PRACTICAL HISTOLOGY

AND

THE MICROSCOPE.

A COURSE

PRACTICAL HISTOLOGY:

BEING AN

INTRODUCTION TO THE USE OF THE
MICROSCOPE.

BY
EDWARD ALBERT SCHAFER,

ASSISTANT-PROFESSOR OF PHYSIOLOGY IS UNIVERSITY COLLEGE, LONDON.

WITH ILLUSTRATIONS ON WOOD.

PHILADELPHIA:

HENRY C. LEA.

1877.

S3

£58

6

PHILADELPHIA ;
COLLINS, PRINTER

7()."» Jnyue Street.

PREFACE.

THE PURPOSE of this work is to afford to those
engaged in the practical study of Histology, plain
and intelligible directions for the suitable prepara-
tion of the animal tissues ; with the object either
of immediate study, or of their preservation as spe-
cimens for future reference. The methods recom-
mended have all been tested by experience.

In an introductory chapter an account is given
of the several parts of the microscope, and the pur-
pose for which they are intended, without entering
into an explanation of its optical construction ; and,
in an Appendix, instructions wrill be found for
measuring and for delineating microscopic objects.

Throughout the book descriptions of tissues have
been purposely avoided, seeing that these are to be
found in systematic works. The order followed is
that of "Quain's Anatomy," 8th Edition.

Vlll PREFACE.

With the exception of Figure 12, which is taken
from Dr. Burdon Sanderson's " Handbook for the
Physiological Laboratory," tLe illustrations have
been prepared expressly for this work ; they are
from the pencil of Mr. Collings.

The methods employed in the practical study of
Embryology have been omitted ; they will be found
admirably given in the " Elements of Embryology"
of Dr. M. Foster and Mr. F. M. Balfour.

UNIVERSITY COLLEGE, LONDON :

November, 1876.

CONTENTS.

INTRODUCTORY.

PAGE

Essential parts of the microscope — Accessory parts — Appli-
ances for microscopic work — General directions for work 13

CHAPTER I.

THE BLOOD.

Examination of human blood — Methods of warming — Ac-
tion of reagents — Mode of applying gases or vapors to a
preparation — Frog's blood — Phenomena exhibited by
white corpuscles — Action of reagents on frog's blood —
Mode of applying electricity to a preparation — Blood-
crystals 25

CHAPTER II.

THE EPITHELIAL TISSUES.

Scaly stratified epithelium — Methods of separating epithelial
cells — Columnar epithelium — Iodized serum — Fixation of
cover-glass — Ciliated epithelium — Ciliary motion — Action
of reagents on ciliary motion . . . . .56

CHAPTER III.

CONNECTIVE TISSUE.

Areolar tissue — Fibres — Corpuscles — Action of acetic acid —
Method of localized oedema — Ground substance and cell
spaces — Silver method — Elastic tissue — Fibrous tissue —
Tendon cells and cell spaces — Adipose tissue ... 70

X CONTENTS.

CHAPTER IV.

CARTILAGE.

PAGE

Articular cartilage — Action of water on cartilage cell — Pre-
servation of cartilage cell — Treatment with nitrate of
silver — Treatment with chloride of gold (Cohnheim's
method) — Warming apparatus — Costal cartilage — Fibro-
cartilage — Staining of cartilage matrix with logwood . 89

CHAPTER V.

BONE.

Mode of grinding sections — Effect of presence of air in
cavities — Modes of decalcifying bone — Structure of
lamellae — Siiarpey's fibres — Process of ossification —
Miller's fluid— Marrow 100

CHAPTER VI.

MUSCULAR TISSUE.

Involuntary muscle — Isolation of cells — Arrangement of
cells shown by silver method — Voluntary muscle of mam-
mals— Action of acetic acid — Frog's muscle — Sarcolemma
— Separation of fibre into disks and fibrils — Isolation of
fibres — Living muscle of water-beetle — Contraction of
muscle — Examination by polarized light — The polarizing
microscope — Sections of frozen muscle (Urban Prit-
chard's method of freezing) — Ending of muscle in tendon 112

CHAPTER VII.

NERVOUS TISSUE.

Medullated nerves — Action of osmic acid — Non-medullated
nerves — Ensheathment of nerve — Silvered nerves — Mode
of isolating cells of spinal cord — Ganglion cells — Pacinian
corpuscles — Motor end-plates 126

CONTENTS. XI

CHAPTER VIII.

THE BLOODVESSELS.

PAGE

Epithelioid lining (silver preparation) — Elastic and mus-
cular layers — Method of preparing sections — Embedding
mass — Process of embedding — Mode of cutting sections —
Treatment of sections — Smaller bloodvessels — Epithelioid
lining — Muscular structure — Study of circulation in frog's
web — In mesentery — In mammals — In lung of toad— In
tongue — Inflammatory changes — Mode of injecting blood-
vessels— Preparation of injection — Preparation of Berlin
blue — Injecting apparatus — Injection of an animal —
Treatment of injected ^issues 138

CHAPTER IX.

LYMPHATICS AND SEROUS MEMBRANES.

Omentum (silver preparation) — Central tendon of dia-
phragm— Lymphatic septum of frog — Injection of lym-
phatics— Mercurial apparatus — Method of puncture in-
jections— Injection of lymphatics of tendon — Treatment
of injected preparation — Lymphatics of diaphragm —
Natural injection — Synovial membranes — Bloodvessels
of synovial membranes — Haversian fringes — Lymphatic
glands 172

CHAPTER X.

THE SKIN, HAIRS, AND NAILS.

Mode of hardening — Method of double staining — Tactile
corpuscles — Bloodvessels of skin — Stirling's digestion
method — Hairs — Sections of hairs, gum method — Nails —
Embedding in gum — Deferred preparations — Bloodves-
sels of muscle — Sections of nerve trunk — Lymph-spaces
of perineurium — Sections of ganglia .... 185

CHAPTER XI.

THE HEART.

t

The cardiac pericardium — Muscular substance — The endo-
cardium— Fibres of Purkinje— Lymphatics of heart . 193

Xll CONTENTS.

CHAPTER XII.

THE LUNGS.

PAGE

The pulmonary pleura — The costal pleura — Mode of hard-
ening lung — Embedding in cacao-butter — Kleinenberg's
logwood — Epithelium of air-cells shown with nitrate of
silver — Mode of injecting lungs — Larynx and trachea —
Thyroid and thymus 196

CHAPTER XIII.

THE MOUTH AND PHARYNX.

Mucous membrane of mouth— The teeth — Sections of hard
tooth — Sections of softened tooth — foentinal sheaths —
Pulp of the teeth — Decalcification with picric acid —
Teeth in situ — Development of the teeth — Staining with
carmine — The tongue — Mode of hardening of tongue —
Conditions of mucous glands — Bloodvessels of tongue —
Palate and tonsils— .Salivary glands — Different condi-
tions of salivary glands — Salivary cells treated with
osmic acid 204

CHAPTER XIV.

THE (ESOPHAGUS AND STOMACH.

Mode of hardening gullet — Embedding membranous viscera,
direction of sections — Vessels of gullet — The stomach —
Distension with chromic acid and spirit mixture — Gastric
glands — Cardiac and pyloric mucous membranes — Stain-
ing of sections with carmine and aniline blue — Cross-
sections of glands: — Cells of glands — Bloodvessels and
lymphatics of stomach 210

CHAPTER XV.

THE SMALL AND LARGE INTESTINE.

Sections of small intestine — Duodenum — Jejunum — Ileum
— Process of fat absorption — Bloodvessels and lymphatics
of small intestine — Nerves of intestine shown by gold
method — Plexus of Meissner — Plexus of Aucrbach —
Large intestine . .. 217

CONTENTS. Xlll

CHAPTER XVI.

THE LIVER.

PAGE

Mode of hardening — Direction of sections — Portal canals —
Capillaries of lobules — Perivascular lymphatics — Inter-
cellular bile channels — Mode of injecting bloodvessels of
liver — Injection of bile-ducts with Berlin blue — Injection
of lymphatics of liver — Study of liver ceils — The pancreas 223

CHAPTER XVII.

THE SPLEEN AND URINARY ORGANS.

Mode of hardening spleen — Washing out of bloodvessels —
Injection of spleen — Teased preparations of spleen — Reti-
form tissue — The kidney — Mode of hardening sections of
cortical and medullary parts — Transverse section of me-
dullary part — Human kidney — Injected kidney — Isolation
of tubules — Fresh kidney — Bowman's capsule and base-
ment membrane of tubules — Suprarenal capsule — The
ureters and bladder — Epithelium cells of bladder . . 229

CHAPTER XVIII.

THE GENERATIVE ORGANS.

Erectile tissue — Prostate and vesicula3 seminales — Scrotum,
labia, and vagina — Human uterus — Uterus of rabbit —
Sections of ovary — Mammalian ovum — Mode of finding —
Artificial impregnation of ovum — Testis — Injection of
lymphatics — Isolation of seminiferous tubules — Basement
membrane of tubules — Tunica vaginalis — The mammary
glands . . . . . . . . . . 237

CHAPTER XIX.

THE CENTRAL NERVOUS SYSTEM.

Preparation with bichromate of ammonia— Use of microtome
— Coloration of sections— Preparation by Sankey's method 243
B

XIV CONTENTS.

CHAPTER XX.

THE ORGANS OF THE SENSES — THE EYE.

PAGE

General directions for hardening in osmic acid and Miiller's
fluid — The eyelids — The lachrymal gland — The sclerotic
— Capsule of Tenon — Lamina fusca — The cornea — Sec-
tions of cornea — Epithelium of cornea— Substantiapropria
of cornea — Corneal corpuscles— Frog's cornea stained
with gold— Prevention of creases in cornea — Rabbit's
cornea stained with gold — Mode of isolating corpuscles
with potash — Nerves of rabbit's cornea— Cell spaces of
cornea prepared with nitrate of silver — Injection of cell
spaces — Nature of " corneal tubes" — Corneo-sclerotic
junction — Ciliary muscle — Lamina suprachoroidea —
Gangliated plexus of nerves — Layers of choroid — Mus-
cular tissue of rabbit's iris — Of human iris — The retina —
Hardening in Miiller's fluid — In osmic acid — Staining of
— Embedding in cacao-butter — Teased preparations of
retina — Preparation with chloral hydrate — Fresh retina —
Hexagonal pigment-cells — Ends of Miillerian fibres shown
with silver nitrate — Retina of lower vertebrata — The
lens — Epithelium of lens capsule — The zonule of Zinn
and hyaloid membrane of vitreous — Bloodvessels of the
eye . . 246

CHAPTER XXI.

THE AUDITORY, OLFACTORY, AND GUSTATORY ORGANS.

Semicircular canals of skate— Cochlea of guinea pig — Sec-
tions of — Teased preparations of — Olfactory mucous
membrane of mammal — Olfactory mucous membrane of
amphibian — Taste-buds of rabbit 275

APPENDIX.

Method of measuring an object under the microscope — De-
termination of magnifying power of microscope — Mode of
drawing microscopic objects — Mode of counting the blood
corpuscles — Microtomes — Microphotographic apparatus
— Employment of eosin as a staining fluid . . . 285

INDEX 297

LIST OP ILLUSTRATIONS.

ARRANGEMENT OF WORKING TABLE . . Frontispiece.

FTG. PAGE

1. Diagram of microscope 14

2. Dissecting lens 18

3. Dissecting microscope 18

4. Scissors, forceps, and mounted needle . . 21

5. Mode of letting down cover-glass 25

6. Simple warm stage 30

7. Simple warming apparatus shown in actual use . . 32

8. Osier's phenomena 33

9. Warm stage with thermometer and gas-regulator . 35

10. Simple moist- chamber 40

11. Gas-chamber 41

12. Clot of frog's blood in capillary tube .... 46

13. Apparatus for passing CO2 over a preparation . . 49

14. Slides for the passage of electric shocks over a prepara-

tion 50

15. Apparatus for passing electric shocks. .... 51

16. Valve of mussel showing gills ..... 64

17. Pravaz syringe ........ 75

18. Method of irrigation 90

19. Warming apparatus with gas-regulator for gold prepa-

rations 95

20. Upper part of gas-regulator ...... 96

21. Outline of paper for embedding trough . . . 143

22. Paper folded to form trough 144

23. Embedding trough of capsule metal, on cork, with tis-

sue pinned in situ . . . . ; . .144

XVI LIST OF ILLUSTRATIONS.

FIG. PAGK

24. Method of cutting sections 146

25. Section-lifter 148

26. Cork for supporting frog ...... 156

27. Structure and position of tongue of toad . . . 159

28. Injecting apparatus ....... 166

29. Canulas for injecting 168

30. Mercurial pressure apparatus for injecting . . . 178

31. Perforated steel needle for puncture injection . . 179

32. Tongue of rabbit ........ 282

33. Ocular micrometer 286

34. Lines of stage micrometer viewed with ocular micro-

meter 287

35. Camera lucida for drawing ...... 288

36. Apparatus for mixing blood and for counting corpus-

cles 289

37. Stirling's microtome . . . . . . .291

38. Hand microtome . . 292

39. Rutherford's freezing microtome ..... 293

40. Microphotographic apparatus 295

PRACTICAL HISTOLOGY.

INTRODUCTORY.

THE practical study of Histology is mainly depen-
dent upon the use of the microscope. The microscope
is a combination of lenses arranged for the purpose
of obtaining and viewing a magnified image of any
minute object. The lenses are set in a tube of vari-
able length — the tube of the microscope (Fig. 1, £, t') —
and this is itself supported in a vertical position, on
a firm, metal stand, which is provided with an
arrangement by which the tube is capable of being
moved, without lateral deviation, in a perfectly
straight, up and down direction. This arrange-
ment is termed the adjustment. Its purpose is to
bring the microscope into that position with regard
to the object in which the latter is most clearly
seen. The object is then said to be in focus.

Two adjustments are commonly provided: one —
the coarse adjustment (adj) — serves to bring the lenses
roughly into the focal position, and is either a tele-
scopic joint or a rack and pinion movement; the
other — the fine adjustment (adjf] — is a fine screw, and
by its means the focus may be obtained with com-
plete exactness even when the highest magnifying
powers are employed. The stand is further pro-
vided with a 'rigidly connected, horizontal table or
stage (st), upon which the object is placed, and which
projects below the tube and is provided with a cir-
cular aperture to admit light from below to the
object, capable of being varied by means of a dia-
phragm (d) furnished with holes of different sizes.
2

14

PRACTICAL HISTOLOGY.

Diagram of microscope.

/, tube seen in section ; t', sliding part of tube ; oc, ocular, with <?, eye glass, and
/. field-glass ; obf, objective ; adj, coarse, and «i.d/', fine adjustment; ,fi, sta^e;
with d, diaphragm; ni, mirror; ft, foot of microscope.

The object i* indicated l.y a small arrow j nst above the aperture in the diaphragm ;
the magnified image by the larger arrow in the middle of the ocular.

THE MICROSCOPE. 15

Diffused daylight is, if possible, employed, and is
reflected, by means of a movable mirror (m) below
the stage, up through the object and through the
tube of the microscope to the eye of the observer.
This is termed viewing an object by transmitted
light. Occasionally, especially when comparatively
large and opaque objects and low magnifying powers
are to be employed, the former are viewed by the
light which is reflected from their surface, whilst
that from the mirror is cut off. In order in such
cases to concentrate as much light as possible upon
the object, a bull's-eye condenser is employed. It is
only in viewing such preparations that the binocular
microscope offers any material advantage inhistology.
The lenses form the essential part of the micro-
scope, and are so arranged that one set of them,
which is placed at the lower end of the tube, pro-
duces a magnified, inverted image of the object at
the upper part. The image thus produced is viewed,
and at the same time still further magnified, by a
lens at the top of the microscope tube. This lens
(e\ placed close to the eye of the observer, is fixed
in the eye-piece or ocular ; but this part includes an-
other glass (the field-glass,/) situate below the first
and fixed in one piece with it, having for its object
the collection of the more divergent rays transmitted
by the lower set of lenses, and serving also to lessen
or obviate any chromatic aberration which might
otherwise be produced. The whole eye-piece (oc)
thus composed is made to slip in at the superior
aperture of the microscope tube. The lower set of
lenses form an achromatic combination which, from
its situation near the object, is termed the object-glass
or objective, and it is upon the perfection of its con-
struction that the useful ness of the microscope mainly
depends. It is seldom necessary to have more than
one ocular of medium strength in use, but at least
two objectives of different magnifying powers are
essential for histological work. One of these, which
in subsequent pages will be spoken of as the low power,

16 PRACTICAL HISTOLOGY.

should, when used with the ordinary eye-piece, give
an apparent linear enlargement of about 75 (75 dia-
meters); that is to say, when a line, the length of
which is known (say y-J-Q- of .an inch), is observed
through this combination, it should appear seventy-
five times as long as it really is (f inch* therefore).
The other, to be mentioned as the high power, should
give an apparent linear magnification of from 300
to 400 times. These two glasses amply suffice for
all ordinary histological studies; but for certain spe-
cial subjects it is advantageous to obtain the use of
a more powerful combination; one that will magnify
1000 diameters or more.

Glasses of this high magnifying power are usually of
the kind known as "immersion-objectives," so-called be-
cause they are made so as to be used with a stratum of
water between the specimen and the lower lens of the
objective. The water (distilled) is applied to this lens
with a splinter of wood or from a pipette before the objec-
tive is screwed on to the tube of the microscope ; the tube
is then lowered by means of the coarse adjustment until
the drop of water comes in contact with the cover-glass,
after which the focus is obtained by cautiously lowering
it further, at first with the coarse, and then with the fine
adjustment.

These comprise the essential requirements of a
microscope, but larger instruments are often pro-
vided with certain adjuncts which render them in a
measure more complete. Thus the stand may be
hinged so that the stage and tube can be tilted
somewhat out of the perpendicular to allow of better
adaptation to the position of the observer. But if
the microscope tube is not inconveniently high, it is
almost as comfortable to work without inclining the
instrument. Moreover, very many preparations, most
of those, for instance, which have to be examined in
fluid, will not admit of inclination. A camera lucida
(one which does not necessitate the tilting of the
microscope) is useful for obtaining an exact sketch
of the outlines of an object.

THE DISSECTING MICROSCOPE. 17

A polarization apparatus is occasionally employed
in investigating the optical properties of the sub-
stances which compose the tissues. (It is also of use
in helping to determine the nature of crystalline
deposits in urine and other fluids.) In connection
with the employment of this the stage-plate and
tuhe of the microscope are, in the best instruments,
made capable of rotating on a vertical axis. In
smaller instruments this movement is, as a rule,
not provided for, and indeed, although convenient,
is by no means essential.

The mechanical stage movement, which is so often
fitted to microscopes of English construction, and
other appliances which tend to mar the perfect sim-
plicity of the instrument when in ordinary working
trim, serve rather to detract from its usefulness for
purposes of histology.

Attention to the foregoing points, of which per-
haps perfect steadiness and rigidity of the stand and
stage is the most important to insist upon, will aid
the student in selecting a useful microscope so far as
the body of the instrument is concerned. The excel-
lence of the objectives can only be competently j udged
of by one who is already somewhat conversant with
the use of the microscope. Preparations of some of
the tissues or fluids of the body (connective tissue,
blood, salivary corpuscles) form the best test objects
for the high powers of an instrument that is to be
employed in histological studies.

Jn addition to the ordinary instrument (which is
generally distinguished as the " compound" micro-
scope), it will be found very convenient to have a
smaller microscope of some sort ; so as to be better
able to follow the needles or other instruments when
the operator is engaged in the separation and manip-
ulation of minute objects. An instrument wrhich is
used for this purpose is termed a dissecting or pre-
paring microscope. Any simple lens which is mount-
ed on a stand will serve (Fig. 2), and even the hull's-

eve condenser, which is generally furnished with the

c o*

18

PRACTICAL HISTOLOGY.
Fig. 2.

Simple form of dissecting microscope, provided with three lenses of
different magnifying power.

Fig. 3.

ACCESSORY APPARATUS. 19

microscope, may be employed as a lens if the marginal
part is covered by a black-paper diaphragm. A very
convenient form of dissecting microscope is shown in
Fig. 3. This consists of a small compound micro-
scope furnished with 'an arrangement for reversing
the image, so that the object appears in its natural
position and not inverted. It is provided also with
a prism for reflecting the light in a direction conve-
nient for the eye, and is placed on a wooden stand
so constructed as to afford support to the arms of the
operator. The ordinary low power objectives may
be used with it.

Besides the microscope, the student who is com-
mencing the practical study of histology will find it
necessary to be provided with the following simple
instruments and appliances : —

Glass-slides and cover-glasses (see Fig. 5). — The mi-
croscopic slides are oblong slips of glass upon which
the object is placed. In this country they are always
cut of the uniform and convenient size of three inches
by one inch. The glass should be quite free from
flaws and specks. The cover-glasses can hardly be
too thin. It is true that when very delicate they are
likely to be broken in the cleaning unless great care
is exercised ; but then, on the other hand, many high
power objectives require to be focussed so close to the
object that a thick cover-glass cannot be used with-
out risk of crushing the tissue, and perhaps of scratch-
ing the objective. Asa rule, the chief difficulty in
cleaning them arises from the fact that their surfaces
are generally covered with a thin film, of grease or
other organic matter which it is almost impossible
to rub off. But this is easily got rid of by placing a
number of them together in a small glass beaker and
pouring a little strong nitric acid upon them. This
quickly destroys every trace of organic matter. The
acid is then poured off, and the cover-glasses are
thoroughly rinsed by allowing water to stream over
them from a tap for two or three minutes. They
are then to be kept in water ready for use, and need

20 PRACTICAL HISTOLOGY.

only be dried when wanted. The drying is effected
by a thin linen cloth, one corner of which is laid flat
on the table, and the cover-glass, having been placed
on this, is gently rubbed, first on the one surface and
then on the other, with another corner of the cloth.
Square cover-glasses should always be used in prefer-
ence to round ones The most convenient size for
general purposes is three-quarters of an inch square.

Mounted needles. — These are fine sewing-needles
mounted in a wooden handle, with about a quarter of
an inch of their point projecting. They are amongst
the most useful instruments which the histologist
possesses, and will be in constant requisition. They
must always be kept clean and sharp. Xeedles may
also be mounted in crochet-needle holders. These
have the advantage that fresh needles can at any time
be readily substituted.

Scissors and forceps. — A small pair of scissors with
short, straight blades, is necessary. Their cutting
edge must always be kept very sharp, especially
towards the point, otherwise they are worse than
useless. A small, curved pair is often useful, but
these are much more difficult to keep sharp. At
least two pairs of small steel forceps are requisite.
The blades should be short, broad at their junction,
so as not to admit of lateral deviation, and towards
the end gradually tapering to a blunt point. They
are slightly roughened at the end, so as to afford a
firmer grasp. The long, slender forceps which are
commonly sold as microscopic forceps are useless for
histological purposes.

Slips of white bibulous paper should always be at
hand. They serve both -for soaking up excess of
fluid from under the cover-glass and for placing the
slide upon, when preparing a tissue that has been
stained, so that it is better seen than it would be
upon a black surface. On the other hand, it is better
to use the black surface for working upon when tis-
sues are unstained. One or two large, shallow glass
dishes, either flat (such as are used by photographers)

INSTRUMENTS.

21

Fig. 4.

Scissors and forceps for histological purposes. Natural size.
7i, end of mounted ueedle.

22 PRACTICAL HISTOLOGY.

or watch-glass shaped, are in constant requisition,
since very many of the manipulations require to be
performed whilst the tissue is immersed and floated
out in fluid; while for applying reagents to a speci-
men under the microscope small pipettes are ex-
tremely convenient.

Reagents. — Two or three small bottles containing
the fluids which are most used for preserving and
mounting preparations should be always on the
work-table. Small bottles provided with a cap and
with a piece of glass tube drawn out to a point and
standing in the bottle are perhaps the best, but small
corked phials with a piece of glass tube passed
through the cork will answer every purpose. One
of these should contain a mixture of glycerine and
water (equal parts of each); another a solution of
Canada balsam in chloroform for fixing the cover-
glass when preparations have been mounted in gly-
cerine (this should have a small brush instead of a
glass tube) ; and another, which will not be used much
at first but will afterwards be often in requisition,
should contain dammar varnish. It is useful also to
have a wash-bottle of distilled water, a flask of salt
solution, a solution of chloride of sodium, containing
1 part of the salt to 150 or 200 of water, and a small
flask of recently prepared logwood alum stain ing-
solution. This is made by taking 5 grammes each
of powdered alum and extract of logwood, and rub-
bing them up thoroughly together in a mortar with
100 cc. of water. The mixture is covered and allowed
to stand overnight, it is then filtered into a bottle,
and a lew drops of a solution of peroxide of hydrogen
are added. The solution will keep two or three weeks
in good condition for staining, but it must always
be refiltered immediately before being used.

Pipettes may be readily made by drawing out in
the flame a piece of soft glass tube at two places
close to one another, so that the intermediate part
remains as the bulb of the pipette. It is well to
make a number at a time, sealing up the ends in the

GENERAL DIRECTIONS. 23

flame while the bulb is still hot ; they are thus made
absolutely dust-tight and may be kept always ready
for use, it being only necessary to break off the
sealed ends when required and to suck the reagent
up into the bulb. A pipette should always be re-
jected after it has once been used, and never em-
ployed for another reagent.

For keeping preparations that have been per-
manently mounted, it is desirable to have a box or
cabinet in which the slides can be put away in a
horizontal position.

General directions for work.— Before com-
mencing see that the table is in order and clean,
and that everything is at hand that is usually
wanted for histological work. Especially look care-
fully to the glasses of the microscope that there is
no dust or other impurity on them. If any glyce-
rine or Canada balsam should have found its way
on to the objective, as is often the case when suffi-
cient care is not taken in placing a preparation
upon or removing it from the stage, they are to be
rubbed off, the former by a cloth wetted with water,
the latter with a little spirit. Before beginning to
prepare a tissue it will be necessary to look over the
description of the mode of making each preparation,
in order to know what vessels and what reagents to
get together. Otherwise many a specimen will be
spoiled by being left too long in one fluid whilst the
one to which it should be transferred is being got
ready. The cover-glass should always be cleaned
and dried before commencing, and placed ready to
hand in some situation where it is not likely to get
broken. It is well always to put the cleaned cover-
glasses in the same place — say, on the foot of the
microscope — otherwise, when wanted in a hurry it
is often difficult to find them at once.

Every specimen that is to be kept must be dis-
tinctively labelled as soon as made ; and if there is
anything of importance to remember about it this
must be at once entered in" the notebook, which no

24 PRACTICAL HISTOLOGY.

one who is working with the microscope should be
without.

Lastly, the student should never trust to the
transient impression of form or structure which the
mere glance at a microscopic preparation conveys,
but should always, whether naturally a good
draughtsman or not, endeavor to perpetuate the im-
pression so obtained by a careful sketch showing
the more important points which the preparation
illustrates. The rough outlines maybe drawn with
a camera lucida or drawing prism, if one be avail-
able, but even without such an instrument a little
practice soon enables a sketch to be produced which
gives a fairly good idea of the appearances seen, and
however rough it may be, serves materially to assist
the memory.

For the most part it is also desirable to note the
results of measurements made with a micrometer.
The use of this, as well as the method of sketching
an object by aid of the camera lucida, will be
described in the Appendix at the end of the book.

THE BLOOD.

25

CHAPTER I.

THE BLOOD.

Preparation 1. — A drop of blood may be most
conveniently obtained for examination from the fin-
ger. It is generally sufficient to give the end of the
forefinger of the left hand a smart prick with a clean
sewing-needle, in the thin part of the skin adjoining
the root of the nail, squeezing firmly with the right
hand above the point pricked, to cause a drop to
exude. If necessary, the finger may first be con-
gested by tying a piece of string tightly round it.
As soon as a small drop of blood has been pressed
out, take up a previously cleaned cover-glass by one
edge with forceps, let the drop come in contact with
the lower surface of the glass near the opposite ed«;e,
so that a little adheres ; and then, letting this edge
come first in contact with the upper surface of the

Fig. 5.

Glass slide and cover-glass, natural size.

The figure shows the mode of letting down the cover-glass (with a drop of blood
on its under surface) gently ou to the middle of the slide.

slide near its middle, gradually lower the other
edge, which is still held in the forceps, on to the
3

26 PRACTICAL HISTOLOGY.

slide (Fig. 5). When the lower blade of the forceps
nearly touches the slide, withdraw the instrument
carefully, so that the cover-glass may now rest evenly
upon the slide by its whole under surface, with the
blood in a uniform thin layer between. It is impor-
tant not to let the cover-glass down too suddenly,
for if dropped carelessly on the slide, many of the
corpuscles will be broken arid destroyed. When the
cover-glass is in its place, there ought to be just
enough blood entirely to fill the space between the
two glasses ; but it is better to have too little than
too much. It might be supposed that the delicate
corpuscles would be crushed between the cover-glass
and slide, but they are for the most part protected
from this by the buoying up of the cover-glass by
the liquid in which they float.

The preparation made, it is to be at once trans-
ferred to the stage of the microscope, and examined
with a power of about BOO diameters. The Held of
the microscope will be seen crowded with corpus-
cles floating in a clear liquid. Probably the first
thing which will strike the beginner is the very faint
color which the so-called red corpuscles present, and
these will also most likely be the only kind of cor-
puscle that he will at first be able to distinguish.
But if at the moment 'of observation there happens-
to be a current in the fluid — produced either acci-
dentally by a shaking of the room, or a draught of
air, or purposely by gently touching the cover-glass
with a bristle — it will be seen that while most of
the corpuscles are carried along by the current, two
or three remain sticking to the glass, whilst the
others are carried past them; and, on close exami-
nation, it will further be clear that these are of a
different nature from the rest, being entirely devoid
of color, and of a pale granular appearance. They
are, in fact, white corpuscles, and once seen, will be
easily recognized again, even when the fluid is at a
standstill.

Another thing that will be made manifest by tiny

THE RED CORPUSCLES. 27

motion in the fluid is the biconcave discoid shape
of the red corpuscles ; for as they roll over it will be
seen that their outline is no longer circular as when
lying flat, but, on the contrary, a lateral view of the
disks is obtained, and the flattening and incurvation
of the surfaces become evident.

When the motion in the layer of blood, in what-
ever way it may have been produced, is subsiding, it
will be seen that whenever one corpuscle comes in
contact with another the two seem to be in some
way attracted to one another, so as to adhere closely
by their opposed surfaces; and other corpuscles com-
ing in the same way in contact with these and adher-
ing, little piles or rouleaux are thus produced, which
form by their junction with one another a network,
extending throughout almost the whole of the pre-
paration. In the cords of this network nearly all
the red corpuscles are involved, and now for the
most part are seen edgeways; but in other parts of
the preparation where the layer of blood is very thin
— the space being too small to allow the corpuscles
to stand edge up, and to combine so completely to
form rouleaux — they may be found still lying flat
and distinct from one another; and these more iso-
lated corpuscles may now be subjected to careful
examination. Keeping a single red corpuscle in
view, if it be brought exactly into focus — that is to
say, if the microscope be so adjusted that the con-
tour of the corpuscle is as distinct as possible — it will
be observed, with the power (800 diameters) which
is at present being employed, that the middle part
appears slightly darker than the rim, whereas if, by
means of the flue adjustment, the objective be now
brought somewhat nearer (lower), the middle part
will come to appear lighter.

The cause of this is probably to be found in the xhape
of the corpuscle, the middle part of which acts upon the
light like a very weak biconcave lens, refracting the rays
of light which nre transmitted through it slightly out-

28 PRACTICAL HISTOLOGY.

wards; so that if the objective is at a certain distance,
all of the rays which traverse the central part do not
reach it, some of them being deflected too much to the
side to impinge upon the lower glass of the objective.
The part in question, therefore, looks a little dimmer than
the somewhat convex marginal part, whereas when the
objective is brought nearer all the rays are intercepted
by it; and the middle part. owing to its greater thinness,
appears lighter than the rim. If a very high power ob-
jective is used, the middle part of the corpuscle will be the
lighter, even when the focus is rightly adjusted, for in
these objectives the focal length is generally very short,
and they approach, therefore, near enough to intercept
the outwardly refracted rays.

With the exception of these differences of shading
(which are merely dependent upon the shape of the
corpuscle), the red corpuscles present a perfectly
homogeneous appearance, and exhibit in the fresh
condition no tendency to separate into the two parts
of which, as the study of the action of reagents will
show, they in reality consist. But there may gener-
ally be noticed, even in a preparation which lias been
made with the greatest care, a red corpuscle here and
there which varies from the prevailing form, having
become more globular, and at the same time rather
smaller in diameter; some of these retain a smooth
contour, whilst others, especially those near the edge
of the preparation, have a jagged or crenate margin,
as if set with little projections, and these may also be
seen on the surfaces of the corpuscles by carefully ad-
justing the microscope. This change of form, which
is very characteristic of the mammalian red cor-
puscles on exposure, seems to be generally caused by
a shrinking of the corpuscles, induced by an increase
in the density of the plasma in which they float; it
may always be produced by adding salt to blood.

Turning now our attention to the white corpus-
cles, not more than two or three of which are to be
seen in each field of the microscope when the ordi-
nary high power is being used, and which, as before

THE WHITE CORPUSCLES. 29

stated, are readily distinguishable from the red cor-
puscles by their want of color and their pale, granu-
lar aspect, we usually notice, if the room is moder-
ately cool, and provided they are not pressed down
by the cover-glass, that they are spheroidal and com-
pletely motionless, exhibiting no indications of vital-
ity. Some of them may be noticed to contain a small
group of well-marked granules, much coarser than
the excessively fine granules .which pervade the
whole substance; and in conformity with this it is
usual to describe two kinds of white corpuscles — the
finely granular, and the coarsely granular — but there
would not seem to be any very essential difference
between the two. As a rule, before the addition of
reagents, no nucleus is visible in either variety,
although, as will be afterwards seen, one or more is
always present in each ; the nuclei are delicate, how-
ever, and readily obscured by the granules of the
protoplasm. If the room is tolerably warm it may
happen that the white corpuscles no longer preserve
their rounded outline, but that from one side or an-
other of a corpuscle a bud -like process extends itself,
to be again retracted into the body of the corpuscle;
spontaneous changes of form being thus effected
which resemble those which are presented by the
common fresh-water amoeba, and are hence termed
a amoeboid;" but in a cold preparation of human
blood, like that under examination, these movements
are seldom extensive, and do not serve to effect an
actual change of place in the corpuscles such as we
shall see to be the case in a preparation which is
artificially warmed.

Further, there may generally be seen in a prepa-
ration of blood a number of excessively minute pale
granules, which, if present in quantity, may be
closely grouped together here and there into masses
or "colonies" of various shapes and sizes (see Fig. 8,
«), which the beginner is sometimes apt to mistake
for white blood-corpuscles. But the objects in ques-
tion have a much fa in tor aspect ; and although under

30 PRACTICAL HISTOLOGY.

certain conditions they — at least, their constituent
granules — may, as we shall presently see, exhibit
indications of vitality, yet nothing resemhling in
nature the amoeboid movements of the white blood-
corpuscles is ever observed in them.

Finally, a few excessively fine and delicate threads
of fibrin may be observed stretching in different di-
rections across the field of the microscope ; but to *see
these distinctly a very high power is needed.

Preparation 2. — In order properly to study the
vital phenomena which are displayed by the white
blood-corpuscles, it is necessary, in the case of man
and warm-blooded animals, to maintain the drop of
blood under observation at or near the temperature
of the body. For this purpose we employ what is
known as a warm stage, of which there are several
forms in use. The simplest consists merely of an
oblong copper plate (Fig. 6), two inches by one inch,
from one side of which a rod of the same metal, four
or five inches long, projects. This plate has a round

Fig. 6.

Simple warm stage, with preparation upon it enclosed between two
cover-glasses.

aperture in the middle, half an inch in diameter,
and is fastened to an ordinary slide by sealingwax.
The preparation is made as follows: Take first a
clean, large-sized (one inch square) cover-glass, which
in this case is to be used instead of a slide, and on it
place a small drop of salt solution. With this mix

SIMPLE WARM STAGE. 31

thoroughly with a needle about an equal amount of
blood obtained from the finger, as in Prep. 1, and
carefully cover the mixed fluid with another cover-
glass, somewhat smaller than the first. If there is
now not enough fluid to fill the space between the
two glasses, add a little more salt solution at one
edge of the smaller cover-glass; but if, on the other
hand, there is too much, soak up the excess with a
small piece of blotting-paper. A very small camel-
hair pencil which has been dipped in olive-oil is now
to be drawn gently along each edge of the smaller
glass; this will prevent evaporation from the ed^es,
which would otherwise quickly ensue on warming
the preparation. The dilution of the blood with
salt solution prevents in great measure the aggrega-
tion of the red corpuscles, while at the same time in
no way interfering with the movements of the white
ones; moreover, it is favorable to the changes which
the above-mentioned masses or colonies of discoid
particles undergo, if any such happen to be present.
The glass slide which bears the copper plate having
been clamped on to the microscope stage, the prepa-
ration thus made is placed upon the copper, and,
having been brought in focus, one or more white
corpuscles are selected for observation — a high mag-
nifying power being used. The rod is now heated
near its end by a small spirit-lamp, and the heat is
conducted by the rod to the copper plate, and from
this is transmitted to the preparation, close to which
a small fragment of a mixture of white wax and
cacao-butter, previously made, and melting at about
30° C., is to be placed upon the copper (Fig. 7). The
lamp is now gradually approached along the rod
until it arrives at a spot the heat transmitted from
which is just sufficient partially to melt the frag-
ment, and it is then left burning at that spot, for
since the fat employed melts at about the tempera-
ture of the body, we know that the preparation will
now be also warmed nearly to the same point.

It will be seen that as the preparation begins to

32

PRACTICAL HISTOLOGY.

get warm the white corpuscles, which were perhaps
previously rounded and inert, begin to throw out
processes and exhibit amoeboid movements, which
become more and more marked as the temperature

Simple warming appirntus, complete, shown in operation.

rises, so that by virtue of these an actual change of
place from one part of the field to another may be
effected. It is well in making this observation to
select a single corpuscle and to sketch its outline and
that of its more immediate surroundings at intervals
of half a minute. As the corpuscles become spread
out in creeping along the glass, one or more nuclei
may sometimes be seen indistinctly in them; more

OSLER'S OBSERVATIONS. 33

often, perhaps, clear spaces or vacuoles are to be seen
in their protoplasm.

The red corpuscles in this preparation may be dis-
regarded, for they show no trace of amoeboid move-
ment, but only become slightly eremite, owing to
the action of the salt solution. The slight shaking
movement which many of them exhibit is the mole-
cular or Brownian movement common to all minute
solid particles floating in a liquid.

The changes which the colonies of discoid particles
(see p. 29) undergo under the present conditions — i.e.,
dilution of the blood with salt solution and warmth —
should be carefully studied. As a rule, the larger the
colony the more actively the changes, about to be de-
scribed, take place. If there are none to be seen in the
blood from the finger, a drop may be obtained from some
other source and prepared in a similar way. They are
common in the blood of some animals — e.g., the rat — and
are generally large and numerous in that of persons who
have been long ill.

The masses in question (Fig. 8, a) are often of consider-
able size, many times larger than a pale blood-corpuscle.
As Osier has shown, the particles which compose them
are free in the circulating blood, and only run together
when the blood is drawn.

Changes seen in one of the masses or colonies of discoid particles from a
drop of human blood, diluted "with salt solution and warmed After Osier.

About half an hour after the preparation has been made
the masses may be seen to have no longer an even, toler-
ably well-defined contour, but to be bristling with exces-
sive minute filamentous projections, each of which has
grown out from one of the minute discoid particles at the

34 PRACTICAL HISTOLOGY.

exterior of the clump (Fig. 8, b) ; and as the filament gets
a little longer and projects more the remains of the disk
may often be seen forming an enlargement near its middle.
Soon some of the projecting filaments begin to oscillate
in the liquid, and at length break away altogether from
the mass, and continuing their oscillatory movement in
the surrounding fluid, become gradually more and more
removed from the clump, until they may eventually .even
pass altogether out of the field of the microscope. Mean-
while other of the disks have grown out into filaments
and taken the place of the liberated superficial ones, and
soon these in their turn become free and give place to
others ; so that in this way the previous solid-looking
mass becomes almost entirely broken up into freely-mov-
ing filamentous particles (Fig. 8, c). These are not all
of the shape above described, but present various forms,
as shown in the accompanying figure.

The above observation shows that the discoid parti-
cles which are occasionally found in theblood^and which
when the blood is drawn tend to become collected into
clumps, are to be regarded as centres of origin from which
minute, filamentous, bacterium-like organisms may under
some circumstances be developed ; but whether this change
ever occurs in the living body, and what further modifi-
cation the filaments may, under conditions more favorable
for their development, undergo, is at present undeter-
mined.

After the observations recorded in the preceding
paragraph are completed, the action of an excess of
heat may be observed; but it is better to use for
this purpose a larger apparatus, in which the degree
of heat can be measured by a thermometer. Such a
one is shown in Fig. 9. In this the preparation is
placed upon the brass box «, which rests on the stage
of the microscope, and is pierced in the centre by a
tubular aperture, to admit light to the object. The
box is connected by India-rubber tubes with a hollow
metal jacket, /, and the whole system thus consti-
tuted is completely filled with water previously
boiled, to the exclusion of air. The water is warmed
at q l>v n small gas- flame, and rising through the tube

t/ •/ ,~ »

MAINTAINING TEMPERATURE.

35

c communicates its beat to the box «, tbe temperature
of which is measured by a small thermometer 6, in-
serted through an obliquely placed tube, quite into
the central hole and immediately under the prepara-

Fig. 9.

Apparatus for maintaining a constant temperature under the microscope.

tion. The cooled water from the stage descends down
the tube c', to pass again round to the flame, and in
this way the water constantly circulates. The bulbed
tube d, tilled with mercury, serves to regulate the flow
of gas, so as to keep the temperature constant at any
desired point. This is effected by turning the steel
screw <?, when this point, whatever it may be, is
reached, so as to raise the mercury in the glass tube,
and thus almost block up the lower end of a small
steel or o-lass tube, which is fixed into the upper end
of the tube d. The gas passes through the small tube,
and then above the mercury and between the two
tubes to be conducted by the side piece h to the
burner below, and it will be understood that if the
temperature now rose higher in the reservoir/, which
surrounds the mercury, this on being warmed will

36 PRACTICAL HISTOLOGY.

expand and tend to cut off more of the gas and thus
reduce the flame, on which the mercury will again
contract, and the flame will rise in consequence, and
so on. It is found that an equilibrium soon becomes
established, and the temperature of the water and
stage remains almost absolutely constant. To raise
or lower the temperature all that is required is to
screw out or in the screw e. The small included
tube is pierced with a minute aperture, to allow a
constant passage of gas, so as to prevent the flame
from being extinguished in the event of the complete
occlusion by. the mercury of the lower end of the
tube in question.

By employing this or a similar apparatus it will
be found that up to and a little beyond the normal
temperature of the blood the white corpuscles become
more active in their movements, but on gradually
warming the preparation still more a point is soon
reached at which they draw in their processes, be-
come spherical, and show no longer any signs of
vitality — the temperature being sufficient to kill the
corpuscles. The red corpuscles remain unaltered
until a temperature of about 55° C. is reached, when
they become altered in shape, and globular ; eventu-
ally their coloring matter is discharged and becomes
dissolved out in the surrounding serum.

ACTION OP REAGENTS UPON THE BLOOD. — The red
corpuscles in the two preceding preparations ap-
peared, even under the highest power, perfectly
homogeneous and structureless, but it can be shown,
by the application of reagents to the blood under
the microscope, that they in reality consist of two
intimately mingled but separable parts — the stroma
(formed of various chemical compounds, such as
cholesterin, paraglobulin, &c.), which is colorless,
and gives the shape to the whole corpuscle ; and the
colored part, which consists chiefly or wholly of a red
crystallizable substance, haemoglobin. The mode of
application of reagents is as follows : A drop of
blood is got ready as in the first preparation, and

ACTION OF REAGENTS ON THE BLOOD. 37

whilst under observation a small drop of the reagent
(which should as a rule be freshly prepared) is al-
lowed to come into contact with the edge of the
cover-glass. Some of the fluid flows under this and
mixes with the drop of blood ; the current produced
by it at first drives the corpuscles before it, but they
soon become stationary, and then the part of the
preparation should be selected for observation where
the reagent is gradually diffusing itself amongst the
corpuscles. In this way every stage in its action
may readily be studied.

Preparation 3. Action of water. — When a
drop of distilled water is applied in the manner
above described, the first efleet is seen to be that the
red corpuscles begin to lose their discoid form, first
one of their sides becoming bulged out, so that they
are cup-shaped, and then the other side, so that they
are now completely globular, as may be seen when
they roll over. Meanwhile the haemoglobin is being
dissolved out of the corpuscles by the water, so that
they are soon quite colorless and hardly to be de-
tected in the now reddish fluid Some seem to offer
greater resistance to the action of the water (and
indeed of most reagents) and to retain their coloring
matter longer than others.

The white corpuscles are also soon affected. They
cease their amoaboid movements, and begin to swell
up by imbibition of fluid, whilst at the same time
with a high power the granules in their interior
may be seen to exhibit the dancing movement which
is characteristic of minute particles floating in
liquid. Often the corpuscles present clear bulgings
at their circumference, or their substance may ap-
pear to burst at one point and become diffused in
the water. As they swell and become clearer, one,
two, or more small round nuclei generally come into
view, and soon these also become swollen, and with
the rest of the corpuscles eventually disintegrate,
nothing being left but a few granules.

Water is thus proved to have a characteristic ac-
4

38 PKACTICAL HISTOLOGY.

tion upon the protoplasmic white corpuscles as well
as upon the very easily alterable red disks, and this
fact must be borne in mind in investigating the ac-
tion of reagents or poisonous substances, both upon
the blood-corpuscles and upon the tissues generally.
If a reagent is to be employed in weak solution,
therefore, it is well to dissolve it either in salt solu-
tion or in fresh serum1 instead of water. Washing
with water tissues which are subsequently to be sub-
mitted to microscopical examination, as is so freely
done in the post-mortem room, is for a similar rea-
son to be deprecated ; but if a trace of bichromate of
potash or of chromic acid, or a little common salt be
previously added to the water, its deleterious effect
upon the protoplasm is in great measure obviated.

Preparation 4. Action of Acetic Acid. — To
investigate the action of dilute acids, it is best, as
just explained, to mix the acid with salt solution
instead of water ; 1 part of glacial acetic acid to 200
of salt solution is an appropriate strength for the
blood. The preparation is made in the usual way,
and the drop allowed to run in at the edge of the
cover-glass. The action of the weak acid upon the
red corpuscles is seen to be quite like that of water;
they are first rendered globular and then decolor-
ized. Upon the white corpuscles it has a somewhat
different action, for although the protoplasm of the
corpuscle becomes partly swollen out into a clear
spheroid, the nuclei are not swollen by the reagent,
but are brought very distinctly into view, as well as
the nucleoli within them, and remain usually at one
side of the corpuscle, with a little granular matter
precipitated around them.

Preparation 5. Action of Tannic Acid. — The
action of tannic acid upon the red corpuscles is pe-
culiar and interesting. Like other acids it tends to
cause the colored part of the red corpuscle to become

1 The scrum employed must be from the blood either of the
same animal or ot'uue belonging to the same species.

MOIST CHAMBER. 39

separated from the stroma ; but as the colored ma-
terial is exuding it becomes coagulated by the astrin-
gent reagent, and in place of being dissolved in the
surrounding liquid, as after the action of acetic acid,
it remains attached to the stroma as a small, bright,
reddish projection.

In the first part of the reaction, viz., the render-
ing the corpuscles globular, it acts similarly to other
weak acids.

The most convenient strength of solution to use
is 1 per cent.; it. should be freshly prepared. It is
well, moreover, first to mix the drop of blood upon
which it is desired to test the action of the reagent
with an equal amount of salt solution ; otherwise
the tannic acid produces such a dense precipitate
with the albumen of the serum that the view of the
corpuscles is greatly obscured, and, indeed, the re-
agent with difficulty reaches them.

Preparation 6. Action of Weak Alkalies.—
A mixture of 1 part .of caustic potash to 500 of salt
solution may be used, a drop being added to the
preparation in the usual way (p. 37). The reaction
takes place very suddenly ; the corpuscles, both
white and red, swell up as soon as the reagent
reaches them, appear to burst, au.d then entirely dis-
appear. The white are affected by a weaker solu-
tion than the red. This is apparent from the fact
that, as the liquid slowly diffuses and mixes with
the blood, the white may be seen to become destroyed
in parts where the red are still unaffected.

Preparation 7. Chloroform. — To observe the
action of chloroform vapor upon blood a moist
chamber is used. This is an apparatus for keeping
a tissue or fluid under examination in its naturally
moist condition, whilst at the same time allowing
its surface to be exposed to air or to any desired gas
or vapor. The simplest form of moist chamber is
made out of a small piece of soft putty or modelling-
wax, which has been rolled out between the fingers
into a round cord about 2 inches long and J inch

40 PRACTICAL HISTOLOGY.

thick ; the ends of the cord are united so as to form
a ring, and this is placed on the middle of a clean
glass slide. A drop of water is put into the centre
of the ring ; this is for the sake of keeping the at-
mosphere of the chamber rnoist ; but the object may
be equally well effected by breathing into the space
as it is being covered over. The object is prepared
on the centre of a clean cover-glass, which is then
inverted over the ring, so that the preparation is
dependent into the chamber, and, whilst freely
exposed to the air in this, is entirely protected from
evaporation and may be readily examined through
the cover-glass, to the under surface of which it
remains adherent (Fig. 10).

Fig. 10.

To investigate the action of chloroform vapor it
is necessary to have some means of passing this into
the moist chamber, whilst the drop of blood is under
observation. For this purpose a slide is employed
(Fig. 11), to which a piece of small glass tubing has
previously been fixed by means of sealing-wax, and
the ring of putty is so placed as to include the end
of this, a small interval being left at the side of the
tube to afford an exit for the vapor. The slide is
then clamped on to the microscope stage (as in Fig.

MOIST CHAMBER. 41

13 6'), and the glass tube is connected by India-rubber
tubing to a bottle containing a few drops of chloro-
form and furnished with a second tube, through
which air can be blown.

Fig.

Chamber for passing a gas or vapor over a preparation under the
microscope.

Before the cover-glass is superposed the blood
should first have been spread out upon it into a thin
layer, so that the chloroform vapor may readily act
upon all parts.

Everything being thus prepared, and some of the
blood corpuscles having bean brought clearly under
observation, air is blown gently into the bottle, and
passing through it becomes charged with the vapor
of chloroform, which is conveyed by the tube into
the moist chamber, where it acts upon the layer of
blood which is on the under surface of the cover-
glass. After a short time it will be seen that the
red corpuscles, as under the action of water and
dilute acids, at first become globular, and subse-
quently their haemoglobin becomes dissolved and
discharged out in the serum. It will further be ob-
served, both in this and in the other preparations in
which this change has taken place in the red cor-
puscles, that to the naked eye the blood has changed
from scarlet to lake, and that whereas when the cor-
puscles were intact even a thin layer of blood pre-
sented a somewhat opaque appearance, it is now

4*

42 PRACTICAL HISTOLOGY.

completely transparent. Hence we may infer that
the opacity of the unaltered blood is due to the
presence of the red particles.
Preparation 8. Examination of Frog's Blood.

— The simplest way to obtain frog's blood for exami-
nation is to cut off the tip of one of the digits, having
previously wiped it dry with a cloth, and to collect
upon a clean cover-glass the small drop of reddish
fluid which exudes. The glass is then inverted upon
a slide and the drop is examined. The blood so
obtained is mixed with lymph, and the corpuscles
are consequently less crowded and better adapted
for observation than when the blood is undiluted.
To procure blood unmixed with lymph the animal
should be pithed, and laid upon its back. The heart
is then exposed and snipped with scissors, and a small
drop of the blood which exudes is taken up with a
glass rod, transferred to a slide and covered. But
before placing the cover-glass down in the usual way
a small length (J inch) of a delicate hair should be
placed in the drop, so that when the cover-glass set-
tles down, the corpuscles, here comparatively large,
may not be crushed by its weight.

When the preparation obtained from either of
these sources is examined, it will at once be seen
that the colored corpuscles are larger and fewer in
number than in human blood, and do not tend to
form rouleaux ; that they have an elliptical outline
when lying flat, but when seen edgeways look quite
narrow and pointed at the ends, with a slight and
gradual bulging at either side; so that, although
disk-shaped, like the mammalian blood-corpuscles,
so far from being biconcave, they are biconvex. The
bulging is due to the presence in the middle of the
corpuscle of another part besides the stroma and the
colored substance found in the mammalian disk.
This, the so-called nucleus, can readily be made out
in most of the corpuscles as a somewhat elongated,
colorless, and slightly granular elliptical body, about
a third the length of the corpuscle, and often lying

FEEDING OF WHITE CORPUSCLES. 43

not quite in the middle but somewhat eccentrically.
Occasionally the nucleus is seen to be round; but
this is an accidental change, and may be brought
about by mechanical injury. Indeed, if the precau-
tions above recommended for avoiding pressure are
not taken, a large number of the corpuscles become
injured, so much so as even to be ruptured and de-
stroyed altogether, in which case the rounded nuclei
are liberated, and may be mistaken by the beginner
for pale blood-corpuscles.

With regard to the white corpuscles, it will be
observed that they are more numerous and larger
than in human blood. Moreover, they soon begin,
even in the cold, to exhibit very distinct amoeboid
movements; and, on account of the greater size of
the corpuscles, both these and the other phenomena
exhibited by them are much more striking, and
tbese corpuscles are therefore much better suited for
observation than those of mammals, which in other
respects they closely resemble. The distinction be-
tween the finely and the coarsely-granular corpuscle
is met with again here; but there is also sometimes
seen a third kind of corpuscle, fusiform or oat-shaped
and devoid of amoeboid properties. It has been sug-
gested that these are cells which have belonged to
and have become detached from the lining membrane
of the bloodvessels.

Preparation 9. Feeding of the White Corpus-
cles.— The white corpuscles of the blood exhibit a
strong tendency to take into their interior any small
particles of insoluble materials which may happen to
be in their neighborhood. This tendency they have
in common with all amoeboid organisms, and it is
nowhere better seen than in the case of the amoeba
itself. Because of their greater size, number, and
activity, it is better to take the frog's corpuscles for
this experiment rather than those of mammalia.

To obtain the white corpuscles in quantity, and
comparatively free from colored ones, take a capil-
lary tube (like those used for preserving vaccine

44 PRACTICAL HISTOLOGY.

lymph in, which can be easily made by heating a
piece of glass tube red-hot in the blowpipe-flame,
and then removing and drawing it out before it has
time to cool), and allow it to till with blood from
the heart, exposed as above directed. Now lay the
tube aside for an hour, and meanwhile the particles
with which the white corpuscles are to be fed may
be prepared. This is done in the following way :
A few drops of salt solution are poured into a watch-
glass, and a cake of Indian ink is rubbed in it until
when thoroughly mixed up the fluid has acquired
a faint grayish color. When left to itself fora time
the coarser particles subside, whereas the finer ones
remain longer suspended. At the expiration of the
hour the blood is taken and its contents are blown
out upon a glass slide, the clot which has formed
within the tube being removed, leaving merely the
exuded serum. This contains a few red blood-cor-
puscles and a large number of white ones, the latter
having by virtue of their amoeboid movements
migrated, during the space of time the tube has
been left, from the clot into the serum. With the
drop of serum a little of the salt solution containing
line suspended particles of the pigment is thoroughly
mixed, and the preparation is then covered, the
edges of the cover-glass being surrounded with oil
or melted paraffin, to prevent evaporation. When
the preparation is observed it will be seen that the
white corpuscles have quite their normal appearance,
whereas the colored ones for the most part exhibit
folds or plaits upon their surface, having undergone
a slight shrinking, owing to the increased density
of the fluid in which they lie. The black particles
from the Indian ink are scattered irregularly over
the fleld ; and although they may here and there
be in contact with the white corpuscles, it is never-
theless to be readily made out that the substance of
the latter is entirely free from pigment. But after
a short time, if the corpuscles are closely observed,
it will be seen that they are gradually faking into

MIGRATION OF WHITE CORPUSCLES. 45

their substance the black particles which were in
contact with them ; and when by their amoeboid
movements from one place to another they are
brought into contact with other particles these also
are in like manner absorbed. On careful watching
it may be made out that the process of intussuscep-
tion commences by the throwing out of processes
which surround the particle to be taken in, and
meet and coalesce beyond it ; once included in this
manner, the grannie afterwards becomes gradually
carried, presumably by the movements of the proto-
plasm, more towards the centre. If, after observing
the preparation in this way for half an hour, it be
laid aside for two or three hours, it will be found at
the expiration of that time that many of the white
corpuscles have taken in a large number of the black
particles, for they do not discharge their cargo, but
carry it about in their movements from place to
place.

This property of the white blood-corpuscles has been
used in pathological investigations to determine the
source of the corpuscles of pus, which resemble the white
corpuscles of the blood. Insoluble particles were in-
jected into the bloodvessels of an animal, and inflamma-
tion was induced at a special part: it was found that the
pus-corpuscles which accumulated outside the blood-
vessels in the inflamed part contained similar particles.
The corpuscles must consequently have come from the
blood, for such particles have not the power of passing
through the coats of the bloodvessels unless carried by
the white corpuscles.

Preparation 10. Migration of White Cor-
puscles.— The process of migration can be quite
easily seen in progress in transparent parts of
animals, and will be afterwards studied when the
methods of observing the circulation of the blood
are described. But for exhibiting the active migra-
tion of the white blood-corpuscles nothing is more
striking than the examination of a capillary tube in

46

PRACTICAL HISTOLOGY.

which frog's blood has been collected and has coagu-
lated. A high power is to be employed, and the
capillary tube must therefore be very line indeed ;
and in order that the wall of the tube should be as
thin as possible, it must be drawn out from a piece
of glass tubing half an inch or more in diameter.
The capillary tube is filled with frog's blood, except
near the ends ; these are then sealed by holding them
successively in the flame for a second or two ; the
tube is then placed in a drop of glycerine on a slide,
covered with a thin glass, and at once examined.
The object of the glycerine is to correct in some
measure by its high refracting power the effect

Fig. 12.

White corpuscles of frog's blood migrated from clot. Highly mngnified.
The clot has shrunk considerably from the sides of the capillary tuba.

upon the light of the cylindrical glass tube. After
a few minutes the clot is seen to be getting smaller,
and a layer of clear serum collects between it and
the glass; the quantity of this gradually increases,

ACTION OF REAGENTS. 47

and soon one or two white corpuscles begin to pro-
ject from the surface of the clot. These protrude
more and more, and others make their appearance,
and all begin to throw out numerous amreboid pro-
cesses, which are actively advanced and retracted,
and by aid of which the corpuscles free themselves
entirely from the shrinking clot, and eventually
become free in the surrounding serum (Fig. 12).

Preparations 11, 12, 13, 14. Action of Re-
agents—Water, Acetic Acid, etc,— upon the
Frog's blood-corpuscles. — The action of reagents
upon the white blood-corpuscle of the frog is ex-
actly the same as upon the mammalian white cor-
puscle; upon the red corpuscle it is in the main
similar, but in some cases a little different, the diffe-
rences partly depending upon the presence of the
nucleus. Thus water causes both the substance of
the corpuscle and the nucleus to swell up and become
spheroidal, and extracts the colored part of the
corpuscles; dilute acetic acid brings the nucleus
strongly into view, and decolorizes the rest of the
corpuscle, which, however, usually retains its oval
shape ; tamric acid causes the colored part to be
exuded from the stroma, to which it generally re-
mains attached as an irregular curdled mass (some-
times the colored part is precipitated around the
nucleus, and then the two may be ejected from the
stroma together) ; chloroform vapor causes the red
corpuscles to become decolorized, and arrests the
movements of the white corpuscles; but these, if not
acted on for too long a time or by too strong a mix-
ture of chloroform and air, resume again on replac-
ing the vapor by pure air. All these reagents are
applied in precisely the same manner as with the
mammalian blood, to the description of which the
reader is referred.

The observations may be made with as great or
with greater advantage upon the blood-corpuscles of
the newt, which are larger than those of the frog.
The blood should be obtained directly from the

48 PRACTICAL HISTOLOGY.

heart, and not by merely snipping a piece off the tail
and collecting; the drop which exudes, for in this case
it is very apt to become mixed with the acrid secretion
from the cutaneous glands. There are, in addition,
two reactions for which newt's blood is particularly
well adapted, viz., the actions respectively of boracic
acid and carbonic acid upon the red corpuscles.

Preparation 15. Action of Boracic Acid.—
The boracic acid is used in solution in water (two
parts per cent.), and the preparation of blood having
been made in the usual way, with or without addi-
tion of salt solution, a drop of the boracic acid solu-
tion is placed at the edge of the cover-glass and
allowed slowly to mingle with the blood. If the
first stages of the reaction are fortunately observed
it will be seen that the colored part of the corpuscle
is becoming collected towards the centre of the cor-
puscle and accumulated around the nucleus, often
remaining, however, at first adherent here and there
to the circumference of the corpuscle, and shrinking
away at the intermediate points, so as to present
somewhat of a stellate figure.1 But soon it is en-
tirely withdrawn and collected around the nucleus,
which has become rounded, and is nearly concealed
by the coloring matter. The corpuscle, now decolor-
ized, has also in many cases become circular, and the
colored nucleus is generally shifted to one side, and
eventually altogether extruded.

Preparation 16, — To investigate the action of
carbonic acid gas the blood must be prepared in a
moist chamber, like that used for chloroform vapor
(Fig. 11). If the preparation is very quickly made
the nucleus in many of the red corpuscles cannot at
first be distinguished, for in the entirely unaltered
state it possesses as nearly as possible the same index
of refraction as the rest of the corpuscle. But when
carbonic acid gas, generated in a suitable apparatus

1 Water and various other reagents may, in the first instance,
have a somewhat similar effect.

WARMTH ON WHITE CORPUSCLES. 49

(Fig. 13, 6, b'\ is allowed to pass into the moist
chamber, a precipitate of one of the constituents of
the stroma (probably paraglobulin) occurs around
the nucleus, and the outline of this is brought into
view, whereas if the carbonic acid is speedily replaced
by air, which may be effected by disconnecting the
tube t from the wash-bottle 6' and drawing air
through it by the mouth, the precipitate is redis-
solved, and the nucleus is again made to disappear.

Fig. 13.

Apparatus for passing carbonic acid gas over a preparation under the
microscope.

ft, bottle containing marble and hydrochloric acid ; b', wash-bottle: t, India-
rubber tube conducting the gas to the stage, s.

It is of advantage in performing this experiment
to add a trace of moisture to the blood before the
observation; this may be most conveniently done
by breathing two or three times on the preparation
before placing it over the ring of putty.

Preparation 17. Influence of warmth on
white corpuscles of Amphibia. — The action of
gentle warmth in accelerating the movements of the
5

50

PRACTICAL HISTOLOGY.

pale corpuscles of the frog or newt may be investi-
gated with the same apparatus as was used for the
observation of mammalian blood at the temperature
of the body. But it will be found that if the tempe-
rature be allowed to rise so high as 38° 0. the move-
ments of the corpuscles of these cold-blooded animals
will soon permanently cease, the corpuscles being
killed ; so that unless this result is desired, some fat
of lower melting-point must be employed to indicate
the temperature limit which is not to be exceeded.

Preparation 18. Action of Electric Shocks.
—For this a slide must be specially prepared by
cementing on to its upper surface with sbellac var-

Glass slide, with two strips of tin-foil, one of which pusses round to the
under .surface, where it rests on the brass stage of the microscope ;
the other strip is isolated from the stage, and may be connected to
the outer coating of a Leyden jar, the charge of wh'ch is ma.de to
pass between the points by connecting the knob of the jar with the
brass-work of the microscope. Opposite a, a small piece of the foil
is fixed to the under surface of the slide, so that this end shall be
level with the other.

ACTIOX OF ELECTRIC SHOCKS ON BLOOD. 51

nish two slips of gold-leaf or tin-foil, with pointed
ends which almost meet in the middle of the slide
(Fig. 14, A). A drop of blood is put here, the cover-
glass is placed over it, and the portion of blood which
lies between the points is brought under observation.
Or a moist chamber may be employed, the cover-
glass used having previously had two strips of tin-
foil cemented to^it (Fig. 14, B). The drop of blood,
being spread out in a thin layer between their
points, is quickly inverted over the ring of putty and
brought under observation. The tin-foil slips are
kept isolated from the brass-work of the microscope,

Fig. 15.

Apparatus for passing electric shocks through a drop of blood, which is
to be examined in a moist chamber. The tin-foil slips are cemented
near their points to the under surface of the cover-glass, and their
free ends are clamped to isolated metal supports, connected by wires
to an induction coil. The tin-foil slips are isolated from the brass
stage of the microscope by the glass slide on which they rest.

and are so arranged that the charge of a small
Leyden jar, or an induced current of electricity, can
be passed through them at any moment (Fig. 15).

52 PRACTICAL HISTOLOGY.

One or more amoeboid white corpuscles which happen
to be in the path which the spark must take in
traversing the interval between the points are kept
in view, and the spark is then allowed to pass. The
white corpuscles immediately cease moving, with-
draw their processes, and become rounded in shape;
in fact, undergo general contraction. But if only
one slight shock be given they soon recover and
resume their movements, although these are often
somewhat altered in character. The red corpuscles
are but slightly if at all affected ; but if a succession
of shocks be transmitted from an electric machine
or an induction-coil, electrolytic action is set up in
the fluid, bubbles of gas are developed, the effects
respectively of acids and alkalies are set up in the
neighborhood of the tin-foil points, and the red
corpuscles undergo changes brought about by these.

Preparation 19. Presence of Glycogen.—
Many of the white corpuscles contain a certain
amount of glycogen, or animal starch, either in dis-
tinct granules or in a more diffused form. This
substance becomes stained of a reddish mahogany
color by solution of iodine, and may thus be readily
detected, both here and elsewhere. The solution to
be used is made by dissolving 1 gramme of iodine
dn 100 cc. of water, which contains 2 grammes of
iodide of potassium in solution.

The preparation, preferably of frog's or newt's
blood, is made in the usual way, and the iodine
solution added at the side of the cover-glass. The
red corpuscles are stained of an intense yellow, but
are otherwise little altered, except that the nucleus,
which remains unstained, becomes globular, and
bulges out at either surface of the corpuscle. The
white corpuscles are instantly arrested in their
movements and killed, preserving exactly the form
which they exhibited when reached by the iodine
solution. Being of less specific gravity than the
latter, they tend to float on it ; and, if the layer of
fluid is thick, must be sought by focussing upwards

HEMOGLOBIN CRYSTALS. 53

in the stratum immediately under the cover-glass.
The main substance of the corpuscle is uniformly
stained of a deep yellow, but many contain groups
of mahogany-stained granules, and from others are
seen to exude after a time pellucid drops of varying
size, which become tinted of a mahogany or port-
wine color, and no doubt contain glycogen.

Preparation 20. Crystals obtainable from the
Coloring Matter of Blood. — The hemoglobin or
coloring matter of the blood may be obtained in
definite crystals, but the form of the crystals varies
in different animals. It is difficult to induce the
crystallization in human blood ; and to obtain the
crystals readily it is best to employ the blood of the
rat.

A drop of this is mixed with an equal amount of
distilled water, covered, and observed under the
microscope. The water has the effect of extracting
the hemoglobin from the corpuscles; until this is
done no crystallization takes place. As the excess
of water begins to evaporate at the edges of the cover-
glass small needle-shaped crystals of hemoglobin
begin to appear, either singly or in bunches, which
become larger and larger until they may attain a
very considerable size.

Preparation 21. — Crystals may also be readily
obtained from guinea-pig's blood, either by the same
method or more readily perhaps in the following
way: —

The animal is decapitated, and the blood as it
flows from the divided vessels is vigorously stirred
with a small bundle of wire, to remove the fibrin.
A small quantity of the whipped blood is then
mixed with about one-third its volume of water,
and, a drop of chloroform being added, the mixture
is thoroughly shaken up for a minute or two. This
has the effect of discharging the hemaglobin from
the corpuscles into the surrounding fluid. A small
drop is now placed upon a slide and left exposed to

5*

54 PRACTICAL HISTOLOGY.

the air for a few minutes. It becomes thickened by
evaporation and dried at the edges, and crystals,
tetrahedral in form, may be detected in it with a
low power of the microscope. The preparation may
then be covered and examined with a higher power.
The crystals increase in size for a time, and new
ones continue to be formed.

Haemoglobin crystals will not keep unaltered for
any length of time.

Preparation 22. — The name " hsemin" has been
given to certain very characteristic crystals which
are formed at the expense of the coloring matter of
the blood, and the production of which serves as an
exceedingly trustworthy test of the presence of
blood, although yielding no indication of the kind
of animal from which the blood has been obtained.
To see them a very small quantity of blood obtained
from the finger or elsewhere is smeared opon a glass
slide and allowed to dry. A cover-glass is then
placed over it, and a drop of glacial acetic acid is
applied from a pipette to the edge of the cover-glass
and allowed to run under by capillary attraction.
The slide is then held by one end in the fingers, and
the middle is gently warmed over a small flame.
As soon as bubbles begin to appear in the fluid the
warmth is discontinued, and the preparation is ex-
amined with a high power. If no crystals appear
as the slide cools a little more acid is added, to re-
place that lost by evaporation, and the slide is
warmed as before, and on cooling again examined.
It will be found that almost all over the preparation
reddish-brown short prismatic crystals, disposed
singly or in groups, will have made their appear-
ance ; most of them are very minute, but they may
be obtained of considerable size by re-warming the
preparation with glacial acetic acid once or twice.

The presence of a chloride is necessary for the for-
mation of these crystals; in the case of recent blood
the chloride of sodium which it naturally contains

HJSM IN CRYSTALS. 55

is sufficient for the purpose; but if it were an old
blood-stain whicb one had to deal with, in which
the chlorides may have been washed away, it would
be previously requisite to mix a minute quantity of
common salt with the stain to be tested in order to
supply the deficiency.

56 PRACTICAL HISTOLOGY.

CHAPTER II.

THE EPITHELIAL TISSUES.

THE epithelial tissues are studied with regard both
to the character and form of the individual elements,
and the relations these bear to one another and to
the membranes they cover. The latter class of ob-
servation can only be properly made by the study
of sections of the various organs and parts where
epithelium is found, and will therefore be left until
the method of making these is explained. The
modes of isolating and studying the individual cells
will, however, be best described in this place.

Preparation 1. Scaly Epithelium, Superfi-
cial Layers. — If a little of the saliva which moistens
the inside of the cheek be gently scraped off with a
small spatula or with the finger-nail, a number of the
superficial cells of the thick stratified epithelium
which is here met with will be brought away, with
it. The material thus obtained is placed upon a
slide and a cover-glass put over. On examining the
preparation numerous flattened scaly epithelium
cells will be seen, either entirely isolated or in little
patches, the cells in a patch being connected together,
with their edges overlapping. The cells are of con-
siderable size, each with a nucleus near its middle,
small in comparison with the size of the cell ; and
the substance of the cell, although clear, yet contains
a number of scattered granules. Moreover, lines
may often be seen running in various directions over
the surface; these are for the most part due to folds
or creases of its substance. Some of the cells may
be seen edgeways, and then, being flattened, will
appear narrow and linear; but on touching the

STEATIFIED EPITHELIUM. 57

cover- glass with a bristle their true form will be
apparent as they tarn over.

In addition to such cells as these a certain number
of much smaller rounded cells may generally be seen
in the saliva, which somewhat resemble the white
corpuscles of the blood, and, like them, frequently
exhibit amoeboid movements. They are, in fact, to
be regarded as white blood-corpuscles which have
either migrated from the bloodvessels into the sali-
vary ducts and have been carried into the mouth
with the saliva, or have corne from the mucous
membrane covering the back of the tongue and the
tonsils, which is very rich in lymph-corpuscles. The
saliva being a watery fluid they are swollen out by
it, and with a good microscope it may be observed
that the granules in the interior of the corpuscles
exhibit the Brownian molecular movement, a phe-
nomenon which, it will be remembered, was exhi-
bited by the white corpuscles of the blood, as a first
result of the imbibition of water.

Preparation 2. Deeper Layers. — There are
several layers of the above-described large flattened
epithelial cells in the epithelium of the mouth.
Below them are other cells smaller and of a more
spheroidal or polyhedral shape, and many of them
having ridges and spines with intervening furrows
upon "their surface. To obtain these isolated it is
necessary to macerate a piece of any membrane
which is covered by a similar scaly stratified epithe-
lium in some fluid which, while it softens and dis-
solves the intermediate substance which cements
the cells together, may preserve their natural form,
and, at the same time, prevent putrefaction from
appearing in the tissue which is undergoing macer-
ation. The best liquid for this purpose is a weak
solution of bichromate of potash, 1 part to 800 of
water (£ per cent,). The portion of tissue must be
small, and the quantity of liquid used comparatively
large. A piece of the mucous membrane of the
mouth, pharynx, or gullet of any mammal may be

58 PRACTICAL HISTOLOGY.

used ; it will require at least two or three weeks'
maceration (for in this kind of epithelium the cells
are very closely united), and the bichromate solution
should be changed every third day. At the expira-
tion of the time stated a small portion of the epi-
thelium is scraped oft' with the point of a knife and
placed in a drop of water upon a slide. It is then
broken up as finely as possible with a pair of mount-
ed needles, a piece of a hair is cut off and placed in
the drop, and the cover-glass is superposed. By far
the majority of the cells seen are the superficial ones
already described ; but others may be found which
are less flattened and smaller in diameter, and have
many of them an irregular toothed margin, their
surface also, as may be seen by altering the fine
adjustment of the microscope, having on it linear
or punctated markings. These are the cells with
ridges and spines above . mentioned ; they will be
again studied in sections of the skin, as well as in
sections of these and other mucous membranes which
are covered with similar epithelium.

Preparation 3. Horny Layer of Epidermis.
— If a very small shred of the superficial part of the
epidermis is taken from any part, the palm of the
hand, for instance, and examined in water under
the microscope, no indications of cellular structure
are visible — nothing but a hard horny confused mass
is to be seen. Remove the cover-glass and place the
shred of epidermis in a drop of liquor potassse. It
will soon swell up and become soft. When this is
the case return it to the water, and, after breaking
it up as finely as possible with needles, cover and
examine. Numerous spheroidal cells are now seen
loose in the preparation, with a distinct contour, as
if inclosed by a membrane, but without a nucleus.
They are, in fact, the scaly cells, which have become
swollen out by imbibition of water, and at the same
time, in consequence of this swelling of their sur-
faces, correspondingly diminished in width.

The same result may be obtained with the cells

COLUMNAR EPITHELIUM. 59

which form the nails; and also with the flattened
cells from the mucous membrane of the mouth.

Preparations 4, 5, 6,-- -The Columnar Epithe-
lium is most characteristically seen, and is best
studied as met with in the intestinal canal. It must
be taken from an animal (rabbit) quite recently
killed, as it rapidly undergoes destructive changes
if left after death in contact with the intestinal con-
tents. When the intestine is cut across at any
part in a recently killed animal the cut edges curl
outwards, and a little of the mucous membrane is
thus exposed. Two very small portions may be
snipped off this and placed, one in a few drops of a
one per cent, solution of osmic acid, and a second
in iodized serum.1

These two portions may be put aside for the
present in their respective fluids, whilst a prepara-
tion of the epithelium in the fresh condition is
made and examined. For this purpose slit open a
piece of the intestine, wash away the mucus and
intestinal contents by allowing a-little of the serum
from the animal's blood to drop upon the inner
surface, and then, with the end of a clean scalpel,
gently scrape the washed surface and transfer
what is brought away on the scalpel to a drop of
fresh serum upon a clean glass slide, and cover the
preparation, averting the pressure of the cover-
glass by means of a piece of hair. On examining
with a high power the specimen so obtained, nume-
rous columnar epithelium cells will be seen, some

1 This is prepared from the amniotic fluid of the cow, by add-
iug to it a crystal or two of iodine, and allowing it to stand for
a few days, with frequent agitation, and then filtering oft' the
clear fluid from any precipitate that may have formed. Almost
any other fresh serous fluid can be used instead, but the amniotic
fluid is generally the most readily obtained in sufficient quantity,
and at the same time perfectly clear and pellucid. The serous
fluid serves to macerate tissues which are immersed in it, whilst
preserving as nearly as possible the original form of their elements.
The purpose of the iodine is to prevent putrefaction ; it serves
also at the same time to render the tissue-elements slightly firmer.

60 PRACTICAL HISTOLOGY.

separate, others in groups. (It will be found advan-
tageous in examining the object to moderate the
illumination by the employment of one of the smaller
holes of the diaphragm : in this way the somewhat
indistinct outlines of the tissue-elements are rendered
plainer, and any details of structure can generally
be more readily made out ; this is the case, indeed,
with most preparations.) In the separated cells, the
conical form may be seen, the cell either tapering at
one end to a fine point, which may be bifid, or
terminating in a rounded extremity. The general
substance of the cell has a faintly granular appear-
ance in the fresh condition, without a distinct out-
line, except at the larger end, which is bounded by
a strongly refracting thickened margin, in which a
few faint striae passing from without inwards may
with a high magnifying power be made out. Near
the centre of the cell is the clear oval nucleus,
bounded by a distinct line, and containing generally
one nucleolus. Globules of fat of varying size may,
if the animal were- killed during digestion, be seen
within the cell ; they are recognized by their strong
refractive power.

Of the groups of cells some may occur in which
the cells are seen from above ; the collective bases
will then present an appearance of polygonal areas
intersected by lines of intercellular substance; in
other groups, where the cells are seen laterally, their
arrangement with regard to one another will be
observed.

Some cells will probably be seen which have
acquired a peculiar chalice-like shape, owing to the
part of the cell near the free surface having become
swollen out, often to the extent of bursting away
and destroying the thickened border. These are
the so-called u goblet cells."

The other two portions of tissue will not be ready
for examination for some days. That in the iodized
serum must be put aside in a cold place, in a well-
stoppered bottle. At the expiration of a week a pre-

MODE OF FIXING COVER-GLASS. 61

paratiori may be made from it by scraping the epi-
thelial surface as before and mounting the product
in iodized serum. The preparation so obtained is
similar to that already seen of the fresh epithelium,
but the separated cells are much more numerous,
and at the same time more perfect, for, the inter-
cellular substance which unites them being softened
and dissolved by maceration in the serum, the cells
are more readily obtained free.

The snip which was placed in osmic acid must be
left for two days ; it is then placed in water for an
hour or two to remove the excess of the acid. To
examine the tissue remove a minute portion and
break it up as finely as possible with needles in a
drop of glycerine on a slide. The cells will be found
to exhibit the same general characters as regards
form and appearance, but they are stained of a dark
gray color, and in consequence appear for the most,
part much more distinct. Any fatty particles which
they may contain are colored intensely black. This
preparation possesses the advantage over the others
that it can be preserved. All that is further neces-
sary is to apply a little fixing cement of some sort
around the edges of the cover-glass.

The kind of fixing cement used for preparations
mounted in glycerine is immaterial; a solution of hard
Canada halsam in chloroform, dammar varnish, gold-size,
or Brunswick black may be employed with almost equal
advantage; but it is important to see more particularly
to two points before applying it. The first is that there
shall be just enough glycerine in the preparation to fill
up the space between the cover-glass and slide, neither
too much nor loo little; any excess must be removed
with blotting-paper. At the same time one or two small
air-bubbles in the glycerine do no harm, unless they are
so situated as to obscure the tissue; on the contrary,
by yielding and becoming somewhat compressed when
the glycerine tends to expand from being exposed (in
summer, for instance) to a warmer temperature, they
may tend to prevent that fluid from bursting its bonds
6

62 PRACTICAL HISTOLOGY.

and exuding through the inclosing cement. The second
point to be attended to is that the slide where the cement
is to be applied is not wetted with glycerine, for this
would prevent the adherence of the cement.

A word may here be said as to the course to be adopted
should any glycerine have accidentally got on to the
upper surface of the cover-glass. No attempt should be
made to wipe it off' at once, for the cover-glass will be
pressed upon and moved, and the preparation will pro-
bably be crushed and spoiled ; but time should be allowed
to the fixing cement to become thoroughly hard, and
then the glycerine can be removed without danger to the
tissue by means of a camel-hair pencil moistened with
distilled water.

For the present it will be sufficient to conclude
the study of epithelium with the description of the
best modes of viewing ciliated epithelium, and of
studying the action of various reagents upon the
ciliary motion. The other more specialized forms
of epithelium, which are found in glandular organs
and elsewhere, will he seen and studied when the
several organs and parts of which they occur are
prepared.

Preparation 7. Ciliated Epithelium in its
living state may he readily obtained from the mouth
and gullet of the recently killed Irog. A drop of
the aqueous humor should first be collected by
passing a capillary glass tube through the cornea
into the anterior chamber of the eye; the drop is
placed upon a slide, and then, the frog's mouth
being held open by an assistant, the roof is gently
scraped with the point of a clean scalpel, so as to
remove the adherent mucus. A little of the epithe-
lium will be brought away with this, and on placing
it in the fluid, and covering the preparation (taking
the precaution of previously placing a hair in the
drop), the cells may besought for with a high power.
For the most part they will be collected into little
groups of three or more, the cilia being in active
movement and producing currents in the liquid, so

CILIA OF MUSSEL. 63

that free particles, blood-corpuscles, and other small
objects are moved along in it. But if the group is
small, or especially if entirely isolated cells are seen,
it will generally be found that the cilia act upon the
pieces to which they are attached like little paddles,
moving them about in the fluid. The cells, it may
be observed, are either shortly columnar or are
spheroidal ; the nucleus is seldom distinct, because
concealed by the granular nature of the cell-proto-
plasm. The cilia themselves can best be seen when
they are moving languidly or when their motion has
altogether stopped; they are very fine, and spring, a
number together, from the base or free surface of the
cell.

Preparation 8. Cilia of Mussel.— But by far
the most convenient object for the study of ciliary
motion is to be found in the gill of the common sea-
water mussel (Mi/stilus editus). Here the cilia are
very large, and their motion will go on unimpaired
for many hours. Hence they are particularly well
suited for the observation of the action of most of
the reagents which affect ciliary movement.

One or more mussels may readily be procured at
any fishmonger's; those only should be chosen which
remain tightly closed, for those with open valves are
in most cases already dead. One of the shells may
then be forcibly separated by means of a knife, when
the gills (Fig. 16, br) will come into view, as flattened
expansions of a yellowish color, covering a consider-
able part of the shell, inside its lining membrane m£
By observing carefully it may be noticed that they
have a striated aspect, the markings passing trans-
versely to their length, and by taking up a small
portion with a forceps it will further be seen that
this striation is due to the fact that the gill is made
up of a number of little bars which are distinct from
one another for the greater part of their length.
Take now a small piece of the gill, including three
or four of the bars, and placing it upon a side in a
drop of the sea- water which the shell always contains,

64 PRACTICAL HISTOLOGY.

separate the bars one from another by means of
needles ; the preparation may then be covered and
observed.

Valve of mnesel. showing br, br, the expanded gills or branchiae, which
owing to the little bars of which they are composed, present a striated
aspect.

ml, mantle ; m, cut adductor moscle ; i, mass of viscera ; the dark pro-
jection just above is the foot.

Each of the bars in question will be seen to be
fringed with large cilia, which are set at an appre-
ciable distance apart along nearly its whole length,
but at the free extremity of the bar are much more
densely arranged. Those in this situation resemble
in appearance the cilia of the frog's mouth, with the
exception that they are very much longer ; and like
them they appear to spring a large number from
each cell, whereas the others are stiff-looking and
obviously thicker, and are connected at their base
each to a single, comparatively small epithelium
cell. In spite of these differences of appearance and
attachment the two kinds seem to be essentially
alike in nature, for the mode in which they move
is similar, and they are similarly affected by re-
agents.

It will be found that after a preparation such as
that just described has been made for several hours

ACTION OF REAGENTS OX CILIA. 65

the movement will have become somewhat languid,
and then the manner in which the individual cilia
move can be more clearly made out. The prepara-
tion can be used also for the study of those agents
which tend to revive and stimulate ciliary motion,
and it will be seen that it is precisely the ones which
most accelerate the amoeboid movements of the white
blood-corpuscles that have the most marked effect
upon the cilia also.

Preparation 9. Action of Warmth upon
Ciliary Motion. — The same mode of applying heat
to a preparation of cilia is to be used as was employed
for observing the effect of warmth upon the blood
(p. 30). It is well, after inclosing the preparation
with oil (or, what is better, with melted paraffin) in
the manner there detailed, to put it aside for some
time, when it will probably he found, as just stated,
that the movement of the cilia is languid or alto-
gether arrested. On now gradually warming the
preparation the motion becomes revived, and as the
heat is raised becomes, pan passn, faster and faster,
until a point is reached at which the tissue is injured
by the high temperature, when the movement stops
altogether, and is not again resumed. But if the
experiment be stopped short of this point and the
source of heat removed, it will be seen that, con-
versely, as the temperature of the stage falls, the rate
of movement also diminishes, until, when again
quite cold, the cilia may again almost stop, although
they can be made to resume their active motion on
again applying warmth.

"Preparation 10. Action of Weak Alkalies.
— A very weak solution of caustic potash in salt
solution, like that which was used in investigating
the effect of weak alkalies upon the blood, is to be
applied to a preparation of cilia which have become
somewhat languid, in exactly the same manner as
in the case of the blood — by allowing a little of the
fluid to pass in at the edge of the cover-glass and
diffuse itself with the "sea-water, so as to come

6*

66 PRACTICAL HISTOLOGY.

gradually in contact with the slowly-moving cilia.
The action is immediate ; the cilia revive and vigor-
ously lash the liquid into which they project, but
the effort is soon exhausted, for the alkaline liquid
penetrating the cells destroys their vitality, and the
motion of their cilia stops beyond recovery. The
stimulant action is not, however, peculiar to weak
alkalies, for it is exhibited also by acids and by
many other substances which, applied concentrated,
would instantly destroy the tissue, but when much
diluted tend to revive and for a time maintain accele-
rated the ciliary motion.

Preparation 11. Action of Carbonic Acid
Gas. — This reagent is to be applied to a cilia pre-
paration in the gas-chamber in the manner directed
for the investigation of its action upon the newt's
blood-corpuscles (p. 48). Everything being ready,
choose a part of the preparation where the cilia are
not acting very vigorously, and whilst still watching
allow the gas to pass over the preparation. Its im-
mediate action is seen to be that of a weak acid —
that is to say, the rate of movement, if not already
at its fastest, becomes accelerated — but as soon as
the oxygen of the air in the chamber is entirely dis-
placed by the continued stream of carbonic acid the
motion ceases altogether. As soon as this result is
obtained cut off the stream of C02 and reverse the
experiment by blowing air in at the side-tube, and
thus displacing the carbonic acid from the chamber.
The motion will almost instantly recommence. This
shows that it was the absence of oxygen and not the
presence of C02 which produced the stoppage of
movement ; for there is, of course, an appreciable
quantity of carbonic acid in the air which is thus
blown from the mouth into the chamber.

Preparation 12. Chloroform.— The gas-cham-
ber is again used for this reagent, the apparatus
being arranged in the way previously recommended
(p. 39). Choosing a part of the preparation where
the ciliary motion is vigorous, gently blow a stream

CILIATED EPITHELIUM. 67

of the mixture of air and chloroform vapor from the
bottle into the moist chamber. The cilia become
gradually slower and eventually stop. Now slip
the India-rubber tube off the bottle, and gently
blow air through the chamber, to displace the
chloroform vapor. The cilia will slowly revive on
the readmission of air, and will soon be found to
work as vigorously as ever. Like that with the
carbonic acid this experiment can be repeated a
number of times with a like result, if the chloroform
vapor is not allowed to remain too long in contact
with the preparation.

-Preparation 13. — To study the characters of the
individual cells a portion of membrane which is
covered by ciliated epithelium is macerated in some
fluid which softens and dissolves the intercellular
substance, whilst preserving the cells themselves.
The best for this purpose is a weak solution of bi-
chromate of potash (1 in 800). A large quantity of
this must be employed, and the tissue — a piece of
the trachea of a rabbit or other animal, for instance —
is left in it for about forty-eight hours. With the
point of a scalpel a little of the epithelium is then
gently scraped from the inner surface, and being
placed in a drop of distilled water on a slide, is
broken up with needles as finely as possible. A
small piece of hair is placed in the drop, to prevent
the soft, delicate cells from being crushed by the
weight of the cover-glass. This is now superadded,
and the preparation carefully examined with a high
power.

Numerous completely isolated cells are seen float-
ing in the liquid, and these preserve for the most
part their natural form and retain their cilia, al-
though the latter are, of course, no longer in motion.
The bright border through which the cilia appear
to pass, the faintly granular cell-substance, the clear
nucleus with bright nucleolus in the middle, and the
tapering, branched extremity of the cell are, in most,
clearly visible. Besides these single cell*, others are

68 PRACTICAL HISTOLOGY.

present which are still united in groups or patches,
in which, when viewed from the surface, the bases
of the cells have a mosaic appearance. Moreover, a
few "goblet" cells may here and there be met with,
no doubt produced in a similar manner to those
found amongst the columnar cells of the intestine
and elsewhere, viz., by a swelling up of the mucus
contained within the cells when these come in con-
tact with a watery fluid. In these cases the base of
the cell is ruptured and the cilia are destroyed.

Besides the ciliated cells certain others may be
seen in the specimen which are of an irregular and
usually elongated shape, with pointed and often
branched extremities, and are destitute of cilia.
They lie, in the natural state, between the ciliated
cells, but their position can only be properly studied
in sections of the mucous membrane.

If it be wished to permanently preserve such a
preparation as that now under description, it is
necessary first to stain the cells somewhat and then
to substitute glycerine for the staining fluid. Either
dilute logwood or one per cent, osmic acid solution
may be used for staining the cells. The former
colors their nuclei very intensely, the latter gives a
uniform gray tint to the cells. The coloring fluid
is applied in the following manner : A little is taken
up into a pipette and a drop is then brought in con-
tact with one edge of the cover-glass. The pipette
is then removed, and at the other edge a small piece
of filter-paper, torn into a triangular shape, is placed,
with the apex of the triangle touching the cover-
glass, so as to draw the fluid slowly through. When
the staining fluid has replaced the water in which
the preparation was made, the filter-paper is removed,
and the preparation is left until the cells appear
sufficiently colored, a little more fluid being occa-
sionally added if there seems any danger of the
specimen becoming dry. With a solution of log-
wood, even though very dilute, a few minutes

CILIATED EPITHELIUM. 69

suffices ; the osmic acid solution should be allowed
to remain an hour in contact with the cells. The
staining fluid is replaced by water in the same way,
and finally, the filter-paper being removed, a small
drop of glycerine is applied at one border of the
cover-glass and gradually takes the place of the
water as this evaporates at the edges. In a day or
two the cover-glass may be fixed with chloroform
balsam.

70 PRACTICAL HISTOLOGY

CHAPTER III.

CONNECTIVE TISSUE.

AREOLAR TISSUE. — In the areolar tissue and in
connective tissue generally there are several parts
which present themselves for study ; and in order
to observe each to the greatest advantage different
modes of preparation are, for the most part, requi-
site.

Preparations 1, 2. The Fibres of Areolar
Tissue. — For the observation of the fibrous elements
simply, without special regard to their arrangement
or relation to the other elements, all that is necessary
is to place a small portion of areolar tissue, taken
from any part, on the centre of a glass slide, just
moistened with salt solution, and with clean, sharp
needles, separate it as finely as possible into filamen-
tous shreds. Then, before there is time for the pre-
paration to become dry, place a drop of salt solution
on a cover-glass and invert this over the tissue. The
object of using but" very little fluid to prepare the
tissue in is to prevent the filaments from running
together and becoming entangled when released from
the needles.

In a preparation so made, nothing is, as a rule,
apparent save the wavy bundles of connective tissue
fibrils, these when much developed obscuring, by
their effect upon the light, the elastic fibres and cor-
puscles of the tissue. But if now a second prepara-
tion be made in precisely the same way, except that,
in place of salt solution simply, salt solution con-
taining one part of acetic acid in 200 is placed upon
the cover-glass, and if then the object is immediately
examined with a high power, it is seen that the

CONNECTIVE TISSUE CORPUSCLES. 71

fibrils which compose the bundles have become in-
distinct, whilst the bundles are much swollen, except,
it may be, at intervals here and there. At the same
time certain other fibres, almost equally fine but
more sharply defined than the white fibrils, and
always running singly, never in bundles, come into
view. These are the elastic fibres. If one of them
be followed for a short distance, it will probably be
seen that it sooner or later gives off a branch which
unites it with a neighboring fibre,whereas the white
fibrils never show any disposition to branch or unite
with one another, but those in each bundle maintain
from end to end a perfectly parallel course. The
elastic nature of the filaments which are brought
into view by acetic acid is shown, in such a prepara-
tion as we are describing, by the fact that wherever
in the process of teasing the tissue they have become
broken across, the fibres have, by the recoil from the
stretching to which they were submitted before the
rupture occurred, been thrown into bold curves, es-
pecially marked near the broken extremities, which
are often recurved. That this curved or coiled ap-
pearance of the elastic fibres, although highly char-
acteristic, and always observable when the tissue is
thus prepared, is, however, not a natural one, is
shown by the fact that, as will immediately be de-
scribed, when precautions are taken to preserve as
much as possible the normal arrangement of the
tissue elements, the elastic fibres are seen to pursue
a rectilinear course.

Preparation 3. The Corpuscles of the Areolar
Tissue. — To demonstrate the cells or connective
tissue corpuscles the preparation is made more
methodically. A film as thin as possible must be
obtained for observation, so as to avoid the necessity
of tearing the tissue. Such films are naturally pre-
sent in the areolar tissue of most parts, and may be
seen when the organs which it connects are gently
drawn asunder from one another, as, for instance,
when the skin is raised and reflected from the sub-

72 PRACTICAL HISTOLOGY.

jacent fascise and muscles. The most convenient
source of such a delicate film is to be found in the
exquisitely thin and transparent tissue which invests
and lies between the muscles of the fore-limb of the
rabbit and guinea-pig. The tissue in this situation,
especially if taken from a young animal, is devoid,
of fat and not so completely overridden by the bun-
dles of white fibrils but that the elastic fibres and
the connective tissue corpuscles can be made out
even without the addition of reagents. The mode
of preparation is as follows :—

The animal having been killed by bleeding, the
skin is snipped through around the upper part of the
fore-limb and is then forcibly reflected from the limb.
In this operation care must be taken to avoid be-
sprinkling the subjacent parts with the cut hairs of
the animal. A piece of the tissue over or between
the muscles is then seized with the forceps and
snipped off with sharp, fine scissors.. The snipped-ofi
tissue shrinks immediately around the end of the
forceps and appears very unsuited for microscopical
examination. But place it on a clean slide, without
the addition of any fluid, and with a pair of mounted
needles endeavor, by drawing out first this corner
and then that, to again reduce the gelatinous-looking
piece to the condition of a thin film, and it will be
found that this can be effected without much diffi-
culty, for when not floated up by fluid the thin edges
of the film tend to stick to the glass, and cease to
shrink away from the position to which they are
drawn by the needles. At the same time, whilst it
is important not to add fluid to that which naturally
moistens the piece of tissue, it is equally important
never, during the whole process of stretching, to let
the film become actually desiccated, for this would
altogether ruin the tissue for microscopic purposes.
The best way to prevent such an untoward result
from happening is to breathe now and then on the
object whilst it is being prepared; by so doing need-
less haste will be avoided and more time and pains

CONNECTIVE TISSUE CORPUSCLES. 73

can be taken for the complete display of the film.
This being effected, a cover-glass (which should have
been previously cleaned and placed in readiness) is
taken, wetted on its under surface with a drop of
salt solution, and quickly superposed over the film
of tissue, which is thus prevented from shrinking up
again into a shapeless mass. The fibres, both white
(in wavy bundles of various sizes) and the elastic,
and corpuscles may now be carefully observed, at
first with the usual high power and afterwards with
the highest obtainable, and some of the corpuscles
should be sketched. Moreover, search may be made
for lymph or pale blood-corpuscles, a very few of
which are generally to be found in the connective
tissue; they are readily distinguished from the fixed
corpuscles of the tissue by their small size — small,
obscure, and generally multiple nuclei — and espe-
cially their amoeboid movements, of which it is pro-
bable no trace will be apparent even to the most
assiduous observation in the connective tissue cells
proper.

Preparation 4. — Although both corpuscles and
elastic fibres may be seen in a preparation of this
kind made with an indifferent fluid, they are better
seen if the white fibres are acted upon by acetic acid,
and still better if this action is combined with that
of some staining reagent, so that the corpuscles are
brought more prominently into view. Moreover,
the preparation admits of being permanently pre-
served in glycerine after such a method of treat-
ment. Up to a certain stage the procedure is the
same as that above described, but instead of placing
salt solution upon the cover-glass, before inverting
it over the film, a solution of acetic acid (one per
cent.) colored by the addition to it of one-third its
volume of logwood-alum solution, is employed. The
object is examined as quickly as possible after the
application.

The connective tissue corpuscles can probably be
made out already in the thinner parts of the prepa-

74 PRACTICAL HISTOLOGY.

ration, with their clear oval nuclei and the flattened
irregular area occupied by their cell substance. In a
few minutes the nuclei will be tinted by the logwood,
and will then show up much more prominently ;
but to get the cell-bodies sufficiently colored it will
be necessary to leave the staining solution half an
hour or more in contact with the preparation. Mean-
while, to obviate the effects of evaporation, a con-
siderable drop of the coloring fluid should be placed
on either side in contact with the edges of the cover-
glass. The excess of fluid, moreover, has a tendency
to raise the latter slightly from the lilm of tissue,
and in this way a more ready access of fresh coloring
fluid is permitted. When it is found on examina-
tion that the corpuscles are properly stained, the
solution may be drawn off by a slip of filter-paper
applied to the edge of the cover-glass on one side,
whilst to the other a drop of distilled water is applied,
the logwood being for the most part in this manner
rinsed away. The filter-paper is then removed, and
a drop of glycerine placed in contact with the edge;
this, as the water evaporates, will slowly diffuse it-
self under the cover-glass and take its place. It is
well to put the preparation aside until the following
day, when the process will be completed ; all that is
then necessary is to draw a camel-hair pencil wetted
with chloroform balsam around the edge of the
covering-glass, so as to fix it securely in its place.

In specimens treated with acid there may be observed
a constricting ring at intervals along the course of
some of the connective tissue bundles, an appearance
which has long been familiar to histologists, but the
cause of which is not yet clearly determined, some sup-
posing it, from its resistance to the action of acids, to be
of the nature of elastic tissue ; others that the appearance
is caused by the process of a corpuscle enwrapping the
bundle ; the latter opinion being mainly founded on
the fact that the ring becomes stained by certain re-
agents which also color the cells. For example, as shown
by Kanvier, the constricting ring is tinted red by picro-

METHOD OF LOCALIZED (EDEMA. 75

carminateof ammonia, whilst the elastic fibres are colored
yellow.

Preparation 5. — In most of the larger animals
(e.g. the dog), the connective tissue is, in the adult,
so densely pervaded by the bundles of while fibres
as to render it impossible to obtain a film delicate
enough for easy observation without tearing the
texture with needles, and thereby distorting the
cellular elements. Or, on the other hand, it may be
desired, even in those animals in which such delicate
tissue as that the preparation of which has just been
described is found, to obtain a specimen from a part
where the connective tissue is not naturally extended
in so advantageous a manner for preparation and
observation. In these cases the following method
may be employed with advantage : —

In a recently killed dog a flap of skin is dissected
back, and a Pravaz or 'other hypodermic syringe
(Fig. 17), provided with a fine canula, having been

Fig. 17.

Pravaz syringe for interstitial injections; c, the cauula. The syringe is
glass with ebonite fittings ; the canula is a fine perforated steel
needle set in ebonite. Natural size.

previously filled with salt solution, the point of the
canula is inserted underneath the layer of connec-
tive tissue which is most superficial on the reflected
portion of skin, and a little of the fluid is forced out.
This does not immediately diffuse itself uniformly
through the loose areolar tissue, but remains for a
short while at the same place, forming a little bulla
of liquid bounded and covered in by a film of tissue,

76 PRACTICAL HISTOLOGY.

the thickness of which depends upon the depth to
which the canuhi was inserted. If it does not
appear thin enough a second attempt should be made
at another spot. Then, before the bulk has time to
subside — that is to say, before the fluid has time to
diffuse itself through the meshes of the tissue — snip
off the whole projection with a single cut of a pair
of scissors, which for this purpose should be particu-
larly sharp and clean, and transfer the snipped- oft'
portion to a clean slide. Here it may either be at
once covered in salt solution and examined without
reagents, or may be treated with acetic acid, logwood,
&c., as in the mode of preparation just described.

Preparation 6. — A modification of the above
method consists in injecting into the tissue a solu-
tion of gelatine instead of a salt solution. The gela-
tine solution is made by taking some very clear
French gelatine, allowing it to soak for an hour in
water, and then, after pouring off' all the excess of
that fluid, placing the soaked gelatine in a beaker
over a water bath until it is entirely melted in the
water which it had imbibed. The syringe is then
warmed by immersing it for a minute or two in
warm water, and is filled with the gelatine solution,
and a little of this is injected into the subcutaneous
connective tissue, so as to produce a bulla like that
made by the salt solution. In cold weather the
gelatine will set almost immediately ; in warm
weather the process may be accelerated by surround-
ing the bulla with small lumps of ice.

When the gelatine is quite firm sections of it are
to be made with a razor. As they are cut they may
be placed in salt solution.

Before mounting them it is well to stain the
specimens, and one of the best staining fluids for
the purpose is a watery solution of magenta. This
colors the elastic fibres strongly, the corpuscles dis-
tinctly, and the bundles of white fibrils slightly,
while the gelatine which was injected and of course
occupies all the interstices is hardly stained at all.

METHOD OF GELATINE (EDEMA. 77

The time of immersing the sections varies of course
with the strength of the fluid, but this should not
be too highly colored, and it can be then seen with-
out much difficulty when the sections are sufficiently
stained. They are subsequently placed in water for
a minute or two to remove the excess of magenta
prior to transferring them to a slide. A drop of
glycerine is now added and the cover-glass laid on,
after which the slide is gently warmed over a small
flame or otherwise until the gelatine in the sections
just melts so as to allow the cover-glass, which was
probably tilted up somewhat owing to the thickness
of the sections, to settle down. The specimen may
then be examined, and if satisfactory, may be pre-
served, the preparation being completed after a day
or two by fixing the edges of the cover-glass with
chloroform balsam.

By the modes of preparing connective tissue already
described the bundles of white fibres, the elastic fibres,
and the corpuscles are brought under observation, and
it would seem at first sight that these of themselves
entirely constitute the tissue. But in considering the
nature of the films obtained — that they are, namely, con-
tinuous over a greater or less area — it is clear that the
presence of fibres and cells is not alone sufficient to
account for the lamina? which are spread upon the slide.
And, indeed, by closely observing the preparation it will
be apparent that there is pellucid substance uniting every-
thing together, through which the fibres run, and in which
the corpuscles lie embedded. There is, it is true, a diffi-
culty in making this out in most parts, in consequence
of its extreme clearness, and the fact that its refractive
index is little different from that of the watery fluid the
tissue is examined in ; moreover, in the logwood pre-
parations the intermediate substance remains entirely
unstained. Nevertheless, towards the edge of the pre-
paration, where a comparison can the better be made
with the surrounding fluid, the fact that such a clear
intermediate substance does really exist will be suffi-
ciently evident ; and the more so if the cover-glass be
slightl}' moved or one edge be gentlv pressed down with

7*

78 PRACTICAL HISTOLOGY.

a needle. But we possess in the silver method of Reck-
linghausen a ready means of demonstrating its existence
in an obvious manner; for the ground substance (or
intercellular substance) of the connective tissue, and,
indeed, of almost every other tissue in the body, possesses
the distinctive property of reducing the salts of silver
under the action of light, so that the metal is deposited
in it either in a free state or, more probably, in combina-
tion. The effect of this deposition is, that the ground
substance becomes stained of a color varying with the
intensity of the light employed, and with oilier conditions,
from a light brown to a brownish-black. The iibrous
elements participate for the most part in this staining,
and are frequently, especially when the preparation has
been, as is usual with silver preparations, mounted in
glycerine, indistinguishable from the ground substance
through which they course, and which also unites the
white fibres into the bundles which they form. The
cellular elements, on the contrary, remain absolutely
unstained, and, moreover, after the action of the silveV
salt are no longer affected by those staining fluids which
otherwise have a particular aSinity for them ; it is there-
fore no longer possible to bring them into view. Where-
ever, then, a cell is situated, there appears after the
reduction of the silver nothing but a white patch upon
or in the brown ground; and if, as is not unfrequently
the case, several flattened cells may have occurred
together with their edges in juxtaposition, the group
appears as a larger white patch intersected by dark lines,
these representing a small amount of intercellular sub-
stance between the individual cells. The appearance is
similar to what is observed in an epithelial tissue after
the silver treatment, for in this the intercellular substance
is always very small in amount. Such an arrangement
of connective tissue cells is on this account designated
" epithelioid." The white patches, then, in the silvered
preparation of connective tissue represent either depres-
sions on the surface of, or actual cavities within, the
matrix or ground substance, containing cells, which
themselves are not visible, so that the white patches are
termed the cell-spaces, or (recalling the analogous case
of bone) the lacuna of the connective tissue.

It is the more appropriate to give them a special desig-

NITRATE OF SILVER PREPARATIONS. 79

nation, because, as may be made out by a careful compari-
son of specimens of connective tissue from the same part,
some prepared with logwood or chloride of gold, to show
the cells, others with silver, to show the cell-spaces, the
cell-spaces are in many cases distinctly larger than the
cells, and may also he of a somewhat different figure;
they are not necessarily, therefore, as has sometimes been
supposed, and as is no doubt the case with the clear parts
of a silvered epithelium, merely the cells left white. The
difference in the relative size of the cell-spaces and the
contained cells obtains no doubt more frequently, or at
least can be more readily made evident, in the firmer
varieties of connective tissue, where the ground substance
is everywhere pervaded with fibrous bundles, and has in
consequence lost its soft and yielding nature, which other-
wise permits it to adapt itself more readily to the shape
of the cells. Moreover it must be remembered that the
natural course of the lymph in the tissue is around the
cells, between them and the inclosing ground substance;
and when from any cause that fluid is in excess the effect
will be that the cell-spaces are more distended and appear
preternatu rally large.

This much having been said in order to explain
the appearances produced by the silver method of
treatment — appearances which have been called in
question, and their value as yielding evidence of any
constant structure in the tissues altogether denied
by histologists of considerable eminence, but, as it
must now be admitted, without sufficient grounds —
the best mode of applying it to ordinary connective
tissue, such, for instance, as the subcutaneous, may
now be described.

Preparation 7. — The skin of a recently killed
rabbit or guinea-pig1 having been stripped off one of

1 These ammals are selected because there is likely to be less
fat in the subcutaneous tissue than in that of the cat or dog or
other animals commonly used in the laboratory. It is important
to remember that any tissue which is to be submitted to the silver
method must be fresh and unacted upon previously by any re-
agent whatever, since this would entirely prevent the desired
effect; moreover, if blood have accidentally got on to the part, it
must first be rinsed away by distilled water.

80 PRACTICAL HISTOLOGY.

the limbs, this is disarticulated at the proximal joint,
and is rinsed for a second or two in a beaker of dis-
tilled water, in order to wash away any blood or
lymph, which might happen to be on the surface,
and which would cause a granular precipitate with
the nitrate of silver solution The latter, a solution
of 1 part of the salt to 200 of distilled water, is
then either poured over the surface or dropped on it
from a pipette. After two or three minutes the
silver solution is quickly washed off by a stream of
distilled water, and the limb is then at once placed
in a beaker of spirit, and exposed to direct sunlight,
or, failing this, to bright diffused daylight. In a
few minutes in the sunlight, and after a longer time
in diffused daylight, the silvered surface will have
acquired a uniform brownish tinge to the naked eye.
When the color is strongly marked, it is as well to
remove the beaker from the light, lest the prepara-
tion become too darkly stained. The limb should
be allowed to remain in the spirit twenty-four hours;
at the expiration of this time it is placed in a dish,
and, by the aid of fine forceps and scissors, a piece
of the superficial stained layer is dissected off under
spirit. In doing this, care must be taken not to
drag at all upon the membrane which is removed,
so as to throw it into creases. The piece is then
transferred to water, and floated on to a clean glass
slide writh the browned surface of the membrane
uppermost. The slide is then carefully removed
from the water, the portion of tissue being kept flat
on its upper surface ; and most of the superfluous
water having been wiped away, a cover-glass, on
which a drop of glycerine has been placed, is in-
verted over the preparation. Before putting on the
cover-glass it is well to examine the object under
a low power, in order to make sure of the absence
of folds and creases or specks of dust upon it : if
any such be seen they must be carefully removed
with a needle. Indeed, it may be recommended as
a golden rule in making histological preparations

CELL -SPACES OF CONNECTIVE TISSUE. 81

never to put the cover-glass in its place until a
glance at the ohject under a low power of the micro-
scope has certified the absence of any marked imper-
fection : if this be attended to, the time will often
be saved which would otherwise be spent in mount-
ing worthless specimens.

These silver-preparations of the subcutaneous tissue
vary considerably in appearance according to the part of
the limb from which they are taken, and it is in man}7
cases difficult full}7 to comprehend the meaning of what
is seen, so that it will be better perhaps to omit for the
present this preparation, especially as the one following
furnishes a very ready and clear means of demonstrating
the cell spaces of connective tissue.

Preparation 8. — The connective tissue which
covers the tendons of the superficial flexor digitorum
of the ox's foot as they run through sheaths formed
by the tendons of the deep flexor is much more easily
prepared by the silver method than the looser kinds
like the subcutaneous just described. A piece of
such a tendon is taken, rinsed in distilled water to
remove the synovial fluid which covers it, and treated
with silver solution in the way just described; and
is then, after washing., placed in the light in strong
spirit. It soon becomes brown, when it may be re-
moved from the light; and after remaining twenty-
four hours in the spirit it is easy, with a sharp knife
or razor wetted with spirit, to obtain a thin surface
section. This, after being immersed for a minute
or two in water to get rid of the spirit, is mounted
in glycerine with the browned surface uppermost.
It should present, if successful, an extremely charac-
teristic and beautiful image of white branched cell-
spaces, single or in groups, upon a brown ground.

In both the preparations last described it is possi-
ble to show the nuclei of the corpuscles which lie
in the cell-spaces by subsequent staining with log-
wood. Sometimes they are visible even without
this treatment.

82 PRACTICAL HISTOLOGY.

Preparation 9. Elastic Network.— The pro-
portion of elastic fibres varies considerably according
to the part from which the connective tissue under
examination is taken. Some serous membranes con-
tain a large number of elastic fibres; and since they
are readily spread out in their natural condition the
network which these fibres form by their branchings
and conjunctions is easily made evident. One of
the best objects for this purpose is to be found in the
rabbit's mesocolon. A piece of this, moistened with
a little salt solution, may be spread out as flat as
possible upon a slide, and a drop of dilute acetic acid
(one per cent.) having been placed on a cover-glass,
this is inverted over the tissue. The white fibrils
quickly swell up and become indistinct, whilst the
elastic network comes clearly into view. The pre-
paration may be made permanent by putting, as in
former preparations, a drop of glycerine at the edge
of the cover-glass, and after this has had time to dif-
fuse itself, cementing the edges with chloroform bal-
sam as usual.

Preparation 10. Elastic Tissue.— The elastic
ligaments are to be regarded merely as connective
tissue structures in which the elastic elements of the
tissue preponderate. There is always a quantity of
ordinary areolar tissue amongst the elastic fibres
even here, but not sufficient to obscure them, espe-
cially as they are generally of larger size as well as
in greater number than elsewhere. It is sufficient,
in order to see them, simply to teaze out a portion of
the ligamenta subflava of the vertebrae or other
elastic tissue in water or salt solution. If it be
desired to keep the preparation it can be mounted
in glycerine.

Preparation 11. Section of Elastic Fibres. —
To observe the shape of the fibres a transverse section
may be made of a piece of ligamentum nuchae of the
ox, in which the fibres are extremely large.

In order to obtain the requisite firmness for cut-
ting, place a small piece of the ligament in a quantity

PREPARATION OF TENDOX. 83

of two per cent, solution of bichromate of potash for
fourteen days ; then place in water for two or three
hours, and transfer to spirit ; in. a day or two sec-
tions across the direction of the fibres may be made
with a razor, the surface of which should be wetted
with spirit. The sections are to be placed in water
for a minute or so, and finally mounted in glycerine.

Preparation 12. Fibrous or Tendinous Tis-
sue.— This may be examined by separating a small
shred from a tendon or ligament, and teazing it out
as finely as possible into its constituent bundles.
The operation is conducted with the aid of needles,
the tissue being placed in a drop of salt solution, and
it is first examined in this, being afterwards treated
with dilute acetic acid and logwood (see Prep. 4).
But it is a troublesome matter to make the separa-
tion fine enough without disturbing too much the
arrangement of the cellular elements of the tissue.
Fortunately we can obtain, from the tail of the mouse
or rat, tendons which are, FO to speak, naturally dis-
sociated ; for excessively fine tendons run along the
whole length of the tail, and can readily be drawn
out, needing no further manipulation than is neces-
sary to place them advantageously under the micro-
scope. The following is the mode of procedure : —

Preparation 13. — In a recently killed mouse the
tail is seized about half an inch from the tip between
the thumb nail and fore-finger of the right hand ;
and the delicate skin being partially nipped through
and the vertebral column broken at this point by the
pressure of the nail, it will be found quite easy, the
base of the tail being fixed by the left fore-finger
and thumb, to separate the end altogether and drag
it away from the remainder of the tail. In doing
so it will be found that the minute tendons which
are attached near the tip, owing to their compara-
tive toughness and strength, are not broken through
at the spot in question, but are dragged out of the
channels in which they run, and may in this way
be obtained in the form of a bundle of exquisitely

84 PRACTICAL HISTOLOGY.

fine silky threads, which are to be immediately im-
mersed in a glass dish of salt solution. Now cut
away two or three of the fine threads with sharp,
clean scissors, and seizing them by one end with fine
forceps, or leading them with a needle-point, float
them on to a glass slide which is held immersed in
the fluid, and is then carefully lifted out. After
arranging the minute tendons as nearly straight as
possible on the slide, and blotting up must of the
superfluous salt solution or allowing it to run oft',
place a short piece of hair beside them, to avert the
pressure of the cover-glass, which is now placed over
the middle of the threads in such a way that, since
they are considerably longer than the width of the
cover-glass, their ends project beyond on either side.
The object of this is to permit them to be drawn
straight with needles should the superposition of the
cover-glass have displaced them. These ends, more-
over, since they are exposed to the air, soon dry and
stick to the slide, so that subsequent treatment with
reagents does not tend to displace the tendons, which
are thus maintained in an extended condition. Ex-
amined thus in salt solution, little is visible beyond
the slightly wavy, closely packed white iibrils, col-
lected, as longitudinal streaks seen here and there
indicate, into a few indistinct bundles. But allow
a little dilute acetic acid (1 part of the glacial acid
to 200 of salt solution) slowly to pass under the
cover-glass, and a remarkable change becomes appa-
rent. "As the acid reaches the tendons, they slowly
swell up and become more transparent, the fibrils
becoming indistinct ; and now chains of small oblong
faintly granular cells, each with a clear nucleus
situated near one end of the cell, and often opposite
that of a neighboring cell, come into view. These
are the tendon-cells, the corpuscles of the fibrous
connective tissue ; only the central thicker portion
of each, which lies in the interstice between three
or more tendon bundles, is seen at present ; the thin
lamellar prolongations, which extend between two

CELLS OF TENDON. 85

tendon bundles, are too delicate to be made out
without staining. In some of the chains a bright
longitudinal line is to be seen on each cell ; this ap-
pearance is merely produced by a lamellar prolonga-
tion of this sort, which happens to extend vertically
to the plane under observation.

After the action of the acid has been prolonged
for some time, the cells gradually lose their dis-
tinctness, and eventually can with difficulty be
made out, although the iibres are more swollen and
indistinct than ever. But if a little of the colored
acetic acid which was used for the subcutaneous
tissue (Prep. 4) is allowed to run under the cover-
glass, first the nuclei, and then the bodies of the
cells and their prolongations, become colored, whilst
the fibres remain unstained, just as in the parallel
case of the areolar tissue.

When the coloration is sufficiently deep, the
staining fluid may be replaced by water, and this
in the usual way by glycerine ; and finally, the edges
of the cover-glass being cemented, the preparation
can be permanently preserved. Examined with a
high power, the tendon cells now appear in the suc-
cessive horizontal planes as quadrangular flattened
bodies, thickest near the middle, and gradually
shading off at either side, and marked with one or
more dark lines running longitudinally, which are,
in fact, the bright lines to which attention was pre-
viously drawn, and which have now become stained.
To show that these are actual flattened extensions
of the cell, and not mere markings, it is necessary to
compare the appearances presented by a transverse
section.

Another method of displaying the cells of tendon is
that originally employed by Ranvier. One or two of the
small tail tendons are placed on a slide, and their ends
are fixed with paraffin, so as to keep them extended. A
few drops of one per cent, solution of picrocanninate of
ammonia are then placed upon them and left for half an
hour, after which the picrocarmme is washed away with
8

86 PRACTICAL HISTOLOGY.

distilled water mid the tendons are mounted in glycerine,
acidulated with acetic acid.

The picrocarmine is made by adding to a saturated
solution of picric acid a strong solution of carmine in
ammonia, to saturation, evaporating the mixture to one-
fifth part of its bulk, allowing to cool, filtering from the
deposit which occurs, and evaporating the filtrate to dry-
ness over a water-bath ; when the picrocarmine is left as
a crystalline powder of an ochre-red color.

Preparation 14. Transverse section of Ten-
don.— To obtain this, it is best to take a large
tendon, tor it is much easier to get transverse sec-
tions of sucli a one, and in all essential points of
structure it is quite similar to the minute tendons
which, for the sake of convenience, we have just
been employing. A piece, then, of any tendon,
large enough to be grasped by the fingers, is placed
in strong spirit for a day or two. This gives it a
very hard, 'horny consistence, and it is easy, with u
sharp knife or razor wetted with the spirit, to get one
or two thin sections from the end. These are placed
on a slide, a drop of the acidulated logwood is added
and left in contact with them until they are suffi-
ciently stained. It is then carefully washed away
by a drop or two of water applied from a pipette ;
and finally a little glycerine is placed upon a cover-
glass, which is then inverted over the preparation.

It will be seen that the tendon is divided into
fasciculi by septa of areola tissue, the corpuscles of
which (seen edgeways) are brought into view, being
stained by the logwood ; it will further be observed
if the sectional area of one of the smaller fasciculi
is attentively examined with a high power, that it
again is divided (although incompletely) into several
still smaller bundles by the branching processes of
deeply colored stellate bodies situate at the angle of
junction between three or more such bundles, and
extending a greater or less distance between the
neighboring bandies. These stellate bodies, with
their processes, are the tendon-cells with the himcl-

ADIPOSE TISSUE. 87

lar extensions, as seen in section ; the smaller fasci-
culi, which are separated by areolar tissue, corre-
sponding with the whole of one of the caudal ten-
dons of the mouse or rat.

Preparation 15. Cell-spaces of Tendon. — To
show the cell-spaces in which the above-mentioned
tendon-cells are contained: —

As before, break off the end and draw out some of
the tendons of a mouse's tail (that previously used
will still yield a sufficient number). Then hold the
tendons in a shallow dish of distilled water, and
with a medium-sized camel-hair pencil brush them
firmly from end to end. six or eight times. Remove
them now from the water, and immerse them in a
large watch-glass of nitrate of silver solution (J per
cent.) for fifteen minutes; then place in a glass ves-
sel of water and expose to sunlight. As soon as they
are well browned, pieces may be cut off, laid straight
in water upon a slide, covered, and the water then
drawn off and replaced by glycerine; after which
the cover-glass may be fixed in the usual way.

Preparation 16. Epithelioid covering of Ten-
dons.— The object of first brushing the tendons is
to remove the layer of flattened cells which covers
the surface of each, and which, if allowed to remain,
prevents the silver solution from properly acting
upon the deeper parts of the tissue. To show this
layer, another set of tendons may be treated with
the silver solution in a similar way, but with a
minute's immersion and without previously brush-
ing them, when it will probably be found that the
superficial epithelioid stratum is alone apparent.

Preparation 17. Adipose Tissue.— The sim-
plest way of showing the fat vesicles is by teasing
out a small portion of the tissue in a drop of salt
solution; taking the precaution of putting a narrow
slip of blotting-paper on either side to avert the
pressure of the cover-glass.

But a far better way of demonstrating the struc-
ture of the tissue generally, consists in the employ-

88 PRACTICAL HISTOLOGY.

ment of the method of interstitial injection with
gelatine (see Preparation 6, p. 76). The gelatine is
injected into the interior of one of the fat-lumps,
and sections are made and treated in the way as
described. By this method the fat-cells are some-
what separated from one another, and all their parts,
as well as the intermediate tissue and bloodvessels,
are much better displayed.

Preparation 18. Membrane of the fat-cell.
— To show this distinctly Ranvier's method may be
recommended. An interstitial injection is made
with a weak solution of nitrate of silver (1 in 1000),
with a minute portion of the fat-lump thus rendered
cedematous is removed with scissors, transferred to
a slide and covered. Many of the fat cells exhibit
the envelope and nucleus separated by a distinct
space from the fat-drop. The silver solution would
appear to have penetrated by endosmosis and to have
become collected between the fat-drop and its inclos-
ing membrane.

Preparation 19. — To complete the study of adi-
pose tissue, the fat-cells should be observed in pro-
cess of development. For this purpose a preparation
of the subcutaneous tissue from a part where fat is
being deposited may be obtained from the moderately
advanced foetus of any mammal. The new-born rat
is especially to be recommended, since in its subcu-
taneous tissue there are generally to be found, not
only cells which are in every stage of fat-deposition,
but others in addition which exhibit the formation
of bloodvessels and the simultaneous formation with-
in the same cells of red-blood corpuscles. The mode
of preparation is very simple, all that is necessary
being to strip the skin from the back, snip oft' with
scissors a little of the gelatinous-looking tissue from
the borders of a tract where the fat is already partly
deposited, and to place the portion so obtained in a
drop of salt solution upon a slide, and cover it with
a thin glass.

CARTILAGE. 89

CHAPTER IV.

CARTILAGE.

Articular Cartilage. — This is to be studied in
sections made both parallel and vertical to the
surface.

Preparation 1. — From an animal that has just
been killed remove one of the limb-bones, with its
articular ends, and with a clean, sharp scalpel or
razor take a slice, as thin as possible, off' the carti-
lage, and quickly, before it has time to become dry,
transfer the piece to a drop of serum upon a slide,
place a cover-glass over the preparation and examine
with a high power. Turning the attention more
particularly to the cartilage-cells, the arrangement
of these in groups in the faintly granulous matrix
will be noticed. Each cell is seen to be provided
with a clear round nucleus, which in some speci-
mens of articular cartilage is so large proportion-
ately that it may be mistaken by an inexperienced
observer for the whole cell. In reality, however,
the cell-substance is represented by the clear material
(or containing, at most, a few highly refracting
granules) which lies around the nucleus and entirely
fills the cavity or space in the cartilaginous matrix
in which the cell lies. But now replace the serum
by distilled water, drawing the former away by
means of a piece of blotting-paper placed at one edge
of the cover-glass, and allowing a drop of water from
a pipette to run under at the opposite edge (see Fig.
18), and the picture soon changes. Examine the
cells at the borders of the slice, for these are first
reached by the water. It will be seen that the clear
cell-substance begins to be separated from the matrix,

b*

90

PRACTICAL HISTOLOGY.

and acquires a jagged outline, fluid collecting in the
interspace which is now left. This clear fluid is
stated by some to be water which has permeated
the matrix, and p-issing into the cell-space has

Fig. 18.

pushed, as it were, the cell substance away. But
the more probable explanation is that the cartilage-
cells, by virtue of a certain amount of vital contrac-
tility which they retain, shrink on the application
of the water, as they do on the application of many
other reagents and on the passage of an electric
shock, and* that the clear fluid which collects around
them is expressed from their protoplasm as it con-
tracts. "Whatever the explanation may be, the effect
is this: that the now jagged, shrunken cell-body
assumes, instead of the clear aspect which in the

ARTICULAR CARTILAGE. 91

fresh condition it presented, a coarsely granular
appearance, so much so indeed that, the nucleus
which was previously so apparent is now entirely
obscured. Moreover, as already indicated, the car-
tilage-cell no longer fills the cell-space in which it
lies. The cells always undergo this change after
death, unless the tissue has been treated with some
reagent which prevents its occurrence.

In preparing a specimen of cartilage with the
object of permanently preserving it, our aim should
be, as, indeed, with every tissue, to obtain it in a
condition and form as nearly as possible approaching
that which it had whilst living. There are nume-
rous reagents which, in place of acting like water
and causing contraction of the cartilage-cells, fix
them in the form they present during life. Amongst
these osmic acid may be mentioned first, as the most
generally valuable reagent which we possess for this
purpose, since it acts in like manner upon nearly all
the tissues. But it will not here be recommended
for the purpose of preserving the tissue of cartilage
in consequence of its costliness, and from the fact
that other and cheaper reagents serve the purpose
equally well. One special value it certainly has,
however ; namely, in showing that the little granules
in the protoplasm of the cartilage-cell are many of
them of a fatty nature, for they are blackened by
the reagent. A one per cent, solution of alum, and
a saturated solution of picric acid (both recom-
meded by Ranvier), preserve the cells of cartilage
admirably. But one of the best and most conve-
nient reagents for the purpose is a weak solution of
chromic acid (1 part to 600 of water).

Preparations 2 and 3. — The articular head of
one of the long bones is removed from the recently
killed animal, split into two down the middle with
a strong knife or a chisel, and the halves placed in
a large quantity of a solution of chromic acid of the
strength above indicated, and allowed to remain in
it a few days. The exact time is immaterial, but in

92 PRACTICAL HISTOLOGY.

specimens, which are left rather longer in the liquid,
the bone in the neighborhood of the cartilage is
softened, and the cartilage-cells are more colored by
the acid, and consequently more apparent. When
it is desired to prepare the tissue for the microscope,
one of the halves is taken from the fluid, washed in
water for a minute or two, and then, the bone being
held in the hand, one or two thin sections are taken
from the surface of cleavage vertically to the articu-
lar surface and extending through the whole depth
of the cartilage, including, if possible, a little of the
adjacent bone. The sections are placed in water on
a slide and covered, and are examined, first with a
low power to see the general arrangement of the
cartilage cell groups in the superficial, intermediate
and deep strata respectively, and subsequently with
a high power to see the intimate structure of the
cells. These should, as before indicated, present as
nearly as possible the same appearance as during life,
the only difference being that the tissue generally is
less transparent, and slightly colored, and that the
cell-outlines are rather more strongly marked. These
differences become even less obvious when glycerine
has been permitted to diffuse itself under the cover-
glass, for the preservation of the specimen.

Sections are next to be taken parallel to the articu-
lar surface and mounted in the same manner; but
it must be borne in mind that it is only those sec-
tions which include parts of the cartilage near the
natural or artificial surface which will be of value
as respects the preservation of the tissue elements in
their natural condition, at least in the case of thick
articular cartilages of large animals. For the preser-
vative solution naturally takes some time to perme-
ate the cartilaginous matrix, and before it has time
to penetrate to the deeper parts, the cells will have
already shrunk away from the walls of the inclosing
cavities and have become changed in the manner
previously indicated. So that the deeper sections
will exhibit merely the irregular, contracted, and

CELL-SPACES OF CARTILAGE. 93

highly refracting corpuscles lying loosely in their
cell-spaces.

At the thinnest parts of all the sections cavities
may he observed in the matrix which are devoid of
cartilage-cells; these having dropped out in the pro-
cess of preparation.

Preparation 4. — These cavities or cell-spaces of
cartilage may he also demonstrated by the same
method as was employed to show the cell-spaces of
connective tissue, viz., treatment with nitrate of
silver and subsequent exposure to the light. For
this purpose a fresh joint should be opened, and the
articular end of one of the bones (preferably a convex
one) removed with the sawr or bone-forceps. The
end thus removed is rinsed in distilled water and
then quickly transferred to nitrate of silver solution
(J per cent.), in which it is allowed to remain three
minutes. It is then again rinsed in water, and if
necessary gently brushed with a camel-hair pencil
to remove adhering silver precipitates ; after which
it is placed in a beaker of weak spirit and exposed
to sunlight. "When thoroughly browned, sections
are made from the surface with a razor wetted with
spirit, and are placed in water (care being taken that
they become completely immersed), after which they
may be mounted in glycerine.

Some of the sections should be taken from near
the edge of the cartilage and mounted on a separate
slide. These should show7 the branched cell-spaces,
which present a transition to the much more ramified
spaces of the connective tissue of the synovial mem-
brane.

The cells themselves which occupy the spaces are
not shown, for, as in silvered tissues generally, the
cell-protoplasm remains absolutely unstained ; indeed,
it is at first sight difficult to believe that the rounded
cavities which are seen really contain cartilage- cells.
But their nuclei may be brought into viewr by stain-
ing with logwood, subsequently the matrix being
colored by the nitrate of silver. As before men-

94 PRACTICAL HISTOLOGY.

tioned in speaking of the connective tissue, it is,
however, difficult, if not impossible, at the same time
to bring distinctly into view the outlines of the cell-
spaces by the silver method, and the bodies of the
cells themselves by some other method of staining.

Preparation 5. — No doubt one of the best and
most generally useful methods of coloring cell pro-
toplasm is that known as the gold method. This is
applied to cartilage in the following way. Thin
sections are made from the articular end of a fresh
bone and are placed in a few drops of a solution of
chloride of gold (1 part in 200 of \vater). After
half an hour they are transferred to a beaker con-
taining a comparatively large amount of distilled
water which has previously been slightly acidulated
with acetic acid, just so as to be distinctly acid to
the taste. The beaker is then covered with a glass
plate and placed in a window in as warm a place as
possible, and where it will be exposed for some hours
a day to the full sunlight. Here the sections are
left for two days, after which time they should have
acquired a dark violet color, and are ready to mount
in glycerine. But before this is done they should
first be examined with a low power in a drop of
water to see whether there is any precipitated matter
upon the surfaces of the sections. If this is the case
it must be brushed away with a camel-hair pencil,
the sections being held with fine forceps during the
process. When finally mounted and examined with
a high power, the cartilage cells should retain pre-
cisely their natural form and appearance, except that
they are stained of a faint violet tinge, whilst the
matrix remains almost entirely colorless. In sections
taken from the edge of the curtilage, the ramified
transitional cells which occupy the corresponding
cell-spaces shown by the silver method will be ren-
dered apparent.

The coloration by the gold and silver methods appears
in some way to depend on the occurrence of a deposition

GOLD METHOD.

95

of the metal in those parts of the tissue for which it has
the greatest affinity ; in the case of gold this is generally
the protoplasm of the cells (and the nerve-fibres, where
any exist) ; in the case of silver it is the ground substance,
or matrix, or intercellular substance ; so that the results
of the two processes may, at least in the case of the carti-
lage-cells, be looked upon as standing to each other in the
light of a positive and negative image, using the terms as
they are employed in photography.

Fig. 19.

Warming nppnrntns for maintaining portions of tissue, after treatment
by the gold method, at a constant, raised temperature. The lower
part of the apparatus is filled with water, into which the bulb of the
mercurial regulator dips.

If it is the winter time, and especially if there is very
little bright sunlight, it is of the greatest importance to

96

PRACTICAL HISTOLOGY.

keep the beaker of acidulated water in which the tissue
is placed warm, as this materially facilitates the reduc-
tion of the gold. For this purpose it is well to have some
arrangement by which the beaker, or several of them, if
there be much of the same sort of work going on, can be
kept at a temperature of about 30° or 40° Centigrade,
and at the same time freely exposed to the light. Fig.
19 represents a convenient apparatus for this purpose.
It is made of zinc or tinned iron with loose glass sides
and top, and may be fitted with a gas regulator (Fig. 20)
to prevent the temperature from rising too high. When
used for the present purpose the apparatus should of
course stand in a good light — in the window if possible.

n, a, small iuuer tube, capable of being slipped up
or down withia c, the upper part of the bulbed
tube containing mercury, d, small aperture to
allow of the constant passage of a small amount of
gas, so that the flame is not entirely extinguished
if the mercury should rise sufficiently high to
entirely exclude the lower end of the tube a.
The tube b, with the corks d nod e, form a simple
and easily constructed arrangement (suggested by
Mr. F. J. M Page) which serves as a gas-tight
telescopic joiat, and enables the tube a to be
raised or lowered according as the temperature of
the chamber is desired to be higher or lower. In
all other respects the working of the regulator is
similar to that belonging to the apparatus shown
in Fig. 9.

The upper part of the raercural gas-regulator of the apparatus shown in
Fig. 19. Natural size.

Preparations 6 and 7. Costal Cartilage.— The

cartilages of the ribs and those of the trachea and
larynx may be prepared and examined in a manner
similar to that recommended for the study of articu-
lar cartilage. Sections, both parallel and vertical
to the surface, should be made from the fresh tissue
and from pieces which have been a longer or shorter

CELL TERRITORIES OF CARTILAGE. 97

time in chromic acid solution ; those from the latter
being mounted and preserved in glycerine as before.

Preparation 8. — The alum process may also be
advantageously used. Sections, as thin as possible,
of a perfectly fresh rib cartilage are to be mounted
in a dilute solution of alum (j per cent.). This will
be found to preserve for a time the natural appear-
ance of the cells, better almost than any other
reagent.

Preparation 9. Cell Territories of the Matrix.
— Sections are to be made of a piece of thyroid
cartilage that has been preserved in spirit, and are
to be stained with logwood. When sufficiently
colored, they are transferred to water and then
mounted in glycerine. The logwood, besides stain-
ing the nuclei of the cells, gives the matrix also a
deep purple color. But this coloration of the matrix
is not uniform, for some parts become stained much
more deeply than others; those regions more imme-
diately around the cells and cell-groups, and which
therefore are, as commonly considered, the latest
formed portions, having apparently more affinity
for the coloring matter of logwood than the other
and older parts. By this method the whole matrix
appears marked out into what may be termed cell-
territories^ although series of definite rings can by no
means be said to be very apparent around the cell-
groups, such as, it is stated, may be produced in
sections of cartilage by treating them with a mix-
ture of nitric acid and chlorate of potash.

Preparation 10. Transition to yellow fibro-
cartilage, — If the arytenoid cartilage of the ox is
sliced longitudinally, it exhibits to the naked eye in
its lower part the opaline bluish appearance of hya-
line cartilage, but in its upper part the faintly yellow
aspect of elastic cartilage, the two parts being sepa-
rated by a distinct line of demarcation. Take now a
thin section from the cut surface, including a little
of both parts, and mount, if fresh, in alum solution,
or, if the cartilage have been previously hardened in
9

93 PRACTICAL HISTOLOGY.

spirit, at once in glycerine. It is immediately seen
that the cartilage above the line of demarcation
merely differs from that below by the superaddition
of a network of comparatively coarse branching
(elastic) fibres. Just at the junction of the two
parts the fibres are but few in number, and at their
ends often imperfect — i. e., they may appear continued
merely as rows of granules as if not yet fully formed.
Further into the yellowish part they permeate the
matrix very thickly, and give it almost a granular
appearance, owing to many of them being seen in
section. But immediately around each cell there is
always a larger or smaller area of the matrix entirely
free from fibres, and this area is in appearance ex-
actly like the matrix of hyaline cartilage. So that
we may conclude that yellow or elastic cartilage is
merely to be regarded as hyaline cartilage, in the
matrix of which a deposit of elastic substance has
taken place in the form of branching fibres; and,
indeed, the study of its development shows this to
be the case.

Preparation 11. Yellow fibre-cartilage. — A
tissue may now be studied which is composed en-
tirely of yellow cartilage, the human epiglottis or
external ear, for example. A section of either of
these shows what at first looks like a granular
matrix dotted with islands of hyaline substance,
each with one or two cartilage-cells in the centre.
The granular appearance of the matrix, when care-
fully observed, is seen to be due to an excessively
close feltwork of fine elastic fibres; in fact, the
structure is quite like that of the upper part of the
ox's arytenoid, although, owing to their fineness and
closeness, the individual fibres cannot be followed
for any distance.

Preparation 12. White fibro-cartilage is best
studied in sections of any ligament which is com-
posed of this tissue, or of the insertion of any tendon
or ligament which is attached, as in the young sub-
ject many are, to cartilage. The tissue may have

CELL-TERRITORIES OF CARTILAGE. 99

been previously hardened in spirit. Sections should
be made both parallel with and across the bundles
of tendinous fibres. They are to be stained with
logwood and mounted in glycerine. It will be seen
in the longitudinal sections that the cartilage-cells
lie in chains between the tendon bundles, occupying
the place of the ordinary tendon-cells; from these
they are chiefly characterized by their sharpness of
outline and (in transverse sections) the absence of
lamellar extensions, as well as their greater thick-
ness ; but transitions between the two are not unfre-
quent, especially near the tendon insertion.

100 PRACTICAL HISTOLOGY.

CHAPTER V.

BONE.

Preparations 1 and 2. Transverse and longi-
tudinal sections of hard bone. — These are first
cut as thin as possible with a fine saw, and are
afterwards reduced in thickness and polished by
grinding. The bone selected should be thoroughly
macerated and bleached ; it should be absolutely
free from grease. Sections may be made from dif-
ferent bones, flat and long, but for typical specimens
of the compact tissue, transverse and longitudinal
sections of one of the long bones of the limbs — the
ulna, for instance — may be recommended, and a
vertical section of one of the flat bones of the skull,
such as the parietal, should also be prepared.

The first thing to be done is to get as thin a
piece as possible cut with a saw from any desired
part. For the purpose a fret-saw may be used,
unless a circular saw is available, when this, if fine
enough, may be employed with advantage. The
piece so obtained is ground down on a hone wetted
with water. The hone must have been previously
freed from all traces of greasy matter by washing
with soap and water with a little soda. The piece
of bone is pressed down and rubbed to and fro on
the hone simply by the finger, being ground first
on one side and then on the other. The feel will
be almost sufficient to tell when it is thin enough,
and this may be confirmed by placing it on a slide
without covering it and examining with a low
power. Numerous scratches will doubtless be visi-
ble on its surface, produced by the grain or' the stone,
however fine this may be ; but unless very obvious,

SECTIONS OF HARDENED BONE. 101

they may be disregarded, for they become almost
invisible in the subsequent preparation. This con-
sists in well rinsing the piece (when satisfied that it
is thin enough) in clean water by aid of a hair
pencil, placing it upon a slide to dry, and, when
dry, mounting it in Canada balsam. The balsam
to be used for this purpose must not be, as is usually
the case with that sold in the shops, semi-fluid, but
quite hard in the cold — a condition which results
from long keeping, or may be produced by heating a
little of the more recent resin in a capsule over a
sand- or wTater-bath, until all the volatile matters are
driven off. A drop of the melted balsam is then
placed upon a slide^ which is warmed over a flame
until the resin has diffused itself pretty evenly over
the central part of the slide. This is then placed
on the table, and whilst cooling, but before the
balsam has time to become quite hard again, the
thin piece of bone is placed upon it. A clean cover-
glass is then taken up by the forceps, a drop of the
balsam placed upon it also warmed in like manner,
and quickly inverted over the preparation. By
this mode of proceeding the balsam fills up and
renders invisible the scratches on the surfaces of the
section of bone, and some of the Haversian canals
may also be filled by it ; but it becomes solidified
before it has time to penetrate into the lacunae and
canaliculi, which remain, therefore, filled with air,
and present the black appearance which is char-
acteristic of any small cavities containing air when
they are viewed under the microscope by trans-
mitted light.

Most of the structural points with regard to bone
can be seen much better in these preparations of the
hard tissue than in sections of a decalcified bone.
But it is important in mounting the pieces after
they have been ground down to be careful that the
balsam does not remain fluid long enough to have
time to penetrate into the thickness of the section ;
if this should happen, the whole is rendered too

9*

102 PRACTICAL HISTOLOGY.

transparent for any of the details of structure to be
made out. On the whole it is perhaps advisable to
purchase one or two good specimens rather than
devote a large amount of time to the manufacture
of what may after all turn out to be but an indif-
ferent preparation. Sections of bard bone and of
teeth are amongst the very few histological prepara-
tions which are usually better made by those who
make a business of preparing, microscopic specimens
for sale than by the student himself.

Preparation 3. — In addition to sections of hard
bone which are made by grinding in the manner
above described, a portion of a well-macerated bone
may be placed in a solution of hydrochloric acid
(10 parts of the commercial acid to 100 of water)
until all the earthy matter is dissolved ; the piece is
then steeped in a very weak solution of carbonate
of soda to free it from the remains of the acid, and
sections may then be made with a razor or sharp
scalpel and mounted in glycerine. But these decal-
cified specimens in no way illustrate the structure
of the tissue so well as the others.

Preparation 4. — It is, however, quite another
question with regard to bones which have not been
submitted to previous maceration. For it is to be
remembered that in considering the structure of a
bone we have to deal not merely with the bony
matter pure and simple and its included cavities,
but that there are in addition the soft contents of
those cavities — the corpuscles in the lacunae, the
bloodvessels in the Haversian canals, and the marrow
in the medullary cavity and in the interspaces of the
spongy tissue. In order properly to view these
perishable structures, we must employ a reagent
which will at the same time decalcify and soften
the hard matter and preserve and harden the in-
cluded soft tissues. Such a reagent is to be found
in a solution of chromic acid, but ns the decalcify-
ing powers of this are but feeble, the portions of
bone placed in it must be as small as possible, and a

SECTIONS OF SOFTENED BONE. 103

large proportionate amount of the fluid must be
used. For example if the bone be a long bone
about the size of the human metacarpal, a piece not
longer than a quarter of an inch should be sawn
from it and placed (suspended by a thread if pos-
sible) in a beaker capable of containing some 200
c. c. of the chromic acid solution. If the bone be
larger, it is well to split the disk thus sawn off into
three or four smaller pieces, since otherwise the
decalcification will occupy too long a time. The
acid used should be at first very weak (1 in 600) ;
in two days' time this may be changed for a solu-
tion of 1 in 400 ; and this again in another two days
for 1 in 200. Beyond this the strength of the acid
should not be increased, but the fluid should be
renewed every three days at least. In addition to
this frequent changing of the liquid, it should,
throughout the whole time of softening, be stirred
as often as possible: this is of the greatest impor-
tance, for every agitation brings fresh portions of
acid to attack the earthy matter of the bone. By
attention to this particular the time which the
pieces take to become thoroughly decalcified may
be materially shortened. The completion of the'
process is ascertained by passing a needle through
the middle of the piece employed ; if it meets with
no gritty obstruction, all the earthy matter is pro-
bably got rid of. This will most likely be the case
in two or three weeks from the commencement of
the operation. Should the pieces of bone, owing to
their too great thickness or density, be much longer
than this" before they are sufficiently soft, the pro-.
cess may be hastened by adding to the chromic acid
solution a little nitric acid (2 c. c. to each 100 c. c.
of chromic solution). This reagent was not avail-
able at the commencement of the operation, because,
although it would have more readily dissolved the
salts of lime than the chromic acid, yet it would
have softened still more the included soft tissues of
the bone instead of hardening; them like that re-

104: PRACTICAL HISTOLOGY.

agent ; but it may be employed during the later
stages, especially in conjunction with the chromic
acid, since the latter reagent has, by its peculiar
coagulant action upon the animal tissues, so altered
their constitution that their structure is no longer
rendered indistinct by diluted nitric, acetic, and
the ordinary acids, which produce swelling and
maceration of the fresh tissues, and especially of the
connective tissue.

The process of decalcification being completed, the
pieces are placed in water for a few hours to get rid
of the excess of chromic acid imbibed, and are then
transferred to spirit; or sections may be made at once
without placing the piece in spirit at all. In cutting
them, if the bit of bone is too small to be held in the
fingers, it may be placed in a split piece of cork. The
sections should be very thin, but it is not necessary
that they should be large or include the whole thick-
ness of the bone; they are to be stained in the log-
wood solution, washed in water, and finally mounted
in glycerine. The corpuscles within the lacunae, or
at all events their nuclei, are beautifully shown, and
all the soft parts are more or less stained, but the
actual substance of the bone is very slightly colored.
At the same time, any lines of demarcation which
may be present indicating successive deposits of
osseous matter in the formation of the bone, or
absorption at any part and subsequent redisposition,
with the characteristic scalloped edge which such a
junction almost always possesses, are very clearly
shown by a difference in the coloration. Moreover,
if in the section there happens to be any portion
remaining of the (ossified) matrix of the original
embryonic cartilage, this, like cartilage matrix gene-
rally, is intensely "stained by the logwood.

Preparation 5. — Instead of chromic, picric acid
may be used for the decalcification. A saturated
solution of the acid is employed, and care is to be
taken constantly to supply fresh crystals of picric
acid to take the place of that which is used up in

PICRIC ACID METHOD. 105

dissolving the lime salts. In all other respects the
proceeding is much the same as for the chromic acid
method, except that the pieces will require much
longer washing in water, and that the spirit in which
they are placed be changed several times before the
excess of picric acid can be got rid of. The sections
may be stained with logwood and mounted in glyce-
rine in a similar manner, and will show the structure
as well, if not better, than those prepared with
chromic acid.

Preparation 6. Lamellae and Sharpey's per-
forating fibres. — For showing these structures, ma-
cerated bones that have been decalcified by hydro-
chloric acid serve very well. They should before
use soak in water for some time, and subsequently
lie in spirit, so as to get rid of the last traces of the
acid. The point of one of the blades of a sharp pair
of forceps is then inserted obliquely into the bone at
its outer surface, and a small piece is gripped by the
other blade and dragged off in such a manner that it
pulls away with it a very thin strip from the super-
ficies of the bone. A few such strips having been
obtained, either from different parts of the bone or
from the same place (at different depths, therefore),
they are placed in water on a slide with the inner
surface uppermost and examined with a low power.
In some places tapering fibres will be seen projecting
from the surface of the torn-off strip like nails pro-
jecting from a board ; in other parts, round or ovalish
holes, corresponding generally in size with these
fibres, will be apparent; these are apertures in the
lamellae where the latter have been pierced by the
fibres of Sharpey, but those fibres have been pulled
out in tearing off the strips. Further, there may be
made out a faint appearance of decussation in the
lamellae, like the checks on a plaid, but oblique in
direction, best marked near the thin edge of the strip
of bone. These appearances are all more evident
when the preparation is covered and examined with
a higher power. To preserve the preparation it is not

106 PRACTICAL HISTOLOGY.

a good plan to replace the water in which the strips
are mounted by glycerine, since this renders the tex-
ture too transparent and the outlines too indefinite.
But the strips may be mounted and preserved for a
considerable time in water merely if the edges of the
cover-glass are fixed first by melted paraffin, and then
by layer after layer of chloroform balsam, so as to
obviate as much as may be the risk of evaporation.

The bones in which the fibres of Sharpey may best be
demonstrated in the way above described are the flat
bones of the skull. They are also to be seen in similar
preparations, and in sections of the long bones. If a sec-
tion be made of such a bone at the place of insertion into
it of a tendon or ligament, and in the direction of the fibres
of the tendon or ligament, it will be seen that the bundles
of fibres of the last-named structures are continued into
the substance of the bone as perforating fibres or fibres of
Sharpey, so that these in fact almost compose the whole of
the osseous tissue at this place (Ranvier). This shows,
moreover, that the fibres of Sharpey are to be regarded as
bundles of fibrous tissue (connected either with the perios-
teum or with a tendon or ligament) which were intercalated
with the osseous substance proper when this was formed
and have become ossified at the same time. When tendons
undergo ossification, the bony substance which is formed
is wholly of the same nature as the fibres of Sharpey;
this may be characteristically seen in the ossified tendons
which are met with in the legs of birds.

Preparation 7. Development of bone in
membrane. — For the study of the intra-membra-
nous process of .ossification it is best to employ the
flat bones of the skull of sheep's embryos from two
to three inches long. The embryos may have been
preserved in Muller's fluid,1 or spirit, or they may
be employed fresh. A piece (corresponding in posi-
tion with the future parietal, for example) of the
still membranous skull-cap is cut out with fine scis-
sors, placed under water if from Muller's fluid, under

1 A solution containing 2£ parts bichromate of potash, and 1
part sulphate of soda, to 100 of water.

PROCESS OF OSSIFICATION. 107

salt solution if recent, and the skin and muscular
layers are torn away from the outside and the dura
mater and cartilaginous layer (which in these animals
rises up laterally from the cartilaginous hasis cranii)
from the inside. The membrane in which the bone
is being formed is then left. It is held upon a glass
slide with a needle or line forceps, and brushed
firmly with a camel-hair pencil, the hairs of which
have been cut off short so as to render the stump
stiff and resisting. The piece must be kept wet,
and examined from time to time with a low power
to see whether the edges of the newly-formed bone
are sufficiently clear of the membrane and corpuscles,
so that the osseous spicules and their fibrous pro-
longations are readily seen. When this is the case
the piece is held with the forceps and well rinsed in
water or salt solution to free it from loose particles
of the soft tissue ; it is then placed in logwood solu-
tion, and when sufficiently colored (two or three
minutes) is mounted in glycerine. The osteoblasts
will be found stained by the logwood ; the ossified
part is dark and highly refracting, the osteogenic
fibres by which it is prolonged remain clear and
colorless.

The process of intra-membranous development
may also be very advantageously studied in sections
of the lower jaw of the foetus or young animal, as
will be afterwards pointed out in describing the
mode of preparing this to show the development of
other structures, the teeth and hair, for example.

Preparation 8. Development of bone in
cartilage, — To study the mode of ossification in
cartilage it is not necessary to have recourse to the
bones of a fcetal animal, since any which are still in
process of growth will serve the same end, and it is
more convenient for purposes of manipulation if
they have attained a certain size. The long bones
of a new-born kitten may advantageously be em-
ployed, and the best preparations are certainly those
obtained from the recently killed animal, although

108 PRACTICAL HISTOLOGY.

for permanent preservation it is better that they
should have been some days in Mtiller's fluid. The
mode of preparing the fresh specimens is as follows.
One of the long bones — the femur, for instance — is
removed, and, its cartilaginous head having been
cleared of the adherent soft structures, is split down
the middle with a strong scalpel, the split extending
a little distance into the subjacent bone. Then by
a movement of the scalpel one of the halves is broken
away, and the junction of the bone and cartilage is
brought clearly into view. One or two slices as thin
as possible are now taken from the surface thus pro-
duced, including the line of advancing ossification,
and are placed on a slide in salt solution and covered.
These sections are of course parallel with the long
axis of the bone ; but to complete the observation
others should be made across that axis, at and a
little below the level of the line of ossification, the
cartilaginous head being first sliced gradually away
until the line of ossification is reached, and then a,
series of sections taken of the part of the cartilage
which is undergoing ossification and of the newly-
formed bone.

The fresh preparations are by far the most beau-
tiful and instructive if the sections are obtained
sufficiently thin. If it is wished permanently to
preserve a section made in this way, the salt solution
in which it is mounted must be drawn off by filter-
ing paper placed at one edge of the cover-glass, and
at the same time replaced by a solution of osmic
acid (one per cent.) applied at the opposite edge.
The preparation may then be left for an hour (the
filter paper being removed), after which time the
osrnic acid is in its turn withdrawn arid replaced by
water, and finally a drop of glycerine is put at the
edge of the covering-glass and suffered to take the
place of the water as this evaporates.

Preparation 9. — Sections, longitudinal and trans-
verse, of ossifying bones which have been in Mliller's
fluid for a few days, are prepared in exactly the

PROCESS OF OSSIFICATION. 109

same manner, except that the treatment with osmic
acid is here unnecessary. The preparations can be
mounted at once in glycerine.

Preparation 10. — To trace the later steps of the
ossification, it is requisite to obtain sections extending
rather more deeply into the newly-formed bone than is
possible whilst this still retains its earthy salts. For
this purpose, therefore, the hone, which may with advan-
tage be considerably smaller than that of the new-born
kitten — one from a small foatus, for example — is decalci-
fied in chromic or picric acid in the same way as was
employed for the deealcification of pieces of the fully-
developed bone (Preps. 4 and 5). But if the foetal bone
is small, the time necessary for such decalcification will
be very much shorter than was requisite for the dense
adult bone. Jn order to cut a longitudinal section suffi-
ciently extensive and thin, it will be necessary to embed
the bone in the wax mass (chap, x.), and the sections
obtained may be stained with logwood, or successively
with both logwood and carmine, and mounted in dammar
varnish in the way usually employed for treating sections.
A still better plan is to stain the bone entire with an
alcoholic solution of magenta, allow it to become per-
meated first with oil of cloves and then with melted
cacao-butter, and afterwards embed in cacao-butter, treat-
ing the sections with warm oil of cloves to dissolve out
the fat, and then mounting in dammar varnish. Or the
stained bone may be permeated with gurn, placed in spirit
containing one-sixth its volume of water to harden the
gum within the tissue, embedded in wax mass, cut, and
the sections placed in water to dissolve out the gum.
The .object of allowing a tissue of this nature, which con-
sists of hard and soft parts intermingled, to become
permeated by the substance above recommended, is that
the soft parts and cavities may be filled up by a hard
material, so that the tissue may offer a uniform resistance
to the razor and the parts of a section may cohere ;
otherwise the soft parts are very apt to become separated
from the hard.

But since these processes are somewhat complicated,
the student is recommended to defer them for a while
10

110 PRACTICAL HISTOLOGY.

until the methods upon which they depend have been
described and explained.

Preparation 11. Medullary tissue. — Ordinary
marrow, which consists for the most part of adipose
tissue, is obtained from the long bones of most
animals. But the spongy tissue of the bones gene-
rally and the medullary canal of the long bones
in some animals — the rabbit and guinea-pig, for
example — are filled with red marrow, which con-
tains little adipose tissue, but is mainly made up
of the so-called proper marrow-cells, which are in
many respects similar to the pale corpuscles of the
blood, and, like these, exhibit amoeboid movements.
To see the marrow-cells, therefore, in their natural
condition the tissue should be taken quite fresh and
examined on the warm stage. The bone — the femur
of a guinea-pig, for instance, preferably a young
animal — having been removed and cleared of the
surrounding soft parts, is broken across, and a small
piece of the marrow picked out and broken up with
needles in a drop of serum or salt solution on a piece
of thin glass. A small piece of hair is then added,
and the preparation is covered ; a brush dipped in
oil is drawn round the edge of the cover-glass to
prevent evaporation, and the specimen is then placed
on the wrarrn stage (p. 30) and examined. The dif-
ferent sizes and varying forms of the marrow-cells
are to be noted, as also the large clear nucleus which
many of them possess, and which at once distin-
guishes these cells from the ordinary white blood-
corpuscles. But many are also observable which
are in every respect similar to the latter, and may
indeed not improbably belong to the blood, for the
bloodvessels are very large and numerous in the
medullary tissue, and yield the numerous red blood-
corpuscles which are seen scattered over the field.
Transitions may be observed between the proper
marrow-cells and the white corpuscles, and as they
both exhibit amoeboid movements it may be inferred

MARROW. Ill

that they are essentially of the same nature, if indeed
the one is not to be regarded as derived from the
other.

Other cells may perhaps he met with much fewer
in number, flattened in form, and sometimes branched,
with a large clear oval nucleus, and in some instances
containing yellowish -red pigment granules. They
are larger than the proper marrow-cells, and exhibit
no changes of form. They appear to be connective
tissue-corpuscles. There is yet another element to
be found in the marrow — most likely to be met with
in a bit taken from near the inner surface of the bone.
This is the rnyeloplaque or ostoclast, and is charac-
terized by its enormous size — whence the name giant
cell — by its granular appearance, and by containing
a number of clear round or oval nuclei grouped
together in the middle of the cell, or, in some cases,
a single large irregular nucleus with numerous buds
from its circumference.

Preparation 12. — To isolate these various ele-
ments of the marrow better than can be done in the
fresh condition, a piece of the tissue is to be placed
in weak spirit (ordinary methylated spirit diluted
with twice its bulk of water) for a day or two.
After this time a small portion is thoroughly broken
up with needles in a drop of water on a slide, is then
covered, and stained by allowing dilute logwood
solution to flow in under the edge of the cover-glass.
The logwood is replaced in a minute or two by water,
and this again by glycerine. By this means a per-
manent preparation is obtained, which can be studied
at leisure, and which very well exhibits the different
kinds of cells above enumerated, whilst the red
blood-corpuscles are rendered almost completely in-
visible.

112 PRACTICAL HISTOLOGY.

CHAPTER VI.

MUSCULAR TISSUE.

Preparation 1. Involuntary muscle. — It is

very easy to isolate the lanceolate cells of which this
tissue consists. For this purpose all that is neces-
sary is to place a piece of any organ containing plain
muscular tissue — the intestine, for instance — in a
weak solution of bichromate of potash (one part to
800 of water) for forty-eight hours. At the end of
this period of maceration, a small strip of either the
longitudinal or the circular muscular fibres is torn
off with the forceps, placed in a drop of water on a
slide, and separated as finely as possible with needles.
A cover-glass is then laid on, and the preparation is
carefully examined with a high power. The ends
and edges of the larger pieces of the tissue have a
somewhat ragged aspect, due to the projection from
them of the^ tapering ends of the fibre-cells. In
addition to these partially separated elements, others
are to be met with scattered over the preparation
which are wholly free, and in which all the charac-
teristic appearances of this tissue can be distinctly
made out. The elongated nucleus in the middle of
each riband-shaped cell can be seen in those cells
which lie flat, but it is at present rather indistinct.
It may, however, be brought more clearly into
view, as can also the faint longitudinal striation
wliich the cells exhibit, by employing one of the
smaller holes in the diaphragm of the microscope to
admit the light to the object. But to show clearly
the nuclei ol^the plain muscular fibre-cells, nothing
is better adapted than staining the tissue with a
weak solution of logwood-alum. It must be used

INVOLUNTARY MUSCLE. 113

quite dilute, and suffered gradually to diffuse itself
under the cover-glass from a small drop placed at
one edge. It is not a good plan to draw it through
by means of blotting-paper, since in this way many
of the isolated elements of the tissue will be drawn
away at the same time. But after the drop of log-
wood solution has passed in great measure or entirely
under the cover-glass, a small drop of strong glyce-
rine may be added at the same spot as the logwood.
This as it diffuses under the cover will gradually
push, as it were, the logwood solution before it, so
as to cause the staining fluid to traverse successively
every part of the preparation, and eventually to
become collected entirely at the opposite edge, the
water meanwhile evaporating and leaving the glyce-
rine in possession of the field. All that is needed
to complete the preparation is, in the course of a day
or two, to fix the cover-glass by painting a little
chloroform balsam around the edges.

The involuntary muscular fibres will be seen in
section in preparations of the stomach and intestine
and numerous other organs, so that it is not neces-
sary to make special preparations at this stage for
the purpose. It may, however, be instructive to
demonstrate the manner in which the cells are
applied edge to edge in order to make up the
bundles and lamellae of the tissue. This is often
shown sufficiently well in a thin strip which has
been stained with logwood and mounted in glyce-
rine without teasing.

Preparation 2. — But the best preparations for
exhibiting the arrangement of the cells are those
stained with nitrate of silver. As in other tissues,
this reagent stains only the intercellular substance,
leaving the cells themselves uncolored ; their out-
lines are thus brought very distinctly into view.
The way to prepare the muscular coat of the intes-
tine by this method is as follows : A piece of intes-
tine is removed from the recently-killed animal (the
large intestine of the frog answers verv well for the

10*

114 PRACTICAL HISTOLOGY.

purpose), and one end being tied up, a glass canula
is fastened into the other end, and the piece of
intestines having been distended with air, this also
is closed by a ligature and the canula removed. The
outer surface is then brushed vigorously with a
camel-hair pencil wetted with distilled water to
remove the epithelioid cells of the serous membrane ;
a few drops of a solution of nitrate of silver (i per
cent.) are allowed to flow over the brushed surface
and to remain in contact with it for two minutes,
the silver solution is then washed off by a stream of
distilled water, and finally the distended intestine
is immersed in a mixture of equal parts of water
and spirit in a beaker and exposed to the light. The
immersion is effected either by tying a piece of
glass rod or other heavy substance to the gut, or by
filling the beaker to the brim and placing a glass
plate over it so as to press the piece of intestine
under the surface of the fluid. In about an hour's
time the preparation may be removed from the
light (a few minutes will be sufficient if it is bright
sunlight), and a small piece is then to be cut out
and mounted in glycerine with the outer surface
uppermost.

Preparation 3. Voluntary muscular tissue :
cross-striped muscle. — For the examination of
this tissue in mammals the animal from which the
preparation is to be taken should have been killed
some hours previously, in order to obviate the
shrinking and contraction which would otherwise
take place in a freshly-severed portion of muscle
placed in fluid, and would obscure many of the
structural appearances. A small longitudinal shred
is torn or stripped oft' from any muscle of the limbs
or trunk, placed in a drop of serum upon a slide,
and the fibres are slowly and carefully separated
from one another, one by one, for as great a length
as possible. A very small slip of blotting-paper is
then placed by the side of the preparation to avert
the pressure of the cover-glass (a hair is not, in this

VOLUNTARY MUSCLE. 115

instance, thick enough for the purpose), and the
latter is then laid on, and the preparation examined
first with a low power, to make out the shape and
extent of the fibres, and afterwards very carefully
with an ordinary high power, and eventually with
as high a power as it is possible to obtain. In this
preparation almost all that is known of the struc-
ture of mammalian muscle may be made out — the
delicate sarcolemma with the muscle nuclei imme-
diately beneath it, which look clear and oval when
the upper surface of a fibre is exactly focussed, fusi-
form when seen at the edge, the dark cross-stripes
of the muscular substance, seen by careful observa-
tion with a very high power to be pervaded by
parallel rod-shaped particles, the clear stripes bi-
sected by an intermediate dotted line, and finally a
longitudinal striation throughout the fibre, which
is the better seen in proportion as the • transverse
striping is less marked.

Preparation 4. — For the purpose of bringing the
muscle-nuclei more distinctly into view, another
piece may be prepared in a drop of dilate acetic acid
(one part in 200 of water). The acid swells up and
softens the interstitial connective tissue, so that the
fibres are more readily separated the one from the
other, and it is found that, whereas in the former
preparation the muscle-nuclei could only be made
out by exercising the greatest care and attention,
they are now extremely obvious, studding the fibres
at intervals, but all of them quite at the surface of
the muscle under the sarcolemma.

Preparation 5. — When the frog's muscles are
prepared in like manner with acetic acid, the nuclei
are, on the contrary, seen to be embedded here and
there in the thickness of the fibre also.

Preparation 6. — The sarcolemma is extremely
delicate in mammalian muscle, and although it may
with care be made out even in the fresh preparation,
is nevertheless much more easily demonstrated in
the muscles of the lower vertebrata, the fro^, for

116 PRACTICAL HISTOLOGY.

example. With this end a piece of the muscle is
teased in a drop of salt solution. This fluid has the
effect of gradually disintegrating the proper sub-
stance of the muscle, so that the latter tends to
break down into a clear fluid with numerous fine
granules suspended in it, and exhibiting an active
Brownian movement. This process of disintegration
generally begins at places where the fibres have been
touched by the needles in the process of separation,
and if the muscle be fresh, the contractile substance
breaks and shrinks away at these places, leaving the
clear sarcolemma bridging across the interval.

Preparations 7 and 8. Disks and Fibrils.— In
order to exhibit the manner in which muscular tissue
tends to break up into either disks or fibrils accord-
ing to the nature of the reagent to the action of
which it is submitted, two pieces of muscle are taken
for an animal that has been dead some hours, and
are placed for a week, the one in a solution of hydro-
chloric acid (1 in 50), the other in a solution of
chromic acid (1 in 200). Small portions are then
broken up as finely as possible with needles upon
separate glass slides. The fibres from the hydro-
chloric acid are, many of them, found to cleave into
transverse clear disks, some of which will be noticed
lying flat, others seen edgeways ; whereas in those
from the chromic acid there is no tendency what-
ever to form such disks, but on the contrary, the
muscular fibres tend to break up into smaller and
smaller longitudinal fibrils.

Preparation 9. — In order the better to compare
the fibres either of the same or of different muscles
as regards length and diameter, and to see their
general shape, it is necessary to isolate a number of
them in their whole length. For this purpose the
process of separation by the aid of needles is some-
what tedious, and we must turn to reagents which
will dissolve the intermediate connective tissue
which binds the fibres together, whilst maintaining
them intact. Such a reagent is to be found in a

LIVING MUSCLE. 117

solution of sulphurous acid (the liquor acidi slil-
phurosi of the Pharmacopoeia answers very well).
The muscle is placed for a week or more in a well-
stoppered hottle containing a considerable quantity
of the acid, and is kept in a warm chamber (like
that shown in Fig. 19), heated to about #0° or 40°
Centigrade. This very much facilitates the process
of maceration, so that after the time stated a mere
gentle shaking of the bottle is sufficient to cause the
muscle to break up in great measure into its consti-
tuent fibres. Some of these may then be removed,
placed side by side in water or weak glycerine on a
slide, and covered, with the usual precautions to
obviate the pressure of the cover-glass. It will be
found that the muscular substance has acquired, in
consequence of the maceration, a granular aspect,
and that the usual structural appearances are for the
most part indistinct. But this mode of preparation
may be advantageously employed for the purposes of
measurement and comparison of size and form of
fibres from different regions of the body.

Preparation 10. Examination of muscular
tissue in the living condition. Ultimate struc-
ture of a muscular fibre. — The delicacy of the
structural elements of the striated muscular fibres
of the vertebrata, and the difficulty of maintaining
the fibres, when sufficiently isolated, in the living
condition, have baffled all attempts that have hitherto
been made to satisfactorily determine their ultimate
structure before it has become altered either by the
death of the tissue or the action of reagents. But
the muscular tissue of some of the in vertebra! a,
particularly of insects and Crustacea, bears an exact
resemblance to the transversely striped contractile
tissue of the higher animals, while at the same time
the appearances are better marked, and separated
portions of the muscle are readily prepared, and may
be examined by the highest powers of the microscope
whilst still living and freely contractile.

The most convenient of these animals to employ

118 PRACTICAL HISTOLOGY.

for the purpose is the common great water-beetle
(Dytiscus marginalis), and the muscles of the limbs,
especially the forelegs of the males,1 which are strong
and well-developed, should be chosen. A slide and
cover-glass having been cleaned, and a lance-pointed
needle or a scalpel and fine scissors and forceps being
at hand, one of these limbs is cut off at the joint
nearest the body, wiped with blotting-paper to free
it from the acrid, strongly-smelling secretion which
the insect emits, and the chitinous integument of the
limb is then slit longitudinally so as to expose the
pale soft-looking muscle within. Then, the member
being firmly held on to the table with forceps or the
fingers, as much as possible of this muscular tissue
is gouged out with the lance-headed needle or scalpel-
point, and transferred to the slide. The muscular
fibres are quickly separated somewhat from one
another by needles, desiccation being prevented by
breathing once or twice upon the preparation, and
the cover-glass is immediately laid on without the
addition of any fluid other than that which naturally
moistens the tissue. The preparation is to be ex-
amined at once with the highest available power.
It is difficult to make out the details of the structure
with a combination magnifying less than 1000 dia-
meters, and the defining power must be of the best.
If the object has been quickly enough prepared,
numerous fibres will be found which show the suc-
cessive series of minute muscle-rods, corresponding
in position with the darker cross stripes of the mus-
cle, and prolonged into the clear stripes, where they
end with knobbed extremities, the juxtaposition of
Avhich in rows side by side gives the semblance of a
dotted line in each clear stripe, single or double accord-
ing as the fibre is more or less extended. Other fibres

1 The males are readily distinguished from the femnles by their
smooth wing-covers, whereas those of the females are rigid and
furrowed longitudinally, as well as by their short strong fore-
limbs with sucker-like extremities.

LIVING MUSCLE. 119

may be seen in which the dotted line is absent, the
muscle rods not being enlarged at their extremities;
and these being placed end to end in the successive
series give an appearance of long, jointed fibrils ex-
tending in the direction of the length of the fibre.
In such fibres the cross-striped appearance is almost
entirely lost, for there are no intervening clear striae,
the appearance of which in the other fibres is proba-
bly due to the presence of the rows of strongly refract-
ing dots.

That the bright appearance of the clear striae may be
produced by and dependent on the rows of minute globu-
lar ends of the muscle rods is shown by the following ex-
periment : A drop of water is placed on a slide, and a very
small drop of oil is transferred to it by a needle-point,
and is then thoroughly whipped up with it, so as to reduce
the oil to as minute globules as possible. The prepara-
tion is then covered and examined with the same magni-
fying power as was used for the water-beetle muscle. The
smallest of the oil-globules, like the dots in the muscle,
look like mere dark specks, and it will be noticed that
each is surrounded by a bright area, an effect always pro-
duced by highly refracting bodies when examined in a
less refracting medium. And if we imagine a number of
such little oil-specks to be placed in a row, there would
be a bright band on either side of the rows of dots in the
water-beetle's muscle.

Contraction of muscle — In the perfectly fresh pre-
parations of the insect's muscular tissue, spontaneous
waves of contraction may be seen passing from end to end
of many of the muscular fibres, and a general idea of the
phenomena which accompany the contraction, such as
thickening of the part of the fibre at the time the wave is
passing, and approximation of the cross-striae, may be
obtained. But the contraction proceeds too quickly for
the details of the process to be watched, and the fibres
are not sufficiently isolated; moreover, it is impossible to
s&y of the particular fibre under examination whether a
contractile wave is about to pass along it or not.

Preparation 11. — In order to get the fibres more
under command, the preparation must be made rather
differently. For the purpose of inducing the muscular

120 PRACTICAL HISTOLOGY,

tissue under examination to contract at any given moment,
a gas-chamber (see Pig. 11) is made use of, and a trace of
the vapor of alcohol is made to pass over the preparation,
at the moment when it is wished to observe the contrac-
tion of a particular fibre. The mode of obtaining and
preparing the tissue is the same as before, except that the
piece scooped out of the freshly-excised lirnh is placed
upon a cover-glass instead of on a slide, and the fibres are
rather more freely separated. The cover-glass is then in-
verted over the putty ring of the gas-chamber, at the bot-
tom of which a drop of water has as usual been previously
placed. The preparation is searched for a portion of a
muscular fibre which happens to be conveniently isolated,
and which shows well the normal structure of the living
tissue. This having been brought under observation with
the highest magnifying power, a little air charged with
the vapor of rectified spirit which has been previously
poured into the vapor-bottle is blown into the gas-cham-
ber; as soon as the contraction which results is over, the
vapor must be replaced by air again, so that the vitality
of the tissue is not too soon destroyed. If the prepara-
tion is very fresh, the contraction is generally so sudden
that it is impossible to follow the details of the process ;
but after a time the tissue responds less actively to the
stimulus, and then with care it is possible to make out
the changes which are happening in the form of the mus-
cle rods, and in the consequent relative arrangement of
the cross stripes of the fibre. Numerous trials, if neces-
sary on fresh preparations, may have to be made before
a successful result is arrived at ; for various circumstances,
especially the shifting of the fibres during their contrac-
tion, may tend to vitiate the observation.

Preparation 12. Examination of muscular

tissue by polarized light. — The polarizing micro-
scope is nothing else than the ordinary microscope with
the addition of two Nicol's prisms, one placed below the
object and another above the ocular ; the upper one is
generally mounted in combination with a low ocular, so
that it is not necessary to use the ordinary eye-piece.
The light, coming from the mirror, becomes polarized as
it passes through the lower Nicol (the polarizer). If now
the upper Nicol (the analyzer) be slowly turned round
as it is being looked through, it will be found that there

POLARIZING MICROSCOPE. 121

are two positions in which the field is quite dark ; that
is to say, the polarized ra}Ts are entirely cut off. By ob-
serving now the relations of the prisms at these positions
of total darkness, it will be found that their planes of
polarization — as shown by the way in which the prisms
are cut — are at right angles to one another. In all inter-
mediate positions a greater or less amount of light is
enabled to traverse the analyzer. But if any object
which possesses the property of refracting light doubly
is placed upon the stage of the microscope, and examined,
and if then the field is made dark by turning the ana-
lyzer, it will lie found that the doubly refracting substance
remains bright, unless it happen so to lie that its optic
axis is parallel with the plane of polarization of either
Nicol. And if the object be a muscular fibre at rest, the
whole fibre will appear bright and doubly refracting,
whereas if it be in the state of contraction the bright
stripes only will allow the light to pass, the dark stripes
in this condition of the fibre being singly refracting.

These observations are best made upon the living
muscle of the water-beetle by aid of the gas-chamber.
The portion of fibre under observation should be quite
free and not overlaid by other fibres. The change in the
optical condition of the fibres which ensues on contrac-
tion ma}', if due care and patience be exercised, in this
way be made out. The results arrived at by the exami-
nation of portions of the tissue which have been hardened
in alcohol, and mounted in glycerine or Canada balsam,
although more easily seen, are less trustworthy, since
the muscular fibres are liable to undergo considerable
changes after death, and under the action of re-agents.

A pretty modification may be made by substituting a
thin piece of mica for the covering glass. This causes
the field of view to become tinted, the particular color
varying with the thickness of the mica, and the relative
position of its optic axis to those of the Nicols, and any
doubly refracting substance which is now examined
assumes the color which is complementary to that of the
field. The object of the revolving stages with which the
larger microscopes are generally fitted is to enable the
observer to modify the position of the optic axis of the
tissue which is being examined, with relation to those of
the Nicols ; and it also serves when the mica is used to
11

122 PRACTICAL HISTOLOGY.

change in like manner the relative position of the optic
axis of this also, and thus to modify the color of the
field of view.

Transversely striated muscle is not by any means the
only tissue which is doubly refracting, for the property
is possessed by the white fibrils of connective tissue, and
by bone, as well as by the plain muscular fibre cells.
But it is the only one which under certain conditions
exhibits alternate bands of singly and of doubly refract-
ing substance. It lias, however, been pointed out by
Ranvier that it is rather the conditions of growth and
formation of a tissue than differences of structure which
tend to determine differences in the optical properties of
the substance of which it may be composed And he
instances the case of cartilage, the matrix of which,
although undoubtedly composed of the same substance
throughout, is doubly refracting in those parts where the
cells, either from pressure or in progress of growth, have
come to assume either a flattened or elongated shape,
singly refracting where they remain rounded.

Preparation 13. Transverse section of mus-
cle.— In examining the fresh muscle of the water-
beetle, in either of the ways above described, it may
happen that a fibre is seen which is turned up
towards the observer, and in which, therefore, by
focussing the vertical part with the fine adjustment,
a view may be obtained just as if the fibre had been
cut transversely. It is then said to be seen in optical
section ; and for muscular tissue this is the only way
in which a section of the fibres, whilst they are quite
unaltered, can be observed. Nevertheless, when it
is not so much the structure of the fibres but their
shape and mode of arrangement into bundles that is
to be the subject of investigation, the tissue may be
hardened by weak chromic acid (} per cent.), or by
alcohol, and thin transverse sections may be cut
with a razor and mounted in glycerine, with or
without previously staining in logwood. There is
another method, that of freezing^ which has luvn
much recommended for hardening fresh muscular

SECTION OF FROZEN MUSCLE. 123

tissue in order to obtain transverse sections of the
fibres in wbat has been thought a completely un-
altered state. It may be doubted, though, whether
the muscular substance does not undergo some
structural alteration either in the freezing or in the
subsequent thawing. Yet, as it is frequently em-
ployed for this tissue, and is of considerable value
in special investigations into the structure of other
tissues and organs, the method may be here de-
scribed.

Preparation 14. Section of frozen muscle.
— The best and simplest mode of freezing a tissue
is that recommended by Urban Pritchard. A solid
cylinder of copper (1£ inch wide and 3 inches long),
with flat ends, is placed for a few minutes in a
freezing mixture composed of equal parts of pounded
ice and salt, by which it is cooled to considerably
below the freezing-point. The copper cylinder is
then removed, wiped quite dry with a cloth, and a
piece of flannel is wrapped three or four times round
it, leaving one of the flat ends exposed. The muscle,
which may conveniently be taken from the leg of a
frog, is cut across near its middle and one of the
halves is placed with its cut surface on the flat end
of the copper block in a drop of gum arabic solution.
This as well as the tissue very speedily becomes
frozen throughout, adheres to the copper, and then,
the block being held in the left hand, sections are
made of the muscle with a dry razor, which should
have been previously cooled by contact with the
vessel containing the freezing mixture, and the
sections are at once transferred to a slide and
covered, either in serum, or, better, without the
addition of any fluid.

Sections made by freezing possess the advantage
that, since they have not been submitted to any
coagulating reagent, they may be treated with
nitrate of silver, chloride of gold, and other fluids
which are of value only when the tissues are per-
fectly fresh.

124 PRACTICAL HISTOLOGY.

Preparations 15 and 16. Termination of
muscle in tendon. — It' the tendons of a mouse's
tail are forcibly drawn out, after nipping off the end
of the tail in the manner described in the account
of the preparation of tendon (p. 83), it will generally
be found that portions of a number of small mus-
cular fibres are adherent to each tendon, for the
fibres have their insertions into the tendon, and are
ruptured by the force employed. These ends,
mounted in serum, serve conveniently for the study
of the mode in which the fibres of a muscle termi-
nate in a tendon, when the fibres of the latter run
in the same direction of those of the muscle. But,
easy as the tissue is to prepare, the observation is
complicated by the fact that the muscular fibres
form generally somewhat of a clump as they pass to
end in the tendon. The preparation may be im-
proved by being stained with picrocarmine solution.
This colors muscular tissue yellow, tendinous tissue
red, so that the distinction between the two is made
more obvious. It is best to take a few freshly
drawn-out tendons, and to mount their ends in a
drop of the picrocarmine solution, surrounding the
edges of the cover-glass with melted paraffin to pre-
vent evaporation of the liquid.

It is easier to make out the way in which the
fibres of a very thin fiat muscle, such as the subcu-
taneous muscle over the frog's sternum, terminate
in the fibrous tissue to which the muscle is attached.
Here the fibres of the tendon do not take the same
direction as the muscular fibres. A frog, having
been killed by destroying the brain and spinal cord,
is laid upon its back, and the skin over the throat
is reflected downwards towards the sternum. The
flat muscle w^hich is thereby brought to view passing
to the skin is snipped across the middle with scissors,
seized on the cutaneous side of the snip with forceps,
and a square piece, including its insersion into the
skin, is cut out. It is placed as flat as possible upon
a dry slide, the curled-up- edges being turned down

TERMINATION OF MUSCLE IN TENDON. 125

by the aid of mounted needles, and a drop of serum
having been placed upon a cover-glass, this is in-
verted over the preparation. The rounded ends of
the muscular fibres are seen fitting into little depres-
sions in the fibrous tissue, and with prolongations
from this passing around and between them.

The bloodvessels of muscle will be studied later, after
the methods of injecting and of cutting sections have been
described. The mode of termination of nerves in volun-
tary muscle will also be most advantageously deferred
until the preparation of the nerves themselves has been
treated of.

126 PRACTICAL HISTOLOGY.

CHAPTER VII.

NERVOUS TISSUE.

Preparation 1. Medullated nerve-fibres. — For

the study of the medullated nerve-fibres a piece of
one of the ordinary nerves — those of the limbs, for
example — may be cut out from any recently-killed
animal. If the nerve be a large one, a thin strip only
should be used, preferably taken from the interior
after the piece has been torn longitudinally into two
halves by fine forceps. The strip is to be placed on
a slide in a little serum or salt solution, and care-
fully separated as finely as possible. This separation
must be effected, not by seizing the piece anywhere
and tearing it up at random, but by inserting tine
needles into it near one end and gently drawing them
asunder, so that the piece is split into two. Repeating
this process a number of times on the resulting pieces
the nerve will be eventually separated into very tine
bundles of fibres, together with a number of more or
less isolated fibres, which are still nearly straight
and uninjured, except near one end. The prepara-
tion may then be covered, and the general character
and appearance of the fibres investigated. To see
the nodes of Ranvier well, a tolerably large fibre,
free for a considerable part of its length, should be
chosen, and by moving the slide it should be care-
fully followed with an ordinary high power. It
will be found that at definite and not very close in-
tervals along the fibre the double-contoured medul-
lary sheath fails altogether, and the axis-cylinder
alone continues the nerve at these points. It is not
very easy to see the oval nucleus in the middle of
each segment in the fresh, unstained preparation.

SHEATH OF NERVE. 127

Preparation 2, — Osmic acid possesses the pro-
perty of staining fatty matters of an inky black
color. Since the medullary sheath of the nerves is
mainly composed of a phosphuretted fat, this becomes
accordingly stained by the acid, whilst the other
parts are left of a grayish tinge. Moreover, the
regular breaks in the fatty sheath — the nodes of
Ranvier — are by this means brought into promi-
nence, owing to the complete intermission of the
darkly stained medullary substances at those places.
At the same time the axis-cylinder can be distinctly
made out crossing the interval, and the primitive
sheath, or sheath of Schwann, can be often seen both
here and also near the centre of each of the segments
into which the fibre is divided, where it is bulged
out by the presence of the oval nucleus. The method
for preparing these osrnic preparations is as follows :
From an animal which has been quite recently killed,
a small nerve, not larger in diameter than an ordi-
nary thread, is chosen, and a piece about half an inch
long is cut out, but in doing so care must be taken
not to drag upon or injure the nerve more than is
absolutely necessary. The piece is placed for four
hours in a little covered glass pot contaiug a drop
or two of a 1 per cent, solution of osmic acid ; it is
then transferred to water for an hour, and finally
placed in a mixture of glycerine and water (equal
parts). It can either be teased in this at once or
left for a day or two or even longer ; in preparing
it, the same precautions must be used as were recom-
mended for the preparation of the fresh nerve (see
preceding paragraph).

Preparation 3. Fibres of Remak.— To see the
gray or non-medullated fibres, pieces of the sympa-
thetic nerve are taken from the neck of an animal
and prepared and examined both fresh and after
treatment with osmic acid, in the same manner as
the cerebro-spinal nerves.

Mode of union of the nerve-fibres to form
the nervous cords. — In the several teased prepara-

128 * PRACTICAL HISTOLOGY.

tions both of the cerebro-spinal and of the sympa-
thetic nerves there will be seen, especially in those
prepared with osmic acid, besides the actual nerve-
fibres, in the first place, a quantity of connective
tissue, for the most part of the nature of areolar
tissue, which formed a general ensheathment for
the nerve, and sent partitions in between its several
bundles or funiculi ; secondly, the special sheaths of
the funiculi, which become torn and stripped away
in the process of teasing, and which look like flat
bands composed of an almost homogeneous substance,
but pervaded by a network of fine elastic fibres and
with round or ovalish nuclei scattered upon them
here and there. Thirdly, there will be seen running
along close to and surrounding the nerve-fibres them-
selves very delicate, nearly straight fibrils of con-
nective tissue, with here and there the nucleus of
a connective tissue corpuscle. These three forms
of connective tissue represent respectively the epi-
rieurium (cellular sheath); the perineuriurn (neuri-
lemma); and the endoneurium, or tissue within
the funicular ; but their relative situation and
arrangement, as well as the lamellated structure of
the perineurium, can only be properly displayed by
transverse sections of a nervous trunk, the mode of
preparing which will not be treated of until the
general directions for the preparation of sections have
been given.

Preparation 4. — But the cell-outlines on the
lamellae of the perineurium may be shown by the
silver method. For this purpose either a very small
nerve is chosen and a piece of it is removed and
immersed for ten minutes in half per cent, silver
nitrate solution, after which it is washed in distilled
water and exposed in glycerine to the light ; or,
should it be wished to prepare a larger nerve con-
sisting of more than one funiculus, this is carefully
dissociated in a few drops of the silver solution, and
after a like treatment is also to be mounted in
glycerine and exposed to the light. After a few

NERVE -CELLS. 129

minutes' exposure to sunlight the preparations may
be examined. It will be found that the sheath of
each funiculus or nervous bundle is covered by large
epithelioid marking, and if the preparation be very
successful, two, three, or even more layers deep of
such markings may be counted by examining with
a high power and carefully adjusting the microscope.

In addition to this it will generally be found that
the medullated nerve-fibres themselves are marked
transversely at each of the nodes of Ranvier by a
dark line, or rather ring, surrounding the axis-
cylinder. These markings appear to be owing to the
existence of a substance here, between the segments
of the nerves, which, like the intercellular substance
elsewhere, has an affinity for the metal. Finally, at
many of the nodes of feanvier, particularly if the
immersion in the silver solution had been rather
prolonged, the latter will have penetrated to and
become reduced in the substance of the axis cylinder,
which therefore will here have the appearance of a
dark line or band piercing the ring of interseg-
mental substance, the twTo together having the sem-
blance, under a moderate power, of a little black
cross upon the nerve at those points. Sometimes
in such preparations the axis-cylinder, where stained
at the nodes by the reduced silver, presents a cross-
striated appearance, but the meaning of this is not
known.

Nerve- and ganglion- cells ; structure of the
nerve-centres. — To study the exact form and ap-
pearance of the tissue elements of the nervous
centres they must be as much as possible isolated
and examined in teased preparations, although their
position and local relations are best made out in
sections of the several organs in which they occur.
For the present we will confine ottrselves to a descrip-
tion of the best methods for isolating the nerve-cells.
For this purpose the tissue is treated similarly from
whatever part of the brain or spinal cord the piece
to be examined is removed ; but, as a typical example,

130 PRACTICAL HISTOLOGY.

a piece of the spinal cord from the lumbar region
may be taken, for the nerve-cells are here very
numerous and consequently more readily found
than in other regions.

Preparation 5, — The human spinal cord is, if
obtainable, best adapted for the study of the cells,
for they are readily picked out under the dissecting
microscope, in consequence of the little mass of dark
pigment which each contains. If a piece of the
human spinal cord cannot be got, the spinal marrow
of the ox or sheep, or other tolerably large animal,
may be employed. The piece to be macerated
should be about half an inch long, and is placed for
forty-eight hours in weak bichromate of potash
solution (1 in 800). At the expiration of this time
it is taken out of the fluid, and with the point of a
scalpel or of fine scissors a small piece of the gray
matter is dug out from the anterior cornu, placed in
a drop of water upon a slide, and separated with
needles into three or four pieces. These are then
placed under the dissecting microscope and carefully
broken up further ; those parts where the cells are
most numerous being singled out, and the prepara-
tion being examined every now and then on the
ordinary microscope with a low power, in order that
the progress of the isolation may be watched. When
as much as possible has been effected in the way of
separating the nerve-cells a piece of hair is placed
in the fluid and the cover-glass gently superposed.
The cells may then be sought with a low power and
carefully examined with a higher one.

If the preparation is very successful, and it be
desired to preserve it, keeping the cells at the same
time as much as possible of their natural aspect, the
best plan to adopt is to allow a drop or two of a 1
per cent, solution of osrnic acid to flow in at the edge
of the cover-glass, and after it has acted upon the
tissue for an hour, by which time the cells will have
'become stained of a dark grayish tint, to carefully
run through from the same side first a little distilled

GANGLION CELLS. 131

water to wash away what remains of the acid, and
then glycerine to preserve the preparation, after
winch the edges of the cover-glass may be cemented.
Besides the nerve-cells, which in a carefully-prepared
specimen may be very well seen, with their long
branching processes extending in some cases far be-
yond the field of the high power objective, some
points in the structure of the nerve-fibres of the
spinal cord can be well made out in these prepara-
tions. For example, it may be readily seen that the
medullary sheath has broken away in many parts
from the white nerve fibres, and where not actually
removed has become swollen and coagulated in
irregular masses around the axis cylinder, changes
which could hardly have taken place were there any
structure surrounding the nerve fibres such as the
primitive sheath of the peripheral nerves. Further,
where the axis-cylinders are in this manner laid bare,
as they often are for a considerable part of their
length, their fibrillar structure can, with a high
power, be made out without difficulty. A similar
structure can also he seen in the processes of the
nerve cells, and extending from them through the
body of the cell itself.

Preparation 6. — The cells from the ganglia,
whether spinal or sympathetic, are isolated in a
manner similar to that employed in the case of the
spinal cord, except that the period of maceration in
the bichromate solution needs to be longer, owing
to the much larger amount of connective tissue by
which the nervous elements are invested. At the
same time it is quite possible to get a certain number
of the cells sufficiently isolated, even from the fresh
ganglion, without any maceration; for each cell
being loosely contained in a special capsule of flat-
tened cells, it readily falls out when the nerve fibres
with which it is connected are ruptured. But
although the cells themselves of the ganglia are
readily enough separated, it is very difficult, both
in the fresh and in the macerated preparations, to

132 PRACTICAL HISTOLOGY.

get their continuation to the nerve fibres, these being
left behind for the most part. The mode of perma-
nently preserving the specimen is like that employed
in the preceding preparation, except that the cells
may with advantage be colored by logwood instead
of osrnic acid.

Terminations of the nerves. — The description
of the mode of preparing and demonstrating the
terminations of nerve fibres in various special parts
of the body will be deferred until those parts and
organs are severally treated of, but the Pacinian
bodies, in which many of the sensory fibres end, and
the end-plates in which the nerves supplying the
voluntary muscles terminate, may be now prepared.

Preparation 7. — The Pacinian bodies are very
readily found in the cat's mesentery. Here they are
at once seen when the abdomen is opened and the
membrane is held up against the light, as clear, oval
specks, either dotted singly here and there, or form-
ing groups of two, three, or more. There is generally
a considerable group in the meso-rectum, and more-
over they are here usually not so much obscured by
the adipose tissue as in the mesentery proper. By
far the best general idea of their structure and the
relation they have to the nerve fibre entering them
is obtained from their study in the fresh condition,
without the addition of reagents. But it is well to
separate them from the surrounding tissue of the
mesentery, for this is often loaded with fat, and,
when not, the fibrous tissue of the membrane tends
to obscure the structure of the little bodies.

In order to isolate one of them, cut out the piece
of the mesentery containing it, carrying one of the
cuts close along the edge of the corpuscle. Then
place the excised piece upon a slide in a drop of
serum, and without actually transfixing the Pacinian
itself, tear away the investing mesenteric tissue bit
by bit under the dissecting microscope. It will be
found that with a little manipulation the corpuscle
shells out as a lemon-shaped body, with a twisted

THE PACINIAN CORPUSCLES. 133

stalk at one end. It is to be examined with a low
power to make sure that the fat is entirely removed ;
the debris may then be wiped away, a little fresh
serum added, a narrow slip of blotting-paper placed
close to the corpuscle to avert the pressure of the
cover-glass, which is then laid on and the specimen
examined.

In these fresh preparations, owing to the extreme
transparency of the layers of the capsule, the core of
the corpuscle with the central fibre, together with
the mode of passage of the nerve fibre into this at
the stalk, can be made out better than after the
action of reagents. And still more so if the outer
layers of the capsule are removed altogether, as may
readily be done with fine needles under the dissect-
ing microscope, leaving only the core and the closely-
set layers of the capsule which immediately surround
it. In tearing away the outer layers it will often
happen that the perineural or neurilemmal sheath
which surrounds the nerve fibre as this passes into
the corpuscle is torn off along with them, since they
are directly continuous with it.

These fresh preparations of the Pacinians are very
beautiful, but unfortunately they cannot be preserved
in that state. Treatment with glycerine causes the
corpuscles to shrink and become too transparent,
and most of the ordinary staining fluids color the
core too deeply and obscure the termination of the
nerve fibre. If it he desired to preserve any such
preparation as showing some one or more points
particularly well, the cautious employment of osmic
acid prior to mounting in glycerine is most to be
recommended. The serum in which the corpuscle
is mounted must first be replaced by salt solution;
this again by 1 per cent, solution of osmic acid, and
this after being left for an hour or more in contact
with the preparation by distilled water, whilst finally
a drop of glycerine is allowed gradually to diffuse
in from the edge of the cover-glass.
12

134 PRACTICAL HISTOLOGY.

Preparation 8. — The structure of the tunics
which form the lamellated capsule of the Pacinian
body is not well shown in the fresh preparation,
owing to the transparency of the object ; indeed,
it is the lines of contact between successive coats
which look like layers of the capsule ; the substance
of the coat being clear and pellucid, gives the notion
of an intermediate fluid. In order to show their
fibrous connective tissue structure (the white fibrils
running transversely, and collected for the most
part near the surfaces of each tunic, and the elastic
fibrils forming a network through the thickness)
some of the little bodies should be dissected out in
the way above described, -and placed for ten days or
a fortnight in a £ per cent, solution of chromic acid.
They are then placed on a slide in a drop of water,
and with fine and perfectly clean needles are broken
up under the dissecting microscope bit by bit, com-
mencing at one end and breaking off transverse
pieces. It will be found that, owing to the direction
of the fibrils, the corpuscles tend to break across
into disk-like portions. The core does not share
this tendency, but this is of little consequence, for
it is not well displayed in these preparations. A
piece of hair having been added, the preparation is
covered and examined. Small fragments will pro-
bably be found which give a sectional view of the
tunics, and others in which they are seen fiat. If
a little logwood solution is allowed to run under
the cover-glass, nuclei on the surface of the tunics
may here and there be stained.

Preparation 9. — These nuclei belong to flattened
epithelioid cells, which cover both outer and inner
surface of each tunic, and which, seen in profile, are
in reality the well-defined lines seen in the fresh
Pacinian, and long described as the coats themselves.
The outlines of these cells may be brought into view
by staining with nitrate of silver. For this purpose,
as is always the case with silver-preparations, the
tissue must be fresh and unacted on previously by

MOTOR END-PLATES. 135

any other reagent. One or two corpuscles are to
be thoroughly isolated and freed from surrounding
mesenteric tissue and fat ; placed for ten minutes
in silver solution (1 in 200); washed in distilled
water, and exposed in glycerine to the sunlight
until of a grayish color, when they may be covered,
and examined.

Preparation 10 — To complete the study of the
Pacinians, sections should be made of them, but as they
are very small, it will be best to defer this until some prac-
tice has been attained in the art of cutting microscopic
sections. A convenient way to prepare them, in order
to show the various parts to advantage, is as follows :
A very small piece of meso-rectum, containing several
corpuscles close together, is cut out (if such can be
found ; if not, one or more may he isolated as before),
and placed in a small beaker containing 100 cub. cent, of
a weak solution of acetic acid (1 in 200), to which about
5 c. c. of the ordinary (^ per cent.) chloride of gold solu-
tion has been added. The tissue is kept in this, in the
light, for three or four days — until it has become of a
dark violet color; it is then placed for a day in weak
spirit, and then in strong, and two or three days later is
ready for embedding and cutting. In embedding the
piece of tissue, it should be so placed that the corpuscles,
at least most of them, are cut as nearly as possible trans-
versely.1

Preparation 11.— The end-plates, or terminal
expansions of the motor nerves, are difficult to find,
and so soon undergo alteration as speedily to become
unrecognizable. On this account it is necessary, in
searching for them, to employ only muscles which
are absolutely fresh. In mammalia the best muscles
to choose for the purpose are those of a lamellar
shape and with short fibres, such, for instance, as the
intercostals of small animals. The muscular fibres

1 A description and delineations of the appearances exhibited
by the Pacinian corpuscles when prepared in these several ways
will be (bund in the '• Quarterly Microscopical Journal'' for April,

1875.

136 PRACTICAL HISTOLOGY.

are severed close to their attachments, so as to get
them in their whole length, and the small, thin
piece of muscular tissue obtained is quickly trans-
ferred to a slide, and mounted, either without addi-
tion of fluid or in a small drop of serum which is
put on the cover-glass before this is inverted over
the preparation.

This should now be thoroughly searched with a
good immersion objective for the nerve endings.
Branches of the intercostal nerve will be found
running across the direction of the muscular fibres.
Starting from one of these, trace carefully one by
one the single nerves which pass off from it. It will
be found generally that they branch one or more
times, and eventually the resulting twigs pass off to
the muscular fibres, each fibre receiving one of the
nerve-twigs. They retain their medullary sheath
until the muscular fibre to which they are attached
is reached, when the sheath suddenly ceases to be
visible, and it is by following the single fibres until
they come in this way to an abrupt termination,
that an end-plate may be met with. But even if the
place where the nerve-fibre joins the muscular fibre is
arrived at, it is still in most cases difficult to make
out the exact mode of termination, in other words,
the structure of the plate. The utmost that can
generally be seen is a clump of clear, round nuclei
embedded in a granular meterial. The difficulty
arises partly from the readiness with which these
structures undergo alteration after removal in warm-
blooded animals, and partly from the fact that they
are often obscured by super- and sub-jacent muscular
fibres or bloodvessels.

Preparation 12. — In the common lizard (Lacerta
agilis) the end-plates may be much more easily
found and satisfactorily seen, but still the utmost
care must be taken in the preparation. The animal
having been decapitated and the trunk pinned out
upon a cork, a piece of one of the limb-muscles —
including the whole length of the fibres — is removed

MOTOR END-PLATES.

and placed on a slide in a drop of serum. The fibres
are then isolated under the dissecting microscope as
carefully and completely as may be, and a piece of
paper or a hair having been added to avert pressure
on the tissue, it is covered and the fibres are ex-
amined with an immersion along their whole length.
Of course, if no end-plates can be found in the first
specimen another must be taken, but it will gene-
rally not be very long before one is found, either at
the edge of a fibre, and therefore seen in profile, or
on the surface, and seen flat.

Unfortunately, a method has not yet been dis-
covered by which they may be well preserved. That
which on the whole answers the best is to treat the
preparation with 1 per cent, osmic acid, having first
washed away the serum by salt solution. After tho
osmic has been about an hour in contact with the
tissue, it may be washed away and glycerine sub-
stituted. In this way the form of the little end-
plate or prominence is well preserved, and the
sudden termination of the medullary sheath of the
nerve fibre well shown, since this becomes blackened
by the osmic acid, but the substance of the end-plate
becomes dark and granular, so that the nuclei are
with difficulty seen.

12*

138 PRACTICAL HISTOLOGY.

CHAPTER VIII.

THE BLOODVESSELS.

Preparation 1. The larger bloodvessels.—
The epithelioid lining can only be properly demon-
strated in fresh bloodvessels stained by nitrate of
silver. For this purpose a piece of a large vessel-
artery or vein — is obtained, either from a recently -
killed animal or from an amputated limb, and hav-
ing been slit open with scissors it is pinned on to a
cork with the inner surface uppermost. Care must
be taken in doing so not to rub this surface in any
way. A little distilled water is then spirted over
the preparation from a wash-bottle, with the object
of removing any blood or serum that may remain
on the wall of the vessel, and a few drops of half
per cent, nitrate of silver solution are allowed to
flow over the surface. After a minute it is again
washed with distilled water, and is then put at once
into a beaker of spirit and placed in the sunlight.
After a time the surface will have acquired a gray-
ish tinge, with a browner patch here and there ; it
may now be removed from the window, but should
be left in spirit until the next day, when it will be
hard enough to enable thin sections to be made with
a razor from the inner surface. In order to cut them
it will be found convenient to remove the piece of
bloodvessel from the cork, and to hold it by one end
by the thumb and fingers of the left hand, so that
the piece rests by its outer surface on the ball of the
finger; the razor is then dipped into spirit, and as
thin a slice as possible (it need not be very large) is
removed from the inner surface of the bloodvessel
and placed in the spirit, after which one or two

THE LARGER BLOODVESSELS. 139

more may be taken from other parts. In making
sections when the piece is held in this way, it will
be found convenient to cut from the operator.

The slices are taken up on a needle or section-lifter
and placed on a slide with the stained surface upper-
most ; they should be in as little spirit as possible,
but at the same time should not be allowed to be-
come actually dry. A drop of the ordinary glycerine
solution is placed on a cover-glass, and this is quickly
inverted over the sections. On examinig them with
the microscope, the outlines of the epithelioid cells,
and perhaps also their nuclei, will be seen in those
sections which were made from the gray part of the
bloodvessel. In sections, however, which include
any of the patches which look brown to the naked
eye, it will be found that the difference of color is
due to the epithelioid cells having at these parts
become accidentally rubbed or washed away before
the silver solution was allowed to act ; for since the
subjacent tissues (the sub-epithelial connective tissue,
if present, arid the muscular tissue of the middle
coat) contain more intercellular or ground-substance
than the epithelioid layer (where it only occurs in
tine lines between the cells), they assume a browner
appearance after the reduction of the silver, and
show under the microscope in the one "case irregular
white patches — the cell spaces — upon the brown
ground, and in the other transversely arranged
lanceolate white markings — the plain muscular fibre
cells — with a variable amount of ground-substance
between. The latter appearance may be obtained all
over the preparation if the bloodvessel which is to
be treated with silver nitrate is first brushed with
a camel-hair pencil moistened with distilled water,
for by this means the epithelioid cells are removed,
and also, for the most part, the sub-epithelial con-
nective tissue Avhere present, the elastic layer being
the only part of the internal coat which remains,
and since this does not reduce the silver salt the
muscular laver is the one which is seen in such cases.

140 PRACTICAL HISTOLOGY.

Except when the epithelioid cells are first removed,
either purposely or accidentally, even a compara-
tively long exposure to the action of the nitrate of
silver solution will not cause the deeper coats to
become stained. This is the case with all structures
which are coated with epithelioid cells.

Preparation 2. Elastic layers ; fenestrated
membrane ; muscular tissue. — To prepare these
several parts, a piece of artery (or vein) is taken (as
fresh as possible, but this is not so imperative as for
the preparation of the epithelioid layer), and placed
for a week or more in a weak solution of bichromate
of potash (about 1 in 800), the fluid being changed
every other day. The piece is then taken out, pinned
down on a cork, with the inner surface uppermost,
and a thin strip torn off from the inner surface with
fine forceps. This is transferred to a slide, and teased
as finely as possible in a drop of water. It will be
found advantageous to employ only as much water
as will keep the tissue moist, and to add more of
this by placing a drop on the cover-glass before it is
laid on. If the small pieces are then examined, it
will be found that they are for the most part made
up of a close network of elastic fibres of varying
degrees of fineness. Many of them have very broad
fibres and small meshes, so that there may be found
in different arteries every transition to the true elastic
membrane. This will itself in all probability be met
with projecting at the edges of some of the fragments
of tissue, or even entirely separated ; the fragments
are generally curled at the edges, are often striated,
and nearly always exhibit rounded holes. These
fenestrated membranes are more frequently met with
in the inner coat of the smaller or medium sized
arteries (such as the basilar), than in the largest ves-
sels (such as the aorta).

But besides the different kinds of elastic tissue
there is also to be found in nearly every such prepa-
ration a number of plain muscular fibre-cells scattered
about in the fluid; for, in stripping off' the inner

THE LARGER BLOODVESSELS. 141

coat, shreds of the middle coat nearly always adhere
to it, and the muscular cells of this readily separate
after the maceration in bichromate. But the isolated
cells present in most instances such a ragged shape-
less aspect that they would hardly be known for
muscular tissue. A convincing proof, however, is
the addition of a little weak logwood solution at
the edge of the cover-glass. This, as it comes to
each of the cells in question, almost instantaneously
stains their long rod-shaped nuclei of an intense
violet, whilst the body of the cell, if the logwood
solution be sufficiently weak, remains uncolored.
The addition of a little glycerine at the edge of the
cover-glass, and the subsequent cementing, are suffi-
cient to preserve the preparation.

The connective tissue and elastic fibres of the outer
coat (as well as the muscular tissue of the same coat
in certain veins) can be equally well seen in a teased-
out preparation.

Preparation 3. Study of the structure of the
bloodvessels by means of sections. — To form a
correct idea of the relative thickness of the several
coats, as well as to observe the differences in arrange-
ment in different arteries and veins, it is necessary
to study them in vertical sections, f.^., sections made
in a direction at right angles to their surface. Such
sections may be either transverse or longitudinal ; it
will be better perhaps to choose the former direction,
for the middle coat is thereby better displayed. But
the vessel to be cut must tirst be hardened. This
may be effected speedily by immersing it in very
strong spirit for a day or two, and indeed this method
can be employed tor nearly all the tissues and organs.
It is preferable, however, in many instances to effect
the process more slowly, by means of some watery
fluid, such as a strong solution of bichromate of
potash or a weak solution of chromic acid, since in
this way the parts shrink less and consequently
retain their form better; the process should, never-
theless, always be completed by means of spirit. In

142 PRACTICAL HISTOLOGY.

the case of the bloodvessels an immersion for a fort-
night or three weeks in a 1 per cent, solution of
bichromate of potash answers best ; the pieces are
then placed for a day in weak spirit, and finally
transferred to strong methylated alcohol. Here they
may remain without detriment until it is convenient
to prepare the sections.

The method of cutting and preparing sections for
the microscope is in the main the same for nearly all
the tissues and organs of the body, and since, in the
further study of these, it will be in almost constant
requisition, it will be convenient to give in this
place an account of the mode usually adopted, re-
serving any special modifications which it may be
needful to make in the preparations of particular
organs until these come to be treated of.

METHOD OF PREPARING SECTIONS.

Preparation of an embedding mass.— Unless
the piece of tissue is large enough to admit of being
held firmly by the left hand while sections are made
with a razor held in the right hand, it is necessary
to support it in some way. This is usually done by
surrounding it in a mould with some fatty mixture
which requires only a moderate temperature to melt
it; the mixture or mass should, when cold, be of
about the same consistence as the hardened tissue.
Perhaps the most generally useful mixture for this
purpose is one of wax and olive oil. This can, by
varying the proportions of the ingredients, be suited
at will to the consistence of the tissue which it is
desired to obtain sections of. For most purposes the
following is the best proportion to adopt, and it is to
be observed that whenever subsequently the "wax-
mass" is mentioned, this particular mixture is indi-
cated : Sixty cubic centimetres of olive oil are poured
into a measuring-glass, and small lumps of white wax
are added to the fluid until it occupies a hundred
cubic centimetres. It is now placed in a porcelain

METHOD OF EMBEDDING.

143

or tin vessel (a small oil-can answers the purpose
very well), and heated over a water-bath until the
wax is entirely melted. The mixture is then thor-
oughly stirred, and put aside until wanted. In
laboratories where it is likely to be much in requisi-
tion it is well to have a supply always ready melted.
This may be done by keeping it in a sandbath at a
temperature of about 40° C.

Process of embedding, — One or more shallow
oblong moulds of different sizes should be kept in
readiness for embedding pieces of tissue in. They
may be made of thin pliable metal, such as sheet
lead or capsule metal (Fig. 23), so as to permit the
sides to be readily bent back and the wax-mass
removed when set; but if these are not at hand a
suitable mould may always be extemporized out of
ordinary stiff paper. A piece is cut of about the
size and shape shown in the accompanying diagram,
Fig. 21, and folded along the lines there marked out,

Fig. 21.

Outline showing the manner in which a small piece of paper is to be
folded to make an embedding trough.

the small dotted diagonals at the corners being scored
with some blunt-pointed instrument, an ordinary
hard lead pencil, for instance. These corners are
then pinched up between the finger and thumb,
bent round so as to be applied to the ends of the

144

PRACTICAL HISTOLOGY.

oblong (Fig. 22, a), and are fixed there by turning
down the flaps cc (Fig. 22, b).

Fig. 22.

Embedding trough made from a piece of piper of the size shown in Fig.
21, with one side, b, completed; the other, «, only half finished ; so as
to show the manner in which the corners are folded and fixed.

The trough being ready, it is placed upon a flat
cork, and the next thing is to take the piece of tissue
(artery, for example) out of spirit and place it for a
minute or two on clean blotting-paper to remove the
excess of fluid; at the same time its surfaces should

Fig. 23.

Embedding trough of capsule metal placed on a cork, and with a piece of
tissue (artery) in situ All that is further necessary is to fill the trough
with melted wax-mass.

on no account be allowed to become actually desic-
cated. A minnikin pin is then pii^s-jd for a good
part of its length through the piece near the end
furthest from that which it is desired to cut, and

METHOD OF CUTTING SECTIONS. 145

the point of the pin is stuck through the bottom of
the mould into the cork in such a way that the
tissue is placed at one end of the oblong, and sus-
tained in a horizontal position about half way down
in the embedding trough, as shown in Fig. 23. All
is done as quickly as possible, so as not to allow the
tissue time to become too dry (if there is any fear of
this it must be moistened now and then with a drop of
spirit), and without manipulating the piece more than
can possibly be helped. The object to be cut being
thus fixed in the proper position, melted wax-mass
is poured into the mould so as to fill it completely,
and entirely to surround the piece of tissue, and the
mould is set aside to cool. It is important not to
use the mass hotter than just above its melting-point,
as the tissues, even when hardened, are liable to be
injured by too high a temperature. When the wax
is completely set the pin is carefully withdrawn and
the trough removed from the cork and placed in
spirit. Here the cooling is soon completed, and the
paper or capsule metal having been taken away the
embedded tissue is ready for cutting.

Method of cutting the sections. — The sections,
unless a microtome or section instrument is used,
when a special form of knife is often provided, are
made with an ordinary razor, the blade of which
should be somewhat hollow-ground on both surfaces.
Some prefer that the under surface should be ground
flat, and this is advantageous for large pieces of
tissue, but it is not so readily kept sharp. For this
last- mentioned purpose a good strop is absolutely
necessary. The razor is kept constantly wetted
during use with spirit, contained in a lar^e shallow
glass dish, into which the instrument is dipped from
time to time, and in which also the sections are in
the first instance placed.

All then being ready, the cake of wax-mass (pro-
tected from the warm fingers by being wrapped
round with a small strip of bibulous paper) is held
in the left hand with the end in which the tissue
13

146

PRACTICAL HISTOLOGY

lies uppermost, whilst the razor is held in the way
shown in Fig. 24, and the wax-mass is evenly and
gradually shaved off layer by layer until the em-
bedded tissue conies into view. The pieces of wax-
Fig. 24.

Process of cutting sections of the embedded tissue.

w, cake of wax-mass, with the piece of tissue seen in it as a dark patch, com-
pletely inclosed and supported by the wax ; p, bibulous paper wrapped
round the wax-mass, except where this is being cut ; d, dish containing
spirit; c, glass cover with section lifter lying upon it. The flask on the
right of the figure serves to receive the spirit which has been used, for this
may be employed again and again if filtered back after use.

mass which are removed in this way must not be
allowed to fall into the spirit, but are to be put on
one side, and the razor wiped clean. It is then again
dipped in the spirit, and a fresh layer of the cake
removed, including a moderately thin section of the
tissue. This is not to be kept, the only object in
making it having been to prepare a clean even sur-
face to cut from. The razor is again wiped clean —
this must be done before each fresh section — re-
wetted with spirit, and, starting at the edge of the

MODE OF CUTTING SECTIONS. 147

piece of tissue, a section as thin as possible is made,
and removed on the razor blade without bringing
away more of the wax-mass than can be helped.
The section, if it seems thin enough, is floated off
into the dish of spirit, and another and another are
made in the same manner. No difficulty will be
experienced in getting rid of any wax that may be
still adherent to the sections ; a touch with a needle
is generally sufficient.

The attainment of dexterity in cutting thin sections in
this manner with the free hand — that is to say, without
the aid of any special instrument — is in great measure, as
may well be supposed, a matter of practice. However,
one or two hints drawn from experience may not be
superfluous.

In the first place the razor must be always kept very
sharp: this is the main secret of thin sections. It should
also, at the moment of cutting, always have a flood of
spirit upon the upper surface. The object of this is to
keep the section free as it is being made, otherwise it is
apt to adhere to the razor and to become broken. Then,
in cutting, the razor must not simply be pushed forward
over the tissue, but drawn over it with a movement from
heel to point ; but at the same time the whole section
must be completed with one such sweep, for a to-and-
fro movement inevitably produces ridges upon the sur-
face of the section. Except in the case of injected pre-
parations, the sections can hardly be cut too thin ; as a
rule, those sections only are worth keeping, which can
barely be seen as they are being cut. It must not be
forgotten that they will appear considerably thicker after
they are stained.

Two or three sufficiently thin sections having been
in this way obtained, the embedded tissue may be
put aside in spirit for the present, in case any more
should subsequently he required.

The sections have, in the tirst place, to be stained.
This is best effected, in the case of the sections of
artery, by immersing them in the ordinary logwood
alum solution for a few minutes. They are then
put into water for a second or two, to wash away

148

PRACTICAL HISTOLOGY.

the excess of staining fluid, which would otherwise
he precipitated, and are transferred from this to
spirit (absolute alcohol is gene-
Fig'- 25- rally recommended, but strong
methylated spirit is sufficiently
anhydrous) in which they are
allowed to lie until it is certain
that all the water is replaced
(live minutes is generally amply
sufficient), after which they are
placed in oil of cloves for a
minute or two. In this the
sections should almost imme-
diately become clear and trans-
parent, and the change has at
the same time the effect of caus-
ing their color to appear darker.
If any parts of the section do
not participate in the change of
appearance, but remain opaque
and cloudy, it is a sign that all
the water has not been extracted,
either because the sections were
not left long enough in spirit, or
because this was not anhydrous
enough (for oil of cloves will not
mix with water nor with alcohol
which contains more than a trace
of water). But it is not too late
to put them from the oil into
strong spirit again, and when
they have been long enough
there to transfer them once more
to oil of cloves. All these trans-
ferences may be effected by aid
of a section-lifter (Fig. 25), made
from a piece of copper or i>'ernian-

Section-lifter. Natural * .

size. silver wire flattened out at one

or both ends, which are bent

over so as to be hoe-shaped. l>y means of this in-

TRANSFERENCE OF SECTIONS. 149

strument even very thin arid fragile sections may be
readily lifted from one vessel to another without
injury. The staining fluid, which should always be
freshly filtered, and not be used for too many sec-
tions, may be in a watch-glass, which should be
covered over by another watch-glass inverted, but
the other fluids which are constantly in use it is
better to keep in covered glass or china pots, for
they — especially the oil of cloves — may be used for
a considerable number of sections without detriment.
The process of preparation may be arrested at any
stage and completed at another time, if the sections
are left in spirit or in oil of cloves.

In transferring sections* from one fluid to another,
and especially when they are put in the staining
fluid, care should be taken that they are actually
immersed, and do not merely float on the surface.

Instead of the ordinary logwood solution Kleinen-
berg's fluid (see post) may with advantage be used
for staining sections that are to be mounted in bal-
sam or dammar. In this case the sections can be
placed from spirit into the staining fluid, and from
the latter directly into the spirit again without the
necessity for placing them in water at all.

The sections then being in oil of cloves, and the
clearing up satisfactorily effected, all that remains
is to mount them permanently. For this purpose
either dammar varnish or a solution of Canada bal-
sam in chloroform — the same liquid that was em-
ployed for cementing the cover-glass of the glycerine
preparations — is employed.1 These solutions, al-
though perfectly limpid even in the cold, readily
dry when exposed to the air, and thus become hard

1 The Canada balsam used, must be heated over a sandbath
until it is quite hard when cold ; it is then dissolved in an equal
volume of chloroform. Dammar varnish is made by dissolving one
part of gum dammar in two parts of oil of turpentine, and one part
of gum mastic in two parts of chloroform ; mixing the solutions
and filtering. The mixture is apt to become opalescent on keep-
ing, but this disappears permanently on boiling the fluid.

13* '

150 PRACTICAL HISTOLOGY.

and firm. The sections are removed by the lifter
from the oil of cloves and gently placed upon a slide,
the excess of oil of cloves is allowed to drain off or
is soaked up with blotting paper, a drop of the
mounting varnish is placed on the cover-glass, and
this is then quickly inverted over the sections. The
preparation is now complete and permanent, and
ready for examination ; but as it will be some while
before the mounting varnish has become hard to any
distance under the cover-glass, care should be taken
not to let this become shifted in any way, since such
treatment would probably crumple or otherwise
spoil the sections.

Preparation 4. The smaller arteries and
veins and the capillaries. — The elongated epi-
thelioid cells which form the walls of the capillaries,
and which line the arteries and veins, are readily
shown in silvered preparations of any vascular
tissue. It is most convenient to choose a vascular
membrane for the purpose, because more readily dis-
played, and of vascular membranes the mesentery
of the frog or toad is perhaps as easy to prepare as
another. The following is the mode of proceeding:
The animal (a male) having been decapitated and
the spinal cord destroyed, the trunk is suspended
for a few minutes by the lower limbs in order to
allow the blood to drain from the body as completely
as possible. The frog is then placed on its back,
and the abdomen freely opened. A loop of intes-
tine is seized with forceps and gently raised by an
assistant, while with a soft camel-hair pencil, mois-
tened with distilled water, the operator carefully
brushes the mesentery on both surfaces, carrying the
brush in every case from the intestine, not towrards
it. This brushing subserves two purposes — in the
first place, the epithelioid cells of the serous mem-
brane, which would obstruct the passage of the
silver solution to the bloodvessels, are removed ; and

SMALLER BLOODVESSELS. 151

in the second place, much of the blood which
remains in the vessels is driven out of them.

The brushing being completed, the loop of intes-
tine, with its included mesentery, is cut off, dipped
for a second into a capsule of distilled water, and
then at once placed in J per cent, nitrate of silver
solution. Here it is allowed to remain ten minutes ;
after which it is rinsed in distilled water and then
placed in common water in the sunlight. If the
day is bright, the silver is soon reduced, and all
that remains to be done is to place the preparation in
a shallow glass dish, and carefully cut off and remove
the piece of intestine, leaving the mesentery. This
must be done under water, and will require sharp
scissors and delicate handling, so as not to drag
upon the mesentery or throw it into folds.

In order to mount it a slide is held in the water
and the membrane allowed to float over, after which
the slide is carefully lifted out with the membrane
flat upon it, and the excess of water is drained off.
Before covering it the preparation must be examined,
both with the unassisted eye and with a low power
of the microscope, so as to detect any folds or creases
in it. If present they can be got rid of, by gently
drawing out first this corner and then that with a
needle. A drop of strong glycerine may then be
placed on the middle of the preparation, and the
cover-glass laid on and allowed slowly to settle
down. More glycerine may be afterwards added at
the edge if necessary. Should it be found, on exam-
ining the preparation with a moderately high power,
that the outlines of the epithelioid cells of the vessels
are not yet sufficiently marked, it will be well to
leave the preparation out in the light, but covered
over from dust, for a day or two.

If there is but little sunlight the reduction of the
silver may often be better effected by placing the
loop of intestine, with its attached mesentery, after
it has been taken from the silver solution and rinsed
in water, in a beaker of weak spirit (equal parts of

152 PRACTICAL HISTOLOGY.

water and strong spirit, freshly prepared), and expos-
ing it to the light in this for an hour or more. The
cutting off of the intestine must be performed in the
same fluid and the mesentery floated from it on to
the slide. The method has the advantage not only
of effecting the reduction of the metal with greater
surety, but also of rendering it easy to obtain the
membrane free from creases, for the mesentery is
partly hardened by the spirit while in a state of
extension, and continues in this condition when
floated on to the slide, so*that it is seldom necessary
further to extend it by artificial stretching. In
these silvered preparations little but the epithelioid
cells can be made out, for the rest of the tissue
generally remains almost unstained, and becomes
very transparent in glycerine.

Preparation 5. — To exhibit the muscular struc-
ture of the small arteries and veins, and the nuclei
of their epithelioid lining and of the walls of the
capillaries, the vessels are stained with logwood.
This is done by immersing the mesentery or other
vascular membrane, either fresh, or better after hav-
ing lain for a day or two in very weak bichromate
of potash solution, in a dilute solution of logwood
alum, until distinctly colored; then place the tissue
in water, and mount it, with the same precautions
as before to prevent creasing, in glycerine. For the
structure of the small arteries the pia mater from
the human brain may be used. A small piece is
stripped off with forceps ; and as it consists almost
entirely of small arteries and veins, and moreover
a few capillaries are generally dragged out with, it
from the cerebral substance, the structure of all the
vessels is, after staining, very well displayed. The
small veins are here exceptional in being entirely
devoid of a muscular coat, whereas the arteries have
this coat well developed, and it is particularly well
shown in consequence of the staining of the trans-
verse nuclei of the muscular fibres by the logwood.
Within these Tnay be detected, by carefully using

STUDY OF THE CIRCULATION. 153

the fine adjustment of the microscope, in the first
place, longitudinal striae, which are produced by
wrinklings in the elastic layer of the internal coat ;
and in the second place, situated most internally,
elongated oval nuclei, which belong to the epithe-
lioid cells lining the vessel. Of course, two layers
of all these structures are come across in focussing
from above down. The outer coat is represented
merely by a few corpuscles and .fibres of connective
tissue which blend externally with the connective
tissue framework of the membrane.

It will be found in carefully focussing from above
down, that at one position of the focus tlie small
vessel has exactly an appearance as if it had been
cut longitudinally through the middle, and as if the
top of the lower half were being examined. The
lumen is seen in the centre, with possibly a few
blood corpuscles still in it ; on either side of this a
well-marked line representing the inner coat ; out-
side this again what seems like a row of rounded
cells, which are really the encircling fibre-cells of
the muscular coat seen as if cut across; and finally,
here and there, outside of all, small cells presenting
the fusiform aspect of connective tissue corpuscles
seen in profile. All these appearances are exactly
the same as if a section had been made along the
vessel, and result from the fact that only those parts
of an object which lie in the horizontal plane that
happens to coincide with the focal distance of the
objective, are distinctly seen, so that it seems as if
only this particular slice tvere present. An " optical
longitudinal section" is thus obtained of the vessel.

STUDY OF THE CIRCULATION.

The study of the bloodvessels cannot be said to
be in any s.ense of the word complete until they have
been viewed in the living condition and with the
blood still moving through them. Such an observa-
tion can, of course, only be made whilst an animal

154 PRACTICAL HISTOLOGY.

is still alive, and in parts which are transparent
enough to allow the vessels to be distinctly seen.
Membranous parts are those which are naturally
best adapted for such observation, as, for example,
the web of the frog's foot and of the bat's wing ;
the tongue, mesentery, and lungs of the frog and
toad, but especially the latter animal ; and the
mesentery and omentum of small mammals. In such
preparations the surrounding tissues, and especially
the connective tissue corpuscles, may be studied as
well as the bloodvessels ; and the changes due to
commencing inflammation which are exhibited by
the bloodvessels, and the migration from the veins
of the white blood corpuscles, can always be brought
on either by the application of irritants, or, as in
the case of the serous membranes, hy simple expo-
sure to the air. The best methods, therefore, of
observing the circulation of the blood in different
parts will be described in the following preparations.
Preparation 6. Circulation in the frog's
web. — One of the common English frogs (Rana
temporaria), of as light a color as possible, is chosen,
and by means of a Pravaz or other hypodermic
syringe two drops of a very weak solution of curare
(1 to 2000 of water) are injected under the skin of
the back. This is generally sufficient, in the course
of from a quarter to half an hour, to render the
animal completely motionless, whilst the pulsations
of the heart and the circulation proceed unimpaired.
The frog is then laid on a piece of cork or soft wood
of an oblong shape which* has a narrow slit at one
end. One of the interdigital webs is placed over
this slit and fastened on either side by means of one
or two minnikin pins or fine needle-points passed
through the adjoining webs. Care must be taken
that the web under examination is at no part tightly
stretched, since this would tend to arrest -or obstruct
the circulation. A slip of blotting-paper or a piece
of linen rag is placed over the animal and kept
thoroughly wetted with water, and the cork, with

CIRCULATION IN THE MESENTERY. 155

the frog upon it, is then placed on the stage of the
microscope (the head of the animal being away from
the observer, and the web over the aperture in the
diaphragm), and is fixed in this position by the
clamps like an ordinary slide. To observe the web
a low power is used to see the general features of
the circulation, a high power being afterwards em-
ployed to observe the parts more in detail. But
this should not (unless it is an immersion) be of too
short a focal distance, since otherwise the lower
glass is apt to become clouded by moisture from the
web. It is not advisable to put a piece of covering
glass on the latter to prevent the clouding, as the
circulation might thereby be interfered with or
arrested.

The web of the frog's foot is the easiest of the
vascular membranes to prepare, but has the disad-
vantage that, owing to its comparative thickness and
its epidermic covering, it is not always easy clearly
to make out the intermediate tissue. At the same
time, being under almost completely natural condi-
tions, the circulation will continue for an indefinite
time quite unimpaired.

Preparation 7. Circulation in the mesen-
tery.— The mesentery of the frog, and still better
of the toad, is admirably adapted by its thinness
and perfect transparency, as well as it great vascu-
larity, for observations on the bloodvessels and sur-
rounding tissues. It is necessary to have a special
mesentery-plate for this purpose, which can, how-
ever, be readily made from an oblong piece of cork,
like that used for the web observation. A round
hole about half or three-quarters of an inch in
diameter is made at one side with a cork borer,
and a small piece of the same material about half
an inch thick, and with a segment of a similar circle
cut out of its side, is fixed on the first with sealing-
wax or small pins (Fig. 26, b). A piece of glass of the
same shape as this segment may be fitted into it near
the top for the mesentery to rest on. The animal —

156 PRACTICAL HISTOLOGY.

a male — having been rendered insensible by destruc-
tion of the brain, or other means, is curarized as
before, and laid upon its back, and a longitudinal
cut about an inch long: is made with scissors through

O O

Fig. 26.

Flat piece of cork arranged as a frog-stage for viewing the circulation
in the web, tongue, mesentery, or lungs.

Over the small pieces of cork a the tongue cau be fixed ; n can he removed when
the slit below it is wauted for the web ; b, cork with a deep groove cut along
one side ; to this the iute.sliae is fastened by needle-poiuts, while the mesen-
tery rests on a semicircular piece of glass which should fit at the top of the
groove.

the skin of the abdomen about half an inch to the
right of the middle line. Before proceeding further,
the operator should wait for a minute or two to
make sure that there will be no bleeding ; and any
blood that may have already exuded should be dried
up with blotting-paper. The abdominal cavity is
then opened by a corresponding cut through the
muscles and peritoneum, taking care, however, to
avoid any veins that may be in sight. Having
again assured himself of the absence of bleeding,
the operator very gently draws out one of the coils
of the intestine, with its included mesentery at the
aperture. The animal is now to be turned over on
its side, and so propped up against the smaller cork
that the wound is about on a level with the top.

CIRCULATION IN THE MESENTERY, 157

All that remains to be done is to place the extruded
mesentery over the aperture, and to keep it in posi-
tion by two or three fine needle points passed through
the surrounding intestine into the cork. In this
case again, the greatest care must be taken in no
way to drag upon the exposed membrane, or to
allow it to be pressed upon. Moreover, the surface
must from time to time be moistened with a little
salt solution, to prevent its becoming dry. But in
spite of every precaution the mere exposure of the
serous surface to the air is sufficient to produce
before long the changes in the circulation which
are characteristic of the commencement of inflam-
mation.

Preparation 8. Capillary circulation in

Mammals. — It is less easy to study the circulation in
the serous membrane of mammals, for the exposure re-
quired for the purpose is apt to be far more prejudicial to
the maintenance of the normal condition of the tissues
than is the case with the cold-blooded vertebrates. It is
necessary, moreover, to maintain the exposed part at the
body temperature, and to immerse it in fluid, since it
would otherwise become at that temperature rapidly
desiccated. The membrane generally chosen is not the
mesentery but the omentum, which in many animals, e.g.
the guinea-pig, is very extensive, and at the same time
thin, and provided in parts with a sufficient number of
bloodvessels. The animal, whieh should be rather a
small one, is anaesthetized with chloral hydrate, a few
minims of a 50 per cent, solution being injected under
the skin. The warm stage (Fig. 9) is in the meanwhile
got read}', and a glass tray (which can be extemporized
out of a small plate of glass, some pieces of glass rod and
sealing-wax) is placed on it and filled with salt solution,
which is maintained at about 38° C. Then the animal
is supported on a block at a convenient level, and the
abdomen having been carefully opened, a little of the
omentum is drawn out and allowed to float flat in the
warm salt solution, where it can be examined either with
a low power or with an immersion objective dipping into
the solution. If the latter be employed a piece of thin
14

158 PRACTICAL HISTOLOGY.

covering glass nnist be placed over the part of the mem-
brane which is to be examined, so as to sink it in the fluid
and keep it steady. But in spite of every precaution, the
circulation under these conditions retains its normal
character but a short while, and inflammatory congestion
and stasis, or complete stoppage of the flow of blood,
rapidly supervenes.1

Preparation 9. Circulation in the lung of the
toad. — This is readily observed with the aid of the
mesentery board. The animal must, as before, be
first rendered insensible and curarized, but it will
be found that a good-sized toad will require at least
six times as much curare as a frog. An opening is
made at the side of the chest .large enough to allow
the lung, which in the toad almost always remains
distended with air, to protrude. The animal is then
propped up on the mesentery board (Fig. 26) in such
a manner that the lung rests over the aperture ft,
and the circulation can be studied in the part which
is uppermost without further trouble. A frog may
be used in a similar way, but there is much greater
difficulty in keeping the lung distended. In either
case the greatest care must be taken to avoid prick-
ing, or in any way rupturing the wall of the lung.

Preparation 10. Circulation in the tongue
of the toad. — By far the most beautiful object for
studying not only the circulation but also the tissues
in the living animal, is the tongue of the toad, and
in a slightly less degree that of the frog. The tongue
is in these creatures an extremely extensile organ,
which, under ordinary circumstances, lies folded
back on the floor of the mouth (Fig. 27, B), but which
can at the will of the animal be protruded for a con-
siderable distance (c). For the preparation of the
organ the cork plate is again necessary ; a smaller
piece of cork of the shape shown in the figure (Fig.

1 For a detailed account of this method the student is referred
to the original description by Knrdon Sanderson and Strieker,
in the " Quarterly Microscopical Journal" for 1.870.

CIRCULATION IN THE TONGUE.

159

26, a) and about one-eighth of an inch thick, being
fastened with pins over the slit which served for the
display of the frog's web.

Fig. 27.

Structure and position of the tongue of the toad (Dowdeswell).

A. Transverse section through the middle of the organ with the lymph-spaces
fully distended ; a a, thick, papillated mucous membrane ; b, tbin lower
membrane; m, muscular bundle cut across, united to the sides of the tongue
by septa of connective tissue 8 ft; v, position of the larger bloodvessels.

B Profile view showing the tongue in its ordinary position within the mouth.

C. The same when extended.

The toad having been rendered insensible and
curarized as before is laid upon its back with its
nose close to the slit. The lower jaw is then raised
and the folded-back end of the tongue is found, and
drawn gently out of the mouth with forceps. The
end has a pointed projection or cornu on each side ;
these are successively laid hold of by the forceps.

160 PRACTICAL HISTOLOGY.

and fastened with needle points to the small piece
of cork on either side of the slit. Before the rest
of the operation is described, a word or two may he
said with regard to the structure of the organ. It
is not solid throughout as in mammals, but hollow,
the interior being occupied by a lymphatic cavity.
This lymph space is traversed by bundles of muscu-
lar fibres (Fig. 27, m) which pass towards the ex-
tremity of the organ and are connected to the sides
by delicate septa of connective tissue (s s). Above
the lymph space in the present position of the
animal — on its back with the tongue extruded — the
mucous membrane is thick and papillated (Fig. 27,
A, a a). Below is a very thin and delicate mucous
membrane (/>), with numerous bloodvessels, and small
muscular fibres running over it. The former mem-
brane is too thick and irregular to allow the delicate
internal structures to be seen through it ; it is there-
fore slit up longitudinally and pinned to either side.
But to do this without injuring tbe delicate parts
below, it must be separated from them, and this can
best be effected by distending the lymph sac with
salt solution. With this object a Pravaz syringe
(Fig. 17), provided with a fine and sharp canula, is
filled with the fluid, and its point is stuck into the
tongue near the end, passing about half an inch
backwards. It will almost certainly be found that
on pressing the piston down, the salt solution will
readily flow into the lyrnph sac, and as it fills this
will cause the thin mucous membrane at the lower
part to become bagged out, and completely separated
from the muscular bundles, m m, and these again
from the thick layer above. The latter is now care-
fully slit up along its middle by sharp fine scissors,
and first one edge of the wound and then the other
is drawn to the side of the slit in the cork and
fastened there by two or three needle-points. If
everything is carefully done, there will be no escape
of blood over the preparation ; but should any blood

OBSERVATION OF LIVING TISSUES. 161

have exuded, it may be washed off by pouring a
little salt solution over the surface.

There is now brought to view the fan -like group
of muscles which pass through the middle of the
lymph sac, and the bundles of which are, as before
mentioned, connected with the sides and with one
another by delicate septa of connective tissue, tra-
versed by a few bloodvessels ; and it is this delicate
connective tissue, of which two strata can generally
be traced, one superficial to the other, which is better
adapted than any other part for exhibiting both ves-
sels and connective tissue corpuscles (the latter rather
peculiar in form and appearance). Moreover, the
mere exposure of the lymphatic surface soon causes
inflammatory changes, and after the preparation has
been made a few minutes only, the first commence-
ment of these is seen in the sticking of the pale cor-
puscles to the walls -of the vessels, speedily followed
by their migration from the veins into the surround-
ing tissue. Nowhere can the fact be more clearly
established, and the details of the process more accu-
rately followed, than here. Moreover, if the animal
is kept properly moist and from time to time given
an additional dose of curare, it remains for a long
while in a perfectly natural condition, and the in-
flammatory process can be studied for as long as may
be desired. By examining such a preparation at
intervals during several days, the same connective
tissue corpuscles being again and again found and
brought under observation, it has been conclusively
proved that, at all events as regards these corpuscles
of the tongue of the full-grown toad, the connective
tissue corpuscles take no active part in the process
of acute inflammation. The circulation of the blood
among the muscular flbres can also be well seen in
this part of the tongue.

Lastly, by focussing through the connective tissue
septa, or by severing the longitudinal muscular bun-
dles which they serve to unite, the vessels of the
lower mucous membrane are brought into view,

162 PRACTICAL HISTOLOGY.

especially if a slip of glass is fitted into the small
piece of cork, so as to support the tongue and pre-
vent the thin membrane from bulging downwards.

METHOD OF INJECTING THE BLOODVESSELS.

Before leaving the subject of the bloodvessels the
best mode of filling them with transparent material
may be described, especially as in the study of the
several organs it is necessary, in order that the course
and arrangement of the vessels may be properly made
out, that sections of injected as well as of uninjected
preparations should be looked at. It will be conve-
nient in this place to describe the injection of a small
animal entire from the aorta, reserving any special
directions concerning organs which are not thereby
properly injected, such, for instance, as the lungs and
liver, until they are severally dealt with.

Preparation of the injection mass.— This is
almost always a solution of gelatine colored, either
red with finely precipitated carmine, or blue with
soluble Prussian blue. Sometimes, but rarely, when
it is wished to inject two sets of vessels of different
colors, both of these are used, but as a rule all the
bloodvessels — arteries, capillaries, and veins — should
be filled with the same injecting fluid; preparations
in which the arteries are filled with one color and
•the veins with another are pretty to look at, but are
difficult to prepare, and present no practical advan-
tage. The gelatine solution is made as follows:
Ten grammes of clear gelatine are cut into small
pieces, and placed in a beaker of cold distilled water
to soak. In about an hour the gelatine will have
swollen to several times its original volume. The
excess of water is now poured off, a glass cover put
over the beaker, and it is placed in a water-bath and
heated until the gelatine is rendered fluid.

For the red injection four grammes of carmine are
rubbed up in a mortar with eight cubic centimetres
of liquor ammonise, and then fifty cubic centimetres

MATERIAL FOR RED INJECTION. 163

of water are added. When the carmine is as com-
pletely as possible dissolved, the liquid is filtered.
The process of filtration occupies some time — several
hours, in fact — and may be conveniently left over-
night. The filtrate is warmed, and the gelatine solu-
tion is gradually added to it with constant stirring.
The next part of the process is to precipitate the
carmine, for otherwise it would difl'use through the
walls of the vessels and color the tissues ; but it must
he precipitated so finely that the particles shall not
be visible even under the highest power of the micro-
scope. To effect this object a small quantity of a ten
per cent, solution of acetic acid is placed in a burette
and allowed to run drop by drop into the warm car-
minized, gelatine solution, which is all the while
constantly agitated. As its alkalinity becomes neu-
tralized the amrnoniacal odor becomes less and less
strong, and eventually disappears, and is replaced by
the vinegar-like smell of acetic acid. The alteration
in reaction may be shown, in spite of the red color
of the solution, by placing a small drop on a piece of
blue litmus paper ; if the opposite side of the paper be
looked at, it will be found to have assumed the
characteristic bright-red color which acids produce,
and which is quite different from carmine. This
change is owing to the diffusion of the acetic acid
through the paper, whereas the carminized gelatine
sets almost immediately and is thus unable to soak
through.

But it is not sufficient in order to effect the pre-
cipitation of the carmine that the fluid should only
just be acidulated ; there must be an excess of acid.
A few more drops are therefore added, and the car-
mine thrown out of solution. This change from
the soluble to the insoluble state is accompanied by
a very marked alteration in color, for whereas whilst
still in solution the carmine imparted the rich deep
red of an ammoniacal solution to the gelatine, after
the precipitation the color of the latter changes to a
paler red, comparable rather to the tint presented by

164 PRACTICAL HISTOLOGY.

the powdered carmine in the dry state. Even after
the production of this change a few more drops of
the acetic acid may be added, for it will do no harm,
and will tend to counteract the natural alkalinity of
the tissues.

The colored gelatine is next filtered through a
piece of flannel or fine linen, previously soaked in
hot water, and again wrung out, and is collected in
a flask as it runs through the filter, and transferred
to the injecting bottle.

For the blue injection 10 grammes of gelatine are
taken, and after having been soaked in cold water
and dissolved up as before, 50 c.c. of a 2 per cent,
solution of Berlin blue, which has been previously
warmed, is gradually added with constant agitation
to the fluid gelatine. The blue mixture is filtered
and is then ready for use, without the necessity of
precipitating the coloring matter, for ftiis being a
colloid is indiiFusible.

It is sometimes advantageous in cases where the
structure of the walls of the bloodvessels is to be the
subject of observation, to use an injecting mass which
is far less deeply colored. This can of course be
readily obtained by diminishing the proportion of
carmine or Berlin blue which is used.

The soluble Berlin blue is of great value for the
purpose of injecting both the bloodvessels and lym-
phatics. Unfortunately it is very troublesome to
prepare. The following is the method recommended'
by Briicke, to whom we owe its introduction : —

Take of potassic ferrocyanide 217 grammes, and
dissolve in a litre of water (solution A).

Take a litre of a 10 per cent, solution of ferric
chloride (solution B).

Take four litres of a saturated solution of sulphate
of soda (solution c).

Add A and B each to two litres of c. Then with
constant stirring pour the ferric chloride mixture
into the ferrocyanide. Collect the precipitate upon
a flannel strainer, returning any blue fluid which at

INJECTION APPARATUS. 165

first escapes through the pores of the flannel ; allow
the solutions to drain off; pour a little distilled
water very carefully over the blue mass, returning
the first washings if colored, and renew the water
from day to day until it drips through permanently
of a deep blue color. This is a sign that the salts
are washed away, and all that is further necessary
is to collect the pasty mass from the strainer and
allow it to dry.

Apparatus employed for injecting. — This con-
sists, in the first place, of a bottle for holding the
colored fluid ; and secondly, of some means of pro-
ducing a steady, elastic, and readily alterable pressure
on the surface of the fluid so that it may be driven
with any required force into the arteries. The
method formerly employed of forcing the injecting
material from a small syringe directly into the
bloodvessels has been almost entirely given up,
on account of the impossibility of estimating the
amount of pressure which is being exerted, leading
often to the employment of too great a pressure and
the consequent rupture of some of the smaller blood-
vessels. Fig. 28 represents a convenient form of
apparatus for general use. The bottle (i),1 which
holds the injecting fluid, is a moderate-sized, wide-
mouthed phial, with a well-fitting vulcanized India-
rubber cork, through which two glass tubes pass.
One of these goes to the bottom, and from it an
India-rubber tube passes, which will be subsequently
connected with the artery canula (c), but not before
this has been inserted in the bloodvessel, in the
manner immediately to be described. The other
passes only just through the cork, and serves to
maintain communication by means of another India-
rubber tube, with the pressure-bottle p. The injec-
tion-bottle is placed during the process of injecting
in a large beaker (b) of warm water (about 40° C.) ;
a piece of cork is wedged in between the bottle and

1 A simil .r one is better shown in Fig. 30, at c.

166

PRACTICAL HISTOLOGY.

the side of the beaker to prevent the bottle from
floating up as it becomes emptied of injection, and

Fig. 28.

Injecting apparatus. Complete.

s, condensing syringe, fixed to the table ; p, pressure-bottle ; 6, beaker of warm
water in which the injection-bottle, t, stands ; ft', small beaker containing
salt solution ; w, water-bath heated by a ring burner below ; the tempera-
ture of the water is indicated by a thermometer, t, placed in it ; c, arterial
canula connected to an India-rubber tube from the injecting bottle; close
to the canula is a steel clip. The canula rests upon a glass plate, which
may serve either to put the animal which is being injected upon, or to cover
it over, if it is thought necessary to place it in the water-bath.

the beaker is covered with a glass plate (not shown
in the figure). The pressure-bottle is a large glass

INJECTION OF BLOODVESSELS. 167

or earthenware bottle capable of holding two or
three gallons, and tightly fitted with an India-
rubber cork, through which two glass tubes pass.
One of these is connected, as before mentioned, with
the injection bottle, and the other with a condensing
syringe (s), by means of which the air within the
bottle can be brought to any state of tension that
may be desired. "Finally, if the injection is to
occupy a considerable time, a water-bath or sand-
bath (?/?), heated by a ring-burner to about 40° C.,
should be provided for receiving both the beaker
containing the injection-bottle and the animal, and
maintaining their temperature during the process.
Ordinarily, however, if the operation be quickly
and dexterously performed, the whole process will
not occupy more than a few minutes, and will be
over before the natural heat of the body has had
time to become dissipated.

Everything then being in readiness, the animal, a
rabbit, guinea-pig, or rat, for example, is killed by
chloroform inhalation, being placed under a bell-
glass with a sponge wetted with chloroform. The
moment it has ceased to breathe, it is taken out and
held by an assistant, whilst the operator first quickly
reflects the skin from the front of the thorax and
then makes an opening in that cavity just over the
position of the heart. This is then seized near the
apex with blunt forceps, drawn out of the aperture,
and held here by an assistant, The aorta is then
found, the point of a pair of forceps passed under
it close to the heart, and a thread ligature drawn
round it. A snip is now made in the left ventricle,
and an arterial canula (Fig. 29, e') passed through
this into the aorta, in which it is tied by the liga-
ture. Then by means of a pipette a little warm
water or salt solution is passed into the canula so
as completely to fill it to the exclusion of air.

^The next thing to do is to connect the canula
with the India-rubber tube which brings the injec-
tion from the bottle. But this tube must first be

168 PRACTICAL HISTOLOGY.

completely filled by the injection, so that it contains
no bubble of air. To effect this, whilst the India-
rubber tube is kept closed by the strong spring clip
with which it is provided, the air in the apparatus

Canulas for injecting. Natural size.

c.1 c.2, c.*, glass canulas of different sizes ; c.4, metal canula : it is sometimes
more easy to insert than the glass ones, especially into fine bloodvessels, or
into lymphatics ; cl, steel clip for clamping an artery, or a small India-rubber
tube; a and b are intended to illustrate the mode of making the glass ca-
nulas ; a, glass tube which has been heated in the middle in the blowpipe
flame, and drawn out so as to be narrower here ; b, the same tube after
having been again heated (by the tip of the flame), and drawn out at the
paints a; a;, so as to narrow it still more at those places The subsequent
proceeding consists in making a nick at I with the edge of a file, breaking
the tube across here, and with a fine, flat, wetted file grinding the end away
obliquely as far as the doited ring In each. The sharpness of the filed edije
is got rid of by inserting it for a moment or two in (he flame. Two similar
canulas are thus made from the one piece of tubing.

is put under a pressure of about two inches of mer-
cury by working the syringe. The free end of the
India-rubber tube is now held up, and the clip
opened until the colored fluid forced up by the pres-
sure begins to escape, when the clip is immediately
closed and the tube is slipped on to the arterial
canula. The greatest care must be taken throughout
to avoid the introduction of air, since this would
obstruct the smaller vessels and prove fatal to the
success of the injection.

The clip is now permanently opened and the injec-
tion suffered to flow into the aorta, at first under
the low pressure of two inches of mercury ; but the

TREATMENT OF INJECTED PARTS. 169

pressure is gradually increased by working the
syringe until a pressure of four or five inches is
attained. The blood in the vessels gets forced before
the injection into the right cavities of the heart, so
that these are soon much distended ; when this is
the case the right ventricle is slit open and the accu-
mulated blood allowed to flow out. The blood is
soon followed at first by a mixture of blood and the
colored gelatine, but afterwards 'by the latter only ;
after this has been escaping for a minute or two the
slit in the ventricle is closed by placing a clip on,
or tying a tape round the heart, and the injection
being thus obstructed in its outflow, accumulates in
the vascular system, and distends all the vessels to
their fullest extent. The success of the injection
may be estimated by the color of those parts which
are not concealed by the fur ; the paws, lips, nose,
and ears, for instance, and the tongue and interior of
the mouth. After waiting a few minutes longer,
until the vessels may be considered to be com-
pletely filled, a tape ligature is put round the base
of the heart, so as to include all the great blood-
vessels, and is slowly tightened. This will effectu-
ally prevent any escape of the fluid gelatine from
the vessels when the canula is removed from the
aorta, which may therefore now be done, the clip
having been first replaced on the India-rubber tube
which is connected with it, so as to prevent the in-
jection from spirting out.

The animal is now put aside until it has become
quite cold and the gelatine is firmly set. The abdo-
men is then freely opened, and the skull-cap removed
(if the brain is wanted), and incisions having been
made through the skin of the limbs here and there,
the animal is placed overnight in weak spirit (equal
parts of water and spirit), and the next morning
those parts and organs which their color shows to
be well injected are carefully dissected out and
placed in rather stronger spirit. In another day or
two they may be transferred to the strongest spirit,
15

170 PRACTICAL HISTOLOGY.

in which they will soon become hard enough for the
preparation of sections. The object of trms gradu-
ally increasing the strength of the spirit is to prevent
as much as possible the shrinking of the gelatine
which otherwise results.

The blue injection possesses the material disad-
vantage that it is apt to become temporarily reduced
in the smaller vessels and rendered almost entirely
colorless, so that it is difficult to determine whether
a successful injection has been made or not. The
color may, however, be readily restored by pouring
some oxidizing fluid, such as a weak solution of
peroxide of hydrogen, over any part about which
there is doubt ; and in the ordinary course of pre-
paring and mounting sections, the blue color is
always brought back, especially if turpentine is sub-
stituted for oil of cloves.

Most other forms of apparatus which are used for
injecting are more or less like the one above
described, the chief modification being in the mode
in which the pressure is produced, this being effected
in one form by allowing water to flow from a tap
into the pressure-bottle (which in such cases is gene-
rally made of metal), and thus compressing the air ;
in another by allowing mercury to flow from one
vessel into another. But the latter method, al-
though useful when small quantities of injecting
fluid only are required, as with the injection of the
lymphatics (with which the apparatus will be
described), is costly for large quantities ; indeed it
will be found that none are more simple and efficient
in working than the one here recommended.

If a condensing syringe is not at hand, sufficient
pressure may be got in many cases merely by blovring
air into the pressure-bottle through an India-rubber
tube, its escape being prevented by subsequently
clipping the tube.

Injections which are fluid in the cold (of which
the best is a one or two per cent, solution of Berlin
blue) are sometimes used for the bloodvessels, espe-

TREATMENT OF INJECTED PARTS. 171

cially for injecting cold-blooded animals, but they
do not as a rule yield such good results as a success-
ful gelatine injection.

Any of the colored gelatine that may remain over
can be preserved (even for a considerable time) until
again wanted, if the precaution is taken, after dis-
connecting from the pressure-bottle and allowing the
fluid in the canula tube to ran back, to place the
bottle, tubes and all, for a few- minutes in boiling
water, and whilst still hot to stopper up the ends of
the tubes with pieces of glass rod. The whole can
then be put away until wanted ; but it is as well to
heat it up now again in boiling water, to destroy any
germs of fungi which may perchance have entered
the bottle.

172 PRACTICAL HISTOLOGY.

CHAPTER IX.

LYMPHATIC AND SEROUS MEMBRANES.

IT is in preparations of the serous membranes that
the structure and arrangement of the lymphatic ves-
sels can be best demonstrated, and it will on this
account be convenient to combine them here under
one head, especially as the method which on the
whole exhibits the structure of the serous mem-
branes best is the only one which shows at all satis-
factorily the structure of the lymphatic vessels and
their relation to the cell-spaces of the connective
tissue.

Preparations 1, 2, and 3. Preparation of the
rabbit's omen turn. — A rabbit having been killed
by bleeding, the abdomen is opened, and the omen-
turn, which is generally to be found crumpled up
close beneath or to the left of the stomach, is raised
with forceps, cut off as close to the line of attachment
to the stomach as possible, and placed in a shallow
dish of salt solution which is at hand to receive it.
Besides this salt solution there should be ready on
the table a little one-half per cent, solution of bichro-
mate of potash in a bottle, some one-half per cent,
solution of nitrate of silver, a wash-bottle of distilled
water, a flat glass dish containing a mixture of spirit
and water (equal parts), two glass plates about four
inches by six, a large soft camel-hair brush, and two
or three clean capsules and watch glasses.

Two small corners are first to be cut off the omentum.
One of these is placed in the bichromate of potash,
put aside, and examined after two or three days; this
is for exhibiting the arrangement of the connective
tissue fibres. The other is first rinsed with distilled

PREPARATION OF THE OMENTUM. 173

water; then placed for one minute in a watchglass
containing a little of the silver solution ; rinsed again
with distilled water, and exposed to the sunlight in
another watchglass containing water. After from a
few minutes to half an hour of exposure, according
to the intensity of the light, it may be removed, and
a portion or the whole of it cautiously mounted by
being floated upon a slide under water. The excess
of water is removed from the .slide, all creases and
folds are carefully got rid of in the same way as with
the frog's mesentery before described, and finally a
drop of glycerine is added and the coverglass super-
posed. This preparation is for the purpose of show-
ing the epithelioid layer which covers each surface of
the membrane. If only a portion were mounted, the
rest may be placed for a few minutes in weak logwood
before mounting; in this way the nuclei of the cells
may be brought to view.

But while this second corner was being exposed
to the light the preparation of the rest of the omen-
turn can be proceeded with.

In the first place, it is floated on to one of the glass
plates and removed from the fluid, and then by draw-
ing gently first at one place and then at another the
creases and folds are gradually removed, and it is in
this way spread out as an exquisitely delicate mem-
brane which may be made to cover the whole upper
surface of the glass plate, and may be extended round
its edges so as to reach the lower surface. As soon
as all folds are in this way removed from the part
which covers the upper surface of the plate the second
glass plate is applied to the under surface of the first,
and the membrane, or at any rate its greater part, is
thus maintained in an extended state. Next, the
surface is gently brushed all over with the camel-hair
pencil moistened with salt solution; this is for the
purpose of removing the epithelioid layer from that
surface, and enabling the silver solution more rapidly
to penetrate. The brushing is not absolutely essen-
tial, for in many parts, especially those in which the

15*

174 PRACTICAL HISTOLOGY.

lymphatics and bloodvessels are most numerous, the
epithelioid layer is deficient, or at least incomplete
and modified.

The salt solution is now thoroughly washed from
the membrane with distilled water, and without
delay a quantity of nitrate of silver solution is
poured on it and allowed to run over every part of
the exposed surface. After five minutes it is again
washed with distilled water, and the glass plates,
with the membrane of course still upon the surface
of the upper one, are placed in the weak spirit con-
tained in the flat glass dish. The dish is then
covered and placed in the sunlight until the silver,
as evidenced by the change in color, is fully reduced.
Small pieces, each about an inch square, are then cut .
with sharp scissors from various parts, floated on to
slides with the browned surface uppermost, and are
exposed to the air for a few minutes to allow most
of the spirit to evaporate, leaving them in water ;
to this a drop of glycerine is added, and finally the
cover-glass is superposed. Or the cover-glass may be
put on first, and then glycerine placed at the edge
and allowed to diffuse in underneath the cover. The
preparation is completed by cementing the covering-
glass in the usual way.

Both before and after the treatment with nitrate
of silver it may have been noticed that the delicate
membrane is studded all over with patches of thicker
tissue, some quite small and insular, others extend-
ing over a considerable area. These patches, which
are characterized by an accumulation of lymphoid
cells and by the small size of the epithelioid cells of
the surface (there being, in fact, in many cases, a
loss of distinction between them and the other cellu-
lar elements of the membrane), are, at least the
larger ones, provided with numerous bloodvessels,
the epithelioid lining of which is often very well
stained by the silver; and these are always accom-
panied by one or more lymphatic vessels, with walls
formed by the characteristic wavy outlined cells.

TENDON OF THE DIAPHRAGM. 175

Preparations 4 and 5, The central tendon
of the diaphragm with its serous coverings. —

The thoracic and ahdominal surfaces of the dia-
phragm present importantdifferences in the arrange-
ment of the numerous lymphatic vessels which are
distributed upon them. To see both properly it will
be necessary to sacrifice two animals. It is best to
use rabbits, since their central tendon is larger in
proportion than that of most otber mammals.

For the preparation of either side both thoracic
and abdominal cavities must be freely opened, the
ventral attachment of the diaphragm being left in-
tact, so that that muscle remains stretched out. A
double ligature is to be put on the inferior vena cava
in the thorax, and the vessel cut between the two
threads; this is to prevent the blood in the vessel
from getting over the membrane. The pericardium
is now cut away from the upper surface of the dia-
phragm, and the suspensory ligament of the liver
from the lower. The surface which it is wished to
silver is then brushed pretty firmly with a camel-
hair pencil wetted with distilled water, after which
a few drops of nitrate of silver solution are allowed
to flow over it, or are applied with the brush. After
five minutes' contact the silver solution is washed
off by a stream of distilled water, and the central
tendon, including also some of the muscular fibres
which converge to it, is carefully removed, pinned
out upon a loaded cork or cake of wax, with the
silvered surface uppermost, and exposed to the sun-
light either in water or weak spirit. When dis-
tinctly browned it is removed from the window,
and pieces from different parts are cut out and
mounted in glycerine.

In addition to these preparations — which exhibit
more especially the lymphatics and cell-spaces of
the serous membrane, and, on the abdominal side,
the lymphatic clefts between the tendon bundles — it
is useful to make another silvered preparation, un-
brushed, of the peritoneal suiface, a third animal

176 PRACTICAL HISTOLOGY. .

being sacrificed for the purpose. This serves to
show the epithelioid layer of the serous membrane,
with the differences in character of its cells in dif-
ferent parts, the cells being much smaller over the
interfascicular lymphatics than elsewhere. Amongst
the smaller cells, moreover, may be seen here and
there the minute darkly-stained angular patches
known as pseudostomata, which are probably merely
accumulations of intercellular or ground substance;
and also, but more rarely, the true holes or stomata
surrounded by a ring of small cells, and leading into
the lymphatic below.

Preparation 6. — Such stornata or orifices leading
from the serous cavities into lymphatic vessels are
met with occasionally in preparations from most of
the serous membranes in mammals. But they are
especially numerous and well seen in the peritoneum
of the frog.

A male frog should be killed for the purpose, and
the intestines and stomach removed so as to expose
the back of the abdomen, but without cutting the
mesentery too near the spinal column. If the trunk
of the animal is now placed in a dish of salt solution,
and the posterior part of the peritoneum carefully
examined under that fluid, it will be found that it
does not closely cover the vertebral column, great
vessels, and other structures which are found at the
back of the abdomen, but is separated from them
by a large lymph-space, divided from the serous
cavity by a membrane. This is covered on the one
side by the epithelioid cells of the peritoneum ; on
the other by those 'of the lymphatic, and to the
unassisted eye appears to form a complete septum.
Under the microscope, however, it is seen to be
studded by very numerous apertures, which can be
seen if the membrane is removed and examined with
a high power in salt solution merely. But to study
their arrangement with reference to the epithelioid
cells, and also to obtain a permanent preparation,
the septum is to be stained with silver. With this

INJECTION OF LYMPHATICS. 177

object the whole, or a portion only, is dissected off
under salt solution (it may be convenient to remove
with it the elongated kidneys which adhere to it
behind and to cut these away only after the staining
is completed) ; it is then rinsed in distilled water to
remove the salt ; placed in silver solution for one
minute; again rinsed, and exposed in water to the
light. After the metal is reduced the preparation
is floated on to a slide, and with the usual precau-
tions to avoid folds and creases, finally mounted in
glycerine. It is desirable, before mounting, to stain
the tissue with logwood in the way recommended
for the unbrushed silvered piece of the rabbit's
omentum, but this is not absolutely necessary, and
much increases the risk of producing folds in the
membrane.

For studying the structure of the larger lymphatic
vessels, as, for example, the thoracic duct, precisely the
same methods, both for teased preparations and for sec-
tions, are employed as were used for the larger blood-
vessels.

INJECTION OF LYMPHATICS.

The minute lymphatics of a part may, where
numerous, generally be readily displayed by simply
sticking a very fine canula into the tissue, and
forcing a colored fluid through this. The best
apparatus for the purpose of obtaining the requisite
pressure is the small mercury apparatus shown in the
accompanying figure (Fig. 30). The mercury con-
tained in the bottle a compresses the air in the pres-
sure-bottle 6, according to the height a is raised
above 6, this height being regulated with the great-
est nicety by the screw d. The bottle c containing
the injection fluid communicates by one tube with
the pressure-bottle, and by another (which passes to
the bottom) with the injection canula f. Gelatine
is not used for injecting the lymphatics, but almost
always injections which are fluid iu the cold. Berlin

178

PRACTICAL HISTOLOGY.

blue solution (2 per cent.) is often employed, but the
best fluid for the purpose is a solution of alkanet in

Fig. 30.

Mercurial-pressure apparatus for injecting lymphatics.

a, reservoir-bottle containing mercury ; b, pressure-bottle into which this tends
to flow; (?, injection bottle containing solution of Berlin blue, connected
with the pressure-bottle by one India-rubber tube, with the canula /by a
second, and with a small manometer by a third; d, handle of screw, by
turning which the stage on which the bottle a rests is raised or depressed,
and the pressure increased or diminished in b ; e, screw clip (opened); g,
spring clip (closed).

turpentine, which readily flows into the lymphatics.
The canula can be made from a piece of glass tube
drawn out to a capillary point, but the best are long
perforated steel needles like those supplied with the
I'nivaz syringes, and as fine as it is possible to pro-

INJECTION OF LYMPHATICS. 179

cure them (Fig. 31). The India-rubber tube con-
nected with the canula is closed by the clip g.

Fig. 31."

Very fine perforated steel needle for injecting the lymphatics of a part.

Preparation 7. — the mode of injecting the lym-
phatics of a tendon may be here described as an
example, especially as the subject was omitted when
studying the minute structure of tendon. One of the
best tendinous structures to choose for the purpose is
the fibrous aponeurosis covering the tendon of the
triceps extensor femoris of the dog. Two sets of lym-
phatics are here met with — one in the substance of
the tendon, consisting for the most part of vessels ar-
ranged conformedly with the direction of the fibres
and connected at intervals by transverse branches,
so as to form elongated and oblong meshes ; and a
superficial one in the areolar sheath which covers
the aponeurosis, consisting of vessels forming a close
plexus with polygonal rneshes. The latter plexus
should first be attempted. Both tube and canula
being completely filled with the injecting fluid to
the exclusion of air-bubbles, the clip g is closed, and
the canula is inserted obliquely for half an inch or
more into the areolar sheath, care being taken not
to mess the surface of the tissue. This will proba-
bly be effected after one or two trials, provided the
canula is sufficiently fine and sharp. The clip is
then removed, and by turning the handle d, and
thus raising the bottle a, the pressure is put on to
about an inch of mercury, as indicated by the gauge
attached to the injection-bottle. If the insertion of
the canula have been fortunate, the blue or red
fluid will almost immediately begin to pass into the
lymphatic plexus, but should there be no result the
pressure may be gradually raised to about two inches ;
higher than this it is not as a rule advantageous to

180 PRACTICAL HISTOLOGY.

go. If there is still no result the canula may be
pushed a little further in the sheath, and perhaps
moved a little to one side or the other in the hope of
thus rupturing a lymphatic and gaining an entrance
into the plexus. Should these and other devices
which experience may suggest still fail, the clip must
be replaced and another insertion tried elsewhere.
It very frequently happens that the injection which
escapes from the end of the canula, instead of pass-
ing into the lymphatics, forms merely a bulla of ex-
travasated fluid in the interstices of the tissue. This
can sometimes, by passing the handle of a scalpel
over it with moderately firm pressure, be induced
to find its way into the absorbent vessels, but if not
the canula must be withdrawn and re-inserted as
before.

For the lymphatics in the fibrous substance of the
aponeurosis the canula must be inserted obliquely
into the tendinous tissue, and the injection forced
in with the same precautions. The pressure may, if
necessary, be raised somewhat higher, for, owing to
the firmness of the tissue, there is less liability to
the occurrence of extravasations.

For displaying these injected preparations they
may, if injected with Berlin blue, be first placed in
spirit to remove all water and precipitate the color-
ing matter in the vessels (the process being completed
by putting the injected part into absolute alcohol),
after which the preparation may be placed in turpen-
tine and mounted in dammar varnish. Another
method, and one which succeeds very well, especially
with the alkanet injection, is to stretch the injected
aponeurosis over a ring of cork and allow it slowly
to dry by exposure to the air. When completely
dry the injected part may be at once mounted in
dammar or in glycerine. By this mode of proceed-
ing the injection is, as pointed out by Bowditch,
rendered more complete, for the fluid which may
have been extravasated in the interstices of the tissue
is apt to become drawn into the lymphatic vessels

INJECTION OF LYMPHATICS. 181

to supply the place of the watery fluid which becomes
lost by evaporation.

Although in many cases it is better to use an ap-
paratus of the kind above described, which enables
the pressure which is being employed to be exactly
estimated, yet it may be stated that with a little
experience the lymphatic vessels, especially those in
the firmer tissues and organs, may often be injected
with great success by using simply an ordinary
Pravaz subcutaneous syringe provided with a line
canula, driving the injecting fluid into the tissue
merely by the pressure of the finger. It is true that
extravasations are very apt to be produced opposite
the point of the canula, but these can often be util-
ized in the manner before mentioned by gently
pressing on them and endeavoring to induce the in-
jection to pass into the lymphatic vessels.

In rare cases a vein is pierced by the canula, and
the system of blood capillaries of the part is then
apt to become filled, but both the vessels themselves,
and the meshes they form, are much smaller than
the lymphatic capillaries, and a knowledge of their
general appearance and mode of arrangement in the
particular tissue will prevent any error from arising
in this way.

Preparation 8. — The lymphatics of the dia-
phragm may be injected with Berlin blue during
life. A young rabbit is chosen, and enough chloral
hydrate is injected under its skin to anaesthetize it
completely (about 8 minims of a 50 per cent, solution
will suflice). The skin of the belly is then cut
through for a couple of inches close to the ensiform
cartilage, and the edges are held aside by an assistant,
and the muscular wall having been pinched up a.
blunt-pointed canula is passed obliquely into the
cavity of the peritoneum at its upper part, due care
being taken to avoid the liver and stomach. About
five cubic centimetres of a saturated solution of
Berlin blue, previously warmed, are now injected
through the canula, which is then withdrawn and
16

182 PRACTICAL HISTOLOGY.

the animal put aside in a warm place. After four
hours, during the whole of which time it remains
under the influence of the chloral, it is killed by
bleeding. The abdomen is then opened, and the
viscera having been drawn aside, the under surface
of the diaphragm is exposed, and the blue which
covers it is washed off by a stream of water. If the
experiment has been successful, it will be found that
the whole network of lymphatics of the central ten-
don is filled with the blue fluid ; for this, assisted
by the constant respiratory movements of the dia-
phragm, has passed from the peritoneal cavity
directly through the open stomata into the lymphatic
vessels. The tendon may be cut out and placed in
alcohol, and eventually, after passing through tur-
pentine, mounted whole in Canada balsam, between
two glass plates, and used for examination with a
low power of the microscope.

THE SYNOVIAL MEMBRANES.

These structures, which are to be regarded as free
surfaces of the ordinary areolar tissue, and present
no essential differences in structural appearance from
this, may be prepared for microscopical examination
by the same methods. The preparations which are
of greatest value are those stained with nitrate of
silver.

Preparations 9, 10, 11. — Since, as is always the
case with the silver method, parts should be as fresh
as possible, and since, moreover, it is convenient to
have large joints to work with, a neat's foot should
be procured from the butcher's unless a freshly
amputated limb is available. In the neat's foot all
three kinds of sy no vial membranes may be found
and prepared. The mode of silvering the synovial
bursee is quite simple, and need not here be detailed ;
the preparation of the vaginal synovial membranes
was described under Connective Tissue (p. 65), this
being taken as typical of the structure of that tissue ;

SYNOVIAL MEMBRANES. 183

and the preparation of the synovial surfaces of the
joints was given under Articular Cartilage (p. 93).
But in the last-mentioned place nothing was said
as to the mode of demonstrating the synovial mem-
brane proper, for we had there to do only with the
cells and cell spaces of the cartilage, and the transi-
tions met with to those of the connective tissue of
the membrane. The appearance presented by the
membrane itself is shown in surface sections made
in a similar way but from the inner surface of the
capsule of the joint. It will be seen that in the ox
the cells, or rather in the silvered preparations the
white cell-spaces, form a close irregular network by
the union of their processes ; in fact, so completely
have the cells become extended into branches that
it is difficult in many cases to make out where the
body of the cell has been. In the human synovial
membranes this is not the case ; in fact, the appear-
ances are quite characteristic of ordinary areolar
tissue. The arrangement of the corpuscles into epi-
thelioid patches is not frequent, but there is no con-
tinuous epithelioid covering, as in the serous mem-
branes. Moreover, the lymphatics, which were so
numerous in those, are not to be seen in the synovial
membranes, although capillaries are present and in
many places approach close to the surface.

Preparation 12. — But to study the characteristic
arrangement of the bloodvessels of the synovial
membranes and to show the drcuhis articuli vascu-
losus, a preparation must be made from one of the
joints of a limb that has been fully injected. Sur-
face sections are then made of the transitional region
where the synovial membrane terminates on the
cartilage, and including also a part of the membrane.
They are mounted in the usual way in dammar
varnish, but without being stained.

Preparation 13. — Fnally the Haversian fringes,
with their secondary processes, may be prepared.
They may be examined fresh in salt solution, and
may also be obtained from the joint which was

184 PRACTICAL HISTOLOGY.

stained with nitrate of silver. To find them it is
best to immerse the joint in fluid, for by this means
they are floated up, and may then be snipped oif
and mounted.

THE LYMPHATIC GLANDS.

Preparation 14, — These are chiefly studied by
means of sections. They are hardened in strong
spirit, into which they are put immediately after
removal from the animal ; in two or three days they
are sufficiently firm to cut, but improve if left longer.
The lymphatic glands of the dog may be recom-
mended for demonstrating the structure of these
organs, especially for showing the lymph-sinuses
and the cortical nodules. The gland is split after
hardening into two equal parts by a longitudinal
cut through the hilus, and one of these halves is
embedded in wax-mass in the usual way (pp. 143,
144), and with the artificial surface near one end of
the wax cake. The sections, which must be very
thin indeed, and should include both cortical and
medullary substance, are transferred from the spirit
to water, and thence to a slide, and mounted in
glycerine, without staining. If the lymph-paths
appear filled up with lymph corpuscles, so that the
retiform tissue which traverses them is not well seen,
but the whole section appears more or less uniform
in structure, these corpuscles may often be in great
measure removed by vigorously shaking up the
sections with water in a test tube, or by gently
brushing them under spirit with a soft camel-hair
pencil. Unfortunately both these methods tend to
break up the sections, and indeed it is not necessary
to employ them, if the sections are made sufficiently
thin.

These organs are amongst the most difficult to
demonstrate the structure of satisfactorily, so that it
may be well to defer their preparation until some
practice has been obtained in making sections of
other parts.

THE SKIN. 185

CHAPTER X.

THE SKIN, HAIRS, AND NAILS.

Preparations 1-6. Sections of the skin.—

Portions should be selected for examination from
different parts of the body ; the palms of the fingers
or toes, the scalp, and a piece from some part of the
general surface ; e. g. the extensor surface of the fore-
arm. The skin of the scrotum may also be prepared,
to show the bundles of plain muscular tissue in the
subcutaneous tissue or dartos, and a small piece of
the ala of the nose, for the sake of the well-marked
sebaceous glands, which open into the follicles of the
minute hairs found in this situation.

The following method of hardening the tissue can
be recommended : A small piece only is removed,
being obtained with as little of the subcutaneous
tissue as possible adhering to it. At the same time,
if it is desired to examine the larger sweat-glands,
this tissue must not be removed too freely, since
those bodies extend down into it. It will be found
that the fresh skin has a tendency to curl in at the
edges ; this should be prevented by pinning the piece
out on a piece of cork. The latter is then inverted
into a beaker containing a mixture of spirit and
chromic acid (equal parts of spirit and of half per
cent, solution of chromic acid). Here, while the
chromic acid hardens the tissue pretty uniformly,
the spirit tends to prevent the epidermis from break-
ing away, as it is apt to do when placed in chromic
acid alone. (Simple hardening in spirit also answers
in many cases fairly well.) After having been a
fortnight in this mixture, the piece of skin is trans-
ferred to strong spirit, and after two or three days

186 PRACTICAL HISTOLOGY-

in this is ready for embedding and making sections.
The piece from the palm of the finger is to be cut in
two directions, viz. (1) across and (2) parallel with
the ridges formed by the papillae. It will be easier
to cut a thin section if the razor is made to travel
from the corium towards the epidermis, rather than
in the opposite direction. The pieces of skin which
contain hairs should be so embedded as to carry the
plane of the vertical section in the direction of in-
clination of the hairs, so as to gain a view of the
hair follicles along their whole extent, and to show
the erectores pili if possible.

The sections are in each case to be placed in log-
wood solution, and when sufficiently stained to be
washed in water and transferred successively to
spirit, oil of cloves, and dammar in the usual way.

The preparations may be much improved, so far
as the exhibition of the epidermis is concerned, by
being placed in a saturated solution of picric acid
for about half an hour before being stained with log-
wood. In this way the horny parts acquire a bright
yellow color, which contrasts strongly with the violet
staining of the Malpighian layer. Owing to the num-
ber of nuclei which become stained in it, this part
of the epidermis is much darker than the papillary
part of the corium which is in contact with it. In
the papillae of the skin of the finger the tactile cor-
puscles may be sought for. They are generally
situated quite near the apex of the papilla, and to
see them well it is important to cut the papillae
exactly vertically, so as to include their length. In
sections which have been somewhat obliquely, and
in sections cut parallel instead of vertical to the sur-
face, the transversely or obliquely cut papillae appear
as round or oval islands in the midst of the deeper
cells of the epidermis. The dentated appearance
presented by most of the cells of the Malpighian
layer can readily be seen with a high power immer-
sion objective,

Of the sections made from the skin of the finger

THE SKIN. 187

one or two of the thinnest should be mounted in
glycerine as soon as cut, without staining, merely
placing them first in water, to remove the spirit.
The fibrous-looking tactile corpuscles can generally
be made out better in these than in the preparations
which have been mounted in dammar.

Preparation 7. — To show the arrangement of the
bloodvessels, sections of skin from a limb which has
been minutely injected may be. made and mounted
in dammar by the usual process. These must either
be left entirely unstained or the staining must be
very slight indeed. These sections will generally
include clusters of fat-cells, with their vessels.

Preparation 8. — The following method will serve for
the demonstration of some points in the structure of the
corium, as well as the arrangement of the bloodvessels.1
One of the limbs of an animal, preferably of a dog, is in-
jected with a solution of Berlin blue, the injecting canula
being- placed in the principal artery of the leg, and a band
being tightened firmly over the upper part of the limb
after the injection has been flowing for a minute or two,
so as to compress everything except that vessel and pre-
vent the further escape of the injection from the veins of
the limb. A pressure of from four to eight inches of mer-
cury is then maintained for several hours. In this way
all the vessels become completely distended with the blue
fluid, the watery part of which in large measure exudes,
so as to render all the tissues cedematous. If colored
gelatine is employed the limb is all the while kept
thoroughly warm by placing it over a large water-bath
and covering it with a glass plate or bell-jar; and the
gelatine in the injecting-bottle and supply-tube must also
be kept fluid by similar means. After the time mentioned
the artery may be tied and the limb, if gelatine has been
employed, removed to a cool place. When it is considered
that the gelatine is entirely set, a piece of the skin is cut
out, stretched over a dialyzing glass and placed in a
beaker of digestive fluid, which has been previously pre-
pared by adding a few drops of a glycerine extract of the

1 W. Stirling in "Journal of Anatomy and Physiology," vol.
x. pp. 185 and 465.

188 PRACTICAL HISTOLOGY.

gastric mucous membrane to five hundred c. c. of a 0.2 per
cent, solution of hydrochloric acid. It is then maintained
for five or six hours at a temperature of 38° C., when the
piece of skin is removed and placed in water for twenty-
four hours. Sections may then be cut in any desired
direction, stained with logwood, and mounted in glyce-
rine ; and although not obtainable very thin, yet, owing
to their clearness and transparency, the arrangement of
the bloodvessels, and of the little muscles attached to the
hairs can be traced with comparative facility.

Preparation 9. Hairs. — To examine a hair, all
that is necessary is to place it on a slide in a drop
of water, cover with a thin glass, and examine with
a moderately high magnifying power. By careful
focussing the cuticular scales can often be made out
on the surface and at the edges of the hair, especially
on the small hairs of the general surface of the body.
The medulla is often absent in hair of the head, but
may generally be found in those of the beard and
whiskers. Many of the black particles which are
seen in a hair by reflected light, and especially in
the medulla, are merely small globules of air in the
interstices of the tissue. That this is so may be
proved by cutting off the light which comes from
the mirror of the microscope and viewing the object
by reflected light, only a moderate power being used.
The black particles, if really due to the presence of
air, will then appear silvery white, just as in the
parallel case of the air which fills the lacunas in a
section of hard bone.

It will be useful to compare the appearances pre-
sented by human hair with those exhibited by the
hairs of some of the common domestic animals.
These are many of them characterized by the regular
arrangement of the medulla (this is nearly always
present in the hairs of quadrupeds), which forms
different patterns in different kinds of animals, so
that the species to which the hair belong may often
be determined.

HAIRS — NAILS. 189

Preparation 10. — The fibrous part of a hair can
be broken up into its constituent fibres and cells, if
it be first steeped for a time in strong sulphuric
acid.

Preparation 11. — The relative proportion of the
three constituent parts of a hair to one another is
best shown in transverse sections.

To obtain sectio'ns of hairs, the simplest plan is to
tie a number together and dip the bunch into a
strong gum, and when this has thoroughly soaked
in amongst the hairs to remove the bunch and either
let the gum dry and harden by exposure to the air
or plunge it into spirit containing a little water, by
which in a few hours the whole mass is rendered
hard. Sections are then made with a sharp scalpel,
and are placed on a slide in water and covered. The
water dissolves the gum away, and all that is now
necessary in order to make the preparation complete
is to. allow a drop of glycerine to diffuse under the
cover-glass, which may then be secured as usual.

The hair-follicles and roots of the hairs are seen
in the sections of skin.

Preparation 12. — The nails are studied by means
of vertical sections made both longitudinally and
transversely. The finger (or toe) should if possible
be previously injected, and the nail with the matrix
and surrounding skin, having been removed, may
be hardened either in spirit or by the spirit and
chromic mixture. When ready for cutting, the
piece should first be bisected longitudinally ; and
one of the halves having been embedded in a wax
and oil mixture, with rather an excess of wax, so as
to render it harder than usual, one or two longitu-
dinal sections (which need not be very thin) a'long
the whole length, to show the general relation of the
nail to its matrix and to the epidermis, are to be
made with a sharp strong scalpel. These need not
be stained ; they may be mounted either in glycerine
or, if injected, in dammar; they are intended only
for examination with a low power. The other piece

190 PRACTICAL HISTOLOGY.

may also be embedded in the same mixture, but in
such a way that the laminae, which in the matrix
represent the papillae of the skin, are cut transversely.
The sections must be as thin as possible, and stained
either with logwood alone or with picric acid and
logwood in the same way as was recommended for
the sections of skin, before being mounted in dam-
mar.

But, owing to the substance of the nail being so
much harder than the subjacent matrix, it is very
difficult to get both parts equally thin. They can,
however, he got of much the same degree of hard-
ness by means of the gum method. The piece to be
cut, which should be quite small, is placed in syrupy
solution of gum and left over-night ; it is then
transferred to a mixture of spirit with one-sixth of
its volume of water. After a few hours the gum
which has penetrated into the substance of the tissue
will be hardened throughout, and the mass can be
embedded and cut in the desired direction, the knife
being wetted with some of the same spirit- mixture.
Strong spirit should not be used, since the gum is
entirely dehydrated by this, and becomes so hard as
to turn the edge of the knife. Instead of embed-
ding the piece in wax-mass it will be found a cleaner
and more convenient method to make a slit in a
winebottle cork, insert the hardened guru-impreg-
nated tissue in the proper position in the slit, and
maintain it in place whilst cutting the sections
by the pressure of the finger and thumb. The sec-
tions are transferred from the spirit to water, which
dissolves out the gum ; when quite free from this
they are stained and mounted as before.

It will be well, before commencing the descrip-
tion of the mode of preparing the several viscera
for microscopical examination, to revert to one or
two preparations which were purposely deferred

LYMPH SPACES. 191

until the mode of preparing sections and of injecting
the bloodvessels had been explained.

Injected muscular tissue is obtained from any
injected limb, and should be hardened in strong
alcohol. If the piece is not large enough to hold
in the fingers, it must be embedded, and moderately
thin sections made both longitudinally and trans-
versely, and mounted, unstained, in dammar.

Transverse sections of a nerve-trunk may be
made from any large nerve that has been hardened
in picric acid (forty-eight hours), and subsequently
in alcohol. The sections are to be stained with
picro-carmine (twenty-four hours), and may be
mounted in glycerine. By this method the medul-
lary sheath of the nerves and the elastic tissue of
the perineurium and epineurium are stained yellow,
the connective tissue lamellae and bundles pale red,
and the corpuscles and nuclei and the axis cylinders
a darker red.

Longitudinal sections of a nerve-trunk from an
injected limb may be prepared in the same way as
those of injected muscle.

The lymph spaces which lie between the lamel-
lae of the perineurium of the nerve, and extend also
amongst the fibres within the funiculi, can very
readily be injected with 2 per cent, solution of
Berlin blue by merely sticking a very fine injecting
canula into a funiculus, and employing moderate
pressure. The injection runs along the course of
the nerve almost as freely as if it were an open tube.
A piece of nerve which has been injected in this
way is to be cut out, hardened in spirit, and trans-
verse sections prepared and mounted in dammar,
without staining, to show the course taken by the
injection. At the entrance of the nerve-roots into
the spinal canal the perineural clefts communicate
with the sub-arachnoid space, so that the nerves can
be injected by merely forcing the injecting fluid into
this.

192 PRACTICAL HISTOLOGY.

Longitudinal sections of ganglia, both spinal
and sympathetic, may be prepared from specimens
that have been hardened either in picric acid and
spirit or in 2 per cent, bichromate of ammonia
solution for five days, then in weak spirit (half
water) for twenty-four hours, and then in strong
spirit. The sections are to be stained in logwood,
and mounted in dammar.

The sections of developing bone (p. 108) (instruc-
tions for embedding which by the cacao-butter pro-
cess, were previously given) may also be now pre-
pared.

THE HEART. 193

CHAPTER XI.

THE HEART.

Preparation 1. The cardiac pericardium.—

The pericardium which covers the surface of the
heart is prepared by the same methods as other serous
membranes. Of these the only one which need here
be described is that by nitrate of silver. This is as
follows : In an animal which has just been killed the
thorax is freely opened, and the pericardium having
been torn open, the base of the heart is secured by a
tape ligature, the great vessels being then cut
beyond the ligature, and the organ removed without
allowing its surface to be smeared with blood. A
part of the surface is now brushed firmly with a soft
camel-hair brush moistened with distilled water,
with the object of removing the superficial layer of
epithelioid cells. The heart is next dipped for a
moment in distilled water, and then nitrate of silver
solution is poured over the whole surface, and al-
lowed to be on it for three minutes, after which the
organ is rinsed again in distilled water, and finally
placed in spirit, in the sunlight. AVhen sufficiently
browned it is removed from the window, and left
for some hours until the surface is hardened by the
alcohol. Surface sections are then made of both un-
brushed and brushed parts, and after soaking in
water for a minute or two are mounted separately
in glycerine. The unbrushed specimens will show
the epithelioid layer ; the brushed ones should
exhibit the subjacent connective tissue, with its
cell-spaces, lymphatics, bloodvessels, and nerves.

Preparation 2.— The muscular substance of
the heart is studied in teased preparations and in
17

194 PRACTICAL HISTOLOGY.

sections. For the teased preparations the heart of a
young animal should be chosen, since in these the
fibres separate more readily into their constituent
cells. A very small shred is placed in a compara-
tively large quantity of TV per cent, osmic acid for
ten days or a fortnight ; it is then broken up in
water as minutely as possible, and the preparation
covered and scanned with a high power. Numerous
little fragments of varying shapes will be found
scattered over the preparation. On careful exami-
nation it will be apparent that each possesses a
nucleus, which can be made more conspicuous by
allowing a little dilute logwood solution to run
underneath the cover-glass. These- little fragments
of the cardiac muscular tissue, which have the char-
acteristic indistinct striation of that substance, are
the cells which by their union end to end form the
fibres.

Preparation 3. — To show the arrangement of the
fibres, and the interstitial tissue and vessels, a piece
of the muscular substance is to be placed in strong
spirit. In two or three days it will be firm enough
to cut. Sections are to be made both parallel with
and across the direction of the fibres ; they are to be
stained with logwood and mounted in dammar.

Preparation 4. The endocardium. — To dis-
play the endocardium the silver method again comes
into requisition. That part of the lining membrane
covering the septal wall of the right ventricle is the
best to prepare, on account of its relative smoothness.
The right ventricle is opened in a fresh heart, and
the outer wall removed entirely, and then a large
piece of the smoothest part of the exposed surface of
the septum is sliced oft' with a razor. A part only
of the endocardium of the detached piece is brushed,
as in the case of the pericardium, and the whole is
then washed and treated with silver solution.
After three minutes it is put into spirit as before,
and when browned and hardened surface sections
are cut and mounted in glycerine.

THE LYMPHATICS OF THE HEART. 195

Preparation 5, — In addition to these silvered
preparations, the endocardium should be examined
in the fresh state. This is done by dissecting off a
piece of the membrane in salt solution and examin-
ing it both with and without the addition of acetic
acid. Other portions may be teased out with a view
to the demonstration of the elastic muscular tissue.
The methods for making and preserving these pre-
parations are the same as were employed for showing
the structure of the coats of the bloodvessels, to the
description of which the student is referred (p. 140).
It may be noted that in some animals — the sheep,
for instance — the peculiar, large, cubical or oblong
cells which form, in series, the fibres of Purkinje will
be found in the endocardium. They are about the
size of fat- vesicles, which are also found in the endo-
cardium in this animal, but the two could hardly be
confounded, for the cells forming Purkinje's fibres
have a clear or slightly granular nucleated central
portion which does not strongly refract the light, and
a striated circumference, which is apparently con-
tinued into that of the neighboring cells; whereas
the fat-cells, although they may also occur in rows,
and may be of much the same size as the cells in
question, present, by virtue of their strong refracting
power on light, a totally distinct appearance.

Preparation 6. Lymphatic system of the
heart. — If the fine canula of a Pravaz syringe filled
with Berlin blue solution is stuck into the muscular
substance of the fresh heart at any part, and the fluid
is forced out at the point, the injection will pass
freely into the lymphatic interstices between the mus-
cular fibres, and if the tube is inserted near the outer
or inner surface, will find its way into the lymphatics
of the pericardium or endocardium, which can in
this way be readily displayed.

196 PRACTICAL HISTOLOGY.

CHAPTER XII.

THE LUNGS.

Preparations 1-2. The pulmonary pleura.—

The serous membrane which covers the surface of
the lungs as well as that which lines the wall of the
thorax is prepared hy the silver process. For the
pulmonary pleura, the lungs of a small animal that
has just been killed are to be removed entire, and
moderately distended with air through the windpipe,
the bronchi being then tied and the two lungs sepa-
rated. One is rinsed for a moment in distilled water,
and a little nitrate of silver solution is allowed to
flow over the surface; after the lapse of a minute
this is washed off again with distilled water, and
the organ is then immersed in a beaker of spirit and
exposed to the light.

The surface of the other lung is to be firmly
brushed with a wet camel-hair pencil, to remove the
epithelioid cells of the surface before treating it with
silver solution. This may be suffered to remain a
minute or two longer in contact with it than with
the other lung; in other respects the treatment is
similar. Both preparations are left in the light until
they appear sufficiently stained, after which they are
to be placed on one side in the spirit for twenty-four
hours. They will then be sufficiently hard to render
it possible to shave off a thin slice from the surface.
The sections so made are to be placed in water, and
subsequently mounted in glycerine, with the outer
surface uppermost.

Preparation 3. The costal pleura is to be pre-
pared in situ after the removal of the lungs and heart.
That of one side may be brushed, the other not; on

THE LUNGS. 197

the latter the silver solution is, as before, to be
allowed to remain a shorter time than on the brushed
part, where the fluid has to penetrate into the
lymphatic vessels, and into the substance of the tis-
sue. The whole thorax may then be cut off from
the rest of the trunk and exposed under water to the
light ; or, if it be too large to do this conveniently,
a piece only of the thoracic parietes on each side is
to be removed and pinned out on to a loaded cork,
which is then placed in a dish of water in the sun-
light. When stained pieces of the membrane must
be carefully dissected off, without pulling upon or
injuring the tissue in any way, floated upon a slide,
the excess of water poured off or soaked up with
blotting-paper, all creases removed from the mem-
brane, and finally the cover-glass superadded, with
a drop of glycerine.

Preparation 4. The lung tissue. — The struc-
ture of the lungs themselves is best shown by means
of sections. The tissue is hardened in the following
way:—

The organs having been removed from the chest
of a recently killed rabbit or cat, care being taken
not to scratch their surface with the broken ends of
the ribs, a glass canula is tied into the end of the
trachea (or into either bronchus). The canula is
then connected by an India-rubber tube with an in-
jection bottle, which is filled with a weak solution
of chromic acid (£ per cent.). By blowing air into
the tube this solution is made to flow into the lungs
so as to distend them moderately. The trachea or
bronchus is now tied up, the canula removed, and
the lungs are immersed in a large quantity of a solu-
tion of chromic acid of similar strength. After two
days the fluid is changed, \ per cent, solution being
substituted, and the organs are cut into pieces, to
enable the fresh fluid more readily to penetrate.
After a week more in this the pieces are placed first
for twenty-four hours in weak spirit and then in
strong spirit.

17*

198 PRACTICAL HISTOLOGY.

The bits, though small, will probably be large
enough to hold in the hand and cut without embed-
ding. Sections may be made both across and along
the course of the bronchial tubes, stained with log-
wood (it will be found that the sections must be left
for a considerable time in logwood, for they stain
with difficulty), and, after going through the usual
processes, mounted in dammer.

Preparation 5. — But, owing to its spongy nature,
it will be found almost impossible to cut very thin
sections unless the interstices of the tissue are filled
with some firm material, and the following directions
may accordingly be observed, if thinner sections than
can be obtained in the ordinary way are desired. A
small piece only (not larger than a kidney-bean) of
the lung hardened as above described is placed for
two days in alcoholic solution of logwrood Kleinen-
berg's.1 The piece of .tissue will have been stained
throughout of an intense dark violet color, and will
look almost black. On removal from the staining
fluid it is transferred through alcohol to oil of cloves.
After an hour in this, by which time the oil will
have had time to penetrate its whole thickness, it is
put into melted cacao-butter, which is kept in the
fluid condition by a temperature of not more than
42° C., and allowed to lie in this for four hours.
An oblong cake of the cacao-butter having been pre-
viously made by pouring some of the melted fat into
a paper mould, a little pit is scooped in it near one
end, and the piece of tissue, now soaked through

1 Kleinenberg's solution is made in the following way (Foster
and Balfour) : —

(1) Make a saturated solution of crystallized calcium chloride
in 70 per cent, alcohol, and add alum to saturation. (2) Make
also a saturated solution of alum in 70 per cent, alcohol. Add
(1) to (2) in the proportion of 1 : 8. To the mixture add a few
drops of a saturated solution of haimatoxylin in absolute alcohol.

This solution may be used in very many csiscs for staining sec-
tions, in place of the ordinary watery solution of logwood alum.
Jt may, if required, be diluted with the mixture of 1 and 2. The
stained sections are placed at once in strong spirit.

THE LUNGS. 199

and through with the cacao-butter, is placed in the
pit in a position convenient for making sections, and
the pit is then filled with the melted cacao-butter.
This as it hardens adheres firmly to and sets into
one piece with the cake. When quite hard — a pro-
cess which takes a considerable time — sections of
the tissue are made with a razor wetted with spirit.
Since all the cavities are filled by and the tissue is
thoroughly impregnated with the cacao-butter, the
whole cuts like a homogeneous piece of this substance,
and sections can be made as thin as desired. As they
are cut they are placed in oil of cloves, which removes
the cacao-butter in a few minutes, even in the cold ;
but in the winter season it is better to accelerate the
solution by slight warmth. The sections can then
be mounted in dammar.

These stained sections of lung may be first ex-
amined with a moderate power, but afterwards a
power of 400 or 500 diameters should be employed,
in order to see the details of structure ; the ciliated
epithelium, muscular layer, and cartilaginous plates
of the bronchial tubes, with the mucous glands,
nerves, lymphatics (seen in section as mere clefts),
and patches of lymphoid tissue in their walls ; the
branches of the pulmonary artery accompanying
them ; the mode in which the terminal air-tubes
dilate into the infundibula ; the air-cells or alveoli,
almost covered with a network of capillaries which
are seen also on the septa between the alveoli, pro-
jecting fii;st into one and then into the other of two
neighboring air-cells. Where they run vertically the
capillaries appear in optical section as circular spots,
looking not unlike nucleated cells. But the exces-
sively delicate epithelium of the air-cells cannot be
well seen in these preparations, for the epithelium
cells remain almost unstained, and it is not easy to
differentiate their nuclei from those of the closely
subjacent capillaries.

Preparation 6. Epithelium lining the air-
cells. — In order to demonstrate the epithelium cells

200 PRACTICAL HISTOLOGY.

I

we make use of the nitrate of silver, but the mode
of proceeding is somewhat different from that ordi-
narily employed. A gelatine mixture is made by
taking ten grammes of gelatine, and, after soaking
it in cold distilled water, melting it, and adding if
necessary more warm distilled water until the mix-
ture measures 100 c.c. A decigramme of nitrate of
silver is dissolved in a little distilled water and
added to the gelatine, and the mixture is transferred
to a glass syringe, which is kept warm over a water-
bath. An animal — preferably a young one — having
been killed, the lungs are removed, a glass canula
with a small piece of India-rubber tube attached is
tied into the trachea, and then the point of the
syringe is slipped into the open end of the India-
rubber tube, and enough of the gelatine mixture
injected into the lungs to distend them pretty com-
pletely. The trachea is now tied and the canula
removed from it. The lungs are then put aside into
a cold place until the gelatine within them has fully
set, when sections, which should be as thin as possi-
ble, are made with a razor, either not wetted at all
or with distilled water only. The sections so ob-
tained are placed on a slide in glycerine, covered,
and exposed to the light. As soon as they seem
sufficiently stained they may be examined with as
high a power as possible, for the purpose of making
out the silver lines between the epithelium cells.

Preparation 7. Bloodvessels of the lungs. —
Lastly, the pulmonary vessels are to be injected and
sections made of the injected lung. The red gela-
tine injection may be used ; this and everything else
is to be got ready just in the same way as for the
injection of the aortic system, but a syringe filled
with melted cacao-butter must be connected with
the trachea in the same way as for the gelatine mix-
ture in the last paragraph ; and moreover the arte-
rial canula is of course to be passed through tlie
right ventricle and tied into the pulmonary artery,
instead of into the aorta. Immediately the gelatine

LARYNX AND TRACHEA. 201

injection has been set flowing through the pulmo-
nary vessels the lungs are pretty fully distended
with the cacao-butter; the pressure is then raised
in the injecting apparatus to about four inches of
mercury. The left ventricle is first slit, to let the
blood out of the pulmonary system, and then
clamped, to prevent the escape of the injecting fluid,
which is allowed to pass in for a few minutes until
it is thought that the vessels must all be completely
filled ; this can be partly made out from the color
which the lungs assume. The trachea and the base
of the heart are then ligatured, and the whole is left
for some time to cool, so that both gelatine and
cacao-butter are fully set. The lungs are then cut
out and placed in weak spirit ; after a day they are
transferred to strong spirit, and in another clay or
two sections may be made (mostly vertically to the
surface of the lung), placed in warm oil of cloves, to
dissolve out the cacao-butter, and then mounted in
dammar. If it is desired to stain the tissue some-
what, so as to show the general structure of the lung
as well as the arrangement of the bloodvessels in
the same preparation, this can be done by placing
the sections, after the cacao-butter has been dissolved
out of them, first in absolute alcohol, to get rid of
the oil of cloves, and then for a few minutes in
Kleinenberg's logwood until they are sufticiently
colored. The transference through absolute alcohol
and oil of cloves into dammar is then proceeded
with, as in the case of other sections.

Preparation 8. Larynx and trachea.— The
trachea and larynx are hardened in £ per cent,
chromic acid (ten days), the hardening being com-
pleted by spirit, and the sections, which may be
longitudinal of the cartilaginous part and transverse
of the posterior membranous part, are to be stained
with logwood (they will require a considerable time),
and mounted in dammar. It will, of course, be
necessary to embed the tissue, and it will be found
advantageous in cutting to pass from the mucous

202 PRACTICAL HISTOLOGY.

membrane outwards, instead of vice versd. But,
owing to the great difference in hardness between
the cartilaginous rings and the rest of the tissue, it
is difficult to get a complete section equally thin
throughout.

Preparations 9-10. Bloodvessels and lymph-
atics of trachea. — In addition to these sections flat
preparations showing the bloodvessels, and others
showing the lymphatics of the mucous membrane,
may be made. The former are got from any animal
that has been injected entire, the mucous membrane
being dissected off and mounted in dammar. The
lymphatics are readily filled by the puncture method
by sticking the point of the injecting canula into
the mucous membrane, and forcing a little Berlin
blue or alkanet-turpentine in. It will hardly ever fail
in finding its way into the numerous lymphatics of
the mucous membrane. The injected portion is dis-
sected off and mounted in dammar varnish or in
glycerine.

Teased-out bichromate of potash preparations to
show the separated epithelial cells have already
been made (p. 67).

Preparations 11-13. Ductless glands of the
larynx and trachea. — The thyroid and thy m us
are studied chiefly by means of sections, for facili-
tating the preparation of which the glands are
hardened in alcohol. They should not be put
entire into this, but either cut into pieces or deep
cuts should be made into their substance, so that
the preservative fluid may penetrate rapidly. It is
well to place them at first in weak spirit (half water)
for twenty-four hours, and then to transfer them to
the strongest possible, in which they are allowed to
remain for a few days until hard enough to cut thin
sections from. Like most organs which have been
hardened in alcohol alone, they stain very readily.
The so-called "colloid" which is met with in the
vesicles of the thyroid is colored by the logwood.
Both glands may be advantageously obtained from

LARYNX AND TRACHEA. 203

the body of a newborn cbild, which should if possi-
ble be injected, so that the mode of distribution of
the bloodvessels, especially those of the thynms, may
be displayed.

The concentrated corpuscles jf Hassall which are
met with in the thynms can be seen in sections of
that organ, but may also be studied isolated in pre-
parations of the fresh part teased-out in salt solution.

204 PRACTICAL HISTOLOGY.

CHAPTER XIII.

THE MOUTH AND PHARYNX.

Mucous membrane of the mouth. — Portions of
the lining membrane of the mouth are best prepared
in the way recommended for the skin, viz. by being
pinned out upon a cork and immersed for two or
three days in a mixture of equal parts of spirit and
half per cent, chromic acid solution. Sections of the
cheek or lip may also be readily prepared.

THE TEETH.

Preparation 1. Sections of hard tooth. — No

preparations exhibit the structure of the teeth better
than these. The hard tooth is ground down first on
one side and then on the other, until a thin section
only remains, and this is mounted in hard Canada
balsam in such a way that the air still remains in
the dentinal tubules, the lacunae of the cement, the
interglobular spaces, and other minute cavities that
may be present. The preparation is similar to that
of bone, but presents greater difficulty. Such speci-
mens may advantageously be purchased, for their
preparation involves the expenditure of a large
amount of time and labor; unless the use of a lapi-
dary's wheel can be obtained, when the process is
much facilitated. They should in every case be
studied first with a low power, and afterwards with
a high power objective.

Preparation 2. Sections of softened tooth.
— But, in addition to the facts which the hard spe-
cimens will show, various others may be made out

THE TEETH. 205

in teeth which have been softened by immersion in
an acid.

The acid generally used when the structure of the
(decalcified) dentinal substance only is to be investi-
gated is the hydrochloric. A 10 per cent, solution
may be employed, and the tooth is steeped in this
until entirely soft, after which it may be preserved
in spirit. Sections are to be made in planes both
parallel with and across the direction of the dentinal
tubes.

Preparation 3. Dentinal sheaths. — To show
the sheaths which line the dentinal tubules, a piece
of such softened tooth is transferred to strong hydro-
chloric acid (contained in a watch-glass, which is
covered by another, inverted). In this it may be
left for about an hour, after which time all that
will be found is a tenacious' soft mass occupying its
place. If some of this be removed with a small
pointed piece of wood, placed on a slide, covered, and
examined, it will be found to consist wholly of fine
tubular threads — the dentinal sheaths. These, being
composed of a substance which resists the solvent
action of strong hydrochloric acid longer than the
other animal tissues, remain for a time visible after
the rest of the dentinal substance has disappeared.

Preparation 4. Soft tissues of the teeth.—
But to study the soft tissues — that is to say, the
pulp and odontoblasts with the processes which
these send into the dentinal tubules — we must, as
in the parallel case of bone, employ a reagent which,
whilst softening the hard parts, at the same time
preserves and hardens the soft parts. Of these, picric
acid is the best to employ. The freshly- extracted
tooth is placed in a saturated solution of the acid,
and crystals of the latter are from time to time
added as required ; the solution being stirred as fre-
quently as possible with a glass rod. It is well to
break the tooth first, so as to expose the pulp cavity,
if this can be done without disarranging the con-
tents too much. When softened throughout — this
18

206 PRACTICAL HISTOLOGY.

can be tested by attempting to pass a fine needle
through it — the tooth is placed in spirit, which
should be changed day after day until it ceases to
become much colored by the excess of picric acid.
All that remains to be done is to cut the tooth in
halves vertically, and embedding one of the halves
in a hard wax-mass, cut thin slices from the artificial
section, stain these with logwood, and mount them
in glycerine.

Preparation 5. Study of the teeth "in situ."
— Still more instructive preparations are obtained by
softening a portion of the lower jaw with the teeth
in situ, and making sections through the whole struc-
ture. It is best to take the jaw of a small animal —
a rat, for instance. The flesh having been cleared
away, the softening is effected with picric acid in
the way above described ; 'and then, after due immer-
sion in spirit, the piece is imbedded, cut, stained with
logwood, and mounted in glycerine as before.

Besides showing the teeth and the way in which
they are inserted into the lower jaw, the structure
of this bone is itself well demonstrated. At the
lower part the constantly growing incisor, which
extends in the rat below the molars to the back part
of the jaw, exhibits the large elongated odontoblasts
of a developing tooth, with their well-marked denti-
nal processes (fibres of Lent), which in some parts
project like harp-strings across a small space which
intervenes between the cells and the dentinal sub-
stance. It will be remarked, also, that in these teeth
the most newly-formed layer of dentine becomes,
especially near its junction with the older parts, very
intensely stained by the logwood. This is the case
with all teeth which are still in process of develop-
ment. Carmine has not the same action.

Preparation 6. Development of the teeth. —
For the study of the development of the teeth sec-
tions are made of the jaws of embryos and young
animals. Perhaps the most convenient to choose are
newborn rats, since sections of their jaws exhibit not

THE TONGUE. 207

only the mode of development of the teeth, but also
that of the hair, the bone of the lower jaw (which
ossifies in the connective tissue around Meckel's car-
tilage), the tongue, and many other parts. The pre-
paration is as follows: The foetuses or young animals
are decapitated, and the heads dropped into a large
beaker of one-sixth per cent, chromic acid. After a
week's time, during which the liquid is now and
then stirred, they are transferred to weak spirit, and
in twenty -four hours to strong spirit. After being
in this a day or two they are ready for cutting.
Either the lower jaw is removed and imbedded sepa-
rately, or the whole head is placed in the mould, and
both jaws are cut simultaneously. The sections are
to be stained, some with logwood, some with carmine
solution (made by dissolving two grammes of carmine
in a few drops of ammonia, and diluting with water
to one hundred cubic centimetres). The stay in
chromic acid may not have been long enough to
remove all the earth from the partly developed bones
and teeth, but what still remains is so small in
amount that it will not prevent a thin section being
made. The earlier stages in the development of the
teeth may be perhaps seen in the molar region ; the
later stages comprising the development of the dental
tissues, especially the dentine and enamel, may be
studied in the much more advanced incisors, wrhich,
as just pointed out, extend backwards in these ani-
mals through the greater part of the length of the
jaw.

THE TONGUE.

Preparation 7. — Small portions of this organ
from different parts are hardened in two per cent,
bichromate of potash (fourteen days), and subse-
quently in spirit, and are imbedded, so as to cut
vertically to the surface of the mucous membrane.
The sections are stained with logwood, and mounted
in dammar varnish. A double staining with picric

208 PRACTICAL HISTOLOGY,

acid and logwood may also be employed in the same
way as with the sections of skin (p. 186). The strati-
fied epithelium, the papillae of the mucous membrane,
and the arrangement of the muscular fibres as well
as the mode of termination of the superficial mus-
cular fibres in the connective tissue of the mucous
membrane, may be studied in these sections. Some
of the sections include mucous glands, which differ
in appearance according to their condition at the
time of death. If they had not been recently stimu-
lated by the ingestion of food or otherwise, the cells
of the gland will still be filled with mucus, as shown
by their swollen appearance, and by their becoming
strongly stained by the logwood; whereas, on the
contrary, if they have recently discharged their
secretion, the cells will be granular and almost color-
less, and there will be indications of mucus in the
lumina of the ducts.

Besides the mucous glands others may be seen in
the neighborhood of the papillae vallatas, which
secrete no mucus, and consequently do not present
the above differences of staining. Their ducts, if
the section pass in the direction they take, will be
found to open almost without exception into the
fossae of the circum vail ate papillae, or, at all events,
near those parts in which taste-buds have been
found. The taste-buds themselves may possibly be
seen in the epithelium on the sides of the papillae
vallatae, and also in that which covers the mucous
membrane- near the root of the tongue on each side.
The description of other modes of preparing them
will, however, be deferred until the organs of special
sense are treated of.

Preparation 8. Vessels of the tongue. — Sec-
tions should also be made of an injected tongue.
These will show not only the numerous vascular
loops in the more obvious papillae of the mucous
membrane, corresponding in number with the micro-
scopic secondary papillae, but also the arrangement
of the vessels in the muscular substance of the organ.

THE SALIVARY GLANDS. 209

The injected specimens are much improved by slightly
staining them with logwood.

Preparation 9. Palate and tonsils.— The soft
palate and the tonsils may be hardened in the same
way as the tongue, or, preferable, by immersion for
a week in £ per cent, chromic acid solution, and sub-
sequent placing in spirit. The sections are stained
with logwood and mounted in dammar.

THE SALIVARY GLANDS.

Preparation 10.— These organs are prepared by
placing small pieces of them as soon after death as
possible in a mixture of spirit and J per cent, chromic
acid solution, equal parts of each. After two or
three days they are transferred to spirit, and in a
day or two more will be ready to cut. They may
also be prepared by merely being placed in strong
spirit for three or four days. The embedding, stain-
ing with logwood and mounting, are effected in the
ordinary manner, the chief difficulty being met with
in the fact that, owing to the loose way in which
the lobules are held together by the intermediate
connective tissue, the sections are very apt to become
broken up by the agitation which ensues from the
mixing of the fluids, particularly when they are
transferred from spirit to water or to a watery solu-
tion of logwood. An alcoholic logwood (Kleinen-
berg's) may, however, be used, and in any case it will
be found that the small pieces exhibit all the details
of structure quite as well as larger ones ; thin sec-
tions which have thus become broken up need not
therefore be rejected.

Preparation 11. — The difference in the structure of
the salivary glands previous and subsequent to the state
of secretory activity is best studied in the submaxillary
of the dog, the animal being killed in the one case after
some hours' fasting, in the other a short time after food.
The glands are hardened and sections are prepared in
the manner above described, and the differences in the

18*

210 PRACTICAL HISTOLOGY.

appearance of the aveoli in the charged and discharged
conditions respectively of the glands are noted.

Preparation 12. — Most of the ordinary hardening
solutions (alcohol and chromic acid, for instance) con-
siderably alter the salivary cells, so that in sections of
the glands the cells are scarcely ever seen of their normal
form and appearance. Osmic acid preserves them in a
more natural condition than most reagents, so that, to
study the individual elements, a very small portion may
be placed in a 1 per cent, solution of this reagent, and
after forty-eight hours broken up finely in a drop of
water on a slide. The preparation may be preserved in
glycerine.

THE (ESOPHAGUS AND STOMACH. 211

CIIAPTEE XIY.

THE (ESOPHAGUS AND STOMACH.

Preparation 1. — The oesophagus is best hardened
for the preparation of sections by a mixture of equal
parts of chromic acid (J per cent, solution) and spirit.
After three or four days in this the tissue may as usual
be transferred to spirit. Before putting it into the
mixture it should, if but a small piece be employed,
be pinned out upon a piece of cork, so as to stretch
it slightly and avoid folds. But if a tubular piece
be available this object may be effected more satis-
factorily loy distending the organ with the preserva-
tive solution through a glass canula tied into one
end, the 6ther end having been secured by a ligature
before the distension ; the piece is then immersed in
the mixture for twenty-four hours, after which it
may be cut open. In embedding the gullet and
other membranous parts, of which it is desired to
obtain sections vertical to the surface, it is well to
proceed in a way somewhat different from usual, the
transfixion with a pin being discarded. A layer of
the melted wax-mass is first poured into the mould
so as to fill it nearly half-full ; the piece of gullet to
be embedded is deprived of all superfluous moisture
by placing it on blotting-paper for a few seconds,
and then, as soon as the wax-mass in the mould is
hard enough to support its weight, it is placed in the
desired position near one end of the box, which is
filled up with more wax-mass. This, especially if a
few degrees above its melting-point, adheres firmly
to the portion of the wax-mass which was first
poured in, so that the whole forms a uniform cake,
with the tissue embedded in it.

212 PRACTICAL HISTOLOGY.

Moreover, in embedding a piece of one of the
membranous viscera it is well to place it, as a general
rule, so that the sections shall be exactly transverse
to the axis of the viscus, following, therefore, the
direction of the circular muscular fibres and cutting
the longitudinal across. When the direction of the
section is known, it is easier to understand the ap-
pearances wrhich the various parts present when cut.
Sections cut parallel to the axis of the viscus, and
taking therefore the direction of the longitudinal
muscular fibres, may be made with equal advantage,
but oblique directions should be avoided.

Preparation 2. Bloodvessels of the gullet—
The arrangement of the vessels is best shown in a
flat preparation. A small piece, obtained from an
injected animal, is transferred, without staining,
from the spirit to oil of cloves, being subsequently
mounted in dammar, with the inner surface upper-
most. Such a preparation is only useful for exami-
nation with a low power, but by this the arrangement
of the vessels in the successive strata can be well
made out.

Preparation 3. The stomach.— The stomach
should always be prepared as soon as possible after
death, for, in the first place, the columnar epithelial
cells covering the inner surface soon become altered ;
and secondly, if digestion were proceeding in the
organ at the time of death, the mucous membrane
itself becomes attacked by the gastric juice in a very
short time.

The abdomen, therefore, is to be opened as soon
as the animal (a cat or dog) is dead, and the oesopha-
gus cut as near the diaphragm as possible, and the
duodenum about two inches beyond the pylorus ;
the folds of peritoneum connecting the viscus to the
liver and neighboring parts are also severed, and the
stomach is removed. If the organ is empty or if
the contents are fluid enough to admit of being
poured out through the pylorus, it may be prepared
as a whole by distension with the spirit and chromic

THE GASTRIC GLANDS. 213

mixture ; spirit alone also answers very well. The
duodenal end is tied up and a glass canula is fast-
ened into the oesophageal end. This is connected
by an India-rubber tube with a glass tube which
passes to the bottom of a bottle containing the "hard-
ening fluid. A second tube passes just through the
cork of the bottle, and by blowing through it the
fluid is forced into the stomach. When the organ
is moderately distended the India-rubber tube is
clipped, to prevent any of the liquid being forced
back into the bottle by the contraction of the mus-
cular walls of the stomach ; the gullet is then secured
by a ligature, and the whole organ is immersed in a
large bottle or covered beaker filled with the same
mixture of chromic acid and spirit. After twenty-
four hours it should be opened and put into fresh
fluid; or, if it is not desired to keep the whole of
the organ, small pieces only from different parts are
so transferred. (Indeed, if the stomach be too large
to harden as a whole, or difficult to be cleared of its
contents, small pieces may be cut out from the fresh
organ, pinned out on a piece of cork or cake of wax,
and thus immersed in the spirit and chromic mix-
ture). In two or three days more the tissue is hard
enough, if the spirit be of the strongest, to cut sec-
tions from, and small pieces may accordingly be
embedded with this view. The sections, "which
should comprise all the coats of the organ, are to be
stained with logwood and mounted in dammar as
usual. They will show well enough the relative
thickness of the several layers and many of the
structural points, but for making out distinctly the
structure of the mucous membrane and the characters
of the cells which occupy the gastric glands it is
necessary to make thinner sections than are easily
obtainable in conjunction with the muscular coat.

Preparation 4. Gastric glands. — With this
object, then, small pieces of the fresh mucous mem-
brane are taken from two distinct parts, one from
near the pylorus and the other from the cardiac

214 PRACTICAL HISTOLOGY.

fundus, and placed at once in absolute alcohol.
When hardened they are embedded separately, and
vertical sections, as thin as possible, are made.
Those from the pyloric piece are to be stained with
logwood and mounted as before in dammar, but one
or two of the thinnest may be selected and mounted
in glycerine. Those from the cardiac piece are to be
stained and prepared in three different ways. In
the first place, two or three may be treated as just
recommended for those from the pyloric piece.
Secondly, other two or three are to be placed for
twenty-four hours in a weak solution of carmine
(the carmine solution, p. 207, diluted five times).
Thirdly, one or two others are to be stained with
aniline blue. There are different kinds of this sold
in the shops ; that known as Nicholson's No. 1 gives
good results. A one per cent, solution is used, and
the sections are left in for about thirty minutes.
They are then placed in a watchglass containing a
mixture of glycerine and water, equal parts, and are
finally mounted in glycerine. The carmine-stained
sections are also mounted in glycerine, after having
been rinsed in water.

These different modes of staining bring out dis-
tinctly the differences between the various kinds of
cells found in the peptic glands. In the logwood
preparations the peptic cells will be found stained
rather less than the rest, whereas in the sections
stained by carmine and aniline they are colored
more deeply. This is especially the case in the ani-
line preparations, where the peptic cells are stained
of a deep blue, whilst the other cells remain almost
colorless.

Preparation 5. Horizontal sections. — Besides
the vertical sections of the mucous membrane others
are to be made parallel to the inner surface, and
therefore so as to cut the glands across. To effect
this a small piece of each region is embedded, with
the part which corresponds to the inner surface of
the stomach placed opposite the end of the mould.

BLOODVESSELS OF THE STOMACH. 215

It is then gradually cut into slices, which will, of
course, comprehend in succession first the mouths of
the glands, then their necks, and finally the deeper
parts. The sections so obtained are to be stained
and mounted in the same way as the vertical sec-
tions.

Preparation 6. Cells of the glands, iso-
lated.— In addition to studying them in sections
in this way the cells of the glands are also to be
studied in teased-out preparations of the fresh mu-
cous membrane. If prepared in serum they will
showT better than by any other method the characters
of the different kinds of cells. The cells will be
more readily obtained separate if a small piece of
the membrane is placed in J per cent, bichromate of
potash solution for twenty -four or forty-eight hours,
but the cells are apt to be somewhat altered, and the
columnar cells of the general surface and mouths of
the glands to become transformed, by the swelling
and escape of their contained mucus, into goblet-
cells.

Preparation 7. Bloodvessels of the stomach.
— But vertical and horizontal sections of an injected
stomach are to be made. This may be obtained
from the animal which was injected entire: If it
were a rat, the preparations are to be made from the
pyloric half of the organ, since in this animal the
cardiac part has a non-glandular mucous membrane
with stratified epithelium like that of the gullet.
The vertical sections need not be very thin ; they
are improved by being placed for a few minutes in
dilute logwood, so as to become slightly colored,
before being mounted in dammar by the ordinary
process. Instead of cutting horizontal sections a
small piece of the injected stomach may, if from a
small animal, be simply mounted flat with the inner
surface uppermost, without staining.

Preparation 8. — An attempt may be made to inject
the lymphatics of the gastric mucous membrane with

216 PRACTICAL HISTOLOGY.

Berlin blue, but the process requires considerable care
and experience, since it presents unusual difficulties.
Indeed, it is only quite recently that attempts to fill any
lymphatics except those in the deepest part of the mem-
brane have been successful. If a successful result is
obtained the injected portions are hardened in alcohol,
and vertical sections, which may be tolerably thick, made
and mounted in dammar.

THE INTESTINE. 217

CHAPTER XV.

THE SMALL AND LARGE INTESTINE.

Preparation 1. Sections of small intestine.

— Pieces of the small intestine are to be prepared in
exactly the same way as the stomach, the mixture
of alcohol and chromic acid solution being employed
to distend the gut, which is then immersed in the
fluid. After a few hours the intestine is opened arid
the fluid changed, and in three or four days the tissue
is transferred to spirit, to complete the hardening.
Three pieces of the small intestine are to be pre-
served in this way, viz., one from the very com-
mencement of the duodenum (this will probably have
been included in the stomach preparation) ; a second
from the jejunum ; and the third from the ileum, in-
cluding one of the patches of Peyer. The pieces
may be obtained from a cat, dog, or rabbit, the con-
tents of the intestine being first washed out by forc-
ing a rapid stream of the alcohol and chromic fluid
through them before tying up the further end.
Instead of distending it with the preservative fluid,
the gut may be opened and kept in an extended
state by pinning it on a cork or cake of wax, which
is then inverted into the fluid.

In embedding the small intestine in wax- mass
care should be taken that the inner surface does not
retain too much spirit between the villi, for this
would prevent the melted wax-mass from penetrat-
ing between them, so that they are thus left without
a support whilst being cut. At the same time the
surface should on no account be allowed to become
quite dry.

It is necessary, in order to see the structure of the
19

218 PRACTICAL HISTOLOGY.

villi, that the sections should be very thin indeed —
so thin, in fact, as to include not the whole thick-
ness of a villus, but only a longitudinal slice ; other-
wise the epithelium on its surfaces interferes with
the view of the internal structure. With a very
sharp razor and considerable dexterity, this may be
effected even when the intestine is embedded by the
ordinary process, but the thin sections so obtained
are apt to become broken up during the process of
staining and subsequent washing. It is therefore
well to adopt for this tissue the cacao-butter process,
described at p. 198. A small piece is stained with
Kleinenberg's logwood, as there recommended, and
after passing through alcohol and oil of cloves is
impregnated with melted cacao-butter, and em-
bedded in a cake of the same material. There is
hardly any limit to the thinness with which sections
from a piece so embedded may be obtained, and all
that is further necessary is to dissolve away the in-
cluded cacao-butter from the sections with oil of
cloves and to mount them in dammar.

Preparation 2. Fat absorption. — For the pur-
pose of studying the course which fatty particles take in
passing from the cavity of the intestine into the central
lacteals of the villi, an animal is killed three or four
hours after a meal composed almost exclusively of fat (it
should previously have been allowed to fast for several
hours). On opening the abdomen the lacteals in the
mesentery will be found filled with chyle, and the cavity
of the small intestine occupied by emulsified fat which is
undergoing absorption. The intestine is opened at once,
and two or three very small pieces of the mucous mem-
brane are snipped off and placed in 1 per cent, osmic acid
solution Another minute piece is placed in a drop of
serum or aqueous humor and is quickly teased-out with
needles ; a piece of hair is added, and the preparation is
covered and examined. One of the portions in osmic
acid is allowed to remain forty-eight hours in the solu-
tion, and is then broken up in water. The others are
transferred to dilute Kleinenberg's solution, and when
stained throughout are embedded by the cacao-butter

NERVES OF INTESTINE. 219

process. The sections are placed, after the cacao-butter
has been extracted from them by warm oil of cloves, first
in spirit, and then in water, and are finally mounted in
glycerine.

In the two teased preparations — serum and osmic —
many of the columnar epithelium cells will be founi to
contain fatty globules of various sizes (stained blaok in
the osmic preparation). Similar, but for the most part
smaller particles will also be found in the numerous
lymphoid corpuscles which are set free from the retiform
tissue of the mucous membrane by the process of teasing.
In the sections the epithelium cells and the lymph cor-
puscles will be observed, in situ, in the same condition,
viz., containing blackened fatty particles, and moreover
the cleft-like central lacteal in the middle of each villus
will be found to contain similar globules. Hence we infer
that the tatty matters are first taken up from the cavity
of the intestine by the columnar epithelium cells ; that
they are transmitted in some way from these to the
amoeboid lymph cells, and that these again convey them
to and discharge them into the central lacteal.

Preparation 3. Vessels of the small intes-
tine.— The bloodvessels of the small intestine are to
be studied by aid of vertical sections of the injected
gut. The sections may be lightly stained with
logwood.

The lymphatics (lacteals) may perhaps be seen in thin
sections of the uninjected preparations as cleft-like spaces
in the villi and in the substance of the mucous membrane,
and surrounding the bases of the lymphoid nodules which
make up the Peyerian patches. It is not an easy matter
to inject those of the mucous membrane, although the
larger plexuses of the submucous and muscular coat can
be more easily demonstrated.

Preparation 4. Nerves of the intestinal
wall. — The nerves of the intestinal canal form a
very interesting subject of study, comprising two of
the closest and most richly gangliated plexuses of
pale fibres which are met with in the animal body.
They may, moreover, by following the method here
to be described, be shown without any great diffi-

220 PRACTICAL HISTOLOGY.

cultj in all parts of either the small or the large
intestine. It is preferable to choose an animal (e.g.
rabbit or guinea-pig) in which the intestinal coats
are not very thick. The following is the mode of
procedure: A piece of glass tubing about a quarter
of an inch in diameter and five or six inches long
is taken, and one end is drawn out into a canula,
whilst to the other a small piece of India-rubber
tube, furnished with a spring clip, is attached.
Chloride of gold solution (J per cent.) is drawn up
into the tube so as almost to fill it, and the clip
is then closed, to prevent the escape of the fluid.
Care should be taken not to suck any of the
gold solution into the mouth. A piece of intestine
about three inches long is removed from the dead
animal, and if not already empty its contents are
washed out by a stream of salt solution. The intes-
tine thus emptied and cleaned is ligatured firmly at
one end, whilst into the other is tied the canulated
end of the glass tube containing the gold solution.
When thus secured the clip is opened and the fluid
is allowed to flow into and distend with moderate
force the piece of gut, the action of gravity being
assisted by gently blowing through the India-rubber
tube. As soon as the intestine is filled with the
gold solution the clip is again allowed to close, and
then, while an assistant holds the glass tube in a
vertical position, the operator ligatures the gut just
beyond the end of the canula, which may not be cut
away. The piece of intestine, thus filled with the
gold solution, is immersed for an hour in more of
the same liquid. It is then placed in a dish of
water and cut open longitudinally with scissors, so
as to allow the contained fluid to escape, after which
the puckered, ligatured ends may also be removed.
The tissue being hardened by the gold solution, the
piece of gut which remains retains its cylindrical
shape. It is well to halve it by another longitudinal
cut, so that both inner and outer surfaces may be
freely exposed to the light. The pieces are now
placed, with their outer surfaces uppermost, in a

NERVOUS PLEXUSES OF INTESTINE. 221

glass vessel of water containing just enough acetic
acid to be sour to the taste, and the vessel is covered
and allowed to stand in a warm place freely exposed
to the sunlight (see p. 96). After two days its color
will be found to have changed to a dark violet. A
few drops of methylated spirit may then be added
to the fluid ; this serves to aid the reduction of the
gold and to prevent the growth of fungi. In another
day or two the tissue will be so dark as to appeal-
almost black. A portion is then removed to a glass
dish of water and prepared in the following way :
In the first place, the glandular mucous membrane
is separated from the rest of the intestinal wall
either by tearing it off with forceps or by scraping
it away with the end of a blunt scalpel. There now
remain the serous and two muscular layers, together
with the submucosa. To the inner surface of the
latter the muscularis mucosae may be still adherent.
The separated fragments of the mucous membrane
are got rid of by pouring away the water first used
and substituting fresh, and then an attempt must
be made by aid of two pairs of forceps, to peel the
submucosa off from the inner surface of the muscular
coat. Of course if the muscularis mucos?e has been
left, tluit will form a part of the layer which is thus
removed. The separation must be done slowly and
carefully, so as to get as large a piece as possible
intact. When this is accomplished satisfactorily a
slide is immersed in the wrater, and the portion of
submucosa so detached is floated on to it, and
re'moved from the water. Its further preparation
consists in allowing the excess of water to run off,
applying a cover-glass, making sure first of all that
the layer is free from folds, and then allowing gly-
cerine to pass under the cover-glass and replace the
water as this evaporates.

Returning to the remainder of the piece of intes-
tine, the next process consists in picking away bit
by bit with forceps the comparatively thick layer of
circular muscular fibres. This is not a difficult ro-

222 PRACTICAL HISTOLOGY.

ceeding, and when it is finished all that remains is
the thin serous coat and the longitudinal muscular
layer, to the inner side of which the nervous plexus
of Auerbach, the intermuscular plexus, is adherent.
~No further separation is required, all that is neces-
sary being to float the piece of tissue on to a slide
with the (concave) inner surface uppermost. But
before applying the cover-glass the preparation is to
be examined with a low power, to see that the sur-
face of the serous membrane is free from a finely
granular precipitate which is apt to be deposited in
the acidulated water. If this is present, the piece
must be replaced in the water and the precipitate
gently brushed oft' with a soft camel-hair pencil.
The preparation is completed in the same way as
that of the submucosa. The latter shows Meissner's
plexus, the cords of which are much finer than those
of Auerbach's. In both plexuses the nervous cords
are stained of a violet color by the reduction of the
gold ; at the points of junction of the nervous cords
are groups of small ganglion cells, the nuclei of
which are hardly stained at all, and consequently
look clear in the midst of the darkly-stained cell-
bodies. The distinction between the individual
cells is difficult to make out. Branches may perhaps
he traced passing from the plexus of Auerbach
amongst the muscular fibre-cells : from that of
Meissner to the muscularis mucosoe, if this is present,
and perhaps also to the small bloodvessels, which are
particularly well seen in the preparation of the sub-
mucous coat.

Preparations 5-7. Large intestine. — For
hardening the tissue and preparing sections of the
large intestine the same methods are employed as for
the small intestine, so that it is unnecessary to re-
capitulate them.

The injected large intestine is prepared, like the
stomach, by means of vertical and horizontal sec-
tions.

The lymphatics are not easy to inject, but present
' 'Acuity than those of the stomach.

THE LIVER. 223

CHAPTER XVI.

THE LIVER.

Preparation 1. ITninjected liver. — To prepare
sections of the liver small pieces are placed in 2 per
cent, bichromate of potash solution for ten days,
transferred from this to weak spirit, and in twenty-
four hours are placed in strong spirit, to complete
the process of hardening. The tissue will be hard
enough to cut thin sections from in another day or
two. The sections are stained with logwood, and
mounted in dammar varnish. They should be made
in two directions, viz. (1) in a plane near arid parallel
to one of the surfaces of the liver, and (2) vertical to
the surface. Those made in the direction first named
will for the most part cut the central or intralobular
veins across, those in the second direction may take
them along their length ; the apparent arrangement
of the blood capillaries and liver cells in the indi-
vidual lobules will differ, both, in accordance with
this difference of direction and also according as the
lobule is cut exactly through its centre or at some
part more or less removed from this. Between the
lobules are seen the branches of the portal vein,
always accompanied by a branch of the bile duct,
the columnar epithelium of which is very well seen
in these preparations, and by a branch of the hepatic
artery. All three are included in a mass of connec-
tive tissue, a prolongation of Glisson's capsule, en-
closing them in a so-called portal canal. In this
connective tissue cleft-like spaces may generally be
seen — two or three in the section of a portal canal —
not merely breaks in the connective tissue, but with
quite a definite wall. These are the accompanying

224 PRACTICAL HISTOLOGY.

lymphatics. Other lymphatics accompany the
branches of the hepatic veins, but are not so easily
seen in the sections, although they can be injected.
The branches of the hepatic veins are readily dis-
tinguished from those of the portal vein, by the fact
that they run unaccompanied by branches of the bile
duct and hepatic artery. The blood capillaries of
the lobules look like spaces (tilled with round clear
bodies, the altered blood corpuscles) between the
rows of cells (in the sections these appear arranged
simply in rows) ; their walls are very thin, and the
hepatic cells appear for the most part to come in
contact with the wall. But in reality there is a
second delicate membrane around many of the capil-
laries, and between it and the epithelioid wall of the
vessel is a space for the passage of lymph (perivascu-
lar lymphatic) ; it is difficult to make this out, how-
ever, in preparations in which the lymphatics are
not injected. The round nuclei of the liver cells are
deeply stained by the logwood, and the cells them-
selves slightly. In the thinnest parts of the sections
the lines of junction between neighboring cells can
be well made out, and not unfrequently the small
capillary passage for the bile which intervenes be-
tween the adjacent sides of the cells can, according
to the direction in which it runs, be recognized with
a very high power either as a horizontal line or as a
minute aperture. To obtain the best results the
pieces of liver, which are not to be more than an
inch or so square and a quarter of an inch thick,
should be placed in the bichromate solution quite
fresh, from an animal killed only a short time pre-
viously.

Preparation 2. Injected liver. — The vessels
of the liver seldom get tilled when the rest of the
body is injected from the aorta. It is generally
necessary to make a special injection of this organ
from the portal vein. For this purpose the usual
red or blue gelatine injection is used, the apparatus
being arranged as described at p. 14t>. The operation

INJECTION OF LIVEK. 225

is conducted as follows : The animal (rabbit) having
been killed by bleeding,1 the thorax is opened ; and
the pericardium being torn away, the heart is raised
and two thread ligatures are passed round the inferior
vena cava. One of these is tightened as near the
heart as possible, and then a snip is made in the vein,
so as to allow the blood to escape freely. Next, the
abdomen is opened, and the intestines 'and stomach
being gently drawn to the left side, the peritoneum
at the back of the abdomen is torn through, and a
ligature placed around the vena cava above the ac-
cession of the renal veins. The portal vein is then
found in the fold of peritoneum which connects the
under surface of the liver with the stomach, and a
ligature, in the noose of which the hepatic artery
may be included, having been passed round it near
the liver, a snip is made in the vessel, and the in-
jecting canula is tied in. This canula is now filled
by means of a pipette with warm salt solution, and
the supply tube (from the injecting bottle), having
been completely filled by the injecting fluid to the
exclusion of air in the same way as in the first in-
jection (p. 167), is slipped over the open end, and
the injection at once allowed to flow. As it passes
by the portal system of veins through the lobules of
the liver into the hepatic system, it forces whatever
blood is still contained in the bloodvessels of the
organ out into the vena cava, whence it can freely
escape into the thorax through the snip which was
there made in the vein. As soon as all the blood is

1 In injecting the whole body it was recommended to kill the
animal by chloroform. This was for the purpose of having the
bloodvessels as much dilated as possible. When an animal is
killed by bleeding, the arteries contract very considerably, and,
remaining contracted some little time after death, offer a con-
siderable resistance at first to the passage of the injection, and
this may tend to spoil the result altogether. In the liver, how-
ever, the case is different, since it is not injected through arteries,
but through veins, which possess little contractility. Any blood
which remains in the vessels does not, so long as it remains fluid,
impede the passage of the injection, but it is driven before it.

226 PRACTICAL HISTOLOGY.

thus driven out, and only pure injecting fluid begins
to pass, this vein is occluded near the diaphragm by
the second thread. The pressure in the injecting
bottle is then slowly raised, but should not even at
the utmost exceed three inches of mercury, for this
amount of pressure will cause all the bloodvessels to
be quite fully distended, and will effect a very con-
siderable consequent enlargement of the organ ; more
might cause rupture and extravasation. After the
lapse of a few minutes, to allow of the complete fill-
ing of all the bloodvessels, a second ligature is tied
round the portal vein close to the liver to prevent
the return of the still fluid injection, and the canula
is cut out from the portal vein (the pressure in the
apparatus having first been removed), and the body
put into a cold place so as to permit the gelatine to
solidify. It is well to hasten the process by pouring
cold water — iced if possible — over the liver. When
the injecting material is entirely set, the organ is
removed and cut into pieces, which are placed in
weak spirit (half water) for twenty-four hours. They
are then put into stronger spirit, and in forty-eight
hours more in the strongest. In three or four days
sections may be made (in two directions as with the
uninjected organ), and mounted, after passing
through oil of cloves, in dammar. It is better not
to stain them.

During the whole process, the greatest care must
be taken not to handle the liver more than can
possibly be helped, for it is very readily scratched
or ruptured, and any such accident would tend to
permit the escape of the fluid injection. This warn-
ing applies with equal, if not greater, force to the
operation next to be described ; that namely, of
filling the bile-ducts.

Preparation 3. — The bile-ducts are injected with
Berlin blue solution, 2 per cent., the mercury appa-
ratus (Fig. 30) being used. The solution, although
fluid in the cold, should nevertheless be employed
warm, as it is then less likely to excite contraction

LYMPHATICS OF THE LIVER. 227

of the biliary ducts. A rabbit is killed by bleeding,
the abdomen opened, and the common bile-duct
sought for close to the portal vein ; a ligature is
passed round it and a small piece of card being
placed under as a support, and to separate it from
the accompanying vessels, a snip is made into it,
and a glass canula is inserted, and having been
passed along the duct as near to the liver as possible
is tied in. The cystic duct is ligatured to prevent
the injection from passing into the gall-bladder. In
the next place the canula is filled with warm Berlin
blue solution by means of a fine pipette ; the (pre-
viously filled) supply tube is attached, the clip on
this opened, and the pressure gradually raised to
about two inches of mercury. The blue fluid, driving
whatever bile there happens to be left in the ducts
before it into the lobules, penetrates first into the
interlobular bile-ducts, and from these into the outer
parts of the lobules, forcing the bile more and more
towards the centre ; here of course there is no escape
for it, except that a little may pass into the lym-
phatics and bloodvessels through their walls. Hence
it will be understood that the injection can only be
made to fill the intercellular biliary passages in the
outer part of each lobule. The injection should be '
persevered with for about half an hour ; the bile-
duct may then be tied and the injecting apparatus
removed ; after which the liver is cut out entire,
without injuring it in any way, and placed in strong
spirit. In twenty-four hours it is cut in pieces, and
the spirit changed, and in less than a week the
pieces will be hard enough to cut. The sections
may be stained slightly with logwood.

Preparation 4. Lymphatics of the liver. —

The lymphatics of the liver are injected through a fine
canula stuck obliquety into the superficial part of the
organ immediately beneath the capsule. Either solution
of Berlin blue or alkanet may be used. The part should
be quite fresh. If the injection be persisted in for a long
while, the fluid may flow out both by the lymphatics

228 PRACTICAL HISTOLOGY.

accompanying the portal vein and those accompanying
the hepatic veins (Ludwig and Fleischl). Very fre-
quently, however, the injecting fluid finds its way into
the blood-system instead of the lymphatics. The injec-
tion of the lymphatics may be accomplished in another
manner, viz., by seeking those lymphatics which accom-
pany the hepatic veins at the back of the liver, and tying
a canula into them. After a time the fluid will be found
to pass out by the vessels which accompany the portal
vein.

The investigation of the lymphatics of the liver is,
however, very difficult, and our knowledge of their course
and arrangement is still by no means satisfactory.

Preparation 5. Hepatic cells.— In addition
to what may be learnt from sections of the organ,
teased-out preparations afford much useful infor-
mation, both of the characters of the liver cells and
of the connective tissue of the lobules. For this
purpose small portions of the perfectly fresh and
warm liver are broken up in serum or salt solution,
and other portions are macerated for a day or two
in weak bichromate of potash, and subsequently
teased out in water.

The pancreas is prepared in the same manner as
tbe salivary glands, to the description of which the
student is referred.

THE SPLEEN. 229

CHAPTER XVII.

THE SPLEEN AND URINARY ORGANS.
THE SPLEEN.

Preparation 1. The uninjected spleen. — The

spleen is hardened in the same manner as the liver,
by placing small pieces of it in 2 per cent, solution
of bichromate of potash, and in about a week or ten
days transferring them first to weak and then to
strong spirit. The sections, which cannot be too
thin, are to be stained deeply with logwood, and
mounted by the ordinary mode of precedure in
dammar varnish.

In these preparations the Malpighian corpuscles'
(or nodules of lymphoid tissue) are very strongly
colored, as are also the trabeculse which traverse the
pulp, especially in those animals in which they are
largely composed of plain muscular tissue ; the sub-
stance of the pulp is but slightly colored by the log-
wood, only the cell-nuclei, and, to a much less extent,
the branching cells of the retiform tissue being
stained. The prevailing color of the pulp is yellow-
ish, owing to the blood (altered in color by the action
of the reagent) which at the time of death remained
in the interstices of the tissue. Moreover, here
and there a speck of coarsely-granular reddish-yellow
pigment may be detected, lodged in one of the cor-
puscles of the spleen pulp. But this will be better
made out in the teased-out preparations subsequently
to be described.

Preparation 2. Irrigated spleen. — By another
mode of preparing the spleen all the blood is first washed
out by a stream of salt solution, injected through the
20

230 PRACTICAL HISTOLOGY.

splenic artery, and the organ is then hardened in the
same way as before. It will much facilitate the process
of hardening if the salt solution is followed by a stream
of the bichromate. This may even be made to distend
the organ somewhat, the distension being maintained by
ligaturing the vessels near the hilus. In such a case the
organ is to be placed entire in 2 per cent, bichromate, and
only cut into pieces after forty -eight hours. For a spleen
which has been thus prepared, the cacao-butter method
of embedding may be employed, after a thin piece has
been stained by alcoholic logwood. By thus removing
the blood corpuscles the retiform tissue of the pulp is
better seen. Klein (Quarterly Journal of Microsco-
pical Science, October, 1875 , relying on appearances
presented by preparations made in this way, describes
the retiform tissue of the spleen as entirely made up of
flattened cells forming by their junction a "honeycomb
of membranes." That such a description is far too ex-
clusive, the study of teased preparations amply demon-
strates, for the network of branched cells, described by
almost all previous observers, is readily seen in them
(see below, Prep. 4).

Preparation 3. Injected spleen.— The spleen
may have been injected in the animal which was
injected entire ; if this is not the case, a special in-
jection is to be made from the splenic artery. When
successfully accomplished, the vessels are as usual
ligatured to prevent the escape of the injection, and
the organ is immersed entire in spirit, at first weak
but with the strength afterwards gradually increased,
as in the case of the liver. The sections will show
what at first sight look like accidental extrava-
sations, large patches namely of injection distributed
all over the organ, with the exception of large round
white patches here and there, pervaded by a few
capillaries. The white patches are sections of the
Malpighian corpuscles, and the part permeated by
the injection is of course tbe pulp, into which the
arterial capillaries freely open.

Preparation 4. Splenic cells. — To obtain
specimens of the spleen substance, which will show

THE KIDNEYS. 231

in a separated condition the cellular elements which
it contains, and of which it is composed, a small
portion of the fresh organ may be teased out with
needles in a little salt'solution or serum. But it
will be found that so much blood is incorporated
with the spleen substance (it forms, in fact, most of
the soft matter which can be expressed from the
fresh section) that the view of the other parts is
obscured by innumerable red blood corpuscles.
Hence before teasing a piece it should be placed for
forty-eight hours in J per cent, bichromate of potash
solution. This destroys the red corpuscles whilst
preserving the character of, and at the same time
macerating somewhat the proper substance of the
spleen, so "that the cells are now readily separated
and seen. By far the greater number are lymph
corpuscles from the lymphoid tissue of the Malpig-
hian nodules and of the arterial adventitia. But,
besides these, other cells are met with ; larger, often
flattened, and many of them with fine branchings.
These are cells of the retiform tissue ; some of them
contain pigment granules, as already intimated.
They may be found^either entirely isolated, or form-
ing a fine network by the intercommunication of
their branches. Their nuclei, as well as those of the
lymphoid cells, are well brought out if a little log-
wood solution is permitted to pass under the cover-
glass. In the fresh preparations, not treated by bi-
chromate of potash or any other reagent, but made
in serum, some of the cells may perhaps be found
containing red blood corpuscles in their interior, and
transitions from these to those containing pigment
are met with.

THE KIDNEY.

Preparation 5. The uninjected kidney. — The

kidney is hardened in the same way as the liver and
spleen, viz., by a strong solution of bichromate of
potash (two per cent.). The piece or pieces that are

232 PRACTICAL HISTOLOGY.

employed should include both cortical and medul-
lary parts; but at the same time should not be
thicker than from a quarter of an inch to half an
inch, otherwise the preservative fluid will not pene-
trate rapidly enough to the deeper parts. They are
to remain in the bichromate solution (a relatively
large quantity) for three weeks; are then placed in
weak spirit, and in twenty -four hours transferred to
strong spirit. In three or four days more the pieces
are firm enough to cut. They will perhaps be large
enough to hold in the hand, and thus the necessity
of imbedding will be avoided; sections as thin as
possible are to be made in a plane vertical to the
surface of the organ, and large enough to include
both cortical substance and Malpighian pyramid.
One such section is first -transferred from the spirit
to water, and is then simply mounted in glycerine;
another is stained with logwood, and after treatment
by the customary process is mounted in dammar
varnish. These sections will show: in the cortical
substance the Malpighian corpuscles, the convoluted
tubules variously cut, and the prolongations of the
straight tubules of the medullary substance and of
the tubules of Henle ; in the pyramidal part the two
last-named tubules, and the collecting and excretory
tubules, seen longitudinally, with a large number of
bloodvessels running parallel to and between them.

Preparation 6. — A transverse section also should
be obtained of the medullary substance. With this
object the end of a pyramid is cut off with a razor,
and, as it is too small to hold in the hand, it is im-
bedded in wax-mass in such a position that the
tubules will be cut exactly across. Sections from
this are mounted like the others in glycerine and in
dammar varnish.

The kidney of the dog or of almost any other ani-
mal may be used for the above preparations, and has
the advantage over the human organ, that it is ob-
tainable in a fresher condition. But since the epi-
thelium of the tubules, at all events of the convoluted

THE KIDNEYS. 233

tubules, differs somewhat in appearance in the human
kidney as compared with that of the lower animals,
a portion of a perfectly healthy organ should be pro-
cured from the post-mortem room as fresh as possible,
and prepared for observation in the same manner.

Preparation 7. The injected kidney.— The
bloodvessels of the kidney will very probably be
filled in injecting an animal entire ; but, if this
should not have been the case, it is not difficult to
make a special injection of the separated organ from
the renal artery. The red gelatine injection may be
used, and the kidney is kept -warm, and the injection
maintained for a considerable time, in order that the
vessels of the glomeruli and the network of capilla-
ries in the cortical substance supplied by their effer-
ent vessels may be completely tilled. The organ is
then set aside in a cool place (surrounded by ice, if
possible), and, when the gelatine is completely set,
is cut into three or four pieces and hardened gradu-
ally, as usual, with alcohol. The sections, which
need not be very thin, but should be quite even, and
comprise the whole thickness of the organ, are to be
mounted, unstained, in dammar varnish.

Preparation 8. Uriniferous tubules — The uri-
mferous tubules may be injected from the ureters for a
considerable part of their length simultaneously, if it be
desired, with the injection of the bloodvessels by a solu-
tion of Berlin blue. But even if well filled they are too
densely arranged to render it possible to trace individual
tubules along their whole extent. This may be better
accomplished by making teased preparations of the kid-
neys of small animals, which have undergone some pro-
cess of preparation, having Cor its object the solution or
softening of the intertubular substance. Several such
processes have been proposed, but none yield entirely
satisfactory results. The best, perhaps, that has yet been
tried consists in digesting tolerably thick slices of a small
kidney in a mixture of four parts of spirit and one of
hydrochloric acid, kept boiling fur three or four hours.
The boiling is performed in a flask fitted with a cork,
through which M louo- vertical tube passes; in this much

20*

234 PRACTICAL HISTOLOGY.

of the vapor which is driven off by the boiling becomes
condensed, and flows down again into the flask. After
the time mentioned the slices are removed and placed in
water; and, after lying in this for a few days, minute
shreds, comprising the whole depth from external surface
to papillae, are split off' with needles, placed on a slide,
and unravelled as much as possible by aid of the dissect-
ing microscope. The preparation is covered with a spe-
cially large piece of covering-glass (a hair being first
added to avert the pressure of the glass on the soft
tubules), and stained by drawing picric acid solution
under the cover-glass. This soon colors the tissue in-
tensely yellow; glycerine may then be allowed to pass in
at the border in order to complete the preparation. Some
of the tubules will be found isolated for a considerable
part of their length, and the passage of the convoluted
tubules into the looped tubes of Henle may especially be
well seen. The epithelium of the tubules is for the most
part well preserved, but that of the convoluted ones has
become very granular, and so swollen out as to completely
fill up the tubules.

Preparation 9. Examination of the fresh
kidney. — When by these various means sufficient
acquaintance ha*s been gained with the various
tubules and their contents in situ, the examination
of the fresh tissue in serum may be attempted.
With this object small snips are to be made from
different parts of a freshly-cut surface with a pair of
curved scissors, and teased out in a drop of serum,
with the aid of the dissecting microscope, so as to
separate as many of the tubules as possible. In
doing this much of the epithelium will become
detached, and the characters of the individual cells
in the fresh condition may be studied.

Preparation 10. — The epitheloid cells of the capsules
of Bowman and of the basement membranes of the tubules
may be shown by nitrate of silver. For this purpose a
fresh kidney is sliced in half by a single cut with a sharp
razor in the direction of the tubules. One of the halves
is thoroughly washed with distilled water, and solution
of nitrate of silver (one-half per cent.) is poured over the

THE URETERS. 235

cut surface. A fter a minute and a half the silver solution
is rinsed off with distilled water, and the piece of kidney
is placed in a beaker of strong spirit, with the silvered
surface exposed to the sunlight. When brown it may be
removed from the light, but is left in the spirit for twenty-
four hours; one or two sections are then made from the
brown surface, clarified in oil of cloves, and mounted in
dammar.

THE SUPRARENAL CAPSULE.

Preparation 11. — To prepare the suprarenal cap-
sule it is separated from the surrounding fat, divided
into two or three pieces by transverse cuts, and placed
in two per cent, of bichromate of potash solution for
fourteen days, when the hardening may be completed
in spirit in the usual manner. Hardening the organ
in spirit alone also gives very good results. The
mode of preparing the sections, which should include
both cortical and medullary substance, calls for no
special description.

If the human suprarenal is not obtainable in a
fresh condition (the medullary substance very readily
softens and breaks down after death), that of the
guinea-pig, which is large comparatively to the
size of the animal, and has the distinction between
cortical and medullary substance well marked, may
advantageously be employed.

Preparation 12. — In a teased-out preparation of
the fresh organ the cellular elements of the cortical
and medullary substance may respectively be studied,
and the effect of a solution of yellow chromate of
potash in coloring the medullary cells brown may
be observed.

THE URETERS.

Preparation 13. — The ureters are prepared in
the same way as the intestine — by moderately dis-
tending an excised portion with a mixture of equal
parts of spirit arid | per cent, chromic acid solution,

236 PRACTICAL HISTOLOGY.

and placing the piece thus distended in a beaker
containing some of the same mixture. After twenty-
four hours the tube is slit open, and transferred to
spirit for two or three days. The sections are to be
made across the length of the tube, and stained and
mounted in the ordinary manner.

Preparation 14, Epithelium of ureter. — To
study the separated epithelial cells a piece as fresh
as possible is cut open, pinned out on a cork with
the inner surface uppermost, and immersed in J per
cent, bichromate of potash solution for from twenty-
four to forty-eight hours. Some of the epithelium
is then scraped off with a spear-shaped needle or the
end of a scalpel, and is broken up in a drop of water.
After the addition of a piece of hair to the fluid
the cover-glass may be applied, and the preparation
examined with a high power. If it prove successful,
with many of the epithelial cells fully separated, it
may be permanently preserved. With this object
the cells should first be stained, by allowing weak
logwood solution to run under the edge' of the cover-
glass. The logwood is to be followed by a drop of
glycerine applied at the same edge; and, when the
glycerine has become diffused underneath, all that
is necessary is to cement the cover-glass.

THE BLADDER.

The urinary bladder, both for sections and teased-
out preparations, is prepared by exactly the same
methods as the ureters. To distend it a glass canula,
connected by an India-rubber tube with a bottle
containing the chromic fluid, is tied into the urethra.
The organ must not be over-distended, but only
moderately filled. Any urine which it may contain
should first be allowed to run out through the
canula.

THE GENERATIVE ORGANS. 237

CHAPTER XVIII.

THE GENERATIVE ORGANS.

Preparation 1. — Those parts which contain erec-
tile tissue will be best studied after having been
injected. Their bloodvessels and sinuses may have
been tilled in the animal which was injected entire
from the root of the aorta; but, if not, a special
injection from the lower end of the abdominal aorta
is to be made, the arteries supplying the lower limbs
being first tied to prevent waste of the injection.
The hardening of the parts in spirit must be effected
very gradually (the spirit being daily made stronger),
since otherwise the gelatine shrinks away from the
walls of the venous sinuses, and the preparation
becomes in great measure spoiled. The sections
which are made should some of them be mounted,
unstained, in dammar, others after being lightly
stained with logwood, so that the plain muscular
and fibrous tissue, and also, in sections including
the urethra, the epithelium of that tube may be
exhibited as well as the vessels.

Preparation 2. — Parts which have not been
injected are hardened in 2 per cent, bichromate of
potash solution (fourteen days), or £ per cent, chromic
acid (seven days), followed by spirit in either case.

Preparation 3. — The glandular organs, such as
the prostate and vesiculse seminales, are prepared
either with the £ per cent, chromic acid solution
followed by spirit, or with spirit alone. The subse-
quent processes of staining and mounting are the
same for all, except that it will be found, as a rule,
that those which have been in chromic acid stain

238 PRACTICAL HISTOLOGY.

less readily than those which have simply heen
hardened in spirit.

Preparations 4-6. — The scrotum, and labia, and
the vagina, are prepared in the same way as the
skin (see p. 187).

Preparation 7. — The human uterus is best
hardened in the 2 per cent, bichromate of potash ;
its cavity should be freely laid open. In animals
(the rabbit, for instance), where it is more membra-
nous, the uterus and the upper part of the vagina
may be prepared together by distending them with
the spirit and J per cent, chromic acid mixture
through a canula tied into the lower part of the
vagina. The vagina is then tied, and the organs
are cut out and placed in a quantity of the solution ;
in twenty-four hours they are laid open and the
fluid renewed, and in another day or two are ready
to be put into spirit. The sections are stained with
logwood, and mounted in dammar.

Preparation 8. Section of ovary. — The ova-
ries are prepared by placing them — with as little
handling as possible, so as to avoid rubbing off the
columnar epithelium which covers the surface — in
£ per cent, chromic acid (whole if taken from a
email animal, such as a rabbit or cat; cut into two
or three pieces if from a larger one). In most of
the lower animals they must be sought much higher
in the abdomen than in the human female; in the
rabbit they occur as small elongated bodies, dotted
all over with little projections (the Graatian follicles),
and situated just below the kidneys. They are
left in the chromic solution for seven days, and then
placed in spirit, and in two or three days more are
ready for cutting. The hardening is effected still
more readily by using the chromic and spirit mix-
ture. The sections are to be stained in carmine
solution ; for logwood sometimes colors very deeply
the coagulated fluid in the Graafian follicles, so that
the epithelial contents are obscured ; this coloration
is probably owing to the presence of mucus (or rather

THE OVUM. 239

mucin) in the secretion of the follicle. After having
been stained the sections are passed through alcohol
and oil of cloves, and mounted in dammar.

Preparation 9. The ovum.— The ripe mam-
malian ovum, although it can be seen within the
larger Graafian follicles in the sections of the
hardened organ, forms a much more beautiful ob-
ject when obtained isolated from the ovary of a
recently-killed animal. A full-grown doe rabbit,
not pregnant, is to be sacrificed for this purpose.
One of the ovaries having been removed, it is held
firmly between the finger and thumb over a clean
glass slide, in such a position that the largest and
most prominent Graafian follicle is almost in con-
tact with the middle of the slide. The follicle is
then picked with a sharp-pointed scalpel, so as to
cause the fluid contents of the follicle to spirt out,
carrying with them the ovum, surrounded by a
number of the epithelium cells. The ovum is rather
too small to be detected with the naked eye, but its
presence may be suspected if, on glancing at the
slide in such a manner that the light is reflected
from the surface of the fluid to the eye, a slight pro-
minence is observed on the otherwise flat surface.
Its presence here is confirmed by examination with
a low power, and it may then be carefully observed
with the ordinary high power. It is better, if pos-
sible, not to apply a cover-glass, for the zona pellu-
cida is apt to become broken ; and, moreover, even
slight pressure spoils in great measure the natural
appearance of the object But if the objective
becomes dimmed by its proximity to 'the fluid, or if
it is desired to employ an immersion, then a thin
cover-glass must be used, and to protect the ovum
from pressure a narrow slip of thick paper (an ordi-
nary hair is not thick enough) is to be put on either
side before the cover-glass is applied. If the fluid
which accompanies the ovurn from the Graafian
follicles is not in sufficient quantity, a drop of aque-

240 PRACTICAL HISTOLOGY.

ous humor may be added to it. It is not possible to
preserve this preparation permanently.
Preparation 10. Artificial impregnation. —

If a preparation similar to that just described is ex-
amined on the warm stage at the temperature of the
body, and a little of the seminal secretion of a male
rabbit with the spermatozoa in full activity is added,
the penetration of these into the zona pellucida ot"
the ovum can be observed.

Preparation 11. Section of uninjected testis.
— The testis is one of the most difficult organs to
prepare, owing to the looseness of its structure. It
is firmest in those animals (cat, pig) in which the
peculiar, granular, polyhedral cells, which accom-
pany and surround the bloodvessels, are most nume-
rous. To harden it two or three cuts are made
almost through its whole thickness, and it is then
placed for ten days in 2 per cent, bichromate of pot-
ash solution, and afterwards in spirit. Sections of
both the body of the testis and the epididymis are
to be made.

Preparation 12. Lymphatics of testis. — In
the sections made as above there will be observed in
the interstices between the seminiferous tubules
large, cleft-like spaces, looking almost like acciden-
tal clefts in the loose connective tissue uniting the
tubules. They are in reality, however, the lacunar
commencements of the lymphatics. To show this
the following simple experiment may be performed :
In a recently-killed dog the fine canula of a Pravaz
syringe, filled with Berlin blue solution, is stuck
through the scrotum into the middle of the sub-
stance of one of the testes, and the solution is slowly,
and without exerting any considerable pressure, in-
jected into the organ. If the abdomen is opened the
blue fluid will soon be seen passing along the lym-
phatics which run in the spermatic cord, and from
these into those of the back of the pelvis and abdo-
men, at length reaching the thoracic duct. If now
the testis is removed and hardened in spirit, and

TUNICA VAGINALIS. 241

sections are made of the hardened organ, it will be
found that the intertubular spaces are occupied by
the blue substance, and, since they are proved by
the injection to be in free communication with the
lymphatics which leave the organ, the spaces are
to be looked upon as giving origin to the lymphatics.

Preparation 13. Isolation of the seminife-
rous tubules. — For obtaining the tubules isolated
for a considerable length, pieces of the testis (pre-
ferably human) are placed for a day or two in
hydrochloric acid, diluted with -J its volume of
water, and maintained at 30° C. They are then
allowed to lie in water until the tubules can be
readily separated with needles (Mihalkovics, Lud-
wig's "Arbeiten," 1874). Teased-out preparations
of the fresh testis-substance are also to be made in
serum, to exhibit the form, stages of development,
and movements of the spermatozoa. For the object
last named the preparation should be examined on
the warm stage.

Preparation 14. Epithelioid cells of semi-
niferous tubules. — To exhibit the fact that the
apparently structureless basement membrane of the
seminiferous tubules is in reality composed of layers
of flattened epithelioid cells, a portion of the testi-
cular substance is partially unravelled in distilled
water, and some of the tubules which are thus
isolated are dipped into nitrate of silver solution
for a minute, and, after being again rinsed in water,
are mounted in glycerine and exposed to the light ;
the lines of junction between the flattened cells are
by this means made evident.

Preparations 15, 16. Tunica vaginalis.— The
tunica vaginalis is to be prepared in the same way
as the other serous membranes (with nitrate of
silver), partly unbrushed to show the epithelioid
covering, and partly brushed tor the sake of exhibit-
ing the parts beneath. For the preparation of the
visceral part, the process is similar to that adopted
for the pericardium covering the surface of the heart
21

242 PRACTICAL HISTOLOGY.

(p. 193), and need not here be more particularly de-
scribed.
Preparation 17. The mammary glands,—

Small pieces of these organs are hardened by being
placed in ^ per cent, chromic acid solution for a
week or ten days, subsequently transferring them
to spirit. The mixture of equal parts of spirit and
J per cent, chromic acid may also be used and pro-
duces the desired result more rapidly (in two or
three days); moreover the sections stain more readily
with logwood. They may be mounted either in
dammar or in glycerine. The appearances presented
by the cells of the alveoli vary considerably accord-
ing to the state of functional activity of the gland.
(See Creighton, Report of the Medical Officer to the
Privy Council, 1875, and Journal of Anatomy and
Physiology, October, 1876.)

THE BRAIN AND SPINAL CORD. 243

CHAPTER XIX.

THE CENTRAL NEVOUS SYSTEM ; THE BRAIN AND
SPINAL CORD.

SOME experience has already been obtained of tbe
methods which are employed for studying the cellu-
lar elements of the central nervous system in an
isolated condition (see p. 130). The spinal cord was
the part then under investigation, but the nerve-
cells found in the gray matter of the cerebrum and
of the cerebellum may be observed in the same
manner with equally satisfactory results. Without
delaying, then, to repeat the directions there given,
we may pass on to describe the best methods of pre-
paring sections of the parts in question.

Preparation with bichromate of ammonia. —
To harden any part of the central nervous system
the most generally useful reagent is the bichromate
of ammonia (2 or 3 per cent, solution). This will of
itself render the tissue sufficiently firm for obtain-
ing thin sections, but it is always best to finish first
with weak and then with strong spirit, especially as
this fluid must be used to wet the razor. The pieces
to be hardened should not be too large, or at all
events not to thick, but the solution has consider-
able power of penetration (differing in this respect
from chromic acid), and the whole length of the
spinal cord of any of the smaller quadrupeds, and
even that of man, may be hardened in it intact if
put in perfectly fresh. It is always better, however,
to cut it into short lengths. The pieces are ready
to be transferred from the bichromate of ammonia
solution to dilute spirit in three or four days if
small ; in a week if rather larger: after twenty-four
hours they are placed in strong spirit.

244 PRACTICAL HISTOLOGY.

In. this way a piece of the cerebellum two or three
small pieces from different parts of the convoluted
surface of the cerebrum, the medulla oblongata, and
pieces of the spinal cord from the middle of the three
regions (cervical, dorsal, and lumbar) are to be pre-
pared. They should be taken from the human sub-
ject if possible, although it is useful to study the
parts, especially the spinal cord and medulla, in the
lower animals as well. The sections, at least those
of the spinal cord and medulla, may be made with
the aid of a microtome (see Appendix), and, after
being stained, are treated in the usual manner with
alcohol and oil of cloves, and mounted in dammar
varnish. Logwood may be used for the purpose of
staining the sections, and also carmine; but, on the
whole, for sections of the central nervous system,
some of the aniline dyes (which may for the most
part be employed in either alcoholic or watery solu-
tion) give better results. One of the best is that
known in commerce as aniline-blue-black, which
stains the nerve-cells and the axis-cylinders of a dark
slate-blue color. For the cerebellum a solution of
aniline blue is recommended.

A double coloration by logwood and eosine (an aniline
dye, possessing a red color) has been recently advocated.
The eosine is to be used in solution in alcohol (see Appen-
dix). The sections are first stained with logwood, and
then placed in eosine. When sufficiently colored by this
they are passed through oil of cloves and mounted in
dammar.

Preparation by Sankey's method, — For the

preparation of large sections of different parts of the
brain (particularly of the cerebral convolutions and
of the cerebellum), destined more particularly to
show the nerve-cells and the course and connection
of their offsets, there is no better method than that
described by Sankey (''Quarterly Journal of Micro-
scopical Science," April, 1876). It is shortly as fol-
lows: With a large and long knife wetted with
spirit, slices of the fresh brain (of the cerebellum

THE BRAIN AND SPINAL CORD. 245

across the laminae, for example) are made, as thin as
possible under the circumstances — one-tenth of an
inch thick perhaps — and are placed in a strong
watery solution (seven per cent.) of the aniline-blue-
black. After three hours the staining solution is
poured off and water substituted, and in a few
minutes this, which will still be intensely colored,
is replaced by fresh water, and so on until the excess
of the staining fluid is washed away from the slices.
If one is cut in half it will be seen that only the sur-
faces are stained, the aniline solution not having
penetrated to the interior. When the washing is
completed the slices are transferred from the water
to glass slides. This can be effected, without risk
of breaking the sections, by floating them on to an
immersed slide, and raising both together out of the
water. The excess of water is then allowed to drain
off', and the pieces are left in a dry place exposed to
the air, so that after twenty-four or at most forty-
eight hours, they will be found firmly dried to the
glass. In the process of dry ins; they will have lost
a good deal in thickness, and this is now still further
reduced by planing off the upper stained surface with
a razor, or other suitable instrument, without scrap-
ing away at any place, if it can be helped, the lower
stained part which is adherent to the slide. The
further preparation consists in covering the section
writh a thin layer of dammar varnish (oil of cloves
is not requisite); after which it may be examined
with the microscope, and, if it appears satisfactory,
a cover- glass added.

There is hardly any limit to the size of which a
section of the brain may be obtained by this method,
and, if due care be taken with the planing down of
the section, it is in nearly every case attended with
success. In addition to exhibiting nerve-cells, the
method is useful for following the course of tracts
of nerve-fibres in the nervous centres, since, owing
to the depth of staining of the axis-cylinders, the
nerves may be traced for considerable distances.

21*

246 PRACTICAL HISTOLOGY.

CHAPTER XX.

THE ORGANS OF THE SENSES — THE EYE.
THE EYELIDS, SCLEROTIC, AND CORNEA.

THE study of the eye should be made as much as
possible from that of the human subject, for there
are slight differences in the structure of some of the
parts in man and animals, and, moreover, it is on
the whole easier to demonstrate the structures in
the human eye. On the other hand, there is no
organ which it is so absolutely essential to obtain
in a perfectly-fresh condition. For this and other
reasons it is scarcely possible to get material from
the post-mortem room, and the only opportunities
that usually present themselves occur when an eye
is removed on account of some injury or disease
which is confined to a particular part ; the other
intact portions may in such cases be available for
histological purposes. In rare instances an entire
healthy eye has to be removed (in operating for the
removal of extensive rodent ulcers of the brow and
face this may be necessary), and if the student should
be able to procure such an one, the following would
be perhaps the best way to deal with the excised
organ with the view of making the best use of it :
As soon after removal as possible separate the eye,
by an oblique cut with a very sharp knife or razor,
into two halves, an anterior and a posterior ; the
cut to start from just behind the attachment of the
iris anteriorly and superiorly, and pass downwards
and backwards towards the posterior part of the
organ, coming out a little below the yellow spot
and optic nerve. Then put the posterior part, after

THE EYE. 247

allowing the vitreous humor to fall away from the
retina, into 2 per cent, solution of osmic acid, and
the anterior part into Miiller's fluid. The cornea
is to be cut through at one place with a sharp scalpel,
so that the preservative fluid may get freely into
the anterior chamber.

The piece in osmic acid is left there for eight
hours ; it is then placed in water for two hours, and
finally transferred to a mixture of equal parts of
glycerine, alcohol, and water ; in this it is to remain
for a week or more, until wanted.

The other piece is to lie in Miiller's fluid a fort-
night, changing the fluid one or twice ; it is then
placed in water for two or three hours, then 'in weak
spirit for a day or two, and finally preserved in
strong spirit. Of the lower animals, the eyes of the
pig serve best for exhibiting the minute structure,
especially of the retina. In this animal the eye
corresponds more closely in point of size, and ap-
proaches more nearly in structure to the human eye
than that of the ox or sheep, the other animals the
eyes of which are usually readily procurable.

Preparation 1. The eyelids. — These are studied
by making sections of the hardened lid across its
long axis and vertically to its surfaces. The lid
may be obtained from a still-born child, preferably
one the bloodvessels of which have been injected.
It is to be hardened in spirit and embedded, and
the sections — which present no unusual difficulty —
stained with logwood and mounted in dammar. In
this way almost all the parts are well displayed ;
the skin with its epidermis on the outer side, and
with a few small hairs and sweat glands seen here
and there; the mucous membrane (conjunctiva) on
the inner side ; the Meibomian glands cut along the
length of their wide, straight duct, with their round
saccules lined with epithelium cells (of a whitish
glistening appearance, due to the fatty secretion
they contain, and which also fills the duct); the
eyelashes with their large hair-follicles and seba-

248 PRACTICAL HISTOLOGY..

ceous glands ; the cut ends of the very small muscu-
lar fibres of the orbicularis arranged in groups, and
separated by connective tissue ; and the general con-
nective tissue which serves to unite all the parts
together, and, becoming denser towards the inner
surface, forms the so-called u tarsal cartilage," long
described as composed of fibro-cartilage, in reality
containing no cartilage-cells.

Preparation 2. The lachrymal gland is pre-
pared in the same way as the salivary glands.

Preparation 3. The substance of the scle-
rotic.— The sclerotic is studied by means of sections
made from an eye that has been hardened either in
spirit or in chromic acid and spirit, the sections
being stained with logwood. There is nothing spe-
cial about these preparations, and they present no
particular interest. But covering the outer surface
of the globe is a loose connective tissue membrane,
the capsule of Tenon, composed of two apposed
layers, lined by epithelioid cells which bound a
lymph space, and covering the inner surface of the
sclerotic is another delicate lamella of loose connec-
tive tissue, closely adherent to the fibrous substance
of the coat, and of a brown appearance, due to the
presence of pigment. This layer (the lamina fusca)
is also bounded internally by a layer of epithelioid
cells, and is separated from a similar lamella (the
lamina suprachoroidea) on the outer surface of the
choroid coat by another lymph space, traversed here
and there by the vessels and nerves as they pass ob-
liquely across it from the sclerotic to the choroid.

Preparation 4. Capsule of Tenon.— To exhibit
the epithelioid cells of the capsule of Tenon a fresh
eye is taken, and the adhering orbital fat, and every-
thing but the insertions of the eye-muscles, removed
from the globe, leaving the loose connective tissue
membrane. The eye thus cleared is rinsed in dis-
tilled water, and a few drops of nitrate of silver
solution are poured over the posterior part. After
three minutes the silver is rinsed off again by a

THE EYE. 249

stream of distilled water, and the eye is placed in
water in the sunlight. When sufficiently stained it
is removed from the window, fastened under water
to a loaded cork by a long pin passed through the
cornea, and a piece of the capsule of Tenon is dis-
sected off the globe, floated flat on to a slide, and
removed from Ithe fluid. After the excess of water
has been got rid of, the piece is covered in glycerine
and examined for the epithelioid markings.

Preparations 5 and 6. Lamina fusca.— The
epithelioid layer lining the lamina fusca is also pre-
pared by nitrate of silver. A square piece of the
sclerotic is dissected off from a fresh eye ; the convex
outer surface of the piece is then pressed in and made
concave, the previously concave inner surface being
made the convex one, and the piece is first dipped
in water, then placed for two minutes in silver solu-
tion, then, after being again rinsed in water, trans-
ferred to spirit and placed in the light, with the
inner surface or lamina fusca uppermost. After half
an hour, by which time even in diffused daylight
the silver will probably be reduced, although owing
to the natural brown color this cannot well be seen,
it is removed, and in twenty-four hours, or when
hard enough for the purpose, sections are made from
the brown surface, placed in water, and mounted in
glycerine. The pigment cells usually obscure the
silver markings to a certain extent. This inconve-
nience can be obviated by using the eye of an albino
rabbit. Here, moreover, the sclerotic is not too
thick to admit of the piece being mounted entire
in glycerine ; the immersion and hardening in spirit
are then not necessary, for the piece may be exposed
in water to the light, and mounted without further
preparation, two or three radial slits being made in
it if necessary with the object of causing it to lie
flat on the slide.

Besides this preparation of its epithelioid layer
the lamina fusca may itself be displayed in an eye,
or portion of an eye, that has been prepared with

250 PRACTICAL HISTOLOGY.

Miiller's fluid. To obtain it a small piece of the
sclerotic is pinned to a cork or wax-cake under weak
spirit (equal parts of water and spirit) ; and the
lamina fusca is dissected oft' from its inner surface,
and floated on to a slide, the spirit being then al-
lowed to evaporate so as to leave the delicate mem-
brane moistened only with water. The preparation
may now be covered, and glycerine added at the
edge of the cover-glass.

Preparation 77 Sections of cornea,— The seve-
ral layers of which the cornea is composed, and their
relative thickness, should first be studied in sections
made vertically to its surfaces. For this purpose
the anterior part of an eye (pig's or ox's if a human
eye is riot procurable) is placed in 2 per cent, solu-
tion of bichromate of potash for fourteen days.
(Miiller's fluid may be used instead, but, for the
cornea, possesses no advantage over the simple bi-
chromate, although, if the retina is to be examined,
Mailer's fluid should be employed.) It is as well to
remove the lens so that the solution passes freely to
the posterior surface of the cornea. After the time
specified the tissue is put into weak spirit for twenty-
four hours, and then transferred to strong spirit.
In two or three days more it will be ready for mak-
ing sections. For this purpose a piece of the cornea
is cut out and embedded in wax-mass. Very thin
sections vertical to the surface are to be made,
stained in logwood, and mounted in dammar. In
this process a source of difficulty is sometimes met
with, in the curling up of the posterior part of the
section when transferred from spirit to oil of cloves,
after having been stained. This can sometimes be
got rid of without spoiling the section, by careful
manipulation with needles ; but, if it be found im-
possible to obviate it in any other way, the plan
may be adopted of placing each section, after it has
as usual been stained with logwood, and rinsed in
water, in absolute alcohol for a few minutes, trans-
ferring it to a slide, and immediately covering it with

THE CORNEA. 251

the thin glass. Oil of cloves is then allowed to run
under and clarify the specimen, which is prevented
from curling up, in consequence of the pressure of
the cover-glass.

Preparation 8. Epithelium of the cornea, —
The stratified epithelium covering the front of the
cornea is well seen in the vertical sections, but the
characters of the individual cells which compose it
must be studied in a teased preparation. For this
purpose a piece of the cornea is placed in a compara-
tively large quantity of J- per cent, bichromate of
potash solution, and allowed to remain in this for a
week, changing the fluid once or twice during that
time. Then with the point of a scalpel, or spear-
headed needle, a small portion, including however
the whole thickness of the epithelium, is scraped off
the front, placed in a drop of distilled water on a
slide, and broken up with needles as finely as pos-
sible. A piece of hair is added, and lastly the
cover-glass, and the specimen is then ready for ex-
amination. The cells of the various layers will be
recognized by the characteristic forms they present ;
those of the deepest layer being in shape like a rifle-
bullet, those next above cupped to receive the
rounded or conical ends of the deeper cells, and the
superficial layers being more flattened as they are
nearer the surface. The fine ridges and furrows on
many of the cells can be distinctly made out with
a high power, and give a jagged contour to the cell.

To preserve the preparation permanently in glyce-
rine it should first be stained with logwood. This
is readily done by applying a drop of a very weak
solution to the edge, and allowing it to diffuse under
the cover-glass; after a short time glycerine is added
at the same edge, and gradually replaces the log-
wood solution, the water from which evaporates
meanwhile at the other borders of the cover-glass.

Preparation 9. The substantia propria of
the cornea, — The fibrous structure of the sub-
stantia propria of the cornea can readily be seen by

252 PRACTICAL HISTOLOGY.

teasing out either a fresh cornea, or one which has
been macerated for a while in weak bichromate of
potash, or in picric acid. The lamellse which the
fibrous bundles form are apparent when an attempt
is made to tear the corneal tissue, and they are well
seen, cut in different directions, in the vertical sec-
tions previously made.

The corneal corpuscles are visible in the sections
as mere lines, each with an enlargement in the
middle, stretching across the containing cell-space,
which is fusiform in section, and is seldom filled by
the included corpuscle. These appearances are best
observed in the human cornea, but may also be
made out in that of the pig and those of other ani-
mals. But although they look like mere lines in
vertical section, they are, like most other connective
tissue cells, in reality flattened out conformably to
the surfaces of the lamellae, and present when viewed
flat great irregularities of form, and numerous
branching processes with which they come in many
cases into connection with one another. They are
best brought to view by the gold method, and,
since this also serves to show the nerves, the two
structures may be studied in the same preparation.

Preparation 10. Corpuscles and nerves of
the frog's cornea. — The brain and spinal cord of
a frog having been destroyed, the animal is laid on
the table or held by an assistant, and the membrana
nictitans of the eye is seized with forceps, and en-
tirely removed by two or three snips with fine,
sharp-pointed scissors. The animal is then taken
up and held in the operator's left hand, the thumb
pressing upwards under the lower jaw, so that the
eyes are made as prominent as possible, and the
point of one of the scissor blades is inserted into the
globe of the eye, just behind the insertion of the
glistening, yellowish iris. By a series of snips made
round the eyeball at this plane, the anterior part,
with the cornea, iris, and lens, is severed from the
posterior, and removed to a watch-glass containing

THE CORNEA. 253

salt solution. Then whilst the edge of the cut scle-
rotic is held by the one pair of forceps, with another
pair the iris is seized close to the same spot, and is
easily torn away from the sclerotic, bringing the lens
with it. So that only the cornea, together with a
narrow rim of sclerotic, is now left, and since it is
floating in fluid it retains its convexo-concave form,
and all crumpling of the tissue is avoided. The salt
solution is now poured ofl', leaving, however, just
enough to float the cornea in, arid the watch-glass is
filled up with one-half per cent, of chloride of gold
solution. The cornea is allowed to remain in this a
full hour; it is then removed to a beaker of water
acidulated with acetic acid, and is placed in a warm
place in the sunlight (see p. 96). After two days
the fluid in the beaker is renewed, a teaspoonful of
methylated spirit being added to prevent the growth
of fungi, and in two days more the cornea may be
taken out and prepared for the microscope. It is
first placed in a flat dish of distilled water, and the
epithelium, which is very dark and opaque, is gently
scraped off the anterior surface. This done, the
sclerotic rirn is cut ofl' with scissors. It is as well
to change the wrater at this stage, so as to get rid of
the debris of epithelium. The next process consists
in the separation of the corneal substance into two,
three, or more thin lamellae. With a little practice
it is not at all difficult, thin as the object already
seems, by holding it down at one edge with a pair
of forceps and working from the same edge with an-
other pair, to separate a very thin lamina from the
concave posterior surface, consisting of the membrane
of Descemet and a delicate layer of the proper sub-
stance of the cornea with its corpuscles. This poste-
rior lamella is not only the easiest to obtain, but is
also, in the frog's cornea, the most important, for it
contains the closest and finest plexus of nerves. To
mount it, all that is necessary is to float it on to a
glass slide, to cover the preparation, and add glyce-
rine at the ed«;e of the cover-glass. But. since the
99

254 PRACTICAL HISTOLOGY.

membranous layer thus obtained has naturally the
convex shape of the cornea, and it is of course de-
sirable that it should lie flat upon the slide with-
out creases, it is well before mounting to
make three or four radical snips in it in
the way shown in the adjoining cut ; these
will enable it to flatten out without fold-
ing, when placed on the slide and covered.
Moreover it is important to examine the
object with a low power previously to covering it,
so that any folds of the membrane, or any foreign
matter or remains of epithelium adhering to it, may
be detected and removed.

The remaining anterior part of cornea may be
further separated in the same way into lamellae,
which are to be mounted with the same precautions
as the posterior lamella. It is not always easy to
get them in quite so complete a layer, but for most
purposes a small shred will, if equally thin, show
nearly as much as an entire lamella.

In all these specimens the corneal corpuscles are
stained of a violet color, varying in tint according
to the success of the preparation, their nuclei being
left nearly unstained. The nerves are colored almost
black, the fibrils looking like fine wires running
singly and in bundles, and provided along their
course with numerous minute varicosities.

Preparation 11. The corpuscles and nerves
of the rabbit's cornea. — The cornea of the rabbit,
or of any other mammal recently killed, is prepared
with chloride of gold in the same way as that of
the frog. The eyelids are first removed, care being
taken in doing this not to get the hair on to the
surface of the cornea. The eye is then to be made
prominent ; either by an assistant who holds it
firmly with forceps thrust back in the orbit, so as
to seize one or other of the eye muscles near their
attachment ; or without an assistant by clamp-for-
ceps, which are inserted in like manner, and by their
weight force the eyeball forward without unduly

THE CORNEA. 255

compressing it. Then the cornea, iris, and lens are
removed together, after cutting round the sclerotic
with scissors, and are placed in salt solution, and
finally the iris and lens are removed in the same way
as in the preceding preparation, and the cornea is
immersed in gold solution. Since it is much thicker
than the frog's cornea, it should remain in the chlo-
ride of gold — which does not penetrate very rapidly
— a full hour and a half, after which it is placed in
acidulated water in the light, and otherwise treated
in the same way as the frog cornea. But as it is
not so easy to separate the mammalian cornea into
lamellae, it is better after four days to place the
stained cornea in spirit for twenty-four hours, when
thin sections parallel to the surface may be cut, and
mounted in glycerine. Embedding in wax-mass is
not necessary if the cornea is held in the left hand,
and allowed to rest over the end of the forefinger,
the razor being directed from the operator.

Preparation 12. Isolation of corneal cor-
puscles.— After the corneal corpuscles and nerves
have been stained with gold in this way, they can
be isolated by dissolving away the intermediate
substance by caustic alkali, the action being arrested
before the corpuscles and nerves, which are more
resisting than the connective tissue bundles, are
are also destroyed. With this object a part or the
whole of a gold-stained cornea — whether of frog or
mammal — is placed, divested of epithelium, in a
watch-glass containing a strong (20 per cent.) solu-
tion of caustic potash or soda, and this is then put
into a warm chamber at 40° C. At the expiration
of three-quarters of an hour the tissue, which is now
quite soft and pulpy, is removed with a section lifter,
and placed in a vessel containing a large quantity
of water faintly acidulated with acetic acid. Small
portions may then be taken up and mounted, with
or without further breaking up, in glycerine. The
corpuscles are beautifully displayed, forming a con-

256 PRACTICAL HISTOLOGY.

tinuous network by the junction of their branches ;
and nervous fibrils may be seen intercalated amongst
the corpuscles, but never actually joined to, or con-
tinuous with, the cells.

Preparation 13. Nerves of the rabbit's cor-
nea.— For exhibiting the nerves of the cornea with-
out at the same time staining the corpuscles and
the epithelium, the following modification of the
gold method may be used (Klein). The cornea of a
rabbit or guinea-pig is put into half per cent, solu-
tion of chloride of gold for an hour and a half. It
is removed from the gold into distilled water and
placed in the light (without warmth) for from
twenty-four to thirty hours, or until the larger
nerve trunks begin to be visible near the circum-
ference, converging towards the centre as irregular,
branching lines. When the staining has arrived
at this stage, and before the corneal substance gene-
rally begins to acquire a violet appearance, the
cornea is removed from -the water and placed in a
mixture of glycerine and water (one part glycerine
to two parts distilled water). After it has been in
this for twenty-four hours, or longer, the corneal
substance should be very little darker than before,
but the nerves much more distinct, and on holding
the cornea between the finger and thumb, and mak-
ing sections from the anterior surface, including the
epithelium, and a little of the substantia propria,
these, when covered in the glycerine mixture, will
show not only the fine and close plexus of nerves
which lies immediately underneath the epithelium,
but also the far more minute network of varicose
ultimate tibrils which extends between the epithe-
lium cells almost to the anterior surface of the epi-
thelium. If not at first sufficiently evident, these
intra-epithelial nerves may generally be brought
more clearly into view by placing a section for a
few minutes in the strong caustic potash solution.
From this it is transferred by a section lifter to

THE CORNEA. 257

water, floated on to a slide, removed from the water,
and covered, glycerine being afterwards added at
the edge of the cover-glass. To prevent the latter
from pressing on the softened tissue, two narrow
slips of thin glass, which may be cut with a writing
diamond, are to be placed one on either side, before
placing the cover-glass over.

Preparation 14 and 15. The cell-spaces of
the cornea. — These are shown in two ways ; by the
silver method, and by the method of puncture injec-
tion.

The demonstration of the cell-spaces by the silver
method may be attempted in the cornea of the frog.
The animal is decapitated and the brain destroyed.
The eyelid is then removed, and the eye having been
made prominent by the pressure of the thumb in
the manner previously recommended, the epithelium
is scraped off the front of the cornea with a sharp
scalpel. The cornea is then rubbed with a stick of
fused nitrate of silver (lunar caustic). After live
minutes the surface is thoroughly washed with a
stream of distilled water from a wash-bottle. The
head is now placed in spirit in the light ; in a short
time (from a few minutes to an hour), when the
cornea is browned, the vessel containing it is re-
moved from the window and left in a dark place for
twenty-four hours. The cornea is then sliced off,
placed in water, where any remaining patches of
epithelium are now removed, slit in a triradiate
manner, so that it may lie flat on a slide, and finally
mounted in glycerine.

The cornea of mammals may be prepared with
silver in a similar manner, but, being generally
thicker than in the frog, it is necessary to allow the
caustic a longer time to penetrate, and, in the final
preparation, to prepare sections parallel to the
surface, instead of mounting the cornea whole.

To inject the cell-spaces of the cornea by the punc-
ture method, the eye of the pig or guinea-pig may

be used, if a human eve is not procurable for the

go*

258 PRACTICAL HISTOLOGY.

purpose. A solution of alkanet in turpentine is the
fluid which should be chosen, and the mercurial
pressure apparatus (Fig. 30) is used. The tube and
fine steel canula having been filled with the alkanet
solution to the exclusion of air-bubbles, the canula is
inserted obliquely into the substance of the cornea,
without allowing the point to pass through into the
anterior chamber. The pressure is then gradually
raised to about two inches of mercury, when the red
fluid should begin gradually to fill the cell-spaces,
and to spread through them over a considerable part
of the cornea. Indeed, if the injection is long enough
continued, the fluid may extend itself even beyond
the cornea 1 margin, and may penetrate into the cell-
spaces in the anterior part of the sclerotic coat. The
operation may with care be successfully performed
without the mercurial apparatus, using merely a
Pravaz syringe. But it is difficult to avoid the pro-
duction of extravasation near the point of the canula.
This does not, however, always militate against the
success of the experiment, for beyond the limit of
the extravasation the fluid may slowly penetrate
into the cell-spaces of the tissue, and this may go
on even after the syringe has been removed, espe-
cially if the cornea is cut out, laid flat on a slide,
and allowed to dry. As watery fluid becomes with-
drawn from the cell-spaces in the process of drying,
the alkanet solution tends to pass in to occupy its
place.

Another very successful plan of inducing the in-
jecting fluid to pass from such an extravasation into
the neighboring cell-spaces consists in gently stroking
from the extravasation towards the margin of the
cornea with a smooth instrument, such as a glass
rod or the ivory handle of a scalpel. But if too
great pressure is exerted upon the fluid it will be
found that, in place of taking the closely reticulating
course which it would pursue if it merely occupied
the cell-spaces, the injection tends to shoot through
the tissue in straight lines, which in successive planes

THE COKNEA. 259

of the cornea! tissue run at right angles to one an-
other. These lines represent the " corneal tubes" of
Bowman. Their appearance is due to the fact that
the pressure exerted has been sufficient to force the
injecting fluid into the interstices between the con-
nective tissue bundles, pushing these asunder, and
burrowing its way through the soft ground substance
which unites the bundles and lamellae. And, since
the cell-spaces occur in this ground substance, the
existence of a slight enlargement or fusiform swelling
here and there on the tubes is accounted for.

These corneal tubes then are to be looked upon as
purely artificial products, not corresponding with
any pre-existing channels in the tissue (except per-
haps when the fluid passes along the sheath of a
nerve). They are always obtained when any fluid
which is not able to penetrate into the cell-spaces is
forced into the substance of the cornea. They are
seen when mercury is injected by the puncture
method, and this is how they were first obtained by
Bowman; and if air be forced with a syringe into
the tissue a similar effect is produced. In all cases
the tubes cease abruptly at the corneo-sclerotic junc-
tion, where the connective tissue becomes denser and
has a less regular arrangement.

Preparation 16. Parts at the junction of
the cornea with the sclerotic. — The corneo-scle-
rotic junction, the ciliary muscles, and the iris are
all well seen in their relations to one another in a
meridional section of the part of the eye where they
are situated. The section may be made from the
anterior segment which we have assumed to have
been hardened in M tiller's fluid and in spirit (p. 247).
It is not necessary to embed this entire, but sufficient
to cut out with sharp scissors under spirit a piece
which includes all the parts above enumerated. The
piece is first placed in oil of cloves for a few minutes;
it is then transferred to cacao-butter, which is kept
just melted over a sand-bath, and is allowed to re-
main in this for fully two hours, so that the butter

260 PRACTICAL HISTOLOGY.

may soak into every part of the tissue. Meanwhile
a paper mould of the usual form is filled with the
melted cacao-butter, which is allowed to become
solid, and in the cake thus produced an excavation
is made near one end large enough to receive the
piece to be embedded which is placed in the hollow
in such a position that radial sections vertical to the
surface may be made, and the excavation is then
filled up with melted cacao-butter. This in harden-
ing sets into one piece with the rest of the cake, and
in cutting the sections the advantage is gained that
all the parts have exactly the same density and offer
a uniform resistance to the razor. Much thinner
sections may therefore be made by this method than
by any other. The sections, as they are obtained,
are placed in oil of cloves, which slowly dissolves the
cacao-butter out from the tissue ; the process can be
accelerated by slight warmth. They are next placed
in alcohol to remove the oil of cloves ; then for a few
minutes in Kleinenberg's logwood; and are finally
passed again through alcohol and oil of cloves, to be
mounted in dammar varnish in the usual way. Few
specimens better repay the trouble of preparation
than these. The cornea, sclerotic, iris, choroid, liga-
menturn pectinatum, canal of Schlemm, ciliary mus-
cle, both radial and circular, and even the ora serrata
and pars ciliaris of the retina are all exhibited with
the greatest clearness in a successful section, and
their structure arid relations may be advantageously
studied.

THE CHOROID AND IRFS.

The choroid coat is to be prepared from an eye
hardened in Miiller's fluid. Besides the main sub-
stance of the coat containing the larger bloodvessels,
the lamina suprachoroidea, thechorio-capillaris, and
the membrane of Bruch, should all be separately
displayed. The posterior attachment of the fibres
of the ciliary muscle, and the gangliated plexus of

THE CILIARY MUSCLE. 261

nerves which is found in the neighborhood of its
posterior attachment, will be exhibited with the
lamina suprachoroidea.

Preparation 17. Ciliary muscle and lamina
suprachoroidea, — To prepare these, the anterior
part of the eye is to be pinned down under spirit,
and the cornea and sclerotic cut away at one part,
when the radiating fibres of the ciliary muscle will
be seen passing meridionally from their origin
opposite the attachment of the iris, and forming a
layer which becomes gradually thinner as it extends
backwards and finally ceases in the superficial part
of the choroid. A small piece of the muscle is
seized with sharp forceps near its origin, and, by
carrying the instrument slowly backwards, is gradu-
ally torn away from the rest. It will be found that
the shred which comes away generally spreads out
posteriorly into a very thin membranous lamina,
this being in fact a piece of the lamina suprachoroi-
dea into which the superficial fibres of the ciliary
muscle are inserted. A considerable length may be
torn off in this way, and the piece so obtained is to
be floated directly on to a slide, Avhich is dipped for
the purpose into the spirit. It must be moved with
great care, so as to avoid folds or creases. The slide
is quickly wiped free of spirit, with the exception of
that which immediately moistens the specimen, and
a drop of freshly-filtered logwood placed upon the
tissue, and allowed to remain on it for ten minutes.
The staining solution is then poured off,, and the
remains of it are removed by allowing a drop or two
of water to flow gently over, without disturbing the
position of the membrane. Finally a cover-glass is
laid on, and a drop of glycerine allowed to run in at
the edge of the cover-glass.

The preparation so obtained is, if successful, a
very striking one. Besides the branched pigment
cells of the choroidal tissue, and a certain number of
cells, similar to white blood corpuscles, 911 the sur-
face of the membrane, a number of large, round or

262 PRACTICAL HISTOLOGY.

oval nuclei are seen in the lamina, which are appa-
rently devoid of cell-body. These are in reality the
nuclei of epithelioid cells which bound the lamina
suprachoroidea externally, and serve as part of the
lining of the lymphatic space which lies between
this and the lamina fusca of the sclerotic. Their
outlines cannot be brought to view without the aid
of nitrate of silver, and the cell-bodies are too deli-
cate and transparent to be shown by the present
mode of preparations. A large number of elastic
fibres are also seen on the membrane, especially at
the terminations of the fibres of the ciliary muscle,
where they appear to come in relation with the ends
of these, an elastic fibre passing for a certain dis-
tance along each side of the muscular fibre, and
seeming to serve in this way for its attachment.
The involuntary fibres are particularly well shown,
their nuclei being conspicuously stained by the log-
wood ; many of the bundles terminate in peculiar
tufts, from which the fibres radiate in all directions.
It may happen that the preparation includes one of
the long ciliary nerves ; this, as it coursed forward
to enter the ciliary muscle, having been torn away
with the shred of membrane. If so, it may be fol-
lowed with the microscope and its branches traced
amongst the bundles of muscular fibres, forming a
plexus with those of the other nerves. In tracing
the branches characteristic ganglion-cell enlarge-
ments will here and there be found interpolated in
the course of a nerve fibre.

Preparation 18. Vascular layers of choroid
and membrane of Bruch. — As seen in the prepara-
tion just described, it is easy to detach the lamina
suprachoroidea from the rest of the choroid. The
other three parts are more difficult to separate, and
their complete isolation may require considerable
time and patience. But for demonstrating their
structure it is not absolutely necessary for them to
!><• completely separated as distinct membranes ; it is
sufficient if, in a piece which includes all, one or

THE IRIS. 263

other is left projecting at the edge, so as in this way
to be seen distinct from the other layers. But before
commencing the attempt at separation, the hexago-
nal pigment cells, which belong to the retina but
frequently adhere to the inner surface of the choroid,
must be entirely removed by gently brushing that
surface with a hair pencil. The separation and
brushing are performed under fluid (spirit), and will
be much facilitated by the use of a dissecting lens.

Preparation 19. The musculature of the
iris. — The circular and radiating plain muscular
fibres of the iris may be demonstrated in the albino
rabbit. The eye is cut in half and the anterior part
placed in spirit for a day or more. Then the lens is
removed, and the segment of the iris — including its
whole width, from the pupillary aperture to the
ciliary processes of the choroid — is cut out and
placed in dilute logwood. When sufficiently but
not too deeply stained, it is put into water for a
minute or two to remove the excess of staining fluid,
then passed through spirit and oil of cloves, and
finally mounted in dammar, with the posterior sur-
face uppermost. The thick ring of the sphincter is
easily seen in these preparations, and also the inter-
lacing bundles of plain muscular fibres of which tjrie
dilatator is composed ; they may be observed to bend
round near the pupil, and take the direction of and
blend with those of the sphincter. At the circum-
ference of the iris also, a similar bending round of
the radiating fibres is observed.

Preparation 20. Human iris. — Although, in
consequence of the presence of the uveal pigment,
more troublesome to prepare, it is nevertheless desir-
able to make a similar preparation of the human
iris, for the musculature is somewhat different, the
dilatator forming a uniform thin expansion, which
covers the posterior surface immediately under the
pigmented epithelium, and not distinct radially ar-
ranged interlacing bundles with intervening meshes
as in the rabbit. The specimen may be made from

264 PRACTICAL HISTOLOGY.

an eye that has been in Miiller's fluid or bichromate
of potash (2 per cent.), and subsequently in spirit.
A piece is cut out as before, and is treated in a simi-
lar way, except that before being stained the pigment
is brushed completely off the posterior surface with
a stiff camel-hair brush. This must be done under
spirit, and of course very carefully so as to avoid
tearing the tissue; during the operation the iris is
examined now and then with a low power, to deter-
mine when all the pigment is removed. It is diffi-
cult to prevent some of the pigment granules from
still sticking to the surface, but, as they tend for the
most part to adhere along the lines of junction of
the fibre cells, their presence does not spoil the object
of the preparation, for the dilatator fibres are if any-
thing better displayed.

THE RETINA.

Preparations 21 and 22. Sections of the
retina. — It will be well to study the general ar-
rangement of the several layers of the retina by means
of sections, before its constituent elements are ob-
served isolated. Two methods of hardening may be
recommended, viz. by Miiller's fluid and by osmic
acid, the process being in both instances completed
by spirit. If possible to obtain it perfectly fresh and
healthy, the human retina should always be used ;
if not so obtainable, that of the pig is preferable to
the retina of most other animals, and in the follow-
ing preparations it will be assumed that the eyes of
that animal are employed.

To harden the retina in Miiller's fluid it is better
to keep the bulb whole, so that the membrane re-
mains supported by the vitreous humor. But to let
the fluid in readily two or three cuts must be made
in the sclerotic with a razor, or a piece even be cut
out at one or two places. The eye is then dropped
into a relatively large quantity of Miiller's fluid, and
allowed to remain in it for a week, the fluid being

THE RETINA. 265

stirred at intervals ; the globe is then cut across, and
left for another week in the liquid, after which time
it is transferred first for twenty-four hours to weak,
and then to strong spirit. In another day or two
it is ready for the preparation of sections. Small
pieces from different regions may be taken, but as
they are all treated in the same way, they may be
described as one: —

The staining of the tissue is first effected by
placing the piece for twenty-four hours in alcoholic
logwood (Kleinenberg's). From this it is transferred
to spirit, then embedded in wax-mass. It is better
so to place it in the embedding tray that the sections
shall be both vertical and meridional ; since, made
in this way, they will take the general course of the
fibres of the optic nerve. The sections cannot be
made too thin, but they should be complete, that is
to say, they should include all the layers of the
membrane. One or two of the thinnest are to be
mounted in glycerine ; the others may be transferred
from the spirit to oil of cloves, and mounted in
dammar. In these preparations the nerve fibres,
the Mullerian fibres, the inner and outer granules,
and in fact the layers generally, are all well dis-
played, but the rods arid cones are much altered, and
the natural form and appearance .of the other elements
is by no means well preserved.

In this respect the osmic method yields far better
results. The bulb in this case is cut open, and either
the whole or a piece only of the retina is taken arid
placed in 2 per cent, solution of osrnic acid. Here
it is left for several hours, and then, after being an
hour or two in water to get rid of the excess of
osmic acid, it is transferred to alcoholic logwood for
twenty -four hours. From this it is placed in strong
spirit, where it may be left until wanted. It is well
to embed the piece by the cacao-butter process, since
by hardly any other method can sufficiently thin
and complete sections of a retina that has been
hardened in osmic acid be obtained. For this pur-
23

266 PRACTICAL HISTOLOGY.

pose it is as usual first placed in oil of cloves, and
then allowed to soak for half an hour or more in
cacao-butter, which is kept just melted by a gentle
heat. When completely permeated the piece of
membrane is placed in the proper position in a
cavity scooped out in a cake of cacao-butter, and
after it has firmly set, sections mny without diffi-
culty be made of exquisite fineness. They are placed
in oil of cloves — in winter it must be very slightly
warmed — which speedily dissolves out the infiltrated
cacao-butter, and they may then be mounted in
dammar. But it is better to transfer them from
the oil of cloves to spirit again, and afterwards to
mount them in glycerine, since in this the details
can be made out with greater readiness than in the
more highly refracting dammar. For the same
reason a saturated solution of acetate of potash is
sometimes recommended as superior to glycerine
for mounting osraic preparations, especially of the
retina.

Preparations 23, 24, and 25. Isolation of the
retinal elements. — Various processes are employed
for macerating portions of the retina in order to
obtain its elements, either in a completely isolated
condition or still partially connected with one
another. It will be best, in the first place, to at-
tempt the separation with a piece of retina which
has been, in 2 per cent, osmic acid for six or eight
hours. It must of course be put in perfectly fresh,
and after the time mentioned it is placed in water
for twenty-four hours. It is then allowed to mace-
rate for a few days in a mixture of glycerine, alco-
hol, and water (glycerine one part, alcohol one part,
water two parts), after which a minute portion is
to be carefully broken up with fine needles in a
drop of weak glycerine, and, a piece of hair having
been added, the cover-glass is superposed, and the
preparation examined with a dry objective and,
after fixing the cover-glass, with an immersion.
The effect of the osmic acid is to preserve the more

THE RETINA. 267

readily alterable elements, such as the rods and
cones and their nuclei, in a condition as nearly as
possible approaching that which they possess during
life.

Other portions of fresh retina are to be placed, one
in one-eighth per cent, of bichromate of potash solu-
tion (for a week), and the other in ten per cent, of
chloral hydrate solution (for two or three days). The
portions so macerated are to be teased out in a drop
of their respective solutions, the usual expedient
beinoc adopted of obviating the pressure of the cover-
glass" by a hair. In the one treated with bichromate
the Miillerian fibres, and in fortunate preparations
the ganglion cells with their processes, may be well
seen'and isolated. The granules of the inner nuclear
layer are also frequently obtained with one or both
processes extending from their opposite poles, but the
rods and cones are for the most part much altered
and granular in appearance, so as hardly to be recog-
nizable.

In the chloral hydrate preparation, the last-named
elements are much better preserved. The external
segments of the rods, which even in the osmic pre-
paration tend for the most part to become altered,
may by this method be frequently seen almost
unchanged, except that the transverse striation,
which indicates their discoid formation, is often
well marked. Most of the other structures are also
well preserved, and the Miillerian fibres and ganglion
cells are sometimes better seen than in the bichro-
mate preparation. Unfortunately neither the bichro-
mate nor the chloral specimens can be well preserved
without deterioration.

Preparations 26 and 27.— The study of the
retina cannot be considered complete until the ele-
ments have been examined in the fresh, unaltered
condition. A small piece, taken from an eye still
warm from the animal, should accordingly be broken
up as rapidly and finely as possible in a drop of
serum, or, failing this, in a little vitreous humor.

268 PRACTICAL HISTOLOGY.

A modification of this very simple method consists
in allowing a small piece to macerate for two or
three days in weakly iodized serum (see p. 45) before
attempting the dissociation, which can then be more
readily effected.

Preparation 28. — The hexagonal pigment of the
retina is seen in most of the teased preparations
above described. In eyes that have been hardened
in Muller's fluid the layer often separates in flakes
of varying size, and nothing is simpler than to re-
move such a piece with a section lifter, and mount
it in glycerine, so as to exhibit the pavemented
appearance which the cells present.

Preparation 29. — On the opposite surface of the
retina also a mosaic-like appearance can be demon-
strated by the aid of nitrate of silver, but it is much
more irregular, and does not depend upon the pre-
sence of epithelium cells, but upon the flattened-out
ends of the Miillerian fibres. To show this appear-
ance a fresh eye is cut in half transversely, and the
vitreous is shelled out from the posterior half; this
is then rinsed in distilled water and transferred to
nitrate of silver solution (one-half per cent.). After
a minute in this it is again rinsed in distilled water,
and exposed in water to the light. When the sur-
face is browned it is removed, a piece of the retina
is cut out under water, floated on to a slide with the
inner, brown surface uppermost, and the water is
drained off and replaced by glycerine, when the pre-
paration can be covered and examined, care being
taken not to let the cover-glass press upon the speci-
men.

Preparation 30. The Retina in the lower
Vertebrata. — The structure of the outer segments of
the rods, which it is difficult to make out in mammals,
can be seen easily, even with an ordinary high power, in
the retina of amphibia. With this object the eye is re-
moved from a recently killed frog, cut across, and a small
portion of the retina is quickly broken up in vitreous
humor. A piece of hair having been added, the prepara-
tion is covered and examined. Almost everywhere the

THE LENS. 2G9

field of view is strewed with large, clear, rod-shaped struc-
tures, some straight, but many of them bent and curved in
different directions, and exhibiting a distinct transverse
striation, or even a tendency to split up into a number of
superimposed disks, this tendency increasing as the pre-
paration is longer made. Some of them have what looks
like a small appendage jointed on at one end, but this is
really the comparatively small inner segment of the rod.
The cones are also very small in comparison, and on that
account may at first be" missed ; they are distinguished by
the possession at the apex of the inner segment of a small,
bright, fatty globules, often of a yellow color. Most
likely a portion of the hexagonal pigmented epithelium
will have come away with the rest of the retina, and in
consequence of the rupture of some of the cells the prepa-
ration will be strewed with pigment granules which, like
all minute granules suspended in fluid, exhibit very strik-
ingly the Brownian molecular movement. Some of the
pigment cells may be observed intact, either isolated or
in patches. If seen in profile, it may be noticed that near
one surface (the outer) the cell is almost entirely free from
black pigment, while from the other fine streamers of the
cell-protoplasm, dotted with pigment granules, extend.
In their natural position these pass between and amongst
the outer segments of the rods.

Preparations 31, 32, and 33.— The retina of a bird,
of a reptile (tortoise), and of a fish are to be teased out
fresh in vitreous, in the same way as that of the frog.
The chief points of interest in these preparations are the
ellipsoid or lenticular bodies in the inner segments of the
rods (bird and amphibian) and cones (bird, reptile, and
amphibian), the bright, fatty globules of different colors
in the inner segments of the cones in the tortoise and
bird, and the twin or double cones, especially large in the
fish's retina. The various other points in which the retina
in these animals differs from that of mammals, may be
studied by employing the same methods of preparations
as for the mammalian retina.

THE LENS AND VITREOUS HUMOR.

Preparations 34. The lens fibres.— The fol-
lowing will be found the best mode of isolating the

23*

270 PRACTICAL HISTOLOGY.

fibres of the lens, as well as for showing their ar-
rangement. Take the fresh eye of any animal —
that of the ox or sheep for example — and cut it
across into an anterior and a posterior half. Place
the anterior part, having removed what remains of
the vitreous humor, in J per cent, solution of bi-
chromate of potash. Then scratch through the
posterior capsule, which is readily ruptured and
curls away from the lens proper. This can easily be
shelled out, and is left in the fluid, the remainder
of the eye being rejected. The lens is allowed to
remain in the bichromate for two or three days,
being merely turned over once or twice. It will be
found that its substance tends both to separate along
the radiating lines which mark the planes of junc-
tion of the ends of the fibres, and also to peel into
concentric lamellae like the coats of an onion ; and,
if a piece of one of these lamellae is taken up with
the forceps, it will tear in the direction of the fibres
from one of the planes of junction of the anterior
surface to the corresponding plane of the posterior.
The fibres can be readily separated with needles in
a drop of the bichromate solution. For this pur-
pose portions should be taken both from the super-
ficial and from the more central parts of the lens.
In many of the superficial fibres a round or elon-
gated nucleus may be detected at one part, and since
the nuclei of adjacent fibres are met with in about
the same region, when a number of fibres are seen
together the nuclei lie in an irregular row. The
riband-like shape of the fibres may be made out at
parts where they are turned over so as to be seen
edgeways.

Preparation 35. Sections of the Lens, — For
cutting sections of the lens it is best to harden it in
Muller's fluid. The whole anterior half of an eye
should be put in this, the cornea having been partly
removed so as to enable the fluid to get freely to the
front as well as to the back of the lens, but the
capsule is not to be ruptured. After two or three

THE VITREOUS HUMOR. 271

weeks, vertical sections may be made, one of which
should pass through the centre from before back, and
should be as complete as possible. They are to be
mounted in glycerine. The lens must not be put in
spirit, or at all events not in strong spirit, to com-
plete the hardening, for this renders the tissue,
especially the central parts, quite hard and horny,
and the outlines of the fibres become obliterated.

Preparation 36. The epithelium of the lens-
capsule, — This may have been seen in the antero-
posterior section as a. single row of nucleated cells,
lying immediately behind the anterior part of the
capsule. To show it on the flat it is to be stained
with nitrate of silver. With this object a lens still
inclosed in its capsule is removed from a fresh eye,
and, after having been rinsed in distilled water,
transferred for five minutes to J per cent, nitrate of
silver solution. It is then again washed with dis-
tilled water, and placed in the light in weak spirit
(equal parts spirit and water). When brown it is
removed from the light, and after twenty-four hours
the substance is hard enough to allow tangential
sections to be made from the anterior surface, which
shall include the capsule, the epithelium and the
parts of the lens substance immediately subjacent to
this. The sections are mounted in glycerine with the
brown surface uppermost: and through the elastic
capsule, which is not distinctively stained, the out-
lines of the epithelium-cells are clearly seen. At
places the silver solution may have penetrated to the
superficial lens fibres, and will be found to have
stained the cementing substance between them.

Preparation 37. The zonule of Zinn and the.
hyaloid membrane of the vitreous humor.—
Take the anterior half of the eye (preserved in spirit)
of an albino rabbit, and having pinned the cornea
downwards on a loaded cork under spirit, and re-
moved the remains of the vitreous humor, gently
seize the lens with fine forceps, and draw it away
from the iris. In doing this it will drag with it tie

272 PRACTICAL HISTOLOGY.

suspensory ligament, the zonule of Zinn, and the
part of the hyaloid membrane continuous with this,
so that the separated lens appears girdled by a deli-
cate, somewhat crumpled-looking, membranous zone,
closely adherent at its inner border to the equator of
the lens, and bounded outwardly by a ragged mar-
gin— the torn edge of the hyaloid. Cut out with
fine scissors a segment of this zone, including its
whole breadth, and with a section-lifter transfer the
piece so removed to logwood solution (Kleinenberg's).
When sufficiently stained — and it stains very readily
— transfer it to a dish of water, and from this float
it on to a slide, avoiding all creases except of course
the natural ones of the zonule. It may then be
covered, and the water in which it is mounted re-
placed by glycerine. Or, instead of placing it in the
water, it imiy be transferred from the logwood to
spirit, and then passed through oil of cloves and
mounted in dammar. These preparations exhibit
well the folds and striations of the zonule, and the
rounded corpuscles, like white blood corpuscles,
which are dotted here and there over the surface of
the hyaloid.

THE BLOODVESSELS OF THE EYE.

For the demonstration of the bloodvessels the head
of an albino rabbit should be injected, a canula being
placed in each carotid, and the two canulas con-
nected to the arras of a Y-shaped tube, the stem of
which is brought into communication by an India-
rubber tube with the injection bottle. After the
blood has been driven out of the vessels before the
flow of injection fluid, the neck of the animal, just
below the place where the canulas are inserted, is
surrounded by a loop of wire, which is drawn as
tightly as possible to prevent the escape of the injec-
tion ; and the pressure is then raised to about four
inches of mercury and kept there for some minutes,
so as to make certain that all the bloodvessels shall

BLOODVESSELS OF THE EYE. 273

be completely filled. The whole is then allowed to
stand and become cold, that the gelatine may set,
after which the eyes are to be carefully excised, and
placed in weak spirit. In twenty-four hours this
may be increased in strength, and in another twenty-
four or forty-eight hours they are to be placed in
strong spirit. When they have been in this a day
or two the following parts may be prepared : —

Preparation 387 The conjunctival vessels,
and the subeonjunctival vessels of the sclero-
tic.— By making a tangential section from the region
of the corneo-sclerotic junction, and after passing the
piece so obtained through oil of cloves, mounting it
in dammar with the outer surface uppermost, the
distribution of the vessels, both in the conjunctiva
and, by focussing more deeply, those in the sclerotic
at and near the margin of the cornea, is exhibited.
Another plan consists in cutting away a small piece,
including the whole thickness of both cornea and
sclerotic, and mounting in a similar way. The
thickness and irregularity of the piece so obtained
is a disadvantage, but, on the other hand, the canal
of Schlemm and the other venous sinuses may be
observed, if the injection has been a successful one,
by focussing still lower than for the looped vessels
of the sclerotic.

Preparation 39. Vessels of the choroid and
iris. — One of the two injected eyes is to be divided
by an antero-posterior cut with the razor into a right
and a left hand. One of the two halves, the one
which does not include the attachment of the optic
nerve, is first taken, and the vitreous, retina, and
lens removed, so as to clear the inner surface of the
choroid and iris. The last-named are next to be
separated as one piece of membrane from the scle-
rotic. The piece so obtained is then to be again
divided into two, by another antero-posterior cut
with the scissors, and the resulting halves are to be
mounted, after passing as usual through oil of cloves,
in dammar, the one with the inner and the other

274 PRACTICAL HISTOLOGY.

with the outer surface uppermost. Each includes,
of course, the fourth part of the choroid coat with
some of the ciliary processes, and a piece of the iris;
and with a low power the course and disposition of
the bloodvessels in these parts can be readily followed.
Besides these comprehensive preparations, separate
ones may be made from the other half of the eye of
a portion of the iris (this is rendered more instruc-
tive by lightly staining it with logwood), and one
or two of the ciliary processes snipped off with sharp
scissors, and mounted so as to be seen in profile.

Preparation 40. The vessels of the retina. —
If the other injected eye be cut into an anterior and
a posterior half, and the posterior part is examined
after removal of the vitreous humor, the bloodvessels
will be seen spreading out from the centre of the
colliculus of the optic nerve. To exhibit their finer
distribution in the retina, a piece is mounted fiat in
dammar without previous staining, while to show
the extent of their distribution in the retinal layers,
vertical sections, which need not be very thin, may
be made from a piece embedded in wax-mass in the
ordinary way, and similarly mounted, without stain-
ing, in dammar.

THE EAR. 275

CHAPTER XXI.

THE EAR.

THE only parts of the ear which require special
directions for their preparation are the semicircular
canals and the cochlea.

Preparations 1, 2, and 3. The semicircular
canals. — To study the structure of the membranous
semicircular canals, those of the cartilaginous fishes,
e.g. the skate, are chosen. • The skull, which can he
readily cut with a scalpel or strong pair of scissors,
is opened quite anteriorly, where it is occupied
merely by a quantity of cerebro-spinal fluid, and
the opening is extended backwards by removing
the roof bit by bit, until the whole of the upper
surface of the brain is exposed. Two thick carti-
laginous masses will be seen, one on either side, near
the posterior part ; the large auditory nerves pass
through a foramen in each into their interior.
These masses inclose the membranous labyrinth,
consisting in these animals of utricle, 'saccule, and
semicircular canals, all of large size, and contained
in corresponding cavities and canals, in the sub-
stance of the cartilage, but of which no trace can
at present be made out. If, however, horizontal
slices are made with a scalpel, one of the canals will
soon be exposed, and this can then be followed in
both directions, cutting the cartilage away so as to
expose the included membranous canal in its whole
length. It will be found to lead at either end into
a large membranous bag — the utricle — with which
the two other canals also communicate, and from
which they can be traced in the same manner.
Besides the utricle, there is another smaller mem-

276 PRACTICAL HISTOLOGY.

branous bag — the saccule — and both contain a white,
pasty, cretaceous, otolithic mass, which lies over the
part to which the nerve proceeds. ISTear one of the
attachments of each semicircular canal to the utricle
is its dilated part, or ampulla, and a branch of the
auditory nerve may be seen proceeding to each of
these, and terminating abruptly in a forked thicken-
ing, which indents the membranous wall and lies
transversely to the axis of the ampulla.

The three ampullae, and the adjacent portions of
the semicircular canals, are now to be removed from
the cavities containing them, and are to be placed,
one in a weak solution of chromic acid (-J- per cent.),
one in osmic acid (2 per cent.), and the third on a
slide in a drop of endolymph obtained from the
cavity of the utricle. The piece in chromic acid is
transferred to weak spirit after three days, and in
twenty -four hours more to strong spirit. After
another day or two it may be placed in Kleinen-
berg's logwood for several hours, and then embedded
either in wax-mass in the ordinary way, or by the
cacao-butter process. Sections are to be made both
of the semicircular canal proper, and of the ampulla,
opposite to and including the entrance of the nerve ;
and the sections are passed through oil of cloves,
and mounted in dammar. The piece in osmic acid
is transferred after twenty-four hours, first to water
for two or three hours, and then to spirit ; after a
day or two in this it may be placed in oil of cloves,
and subsequently permeated with and embedded in
cacao-butter. The sections, after the cacao-butter
has been removed by oil of cloves, and this again
by spirit, are finally mounted in glycerine. The
third piece, especially the part where the nerve
enters, is broken up at once in the drop of endo-
lymph, and examined with a high power, with the
view of observing the two kinds of epithelium cells —
columnar and spindle-shaped — which occur here, and
the stiff, hair-like projections which are attached to

THE COCHLEA. 277

them. The demonstration of these structures pre-
sents, however, the greatest possible difficulty.
Preparation 4. Sections of the cochlea.—

On account of the thinness of its osseous parietes,
the ease with which it may be obtained separate
from the surrounding bone, and its comparatively
large number of spiral turns, the cochlea of the
guinea-pig offers far greater facilities for study, and
especially for the preparation of sections, than that
of any other animal. The following is the mode of
finding and procuring it: In the recently-killed
animal the aperture of the mouth is prolonged back-
wards on either side, by cutting through the cheeks
and temporal muscles with strong scissors. The
lower jaw is then seized and forcibly torn away
from the rest of the head, so that the base of the
skull is exposed. Here will be seen on either side,
just behind the fossa for the articulation of the con-
dyle of the jaw, a large white bony projection — the
tympanic bulla. This is not yet to be opened, but
the cartilaginous external auditory meatus is first cut
tli rough, and with the aid of bone-forceps or strong
scissors, the bulla in question, together with the
petrous bone to which it is attached, separated from
the rest of the skull. In a young animal this can
be readily effected, simply by inserting a strong
blunt instrument into the base of the skull just in
front of the bulla, and using it as a lever, raising
the bone and forcing it away from its attachments.
The bones of either side being thus removed, the ad-
hering soft parts are cleared away, and the bulla is
broken open at its most prominent part. On now
looking into the cavity there will be noticed, on one
side the delicate tympanic membrane stretching over
the end of the external meatus, with the handle of
the malleus attached to it, and on the opposite wall
a well-marked conical projection, indeed, its bony
wall is so thin that it is possible to count the number
of turns (four) which it presents. By cutting the
bulla round with strong scissors, the two parts — one
24

278 PRACTICAL HISTOLOGY.

including the tympanic membrane, and the other
the cochlea — are separated from one another, and the
membrane part may at once be dropped into weak
spirit and put aside to be subsequently stained and
mounted. From the other part as much as possible
of the substance of the petrous bone is snipped away,
bit by bit, from around the base of the cochlea with
scissors or bone forceps, but great care should be
taken in approaching the cochlea itself, as this is
very readily split. When the surrounding bone has
been in this way removed, the cochleas are dropped
into Miiller's fluid. In this they are to be left for a
week or fortnight — even a longer immersion will do
them no harm — until the sort structures in the in-
terior are somewhat hardened. The process is then
completed, whilst the bone is at the same time
softened by transferring the cochleae to a saturated
solution of picric acid. When the bone is com-
pletely softened, a process which is much facilitated
by frequent disturbance of the fluid, the cochleas are
transferred to weak spirit (half water), and in
twenty-four hours more to strong spirit. After
beini>; in this for two or three days they are ready for
embedding. The best mass to use for this purpose
is a mixture of wax and cacao butter, equal parts of
each. One of the prepared cochleas is fixed by a pin
in the embedding box in such a position that the
plane in which the sections are made shall be exactly
parallel with the axis of the cone which the cochlea
forms, and the cacao-butter and wax, previously
melted and thoroughly mixed, are poured into the
mould. When the mass has become hard, sections
may be made ; the first ones will include only the
large basal turn of the cochlea, then the higher turns
will all be included in succession, until at last the
modiolus is reached. All the sections which have
been made up to this point may be rejected. Great
care must now be taken to make thin and complete
sections of this central part. They will of course be
triangular in shape, with a rounded apex; the sec-

THE COCHLEA. 279

tion of the modiolus occupies the axis from the base
to near the apex of the triangle, and on either side
are the sections of the successive spiral turns. Not
more than three or four complete axial sections can
l>e obtained from each cochlea. The razor and the
surface of the cake should be wetted with oil of
cloves instead of spirit. It may be applied by means
of a large camel-hair brush, and its use is that it
renders the parts more coherent, and tends to prevent
the organ of Corti from breaking away from the
basilar membrane, which it is otherwise apt to do.
The sections are examined, unstained, with a low
power, and if the organ of Corti is sufficiently com-
plete they are at once transferred to an alcoholic
staining fluid. One of the best is a weak solution of
acetate of rosaniline, but if this is not at hand, some
other aniline dye, such, for example, as eosin (see
Appendix), may be employed. When stained the
sections are again placed in oil of cloves, and finally
mounted in dammar.

Preparations 5 and 6. Teased preparations
of the cochlea. — Successful sections will show
the general position and relations of the rods and
other parts, and to a certain extent the individual
elements. But only a profile view can in this way-
be obtained, and since the minute structure of the
elements composing the organs of Corti can only
be properly seen when isolated, it is necessary to
prepare other cochleas with this object in view.
Another animal is accordingly sacrificed, and the
cochleas removed as before. One placed in a 2 per
cent, solution of osraic acid ; the other in a J per
cent, solution of bichromate of potash ; but, before
dropping them into their respective fluids, the bony
wall must be scraped through here and there with
a scalpel, so that the fluid "shall at once penetrate
to the interior of the turns. After two days the
cochleas may be further prepared in the following
way : —

The uppermost turn is broken or snipped off with

280 PRACTICAL HISTOLOGY.

scissors, placed in a drop of water on a slide, the
shell of bone which forms the cupola and outer wall
removed, and the piece of lamina spiralis examined
with a low power (without covering the preparation)
in order to learn to recognize the structures which
lie on it. The glass slide is then removed to the
dissecting microscope, and with very iine needles
the lamina spiralis is separated from the columella,
which is then rejected. Next all the parts on the
lamina, but especially the row of rods of Corti, to
which the hair-cells as a rule cling, are broken up
finely, -but at the same time slowly and carefully,
the preparation being examined now and again with
the highest power which it is safe to use without a
cover-glass. One of the chief difficulties is apt to
arise from portions of the tissue sticking to the
needles ; if this is the case, pieces of glass rod drawn
out to a fine point may be substituted. When the
more important parts have been broken up pretty
completely, any thick pieces of tissue unimportant
to the present observation, such as bits of bone, or
periosteum, bundles of rnedullated nerve fibres, &c.,
should be picked out, and then a cover-glass laid on
and the preparation examined. To preserve either
preparation permanently glycerine may be allowed
to diffuse in at the edge of the cover-glass ;' but the
bichromate specimen should first be treated with a
drop of carmine solution, so that the elements are
somewhat stained, otherwise they will be rendered
too transparent by the glycerine.

In this way a number of specimens may be obtained
from each cochlea — proceeding from above, down,
and preparing turn after turn ; and careful sketches
should be made of the different structures met with,
and their arrangement with regard to one another.
It will be found that the osmic preparations serve
best for showing the lamina reticularis and the
lamina basilaris, and the bichromate preparations
for the hair-cells and the membrana tectoria ; the
other structures are almost equally well seen in both

THE OLFACTORY ORGAN. 281

kinds of preparations. The large fat droplets in
some of the epithelium cells of the uppermost turn
are peculiar to the guinea-pig, as is also the arched
projection — seen in the sections — at the part where
these cells are found. The fat drops are stained
black in the osmic preparation.

THE OLFACTORY ORGAN.

Preparations 1-3.— Small pieces of the upper
turbinate bones, or from the upper (olfactory) region
of the septum nasi,from the dog or rabbit, are placed,
one piece in one-sixth per cent of chromic acid solu-
tion, a second in one-fourth per cent, of bichromate
of potash, and a third in one per cent, of osmic acid.
The one in chromic acid may remain a week, when
it is transferred to weak spirit and in twenty-four
hours more to strong spirit. After a day or two in
this, vertical sections are prepared from it.

The other two pieces are examined after forty-eight
hours' maceration, small pieces of the mucous mem-
brane being teased out so as to isolate the epithelium
cells (both columnar and spindle-shaped), and if pos-
sible, especially in the osmic preparations, to study
the connection of their branching lower ends with
subjacent structures. These preparations can be pre-
served with glycerine, the bichromate one being
stained with logwood.

Preparations 4 and 5. —Teased preparations
should also be made of the olfactory mucous mem-
brane of the frog or newt. Having cut oft' the head
of the animal, and slit up the nostrils with fine scis-
sors, place it in a quantity of one-fourth per cent,
solution of bichromate of potash. After two days'
preparations of the epithelium from both the anterior
and posterior part of the passage may be made. The
cells are obtained with the greatest ease, by scraping
the mucous surface with the point of a scalpel, and
shaking out the material in a drop of water on a
slide. A piece of hair is added, and the preparation

24*

282 PRACTICAL HISTOLOGY.

covered and examined. In the portion obtained from
near the anterior nares ordinary, columnar, ciliated
epithelium cells will be seen. In that form the true
olfactory part the cells, although many of them are
destitute of cilia, and in addition to the columnar,
elements spindle-shaped (olfactory) cells are met with
which are provided with a bunch of stiff-looking,
hair-like processes, resembling the similar appen-
dages of the auditory epithelium.

THE GUSTATORY ORGANS.

For studying the taste-buds, the foliated papillae
which are found on either side of the base of the
rabbit's tongue are used. To obtain them the tongue
is cut out entire from the recently-killed animal,
when the little .oval patches marked with transverse
ridges may readily be found (Fig. 32, p, p). They

Fig. 32.

\
P

Tongue of rabbit, seen from above.
p, p, Papillae folialse.

are snipped off with curved scissors, and one is
dropped into a mixture of equal parts of spirit and
one-half per cent, of chromic acid, and the other is
placed in one per cent, of osmic acid. After two
days the spirit and chromic mixture is to be ex-
changed for strong spirit, and in twenty-four hours
more, the piece of tissue may be embedded so as to
cut sections, which should be as thin as possible,
vertical to the surface of the mucous membrane,

TASTE-BUDS. 283

and across the direction of the ridges. The sections
are stained and mounted as usual.

The piece that was placed in osmic acid may be
used at the end of forty-eight hours. In the first
place two or three sections are to be obtained like
those made from the other piece, without however
embedding the tissue, but simply holding it in the
fingers or in a piece of split cork. One such section
is to be placed in a drop of water on a slide, and an
attempt made with needles, under the dissecting
microscope, to separate some of the taste-buds from
the surrounding epithelium. For this purpose the
needles must be very fine, sharp, and clean, and the
lens used as high as is consistent with convenience
of manipulation. When one or more taste-buds have
been thus separated, the rest of the section is re-
moved, and the isolated buds are broken up as com-
pletely as possible into their constituent cells. The
specimen may then be covered, and a drop of glyce-
rine allowed to diffuse in under the edge of the
cover; after which an examination of the [(repara-
tion may be made, at first with the ordinary high
power, and afterwards with an immersion objective.

APPENDIX.

Method of measuring an object under the
microscope. — If while the one 63^6 looks down the tube
the other is allowed to remain open, an image of the
object will appear projected on the table at the side of
the microscope, and it is not difficult to mark off, upon a
sheet of paper placed here, the points between which the
measurements are to be taken. The preparation is then
removed, and a stage micrometer is substituted for it,
the parts of the microscope being left in the same condi-
tion as before. The stage micrometer is a glass slide on
which fine equidistant parallel lines have been ruled with
a diamond. The distance between the lines is marked on
the. slide ; it is generally either the T<jotn an(* Wo otn Parfc
of an inch, or the y^th and T^oth part of a millimetre.
The lines are observed with the microscope in the same
way as the object, and their image can of course be simi-
larly projected upon a sheet of paper and there marked
down. The distances between the lines being known, it
is easy, by comparison of the two markings, to find out
the distance between the opposite points of the object.

Some microscopes are provided with an eye-piece
micrometer (Fig. 33). This is an ordinary ocular witli
a flat piece of glass (m) having a scale ruled upon it by
a diamond, inserted between the field-glass and eye-glass.
The value of the divisions of the scale should lie deter-
mined once for all for each objective by observation of a
stage micrometer (see Fig. 34), the tube of the micro-
scope being fully drawn out, and should be marked on
the ocular; and in subsequently using it for measurement
all that is necessary is to see how many divisions of the
scale the object under examination covers. Thus, sup-
posing it had been found by examination of a stage
micrometer that with the high power objective and the

286 APPENDIX.

tube drawn out each division of the eye-piece micro-
meter was worth TsVu inch, any object which when viewed
by the same objective and length of tube took up three
divisions of the eye-piece micrometer would measure
i5Svffths or gjtfth of an inch.

The advantage of the ej^e-piece micrometer is that
when its values are once ascertained the size of an object
can be read off at once.

Fig. 33.

Ocular micrometer, natural size.

Part of the side is represented as broken away to show the field-glass at the
bottom, and the micrometer-glass, rn, a little below the middle The collar,
r serveH to vary the distance of the eye-glass from the microniPter.

Determination of the magnifying power of a
microscope. — The magnifying power of a microscope
is determined by comparing the distance between the
lines of the stage micrometer, as they appear imaged
upon the paper, when this is exactly ten inches1 from the
eye, with the known interval between them. For instance,
if, with the high power objective and the ordinary ocular,
the interval of ^1 of an inch of the micrometer was

1 The ordinary distance of distinct vision.

APPENDIX.

287

represented on the paper by a space of half an inch, this
interval is magnified as many times as the j^V^th of an
inch will go into half an inch, that is to say, 500 times ;
and every other object under similar conditions is magni-
fied to a like extent.

Fig. 34.

Lines of stage micrometer viewed with an ocular-micrometer.
The finer lines are those of the stage micrometer ; about eighteen of the lines of
the ocular micrometer are comprised in oue of the larger intervals between
them, so that if these intervals represent _^_ inch, the subdivisions of the
ocular micrometer will represent l_._ inch.

The enlargement thus obtained may be determined
once for all for each objective, the same ocular being
used and the tube being drawn out to the same extent —
say, to the full length — in each case, and scales may be
made representing the intervals between the micrometer
lines under the different powers. For purposes of measure-
ment it will then only l)e necessar}7 to compare the pro-

288

APPENDIX.

jected image of an object with the scale which was made
under like conditions, without again making use of the
stage micrometer.

Mode of drawing microscopic objects. — The
most convenient and ready way of delineating an object
is to sketch it with the free hand on paper placed at the
right of the microscope, the left eye being applied to the
tube. But if perfect accuracy of size and relations of
parts are desired, the outlines are first traced with a
camera lucida. The simplest for ordinary use is that of
Zeiss (Fig. 35), which is a combination of two prisms so

Fig. 35.

Camera lucida, for tracing the outlines of an object without tilting or
otherwise disturbing the microscope.

The metal ring fits on to the upper end of the microscope tube, and the aperture,
a, is placed immediately over the eye-glass, this part of the camera being
somewhat more depressed than is represented in the figure.

arranged that when placed above the ocular the surface
of the table in front of the microscope is seen at the same
time as the object, the image of the one being superposed
on that of the other. The paper for drawing is placed on
this part of the table, supported upon an inclined plane
of wood, and the main outlines are traced out on it, the
details and shading being afterwards filled up without
the camera.

The lines of the stage micrometer can very easily be
traced in the same way by aid of the camera lucida.

APPENDIX.

289

Mode of counting the blood corpuscles. — In

order to separate the corpuscles and prevent coagulation,
the blood used is first diluted to a definite extent— say a
hundred times — with a 10 per cent, solution of sulphate
of soda. The mixing can be performed in a measuring
glass if the blood is in sufficient quantity, but if only a
small drop is obtainable, snch for example, as is got by
pricking the finger, the mixer shown in Fig. 36, B, may
be used. This consists of a capillary tube terminating

Fig. 36.

Malassez's apparatus for counting the blood corpuscles.

A, Capillary tube for receiving the diluted blood ; B, pipette for m'.xing a
small quantity of bio >d with the diluting fluid

in a bulb, the capacity of the bulb between the marks I
and 101 being exactty 100 times that of the tube from its
point to the mark 1. A small glass ball is inclosed in
the bulb, and serves by its movements to facilitate the
mixing. The capillary tube is allowed to fill with blood
as far as the mark 1 ; sulphate of soda solution is then
sucked up as far as the mark 101. As it passes in, it of
course pushes the blood before it into the bulb, and the
two are there thoroughly mixed by gentle agitation.
The next thing is to count the corpuscles in a known
25

290 APPENDIX.

quantity of the mixture. The most convenient plan is
that of Hay em and Nachet. A slide is used having a
glass ring, 1 millimetre in depth, cemented on to its upper
surface. A drop of the mixture, not enough to fill the
cell so formed, is placed in the middle of the ring, and a
perfectly flat cover-glass is so laid on that the drop touches
and adheres to it without reaching the sides of the cell.
The slide is placed on the microscope, and as soon as the
corpuscles have settled down to the bottom of the drop
the number in a definite area is counted. If the area
chosen is J of a millimetre square, this will give the num-
ber which were contained in £ millimetre cube of the
mixture, and multiplying this by the number of times the
blood was diluted, the result will be the number of cor-
puscles in J millimetre cube of blood.

Areas of J millimetre square might be marked with a
diamond (like the lines of a stage micrometer) upon the
centre of the slide, and subdivided into smaller squares
to facilitate the counting. But it is found more conve-
nient to measure them off by using an ocular micrometer
similar to the one shown in Fig. 33, but with a large
square subdivided into smaller squares substituted for
the scale on the micrometer glass, m. To obtain the de-
sired value for the large square, a stage micrometer, ruled
in parts of a millimetre, is placed under the microscope,
and by adjusting the draw-tube, the sides of the square
on the ocular are made exactly to subtend the interval of
\ millimetre on the stage micrometer. A mark is then
made on the tube to indicate the extent to which it is
drawn out, and whenever an examination is made the
draw-tube is always adjusted to this mark, the same ob-
jective (one of moderate power) being of course used in
every case.

By another method — that of Malassez — a little of the
mixture of blood and sulphate of soda is transferred to a
very fine flattened capillary tube (Fig. 36 A) the capacity
of a given length of which is ascertained previously and
marked on the slide to which the tube is fixed. Thus in
the capillary tube shown in the figure, a length of 400
micro millimetres1 represents the TFV.s part of a cubic

1 A micro-millimetre (/*) is the one-thousandth part of a milli-
metre.

APPENDIX. 291

millimetre of the mixture. The counting is performed
with the aid of a squared ocular micrometer, the micro-
scope as before having been so arranged by observation
of a stage micrometer that the side of the square shnll
have the value of one of the lengths (400 ,«, for example)
marked on the slide. The result of the counting gives
the number of corpuscles in a known quantity (y^.-g- cub.
mill.) of the mixture, and the number in the same volume
of blood can readily be deduced.

Microtomes. — When it is desired to obtain a number
of perfectly even, consecutive sections of an organ, of the
spinal cord for instance, a section-instrument or micro-
tome may be employed. One of the first of these to be
introduced was that devised by Mr. Stirling (Fig. 3t),

Stirling's microtome.

and most of the other instruments are modelled on the
same principle. It consists of a hollow brass cylinder
with a broad metal plate (p) fitted over the top, and a
long, finely cut screw (.s-) working in the lower end, and
serving to push a brass plug upwards in the tube. The
instrument is clamped at the edge of the table by the
screw cs.

292 APPENDIX.

The tissue to be cut is embedded in the brass cylin-
der in much the same way as in an ordinary embedding
trough. A little of the embedding mass is first poured
in ; when this has begun to set the tissue is placed upon
it, and the cylinder is then filled up with more embedding
mass so that the object is completely inclosed. It is
better to use a mixture of paraffin and hog's lard (5 parts
paraffin to 1 part lard) as an embedding mass, since the
ordinary wax and oil mixture tends in cooling to shrink
away from the sides of the tube.

In cutting the sections the metal plate p serves to
direct the razor, so that it moves in a perfectly even plane.
By turning the screw .s the plug of embedding mass with
the included tissue is caused to project very slightly
above the plane of the plate, so that when the razor, wetted
as usual with spirit, is carried over this, a section is ob-
tained varying in thickness according to the extent to
which the screw was turned. In like manner a large
number of consecutive slices may be made. But it is
doubtful whether, with this or any other microtome, it is
possible to get sections as thin as they can be made with
the free hand.

Simple form of microtome for holding in the hand whilst cutting sections.

iMjnire 88 shows a smnller instrument (Ranvier's),
differing from the other one chiefly in the fact that it is
held in the hand whilst the sections are cut. in place of

APPENDIX. 293

being damped to the table. Its smaller size renders it
more handy to work with, and if it is necessary to inter-
mit the slicing for any length of time, the embedded tis-
sue can be preserved without injury by inverting the in-
strument into a beaker of spirit.

Dr. Rutherford's freezing microtome (F"ig. 39) is
designed to enable sections to be made of a tissue
hardened by freezing. The instrument can also be used
as an ordinary microtome. It may be described as an

Fig. 39.

o
Rutherford's freezing microtome.

B, Plate of gun- metal, with aperture, A. leading into well of micro tone; 0, lower
t'nd of cylinder in which the screw, D, works, moving a brass plug up or down ;
E, indicator for showing the fraction of a revolution imparted to the screw; 0,
trough to hold the freezing mixture, with H, tube to c induct away the water
produced by the melting of ihe ice ; F, clamp to fix the instrument to a table.

improved form of Stirling's microtome with the addition
of a capacious metal trough which surrounds the upper
part of the cylinder. The trough is filled with a freezing
mixture (snow or pounded ice and salt, equal parts).
Into the well or tube of the microtome is poured a thick
solution of gum, which soon begins to freeze at the peri-
phery. The tissue to be cut (which may be either fresh,
or hardened by one of the ordinary methods, and should
preferably be soaked in gum for some hours previously),
is placed in the gum and held there until fixed by the
advancing congelation, and when the whole is uniformly

294 APPENDIX.

hard, sections are cut as with the ordinary instrument
except that the razor Used must be dry, not wetted with
spirit. The sections are placed in weak spirit (1 part
spirit 2 parts water) if a hardened tissue, in salt solution
if fresh, to dissolve out the gum, and they can then be
mounted in any desired manner.1

Gum is useful for the freezing process because it ac-
quires a cheesy consistence when frozen, in place of be-
coming hard and crystalline (Urban Prit chard),

Microphotographic Apparatus. — Photography is
every day coming more into use for obtaining images of
microscopic objects. The mode of application consists
in the adjustment of a photographic camera to the tube
of the microscope (which it is better to divest of its eye-
piece); the image formed by the objective is received and
focussed upon the ground-glass plate of the camera, the
objective of the microscope representing in fact the lens
of the ordinary camera. Either wet or dry sensitized
plates are employed, and the mode of exposing the plate
and all the subsequent processes of developing, fixing,
and printing are in every respect the same as in ordinary
photography. Jt will be sufficient, therefore, in this place
to describe a convenient form of apparatus (Fig. 40), and
the manner of obtaining an image of the microscopic ob-
ject; an account of the subsequent procedure, and the
precautions it is necessary to take, will be found in recog-
nized standard works on photography.

Since it may often be required to photograph wet pre-
parations, the microscope should be kept vertical, and the
camera must therefor be supported above the microscope.
This is done by means of a strong mahogany frame con-
sisting of a stand, .?£, upon which the microscope is placed
and four vertical pillars rigidly connected below to the
stand, and above to one another by cross-pieces. Two
brass rods, r, one on either side, run vertically between
the stand below and the cross-piece, and serve more imme-
diately to support the camera, r, which is capable of slid-
ing up and down on the rods and can be fixed by screw-
clamps at any height.

The camera should be lightly constructed, with a bel-

1 For a more complete description sec Rutherford,
Practical Histology." 2d edition, L87G.

295

Microphotographic apparatus. One-seventh the natural size.

lows-adjustment gradually tapering, and fitted below with
a cloth-lined tube which exactly fits over the microscope

296 APPENDIX.

tube, to which it is clamped by the screw d. Just above
the tube is placed the "stop" for shutting the light off' at
any moment from the camera plate; it consists of a light
hinged flap, which, by turning a small handle (not shown
in the figure), may be made to fall over the aperture.

The microscope is put in the centre of the stand, being
held firmly in place by three projecting blocks provided
with strong wooden buttons for clamping the foot, and
there is a round hole in the centre of the stand st, so that
the apparatus can, if desired, be used in the horizontal
position, the light being sent directly through the hole.
It is well to unscrew and remove the whole of the upper
part of the microscope tube (Fig. 1, £'), the cloth-lined
tube at the lower end of the camera being made to fit
over the lower and larger part (t).

When the apparatus is in the position shown in the
figure, the ground-glass camera plate is horizontal, and it
would be extremely awkward to lean over and observe
the image upon the plate and at the same time adjust the
focus. To obviate this difficulty the plane mirror m is
provided; it is inclined at a convenient angle, so that the
image on the plate is reflected towards the observer stand-
ing behind the microscope. When focussing, the upper
part of the apparatus as well as the head of the observer
may be enveloped in a loose black cloth.

It is necessary to employ bright sunlight (or some other
intense and actinic source of light), which should be
allowed to pass through a glass vessel containing a solu-
tion of amrnonio-sulphate of copper, so as to render the
light mono-chromatic. It is then received on the mirror
of the microscope (supposing the apparatus to be vertical)
either directly or after traversing a condensing lens, and
is reflected by the mirror up through the object and the
tube of the microscope in the ordinary way. An objective
of any magnifying power, even an immersion may be em-
ployed, provided there be light enough'. With the same
objective greater or less magnification is obtained accord-
ing as the camera is raised or lowered.

The object is focussed upon the ground-glass with the
utmost exactitude. The light is then cut off, the mirror,
w, removed, and the sensitized plate substituted for the
ground-glass plate as in the ordinary process.

Mr. G. Giles has devised a simple mode of adapting

APPENDIX. 297

an ordinary photographic camera to the microscope, of
which a brief description will be found in the u Quarterly
Journal of Microscopical Science," 1876, p. 111. It ap-
pears to have yielded good results, but for wet prepara-
tions has the disadvantage that the microscope must be
placed horizontally.

Employment of Eosin as a staining fluid,—
Dilute solutions of eosin, an aniline-preparation newly
introduced into commerce, have recently been much re-
commended for coloring the tissues. The dye can be
used dissolved either in water or alcohol. For the watery
solution Dreschfeld recommends a strength of about
1 per 1000; this takes from a minute to a minute and
a-halfto stain sections; they are subsequently put for
a very short time into water slightly acidulated with
acetic acid, and then either examined in glycerine or
mounted in dammar. For portions of tissue which are
€to be hardened in alcohol, the process of hardening and
staining can be effected simultaneously by the employ-
ment of an alcoholic solution of eosin. The color im-
parted by eosin is a rose-red. (E. Fischer, u Arch. f. Micr.
Anat." 1875, p. 349 ; J. D resell leld, "Journal of Anatomy
and Physiology," Oct. 1876.)

INDEX.

A BSORPTION of fat, 218
XI Adipose tissue, 87

development of, 88
Alkanet for injecting lymphatics,

177

Ammonia bichromate, for harden-
ing nervous tissue, 243
Aniline blue-bl;ick, for staining

nervous tissue, 244
blue, for staining peptic cells,

214
for staining cerebellum,

244

Appliances for microscopic work, 19
Areolar tissue, action of acetic acid

on, 70
corpuscles, 71

stained with logwood,

73

constrictions on fibre bun-
dles, 74
elastic fibres, 71

stained with ma-
genta, 76
fibres of, 70
interstitial injection of

gelatine into, 76
preparation by method of

localized oedema, 77
prepared with nitrate of

silver, 80

Arytenoid cartilage of ox, 97
Axis-cylinders in spinal cord, 130

BERLIN-BLUE, mode of prepar-
ing, 164

reduction of, in tissues, 170
solution for injecting lym-
phatics, 177
Blood, action of electric shocks on,

51

human, action of reagents on,
36

Blood, human, action of —
acetic acid, 38
alkalies, 39
chloroform, 39
tannic acid, 38
water, 37

mode of obtaining, 25
on warm stage, 30

effect of super-
heating, 36

Blood corpuscles, human, red, ap-
pearance varies with
distances of objec-
tive, 27

effect of salt upon, 28
observation of, 26
structure of, 36
mode of counting, 289
human, white. 28

amoeboid movements

of, 32

development of, 88
of frog, 42

action of reagents on, 36

iodine, 52

feeding of white corpus-
cles, 43

migration of white corpus-
cles, 45
influence of warmth on

white corpuscles, 49
of newt, action of boracic acid,

48

action of carbonic acid, 48
Blood crystals, 53
Bloodvessels, development of, 88
injection of, 162
larger, epithelioid lining of,
shown by nitrate of
silver, 138

elastic layers of, 140
fenestrated membrane of,

140
muscular tissue of, 140

300

INDEX.

Bloodvessel?, larger —

mode of hardening, 141
sections of, 141
sub-epithelial layer of, 139
smaller, epithelioid cells of, 150
muscular structure and

nuclei, 152

Bone, corpuscles in lacunae, 104
hard, grinding section of, 100
precautions in mounting,

101
softened in hydrochloric acid,

102

in chromic acid, 102
in picric acid, 104
lamellae and Sharpey's

fibres, 105
Brownian movement, 35

CABINET for keeping specimens
\J in, 23

Camera lucida of Zeiss, for deline-
ating objects, 288

Canada balsam, solution in chloro-
form, 149

Canulas for injecting; mode of pre-
paring, 168

Capillary tubes, mode of making. 43
Carmine gelatine injection, 162

solution for staining tissues, 207
for staining sections of sto-
mach, 214

Cartilage, articular, in fresh state, 89
vertical and tangential

sections of, 92
cell spaces of, 93
Cartilage-cells, action of water on,

89

preservation of, 91
stained by chloride of gold, 94
Cartilage, costal, 96

matrix, cell territories of, 97

stained by logwood, 97
transition between hyaline and

yellow, 97

Cell spaces of connective tissue, 78
Central tendon of diaphragm pre-
pared with nitrate of silver, 175
Cerebellum, 243
Cerebrum, 243
Chloral hydrate for preparing retina,

267
Choj-oid coat of eye, 260

bloodve.-sels of, 273
lamina suprachoroi-
dea, 261

Choroid coat of eye —

layers of, 262
Ciliary motion, action of reagents

on, 65

carbonic acid on, 66
chloroform on, 66
warmth on, 65
weak alkalies, 65
Ciliary muscle, 261
Circulation in otnentum of guinea-
pig, 157

in frog's web, 154
in lung of toad, 158
in mesentery of toad, 155
in tongue of toad, 158
Cochlea, mode of procuring, 277
precautions for embedding and

cutting, 278
softening of osseous parietes,

278

teased preparations of, 279
Cohnheim's gold method, 94
Condenser, bull's-eye, use of, 15

used as dissecting lens,

17

Conjunctiva, bloodvessels of, 272
Connective-tissue corpuscles in

areolar tissue, 71
tongue of toad, 159
Cornea, cell spaces of, 257

mode of injecting, 257
epithelium of, 251
mode of hardening, 250
precautions to avoid curling up

of sections, 250
substantia propria of, 251
of frog, corputcles and nerves

of, 252
of rabbit, corpuscles and nerves

of, 254

isolation of corpuscles, 255
nerves of, 256

Corneal tubes of Bowman, 259
Corneo-sclerotic junction, 259
Covei -glasses, mode of averting

pressure of, 42
mode of cleaning, 19
fixing, 61

by paraffin, 106

DAMMAR varnish, 149
Delineation of microscopic ob-
jects, 238
Diaphragm of microscope, use of,

13
Directions for work, 23

INDEX.

301

ELASTIC fibres in areolar tissue, '
71

transverse section of, 82
networks of serous membrane,

82

of artery, 140
tissue, 82

Electricity, mode of applying, 50
Embedding in wax-mass, 142
moulds for, 143
by gum method, 190
membranous viscera, precau-
tions for. 212
Endocardium. 194

fibres of Purkinje in, 195
End-plates of mammals, 135

of lizard, 136
Eosin, as a staining fluid, 297

for staining nervous tissue,

244
Epidermis, cells of horny layer j

shown by potash, 58
Epithelioid-cells, 78

covering tendon, 87
Epithelium, ciliated, from frog's >

mouth, 62

from gills of mussel, 63
study of separated cells, j

78

columnar, 59
scaly, of mouth, 56

deeper layers of, 57
Erectile tissue, mode of hardening,

237 .
Eye, bloodvessels of, 272

general mode of preparing, 246
of pig as a substitute for human

eye, 247

Eyelids, sections of, 247
Eye-piece of microscope, 15

FAT-CELLS, 87
membrane of, 88
development of, 88
Fibrine in blood, 30
Fibro-cartilage, yellow, of epiglot-
tis, 98

transition to hyaline, 97
white, 98
Fibrous tissue, 83
Field glass, 15
Forceps, 20
Freezing method of hardening —

Dr. Rutherford's, 293
Dr. Urban Pritchard's,

123
26

GANGLIA, sections of, 192
Ganglion cells, 131
Gas, carbonic ncid, mode of apply-
ing to a preparation, 49
chamber, 40
Gastric glands, 213

cells of isolated, 215
Gelatine injecting fluid, 162

mode of preserving,

171
Glycogen, its presence in white

blood corpuscles, 52
Goblet cells, 60
Gold chloride, method of staining

with, 94
Granules, masses of, in blood, 29

Dr. Osier's observa-
tions upon, 33
Gullet. See (Esophagus.

TJ^MATOXYLIN. See Logwood.
JLl Haemin crystals, 54
Haemoglobin crystals, 53
Hairs, 188

development of, 207
Hayem and Nachet's- mode of count-
ing blood corpuscles, 290
Heart, muscular substance of, 193

lymphatics of, 195
Hepatic cells, 228

IMMERSION objectives, mode of

JL using, 16

Inflammation changes in mesentery

of toad, 157
in ti ngue of toad, 161
Injected parts, mode of preparing,

169
Injection apparatus, 165

of an entire animal, 167
of bloodvessels, 162

mode of killing an animal

for, 225

of lymphatics, 177
mass, mode of preparing, 162
Injections fluid in the cold, 170
Instruments required for micro-
scopic preparation, 20
Intestine, large, 222

small, bloodvessels of, 219
mode of hardening, 217
nerves of, 219
precautions for embedding,

217
Iodized serum, 59

302

INDEX.

Iris, bloodvessels of. 273
muscular tissue of, 263
sections of, 259

KIDNEY, bloodvessels of, 233
examination in fresh con-
dition, 234

mode of hardening, 231
tubules, isolation of, by Lud-

wig's method, 233
epithelium of, 232
basement membrane of,
stained with nitrate of
silver, 234

transverse section of, 232
Klein, Dr. E., on structure of
spleen, 230

LABIA, mode of preparing, 238
Lachrymal gland, 248
Larynx, 201

Lens, isolation of fibres, 269
sections of, 270
suspensory ligament of, 272
capsule, epithelium of, 271
Liver, bloodvessels of, 223
lymphatics of, 227
mode of hardening, 223

of injecting bile-ducts, 226
Logwood solution for staining tis-
sues, 22

Kleinenberg's, 198
Lung, embedding by cacao-butter

process, 198

epithelium of air-cells, 199
injection of bloodvessels, 200
mode of hardening, 197
Lymphatic glands, 184
Lymphatics, injection of, 177

of diaphragm, natural injec-
tion of, 181
of tendon, mode of injecting,

179

larger, 177

smaller, in omentum and cen-
tral tendon, 172-5

MAGNIFYING power of micro-
scope, estimation of, 286
Malassez's mode of counting blood

corpuscles, 290
Mammary glands, 242
Marrow, red, on warm stage, 110
giant cells of, 111

1 Measurement of an object under

the microscope, 285
Mesentery, stained with nitrate of

silver, 151
circulation in, 155
Micrometers, 285
Microphotography, 294
Microscope, biuoc»lar, use of, 15
best test-objects for, 17
eye-piece of, 15
for dissection, 17
parts of, 13
powers of, 16

selection of, by student, 17
Microtome, Stirling's, 291
Ranvier's, 292
Rutherford's, 293
Migration of white corpuscles from

veins, 161

Moist chamber made with putty, 39
Mouth, mucous membrane of, 204
Mucous glands of tongue, 207
Muscle, bloodvessels of, 125

ending in tendon in mouse's

tail, 124
endingin tendon in frog's neck,

124
examined by polarized light,

120

section of, in frozen state, 123
transverse section of, 122
of water-beetle, 118

mode of production of
the transverse
striae, 119
studying contrac-
tion of, 119
involuntary, mode of isolating

cells of, 112
showing nuclei, 115
prepared with nitrate of

silver, 113
voluntary, action of acetic acid,

115
demonstration of sarco-

lemma in, 115
mammalian, 114
isolation of fibres, 116
separation of, into disks

and fibrils, 116
Muscle-rods, 118

N

AIL, cells of, separated by pot-
ash, 58

embedded by gum method, 190
sections of, 189

INDEX.

303

Needles, mounted, 20
Nerve cells of ganglia, 131

of spinal cord, 130
Nerve fibres, medullated, 126

in spinal cord, 130
treated with osmic

acid, 129
stained with nitrate of

silver, 129
non-medullated, 127
Nerve trunk, perineurium of, shown

by silver method, 128
sections of, 191
structure of, 128
Nerves, motor, ending of, 135
Nervous plexuses of intestine, 219,

222

Nervous system, central, mode of
preparing, 243

OBJECT-GLASS, or objective, 15
Objective, immersion, 16
Ocular of microscope, 15
(Esophagus, 211

bloodvessels of, 212
Oil of cloves for clarifying sections,

148

Oil-globules, effect on light, 119
Olfactory mucous membrane, 281
Ornentum, prepared with nitrate of

silver, 172
circulation in, 157
Optical section, 153
Organ of taste, 282
Osmic acid colors fatty substances

black, 61
for nerve, 129
for retina, 264-66

Ossification, intracartilaginous, 107
intramembranous, 106
of lower jaw, 206
Ovary, 238

Ovum, mode of obtaining, 239
impregnation of, 240

PACINIAN corpuscles from cat's
mesentery, 132
sections of, 135
treatment with chromic

acid, 134

nitrate of silver, 134
osmic acid, 133
Palate, 209
Pericardium, 193
Perineurium, lymph spaces of, 191

Pia mater, vessels of, 152
Picrocarminate of ammonia, 85
Pigment, hexagonal, of retina, 268
Pipettes, mode of making, 22
Pleura, 196

Polarization apparatus, 17
Polarizing microscope, 120
Pritchard's, Dr. Urban, mode of

freezing tissues, 123
Prostate gland, 237
Pulmonary vessels, injection of, 200
Purkinje, fibres of, 195

"REAGENTS in common use, 22
XX action of, upon the blood,

36

bottles for, 22
mode of applying, 36
Recklinghausen's silver method, 78
Remak, fibres of, 127
Retina, bloodvessels of, 274
fibres of Miiller, 265, 267
fresh, in vitreous humor, 267
in iodized serum, 268
isolation of elements, 266
methods of hardening and cut-
ting sections, 264
of bird, reptile, and fish, 269
of frog, 268

SALIVARY corpuscles, 57
glands, 209
Salt solution, 22
Sankey's, Mr. H. R. 0., method of

preparing sections of brain, 244
Sclerotic coat of eye, 248

bloodvessels of, 272.
lamina fusca, 249
Scissors, 20
Scrotum, 238
Section -lifter, 148
Sections, mode of cutting, 145
staining, 147

with Kleinenberg's

logwood, 149
mounting, 149
Semicircular canals, mode of finding

and preparing, 275, 276
Seminiferous tubules, epithelioid

cells of, 241
isolation of, 241
Silver nitrate, method of staining

with, 78
Skin, 185

bloodvessels of, 187

30 i

INDEX.

Skin-
double staining of, 186
preparation of, by Dr. W. Stir-

ling's method, 187
Slides for microscopic purposes, 19
Spinal cord. 243

isolation of cells of, 129
Spleen, 229

demonstration of retiform tissue

of, 230

injection of, 230
Stomach, 212

bloodvessels of, 215
glands of, 213
horizontal sections of, 214
lymphatics of, 215
Stomata in lymphatic septum of

frog, 176

Suprarenal capsule, 235
Synovial membranes, 182

bloodvessels of, 183
Haversian fringes of, 183

TACTILE corpuscles, 186
Tarsal cartilage of eyelid, 248
Taste-buds in papillae foliatae of rab-
bit, 282

circumvallatae, 208
Teeth, in situ, 206

development of, 206
dentinal sheaths of, 205
sections obtained by grinding,

204

sections of, softened, 204
Teeth, soft tissues of, 205
Tendon of mouse's tail, 83

action of acetic acid

on, 84

cell spaces of, 85
transverse section, 86
cells, 85

i Tenon, capsule of, 248
Testis, 240
Thymus gland, 202
Thyroid body, 202
Tongue, 207

bloodvessels of, 208

of toad, 158
Tonsils, 209
Trachea, 201

epithelium of, 67
Tunica vaginalis, 241

DRETERS, 235
Urinary bladder, 236
Uriniferous tubules. See Kidney.
Uterus, 238

VAGINA, 238
Vaginal synovial membranes,
182

Vesiculae seminales, 237
Vitreous humor, hyaloid membrane
of, 271

WARMING apparatus, with gas
regulator, for chloride of gold
preparations, 96
Warm stage, simple, 30

mode of estimating

temperature, 31
with gas regulator, 34
Water, action of, on fresh tissues,

37
Wax-mass for embedding, 142

70NULE of Zinn, 71

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