . . . . 20
Cutting the Sections . . . . . • .20
vi Contents.
CHAPTER IV.
ON STAINING.
PAGE
List of Staining Agents ..... 22
Logwood Stain ... . .23
On Staining Sections with Logwood that have been prepared
with Chromic Acid .... 24
Carmine - .24
Indigo-carmine • • 25
Purpurin . 25
Anthra-purpurin • 26
Eosin . • 26
Picro-carmine • 27
Anilin Dyes
Anilin Dyes soluble in Spirit
CHAPTEE V.
ON DOUBLE STAINING.
Picro-carmine and Logwood .
Carmine and Indigo -carmine . 37
Treble Staining . - 39
Chloride of Gold and Anilines • 42
CHAPTER VI.
ON MOUNTING.
Slides .... 44
Cover Glasses . 44
On measuring Cover Glasses • 45
On cleaning Cover Glasses 45
Contents. vii
PAGE
Mounting Fluids ... . 46
To mount in Canada Balsam . . . . 48
On breaking down old preparations . . -52
Mounting Large Sections ... 53
Thin Slides - . . 55
CHAPTEE VII.
Method of obtaining Animal Tissues for Examination . 56
Dissection of Frog • . 58
Dissection of Newt ...... 59
PRACTICAL HISTOLOGY.
Blood . ..... 60
Epithelium . • «... 62
Cartilage . ... 69
Bone . «... 71
Muscular Tissue . . . . . -72
Nervous Structures 75
Blood Vessels . ..... 79
Salivary Glands, Pancreas .... 81
Teeth . 82
Alimentary Canal . ... 82
Liver . • . . . . . . .84
kung . 86
Kidney . .... 86
Genital Organs, Male ..... 33
Genital Organs, Female .... -90
Spermatozoa • • • . . 90
Special Senses • . . . . . . .93
Nasal Organ . . -95
Eye ....... .95
viii Contents.
PRACTICAL PATHOLOGY.
PAGE
On preparing and mounting Pathological Specimens . . 99
To make permanent preparations of a Cancer in a short time • 99
On Double and Treble Staining Morbid Growths • • 101
Large Sections of Pathological Specimens . . • 101
Amyloid Degeneration . . . 102
Hydatids • . 102
Short history of the manner in which a portion of the Morbid
Growth is prepared by the Chromic Acid method • 103
PEACTICAL HISTOLOGY AND PATHOLOGY.
CHAPTEE I.
INTRODUCTION.
HISTOLOGY or the minute anatomy of healthy tissue as
revealed by the microscope, has made vast strides in
the last few years, and now forms a most important
part of Medical education ; a thorough knowledge of
the normal structure of the animal tissues being ab-
solutely necessary for the appreciation of pathological
change.
This subject is now taught at the Medical schools,
and there is no excuse for a student who has finished
his curriculum without some knowledge of practical
histology, and of the minute anatomy of the human,
body.
There are many, however, who qualified some years
ago when little attention was paid to this subject, who
have consequently no practical knowledge of the methods
required to prepare any morbid growth they may meet
in their professional career, or how to set about a micro-
scopical examination of the same.
With a view to help those who have not been able
to get the necessary knowledge during their student
career, and also those students who wish to form a
Laboratory at home, the present work has been written,
Introduction.
giving only the ordinary methods used by the author in
his own Laboratory.
When all these methods have been thoroughly worked
out, the student will find himself competent to try any
of the various processes mentioned in larger works, and
to judge of their utility.
Many men on reading the different hardening, cut-
ting, staining, and mounting processes which any tissue
has to undergo before it can be examined with the
microscope, will be inclined to think it very tedious
work. It is, however, a mere matter of routine, and
when once this routine is established, the whole thing
is comparatively simple. It takes very little time to
change the hardening fluid, and if the student gets into
the habit of looking over the bottles on the shelf every
morning, where he keeps those tissues in the process of
hardening, a glance at the labels will show those re-
quiring a change. When the sections are mounted and
examined under the microscope, he will find himself
amply repaid for all his trouble if he has faithfully
carried out the different processes in every detail.
It is always better to have one or two shelves devoted
to those preparations which require changing ; and those
such as chromic acid, which require fresh fluid often,
should be kept by themselves. Each bottle should be
labelled, and the tissue, date, and hardening fluid,
clearly written on the label. Every morning this
shelf should be examined, and those requiring it,
changed; the date being each time written on the
label, so that it may be seen at a glance how long the
tissue has been in the fluid, and whether the hardening
Introduction.
agent ought to be renewed. Miiller's fluid, and bichro-
mate of potash preparations, may be placed by them-
selves, and need only be looked at occasionally.
A large outlay is not required for a course of Histo-
logical investigation, and the following list will show all
those articles, re-agents, &c., which the student will find
absolutely necessary.
1. Microscope. 1 Eye-piece. 2 Object glasses.
2. | gross ground edge, 3+1 slides.
3. 1 oz. No. 1, f square cover glasses.
4. 1 oz. No. 1 „ „ „
5. 1 hollow ground razor.
6. Needles in handles.
7. 2 pair sharp pointed forceps.
8. 1 pair broad pointed forceps, not roughed, for taking up clean
cover glasses.
9. Copper lifter.
EE-AGENTS.
f per cent. Salt solution.
\ ,, Solution Nitrate of Silver.
I „ „ Chloride of Gold.
5 ,, ,, Bicarbonate of Soda.
Glycerine "\
Canada Balaam > In drop bottles.
Dammar Yarnish J
A Bottle of Hollis' Glue.
A Williams' Microtome.
Watch Glasses.
Glass Capsules.
Dissecting Case.
Curved Scissors.
B2
The Microscope.
THE MICKOSCOPE.
THE most expensive as well as the most important arti-
cle required is the microscope, and a good one should
be procured.
In purchasing a microscope, it is necessary to goto
an optician who makes his own instruments to get one
that is worth anything, as a large number are made by
wholesale manufacturers and sold to various traders
who put their names on them and sell them as their
own make. It is an easy matter to find out a bona
fide maker, as there are very few of them, and it is their
interest to sell a good instrument. A microscope
such as that required by anyone beginning a course of
histology, can be procured for £5 5s., and nothing worth
having can be bought at a lower price. If the instru-
ment be procured from a bona fide maker it will last a
lifetime with ordinary care.
The compound microscope consists of the stand, eye-
piece, and object glasses. The stand should be a tripod
having a stage of blackened glass, and a draw-tube
lined with cloth. The tripod foot gives perfect steadi-
ness, and for this reason the Continental models with
horse- shoe foot have been given up by many of the best
microscopists.
The price of these stands, with two object glasses,
ranges from £5 5s. to £6 12s. 6d.
The student should ascertain by looking down the
tube with the eye- piece removed, whether the hole in
the stage is concentric with the tube, and then try the
different holes in the diaphragm in the same way. The
The Microscope.
diaphragm is an important part of the stand, as it is
very often necessary to cut off some of the rays of
light to get the best effect with high powers ; and for
this purpose the tubular diaphragm which is slipped
into a fitting under the stage, is the best; it has a
very small hole, and can be removed altogether when
using a low power ; a good plan is to have no fixed dia-
phragm at all, but a small piece of metal with a milli-
metre hole in the centre, made so that it can be slipped
into the hole in the stage from above, where it will lay
flush with the glass plate. It must be carefully cen-
tred with the tube.
Having selected a maker, the student should get him
to explain the working of the different parts, as all that
is necessary for a beginner can be learnt in ten minutes
in that way.
This small student's stand is all that will be required
by the student for a long time, as with it high powers
can be used and whatever work may be done in the
future, it will always be the working stand.
Good students' microscopes are made by Mr. Crouch,
66 Barbican; Messrs. Swift and Son, University Street.
The Object Glass. The object glass or objective, is
the most important part of the microscope, and it is
necessary to have good glasses to do satisfactory work.
The most useful for the student are the f or |- for a
low power, and the J, 1, or ^ for a high power ; with
a f and ^ the student can do all the requisite work for
a long time, and then with the addition of a J, and oil
immersion ^ he would be set up for life.
The student should get some good microscopist to
test the object glasses for him before purchasing them,
The Microscope.
and he should see that they are tested on some histo-
logical object, and not on diatoms, as the wide angle
necessary for resolving test diatomaceae are the reverse
of useful to the young Histologist. The most important
glass for the student is the high power, and it is neces-
sary that this should be a good one.
High Powers. The best high powers are made by
Messrs. Powell and Lealand, they are of course expen-
sive but pay well in the end; they are made with
correction collars and are more adapted to large stands
with mechanical stage. Zeiss' E = j- and F = ^ are
very good glasses, and well adapted to the students'
stand and are not expensive ; they have no correction
collar, and the cover glasses should be measured to get
the best effect ; -006 will do very well for D and E, and
•004 for F, the same applies to Mr. Crouch's ± which is
corrected to a cover glass of -006.
Oil Immersion Lenses. These glasses are taking the
place of water immersion high powers in Histologi-
cal research, as they have no correction for thickness of
cover glass, and are consequently much easier to use,
the only drawback is that the essential oil used will
dissolve Canada balsam, Dammar varnish, and many of
the other sealing fluids, and it is necessary to cover them
with Hollis' glue which is not acted on by cedar oil.
These glasses were first made by Zeiss of Jena, and
since by Messrs. Powell and Lealand. The first glass
made by Zeiss was the J-, he then brought out a T^-
and afterwards a J^, of these the T^ is unquestionably
the best glass. Messrs. Powell and Lealand have made
_3^? _!_, _i_y and -^3- on this principle, and have succeeded
in removing a great objection to oil immersion lenses ;
The Microscope.
they have made their glasses perfectly homogeneous,
that is, they require no change of the oil when using
oblique light on an object, and no correction with the
draw tube is necessary when using a very thin cover.
With Zeiss' -^ it is necessary to draw out the tube two
inches with a '004 cover glass, and for central light a
mixture of fennel and olive oils is required, which must
be changed to cedar oil for oblique light.
With Messrs. Powell and Lealand's glasses cedar oil
is used indifferently for central or oblique light, and no
correction is necessary for a thick or thin cover glass.
In using oil immersions, all that is required is to
place a very small drop of oil on the front lens, screw
the glass into the body, and lower it on to the slide until
contact is made, which can be seen by bringing the eye
to a level with the surface of the slide. The glass is
then focussed by the fine adjustment until the object is
seen sharply defined. With these glasses the best effect
is obtained the moment the glass is in focus, and they
have an enormous superiority over a dry glass of the
same power in sharpness of definition and brilliancy.
All slides intended for use with high powers should be
sealed with Hollis' glue, and then the cedar oil left on
the cover glass can be wiped off without the least dan-
ger to the preparation.
Eye- Pieces. Students' microscopes are generally sold
with two eye-pieces, nos. 1 and 3, or A and C, but the
no. 1 or A is the only one required, as no good result is
obtained by using an eye -piece with a higher magnify-
ing power, the higher eye-piece only magnifying the
image seen with the object glass.
Achromatic Oondensor.
Higher eye-pieces are, however, useful in testing an
object glass, as very many of those sold, while giving
fairly good results with a no. 1 eye-piece, become blurred
and indistinct when a no. 3 or 4 is used.
Illumination. Daylight is the best light to use for
Histological work, and in the summer time there is
generally enough light for ordinary work. In the win-
ter, however, a lamp is often required even in the day-
time, as there are many days when sufficient light
cannot be obtained.
It is necessary, therefore, to be provided with a lamp
burning either gas or parafine oil. The best gas lamp
is made by Baker, it is a modification of the Highley
microscopic lamp and is fitted with a Sugg's burner, it
will be found very useful. An ordinary parafine lamp
which can be purchased for 2s., with a f inch flat wick,
will be found all that is necessary by those who do not
wish to get the more expensive gas lamp. It has also
this advantage, the wick being flat the edge of the flame
can be turned to the microscope, giving an intensity of
light very useful in the investigation of fine structures
under very high powers.
ACHKOMATIC CONDENSER.
A SMALL condenser made to go into the fitting under the
stage will be found very useful, but for high power work
with a large stand a wide angled condenser is necessary,
having a series of stops for oblique light, and a graduated
diaphragm by which the rays of light can be gradually
cut off until the best definition is obtained.
On Large Stands for High Powers. 9
STAND CONDENSEK.
It is better to use the direct light of the lamp without
the intervention of a stand condenser, as by this instru-
ment the light is diffused and the sharpness of definition
impaired. A stand condenser is required to throw a
light on opaque preparations, but as these are not often
used by the student of Histology, it is not required by a
beginner.
ON LARGE STANDS FOR HIGH POWERS.
THE advanced student will require a Stand with me-
chanical stage and sub- stage when he makes investi-
gations into minute structures with high powers, and
for this work it is most important that the mechanism
of the Stand should be of the very best description. To
get this he must go to the best makers, and of course be
prepared to pay a high price.
The best Stands are made by Messrs. Powell and
Lealand, and Messrs. Boss and Co.
The author has used a No. 2 stand of Messrs. Powell
and Lealand for a long time, and found it all that
could be desired.
ON THE BINOCULAR MICROSCOPE.
THE binocular microscope has not met with much favor
from Histologists as yet, from the fact that no power
above a % inch could be used without a special stand or
apparatus costing a large sum. As, however, it has
been shown by the author* that the binocular can be
* See Quarterly Journal of Microscopical Science for July, 1880.
10 On the Binocular Microscope.
used with the T^ oil immersion, the student should try
it, and see what a different view it gives of various
structures and their relation to one another. Any or-
dinary stand made on the Jackson -Lister model will do,
so that the body can be brought almost into contact
with the stage. A Zeiss' D can be made to work per-
fectly well by fitting the front part into an adapter, so
as to bring the lenses close to the prism. The relief
to the eyes is very great.
For a higher power Messrs. Powell and Lealandmake
their T^ oil immersion with a screw cut on the outside,
so that the front part containing the lens can be screwed
into an adapter, which they supply with the glass. By
this means perfect stereoscopic effect is obtained, and
the observer is enabled to realise the precise relations of
the different structures he is looking at to one another.
CHAPTEE II.
ON PEEPABING TISSUES FOE EXAMINATION.
THE most essential point in microscopic investigation is
the proper hardening of the material to be examined,
and this must be done gradually, as if any tissue is
placed in a strong solution, the elements of which
it is composed at once shrink, and it is impossible to
form any correct idea of their nature.
1. Chromic Acid Mixture. The most useful harden-
ing agent is a mixture of chromic acid and spirit. Make
a solution of chromic acid in water 15 grains to the
pint, this is about ^ per cent. Take of this 2 parts ;
methylated spirit 1 part.
The material must be cut into small pieces about half
an inch square, and a large quantity of fluid used, a wide
mouthed stoppered bottle holding from 6 — 10 ozs., ac-
cording to the quantity of material, is best ; change the
fluid at the end of 24 hours, and again every third day,
and the material will be hardened in from 8 — 12 days,
this can be easily proved by taking out a piece and feel-
ing it. If allowed to remain too long it gets brittle.
When it is found to be moderately hard, usually after
about 9 — 10 days, pour off the chromic acid mixture,
and wash well, replace it by dilute spirit made thus : —
Take
Methylated Spirit 2 parts, and
Water 1 part.
12 On Preparing Tissues for Examination.
Let the material remain in this for from 24 — 36 hours,
never longer than three days, and then replace it by
pure methylated spirit, it may remain in this for an in-
definite time, but it will often be found that the spirit
becomes cloudy and full of deposits in a few days ; in
this case it is only necessary to change the spirit until
it remains clear.
A large quantity of i per cent, solution of chromic
acid should be kept on hand, and it should be mixed
with the spirit as required, it will be found the most
useful of all the hardening agents, if it is changed at
the proper times, and it should not be used stronger
than is given above, it will even be found beneficial to
use it weaker in some cases.
2. Muller's Fluid is a good hardening mixture, but
requires a much longer time, taking weeks to do what
the chromic acid mixture will do in days. It is made
thus : —
Take
Potass. Bichrom. 2 parts.
Sodse Sulph. 1 part.
Water 100 parts.
The advantage of this mixture is that larger pieces
can be hardened in it, and it does not require
changing after the first week or two, but it will take
from 5 — 7 weeks to harden anything according to
its size. The material, when sufficiently hardened,
should be well washed and then placed in dilute spirit
in the same manner as recommended after hardening
in the chromic acid mixture.
On Preparing Tissues for Examination. 13
3. Dilute Spirit. Many tissues can be hardened in
spirit alone if they are placed in dilute spirit at first,
so that the elements of which they are composed are
not shrunk. This process is also used after hardening
by any of the others.
Dilute spirit is made by adding 1 part of water to
2 parts of methylated spirit.
The material to be hardened should not be left in
this mixture more than from 24 — 48 hours.
It is then transferred to pure methylated spirit.
4. Bichromate of Potash. Make a 2 per cent, solution
and keep it on hand, as it is very useful for many tis-
sues that require slow hardening. A solution can be
made much more quickly with warm water than cold.
This solution is also very useful to place portions of
morbid material in, on their removal from the body in
the post-mortem room, they can afterwards be trans-
ferred to the chromic acid mixture for more rapid hard-
ening. This solution takes from three to seven weeks
to harden according to the size of the specimen, and the
frequency with which the solution is changed.
5. Bichromate of Ammonia. A 2 per cent, solution is
used in precisely the same manner as the former, and
is applicable to the same tissues.
6. Chr ornate of Ammonia. Make a 5 per cent, solu-
tion, that is 1 oz. of the salt to 20 oz. of water, and
filter. Keep it in a stoppered bottle. When this harden-
ing agent is used for fresh tissue, such as mesentery, a
small quantity is placed in a glass vessel and the tissue
immersed in it for 24 hours, it is then washed until no
more colour comes away and mounted in glycerine.
14 On Preparing Tissues for Examination.
For other tissues it is necessary that the material
should be cut into small pieces and left in the solution
for 24 — 48 hours. It is then placed in distilled water,
which must be changed several times until it is no
longer tinged. The hardening is completed by the spirit
process (page 13).
7. Chloride of Gold. Half per cent, solution. This is
sold in small glass tubes, each containing 15 grains of
the chloride, equal to 7 grains of pure gold. Take one
of these tubes and file a ring round it above the bulb,
it can then be easily divided into two parts, empty the
gold chloride into a 6- ounce bottle, and wash out any
particles that remain with distilled water, fill up the
bottle. This will be under a half per cent, solution, but
answers very well. Place a small quantity of this solu-
tion in a watch glass and immerse the tissue, which
must be perfectly fresh, in it, let it remain in the dark
for from half to one hour or more, then place in dis-
tilled water, which must be changed several times, and
expose to diffuse daylight until it becomes a violet
brown ; about 24 hours will do in summer.
The tissue can then be mounted in glycerine, if it is
a small thin substance, such as a Tadpole's tail.
If, however, larger portions of any tissue are stained
with gold chloride, the hardening will not be sufficient,
and they must be further hardened by the spirit pro-
cess (page 13).
Mouse- tail, stained and hardened by the gold process,
may be decalcified by placing it in a half per cent, solu-
tion of chromic acid or a saturated solution of picric
acid for a few days.
On Preparing Tissues for Examination. 15
For another gold process, see Cornea. Gold chloride
has a staining as well as a hardening action.
8. Picric Acid. A saturated solution of picric acid
will decalcify small bones. It is also used in some
cases as a hardening agent by adding 1 part of water
to 2 parts of a saturated solution ; but it does not
seem to give such good results as the chromic acid
mixture.
9. Osmic Acid. This can be procured as a 1 per cent,
solution in water, and it is then diluted to various
strengths as required. It blackens fat and the medul-
lary sheath of nerves.
A piece of mesentery placed in a weak solution for
half an hour will show the fat cells lying along the
course of the blood-vessels, as round black bodies.
It is also used for hardening the internal ear.
CHAPTEE III.
ON CUTTING SECTIONS.
SECTIONS may be cut either by hand with a razor, or
with a microtome.
1. In cutting sections by hand it is necessary to im-
bed the tissue in some material which will cut easily,
and at the same time hold it firmly.
The best substance for this purpose is a mixture of
wax and olive oil.
Take equal parts of white wax and olive oil by weight
and melt them together, pour into a shallow vessel and
when cold cut into small blocks.
Small tin-boxes with a removable bottom are re-
quired to hold the mixture while the tissue is being im-
bedded, and the best size is 2 inches long, by f of an
inch wide, and J of an inch deep.
It will be necessary also to have a small porcelain
ladle and a stand to raise it above a spirit lamp or gas
jet. Melt some of the wax mass in the ladle and be
careful not to make it too hot.
Prepare the tissue so that the face which is to be cut
can be easily recognised, stick a needle into it away
from the part which is to be cut, drain off most of
the spirit by laying it on filtering paper, and then im-
merse in the melted wax mass, so that it is perfectly
covered, take it out and let it cool. Take a small piece
of filter paper and place it over the removable bottom
On Cutting Sections. 17
of the tin box, and then fix it in its place, the filter
paper will prevent the wax from running out if the bot-
tom fits loosely. It is also useful to leave a little of the
paper projecting on which to write the name of the
material imbedded. Then half fill the box with melted
wax mass and hold the material in it, keeping it quite
steady until the wax hardens, then by gently screwing
the needle round it can easily be removed, and the box
filled up with wax mass. It will be found a saving of
time to imbed a portion of material at each end of the
box. When the wax mass has become thoroughly
hard, which will take some time, especially in warm
weather, pull off the bottom and push the wax mass,
with the filter paper adhering out of the box. It can
then be laid by until wanted, the name of the material
imbedded being written on the paper.
For cutting sections thus imbedded, a hollow ground
razor is necessary ; a very good one for this purpose can
be procured from Baker, High Holborn ; the razor must
be very sharp. A small glass capsule about J of an
inch deep filled with methylated spirit is also required
to put the sections into when cut, and to moisten the
razor in.
Take the wax mass and with a scalpel carefully
remove small slices from one end, until the imbedded
mass can just be seen, then take the razor and dip it
into the capsule, taking up a little spirit, let this run
along the edge so as thoroughly to moisten it, and com-
mence cutting as thin sections as possible, by drawing
the razor diagonally across the mass with a steady
sweep ; this must not be done too quickly, and the
c
18 The Freezing Microtome.
amount of pressure to be put on the razor will depend
on the tissue imbedded and can only be learnt by prac-
tice. As each section is cut dip the razor into the cap-
sule of spirit and wash it off. Wipe the razor occa-
sionally and remove adhering portions of wax mass,
and always keep the edge wetted with spirit. When a
sufficient number of sections have been cut, the thin-
est should be selected and removed to a watch glass
containing clear spirit.
Great care is required in cutting sections by hand, to
hold the razor firmly yet lightly, so as to cut them thin
and at the same time even, and this cannot be done
without a great deal of practice.
For larger sections the boxes must be proportionately
increased in size, and it will be found convenient when
the wax mass is as wide or wider than the razor to cut
off slices from each side so as to reduce the surface to
be cut as much as possible without interfering with the
stability of the imbedded material.
A small flat spear-headed needle will be found useful
for taking up very small sections.
THE FREEZING MICROTOME.
A much easier method of cutting sections is by using
a microtome. Of these there are a number made, in
some of which the material is imbedded in wax mass,
or a mixture of paraffin and lard, and raised gradually
by a screw, while a razor is worked on a flat plate
shaving off sections ; these are also made with a cham-
ber to contain a freezing mixture, so that the material
can be imbedded and frozen.
To prepare the Material for Freezing. 19
Only one microtome will, however, be described in
this work, as it is very simple and does the work well.
This is Williams' microtome, made by Swift of Univer-
sity Street. It consists of a tub to contain the freezing
mixture, with a brass standard into which screw the
brass circular plates on which the material is frozen.
A top with a glass surface fits on to this, having a hole
through which the circular plate projects. The knife
is fixed into a triangular frame, having screws at each
angle by which it is raised and lowered.
To prepare the microtome for use :
1. Have the knife as sharp as possible.
2. Pound some ice finely in a cloth.
3. Scrape some salt into a fine powder.
With a spoon, put a layer of ice into the tub and then
some salt on it, mix with the spoon, and so on, until the
tub is about half full, then ram it hard with a stick and fill
again, put in the salt and ice in about equal proportions,
leave room for the top, wipe off the salt and ice from
the edge, put on the top and fix it with the screw for
that purpose. Screw the circular plate into its place,
and the microtome is ready for use.
An India-rubber tube must be fixed to carry away
the drainings as the ice melts.
To PREPAKE THE MATERIAL FOR FREEZING.
Any tissue which has been preserved in spirit,
must be soaked in water for 24 hours to remove the
spirit, and then placed in mucilage for another 24
hours.
c2
20 Cutting the Sections.
It will be found a great saving of time, when a num-
ber of specimens are to be cut at one freezing, not to
have the material too thick, as a piece a quarter of an
inch thick will give an enormous number of thin sec-
tions, and take only a short time to freeze.
To MAKE MUCILAGE.
Pour warm water on picked gum Acacia and make a
solution rather thicker than the mucilages sold in the
shops.
CUTTING THE SECTIONS.
Eemove all the specimens to be cut, from the gum,
and place them in a small saucer ready at hand. Take
up one with a pair of forceps and lay it on the circular
plate of the microtome, drop some gum solution on it
with a small brush, and see that it runs down on to the
plate all round the specimen so as to fix ib firmly.
When it is thoroughly frozen adjust the razor so that
ib will just pass over without touching, and then lower
the screw at the apex of the triangle by giving it a
slight turn to the left and push the knife across the
material in a diagonal direction. Have a small vessel
ready containing warm distilled water. It is necessary
to use disbilled water, as in ordinary water the lime in
solution is precipitated by boiling and the specimen will
be covered by fine particles of carbonate of lime and
utterly ruined. Moisten the upper surface of the razor
with gum solution, and the sections as they are cut will
slip up on it without curling, carefully remove them
with a camel-hair brush and place them in the warm
Cutting the Sections. 21
distilled water and let them remain for 10 minutes or
longer, until the gum is dissolved out ; this will take
longer with some material, such as testicle, than others.
With this microtome the most beautiful sections can
be cut perfectly even throughout, surpassing anything
that can be done with the hand. With care very large
sections may be cut quite as readily as smaller ones,
but to cut them well the razor must be very sharp, and
the material not too hard ; those hardened in chromic
acid mixture, No. 1 (page 11) seem to do the best.
Very little force is required in pushing the knife across
the material, and if it is sharp a very slight turn
of the screw each time will cut a section ; these ought
to be so thin as to be almost invisible, as the gum melts
on the razor.
In cutting some material, such as retina, it is advis-
able to stain it en masse before freezing, otherwise the
sections cannot be seen when placed in water.
CHAPTEE IV.
ON STAINING.
LIST OP STAINING AGENTS.
1. Hsematoxylin or Logwood 5. Anthra-purpurine
2. Carmine 6. Eosin
3. Indigo-czvrmine 7. Picro- carmine.
4. Purpurine
Aniline Dyes Soluble in Water.
8. *Soluble Aniline or China 13. Dahlia, Eosanilin and Me-
Blue thylanilin Violet
9. *Pure Soluble Blue 14. Malachite Green
10. Serge Blue 15. *lodine Green
11. Tyrian Blue ] 6. *Bismarck Brown.
12. *Safranine
Aniline Dyes Soluble in Spirit.
17. Spiller's Purple 21. Citranine
18. *Rosein 22. Aurine
19. Anilin Red 23, *Pure Opal Blue.
20. *Anilin Violet
Selective Stains.
Osmic Acid Chloride of Gold Nitrate of Silver.
To demonstrate the minute structure of any tissue, the
sections require to be stained with some colouring
agent which will show the different elements more
plainly by their absorption of the colouring matter and
bring out very transparent parts which otherwise would
be hardly discernible.
Of all the staining agents, logwood is the most useful,
and a solution prepared in the following manner will be
found the most efficient.
To Stain with Logwood. 23
LOGWOOD STAIN.
1. Take of
Extr. Hsematoxyl, grms. 6
Alumen, grms. 18.
Mix thoroughly, while mixing add 28 c.c. of distilled
water. Filter and add to the filtrate 3 j of spirit of
wine. Keep in a stoppered bottle a week before using.
What remains on the filter can be mixed with 14 c.c. of
distilled water, and left soaking in it for an hour or so,
then filter and add to the filtrate 3 BS of spirit of wine.
This second solution is as strong as the first.
To STAIN WITH LOGWOOD.
Make a cone with a small round filter paper, and
pour some of the staining fluid into it, let from seven to
ten drops fall into a watch glass and dilute with dis-
tilled water. Let the sections remain in the solution for
about a quarter of an hour, the time will depend on the
tissue and the manner in which it has been hardened.
Some tissues take in the stain very rapidly, others
slowly. Take out a section from time to time, and place
it in a watch glass of ordinary filtered water to see if it
is stained deeply enough.
When the sections appear to have stained thoroughly
remove them to a watch glass of ordinary water, and
wash them to remove the excess of colouring matter.
In staining with logwood it is necessary to be careful
that too many sections are not placed in the solution
at once, as they will lie thickly one on the other, and
the staining will not be uniform ; it is also necessary to
24 On Staining.
dilute the logwood stain with distilled water, as or-
dinary water will not give the same result, owing to
the different matters held in solution ; but it is better
to use ordinary water for washing the sections after
staining as it helps to fix the color.
The solution should not be too strong, as better re-
sults are obtained from staining the sections slowly
than from doing it rapidly, and it will always result
in a loss of time if an attempt is made to stain a large
number of sections in a strong solution.
ON STAINING SECTIONS WITH LOGWOOD THAT HAVE BEEN
PREPARED WITH CHROMIC ACID.
It is necessary to remove the chromic acid from sec-
tions hardened in that fluid, and this is done by taking
some of the 5 per cent, solution of bicarbonate of soda
diluting it slightly, and soaking the sections in it for
some time, ten to twenty minutes will generally be suf-
ficient.
Then remove them to plain water and wash well.
Prepare two watch glasses of dilute logwood stain
and place the sections in one of them, let them remain
for a minute and then place them in the other ; there
they must stay until stained deeply enough.
If the first watch glass be now examined, the log-
wood stain will be found to have become quite granular,
and if this precaution had not been taken it would have
been deposited as minute granules all over the sections.
2. Carmine. This stain, formerly so much used, is
now almost given up by Histologists, as a general stain,
in favor of logwood which is found to differentiate the
On Staining. 25
tissues much better while it has not the deleterious
effect of carmine on the eyes when used with lamp
light.
Carmine is, however, valuable in double staining,
either as a carmine solution or in conjunction with
picric acid as picro- carmine.
The carmine solution is prepared by mixing :
Carmine, 3 ss.
Borax, 3 ij.
Aqua, ^ iv.
and pouring off the clear supernatant fluid. It must
not be filtered.
3. Indigo- Carmine or Sulphindigotate of Soda. This
is a useful stain in conjunction with carmine, especially
for pathological specimens.
First make a saturated solution of the powder in dis-
tilled water and filter, take some of this solution and
pour it into methylated spirit until it has attained a
moderately deep color ; a good deal of the colouring
matter will be precipitated, and it must be carefully
filtered to remove this, it is then ready for use. The
solution should not have a very deep blue color and
when held to the light, should show a purplish tint. It
does not require long to stain the sections in it, and
they should be allowed to remain long enough to stain
them evenly, as it will be found that they stain first at
the edges, after having been passed through dilute hy-
drochloric acid.
4. Purpurine. True purpurine is a red colouring
matter extracted from madder, it is rather expensive
26 On Staining.
and not easily obtained. It is soluble in spirit with
the addition of slight heat.
Place some of the powder in a test tube and add
some rectified spirit, warm gently over a spirit lamp.
When dissolved, filter and keep in a stoppered bottle.
It stains readily and brings out some tissues such as
muscle and mucous glands well.
It does very well in double staining with aniline
blues.
5. Anthra-Purpurine. A brown powder which is
often sold for purpurine. It is isomeric with it, but
is found as a secondary product in the preparation of
alizarin from anthracene.
It is very slightly soluble in water and may be re-
moved entirely from aqueous solutions by means of
ether.
A solution may be made by first dissolving it in
spirit and then adding a warm solution of alum in
water, the color will then become reddish. It stains
muscle and epithelium but not connective tissue.
It does not show up minute structures as well as
logwood.
It does not combine with picro- carmine in double
staining, but the difference in the two colors is so slight
as to make this of little value.
6. Eosin. Is not an anilin color but is a potash salt
of resorcin. Kesorcin is obtained by the action of
melting potash on galbanum. Eosin is largely soluble
in water and has a beautiful garnet red color. In using
it a strong solution is required and the sections must
be well washed in water after staining. It is a very
On Staining. 27
delicate color when used alone and almost too trans-
parent, but in combination it becomes opaque.
This is the ordinary eosin, there is also an alcoholic
eosin called primrose which is much used by silk dyers,
it is soluble in a weak solution of spirit of wine, it fixes
readily and gives a more brilliant color than ordinary
eosin, and is not affected by light. There is also a blue
shade of eosin which is said to have a great affinity for
wool, and can be fixed readily by a solution of soda
hypophosphis ; this might be useful for investigations
in diseases of the hair.
7. Picro- Carmine. This useful stain is difficult to
make and the process is very tedious, it is better there-
fore to buy it prepared for use. It can be procured
from Martindale, New Cavendish Street, at a reason-
able price.
Filter about 10 drops into a watch-glass and dilute
with distilled water. The sections must remain for
some time, from twenty minutes to half an hour, and
if at the end of that time they have not stained suffi-
ciently, a little more picro-carmine may be added.
They are then placed in water acidulated with a few
drops of acetic or picric acid and left for an hour.
When making experiments in treble staining a num-
ber of sections may be stained in picro-carmine and
then placed in methylated spirit ; there they may re-
main until required, as the spirit does not affect the
stain, which forms a very good ground color, on which
to try combinations of different anilines.
It is also a good stain for fresh tissues, such as
mesentery when used with logwood. Also in sections of
28 On Staining.
skin it is very useful as a ground color, and by staining
a number of sections of the same tissue with picro-
carmine and then with two other colors, a variety of
results will be obtained.
In some the tissue for which picro- carmine has a
special affinity, such as connective tissue will be found
unaltered, while the surrounding tissue has taken on
the new colors. In others the picro- carmine has com-
bined with one or both, and a new color is formed.
Or again, it may be entirely supplanted by one of the
other colors.
ANILIN DYES.
These may be divided into two classes, those soluble
in water and those soluble in spirit ; and this classifica-
tion will be found very useful for experiments in double
staining.
There are an immense number of anilin dyes now
manufactured, but a great many of these will be
found precisely similar in their colour and action,
although bearing different names, while others are at
the most only different shades of the same colour.
The foregoing list gives a selection of the most useful
of these dyes, and those marked with an asterisk will
be found to give the best results. Many of the others
would, however, be valuable stains if any means could
be discovered for fixing them so that they could be
passed through spirit without becoming obliterated.
8. Soluble Anilin Blue or China Blue. This is a use-
ful stain for some tissues, such as stomach and spinal
cord. It is made very simply by taking some of the
On Staining. 29
granules, and placing them in a large test-tube, and
adding distilled water : the solution ought to be strong,
nearly saturated. When all the granules have dis-
solved, filter the solution, and keep it in a stoppered
bottle.
Sections that are to be passed through spirit require
to be rather deeply stained, as a good deal of the colour-
ing matter will come out in the spirit, and it is better
to remove them at once from the water to absolute
alcohol, as the colour is not affected so much by it.
9. Pure soluble Anilin Blue. This is a very good blue
colour, soluble in water : it is more brilliant than the
former, and more expensive ; the China blue is, in fact,
prepared from it. A saturated solution must be made
and great care used in staining with it, as its action is
very rapid. Like all anilin colours, its brilliancy is
impaired by a long immersion in spirit.
This colour has not been much used as yet, but it
will probably become a favourite when it is better
known. To use either of these solutions, a few drops
are placed in a watch-glass, and diluted with water,
one or two drops are quite sufficient, the sections are
then washed well in water.
10. Serge Blue. This is another shade of blue ; a
good colour, and stains well, but will not stand immer-
sion in spirit.
11. Tyrian Blue. A much less brilliant colour than
any of the others, more inclined to purple : it does not
stand spirit, and cannot be much used until some
method is found for fixing it.
12. Safranine. Is a good deep colour, and stains
30 On Staining.
gland tissue, such as mucous glands in the tongue, it
does not, however, stand immersion in spirit well, and
the sections require to be deeply stained. It has been
recommended for staining amyloid degeneration, but
does not excel several other anilines which can be more
easily fixed.
13. Dahlia. Bosanilin, Methylanilin Violet. These
three, differing but little in colour, may be con-
sidered together ; they can be partially fixed by
passing the sections through a 1 per cent, solu-
tion of hydrochloric acid, but even then they will
not stand prolonged immersion in spirit, and it is
better to remove them from the water at once to abso-
lute alcohol. The solutions must be rather strong, and
the sections well stained before they are removed to the
acid solution.
14. Malachite Green. A very beautiful colour, but
will not stand well : it is almost entirely removed by
spirit, and is therefore not of much use to the Histolo-
gist.
15. Iodine Green. A darker green than the former,
and very durable, standing spirit well and not fading,
as far as two years experience goes. It is an invalu-
able colouring agent in double staining, as it is not so
opaque as anilin blue. Make a saturated solution in
water and filter, place a few drops in a watch-glass
and dilute : it is very strong, and the section, when
taken from spirit, will float on the surface, when it may
be seen taking in the colour : if a light stain only is
required it will be sufficient to let it remain on the
surface ; but if a darker stain is wanted it must be
On Staining. 31
wholly immersed, and then it must not be left long
or the stain will be too deep, and it cannot be after-
wards removed.
This is one of the most useful of the anilins soluble
in water, and the results when it is carefully used are
very beautiful ; it picks out all the nuclei, and in grow-
ing bone it colours the unossified cartilage, giving a
very striking result. It is also a most valuable anilin
in double staining ; its action will be described under
that head.
16. Bismarck Brown. This is a good colour, a deep
rich brown. It is easily soluble in spirit but does not
make a very deeply coloured solution, it stains well.
An aqueous solution can be made by the addition of a
little dilute acetic acid, this solution also stains well
but it remains to be seen whether it will fade or not.
The aqueous solution stains gland tissue well, and in a
section of tongue where the mucous glands are deeply
stained with the aqueous solution, the colour has not
faded after an exposure of two months. It is doubtful
as yet which will prove to be the best solution, the
aqueous is decidedly the richest colour to the eye but
must be kept for a week, after making, before it is
used.
ANILIN DYES SOLUBLE IN SPIRIT.
17. Spiller's Purple. This is a very good colour for
use in double staining, but it is very difficult to fix it
and if the section is passed through methylated spirit,
it will be almost all washed out ; it is not quite so easily
acted on by strong absolute alcohol, '795 sp. gr., but
32 On Staining.
the section must be very deeply stained to get enough
left in it to make a satisfactory specimen.
There are several other colours soluble in spirit which
are much easier to use and consequently this has not
been much tried, but it would well repay the trouble
if some means were found to make it more permanent.
A saturated solution must be used, and it is better not
to wash the section too much in spirit, before putting
it in the aqueous solution in double staining.
18. Rosein. This is the best of all the anilin colours
soluble in spirit and must not be confounded with ros-
anilin. It is easily soluble in spirit, the ordinary
methylated spirit will do, its action is rapid, and it is
better to use a strong solution and immerse the section
for a very short time.
To make the solution, place some of the granules in
a large test tube and nearly fill with spirit, cork and
shake it and let it stand, when all the granules are
dissolved, add some more until the solution appears
to be saturated, then filter and keep in a stoppered
bottle. Place some in a watch glass and dilute with
about an equal quantity of spirit ; this will do for all
ordinary purposes.
19. Anilin Eed. This is another shade of red and
does not differ much from rosein in colour while it has
not its universal applicability, and does not fix so well.
It has not been much used, and experiments might
bring out some valuable points in it as a dye for special
subjects. It is prepared and used in the same manner
as rosein.
20. Anilin Violet. This colour comes next to rosein
On Staining. 33
in utility while it surpasses it in beauty ; it is not quite
so easily fixed, otherwise it can be used in the same
manner and as readily as rosein. It has this advan-
tage over rosein, it is a much softer colour to the eye
and makes a really beautiful combination with some of
the aqueous solutions in double staining.
It is readily soluble in spirit, and even diluted makes
a very opaque solution, rendering it a matter of diffi-
culty to find the sections in it ; they may be seen, how-
ever, by holding the watch glass against a lamp or
bright sky.
Good results are often obtained with this colour by
removing sections from water and letting them spread
out a little on the needle in a strong solution and
then removing them quickly to an aqueous solution of
another color.
21. Citranine. A yellow substance insoluble in water
and only soluble in spirit by the application of heat.
It does not seem to give a deep enough colour to be a
useful staining agent in microscopical investigations,
but a more extended trial may bring out some valuable
peculiarities, as it seems to differ a good deal from the
other anilins.
22. Aurine. This is another colour of which there
is not much known of its action on animal tissues ; as
far as it has been used, it has not given any satisfactory
result.
23. Pure Opal Blue. This is the best blue soluble in
spirit, and is therefore useful in cases where an aqueous
stain is not admissible. It is made by dissolving the
powder in spirit and then filtering it. Methylated
34 On Staining.
spirit will do if strong, but spirit of wine is better. A
few drops are taken and diluted with spirit, and the
sections immersed from three to five minutes, and then
washed in spirit.
SELECTIVE STAINS.
These will be found under their respective headings.
CHAPTEE V.
DOUBLE STAINING.
BY double staining is meant a process in which two
colours are taken, which have affinities for different
elements in the tissues to which they are applied.
Thus while one colour will stain the connective tissue
and protoplasm of cells, the other will colour all nuclei
and so differentiate the different elements as to make
them more easily discernible. Others again will stain
different glands according to their secretions. Thus
showing a distinct chemical reaction between glands
differing in their functions.
In other cases the duct of a gland can be stained of a
different colour to the surrounding tissue and its own
secreting substance, by which means it is easy to distin-
guish it, and thus show if it is implicated in any mor-
bid change, and also in some cases prove whether the
morbid change is primary in it or has extended from
surrounding tissues, in which case all the ducts would
not probably be similarly affected.
Double staining is a subject that requires to be very
much more worked out than it has been hitherto, and
in the present work, those processes only will be given
in detail, which have been fairly well tried ; many other
combinations of staining agents will be apparent to the
student, which have not yet been worked out, for
want of time, and there is no pursuit in which patience
D2
36 Picro- carmine and Anilin.
and time for experimenting are more required, than in
double staining.
The student must not be discouraged by many
failures as there is always some new fact to be learnt
and noted, and in the application of these to future
experiments, some brilliant results are sure to be ob-
tained.
PlCKO-CAKMINE AND LOGWOOD.
This combination has been used for a long time, and
gives very satisfactory results. The student should
begin with sections of scalp, skin, or tongue, and the
result, if the process be carefully carried out, will be
found very satisfactory.
The sections must be first stained in picro- carmine
and then in logwood. Make a dilute solution of picro-
carmine in distilled water, about 10 drops to the watch
glass, and let the sections remain in it for from twenty
minutes to half an hour, then wash in water and place
in distilled water acidulated with 1 or 2 drops of acetic
or picric acid. Let them remain in this for about an
hour. Eemove the sections from the acidulated water
and place them in dilute logwood stain ; this should not
be too strong, from 5 to 7 drops to the watch glass of
distilled water; do not let them stain too deeply.
When sufficiently coloured, which will be shown by
their becoming a faint lilac colour, they must be
washed to remove the excess of logwood, and mounted
in the usual manner.
This double stain is very effective when used with
fresh tissues, such as serous membranes ; it brings out
Staining Process. 37
the connective tissue corpuscle in the mesentery of the
newt, and at the same time shows the non-striped
muscle tissue very well. It is also useful in bringing
out the delicate tissue in the tubuli seminiferi of the
testis and showing the developing spermatozoa there.
In any tissue when there are elements of different
kinds, such as scalp or developing bone, it will be
found to give very good results. The logwood stain
must not be too deep, as it is a very opaque colour.
CARMINE AND INDIGO- CARMINE.
This is a very useful double stain, and is especially
applicable to sections made from material hardened in
chromic acid mixture as they do not require to be
passed through a solution of bicarbonate of soda, but
can be placed at once in the carmine solution.
In this staining process three solutions are necessary.
I. Carmine solution (page 24).
II. Hydrochloric acid, 1 part ; absolute alcohol '795,
9 parts.
III. Indigo carmine solution (page 25).
STAINING PROCESS.
Take a few drops of No. 1 in a watch glass and im-
merse the sections, let them remain for two or three
minutes and then remove them to a watch glass con-
taining a small quantity of No. 2. Let them remain
in this until they take on a bright rose colour, which
will be in a few seconds, then wash them in methyl-
ated spirit to get rid of the acid. They must be washed
in several changes of spirit. When the acid has been
38 Pier o- carmine and Anilin Colours.
thoroughly removed, place the sections in a little of No.
3 solution undiluted, and let them remain in it until
they show a distinct blue tinge : the proper depth of
this staining will be learnt by practice.
When carefully used this staining process is an ad-
mirable one, but there are one or two points that
have to be attended to, or the two colours will not
be sufficiently differentiated. If the section is left
too long in the acid mixture the carmine will be
taken out of the edges and these parts will after-
wards take on the blue stain deeply, and so give a
result the very opposite of that intended, as the
whole value of double staining depends on one co-
lour picking out the whole of one particular tissue
throughout the section, and if this is not done the spe-
cimen is of no use. If the carmine stain is only just
sufficiently acted on by the acid, so as to change the
original dull purple colour to a bright rose, and the
edges of the specimen are not bleached, it will, when
put in the indigo -carmine solution, stain evenly through-
out.
If the acid solution be too strong it will have the
same effect as too long immersion in a weaker solu-
tion, and a few seconds will bleach the edges. This
process will be found very useful in pathological inves-
tigation, as the carmine picks out very distinctly all the
new growths.
PlCRO-CAKMINE AND ANILIN COLOUES.
Some very good results may be obtained by staining
sections first in pier o- carmine, then letting them remain
Treble Staining. 39
in acidulated water for an hour, and afterwards stain-
ing them with various solutions of anilin colours.
Safranine, after picro-carmine, gives a good double
stain, as the picro-carmine colours all the connective
tissue and nuclei, while the safranine stains muscle,
epithelium, &c. ; but the two colours are not sufficiently
different to give as good a result as logwood and picro-
carmine, although they will be found useful where great
transparency is desired.
Picro-carmine and iodine green give a very beautiful
effect when it is wanted to isolate gland tissue ; such
as Peyer's patches, or the glands in the tongue, oeso-
phagus, or solitary glands in the large intestine. The
picro-carmine staining everything but the glands, which
remain a bright green.
Eosin and anilin blue give good results, but require
to be used cautiously, as if the staining is too deep the
section becomes opaque. To get the best effect, the
section should be very thin, and must be well washed
after staining with eosin, and then just immersed for a
few seconds in the anilin blue.
A great many other combinations will suggest them-
selves to the student, and he will be amply rewarded by
experimenting further with the various staining agents
mentioned.
TREBLE STAINING.
The combination which has given the best effect so
far in treble staining is : picro-carmine, rosein, and
iodine green. Stain the sections well according to the
process already described for picro-carmine, and soak
them in acidulated water. Then take a few drops of
40 Treble Staining.
rosein solution, No. 18 dilute it with spirit, and im-
merse the sections for two or three minutes, remove
them to methylated spirit and wash off the excess of
colouring matter. Then place them in a dilute solution
of iodine green. Coming from spirit they will float on
the top of the watery solution, and this in many cases,
when the green stain is not required to be very deep, is
quite sufficient. When a deeper stain is required, im-
merse them altogether, and let them remain a minute
or two ; but it must be borne in mind that this colour
cannot be washed out again if too deep, which the
spirituous stain can, so that it is better to have a sec-
tion apparently over-stained in the rosein solution,
while it is even under- stained in the iodine green.
After washing, the sections are mounted in the usual
manner. It will be found, however, that a good deal
of the rosein will come out in the second immersion
in spirit, and it is necessary to change it until no
more colour comes away ; otherwise the oil of cloves
will become coloured, and from it the Canada balsam,
in which the specimen is mounted.
With the above mentioned three colours, the most
beautiful effect may be obtained, but it will take some
time and practice to get the process exactly right, and
this is a matter which can only be gained by experi-
ence. The results will be found to vary with the
length of time the section is immersed in each of the
two last colours, and also with the strength of the
solutions.
The section should be uniformly and deeply stained
with picro-carmine. The other two solutions should be
Treble Staining. 41
saturated in the first instance, and then diluted one-
half at least. If they are to be laid aside for some time
before mounting they should not be left in spirit, but in
oil of cloves. Only a few sections should be stained at
one time or some will be found much more deeply
stained than others. The best results will also be ob-
tained with material that has been hardened in chromic
acid.
This staining process is well shown in a section of
the base of a cat or dog's tongue, cut through one of
the circumvallate papilla?, the section should be suffi-
ciently large to include some of the mucous glands, of
which there are a large number in that region.
If the staining is well done it will show all the
muscle fibres stained with pier o- carmine, the connec-
tive tissue, protoplasm of cells, &c., stained with rosein ;
while all the nuclei in the superficial epithelium, serous
glands, non-striped muscle tissue in the vessels, and
elsewhere, are stained a brilliant green.
The most important fact demonstrated by this pro-
cess is the different chemical reaction shown by the
various glands. In the mucous glands, while the epi-
thelium lining the duct is stained in precisely the same
manner as the superficial epithelium of the organ, it
will be found that the moment the secreting epithelium
is reached a new colour presents itself, which differs in
toto from either of those employed in the process ; thus
showing that the secretion has the power of causing
these two colours, green and red, to combine, forming
different shades from purple to blue,* according to
* Iodine green is a very blue green.
42 Chloride of Gold and Anilines.
which colour predominates. In the serous glands, how-
ever, quite another aspect is presented; there is no
combination as in the mucous glands, but the proto-
plasm of the cells is stained more or less deeply with
rosein, while the nuclei have taken on the green ; the
colour differs, however, from that of the surface epithe-
lium, and appears to have taken on picro-carmine to
some extent, which, with the rosein, gives a dull red
colour.
In many places will also be seen small masses of
adenoid tissue which have stained a bright green
throughout.
Altogether this makes one of the most brilliant speci-
mens in the whole range of histology, and although the
process is rather troublesome, and requires a certain
amount of practice to determine the time required for
each immersion, it amply repays when once properly
done.
Take only a few sections at a time, and do not hurry
over the different processes, and after a few trials the
exact time of immersion will be hit on, and should be
recorded.
CHLORIDE OF GOLD AND ANILINES.
Some very striking results may be obtained by first
staining fresh tissues, especially growing bone, in chlo-
ride of gold solution (page 7), and then decalcifying
and hardening in spirit. After the material has har-
dened sufficiently, sections may be made and stained
with two colours. It is not quite clear what action the
gold chloride has on those parts it does not stain, but
Chloride of Gold and Anilines. 43
that it has some, is evident from the difference of the
action of anilin dyes on those specimens prepared in
gold, from those hardened in any other manner.
A very good material for the purpose is the tail of a
young rat or mouse, placed in half per cent, solution of
gold chloride for an hour or two, and then decalcified
and hardened in the usual way. Very thin transverse
sections should be cut, and stained first in rosein and
then in iodine green.
On examining the specimen the gold staining will he
seen on the periphery, bringing out the tendon cells,
and giving a dark hue to everything for a certain dis-
tance from the outside ; but within this a great variety
of colour will be found, the different tissues being
stained in a most gorgeous manner. In the middle
the bone trabeculse will be seen faintly stained, while
the calcified cartilage, in their centres, is stained a
bright colour, totally different. All these colours may
be varied by using different anilin solutions, and a very
pretty result may be obtained by simply staining with
iodine green. In the above instance the true bone is
only faintly stained, while the calcified cartilage takes
the colour deeply ; this may be reversed by using the
carmine and indigo -car mine process after the gold, when
the bone will be found deeply stained, while the calci-
fied cartilage is not stained at all, and looks like a clear
space in the bone trabeculas. These two processes will
be found invaluable in any investigation into the deve-
lopment of bone.
CHAPTEE VI.
ON MOUNTING.
SLIDES.
Glass slides, 3x1, must be cleaned before using,
and a good plan is to keep some cleaned, ready for use,
in a two-dozen box with rack work, where they stand
on their edges and do not get dusty. The ordinary
slides sold at the shops at 6s. a gross, are easily cleaned
with a chamois leather. Sometimes, however, especially
when using slides for the second time, they cannot be
cleaned so readily, and they must be soaked in a decoc-
tion of oak galls for some hours ; this is made by pour-
ing boiling water on bruised oak galls and straining.
COVER GLASSES.
The usual size of these is f of an inch square, but
larger ones will be required, and some of | of an inch
should be obtained. For practical men square cover
glasses are the best ; but where aesthetic mounting is
the order of the day, round cover glasses and variously
coloured cements for sealing, with the help of a turn-
table will be required ; these, for obvious reasons, will
not be considered in this work.
Thin glass is made by Messrs. Chance, of Birming-
ham, in three thicknesses, designated by the numbers
1, 2, and 3 ; of these No. 1 is the thinnest. This is the
best to use. It varies in thickness from '004, or even
thinner to -008.
On Mounting. 45
ON MEASURING COVEK GLASSES.
For ordinary students' work the No. 1 cover glasses
will do perfectly well, as they are very near the thick-
ness to which object glasses, without a correction collar,
are adjusted ; but when high power glasses are to be
used, it facilitates the work very much, to know the
thickness of the cover glass under which the specimen
is mounted, and with very high powers, or those with
wide angle of aperture, the cover must be at least '004
to enable the glass to work through it. Powell and
Lealand's -^ water immersion requires a cover glass
•003 of an inch.
To save expense, as dealers charge a high price for
very thin measured glass, the student will find it a
great advantage to purchase a small American steel
gauge, sold by Buck, Holborn ; it is made for measur-
ing the thickness of sheet brass in fine work, but does
admirably for measuring cover glasses ; the way to use
it will be at once apparent. With this instrument let
the student measure an ounce of No. 1 glass, and select
all those that are '004 and under, they may be put
away for future use. This is not much trouble, and a
few of these very thin glasses will last a long time, as it
is only in special work that they will be required. The
oil immersion lenses have also done away in a great
measure with the necessity of using measured cover
glasses.
ON CLEANING COVEE GLASSES.
The following plan will be found a very good one,
both in the saving of time and breakage.
46 On Mounting.
Place the cover glasses to be cleaned in a glass
vessel containing strong sulphuric acid, and agitate
gently until the acid has penetrated between the
glasses and driven out the air-bubbles, let them remain
in this for an hour or two and then wash well in water
until no acid is left. Eemove them to a capsule con-
taining methylated spirit. Take out each one separ-
ately with a pair of broad pointed forceps, and wipe dry
with a silk or soft linen rag.
With very thin cover glasses, such as -003, each
glass may be dipped in absolute alcohol when taken out
of the methylated spirit and then carefully dried with
an old silk handkerchief.
MOUNTING FLUIDS.
For fresh tissues :
Glycerine .
For hardened tissues :
Canada balsam solution.
Dammar varnish.
MOUNTING FKESH TISSUES,
Place the tissue to be mounted in a capsule of water
of sufficient depth to cover more than half of an ordi-
nary glass slide, when placed in it with one end on the
bottom and the other resting on the opposite side.
With a needle, bring the tissue over the middle of the
slide and hold it there, at the same time raise the upper
end of the slide very gently, so that the tissue will
adhere to it and be raised out of the water. See that it
On Mounting. 47
is not folded in any part. Lay the slide on some filter
paper, and with needles spread out the tissue to its
fullest extent, without stretching it. It is necessary to
be very careful of this, as if the tissue be a serous mem-
brane, stained with silver, the outlines of the cells will
be completely destroyed wherever it has been stretched.
In the same way, non- striped muscle fibre in the me-
sentery of the newt, will be broken up and quite ruined.
When the tissue appears to be extended in a natural
manner, without folds, take up the slide and wipe off
all moisture from it with a clean cloth, if there is a
large quantity on the specimen, some may be removed
with a bit of filter paper, but great care must be taken
not to touch the specimen itself with the paper as it
will adhere to it ; at the same time it must not be
allowed to become dry, and if this seems probable, it
can easily be moistened by breathing on it occasionally,
until the cover glass is ready. Take up a clean cover
glass and place a drop of glycerine on the centre, invert
and place it horizontally on the specimen, leaving the
weight of the cover glass to spread out the glycerine.
If there is an excess of glycerine round the edges of the
cover glass, it must be removed by placing small pieces
of filter paper in contact, which will soon absorb the
superfluous fluid, but must not be left too long or they
will drain it from under the cover glass. When suffi-
cient has been taken up by the pieces of filter paper,
remove them and wipe the slide with a dry cloth, tak-
ing care to clean off all glycerine without touching the
cover glass.
When this is done the preparation must be sealed, by
48 On Mounting.
painting round the cover glass with either Dammar
varnish or Hollis' glue, taking care that only the ex-
treme edge of the cover glass is included. It will be
necessary to give a second and third coat if Dammar
varnish is used, at intervals of a few days.
It will be found a good plan to seal first with Dam-
mar varnish, and afterwards to cover this with Hollis'
glue, as it makes the preparation more secure, and it is
absolutely necessary to have them sealed with Hollis'
glue when oil immersion lenses are to be used, as the
cedar oil does not touch it, while it dissolves Dammar
varnish at once.
Specimens carefully prepared in the above manner
may be kept for years without deteriorating.
MOUNTING IN CANADA BALSAM OR DAMMAR.
Canada balsam mounting fluid is prepared by mixing :
Canada balsam, 105 parts.
Turpentine, 35 parts.
Chloroform, 35 parts.
Dammar varnish is prepared thus :
Take of
Gum Dammar in powder, i oz., and dissolve it in
turpentine, 1-J- oz. Filter.
Gum mastic, i oz., and dissolve it in chloroform,
2 oz. Filter.
Mix the two solutions and filter again.
Put in stoppered bottles, and see that they are per-
fectly free from moisture before using. A small drop-
bottle of each of these fluids must be kept for daily
On Mounting. 49
use, and when they get thick from evaporation a little
chloroform can be added.
Both of these mounting fluids are used in the same
manner and one description will apply equally well to
each. Canada balsam is the one commonly used, as
the materials of which it is composed are very cheap,
while gum dammar is rather expensive. The Dammar
varnish is also sometimes apt to become cloudy after a
time and it is difficult to make.
To MOUNT IN CANADA BALSAM OK DAMMAR YARNISH.
The sections having been properly stained and
washed, are placed in methylated spirit to remove
some of the water, and then immediately transferred
to a small quantity of absolute alcohol in a watch
glass, and covered with another to prevent evaporation.
They should be left in this for about 10 minutes.
The absolute alcohol, which should be the strongest
sp. gr. -795, has a great affinity for water, and will
remove all that is in the sections.
"When ready remove the sections one by one from the
absolute alcohol with a needle, and drain off as much
alcohol as possible by touching the section on the back
of the hand or on a piece of clean filter paper, the back
of the hand is the best, as some fibres from the filter
paper may adhere to the section, which when seen
under the microscope, will not improve the beauty of
the preparation; when sufficiently drained, without
being allowed to become absolutely dry, they are placed
in a vessel containing oil of cloves ; they will spread
out on the surface of the oil, and as the spirit evapo-
E
50 On Mounting.
rates they will become completely permeated with it
and very transparent. If there are any folds these
should now be straightened out carefully with needles.
Having placed a drop of Canada balsam on the slide,
spread it out slightly with a needle, select a section in
the oil of cloves, and pass the copper lifter under it,
raise the lifter and hold the section in position with a
needle by its upper corner, and having made sure there
are no folds, remove the lifter with the section on it
from the oil of cloves, let as much oil drain off as possi-
ble against the side of the vessel, and remove the rest
by placing the edge of the lifter on a piece of filter
paper. Place the edge of the lifter on the slide in the
drop of Canada balsam, and gently draw down the
section with a needle as soon as a corner projects from
the lifter on to the slide, hold it there lightly with the
needle and slowly draw away the lifter ; if this is care-
fully done the section will lie in its place in the middle
of the slide without any folds.
A lifter is made by beating out the end of a copper
wire, filing it smooth, and then turning up the broad
portion slightly.
Take up a cover glass with the broad pointed forceps
and hold it between the thumb and fore-finger of the
left hand, place a small drop of Canada balsam on its
lower edge, transfer to the right hand and gently lower
it on to the section, keeping the left thumb against one
corner to prevent its slipping, and gradually lower it by
removing the right hand very slowly, watching all the
time to see that no air bubble is entangled in the
section.
On Mounting. 51
With a little practice this can be done very neatly
without an air bubble in any part of the preparation ; it
requires patience, however, and it is no use to try air
pumps or any dodges, to remove the bubbles, as they
are useless ; the only thing to be done when an air
bubble lodges in a cavity of the section and refuses to
move in any way by gentle pressure, is to lift the cover
glass, and transfer the section to oil of cloves, and then
remount it.
When several sections are to be mounted on one
slide, a slight pressure on each with the needle will
generally retain it in its position, if too much of the
mounting fluid is not used.
It will often be found on examining preparations
after they have been mounted some little time, that
the fluid has evaporated and left a vacuum under the
cover glass ; in this case a drop of the mounting
fluid must be placed on the slide in contact with the
cover glass, and it will immediately run in and fill up
the empty space, provided always an egress has been
allowed to remain for the contained air, when this is
impossible from the small size of the hole at the edge
of the cover glass, the only thing to be done is to wait
until some of the material, of which the mounting fluid
is composed, has been dissolved by the fresh fluid.
Applying heat will effect it, and at the same time in
all probability ruin the specimen.
Each preparation should be examined under the mi-
croscope and if found to be worth keeping, labelled.
On the label should be noted the tissue, date of its pre-
paration, mode of hardening and staining, thickness of
E2
52 On Mounting.
cover glass if it has been measured, and anything of
note which may be seen at the time it is examined.
Exceptionally good sections should always have a pri-
vate mark to show that they are not to be given away
or exchanged.
They should be kept in a cabinet where they may lie
flat.
ON BKEAKING DOWN OLD PKEPAEATIONS.
It is often necessary to break down an old prepara-
tion and remount it. The cover glass may be broken,
the staining faded, or the cover glass may be too thick,
and preparations should never be discarded for these
reasons, as it is quite easy to remount them. When a
specimen has been mounted in glycerine, it is an easy
matter to remove the cover glass, all that is necessary
being to cut round the cement with a sharp knife, lift
the cover glass carefully with a needle, and float off
the section in water ; if it is very delicate the cover
glass had better be removed under water. The section
can then be washed, to remove the glycerine, and re-
stained if required ; it will then be ready for mounting
in the usual manner.
To break down a specimen mounted in Canada bal-
sam or Dammar varnish is more difficult, especially if
it has been mounted long enough to allow the balsam
or Dammar to become hard. It must be placed in a
bath of chloroform until it becomes soft enough to re-
move the cover glass, and this may be facilitated by
passing the slide over the flame of a spirit lamp so as
to heat it very slightly, but this requires care as the
section may be easily ruined.
On Mounting. 53
After the cover glass has been removed the section
must be floated off into chloroform until all the balsam
or Dammar has been dissolved out of it, and then placed
in alcohol for a short time ; it may then be re- stained if
necessary and mounted again.
MOUNTING LAKGE SECTIONS.
In manipulating large sections it is rather difficult to
pass them through the different processes without in-
jury. This may generally be done with care, and they
may even be double stained.
There are some tissues, however, so fragile that they
cannot be lifted on the needle without tearing, and
these must be left in one vessel and the different pro-
cesses applied to them there. This is not a very satis-
factory method, as the staining cannot be so well done
unless a large quantity of fluid is used, and every section
carefully separated from its neighbour, they are also apt
to be injured in pouring off the different fluids. This is
only required in exceptional cases. It is when the mount-
ing from the oil of cloves comes, that the difficulty is
experienced ; as, even if a lifter is specially made large
enough to take up a whole section, the adhesion of the
section to such a large surface is so great, that it is im-
possible to get it off without tearing, if the section is as
thin as it ought to be. It may, however, be done by
using the cover glass as a lifter in the following man-
ner.
Take as an example a longitudinal section of kidney
of large dog or man, having been safely stained, it lies
in the oil of cloves ready to be transferred to the slide.
54 On Mounting.
The section measures say about 1J inches by 1 inch,
some slides must be procured 3 by 2 inches and some
cover glasses 2 by 1£ inches, these had better be of No.
3 thin glass.
Having cleaned one of the slides, place some Canada
balsam on it and spread it out with the needle, to some-
thing near the size of the specimen, then take the cover
glass and pass it into the oil of cloves under the speci-
men, in the same way the copper lifter is used to smaller
sections ; lift the cover glass and keep the section in its
place, then drain off the superfluous oil by holding the
cover glass on filter paper, on lifting it fresh from the
oil it should be allowed to drain slowly from one corner,
then invert the cover glass with the section on it, place
a little Canada balsam at the lower edge, and lower it
gently into the Canada balsam on the slide ; this must
be done very carefully, as bubbles will be found here
and there, and the cover must be lifted a little and
lowered again, until they have all been driven out. It
is a tedious process but amply repays the trouble.
The great drawback in this method is that the front
of the cover glass is covered with oil of cloves and can-
not be cleaned until the balsam sets, a matter of time
with such a large surface. It can certainly be sealed
up with Hollis' glue, but even then it is not safe and
requires a great many coats before the glue is suffi-
ciently strong to resist such pressure as is required to
clean the cover glass.
With some tissues it is possible to use a large cover
glass as a lifter, and by allowing a large quantity of oil
of cloves to remain to draw it off on to the slide and cover
On Mounting. 55
in the usual way, but with other tissues such as testis
this is utterly impossible if the sections are thin, and
they can only be mounted in the manner first men-
tioned.
THIN SLIDES.
For preparations to be examined, under very high
powers, thin slides made from glass called 9 or 10 oz.
crown are useful, as they allow the condenser to come
close to the object.
CHAPTEE VII.
METHOD OF OBTAINING ANIMAL TISSUES FOE EXAMINATION.
THE animals required will be a cat, rabbit, and guinea
pig ; and for some special tissues, a frog, salamander,
and newt.
To kill the first three animals, place them in .a
box with a tight- fitting lid, having previously intro-
duced a sponge saturated with chloroform. In this
confined space the chloroform will render the animals
insensible in a very short time, and then kill them
without inflicting the slightest pain.
The other three animals may be chloroformed under
a small bell glass on a plate.
Having killed either of the first animals, make an in-
cision through the skin, from the chin to the anus, and
reflect it on each side, open the abdomen and remove
some of the mesentery and omen turn for preparation by
the silver process ; open the thorax and make an in-
cision into the heart and drain off the blood before it
coagulates. Before opening the heart a small piece of
the centrum tendineum of the diaphragm may be care-
fully cut out to be prepared by the silver process. Then
open up the thorax and abdomen thoroughly. Take out
the lungs with a small portion of the trachea attached.
Kemove the heart, open up the ventricles. Eemove the
liver and cut it into small pieces. Take out the sto-
mach with a piece of oesophagus and duodenum at-
tached, open it longitudinally and wash it in dilute
Animal Tissues for Examination. 57
solution of chromic acid to remove particles of food, &c. ;
this must be done gently, and it must not be rubbed.
Open the intestines with scissors, and wash in dilute
chromic acid ; cut into short lengths. Cut out care-
fully the ilio-coecal valve with a portion of intestine on
either side. Eemove the kidneys, and open one longi-
tudinally, the other transversely, in two or three places.
Take out the spleen and pancreas and cut them in
small pieces. Take out some of the mesenteric glands ;
remove carefully the uterus and ovaries if a female.
Open the scrotum and remove the testes, in many cases
these can be got out more easily by pushing them up
into the abdomen. Dissect out the penis and remove
it. Then cut round the skin of the neck and disarticu-
late the skull, taking care to leave the remaining por-
tions of the trachea and oesophagus. Disarticulate the
lower jaw and remove the tongue with the trachea and
oesophagus ; cut off these at the base of the tongue,
then divide that organ longitudinally, leaving one side
whole, make transverse cuts in the other. If large it
will be necessary to make some longitudinal incisions in
the first for hardening with chromic acid ; but this
should be done so as not to interfere with making longi-
tudinal sections of the whole of one side. Take out the
submaxillary glands. The eyes and brain will then
only remain, and great care must be exercised in taking
them out.
The best plan is to remove the brain first ; for this
purpose take off the covering of the nasal organ with a
pair of bone forceps, and snip off pieces of bone from
the upper part of the skull, taking care not to touch the
58 Dissection of Frog.
brain underneath, at the same time care must be used
not to squeeze the eyes in holding the skull. When all
the upper part of the brain is laid bare, the bone should
be nipped through in front of it with the forceps, and
the base of the skull divided ; by gently cutting away
the bone by degrees and dividing the nerves proceeding
from the brain, it can be completely freed with the
cerebellum, pons, and medulla intact.
• It requires some practice and care to do this well,
but even if the brain is a little torn in removing it,
there will still be plenty to harden and make sections
of. The bone can now be cut through, round, and in
the orbit the optic nerve and recti muscles divided, and
the eye removed whole.
Portions of nerve can be taken from different parts,
the optic, sciatic, &c. Portions of muscle may also be
cut out. The aorta and some of the larger arteries
should be removed and cleaned from the surrounding
tissues.
DISSECTION OF FROG.
The whole eye may be removed from a frog and
placed in Muller's fluid, but the cornea is required to
make a gold preparation.
Having killed a frog, wrap it in a cloth and render
the eye tense with the thumb of the left hand. Insert
one of the points of a fine pair of curved scissors at the
edge of the cornea and cut it round carefully, separate
it from the rest of the eye, and place it in gold chloride.
The mesentery and meso-gastrium may be prepared
by the silver process.
Dissection of Newt and Salamander. 59
DISSECTION OF NEWT AND SALAMANDEK.
Kill a newt and cut off the head, fix it to the table
on its back, by a needle passed through the neck and
another through the tail. Make a longitudinal incision
down to the anus, take up the upper end of the
stomach with a pair of forceps, and by carefully cutting
away the connective tissue attaching it to the cavity,
the whole of the contents of the body can be removed
en masse.
Cut away the lungs and liver, which are not wanted.
Make an incision through one side of the stomach in its
whole length, and then put the whole in solution of
chromate of ammonia. In Triton cristatus there will
generally be found in the male, two testes on each side,
but sometimes three or four ; some of these may be re-
moved for the purpose of making a fresh preparation
of Spermatozoa.
Salamander is treated in the same way for the same
tissues.
PRACTICAL HISTOLOGY.
BLOOD.
Take a small drop of newt's blood, cover and ex-
amine : notice the difference in shape and number
between the coloured and white corpuscles. Prick
the finger and examine human blood in the same way.
AMOEBOID MOVEMENT.
A warm stage is required to show the amaeboid
movement of the white blood corpuscles.
With a camel's hair pencil apply a little oil round
the edge of a cover glass, place a small drop of per-
fectly fresh newt's blood in the centre of the glass,
and cover with another ; lay the preparation on the
warm stage over the central hole, and apply the spirit
lamp to the wire. The thermometer should rise to
30° C.
Select one of the large colourless corpuscles, and
sketch the different movements shown by it at distinct
intervals. Make a similar preparation of human blood,
and examine in the same manner. The cover glass
must be warmed for human blood, and the top cover
glass should be touched on the spot of blood coming
from the pricked finger. The coloured blood corpuscles
will form rouleaux if properly prepared.
A preparation of fresh newt's blood should be ex-
amined with a high power on the warm stage to see the
Feeding Blood Corpuscles. 61
beautiful intra-nuclear and in tra- cellular network of
fine fibres in the red corpuscles.
FEEDING BLOOD COKPUSCLES.
Eub up vermilion cake in f per cent, salt solution,
add a very small drop of this to the blood on a slide,
cover and examine on the warm stage, paint a little
oil round the edges to prevent evaporation.
In the case of newt's blood the thermometer should
not rise above 30° C., and in that of human, not above
40° C. After a time the white blood corpuscles will be
found to have enclosed some of the vermilion granules.
Newt's blood from the larger size of the corpuscles will
show this best.
IRRIGATING BLOOD CORPUSCLES.
Make a f per cent, saline solution of newt's blood on
a slide, cover and examine : notice the red corpuscles
gradually becoming crenated. Try the effect of irriga-
tion on this preparation, place a small piece of filter
paper in contact with the edge of the cover glass, and
with a capillary tube place a small quantity of the fluid
to be introduced on the opposite side ; as the filter
paper withdraws fluid from one side, the new fluid will
flow in from the other. When thicker fluids, such as
glycerine are to be removed, a capillary tube is neces-
sary on one side to remove it by suction with the mouth.
The fluids to be tried by irrigation are :— Dilute
acetic acid ; distilled water ; 2 per cent, solution Tannic
acid; 2 per cent, solution Boracic acid. For their
effect the student is referred to the Atlas of Histology
by Dr. Klein.
62 Squamous Epithelium.
HJEMIN CRYSTALS.
These are interesting as demonstrating the presence
of blood, and they are very easily prepared, and can
be sealed up and kept as a permanent preparation.
Glacial acetic acid decomposes the blood pigment, and
forms a hydrochlorate of haematin in the presence of
sodium chloride.
Place some ordinary table salt in a watch glass, and
hold it over a spirit lamp until it is thoroughly dried.
Take equal parts of any dried blood and this dried
salt on a slide, put on a cover glass, and with a capil-
lary tube run in glacial acetic acid, warm it gently over
a spirit-lamp until a good deal of the acid has evapo-
rated, and then examine under the microscope. To
mount this as a permanent preparation, wash out with
distilled water all the glacial acetic acid, and seal with
Hollis' glue. It is better to use some rather largish
masses of blood, or when the acid is being washed out
all the crystals will go as well, the larger masses will
be held by the weight of the cover glass, and their
margins wi]l be found covered with Hsemin crystals.
EPITHELIUM.
SQUAMOUS EPITHELIUM.
With a blunt knife scrape a little saliva from the
back of the tongue or inside of the cheek, cover and
examine. Look for squamous epithelium, the so-called
salivary corpuscles showing Brownian movements of
the granules they contain, micrococci and in many
cases bacteria. «
Columnar Epithelium. 63
Shed skin of newt. This makes the best preparation
of surface epithelium. Place a newt in a glass jar of
water, and in three or four days it will he found that
it has shed the entire outer layer of epithelium as a con-
tinuous skin ; unroll it carefully, and cut into small
pieces, stain with logwood and mount in glycerine.
The staining will be facilitated by first placing it for
a short time in slightly acidulated water. Seal with
Hollis' glue, and examine with a low power. The
outlines of the cells and their nuclei will be very well
shown.
Make a vertical section of skin prepared in chromic
acid mixture (wherever this mixture is mentioned, solu-
tion No. 1, p. 11 is meant). Stain in logwood and
mount in Canada balsam.
Observe the different layers of cells, and with a high
power seek for the prickle cells of the Eete Malpighi.
These are, however, better seen in some epithelial
Cancers.
COLUMNAE EPITHELIUM.
Take the stomach of a newt or salamander prepared
in solution of chromate of ammonia (page 13), and wash
well until no colour comes away, place the whole in a
dilute solution of picro- carmine, about 15 drops to a
watch glass of distilled water, and let it remain until it
has taken on a deep red colour, then remove and wash
off the excess of colouring matter ; scrape off a little of
the surface material from the inside of the stomach,
and tease out gently on a slide in a drop of glycerin,
cover and examine.
64 Ciliated Columnar Epithelium.
With a high power the network in the cells and their
nuclei will be seen as depicted in the Atlas.
Make a longitudinal section of the large intestine of cat,
dog or rabbit, prepared in chromic acid mixture, stain with
logwood, mount in Canada balsam, and examine. The
columnar cells will be seen in rows, some of them having
become goblet shaped, that is distended by their secre-
tion, and if the animal has been killed some time after
feeding, when digestion is going on, these goblet cells
will stain deeply with logwood, and the mucin will be
seen poured out from the cell in deeply-stained masses.
If, however, the animal has been killed within a short
time of feeding, that is before the mucigen has been
changed into mucin ; these goblet cells will not stain
with logwood but will stain with anilin colours.
CILIATED COLUMNAR EPITHELIUM.
Take a portion of the trachea of cat, prepared in
2 per cent, solution of bichromate of potash, wash well
in water, and stain the mass in logwood ; it must be
put in a strong solution and left in for several hours ;
when deeply stained remove it, wash well until no more
colouring matter comes away. Then with a small
knife scrape away a little of the inner surface, and
place it in a very small drop of glycerine on a slide,
pound it with the rounded end of a needle holder until
the whole drop is seen to be permeated with fine par-
ticles, and no large ones are left ; place the cover glass
on gently and allow the fluid to spread under it ; seal
and examine.
Endothelium. 65
The ciliated cells will be found isolated and can be
readily examined. Two other varieties of cells will be
found, which are the cells of the deeper layers and they
are not ciliated ; try with the J to make out the stria-
tion of the line running across the cell at the base of
the cilia ; a good glass will show this ; the T^ oil im-
mersion will show that this striation is caused by the
cilia, which are continuous with the longitudinal stria-
tion in the body of the cell.
Make a transverse section of the epididymis of man
or dog prepared in chromic acid mixture and stained
with logwood. Here the ciliated cells will be beauti-
fully shown, the cilia being much longer than in the
trachea ; they can be traced through the striated
border, with a high power in the same manner, as in
the cells of the trachea.
ENDOTHELIUM.
Take a portion of mesentery of cat and prepare by
the silver process. When it has been left long enough
in distilled water, stain it with logwood and mount in
glycerine ; seal and examine.
The nuclei will be well shown by the logwood stain,
but the outlines of the cells will not probably be very
distinct at first, but will become more so by the action
of light. It will then be seen that the silver has been
deposited in the intercellular substance between each
cell, giving it a dark border. By focussing carefully a
second layer of cells will be brought into view, whose
outlines do not correspond with the first ; these are the
endothelial cells on the other surface of the membrane.
66 Connective Tissue Corpuscles.
In some places masses of small cells will be found
deeply stained, these are germinating cells.
Other serous membranes should be prepared by the
silver process, the centrum tendineum of the diaphragm
makes a very good preparation, showing the groups of
germinating cells, and the difference in shape of the
endothelium on each surface.
The lymphatic capillaries will also be seen by the
different shape of the endothelium, giving them the ap-
pearance of trabeculse running through the membrane.
A silvered preparation of the septum cisternae lym-
phaticae magnae should be made.
Eemove the viscera from a recently killed frog, and a
large lymph sac will be found on each side of the spinal
column behind the stomach, the septum separates each
sac from the peritoneal cavity. It may be stained in
situ by pouring a little i per cent, solution of nitrate
of silver over it and allowing it to remain a few minutes,
or the septum may be carefully removed and placed in
silver solution. The septum will show the germinating
cells round the opening of the stomata. In all cases
where preparations of serous membrane are to be made,
the animal should be bled first.
CONNECTIVE TISSUE COEPUSCLES.
Make a gold preparation of Tadpole's tale.
This will show the connective tissue corpuscles very
well with their branched processes. Pigment cells are
also numerous. Several of these preparations should
be made, as they show a great many different struc-
tures.
Tendon. 67
Make a preparation of Newt's mesentery in 5 per
cent, chromate of ammonia (page 13), and double stain
with picro-carmine and logwood (page 36). In this
preparation very large branched corpuscles will be seen
having the hyalin ground plate stained with logwood ;
these make most beautiful objects for examination with
high powers.
The corneal corpuscles will be mentioned in another
place.
TENDON.
To show the tendon cells which lie in the interfasci-
cular lymph spaces, take a young mouse just killed and
remove the skin of the tail, then with the fore- finger
nail separate two of the caudal vertebrae and forcibly
remove the distal portion. Several white threads will
be left, these are the tendons. Take a small bit of one
of the finest and place it in slightly acidulated water
for a short time, then remove to half per cent, gold
solution, let it remain about twenty minutes. Then
place it in distilled water, which must be changed once
or twice, until it becomes a bronze colour. Take a
small bit and place it in a drop of glycerine on a slide,
and separate it into as many fibrils as possible. Cover
and examine.
Take the tail of a young rat and prepare it in gold
chloride (page 14). Make .transverse sections and
double or treble stain them. The tendon cells will be
seen darkly stained, with the gold lying between the
bundles of fibrous tissue forming the tendons.
Tendon should also be examined in the fresh state,
F2
68 White Fibrous Tissue.
by taking a small portion from the mouse's tail and
mounting in salt solution, then irrigating it with very
dilute acetic acid, and watching the change that takes
place, as the fibrous tissue swells up and becomes in-
distinct, the cells becoming granular.
The acetic acid should be only just sour to the taste.
After a time the whole of the fibrous tissue will have
disappeared, leaving a very few elastic fibres which are
untouched by the acid.
Take also some of the fresh tendon and place it in
logwood stain, to which a few drops of glycerine have
been added ; let it remain until deeply stained. Tease
out small portions in glycerine on a slide ; the tendon
cells will be well shown by this process.
ELASTIC TISSUE.
Make a preparation of mesentery of frog and mount
in glycerine ; a very fine network of elastic fibre will be
found throughout the whole structure.
Take a small slice of the ligamentum nuchse of the
ox, which can be readily procured from the butcher's.
Place it in dilute acetic acid for some little time, until
it swells up, then tease a small portion in a drop of
glycerine on a slide. Cover and examine.
WHITE FIBKOUS TISSUE
Is well shown in many of the preparations of serous
membranes. A special preparation should, however,
be made, by hardening omen turn in 1 to 2 per cent,
bichromate of potash and staining with logwood; it
will show the large amount of fibrous tissue present in
Cartilage. 69
a serous membrane, forming the greater part of the
framework.
White fibrous tissue is also well seen in sections of
skin ; also in submucous tissues.
ADIPOSE TISSUE.
Well seen in some of the preparations of serous
membrane, when the fat cells lie thickly along the sides
of the blood vessels. Also seen in cutis vera, and in
many other parts. It is not necessary to make a spe-
cial preparation of it.
A serous membrane placed for a short time in dilute
osmic acid, and mounted in glycerine, will show the fat
cells differentiated from the surrounding tissue, as they
have all become blackened by the action of the osmic
acid.
CAETILAGE.
HYALINE CARTILAGE.
The thin cartilaginous expansions from the sternum
of the newt, prepared by the gold process, make very
good specimens of hyaline cartilage. Thin sections
may be cut by the microtome, or by hand. In this
preparation the lymph canals will be seen looking like
dark processes proceeding from the lacuna, in which
the cell lies, into the hyaline matrix.
Sections of the nasal cartilages of small animals,
growing bone, &c., will all give good examples of hya-
line cartilage.
In the fresh state the cells will be seen to fill the
10 Cartilage.
lacunae ; but in hardened specimens they have all
shrunk, more or less, leaving a space.
Specimens of cartilage should also be hardened in
chromic acid mixture, and thin sections stained with
logwood.
FlBKO- C AKTILAGE .
Make a longitudinal section of mouse's tail, and
notice the intervertebral cartilage and its gradual tran-
sition into hyaline cartilage on the bone.
The fibres will be seen in various aspects as they
cross one another, and several sections should be ex-
amined; the ceUs will be seen lying between the fibres.
Make a section of the intervertebral disc of sheep or ox,
hardened in chromic acid mixture, and stain with log-
wood. Mount some sections whole and tease out others
on the slide. Cover and examine. It will be difficult
to make out the fibres in some sections.
ELASTIC CARTILAGE.
This can be well shown in the lobe of the ear or in
the epiglottis. Procure the epiglottis of a sheep and
harden it in the chromic acid mixture, cut sections and
stain them with logwood.
The ear of a child prepared in the same manner, sec-
tions cut and stained with logwood.
The ear lobe of a pig also prepared in chromic acid
mixture and stained with logwood. Sections of these
must be thin and they must not be hardened too much
or it will be difficult to cut them.
The pig's ear when well prepared makes a very use-
ful specimen, as it shows a great many tissues.
Bone. 71
BONE.
PREPARING HARD BONE.
Bone must be examined in two forms, first in its dry
state, and secondly when it has been decalcified or had
its earthy salts removed. In the case of dry bone a very
few sections will suffice as it is a difficult and laborious
task to get them well made.
The bone is fixed in a vice and sections as thin as
possible are cut with a fine saw, these are rubbed down
with fine emery on a stone, and finally polished on a
hone ; they must be well washed to remove all debris
and are better mounted dry, as they are apt to become
too transparent when mounted in glycerine or other
media.
DECALCIFYING BONE.
It is a very different matter to make sections of a
bone after the earthy salts have been removed, as it
can be cut, as easily as any other tissue, with the freez-
ing microtome or razor.
To macerate small bones, such as mouse tail, half per
cent, chromic acid will be sufficient ; they should not be
left in too long. Larger bones must be cut into small
pieces and placed in half per cent, solution of chromic
acid for a week or ten days, and then one-twentieth of
the volume of hydrochloric acid added to the original
fluid. First harden then soften them, five or six days
after the addition of the acid will be enough according
to the size of the bones. They should then be tho-
roughly washed in water for several days or a week,
72 Muscular Tissue.
according to size, to get rid of the lime salts, and then
preserved in spirit.
Bone may be macerated in a saturated solution of
picric acid, but it does not act so well as the above.
It must be kept saturated by the addition of fresh crys-
tals.
Make longitudinal sections through the head of a
long bone such as the femur of a small kitten, double
stain with picro- carmine and logwood and mount in
Canada balsam. Take a portion of the lower jaw of a
very young kitten near the condyle and decalcify it.
When prepared cut transverse sections and double
stain them with picro- carmine and logwood and mount
in Canada balsam. Sections also through the carpus
and tarsus of a foetal child or kitten may be made and
stained in the same manner.
The easiest specimens of growing bone to be got are
from kittens just born, they should be decalcified in
chromic acid as mentioned before.
MUSCULAE TISSUE.
NON-STKIPED, STRIPED, HEART MUSCLE.
1. Non-striped Muscle. Make a preparation of the
mesentery of newt or salamander in chromate of
ammonia, page 13, and mount in glycerine. This
specimen shows better when double stained with picro-
carmine and logwood, page 36.
Examine first with a low power and see the distri-
bution of the muscle fibres through the mesentery, and
then with a high power to see the structure of the in-
Muscular Tissue. 73
dividual fibres ; with the one- sixth the network in the
nucleus can be seen but it will require a one- eighth
or Zeiss' E to make out the fibrils passing out of the
ends of the nucleus into the body of the cell, look care-
fully also for the transverse markings on the cell and
the difference of its diameter in some places, compare
their appearance with the plate in the Atlas of histo-
logy.
These fibres are very large and give a good idea of
the structure of a non- striped muscle fibre.
In the large area in which this tissue is distributed
throughout the body of a mammalian animal, the in-
dividual cells are very much smaller and their struc-
ture cannot be made out without a high power,
especially as they he very thickly together. In the
intestine, however, a thin section will show a few
fibres running up from the muscularis mucosae to the
basement membrane, and in these when well prepared
and stained the same structures can be seen as in the
much larger ones, in the mesentery of newt.
To see the intercellular substance by which the mus-
cle fibres are held together, take a small portion cut
longitudinally from the intestine and prepare it in
chromate of ammonia, page 13, and stain in logwood.
Cut longitudinal sections so that the circular muscle
coat of the intestine is cut transversely, and it will be
seen that each fibre is separated from the others by
a homogeneous substance — the intercellular cement.
There will be only a few nuclei cut through and these
will be seen deeply stained.
2. Striped Muscle. Striped muscle is best shown
74 Muscular Tissue.
in one of the large water beetles, Hydrophylus
piceus. There is another water beetle often sold
for this purpose which does not answer so well, the
Dytiscus marginalis, it may be distinguished by being
smaller and having a yellow line round the margin of
the upper surface.
Fresh Preparation of Muscle to show the Sarcolemm,a.
Kill the beetle and remove one of the legs, open the
chitinous covering and snip off a bit of the muscle,
place it on a slide in a drop of distilled water, tease it
out, cover and examine. The sarcolemma will be seen
in places raised from the muscle substance.
Irrigate the same preparation with dilute acetic acid
and the nuclei of the muscle corpuscles will soon come
into view.
To make a permanent preparation stain a small por-
tion of the leg muscle with logwood, tease it out care-
fully into as fine fibrils as possible in a drop of glycerine
on a slide, cover and seal up with Hollis' glue. Dissect
out some of the muscles of the thorax and mount in the
same way. Examine the muscles in a transverse sec-
tion of the tongue of any animal prepared in chromic
acid mixture and double stained with picro- carmine
and logwood.
3. Heart Muscle. Sections made from the heart of
any small mammal will show the peculiarities of this
variety of muscle fibre. Make transverse and longi-
tudinal sections, and stain them with logwood. Note
the position of the muscle corpuscles in the transverse
sections, and the anastomosing of the fibres in those
cut longitudinally.
Nervous Structures. 75
NEKVOUS STEUCTUEES.
MEDULLATED NERVE FIBRES.
Dissect out the sciatic nerve of a frog and stain for a
few minutes in half per cent, solution of nitrate of
silver, wash well and expose to the light in distilled
water until it has become of a brown colour ; cut small
portions and gently tease out so as to separate the
fibrils, this must be done carefully in a drop of gly-
cerine on a slide ; cover and examine.
NODES OF EANVIER.
These will be found marked by black crosses in the
course of the nerve fibre, the silver being heavily de-
posited at the node and penetrating a short distance
either way giving the appearance of a cross. These
nodes can be seen in the fresh nerve or in nerve hard-
ened and stained in the ordinary manner.
HARDENING NERVES.
Take any of the larger nerves from the animals used
for material and harden some in 2 per cent, bichromate
of potash, page 13 ; others in chromic acid mixture.
Cut longitudinal and transverse sections of each of these
and stain with logwood, mount in Canada balsam.
76 Nervous Structures.
NON-MEDULLATED NERVE FIBRE.
This is best seen in a gold preparation of the tad-
pole's tail ; it will require a high power to trace the
finest fibres, Zeiss' E or F, but the one-twelfth oil im-
mersion of Powell and Lealand with the binocular will
enable the student to form a more correct idea of the
structures he is looking at and their relations to one
another.
SPINAL CORD.
Procure a fresh spinal cord of calf from the butcher
and cut it into pieces corresponding to the different
regions. Prepare one half of each of these pieces in a
2 per cent, solution of bichromate of potash, page 13,
and the other half in chromic acid mixture ; those pre-
pared in bichromate of potash may be put in as they
are, but those in chromic acid mixture must be cut into
lengths of about half an inch. The cord must be care-
fully handled care being taken not to squeeze it, and it
should be cut with a sharp razor. Transverse sections
of these must then be cut and stained with logwood,
some may be stained with anilin blue, page 29, others
double stained with carmine and indigo- carmine, page 37.
Sections can be cut by the microtome, but the razor
must be very sharp and the cord well hardened. Very
thin sections can also be cut by the hand and each
section should be washed off the razor into spirit as
it is cut. This should not be done with a brush but
by dipping the razor into spirit. The cord prepared in
bichromate of potash will show the nerve structures,
that prepared in chromic acid mixture the neuroglia.
Nervous Structures. 77
To SHEW THE LARGE MULTIPOLAB NERVE CELLS IN THE
SPINAL CORD.
Cut a piece of spinal cord through the middle longitu-
dinally, take one side and holding it in the hand cut
out with a sharp razor as thin a slice as possible of the
anterior column, stain this deeply in picro- carmine.
Take a small portion and place it in a drop of gly-
cerine on a slide and tease out very carefully with two
needles, this must be done gently and continued for
some time until the whole piece is reduced to very
minute portions, hardly discernible except by their
colour ; cover and seal up.
If this process has been carefully done, the large cells
will be seen stained with picro-carmine and completely
isolated with processes of different lengths, according to
the care which has been exercised in teasing.
Several sections of the anterior column should be
made and after staining they should be placed on a
slide and examined with a low power ; those which con-
tain a large number of multipolar cells, will be readily
seen.
BRAIN.
Small brains, such as rabbit's, harden very well whole
by the spirit process, page 13, they must then be placed
in absolute alcohol, before doing this the different parts
should be separated, viz : frontal lobes, cerebellum,
pons, &c.
After remaining in the alcohol a few days, sections
may be cut of any of the parts to be examined and
78 Nervous Structures.
stained with logwood, which shows up the structural
elements better than any other stain.
Human brain is best hardened in 2 per cent, solution
of bichromate of potash, page 13 ; it must be well
washed until no more coltor comes away, before sections
are cut. This method is equally applicable to all large
brains.
Large sections of brain may be cut with the micro-
tome, but for Histological work small sections will be
found to show all that is required. It is a compara-
atively easy matter to cut a large section, but to pass
this section through all the different re-agents and
finally to get it laid out smoothly on the slide, without
tearing, is a very difficult matter indeed.
Bichromate of ammonia (p. 13), may be used in the
same manner as bichromate of potash, for all nervous
structures.
To show the neuroglia of the brain, it must be cut in
small pieces and hardened in the ordinary chromic acid
mixture.
PACINIAN COKPUSCLES.
These are best seen in the meso-rectum of cat, where
they are visible to the naked eye as oval bead-like
bodies. Cut out a portion of the meso-rectum and
spread it on a flat piece of cork, fasten it at the sides
by a few pins, and invert the cork in a vessel containing
a 2 per cent, solution of bichromate of potash ; let it re-
main in this for a few days, then cut it into small pieces
and wash well, place them in a very dilute solution of log-
wood, one drop to a watch glass of distilled water will
Blood Vessels. 79
be enough. They must be allowed to stain very gra-
dually, as the logwood takes some time to penetrate the
capsules. The solution of logwood must be changed
several times as it is apt to become granular. It will
take about 48 hours or longer, to stain the corpuscles
thoroughly. When they have taken in the colour suf-
ficiently, wash them well in plain water and mount in
glycerine.
Vertical sections should also be made of the pad of
a cat's foot, hardened in chromic acid mixture and
stained in logwood.
BLOOD VESSELS.
CAPILLAKIES.
Take the tail of a half-grown Tadpole or common
frog, and place it in a 5 per cent, solution of chromate
of ammonium for 24 hours to remove the epithelium,
then wash well until no colour comes away in the
water, and double stain with carmine and indigo- car-
mine (page 37). Mount in Canada balsam.
By this process the capillaries will be deeply stained
with carmine, and can be seen in their natural con-
dition.
Examine the capillaries, in a gold preparation, of
a Tadpole's tail stained with logwood.
Either of these preparations will show them in pro-
cess of development from branched connective corpus-
cles. Examine them carefully for the nuclei of the
walls, and observe in many the contained blood cor-
puscles.
80 Blood Vessels.
ARTERIES AND VEINS.
Take the aorta of the dog or cat and prepare either
in chromic acid mixture or spirit mixture (page 11).
Make longitudinal and transverse sections, stain some
with logwood and double stain others with pier o- car-
mine and logwood (page 36). Mount in Canada bal-
sam. Also make transverse sections of the whole aorta
of rabbit and of smaller arteries and veins of the same
and other animals, prepared as above. Stain with
logwood and mount in Canada balsam. Examine these
carefully to see the different amount of elastic tissue
and non-striped muscle in the different arteries.
Veins are prepared in the same manner as arteries,
but as they are so much slighter in structure it is easier
to examine them in situ in such sections as tongue,
kidney, &c.
ENDOTHELIUM OF BLOOD VESSELS.
To examine the endothelium of a blood vessel it
should be opened and then placed for two or three
minutes in half per cent, solution of nitrate of silver,
and exposed to the light in distilled water until it has
become a brown colour. Then pin it out on a cork
and tear off with the fine pointed forceps thin strips
of the intima. Mount these in glycerine.
To examine the different coats of a blood vessel
separately, it must be macerated for a few days in a
2 per cent, solution of bichromate of potash, well
washed, and strips torn off from the different coats ;
these can be then stained and mounted.
Salivary Glands — Pancreas. 81
LYMPHATIC GLANDS.
The lymphatic glands of the cat are very good for
examination, and should be perfectly fresh ; they are
best hardened in chromic acid mixture.
Thin sections should be made with the microtome
and stained with logwood. After washing, some of the
thinnest should be placed with some water in a test
tube and shaken for half an hour or more, to detach
the corpuscles from the adenoid reticulum. They must
be shaken steadily or they will be knocked to pieces.
Two or three sections only should be shaken at one
time. They are afterwards mounted in the usual man-
ner in Canada balsam.
It is a good plan to inject a solution of Berlin blue
into the lymph channels of a lymphatic gland, to de-
monstrate the passage of lymph through it. It is done
by inserting the point of a hypodermic syringe, filled
with a solution of Berlin blue, through the capsule
of a fresh gland, and slowly injecting the colouring
matter ; the gland is then prepared in the usual man-
ner, and sections cut and stained with logwood.
THYROID GLAND.
This may be prepared in precisely the same manner
as a lymphatic gland.
SALIVAKY GLANDS— PANCEEAS.
May be hardened in chromic acid mixture or in
spirit mixture and care must be taken that they are
fresh and not over hardened. Sections may be cut by
the microtome or by hand and stained in logwood.
82 Alimentary Canal.
TEETH.
Sections of hard teeth are prepared in the same way
as bone. Teeth may be decalcified by the same pro-
cess as that used for bone, page 71. Good sections of
teeth in situ may be made by removing the lower jaw of
some small animal as rat or mole and decalcifying it,
after the lime salts have been thoroughly washed out,
it should be soaked in gum for 24 hours and then cut
with the freezing microtome.
Sections may either be mounted without staining in
Canada balsam or they may be double stained with
picro-carmine and logwood.
ALIMENTAKY CANAL.
STOMACH.
The stomach may be prepared in several different
ways.
1st Method. Eemove and open a fresh stomach of
dog, cat or rabbit, and wash it slightly in dilute chromic
acid, then place it in the ordinary chromic acid mixture
and proceed as described at page 11.
2nd Method. To show the peptic cells take fresh
stomach and wash quickly, then plunge into pure me-
thylated spirit.
3rd Method. Put the stomach in Muller's fluid un-
washed, for 48 hours. Then cut narrow strips of the
mucous membrane about half an inch long by one
eighth of an inch wide and wash these in one-tenth per
cent, osmic acid, then place them in half per cent.
Alimentary Canal. 83
osmic acid from one to two hours to stain, then place
them in one- sixth per cent, chromic acid and complete
the hardening in the usual manner. The one- sixth
per cent, chromic acid is here used without the addi-
tion of methylated spirit.
Some sections of stomach should be taken from the
different parts, pylorus, cardiac end, &c., and these
should be stained in logwood ; those which are required
to show the peptic cells should be stained in anilin blue.
Pyloric end of stomach with commencement of duo-
denum should be hardened in chromic acid mixture or
if the whole stomach has been hardened a portion show-
ing the junction of these two parts should be cut with
the microtome and double- stained, a few sections also
should be stained with logwood. Examine these sec-
tions for the gradual change in the epithelium as the
one organ passes into the other.
DUODENUM
May be hardened in chromic acid mixture and sec-
tions stained with logwood.
Notice Brunner glands cut in different sections, also
goblet cells amongst the columnar epithelium and the
fine non- striped muscle fibres running up from the
muscularis mucosae to the basement membrane.
ILEUM.
The whole of the intestine may be hardened in
chromic acid mixture or in spirit mixture, page 11, it
must not be much handled and should be first slightly
washed in very weak solution of chromic acid. Sec-
G2
84 Liver.
tions are best cut with the freezing microtome and may
be stained in a great many different ways.
In a section containing a portion of Peyer's glands,
the treble staining process, page 39, may be used, and
the result will be very good as the Peyer's glands take
on the green alone, without combining another colour
with it, as all the other elements in this specimen do,
so that they are brought out as brilliant light green
bodies.
ILIO-CCECAL VALVE.
A section should be made through the ilio-coecal
valve with a little of the intestine on either side of it,
and this should be trebly stained by the process men-
tioned at page 39. Some sections should, however,
always be stained with logwood to compare with the
others, as although double and treble staining differ-
entiate the various tissues, logwood brings out the
structural element better than any other stain.
SOLITARY GLANDS.
Sections should be made through a piece of large
intestine containing a solitary gland and this will be
weU brought out by the treble staining process.
LIYEE.
The liver may be prepared for examination in three
ways : —
1. By the ordinary chromic acid mixture, page 11.
2. By dilute spirit, page 13.
3. By Muller's fluid, page 12.
Liver. 85
1. The Chromic Acid method. The liver must be per-
fectly fresh and cut into small pieces about half an inch
square, these should be placed at once in the fluid
without washing. A large quantity of blood will exude
from them after being in the hardening fluid a short
time, and it will be necessary to change it in many cases
at the end of 12 hours. It is a good plan to shake the
bottle containing the specimen in process of hardening
occasionally, this must be done gently, so as to just alter
their position, and when there is a quantity of sediment
at the bottom of the bottle, and the fluid has lost its
yellow color and begins to look muddy, it is time to
change it. Portions of liver require changing a little
oftener at first than other normal structures.
2. The spirit method is used in the ordinary manner
for liver, but the dilute spirit will generally require
changing once before using the pure methylated spirit.
3. Midler's Fluid. This may be used when large
portions of the organ are to be hardened and when
time is no object.
When the material is well hardened by either of these
processes, beautiful sections may be cut with the freez-
ing microtome, and they show best when stained with
logwood.
The intra-cellular and intra-nuclear network is seen
very well in the cells of the liver, and makes an in-
teresting object for a moderately high power. The
specimens should be searched for bile ducts cut trans-
versely, looking like minute triangular openings between
the cells.
86 Kidney.
LUNG
May be hardened in either chromic acid and spirit,
or Muller's fluid, but to harden it well the fluid must
be injected into the lung through the trachea. This is
very easily done : the lung having been removed with
a portion of the trachea attached, an ordinary brass
syringe with ivory nozzle is filled with the hardening
fluid, the nozzle inserted in the trachea, and the lungs
gently distended with the fluid ; when sufficiently full,
the trachea is tied and a weight attached. The lungs
are then placed in a tall vessel containing the harden-
ing fluid, which is changed as often as necessary.
To SHOW THE EPITHELIUM OF THE ALVEOLI
Inject through the trachea a f per cent, solution of
nitrate of silver, and then harden the lung by the spirit
process (page 13), and make horizontal sections, these
must be rather thick to get a correct idea of the epithe-
lium as lining a cavity. These specimens will show the
stomata between the epithelial cells.
Lung is best stained with logwood. Sections should
be made through a bronchus and the small masses of
ganglionic cells examined, these same sections may also
be double or treble stained to differentiate the glands
of the bronchi.
KIDNEY.
This organ is very well hardened in chromic acid
mixture.
Remove the kidneys from a freshly killed animal,
Kidney. 87
take one and divide it transversely into several pieces
and place them in the fluid. The other may be divided
longitudinally by one cut, and large kidneys may be
hardened in chromic acid in this way. Sections
may be cut by the freezing microtome, and are best
stained in logwood, or double stained in carmine and
indigo- carmine (page 37). To show the minute struc-
ture of the cells in the collecting tubes, fine striation of
epithelium, &c., Heidenhain's method is the best.
Cut the kidney into small pieces longitudinally in the
direction of the pyramids, and place them in a 5 per
cent, solution of chromate of ammonia, from 24 — 48
hours in a stoppered bottle, then wash for several hours
until no more color comes away, changing the water
several times, and place in dilute and then in strong
spirit in the ordinary way (page 13).
Sections should be made both vertical and transverse
of the cervical and medullary portion of the kidney, as
well as large sections longitudinal and transverse of the
whole organ ; by this mean ' the general structure of
the organ and the arrangement of the tubes can be
examined, while in the smaller sections, some of which
should be mounted under thin covers, the minute struc-
ture can be studied ; for this no stain succeeds so well
as logwood.
Examine sections of the cortical part for the striation
of the epithelial cells, and notice the imbrication in
some parts.
BLADDER — URE TER .
Cut into small pieces and harden in chromic acid
mixture.
Genital Organs — Male.
To SHOW GANGLIA OF BLADDER.
Eemove the bladder, empty it, and place in \ per cent,
solution of gold chloride 2 hours in the dark. Then
place it in water well acidulated with acetic acid, until
it has become swollen up to a good size, then preserve
it in glycerine.
The gold will be seen deposited in small patches here
and there on the surface, one of these is removed with
a pair of curved scissors and mounted in glycerine.
GENITAL OEGANS— MALE.
TESTIS.
The best hardening fluid for the testis is undoubtedly
the chromic acid mixture. The organ is cut into small
pieces, or deep cuts made into it with a sharp razor,
according to the size. It must not be washed and
should be handled as little as possible.
Sections are made in different directions through the
various parts, and it is advisable to make some large
sections through the corpus Highinori and globus major
to show the structure and relation of the different
parts ; some of these sections will show the epididy-
mis as well. These sections if at all large are very
difficult to mount, as they break to pieces on the lifter,
and it is often impossible to get them on to the slide
whole ; in this case the best plan is to use the cover
glass as a lifter, and clean it afterwards, when the
Canada balsam has set.
Sections of testis are best stained in logwood, or
double stained in picro-carmine and logwood (page 86).
Genital Organs — Male. 89
Some of the thinnest sections should be mounted
under *003 cover glasses, as the developing sperma-
tozoa form very interesting objects for the highest
powers.
EPIDIDYMIS AND VAS DEFEKENS
Are hardened in the same way as testis, and epididy-
mis is generally hardened and cut with the testis.
Stain with logwood.
Examine the columnar epithelial cells of the epididy-
mis with very long ciliary processes. With a good
object glass of moderate power these processes can be
seen to be continuous with the longitudinal striation in
the body of the cell, and can be traced by careful
focussing through the striated line at the margin of the
cells.
PKOSTATE, GLANS, ETC.
These can all be hardened in chromic acid mixture
in the usual manner. The glans may be placed in gold
chloride for two hours and then hardened in spirit,
after which longitudinal sections will show the nerve
structures. The glans of a small animal hardened in
this way and cut longitudinally, will show the differ-
ence between the epithelium of the mucous surface and
that in the meatus and commencement of the urethra ;
it will also show large bundles of medullated nerves
arranged in a peculiar manner, and many other things
worth studying.
90 Spermatozoa.
GENITAL OKGANS— FEMALE.
UTERUS, FALLOPIAN TUBES, VAGINA.
Can all be hardened in chromic acid mixture, and
should not be washed unless absolutely necessary.
To examine the glands of the uterus it is better to
use an animal that has borne young.
These organs may also be hardened whole in Muller's
fluid or bichromate of potash. The sections are best
stained in logwood.
OVARY
Is best hardened in chromic acid mixture and should
not be handled more than is absolutely necessary. The
whole organ may also be hardened in Muller's fluid.
MAMMARY GLAND.
Cut small pieces and place in chromic acid mixture,
when hardened stain with logwood.
PLACENTA.
The placenta of guinea-pig is the best for examina-
tion, and should be taken a little after half the period
of gestation has passed. It may be prepared in chro-
mic acid mixture or in spirit mixture. Vertical sections
should be cut as thin as possible, and stained with
logwood.
SPEBMATOZOA.
The living spermatozoa of Triton cristatus make a
most beautiful preparation and are readily procured.
Spermatozoa. 91
Take a large male newt which may be known by the
serrated crest or fin along the back, and kill it, quickly
remove the viscera, and the testes will be found, gen-
erally two or three each side, as small round bodies
which cannot easily be mistaken. Take one of these and
make a small cut in it, remove some of the milky fluid
which exudes to a slide, and add a little salt solution or
distilled water. Cover and examine. A power about
i will be required to see the spiral filament well.
A large number of spermatozoa will be seen in the
field making slight lashing movements with the long
filiform body, and on closer examination the filament
will be seen in rapid movement ; this movement com-
mences at the elliptical body at the base of the head,
and gives the idea at first sight that the filament is
being poured out from it. After watching for some
little time, the movement will become slower, and it
can then be seen that the filament is attached to the
body by a membrane, and that it is waved rapidly
from side to side, by carefully watching it as the
motion gets slower and nearly stops the -membrane
connecting the filament to the body, can be clearly
seen.
To MAKE A PERMANENT PREPARATION OF NEWT'S
SPERMATOZOA.
Place the testes in 5 per cent, solution of chromate of
ammonia for 24 hours ; wash until no colour comes
away in distilled water, then divide one of the testeg
in two, and taking one half in a pair of forceps press
the cut surface on a glass slide, a small quantity of
92 Spermatozoa.
fluid will adhere to the slide, to this add a small drop
of glycerine and gently mix the two fluids. Cover and
examine.
To STAIN NEWT'S SPEKMATOZOA.
After having washed away all traces of the chromate
of ammonia, make an incision into the testis nearly
dividing it, and place it in undiluted logwood stain for
an hour or more, then wash away all superfluous stain
and mount in glycerine. Spermatozoa may be double
stained, but it is a very tedious process, as a little too
long immersion in either stain spoils the whole pro-
cess. The best way is, after washing off the super-
fluous logwood stain, to dip a portion of the testis in
undiluted spirituous solution of rosein ; a little must be
mounted to see if the stain is deep enough, if not it
must be dipped again.
It is possible in this way to get spermatozoa of newt
or salamander with the long pointed head stained with
rosein, while all the other parts are stained with log-
wood.
MAMMALIAN SPEKMATOZOA.
To make preparations of mammalian spermatozoa,
a little glycerine is placed in a watch-glass, and one or
two drops of absolute alcohol added. A cut is then
made into the globus major of a fresh testicle, and a
little of the fluid removed on the point of a knife and
placed on a slide ; a small drop of the glycerine is then
mixed with it, it is then covered and sealed with HolhV
glue.
Special Senses. 93
HUMAN SPERMATOZOA.
A small drop of semen is mixed with glycerine, to
which a little absolute alcohol has been added ; it is
then covered and sealed up. It is best in this case to
use thin covers that have been measured, and to note
their thickness on the label, as the human spermato-
zoon is so very minute it requires the highest powers
to make out the filament.
SPECIAL SENSES.
INTERNAL EAR. COCHLEA.
The guinea-pig is the best animal, as the large tym-
panic bulla is easily exposed. Eemove the periosteum
from the bulla, and open it carefully with the point of a
pair of straight scissors ; as soon as a small opening
has been made it can be enlarged, and the cochlea will
be at once seen : as much of the surrounding bone
must be removed as can be done without injury to the
parts required, and it will be ready for preparation.
This may be done in several ways, but the two fol-
lowing are the best.
1. Place it at once in absolute alcohol, and let it
remain 24-48 hours, then place it in ^ per cent, solu-
tion of osmic acid for 24 hours. After this place it
in a half per cent, solution of chromic acid, to which
1-2 drops of hydrochloric acid have been added. Let
it remain in this until the bone is softened throughout.
2. Place the piece of bone in the ordinary chromic
acid mixture for a week, and then remove it to £ per
cent, chromic acid, to which 1-2 drops of hydrochloric
94 Special Senses.
acid have been added. Kemove when the bone is
softened.
When the bone is decalcified by either of these pro-
cesses, it must be washed for several days, to remove
the lime salt. It is then ready for cutting sections.
In the case of the internal ear, sections are much better
cut by hand, as the fine structures such as the rods of
Corti are quite disarranged by freezing, and cutting
with a microtome.
The bone must now be placed in gum solution, for
24 hours, and then removed to spirit slightly diluted
with water. If the spirit is too strong, the gum will
form a substance like chalk, and quite as hard ; this
can, however, be softened by placing it in water. When
the gum is sufficiently hardened by the spirit, it can be
embedded in wax mass, and sections cut by hand in the
ordinary manner. These sections must be very gently
handled, and should not be lifted with a needle but with
a fine camel hair pencil. They are best stained in log-
wood. It is a good plan to stain the whole bone in
logwood before placing it in the gum solution, but care
must be taken that it is not too deeply stained. The
undiluted logwood should be used, and it will require
about 6 hours or more to stain it thoroughly.
Sections must be cut through the semi-circular canals
and also through the cochlea, and the cochlea should
be so embedded that sections will be cut through its
whole length.
Eye. 95
NASAL OEGAN.
The nasal organ is prepared in the same manner as
the internal ear, and can be conveniently removed and
hardened with it. Transverse sections should be made
through the anterior part to show the membrane of the
respiratory part and transverse sections further back,
and show the olfactory membrane with its peculiar
epithelium.
The septum of the nose should also be carefully re-
moved from a specimen, and longitudinal sections made
of it : if these sections are very carefully handled, they
will show both the respiratory and olfactory epithelium
very well. They are best stained in logwood and
mounted in Canada balsam.
EYE.
The eye may be hardened in chromic acid mixture
or in Muller's fluid. It must be removed without
squeezing, and a few incisions made, it can then be
hardened whole. When sufficiently hard, divide the
eye longitudinally with a sharp razor.
RETINA.
The retina will be found lying on the inside of the
posterior part of the eye, from which it may be gently
detached by a spear-shaped knife, it may be then frozen
and sections cut ; it is better, however, to stain it first,
as thin sections are so transparent it is difficult to see
them. When prepared in Muller's fluid the retina is
96 Eye.
very brittle, but in chromic acid mixture it is much
tougher and can then be cut in strips and several frozen
together in the microtome. Muller's fluid shows the
nervous structure best, chromic acid the connective
tissue. Good sections of the retina may be obtained
by cutting the whole eye of the frog, and it may be
double stained first.
To do this, first place the whole eye in a strong solu-
tion of rosein until it is deeply stained, then wash away
the superfluous stain in methylated spirit, next place
the eye for a short time in strong solution of iodine
green and wash it well, soak in gum solution and freeze.
By this means very good sections can be obtained, the
granular layers having stained with the iodine green,
the others with rosein.
They must be mounted in Canada balsam and should
not be left long in spirit.
COKNEA.
The cornea may be removed from an eye hardened
either in chromic acid mixture or in Muller's fluid, and
sections made with the freezing microtome ; they are
best stained with logwood.
To demonstrate the Corneal Corpuscles and Nerves.
Eemove the cornea from an animal just killed : this
is done by cutting round the margin with fine curved
scissors. Place the cornea in i per cent, solution of
gold chloride in the dark. Let it remain for from one
hour to one hour and a half for a guinea-pig, an hour
Eye. 97
and a half to two hours for a rabbit. Then place it in
distilled water, which must be changed once or twice,
for 24-36 hours exposed to the light, it will then have
become a violet colour.
It is now placed in a mixture consisting of
Pure glycerine, 1 part.
Distilled water, 2 parts.
Let it remain in this for two or three days in the
dark. It is then taken out, and gently washed and
placed in a wide mouthed vessel containing a filtered
nearly saturated solution of tartaric acid. As it ab-
sorbs this liquid, the colour will become darker and it
will sink to the bottom of the vessel. The vessel is
now plunged into water at a temperature of 40° to 50° C.
to such a depth that the two fluids will stand at the
same height.
Sections may now be made with a very sharp razor
by holding the cornea between the finger and thumb of
the left hand. This requires great care, and cannot be
done without a good deal of practice. The sections
are mounted in glycerine and sealed with Hollis' glue.
Before cutting the sections, while the cornea is still
in the distilled water, it is well to pass a camel's hair
pencil gently over the surface, to remove the gold de-
posited there.
IBIS AND SCLEROTIC.
A portion of the eye containing part of the cornea
sclerotic and iris may be cut out, and frozen. Sections
of this stained with logwood, show the junction of the
cornea and sclerotic, and the structure of the iris.
H
98 Eye.
LENS.
In cutting sections of the whole eye of frog, good
sections of the lens can be obtained. In the whole eye
prepared by either of the methods given, the lens will be
found hardened, when the eye is opened ; and sections
may be cut by the freezing microtome, but they gene-
rally break up if thin ; enough, however, can be
obtained to enable the student to examine into the
structure.
PRACTICAL PATHOLOGY.
ON PREPARING AND MOUNTING PATHOLOGICAL SPECIMENS.
VERY little need be said about preparing pathological
specimens, as most of the processes already mentioned
will apply equally well to morbid tissues. It is often
necessary to find out at once what a tumour or new
growth consists of, and for this purpose it is necessary
to examine the fresh tissue. In some cases this is
sufficiently hard to be frozen and sections made, while
in others a small portion can be teased out on a slide
and examined.
This may give a rough idea of what the morbid
growth is composed, but for a thorough examination
and when it is desired to keep preparations of any
disease, a complete process of hardening must be
adopted, and sections cut and stained as in normal
tissues, and for this purpose it is necessary to have the
material as fresh as possible.
To MAKE PERMANENT PREPARATIONS OF A CANCER IN A
SHORT TIME.
This method may be applied to most of the sarco-
mata and carcinomata, and is valuable, as it can be
used on portions of morbid tissue excised from the liv-
ing body. Snip off a small portion of any morbid
growth, such as cancer, with a pair of curved scissors.
H2
100 Morbid Growths.
Place it in a mixture of dilate spirit (page 13) for 12
hours, then remove it to pure methylated spirit for 12
hours and finally to absolute alcohol for 12 hours. It
will then in all probability be fit to cut sections from.
Imbed in wax mass and cut some sections by hand,
stain with logwood and mount in Canada balsam.
There are some morbid growths, such as medullary
carcinoma, which cannot be dealt with in this way, and
they must be hardened in the chromic acid mixture in
the usual manner.
In all morbid growths where there is a large epi-
thelial element, they can be best hardened in spirit
mixture. When large masses are to be hardened,
Muller's fluid or 2 per cent, bichromate of potash are
necessary, and must be used in the manner described
for normal tissue at page 12.
It is quite as important that pathological specimens
should be properly hardened as normal tissues, but
how seldom is this done. In the first place it is diffi-
cult to get the morbid tissues fresh enough, and yet
they are often put on one side or at most placed in the
lump in a very small quantity of methylated spirit for
some time before being hardened, and it is expected
that good sections can then be prepared from them.
Nothing is more erroneous than this idea ; the sub-
ject has been probably dead 24 hours at the least when
the post mortem is made, often longer, and in summer
especially, this means utter ruin to such organs as the
spleen. How important is it therefore that such organs
as are fresh should be put in the hardening medium at
once. For this purpose a wide-mouthed bottle of chro-
Morbid Growths. 101
mic acid mixture should be taken to every post mortem
examination, and small bits of any organ that may
seem interesting on any account may be put in. A
small paper label may be tied on and they can be
separated afterwards.
Almost every morbid growth can be hardened in the
chromic acid mixture (page 11), in the same manner
as normal tissues. Brain and spinal cord, however,
are better prepared in 2 per cent, solution of bichro-
mate of potash (page 13).
ON DOUBLE AND TREBLE STAINING MOBBID GEOWTHS.
Some very good results will be obtained if the differ-
ent staining processes mentioned, as well as any others
that may suggest themselves, are tried on different
morbid growths.
For instance, well hardened sections of rodent ulcer
and epithelioma may be stained by the indigo- carmine
process (page 37) and carefully compared. Other sec-
tions of the same material should be then stained with
rosein and iodine green (see treble staining) and again
compared. In this way, some definite result may be
worked out, which by using other specimens of the
same disease may be confirmed.
LAUGHS SECTIONS OF PATHOLOGICAL SPECIMENS*
If a large section is wanted of any morbid growth to
show the distinction between the healthy and diseased
parts, such as a section through a cancer and the side
of the uterus from which it is growing, it is better to
102 Hydatids.
cut a moderately thick slice, say about a quarter of an
inch thick, and harden it in chromic acid mixture, than
to harden the whole mass in spirit, as it will be found
a very difficult matter to cut large sections of spirit
hardened material ; they become so hard that they
make the knife jump, and the section is consequently
uneven.
AMYLOID DEGENEKATION
Is best hardened in chromic acid mixture, and in
kidney especially is very well shown by this method.
To show the amyloid substance well it must be stained
a different colour to the surrounding tissue, and this
may be done by several of the double staining pro-
cesses ; as the anilin dyes seem to have a special af-
finity for amyloid degeneration, the following should
be tried :
Carmine and indigo carmine (page 87).
Eosein and iodine green.
Eosin and anilin blue.
and many other combinations.
HYDATIDS.
To make a preparation of hydatid cysts, take a por-
tion of the wall of a large cyst and scrape off some of
the gelatinous matter adhering to it. Place a little of
this on a slide and tease it gently in a drop of glycerine,
cover and examine. If there are any small cysts show-
ing booklets, &c., well, seal it up with HolhV glue.
Morbid Growth. 103
SHORT HISTORY OF THE MANNER IN WHICH A PORTION OF
MORBID GROWTH is PREPARED BY THE CHROMIC ACID
METHOD.
1st day. Small pieces placed in chromic acid mix-
ture (page 11).
2nd day. Fluid changed.
5th day. Fluid changed.
8th day. Fluid changed.
9th day. Spirit mixture (page 13).
10th day. Pure methylated spirit.
14th day. Plain water.
15th day. Mucilage.
16th day. Section cut, stained, and mounted.
INDEX.
Absolute alcohol, 49
Acetic acid, 27, 36, 68
Achromatic condenser, 8
Adenoid reticulum, 81
tissue, 69
Air bubbles, 51
Alimentary canal, 82
Amoeboid movement, 60
Amyloid degeneration 102
Anilin blue, 39, 83
red, 32
violet, 32
dyes, 28
list of, 22
soluble in spirit, 31
soluble in water, 28
Anthra-purpurin, 25
Arteries, 80
Aurine, 33
Beetle, 74
Berlin blue, 81
Bicarbonate of soda, 24, 37
Bichromate of ammonia, 13
— potash; 13, 68
Binocular microscope, 9
Bismarck brown, 31
Bladder, 87
Blood, 60
vessels, 79
Bone, 71, 72, 94
decalcifying, 71
Boracic acid, 61
Brain, 56, 77, 101
human, 73
Bronchus, 86
Brunner's glands, 83
Cabinet, 52
Canada balsam, 48
Capillaries, 78
Carmine, 24, 37
Cartilage, 69
Cells, tendon, 67
Cerebellum, 77
China blue, 28
Chloride of gold, 14, 42
Chloroform, 49
Chromate of ammonia, 13
Chromic acid, 11, 24
Cilia, 65
Ciliated epithelium, 64
Citranine, 33
Cleaning cover glasses, 45
slides, 44
Cochlea, 93.
methods of hardening,
93
Columnar epithelium, 63
Condenser, 8
stand, 8
Connective tissue corpuscles, 66,
79
Cornea, 96
Corneal corpuscles, 96
Corpus Highmori, 88
Cover glasses, 44
Cutis vera, 69
Cutting sections, 16, 17
with micro-
tome, 19.
Dahlia, 30
Dammar varnish, 48
Decalcifying bone, 14, 42
Dilute spirit, 13
Distilled water, 20, 23
Double staining, 34, 36, 37
Duodenum, 83
Dytiscus marginalis, 74
Ear, 70
Elastic tissue, 68
Endothelium, 65, 80
Eosin, 26, 39
Epididymis, 65, 88
106
Index.
Epiglottis, 70
Epithelioma, 101
Epithelium, 62
Epithelial cells, striation of, 87
Eye, 58, 95
Eye-piece, 7
Fallopian tubes, 90
Feeding blood corpusles, 61
Fibro-cartilage, 70
Freezing microtome, 18
— mixture, 19
Labels, 2
Lamp, 8
Large slides, 54
Lens, 98
Lifter, 50
Ligamentum nuchse, 68
List of apparatus, 3
Liver, 84
Logwood stain, 23, 37
Lung, 86
Lymphatic gland, 81
Malachite green, 30
Mammary gland, 90
Measuring cover glasses, 44
Medullary carcinoma, 100
Medullated nerves, 89
Mesentery, 27, 37, 58, 68
Meso-rectum, 78
Methyl- a nilin violet, 30
Microscope, 4
Microtome, 18
Mounting, 50
fluids, 46
Frog, 58
Ganglia of bladder, 87
Germinating cells, 66
Glans, 89
Glass capsules, 17
Globus major, 88
Glycerine, 46
Gold chloride, 88
Gauge for cover glasses, 45
Guinea pig, 90, 93
Gum solution, 94
Hardening, 11
Hsemin, 62
Heart, 56
f1 yj 5I^°r^gn^'
large sections, 53
Mouse tail, 14, 43, 68, 71
Mucilage, 20
Muller's fluid, 3, 12, 85
Multipolar nerve cells, 77
Muscular tissue, 74
Muscularis mucosse, 73
Muscle corpuscles, 74, 75
Nasal organ, 95
Nerves, 58, 75
High powers, 6
Hollow ground razor, 17
Hydatids, ]02
Hydrochloric acid, 37, 71
Hydrophylus piceus, 74
Hypodermic syringe, 81
Ileum, 83
Ilio-coecal valve, 84
Illumination, 8
Imbedding, 16
Indigo-carmine, 25, 37
Intestine, 56, 73, 83
Intercellular substance, 73
Internal ear, 93, 94
Intima, 80
Intr a- cellular network, 85
Iodine green, 30, 39
Iris, 97
Kidney, 57, 86
Neuroglia, 76, 78
Newt, 91
Nodes of Ranvier, 75
Non-striped muscle, 72
Object glass, 5
Oil immersion lenses, 6, 10, 76
Olfactory organ, 95
Umentum, 68
Osmic Acid, 15, 69, 83, 95
Ovary, 90
Killing animals, 56
Index.
107
Pacinian corpuscles, 78
Pad of foot, 79
Pancreas, 81
Pathology, 99
Penis, 57
Peyer's glands, 39, 84
Picric acid, 14, 27, 72
Picro- carmine, 27, 36, 39
Placenta, 90
Pons, 77
Preparing cancer, 99
morbid specimens, 103
Prickle cells, 63
Prostate, 89
Pure opal blue, 33
soluble blue, 29
Purpurine, 25
Pylorus, 83
Reagents, 3
Eespiratory epithelium, 95
Retina, 21, 95
Eodent ulcer, 101
Rods of Corti, 94
Rosanilin, 30
Rosein, 32
Safranine, 29, 39
Salamander, 59
Salivary gland, 81
Sarcolemma, 74
Sclerotic, 97
Sealing preparations, 48
Selective stains, 34
Semicircular canals, 94
Serous membrane, 69
Serge blue, 29
Silver process, 65
Skin, 27
Slides, 44
Soluble anilin blue, 28
Solitary glands, 84
Spermatozoa, 59, 89, 90
Spiller's purple, 31
Spinal cord, 28, 76, 101
Staining, 22
Stand condenser, 8
Stomach, 28, 56, 59, 82
Striped muscle, 73
Tadpole's tail, 14, 66, 74, 76
Tannic acid, 61
Teeth, 82
Tendon, 67
. ~Dna an
Testis, 57, 59, 88
— - nf-no-ctrf O1
Thin slides, 55
Thyroid gland, 81
Treble staining, 27, 39
Triton cristatus, 90
Tubuli seminiferi, 37
Tympanic bulla, 93
Tyrian blue, 29
Urethra, 89
Ureter, 87
Uterus, 90
Vas deferens, 89
Vagina, 90
Veins, 80
Warm stage, 60
Wax and oil mixture, 16
White fibrous tissue, 68
Printed by H. K. Lewis, 136 Gower Street, London, W.C.
On one sheet, gummed at back, price 6d.
HISTOLOGICAL LABELS
.FOR
MICEOSCOPE SLIDES.
About 100 Printed Labels, being those most required by Students.
LONDON : H. K. LEWIS, 136 GOWER STREET.
MEMOEANDA AND FOEMULJi.
DEC 3 1930
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