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COLLEGE OF PHYSICIANS
AND SURGEONS

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MANUAL FOR THE
PHYSIOLOGICAL LABORATORY.

BY

VINCENT DORMER HARRIS, M.D. Lond., F.R.C.P.,

Examiner in Ehiiientary Phyxiologij on the Conjoint Board in England of the

Royal ColUiH' of Physicians and Surgeons; Physician to the City of Lomlon

Hospital for Diseases of the Chest, and to the Metropolitan Dispensary ;

Demonstrator of Physiology at St. Bartholomew's Hospital, etc.,

AND

D'ARCY POWER, M.A., M.B. Oxon., F.RC.S. Eng.,

Surgeon to the Metropolitan Dispensary ami to the Victoria Hospital for Children:

Curator of the Museum and late Assistant Demonstrator of Physiology at St.

Bartholomeio's Hospital, etc.

FOURTH EDITION,
WITH ILLUSTRATIONS.

LONDON :
BAILLIERE, TINDALL, AND COX,

20, KING WILLIAM STREET, STRAND.

[PARIS AND MADRID.]

1888.

[All Rights Resened]

PREFACE TO THE FOURTH EDITION.

The present edition is considerably altered. The Introduction
has been enlarged, the Histology revised, and the Chemical
Physiology considerably added to and improved.

The Physiology Proper has also undergone some modification.
In each division of the book much new matter has been intro-
duced.

The object of the authors in this edition, as in the former
ones, has been to make the book of real practical value to the
student, and not to burden him with descriptions of methods
which are not within his scope. The alterations have been
suggested by the experience of many years' teaching, and we
think that they will be found to add to the usefulness of the
book.

To our colleagues, Dr. Lewis Jones, Dr. Tooth, and Dr. Shore,
we would tender our most hearty thanks for the help which they
have at all times so readily afforded to us ; and more especially
for the assistance which they have rendered in re-casting

IV PREFACE TO THE FOURTH EDITION.

Parts II. and III. Dr. Jones has further increased our obliga-
tion to him by the care and time which he has expended in
reading the proof-sheets of the present edition, thereby correct-
ing many errors and inaccuracies into which we should otherwise
have fallen.

To Mr. John Murray and to Messrs. Churchill, as well as to
several scientific instrument-makers who have allowed us the
use of their illustrations and plates, we also beg to tender our
best thanks.

CORRIGENDA.

Page 24, line 1, read 'Paraffin.'
„ 163, „ 31, for ' sulphindigotate of carmine ' read ' sulphindigotate
of soda or indigo carmine. '

PART I.
PRACTICAL HISTOLOGY.

CHAPTER I.

THE MICROSCOPE AND MICROSCOPICAL
METHODS.

The subject of practical histology may be conveniently intro-
duced by an account, firstly, of the microscope and its accessory
apparatus, and, secondly, of the methods of preparing tissues in
order that their structure may be adequately studied with the
aid of that instrument.

The present chapter, therefore, will be devoted to a descrip-
tion, as brief as possible, of these two parts of the subject in
turn.

THE MICROSCOPE AND ITS ACCESSORY APPARATUS.

The form of microscope which we select for description is
the one generally known as the Continental Model (Fig. 1).
We choose it because of its simplicity. Although originally it
was manufactured by foreign makers only, and differed in plan
from most of the English microscopes, at the present time
instruments very similar in construction, although differing
somewhat in detail, are made by nearly all the best English
makers.

The model is often called the Hartnack Model, as it was
chiefly by means of the instruments of that make that the
small continental microscope became popular in this country.

1

PRACTICAL HISTOLOGY.

In this model the stand or body uf the instrument consists of
two parts. Of a base of solid brass, into which is fixed vertically
a substantial brass pillar about two inches high, and to which is
attached by a hinge the second portion of the stand. This
latter is made up of a hollow cylinder of brass, carrying below
at right angles the stage of the microscope, and above, by a
projecting arm, a long brass collar, in which the tube of the

Tube with Ocula:

Draw-tul>

Collar in whic''
tube work

Fine adjustmen

Hincje connectiii,'
stand with bocV

L'oudeuser

)bjective
jtage

Mirror

Fig. 1. — Contiueiital Model Microscope.

microscope slides. At the top of the hollow cylinder is a screw
with a milled head which works the fine adjustment ; when the
screw is turned the tube of the microscope is moved very
slightly to or from the stage. In the simpler forms the coarse
adjustment, or the freer movement of the microscope tube up
and down, is effected by the hand, whilst in the more expensive
models it is performed by a rack and pinion. The stage of the
microscope, upon which the object-glass is placed, is a flat plate

PRACTICAL HISTOLOGY. 3

of brass blackened upon its upper surface, or of glass cemented
on to a (lark background, and perforated by a central aperture.
Through the aj)crturc light is reflected from a mirror ; the
amount of light admitted to the lens is regulated by a small
cylinder which fits into the central aperture; this, when withdrawn
from below, will receive the diaphragms ; these are perforated
by apertures of different sizes ; the smallest is a mere pinhole,
and is for use with the strongest magnifying ])0wer. One of
the diaphragms should be inserted into the cylinder, and re-
placed in the centre of the stage. When in position it is exactly
flush with the upper surface. In some microscopes this form of
diaphragm is replaced by a blackened disc of metal perforated
near its circumference by holes of various sizes, and revolving
round its centre. At the back of the stage is a pair of brass
clips for holding the slides in position when the microscope is
tilted. In some instruments the movements of the stage in
different directions are effected by screws; but in this the stage
is fixed, and the finger and thumb suffice to move the slide
upon it.

Beneath the stage is a movable mirror with two faces, one
concave, the other plane ; the concave mirror is the more com-
monly used, as it condenses the light upon the object and thus
affords a better illumination. The tube of the microscope
consists of a hollow brass cylinder from five to eight and a half
inches in length. It contains a second or draw-tube, which
can, if necessary, be drawn out, in order to increase the magni-
fying power. The ujjper extremity of the tube receives the
ocular or eye-piece : the oculars vary in magnifying power.
It is better not to change the oculars, but always to w'ork with
one of moderate power.

Into the lower end of the tube the objectives, powers, oi
lenses should be screwed. These also vary in magnifying
power.

In using the microscope, i)ut on a low power first ; then
adjust the mirror in such a way as to get a full illumination of
the field, so that the eye applied to the ocular sees a circle
of light of equal intensity in all its parts. Never use the
direct rays of the sun. Next put the object to be examined

1—2

4 PRACTICAL HISTOLOGY.

upon the stage in such a Avay as to be over the centre of the
aperture : the diaphragm with largest aperture is to be used.
Bring the lens to within half an inch of the slide, and carefully
employ the fine adjustment until the object is distinctly seen ;
then alter the focus so as to observe it at various depths.
After examining "svith the lower power, remove this and replace-
it by higher powers, at the same time changing the diaphragm.

Fig. 2. — English Model of Stutleuts' Microscope : the nose-piece
carries two objectives.

for a smaller one : bring the lens down until it almost touches:
the object, and then focus upward by means of the fine adjust-
ment.

Students are apt to leave a portion of the lens attached to.
the tube when they change the powers ; or perhaps the field'
may be obscured by drops of glycerine, Canada balsam, etc.,.
adhering to the surface of the lens. Attention may be directedl

PRACTICAL IIISTOLOCY. 5

to these points when it is found tliat the object cannot be
focussed clearly. A drop of methylated spirit upon a soft
handkerchief is sufiicient to remove Canada l^alsam from lenses.
A condenser, or convex lens fitted into a jointed lever which
slides up and down the tube of the microscope, is provided ; it-
is employed for condensing light upon opaque specimens ; it
may, however, be removed, since it is scarcely ever needed inj
modern histology, as the majority of specimens are prepared for
examination by reflected light. j

It will be seen by an examination of Fig. i^, which represents
u student's microscope of English make, that it differs but little
from that which we have described. The body or stand is,
however, a tripod arrangement, which is the form at present in
vogue in this country.

Before giving a brief description of the optical plan of a
compound microscope, into the construction of which many
lenses enter, we may mention, in passing, the simple or dis-
secting microscope, which consists essentially in a stage
illuminated from below by a movable mirror. Above the stage
is an arm which can be raised or lowered by a rack and pinion.
The arm carries a lens similar to that employed by watch-
makers. The dissecting microscope is serviceable in certain cases
Avhere it is necessary to tease or otherwise manipulate very
delicate tissues. It is chiefly, however, of use to biologists.

The Mode in which the Compound Microscope Magnifies.
— The rays of light (Fig. o) which proceed from the object under
examination are focussed behind the objective at a point in the
lumen of the tube of the microscope the position of which is
dependent upon the strength of the magnifying power employed.
Thus the higher the objective, the nearer is the object focussed to
the lower end of the tube ; and the less the magnifying power of
the objective, the higher in the tube is the focus. After the
various rays have been brought to a focus they cross each other
and become divergent, until they are a second time gathered up
by the front lens of the ocular, which focusses them within the
eye-piece at the level of the diaphragm. An enlarged but
inverted image of the object in front of the objective is thus
formed, which is a second time magnified by the lens of the

6

PRACTICAL HISTOLOGY.

ocular nearest

to the eye. The magnifying power of the
microscope is increased within certain limits by
increasing the distance between the objective
and the eye-piece, and it is for this reason that
most of the modern instruments are provided
with a 'draw-tube.' In using the draw-tube, how-
ever, as well as in viewing objects with highly
magnifying eye-pieces, it must be borne in
mind that it is only the image formed by the
objective which is enlarged, and not the object
itself, and with such increased magnification
any defect or distortion on the part of the
objective will be further amplified, and it is
therefore best only to employ eye-pieces of
moderate strength.

Lenses or Objectives— In Fig. 3, the
objective, although one of low magnifying
power, is seen to be made up of combinations
of lenses instead of single plano-convex glasses.
This is the plan always adopted nowadays
with the compound microscope. Objectives
of high magnifying power are indeed always
made up of more than two such combinations.
The explanation is not far to seek.

Single plano-convex lenses are subject to

two great defects, of which the first is

spherical aberration. This results from the

fact that rays of light which pass through the

circumference of the lens are brought to a

focus at a point nearer to the lens than the

rays which pass through the centre. The

second defect is that of chromatic aberration^

^. consequent upon the complex nature of white

i^ ig. y. —Diagram , . , „, . . , , i . i i

of the Method iii light. The rays m passing through the lens

which a Compound ^re split up into their component parts, and

fies. ° thus the colours of the spectrum are obtained

in the same way as when light passes through a prism. Each

colour of the spectrum is focussed at a different point, and the

PRACTICAL HISTOLOGY. 7

image is therefore surrounded by a coloured fringe. It has
been found, however, that by a skilful combination of different
kinds of glass, objectives can be manufactured in which the
spherical and chromatic aberrations are practically done away
with. Objectives, therefore, are usually composed of com-
binations of lenses, varying in number from two to eight, such
as we have above-mentioned, and they are composed of flint
and crown glass ; the flint-glass lenses being concave, and
ground so as to fit the opposed convex surfaces of each crown-
glass lens. In the better class of objectives the lenses farthest
away from the object are ground in such a manner as not only

Fig. 1. — Old Form of Combination
of Lenses.

Fig. 5. — New Form of Combination
of Lenses.

to correct their own spherical aberration, but also that of the
front lens. By this means an objective is obtained in which
the front lens consists of a single glass, and not. as in the older
forms, of an achromatic combination of convex and concave
glasses, which only allowed of a small angular aperture.

By the angular aperture of a lens is meant the number of
rays of light which it is capable of transmitting. Such aper-
ture is measured by observing, with a special instrument, the
angle subtended from the centre of the objective by the most
oblique ray of light transmitted on either side. For ordinary
purposes an objective whose focal length is ^ inch should not

8 PRACTICAL HISTOLOGY.

possess a greater angular aperture than 100" ; whilst ^-inch
should not exceed 130', for beyond this point the larger amount
of light entering the objective is more than counterbalanced by
the increased proximity of the lens to the object, and the
diminished amount of penetrating power which such a glass
possesses. This statement does not hold true for immersion
lenses, in which the angular aperture can be increased to a
very great extent without detriment to the utility of the
objective.

Immersion Lenses.— The development of modern histology,
especially in the direction of the recognition and cultivation of
the exceedingly minute forms of vegetable life known as micro-
organisms, has led to a corresponding demand for higher micro-
scopic magnifying powers. This demand was at first met by the
manufacture of the more powerful objectives which have already
been described, in which an interval of air intervenes between,
the front of the lens and the surface of the glass covering the
preparation. It was soon found, however, that such higher
objectives focussed inconveniently near the cover-glass, whilst
at the same time they had a very limited field, and did not
admit of the passage of sufficient rays of light to yield a good
illumination, A new form of objective has therefore been con-
structed of such a character that it focusses an object when the
interval between the front lens and the cover-glass is occupied
by some fluidiwhich reduces to a minimum differences in the index
of refraction. Such objectives are known as ' immersion ' lenses,
to distinguish them from the old;form of ' dry ' lens. Water,
ghcerine, and oils have been successively employed in connection
w^ith these lenses ; but cedar-oil condensed until it has a somewhat
higher specific gravity than usual, is now most commonly used for
the purpose, because its index of refraction is found to be very
nearly equivalent to that of crown glass, and therefore nearly all
the rays of light which are focussed upon the object pass through
the objective into the body of the microscope.

To use an immersion lens the mounted specimen must be
.sealed with marine glue, gold-size, or other cement which is not
soluble in cedar-oil. A small drop of the clear oil is then
lightly placed, by means of a clean glass rod, upon that portion

PRACTICAL msTOLOCY. ^

of the cover-plass which is immediately over Ihe part of the
preparation to be examined. The lens is afterwards brought
down carefully until its surface is in actual contact with the
oil, and it is then focussed by means of the fine adjustment.
The immersion lenses employed in histological research are
usually without correction for the thickness of the cover-glass,
as they are more readily employed than when a different
correction of the lens has to be made for each thickness of
cover-glass employed. Before using the immersion lens it will
usually be found expedient to find the particular spot in the
preparation with an ordinary high power, and if the specimen
is permanent the part required should be marked out for future
reference by drawing round it upon the cover-glass a small ring
of cement or ink.

In the majority of cases a special form of microscope will be
required, as it will be necessary, in order to obtain a sufficiently
powerful illumination, to attach an achromatic condenser
beneath the stage. After using the immersion lens it should
be at once unscrewed, its front surface should be gently wiped
with a soft handkerchief or piece of chamois leather kept for
the purpose, and it should then be replaced in its box.

Oculars or eye-pieces are of two great types. The Huy-
ghenian is in common use, and consists of two plano-convex
lenses, separated by a distance equal to half the sum of their
combined focal length, with a diaphragm placed between them.
The Kellner, or orthoscopic ej^e-piece, is chiefly employed in
micro-photograjihy, and is composed of an upper achromatic
lens, and of a lower bi-convex lens placed in the focus of the
upper or eye lens. It has no intervening diaphragm, and there-
fore has nearly double the field of view possessed by the Huy-
ghenian eye-pieces.

Apockromatic Objectives. — Zeiss has recently introduced a
new series of lenses, which he has termed Apochromatic. The
particular advantages claimed for them are that in consequence
of an improved method of correctiou, and the use of new kinds
of glass, the spherical aberration is entirely abolished. The
lenses can be employed with much higher eye-pieces than has
hitherto been the case ; and the natural colour of objects, even

10

PRACTICAL HISTOLOGY.

in the more delicate tints, are reproduced unaltered. To obtain
the best results with these objectives, they should be employed
with the compensating eye-pieces manufactured by the same
firm ; when this is done an image is obtained which is uni-
formly free from colour throughout the whole field of view.
Drysdale, after carefully testing this combination of ocular
and objective, has come to the conclusion that results may be
obtained with them which are equal but not superior to those
yielded by the immersion lenses of the best English makers.

Light. — The best light for microscopic work is that afforded
by the sun, when its rays are not too powerful. A fine sunny
spring day affords what may be called a
tj^pically favourable light. The light
should be considered best when reflected
from white clouds in a blue sky. In
England, however, only too frequently
artificial light has to be made use of.
Various forms of gas and oil lamps have
been suggested. A drawing of a con-
venient form is given. As a rule we
employ gas argand burners, with blue
glass funnels. The light is generally
transmitted to the object by a slightly
concave mirror through a diaphragm.
The illumination of objects when opaque
is done by means of condensers of various
kinds, which focus the light upon the
object from above, or condense it laterally
by parabolic side - reflectors. Opaque
objects, however, as before mentioned,
seldom need to be examined in histological work.

In using immersion objectives, the ordinary mirror is supple-
mented by some form of achromatic condenser, of which perhaps
the illuminating apparatus designed by Abbs may be considered
as the best. It consists essentially of two or more lenses, so
combined as to transmit a large pencil of achromatic light,
which is reflected either from a concave or plane mirror. The
amount of light entering the instrument may be regulated by

Fig. ti. — Microscope
Lamp.

PRACTICAL HISTOLOGY.

11

introducing diaphragms ; whilst the direction of the rays can
be varied at will by turning a milled head. The top lens, when
the apparatus is in position, fits accurately into the central
aperture in the stage of the microscope, and is flush with itn
upper surface.

Of Drawing Microscopical Objects. — If the student is a
good draughtsman, practice is the only thing required before he
can make good drawings of microscopic tissues. In others prac-
tice will very possibly bring out latent talents. Some, however,
whatever their perseverance, will need aid ; and this is given them

Fig. 7, — Diagram of the Positiou of the Microscope, etc., when using
the Camera Lucida .

by an instrument called the camera lucida. This consists of
a prism arranged in such a manner that, when attached to the
microscope tube by a holder, and placed exactly over the eye-
piece, the image of the object is thrown upon some part of the
table where a sheet of white paper may be laid, and the outline
of the object can then be traced upon it. An instrument con-
structed for a similar purpose is Beale's neutral tint reflector,

12 PRACTICAL HISTOLOGY.

"which is thus used : The cap of the eye-piece is removed,
and the reflector is applied in place of it. The microscope
should then be inclined to a horizontal position, and at
ten inches from the table, and the paper is placed exactly
underneath the reflector. After the object is focussed and
properly illuminated, the eye should be brought close to and
exactly ovn- the reflector, and the image will then appear to be
thrown upon the paper, and may readily be traced.

Directions for Choosing a Microscope.— Great care is
necessary in the choice of a microsope, and the student is
recommended not to buy one without asking the advice of
some one who is well acquainted with modern instruments.
The supply of different kinds is now so extensive, that a
description of what should be chosen is difficult and might be
invidious. We may, however, tell him roughly irhat not to (jet.
Let him not buy one of those large constructions of brass which
are so often strongly recommended by dealers, or one which has
a complicated arrangement of screws and buttons to move the
object-glass. This would be simi)ly paying money for useless
material. Binocular microscopes cannot be recommended, nor
those in which the fine adjustment tilts the draw tube forward.
Second-hand instruments, except modern instruments of well-
known makers, are to be avoided, as are also old instruments, of
whatever kind. The following points of advice may be of
some use :

The stand must be small, and at the same time firm ; the fine
adjustment delicate and steadfast. The oculars ought to be
clear and achromatic, free from flaws, scratches, and spherical
aberration.

The lenses should also be free from any such faults, and
should exhibit a flat field, the whole of the field being in focus
at once ; should have a fair power of penetration — i.e.^ should be
capable of showing the parts beyond the exact focus ; and should
also possess what is technically known as resolving ])ower — /.e.,
should be able to focus clearly a number of fine lines, close
together, in an object.

Every part should be carefully tested, and if found defective
should be returned to the maker after a fair trial, even if paid

practic;al histolo(jy.

13

for ; and, indeed, the best makers are anxious that no imperfect
lenses bearing their names should be in current u^e.

Of course the question of jn-ire is a very important one —
how much money is to be expended upon the purchase of an
instrument ? It is seldom necessary to spend more, to begin
with, and not expedient as a rule to spend les^^, than //tr riit'mean.
For this money the student obtains a good stand, two eye-pieces,,
and two objectives, ^-inch and 1 or ^, in a stout case ; in fact,
all that he will be likely to require for a considerable time.

The accompanying figure of a Student's Microscope shows such
an instrument. It may be
obtained from one of the
following makers :

Beck, G8, Cornhill ;

Crouch, 66, Barbican ;

Swift, 81, Tottenham
Court Road ;

Pakkes, 5, St. Mary's Row,
Birmingham ; or

Stanley, London Bridge.

Several makers have in-
troduced microscopes even
cheaper than the ones above-
mentioned. Beck's Star
Microscope, at three to four
guineas, is a marvel of cheap-
ness. Crouch and Parkes
also make microscoi)es at
£3 10s. to £4. It is requisite
also to mention here, that
Leitz, of Wetzlar, supplies a
microscope at £5 5s. (stand
No. IV., with lenses 3, 5, 7, oculars I. and III.), and one at £3 lOs.
(stand No. V., lenses 3 and 7, and oculars I. and III.), quite
suitable for students' use.

Not a few prefer to purchase an English stand, with certain
favourite German lenses. Those who propose to follow this
course will find Baker's stand, and Zeiss's A and D lenses, a good

Fig. 8. — Student's Microscope.

u

PRACTICAL H1ST0L0(JY.

combination. Messrs. Baker (High Holborn) are also agents

for Zeiss, Reich ert, and other German firms.

It may appear unfair to mention the names of certain makers,
and to omit the names of others. In a
pi-actical book, however, it is evident that
definite advice should be tendered. "We
recommend the microscopes the perform-
ances of which we have had opportunities
of judging. Other microscopes, of which
we have had less experience, may possibly
be as good as those recommended.

Of more expensive microscopes, in addi-
tion to several made by the before-men-
tioned firms, the following are recommended
as suitable for advanced students :

Zeiss (Jena). — Stand No. V. a, with

objectives A and D, and No. 3 ocular,

costs £8 8s. Stand No. YIII., with

similar ob j ecti ves and ocular, costs £ 6 5s.

Stand No. II., with objectives 3 and 7,

and two oculars, costs £7 10s. ; and with No. 8 objective

as well, £9 5s.

Reichert (Wien). — Stand No. XI. f, with objectives Sand G,

and one ocular, costs £7 19s.
Nachet (17, Rue Saint Severin, Paris). — No. IX., with
objectives 3 and 6, and two oculars, is an excellent
instrument, and costs £G 10s. We are very ])leased with
his No. XYIII., microscope portatif de cojage^ the cost of
which is £8.
Terick (2, Rue de la Parchemenaire, Paris) microscopes

are good, but expensive.
Hartnack's microscopes do not seem to be so popular as
they formerly were in this country, since the business of
the old firm has passed into other hands. We have not
had much experience, of late years, of this model,
which was formerly so excellent.

As regards the higher class microscopes of many of the

Fig. 9.— Star Micro-
scope.

Leitz (Wetzlar).-

PRACTH AI, HISTOLOGY. 15

English makers, the prices appear to be too high, and there is a
tendency to make the instrument eninbersorae.

The more expensive Stands and Lenses. — A few words about
Zeiss's stands may be useful. Nos. I., II., III., and IV., price
£15, £12 10s., £10 10s., and £10 5s., respectively, are provided
with an Abbe's condenser, rack and pinion coarse adjustment,
and all requisites for fine work. The eye-{)ieces are numbered
1 — 5, from two inches to seven-tenths of an inch focus, and
cost seven shillings each : the most useful numbers are 2 and 4.
The objectives, A — F (twelve in number), vary in price from
six shillings to £4 4s.; and oil immersions, i^-inch, £12 ; ^Ij inch,
£10 ; and i^s-inch, £20.

Leitz's large stands are good and less exjjensive, about a
third less in price than those described above. His oculars cost
five shillings each ; and his oil immersions, ^^^-iuch, £5 ; j\j-inch,
£7 10s.; and .^-inch, £10.

Reichert's stands and oculars are much the same in price as
Zeiss's. His oil immersions, j^^-inch, £8 ; 7,\^-inch, £13 ; and
...\,-inch, £18.

Powell and Leland's lenses are said to be the best in the
trade, but are expensive. Their apochromatic homogeneous
immersion object-glasses, ^-inch, ^^-inch, jV-inch, 1"40 numerical
aperture, cost £25 each ; and a set of three compensating eye-
pieces costs £5.

A nose-piece is a convenient appliance screwed to the
microscope tube for carrying two or more objectives, so that one
may be exchanged for another without the trouble of unscrewing
it from the tube. The nose-piece, represented as attached to the
microscope in Fig. 2, is for two powers. It is fixed to the tube
of the microscope by a screw. The arm carrying the objectives
revolves on a centre, which also attaches it to the upper portion or
framework of the nose-piece, which is screwed into the micro-
scope tube at one end, and is bent away from the stage on the
other.

By carefully pushing one objective away from the microscope
tube, the second one is brought into its place.

16 PRACTICAL IIISTOLOGV.

MICROSCOPICAL METHODS.
The second portion of our subject treats of the methods by
"which tissues should be prepared for microscopical examination.
These methods will be described under two heads :

1. The Examination of Fresh Tissues.

2. The Examination of Hardened and Prepared Tissues.

I. The Examination of Fresh Tissues.

The simplest example of examining a fresh tissue would be
afforded by the examination of a drop of blood. The drop
is placed upon a thin piece of glass, about {' inch in diameter,
or cover-glass, which is then inverted upon a glass slide or
object-glass. The specimen of blood is at once fit for examina-
tion, and continues to be so until it dries up.

Similarly, one may examine fluid drops of any kind which
are supposed to contain solid particles, the nature of which it
is necessarj" to make out with the microscope ; r-.^., sediments
of urine, scrapings of fresh tissues or organs, or micro-organisms
when suspended in water. The only requisite to the successful
examination being, that the particles should be thin enough to
be transparent.

In some cases, again, it is requisite to examine some
peculiarity of the structure of a tissue unaltered by reagents, or
to observe some action, such as the movement of cilia, which
speedily ceases after removal of the tissue from the body.
Under such circumstances a small piece may be snipped off
with scissors, teased (p. 40) with needles, and mounted in one
of the following solutions, when sufficient of its structure can
generally be made out — drying of the specimen being prevented
by edging the cover-glass with oil or paraffin-wax :

iSTormal saline solution — a 0"G per cent, solution of sodium

chloride.
Blood serum.
Aqueous humour.

Iodised serum, — i.e., serum or liquor amnii to which a little
iodine has been added as a preservative.
Muscle may be examined without the addition of any

rRA< TI< AL HISTOLOGY. 17

reagent ; but its evaporation must be prevented by the employ-
ment of appropriate means.

Other exami)les of a like nature will be treated of under
their proper headings.

2. The Examination of Hardened and Prepared Tissues.

Speaking generally, it is impossible to make out the structure
of a solid tissue thorouglily until a thin section of it has been
obtained. The consistence of the tissues in the recent state, as a
rule, will not allow of such sections being cut, and it is therefore
necessary to harden them before attempting that process.

The hardening of tissues may be accomplished either by
freezing them or by various chemical reagents. The latter
act by coagulating the albumen, by withdrawing the water, or
in some instances, perhaps, by combining with the albumen
to form a harder compound — in a manner comparable to the
process of tanning.

Hardening reagents. -It is of the utmost consequence that
the process of hardening be attended to with the greatest
attention. The choice of a suitable reagent in each case will be
found, in spite of anything said to the contrary, a matter of con-
siderable moment, and its mode of use equally important. We
mention the hardening reagents we have found most service-
able.

Chromic Acid and Spirit. — A mixture of chromic acid and
si)irit is the fluid recommended for general use, and can be
employed almost universally. It is thus prepared : Chromic
acid }. per cent, solution {i.e., containing 1 grm. in GUO cc.
distilled w^ater), 2 parts ; methylated spirit, 1 part.

Another way of making a solution is by taking equal parts
of 0*5 per cent, solution of chromic acid and of methylated
spirit. The mixture produces its effects in about seven to ten
days.

Chromic acid, without the addition of spirit, in "lb to 'o per
cent, solutions, is a rapid hardening fluid. The tissue is suSi-
ciently hard in a week for sections to be made ; if, however, it
remains in the mixture for a longer time, it tends to become
brittle.

18 PRACTICAL HISTOLOGY.

Potassium bichromate in solutions, the strengths of which
vary from 1 per cent, to '> per cent. : the best is the 2 per cent.,
and this, if changed every four days, hardens tissues in a
fortnight.

Ammonium chromate in sohitions 2 per cent, to 5 per cent,
is superior in some ways to the similar potassium salt. Tissues
should not remain in it for more than a day or two.

Ammonium bichromate is specially recommended for hard-
ening the brain and spinal cord, in solutions of 5 per cent.
strength. It must be prepared fresh when wanted.

Muller's fluid is made by taking potassium bichromate.
2 grms., and sodium sulphate 1 grm., and dissolving them in
100 cc. of distilled water. When this reagent is emploj-ed, the
process of hardening is slow, but efficient. The fluid has the
advantage of being exceedingly penetrating, and so will harden
satisfactory V larger pieces of tissue than other similar agents.
Half the quantity of copper sulphate may be substituted for
the sodium sulphate.

Picric acid, in saturated solutions, or in mixture with an
equal quantity of methylated spirit, or with an equal quan-
tity of o to 10 per cent, hydrochloric or nitric acid, is a useful
and quick-hardening fluid.

Mercuric chloride, in saturated or weaker solutions, or
mixed with acetic acid, is becoming a popular hardening reagent.
Its action is rapid.

Nitric acid, 3 per cent, to 10 per cent., is recommended for
hardening the brain. It requires for its action two or three
weeks.

Methylated spirit is a hardening reagent in very common
use, and may be employed with advantage in the preparation
of salivary glands, stomach and intestine, etc.

Absolute alcohol of specific gravity 0"795 is the most rapid
hardening fluid. It is not often used, on account of its expense,
and because it is said to have a greater tendency to produce
shrinking of the tissue hardened, than preparations of chromium.
The pancreas is an organ, at any rate, which must be immersed
in this fluid, in preference to any other, for the purpose of
hardening.

rilACTK AI, HISTOLOCV. 1 !♦

Osmic acid. 01 to 1 per cent. \ All of these solutions

Gold chloride. •-'•'' to •.'> per cent. | harden, and at the

Palladium chloride, '25 to '5 1 same time stain tis-

per cent. J sues.

Directions for Hardening. — Never use anything but fresh
tissue. Nearly all the material used, therefore, must be taken
from the lower animals just killed, as it is seldom possible to
obtain specimens from the j)ost-mor(eiu room sufficiently fresh to
give satisfactory results.

Cut ihe tissue into ]»ieccs with a sharp knife or razor. The
size of the pieces "vvill vary with the reagent used. "When
chromic acid is the hardening fluid, the pieces should not be
larger than a hazel-nut. "When alcohol, potassium bichromate,
or Mailer's fluid is employe J. they should not exceed twice that
size.

Do not wash with water : but if it be necessary to get rid
of any foreign body, allow a small stream of saline solution, or
of dilute spirit, or of a weak solution of potassium bichromate,
to run upon the tissue from a wash-bottle.

Place the cut pieces in a large excess of the hardening reageit
in a stoppered bottle.

Change the hardening reagent frequently — ■?.//., the chroncic
acid and si)irit solution on the second, fourth, and seven :h
days.

In all cases, in a week to ten days remove the specimens to
spirit, or, if that fluid be contra-indicated, to a "5 per cent.
potassium bichromate to complete the hardening,

SECTION CUTTING.
Having hardened the material, the next thing is to cut thin
sections from it ; for unless the sections be thin, no amount of
after preparation will make them fit objects for microscopical
investigation. The methods which have been proposed from
time to time to effect this object are numerous, but they may
be divided into two classes — viz. :

(1) Methods of cutting by hand.

(2) Methods of cutting with machines called microtomes.

2—2

20

PRACTICAL HISTOLOGY.

I. Section-cutting by Hand.

In cutting a small piece of tissue it is customary to embed it
in some other tissue, or in a wax mass of some kind. For the
former purpose, the tissue which is to be cut is placed between
two pieces of hard liver,* or material of similar consistence, and
held tightly in place between the finger and thumb, and cut
with a razor in the manner to be described below. Instead of
the liver, pieces of turnij), carrot, or potato may occasionally be
substituted.

The usual method, however, is to embed the specimen in a
wax mass.

Embedding materials are wax masses of some kind, modified

\

E

A

/

/

C

i)|

B

B'

A N

C

j:

A

D^

Fig. 10. — Diagram to show Fonnatioii of Embeclcliiig Box.

according to the state of the weather and the material to be
cut. The following are those most commonly employed :

"White wax and olive oil, equal parts ; melted and well
mixed. This mass may be varied in consistency by
varying the amount of the olive oil used. Japanese wax
(8d. per lb.) is about one- fourth the price of English,
and answers well.
Paraf&n and Lard.- -Take five parts by weight of solid
paraffin (a paraffin candle will do very well), and one part
by weight of hog's lard and of paraffin oil ; melt at a
gentle heat, and mix thoroughh*. Paraffin wax (Is. 4d.
per lb.) of two kinds, melting at about \o and 55' C, may

PRACTICAL 1IIST0L0C;Y.

21

be obtained ; and, by niixint,' in v:ui(/ns propoiti< iis, will
supply wax of required melting-points for the different
seasons of the year, witliout further admixture of lard.

Spermaceti and Castor Oil. -Take four ])arts of sper-
maceti and one ])art of castor oil.

Cacao butter may be used alone or combined with paraffin,
wax, and oil, or with spermaceti and parathii.

To melt the Wax Mass.— Tiie wax mass is melted in a small
porcelain capsule provided Avith a handle, over the flame of a
Bunseu's burner or spirit-lamp. Care must be taken that the
material is not burnt. It is usual in laboratories to place the
capsule on a piece of fine iron gauze on a tripod, and to place a
gas flame from a Bunsen's burner of the smallest size beneath
it : a glass rod may be used as a stirrer.

To embed the Specimen in Wax Mass for Cutting.— A piece
of stout pa})er is taken, six inches long and three broad. This

Fig'. 11. — Paper Box for Embedding.

is doubled into three longitudinal folds ; after this from each
end folds of two inches long are marked off. The paper is then
opened out, and of the three longitudinal folds the middle one
forms the bottom and the lateral ones the sides of the paper
box. The ends are made from the middle part of the end folds.
The ends of each flap are marked off into two equal squares,
]•: c, c D, E^ c\ ('^ 1)1. The squares e b a c and e' ij^ a^ c^ are
doubled into two parts across the diameters A B, A^ h\ and these
triangular folds thus made are pinched up and pressed against
the end of the box to support it ; they are retained in position
by the remainder of the end fold represented by a a^ d d^ being
turned back over them.

Having made a paper box in the manner above described, and
having melted the wax mass, take the specimen to be embedded

22 PRACTICAL HISTOLOGY.

upon a needle mounted in a holder, and having removed the

superfluous absolute alcohcl i i which the tissue ought to have

been immersed for at least ten or fifteen minutes before the

operation is commenced) with blotting-paper, half fill the paper

box with the melted wax mass, and dip the specimen into it

several times, until it is thoroughly enveloped in wax. Allow

the wax to ccol, and place the tissue on it in the box at one

end ; then fill the box with melted wax, and after it has

hardened, mark on the outside the i)osition of the tissue. AVhen

quite hard, turn out the wax and the embedded tissue by opening

the ends of the box, and place for a few minutes in methylated

sjjirit.

Instead of the simple paper box, tin boxes with removable
bottoms may be substituted, or hollow brass cylinders of various
diameters, placed upon a glass plate.

The tissue is now ready for cutting. It is as well to shave off
the corners of the wax. and also to cut off several thin slices
from the end near which the specimen is with a sharp knife, in
order that when the cutting of the specimen is to be done, the
razor may not be blunted by having to cut much wax.

Razors. — As a good deal of the success of hand-cutting
depends upon the sharpness of the razor, one cannot be too par-
ticular in choosing the instrument. It should be of good steel,
capable of easy sharpening on a strop. It must also be broad
bladed and hollow-ground. The original ' Army Razor '
answers all these requirements. It should always be stropped
in one direction. Section-cutters of various forms have been
introduced, but do not f ossass any advantage over razors.

Directions for Hand Section-cutting.— Be sure the razor is
very sharp.

Hold the razor firmly in the right hand. M'iih the fingers
closed above the handle : take the wax mass between the index-
finger and thumb of the left hand, support the back of the
razor on the former, and cut from left to right and from heel
to tip of the razor.

Let the handle be kept in line with the blade.
Keep the blade well wetted with spirit, into a glass capsule
half full of which also the cut specimens must le floated off with

riiAcTiCAL ]1istolo(;y. 23

a carael's-hair brush .if ter each sweep of the razor ; unless the
specimen has been aheady stained and dehydrated, in which case
<love oil is to be used instead of spirit for wetting the razor and
preserving the sections.

2. Section-Cutting with Microtomes.

It must be confessed that hand section-cutting is very much
going out of fashion.

The reason is not far to seek. The skill required for hand
cutting in order that sections uniformly thin may be cut with
certainty is very rare. Microtomes by various mechanical
means in cutting sections do away with the uncertainty of
hand cutting. They are invariably used when a large number
of sections is required, and are invaluable for cutting for a large
class. Some histologists still prefer hand cutting for all delicate
materials ; indeed, for some organs few microtomes can be
employed with satisfactory results.

The simplest microtome is Ranvier s, which consists of three
hollow brass C3linders fitting one inside the other ; to the most
external a flat circular brass i)late is fixed at one end, and to the
other a cap which can be screwed on, carrying through its centre
a fine screw. The tissue having been embedded in paraffin-
wax in one of the cylinders and allowed to cool, the cap is
adjusted, the microtome held in the hand, and the screw turned
until the wax is carried up sufficiently high for the embedded
specimen to be cut ; the flat plate guides the razor. After each
section is cut the screw is turned slightly, thereby presenting
sufficient of the specimen for another section,

Stirling's microtome is on the same principle as Ranvier's,
but is larger, and is fixed to a table by means of a screw. Only
one cylinder, however, is fitted to the instrument,

Rutherford's microtome provides a trough which may be
used to contain a freezing mixture of ice and salt. AVhen this
is used, the tissue is embedded in thick gum, which, on freezing,
becomes quite solid, and may be readily cut.

Both these microtomes may be provided with glass plates in-
stead of brass, to guide the razor.

These three microtomes raise the tissue embedded in its

24

PRACTICAL IIISTOLCGY.

Fig. 12. — Zeiss' s Microton

dara ffin or gummy cylinder to the knife. This is. in our opinion,

a mistake. The tissue should
be firmly fixed, or at any rate
should be exposed to as little
disturbance as possible, and the
razor should be lowered to it,
and not vice versa.

In Zeiss's microtome (Fig. \'l)
our objection is partly met by
the cylinder containing the wax
being moved upwards bodil}-,
^ and not the wax only. For many
^g reasons, indeed, we strongly re-
^ commend this form to student?.
The instrument is well made,
and the screw is so fine that sec-
tions of extreme thinness may be cut by its aid. The price of
it is £2.

Of microtomes upon the same principle, but in which the knife
is fixed and works in a plane, moving backwards and forwards,
and cutting a section at each sweep, the tissue embedded in wax
or frozen in gum being raised by means of a fine screw, we may
mention the following :

Rivet, the £2 10s. form is the best.

Jung (£7, £4 15s. and £2 15s.), Heidelberg, of extreme

delicacy and accuracy.
Leitz, Support-Mikrotom (£6).

Rsichert, Schlitten-Mikrotom (£5 12s., £7 10s., and £0 5s.).
Bcecker, Wetzlar (£9 Os.).
Zeiss (£5 10s.).

Beck, Universal Microtome (£15 15s.).
Caldwell's Microtome, and the rocking-microtome, made by

the Cambridge Instrument Company.

All of these are good. Special advantages are usually claimed
for each variety— some automatic action of turning the raising
screw, some extra steadiness, a ribbon arrangement by which the
sections when cut are removed in a continuous series, or what not.

PRACTICAT, lirsT< H.0( ; V

25

At the same time it should be recollected they are very expensive
— Caldwell's microtome originally costing, wc believe, about £30.
The ribbon microtomes are indeed triumphs of mechanical con-
btruction, but are, of course, not applicable for students' use.

The form of microtome, however, which we believe from long
experience to be the best for all practical purposes is Williams's.
In it the specimen is fixed, and the knife is approached to it.
It consists of a circular wooden box about eight or nine inches
in diameter. Into the centre is fixed a circular solid brass
jtillar, -with a brass plate at the top. This box contains a
freezing mixture of ice and salt, and is provided with a waste
tube to get rid of the water as the ice melts. The cover of the
box is wooden, but has a plate of glass fixed upon its upper
surface ; it also has a central aperture to admit the top of the
brass cylinder. This cover is fixed down after the freezing
mixture has been placed in the box, and fastened with a lateral
screw. The specimen, which should first be left for twelve
hours in water (to get rid of the spirit), is placed on the cylinder
plate in a little gum. The gum is soon frozen, and the specimen
is fixed b}' this means. The cutting is effected by means of a
razor, which is fixed in a movable triangular brass frame,
supported on three screws. By the adjustment of the front
screw the thinness of the

section is regulated. The
frame works smoothly
over the glass table. The
upper surface of the razor
should be slightly moist-
ened with gum. As the
sections are cut they
should be swept off the
razor with a camel's-hair ^^S- 13.— Williams's Microtome.

brush, and should be washed in warm water before staining.

This microtome adapted for use, with ether as the freezing
agent, may be obtained in various forms. The instrument costs
about £2 10s.

Freezing mixtures are made by taking pounded ice, adding
«omc rough bay-salt, and thoroughly incorporating the one with

•2Q

PRACTICAL HISTOLOiiY.

the other. In very cold ^veather snow may be obtained and
used in place of the ice.
A strong solution of pure gum arable in boiling distilled

water is made, and

A

Fi?. 1-1. — Catlicart's Microtome.

filtered through calico.
To this are added a few
drops of melted i)ure
carbolic acid. The pro-
portion of gum is about
a pound to a quart of
water. It should be
kept in a stoppered
bottle, and boiled occa-
sionally, to prevent de-
composition.

"We may mention a
convenient and cheap
form of ether-freezing
microtome, viz., Cath-
costins: l.'js. to

A Metal i^late upon -uliicLi the tissue is
l)lace(l ill guin ; b Bottle coutainiiig ether ; c

ether sprav apparatus ; d Screw to raise carts,

tissue when frozen; e Screw to fix micro- .>,, fianrf of which

tome to table. The cutting is done by a -'-'^•' ^ ^^^^^ ^^ wuicn

plane which rests on the supports on either we annex.

''"^^°^-^- We now adapt the

principle of the knife-carrying triangle in section-catting
to nearly [0\ our histological work. It was found in
practice that the small brass caps for use with specimens
embedded in wax, to be screwed on to the central brass pillar in
Williams's microtome, did not work well. Instead of this, we
use a sheet of plate-glass, upon which we place the specimen to
be cut thoroughly infiltrated with paraffin- wax. When the wax
is cold the specimen is fixed upon the plate. The triangle in
use in this case is of much heavier make, and the screws are
larger, and of finer worm, than usual. With a simple appa-
ratus such as this, sections of every kind of tissue which
Avill bear staining in mass and soaking in melted wax may be
luade with ease. We have had much success with embryo-
logical specimens treated after this manner. Mr. Stanley has
made these heavier triangles for us. Their cost is about £1 5s.

I»RACTICAL lUSTOLOiiY. *J7

As regards the preparation of tissues for this method of
cutting, see p. 41.

Having cut thin sections of the tissue to be examined with
the microscope, and having washed them in water to get rid of
the gum, or in spirit in which the adhering wax drops off if the
sjjecimens have been embedded in that material, it is necessary
to proceed to the next operation, viz., that of Staining.

STAINING REAGENTS.

Almost all tissues require to be stained with some dye, in
order that certain elements of their structure, which would
otherwise be indistinct, may be more clearly shown. The follow-
ing dyes are those which are usually employed :

Aqueous Haematoxylin or Logwood is the simplest and
best. It is a highly selective and clear dye. It is made by
taking dried extract of haematoxylin ('»0 grms., alum in powder
ISO grms., and rubbing thoroughly together in a mortar, and
adding slowly 300 cc. distilled water ; mixing carefully and
filtering ; to the filtrate adding :!() cc. of absolute alcohol, or
50 cc. of a 1 per cent, solution of thymol, and preserving in a
stoppered bottle.

The solution should be kept in a cool place for at least a
fortnight before using. The older it is the more excellent it
becomes. A second but weaker solution may be made by add-
ing a second ij'Ml cc. of distilled water to the same logwood
alum, and proceeding in a similar manner.

Alcoholic Haematoxylin is prepared by making saturated
solutions of crystallized calcium chloride and of alum in proof
spirit, mixing the solutions in the proportion of one to eight,
and adding to this mixture a saturated solution of haematoxylin
in absolute alcohol, drop by drop, until the whole becomes a
dark purple. This solution may be used at once, but is greatly
improved by keeping.

Carmine was formerly used much more than it is at present ;
it has the disadvantage of staining specimens a colour trying
to the eye, whilst it has not the great selective power of haema-
toxylin. There are many ways of making carmine solutions.

28 PRACTICAL HISTOLOGY.

('/; Take 2 grms. of carmine, and rub thoronnjhly in a
mortar with a few drops of water ; then add 4 cc. liq.
ammoniac and 48 cc. distilled water : filter into a bottle,
which s^hould be left unstoppered for a day or two for
the excess of ammonia to evaporate. This forms a
strong solution, w^hich must be diluted before using
(Klein).

('^) Beale's solution is thus prepared : Dissolve carmine
grm. j. in liq. ammon're fort, o cc. warm, add aq. des-
tillat. 120 cc, and filter. Then add glycerine liO C3., and
ppir. vini rectif. 120 cc, and keep in a well-stoppered
bottle.

((•) Borax carmine is made by thoroughly mixing carmine
(grms. ij.) and borax (grms. viij.) in a mortar, and
dissolving in warm water for twenty-four hours. The
supernatant fluid, which should be decanted, is then
ready for use.

(r7) Alum carmine. Boil an aqueous solution of ammonium
alum with excess of carmine for ten to twenty minutes ;
filter, and dilute the filtrate until it contains 1 to 5 per
cent, of alum. Add a few drops of carbolic acid to pre-
vent the growth of fungus.

{e) Alcohol carmine. A teaspoonful of carmine dissolved by
heating for about ten minutes in oO cc. of 00 to 80 per
cent, alcohol, to which three or four drops of hydrochloric
acid have been added, and then filtered,

Picrocarmin, oi- solution of picvocarminate of ammun'ia, is now
much used, in combination with another dye when it is expedient
to ' double stain ' tissues in order to bring out certain special
features in their structure. It is prepared by adding a saturated
ammoniacal solution of carmine to a saturated solution of picric
acid until a precipitate begins to form, evaporating in a water-
bath to one-third its bulk — filtering and evaporating the filtrate
to dryness : a crystallized mass, easily soluble in water, is
obtained, which is picrocarmin. The strength of the solution
should be about 1 per cent, to ?> per cent. During preparation
the ammonia should be kept in excess.

It may also be made by taking Beale's carmine without

rRACTICAL IIlSTol.OCjy. '20

alcohol, and adding the picric acid in a similar niarincr. The
glycerine prevents burning, which is not unlikely to occur.

Many other formuhc have been given for mailing this useful
dye, which we do not find superior to those above given.

Cochineal. — (a) Take 7 gnus, cochineal and 7 grins, alum in

powder, thoroughly rub together in a mortar, and add

700 cc. distilled water; evaporate to 4UU cc, filter /iricc^

and afterwards add 5J.or5ij. absolute alcohol, or of 1 per

cent, thymol solution.

(b) Powdered cochineal 10 grms., alcohol 70 per cent. loO cc.

Macerate for a week, in a stoppered bottle, shake up

frequently'. The filtered solution will be ready for

staining. AVash stained sections in alcohol of above

strength.

Purpurin (Ranvier).- The formula of a solution of this dye

first introduced by Ranvier, which is most stable, is Grenacker's :

Glycerin 50 cc, powdered alum 2 grms. Add a knife-pointful

of purpurin, and boil. Let the solution stand for several days,

and filter. Staining in this solution takes 10 to 30 minutes.

Ink-stain. — Excellent results may be obtained by staining
nervous tissue, especially the spinal cord, in diluted solutions of
Stephens's blue-black ink.

Eosin, CjoHgBrjOrjKo, a rose-red i)henol dye, is used chiefly
to stain nervous tissue and blood, and in combination as a
double stain. It is very soluble in water, and requires to
be fixed. In practice it is advisable to leave the tissue in a
'01 per cent, solution for twenty-four hours, and then to pass it
through acidulated water. It is unnecessary to keep it in solu-
tion, as it can be made so easily.

Some, however, recommend a 1 per cent, solution in alcohol,
others in ammonia, and others 1 per cent, in alcohol with alum.
As far as our experience goes w'e have obtained the best results
from a simple aqueous solution.

Aniline Dyes. — An immense number of aniline dyes are now
manufactured, and most of them have been tried in histological
work.

Man}' of them stain quickly and penetrate well, but their use-
fulness is diminished by their tendency to wash out.

30

PRACTICAL HISTOLOGV.

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vviiter, sol. in
dilute spirit.

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PRACTICAL HISTOLOGV^ 31

In usini,' .iniline dyes, therefore, it is necessary t.» ^tain speci-
mens deeply, and to pass them through the desiccating and
clearing fluids quickly, otherwise the colour passes into the
solutions ; for at jnesent no one fixing fluid has been made
which produces certain results ; in son'e cases, however, a weak
solution (about •'> per cent.) of hydrochloric acid is effectual.
Solutions of mercuric chloride, of potassium acetate, of sodium
xanthate,and of tannin. havealso been recommended as mordants.

The table on p. 30 includes most of the aniline dj-es which
have been employed in microscopical work.

Speaking roughly, the dyes are most useful which will dis-
solve without the aid of spirit. In nearly every case slow
is to be preferred to (^uick staining ; this is especially true with
regard to iodine green, aniline black, and Bismark brown.

Some information will be given later on as to the use of these
anilines in bacteria staining.

Silver Nitrate is used when it is required to demonstrate the
endothelial cells of serous^membranes. The'salt is taken up by
the intercellular substance when fresh, and is reduced as a black
precii»itate under the action of light, which maps out the cells.
in black lines. The fresh tissue should be placed after removal
from the body in a '5 or '25 per cent, solution for ten or fifteen
minutes ; it should then be washed carefully in distilled water,
and exposeJ to the light in glycerine diluted with three times its
bulk of distilled water. Silver nitrate is also used to stain nerve-
fibres.

Preimratlon of the Solution of Silver Xitratc. — Powder 5 grms.
of crystallized silver nitrate finely in a mortar, and add
gradually lOOU cc. cold distilled water. After the salt has dis-
solved, preserve in a stoppered bottle of dark glass, or in one
around which some black paper has been pasted, and keep in a
dark cupboard. The use of the solid silver nitrate in bringing inta
view the cell spaces of the cornea will be alluded to farther on.

Ammonium-molybdate produces a bluish-gray general stain,
which acts well as a base for double staining. A 5 per cent,
solution in water may be used, and the specimens should be
exposed to the light for twenty-four hours. The salt is expen-
sive, and the advantage of its use not very marked.

32 PRACTICAL HISTOLOGY.

Gold chloride selects and stains certain tis^sues, principally
the nervous ; it also brings out the cells of fibrous connective
tissue, cartilage and cornea.

Meiliod of Gold Staining. — Of the many Avays we have tried
of using this salt we recommend the following. The tissue is
removed from the animal immediately after it has been killed,
and is placed in "5 per cent, solution of gold chloride for half an
h'our to an hour ; it should then be washed in distilled water,
kept in a warm, dark place in a saturated solution of tartaric
r.cid for three or four hours, and afterwards exposed to the
light in equal parts of glycerine and water. Immersion in
filtered lemon-juice, in citric acid, or in dilute formic acid for
about five to ten minutes previous to the bath of gold chloride
is advised by some. Various acids, too, are recommended in-
stead cf tartaric, as citric (saturated solution), formic ("20 per
cent.), acetic (4 per cent.), oxalic (^ per cent.). The double
chloride of potassium and gold may be substituted for the gold
chloride.

Method of pveparinrj the Solution. — The gold salt is sold in
sealed glass tubes, containing about 1 grm. ; the tube should be
broken, and the salt should be dissolved and preserved in a
manner similar to that described above under the heading of
Silver-nitrate Staining.

Palladium chloride, an irregular general stain, yellow to
black, in solutions varying from "1 to •05 per cent., maybe used ;
it has the same effect as gold chloride in hardening tissues and
staining them at the same time. It is very expensive, and is
not recommended.

Osmic acid, as well as the two preceding salts, possesses the
property of hardening as well as staining tissues placed in it.
It is usually sold in 1 per cent, solutions, which may be diluted
with distilled water at pleasure. The solution must be kept in
a dark glass bottle. Osmic acid stains fat-globules black, and
Irings out the medullary sheath of nerves. S])ecimcns to be
stained with this reagent must remain in it for about an hour,
and should then be removed to spirit.

Method of using the Staining Reagent. — Unless another
reagent is specially advised, the student should, as a matter of

rHACTICAL HISTOLOGY. 33

course, employ haimatoxylin, as it is the easiest to use and the
best of all stains.

Such specimens as have been already selected as sufficiently thin
should be thoroughly washed in distilled water or in methylated
spirit, and placed in spirit in a watch-glass of suitable capacity.

Sections of tissues, hardened in any chromium preparation,
should then be transferred to a watch-glass containing a solution
of sodium bicarbonate 1 per cent., and should be allowed to
remain in it for five minutes, in order that the effect of the
hardening reagent may be neutralized. They should then be
well washed in warm distilled water (iiO^ to 40^ C).

To prepare the staining solution, it is as well to take a large
watch-glass, fill it three-quarters full of distilled water, and then
add to it from five to ten drops of aqueous haimatoxylin. The
hoematoxylin solution is kept in a bottle provided with a funnel
iind filter-paper, as the reagent must be filtered before it is used.

Having made the solution, and thoroughly mixed it, place the
sections in it carefully one by one with a broad needle, and
if they float, press them down and leave them in the fluid for
some minutes. The time required varies, as some tissues stain
much quicker than others, and it is therefore necessary to take
out a section from time to time, and to place it in a watch-glasi;
full of distilled water, so that the staining may be regulated.
Great care is necessary, in order that the specimens be stained
neither too much nor too little. The examination of the
specimen in the watch-glass should be done with a white
background, which maybe obtained by placing the gla-^s on a
white filter-paper, or still better on a white glazed tile. This
tile may easily be obtained, and will be foand a great con-
venience.

It may not be out of place to notice here that stained specimens
should always be examined during manipulation in this manner,
and that unstained specimens can be most conveniently examined
if placed in a watch-glass upon a black plate. This black plate
is generally a square piece of glass with the back blackened and
protected with varnish. A dark-coloured glazed tile will answer
the purpose.

When the sections are sufficiently stained, they must be

3

34 PRACTICAL HISTOLOGY.

washed in distilled water and placed in methylated spirit, ready
for the next process (pp. 35-40).

Instead of the washing in the warm distilled water previous to
staining, washing in a very dilute solution of hasmatoxylin
(three or four drops to a watch-glassful of water) may be sub-
stituted.

If the sections le accidentally overstaiued, place them in a
'5 per cent, solution hydrochloric acid for a few seconds.

Watch-g' asses and capsules of various sizes should be
obtained. The ^mall thin shallow glasses are of no use;
those recommenclel Q.vi large, thick, and deep. The capsule
which is used to contain spirit for hand-cutting must be large
and capacious — 2^ to 3 inctes in diameter, and li inches deep.

Staining ^vith Carmine, Eosin, and Aniline Dyes.— The
method of staining with these reagents is very similar to hajma-
toxylin staining, but specimens should remain in the solution
for a much longer time (twenty-four hours or more), and the
dye should be fixed by passing'^the sections through acidulated
water. Sodce bicarbonate solution is not necessary. When a
spirit solution of any dye is used, pass rapidly on to the next
process after staining. "VVe cannot, however, give any other
than general directions for staining with these dyes, and the
operator will soon find out the best methods of using each.

Double Staining. — To demonstrate the structure of some
tissues satisfactorily, it is necessary to make use of two or more
staining reagents. This process is called, double or multiple
staining, according as two or more dyes are used. The simplest
form of double staining is that adopted for tissues which are
first stained with silver nitrate, gold chloride, or similar reagent,
and then with hiematoxylin, carmine, or aniline. Other com-
binations may be made, of which the following are useful :

Picrocarnun and luematoxulhi.

Eosin and hcematoxyl'm.

Eosin and aniline green, etc.

Picroearmin and anilines, etc.

Euchsine and methylin blue,

Lo.-yin and niethylin blue.

A ndine rose and aniline green.

Lismarh brown and aniline green.

PRACTICAL IIISTOLOflV. 35

It is unnecessary to do more than mention Irehh staining. If
this be ever required, the combination recommended is, {])p{rro-
cannin ; (2) rosein : (3) imJine fjrrcv.

As regards staining with four colours, one of the authors has
found that it may be done with (1) picrocarniin or ens'in ; (2)
f Of) (rood ; (3) aiiiliiif rose : (4) aniline green. If the tissue have
been already stained in gold chloride, five stains will have been
used. However, the processes are tedious, and not of any great
practical value.

Dehydration and Clearing. — AVhcn the sections of the
tissue have been stained, or double stained, as the case may be,
unless they are to be mounted in glycerine, or one of its substi-
tutes, they require to be dehydrated and cleared.

Dehydration. — This process is effected by passing them
through methylated spirit and absolute alcohol. After staining
they should remain in each of these fluids for five minutes.

Clearing. — After immcr.-ion in alcohol, the sections have to
be placed in a fluid which will render them transparent. The
fluid most extensively employed is clove oil : it is a hydrocarbon
isomeric with oil of turpentine, and possesses the advantage
over the latter of being more agreeable to manipulate. Oil of
turpentine may, however, be used, as also some of the other
oils which are isomeric with it. A clearing fluid we have found
a cheap substitute for clove oil is, turpentine 4 parts, crea.sote
pure 1 part, mix and filter. These fluids penetrate the tissues,
and render them fit to be mounted in Canada balsam.

MOUNTING.

For this process the operator must have —

(1) Glass slides,* which are slips of glass three inches long
and one inch broad, about the thickness of ordinary window -
glass, with or without ground edges. They arc .sold by the
makers of microscopic requisites, at from 2d. to Od. a dozen. It

* Tliese, as well as cover glasses, may be obtained of Tate, Holl)oni ;
Stanley. London Bridge ; Beck, Coruliill ; Mediand, Loudon Bridge ;
Baker, Holborn ; Crouch, Barbican ; and of many othtrs.

3—2

36 FRACTICAL HISTOLOGY.

is rs well to keep a good stock on hand ; and, as a lule, within
reasonable limits the thinnest are the best.

(2) Cover-gIasi<:es, which are made of extremely thin glass,
circular or square, ^ to | inch in diameter. There are several
kinds sold, usually known as ordinary, thin, and extra thin.
The ordinary are quite thin enough for the student, but some-
times it is ai well for him to provide himself with each kind.
The ordinary cover glass measures from '004 to '008 inch in
thickness.

To measure Cover-glasses. — Thin glass may be placed edgewise
in the stage forceps of the microscope and measured very accu-
rately with the micrometer. The student maj^, however, neglect
this operation.

To cut Cover-r/Iasses. — Thin glass may be bought in sheets, and
cut into squares or circles by the operator with a diamond, but
this is not advised.

To clean Sikhs and Cover-glasses. — For the former it isgeneral'y
only necessary to wash them in soft water or weak soda-water,
d ying with a clean cloth, and polishing with chamois leather.
A mixture of ether and alcohol, caustic potash, infusion of nut-
galls or alcohol, however, has to be used sometimes. Covei-
glasses may be washed in strong potash, or in infusion of nut-
galls, or in alcohol, and should be dried and polished with a fine
cambric pocket-handkerchief.

(3) A sect/'on-Iiftn', which may be easily made by beitin^ out
flat one end of a thick copper wire, four or five inch( s in length.
The flattened portion should afterwards be filed at the edges,
and rubbed smooth with sand-paper.

Fig. 15. — Section-lifter.

Merj carefully finished section-lifters may be obtained of
instruaaent-makers ; the annexed figure represents one form

PRACTICAL lIISTOLOf;Y. .'^7

of lifter suggestetl by the authors for mounting large sec-
tions.

Mounting Fluids. — Fresh tissues may 1 e mounted in any of
the reagents mentioned at p. 1(). The following fluids may
also be used :

Potassium acetate, in saturated solution, employed chiefly

for mounting; vetjctable tissues.
Glycerine, one of the most useful of mounting fluids. It
may be em.ploycd for fresh tissues, as well as for tho.^e
■which have been hardened and cut into sections. The
fluid should be of high specific gravity ; some micro-
scopists advise dilution with a third of distilled water,
but we cannot endorse this recommendation.

It must be remembered that in this reagent some

tissues swell up— i.e., fresh tendons — and so lose their

characteristic structure, and for such the reagent is

contra-indicated.

Directions for Mounting in Glycerine.— Place the tissue for

a quarter of an hour in distilled water, transfer to the slide,

spread out, and remove the excess of water. Then place a drop of

strong glycerine on a thin cover-glass, and invert it over the

specimen, taking care to keep the drop in the centre of the

cover until its lowest point touches the centre of the specimen,

and then allow the cover to fall gently on it. If the glycerine

entirely fills the space under the cover, and is not in excess, the

edges may be painted round with some cementing varni>h. If

too much have been taken, the excess may be removed with a

capillary pipette, or with filter-paper.

Farrant's solution is a useful substitute for glycerine, to be
employed for the mounting and preserving of sections and
teased specimens. It does not render the tissues so transparent
as does glycerine ; whilst the cover-glass becomes fixed to the
slide as the solution hardens.

It is made by adding an equal weight of powdered gum
arabic to a mixture of equal parts of glycerine and a satuiated
aqueous solution of arsenious acid.* The mixture is then

* The arsenious acid may be omitted, and a piece of camphor may be
ii.troduced in its place.

38 PRACTICAL HISTOLOGY.

allowed to stand for six weeks, being stirred at intervals. Any
gum which remains undissolved is then filtered oft" ; and the re-
Fulting clear filtrate is Farranfs solution.

Glycerine jelly is made by taking pure gelatine 8 parts,
soaking it in cold water for Feveral hours, pouring off the water
and warming the gelatine until ]melted, adding 1 part of egg
albumen, boiling until the albumen is coagulated and the gela-
tine is clear, filtering through flannel, and finally adding <>
parts of a mixture of 1 part of glycerine to 2 of cam]>hor
water. [See aLo Appendix.] It is advisable to buy this
reagent, as the making of it is difficult.

Carbolic acid solution (one in forty) may sometimes be ufed,
or a mixture of this with alcohol and arsenious acid.

Castor oil is employed to mount crystals, etc., \\hich aie
soluble in Canada balsam.

Directions for Mounting in Canada Balsam, etc.— It may
le as well here to recapitulate the processes to be gone through
before the sections are fit for mounting in Canada balsam. If
the section be cut in f-pirit or water, it is passed through —

If hardened in any pref aia-

\

tion of chromium to neu-
Scdas licarhcn, 1 per cent. ' , ^■ .■> u a ■

' '■ I tralize the hardenmg re-

agent.

sh away excess of the
icarbonate.

5 To wa
( bica:

Distilled -water.

Staining fluid.

Methylated spirit. ) ^^ ^^^ j,,^j^_

Absolute a-conol. 3

Clove oil, or ) rr^ 1 X i.

„ , .' , „ X ^ I' To render transparent.

Turpentine and Creasote^etc J

Piejxn-ation of Canada Baham Sole. I ion. — The best method is
to take the commercial balsam, expose it to a tem])erature of
70° C. for twelve hours, to dry it and render it quite hard, and
then to dissolve in benzol and filter. It should be preserved in
a stoppered bottle.

Another metbod is to mix equal parts of Canada balsam and
chloroform and warm. Thi balsam is entirely dissolved.
Filter.

rRACTK'AL HISTOLOGY. 39

Preparation of Dammar Solution. — Dissolve gum dammar in
powder 50 grms. in 150 cc. turpentine, and filter : gum mastiche
l>{) grms. in 'JOO cc. chloroform, and filter. Mix the solution,
and again filter. This solution can be employed in place of
Canada balsam. Benzol may be employed instead of turpentine
and chloroform.

Drop-hottlexfor Canada Baham, Dammar^ etc. — Mounting fluids
such as Canada balsam are usually kept in bottles with narrow
elongations of the stoj^per-, which dip into the fluid, and M'ill
deposit it on the cover-glass in drops (Fig. IG).

Having placed the sections in clove oil in a watch-glass or suit-
able glass dish upon a white plate, as above directed, take a clean
slide and place it upon a white filter-paper. Next, by means of a
section lifter and a needle, withdraw the se3tion from the clove
oil, and bring it down upon the centre of the slide, and remove
the excess of clove oil with filter- paper. Then take a clean
cover glass, and p'ace a drop of the Canada balsam solution on the
centre of it, and proceed in the manner described under the
head of Glycerine Mounting (p. 37), or take hoM of a corner
with a pair of forceps, and gradually incline it over the speci-

Fig. 16. — Drop-bottle for Cuuatl.i Balsam, etc.

men, allowing the opposite edge to touch the slide first outside
the specimen, and then allowing the other part held with the
forceps to fall gently on the specimen. Either of these
methods will prevent the formation of many air-bubbles under
the cover-glass. It is almost impossible not to have some, but

40 PRACTICAL HISTOLOGY.

these will disappear if the specimen be kept in a fairly -wann
place for a few hours.

Cementing Reagents.— Sections mounted in glycerine and in
similar fluids must have, and those mounted in Canada balsam
or Dammar varnish may have, their cover-glasses secured with
cementing material which is painted on with a camel's-hair
brush. It would be useless to mention more than a few of
these cementing fluids. The most useful are :

Dammar varnish, prepared according to the above formula.
Canada balsam, made with benzol.

Brunswick black, which is asphalte dissolved in turpen-
tine or naphtha.
Gold-size.

Marine Glue (Hollis's).— The authors find this the most
convenient cement, as it dries very quickly, and is very in-
soluble.

Before concluding this portion of the introduction, we must
not omit to mention certain additional operations of use to the
microscopist.

TEASING

Is done with needles mounted in some kind of handle. These
may be made very easily by taking a fine-pointed needle, heating
the eye red hot in a flame, and pushing it into a penholder
or suitably cut piece of wood. More elaborate needle-holders
may be bought of any maker of microscopic requisites.

Directions for Teasing. — Cut off a small portion of the tissue
to be teased with a sharp knife or pair of scissors, and place
it on a clean glass slide, in a small drop of the fluid in which the
object is to be mounted. Place the slide upon a firm support as
close to the eye as convenient, and with a couple of mounted
needles separate the tissue into, as nearly as possible, parallel
fibres. Some tissues cannot be so separated, and in that case
they must be broken up with needles into minute pieces.
Sometimes the aid of a lens or of a dissecting microscrope has
to be called in.

A rougher method than teasing may occasionally be used
when epithelium cells have to be examined. It consists of

ritACTlCAI, HISTOLOGY. 41

taking a scraping of the epithelial surface on a slide, adding a
drop of glycerine, and gently t:ip[)ing the cells with the smooth
end of an ivory or hone ])enholder until they are seen under the
microscope to he separate. This method i.«, however, almost
sure to damage a certain proportion of the cells.

SOFTENING, OR DISSOCIATING.
This process is employed for two purposes — either to facili-
tate the teasing of tissues into their elements, or in order that
sections may be conveniently cut of tissues otherwise too hard
for the knife. Under these circumstances small pieces of the
given tissue should be allowed to soak in one of the following
solutions :

Potassium Bichromate, 2 per cent.— This is particularly
useful for the purpose of dissolving up the cementing
material between the fibres of tendon.
Baryta solution, for a similar purpose.
Iodized serum.
Weak methylated spirit, for lymphatic glands, spleen, and

testicle.
Chromic acid, 5 per cent., to which a few drops of sul-
phuric and nitric acid have been added, for bone and teeth.
Hydrochloric acid, 2 per cent, to 3 per cent., or a mixture
of this with nitric acid, 1 per cent., for bone, teeth, and
cartilage.
Picric acid, a satuiatcd solution, especially for teeth.

Of Preparing S actions of Brittle or Specially Delicate
Material. — AVhen tissues are too brittle and too delicate to be
cut into sections, stained and subjected to the risk of damage
by transference from one reagent to another in the ordinary
way, it is advisable to stain them en masne, and so to prepare them
that the sections when cut may be at once mounted. This
method is specially to be adopted with embryological specimens,
with the internal ear, and sometimes with diseased tissues. The
process may be thus summarised. After hardening, the speci-
men is placed in some staining reagent which possesses the quality
of penetrating as well as that of being stable — carmine, cochineal,
or picrocarmin being the most suitable for the purpose. In this

42 PRACTICAL HISTOLOGY.

solution it is kept for t\vent3--four to forty-eight hours, at a
temperature of about 32' to 35° C. It should then be washed
in water, next in methylated spirit, and afterwards in absolute
alcohol for an hour at least. From this reagent it must be
removed to turpentine for a variable time, according to the size
of the specimen, for at least an hour. From turpentine, the
excess of that reagent having been removed with blotting-
paper, it should be placed in melted paraffin. The paraffin
should be kept just melted at a temperature, say, of 45° to 55° C.
The tissue must remain in melted paraffin for at least two
hours, but a longer time is necessary if the size be large, and
twenty hours is recommended if the time can be spared. After
this the tissue may be placed upon the glass plate described on
p, 2G, and cut with the microtome there mentioned. After
tome practice ribbons of sections may be cut, and they may be
mounted according to the useful ' shellac plan.' This consists
in painting the slide upon which the sections are to be mounted
with white shellac dissolved in creasote with a camel's-hair
b.'ush, and placing upon it the ribbon of sections. On slightly
warming the slide the sections sink into the shellac, and by
these means are firmly fixed to the fclide. The paraffin can then
bs dissolved away by leaving the slide in turpentine for some
time at the ordinary temperature. After the last operation has
been completed the sections may be mounted in Canada balsam.

INJECTION.

In order to show the distribution of the blood-vessels
in an organ, or of the l^^mph-vessels or channels, or to demon-
strate the bile-ducts in the liver, methods of injecting these
vessels, etc., are adopted. The material used is such that when
the operation is over, it remains in s'tia within the vessels.

In carrying out this process, the injection material should
first be prepared. It consists of a mixture of carmine and
gelatine, made as follows : Suspend 4 grams of carmine in a
minimum of water, adding 8 cc. of liq. ammoniae, and afterwards
48 cc. of distilled water ; filter the solution. Dissolve 13
grams of the best gelatine in 100 cc. of water by the aid of beat
derived fi^om a water-bath, and filter. Add the carmine solu-

PRACTICAL IIIST0L0(;Y. 4 3

tlon to 7"2 cc. of the warmed gelatine filtrate. Then add, with
constant stirring, 4 or .') cc. of glacial acetic acid to each IHcc. of
the carmine and gelatine solution, kept at a temperature of 40' C.

A solution of gelatine prepared as above, witii the substitution
of Berlin blue for the carmine, forms an equally good injecting
fluid. The Berlin blue is an aqueous solution of a strength of
2 per cent., made by dissolving 10 grams of Briicke's soluble
blue in 500 cc. of distilled water.

Methods.— The animal to be injected should be killed by
chloroform, in order that the arteries may be dilated to their
utmost extent, and an incision into one of the larger blood-
vessels should then be made. Whilst it is still warm it should
be immersed in a bath of water at a temperature of 40" C.
Tie a nozzle provided with a stopcock into the vessel previously
opened— carotid femoral, or crural, as the case may be— fill the
nozzle with saline solution by means of a pipette. Then attach
it to a strong brass syringe, ])reviously filk-d with the injection
material, which has been rendered fluid by warming it, and push
down the piston so as to drive the injection material into the
vessel. This must be done very slowly indeed, and the progress
of the injection should be ascertained from time to time by
examining the more vascular organs of the body ; ejj.^ the
tongue or ear.

The injection of particular organs, such as the kidney, is
effected by inserting the nozzle of the sjringe into the main
artery leading to it ; great care and long experience are,
however, necessary to obtain successful results.

Instead of the pressure of a syringe worked by the hand the
more easily regulated pressure of a column of mercury exerted
upon the injection mass contained within a suitable bottle may
be substituted, or of air compressed with an air-pump into
a bottle communicating with that containing the injection.

The lymphatics can readily be demonstrated in the intestine
by injecting them with a watery solution of Berlin blue. This
is done by inserting the point of an ordinary hypodermic
syringe, filled with the staining solution, into the coats of the
intestine in the neighbourhood of a Peyer's patch, and then
gently pressing down the piston.

44

PRACTICAL HISTOLOGY.

After injection in the case of the entire animal, the body
should be transferred to a large vessel, containing ice-co^d
methylated spirit, without removing the nozzle from the artery.
When isolated organs have been injected, they should be sus-
pended in ordinary alcohol in a beaker.

ACCESSORY APPARATUS.
Boxes and Cabinets. — Having prepared and mounted perma-
nent specimens of any tissue, it is necessay to label them care-
fully, noting the method of preparation, the mounting material
and date, and to set them aside in a tray box, such as is seen ia

Fig. 17.— Tray Box for Mcjiitecl Specimens,
the figure ; these may be obtained of the instrument -maker?.
The boxes are arranged to hold from one to six dozen slides; when

Fig. 18. — Kuives for microscopic work.
a larger collection has been made, the worker will find a cabinet
very convenient.

Knives.— For the various operations of dissecting tissues.

l'KA( TH'AL IIISTOLOCJY.

45

cutting ill pieces and scraping, ordinary scalpels are very useful.
For more delicate dissections, etc., other knives are used — e.g.,
those jirincipally employed by ophthalmic surgeons, and figured
above (Fig. 1^). It is hardly neccs^ary to add that knives must
always be kept very diurp.

Scissors.— Must be fiu3 and sharp, and of several sizes when
possible ; the diagram below will indicate a useful kiud.

Fit;. 10.— Scissors for microscopic work.

Fif. 20. — Cabinet for microscopic woi'k.

Cabin 3ts for microsiopic mounting have been arrarged by
fi-veral in^trament-mikeis containiug all the requidtss for the

4G

PRACTICAL HTSTCLOOY.

student in a compact compass. The prices vary from £1 Is. to
£1 7s. Gd. They save the beginner some trouble.

Forceps. — Should be fine-pointed and slender, if choice be
allowed, but dissecting forceps are quite suitable for ordinary
work.

Fig. 21. — Forceps for microscopic work.

Needles. — As before mentioned, a sewing needle inserted
into a piece of wood, leaving about ^ or ^ inch of the pointed
end uncovered, is quite sufficient for all purposes. Broad
n'^edles are also very useful.

It may be useful now to add an epitome of the apparatus and
reagents which mav have to be used in microscopical work.

Apparatus :

Microscope.

A pair of scissors.

A pair of fine forceps.

Two scalpel^;.

Xeedles mounted in handles.

Razor.

Glass slides and thin cover

glasses.
Watch-glas«es fO).
Glass capsules.

Reagents. — Those in most
Potassium bichromate, 1 per

cent, and 2 per cent, s^o-

lution.
Ammonium bichromate, 5 per

cent, solution.
Ammonium chromate, 5 per

cent, solution.
MiUler's faiid.
Chromic acid, ^ per cent,

solution.
Picric acid.

Osmic acid, gold chloride, etc.
Methylated spirit.
Absolute alcohol.
love oil, or creisote mi.x:tur3.

Section-lifters ("2).

Labels.

Filter-papers.

A box or cabinet for mounted

specimens.
Paraffin or White AVax and

Olive Oil mixture.
Gum solution.
Shellac and creasote.

common use are :

Hcematoxylin solution.

Picroc?rmin solution.

Carmine.
' Aniline colours.

Eosin.

Sodse bicai b., 1 percent., solution.

Acetic acid, ,, ,,

Saline solution, OG per cent.

Tannic and boracic acids.

Glacial acetic acid.

Sodium chloride.

Glycerine.

Dammar varnish.

Canada balsam.
Distilled water .

CHAPTER IT.

THE BLOOD.

Human Blood. — Prick the finger with a needle, and apply a
clean cover-glass to the drop of blood which issues, so that a
little is deposited upon it ; then mount on a perfectly clean
glass slide, and examine with a 1th or ,Uh objective. Notice that
there are various kinds of corpuscles floating about in a f.ui.l
medium.

A. Coloured Corpuscles.— These are circulardiscs ^Jon^^ jr^oa
of an inch indiameter,and io»jJ(7 of an inch in thicknes?, depressed
a little on each side. When seen sideways, biconcave or dumb-
bell shaped : cf a pale butf colour ; but when aggregated, of a
reddish tint. They have a tendency to run together, collecting
in rolls or rouleaux, Xotico a corpuscle as it rolls over, and
observe the change in its form, that it is alternately circular and
biconcave. The corpuscle has no nucleus; the false appearance
of a nucleus is occasioned by the refraction of light in passing
through a biconcave disc. Prove this by slowly altering the
focus. The centre of the corpuscle is seen to become lighter
than the periphery when out of focus. The corpuscles consist
of two parts : a stroma, which is colourless, and the coloured
part, a red crystallizable substance, hrcmorjlolAn.

B. Colourless Corpuscles. — Their proi>ortion to the coloured
varies from 2 to 1<> in l'A>". When perfectly fresh they are
spherical and faintly granular ; they quickly alter, and become
markedly granular. They are about ^^^C) inch in diameter
nucleated, the nucleus not often apparent without the addition
of weak acetic acid. They are nearly always isolated ; do not

48 TRACTICAL HISTOLOGY.

collect together or mix with the coloured discs. They are en-
dowed with the power of spontaneous motion, the so-called
wiKuhoid uioveinent.

C. Notice that here and there are small more or less rounded,
or slightly oval, granules, about one-third the size of the ordinary
coloured corpuscles. These are the Hood plates of Bizzozero,

Action of Reagents. — Make another preparation of a drop of
blood on a slide, and add to it a drop of saline solution. Cover
it with a cover-glass. Any reagent may now be made to act
upon the blood by placing a drop of it on one side of the slip,
and applying a piece of filter-paper to the opposite side. A
stream of the fluid passes under the cover-glass. This is called
Irrigation. Irrigate specimens of blood with the reagents
mentioned below. The structure of the corpuscles may be thus
demonstrated.

(1) ^yater: the coloured corpuscles become smooth and pale ^
and disappear : their colouring matter being discharged, leaving
a colourless stroma.

("2) Dilute acetic acid : the same changes take place : the-
colourless corjiuscles swell up. b-?come more distinct, and their
nuclei more apparent.

CS) Tannic acid : the colouring matter collects in small lumps
at the sides of the coloured corpuscles, and is, after a time, dis-
charged.

(4) Saline solution, 1 per cent., causes the coloured corpuscles to
become crenate and spinous, from shrivelling up of their proto-
plasm! Syrup and other fluids of considerable density, and
slow drying, also produce a similar appearance.

The action of other reagents may also be studied :

(5) Carbonic acid gas: a gas-chamber maybe made by taking
a slide and placing upon it a circle of putty ; beneath the putty
two small glass tubes are fixed, so that their ends extend into
the cells thus formed. The blood is placed upon a cover-glass
^vhich is inverted upon the ring of putty. One of the glass
tubes is then connected by means of an india-rubber tube with
the bottle in which the gas is generated. The stream of gas is
allowed to pass into the chamber, and its action upon the blood

PRACTICAL HISTOLOaV. 49

corpuscles is observed. The gas passes out of the chamber
through the second tube.

The preparation is brouglit into focus, and rar/ionlc tirid gtis is
allowed to pass through the chamber. The coloured blood cor-
puscles, which had l)ecome crenatc from the action of the salt
solution, again acquire a smooth outline, owing to the swelling
up of the parts between the projections. On admitting air to
the chamber the corpuscles again become crenate. The nuclei
of the colourless corpuscles become more distinct. If frog's
blood has been taken instead of human the nuclei of the coloured
corpuscles appear more distinct, owing to the coagulation of the
substance surrounding the nucleus.

(T)) Cldorofurm. — Preparation as for gases. The coloured cor-
puscles become globular, the haimoglobin being finally dissolved
and discharged into the plasma ; the blood, when seen with the
naked eye, being transparent (lake) and no longer opaque,

(7) Elcelnrltij. — The blood is placed on a slide in such a posi-
tion that when it is covered it spreads between two poles of
tinfoil situated six millimeters apart, which are connected with
the secondary coil of an induction apparatus. After a succession
of induction shocks the coloured corpuscles lose their smooth out-
line, become crenate, then like mulberries, and finally horse-
chestnut-shaped. They swell up, and ultimately become de-
colourized. The colourless corpuscles sw^ell up and disintegrate,
their granules exhibiting molecular movements. With a constant
current from a single Bunsen's cell the coloured corpuscles at the
positive pole undergo changes which correspond to those exhi-
bited under the action of an alkali, and at the negative pole of
an acid. The colourless corpuscles assume a spheroidal form, the
amoeboid movements being resumed as soon as the stimulus has
passed.

(8) Alhdies. — A mixture of 2 cc. of caustic potash in 1000 cc.
of saline solution causes both coloured and colourless blood cor-
puscles to sw^ell up, burst, and disappear. The coloured cor-
puscles appear to be more rapidly affected than the colourless.

(9) Alcohol. — A mixture of one-third spirit and two-thirds
water, acting upon amphibian blood, causes the nucleus to swell
and brings into view the nucleolus in the coloured corpuscles.

4

50 PRACTICAL HISTOLOGY.

It also renders the nuclei of the colourless corpuscles more evi-
dent, whilst one or more delicate and clear ' blebs ' grow from
the periphery of the colourless corpuscle. These ' blebs " appear
to consist of some colloid substance, into which endosmosis
rapidly takes place,

(10) Carbolic Acid. — With dilute solutions the coloured cor-
puscles shrink, and lose their regular contours ; after a time
they swell up, become pale, and disintegrate ; the colourless cor-
puscles in a dilute solution of carbolic acid in saline solution
(1-800 to 1-lGOO) do not continue their amoeboid movements for
so long a time or so rapidly as they otherwise would, and the
movements are generally of simple extension and retraction of
processes, no locomotion taking place. "When the corpuscles
cease to move, they do not disintegrate as rapidly as when the
carbolic acid is absent. AVith stronger solutions the coloured
corpuscles become granular, and the stroma breaks up to form a
homogeneous brownish-red material of a high refractive index,
which may appear either as an irregular network within the
cell, or in the form of globules which tend to coalesce, and are
insoluble in water, but are dissolved by carbolic acid. "With
solutions of ^(j to -.}^J, the movements of the colourless corpuscles
cease instantly, the cells shrink, and become coarsely granular.

(11) Of Feeding the Colourless Corpuscles. — The preparation of
blood on the warm stage is iiTigated with carmine, vermilion, or
aniline blue, in a finely divided state, or with milk. The colour-
less corpuscles will be found after a short time to have ingested
some of the finer particles. The particles are taken into the
substance of the corpuscle, by the union around it of two of the
protoplasmic processes, and they thus lie at first close to the
periphery of the cell, being carried at a later period nearer to its
centre.

Of Counting the Blood Corpuscles. — This operation may be
accomplished by means of an instrument called the Iltvmcujto-
meter (Gowers).

It consists of (i.) a metal tray with a central aperture and a
pair of cli} s ; the tray is larger than (ii ) a glass slip which it
ho'.ds. The slip is of the ordinary size ; it is provided at its
cent--e with a cell which is exactlv one-fifth of a millimeter in

PRACTICAL HISTOLOGY.

51

depth, and whose floor is ruled in such a way as to form a series
of s(iuaie<, each measuring one-tenth of a millimeter (c). (iii.)
A tube (u) with a bore like that of a thermometer, graduated
to contain five cubic millimeters, (iv ) A pipette (a) of 'Jltr>
cubic millimeters capacity, with a fine aperture, (v.) Elastic
tubes with mouth-pieces of glass, (vi.) A small glass jar (d).
(vii.) A glass stirring rod in the form of a paddle (e). (viii.) A
guarded needle (f). (ix.) Cover-glasses,wash-leatber, and brushes,
(x.) A bottle of sulphate of soda in solution of sp. gr. 1025.

Fig. 22. — Hicmacvtometer.

A. Pipette for measuring tlie saline solution.

B. Capillary tube for measuring the Itlood.

c. Cell of glass surrounding the place where the divisions are marked
on the slide, mounted on a perforated metal slide with side springs
to keep down the cover-glass.

D. Jar in which the blood is diluted.

E. ^Mixcr.

F. A guarded needle.

Fix the elastic tubes to the pipette and to the thermometer
tube, draw up bv suction 005 cmm. of the sulphate of soda solu-

4-2

52 PRACTICAL HISTOLOGY.

tion, and expel it into the glass jar. With the guarded needle,

which should be perfectly clean, draw a large drop of blood

from the palmar surface of the last phalanx of the left middle

finger. The blood will be more easily obtained, and with less

pain, if a handkerchief has been wound tightly round the finger

from below upwards ; the blood should be drawn by a single

rapid prick of the needle. Suck up the blood into the capillary

tube, until it extends slightly beyond the five cubic millimeters

mark ; remove the excess by means of a piece of clean blotting

paper applied to the end of the tube : and when exactly five

cubic millimeters of blood are left, expel them into the solution

of sodium sulphate which has already been measured out. Mix

the blood and the sodium sulphate together by a light but rapid

rotatory moyement of the paddle. Place one or more drops in

the cell upon the slide ; coyer it with a thin cover-glass, and

replace it upon the tray, where it should be allowed to remain

for three minutes before the examination is commenced, in order

to allow the blood corpuscles to settle. The solution of blood

should exactlj^ fill the cell, neither more nor less. In cleaning-

the cell, it is important that it should not be rubbed, or the

micrometer lines will soon be effaced. A stream of distilled

water from a wash-bottle, and the subsequent use of the Eoft

camel's hair brush, will be found to be effectual. Examine the

blocd with a high power ; the corpuscles will be found lying in

the squares of the micrometer. Count the number of coloured

corpuscles in ten squares, putting down each as they are counted

upon a piece of paper ; add up the total and divide by ten : an

average cf the corpuscles for each square will thus be obtained.

In the case of corpuscles which are upon the boundary lines of

th3 squares, add or omit them according as the centre is or is

not on the line bounding the square under notice at the time.

In the typically healthy blood of man, each square should contain

on the average fifty coloured corpuscles ; in a woman the

number is somewhat less. Examine the same ten squares, and

count the number of colourless corpuscles ; one or two should

alone be present.

Of Prepaiing Permanent Specimens of Stained Human
Blood.— It is found in practice to be anything but easy to pre-

PRACTICAL HISTOLOGY. 53

pare specimens of liuni;iu blood, so that the corpuscles may
retain their shape and may he at the same time well stained.
After the trial of a large number of ditl'ereut methods, the fol-
lowing is recommended as giving the most satisfactory resultF.
The finger is jiricked and a large drop of blood allowed to exude ;
a perfectly clean cover-glass is lightly drawn upon the top so
that a very thin layer of blood adheres, so thin as hardly to be
evident until it is dry. It is then dried in the air or put at
once without drying into one of the following solutions, viz.,
chromic acid jl^ per cent. ; bichromate of potassium I per cent. ;
methylated spirit or absolute alcohol for five or ten minutes,
washed in water and again dried. The specimen is now ready
for staining. The best dye for this purpose is a recenthi
prepared 1 per cent, solution of Spillers purple in water
to which a few drops of alcohol have been added, or a weak
spirit solution of rosein. A few drops of one or other dye having
been filtered into a watch-glass, the cover-glass is placed upon
the surface of the solution blood downwards, and allowed to
remain so for from five to ten minutes. It is then removed,
washed for some time in a gentle stream of distilled water, dried
thoroughly, and mounted in Canada balsam, with or withou
previous treatment in clove oil for a minute or two. On exami
nation of the specimen the coloured corpuscles should be founa
of normal shape and coloured purple or red, according to the dye
used, whilst the colourless corpuscles are correspondingly stained.
The method with Spiller's purple will be found especially useful
when blood is examined in diseased conditions in which the exis-
tence of micro-organisms is suspected, and is superior to those
obtained with many of the aniline dyes (such as methyl-violet).

Amoeboid Movements of the Colourless Corpuscles.

In order to demonstrate the amoeboid movements of the colour-
less corpuscles, it is necessary to make use of the irann stay. Of
this there are various kinds in use. The simplest is a glass
slide, to which a perforated circular plate of copper is cemented ;
this is joined to a projecting rod of the same metal. The rod
communicates heat from a spirit-lamp to the plate, upon which

54

PRACTICAL HISTOLOGY.

is placed the specimen of blood to be examined. The tempera-
ture is regulated by placing a small piece of cacao butter, which
melts at 35' C, upon a flattened portion of the rod near the
copper disc ; when the butter begins to melt the spirit-lamp
should be removed.

The warm stage of Strieker consists of a metal box with
central aperture. It is heated either by a copper rod, which can
be fixed to it, and which is warmed in the flame of a spirit-lamp,
as in the simpler apparatus, or by passing a current of hot-water
through the stage itself, or by means of electricity. A thermo-
meter, whose bulb encircles the central aperture, registers the
emperature. If hot water be used, an india-rubber tube should
be attached to the two brass tubes which project from the sides.
One of these tubes should then be placed,
in connection with a jug of hot water, on
a higher level than the stage of the micro-
scope, whilst the other tube acts as a
waste pipe. A syphon action may thus
be established, and the water will circulate
through the stage.

To use the warm stage, place it upon the
stage of the microscope, in such a position
that the central aperture corresponds with
the centre of the largest diaphragm, while
the copper rod projects beyond the stage.
Place a spirit lamp beneath the rod so that
Fig. 23.— Wana Stage, it is heated ; watch the effect of the heat
upon the thermometer, and take care that the mercury does not
rise above 39'C. When it gets near this point, move the spirit
lamp further away. "Whilst the stage is being heated, prepare
the specimen of blood to be examined. A drop of blood drawn
from the finger, or from the tail of the newt, should be diluted
with an equal bulk of normal saline solution, and should be
received upon a large, square, and perfectly clean cover glass ;
a second cover-glass of the same size and shape should also be
ready. A little olive oil or melted paraffin should be evenly
applied with a camel's hair brush along the edges of the cover-
glass holding the blood ; the second cover-glass is carefully put

PRArTir'AL iiiSTOi.oriY. 55

over it, and ti.e biroil sp?ea<ls out to f or n atliin uniform layer ;
a moist chamber is thus formed by the oil preventing evapora-
tion. The two oovcv-glasses containing the blood between
them are then put over the apjrturc in (he warm stai^'e, ai 1

Fig. '24. — Alterations in the Shape of a Colourless Corpuscle observed for

Un minutts.

examined ■with the highest available power, the movements of
a single colourless corpuscle being noted and recorded by drawing
it at intervals of a minute (Fig. 24).

Strieker's stage is expensive ; but a less costly modification,
■which answers the purpose "well, is shown in the annexed
diagram (Fig. 2o), It is ])rovided with a thermometer which
accurately registers slight variations of temperature. With the
■warm stage thus described is combined a gas chamber less rough
than the simple form already described.

Gas Chamber — It may be made by putting the tube leading
from the gas generator into connection with either of the
tubes which project from each side of the solid projection to
which the copper rod is fixed. These tubes are on the side
opposite to the thermometer ; they must not be mistaken for
the tubes "which have been previously described as for the
passage of hot ■water. To use the gas chamber, it is necessary
to encircle the aperture with a ring of putty ; the cover-glass
containing the preparation to be examined is then placed upon
the putty with the tissue downwards, that is to say, in the
chamber. The clamp upon the gas tube is then relaxed, and
the gas passes into the chamber, when its effect upon the tissue
is noted. Air is re-introduced into the chamber by disconnecting
the tubing, and sucking through or otherwise passing air into it.

Hsemoglohin Crystals. — These crystals may be prepared as
follows : Take a drop of blood from a guinea-pig, rat, or dog,
and let it coagulate on a slide ; add a little water, then take
up the clot with the forceps, and let several small drops fall
from it upon another slide. Cover with a cover glass. As

56 PHACTICAL HISTOLOdY.

these drops evaporate, hgemoglobin crystals of various sizes shoot
out from the edges, separately and in bundles, varying in shape
according to the animal from which the blood had been taken.
In the case of the blood of the three animals mentioned, an easy
way to form the crystals is to shake up some drops of their
defibrinatcd blood in a test tube half filled with distilled water ;
in the course of an hour or so the sediment will be found to be
chiefly composed of haemoglobin crystals. Ether, or chloroform,
also appears to aid the separation of the haemoglobin from the
stroma, and must be used instead of water in the first method
of preparation.

Haemin Crystals consist of the hydrochlorate, or hydrochlo-
ride of Jiaiiiafin — heematin is a derivative of haemoglobin, which
easily splits up into Juematiii and globulin. The crystals of
haemin are thus prepared : A drop of blood is dried on a glass
slide. Two or three granules of common salt are added. The
blood is powdered and thoroughly mixed with the salt. \Yith a
capillary pipette a drop of glacial acetic acid is added, and then
the preparation is covered with a cover-glass. The blood and
salt should be thoroughly dissolved in the acid. The tempera-
ture is gradually raised to the boiling-point over a spirit-lamp,
until the greater part of the acid has evaporated. On ex-
amination of the residue with the microscope a number of
small, reddish-brown rhomboidal crystalline plates are seen.
The specimen should be irrigated with distilled water until the
excess of salt is washed away, and then dried and mounted in
Canada baliam.

Blood Corpuscles of Vertebrates other than Man.

In nearly all mammaJki the coloured corpuscles are round, disc-
like, non-nucleated bodies, similar to those of man, but differing
in size. In this respect they varj^ considerably. In h'lnh., reptiles,
amphihia, and jiah, the corpuscles are oval and nucleated, the
nucleus presenting a central elevation on each surface. These
corpuscles are larger in birds than in mammalia ; still larger in
fish ; and of a yet greater size in amphibia.

The general characters of the colourles-; corpu£C-e3 are similar

PRACTICAL HISTOLOGY. 0<

in all animals, but are found in much larger proportion in tlic
blood of fish and am[»hibi:i th:ui in that cf nuimmalia and birds.
Examination of the Blood of Frogs and Newts.— The blood
of newts and frogs affords excellent opportunity of studying the
properties of blood corpuscles on an enlarged .scale as it were.
The coloured corpuscles of the newt are larger than those of a
frog, but in each case are oral in shape, with a distinct oval
nucleuit, showing a fine intranuclear network ; the nucleus is
colourless, has a tendency to escape from the body of the cell,
but bulges out the central portion of the corpuscle on each side
when it is seen sideways. The colourless corpuscles vary greatly
in size, thelargest not being larger than the coloured corpuscle, and
the smallest of the size of its nucleus. Note that some are more
granular than others, and some are much more active in their
amoeboid movements. They possess one or more nuclei. Re-
agents act upon amphibian corpuscles much as in the case of
human blood. Specimens should be irrigated with (I) dilute
acetic acid, (2) tannic acid, (3) boracic acid, 1 per cent., and the
effects of the re-agents upon both coloured and colouiless cor-
puscles should be watched. The last-mentioned re-agent causes
the colouring matter to collect around the nucleus of the coloured
corpuscle, which may then be discharged from the stroma.

To Double-stain the Blood- Corpuscles of Amphibians.—
Blood in very thin films should be allowed to dry in the air upon
thin cover-glasses. These should then be placed in absolute
alcohol for an hour and dried. A few drops of a dilute spirit
solution of fuchsin should then be poured upon the dried blood,
and should then be at once washed off, and the specimen
thoroughly washed with a gentle stream of water from a wash-
bottle. The cover-glass should then be dried in the flame of a
spirit-lamp and allowed to cool. "U'hcn ready for the second
dye, a small quantity of methylene blue solution should be
dropped upon it and allowed to remain for two or three minutes.
A second washing with a stream of distilled water should then
follow until the washings are all but colourless ; and the pre-
paration should finally be dried and mounted in Canada balsam.

CHAPTER III.

EPITHELIUM.

^Iethod.^ of Examinatiox. — Examples of the various kinds of
epithelial cells may be studied either by taking scrapings from
THE FRESH TISSUES^ or in SECTIONS cut from the hardened
material. Where the cells are arranged in layers, or strafified, in
order to see the relation of one layer to another, the latter method
must be adopted : but the shape and size of the individual cells
and their nuclei may be examined according to the first method.

To obtain epithelial cells of various kinds, -with their nuclei
STAINED, it is advisable to subject the tissues from which they
are to be prepared to the following process : Immerse in 2 per
cent, solution of potassium bichromate for twenty-four hours
after removal from the body, and then wash with distilled water
until the washing? are no longer yellow. Afterwards transfer
to a mixture of equal parts of aqueous heematoxylin and gly-
cerine for a day, and preserve in glycerine.

A. Squainous Epithelium. — [a) "With a blunt knife, or with
the finger nail, scrape off a thin shred from the mucous mem-
brane of the rheeh; mix it with a drop of normal saline solution
on a slide ; place on it a cover-glass, and examine with a one-
fifth objective.

The cells are large, flat, ronndish, or irregularly polyhedral
bodies of various sizes. Their substance is more or less trans-
parent, containing granular matter. Tbe nuclei are small and
oval, frequently granular ; they are sometimes missing.

(li) Tease a scraping from the inner surface of the (esophagus of
a cat or dog, prepared after the above method, in a small drop

pRArTrrAi. histoi.ocjv. 59

of glycerine. The nuclei of the cells are seen to be stained with
the dye.

B. Columnar. — Take a s^mall scraping from the mncons mem-
brane of the inteatineot some animal (cat, rabbit, ordog),pre}»ared
as above, and tease it up with needles in a drop of glycerine on a
glass slide.

The cells are cylindrical or conical in form, with a fairiy well-
defined outline ; protoplasm finely granular ; nucleus clear, oval,
well-defined. "When an aggregation of cells is seen from above,
as on the surface of a villus, it ha> the appearance of a regular
mosaic.

C. Transitional. — This type of epithelium is best seen in the
Uathler, from which it may be prepared in manner similar to
the above. The shape of the cells should be noted : some are
tailed, others concave on one side, spindle-shaped, or caudate.
The nuclei are very large.

D. Ciliated. — Scrape lightly the mucous surface of a prepared
trachea, tease out in glycerine, and examine in a similar manner.

The free border of each cell is provided with cilia, whilst the
deeper portion is prolonged into a fine process or tail.

Ciliary Motion.

The movement of cilia may be studied by cutting off
with a sharp pair of scissors a small fragment of one of the
gills (branchiae) of a living oyster or mussel, teasing it with
needles in a dro]) of saline solution, mounting and examining in
the same fluid. The epithelium scraped from the roof of a frog's
mouth, or that obtained from the mucous membrane of the
nose, or that lining the alimentary canal of the earthworm, is
also well adapted for the demonstration of ciliary motion. The
highest available magnification should be used after a general
survey has been taken under a low power. Ciliary movement is
seen at first to be very rapid ; but it soon becomes slower, and
finally ceases.

Efectis of Reagents. — Dilute alkalies retard, and then stop the
movements. If the ciUa are working slowly, or have stopped in
a preparation which has just been put up. the cireful addition
of a very dilute solution of caustic potash or dilute acetic acid,

CO rRACTICAL HISTOLOGY.

or the passage over it of carbonic aciJ, or an electric shock, will
generally renew or accelerate the movements for a short time —
the ultimate effect, however, being to destroy the cilia.

Carbonic acid first accelerates, then retards, and finally stops the
ciliary action, the movements recommencing if air is allowed to
take the place of the carbonic acid.

Chloroform retards and finally stops ciliary action ; the move-
ments recommencing on the admission of air, if the vapour has
not been applied for too long a period.

War/ntJi accelerates the action of cilia which were previously
moving slowly, the movements ceasing at a temperature which
is sufRcient to destroy the vitality of the cells.

E. Glandular. — It is advisable to postpone the study of this
form of epithelium until sections of glands, such as the liver or
kidney, have been cut and prepared. The calls may, however,
be seen by taking a scraping from the freshly-cut surface of a
kidney- or liver, and preparing it in salt solution.

The cells vary in shape and size, are pale, and fairly well defined.

F. Pigment can be studied in scrapings from the clwroid, iris,
etc., teased and mounted in glycerine. The cells are either
irregular and wide-branching with clear nuclei, or flattened and
polygonal.

Endothelium.

Endothelium is a variety of epithelium which, as a single
layer, lines serous membranes, blood and lymphatic vessels, etc.

Preparcdion. — Open the abdomen of a cat, dog, or rabbit
which has been killed, a few minutes previously, by bleeding,
and carefully remove the omcntcm and portions of the meseii-
tjri/, so that no hair or blood adheres. Place them at once in a
solution of silver nitrate (| per cent.), gently shaking out the
folds in the membrane so that no part escapes the action of the
reagent. Allow them to remain in the silver solution for ten
minutes and then remove to a vessel of distilled water and well
wash them, changing the water after the washing has been com-
pleted. Xext exjtose them to sunlight, in the water, until they
assume a brownish tint. The tissue is now ready for mounting.

PRACTICAL HISTOLOGY. 01

Small pieces should be cut off with a pair of sharp scissors,
spread out upon a slide, and mounted in glycerine.

Yer}' excellent specimens of endothelium may also be obtained
from the pleura jw^vVorf?*/ of the same animals, after treatment
in a similar way.

In order that not only the outlines of the cells may be traced,
but also that their nuclei may be evident, the tissue should be
further stained in hrematoxylin, carmine, or one of the aniline
dyes.

A good preparation of endothelium, showing numerous ?to-
MATA, may be obtained from the frog's anterior lymph-?ac
{ciaterna lymphatica magna) by filling it with the above-men-
tioned solution of silver nitrate by means of a capillary pipette,
and, after ten minutes, removing the anterior wall, washing in
distilled water, and mounting in glycerine.

On examination, the surface of the membrane is found to
consist of a single layer of flattened polyhedral cells, variously
modified, and forming a mosaic. The nuclei are generally
single, and, except in doubly stained specimens, appear as bright
and almost colourless oval bodies within the cells. When seen
in profile, they occasion projections from the surface.

CHAPTER lY.

THE CONNECTIVE TISSUES.

These comprise (1) white fibrous tissue, (2) areolar tissue,
(d) elastic tissue, (4) adenoid tissue, ('>) gelatinous or embryonal
tissue, (()) adipose tissue, (7) cartilage, and (s) bone.

1. White Fibrous Tissue.— Typical examples of this tissue
are found in tendons. Those of the tail of the mouse or rat are
exceedingly fine, and may easily be obtained. The tail is cut oif
close to its base. The skin is removed, and a small piece of the
extremity is pinched off between the nails, and is drawn away
from the rest of the tail. In separating this piece, a number of
fine threads, the tendons, will be noticed. Some of these of
moderate size are selected and teased out in saline solution.

Teazing in this manner will show ihejihrous humUes of which
the tendon is made up. Acetic acid may now be added, and the
tendons will be seen to swell up and dissolve, leaving their
elastic sheaths unaffected.

To demonstrate the t/^ndon corpuscJes several methods may be
adopted. («) The first method is to place the fresh tendons in a
mixture of equal parts of aqueous hcematoxylin and glycerine
for two days, tease in glycerine, and press the cover-glass slightly
after mounting, (h) Another method is to stretch the tendons
whilst still perfectly fresh upon a glass slide. The extremities
of the tendons are allowed to dry, and by this means they are
maintained in an extended condition. A few drops of picro-
carmin are placed upon the centre, and are washed away with
distilled water after the ex})iration of half an hour. A drop of
glycerine acidified with acetic or formic acid is then added, a

PRACTICAL HISTOLOGY. 03

hair is placed by the side of the tendon to obviate pressure, and
a cover-glass is put on, the preparation being sealed up in the
usual way. (r) Thirdly : (lood results can also be obtained by
mounting the isolated tendons in a 1 per cent, solution of
acetic acid, to which one-third its volume of logwood alum solu-
tion has been added. The preparation must be examined as
soon as })Ossible. (d) Fourthly : By treatment with a ^^ per
cent, solution of osmic acid for an hour, washing in distilled
M-ater three hours, and subsequent staining with picrocarmin
for twenty-four hours.

To demonstrate the lumpha'ic sjxtces, the tails of mice, after the
removal of the skin, are placed in gold chloride in small pieces,
left in the solution an hour, and treated in the usual manner (see
p. 32) to reduce the gold. After this has been thoroughly effecied
they should be placed in a solution of acid (HCl, 2 per cent.) to
soften the bone. From this material very thin sections should
be cut, stained in hasmatoxyliu, and mounted in Canada balsam.

Stuuctuue. — From these various preparations the structure
may be made out. The tissue is seen to consist of parallel bundles
of fibres, w^hich vary in thickness and are held together by a homo-
geneous and albuminous cement substance. The individual fibres
forming the bundles are straight or w^avy, and are extremel}' deli-
cate. The acetic acid added to the tendons causes tlie fibres to
swell lip and to disappear, owing to the presence in the tissue of a
substance which is readily convertible into gelatine. The bundles
of fibrils are surrounded by a more or less complete sheath of
elastic tissue, which is not acted upon by dilute acids ; hence the
constricted appearance seen in the tenJons to which acetic acid
has been added.

On examination of the tendons treated to show the corpuscles,
they will be found to consist of parallel bundles of fibres, whose
substance is almost colourless, arranged in groups. Between
each two groups is a lymph-channel, in which lie nearly parallel
layers of delicate stained cells — the conneciicetissue ov tendon cdls
— forming for each channel a single continuous row of irregular
angular plates. Each plate is provided with a more deei)ly
staining nucleus. The cells are separated from each other by a
cementing substance, and they possess fine processes. Each cell

64 PRACTICAL HISTOLOOY.

presents a straight ridge — the elastic stripe. This ridge is formed
by the union of two or three concave portions of which the
cell is composed, to enable it to adapt itself to the curved sur-
faces of the tendon bundles. The hjmphatics are demonstrated
in transverse sections of the tail, stained in chloride of gold ;
dark masses are seen in the tendon, corresponding to the
hpuphatic channels, filled with an albuminous fluid plasma.
Radiating from these masses are fine septa — the cement substance
— binding together the contiguous bundles,

2. Areolar Tissue. — This form of tissue is really a variety
of fibrous tissue, and is best seen in specimens of intestine and
skin. By the injection into the subcutaneous tissue of a rat which
has just been killed of a 0"2 per cent, solution of nitrate of silver or
osmic acid, a small artificial bulla is formed. This is allowed to
remain for ten to thirty minutes, and is then opened with a pair
of fine curved scissors, and the delicate subcutaneous tissue is
rapidly removed and spread out on a glass slide. It is imme-
diately covered with a thin glass, and the preparation is stained
for twenty-four hours with picrocarmin. Glycerine is passed
through until all the superfluous staining material is removed,
after which the preparation is sealed up.

Structure, — The tissue is composed of delicate bundles of
ordinary white fibrous tissue, some of the fibres arefibrillated, and
all interlace with each other ; the meshwork thus formed con-
tains a few very fine fibrils of elastic tissue^ which become still
more evident if dilute acetic acid have been allowed to act upon
the preparation. The interspaces are filled with lymph, con-
taining hjmph corpuscles. 'L?iVgQ plate-like cells, which appear to
lie upon the surface of the bundles of fibres, are also seen.
When viewed sideways, these cells have a branched appearance,
and form the plasmatic cells. Fat cells are also present.

3. Elastic Tissue.— The coarse and larger fibres may be
demonstrated by teasing out a small piece of the ligamentum
nuchas of an ox in glycerine, and the finer fibres by mounting a
piece of the omentum of a rabbit or cat on a slide, irrigating it
freely with dilute acetic acid, and then staining with hsema-
toxylin, or with an aniline dye, preferably a magenta or fuchsine,
and preserving it in glycerine.

PRACTK Af. HISTOLOGY. G5

Stkkti Ki.. — Oil examination elastic fibres are either tkhk,
well-defined, and form bundles ; or they are /?//'% shining, and
not in bundles. They hrmicJi dichotomou><ly, and anaa-
tninose with each other to form a real network ; when torn,
they curl up at the ends. They do not swell up when treated
with acids, and on prolon^^^ed boiling they yield elastin and not
gelatine as do white fibres.

4. Adenoid Tissue, retiform or lymphoid tissue, can be de-
monstrated by cutting and staining sections of a lymphatic gland
which has been kept in dilute spirit. After staining with hjenia-
toxylin the sections should be shaken in a test tube half filled
with water for an hour or more. The portions of the broken-
up sections should then be dehydrated, cleared, and mounted in
Canada balsam.

Adenoid tissue forms the basis of the spleen, the lymphatic
glands, the tonsils, thymus, Peyer's glands, etc.

Stkuctuuk. — It consists of a fine reticulum composed of the
branchings of corpuscles, which either retain or do not retain
their nuclei. In the reticular spaces are contained, closely packed
together, very small corpuscles with nuclei almost entirely filling
them— the hjmpho'id corpuscles. In order to study the reticulum
the corpuscles must be removed, and this is the object of sub-
jecting the sections to the treatment by shaking. Chemically,
the fibres of the reticulum differ from both yellow elastic and
white fibrous tissue.

5. Gelatinous, Embryonal, Mucous, or Wharton's Tissue
is present in the umbilical cord and in the foetal skin. It is
obtained from a bulla formed by the injection of a dihite solu-
tion of gold chloride, in a stronger solution of which it is subse-
quently stained.

Strlxtl'KE. — In the youngest condition, it will be found to
consist of a transparent jelly-like substance containing a hyaline
mucous substance wilhin a reticular framework. At a later
period bundles of fhrous connective tissue are apparent, as well as
branched cells, blood-vessels^ and /(// rells in an early stage of de-
velopment. The tissue yields mucin on boiling. Tlie vitreous
humour appears to be a variety of this tissue in which the
branched cells have lost their processes.

5

66 PRACTICAL HISTOLOGY.

6. Adipose Tissue may be seen best in sections of scalp, or in
pieces of omentum stained in lioematoxjlin. A small piece of
fat, which has been partially teased, may be placed in ether for
twenty-four hours, and the fatty portion will be dissolved out.
Examine also the preparation of areolar tissue formed by the
injection of nitrate of silver as well as sections of fatty tissue
stained withosmic acid in order that the action of that reagent
upon the fat-cells may be observed.

Steuctcre. — Adipose tissue consists of a matrix or net-
work of areolar tissue containing fat-cells. Fat-cells are clear,
well-defined, rounded vesicles of varying size, filled with an
oily fluid, which often gives rise after death to crystalline
needles^ radiating from the centre of the cell. In successful
preparations, especially of the omentum, a fine zone of j^i'oto-
plasm, with a nucleus at one pole, can be seen surrounding the
cell more or less completel}-. The fat-cells may either form
compact masses, with only a small amount of connective tissue,
or they may be more or less isolated. The tissue possesses a
capillary network of Mood-vessels. Between the fat-cells, flat-
tened nucleated connective-tissue cells may be demonstrated.

7. CARTILAGE.
Cartilage consists of two parts : (1) Cells ; and (2) Matrix, or
intercellular material. According to the nature of the matrix,
cartilage is classed as :

a. Hyaline cartilage.

h, Fihro-cartilage.

c. Elastic cartilage,
a. Hyaline Cartilage is found in various localities, from
which the names costal, tracheal, articular, ossifjing or inter-
mediary, and embryonal, are derived. Portions of each of these
cartilages should therefore be examined. The cartilages may be
prepared in a sc.lution of chromic acid 1 in GCO, in a saturated
solution of picric acid, or by the gold method. In every case the
sections, which must be very thin, should be stained with car-
mine or hasmatoxylin. It may be as well, also, to stain some
portion of the nasal cartilages in osmic acid.

Steuctuee. — All cartilage, with the exception of the free ex-

I'lIACTICAT. HISTOLOGY. G7

treraity of articular cartilage, possesses a delicate vascular con-
nective-tissue sliealli — ilia pcrirJioufh-iuin. The, cartUufjc-cclls are
spherical or oval protoplasmic bodies, generally containing a
single nucleus. The cell protojla^jra forms a fibrillar meshwork
which is contracted in embryonal and articilar cartilages. Each
cell is placed in a laciin;i, enclosed by a firm, structureless, but
transparent inatr'ix. yii4di(ig chondi-in on prolonged boiling. In
growing cartilage, a special layer — th • iiuiifhir/ memhrane — can be
distinguished between the lacuna an 1 the gi"ound substance. In
some cases a single lacuna may contain more than one cell, due
to reproduction of the cartiiage-ctll by fission ; and the various
stages in the division of cells may often be well seen. Near the
perichondrium the cartilage-cells become flattened and smaller ;
near the articular surface they arc branched ; in ossifying carti-
lage they are arranged in parallel row-s. The matrix possesses an
anastomosing system of lacunas and canals in connection with
the lymphdlic system.

h. Fibro-Cartilags. — White fibro-cartilage occurs in the inter-
vertebral substance and in sesamoid bones, and may be well seen
in longitudinal sections of a mou^^e'stail stained in gold chloride.

Sthucture. — It consists of groups of slightly flattened cells^
each with a round nucleus, and enclosed in a distinct capsule.
The matrix is composed of bun lies of fibrous tissue, which some-
times form lamellce with occa>*ionally a concentric arrangement.

c. Elastic Cartilage. — This form of cartilage occurs in the
lobe of the ear, in the epiglottis, in the cornicula laryngis, and
iu the Eustachian tube. Sections should be cut of the pig's
car, in wliich it is seen very well, stained in hoematoxylin and
mounted in Canada balsam.

Sthucture. — Elastic cartilage in the adult is hyaline cartilage
permeated by elastic fibrils. Tlie fibriln are arranged so as to
form the trabeculas of a reticular framework ; they branch and
anastomose very frequently. The meshes contain fusiform
groups of large nucleated celh^ surrounded by a larger or
smaller amount of hyaline cartilage substance.

Ossifying or Calcifying Cartilage.— In order to show ossi-
fying cartilage, a foetal femur oi- other long bone is decalcified
with the chromic acid and hydrochloric acid mixture, and sec-

68 PRACTICAL HISTOLOGY.

tions both transverse of the shaft and longitudinal of the
extremities should be cut. The sections may be doubly stained
in picrocarmin (or eosin) and ha^matoxylin. This variety of
cartilage is found at the junctions of cartilage with spongy bono
in the epiphyses and ends of the shafts of long bones, arid repre-
sents the method by which bone increases in length. It will be
seen that the stages of the formation of bone in this vfay corre-
spond almost exactly with the so-called development of bone in
cartilage (p. 70). In a longitudinal section through the end of
a groYv'ing long bone the following layers can be seen at the
junctions of the cartilage and bone :

(1) Ordinary hyaline cartilage covered with perichondrium iii
the diaphysis of a long bone at the junction of the cartilage with
the spongy bone, a characteristic arrangement of the cartilage
cells in longitudinal columns. The cells are also seen to be
conical in shape, pressed together and flattened transversely.

(2) A transparent layer, in which the lacuna) of the cartilage
cells are seen to be enlarged, the matrix diminished, the cells
enlarged and transparent, and their nuclei swollen.

(3) The lacunfE are becoming confluent, and the matrix:
calcified,

(4) The enlarged lacunae are seen to be filled with marrow,
and the trabeculte of calcified cartilage are covered with layers
of marrow-cells, amongst which are enlarged multi-nucleated
mother-cells (giant-cells).

(5) The marrow-cells (osteoblasts) are seen to have deposited
layers of low ossific material upon the calcified trabeculfe. and at
the same time the calcified centres have become less free from
absorption.

(G) The calcified centres of the trabeculas have disappeared,,
leaving ossific trabeculas which form the spongy bone.

8. BONE.

The fresh" bones of any small animal, well cleared of the-
surrounding tissues, should be placed for tM'o or three weeks
in a large quantit}^ of h per cent, solution of chromic acid,
containing five drops of hydrochloric or nitric acid to each
ounce of the solution. When the whole of the earthy matter is-

I'KArTKJAL IIISTOLOOY. <iO

ilissolved out, sections sboukl be cut with a i;i/.)r in various
directions, and examined in glycerine.

As the i)reparation of specimens of hard bone requiies much
time, the student is advised to buy both transverse and longitu-
dinal sections of that material.

The structure can be le^t made out in a specimen of hard
bone.

a. Compact Bone. — In transverse sections of the compact tissue
of long bones are seen Haversian systems, more or less perfect,
and Haversian interspaces. Each system consists of the central
Haversian canal (which is generally round or oval, with an
average diameter of ,.,',y in., and is lined with a delicate mem-
brane continuous with the periosteum), surrounded by concentric
lamellne of bone, between and in which are the lacunai and canali-
culi. Lacuna! -(/ou ^"' ^" length, generally well-marked, contain
shrunken bone corpuscles. Canalicidi usually indistinct, but,
when seen plainly, forming a complete system of communication
between the lacunar of the same and neighbouring Haversian
systems and interspaces. They contain in the fresh condition
prolongations from the bone corpuscles. Each Haversian system
is more or less isolated from its neighbour by a layer of bone
which contains but few canaliculi. The Ilacerslan intcmpaces are
the portions of bone filling up the interval between one or more of
the circular Haversian systems. They do not contain any central
canal ; their general characters are otherwise similar to the
systems.

In longitudinal sections the Haversian canals which run longi-
tudinally are seen to anastomose freely by transverse or oblique
channels. The lacunae and canaliculi present much the sams
characters as in transverse sections. The Haversian canals
which run near the circumference of the bone may open on the
outer surface so as to admit blood-vessels from the periosteum,
whilst those opening into the medullary canal receive blood-
vessels, and in the case of the larger ones, medulla from the
interior of the bone.

In preparations of calcified bone it may be seen that the
lamella3 are bolted together by the pevforating jibres of Sliarpey.
Bone situated immediately beneath an articular cartilage differs

70 PRACTICAL HISTOLOGY.

in not possessing Haversian canals, in the lacuna) being larger
than in ordinary b.)ne. and in being dtstitute of canaliculi. The
periosteum covering the free surface of bones consists of an
external layer of dense fibrous tissue supplied by capillary blood-
vessels, and of an internal osteogenetic layer containing a plexus
of delicate connective-tissue fibrils ; in themeshwork formed by
these fibrils are capillary blood-vessels and a number of nucleated
cells. The mecluUa is of the yellow kind, and is chiefly composed
of fat-cells, with intervening membranes of flattened connective-
tissue cells : it also contains numerous cells possessing one or two
nuclei.

h. Spongy or Cancellous Bones. — In spongy bones, f./7.,averte-
bral, carpa], or tarsal bone, the tissue consists o£ boneitrabecula?,
forming a more or less open framework, in which is embedded
the medullary substance. Bone trabeculse contain lacunae with
bone corpuscles and ill-developed canaliculi. The medullary
substance is of the red kind ; it is rich in blood-vessels,
and in cells having the characters of lymph corpuscles, fat-
cells, etc.

The Development of Bone. — It is usual to describe two ways
in which boue is developed, viz., (1) in cartilage, (2) in membrane ;
but in reality the shaft of all permanent bones is developed in
membrane, as will be seen in the following description. The
so-called development in cartilage is really a preliminary stage,
and all bone which is so formed, except at the growing ends,
does not remain a part of the permanent bone, but it is
reabsorbed nearly as soon as it is made.

In the account of the formation of bone in cartilage, the
stages under which the process is described are more or less
arbitrary, being inserted for the convenience of description.

In Cartilage. — This must be studied both in longitudinal and
transverse sections of foetal bones in various stages of develop-
ment, doubl}' stained in the manner above described. Stage i.
Hyaline cartilage covered by perichondrium. Tlte perichondrium
consists of an outer layer of embryonal connective tissue, and
an inner osteogenetic layer containing spherical cells — the future
osteoblasts and blood-vessels. Stage ii. Th: hr.icr hujrr of j^eri-
chondrium ptenelrates th" cartihr/". forming for itself channels by

rilACTK AL HISTOLOGY. 71

absorption, and carrying with it blood-vessels and cells. The
growth of the perichondrium inward starts at the centres of
ossification. Stage iii. T/ie primari/ marrow cavifies arc formQd
by the appearance of lacunas near the cartilage channels, which
then become confl;jent, whilst the trabeculaj separating nci^'h-
bouring lacuna) become calcified. The primary marrow filling
the marrow cavities is the periosteal ingrowth containing the
vessels and cells. Stage iv. T/ie calcified trahecuhr become en-
shcafJicd with osseous material, and are then absorbed. A net-
work of osseous trabcculie instead of a network of calcified
cartilage is thus formed, whilst the whole tissue resembles
spongy bone (endochondral bone). The surfaces of the osseous
trabeculic are covered with osteoblasts, whilst the cavities
separated by the trabecula: are filled witli marrow rich in vessels
and cells. So far we have almost the same stages as described
above, under the head of ossifying cartilage. Stage v. The
endochondral spongy bone is absorbed, from the centre outwards :
the large jnedtd/ar// cavifi/ is thus formed. Bone from the peri-
osteum is also simultaneously developed round the endochondral
bone. The osteohlas's multiply and become converted into the
osseous matrix and into bone corpuscles. The meshes of the
spongy periosteal bone are tlie Haversian spaces; they contain
marrow from which a series of concentric lamcllfe are formed.
The spaces are thus gradually reduced to Haversian canals.
Stage vi. All the endochondnd bone is absorbed, and the ossified
trabeculas are represented by the interstitial substance separating
the concentric Haversian lamella?.

As the bone increases in thickness, though continued by the
formation of new bone on the outside by the periosteum, the
primary periosteal bone is pushed towards the centre and is
absorbed, first of all becoming more spongy in its nature ; the
bone formed later from the periosteum being much denser and
more compact in structure than that first formed.

In Membrane. — This is best studied in stained and prepared
sections of fceUil lower jaw. The membrane corresponds to the
future periosteum ; it consists of two parts, as above. Stage i.
The cells of the osteo-genetic layer— ^Ae osteoblasts — increase
and form the osseous matrix by excreting ossein around them-

72 PRACTICAL HISTOLOGY.

selves, thus forming ossified trabeculte which start from the
centres of ossification. Stage ii. Portions of the trabeculie are
absorbed, whilst, as in endochondral bone, concentric lamellie
are formed by the marrow in the Haversian canals.

The formation of intramembranous bone is identical with
the formation of periosteal bone. The absorption of osseous
substance is in nearly every case associated with the presence
of multinucleated giant cells — the osteoclasts.

CHAPTER \.

MUSCLE.

TiiEUE are Itco caridies of muscular tissue, (1) striped and
(2) unstriped, gr plain. Of the striped muscle, that which is
found in the heart differs somewhat from the rest.

In order to study the structure of muscular tissue the follow-
ing preparations should be made :

(«) Of Fresh Maude. — (i.) With a couple of needles open the
chitinous shell of the leg of a water-beetle or of a cockroach, turn
out the muscle on a slide tease and mount in saline solution ;
edge round the cover- glass with dammar, or paraffin, to avoid
evaporation, and examine at once. The striations are very-
marked and coarse. If examined whilst the muscle is still
irritable it may be that the wave of contraction will be seen
in the fibre, (ii.) Tease a piece of fresh muscle from the
crab, or from the thin muscles attached to the jaw of the frog.

(iii.) To see the sarcolemma, treat fresh muscle with dilute
acetic acid.

(iv.) To see Cohuhelni's fields, make sections of frozen fresh
muscle and examine in salt solution.

(v.) To see the nerve terminations, treat muscles from the frog
or lizard, about five hours after death, according to the chloride
of gold method.

(vi.) To see muscle terminations in tendon, take a thin, straight
muscle, ending in a tendon, from a frog, put in a '5 osmic acid
solution for several hours, and tease in glycerine — and macerate
another in caustic potash, 40 per cent, solution.

74: PRACTICAL HISTOLOGY.

(vii.) To see transverse muscle di>>cs, immerse in 1 per cent,
acetic acid or 0"1 hydrochloric acid, and tease in glycerine.

{b) Of Hardened Muscle. — Voluntary and heart muscles from
various animals should be hardened in chromic acid and spirit ;
pieces should be taken and teased in glycerine, with or without
previous staining in hsematoxylin.

To see the j^f^if^'-^'tive fibrilhe, macerate the muscles of the lower
jaw of a frog, or the tail of a tadpole, in a saturated solution of
picric acid for a week, or in 5 per cent, chromic acid for a few
days, and tease in glycerine.

To see the general relations of muscle fibres, the position of the
nuclei and the appearance of sections of muscle cut in different
directions, thin sections should be made (a) of the tonr/ue of a
rabbit, and [b) of the heart of a rabbit, or guinea-pig, which
should be siained in hajmatoxylin, prepared, and mounted in
Canada balsam.

(c) Of Unstriped Muscle. — Unstriped tissue may be demon-
strated by distending a piece of rabbit's intestine with saline
solution, leaving it in a 1 per cent, solution of anilin black for
twenty-four hours, and stripping off the outer coat with forceps.
Small pieces may be mounted in glycerine.

The muscular-fibre cells may also be seen by stripping off
piecas of the outer coat of the intestine, which has been
macerated in a solution of chromic a^id or potassium bichromate ;
they may be washed, stained, and teased in glycerine.

The tissue is well seen in sections of the stomach of the newt
or frog which have been hardened in chromic acid and stained
in hEeaiatoxylin ; verj- large individual cells are well shov\'n in
the mesentery of the newt, which has been put fiesh into
ammonium bichromate 5 per cent, for twentj'-four hours,
stained in h^ematoxylin and mounted in glycerine.

With the aid of the preparations indicated above, the general
structure of the muscular tissue can be made out. (1) It will be
as well to consider, first of all, the striped muscle of the voluntary
or skeletal type. It consists of \owg fibres, which are cylindrical,
but appear in transverse section as rounded polygons. Each
fibre is made up of a number of exceedingly fine and delicate

PRACTICAr. niS'R>L(X;Y. iO

filaments, the Jibrilhr^ enclosed within the sarcolemma. The
fibres are agi^regated into hundUs ; several bundles forming
ytf>tiV»//, and these the anatomical muscle. Periini/aiurn or fibrous
connective-tissue surrounds the bundles ; from it pass off be-
tween the muscle fibres small processes of connective-tissue,
with cell plates and plasma cells — the endomysium.

So far, with the exception of the fibres and of the endomy-
sium, the appearances may be made out with the naked eje,
or with a very low power of the microscope. But now the
higher powers are required.

Each fibre will be seen to consist of broad dim bands of highly
refractive substance, representing the contractile portion of the
muscle-fibre — the contractile discs — alternating with narrow
bright bands of a less refractive substance — the interstitial discs.
After hardening, each contractile disc becomes longitudinally
striated, the thin oblong rods thus formed being the sarcous
eUntents of Bowman. The sarcous elements are not the optical
units, since each consists of minute doubly refracting elements
— the disdiaclasts of Briicke. When seen in transverse section,
a muscular fibre appears to be subdivided by clear lines into
polygonal areas— Cohnheini's field.-, each corresponding to several
sarcous-element prisms. The clear lines arc due to a transparent
interstitial fluid substance pressed out of the sarcous elements
when they coagulate. The sarcolemma is a transparent struc-
tureless elastic sheath of great resistance, which surrounds each
fibre. From the sarcolemma, transverse membranous septa —
the membranes of Kravse — extend inwards across the muscle at
regular intervals. By these septa the muscle-fibre is divided
into equal-sized muscle compartments, each containing one con-
tractile disc. The membranes of Krause are so placed that each
passes across the middle of an interstitial disc, which is thus
divided into two lateral discs.

A thin transverse median disc — the disc of Hensen — is occa-
sionally seen to divide the contractile disc. In some fibres,
chiefly those from insects, each lateral disc contains a row of
bright granules forming the granular layer of Flogel. The fibres
contain nuclei, which are roundish, ovoid, or spindle-shaped in
different animals. These nuclei arc situated close to the sarco-

76 rRArnCAL HISTOLOGY.

lemim. their long axes being parallel to the fibres which contain
them. Each nucleus is composed of a uniform network of
fibrils, and is embedded in a thin more or less branched film of
protoplasm. The nucleus and protoplasm together form the
muscle-cell or uiusde corpuscle of Max Schultze.

In injected-, specimens it will be seen that the arteries and
veins are in the- perimysium : that the capillaries are in the endo-
mysium, between the fibrillin ; and that the capillary plexus
is elongated. The individual capillaries run parallel with the
muscle-fibres, and anastomose with each other by short branches.

Heart Muscle. — The fibres seem to be very small and finely
striated. The striae are generally indistinct, often showing only
as coarse granules. There is no sarc^lenima. Many of the fibres
anastomose and branch. The nuclei are in the centre of the
fibres.

Unstriped Muscle. — Is made up of bundles of cells,
bound together by an albuminous cementing substance — the
endomysiam — in which lie connective-tissue cells and a few fibres.
The periini/siam continuous with the endomysium is the fibrous
connective-tissue surrounding and separating the bundles of
muscle- cells. Fibres, fusiform, band-like, or spindle-shaped,
containing elongated or staff- shaped nuclei, are placed midway
in the fibres. The ends may be split into two or more parts, as
may frequently be seen in the cells obtained from arteries and
veins. Each muscle-cell consists of a fine sheath, probably elastic :
of a central btmdle of fibrils representing the contractile sub-
stance ; and of an oblong nucleus, which includes within a mem-
brane a fine network anastomosing at the poles of the nucleus
with the contractile fibrils. The ends of the fibres, which are
usually single, may sometimes be divided. There is no sarco-
lemma.

CHAPTER YJ.

NERVOUS TISSUE.

Xkhvous tissue consists of nerve-Jihres and nerve-celh, together
with support inq connrrfiveiissxe. Nerre-fibres should be studied
first of all in the trunks of cerebro-spinal and sympathetic nerves,
and in the olfactory, optic, and auditory nerves. Afterwards nerve-
fibres, as well as nerve-cells, should be examined in the central
nervous system— in the spinal cord and brain.

NERVE FIBRES.

Preparation.— A medium-sized nerve-trunk, c.r/., the sciatic
nerve of a rabbit, guinea-pig, or frog, should be removed from
the recently-killed animal, and cut into short lengths. One or
more of these lengths should then be placed in the following
solutions: (a) Oaniic acid. 1 percent., (h) Gold chlori'le, "5 per
cent., (c) Silver nifrafe, *25 per cent., (d) Ilfcmatoxjjh'a or car-
ni'nw, and teased with needles whilst in the reagent in order that
it should penetrate properly.

The osmic acid should be allowed to act for an hour or two,
and the fibres, when teased, will be found to show the medul-
lary sheath, the nodes of Ranvier and the sections of Lanter-
mann — the first being stained black.

The gold chloride method will demonstrate the external
nerve sheath and the axis cylinders.

The silver nitrate requires about a quarter of an hour for its
action ; it will show the endothelium covering the nerve-bundles,
and also, by more prolonged action, the nodes of Ranvier.

The hsematoxylin and carmine will show, in a good prepara-

78 PRACTICAL HISTOLOGY.

tion, the nucleated external nerve sheath and the axis cylinder ,
A considerable time is necessary, however, to accomplish this
action — twenty-four hours or more— and very frequently the
stain -will not penetrate to the axis cylinders even then,
unless the fibres have been placed first of all in absolute alcohol,
ether, or chloroform.

The fibres should in all ca.^es be teased and mounted in
glycerine.

Xerve-trunks of a rather larger size should be pinned out
straight on a piece of cork, and hardened in chromic acid and
spirit solution. Of these transverse sections should be cut,
f-tained in hematoxylin, prepared and mounted in Canada
balsam.

The auditory, sympathetic, and optic nerves niav also be
examined by teasing, and in transverse section after hardening
and staining.

Structure. — Xerve-fibres are of two kinds, (a) MctlulJated^ or
(Z/) Xon-meduUated. The former chiefly make up the cerebro-
spinal, and the latter the sympathetic system.

(«) The cerebro-spinal nerve-trunks, with two or three excep-
tions, are composed of a variable number of bundles of nerve-
fibres {funiculi), each of which has a special sheath {pfrhinirium
or neurilemma). These bundles are inclosed in a firm fibrous
sheath (^jj/«ei/nw/n), which also sends in processes of connective-
tissue supporting and connecting the bundles together, "Within
the funiculi, between the fibres, is a delicate supporting tissue
[the endoneunufii). Each medullated nerve-fibre is made up of
the following parts : (1) Primitive nerve sheath, or nucleated
sheath of Schwann ; (2) Medullary sheath, or white substance of
Schwann ; (3) Axis cylinder, primitive band, axis band, or axial
fibres.

(1) The nucleated sJicafh of Scliwann is the external layer of
the fibre. It appears to be a simple, transparent, colourless,
homogeneous structure, containing a varied number of oval
nuclei embedded in protoplasm.

(2) The medullary sheath^ or the intermediate laj'er, forms the
greater part of the nerve-tube. It surrounds the axis cylinder,
nnd has a double contour : and at regular intervals aie constric-

ri;.V(TRAL llI.sloLUCJY. 79

lions in tlic nKHlull.uy shciilh {l>\iiiri( r's njiishic/iuns-')^ caused liy
the thimiini,' or interruption of the medullary substance. In it
may be dcm<Histrated, especially by the action of osmic acid, Iho
sediona of Lanlcniutiin, of ■\vliieh its structure is said to be
made up.

(;») Tlie au'i.^ ci/Iiinhr is situated in the centre of the norve-
tube, and appears as a fainlly-marked band, Avith an exceedingly
fine and even outline. It is made up in the prepared specimen
of a number of most delicate fibrils (jirimilire /ibrils).

Between the axis cylinder and the medullary shealh Ihrro is
said to be a small space containing, in the recent state, albumi-
nous fluid. At the constrictions df llanvier, in nerves stain( d
with silver nitrate, a black cross is seen, due to the action (=f the
silver on the albuminous cement substance between the con-
stricting folds, and to its penetrating and staining the cement
substance surrounding the axis cylinder for a variable distance.

(/>) In the oi>tic and auditory nerves the nerve trunk is not
made up of distinct bundles, and consequently there is no pe'i-
nenrium, but the trunk may bo hoked upon as a single bundle
of fibres, and if so, the epineurium must be said to be abs< nt.
The nerve-fibres in the trunk h?ve no extrrnal nerve f-heath,
but, instead, the endoneurium, which may also be called n^u-
rogl'a, U more mnrked than in the other ccrebro-spinal nerves.

(c) In the sympathetic system, as well as in the olfnctory
nerve, the nerve fibres possess no medullary sheath, and so are
called uon-medullatcd nerve-fibres.

SPINAL CORD.

Prpparaticn.— Short lengths (about ^ to ^ inch) of the spinal
cord of the cervical, dorsal, and lumbar regions of a calf, shcfp,
or pig, should be hardened in a 5 per cent, solution of bichromate
of ammonium or the usual mixture of chromic acid and spiiit
for a W( ek, and should then be transferred to spirit.

A solution of eosin is recommended for staining the sections,
and carmine or picrocarmin and ha^matoxylin act well. Stephens'
blue-black ink diluted one half or two thirds, or anilin black,
1 per cent, solution, stains the ganglion cells excellently.
Double staining with eosin and hoematoxylin produces good
results.

80 PRACTICAL HISTOLOGY.

In order to study the areas or tracts in the spinal cord it i?
necessary to obtain pathological material from the human sub-
ject in which tracts of degeneration may be demonstrated. The
following method is recommended for the pnrpose. It is called
the Weigert'- hfematoxylin method.

Sol lit ions required arf :

1. Hoematoxyliu ext 1 grm.

Absolute alcohol 1<> cc.

Distilled water 'JO cc.

2. Ferricyanide of potass. ^\ grra.

Borax 2 grm.

Water 100 cc.

Grind the hfematoxylin extract in a mortar with a little alcohol,
then add the alcohol and water. The solution is better if kept
a day or two before using.

Melhod of Ui^iruj. — The sections of brain or cord are placed in
solution 1, till they are stained a d^epand uniform black. Tbi>
may take any time from one hour to twenty-four. The opeia-
tion of the stain is much helped by keeping the preparation
in an incubator at a temperature of 40' C. Overstaining is
scarcely possible. The sections are then taken out, shghtly
washed in clean water, and placed in solution 2. A slight brown
cloud is then seen to come from them, and the colour
gradually disappears from the grey matter, or from areas of
deo'eneration if there be any. This operation is complete
when no more brown colour appears in the yellow fluid. The
medidlnjejl nerre-nhres will now be found to be deeply stained a
rich purple, all the rest of the section being a brownish-yellow.

Thus the brain or cord may be stained in toto by this method.
but the operation, of course, takes much longer. An average
time, for instance, for the penetration of the stain into a slice of
cord or medulla -^V i'lch thick would be three or four days, and
about the same time would be required for the developing pro-
cess in solution 2.

Precautions. — The material should be hardened in MiUler's

fluid, or bichromate of potash. If bichromate of ammonium or

chromic acid have been used it should be entire!)/ removed by

* JVei'/ert, Fortscliritte cTer Mediciii, 1SS4, p. 190. TVe are indebted to
Dr. Tooib for this accomit. He has used the method with much success.

PKALTILAL lIISTOLOliY. 81

CDn<?tant wa'^bing. The best results are obtained from material
from which every trace of the hardenini,' flui-l, whatever it may
be, except alcohol, is removed. If the material has been pene-
trated with wax or paniflia for the purpose of section cutting,
it will never take the stain satisfactorily or with certainty, how-
ever well the wax may hive been removed.

Tracts of Degeneration. — By means of disease various tracts
are mapped out in tLe cord, s-howing in many cases the courses
of the fibres. These tracts, as seen on transverse section, are as
follows : In the posterolateral column, a little nearer the grey
matter than the exterior, a somewhat oval or wedge-shaped
area, consisting of fibres which have decussated in the anterior
pyramid of the medulla and passed over to the lateral region.
This area forms the crossed pyramidal tract, which diminishes
in size from above downwards, and it can be traced as low as the
third or fourth saci-al nerve. Outside this a very narrow area —
the direct cerebellar tract — can be traced down as far as the second
lumbar. On either side of the anterior fissure the uncrossed, or
direct anterior pyramidal tract, diminishes downwards until it
disappears about the middle of the dor^al region. On either
side of the posterior fissure is the posterior median column, or
column of Goll, traceable as low down as the middle of the
dorsal region ; outside this, the posteroexternal colunm, or
column of Bardach, can be distinguished. Between the anterior
cornu and the exterior of the cord anterior to the direct
cerebellar tract is Gofcers tract o/ ascending degeneration.

In Structure the spinal cord consists of (1) ivhite matter, (2)
grey matter, and (.'») neuroglia. Observe the structure of the
spinal cord by means of a transverse section taken from the
region of the cervical enlargement. In the first place examine
the stained section mounted in Canada balsam under a two-
inch objective, or el>e with a hand magnifying-glass. Xotice
the roughly oval shape of the section.

The central and more deeply-stained portion is the grey
matter. It is less granular than the white matter seen round
the circumference. It offers for examination two crescentic
masses with their concavities turned outwards, joined across the
jniddle by a transverse portion (the grey commissure). In the

G

82 PRACTICAL HISTOLOGY.

centre is a small canal lined by a layer of coUininar cells, which
in young animals are ciliated. Each crescent presents (a) the
anterior horn, or cornu, which is short and thick and extends
towards the anterior roots : (?>) the posterior horn is longer and
more slender. In the concavity of each crescent the grey matter
sends out processes which enclose portions of the white
substance.

Surrounding the giey matter on all sides is the granular white
matter. It is traversed by delicate bands of medullated fibres,
which extend from the extremities of the gre}' matter towards
the outside of the cord. These fibres are the anterior and pos-
terior roots respectively of the spinal nerves. A delicate coat
of fibrous tissue — the ])ia-mater — encircles the cord and sends
into it numerous processes. The white substance is cleft ver-
tically by a space — the anterior inrdian fissure — which extends
nearly down to the transverse portion of the grey matter. It
divides that portion of the cord which is nearest to the bodies
of the vertebra}. The white matter on the posterior aspect of
the cord is divided by a somewhat similar space — the j^osterior
media.') fissure. This fissure differs from the anterior in the fact
that it is narrower, that it is deeper as it extends down to the
grey commissure, and that it is usually occupied l)y the delicate
connective-tissue forming the basis of the cord called thr
neurof/Iia. Laterally the surrounding pia-mater penetrates the
white matter at various points ; these ingrowths are somewhat
more marked in the neighbourhood of the anterior and posterior
roots. This portion of the cord ia therefore divided by antero-
lateral and postero-lateral fissures into antero-median, antero-
lateral, lateral, postero-lateral, and postero-median columns. At
the bottom cf the anterior fissure is a transverse band of white
substance called the anterior white commissure ; it separates the
fissure from the anterior grey commissure, which in turn is
separated from the posterior grey commissure b}' the central
canal. The posterior grey commissure lies immediately above
the posterior median fissure.

Under a somewhat higher power, such as a half -inch objective,
it will be seen that the grey matter is composed of a finely
crranular matrix enclosing numercus polygonal bodies which,.

im:a<ti(AL histology. 83

in a well-pivi>;irc'(l speoiineii, have many delicate b"aiiclies.
These bodies are the ganglion cells. They are largest and most
numerous in the anterior and posterior extremities of the horns
as well as round the periphei-y. They are least numjroiis in the
centre and in the comn\issnres. The granulai' appearance of the
white matter is due to the section of a large number of medul-
lated noi've-fibres whicli vary considerably in size and are em-
bedded in a matrix of neuroglia. In transverse section these
fibres consist of circular masses with a more deeply stained
centre, representing the axis cylinder. In the anteiior region
the white matter is traversed by several bundles of non-medul-
lated fibres, which form the antei'ior motor roots, whilst in the
posterior region, the posterior or sensory root passes through it
as a single l)and.

Under a still higher power, such as a ^l inch objective, the
various constituents of the cord may be considered in greater
detail. (1 ) The white matter consists of longitudinal fibres
which in transverse section measure y.-^^io to j^luw ^^ '^''^ inch in
diameter. They are finer in the posterior and po-itero-lateral
columns than elsewhere. Each fibre can now bo seen to consist
of an external coating derived from the neuroglia which stain*:,
and whicli surrounds an unstained portion, which again encloses a
small central and deeply staining portion. The unstained part is
often arranged in concentric rings. Connective-tissue corpuscles
belonging to the neuroglia are sometimes found between the
nerve-fibres. (2) The grey matter is made up of small non-medul-
lated fibres, which chiefly form a dense network, continuous with
the roots of the nerves ; part of these fibres are derived from the
branches of the nerve-cells embedded in the network. The cells
are of two kinds : (i.) Largp^ hranched^ and nucleated, which are
most numerous in the anterior cornua, especially at their upper
and outer parts. They are also found at the inner part of the
base (cervix cornu) of the i)Osterior horn, forming ih.Q posterior
vesicular column, which is best marked in the lumbar enlarcre-
ment of the cord. Lastly in the concavity of the crescent is a
group of cells, occupying a projection of grey matter there and
called the ^y«c/?rs- internte lio-lateralis ; it is best marked in the
dorsal region, (ii.) Smaller cells scattered throughout the grey

6—2

84 PRACTICAL HISTOLOGY.

matter, but chiefi}' at the tip (caput cornu) of the posterior horn,
in a finelysfranular basis, and among the posterior root fibres (s ?//..§-
tnnt'ia fjelafinosa cinerea of Rolando), (iii.) Tlr nfurofjlJa forms
the delicate connective-tissue supporting the grey and white
matter of nerve structures. Its general characters are excellently
seen in some parts of the pineal body. It consists of a trans-
parent semi-fluid matrix, interspersed with delicate fibrils
resembling those found in elastic tissue. The fibrils form a
network, in the meshes of which lie small branched neuroglia
cells. A few larger branched cells of Deiters are also present.
The neuroglia forms a perivascular lymph-space for the blood-
ve?«^els. which are numerous but smnll.

The Peculiarities of Different Regions of the Spinal
Cord.— The outline of the grey matter and the relative propor-
tion of the white matter varies in different regions of the spinal
cord, and it is therefore possible to tell approximately from
what region any given transverse section of the spinal cord has
been taken.

In the cfrvicaJ enJavgemrnf the grey matter occupies a large
proportion of the section, the grey commissure is short and
thick, the anterior horn is blunt, whilst the posterior is some-
what tapering. The anterior and posterior roots run some dis-
tance through the white matter before they reach the periphery.

In the dorsal ref/ion the grey matter only bears a small relation
to the white, and the posterior roots in particular run a long
course through the white matter before they leave the cord ;
the grey commissure is thinner and narrower than in the cervical
region.

In th" himhar enlargement the grey matter again bears a very
large proportion to the whole size of the transverse section, but
its posterior cornua are shorter and blunter than they are in the
cervical region. The gre}' commissure is short and extremely
narrow.

^1/ the 'I'pper part of the conns meduJlarh^ which is the portion
of the cold immediately below the lumbar enlargement, the grey
substance occupies nearly the whole of the transverse section,
as it is onl}' invested by a thin layer of white substance. This
ihin layer is wanting in the neighbourhood of the posterior
nerve-roots. The grey ccmmissure is extreme! v thick.

IMIACTICAT. IIISTOI.OCV. 85

At the heel of the fyUi sacral lerlebra the grey matter is again
in excess, and the central canal is enlarged, appearing T shaped
in section, whilst in the upper portion of the /Hum tcrminah' the
grey matter is uniform in shape without any central canal.

MEDULLA OBLONGATA OR BULB.

Preparation, as for the spinal cord.

Structure. — Under a low power a transverse section through
the bulb (i.) a little helow the apex of the calamus scriplorio.s
shows the great depth of the anterior median fissure, with
the comparative shallowness of the posterior fissure. The latter
fissure is bounded on cither side by the fascicules f/racilis, which
is the continuation upwards of the posterior median columns of
the spinal cord. The upward prolongation of the postero-
lateral column which has here expanded into the wedge-shaped
cuiteate fasciculus is situated more anteriorly than the 'fasciculus
gracilis, and still more in front is a new band of fibres known as
the tubercle of Rolamlo. The olicary ho(hj, which is dentate in
section, is yet more anterior, and is separated from x\iq pyramid
by a bundle of fibres, which represents tlte root of the lii/poglosf^al
nerve.

The higher power will show that the anterior and lateral
regions of the grey matter are here arranged in a reticular for-
mation caused by the intersection of nerve-fibres running in
longitudinal and transverse directions. Numerous multipolar
ganglion-celLs are scattered throughout the reticular formation.

(ii.) A section through the bulb at the level of the fourth ven-
tricle shows that the anterior median fissure still remains as a
deep cleft, whilst the posterior median fissure has expanded
into the space which represents the fourth ventricle. A layer
of grey matter, containing numerous ganglion-cells, forms the
floor of the ventricle. The group of ganglion-cells nearest to
the middle line upon either side is the nucleus of the, fasciculus
teres. A little external and anterior to it, i.e., deeper in the
grey matter, is a collection of large ganglion-cells, forming th;
nucleus of the liypoglossal nerve, from, which the nerve-roots may
be seen passing through the reticular formation to the point
where they leave the bulb, between the pyramid and tlie olivary

SQ PRACTICAL HISTOLOGY.

nucleus. Still more externally are other groups of ganglion-
cells, from which tJw vagus arises. This group of cells is partially
subdivided by a bundle of fibres known as the fascicuhis sol'i-
iarius. which appears circular in transverse section ; of the two
groups, the more central and deeper is the larger. The root of
the nerve passes outwards through the reticular formation. The
nucleus gracilis and the nucleus cuncatuslie still more externally ;
they are portions of grey matter which extend respectively into
the fasciculus gracilis and fasciculus cuneatus. The ascending
root of thejifth ncrce is situated laterall}', as a well-defined bundle
of white fibres.

CEREBELLUM.

The cerebellum is hardened in the usual bichromate mixture.

To stain specimens of it, the following method is recom-
mended. Thin sections should be left in a "01 per cent, solution
of eosin for twelve hours, and then, after washing in distilled
and slightly acidulated water, should be placed in a weak solution
of aniline green for fifteen or twenty minutes, being afterwards
passed rapidly through the ordinary reagents, and mounted as
for cerebrum.

Structure.— It is divisible into (a) Cortical grey; and (b)
Internal white substance.

(a) The Cortex is composed of (1) llie nwlecular lat/er, the
most external, consisting of a nerve network containing small
multipolar ganglion-cells. The fibres of tlie network in the
more superficial portions are nearly vertical to the surface ;
they are deiived })artly from the neuroglia, partly from the pro-
cesses of the cells of Purkinje. (2) A single layer of large,
spindle-shaped ganglion-cells ^Jj to j^W inch in diameter,
Purlhije's cells. Each cell posseiises one branched process which
extends into the molecular la} er, where it branches dichoto-
mously, seme of the finest ramifications looping backwards to
terminate in the granular layer, and an unbranched axis-cylinder
process passing downwards. The cells lie in a pericellular
space, and each consists of a minute network of fibrils extending
into the branched processes. The nucleus is sj)herical and oval.
(.3) The granular lager, containing a network of minute fibrils

PRACTICAL HISTOLOCjy. 87

and dense groups of granule-like corpuscles. These corpuscles
average n,\)o ^^ ^jjVo ^°^^^ ^^ diameter.

(i?) The inter iKil ii'liite suhstance^ or Medullary Centre, con-
.sists of nerve-iibres arranged in parallel or interhicing bundles.

The neuvo<jl\(i of the white matter contains rows of small
cells, each Avith a spherical nucleus, between bundles of nerve-
fibres.

The blood-vessels of the grey matter pass from the pia mater
in a vertical or oblique direction, and anastomose into a uniform
network. The blood-vessels of the white matter form a net-
work with longitudinal meshes. The vessels lie in lymph
channels, the /xrlntscular lijhipliatlcx of His.

CEREBRUM.

Preparation.-- Having carefully removed the pia mater, place
small pieces from different parts of the human cerebrum in a
2 per cent, solution of ammonium bichromatefor two days, after
which transfer to weak, and finally to strong spirit. Take care
to get vertical sections. Stain in aniline blue-black.

Structure. — Get a general view of the structure with a |^ or
I inch lens, and then use a I objective. With the former it will
be seen that the cerebral convolutions are divisible into (a)
Cortical grey portion, and (i;) Medullary white substance.

With the high power the (a) Cortical Grey portion is seen
to be composed of :

(1) An external layer containing a few small cells with fine
processes embedded in a considerable quantity of neuroglia.
This layer composes about /^ of the whole thickness of the
grey substance.

(2) A second lai/er of small, densely aggregated, pyramidal
cells, provided with branching processes. This layer is of nearly
the same extent as the previous one.

(H) A third Icujcr of greater width, somewhat paler than the
first and second layers ; it is composed of large and small
pyramidal cells, arranged with their apices turned towards the
surface of the convolution. The larger cells average \-^C)o i^^^
across their base. The cells are arranged in groups, and are

88 PRACTICAL HISTOLOGY.

separated from each other b}^ bundles of radiating nerve-fibre?,
each bundle being about y/^u inch in diameter. The pyramidal
cells send downwards three processes, of which the middle one
forms an axis cylinder. Both cells and processes are striated
longitudinally, and generally contain a little yellowish pigment.

(4) A fourth laijer is somewhat narrower than the preceding ;
it consists of small, irregularly placed, granule-like corpuscles,
with delicate processes The cells are less distinctly separated
into groups.

(5) The lowest Jajjer is of considerable width ; it contains, in
addition to cells resembling those of the fourth layer, fusiform
cells arranged vertically at the summit of a gyrus, but parallel
to the surface of a sulcus. This layer gradually blends with

(b) The White Substance, which is composed essentially of
white nerve-fibres, smaller than those of the spinal cord, and
with an average diameter of yo^^yQ inch. In the neighbourhood
of the corteXj a few non-medullated fibres can be seen.

(c) The Jieurof/liajfoTuied of a homogeneous matrix, in which
lie numerous elastic fibrils, connected into a network. "With
this network the branched nucleated cells of Deiters are con-
nected.

SYMPATHETIC SYSTEM.

Harden (Ij portions of the Gasserian ganglion from the sheep
in chromic acid and spirit. (2) Pieces of the sympathetic nerve
from the neck of the ox, in the manner recommended for medul-
lated nerve-fibres. (3) Paccinian corpuscles by snipping out
pieces of the mesentery or meso-rectum of the cat, in which
they may be seen as small bodies embedded in the fat : isolate
them with needles, treat with osmic acid, stain in picrocarmin.
and mount in glycerine. (4) Meissner's and Auerbach's plexuses,
situated the one in the submucous coat, and the other between
the muscular coats of the intestine, are best prepared from the
rabbit or guinea-pig. A piece of intestine, three inches in length,
is distended with the juice of a fresh lemon, the ends being
ligatured ; it is allowed to remain in tbe lemon juice for about
five minutes. The ligatures are then removed, and it is washed
thoroughly in water, and filled with a 2 per cent, solution of gold

I'KA* rnAF. insT«ti.(j(.Y. 89

chloride : it is again ligatured, and is then suspended for h ilf
an hour in a 1 per cent, solution of gold chloride, washed
thoroughly, and transferred to a 24 per cent, solution of formie
acid to reduce the gold, the preparation being kept in the dark.
After reduction of the gold, the intestine is of a rich reddibh-
brown colour : it should be again washed. Peel off strips of
the outer muscular coat with forceps, and mount in glj-ceiine.
Auerbach's plexus will be seen. The plexus of Meissner is de-
monstrated by treating the intestine as before, and afterwards
inverting it and removing the mucous coat, when portions of
the submucous coat may be picked off from the muscular coats,
and mounted in glycerine.

General Characters.— r/«' fjanfjUon-ceUs are of various shapes
and siziis, gentrallv smaller than the cells of the cerebro-spinal
ganglia ; they possess a capsule, and consist of a network of fibres.
There is usually one large, excentric, oval nucleus, which may,
however, be double. The cells possess one or more processes,
which are continuations from the cell substance, and are
invested by a prolongation from the hyaline sheath. According
to the number of processes, the cells are unipolar, bipolar, or
multipolar. In the frog the bipolar cells arc peculiar, since one
of the processes appears as a ' spiral fibre * twisted round the
other process, or ' straight fibre.'

The nerve-trunks contain medullated fibres similar to those
already described, and non-meduliated, or fibres of Remak.
Remak's fibres are pale, finely fibrillated axis cylinders,
invested with a hyaline sheath of Schwann provided with nerve-
corpuscles.

The Paccinian bodies are oblong corpuscles connected with a
medullated nerve-fibre which represents its stalk. The cor-
puscle consists of a number of concentric capsules. Each capsule
is composed of a hyaline basement membrane, which is probably
elastic, and in which are embedded fine connective-tissue fibres.
On the inner surface of the basement membrane is a layer of
flattened nucleated endothelial cells, which are visible after
treatment with nitrate of silver. In the centre of the corpuscle
is a clear mass in which lies the axis cylinder of the perforating
nerve-fibre. The axis cylinder generally breaks up into two or

^0 PRACTICAL HISTOLOGY.

three branches, or it may terminate in a bud or in a pointed or
fringed extremity. The corpuscles contain, between the capsules,
caiiillary blood-vessels and a few plasma-cells.

The nerve plexuses consist of a meshwork of flat nerves, each
of which is ensheathed in a delicate endothelial membrane ; at
the nodal points are groups of ganglion-cells, which vary in size
and shape ; the larger ones possess a capsule and processes. The
plexus of Meissner consists of a larger and less regular mesh-
w^ork than that of Aaerbach. The two plexuses are connected
by branches which pass through the circular layer of muscle.
Each plexus gives off branches which supply the surrounding
tissues.

CHAPTER VII.

BLOOD-VESSELS.

Of three kinds, (a) Arteries, (b) Veins, and (c) Capillaries,

(a) Arteries. — Longitudinal and transverse sections of a
medium-sized artery (or vein), which has been hardened in a 1
per cent, solution of potassium bichromate, should be stained in
logwood, prepared, and mounted in Canada balsam.

Good transverse sections of small arteries and veins may be
.seen in sections of various organs, e.g., the tongue, liver or heart.
Structure. — Arteries (except those of minute size) have three
coats :

1. Internal coat, consisting of (a) An enJotlielial layer, form-
ing the lining of the vessel, of thin elliptical or irregularly
polygonal cells, often lanceolate, with nuclei and nucleoli :
(Jj) A suh-endotheUal layer of delicate connective- tissue, with
branched corpuscles ; (c) Elastic layers of longitudinal elastic
networks and ' fenestrated ' membrane.

2. Middle coat chiefly consists of circular unstriped muscle
Jihres, mixed with elastic fibres, and a sparse amount of connec-
tive-tissue.

3. External coat {tunica a(/f£'«^<ViV/.) chiefly consists of fine and
closely felted bundles of connective-tissue, together with longi-
tudinal elastic tissue between them.

In the largest arteries, the middle coat consists of alternate
layers of elastic tissue and uustriped muscle. In the smallest
arteries (arterioles) the coats are reduced to a muscular, prin-
cipally of circular fibres, and a lining endothelium.

(u) Veins.— Structure. — As of the arteries, with these dif-

92 PRACTICAL HISTOLOGY.

ferences : The elastic tissue of tho internal coat seldom occurs
in the form of fenestrated membranes. The middle coat is
thinner, contains less muscular tis=;ue and more white connec-
tive-tissue. The external coat of some veins has a considerable
amount of unstriped muscular fibre. The valves, when they are
present, consist of folds of the internal coat covered on each
surface with endothelium. Each fold contains a few muscular
fibres and. a little connective-tissue derived from the middle
coat.

(c) Capillaries. — These are tobeobtainedfrom thepia-mater.
The brain of some animal, such as a cat or dog, should be left
for two days in a 2 per cent, solution of potassium bichromate,
then the pia-mater may be stripped olf in pieces, stained, and
mounted in the usual manner. Capillaries are well seen also in
the mesentery of a cat or other animal.

Structure. — The walls of the capillaries proper are formed
entirely of a simple epithelial layer of flattened lanceolate cells,
joined edge to edge, and continuous with the layer which lines
the arteries and veins. The larger capillaries have an outside
structureless or finely fibrillated coat. In rather larger vessels
(small arteries and veins) there is added, outside the epithelium,
a thin layer of unstriped muscular fibre.

CHAPTER YIIl.

ALIMENTARY CANAL AND ITS APPENDAGES.

TONGUE.

Vkktral sections of a rabbit's or cat's tongue, which haa
been hardened in equal parts of ^ per cent, chromic acid and
spirit, should be stained and treated in the usual manner.

Sections of the papilla follata which is found on either side of
the base of the rabbit's tongue should also be treated in a similar
manner. The tongue furnishes excellent material for double or
triple staining.

Structure. — The tongue, for histological purposes, may be
considered to consist of a mucous membrane covering voluntary
muscles and glands.

TJie mucous membrane of the dorsal surface is elevated into
numerous papillie, which are of varying shape. Each papilla is
covered by stratified epithelium, the superficial layers of which
in the carnivora are converted into dense plates of chitinous
material. The papillte, according to their shape, are known as
the filiform, the fungiform, and the circumvallate.

((/) The filiform papilhn are the most numerou'', and are found
upon the anterior and lateral surfaces of the organ. The}- are
minute elevations of the corium covered by epithelium, their
apices being frequently subdivided into secondary papillae.

{h) The fuufjiform pap'ilhe resemble in shape unexpanded
mushrooms or ' puff-balls.' They consist, like the filiform papillae,
of areolar tissue enclosing a loop of capillaries, and they are
covered with a layer of squamous epithelium. These papillae
are most numerous at the apex and near the margins of the
tongue.

04 PRACTICAL HISTOLOGY.

(r) The clrcninvnllnte pajnlhu are situated at the posterior
portion of the dorsal aspect of the tongue, and are much larger
than either the filiform or fungiform papillae. In section they
appear as flat-topped elevations of the mucous membrane, with
a deep depression upon either side. The duct of a serous gland
frequently opens into the bottom of the depression. They arc
covered with flattened epithelium like the other varieties of
papillae, but at their sides some of the epithelial cells have become
modified into taste-hucls.

(d) Tlie iKi.pUhi foViaUt is an elongated oval patch seen upon
either side of the rabbit's tongue. It consists of a number of
parallel invaginations of the mucous membrane. The epithelium
thus invagiuated contains numerous ovoidal or flask- shaped
bodies identical with those found in the human circumvallate
papillae : they are the tm^te-hiuh. Each of these bodies con-
sists of a layer of flattened epithelial cells, arranged round, and
forming a covering for, a bundle of central cells. The central
cells are spindle-shaped, one extremity projecting through the
aperture left by the external cells, whilst the other extremity
is branched and extends into the submucous layer, where
it probably becomes connected with a terminal fibre of the
glosso- pharyngeal nerve.

Numerous mucous secreting glanda are found in the tongue.
Of these the largest are the glands of Nuhn, situated at the tip.
Serous glands are also present, they open into the trenches of the
circumvallate papillEe, Lgniphoid tissue is found in large quantity
in the submucous tissue at the posterior part of the tongue, in
some cases forming follicles ; numerous crypts and recesses, too,
are found, the walls being studded with nodules of lymphoid
tissue.

The mucous memlrane on the under surface of the tongue con-
sists of a thin layer of stratified pavement epithelium, with a
somewhat dense submucous tissue. On the dorsum of the tongue
the submucous coat is incomplete and scanty, though it contains
a large number of blood-vessels.

The muscular xwrtion of the tongue consists of longitudinal,
transverse, and vertical fibres of striated muscles ; considerable
quantities of fat and gland tissue are found between the bundles
of muscle.

|'i;a( IK Ar. nisTui.oiiv. 95

THE TEETH.

These structures may l»c examined [tii By means of sections
of the hard t(»()th : (rijnd down the tooth on both sides till it
is quite thin, then mount in hard Canada balsam, so as to
retain the air in the various cavities.

(A) By means of sections of the softened tooth : Place the
tooth in 10 i»er cent, solution of hydrochloric acid till it is quite
soft, then immerse in spirit : by this means the structure of the
dentinal substances may be investigated : or place the tooth
(preferably broken across) in a saturated solution of picric
acid until it is quite soft. Complete the hardening in spirit,
changing the spirit so long as it becomes tinged with the
picric acid. This method cf preparation "will preserve ih^
pulp and odontoblasts.

To demonstrate the pulp, break a freshly extracted tooth, and
immerse it in osmic acid 1 i)er cent, for twenty-four hours.

To show the development of teeth, place the lower jaw of a
foetal rat, dog, or kitten, from which the muscles have been
removed, in ^V per cent, chromic acid for seven days, then remove
to weak spirit for twenty-four hours, and finall}- to strong
spirit till required.

(c) The dentinal sheath lining the tubules may be isolated by
boiling for ten minutes in strong sulphuric acid. In each case
stain in htematoxyliu.

iXoie. — It is best to buy prepared sections of teeth.]

Structure.— (1) A tooth consists most externally, and above
the gum, of enamel. In the recently cut tooth, which has not been
much used, there exists above the enamel a covering of epithelial
or horny nature (Xasmyth's membrane), which is structureless,
and has a thickness of tjtjooo ^^ tooott liich. The enamel covers
the crown and neck of the tooth ; it is an epithelial product,
consisting of closelv aggregated polvhedral cvlinders forminsr
the enamel fibres (prisms or columns). The set fibres are crossed
by a number of darker lines, arranged in concentric layers,
' contour lines.' In transverse section, the enamel fibres are seen
to be six-sided prisms, with an average diameter of 5,^7^7 inch.

(2) The Dentine^ or ivory, covers the body and root of the

06 PR ACTIO A L HISTOLOGY.

tootla ; on the surface it lies immediately below the enamel.
The dentine consists of a compact bone-like substance, ■which
contains no bone corpuscles, and is permeated by dichotomously
dividing canals, the dentinal-tubules, which average x/oo i^^^
in diameter. The dentinal-tubules run perpendicularly to the
surface of the pulp cavity, into which they open bj- their lower
extremities. The tubules present a proper wall, consisting of a
membranous tube, and each contains a process of protoplasm
from the superficial layer of the pulp-cells. Examined under a
lower power, the tubules are seen to form two or three gentle
curvatures, which give rise, when a number of tubules are seen
together, to a scries of concentric lines (lines of Schreger).
Certain interglobular spaces, due to imperfect deposition of salts,
are also frequently seen in the dentine.

(3) The crusta petrom. or cement, invests the portions of the
tooth which are not protected by enamel. It closely resembles
bone in its histological appearances, except that the lacuuje and
canaliculi are larger and more irregular. "When the cement is
very thick, it may contain vascular channels, which are com-
parable with Haversian canab. The perforating fibres of
Sharpoy are present inconsiderable numbers in the cement.

(4) 77/e^Jz;//) occupies the central cavity of the tooth ; it con-
sists of jelly-like connective-tissue, in which run nerves and
blood-vessels. The outermost layer of cells, forming the pulp,
are elongated in form, the bodies somewhat resembling columnar
epithelium cells. This layer forms the membrana eboris of
which each cell is an odontoblast. The odontoblasts send off
one or more processes, which run in the dentinal-tubules :
processes which connect the cells together laterally, and pro-
cesses which unite the cells to others, lying more deeply.

(5) Osteodentine, or secondary dentine, is the hard substance
deposited on the inner surface of the dentine, which is produced
by the graduai conversion of the pulp.

Development. — (1) The first rudiment of a tooth appears as
a solid prolongation of the stratified epithelium, which grows
downwards from the stirface into the mucous membrane. This
process of epithelium is i\iQ primary enamel organ.

(2) The enamel organ becomes invaginated at its deep end by

rilACilCAL lIISTuI.Oe;Y. 97

a mass of tissiio derived from tho inucoiis membivuio, called the
embryonal tooth papilla. Tho primary enamel organ is thus con-
verted into the enamel cap covering the tooth papilla.

(3) rAf^ja^^iV/a is vascular, and is composed of a network of
nucleated cells; it forms the pulp, and by means of its odonto-
blasts forms the dentine.

(4) TJie odontoblasts appear on the papilla as a peripheral
stratum of large cells arranged vertically.

(5) The dentine is formed by the calcification of the substance
which surrounds, and is perhaps derived from, the distal ex-
tremities of the odontoblasts, whilst

(G) The dentinal fibres are processes of the odontoblasts.

(7) 'The tooth sac, or the mucous membrane which immediately
surrounds the enamel cap and tooth papilla, gradually grows
over the former, and separates it from its connection with the
surface epithelium.

(8) The enamel cap consists externally of («) columnar cell<,
more internally of (/>) polyhedral cells, followed by (c) flattened
epithelial cells in the centre, and again of {d) polyhedral, with
((?) columnar cells most internally — i.e , nearest to the tooth
papilla.

(9) The enamel cap is limited both externally and internally
by a monbrana propria.

(10) The enamel cap becomes divided into an inner and outer
membrane by the transformation of the middle layer (8c) into a
transparent tissue. 2'he inner membrane is composed of columnai
cells, the enamel cells, in contact with the dentine ; each is a long
hexagonal prism, and is nucleated at its lower part. Outside the
layers of enamel cells are one or more rows of small polyhedral
cells, forming the stratum intermedium. The outer membrane is
composed of several layers of epithelial cells.

(11) The enamel is formed by the enamel cells of the inner
membrane elongating at their distal extremities ; the elongated
portion is transformed directly into enamel,

(12) The cells of the stratum intermedium are used for the
regeneration of the enamel.

(13) The cells of the outer epithelium produce the enamel
cuticle.

7

98 PKACTICAL HISTOLOGY.

(14) The cement is formed from the tissue of the tooth sac in
exactly the same way as sub-periosteal bone is developed.

(15) During the stage of the primary enamel organ (a) a
lateral process grows out from the epithelial cells, which repre-
sents the rudiment of the enamel organ of the permanent tooth
{^suc of reserve^.

(IG) The permanent teeth are developed on exactly the same
plan as the deciduous set.

SALIVARY GLANDS.

Sections of the submaxillary gland of a cat or dog, and of the
parotid of a rabbit or dog, should be made in various directions
after hardening in spirit.

Structure. — The salivarj- glands are compound tubular glands
enveloped in an incomplete capsule. The capsule consists of
fibrous tissue which sends septa into the substance of the gland.
The septa support the blood-vessels, lymphatics, nerves, and
ganglia, and they divide the glandular substance into lobes,
lobules, alveoli, and acini. Each lobule is made up of the convolu-
tions of the main division of a duct bound together with connec-
tive-tissue. The convoluted parts are lined by and almost filled
with a single layer of columnar cells {salivary cells) enclosing
a nucleus. These cells, when isolated, are not unfrequently
found to be branched. The basement membrane of the tubes
consists of branched and flattened cells, and between it and the
salivar}' cells are found, here and there, but not in the parotid
granular semilunar bodies, the semilunes cf He'idenhain. The
smallest divisions of the ducts have a relatively small lumen, and
are lined near the convolutions with flattened epithelium, and
then with nucleated columnar cells which present a longitudinal
striation. The larger ducts acquire an external coating of con-
nective-tissue, and are lined with a si^^gle layer of columnar
epithelium. In the walls of the largest ducts are unstripcd
muscular fibres.

The salivary [/lands are divided into (a) Mucous glands, in
which the alveoli are large, and the cells are (1) mucous cells,
transparent and columnar, with their pointed extremities applied
to the membrana propria ; the cells are imbricated ; the nucleus

rUALllCAL lIlSTOLociY. 0!)

is much compressed, and is near the mcnibrana propria. ("2) The
semilunes of lleidenhain, or the crescents of Giannuzzi, semi-
lunar groups of cells situated here and there between the mucous
cells aud the raenibrana propria. The cells are small and pol> -
hedral, with a spherical nucleus. The submaxillary and orbital
glands of the dog and the sublingual gland of man are of the
mucous type.

{b) Serous (/lands, in which the lumen of each alveolus is
small, and the epithelium consists of a single layer of short
columnar cells, each with a spherical nucleus situated at the
periphery of the cell. The parotid gland and the greater part of
the isubmaxillary gland of man and the guinea-pig, as well as
the submaxillary and orbital glands of the rabbit, are serous
glands.

(c) Muco-salivary glanih, such as the submaxillary gland of
man and the guinea-pig, are formed by the mixture of the
mucous and serous types of glands.

TONSILS.

Sections made from a tonsil which has remained for a week
in J- per cent, solution of chromic acid, and subsequently in spirit,
should be stained with haematoxylin and mounted in Canada
balsam. An enlarged tonsil which has been removed from a
child will answer the purpose.

Structure. — A tonsil consists of an elevation of the mucous
membrane presenting upon its surface fifteen orifices leading
into crypts or recesses, in the walls of which are placed nodules
of lymphoid tissue. These nodules are enveloped in a less dense
lymphoid or adenoid tissue which reaches to the mucous surf ace.
The mucous surface is usually covered with squamous epithe-
lium, and may present rudimentary papillte which are then
formed of adenoid tissue. The tonsil is bounded by a fibrous
capsule. Into the crypts open a number of ducts of mucous
glands,

CESOPHAGUS.
Soaall pieces of the oesophagus (both of the upper and lower
parts) of a dog should be hardened in chromic acid and spirit.

7—2

100 PRACTICAL HISTOLOGY.

Structure. — Of three coats : (1) An external or muscular coat
consists of two layers, longitudinal and circular, the former or
external layer at the commencement being disposed in three
fasciculi, one in front and one on each side. At the upper end
of the cesophagus, the muscular coat is red, and consists of
striated muscle ; lower down it becomes paler, and the fibres are
mostly unstriated.

(2) A submucous coat consists of areolar tissue, and contains
mucous glands (which are best seen in sections from the lower
part) and whose ducts pass inwards to open on the mucous mem-
brane.

(3) A mucous coat which is firm and wrinkled, provided with
minute papillae, and covered with a thick layer of stratified
epithelium. It is separated from the submucous coat by a layer
of unstriated muscular fibres, longitudinally arranged, which is
partially imperfect as a layer above, but complete below (jnuscu.
laris mucosa;).

The artcrifs situated in the submucous tissue give off branches
which form a network of capillaries in the upper part of the
mucous coat ; from this network loops are given off to supply
the papillae.

STOMACH.

The stomach of a cat or dog maybe used. After removal
from the recently killed animal, the organ should be turned
inside out, and washed with a gentle stream of weak bichromate
of potash or spirit. Pieces of the mucous membrane from
different parts should be snipped off with a sharp pair of scissors,
and placed in strong alcohol. Pieces of the whole thickness of
the viscus should be cut oft" ; these may be haulened in weak
chromic acid, or in chromic acid and spirit mixture.

Seciions must be cut both vertical and parallel to the surface
at different depths.

To demonstrate the structure of the glands of the mucous
membrane, some sections from each pait shcuJd be stained
in Icgwocd, and olbeis in ar.iline Hue '5 per cent, solution, as the
aniline will slain tie } eptic cells very cltti)]T, acd so differ-
entiate them fiom the cubical central cells. Ihis is especially

PKACTICAL HISTOLOGY. 101

evident in borizontul i^eciiuns of the peptic glands. The
aniline tinted specimens must be passed through slightly acidu-
lated water, as asual in aniline staining, before they are placed
in sjiirit.

Structure. — The stomach is made np of four coats :
(1) The mucous OT internal coat is smooth, soft, pulpy, and pink
in colour, becoming grey soon after death. Thickest at pylorus,
thinnest at the great curve. Loosely connected with the mus-
cular coat by means of the submucous tissue, and so presenting
temporary ridges (rugae) when the organ is contracted. It con-
sists almost entirely of small tubular glands, aiTanged close to
and parallel with each other, varjin^ in diameter from -J,-, to
.j^Q of an inch, and in length from Jj to .\j of an inch, lined to
a variable extent by columnar epithelium, which also covers the
whole of the mucous membrane.

The tubular glands are for the most part simple, except near
the pylorus, where they become larger, longer, and branched.
The orlands consist of a basement membrane formed of branched
stellate cells joined edge to edge, and sending processes on the
one hand to join the retiform tissue of the mucous membrane,
and on the other to support the gland-cells. The glands are
of two kinds, differing chiefly in the character of the cells and
of their secretion. The one, the so-called * /u ucous or pyloric
glands' are often branched, are confined to the pylorus, and
are lined throughout by columnar epithelium ; but towards the
• fundus,' or closed extremity of the gland, the cells tend to
become cubical. The other, the peptic glands, are distributed
throughout the whole of the mucous membrane, except at the
pylorus, butare most typical perhaps towards the cardiacend; they
are less often branched, but two glands generally open into one
duct, which occupies a third of the whole length of the gland.
The lower end, or fundus, is somewhat dilated, and sometimes
slightly curved. The duct is lined with columnar epithelium ;
its middle third contains two distinct kinds of cells : outside,
large granular cells with small nuclei, bulging out the basement
membrane, and making irregular the outline of the tubes, called
the pe^Jtic or parietal cells;- and inside, a layer of smaller finely

* It may be as well to remind the student that the so called ' peptic
cells ■ are no longer thought to secrete the pepsin.

102 PRACTICAL HISTOLOGY.

granular cubical cellj;, central cells^ which bound a small lun:en.
In the lower third, or fundus, the parietal cells do not form a
continuous layer, but occur here and there irregularly ; the
remainder of the tube is jBlled with cubical central cells, which
leave a very small lumen unoccupied. The cubical cells closely
resemble those lining the fundus of the pyloric glands.

Between and beneath the glands is a quantity of delicate
connective-tissue, forming the mucous membrane proper, which
here and there is collected into small masses somewhat re-
sembling the solitary follicles of the intestine. A double layer
(circular and longitudinal) of unstriated muscles (jnuscularis
mucosce) separates the mucous membrane from the submucous
coat.

(2) The submucous coat consists of areolar tissue with some
fat, together with blood-vessels and lymphatics ; small nerve
ganglia and fibres are also found in it.

(3) The muscular coat consists of three layers of unstriated
fibres, externally of longitudinal, then of circular, and internally
of oblique fibres : the circular layer is the only complete one.
Between the layers may be found j^I^^x uses of nerves.

(4) The serous coat is the peritoneal covering of the organ.
The arteries, after penetrating the muscularis mucosae, break

up into capillaries in the mucous membrane, which form a more
or less elongated meshwork around the glands. Near the surface
the meshwork is very dense, and forms a well-marked superficial
layer beneath the epithelium.

SMALL INTESTINE.

To study the epithelium in the fresh state, a scraping from the
mucous membrane of the intestine of a recently killed animal
may be teased and mounted in saline solution.

For the purpose of studying the relations of the various
structures in the mucous membrane — e.[/., villi, Brunner's and
Liebeikiihn's glands, Beyer's patches, etc. — small pieces from
each part of the intestine of the cat, dog, or rabbit should be
hardened in weak chromic acid or chromic acid and spirit mix-
ture, and cut into sections in various directions.

To demonstrate the large lymphatic sinus surrounding the

rilACTICAL HIST0L0(.V. IO.j

follicles com])risin;^' a Poycr's patch, the ileum is used, and the
sinus is injected wiih lierlia blue by the puncture metho«1,
whilst •"> per cent, solution of silver nitrate will demonstrate
the endothelial lining of the vessel.

To demonstrate the absorption of fat by the villi, a part of
the intestine of an animal recently fed on fatty food should Le
ligatured and placed in Miiller's fluid ; in about a week's tirr.c
pieces may be placed in osraic acid for twenty-four horn's, and
should afterwards be replaced in the solution.

For preparation of Mcissner's plexus and the ganglia of Auei-
bach, see p. 88.

Structure. — There are four coats: (1) The mucous coal
posse sst's :

(a) Vdlcuhc connivenfes, which are large, naked-eye permanent
folds or crescentic projections running transversely to the axis
of the inte.>tine, and containing the submucous coat. They first
appear in the duodenum, not far from the pylorus ; are largest
in the duodenum and upper half of the jejunum, and then
gradually become smaller, until they disappear about the middle
of the ileum.

(b) Villi are small processes, closely set on every part of the
small intestine, over the valvulae conniventes, as well as between
them. They are conical and flattened in form, sometimes cylin-
drical, or with the free end clubbed. Largest in the duodenum
and jejunum, in length varying from one-fourth to one-third of
a line ; smaller, shorter, and fewer in ileum. They consist of
projections of the mucous membrane, being covered with
columnar epithelium, enclosing blood-vessels, lymphatics, and
muscularis mucosae, bound together by fine retiform tissue, which
also forms the basement membrane and encloses numerous
lymphial corpuscles.

(c) Crypts of Lieherkuhn are very numerous small tubular
glands, existing everywhere in the small intestine ; they are
lined with columnar epithelium.

{d) Branners glands are smaller compound tabular glands
found in the first half of the duodenum ; Ij'ing in the submucoun
coat, their ducts pass through the mucous coat.

(«) l\'!ipvs patches or glands, or lymphatic follicular glini?

104 PRACTICAL HISTOLOGY.

occur either solitary or collected (' agminated') into oblong
patches. When solitary, they are found everywhere in the
small intestine, both between and upon the valvulae conni-
ventes ; when agminated to form Peyer's patches they occur in
the ileum, especially at its lower part, lying in the long axis of
the intestine opposite the attachment of the mesentery.

(2) The submucous coat resembles that of the stomach, as
does also

(3) The muscular coat, but this has no oblique fibres.

(4) The serous coat of the duodenum is partially incomplete.
The arteries passing through the musciilaris mucosae give off

numerous capillaries which form a network around the crypts
of Lieberkuhn ; the artery which passes into the villus generally
ascends to the apex, and then breaks up into a dense plexus of
capillaries, which spreads over the apex and base. The capil-
laries are alw^ays situated in the periphery next the epithelium.
There are generally one or two veins developed from the capil-
laries of the villus. The JTjmpliatlc of each villus consists of a
single central vessel, or of two such vessels anastomosing with
each other.

LARGE INTESTINE.

The large intestine is similar in structure to the small intes-
tine, but wdth the following differences :

(1) Hie mucous coat has neither villi, Brunner's glands, nor
valvulse conniventes, and its crypts of Lieberkuhn are longer,
more numerous, and are placed more closely together. The
lymphoid follicles are always solitary.

(2) 2Iu.<:culur coat — in the colon and caecum the longitudinal
layer is collected into three fiat bands, which causes the intestinal
wall to be puckered into ' sacculi.'

(3) The serous coat of the colon and upper part of rectum is
developed into small projections containing fat (appendices epi-
ploicce). It is incomplete in some parts.

THE PANCREAS.

The pancreas of a recently-fed dog should be taken, cut
into pieces about the size of a small hazel-nut, and placed at

PRACTICAL IIISTOLOCV. 105

once in abi-olute alcohol to harden. For the hake of con.-
parison, another pancreas from a fastinj,' dog should he
treated in a similar manner. Sections should be stained and
prepared in the usual way. Unless hardened in alcohol, the
inland is very likely to become useless (from self-digestion ?) for
microscopic pui[)oses.

Structure. — The capsule and septa, as well as the blood-
vessels and lymphatics, are arranged as in the salivary glands ;
the gland is, however, looser and softer, and the lubes and lobules
are less compactly arranged.

The larger ducU possess a very distinct lumen, and a mem-
brana propria lined with columnar epithelium cells which are
longitudinally striated, but are shorter than those found in the
ducts of the salivary glands.

In the smaller ducU the epithelium is short and the lumen is
smaller.

The intermediary duels opening into the alveoli possess a dis-
tinct lumen, with a membrana propiia lined with a single layer
of flattened elongated cells.

The alveoli are branched and convoluted tubes, with a mem-
brana propria and a single layer of columnar cells. The cells
consist of an outer part nearest the membrana propria, which
is homogeneous, and stains the more deeply ; and an inner, more
granular, and less readily stained portion. The alveoli do not
contain the semilunes of Heidenhain and have no distinct lumina,
their places being occupied by fusiform or branched cells.

THE LIVER.

Small portions of the fresh liver of a pig, rabbit, or puppy
should be steeped for four or five days in a 2 per cent,
solution of potassium bichromate, and then for one or two days
in methylated spirit. Sections should afterwards be cut and
treated as usual.

It is well, also, to mount sections of a liver which has been
injected through the portal vein with 2 per cent, solution of
Berlin blue, and then hardened in spirit, and also, if possible, of
liver which has been injected through (a) the bile-duct, under
very low pressure, and {b) ihe hei»atic artery.

106 PRACTICAL HISTOLOGY.

Structure. — It has a serous and fibrous coat. The former is
absent from the posterior border, and from the portal fissure,
where the latter, which elsewhere is thin, is most developed. A
strong sheath of areolar tissue (' Glissons capsule ') surrounds
the vessels of the organ as the}- ramify in it, and, at the trans-
verse fissure, becomes continuous with its fibrous coat.

The liver substance proper consists of lobules, which are closely
packed polyhedral masses more or less distinct, arranged around
the sides of the bianches {sullobular) of the hepatic veins, and
connected to them by minute veins which begin in the centre of
the lobules {intralobular ve'in^).

Each lobule consists of a mass of compressed spheroidal or
polyhedral nucleated and nucleolated cells, measuring from
Yo^jj^th to siuth of an inch in diameter, often containing oil-
globules. Surrounding the lobules is a variable amount of fine
connective-tissue, in which is contained a minute branch {inter-
lobular) of the portal vein, a branch of the hepatic artery and of
the hepatic ducc, together with minute lymphatic vessels cover-
ing them. The lobules are distinct when the interlobular tissue
forms complete septa around them ; if the septa are incomplete,
as is usually the case in man. the lobules become confluent.
Fine fibrous tissue surrounds the interlobular vein, and a delicate
supporting network of flattened, branched corpuscles exists
within the lobule between the cells and the blood-capillaries.
Between the columns of the cells run the radicles of the hepatic
vein which open into the intralobular vein, and between the
cells begin the radicles of the hepatic duct. "Whether these
radicles of the bile capillaries have a definite membrana propria is
undetermined. The interlobular bile-ducts are endothelial tubes
with a large lumen, lined with columnar epithelium. The
larger ducts are surrounded with circular unstriped muscle-cells,
and have a distinct mucous membrane of loose connective-
tissue lined with columnar epithelium, and containing mucous
tubular glands. The lymphatics of the lobule originate in the
spaces around the capillaries of the lobules. The branches of
the hepatic artery run between the lobules with the interlobular
veins ; in parts they surround the veins as a plexus ; the arterial
branches frequently anastomose with each other, and give off

PRACTICAL HISTOLOGY. 107

capillaries to supply tlie Funoiinding connectivc-tit:suu and
vessels, the bile-ducts receiving numerous branches. The ulti-
mate capillaries enter the lobules, where they form a plexus.
The blood from the artery is carried away by a special set of
veins which open into the interlobular veins ; none of it passes
into the intralobular vtin«.

The structure of the Gall Bladder is similar to that of the
large hepatic ducts, but the mucous memhraiie is thicker, and is
thrown into folds and villous projections. The muscular coat
also is thicker, and is surrounded bv conneciive-ihsue and an
outer layer of iieyifoneuiii.

CHAPTER IX

RESPIRATORY TRACT.

E PIGLOTTIS.

The hnman epiglottis, it moved as soon asfossible after death,
should he u£ed. It should be cut into small pieces, and placed
in the chrcmic acid and spirit mixture until it is sufiEciently
haidened. Tiansverse and veitical sections should he Tvell
stained in haimatoxylin, and mounted in the ordinary way.

Structure. — The epiglottis consists of a sufforticg cartilage
of the elastic variety, enclosed in a fibrous perichondrium, and
covered on both sides with mucous membrane.

The anterior surface, i.e., the one towards the tongue, is covered
by mucous membiane which haidly differs from that of the
pharynx. This membrane consists of £bious tissue, elevated
towards the suiface, in the foim of ludimentaiy papillae, and
covered with several la^ ers of squamous epithelium. In it ramify
the capillary blood-vessels, and in its meshes are a large number
cf l^mfhatic charnels. Under the mucous membiane in the kss
dense fibrcus tissue, or submucosa, are a number of tubular
mucous glands.

The podfricr cr laiyrigcal tviface is covered by a muccus
membiane which is similar in stiuctuie to the above, but the
epithelial coat is thinner, the strata of cells beirg less numerous.
The papillae are fewer and less distinct. The proper substance
of the mucous membiane appears to be in great part adenoid
tissue, which here and there is collected into distinct masses.
The glands cf the pcsteiior outface are smaller but mcie numer-
ous than those on the anterior surface. In many places the

rR-\.CTICAL HISTOLOGY. 109

glands which are situated nearest to the psrichondriuin are
directly continuous throu;|hap3rturos in the cartilage with those
on the other side, and not unfrequently the ducts of the glands
from one sida of the cartilage piss through and open upon the
mucous surface of the other. Occasionally the epithelium of the
posterior surface is columnar.

LARYNX.

The structure of the larynx closely resembles that of the epi-
glottis on the one hand, ani that of the trachea on the other.

The framework is hyaline cartilage enclosed in a fibrous sheath,
and covered entirely by a mucous membrane. The epithelium
covering this membrane is columnar ciliated, of which some of
the cells are goblet-cclh, except over the upper part of the false
vocal cords, the arytenoid cartilage =!, the true vocal cords and
imn3diately below, where there are stratified squamous cells.
There is a distinct basement membrane under the epithelium.

The mucosa is a dense fibrous tissue containinor a laro-e
quantity of adenoid tissue ; it is here and there separated f i'ooi
the submucosa, in which lie the glands, by a thin layer of elastic
fibres.

The submucosa is scanty near the true vocal cords, and con-
tains no glands ; elsewhere it is distinct, and in it, as is generally
the case with mucous membranes, the large vessels and nerves
split up for the supply of the superficial structures. Taste-
goblets are found in the epithelium on the posterior surface of
the epiglottis, in^that covering the ary-epiglottidean folds, and
the inner surface of the arytenoid cartilage, and also on the true
vocal cords.

TRACHEA AND LUNG.

Distend the lungs of a recently-killed rabbit or cat, through
the trachea, with ^ per cent, solution of chromic acid, tie up the
trachea, and immerse it in a large quantity of chromic acid of
similar strength. Change the solution for one of a :| per cent,
after two days ; in a week cut in pieces, and remove to methylated
spirit.

110 PRACTICAL HISTOLOGY.

Before catting sections it is necessai-y that the embedding
mass shall have thoroughly penetrated into and filled up the
interstices of the tissues, and so it is best to place the piece of
lung to be embedded in the wax mass -when it is quite hot. In
some cases it is as well to stain the lung, as a whole, with
haematoxylin, and pass it through alcohol and oil of cloves
before embedding. Unless the interstices are filled up, it is
almost impossible to cut thin sections.

To free the cut sections from wax, pass them through oil of
turpentine before putting them into oil of cloves.

A better method is to soak the lung in gum, and then to cut
sections of it by means of the freezing microtome ; after-
wards removing the gum b}' ]>lacing the sections in warm
water.

Thin sections of lung injected through the pulmonary artery
with Berlin blue, and through the trachea with h per cent,
solution of silver nitrate, should be made and treated in the
usuil manner. Sections of trachea should also be prepared.

Structure. — (1) Of the trachea, (a) An elastic frameicorh of
incomplete rings or hoops of hyaline cartilages, 16 to 20 in
number ; each presents a curve of rather more than ^ of a circle.
These rings are held together by a strong fibrous membrane,
more or less elastic, which not only occupies the interval between
them, but is prolonged over their outer and inner surfaces ;
behind, where the cartilage is incomplete, the fibrous membrane
is strengthened by a continuous layer of unstriated muscle,
chiefly arranged transversely. (^) ^ submucous coat of areolar
tissue and fat is also present; it contains immediately beneath the
mucous membrane longitudinal fibres of elastic tissue, which are
for the most part collected into bnndles. Tubular mucous glands
are found in this coat, and also upon and beneath it. (c) A mucous
memhrane containing a large amount of lymphoid tissue ; under
the epithelium is a basement membrane of flattened cells, which
send up processes to the epithelium. In the deeper parts are
many elastic fibres. On the surface are several laj'ers of epithe-
lium, of which the more superficial are columnar and ciliated, often
branched below to join the connective-tissue corpuscles. Be-
tween the branched ends of these cells are smaller elongated

ritACTlCAL ]Il.STOLO(^Y. I I I

cells, prolonged upwards towards tlic .suifaco, and downwards
to the basement membrane. Beneath thc^e are one or more
layers of irregularly shaped cells.

(2) Of the hronchi, as of the trachea.

(3) 0/ liiug. The tissue is made up of lobules attached to the
minute divisions of the air-tubes, by which they are held together,
as wull as by blond-vessels and interlobular tissue. The lobules,
although adherent, are quite distinct ; the structureof each repre-
sents that of the entire lung, and consists of a minute air-tuhe
Avitii terminating air-cella, lined with tcselated ejntheliiim, to-
gether with the pulmonary and bronchial blood-vessels, lympha-
tics, nerves, and areolar tissue. The principal divisions o£ the
bronchi divide, generally dichotomously, into branches running
in all directions, which never anastomose, but terminate sepa-
rately in the lobules. Within the lobules each bronchial tube
finally ends in small recesses (air-cells, alveoli, or vesicles), having
previously lost its cylindrical form, from being beset with similar
air-vesicles on all sides ; in this condition the tube becomes what
is called an iufundihulum. The structure of the air-tubes gradu-
ally changes as they become smaller. The cartilages become
irregularly shaped plates of different sizes, scattered over
the side.] of the tubes, gradually becoming fewer, and finally
disappearing before the infundibulum is reached. The fibrous
coat extends to the smallest tubes, by degrees becoming simply
areolar. The mucous membrane becomes thinner, but retains
its former epithelium, the cells becoming very short columnar
in the smallest bronchi. The longitudinal elastic bundles are
traceable into the smallest tubes. The muscular fibres ultimately
form a continuous circular layer inside the cartilaginous plates.
The walls of the infundibula consist of (a) unstriped muscle
arranged circularly ; (Z/) a network of elastic fibres ; (c) fibrous
tis'Sue and connective-tissue cells ; {d) a dense meshwork of capil-
lary blood-vessels ; (^) small polyhedral cells and large flattened
cell-plates. These plates vary in shape and size according to the
amount of distension of the air-vesicles ; they are best seen in
lungs stained with nitrate of silver. (/) Between the cell-plates
psciulo-Hiomata may be found ; i.e., larger or smaller circular or
angular openings similar to the stomata found in serous mem-

112 PRACTICAL HISTOLOGY.

branes ; they lead into the lymph-canalicular system of the
alveolar wall.

Tlie blood-vessels constitute a dense capillary plexus upon the
alveolar septa : in the contracted lung the capillaries are very
sinuous and close together, whilst in the distended lung they are
straighter and further apart. Near the pleura and bronchi the
capillaries anastomose with the capillaries of the bronchial artery.
The larger arterial and venous branches are situated in the inter-
lobular connective-tissue, which is continuus with their outer
coat. Th^ lymphatics are arranged in three systems, (a) The
subpleural lymphatics, forming a dense plexus whose meshes
mostly correspond with the outlines of the alveoli. (&) The
perivascular lymphatics, whose vessels accompany the branches
of the pulmonary artery and vein, (c) The peribronchial lym-
phatics, remaining in the outer coat of the bronchi, and anasto-
mosing freely with the perivascular lymphatics.

CHAPTER X.

SKIN AND APPENDAGES.

Small pieces of skin from various parts— f.^., from palm of
hand, fingers, or toes, seal]), scrotum, and general surface, should
be hardened in equal parts of chromic acid, h per cent., and of
methylated spirit, for a week, changing the liquid on the second,
fourth, and seventh days, and then removing to spirit until
required. Sections may be made in various directions (cutting
towards the epidermis is the easiest way), stained, prepared, and
mounted in the usual manner.

Double staining, with picrocarmin as well as with logwood, is
recommended.

Injected specimens of skin may be prepared by injecting 2 per
cent. Berlin blue solution into the main artery of a limb of a
dog, or one of the upper extremities of a foetus.

Structure. — The skin consists of two parts :

1. Epuhniiis^ or external sJ/in, which is made up of several
more or less distinct layers, (a) The most superficial horny
layer (stratum corneum) varies in thickness, and is composed of
layers of flattened epithelium, w^hich show nuclei only after
treatment with softening reagents, e.g., caustic potash. (6) The
next layer (stratum lucidum) is generally homogeneous and
thin ; it is composed of closely packed scales, (c) A layer of
granular cells (stratum granulosum), flat, spindle-shaped, and
nucleated, which stain deeply in logwood. (rJ) Finally, the
Malpighian layer (rete Malpighii, or rete mucosum), consisting
of stratified epithelium, the deepest layers of which are columnar,
the next more or less cubical ' ridged ' cells, connected together

114 PRACTICAL HISTOLOGY.

by filaments or })rickles, and most superficially are layers of
flattened cells.

2. Internal, or true shin (corium or cutis vera), is made up of
dense areolar tissue, in which is found, lying deeply, a good deal
of fat ; muscular fibres occur in the neighbourhood of hairs ;
they exist as a distinct layer in the subcutaneous tissue of certain
parts, e.r/., scrotum, penis, areola of the nipple, etc. In the
superficial part of the corium are numerous conical elevations or
papillae, which are received into corresponding pits in the epider-
mis ; they are most developed where sensation is most acute.
The subjacent or reticular part of the corium contains hair
follicles, with sebaceous glands, and sweat glands.

Xerves and blood-vessels are numerous : the former, ending in
the Malpighian layer in a delicate network, and supplying certain
of the papilla, form special endings (end bulbs and tactile cor-
puscles) ; the latter form near the surface a dense netwoik of
capillaries with rounded polygonal mei-hes.

GLANDS.

A. Sweat glands are found distributed throughout the skin
generally, and are exceedingly numerous. Each gland consists of
a long duct which passes through the skin in a more or less wavy
manner, to open on the surface, and a coiled gland proper con-
tained in the subcutaneous tissue. The duct of the gland con-
sists of a narrow tube made up of a basement membrane (homo-
geneous), lined with several layers of small cubical epithelial
cells limited internally Vjv an endothelial membrane, which en-
closes a lumen, generally circular in form. The gland proper is
made up of the coils of the duct, differing in number according
to situation. The coils nearest the duct proper differ little in
structure from the above, but the remainder of the gland (distal
portion) is found to have a single layer of columnar cells lining
it. instead of several lavers of small cells : the internal limitinij
membrane is less distinct, and the merabrana propria, or base-
ment membrane, is strengthened and made thicker by an internal
layer of longitudinal unstriped muscular fibres. The glands in
the neighbourhood of the anus are exceedingly large.

rRACTfCAL HISTOLOGY. 1 1 J

B. Cemminous glands are similar in structure to the sweat-
glands elsewhere, but the glaiul proper is throughout like that of
the distal part of the sweat-gland, as described above.

C. Sebaceous glands, as a rule, open into the neck of hair
follicles. Each gland is composed of a short duct, which
branches into several dilated alveoli, which may each be further
subdivided. The duct is lined with two or three layers of small
nucleated cells, and each alveolus is lined with smaller cubical
nucleated cells. The remainder of the alveolus is filled up with
cells increasing in size towards the centre, and filled with fat.
These central cells have been produced by the division of the
lining cells, and, as they reach the centre of the alveolus, pass
into the duct, lose their nuclei, and, discharging their fatty con-
tents externally, shrivel up, and are discharged in the sebaceous
secretion.

HAIR.

Hairs may be scan in sections of the skin or scalp, especially well
in doubled-stained sections which have been placed in picrocarmin
and hfematoxylin. Single hairs may be examined in any reagent,
but best in caustic potash. Transverse sections are made in the
ordinary operation of shaving, and may be examined in saline
solution.

Structure. — The free extremities of hairs above the skin are
pointed, the attached extremities are received into follicles in
the corium ; between ihe extremities is the shaft. The follicular
€nd is bulbous, and cased in a compound sheath.

.1 hair it.^elf is made up of (a) an external covering of thi i
€ca'e3 (cuticle) ; {b) a cortical substance made of coloured horny
matter ; and finally (r) the medulla or pith, which is absent in
some hairs.

The hull) of the hair rests upon and overlies an elevation of the
follicle (papilla), which is composed of undeveloped nucleated
connective-tissue corpuscles and a few fibres.

The sheath of the hair is divided into («) internal, of two
layers of large cells, the external layer consisting of transparent
oval cells without nuclei, and the other ^ajer of polyhedral

8—2

116 PRACTICAL HISTOLOGY.

nucleated cells ; (h) external, of a variable number of layers of
cells, becoming more columnar externally.

The hair follicle consists of an involution of the cutis vera,
forming three layers : (u) external is very thin, made up of
longitudinally arranged connective-tissue bundles, with fusiform
nuclei and elastic fibres ; (b) middle is thicker, and made up of
transverse undeveloped fibrous tissue, -with rod-shaped nuclei ;
(c) internal, of a thin, striated, transparent membrane of endo-
thelial cells. To the outside of the follicles thin bundles of un-
striped muscular fibre are attached, and into the follicle open
the ducts of sebaceous (simple tubular) glands, generally one
on each side.

NAIL.

Sections of a finger-nail in situ and of the subjacent bed or
matrix should be made and double-stained.

Structure. — A nail is composed of flattened epithelial scales,
and is equivalent to the superficial or horny layer of the epider-
mis. The deeper layers of the nail are softer than the more
superficial. Underneath the nail are highly vascular papillae
which form the bed or matrix. Posteriorly it is received into
a groove in the skin (root). The growth of the nail is effected
by constant additions of cells to the root and under surface, so.
that it grows in length and in thickness at the same time.

CHAPTER XT.
GENITOURINARY ORGANS.

(A) KIDNEY.

Tin: kidney should be hardened in the same way as the liver.
Sections should be made in various directions. Sections of an
injected kidney should also be prepared. The best injecting
material is either carmine-gelatine or Berlin blue.

Structure.— There is a distinct fibro-areolar coat, or capsule,
thin, firm, smooth, and easily detached. The proper substance
of the organ is divided into three regions, the cortical region^ the
boundari/ Icujer, and tlie pap'dlaru region.

On a vertical section the cortex is that lighter part nearest
the capsule, whilst the redder portion is the medulla, which is
seen to be made up of a number of pyramidal portions, each
papilla converging to the interior and towards branches (calices)
of the dilated portion of the main duct or 2Jelvis of the kidney.
Each calyx incloses two or three papillae. The part of the base
of the pyramid towards the cortex, between it and the papillary
portion, is called, as above mentioned, the houndary layer. The
pyramid itself is called the pyramid of Malpighi. The papillary
])ortion appears distinctly and vertically striated in consequence
of the vertical direction of both tubules and blood-vessels, of
which the kidney is principally made up. The boundary layer
is also striated for a similar reason ; but the cortex, although
containing vertical columns, from the arrangement of some of
the tubules (medullary rays), no longer contains the blood-vessels
arranged in vertical directions, nor are all the tubules straight.

118 PRACTICAL HISTOLOGY.

l.'ut convoluted, forming the lahyrinfli. From the medullary rays
diminishing in thickness from the boundary layer outwards
towards the capsule, each presents a triangle, with its base at
the boundary layer. These triangles are called the [lyraiuids of
Ferrcrin. The cortical substance separates the pyramids from
each other, and inclcses them everywhere except at the papillse ;
one layer of it. situated immediately beneath the capsule, forms
the most superficial part of the organ. The portion of the
cortical substance intervening between two pyramids is known
as the col u inns of Bertini.

The papillae are studded with minute openings leading into
tubes {tuhul'i ui'tnifen). through which the urine passes out into
a primary division {irfundihuluin) of the pelvis, or dilated part
of the duct ("refer) of the kidney.

The tubes of the pyramids, as they pass up, divide again and
again at very acute angles, until they arrive at the cortical layer,
and then become convoluted. Each tube begins in a spherical
dilatation [MaljyKjliian capnde)^ enclosing a tuft of minute vessels
{MalplgJiian tuft). Arising thus in the cortex, a tube is at first
convoluted, and consists of a basement membrane lined and
almost filled with granular epithelium ; afterwards becoming
smaller, it passes straight down the pyramid towards the papilla,
and returns again, forming a looped tuhe of Ilenh lined with
squamous epithelium, then again becomes convoluted, and finally
joins a branch of a straight tube of the pyramid (collecting/ tuhe).
The collecting tubes are lined with columnar epithelium, and,
joining together, form the excretory tubes or ducts of Bellini,
which open at the papilla.

A renal tube is said to be made up of the following sections :

(1) The JIalpighian capsule^ lined with squamous epithelium.

(2) Tlie nccl:^ a constricted portion joining the capsule, and
lined in the same way.

(3) A portion enlarged and convoluted, called the pro:dmcd
convoluted tuhe, lined with polyhedral or short columnar cells,
with a lumen of about one-third of the diameter of the tube.
The cells are vertically striated.

(4) The spired tuhe, which passes downwards, the structure of
which is similar to the last described section.

PRACTICAL IIISTOLOUY. 110

(5) The constricted portion, called the dtscendinr/ limb of the
loop of Heule, lined with squamous epithelium.

(G) T/n" loop of Ileal €, lined with squamous epithelium,

(7) The ascenling limb, which becomes rather suddenly en-
larged, lined with striated epithelium.

(>^) The s^nral portion of ascending loop is again somewhat
constricted.

(9) The ascending loop again becomes narrower, but is
straight.

(10) The irregxlar t abide has a very irregular and angular
outline, sometimes beiiig three or four limes as thick as at others ;
this is due to the irregularity in the size of the contained epi-
thelium. The cells are striated, angular and imbricated.

(11) The intercalated section (Schweigger-Seidel). or the distal
convoluted tube, is similar in structure to the proximal convo-
Inted tube.

(12) and (13) Curved collecting tubes are thin tube?, lined
with polyhedral cells, or spindle-shai)ed and flattened.

(14) The straight collecting tube, which pa ses into the
boundary layer, and enters —

(15) The large collecting tube, or tube of Bellini.

(16) The tube of Bellini, having anastomosed with similar
tubes, forms the main tub.^ of the pyramid which opens into
the calyx of the pelvis, with a ^ mouth' at the apex of the
papilla.

Blood-veiSels. — The blood-supply of the kidney is furnished
by the renal artery, which divides into branches which lie
between the cortex and the boundary region ; smaller vessels
pass up and enter the cortex, and pass down to supply the
medulla. The vessels of the cortex pass up in the labyrinth
between the medullary rays (interlobular) and give off trans-
verse branches, the afferent vessels of the Malpighian tufts ;
these break up within the capsule into convoluted capillaries,
re-uniting into the efferent veins; these again break up into
capillaries around the convoluted tubes, to be afterwards collected
into small branches of the renal vein. The vessels of the
medulla break up in the boundary region, and send off straight
vessels between the tubes of the papillary region {vam recta) ;

120 PRACTICAL HISTOLOGY.

the vessels decrease in number towards the papilla, as most of
them break up into capillaries around the tubules, which
capillaries anastomose near the cortex with those of that region.
The veins of the papillary region begin simply in the papilla,
increasing in size and number as they pass upwards ; join
with the veins of the cortex to form the main branches
of the renal vein which accompany the main branches of the
renal artery, and lie between the cortex and medulla, as above-
mentioned.

A certain amount of interstitial connective tissue is found
supporting the tubes and blood-vessels.

(B) URETER.

To prepare the ureter for section-cutting tie one end of it,
and distend it with chromic acid and spirit ; leave it for
one day in the same mixture ; then cut it into short lengths,
and remove to spirit for a week. The cells may be shown
by hardening a piece of ureter in bichromate of potash 1 per cent.
solution, staining deeply in logwood, and scraping the inside,
teasing and mounting in glycerine.

Structure. — Consists of three coats : (1) An sterna] Jihrous ;
(2) a middle of two layers (circular and longitudinal) of un-
striped muscular jibres; (3) an internal or 'mucous, lined by
stratified epithelium, the upper cubical cells of which have their
under surfaces hollowed out to receive the second layer of pear-
shaped cells.

(C) BLADDER.

The bladder should be prepared in the same way as the ureter.
Double staining with eosin and haematoxylin brings into view
the differences in the foim of the lining cells.

Structure. — Consists of four coats : (1) An external or serous
— is incomplete, as it is only found at the u]iper and posterior
parts. (2) .4 muscular, consisting of three layers more or less
complete — viz., (a) external longitudinal, (6) circular, (c) internal
longitudinal. (3) A submucous of connective-tissue. (4) .4
mucous, lined with stratified epithelium, the upper layer being

PRACTICAL HISTOLOGY. 121

made up of polyhedral cells, with one, two, or three nuclei,
presenting depressions with intervening ridges for the second
layer of club-shaped cells ; the next layer is made up of more
fusiform cells.

(D) PROSTATE.

The prostate should be immersed in a ^ per cent, chromic acid
for two days, and should then be removed to spirit.

Structure.— In structure the prostate consists of small glands
imbedded in an abundance of muscular fibres and connective-
tissue.

The glands consist of numerous small saccules, opening
into elongated ducts, which unite into a smaller number of
excretory ducts. The acim\ in the upper part of the gland,
are small and hemispherical : whilst in the middle and lower
parts the tubes are longer and more convoluted. The acini are
of two kinds, {a) lined with a single layer of thin and long
columnar cells, each with an oval nucleus in outer part of wall ;
(h) acini resemblincj the foregoing, but with a second layer of
small cortical, polyhedral, or fusiform cells between the mem-
brana propria and the columnar cells. The ducts are lined by a
laver of columnar cells, beneath which is a layer of small poly-
hedral cells.

The tunica adventitia is formed of loose connective-tissue
containing fat.

Large hlood-ye$sd.< pass into the interior of the organ to form
a broad, meshed, capillary system. Xerve-trxnls and numerous
large ganglion cells surround the cortex. Paccinian bodies are
also found in the substance of the prostate.

(E) VAS DEFERENS.

The vas deferens should be prepared by hardening in a 2 per
cent, bichromate of potash solution for fourteen days, after
which it should be placed in spirit.

Structure. — Like the vesiculae seminales (see p. 123) it con-
sists of three coats : (1) An external, of connective-tissue, outside
which longitudinal fibres of unstriated muscles are often seen.

(2) A muscular, two longitudinal layers with an intermediate
circular one.

122 PRACTICAL HISTOLOGY.

(3) A iiiucous, of connective-tissue and elastic fibres ; this
layer is often thrown into three or four longitudinal ridges ; the
epithelium consists of columnar epithelium, ciliated only near
the epididymis.

The nervps form a plexus in the tunica adventitia.

(F) TESTICLE.

Place the testicles, preferably of rat or cat, after making two
or three cuts in them, in equal parts of chromic acid ^ per cent,
and methylated spirit. Change three times in a week, and
remove to spirit, or inject a 1 per cent, solution osmic acid into
the tunica albuginea, then place in strong spirit for several days--,
and afterwards for two days in absolute alcohol previous to
making sections. Stain with hematoxylin or carmine ; prepare
and mount as usual.

Structure. — The outer coat consists of connective-tissue, the
tunica alhufjinea., from which radiate incomjjlete septa uniting
into a thick wedge-shaped body, the corpus Highmori.

The testicle is divided by these septa into lobes, each consist-
ing of small and convoluted tubes, the iuhul'i. seminiferi. These
tubes are composed of a basement membrane of flattened endo-
thelial cells, a single roAV in small animals, more than one in large
animals, within which are a number of cells not arranged in any
definite order — the seminal cells. The outer form a single row.
The tubuli seminiferi have a uniform diameter of jIq to ^J^
inch ; they commence in free closed extremities or in anasto-
mosing arches, and unite to form the vasa recta. In transverse
section the seminal tubules are seen to have a narrow lumen
surrounded by polygonal cells, of which the peripheral ones are
arranged radially. In the interstitial connective-tissue between
the tubuli seminiferi are a number of connective-tissue
corpuscles. From the seminal cells the spermatozoa are
developed.

The ras(( recta^ about twenty in number, are r}^ to ^^j inch in
diameter. They possess very thin walls, and pass upwards and
backwards to terminate in the rete vasculosum testis.

The retc testis is lined with pavement epithelium, and

PRACTICAL HISTOLOGY. 123

opens into twelve to twenty vasa efferantiii, forming the
coni vasculosi.

The roni vaxcuhm arc J„ inch in diameter, and open into the
canal at the epididymis.

The epididymis luul vasa efferentia contain plain muscular
fibres; the lining cells arc columnar and ciliated, elongated in
the epididymis, shorter in the vasa efferentia.

Remove and draw i^permatozoa from the fresh glands, and
notice (a) the head, (/>) the middle portion, (e) the caudal ex-
tremity.

The hi ood -vessels %\xrvo\xnA the convoluted tubules with a long-
meshed wide capillary plexus.

The l^mph passages form an extensive canalicular system.

(G) VESICULiE SEMINALES.

The vesicula) seminales may be prepared either in ^ per cent,
chromic acid for seven days, followed by spirit, or by hardening
in methylated spirit.

Structure. — (1) There is an external connective-tissue coat.

(2) A middle muscular coat of three layers, the internal of
longitudinal fibres, middle of circular fibres, external of longi-
tudinal fibres. (3) A mucous coat thrown into rugae, the epi-
thelium of cylindrical cells provided with striated borders, the
deep layer being polyhedral. The mucous membrane contains
a few muscular fibres.

GcuujVion cells and nerve plexuses are numerous in the outer
coat. According to Leydig, a number of racemose fjlands are
present.

(H) THE PENIS.

The penis of a human foetus should be used, if it can be
obtained, otherwise that of the cat or dog. Itshoidd be injected
from the abdominal aorta, after ligature of the external iliac
arteries, and should then be hardened in \ per cent, chromic
acid for a fortnight. Sections should be made in various
parts.

Structure. — {a) Tlte urethra is lined by stratified pavement
epithelium in the lower part of the prostatic and membranous

124 PRACTICAL HISTOLOGY.

portions ; in the upper half the epithelium is of the stratified
transitional variety ; in front of the bulb the epithelium becomes
columnar, whilst the fossa navicularis is again lined with stratified
pavement epithelium. The mucous membrane consists chiefly of
fibrous connective-tissue, intermixed with which are many elastic
fibres. It is surrounded by muscular tissue of the unstriped
variety. In the membranous portion many large veins run
amongst the bundles of muscular tissue. Many mucous glands
are present.

(h) The corpora cavernosa consists of a matrix, chiefly of un-
striped muscle-fibres, intermixed with which is a little con-
nective-tissue and a few elastic fibres. The matrix is arranged
in bundles, and separates the very large voious sinuses which
constitute the greater part of the substance of each cor-
pus cavernosum. The sinuses anastomose with each other
to form plexuses, and each is lined by a single layer of
flattened endothelial plates. The arteries run in the muscular
trabeculse.

(f) The corpus spongiosum urethra; con.'sisi^ oi an inner portion
or plexus of longitudinal veins, and of an outer or really
cavernous portion identical in structure with that which
has just been described. The hjmpliatlcs of the penis are very
numerous.

The nerves form a dense subepithelial plexus.

Covpers glands resemble the sublingual gland ; they are large
compound tubular mucous glands.

(I) OVARY.

The ovaries of a cat or rabbit are placed, with as little hand-
ling as possible, in a mixture of equal parts of spirit, and i per
cent, chromic acid solution for two or three days, and afterwards
in spirit. The sections should be stained with hsematoxylin or
carmine.

Structure. — The ovary consists of an encapsuled stroma and
embedded Graafian follicles.

The outer coat or capsule consists of low columnar epithelium
cells, beneath which is a firm layer of fibrous tissue.

The stroma of fibrous tissue and elastic fibres containing blood-

PRACTICAL HISTOLOGY. 125

vessels, and in the deci)er portion muscular fibres. The cor-
tical portion contains a large number of closely-set vesicles,
yJ^ inch in diameter. Each vesicle, or primordial ovum, is sur-
rounded by a corona of small nucleated cells. Below this layer
of vesicles are more advanced ova, the deepest being the most
mature.

The Graafian follicle, t?() to ^ inch in diameter, contains a ripe
ovum, and is surrounded by fibrous tissue, and by the tunica
vasculosa, more internally by the tunica granulosa, consisting of
several layers of granular prismatic cells. In a thickened
portion of the tunica granulosa (discus proligerus), the ovum is
embedded on the inner surface and to one side of the Graafian
follicle. The tunica granulosa is separated from the discus pro-
ligerus, except at their point of union, by a space containing a
clear albuminous fluid. The ovum, j^g inch in diameter,
consists ((() of an external, firm, transparent membrane,
which is finely striated radially (vitelline membrane, or zona
pellucida) ; (h) of a mass of granular protoplasm (vitellus,
or yolk) ; (c) of a small clear vesicle, -oty i^^^ i^ diameter
(germinal vesicle), embedded in the vitellus, and which encloses
(d) a dark granular spot (germinal spot, or macula germinativa).
liooo inch.

The corpus luteum is a Graafian follicle which has discharged
its ovum ; it is filled with a reddish-yellow mass of elongated
cells, the colour being due to the formation of pigment, which,
however, is not derived from the slight hsemorrhage which takes
place on the escape of the ovum.

(J) UTERUS.

The uterus of a cat or rabbit should be distended with a mix-
ture of equal parts of \ per cent, chromic acid solution and
strong spirit through the vagina. The openings into the organ
should be tied, and the organ should be removed to a bottle
containing same mixture. The solution should be changed at
the end of twenty-four hours and the uterus laid open,

Stmcture. — The external serous coat is derived from the peri-
toneum.

The muscular coat is intermixed with fibro-areolar tissue

126 PRACTICAL HISTOLOGY.

blood-vessels, lymphatics, and some veins. The muscle is
arranged in three layers : (a) the external longitudinal, tha
weakest coat ; (h) transverse fibres forming the strongest layers ;
((') oblique fibres which become annular to form the sphincter
uteri. The cells constituting the muscular layers are fusiform,
with long tapering extremities ; the nucleus is always single.

The mucous memhrane is smooth in the fundus and body of
the organ ; it is raised into transverse folds in the upper portion
of the cervix ; and forms papillae in the terminal portion of the
cervix. It is lined with columnar ciliated epithelium. The
glands are tubular, often spiral, sometimes slightly branched.
They are found in the fundus and body, and are lined with
ciliated epithelium. Small closed sacs (ovula Nabothi) are also
distributed regularly over the mucous membrane.

The blood-vessels are large and numerous ; the lymphatics
form large plexuses in the peripheral layers of the pregnant
uterus ; the nerves are medullated and non-medullated, a few
ganglion cells being also present.

(K) FALLOPIAN TUBES.

Are prepared in the same way as the uterus.

Structure. — (1) A71 external serous coat, rich in vessels and in
connective-tissue. (2) A longitudinal, and a thicker circular
layer of unstriated muscle. (3) A mucous memhrane thrown
into longitudinal rugce, and lined with columnar ciliated epi-
thelium ; no glands are present, and, as yet, no nerves have been
detected. The mucous membrane contains a layer of muscular
mucosae.

(L) MAMMARY GLANDS.

The gland, cut into small pieces, is placed in a solution of equal
parts of spirit and i per cent, chromic acid solution for two days,
afterwards in weak and strong spirit. It should be stained in
b£ematoxylin. Double staining may also be used.

Structure. — The mammary gland consists of a number of
individual racemose glands united by intervening areolar tissue.

rilACTICAL IIISTOLOUY. 127

The lohes thus formed have a considerable quantity of adipose
tissue between them, whilst the blood-vessels and the small
medullated nerves run in the connective-tissue stroma.

The racemose glands oi)en by means of ducts, tJic Itirti/erona
durts, which unite together until fifteen to twenty excretory
canals are formed,

The galactophorous ducti^, which converge towards the nipple.
Xear the nipple the galactophorous ducts become dilated to
form sinuses, but they undergo constriction again before open-
ing to the exterior.

The gland vesicles consist of a membrana propria with
flattened stellate cells, lined by low columnar ei)ithelium. The
vesicles are filled with fat-globules ; and if the oil be extracted
by immersion of the gland in ether, casein remains behind. The
terminal vesicles are at firet simple, but as the gland develops
they produce buds.

The ducts consist of areolar tissue with a circular and longi-
tudinal layer of elastic fibres ; they are lined with low cylindrical
epithelium, which becomes flattened near the nijjple. Near the
nipple also, and beneath the areola, unstriated muscular fibres
are found.

The blood-vessels form a dense capillary network around the
alveoli, forming a continuous system for each lobule.

CHAPTER XII.
DUCTLESS GLANDS.

THYROID GLAND.

Should be prepared by immersion of the gland for twenty-
four hours in a mixture of spirit and water, then in strong
spirit, till the tissue is suflBciently hard. It may also be
hardened by allowing it to remain for a month in Miiller's
fluid, or in -} per cent, chromic acid for a fortnight. Should
be stained in hsematoxylin.

Structure. — A thin transparent layer of dense areolar tissue,
free from fat, containing elastic fibres. This connective-tissue
frameicorh traverses the interior of the organ in the form of
strong trabeculee ; it incloses rounded or oblong irregular
cavities, the vesicles. The vesicles consist of a thin hyaline
membrane lined by a single row of low cylindrical cells. The
cavities of the vesicles are filled with a coagulable fluid, or more
frequently with a colloidal substance. The colloidal substance
increases with age, and the cavities appear to coalesce.

In the interstitial connective-tissue is a round meshed capil-
Jary pJej:u><, and a large number of lymphatics. The nerves
adhere closely to the vessels.

THYMUS GLAND.

Preparation. — As for the thyroid gland. Some sections of the
fresh gland should be teased in saline solution in order that the
concentric corpuscles may be minutely examined.

rRACTICAL HISTOLOGY. 129

Structure. — A cdjjxule of thin areolar tissue which sends down
processes dividing the gland into lobules. The outer surface of
the organ is covered with a layer of flattened cells.

Each lobe is made up of a number of polyhedral lobules, con
nected by delicate areolar tissue, which are in turn composed of
small /o//<t7<s. The follicles are composed of adenoid tissue or
retiform tissue, the meshes of which are filled up with lymphoid
corpuscles. The follicles are therefore comparable with the
spleen, tonsils, lymphatic glands, and Peyer's patches.

Scattered in the adenoid tissue are the concentric corpuscles
of Hassall, composed of a deeply staining substance, with high
refractive index. Of these there are two kinds — one, the smaller,
simple : the other, the larger, compound. The arteries radiate
from the centre of the gland. The lymphatics are large. The
nerves are very minute, the terminations have not been traced.

PITUITARY BODY.

Should be prepared in the same way as the thjroid.

Structure. — Of two lobes — a small posterior one^ consisting of
grey nerve-tissue ; an anterior larger one, resembling the thyroid
in structure. A canal, lined with flattened or with ciliated epi-
thelium, passes through the anterior lobe ; it is connected with
the iufundibulura. The gland sjjaces are oval, nearly round at
the periphery, si)herical towards the centre of the organ ; they
are filled with granules and nucleated cells. The vesicles are
enclosed by connective-tissue, rich in capillaries.

PINEAL GLAND.

Should be prepared by hardening in alcohol, or by macera-
tion in M liner's fluid ; or, better still, by means of osmic acid.

Structure. — A central cavity lined with ciliated epithelium.
The glandular .substance is divisible into

(rt) An outer cortical layer, analogous in structure to the
jiituitary body ; and

(h) An inner crntral lager, wholly nervous. The cortical layer
consists of a number of closed follicles, containing (a) cells of

9

130 PRACTICAL HISTOLOGY.

variable shape, rounded, elongated, or stellate ; (h) fusiform
cells. There is also present a gritty matter, the acervulus
cerebri, consisting of round particles aggregated into small
masses. The central substance consists of white and grey
matter.

The hlood-vesseJs are small, and form a very delicate capillary
plexus.

SUPRA-RENAL CAPSULE.

May be hardened in bichromate of potash, 2 per cent., for
a fortnight, in Miiller's fluid for a month, or in osmic acid four
hours ; in each case complete the hardening in spirit. The
glands from the guinea-pig, rabbit, and other animals should
be used and compared with the human supra-renal.

Structure. — An outer sheath of connective-tissue sends in pro-
longations, and forms the framework of the gland.

The cortical j^ortion, divided into (a) an external layer of
closed vesicles, the zona glomerulosa. The vesicles contain a
finely granular greyish substance, no fat-globules, but generally
a few small cells. (^) A layer of cells arranged radially, the
zona fasciculata. The substance of this layer is broken up into
cylinders, each of which is surrounded by the connective-tissue
framework. The cylinders thus produced are of three kinds —
one containing an opaque, resistant, highly refracting mass (pro-
bably of a fatty nature) ; frequently a large number of nuclei
are present ; the individual cells can only be made out with
difficulty. The second variety of cylinders is of a brownish
colour, containing finely granular cells, in which are fat-globules.
The third variety consists of grey cylinders, containing a
number of cells whose nuclei are filled with a large number of
fat granules. (<?) The third layer of the cortical portion is the
zona reticularis. This layer is apparently formed by the break-
ing up of the cylinders, the elements being dispersed and isolated.
The cells are finely granular, and have no deposit of fat in
their interior ; but in some specimens fat may be present as
well as certain large yellow granules, which may be called pig-
ment granules.

The medullary suhstavce consists of closed vesicles ; of elements

PRACTICAL HISTOLOGY. 131

of the cortical substance ; of numerous blood-vessels ; and of
an abundance of nervous elements. The cells are poor in fat,
and occasionally branched ; the nerves run throu^'h the cortical
substance, and anastomose over the medullary portion.

Blood-vessels. — The cortical portion is supplied with a rich
plexus of cajjillaries, whose meshes are polyhedral in the outer
and middle zones, and more elongated in the zona fasciculata.
The medulla is supplied with a very rich plexus of wide capil-
laries. In all parts the blood-vessels are embedded in the
trabeculie.

Lijmph spaces and sinuses, best seen between the cells of the
zona fasciculata, but existing in other parts, occupy the inter-
cellular spaces and lacunse : the efferent l3^mphatics provided
with valves lie in the capsule and in the connective-tissue around
the central veins.

9-2

CHAPTER XIII.
LYMP HATICS.

(A) VESSELS.

(1) Trunks. — Preparation. — Make sections of a thoracic duct
which has been hardened in bichromate of potash, and sub-
sequently in spirit, stain with logwood, and mount in Canada
balsam.

Structure. — Lymphatic trunks, such as the thoracic duct and
the lymphatics leading to the mesenteric glands, have nearly the
same structure as veins, and like them consist of three coats.
They are provided Avith valves, especially at their subdivisions.
The endothelial cells lining them are elongated.

(2) Capillaries. — Preparation. — To demonstrate the structure
of lymphatic capillaries, the epithelium covering the central
tendon of the diaphragm of a rabbit or guinea-pig must be
pencilled off with a camel's-hair brush, the tendon should then
be stained with nitrate of silver, and mounted in glycerine.

Structure. — Lymphatic capillaries consist of a single layer of
sinuous endothelial cells, united together by intercellular sub-
stance so as to form a membrane.

(B) GLANDS.

Preparation. — Thin sections of a lymphatic gland which has
been previously hardened in dilute spirit, Miiller's fluid, or in
bichromate of potash, should be stained with logwood, and
shaken in a test-tube half full of water for thirty minutes or
more. They are then to be prepared and mounted in balsam in
the ordinary manner.

rRACTICAL HISTOLOGY. 133

Structure. — Each lymphatic gland is surrounded by a capsule,
which consists of connective-tissue intermingled with unstriped
muscular fibres. From the capsule are given off a number of
trahecuhe, which give support to the blood-vessels, and pass into
the interior of the gland, so as to divide it into a number of
compartments or alveoli, which contain the adenoid tissue or
proper tissue of the gland.

The adenoid tissue is arranged in the form of follicles in the
cortex, and of rounded cords in the medulla. Between the
walls of the alveoli and the proper tissue of the gland are a
number of spaces lined by endothelium — the so-called sinuses of
a lymphatic gland. These sinuses are continuous on the one
hand with the afferent vessels, and on the other with the
efferent vessels.

(C) THE SPLEEN.

Preparation. — Small pieces of fresh spleen are hardened in
2 per cent, solution of bichromate of potash, and subsequently
in spirit, till they are fit for making sections. Injected speci-
mens should also be examined.

Structure. — The spleen possesses two coats, a serous and a
fibrous.

The serous coat is derived from the peritoneum, and covers the
organ almost completely.

The Jihrous coat, or tunica propria, is composed of connective-
tissue, which in some animals is intermingled with a large pro-
portion of unstriped muscular fibres. From its inner surface
processes or trabeculae pass into the interior of the organ,
and interlace freely, so as to form the trabecular franieicork
of the spleen. At the hilum the capsule passes in with the
blood-vessels for which it forms sheaths, which become con-
nected with the trabeculas above described. The interstices
between these trabeculae contain the proper tissue of the
spleen, or spleen pulp. The spleen pulp is composed of an
adenoid reticulum, forming meshes, with endothelial jilates at-
tached ; into these meshes the small arteries pour their blood,
and with these meshes, by their widening out, and by the

134 PRACTICAL HISTOLOGY.

arrangemeut of the endothelial plates into a distinct lining, the
veins are continuous. The spleen pulp contains red blood
corpuscles in all stages of decay and renovation, a large number of
colourless corpuscles and blood pigment. Small arteries pass off
almost at right angles from the branches within the trabeculae,
into the spleen pulp, and exchange their outer coat of con-
nective-tissue for one of adenoid tissue. This adenoid sheath,
which takes the place as it were of the lymphatic vessels
which surround the arteries whilst they lie in the trabeculae,
forms cords of adenoid tissue, with arteries occupying some
position, not necessarily the central. The cords are not every-
where of the same diameter, increasing here, diminishing there ;
and on section appear as more or less circular masses of adenoid
tissue, highly vascular when injected. To these masses seen on
section the term MalpigTiian corpuscles has been applied.

CHAPTER XIV.

THE SPECIAL SENSES.

(A) THE EAR.

Preparation. — Remove the lower jaw from a recently killed
guiuea-pig, iu order to expose the auditory bulla. Carefully
break open the bulla after removing the soft parts from it, and
look for the cochlea. Remove the cochlea by chipping away the
surrounding bone, and immerse it at once in chromic and hydro-
chloric acid mixture until the bone is soft enough to be cut with
ease. Place it then in weak spirit, which should be changed as
often] as it becomes yellow from the picric acid. Finally, put
the cochlea into absolute alcohol for twenty-four hours before
imbedding it. The preparation should be stained in picrocarmin,
dehydrated, cleared and saturated with paraffin, and cut with a
heavy microtome on a glass plate.

Structure. — The cochlea is a gradually tapering spiral tube,
winding round a central column, the modiolas. It is divided
along its whole extent by a spiral lamina, which projects from
the modiolus, into two main portions — the scala tympani and
the scala vestibuli.

The spiral lamina is partially osseous and partially membranous.
The membranous portion, the basilar membrane, is connected
to the outer wall of the cochlea by its union with the spiral
liijamenf, which is a projection inwards of the periosteum and
subperiosteal tissue of the cochlea.

The scala vestibuli is subdivided into scala vestibuli proper,
and du.i-tus cocltUo: or scala metlic, by the membrane of Reissncr,
which passes from the spiral lamina to join the lining periosteum.

The membrane of Reissner is composed of a delicate membrana

13G PRACTICAL HISTOLOGY.

propria, continuous with the periosteum covering the scala
vestibuli. It is lined with a laj-er of flattened endothelium on
the face turned towards the scala vestibuli ; whilst that bound-
ing the ductus cochleae is provided with a single layer of poly-
hedral cells.

The periosteum consists of ordinary connective-tissue, thickened
here and there by retiform tissue.

The spiral Jigament, to which the basilar membrane is attached,
consists of periosteum thickened by the retiform tissue, the cells
being elongated, and radiating from the attachment of the basilar
membrane. At this point there is generally a large blood-
vessel ; whilst between the spiral ligament and the membrane of
Reissner the periosteum contains pigment-cells and a number of
blood-vessels.

The floor of the ductus cocMece is formed of a narrow portion
of the spiral lamina, and of the basilar membrane. This portion
terminates in a border which is C-shaped when seen in section,
the lower limb of the C being prolonged and tapering. This
limb is the end of the osseous lamina ; it is covered by a thin
membrane. The upper portion of the C is the Jimhus of the
spiral lamina, whilst the bay of the C is called the spiral
(jroove.

The limhus has a jagged edge, as it is raised into a number of
tooth-like projections.

The organ of Cortl forms a portion of the epithelium covering
the basilar membrane ; it consists of an outer and inner set of
stiff rod-like bodies. The feet of the rods rest upon the basilar
membrane, whilst they incline towards each other until they
meet at their heads. By the meeting of the rods an arch is
formed over the basilar membrane ; it is filled with endolyinph.
On the inner side of the inner rods, and the outer side of the
outer rods, are epithelial cells with short hair-like prolongations,
the inner and outer hair-cells ; the outer cells are more numerous
and more elongated than the inner cells. The hair-like pro-
longations of the outer hair-cells project through rings which
surround the tops of the cells, and which are bounded by
minute fiddle-shaped cuticular structures— ^//e pihalanges. A
reticular memhrane is thus formed, which covers this part of the

PRACTICAL HISTOLOGY. 137

organ of Corti. Ou either side of the two sets of hair-cells the
epithelium passes continuously into the simple layer of cubical
cells, which is found in the spiral groove, and covering the outer-
most i)art of the basilar membrane. The whole organ of Corti
is also covered by a thick and highly elastic ferlor'ial memhrane.

The inner rods are smaller and more numerous than the outer
rods ; they may be compared to the upper portion of the human
ulna ; whilst the outer rods resemble the head and neck of a
swan. The concavity of the inner rod receives the rounded
portion of the outer rod, which would correspond to the back of
a swan's head ; whilst the beak of the swan becomes connected
to the reticular lamina. Both rods are more slender towards
their middle, and expand again, so as to rest by a widened foot
upon the basilar membrane ; both are longitudinally striated.
In the head of the outer rod — and occasionally, also, in the
inner rod— is an oval nucleus, staining more deeply than the
rest of the cell.

Structure of the loallofthe meinhranous semicircular canaU. — The
wall consists, from without inwards, of (a) an external fibrous
lai/er, containing numerous nuclei, blood-vessels, and irregular
pigment-cells. This layer is especially developed at the ends of
the oval section where it coalesces with the ligamenta labyrinth!
canaliculorum. (/j) The tunica propria^ which present?", after
staining, a delicately striated and granular appearance, {r) Cap'd-
liforni /n'ocesses, which project into the interior of the canal,
except at the part where the membranous canal touches the
bone, (d) The eplthelifon^ a single layer of pavement epithelium-
cells investing the papillje ; it is continued into the depressions
between them.

In the anipulke («) the fibrous hnjcr forms a loose mesh-
work, whilst {b) the tunica propria is so much thickened as to
cause a rounded transverse projection into the cavity — the crista
acustica, or septum transversum. (c) The epithelium^ covering the
crista acustica, consists (i.) of long cylindrical cells, each with a
large nucleus ; these cells support the other nervous and epi-
thelial elements, and rest upon the tunica propria ; (ii.) fusiform
cells which lie between the columnar cells : each cell has a long
stiff cilium, tlie auditory hair, and is in direct connection with

13S PRACTICAL HISTOLOGY.

the ultimate fibrillffi of the auditory uerve. (fl) The nerves,
after passing through the tunica propria, form a very delicate
plexus in the epithelial layer.

(B) THE NOSE.

Preparation.— Small pieces of the upper turbinal bones from
the head of a freshly killed sheep, dog. or rabbit, should be
treated in the chromic acid and hydrochloric acid solution for a
week, or in I per cent, bichromate of potash, or in 1 per cent,
osmic acid, for forty-eight hours. Sections may be made
through the nasal region of a young guinea-pig's head which
has been previously hardened in chromic acid and spirit. The
fresh tissue may also be treated according to the chloride of
gold method.

Structure. — In a vertical section through the septum nasi,
the os.seous portion is seen to be invested by periosteum, which is
immediately covered by a thick layer of elongated tabular
glands, some simple, others more complex— the glands of Bow-
man. These glands have an eitithelial lining of granular
spherical cells at the base ; of a more polygonal and less granular
form near the excretory duct. The ducts open on the surface
between the elements of the external layer. The glands become
less numerous and ultimately disappear at the point where the
olfactory region passes into the ordinary mucous membrane,
being replaced by the mucous glamls. The glands are separated
from each other by ordinary connective-tissue, in the deeper layers
of which are pigment-cells and free pigment masses, as well as
capillaries and ramifications of the olfactory nerves.

The epitheliam, which covers the mucous membrane, consists
of an external finely striated portion and an internal granular
layer. In the newt the epithelium-cells may be separated into
groups, by teasing after maceration for forty-eight hours in
Miiller's fluid. Each group consists of two kinds of cells ; one
kind, which is larger than the other, presents an elongated oval
form, and is situated externally. The smaller of the two kinds,
or olfactory cells, possess a large round nucleus and two very long
fine processes, of which the thicker runs outwards, whilst the
finer is directed inwards. The external process is composed of

PRACTICAL HISTOLOGY.

139

two substances, an outer, which swells up under the iutluonce of
certain reagents, and an internal thread, which remains un-
affected. In man and mammalia generally the olfactory cells
have no cilia. The larger celU are provided with oval nuclei,
and extend through the whole thickness of the epithelial layer.
The external portion of these cells is more or less cylindrical,
and is striated longitudinally. A row of dots can be dis-
tingui^hed upon the e.vternal extremities. The trunks of the
t'l/actori/ nerve run in the glandular layer either obliquely or
horizontally. The ultimate fibrils of the nerve pass into the
epithelial layer and probably into the olfactory cells.
For an account of the Organ of Jacobson, see Appendix.

(C) THE OPTIC APPARATUS.

(Ij The 'Eyelids.— PrejxiraU'on.— The eyelids and the lachrymal
apparatus may be obtained from a pig if human material is not
available. They should be placed in the usual solution of
chromic acid and spirit.

The skin of the eyelids consists of an epidermis of flattened
cells and of a thin corium : the papillae of the latter are small,
and the subcutaneous tissue very loose, containing numerous and
wide lymphatics ; a few groups of fat-cells are present. The
sweat-glands are small, and the hairs fine, with small sebaceous
glands. At the anterior edge of the free margin of the lids the
papillae become larger, and the hairs are converted into eyelashes.
Immediately behind the cilia are the ducts of the glaiuJaof Mohl,
which frequently open into the ducts of the sebaceous glands.
The glands of Mohl closely resemble in structure the sweat-glands.

Xext to the subcutaneous tissue are bundles of striated fibres
of the orbicularis muscle^ separated from each other by loose
connective-tissue, which occasionally contains fat-ceUs.

The tarsal plate is a dense felted mass of fibrous tissue which
does not contain any cartilage : its anterior and posterior
surfaces are intimately connected by bundles of connective-
tissue with the skin of the eyelid and with the conjunctiva.

Near the posterior edge of the free margin are the mouths of
the Meibomian glaiuh. arranged in a single row. The ducts are
embedded in the tarsal plate, and are in direct connection with

140 PRACTICAL HISTOLOGY.

saccular single or branched alveoli ; the glands resemble seba-
ceous glands. At the posterior edge of the lid the stratum
Malpighii becomes modified, and passes into the conjunctiva.
A comparatively thick layer of muscle, the muscuhis c'lliaris
BioJanl^ intervenes between the mouths of the Meibomian glands
and the eyelashes.

The ini'.cosa of tlie conjunctiva paTpehm is a comparatively thick
connective-tissue membrane, which generally contains a variable
amount of diffuse adenoid tissue.

The conjunctiva palpehrcc itself consists of one or twola} ers of
small polyhedral cells, upon which is superposed a layer of longer
or shorter conical or columnar cells, amongst which are frequently
seen some goblet-cells. Small mucous glands are embedded in
the tarsal plate in the neighbourhood of the conjunctiva
palpebral.

{'2) The lachrymal gland is a serous gland ; it is divided into
lobes and lobules by the connective-tissue capsules. The larger
interlobular ducts are lined with a layer of thin columnar epi -
thelium cells.

The Intralobular ducts are also lined with columnar cells,
whose external portion is distinctly fibrillated, whilst the inner
portion is only slightly striated : the nucleus being situated about
the centre of each cell. The intermediate portions of the intra-
lobular ducts, and the parts immediately opening into the alveoli,
consist of fine tubes lined with a layer of flattened cell-plates,
which are often imbricated.

Tlic alveoli are more or less tubular, and are provided with
lateral and terminal tubular or saccular branches.

The memhrana projyi'ia consists of branched flattened homo-
geneous cells, from which septa extend between the cells of the
glandular epithelium.

The glandular epithelial cells form a single la^-.er of polyhedral,
or cubical, granular-looking cells, each provided with a spherical
nucleus. The distribution of blood-vessels is identical with that
of the salivary glands.

(3) Cornea. — The anterior part of a human eye which has been
hardened in '2 per cent, solution of bichromate of potash for a
fortnight, and then in spirit, should be used.

rHACTICAL HISTOLOGV. Ml

To demonstrate the connective-tissue cells and nurvos, the
fresh cornea of a froj^' or rabbit should be cut out, and placed
for about an hour and a half in chloride of gold solution }, per
cent., then in slightly acidulated water, and exposed to the light
for twenty-four hours or more, according to its intensity, and
mounted whole (or in sections in the case of the rabbit's cornea)
in glycerine.

In order to staiti the ground substance, the fresh or living
cornea of a pithed frog should be pencilled with solid nitrate of
silver, well Avashcd in distilled water and mounted in glycerine.

Structure. — The cornea consists of five layers : ( 1 ) The super-
ficial or conjunctival layer is composed of stratified eiiitheJunn^ of
three or four distinct strata of nucleated cell-^, the lowest of
which is columnar.

(2) This layer merges into a thin homogeneous layer (dis-
tinct only in the human eye), the anterior elastic lamina^ which
does not differ from the substance of the cornea proper, except
in its greater density and in the absence of corneal corpuscles.

(3) The proper substance of the cornea, made up of alter-
nating layers of fibrous tissue parallel to the surface. These
are separated from one another by the ground substance, in which
are the cell-spaces, of irregular branched form, which freely
communicate with the cell -spaces of the same, as well as of
other layers. In the chloride of gold cornea, these colls appear
as large and branched granular dark-red or black cells with large
oblong nuclei containing nucleoli. In the spaces, but not filling
them up entirely, are the corneal corpuscles, which are branched
cells of various forms.

(4) Membrane of Descemet, or posterior elastic lamina— a,
firm, structureless, but brittle, transparent membrane covered
by

(5) A layer of endothelial or epithelial cells.

There are no blood-vessels in a healthy cornea, except at
the periphery.

Nerves enter the proper substance of the cornea, and, becoming
transparent, form a plexus, from which finer branches goi no-
forward form another ' sub-epithelial plexus,' from which, again,

142 PRACTICAL HISTOLOGY.

finer fibrils pass among epithelial cells, forming the ' intra- epi-
thelial plexus.'

(4) Retina. — The posterior part of tlie eye of a pig (if no
fresh human eye can be had) is hardened in Miiller's fluid for
a week, and then transferred to alcohol ; pieces of the retina
may then be stained in logwood, and cut. Double-staining with
eosin and aniline green, or with aniline rose and aniline green,
helps to differentiate the layers.

Another method is to place in 2 per cent, solution of osmic acid
for four hours, then in water for one hour, to get rid of the
excess of osmic acid, and finally in logwood. The retina,
thus treated, should be embedded in cacao-butter, instead of the
ordinary wax.

Structure.— It consists of eight layers in the following order,
from within out.vards :

1. A lai/er of nerve fbres, which is wanting at the yellow
spot : the fibres consist of axis cylinders only ; it diminishes in
thickness anteriorlj'.

2. .1 hiyer of )iervc- cells (ganglionic layer), consisting of cells
of a spheroidal or pyriform figure ; one process of each extends
into the first layer, and is doubtless continuous with it. From
the other end of the cell, one or more processes extend outward
for a variable distance into the next layer. In the yellow spot
there are several layers of cells ; elsewhere, only one layer.

3. An inner molecular Jayer^ a dense network of various
fibrils which appear as a thick stratum of granular-looking
substance.

4. An inner nuclear layer con^i^i^ of transparent nucleus-like
bodies, of at least four kinds— (c/) a few connected with the
fibres of Miiller (to be described below) ; {h) the largest number,
like bipolar cells, one pole unbranched, passing inwards, and
being connected with a nerve-fibre— the other, thicker and
branched, running outwards, is supposed to break up into a
plexus in the outer molecular layer ; (f) unbranched cells found
as a complete stratum at the innermost part ; {cl) scattered in
the outermost part, are rounded cells, of large size, with only
one process.

PRACTICAL HISTOLOGY. 143

T). An outer molecular layer, thinner than (3), but otherwise
of the same structure.

G. ^In ouler nuclear la>/rr, consists roughly of two kinds of cor-
puscles embedded in a reticular matrix : (a) those connected with
the rods are most numerous, and may be considered as dilata-
tions situated in the centre of the fine rod fibres, they have an
elliptical striated nucleus, but no nucleolus ; (h) those connected
with the cones are fewer, pear-shaped, not striated, and situated
nearer the outer part of the layer in the thicker cone-fibre.

7. -1 lai/er of rods and cones, is composed of elliptical elon-
gated bodies, ihe rodx, and shorter, thicker, club-like bodies, the
cones ; each consists of two parts, inner and outer, of which the
outer is transversely striated and smaller, and in the cones tapers
to a point, whilst the inner is fibrillated externally, but homo-
geneous internally.

8. The 2^1 f/mentar>j layer, or uvea, consists oi a single stratum
of hexagonal pigment-cells.

The fibres of Midler consist of bands, which pass through all
the layers of the retina, binding them together ; they commence
by a broad base, forming by their union the memhrana limitans
interna; and, at the outside of theretina, the inemhrana limitans
externa. In the inner nuclear layer they give off processes
which contain a clear, oval nucleus. In the outer nuclear layer
they break up into fibrils, and partially enclose the rod and
cone fibres.

Blood-vessels. — The arterial and venous branches are situated
internally under the layer of nerve-fibres. The capillaries are
arranged in plexuses, with large meshes. They occur in the
inner molecular layer, one plexus being situated near the inner
nuclear layer, the other near the layer of nerve-cells. A plexus
also exists in the inner nuclear layer, whilst another lies more
superficially in the outer molecular layer.

(5) Crystalline Lens.— («) Harden the eye of a frog, from
which the cornea has been removed, in a solution consisting of one
part of fuming nitric acid, three parts of water, and one part of
glycerine. At the end of twenty-four hours remove it from this
solution, and allow it to remain for a day in water. Tease a

H^ PRACTICAL HISTOLOGY.

portion of the lens thus prepared in glycerine, and mount it
in Farrant's solution or glycerine.

(Z>) Sections of the lens should be made from eyes which have
been hardened for a fortnight in Miiller's fluid, and afterwards
in Aveak spirit.

(r) The capsular epithelium is best demonstrated by stain-
ing the uninjured lens of the frog in nitrate of silver,
and afterwards mounting portions of the anterior capsule in
glycerine.

Structure. — (a) Of the capsule. The portion which covers
the anterior surface of the lens consists of a thick elastic layer,
immediately behind which is a single layer of granular hex-
agonal epithelium cells, each of which is provided with an
oval nucleus. The elastic lamina covering the back of the lens
has no such lining epithelium, but is in close contact with the
lens-fibres.

(li) The lens itself is composed of (1) 'The I ens-jihre>^ ^vfhich. are
elongated bands running from the posterior to the anterior sur-
face ; they are broader behind than in front. Each fibre contains
a nucleus, which is more distinct in the peripheral than in the
central fibres. Every fibre is hexagonal when seen in transverse
sections, and is serrated along its narrow^ edge, the teeth of one
fibre fitting into the notches of its neighbour. (2) The Interstitial
substance is like that of connective-tissue, it is permeated by
lymph channels.

(6) Iris. — The iris from an eye hardened in chromic acid and
spirit mixture, together with the ciliary processes, can be cut by
means of the freezing microtome ; the operation requires much
care. The whole iris of a small animal may be mounted and
examined in a recent state in saline solution.

Structure. — The iris is principally made up of connective-
tissue and blood-vessels. This forms the middle layer. In front
it is covered by endothelium, which may contain pigment, a
homogeneous basement membrane intervening. Behind there
are similar layers, i.e., an endothelial pigmentary layer, the uvea,
and an intervening basement membrane. Around the inner
border is a circular muscle of unstriped fibres, the sphincter
^Jiipilhe. Under the uvea, i.e.^ between that and the iris proper,

PRACTICAL HISTOLOGY. 146

is a tbin radiating membrane of muscle cells, which jtasses out-
wards from the sphincter. This is the dildtor i^up'ilhe. The
blood-vrs.sels are arranged in dense capillary plexuses in the
tissue proper and on the sphincter. The ^^ervcs form a plexus
near the outer edge, from which pass off medullated fibres,
terminating in the dilator and in the anterior surface of
the iris proper, and also uon-medullated nerves to the
sphincter.

7. Ciliary Processes. — The ciliary processes resemble the iris
in structure, having a similar connective-tissue basis, containing
branched pigment cells, covered by a transparent membrane, the
lamina vitrea, whilst external to this is the uvea, differing in no
way from the uvea of the retina.

The uvea is covered by a single layer of transparent columnar
cells.

The ciliarn muscle is connected -with the outer part of the
ciliary processes, and is made up principally of fibres radiating
outwards, but partly of circular bundles.

8. Choroid. — The choroid consists of the following coats :

(rt) Lam'tnajuxca, a loose connective-tissue, with corpuscles,
both with and without pigment, branched or unbranched ;
the lamina suprachoroidea is a continuation of the lamina
fusca.

{h) Sfratcm vasculosum, in which are the large blood-vessels
embedded in loose connective-tissue.

(r) An elastic layer containing small arteries and veins, covered
on each side by endothelium.

{<l) Memlrana chorio-capillaris, which contains the dense
capillary me-ihwork, covered by cells, spindle-shaped and
flattened, with or without pigment.

(e) Lamina vitrea (as above).

(/) Uvea, or retinal pigment.

1). Sclerotic. — This coat is made up of dense fibrous tissue,
the bundles of which in part cross and interlace. Between the
bundles are connective-tissue corpuscles, almost precisely similar
to those of the cornea. These cells are contained in spaces
which form an anastomosing lymph-canalicular system. Non-
medullated nerve fibres are said to exist in a dense plexus in the

10

146 PRACTICAL HISTOLOGY.

tissue. Near the ligamentum pectinatum iridi?, at the corneo-
scleral junction, is a circular canal, lined Tvitb endothelium ; it
communicates indirectly with the lymph space?[mentioned above,
and is called the canal of Schlemm.

PART ir.

PHYSIOLOGICAL CHEMISTRY.

Apparatus and chief Reagents required.

Test-tubes.

Test-tul)0 stand.

Retort stand.

Platinum foil and wire.

Three Berlin dishes.

Three beakers.

Sand bath.

Filter papers and funnels.

Sulphuric, Nitric, Hydrochlon'c,
Acetic, Tannic, Carbolic, Picric
and Boracic acids.
Solutions :

Magnesium chloride and sulphate.

Copper sulphate.

Sodium carbonate, sodium chlo-
ride, 10 per cent.

Caustic potash, or soda.

Ammonia and ammonium sul-
phide.

*Millon's roafjent. (Mixed mer-
cury nitrate and nitrite?)

Potassium ferrocyanide.

Potassio-mercunc iodide.

Mercuric nitrate and chloride,
nieiTurous nitrate.

Lead acetate.

Calcium chloride.

Litmus, etc., etc.

Alcohol.
Ether.

Ozonic ether.
Lime water.
Tine. Guaiaci.

Solid sodium chloride and magne-
sium sulphate.

This division of the book treats of the chief substances found
in the animal body, also of foods, and the action of the digestive
juices upon them, of the secretions and excretions, and of
calculi. For the sake of convenience the substances are treated
of in the following order : (1) Proteids, or albuminous sub-
stances, which occur in the animal tissues and in food ;
Nitrogenous bodies, other than proteids : (2) Carbo-hydrates ;
(3) Oil and fats ; (4) Healthy urine, and its constituents ; (5)
Blood, Milk and Bile : Gall-stones ; (6) Digestive fluids ; (7)
Abnormal urine and Calculi ; (S) Examination of organic
substances.

* Preparation ofMillons Bear/enf. — Take equal parts by weight of pure
mercury and of nitric acid, add the acid to the mercury, place in a venti-
lated cupboard, and leave until the mercury is dissolved— if necessary,
warming slightly. Then add twice its bulk of water. After a time a crys-
talline white i)recipitate fails, and the supernatant tluid is decanted,

10—2

CHATTER I.

PROTEIDS AND THEIR ALLIES.

Peoteids or albumins are bodies which are found to be present
in all protoplasm, of which, indeed, they constitute the chief
part. They are highly nitrogenous substances, and contain the
elements carbon, hydrogen, oxygen, nitrogen and sulphur in
certain proportions, which may vary (C, from 51 "o to 54*5 ;
H, GO to 7-3 ; O, 20-9 to 23-5 : N, 15-2 to 17 ; and S, -3 to 2)
slightly. Associated with proteids in protoplasm are found
carbo-hydrates and fatty bodies, as well as nitrogenous organic
bodies other than proteids, and some salts — e.g., sodium and
potassium chlorides. According to some the salts are in part
contained in the proteid molecule.

General Properties of Proteids.— They are all amorphous ;
eome are soluble, some insoluble in water ; some are soluble in
saline solutions, some insoluble ; all are soluble, often with de-
composition, in strong acids and alkalies ; are insoluble in strong
alcohol. Their solutions, for the most part, will not pass
through animal membranes, and exercise a left-handed action
on polarised light.

General Reactions of Proteids.— The solutions of proteids,
even if dilute, give the following tests :

(i.) They turn yellow on heating icith strong nitric acid ; the
colour deepens on addition of ammonia (xanthoproteic re-
action).

(ii.) They give icith M'lUon^s reagent a jjinh preciiAtate or mere
colouration^ either directly or en hciUng.

PHYSIOLOGICAL CHEMISTRY. 140

(iii.) They f/'tce loitJi an cxcesn of caustic so hi cr potaah^ nnd
a (Imp of copper mlphate^ a violet cohmrafiai (biuret reaction).

Many of the proteids give, in addition, the following tests :

(iv.) With excess of acetic acid, and potassium ferrocyanide, a
white precipitate.

(v.) AVith excess of acetic acid and a saturated solution of
sodium sulphate, on boiling, a white precipitate. This test is
used to get rid of all traces of proteids, except peptones, from
solution.

(vi.) Boiled with strong hydrochloric acid, a violet red.
(vii.) With cane sugar and strong sulphuric acid a purplish-
violet.

(viii.) They are precipitated on addition of

Citric or acetic acid, and picric acid ; or,

„ „ and sodium tungstate ; or,

„ „ and potassio-mercuric iodide.

Varieties of Proteids. — Proteids are divided into seven
classes, chiefly on the basis of their solubilities in various
reagents. Each class, however, if it contain more than one
substance, may often be distinguished by other properties com-
mon to its members.

(1) Xafive- Albumins. — Soluble in water and in ealine solu-
tions, coagulated, i.e., turned into coagulated proteid, on heating.

(2) Derived-Albumins. — Soluble in acids or alkalies, insoluble
in saline solutions and in water, not coagulated on heating.

(3) Globulins. — Soluble in strong or in weak saline solutions,
insoluble in water, soluble in dilute acids and alkalies, coagulated
on heating.

(4) Fibrin. — Insolubla in water, dilute saline solutions, or
dilute acids or alkalies ; soluble in strong saline solutions
(partly) and in strong acids ; soluble in gastric or pancreatic
fluids.

(5) Peptone.^. — Soluble in water, saline solutions, acids, or
alkalies ; not coagulated on heating.

(6) Coafjulated Proteids. — Soluble only in gastric or pancreatic
fluids, forming peptones.

150 PHYSIOLOGICAL CHEMISTRY.

(7) Amyloid sulsiance, or Larclaccin. — Generally insoluble,
even in gastric or pancreatic fluids at ordinary temperatures.
Gives a brovrn colouration with iodine.

Class I.

NATIVE-ALBUMINS.

(a) Eg fj- Albumin.

(b) Serum- Albumin.

(a) Egg- Albumin :

Prejjuration. — The white of an egg is cut up into pieces with a
pair of scissors, shaken up with four or five times its bulk of
water in a flask, and filtered through muslin.

P roper tie-<. — The solution is a transparent, frothy, yellowish
fluid, neutral or slightly alkaline in reaction.

Tests. — With the solution show the following tests :

(i.) It gives all of the general proteid reactions.

(ii.) Evaporate a portion to dryness in a water bath at a
temperature not exceeding 40° C. The albumen is dried up into
a yellowish, transparent, glassy mass, soluble in water.

(iii.) Heat another portion to a temperature of TO'C. It is
coagulated, i.e.^ changed into a new substance, coagulated proteid,
which is quite insoluble in water.

(iv.) Show that coarjulation also takes place :

(a) By the prolonged action of alcohol ;

{b) By strong mineral acids, especially by nitric acid, also

by tannic acid, or carbolic acid ;
(o) By ether the coagulum is soluble in caustic soda.

(v.) Show that it is prec'qntated^ i.e., forms an insoluble com-
pound with the reagent, soluble on removal of the salt by
dialysis, without coagulation, with either mercuric chloride,
lead acetate, copper sulphate or silver nitrate, the precipitate
being soluble in slight excess of the reagent.

(vi.) Show also Heller s test, which is a delicate one for mere
traces of the albumin in solution. Pour a little strong nitric acid
into a test-tube, and add gradually a dilute solution of albumin ;

PHYSIOLOGICAL CHEMISTRY. 151

the albumin is precipitated at the point of contact with the
acid in the form of a fine white or yellow ring.

(i:) S erum- Albumin :

Preparation. — Blood serum is diluted with water after the
paraglobulin has been removed.

yV.s/.s. — With the solution show that

(i.) It gives similar reactions to egg-albumin, but differs from
it in not being coagulated by ether.

(ii.) It also differs from egg-albumin in not being easily pre-
cipitated by hydrochloric acid, and in the precipitate being easily
soluble in excess of this acid. Serum-albumin, either in the
coagulated or precipitated form, is more soluble in excess of
strong acid than egg-albumin.

DIALYSIS.

Native-albumins are very slightly diffusible. Show this by
taking a solution of egg- or serum-albumin and placing it in a
dialyser, and testing the outside liquid from time to time for
albumen.

Salt should be mixed with solution of albumen, and tested for
in the outside liquid. It is speedily detected ; not so the albu-
men.

Class II.

DERIVED-ALBUMINS.

(a) Acid- Albumin.

(b) Alkali- Albumin.

(c) Casein.

(a) Acid- Albumin :

Preparation. — Acid-albumin is made by adding small quanti-
ties of dilute acid (of which the best is hydrochloric, "4 per cent,
to 1 per cent.), to either egg- or serum-albumin diluted five to
ten times, in a flask, and keeping the solution at a temperature
not higher than 50' C for not less than half an hour.

Acid-albumin may also be made by dissolving coagulated
native-albumin in strong acid, or by dissolving any of the
globulins in acids.

152 PHYSIOLOGICAL CHEMISTRY.

Tests. — With a solution of acid-albumin show that

(i.) It is not coagulated on heating, but that on cxacthj
neutralising the solution, a flocculent precipitate is produced.
This may be done by adding to the acid-albumin solution a little
aqueous solution of litmus, and then dropping a weak solution
of caustic i)otash from a burette, until the red colour disappears.
The precipitate is the derived-albumin. It is soluble in dilute
acid, dilute alkalies and dilute solutions of alkaline carbonates.

(ii.) It gives the proteid tests.

(iii.) It is coagulated by strong acids, e.g., nitric acid, and by
strong alcohol.

(iv.) It is insoluble in distilled water, and in neutral saline
solutions.

(v.) It is precipitated by saturation with sodium chloride.

(vi.) There is partial coagulation on boiling in lime-water, and
further precipitation on addition to boiled solution of calcium
chloride, magnesium sulphate, or sodium chloride.

(b) Alkali-Albumin:

Pre}:iuraiion. — If solutions of native-albumin, or coagulated
or other proteid, be treated with dilute or strong fixed alkali,
alkali-albumin is produced.

Solid alkali-albumin may be prepared by adding caustic soda
or potash, drop by drop, to undiluted egg-albumin, until the
whole forms a jelly. This jelly is soluble in dilute alkalies on
boiling.

Tests. — A solution of alkali-albumin gives the tests corre-
sponding to those of acid-albumin. It is not coagulated on heat-
ing. It is thrown down on neutralising its solution, except in
the presence of alkaline phosphates, in which case the solution
must be distinctly acid before a precipitate falls.

To dmerentmte hetv:een Acid- and Alkcdi-Albunnn, the follow-
ing method is useful : —

(1) Alkali-albumin is not precipitated on exact neutralisation,
if sodium phosphate has been previously added.

(2) Acid-albumin is precipitated on exact neutralisation
whether or not sodium phosphate has been previously added.

(c) Casein :

Preparation. — Casein may be prepared from milk by the

rHYSIOLOGlCAL CHEMISTRY. 153

following process : Dilute the milk with three to four times its
volume of water, add sufficient dilute acetic acid to render the
solution distinctly acid, but not more than a few drops, and filter
off the casein. To purify it, wash with alcohol and then with
ether.

Casein may also be prepared by adding to milk an excess of
crystallized magnesium sulphate or sodium chloride, which
causes it to separate out.

Tests. — Casein gives much the same tests as alkali-albumin.

It is soluble in dilute acid or alkalies.

It is reprecipitated on neutralisation, but if potassium phos-
phate be present, the solution must be distinctly acid before the
casein is depo.-ited.

Class III.

GLOBU LINS.

(a) GJobiiUn. (d) Fibrinogen.

(b) Mf/os'nt. (e) ViteUin.

(c) Parafjlohulin. (f) Gloh'in.

General Properties of Globtdins. — They give the general proteid
tests.

Are insoluble in water.

Are soluble in dilute saline solutions.

Are soluble in acids and alkalies forming the corresponding
derived-albumin.

Most of them are precipitated from their solutions by satm-a-
tion with solid sodium chloride.

Are coagulated on heating.

(a) Globulin or Crystallin :

Preparation. — It is obtained from the crystalline lens by
rubbing it up with powdered glass, extracting with water or
dilute saline solution, and by passing through the extract a
stream of carbon dioxide.

Tests. — It differs from other globulins, except vitellin, in not
being precipitated by saturation with sodium chloride.

(b) Myosin :

PreiKinition. — Myosin may be prepared from dead muscle by

154 PHYSIOLOGICAL CHEMISTRY.

removing all fat, tendon, etc., and washing repeatedly in water,
until the washing contains no trace of proteids. mincing it and
then treating with 10 per cent, solution of sodium chloride, which
will dissolve a large portion into a viscid fluid, which filters with
difficulty. If the viscid filtrate be dropped little by little into a
large quantity of distilled water, a white flocculent precipitate
of myosin will occur.

Tests. — With a solution of myosin show that

(i.) It is soluble in 10 per cent, saline solution.

(ii.) It is coagulated at GO' C. into coagulated proteid.

(iii ) It is soluble without change in very dilute acids.

(iv.) It is precipitated by picric acid, the precipitate being re-
dissolved on boiling.

(v.) It may give blue colour with ozonic ether and tincture
of guaiacum.

(c) Paraglohulin :

Preparation. — Paraglobulin may be precipitated by saturating
serum with solid sodium chloride or magnesium sulphate, as a
bulky flocculent substance, which can be removed by filtration
after standing for some time.

It may also be prepared by diluting blood serum with ten
vols, of water, and passing carbonic acid gas rapidly through it.
The fine precipitate is collected on filter, and washed with water
containing carbonic acid gas.

Tests. — It is very soluble in dilute saline solutions, from which
it is precipitated by carbonic acid gas and dilute acids.

Its solution is coagulated at 70' C.

Even dilute acids and alkalies convert it into acid- or alkali-
albumin.

(d) Fibrinogen :

Preparation. — Fibrinogen is prepared from hydrocele or other
serous transudation by methods similar to those employed in
preparing paraglobulin from serum.

Teds. — Its general reactions are similar to those of paraglo-
bulin.

Its solution is coagulated at h'l^-'oh'' C.

Its characteiistic property is that, under certain conditions, it
forms fibrin.

PHYSIOLOGICAL CIIEMISTIIY. 155

(e) Vitellin:

Preparatujii. — Yitellin is prepared from yolk of egg by wash-
ing with ether until all the yellow matter has been removed.
The residue is dissolved in 10 per cent, saline solution, filtered,
and poured into a large quantity of distilled water. The pre-
cipitate which falls is impure vitellin.

Testa.— It gives the same tests as myosin, but is not precipitated
on saturation with sodium chloride.

It coagulates between 70' and 80^ C.

(f) Globin :

Is the proteid residue of haemoglobin.

C L A ^ S IV.
FIBRIN.

Fibrin :

rreimration.—Yihi'm can be obtained as a soft, white, fibrous,
and very elar.tic substance by whipping blood with a bundle of
twigs, and washing in a stream of water until all the adhering
blood-colouring matter be removed.

Teds. — It differs from all other proteids, in having a filamen-
tous structure. Examine with the microscope.

It is insoluble in water and dilute saline solutions, slightly
soluble in concentrated saline solutions, soluble on boiling in
strong acids and alkalies.

On boiling it is converted into coagulated proteid.

When dissolved in strong saline solution it gives many of the
same reactions as myosin. "When dissolved in acids or alkalies,
is converted into corresponding derived-albumin.

It gives a blue colour with tincture of guaiacum and ozonic
ether.

Class V.

PEPTONE.

Peptone :

Prpparat'ion. — Peptone is formed by the action of the diges-
tive ferments, pepsin or trypsin^ on other proteids, and on gela-
tine (see pp. 185, 18G).

Tests. — Make a solution of commercial peptone in warm
water, filter, and with the filtrate show the following tests :

156 PHYSIOLOGICAL CHEMISTRY.

(i.) It is soluble in water and in dilute saline solutions,
(ii.) It is not coagulated on heating.

(iii.) It is not precipitated by saturation with NaCl, or MgS04,
or by COo.

(iv.) It is not precipitated by boiling with sodium sulphate
and acetic acid.

(v.) It is not precipitated by addition of dilute acid or alkali.

(vi.) It is precipitated from neutral or slightly acid solutions by :

Mercuric chloride, the precipitate being only partl}^ soluble

in excess.
Ai'gentic nitrate.
Lead acetate.
Potassio-mercuric iodide.
Bile salts.

Phosphoro-molybdi3 acid.

Tannin, the precipitate soluble in dilute a^id, not in excess.
Picric acid (saturated solution), precipitate disappears on

heating and partly returns on cooling.
Absolute alcohol {not with dilute). N.B. It is not coagulated.
Ether,
(vii.) The solution gives :

The Xanthoprote'ic reaction easily, but there is very slight,

if any previous precipitation with the nitric acid.
The Biuret reaction — but the colour {•& 2)inh instead of violet

(use a mere trace of copper sulphate).
With MiUon's test — not so easily as with native albumins,
"With Ferrocyanide and. acetic acid — only in cases where the
peptone is impure (Meissner's A and B peptones), is there
any precipitate,
(viii.) It diahjsesfreehj. — Show this with a simple gut dialyser
in the manner indicated p. 151.

Class YI.

COAGULATED PROTEIDS.

Coagulated proteids are formed by the action of heat upon
other proteids ; the temperature necessary in each case varying in
the manner previously indicated. They may also be produ3ed
by the prolonged action of alcohol upon proteids.

PHYSIOLOGICAL CHEMISTRY. 157

Tests.— They are soluble in strong acids or alkalies ; slightly
so in dilute.
Are soluble in digestive fluids (gastric and pancreatic).
Are insoluble in saline solution.

Class VII.

LARDACEIN OR AMYLOID SUBSTANCE.

Lardacein is found in organs which are the seat of amyloid de-
generation.

Tf's^*.— Insoluble in dilute acids and in gastric juice at the temperature
of the body.— Coloured brown by iodine and bluish-purple by methyl
violet.

THE GELATINS OR NITROGENOUS BODIES OTHER THAN

PROTEIDS.

(a) Gelatin. (c) Elaatin.

(i?) Mucin. (d) Chondrin.

(e) Keratin.

(a) Gelatin :

Prepamtlon. — Gelatin is contained in bone, teeth, fibrous
connective-tissue, tendons, ligaments, etc. It may be obtained
by prolonged action of boiling water in a Papin's digester or of
dilute acetic acid at a low temperature (15° C).

Properties. — The percentage composition is O, 23-21, H, 7*15,
N, 18"32, C, 5076, S, 0*56. It contains more nitrogen and less
carbon than proteids.

It is amorphous, and transparent when dried. It does not
dialyse.

It is insoluble in cold water, but swells up to about six times
its volume : it dissolves readily on the addition of very dilute
acids or alkalies.

It is soluble in hot water, and forms a jelly on cooling, even
when only 1 per cent, of gelatin is present. Prolonged boiling
in dilute acids, or in water alone, destroys this power of forming
a jelly on cooling.

Tests. — A fairly strong solution of gelatin— 2 per cent, to
4 per cent.— gives the following reactions :

158 PHYSIOLOGICAL CHEMISTRY.

(a) With protekl tests:

(i.) Xantlwproteic test. — A yellow colour with no previous
precipitate with nitric acid, becoming darker on the
addition of ammonia,
(ii.) Biuret test. — A violet colour,
(iii.) JTillon's test. — A pink precipitate.
(iv.) Potassium ferrocyanide and acetic acid. — No reaction.
(v.) Boiling with sodium sulphate and acetic acid.— Xo
reaction.

(b) Special reactions :

(i.) No precipitate with acetic acid.
(ii.) No precipitate with hydrochloric acid,
(iii.) A white precipitate with tannic acid, not soluble in

excess or in dilute acetic acid.
(iv.) A white precipitate with mercuric chloride, unaltered

by excess of the reagent.
(v.) A white precipitate with alcohol.
(vi.) A yellowish-white precipitate with picric acid, dis-
solved on heating and reappearing on cooling.
Bone consists of an organised matrix of connective-tissue
which yields gelatin and inorganic salts.

Inorganic salts can be removed by digesting bone in hydro-
chloric acid. The gelatinous matter left retains the form of the
bone. By long boiling in water it is converted into a solution of
a gelatin.

When bone is heated, the first action is to decompose the
organic matter, leaving a deposit of carbon. On further ignition
in air this carbon burns away, and only inorganic salts (princi-
pally calcic phosphate) are left.

Take two pieces of bone, calcine one piece, and boil another

in dilute hydrochloric acid in a test tube ; test the ash from

the one for phosphates, and test the undissolved part of the

other and also the solution in which it has been boiled for gelatin.

(b) Mucin :

Preparation. — Mucin is the characteristic component of mucus;
it is contained in foetal connective-tissue, tendons, and salivary
glands.

It may be prepared from ox-gall, by acidulation with acetic

PHYSIOLOGICAL CHEMISTRY. 159

acid and subsequent filtration, or from ox-gall by precipitation
with alcohol, afterwards dissolving in water, and again pre-
cipitating by means of acetic acid.

It can also be obtained from mucus by diluting it with water,
filtering, treating the insoluble portion with weak caustic alkali,
and precipitating the mucus with acetic acid.

Propertie.-i. — ^lucin has a ropy consistency.

It is precipitated by alcohol and by mineral acids, but dissolved
by excess of the latter.

It is dissolved by alkalies and in lime water.

It gives the proteid reaction with Millon's reagent and nitric
acid, but not with copper sulphate.

Neither mercuric chloride nor tannic acid gives a precipitate
with it (V).

It does not dialyse.

(c) Elastin is found in elastic tissue, in the ligameuta subflava, liga-
mentum nucli;p, etc.

Prejmrafion. — Take the fresh ligamentnm nncli;r of an ox, cut in i)ieces,
and boil in alcohol and ether to remove the fat. Remove the gelatin by
boiling for some hours in water. Boil the residue with acetic acid for some
time, and remove the acid by boiling in water, then boil v,-iih caustic soda
until it begins to swell. Eemove the alkali, and leave it in cold hydrochloric
acid for twenty-four hours, and afterwards wash with water.

Properties. — It is insoluble, but swells up both in cold and hot water. Is
soluble in strong caustic soda.

It is precipitated by tannic acid ; does not gelatinize. Gives the proteid
reactions with strong nitric acid and ammonia, and imi)erfectly with Millon's
reagent.

Yields leucin on boiling with strong sulphuric acid.

(n) Chondrin is found in cartilage.

Preparation. — By boihns: small pieces of cartilage for several hour.=?,
and filtering. The opalescent filtrate will forai a jelly on cooling.
Chondrin is jirecipitated from the warm filtrate on addition of acetic
acid.

Properties. — It is soluble in hot water, and in solutions of neutral salts,
e.g., sulphate of sodium, in dilute mineral acids, caustic potash, and soda.
Insoluble in cold water, alcohol, and ether. It is precipitated from its
solutions by dilute mineral acids (excess re-dissolves it), by alum, by lead
acetate, by silver nitrate, and by chlorine water. On boiling with strong
hydrochloric acid, yields gi-ape-sugar and certain nitrogenous substances.
Prolonged boiling in dilute acids, or in water, destroys its power of forming
a jelly on coohng.

(e) Keratin.

CHAPTER IT.

CARBO-HYDRATES.

Cakbo-hydrates are bodies composed of six or twelve atoms of
carbon with hydrogen and oxygen, the two latter elements being

in the proportion to form water.

Thev are arranped into three classes ; the members of each class with
which we have to flo are printed in ordinary type, the most important of
the others in italics.

Amyloses. Saccharoses. Glucoses.

C,H,„0, Ci,H,.,0„ QH-,,0,

Starch Saccharose, or cane sugar Dextrose or grape sugar

Dextrin Lactose Laevulose or fruit sugar

Glycogen ^faltose Inosite

Inv.Un Mellitose Mannitose

Cellulose

Gum

(a) Starch (C^Hj„05) is contained in nearly all j^Icints :

Obtain some starch from a potato, by grating it into a bowl of
water, allow it to settle, and pour off the supernatant water ; a
layer of starch remains at the bottom ; wash several times, each
time allowing the starch to settle and pouring away the water ;
when clean, dry at a moderate temperature.

Or ijova. flour — tie in a bag and wash into a vessel under the
tap : allow to settle and proceed as above.

(Or it may be obtained from many seeds, roots, stems, and wnie
fnrds, by somewhat similar treatment.)

Character.^. — It is a soft white powder composed of granules
having an organised structure, consisting of gramdose (soluble in
water) and cellulose (insoluble in water) ; their shape and size
vary according to the source whence the starch has been obtained.

rilVSIOLOGICAL CHEMISTRY. IGl

Examine starch from potato, rice, and flour, under the micro-
scope, and compare the size and shape of the granules.

Teals. — It is insoluble in cold water, in alcohol, and in ether.

It is soluble after boiling for some time, and may be filtered
(this is due to the granulose swelling up, bursting the cellulose
coat, becoming free and dissolving in water = soluble starch or
amylin).

It gives a blue colouration with iodine, which disappears
on heating and returns on cooling.

It is converted into dextrine and grape-sugar by amylolytic
ferments or by boiling with dilute acids.

(r-) Glycogen :

Preparation. — Apparatus necessary : A solution of potassio-
mercuric iodide, made by precipitating a solution of mercuric
chloride with potassium iodide, washing the precipitate, and
adding it to a boiling solution of potassium iodide till the latter
is saturated. Any precipitate which occurs on cooling is to be
filtered off. Dilute hydrochloric acid. Methylated spirit, a
large bottle ; ether ; absolute alcohol. Large funnel and
Swedish filter- papers. Large knife ; capsule ; several beakers ;
distilled water ; ice. Mortar and pestle ; large Bunsen's
burner.

Glycogen, which is usually obtained from the livers of animals,
is also present to a considerable extent in the muscles of very
young animals.

To prepare glycogen, it is best to use the liver of a large
rabbit. The animal must have been well fed on a diet of
grain and sugar for some days, preferably weeks, previously, and
should have a full meal of grain, carrots, and sugar, about two
hours before it is killed, in order that it may be in full digestion.
The rabbit is killed either by decapitation or by a blow on the
head, the abdomen is rapidly opened, and the liver is torn out
and choi)ped up as quickly as possible with the knife, and is
thrown, with the exception of a piece preserved to show
the presence of sugar, into water which is kept boiling. This
operation must be performed within half a minute of the death
of the animal, and the water must not be allowed to fall below
the boiling-point. The liver is to remain in the boiling water

11

162 PHYSIOLOGICAL CHEMISTRY.

for five minutes ; it is then placed in a mortar, reduced to a pulp,
and again boiled in the capsule for ten minutes. The liquid
is jfiltered, and the filtrate is rapidly cooled by placing the vessel
in iced water. The albuminous substances in the cold filtrate
are precipitated by adding potassio-mercuric iodide and dilute
hydrogen chloride alternately as long as any precipitate is
produced. The mixture is then stirred, is allowed to stand
for five minutes, and is filtered. Alcohol is added to this
second filtrate until glycogen is precipitated, which occurs after
about 60 per cent, of absolute alcohol has been added. The
precipitate is then filtered off and is washed with weak spirit,
strong spirit, absolute alcohol (two or three times), and finally
with ether. It is then dried on a glass plate at a moderate heat,
and, if pure, should remain as a "white amorphous powder. If
the water has not been completely removed, the glycogen will
form a gummy mass : in this case it must again be treated with
absolute alcohol.

Properties. — It is freely soluble in water, the solution looking
opalescent and giving a port-wine colouration with iodine, which
disappears on heating and returns on cooling.

It is insoluble in absolute alcohol and in ether.

It exists in the liver during life, but very soon after death is
changed into sugar. Prove this by boiling the piece of liver
which was put aside in the preparation of ghxogen and which
was not at once thrown into boiling water ; filter and test the
filtrate for sugar.

It is converted into sugar by ferments, or by boiling with
dilute acids.

(c) Dextrine :

Preparation. — Is made in commerce by heating dry potato-
starch to a temperature of 400^ It is also produced in the
process of the conversion of starch into sugar by diastase, and
by the salivary and pancreatic ferments.

Properties. — A yellowish amorphous powder, white when
pure, often with peculiar smell.

It is soluble in water.

It is insoluble in absolute alcohol and in ether.

It corresponds almost exactly in tests with glycogen ; but one

PHYSIOLOUICAL CHEMISTRY. 163

variety (achroo-dextrine) does not give the colouration with
iodine.

(d) Glucose occurs widely diffused in the vegetable kingdom,
in diabetic urine, in the blood, etc.; it is usually obtained from
grape-juice, honey, or carrots.

Properties. — It is easily soluble in water ; not so sweet as cane-
sugar.

It is not easily charred by strong sulphuric acid.

It is soluble in alcohol.

Te>tts. — With a solution of glucose show the following tests :

(i.) Tromniers. — Add an excess of caustic potash and then a
solution of copj>er sulphate, drop by drop, to the solution, in a
test-tube, as long as the blue precipitate which forms re-dissolves
on shaking the tube. Heat the upper portion of the fluid, and a
yellowish- brown precipitate of copper suboxide appears.

(ii.) Moore's. — Heat the solution of sugar in a test-tube with
caustic potash ; a brown colouration appears,

(iii.) Fermentation. — If a solution of sugar be kept in a
warm place for some time after the addition of yeast, the sugar is
converted into alcohol and cai'bon dioxide. (CgH^oO^j = •2C..H5OH

+2CO0).

(iv.) Bismuth or Bottcher's test. — Add a little bismuth oxide
or subnitrate and an excess of caustic potash to the solution in a
test-tube, and heat ; the solution becomes at first grey and then
black.

(v.) Picric acid test. — To the solution add about a fourth of
its bulk of picric acid (saturated solution) and an equal quantity
of caustic potash, and boil : the liquid becomes of a very deep
coffee-brown.

(vi.) Indigo carmine test. — To the solution add a strong solu-
tion of sodium bicarbonate, and then a little sulphindigotate of
carmine. Warm : the blue colour disappears, but returns on
shaking the test-tube.

For the Quantitative estimation of sugar see p. 189.

(e) Lactose is contained in milk (p. 180).

Properties. — It is less soluble in water than glucose.

It has a gritty and slightly sweet taste.

1 1 -1

164 PHYSIOLOGICAL CHEMISTRY.

It is insoluble in absolute alcohol.

It undergoes alcoholic fermentation with extreme diflEiculty.

It gives the tests similar to glucose, but less readily.

(f) Inosite is a non-f ermentible variety of glucose occurring in
the heart and voluntary muscles, as Tvell as in beans and other
plants.

It crystallizes in the form of large colourless monoclinic
tables, which are soluble in water, but insoluble in alcohol or
ether. It may be detected by evaporating the solution con-
taining it nearly to dryness, and by then adding a small drop
of a solution of mercuric nitrate, and afterwards evaporating care-
fully to dryness, a yellowish-white residue is obtained ; on further
cautiously heating, the yellow changes to a deep rose-colour,
which disappears on cooling, but reappears on heating. If the
inosite be almost pure, its solution may be evaporated nearly to
dryness. After the addition of nitric acid, the residue mixed
with a little ammonia and calcium chloride, and again evaporated,
yields a rose-red colouration.

CHAPTER III.

OILS AND FATS.

Oils and fats are neutral substances, with the composition of a
compound ether, glycerin, C3H5(OH).j being the alcohol :
Are lighter than water, sp. gr. '91 to 94 ;
Give a greasy stain on paper ;
Are insoluble in water ;

Are soluble in ether, chloroform, turpentine, or alcohol.
Glycerine. — A sweet-tasted viscid liquid, soluble in water and
in alcohol ; insoluble in ether.

Test. — Heat a little glycerine in a test-tube with some solid
acid potassium sulphate ; acrolein, a body of pungent smell, is
produced {Q.^B.f>.,-2UJd=G^S>)-

Olive Oil (glycerine oleate) | ^^^^^^-^ \

(i.) Shake up a few drops of olive oil in a test-tube with water
and then allow it to stand ; note that no emulsion or mixture
takes place. Add ether, and shake the test-tube ; note the solu-
tion of the oil in that reagent.

(ii.) Add to some oil and water a few drops of caustic potash
and warm. Complete solution takes place, a potassium oleate
or soap is formed, and glycerine liberated. Both the soap and
glycerine are soluble in water.

(iii.) Add some lead oxide to some olive oil and water in a
porcelain basin, and apply heat, stirring the mixture. Lead
oleate is formed and glycerine is liberated (lead oleate is lead
soap).

166 PHYSIOLOGICAL CHEMISTRY.

Fat. — (i.) Heat some fat in a test-tube, with water, until
melted ; add caustic soda. The fat is saponified, stearate of
sodium and glycerine being formed.

(ii.) Add sodium chloride to the solution ; sodium stearate
separates out.

(iii.) Add acid to another portion ; sodium stearate is decom-
posed. Stearic acid separates out.

(iv.) To another portion add a solution of calcium chloride ;
calcium stearate is precipitated.

CHAPTER IV.

HEALTHY URINE AND ITS CONSTITUENTS.

HEALTHY URINE.

Is a perfectly transparent amber-coloured liquid, with a peculiar
and characteristic, but not disagreeable odour, a bitterish taste,
and a slightly acid reaction. The specific gravity varies from
1015 to 1020 ; under exceptional circumstances it may be as low
as 1001, and as high as 1025. Alight cloud of mucus may often
be seen in urine after standing.

Test its reaction with litmus-paper.

Take its sp. gr. with the urinometer.

Constituents. — Its characteristic constituent is

(a) Urea (C0iSr._,H4).— Evaporate urine to half its bulk, filter,,
and add pure strong nitric acid ; impure urea nitrate separates
out. To obtain pure urea from this, purify it by re-crystallisa-
tion with animal charcoal and afterwards decompose it with
barium carbonate into barium nitrate and urea. Evaporate the
mixture to dryness and extract with hot alcohol. The pure
urea will separate out from the alcoholic solution on cooling,
[See also Appendix.]

Properties of Urea. — It is soluble in water and in alcohol.
Make a strong solution of urea in water, and use it for the
following experiments :

(i.) Allow a few drops to crystallise on a glass slide, and
examine with the microscope. Note that the urea crystallises
out in transparent four-sided prismatic needles, terminated by
one or two oblique facets.

168 PHYSIOLOGICAL CHEMISTRY.

(ii.) Either in a test-tube, or on a glass slide, add a few drops
of pure nitric acid to some of the urea solution. Urea nitrate
separates out (COX.H4.2HXO0) in the form of six-sided tables.
Examine the crystals with the microscope.

(iii.) Proceed in a similar manner to ii., but use a concen-
trated solution of oxahc acid. Urea oxalate separates out
(C0X.,H4.C.,H..0J in the form of tabular plates, or prismatic
bundles. Examine the crystals with the microscope.

(iv.) To a concentrated solution of urea in a test-tube add
mercuric nitrate solution : a white precipitate of a compound of
mercuric oxide and urea is formed.

(v.) Proceed as in iv., but previously add some common salt.
The precipitate does not form until a large quantity of the re-
agent has been added : as the sodium chloride causes the mercuric
nitrate to split up into mercuric chloride and sodium nitrate
and until the whole of the sodium chloride has been used up
there is no unchanged mercuric nitrate to produce the precipitate.

(b) Uric Acid (C5H4X4O3) is another characteristic consti-
tuent of urine, from which it may be obtained thus : Add five
or sis drops of pure nitric acid to two ounces of urine in a
narrow urine glass, and allow it to stand for twenty- four hours.
A red brick coloured sediment of uric acid will be observed.
Examine some under the microscope. Brown crystals of various
sizes will be seen, the most characteristic being plates, rosettes,
and lozenge-shaped.

Properties of Uric Acid— It is insoluble in cold water, very
slightly soluble in hot, soluble in caustic alkaline solutions,
forming urates, most easily soluble in solutions of lithium salts.

With uric acid powder show the following tests :

(i.) JIurexide test.— Xdd a drop of strong nitric acid, evaporate
to dryness over water-bath, or at a temperature not exceeding
40' C. Alloxan (C4H0X0O4) is formed, as a reddish-coloured
residue. Add a drop of ammonia solution, and the purple
colour of murexide (CsHgXgOg) is produced.

(ii. ) Schif's test. — Dissolve uric acid in a solution of sodium car-
bonate, and place a drop upon a filter-paper moistened with
solution of silver nitrate ; a black stain will result.

(c) Phosphates (earthy). — Mix equal quantities of caustic

PHYSIOLOGICAL CHEMISIi:V. 169

potash and urine in a test-tube. A transparent flaky precipitate
will separate.

(d) Chlorides. — Add solution of silver nitrate to urine, a
curdy, yellowish precipitate will fall of chloride and phosphate
of silver ; add nitric acid, the phosphate will dissolve, leaving
behind the chloride, which is soluble in ammonia. The phos-
phates may also be shown by adding molybdate of ammonium
and boiling. A yellow colour or precipitate will occur.

(e) Sulphates. — To urine acidulated with hydrochloric acid,
add barium chloride solution. A precipitate of barium sulphate
will occur, insoluble in nitric acid.

(f) Colouring matter may be precipitated with the chlorides,
sulphates, phosphates on addition of lead acetate.

Quantitative estimation of Urea. — (i.) Hupohromite
method. — One of the forms of apparatus* employed in this
method (Russell and West's) consists of («) a water-bath sup-
ported by three iron bands, arranged as a tripod. The bath is
provided with a cylindrical depression, and with a hole, into
which fits a perforated india-rubber cork ; (^) a bulb tube with
a constricted neck ; (c) a glass rod provided with an india-
rubber band at one extremity ; {d) a pipette of five cubic centi-
metres capacity ; {p) a graduated glass collecting tube ; (/) a
spirit lamp ; {(j) a wash-bottle with distilled water ; (Ji) hypo-
bromous solution. The hypobromous solution is made in
the following way : three and a half ounces (100 grm.) of solid
caustic soda is dissolved in nine ounces (250 grm.) of distilled
water. When the solution is cold, seven drachms (25 c.c.) of pure
bromine are to be added carefully and gradually. The mixture is
not to be filtered : it keeps badly, and for this reason it should
be made shortly before it is required ; or the solution of caustic
soda in water may be made in large quantities as it does not
undergo any change, the bromine in the proper proportion beiug
added at the time it is required for use.

Method.— F'lW the pipette to the mark on the stem with the
urine to be examined ; pour the 5 c.c. of urine thus measured out
into the bulb ; fill up the bulb tube as far as the constricted neck

* Made by Cetti, Brook Street, W.C.

170 PHYSIOLOGICAL CHEMISTRY.

with distilled water from the wash-bottle ; iusert the glass rod (c)
in such a way that the india-rubber band at the extremity fills up
the constricted neck ; the diluted urine should exactly occupy
the bulb and neck of the tube, no bubble of air being below the
elastic band on the one hand, whilst on the other the fluid should
not rise above the band ; in the former case a little more water
should be added, in the latter afresh portion of urine must be
used, and the experiment repeated. After adjusting the glass
rod, fill up the rest of the bulb tube with hypobromous solution ;
it will not mix with the urine so long as the rod is in place.
The water-bath having been previously erected, and the india-
rubber cork fixed firmly into the aperture, the bulb tube Ls to
be thrust from below through the perforation in the cork. The
greater part of the tube is then beneath the water-bath, the
upper extremity alone being grasped by the cork. Fill the
water-bath half full of water, fill also the graduated glass tube
(e) with water, and invert it in the bath ; in doing this no air
must enter the tube, which when inverted should be completely
filled with water. Now slide the graduated tube towards the
orifice of the bulb tube, at the same time withdrawing the glass
rod which projects into the bath through the cork. A.t the
instant that the rod is withdrawn the hypobromous solution
mixes with the diluted urine, and a decomposition takes place
represented thus :

CO]S'oH,+3XaBrO-f2NaHO=3NaBr+3HoO+Na2C03+N2.

Urea-f-sodium hypobromite-j-caustic soda=:sodium bromide
-|-water-|-sodium carbonate+uitrogen.
The nitrogen produced is given off as gas, and displaces the
water in the graduated tube, which is held over it. The gas is
at first evolved briskly, but afterwards more slowly ; to facili-
tate its evolution, the bulb of the tube may be slightly icarmed
with a spirit lamp ; as a rule, however, this is unnecessary.
After ten minutes, the amount of water displaced by the gas
should be read off on the tube, which is divided into tenths.
Each number on the tube represents one gram of urea in 100 c.c.
of urine. Normal urine should yield roughly 1 •5-2*5 parts of nitro-
gen by this test. If 5 c.c. of urine gives off more nitrogen than
fills the tube to iii., dilute the urine with an equal volume of

PHYSIOLOCUCAL CHEMISTRY. 171

water, and take 5 c.c. ; read oft' and multiply by two. If the
urine contain albumen, heat it with two or three drops of acetic
acid, filter, and take 5 c.c. of the filtrate.

(ii.) Lieh'ujs MrtJwd—Thh method is of greater accuracy.
The solutions required are (a) hcmjtd mixt tire = 2 yo\s. of saturated
solution of barium nitrate and 1 vol. of saturated solution of
barium hydrate; (h) standard solution oi inerciiric nitrate, such
that 1 c.c. will precipitate "01 grm, of urea, and (r) a solution of
carbonate of aoda.

Met Jwd.— Take 40 c.c. of urine, add 20 c.c. of («), filter off the
precipitate of sulphates and phosphates ; keep the filtrate. Fill a
burette with {b), and take 15 c.c. of the filtrate in a dish. Let
(b) fall drop by drop into the 15 c.c. in the dish, stirring con-
stantly. Have ready a glass plate with several separate drops
of (c), and from time to time add a drop of the urine mixture
by means of a glass rod to one of the drops. When a yellow
colour first appears in a drop of the NaCOg, the mercuric nitrate
is just in excess. Read the burette. Calculate as follows :

1 c.c. of mercuric solution precipitates -Ql grm. of urea .*. the
No. of c.c. usedx -01= amount of urea in 15 c.c. of filtrate, i.e.
in 10 c.c. of urine. But 10 c.c. of urine usually contains enough
NaCl to act on 2 c.c. of mercury solution. Hence, when reckon-
ing the number of c.c, of standard mercury solution used, a deduc-
tion of 2 c.c. must ahcays be made.

Quantitative Estimation of Chlorides :

Liebifs Method.— The solutions required are (a) baryta mix-
ture as above ; and [b) standard solution of mercuric nitrate,
such that 1 c.c. would be capable of decomposing -01 grm. of
sodium chloride.

Method.— Take 40 c.c. of urine free from albumen, and add
20 c.c. of («). Filter. Take 15 c.c. of filtrate and place in a
flask or dish, adding a drop or two of nitric acid. Fill a burette
with (6), and slowly run some of this solution into the filtrate in
the dish, stirring constantly. As soon as a distinct cloud appears
in the diluted urine, and does not disappear on stirring, then all
the sodium chloride in urine has been decomposed. Read
burette. Calculate as follows :

172 PHYSIuLOGICAL CHEMISTRY.

1 c.c. of mercury solution decomposed -01 grm. of XaCl.
/. the number of c.c. usedx "01 grm. =number of grms. of XaCl
in 15 c.c. of filtrate, i.e., 10 c.c. of urine.

Quantitative Estimation of Phosphates :

The solution required are («) solution of sodium acetate. coniRin-
ing 100 grm. of sodium acetate, lOO c.c. of acetic acid, and 0<)Uc.c.
of distilled water ; (b) a solution of uranium acetate or nitrate^
such that 1 c.c. will precipitate -005 grm of phosphoric acid : and
(c) a solution of ferrocyanide of potasdum.

Method. —Take 50 c.c. of urine. Add some (a) solution, and
heat on water-bath to nearly 100' C. Fill burette with (&), and
allow this to fall into the urine slowly. Have ready a glass
plate with several distinct drops of potassium ferro-cyanide
solution. From time to time add a drop of urine mixture to
one of the drops : and when there first appears a reddish-
brown colour in a drop of potassium ferro-cyanide, all the phos-
phates are precipitated. Eead burette. Calculate thus :

1 c.c. precipitates '005 grm. of phosphoric acid, .'. the number
of c.c. usedx '005 grm. =nuaiber of grms. of phosphoric acid in
50 c.c. of urine.

CHAPTER T.

(A) THE BLOOD.

Ci.) Test the alkaline reaction of the blood in the following way,
as recommended by Schafer :— A drop of blood, obtained by
pricking the finger, is placed upon the smooth-coloured surface
of a piece of dry, faintly reddened, glazed litmus-paper, and
after a few seconds is wiped off with the corner of a handker-
chief or clean linen rag moistened with water. The place where
the blood has stood is seen to be marked out as a well-defined
blue patch upon the red or violet ground.

(ii.) Show that the coagulation of blood is retarded by freezing
and by the presence of neutral salts. Draw a few drops of blood
from the finger into a watch-glass previously cooled in a freezing
mixture ; no coagulation takes place at the freezing tempera-
ture. Add a drop of blood to a little saturated solution of
sulphate of soda in a watch-glass ; the blood does not coagulate.
Two c.c. of blood are placed in a platinum capsule, which is
surrounded by alternate layers of pounded ice and salt. The
capsule is allowed to remain until the blood is frozen ; it is then
removed, and the solid mass of frozen blood is gradually thawed.
The blood on again becoming liquid will be found to be darker
in colour and more transparent than it was previous to congela-
tion. It is in the laJ:e condition, owing to the discharge of the
haemoglobin from the corpuscles into the plasma.

The tests for blood are (iii.) the examination by the spectro-
scope when oxyhremoglobin and its derivatives give cbaracteristic
absorption bands, to be presently mentioned (p. 174).
(iv.) The formation of oxyhiemoglobin crystals (p. 55).

174 PHYSIOLOGICAL CHEMISTRY.

(v.) The formation of hsemin cr3-stals (p. 5G).

(vi.) The guaiac test. — A drop or two of tincture of guaiacum
is added, and mixed by shaking with the suspected fluid ; to the
mixture is added some ozonic ether ; if blood be present a
sapphire-blue colour appears at the junction of the fluids, and
mounts upwards through the ozonic ether, bubbles of gas being
at the same time evolved.

Hsemoglobin and its Derivatives.— (i.) Dilute defibrinated
blood with ten or twelve times its bulk of distilled water, place
some of the solution in a test-tube (12 cm. x 2 cm.) or other
suitable vessel, and examine with spectroscope. The whole of
the spectrum will be seen to be cut off except the red. Dilute
some of the above four times, and it will be found that an
absorption band extends from D (sodium band) to a little
beyond E (middle of green) ; but that the red, orange, yellow
and part of the green will be seen. On further dilution (five
times and beyond) the broad band will be found to be replaced
by two narrower bands separated by a green interspace. Of the
two bands, the one to the left next to D is the narrower and
deeper. On extreme dilution with water both of the bands dis-
appear, but of the two the band D persists the longer. Before
the bands disappear they are seen as faint shadows. These
appearances indicate the presence of oxyhcemoghhin.

(ii.) To a solution of blood (1 in 20) add a few drops of
ammonium sulphide* (NH^HS) and thoroughly mix ; the solu-
tion becomes purplish and on spectroscopic examination the
single band of reduced hcernoglohin will be seen. It is fainter
and with less sharply defined edges than those of oxyhsemoglobin,
extends from D to E, none of the green being visible in the
midst. On dilution the band soon disappears, but as long as it
remains, continues single. There is in addition more absorption
to the left and less to the right end than in (i.).

(iii.) Pass CO for some time through blood diluted as in (ii.)
•On examination the two bands of rarhonic oxide IwimogJohhi
may be observed. These bands are almost exactly similar to

* This we find to be the most cojivenieut reducing agent, but Stokes'
fluid, a sehitiou of ferrous sulphate, to -nbidi is added tai-taric acid, and
then XH4HO until reaction is alkaline, or a solution of a stannous salt
treated in the same way, may be used.

PHYSIOLOGICAL CHEMISTIIY. 175

those of oxyhasmogloltin, but ;ire slightly nearer the violet end,
and are l>esides more equal and deeper. The solution resists
reducing agents. Nitric oxide gas has much the same effect.

(iv.) To a further portion of diluted blood add some drops of
acetic acid, and boil. The liquid becomes brown and the spec-
trum shows a distinct band in the middle of the red (to the
right of C), indicating the presence of cu'k] lidinatin. After
cooling, add ether and shake up well. The ether on settHng at
the top is seen to be of a brown colour, and on examination
shows three bands, besides the C band, viz., a very fjunt narrow
band to right of D, a broad band to left of E, also a faint and
third about midway between E and F.

(v.) As in (iv.), but add excess of ammonia : a band is seen in
the red, but nearer D {idhaline luv.mat'iu).

(vi.) To liquid (v.) add a reducing agent ; a spectrum showing
two bands somewhat like those of oxyhtemoglobin, but more to
the right, is seen : Stokes' reduced hnnnatln or ha-rno-chroinaqen.

(vii.) Dissolve some dried blood in saline solution. It will
$how a spectrum of methcemofjlobin, consisting of the two bands
of oxyha3moglobin ; another band in the red near C, like that of
(iv.). On the addition of ammonia the last band is replaced by
one close to the left of D. On addition of ammonium sulphide the
spectrum of (ii.) will appear, and on shaking with air that of (i.)

(viii.) Dissolve a little fresh blood in strong sulphuric acid
and, if necessary, filter through asbestos ; a beautiful deep red-
coloured fluid is the result — Ticematoporphyrin. It gives two
bands, one at D, and a dark well-defined band about midway
between D and E,

Estimation of the Colouring Matter of the "Eloo&.—Hcamo-
{jlohinometerJ' A method of approximately estimating the
amount of the hemoglobin in blood has been devised by Dr.
Oowers, with an instrument called a hannoglohinometer. The
theory of the apparatus consists in comparing a given sample of
the blood to be examined, diluted with given quantities of
water, with a standard colour solution representing the colour
of a normal (1 per cent.) solution of blood. The apparatus con-

* Made by Hawkeslev, Oxfor>l Street.

176

PHYSIULOGICAL CHEMIhTKY

sists of two glass tubes of exactly the same size. One contains
(d) a standard of the tint of a dilution of |20 c. mm. of
blood, in 2 c.c. of water (1 in 100), composed of glycerine
jelly tinted with carmine and picrocarmin. The second tube (c)
is graduated, 100 degrees being equal to 2 c. (100 times 20
c. mm.).

Fig. 25. — The KcTemoglobiuometer (Gowers).

The 20 c. mm. of blood are measured by a capillary pipette
(b) (similar to. but larger than, that used for the hsemacyto-
meter). This quantity of the blood to be tested is ejected into
the bottom of the tube, a few drops of distilled water being
first placed in the latter. The mixture is rapidly agitated to
prevent the coagulation of the blood. Distilled water is then
added drop by drop (from the pipette stopper of a bottle (a)
supplied for that purpose), until the tint of the dilution is the
same as that of the standard, and the amount of water which
has been added {I.e. the degrees of dilution) indicates the amount
of haemoglobin.

Since average normal blood yields the tint of the standard at
100 degrees of dilution, the number of degrees of dilution
necessary to obtain the same tint with a given specimen of

PHYSIOLOCJICAL CHEMISTRY. 177

blood is the percentage proportion of the hfemoglobin contained
in it, compared with the normal. By ascertaining with the hajma-
cytometer the corpuscular richness of the blood, wc are aljle to
compare the two. A fraction, of which the numerator is the
percentage of hemoglobin, and the denominator the percentage
of corpuscles, gives at once the average value per corpuscle. In
using the instrument, the tint may be estimated l)y placing a
piece of white paper behind the tubes ; some light is, however,
reflected from the suspended corpuscles from which the haemo-
globin has Ijcen dissolved. It will be found that during six or
eight degrees of dilution it is difficult to distinguish a difference
between the tint of the tubes. It is therefore necessary to note
the degree at which the colour of the dilution ceases to be
deeper than the standard, and also that at which it is distinctly
paler. The degree midway between these two will represent
the haemoglobin percentage.

Serum :

(i ) Take some clear serum in a large test-tube, and add to it
an excess of solid magnesium sulphate or sodium chloride.
Shake violently. A flocculent precipitate of paraglohulin is
obtained ; separate it by filtration.

(ii.) Dissolve the precipitate by washing the filter containing
it with a small amount of distilled water (it being soluble in
dilute saline solution, which is thus made by the water dissolving
the adhering salt), and with part of the solution ascertain the
temperature at which the globulin coagulates. Retain the
remainder (</),

(iii.) In filtrate from (i.) show presence of smua-alhumni by
heat, noting the temperature at which it coagulates. Filter.

(iv.) Test filtrate from (iii.) for any proteid remainder, also for
cJdor'ide.^ and plw^phates.

(v.) Dilute serum with ten times its volume of water and pass
a brisk stream of C0.> through the solution ; a precipitate of
paraglohulin will a})pear. Separate by filtration, and dissolve as
in (ii.). To one portion of the solution apply the globulin tests ;
retain the second portion (/S).

12

178 PHYSIOLOGICAL CHEMISTRY.

Hydrocele Fluid :

(vi.) Take some hydrocele fluid and treat as in (i.) ; a pre-
cipitate of p'bn'nogcn occurs. Separate it.

(vii.) Dissolxe fihriiiogfii, as in (ii.), and proceed in exactly
similar manner, and taking one portion of solution, find the
temperature at which it coagulates, and retain the other
portion (y).

(viii.) As in (iii.), show the presence of serum-albumin, and
after its precipitation and removal by heat and filtration, test the
filtrate for any proteid residuum, and for chlorides.

(ix.) Precipitate fibrinogen by the CO., method as in (v.) ;
apply the globulin tests to one portion and retain the other (r).

Coagulation :

(x.) Take fresh serum and add it to an equal quantity of fresh,
clear hydrocele fluid (faint yellowish to green in colour, S.G.
lOlG-1022), and keep it at a temperature of about 37' C. After
a variable time a clotting will occur.

(xi.) Add together solutions (a) and (y) ; clotting will most
likely occur (if the fluids from which they have been obtained
were fresh).

(xii.) Add together solutions (/3) and (r) ; clotting does not
occur.

(xiii.) Allow horse's blood to flow into the saturated solution
of magnesium sulphate or sodium chloride and thoroughly mix ;
set aside in ice for a day or two. Draw ofl: supernatant liquor
of so-called salted plasma, dilute one portion with many times
its bulk of water, or place in dialyser ; a clot will after a while
form if kept at about 37 C. Saturate the other with solid mag-
nesium sulphate or sodium chloride ; a precipitate of i>lasmine
will form,

(xiv.) Treat plasmine as in (ii,), and the solution should
clot.

(xv.) Treat plasma with an equal volume of 13 per cent,
solution of sodium chloride ; fibrinogen will be separated ;
remove by filtration and dissolve as above, and then add solid
sodium chloride to saturation, and paraglobulin falls. Remove

i'HVsi(>i,()(ii('Ai, (iii'Misritv. 170

by filtration ami dissolve. Tlic solutions when mixed should
after a time clot/'

Fibrin : See p. 1 ')'>.

(B) MILK.

An opaque white fluid :

(i.) Examine a drop of milk under the microscope with a high
power. See that it consists of fat globules of different sizes in
II clear fluid. Add dilute acetic acid by irrigation, and observe
the coalescence of the globules owing to the excess of acid
dissolving their casein membrane.

(ii.) Test the alkaline reaction oi fresh milk (London milk may
be slightly acid) ; it becomes acid on standing, owing to the
formation of lactic acid by fermentation, whilst in consequence
the casein separates.

(iii.) Take the specific gravity of fresh milk with the hydro-
meter, 1025-30— skimmed milk is higher. To 50 cc. of skimmed
milk add water gradually from a burette till the specific gravity
is brought down to normal. Read off the amount of water
required.

The constitnenis whose presence is to be demonstrated are oU
or fa f, casein, serum-albxmin, lactone or milk Hiif/ar, and sal(.^.

(iv.) Fat. — To a portion of milk, add its own volume of caustic
potash or soda, and warm the solution f/cntli/ ; the oil globules
will be set free from their enveloping casein. Cool, add ether,
shake the mixture, and allow it to stand. The fat will be
dissolved in the ether, and will form with it a clear superstratum.
Remove the transparent top layer with a pipette, evaporate off
the ether ; the oil will be left, and will give the cluiracfcrhlic
f/reasf/ spot vlien drojiped upon paper.

(v.) Casein. — Dilute some of the milk with its own bulk of
water ; add a few drops of dilute acetic acid until a slight
granular precipitate is formed. Warm the solution genii// to
40° C, and a copious flocculent precipitate will appear. Filter
off, and label the precipitate 'A.'

(vi.) a. Serum albtindn. — Boil the clear filtrate (from A); a

* Many other experiments ou coigulation may be done if time permit,
e.g., use of tibrm femient.

12—2

180 PHYSIOLOGICAL CHEMISTRY.

coagaium of albumin will be formed. Filter this off, and label
the precipitate ' B.'

b. Ackl-alhumin. — Exactly neutralise the clear filtrate (from
B), with caustic potash or soda ; a precipitate of albumin which is
soluble in acids may appear.'-'" Filter off precipitate, and label
ic'C

(vii.) Lado^c. — Test the clear filtrate (from C) by Trommer's
test for sugar.

(viii.) To precipitate A in a test-tube add nitric acid, the pre-
cipitate is dissolved ; boil, and when cold add strong ammonia ;
the solution becomes orange coloured {xanthoproteic test).

(ix.) To a second portion of precipitate A add sodium phos-
phate ; the precipitate will be dissolved : add dilute acetic acid
to the solution till a neutral reaction is just obtained ; no pre-
cipitate occurs : add more acetic acid ; a precipitate is thrown
down (since acid albumin is now present) — casein or aJlcil'i-
alhnmin is present.

(x.) To a third portion dissolved in caustic soda or potash add
lead acetate ; a black precipitate of lead sulphide is formed.
Therefore sulphur is present ; consequently casein, and not
artificially produced alkali-albumin, is present.

(xi.) Test the precipitate on filters B and C, by boiling with
JliUon's reagent: a pink colouration will in each case be produced,
sliowing that albumen is present.

(xii.) Test a second portion of the clear filtrate from C for (i.)
phosphates^ by the addition of ammcnio-sulphate of magnesia; a
precipitate is formed : also by molybdate test ; and for (ii.)
chlorides by the addition of silver nitrate to another portion
which has been acidulated with nitric acid ; a curdy precipitate
will fall, soluble in ammonia.

(xiii.) Milk may be curdled in several ways :

(a) By rennet. — Add some extract of rennet to some milk

warmed to about 45^ C. Observe : the coaguhim

of casein.

(Z/) By acid, as above. — Take two poitions of curd

precipitated by acid ; dissolve one in caustic soda

* Tliis acifl albnmm lias been produced by the previous acidification of
the milk iu the precipitation of casein.

PHYSIOLOGirAL CHEMISTRY. 181

and the other in lirae-water, and filter each. Add
some rennet to both filtrates, and observe the result.

Lime solution coagulates.

Soda „ does not coagulate,
(f) By mlt. — Shake some milk in a test-tube with solid
Fodium chloride or magnesium sulphate : filter and
dissolve (Qy.) some of the curd in water and add
rennet. Observe that it coagulates.

(C) THE BILE.

The bile is a more or less viscid fluid of a colour varying from
deep yellow to green or brown.

(i.) Test its reaction. Alkaline or neutral : and its S.G-. —
1018-20.

(ii.) Xeutrali.s3 and boil. There is no precipitate, which indi-
cates the absence of albumins.

(iii.) Dilute with water and acidify with acetic acid. There
will be a precipitate of mucin. Filter. (Dissolve the precipi-
tate on the filter in lime-water, and test with Millon's reagent
and with had acet'de).

(iv.) To a portion of the filtrate from ,'iii.) apply Gmelin's test
for bile pigments.

Gmelins T^st for Bile Pigments. — Place a drop of bile on

a white plate, and add a drop of strong yellow nitric

acid to it. A play of colours is produced. Green,

blue, violet, red. and yellow appear in succession.

(v.) To another portion of the filtrate apply Pettenkofer's test

for bUe salts.

Pettenhofers Test for Bile Salts.— X.M a few grains of
white sugar, or a drop or two of syrup, to a solution
of bile in a test-tube, shake well, add strong sulphuric
acid, and cool. A reddish-purple colour is pro-
duced,
(vi.) To another portion of the filtrate add chloroform and
warm slightly. This extracts the colouring matter (hdiruhin).
Remove the chloroform solution with a pipette and examine
it with spectroscope. Observe : absence of absorption bands
Apply Gmelin's test.

182 PHYSIOLOGICAL CHEMISTRY.

(vii.) Bile shaken up with oil divides it into very small
globules (an emulsion being formed). This can under very slight
pressure pass through animal membranes ; oil alone cannot.

(viii.) Take two filters, wet the one with water, the other with
bile, and pour a small and equal amount of oil on each. Oil passes
slowly through the one wetted with bile, not at all through the
other.

(ix.) Bile precipitates pepsin and peptones. Add a considerable
quantity of bile to a solution of fibrin digested in artificial gastric
juice ; a precipitate of pepsin and peptone will fall, and the
solution will be found to have lost its proteolytic properties.

Bile Acids, Glycocholic (CfiH^aXOg), and Taurocholic
(aeH,5XS0,).

Preparation. — ^lix bile, which has been evaporated to one-
fourth of its bulk, with animal charcoal, evaporate to perfect dry-
ness on a water bath, and extract it, whilst still warm, with
absolute alcohol. The alcoholic filtrate should be colourless; if
this is not the case, more charcoal must be added. The alcohol
is distilled off, and the dry residue is treated with absolute
alcohol. The alcohol is then filtered off, and to the filtrate
anhydrous ether is added as long as a precipitate is thrown
down. The solution and precipitate are to 1j3 set aside in a
closely-stoppered bottle for some daj-s, when crystals of h'liin
will be produced. If the reagents were not perfectly anhydrous,
a gelatinous mass will be formed, but no crystals. Bilin consists
of glycocholic and taurocholic acids, which may be separated
from one another by dissolving it in water, and adding first
solution of neutral lead acetate, and then a little basic lead acetate.
This combines with glycocholic acid to form an insoluble lead
glycocholate. Filter, and add to the filtrate basic lead acetate
and ammonia ; a precipitate of lead taurocholate will be formed,
which may be filtered off. In either case the lead must be got
rid of by suspending or dissolving in hot alcohol, adding hydro-
gen sulphate, filtering, and adding water.

Cholesterin. — It is contained in bile, gall-stones, nervous
matter, etc.

Preparation. — Usually by extracting powdered gaU-stcnes
with ether or boilinsr alcohol. Cholesterin crvstallizes out on

PHYSIOLOGICAL CHKMISTRY. 183

evaporation of the extract. The insoluble residue consists of
bile, colouring matter, and mucin.

Properties and Tenltt. — Crystallizes in rhombic plates. Examine
under the microscope.

It is insoluble in water and cold alcohol ; freely soluble in
boiling alcohol and in ether.

It gives a red colour with strong sulphuric acid and with nitric
acid and ammonia.

It gives a greenish blue to violet with sulphuric acid and iodine
or zinc chloride, and with suliihuric acid and chloroform a play
of colours, beginning with blood-red, and ending with green,
afterwards disappearing.

Analysis of Gall Stones :

(i.) Make an ethereal extract. Allow a few drops to evapo-
rate on a slide, and examine for cholesterin crsstals with a
microscope.

To a portion of the extract add strong sulphuric acid ; a bright
red colour indicates choleMerin.

(ii.) Boil the residue (coloured; in water with a few drops of
acid ; pour oflE the water, and add some warm chloroform.

Eximine the chloroform solution for bilirubin as above.

CHAPTER YI.
THE DIGESTIVE FLUIDS.

(I.) SALIVA.

Procure a copious flow of saliva by touching the tongue
with a crystal of tartaric acid, avoiding, as much as possible,
any mixture of the acid with the saliva.

Salivary Digestion.— Take five test-tubes containing equal
quantities of starch paste, and add to each equal quantities of
saliva, (i.) Boil briskly and place in the incubator ; (ii.) Place in
ice and salt ; (iii.) Keep at the temperature of the room ; (iv.)
Place in the incubator ; (v.) Render distinctly acid and place in
the incubator. At the end of ten minutes test all for sugar by
Trommer's test, (i.), (ii.), and (v.) will contain no sugar ; (iii.)
a little ; (iv.) abundance.

Composition of Saliva :

(a) Inorganic Constituents. — Test qualitatively, small
portions, for the following :

1. Carhonates. — Shake with a small quantity of any strong acid.
Bubbles of CO^ will be evolved.

2. Chlorides. — Acidulate with nitric acid, a precipitate with
silver nitrate, soluble in ammonia.

3. Phosphates. — A yellow precipitate with ammonium molyb-
date on boiling.

4. Sulphates. — A precipitate with barium chloride or nitrate,
which does not disappear on boiling with nitric acid.

5. Potassium. — A violet colour in Bunsen flame, visible through
blue glass.

rHYSIOLCKJUAL CHEMISTUY. 185

(5. Sodium. — Yellow flame.

7. Ctilciuui. — Precipitate by amraonium oxalate.

K. Muf/iifsiuiii. — Add ammonium chloride and ammonia first,
then sodic phosphate ; a precipitate of ammonio-magnesium
phosphate will result.

0. Potas.-<itnn Suljyhoct/anate. — It gives a reddish colour on
adding ferric chloride, which is not altered by hydrochloric acid,
but discharged by mercuric chloride (meconic and acetic acids
give somewhat similar colour, which is discharged by hydrochloric
acid in the former and by mercuric chloride in the latter case).

(l{) OlKJAMC CoNSTlTUKNTS :

1. Acidulate with acetic acid ; a precipitate of i.iucin. Filter.

2. Apply to some of the filtrate the proteid reactions.

;5. Saturate more of the filtrate with solid sodium chloride :
a precipitate of fjlohuUn appears.

(II.) GASTRIC JUICE.

(a) Pepsin. — Show the digestive action of pepsin by means of
glycerine extract of pig's stomach or solid pepsin.

Take three test-tubes containing (i.) hydrochloric acid solu-
tion '2 per cent, and fibrin ; (ii.) pepsin, water, and fibrin ; (iii.)
pepsin, hydrochloric acid "2 per cent, solution, and boiled fibrin:
also (iv.) a flask containing pepsin, hydrochloric acid '2 per cent.,
and fibrin, all in larger quantities than above. Place all in
incubator for fifteen minutes. Xote that in (i.) the fibrin swells
and becomes transparent, but does not dissolve ; in (ii.) and (iii.)
the fibrin is unchanged; in (iv.) the fibrin is dissolved. Filter (iv.).

Neutralise filtrate ; a fairly copious precipitate of syntomn^
imrapeptone or anti-albumose (Kiihne) results. Filter. To filtrate
add strong nitric acid precipitate of so-called A peptone (Meissner)
or heni'udbumose (Kiihne}. Filter again and add potassium
ferrocyanide, a precipitate of B peptone. Filter this, and apply
to the filtrate picric acid and test for C or true peptone^ etc.
(p. loG).

lieactioits of pure Pep-^in :

1. Should not be precipitated by nitric acid, tannic acid,
iodine, or mercuric chloride.

2. Is precipitated by platinum chloride, and neutral lead
acetate.

186 PHYSIOLOGICAL CHEMISTRY.

(b) Ranneb. — The action of the curdling ferment of the
stomach upon milk has already b2en shown.

(III.) PANCREATIC JUICE.

The digestive action of the pancreas may be tested by means
of extracts of the gland of various kinds in many ways. Take
the pancreas of a pig, dog, or ox, and remove all adhering fat,
chop it up into small pieces and treat in the following ways :
(i.) Pound in mortar with distilled or slightly acidulated water,
keep at 25' C, for an hour — strain through coarse muslin ; (ii.)
pound in mortar with powdered glass, slightly acidulate with
dilute acetic acid, leave for half an hour, and then add ten to
twelve times its volume of glycerine ; thoroughly mix. (iii.)
Proceed as in (i.), but use brine instead of water ; (iv.) make
an extract of the fresh gland with water or glycerine ; but with-
out acid or long exposure to the air.

\_Imtead of these extracts certain commercial prej^iaraiions matj
he emploj/ed, e.g., Benger's Liq. Pancreatic ns,'\

1. Add a few drops of one of the above preparations and
some sodium carbonate — 1 per cent, solution — to some starch
mucilage in a test-tube, expose in a water bath at 4U C. for fifteen
or twenty minutes, test for sugar. The intermediate products
(eri/fhro-dextr in, giving a red colour with iodine: and then «(?/<roo-
dextrin, giving no colour with iodine, but precipitated by alcohol)
maybe tested for, if the action is allowed to go on only for a less
time and be stopped by boiling. Experiments proving the con-
ditions under which the ferment acts are similar to those described
for the salivary ferment. Do them.

2. In a similar manner place some of the extracts (i.) or (ii.)
and (iv.) in test-tubes with the alkaline solution as in 1, and add
to each a fragment of boiled fibrin : place them in a water bath
as above for half an hour, neutralise, filter, boil and refilter, and
test filtrate for peptone; the test-tube containing (i.) or (ii.) will
contain more peptone — indicating that extract (iv.) contains less
of active ferment.

'6. As in 2, but boil one solution; no solution of fibrin takes
place ; allow the unboiled digestive fluid to remain in water-bath

PHYSIOLOCilCAL CHEMISTRY. 187

for an hour, and watch, from time to time, the gradual erosion
of the edges of the fibrin, noting that the fibrin does not swell
up and become transparent as in gastric digestion.

4. For intermediate products of pancreatic proteolytic
digestion, viz., Jidni and ai(ti-alhiinio.se {Kiilm'} or ulkali-nlbuui'ia
€tr., neutralise the fluid after half an hour's digestion with weak
acid, and a precipitate occui-s ; and take out the undissolved fibrin,
and show that it is easily soluble in 1 per cent, hydrochloiic acid
and in 10 per cent, saline solution. The solution in the latter is
precipitated by heat and by nitric acid. This shows that the
fibrin has boen converted into a soluble form, of a globulin type.

it. Add some of the most active extract to a larger quantity of
fibrin and sodium carbonate solution in a beaker ; allow the
action to go on for two hours at 40' C, then neutralise with
acetic acid, filter, boil, and refilter. Evaporate the filtrate to one-
third of its bulk, add absolute alcohol while still hot — leave it
for twenty-four hours to precipitate peptones — filter, concentrate
the filtrate ; ///ros/;<^ will crystallise out : pour off mother liquid,
and concentrate still further ; leurin will crystallise out.

G. In a pancreatic digestion fluid, when the action has gone on
for twenty-four hours at least, notice its smell, and test for imlol
— by acidulating with dilute sulphuric acid — boiling, and adding a
dro]) of very dilute nitrous acid ; a reddish-pink colour should
apj)earat once or on standing. If the solution give a purple colour
with chlorine water, the associated hody, naphthalamine^is present.

7. Add some of (i.) neutralised if necessary, to melted lard at
37' C, in proportion of 2 to 1 ; rub together over a water-bath — a
thick creamy emulsion will result.

(X.B. — Saponification and emulsion of oil by pancreatic extract
are neither certain nor satisfactory.)

8. Add some of (iii ) or (ii.)to milk in a test-tube, at tempera-
ture of 40' C. ; notice that the casein is soon precipitated, and
then dissolved, forming peptone ; also in another test-tube do
the same experiment with milk diluted \ : note that precipitation
does not occur, but that in a few minutes after the addition of
the extract the casein can be precipitated on boiling {melacasein)
as well as on addition of dilute acid.

(TV.) THE BILE. See Chapter V., pp. ISl-lS;!.

CHAPTER Yll.
UNHEALTHY URINE AND CALCULI.

Albumen present. — (i.) If the urine be neutral or acid, the
albumen is precipitated on boiling. If alkaline, render slightly
acid with nitric acid or acetic acid, and boil. Albumen is pre-
cipitated.

Care must be taken, in testing for albumen in urine, that the
test-tube which is employed be clean and free from acid. Per-
form the following experiment to show the necessity of this
precaution. To a small quantity of albuminous urine add an
excess of strong nitric acid ; a precipitate of albumen is thrown
down. Pour the contents of the test-tube away, and without
washing it, fill it up with a fresh sample of the albuminous urine.
After allowiog it to stand five minutes, boil the solution, when,
although albumen is known to be present, no coagulum will be
formed, since the acid remaining in the test-tube from the pre-
vious experiment has been sufficient to convert the albumen
into acid albumen. The experiment will frequently, however,
be unsuccessful, unless a large excess of the strong acid has been
first added,

(ii.) .1 more delicate test. — Acidify urine with acetic acid ;
mucus will be precipitated if present. Filter, and add ferro-
cyanide of potassium to clear filtrate ; a precipitate will be
formed in presence of albumen.

(iii.) Heller's test (p. 150).

Bile pigment or acid present. — Adopt tests (p. 181).

Uric acid present. — Employ the murexide test (p. 168).

PHYSIOLOGICAL CHEMISTRY. 1-^9

Urates present. — The dei>osit dissolves on heating ; some-
times reappears on cooling. Urates dissolve in canstic alkalies ;
uric acid is separated on adding strong acids. Apply raurcxide
test to the deposit.

Phosphates present. — The phosphates may be in solution,
or may form a deposit. If in solution the urine is feebly acid
or neutral. On boiling urine, the phosphates are deposited, the
deposit being soluble in weak acid. Deposits of phosphates are
insoluble in caustic alkalies.

The phosphates are either iu the fonn of aniorphons phosphate of lime
(Ca3iP04'ji. crystalHzed pliospliate of lime i'JCaH.PC)4l, or ainmoiiio-maK-
uesiuui pliospiiate or triple i)hosi>hate (Mg(NH4lP04 -i-GHjOj, sometimes
precipitated all together, or the lirst aud third varieties.

The reason why phosphates are deposited on boihng in urine which wjs
before dear is uncertain, but possibly it may be due to the action of beat
exi>elling cai'bon dioxide, or decomposing ui'ea into ammonimu carbonate,
which renders the urine alkaline.

Oxalates present. — The deposit is soluble in hj-drochloric acid,
but insoluble in acetic acid. Examine their crj'stalline form
with a microscope.

Sugar present Diabetes). — Specific gravity generally high,
103<> to 1 <».')•». Apply Trommers test, or any of the others
mentioned (p. 163).

Quantitative estimation of grape sugar.*

1. Fc}dii\ii'.-< Mitlujd. — S'-Autiun r(i2u'u-id = co\^\ieT sulphate and
caustic soda, with some sodic potassic tartrate of such a strength
that 10 CO. of solution contain the amount of cupric oxide which
•05 grm. of sugar can reduce to cuprous oxide. (This solution
should be freshly prepared.)

Method. — Remove any albumen which may be present in the
urine by boiling and filtering. Take 10 cc. of the urine, free
from albumen, and add Ou cc. of distilled water. Place this in a
burette. Put into a flask or dish 10 cc. of the standard solution,
and dilute with four times its bulk of water and boil. Run into
it. from burette, some of the diluted urine, say 20 cc, and boil.
Allow precipitate to settle, and if supernatant fluid is still blue,

* FehUnr/'s sohifion is made as follows : Take of sulphate of copper, 40
grms. : neutral tartrate of potasb, 160 gnus. ; caustic sotla (sp. gr. I'l'l),
750 grms. ; add ihstilled water to 115 i-5 cc. Each 10 cc. contains 05 ^rm.
of sugar.

190 rilYSIOLOGICAL CHEMISTRY.

add, say, 5 cc. from burette, and boil again, and so on, till the
fluid ceases to have a blue tinge, taking care, towards the end of
the process, to add only a few drops each time. If, after add-
ing 20 cc. of diluted urine and boiling, the fluid has been de-
colourized, too much urine has been added, and another 10 cc. of
standard solution must be measured out, running in less than
2'3 cc. (say 10 cc.) in first instance.

When you have thus determined the number of cc. of diluted
urine required to decolourize the solution, that volume contains
the amount of sugar necessary to reduce 10 cc. of standard solu-
tion, i.e., '05 grm. But one-tenth only of this is urine, .'. one-
tenth of number of cc. used contains 'O.^ grm. of sugar. From
this you can easily calculate the percentage.

2. Pacijs Jlodijication of Fehlinr/'.s Method. — By Fehling's
method it is difficult and tedious to judge of the point of com-
plete reduction of the cupric oxide. Dr. Pavy, accordingly, u^es
a strongly ammoniacal solution of the above. A certain amount
is introduced into a small flask, which is then heated till the
vapour of ammonia escapes by a narrow tube. The sugar solu-
tion is then allowed to flow from a burette into the flask until
the blueness has disappeared, the solution being kept boiling all
the time. The blueness is apt to disappear suddenly, and care
should therefore be taken towards the end of the process.

Calculate as in Fehling's method.

3. Esti/nation of sugar bij fermentation. — Take specific gravity of
urine before and after fermentation. Each degree of specific
gravity lost by the urine represents one grain of sugar per ounce
of urine.

4. Sugar may also be estimated by adding yeast to urine, and
collecting the carbon dioxide evolved. The carbon dioxide is a
measure of the amount of sugar present.

Blood present. — Examine the deposit formed on standing,
with the microscope, for blood corpuscles ; add to another por-
tion a drop of tincture of guaiacum, and about a drachni of
ozonic ether : a blue colour will a]>pear at the junction of the
fluids.

Pus present. — Examine the deposit with the microscope.
Add caustic potash ; the urine becomes stringy.

PHYSIOLOGICAL CHEMISTRY. 101

Carbolic Acid in Urine. — The urine is dark olive-green or
black when first passed : on standing a deposit resembling
altered blood often takes place, and the urine becomes lighter in
colour. On the addition of strong sulphuric acid, the odour of
tar is exhaled from the urine. The addition of perchloride of
iron develops a blue colouration.

Salicylic acid in urine gives a purple colour ^vith the per-
chloride ot" iioii.

Chylous Urine.— The urine may be clear or milky -svhen
passed ; on standing it coagulates, forming a tremulous mass
which after a time liquefies. Examine for albumen, molecular
fat, and the nematoid •worm, Jilart'a samjuinis Jtominis.

URINARY CALCULI AND DEPOSITS.

(i.) Ignite a small portion on platinum foil. If it burn away
completely, it is probably uric acid. To confirm this apply
mui*exide test (p. 1(38).

(ii.) Boil the powdered calculus with distilled water, or, if a
urinary deposit, with the supernatant urine.

The powder or deposit may be dissolved wholly or partially —
or undissolved.

(a) Dissolved portion con- (u) Undissolved portion
sists of may consist of

Urates— ^h\ch. are mostly Phosphates,

deposited on cooling. Calcium oxalate^

Test for urate of ammonium Uric acid,

by boiling with potash to de-
monstrate presence of am-
monia ; and by murexide test
for the uric acid. If it is not
ammonium urate it is probably
potassium or sodium urate ; I
for either base, test in the
ordinary manner. ;

192 PHYSIOLOGICAL CHEMISTRY.

(iii.) Take some of (r-), add a few drops of hydrochloric acid,
and boil.

(< ) Dissolved.
Phosphates,
Calcium o.ralate.

(d) Undissolved.

Uric acid.
Confirm bv murexide test.

(iv.) Take some of (c) solution and add excess of ammonia; a
precipitate will fall in either case : add acetic acid in excess ;
the precipitate is

(e) Dissolved. (f) Undissolved.

Phosphates. Oxalates.

Confirm by molybdate test.

If the precipitate in (c) solution is partially dissolved and
panially undissolved, phosphates and oxalates are probably
mixed.

TABLE OF ABNORMAL URINES.

Urines may be abnormal in :

(i.) Colour :

Blood (red, or smoky), "\

carholic acid (h\2,Qk). 'Too Albaminous urineA Tco
Bile (brown), ci/.-<iin ■ dark. Chi/Ious ,, pight.

(yellowish green). j

Excess of nitrogenous constituents (orange).
Effects of drugs, e.g., rhuharh, red.

(ii.) Smell :

Sweet in diabetic urine.

Very ranh in urine containing excess of urea or urates.

Ammoniacal in decomposing urine.

"When cgstin is present, sweethriar.

(iii.) Reaction :

Alkaline, with excess of phosphates.
Stronghj acid, with urates in excess.

(iv.) Specific Gravity :

Too high in diabetes mellitus, and in excess of urea.

Too lov, in chronic Bright's disease, hysteria, and anasmia.

PHYSIOLOGICAL CHEMISTRY.

VXl

Abnormal urines
(a) Contain no
sediment.
All)unic'n.
Phosphates.
Sugar.
Bile.

Carbolic acid.
Blood.
Salicylic acid.

may

(i:) Contain sediment on standing.

Urates.

Phosphates (some-
times).

Pus.

Mucus.

Oxalates.

Uric acid.

Albumen (sometimes)

Carbolic acid (some-
times).

Cystin.

Chylous urine (be-
comes transparent
when shaken up
with ether ; test
for albumen).

(a) If containing no sediment.

Boil.

Precipitated.

Not precipitated.

Albumen.

Sugar.

FhosjyJiates.

Bile.

Blood.

Carbolic acid.

Add nitric acid.

bahcyhc acid.

Precipitate

Undissolved

Apply Gmelins test (p. 181).

dissolved.

Albumen.

Play of No play of

Phosiphatex.

Blood.

sample

colours. colours.
Bile. Sugar.

M It h fresh

of urine

apply

Carbolic acid.

fjuaiacuii

I test

Salicylic acid.

(p. 174).

Apply 2'rommer'x test (p. IG:^).

Blue N

0

Reduction of No reduction.

colour. colon

ration.

copper. Carbolic acid.^

Blood. Alb

I men.

Sugar. Salicylic acid.'^

(Examine w

ith the micros

sCope.)

* Apphj special tests (p. 191).

13

194

PHYSIOLOGICAL CHEMISTRY.

(b) If containing a Sediment :

BoU.
Precipitate or sediment
dissolved.

Urates.

Sediment undissolved.
Fhosphatcs — increased.
A Ibumen — increased.
Pus. C'l/stin.

Mucus.
Oxalates.
Uric acid.
Carbolic acid (see above).

Add nitric add.
Precipitate dissolved. Precipitate undissolved.

Fhoq>hates (soluble in
acetic acid).

Oxalates (insoluble in
acetic acid. Examine
sediment ■with micro-
scope, octahedral crjs-
tals or dumb-bells).

Cystiii (do. hexagonal
plates).

Albumen — increased.

Pus.

Mucus — not increased.
Uric acid.

Add caustic potash to fresh portion.

Dissolved.
Uric add.
Pus (converted into

a glairy mass).
Mucus.

Apply confirmatory
tests.

Undissolved.

Albumen.

CHAPTER YIII.
EXAMINATION OF ORGANIC SUBSTANCES.

The following notes may be useful. They are not by any
means exhaustive of the subject ; and the advanced student is
advised to supplement them by careful manipulation.

(I.) PROTEIDS.

Plan of examination of a solution containing one or more
proteids, with and without other substances.

1 . Notice whether clear : if not, filter,

2. To the filtrate or to the original solution apply the proteid
reactions, with HXO3, with Millon's reagent and with copper
sulphate and caustic potash.

i). Try the reaction of the filtrate or original solution :
May be (a) acid,

(b) alkaline,

(c) neutral.

If (a) or (h) carefully neutralize :

A precipitate indicates acid or alhaU-alhumin, as the
case may be, if, when re-dissolved in dilute acid
or alkali, after filtration, the solution gives the
proteid reactions.

4. Boil some of the original solution. A precipitate indicates
{(l) a ha'ii'e-alhuiii'in^ or {e) a, fjlobidin.
To distinguish {(J) and (e) :
r-. Saturate a portion of original solution with solid XaCl or

MgSO .

13—2

19G PHYSIOLOGICAL CHEmSTRY

Non-precipitated

native-albumin

(Apply special tests for serum
and egg -albumin).

Precipitated
globulin
(Pour some of original solution
into distilled water ; a distinct
precipitate shows [derived-
albumins having been ex-
cluded] that mijoHin is pre-
sent).

If the presence of native-albumin as well as globulin be
suspected, filter, and boil filtrate — coagulation proves
it, unless it be slight, when it may be due to imper-
fect precipitation and separation of globulin.

6. To filtrate from 3, if peptone is suspected as well as acid-
or alkali-albumin, or to filtrate from 4 if it is suspected as well
as native-albumin or globulin, carefully apply the biuret reaction
and other tests (}). 15G) for peptones ; but remember that
gelatin gives almost exactly similar tests, and so put aside some
of the solution to cool, and if gelatin be present in sufficient
amount, it will solidify ; then proceed as in III. 2.

A solution which has been proved to contain one or more
proteids may yet contain other organic substances, and to detect
these, it is necessary to remove the proteids as far as possible
by the above methods and to apply special tests according to II.

If in filtration of the original fluid in 1, a considerable amount
remains on the filter, proceed as in IV.

(II.) CARBOHYDRATES.

A solution from which the whole of the proteids, except pep-
tone, has been removed, by boiling with sodium sulphate and
acetic acid and filtering, or which is known to contain no pro-
teids, may be examined for cr.rbohydrates^as follows :
1. Add iodine aolution.

Blue colouration, whish disappears on heating, and re-
appears on cooling=6''arc/«.
P<.rt'V:\ne co' o\iv:.tion.=[,ljcogen or dextrin.

PHYSIOLOGICAL CHEMISTRY. 107

2. If peptones be present, remove them by the following
method : Evaporate solution to dryness carefully, and add boil-
ing absolute alcohol. Separate the solution from the residue
by decantation and filtration. Evaporate off the spirit. Make
a watery solution and test for sugar.

3. If no peptones be i)resent, apply Trommer's test for
sugar.

4. If starch or sugar be present in the solution, and glycogen
or dextrin is suspected as well, it becomes necessary to add, to a
small portion in a test-tube, rather more than its bulk of absolute
alcohol. A precipitate of glycogen, which may be redissolved
in water and tested with iodine, will occur if it be present.
Concentrate the filtrate and add an excess of absolute alcohol.
If dextrin be present it will be precipitated.

Other organic substances may still be present. Proceed as in
succeeding papers.

(III.) GELATIN, MUCIN, UREA, URIC ACID.

1. Proceed as in I. If the solution give the xanthoproteic
and Millon's tests, but none of the other proteid tests, the
solution may contain mucin or chondrin {tyrosin).

Add some dilute acetic acid to a portion of solution :

'Precipita.ie = mucin or chondrin.
Confirm by obtaining a precipitate with alcohol,

Xo precipitate with tannic acid,

No precipitate with HgCL.
JIucin and cJiondria cannot be distinguished from each

other.

2. If the solution give the above proteid tests, and also the
biuret (purple) reaction, but there be no reaction with ferro-
cyanide of potassium and acetic acid, and also no precipitate
with acetic acid alone, test for gelatin (p. 158), and allow the
solution to cool ; it w411 gelatinize.

(If tjTOsin be suspected, extract with a few drops of hot
dilute ammonia and evaporate a portion to dryness ; allow it
to crystallize, and examine with microscope.)

198 PHYSIOLOGICAL CHEMISTRY.

3. If no reaction has resulted from 1 and 2 and from,
above, the solution may contain urea or uric acid (in the form
of urates).

To some of solution add NaCl and then Hg(N03)o, drop
by drop. If no precipitate forms at first, but af ter-
^-ards appears, urea is probably present.

Confirm by obtaining crystals of urea and of urea nitrate^
and examining with microscope.

4. Evaporate some of the solution and apply the mure.vkle test
for uric acid.

(If uric acid be suspected it is better to acidulate some of
the solution "with HCl, and allow it to stand for
twenty-four hours. Uric acid, if present, falls as a
crystalline deposit, and may be examined micro-
scopically, or by the murexide test.) ]

(IV.) EXAMINATION OF A SOLID SUBSTANCE.

1. Preliminary Examination. — Observe whether it be amor-
phous or crystalline (if the latter, examine the crystalline form
under the microscope), and apply the xanthoproteic, biuret, and
ferrocyanide of potassium and acetic acid reactions. Observe
its odour, if any. Thus gain a clue as to the nature of the
substance. Then proceed as follows :

If a Proteid or Gelatin :

2. Test solubility in water.

SOLUBLE. INSOLUBLE.

Naiive-alhumins. Other proteids.

Peptones.

Gelatin.

If soluble, apply special tests to the solution ; if not, try
solubihty in acid, alkali and salt solutions, and apply special

tests.

PHYSIOLOGICAL CHEMISTRY. 1^^

If not a Proteid :
:\. Test its solubility in water (cold).

SOLUBLE. INSOLUHLE.

Grape-suf/ar, Starch (soluble in HOT water).

Milk-HiKjar. TyroBui (soluble in HOT water).

Inos'it. Uric acid.

Ghjcogen. Cholesferiu.

Dextrin. {Fats.)

Urea.
Leucin.
4. If not a proteid, and soluble in water, add iodine solution
to a portion of watery solution.

Port-wine colouration --=5^/?/co5f{'n or dextrin.
Distinguish glycogen and dextrin by the odour and by their
respective solubility in alcohol.

f). If no result with 4, apply Trommer's test to a portion of
watery solution. If cupric oxide is reduced = ^rai^e-swi/«r.

onilJc-sugar.
Distinguish by adding absolute alcohol to a portion of the solu-
tion (after concentration).

Mill-sugar, white precipitate.
Grape-sugar, no precipitate.
»•) If no result with 4 or 5, apply special tests for urea.
7. If no result with 6, apply the special test for leucin as

f0ll0W« : 11 i:

Heat in a dry test-tube in Bunsen's flame ; a smell ot
amylamine=." t'eacm

(8. If no result, apply special test for inosit as follows :

Add HNO3 to original substance, evaporate carefully,
moisten with CaCl, solution ; evaporate again, a rosy-
red spot=inosit.)
0. If not a proteid and not soluble in cold water, apply heat :

SOLUBLE. INSOLUBLE. MELT.

Starch. Uric acid. Fats (apply

Tyrosin. Cholesterin. ether test).

Urates.

200 PHYSIOLOGICAL CHEMISTRY.

If soluble. TO a portion of the "watery solution apply the
test for starch. If no result, test for tjros'in (see 3),
and for urates (p. 189).
10. If not a proteid and insoluble in water (cold or hot), apply
murexide test for uric acid.

n. If no result, test for cholesterin.

PART III.
PRACTICAL PHYSIOLOGY.

CHAPTER I.

EXAMINATION OF THE CIRCULATION
MICROSCOPICALLY.

Tiip: circulation may be studied («) in the web of a frog's foot
(this is the most easy method, as a frog can readily bs obtained
at all seasons of the year) ; (b) in the tail of a tadpole : (c) in
the caudal fin of a small fish, e.f/., goldfish or minnow ; (d) in the
mesentery of any of the smaller mammals, such as the mouse or
young rat.

To demonstrate the circulation in the web of a frog's foot,
prepare a small stand upon which to rest the body of the animal.
Such a staud is readily made from a block, or by supporting a
thin and flat piece of wood upon props by the side of the micro-
scope stage. In the latter case a hole with a diameter of about
three fifths of an inch should be made near one extremity. At
the end of the board nearest the hole, slits are to be cut for the
l)assage of threads. A light-coloured frog is then to be selected,
and its head wrapped in a damp cloth, whilst its body is arranged
in such a manner upon the stand, that one hind-foot extends
over the hole in the board, the other being tucked up out of the
way. Ligatures should then be passed over the ends of two
adjoining toes and Dulled ticrht. In this way, by a little mani-

202

PRACTICAL PHYSIOLOGY.

pulation, the threads may be fixed so as to allow of a flat surface
of the web between the toes being satisfactorily examined.
Care must be taken not to stretch the web to an excessive extent^
lest the circulation be impeded. In the majority of cases, the
frog will remain perfectly quiet for a long period of time, and
this is especially the case if the nose of the animal be brought
into close contact with the board upon which it lies. Occasion-
ally the frog resists all blandishments of this nature, and
exhibits the greatest restlessness. It will then be necessary ta
subject it to the influence of ether, or to inject beneath the skin
of the back a very weak aqueous solution of urari. The efiPect
of the drug is to render the animal motionless, by paralyzing the
endings of the nerves in muscles, and thus preventing the trans-
mission of motor impulses. Urari requires from fifteen to thirty
minutes to produce its full effect. The web, after a suitable
piece has been obtained for examination, should be brought into-
focus, and should be examined first with a low power of the
microscope, and afterwards with a high power. From time to
time, during the examination, the web should be moistened
with water. The examination should not be commenced until
two or three minutes after the web has been fixed, in order to-
allow the circulation, as far as possible, to return to its normal
condition.

Under the low power notice and draw (a) the black pigment
cflls of iiTegular shape and of varying size lying more superficial
than (/.') the arterioles, in which the blood current is more rapid
than in (e) the venules, (r/) the capiUarles. Observe the altera-
tions in their size. With the low power select a thin piece of
the web, where the vessels can be distinctly seen, and continue
the observation under the high power. Determine {a) the rela-
tive positions of the coloured and colourless corpuscles, the
coloured in the centre, the colourless at the sides of the vessels ; (6>
the diapedesis, or passage of colourless blocd corpuscles through
the walls of the capillaries. This phenomenon can, however,
rarely be observed.

The circulation in the tail of the tadpole is readily examined
if the animal be first placed in a watch-glass full of water, to
which a drop cr two of urari solution has been add3d. When

PRACTICAL niYSIOLOGV. 203

the tadpole has beccme motionless, it should be transferred (o a
slide, and examined at leisure. If it is considered necessary, the
thinner portions of the tail may be covered with a cover-glass.
The same features will be recognised as were described in the
case of the frog's vreb.

Fig. 26 — Glass for examiuiug the Circu atiou iii the Fish's Tail.

For the examination of the circulation in a fish, all that is
required is to place the fish, generally a goldfish, in a suitable
vessel, through which a stream of water is kept running con-
tinuously. This can be done by means of Caton's trough (Fig.
"6), or in a simple glass box partially covered over. Into the
covered part the tail is inserted, the fish lying comfortably in
the trough, which is filled with water, and into which a con-
stant stream flows. The box is placed upon the stage of
the microscope, and the tail can be examined with a low
power.

The arterial schema is an apparatus designed to represent in
a diagrammatic form the main phenomena of the arterial pulse.
It consists of a series of elastic tubes of varying calibre which
are arranged somewhat as they are in the vascular system, the
larger tubes being equivalent to the main vessels, the smaller
ones to the arterioles and venules. The ends of the tubes can
be closed by means of ivory pegs, and in this way and by
clamps the resistance within the .system can be raised to any
required extent, and the varying conditions of the circulation
can be imitated. In the centre of the system is a thick-walled
elastic sac provided with valves, as in the case of an enema
syringe, so that fluid can pass thiough it in one direction only.
"When required for use, the tubes are moderately distended
with water. Three levers similar to those employed with the

204

FRACTICAL PHYSIOLOGY.

sphygmograpb are arranged at intervals of six to eight inches
along the tubes, in such a way that their movements are re-
corded in a vertical series of tracings upon the revolving drum.
The central sac is then compressed rhythmically by the hand,
and the resulting tracings made by the levers are afterwards
carefully noted and compared.

The sphygmograph is an instrument used for representing
graphically the characters of the pulse. In it a small button
rests upon the artery, usually the radial ; this button is attached

Fig. 27— Dudgeon's Spliygmogi-apb.

to the under surface of a steel spring. The movement of the
button is communicated to an upright screw working in an
arm of metal, which, with the spring, is fixed (although capable
of up and down movement) by means of a screw to the frame
of the apparatus. The movement of this adjustable screw is
communicated to a lever of light wood, since the metal arm has
in front a piece of metal projecting upwards, which comes in

PRACTICAL PHYSIOLOGY. 205

contact with the lever near its fulcr(in\ The lever writes on a
smoked glass or card, which is moved by clockwoik along a
groove on the u])per surface of the brass box containing the
clockwork, which is fixed on the flat piece of metal forming the
frame of the instrument. The sphygmograph is bound en to
the wrist with the button on the artery (but not pressing too
hard), and the clockwork backwards. The smoked surface is
arranged in place ; the lever adjusted by means of the screw, fo
that its end writes on the smoked surface by means of a sharp
point. The clockwork is wound up and set going, ?nd the
character of the pulse is represented on the moving surface by
means of a tra,cing on the paper. The clockwork is then stopped,
the tracing removed, the circumstances under which it was taken
noted down, and the paper or glass is varnished. Nearly all the
best sphygmographs are provided with an apparatus for adjust-
ing the amount of the pressure which the spring exercises upon
the artery. The form of the instrument chiefly used in this
country is a modification of the original sphygmograph of
M. Marey.

A small instrument invented by Dudgeon is in considerable
use ; it is represented in Fig. 27. The advantages claimed for it
are : It magnifies the movements of the artery in a uniform
degree, viz., fifty times. The pressure of the spring can be
regulated from one to five ounces. It requires no wrist- rest, and
may be used with equal facility whether the patient is standing,
fitting or lying. "With it a tracing of the pulse can be made
almost as quickly as the pulse can be felt with the finger. Its
sensitiveness is so great that it records the slightest deviation in
the form or character of every beat. Its construction is so simple,
that if accidentally broken any watchmaker can repair it. It
is 80 small ('2h by 2 inches), and it is so light (4 ounces), that it
can easily be carried in the pocket. It is only onc-thiid of the
price of the ordinary instruments.

Method for estimating the Blood Pressure in a Rabbit.^ —
Make a saturated solution of chloral hydrate in "To per cent,
saline solution : inject 15—20 minims of the chloral solution

*A license uudcr the Vivisectiou Act is necessary before this experi-
ment can be legally iierfomied.

206 PRACTICAL PHYSIOLOGY.

beneath the skin of the abdomen of a live rabbit. Leave the
rabbit for half an hour, ani in the interval prepare the rest of
the apparatus. Fill a pressure-bottle with a saturated solution
of sodium sulphate, and suspend it by means of a string and
pulley attached to the ceiling, about four feet above the operat-
ing-table. The bottle should have a hole near its lower part, to
which a long india-rubber tnbe is attached ; the fluid is pre-
vented from running out by means of a clamp attached to the
lower part of the tube. Get ready Czermak's mbbit-holder, and
arrange ligatures upon it for binding the fore and hind legs of the
animal. Arrange the recording apparatus (p. 228) on one side of
the rabbit-holder, and see that the clockwork is wonnd up, and
that it is in working order. Gum a slip of glazed paper round
the recording drum (p. 220). and blacken it by revolving the
drum over the flame of a paraffin lamp. The coating of lamp-
black should be as uniform as possible, and not too thick. If the
kymograph writes with a pencil or with a pen, such blackening is
not requisite ; in the latter case fill the can of the pen with a
few drops of aniline ink. If a continuous tracing is to be taken,
arrange the feeding- roller in a proper position. In the case of
the mercurial, or Ludwig's kymograph, see that the u tube is
jpartially filled with clean and bright mercury ; that the top of
the float is not below the level of the mercury, and that the
'weighted thread presses upon one arm of the pen in such a way
as to keep it in contact with the drum. Block up the shorter
limb of the U tube with a piece of wood. Take a i-shaped glass
tube, connect the vertical portion with the tube descending from
the pressure-bottle : and one of the horizontal rami with the
tube which projects from the shorter limb of the u tube, by
means of a piece of india-rubber tubing (or better still, by means
•of a piece of substantial leaden piping). To the other horizontal
ramus attach a piece of india-rubber tubing, and clamp the end :
open the clamp which has hitherto closed the tube leading from
the pressure-bottle ; the sodium sulphate will pass through the
1-shaped tube, and will fill the tubes in connection with it.
Remove the piece of wood from the top of the shorter limb of
the U tube, and replace it as soon as all the air is expelled, and
the limb is full of sodium sulphate solution. Loosen the clip

rrtACTICAL PHYSIOLOGY. 207

upon the india-rubber tube for an instant, and allow a few drops
of the solution to escape ; no .air should now be present in the
system of tubes thus arranged : if leakage takes place in any
part, and air enters, it must be remedied. Select or make
a cannula large enough for the carotid or femoral artery of
the animal to b3 experimented upon ; this requires consider-
able experience, and reference had better be made to the demon-
strator. Arrange the instruments near at hand which are
requisite for the operation of exposing the vessel : they are a
box of scalpels, scissors, and forceps, fine and coarse, a p:\ir of
each ; a pair oi Krunecker's bull-dog forceps, aneurysm needle,
ligatures of silk, a splinter of wood (a match answers the pur-
pose very well), a 'seeker,' and sponges.

Fix the rabbit upon the rabbit-holder ; it should be insensible
at the time of the operation, and is fixed by laying it upon its
back, unscrewing the bit at the end, and removing the central
steel peg ; pass the peg through the side of the bit, in such a way
that it passes within the mouth, between the lips and behind the
incisors of the two jaws, passing out on the opposite side,
where it fits into a hole in the bit. The animal is thus held by
the teeth. Screw up the end of the bit ; the lower ja.v will
thus be compressed, and the animal will thereby be prevented
from opening its mouth and loosening the central pin ; if need
be, the pressure may be increased from time to time by means
of the screw. Extend the hind-legs, and pass the ligatures over
the thigh on each side ; tighten and fix them. Pass ligatures
over the fore-legs, and fix them in such a way that the first
joints are flexed, the limb being held at the elbow.

Before proceeding further, see that everything is in proper
working order. The points to be chiefly attended to are : (1)
The recording apparatus, that it is wound up, and that it works
smoothly. (2) Tlie hymograph, that the pen or style writes dis-
tinctly, and without too much pressure ; that no bubbles of air
exist in the tube. (3) TJie xysteni of tabes ^ that they are full of
the solution of the sulphate of soda, that they contain no air, and
that they are not kinked. (4) That the pressure bottle is neither
too high nor too low ; and that if need be it can readily be
raised or lowered to equalize the pressure of blood. (5) That

208 PEACTICAL PHYSIOLOGY.

Ihe distal end of the cajunihi ^'s accurately, and can be easily
tied into the proximal clamped end of the india-rubber tubing
which is in connection "with the pressure-bottle and the kymo-
gra])h.

Expose the carotid in the rabbit. First clip away the fur over
one side of the neck, and make an incision along the side of the
trachea for about three inches. Separate the muscles carefully
vith a seeker, taking care not to go too far outwards. The
artery is readily discovered ; it may be distinguished from the
large vein which accompanies it by its more opaque appearance,
.•IS well as by its lighter colour, and by the pulsation which it exhi-
bits. Dissect it out carefully for a short distance, and pass the
aneurysm needle, armed with a ligature beneath it ; withdraw
the needle, leaving a loop of thread round the artery ; cut the
loop and two ligatures will be thus formed. With the bull-dog
forceps, clamp the proximal portion— i.e., the part nearest the
heart— of the artery. With one of .he ligatures tie the distal
portion of the arterj^ as high up as the incision will allow.
Eaise the portion of the artery between the clamp and the liga-
ture by passing the splinter of wood beneath it, and with a pair
of sharp and fine-pcinted scissors make a v-shaped incision into
it. Fill the cannula, by means of a pipette, with the sodium
sulphate solution, and insert it into the incision in the artery ;
the smaller end being directed towards the heart. Pass the
second ligature over the cannula, and tighten it round the artery
in such a way as to tie the cannula firmly into the vessel (to
facilitate this operation the cannula is provided with a shoulder,
behind which the ligature should pass). Fit the india-rubber
tube leading through the i piece to the manometer, on to the
other end of the cannula, taking care that both the tube and the
cannula are quite full of the soda solution, so that no air may
be enclosed at the point of junction. Remove the clamp which
has hitherto prevented the escape of fluid from the india-rubber
tube nearest the cannula. Set the clockwork of the recording
■apparatus in motion ; with one hand open cautiously and gra-
duulhj the clamp upon the tube of the pressure-bottle, whilst
with the other hand remove the bull-dog forceps from the
artery. If everything has been properly arranged, a few drops

ri;A( I icAL riivsioi.cK.v. 200

of blood will pass into the canmila, but thu piessure in tbo
artery will be counteracted by the column of sodium sulphate
.•■olution, which transmits tlic variations in the Ijicod pressure to
the mercury in the [} tube, whence it is tiansmitted to the lever
M hich records it upon the drum. It is possible that the pressure
bottle may be too high, and that the column of liquid is more
than enough to counterbalance the blood pressure ; in which
case the sul^ihate of s-oda will enter the blood, and will not only
vitiate the experiment, but in many cases will actually kill the
animal ; hence it is nccci-sary to be cautious in opening the
clamp. The sulphate of soda arrests, to a certain extent, the
co.igulation of the blood, and it is therefore employed ; but it
often happens that the cannula becomes blocked by a clot, in
which case it will be neccsrary to detach the tubing and clean
out the cannula, or it may be necessary to clamp the artery, and
insert a fresh cannula.

To )nahe a CannuJn fw me during Blood-prc: stive Experiments.
— Take a piece of hard glass tubing, with a bore of about the
size of an ordinary quill pen. Soften the end of the tube in the
flame of a blow^-pipe, and draw it out gently for about an inch :
there will then be a narrower portion of tube between two pieces
of the full size. When the tube has cooled, heat a portion of the
narrower part in the f.ame, and draw it out very slightly ; by
this means the narrower portion will be thicker in the centre
than at one side. File through the middle of the narrower por-
tion in an oblique direction. A cannula with an oblique open-
ing at its smaller extremity will thus be formed. It must be
finished by carefully rounding off its edges in an ordinary gas
tlame, and by filing down the aperture with a three-cornered
file, until it ])resents the necpssary obliquity. The narrowed
portion will have a neck to prevent the ligature slipping off
when it is tied into the artery.

The cardiograph registers in a graphic manner the heart's
impulse. It consists of two portions : (a) A hollow metal disc
whose face is covered with a thin membrane of india-rubber.
The disc is provided with three levelling screws ; from its pos-
terior surface passes off a tube bent at right angles. In front of
the elastic membrano is an ivory knob, which is in connection

14

210 PRACTICAL PHYSIOLOGY.

with a delicate spring arising from the side of the disc. The
extremity of the spring is also provided with a pointed steel
ssrew, resting exactly on the centre of the membrane, (h) The
registering portion (Marey's tambour) consists of a second disc,
whose elastic membrane is in connection with a lever, and from
whose under-surface a tube also proceeds. The tubes of the
two discs are connected with each other by a portion of elastic
tubing, and in this way an air-tight cavity is produced, so that
any movements executed by the membrane of the first disc are
reproduced in the second disc, and are transmitted to the lever by
which they are recorded in the ordinary way upon the rotating
drum. In using the instrument, the patient is made to lie down
upon a couch of convenient height ; the chest is bared, and the
apex beat of the heart is found in the fifth left costal interspace,
somewhat below and internal to the nipple. The first disc is
then applied in such a way that the ivory knob is exactly over
the point at which the beat of the heart is felt. The impulse is
thus transmitted to the lever, which executes certain movements.
If these movements be registered upon the revolving drum, it
will be found that they consist of a sudden ascent at the instant
of the ventricular contraction, and of an equally marked but less
sudden fall.

The Stethoscope. — The sounds of the heart are heard by
means of the stethoscope. The simplest form of this instrument
is a cylinder of wood or metal expanded at one end into a
conical portion, which is applied to the chest wall, whilst the
opposite extremity is provided with a slightly concave disc, to
adapt it to the observer's ear. The room should be perfectly
quiet, the patient should bare his chest, and remain standing.
The finger should then be placed upon the apex-beat of the heart.
The observer, standing in front of the patient, should apply the
conical end of the stethoscope over this point, and his ear to the
opposite end : at the same time he should feel with the fingers
of his left hand beneath the sterno-mastoid muscle of the left
side for the carotid artery. Two sounds will then be heard, one
accompanying the impulse, called the Jirst or systolic sound :
the other following the impulse, and known as the second, or
diastolic sound. The first sound is the longer and more deep-

PUAITICAL PIIYSIOLOCJY. 211

toned ; it is best heard at the apex of the licart. The secoml
sound is sharper and shorter ; it is best heard in the third inter-
costal space, close to the sternum, though it is also audible at
the apex. After the second sound is a pause, so that tlie normal
cardiac cycle is roughly represented by the rhythm Inbh, iirtp—
iuhb, (Jttp. Ill listening to the sounds of the heart, the respira-
tory sounds may be neglected. Care must be taken that the
stethoscope be applied evenly to the chest wall, that the tube is
not touched by the clothes or lingers whilst the examination is
being made, and that the observer does not press so heavily
against the stethoscope as to cause pain to the patient.

The ophthalmoscope, brought into use by Helmholtz, consists
in its simplest form of («) a slightly concave mirror of metal or
silvered glass, perforated in the centre, and fixed into a handle;
nnd (/>) a biconvex lens of about 2.V— :} inches focal length.
Two methods of examining the eye with this instrument are in
common use— the direct and the indirect ; the student should
endeavour to accustom himself to use both methods of investi-
gation with equal facility.

A normal eye should be examined ; a drop of a solution of
atropine'^ (two grains to the ounce) should be instilled about
twenty minutes before the examination is commenced ; the f
ciliary muscle is thereby paralyzed, the power of accommoda-
tion is abolished, and the pupil is dilated. This will materially
facilitate the examination ; but it is quite possible to observe all
the details to be presently described without the use of this
drug. The room being now darkened, the observer seats him-
self in front of the person whose eye he is about to examine,
placing himself upon a somewhat higher level. A brilliant and
steady light is placed close to the left ear of the patient. The
atropia having been put into the right eye only of the patient,
this eye is examined. Taking the mirror in his right hand, and
looking through the central hole, the operator directs a beam of
light into the eye of the patient. A red glare, known as the
rejiex, is seen ; it is due to the illumination of the retina. The
patient is then told to look at the little finger of the observer's
ra*ia/ ^^'^^*^^*''^^'^ bomatropin lijilrobromate, as the effect passes oflf more

14-2

212 PRACTICAL PHYSIOLOGY.

right hand as he holds the mirror ; to effect this the eye is
rotated somewhat inwards, and at the same time the reflex
changes from red to a lighter colour, owing to the reflection from
the optic disc. The observer now approximates the mirror, and
with it his eye to the eye of the patient, taking care to keep the
light fixed upon the pupil, so as not to lose the reflex. At
a certain point, which varies with different eyes, but is
usually when there is an interval of about two or three inches
between the observed and the observing eye. the vessels of the
retina will become visible as lines running in different direc-
tions. Distinguish the smaller and brighter red arteries from
the larger and darker coloured veins. Examine carefully the
fundus cf the eye, i.e., the red surface — until the optic dii-c is
seen : trace its circular outline, and observe the small central
white spot, the phyaiolorp.cal pit : near the centre is the
central artery of the retina breaking up upon the disc into
branches ; veins also are present, and correspond roughly to
the course of the arteries. Trace the vessels over the disc on
to the retina. The optic disc is bounded by two delicate
rings, the more external being the choroidal, whilst the more
internal is the sclerotic opening. Somewhat to the outer side,
and only visible after some practice, is the yellov: spot^ with the
small lighter-coloured /orca centralis,in its centre. This consti-
tutes the direct method of examination ; by it the various details
of the fundus are seen as they really exist, and it is this method
which should be adopted for ordinary use.

If the observer is ametropic, i.e., is myopic or hypermetropic,
he will be unable to employ the direct method of examination
until he has remedied his defective vision by the use of proper
glasses.

In the indirect method the patient is placed as before, and the
observer holds the mirror in his right hand at a distance of
twelve to eighteen inches from the patient's right eye. At the
same time he rests his little finger lightly upon the temple, and
holding the lens between his thumb and forefinger, two or three
inches in front of the patient's eye, directs the light through
the lens into the eye. The red reflex, and subsequently the
white one. having been gained, the obseiver slowly moves his.

I'KACTICAL J'llVSIOLOGY. 213

mirror, and with it bis eye, towards or away from the face of
the patient, until the outline of one of the retinal vessels becomes
visible, when very slight movements on the part of the observer
will suffice to bring into view the details of the fundus above
described, but the image will be an inverted one. The lens
should be kept fixed at a distance of two to three inches, the
mirror being alone moved until the disc becomes visible ; should
the image of the mirror, however, obscure the disc, the lens may
bo slightly tilted.

The laryngoscope is an instrument employed in investigating
during life the condition of the pharynx, larynx, and trachea.
It consists of a large concave mirror with perforated centre, and
of a smaller mirror fixed in a long handle. It is thus used : the
patient is placed in a chair, a good light (argand burner, or
lamp) is arranged on one side of, and a little above his head.
The operator fixes the large mirror round his head in such
a manner that he looks through the central aperture with
one eye. He then seats himself opposite the patient, and so
alters the position of the mirror, which is for this purpose pro-
vided with a ball and socket joint, that a beam of light is
reflected on to the lips of the patient.

The patient is now directed to throw his head slightly back-
wards, and to open his mouth ; the reflection from the mirror
lights up the cavity of the mouth, and by a little alteration of
the distance between the operator and the patient the point at
which the greatest amount of light is reflected by the mirror — in
other words its focal length — is readily discovered. The small
mirror fixed in the handle is then warmed, either by holding it
over the lamp or by putting it into a vessel of warm water ;
this is necessary to prevent the condensation of breath upon its
surface. The degree of heat is regulated by applying the back
of the mirror to the hand or cheek, when it should feel warm
without being painful.

After these preliminaries the patient is directed to put out his
tongue, which is held by the left hand gently but firmly against
the lower teeth, by means of a handkerchief. The warm mirror
is passed to the back of the mouth, until it rests upon and
slightly raises the base of the uvula, and at the same time the

214 PRACTICAL PHYSIOLOGY.

light is directed upon it : an inverted image of the larynx and
trachea will be seen in the mirror. If the dorsum of the tongue
be alone seen, the handle of the mirror must be slightly lowered
until the larynx comes into view : care should be taken, however,
not to move the mirror upon the uvula, as it excites retching.
The observation should not be prolonged, but should rather be
repeated at short intervals.

The structures seen will vary somewhat according to the con-
dition of the parts as to inspiration, expiration, phonation, etc. ;
they are first, and apparently at the posterior part, the base cf
ihe tongue, immediately below which is the arcuate outline of
the epiglottis, with its cushion or tubercle. Then are seen in
the central line the true vocal cords, white and shining in their
normal condition. The cords approximate (in the inverted
image) posteriorly ; between them is left a chink, narrow whilst
a high note is being sung, wide during a deep inspiration. On
each side of the true vocal cords, and on a higher level, are
the pink false vocal corrls. Still more externally than the
false vocal cords is the argteno-epiglotfidean fold, in which are
situated upon each side three small elevations ; of these the
most external is the cartilage of Wrisherg, the intermediate
is the cartilage of Santoinni, whilst the summit of the arytenoid
cartilage is in front and somewhat below the preceding, being
only seen during deep inspiration. The rings of the trachea, and
even the bifurcation of the trachea itself, if the patient be
directed to draw a deep breath, may be seen in the interval
between the true vocal cords.

CHAPTER II.
NERVE MUSCLE PHYSIOLOGY.

CONTRACTILITY OF MUSCLE.

In order to show the excitability of living muscle it is usual to
employ the muscles of a pithed frog, either remaiuing in situ or
removed from the body. Any muscle will answer the purpose
which may be conveniently dissected out, but it is usual to
employ those to which nerves can be easily traced, and to apply
the stimuli to the nerve supplying the muscle, instead of to the
muscle itself directly.

The first operation required is (o {nth a frog, i.e., to completely
destroy its central nervous system. In order to accomplish this
the animal must be held by its two fore-legs in such a way that
its belly is pressed against the dorsal surface of the left index
finger, the head projecting beyond the tip of the finger, and be-
ing pressed downwards by the thumb of the left hand. The skin
on the back is then put on the stretch, and the nail of the right
index finger is drawn down the centre of the head towards the
si)inal column. Immediately below the head, and at its junction
with the vertebrae, a slight depression will be felt, marking the
position of the occipito-atlantoid membrane. A small triangular
snip is made over this spot with a pair of sharp-pointed scissors*
the membrane is then divided, and a wooden match sharpened
at one extremity is thrust upwards into the brain to destroy
sensibility, and then a long blanket-pin is pushed downwards
into the spinal canal to destroy the spinal cord. In performing
this operation no bleeding should occur. Care must be taken that

216 PRACTICAL PHYSIOLOGY.

the pin really enters the spinal canal, and that on the one hand it
does not simply pass beneath the skin of the back, whilst on the
other it does not pass into the abdominal cavity. The sndden
extension of the hind limbs ma}- be taken as a proof that the
destruction of the cord has been properly completed.
A Nerve-Muscle Preparation may be made in this manner :
(i.) Having pithed a vigorous frog, open the abdomen with a
pair of sharp scissors, turn aside and remove the viscera, and
expose the sacral plexus of each side : free the nerves from con-
nective tissue, clamp the head of a frog in a holder, and attach
to a retort stand, allowing the lower extremities to hang about
eight or ten inches above the table. A pair of electrodes may
now be inserted behind the nerves of the plexus. The elec-
trodes are made by fixing with sealing-wax two pieces of copper
wire in glass tubes about an inch and a half long, and allowing
the ends to project, fastening the tubes together with sealing-
wax, and soldering to one end of the projecting wires two
thinner flexible wires, two feet long, covered with cotton. The
ends projecting not quit€ so far at the opposite ends of the tubes
are then scraped with a knife, and bent nearly at right angles, and
the electrodes are complete. Convenient electrodes may also be
made by inserting pins two inches long into a cork, soldering
wires to the heads, and turning up the points as above.

(ii. ) The directions for making the more usual form of nerve-
muscle preparation, however, are as follows : Pith a frog and
remove the skin from the Viack of one thigh ; this will expose
the muscles enclosed in a fine sheath of fascia ; with two pairs
of forceps tear the muscles apart by breaking open the sheath.
Three muscles will be exposed — the triceps, on the outside, the
semi-memhranosus on the inside, and lying between the two, and
partially covered by them, a smaller muscle with tendinous ends,
the biceps. Follow the biceps to its origin, and carefully cut it
through with a pair of scissors : then catching hold of the
divided end. pull it forcibly down towards its insertion, and en-
tirely remove it. By this method of procedure the sciatic nerve
will have l>een exposed. Carefully dissect it out, tracing it up-
wards to spinal column, and downwards to the gastrocnemius —
notice its first and chief division to supply the two heads ; do

PRACTICAL rilVSIOLOGY. 217

not prick the bloodvessels in contact with the ncivc, but gently
separate them : cut through the spinal column with spinal cord,
and divide the part detached vertically into two, and by holding
the ])iece of bone belonging to the side you are dissecting, lift
u]) the nerve, and free it from surrounding tissues. The nerve
will now be ready for succeeding experiments. During the
operation it ought not to have been pinched, piicked, or other-
wise injured.

The nerve must be placed upon the electrodes, and the jire-
paration is ready for the demonstrations of mus^cular con-
tractions under appropriate stimuli. Care should be taken that
the preparation, whichever it be, should not dry up.

Stimuli are aiiher (]) elecirical, {2) mechanical, (?j) thermal,
or (4) chemical.

(1) Of electrical stimuli, the effects of {a) the induction shock
and {h) the galvanic current must be demonstrated.

(^0 Effects of the Induction Shock.— Apparatus required :

(i.) A lattery, the one iu most common use being DanielVs^
which consists of an outer vessel of copper, which forms the
negative plate, full of a saturated solution of copper sulphate in
which is placed a porous earthenware vessel containing a stout
rod of well-amalgamated zinc, which forms the positive plate,
immersed in dilute sulphuric acid (1 in 8). The electrical
current passes from the copper to the zinc outside the battery,
and in the reverse direction inside. The electrode attached to
the copper is the positive, and that to the zinc the negative—
the copper being the positive pole and the zinc the negative.
From the presence of the two fluids the current of electricity is
pretty constant in strength, altering little during long periods
of time, hence called constant or cont'nuous. A supply of solid
copper sulphate is placed upon a ledge attached to the inner
and upper part of the copper vessel, so that the solution of
copper sulphate may be maintained at the point of saturation.

(ii.) Tu:o heys, one of which should be of the form indicated
in Fig. 28, by means of which the current may be made or
interrupted, and the other a mercury key.

(iii.) Wires, insulated by a gutta-percha covering.

218

PRACTICAL PHYSIOLOGY.

(iv.) Electrodes. — Either the glass or cork form mentioned
above. A pair with platinum points would be useful.

(v.) Du Bois Reymond's induction
apparatus (Fig. 29).

Method of Experiment. — (i.) Prepare a
Daniell's cell, connect it by wires with the
top screws (rZ d) of an induction coil (Fig.
29)— these f crews are in connection with
the ends of the coil of wire forming the
primarij coil (c) — interposing a mercury
key, attach electrode wires to screws of
secondary coil (7), interposing a key
(Fig. 28) for short circuiting, place
electrodes behind nerve of a nerve-
muscle preparation of the second form
indicated above. Each time the battery
circuit is completed by closing the
key, and broken by opening the key, an induced current is
momentarily produced in theeeccndary coilin opposite direction

Fig. 2S. Du Bois Eey
inond's Key.

Fig. 29. Du Bois Eeyinond's Induction Coil,
on making, and in the same direction on breaking; and each of
these, if strong enough, Mill produce a single muscular contrac-
tion.

rilACTICAL rilYSlUUHJV. 210

(1) To ahoir tliat ilie hrpak'niij contrad'ion iippairs hejore f/i'^
makimj. — Slide the secondary coil a considerable distance from
the primary coil, open and shut mercury key — no contraction.
Move secondary coil nearer primary coil until a contraction
ensues— note that contraction first appears when key is opened
{hreahinrj coii(ractioii) — note the distance in millimetres — ])ro-
ceed until a contraction appears also on closing key {mak'inff
contraction).

(2) To shoir tliot ill" sf'nmdus is incrraaed as it descends tlio^
neiTC. — In a preparation in which the sciatic nerve is dissected
out, but not cut, proceeding in an exactly similar way to (1)»
note results (in form of a table) with platinum electrodes placed
behind nerve, (a) high up, {h) mid-way, (r) close to muscle, and
also {d) with electrodes upon gastrocnemius itself.

(3) To show that lohen the nerve is divided^ its irritahdily is at
first increased, in the neighbourhood of the section. — Divide the
nerve high up, and repeat experiments ; compare results.

(4) To shore the effect of temperature upon irritahilitij. — In
preparation which has been kept cold in ice, and (5) In another,
which has been warmed moderately, repeat the above experiments.

(6) y'o shoic that irintahiU'y r/radualhj diminishes some time
after section of the nerve has been done.— Ji. time permit, repeat
experiment (2) with the same preparation after an hour has
elapsed, and again after a second hour, and compare results.

N.B. — Always be careful that your battery is working, your wires un-
broken, your contacts secure, and your metallic connections
bright.

The use of the key in the secondary circuit is to cut off the make or
break contraction, or both ; thus if key 2 is open whilst key 1 is opened and
shut, contractions will occur at make and break. If key 2 is open, and then
key 1 is opened, a breaking contraction will occur, but the making contrac-
tion may be cut off by closing key '2 before key 1. And similarly, if key
2 is closed and then key 1 is opened, and opened before key 1 is shut, a
single making contraction will occur. If key "2 is dosed, whilst key 1 is
opened and shut, no contraction will occur.

Effects of a Series of Induction Shochs. — Connect the wires
from the battery with the screws at the bases of the two pillars of
an induction coil (r, a). Interpose a key in the secondary circuit.
The current passes up the outer pillar, along the sjjring, until it
arrives at the point where contact is made by means of a plati-
num-pointed screw {c), adjustable, and in connection with the

'220

PRACTICAL PHYSIOLOGY.

end of the primary coil ; the current passes bj' this connection
through the primary coil, and then through the coils of wire
surrounding two pieces of soft iron, and thence to the battery
through the middle pillar coil. As soon as the current passes
through the electro-magnet, the soft iron is magnetized, and
draws down the hammer. This breaks contact with the spring,
and the current is by this means interrupted, to be again made
as soon as the magnet ceases to act from the stoppage of the
cuirent through the electric coil round its soft iron. In this
way a series of rapid make and break shocks occurs, and these are
represented in the induced current, and therefore in a series of
make and break contractions of the muscle, when the electrodes

Fig. 30. Diagram of the Course of the Current through the In-
ductiou Coil wheu Hebulioltz's Conuectmg Wire is used.

placed under the nerve are connected with the screws of the
secondary coil, and the key is opened : the effect of this is to
send the muscle into a state of tonic spasm, or t.'tanus. The
frog's leg and foot are rigidly extended. The contraction wil^
continue for some time, but will finally give way under pro-
longed stimulation.

The apparatus is known as the magn''iic interrupter. With
the magnetic interrupter thus employed, the break current is
found to be much more powerful than the make, and sd the

PRACTlcAf. PHYSIOLOGY. 221

stimuli are not uniform, as they should be to produce a true
tetanus. A modification, therefore, is neces.sary, the object of
which is to equalize, as far as possible, make and break shocks.
This consists of a stout copper wire to connect the outside
pillar with a binding screw, which is in connection with the
screw at one end of the primary coil ; in this way part of the
current always passes at once to the primary coil and only a por-
tion of it is made and broken by the interrupter, whether or
not the jdate is attracted to the magnet. The point of the
middle pillar must l>e raised by means of the middle screw, and
the screw attached to the end of the primary coil must also be
screwed up away from contact with spring. The current on
entering (sup])Osing the contact between the spring and the
middle ])illar to be made) divides ; one portion passes through
the primary ciicuit and magnet, the other passes through the
contact down the middle pillar and back to the battery
(Fig. 30) ; but as the part of the current which passes through
the magnet is insufficient for the purpose of retaining the
hammer in contact, the current is broken by the hammer spring-
ing away from the magnet; then the whole of the battery current
passes through the primary coil and magnet, and again the
hammer is attracted to the latter, and so on. It will be seen
that by this arrangement only a portion of the current is inter-
rupted.

If time permit, the following further experiments may be
done before proceeding to {b) :

The extra otrrent of Faraday may be clemonstrated by taking a Daniell's
element, two keys, the priiuaiy coil, a nerve muscle preparation, and elec-
trodes. The a]>paratus is aiTauged so that both keys, as well as the induc-
tion coil, are placed in the primnry ciicuit. whilst to the second key the
electrodes are connected. On testing the current \\"itli the tongue, suppos-
ing the key No. 1 he open, on opening key No. 2, as well as on shutting it.
there is an appreciable effect u]ion the tongue. When the coil is cut off
by closing key 1, there is very little or no effect on opening key 2. The
effect is produced by the extra induced current. It may also be shown by
diminisliLug the battery current by a wire directly connecting the poles
imtil no contraction occurs. If key 1 is closed, then, on breaking with the
primary coil, a contraction will occur.

i'iiipolar Excitation. — Arrange the battery and coil for single induction
shocks, and connect one declrode with one of tlie screws at the end of the
secondary coil. Place this under the exposed sciatic nerve of a nerve-
muscle preparation, v.hich should be arranged on a ilate of glass upon a

•0 o •■)

traltical physiology.

frog board; i.e., a flat piece of board covered witb cork. Open and sliut a
key in tlie primary circuit, and there will be no response ; now touch the
muscle with the finger or a pair of forceps held in the hand, and it will
contract.

GalcanVs Experiment. — Take a piece of zinc, thoroughly cleaned, and
coil round one end a piece of copper wu-e, which projects in such a way
that a fork with two equal prongs is made. Insert the zinc behind the
lumbar nerves of a pithed frog, and allow the copper to fall upon the thigh
muscles ; a contraction will occur. If the muscles he very excitable there
will also baa contraction when the copper is removed fi-om the muscle.

Galvani's Experiment.

Intermption hjf means of a Sprim. — Bring one of the wires from a
Daniell to the end of a steel spring which is fixed in a sujjport in such a
way that when it is set into vibration the opposite end dips into a mercury
cup in metallic connection with the primary coil, and which is also con-
nected with the battery (the spring and cup takmg the pla2e of a key),
ooiuiect the electrodes with the secondary coil, and place them under the
sciatic nerve of a pithed frog. Make the spiing vibrate, and notice that if
the spring is short, tetanus is at once set up, and if long, that one con-
traction is distinctly piled on the top of another at first, and that a more
gi-adual tetanus is produced.

Iitten-v.pjtion hij means of a Metronorae. — Insert a vibrating metronome
into the primary circuit of an induction coil, and allow the series of induc-
tion shocks thus regulated to break into a nerve-muscle prepai*ation. The
limb will gradually pass into tetanus.

{h) Effects of the Galvanic Current. — Prepare a nerve-
muscle preparation, and get ready a Daniell's cell. Bring the
■wires of the latter to the inner binding screws of a Du Bois

PRACTICAL niYSIOLOriY. 223

lleymond's key (Fig. -8). Bring tho electrode wires to the
outer binding screws ; arrange the electrodes behind the nerve
of the preparation. Open the key by raising the handle, and
after a few seconds close it again ; a contraction of the muscle
will occur at the opening and at the closing, that is to say, at
the make and at the break, and during the intercal there icill he
no co)itradiun, although the current is passing through the nerve
all the time. The use of the kej- is to shut off the current from
the nerve, as whilst it is closed the brass plate affords much less
resistance to the current than the nerve ; and as the current will
pass in the direction of least resistance, the whole of it passes
through the key to the battery again. On opening the key the
current is bound to pass through the nerve, which it excites, and
so produces contraction in the muscles supplied by it. The
electrode connected with the copper, or the xmsitive electrode^ is
called the anode, and that connected with the zinc, or the nega-
tice electrode, is called the lathode. As the current passes fron?
the positive to the negative, when the kathode is nearer the
muscle than the anode, the current is said to be descending ; and
when, on the other hand, the kathode is above the anode, the
current is called ascending.

If the key be opened for some time, and then the battery wire
be removed, a contraction will occur several times on closing the
key, without the battery current ; this is due to the jwlari'jaiion.
of the electrodes by the current : as many as twenty contractions
may be shown under favourable circumstances in this way.

Regulation of the Strength of a Galvanic Current. The
Rheochord. — This is done by interposing in the circuit a
graduated wire, the resistance of which is regulated. This wire
is arranged to form the rheochord (Fig. 32). The one in use is
that of Du Bois Reymond.

The instrument consists of a long box or board, on which the
resistance wires are stretched. At one end are fixed several
brass blocks, separated from one another, and disconnected
except by fine German-silver wire. At each corner of this end
are binding screws, a and r., each connected with the brass block
nearest. Beginning at the block nearest a, a wire passes a con-
siderable distance up the board, passes round a peg and returns

224

TRACTICAL PHYSIOLOGY.

to the second block, from which a wire, in a similar manner,
considerably shorter, connects it with the next block, and so on.
From the two blocks at the end of the row two thin platinum
wnres pass to the opposite end of the board (c), and are there
insulated ; they are, how^ever, connected by means of a slide,
formed of cups of mercury. This slide is capable of being
moved up and down the wires. The brass blocks are, moreover,
capable of direct connection (except the two at the corner (b) of
the board, between which are the travelling mercury cups) by
the insertion of plugs. Supposing the rhcochord is interposed
in a continuous current in place of a key, and to the binding
screws the wires of the battery are attached, as well as the elec-
trode wires : if the slide be close to the brass blocks and all the
plugs in, the rheochord, offering no resistance to the current,

Fig. o2. Klieocliorcl of Du Bois Eeyii.oucl.

allows it to return to the battery, and none of it passes into the
nerve. If the slide be pushed a short distance down the wires,
the current meets with a certain amount of resistance, and so a
fraction of the whole battery current Avill pass into the nerve.
In like manner a greater and greater current passes into the
nerve, if the slide be pushed farther away from the blocks, and
if the plugs be removed one by one, as in this way the resist-
ance offered to the passage oE the battery current through the
rheocord is more and more in3reas3d.

Effect of varying the Strength and Direction of the
Current.— Take one or two Daniell's elements, and connect the
wires from them with the rhcochord binding screws, which
should also be connected with a reverser or a commutator
(Figs. 33, 34) ; if the latter, to the upper screws : if the former,
to the middle S3iews. The electrodes are connected to the
lower screws of the commutator, and to the end screws of the

PRACTICAL PHYSIOLOGY.

225

reverser. The handle of the commutator can be raised or
lowered, and thus the direction of the current is changed.

Fig. 33. Commutator.

When horizontal the current is cut olf. In the reverser the
current is changed by rotating the arch of wire, a b in the fig., to
the right or left alternately.

A

Fig. 34. Keverser. Brass wires (not shown in fig.) connect
mercury cups corresponding to A' and B", and A" and B'.

First of all put all the plugs in the rheochord, and push the
mercury cups close to the blocks. No excitation occurs on rais-
ing or depressing the handle of the commutator. Gradually
increase the strength of the current, and note the point at which

15

226 PRACTICAL PHYSIOLOGY.

a contraction takes place as well as the direction, and whether it
occurs at the make or break, or both. Make a table of the results
and it will be found that the make contraction of the descend-
ing current is the first to occur, then the make of the ascending.
Then, as the current becomes moderately strong with the make
and break of both, and with a very strong current with the make
of the descending and the break of the ascending.

The Rheoscopic Frog. — Prepare two nerve-muscle prepara-
tions, in the one case exposing the sciatic nerve throughout its
length, and removing the skin from over the gastrocnemius only,
but in the other case removing the leg with a long length of
nerve. Insert the electrodes beneath the sciatic nerve of the
first preparation ; place this on a glass plate, in order that ii may
be insulated. Place the sciatic nerve of the second preparation
over the thigh muscles of the first, and excite the muscles of
the first with a single induced current ; the muscles of the
second will contract. Repeat with a series of shocks ; the second
preparation will be thrown into tetanus, as well as the first.
Show that this is not due to escape of the current by ligaturing
the nerve. It is caused by the contraction of the muscles of the
first producing a variation in their natural current (p. 236).
This acts as a single stimulus to the nerve of the second, and so
causes a contraction. Instead of passing a current through the
nerve-muscle preparation, the nerve of the second may be
dropped upon the muscle of the first preparation in such a way
that one part of it falls upon the equator, whilst another part
falls upon the muscle near its insertion into the tendon, or upon
a transverse section of the muscle. The instant that the nerve
falls upon these two points, the muscle of the second prepara-
tion will give a single contraction. The experiment may also
be demonstrated by allowing the nerve of the limb to rest in a
curve upon the exposed heart of the frog ; at each systole of the
heart the muscles of the limb will contract.

Effect of Urari. — Carefully destroy the brain of a frog, with-
out allowing the escape of any blood, inject a drop of a standard
solution of urari, 0"1 per cent., into the posterior lymph-sac,
after having dissected out the sciatic nerve of one leg, and liga-
ture the limb tightly, in order to arrest the circulation in it,

PRACTICAL PHYSIOLOGY. 227

excluding the nerve. Place aside under a glass shade for an hour,
and test the excitability of both limbs. It will be found that the
muscles of the ligatured limb will respond to stimuli applied both
directly and also through the nerve, whereas the other limb will
not respond to nerve stimulation, although it does so when the
stimuli are applied directly to the muscle. This experiment shows
that the poison has acted U])on the nerve terminations, as it has
affected neither nerve nor muscle.

2. Mechanical stimulation.— Prepare a nerve-muscle pre-
paration : pinch or prick the nerve ; contraction will occur.

3. Thermal stimulation.— Touch the nerve or muscle of a
nerve-muscle preparation with a hot needle ; a contraction will
result.

4. Chemical stimulation. — Allow the nerve of the same or
of a new nerve-muscle preparation to dip into a watch-glass full
of strong saline solution ; flickering contractions which may pass
into tetanus will result. A similar experiment may be per-
formed with glycerine. Ammonia will stimulate muscle, but
not nerve ; glycerine has the reverse effect.

GRAPHIC METHOD.

This consists in arranging the muscle-nerve preparation in such
a way that, on contracting, the muscle raises or moves a lever,
which lever is made to mark on a rapidly travelling surface.
Yarious methods satisfy these requirements. In the first place,
the muscle and nerve may be removed from the body, or may be
retained in situ, and, again, the recording apparatus may be a
revolving cylinder covered with blackened paper, or it may be
a pendulum myograph or a spring myograph ; of all of which
apparatus there are many varieties.

The Recording Cylinder (Fig. 35).— This apparatus con-
sists of a cylinder or drum, which is arranged to move upon
axes, revolving at definite and different rates, by means of a
■clockwork mechanism contained in a brass box, firmly resting
upon supports. The velocities of the movement are (1) slow ;
(2) medium ; (3) fast. The axes of 1 and 3 move in the same
direction, viz., from right to left ; 2, on the other hand, moves

15—2

228

PRACTICAL PHYSIOLOGY.

in the reverse direction. By means of a screw in the axis of the
drum itself the drum maybe raised or lowered at the will of the

Fig. 35. AiTaugement of Apparatus for recordirig with a revolving
cylinder the Contractions of Muscle.

A. The cylinder or di'uni which is situated on the most anterior and
most rapidly revolving axis. It is in connection with the brass
box containing the clock-work. Two other axes ai'e seen behind,
and on either side of that bearing the drum.

B. Frog ou a support ; its muscle is connected by a thread with the

Myograph : the whole being supported upon an upright springing
from a triangular steel rod.

C. Du Bois Pieymoud's induction coil.

D. Du Bois Pieymond's key.

E. Key in x>rimary circuit.

F. Battery.

{After Kirhes Physiology.)

operator. The apparatus works as well when placed in a hoii-
zontal as in a vertical position. In the front of the box is

PRACTICAL PHYSIOLOGY. 229

arranged a fan in a metal frame, which regulates the clockwork
movement. The movement may be stopi)ed by means of a metal
clip and handle, which are fixed so that when the handle is
jiressed down the clip catches a steel axis about which the fan
revolves, and the clockwork is stopped. Underneath the case of
the clockwjrk ia a handle by which it is wound up. For the
sake of convenience, in the front of the brass box of the clock-
work are two screws, by which is fixed a metal frame, carrying
a long, stout, triangular steel bar, which can be adjusted at
different positions and lengths, by means of screws in the frame.
On this triangular bar various metal uprights for carrying
apparatus can be arranged. Also at one point of the lower edge
of the drum is attached a metal catch for the purpose of opening
a key placed in its way, called a turn-over or trigger key
(Fig. 36), the use of which is to have the muscular contraction
recorded exactly at the same place on the cylinder.

Having mastered the mechanism of the apparatus, the student
will then arrange it for work. The drum must be evenly covered
with glazed paper, which is generally kept ready cut in strips of
the size of the drum. A strip is placed round the drum, care
being taken that the join should not be near the metal catch
mentioned above, as in that case the tracing of the lever will be
over the join, and so be spoiled. Having firmly and evenly
covered the drum, it must be blackened over the smoky flame
of a small paraffin lamp or a spirit lamp, in the spirit of which
camphor has been dissolved. For these operations the drum
will have been removed from the clockwork ; it may now be
replaced, the clock wound up, the metal bar firmly fixed, and
everything arranged in readiness for the recording operation.

Method. — As above mentioned the muscle and nerve may
remain in situ, or be removed from the body. First of all use
the apparatus already described. This consists of a triangular
piece of wood, covered to a certain extent with cork, and
with an upright cylinder of the same material fixed at the
side. In front is a lever arrangement, by which the move-
ment of the muscle in contraction is communicated to the
recording apparatus. On one side is fastened a small collar,
which can slide up or down an upright fixed to the triangular

230 PRACTICAL PHYSIOLOGY.

rod above described, and by means of a screw can be secured at
any height which may be required.

In a nerve-muscle preparation, such as ii. (p. 216), remove the
skin from the foreleg of the frog, and having cut through the
tendon of the gastrocnemius at the os calcis, turn it up and cut
off the remainder of the leg just below the knee-joint. Attach
a strong ligature of silk or thread to the tendon, place the frog
on the cork plate, firmly fix the limb by passing a long pin
through the knee-joint, and then attach the ligature from the
tendon to the metal at right angles to the marking lever, care-

C 3 0 7

fulh' noticing that the ligature is taut, and that the muscle is
reall}- pulling on the lever ; load the lever with a 10 or 20 grm.
weight, and fix the myograph on the upright before spoken of,
which slides along the triangular steel bar, so that the lever
touches lightly the blackened surface of the recording cylindei'.
with the point writing the proper way. The apparatus is now
arranged.

Moist Chamber. — This consists of a moveable platform, slid-
ing up and down the upright of a stand not unlike a medium-
sized retort stand, and capable of being fixed by means of a
collar and screw. The platform is made of hard wood or of
vulcanite, about 3i or 4 inches in diameter, and is furnished with
two sets of binding screws for electrical purposes. These screws
are continuous with wires which pass through the platform and
project below, so that battery or other wires may be attached to
them. In the front of the platform is an opening about one inch
square ; upon the same upright slide, (1) an electrode holder, made
very simply by fixing two copper wires in a small block of wood
with the centre hollowed out, the wires being stretched across
the hollow, and the end fixed in the wood ; they must be about
one third of an inch apart, and are to be separated by a piece of
cork. This wooden block is fixed upon a holder fitted with a
collar and screw to move up and down the upright. (2) A brass
rod, with the circular brass holder of a screw-clamp working
easily in a collar at its end. The clamp holder can be fixed by a
screw. A tall glass shade, large enough to cover the whole of
the above, fits into a groove which runs round the platform a
quarter of an inch from the edge. "When in use, pieces of blot-

PRACTICAL PHYSIOLOGY.

231

ting paper, wetted with water or saline solution, are inserted
beneath the shade, to keep the contained air moist ; hence the
apparatus is called a moist chamber.

Under the platform of the moist chamber is attached a metal
screw collar apparatus, similarly capable of movement up ami
down the upright of the stand, to which is attached a fine meti.l
spring and lever of light wood, capable of movement up and
down about a fulcrum near the collar. Having prepared a nerve-
muscle pre])aration in a manner similar to that described above,
but in addition having divided the sciatic, and turned it down
over the muscle, clear the femur entirely of muscle, and divide at
its lower third ; then fix the femur in the clamp of the moist
chamber, attach a ligature to the tendon {teudo achillis), and
carry it through the opening of the stage to the lever below,
which may be weighted in the same way as in the other appara-
tus, with ten to twenty grams ; place the nerve on the electrode?,
and bring the point of the lever to write on a cylinder as before.
In the following experiments one or other of the above arrange-
ments may be used.

a. Single Imluced Currents. — Arrange the induction coil,

battery and electrodes as described

(p. 217), interposing the turn-over

or kick-over key in the primary cir-
cuit. Cause the cylinder to revolve

on the middle axis, with the key

open. Before allowing the lever to

touch the drum, find out the point

at which the induction apparatus

will give a sufficient stimulation ;

allow the drum to reach its proper

rate of velocity ; then, by means of

a tangent screw, or some other

delicate adjusting arrangement,

make the lever touch the paper ;

let the catch pass the key, and then

close it. In the next revolution the Fig. 36. Trigger or kick-over key.

catch will open the primary circuit, and a contraction will

occur. Mark the exact period of excitation by allowing the

232 PRACTICAL PHYSIOLOGY.

cylinder to make nearly another revolution ; close the key, and
gradually advance the cylinder, until the catch touches it, then
slightly raise the lever, and allow it to mark the paper.

h. Faradization. — Arrange the key in the secondary circuit,
and the apparatus as usual for this purpose (p. 219). When the
hammer is properly working, allow the cylinder to revolve
rapidly, and open the key. A curve of tetanus will be recorded.

c. Records of tetanus curves with spring, and also with metro-
nome, and with vibrating reed, may be taken in a manner similar
to the above, but use the medium speed axis.

d. Effects of Heat and Cold. — Arrange the cylinder on the second
axis, allow the lever in connection with a nerve-muscle prepara-
tion to mark on the smoked paper, and set the clockwork in
motion ; a straight abscissa line will be drawn. Interpose the
kick-over key in the primary circuit, and by allowing the catch
to open the key, record upon this line a single muscular contrac-
tion, and mark the latent period. Now cool down the muscle by
fining a test-tube full of small pieces of ice, and bringing it for
ten minutes into close proximity to the muscle. Then set the
cylinder in motion ; and when everything is in readiness, remove
the test-tube and close the key. On its^next revolution the
cylinder will open the key, and a muscular contraction will
occur at the same point as before ; the curve will be found to
be less sudden and more prolonged. By filling the test-tube
with water of gradually increasing temperature, a series of
curves may be taken on the same line, which will show that up
to a certain point the curve will be more sudden and of shorter
duration.

e. Effects of Poisons.— ln]Qci to Jg mgrm. of veratria into
the posterior lymph-sac of a frog, and record the contraction of
the gastrocnemius at various intervals after injection. It will be
found that the full effect of this drug is to enormously lengthen
the curve. Use medium speed. Other poisons may be tried in
a similar manner.

/. Eff^ect of Fatigue. — After using a muscle to demonstrate a
single twitch, tetanus, and for other experiments, it will be
found that the contraction will after a time alter, and that when

PRACTICAL PHYSIOLOGY. 233

a single curve of a fatigued muscle is compared with that of a
fresh one, it is found to be much more prolonged, and possibly
less high, and that the latent period is longer.

g. Relafion of the Cuntractiun to the Load. — By loading a
muscle with different weights, it will be found that with the
same stimulus the contraction first of all increases as the load is
increased from zero upwards by small increments. As the load
continues to be increased, the increment diminishes, and finally
gives place to a decrease. The initial increase of contraction
is most prominent when its stimulus lies within a certain range
of intensity.

h. Time Measurement. — This is done by the vibrating tuning
fork, or by a reed made to vibrate a definite number of times
in a second. The tuning fork of large size has on one prong a
small style attached ; and after the prong has been smartly
tapped by means of a mallet or similar instrument covered with
felt, the style, if applied to the revolving cylinder, will mark the
number of times the fork vibrates in a second. Another way is
to place the tuning fork in a battery circuit, and allow its vibra-
tions to be communicated to a small chronograph, which writes
on the recording surface. This instrument consists of a small
electro-magnet. Each time the iron is magnetised it draws
down a piece of metal, arranged on a frame in such a way that
it can move to or from the magnet ; at the other end of the
frame is a small pedunculated hook, to which is fastened an
elastic counterpoise. To the frame is fixed a style, capable of
writing on a drum. Each time the rod vibrates, the current is
made, the magnet draws down the piece of metal, and so the
style makes a stroke on the smoked paper. At the break the
elastic raises the style, and so on.

The Pendulum Myograph. — In this instrument, the clock-
work movement, which is frequently unreliable, is replaced by
the force of gravity. The recording plate is attached to a
pendulum. On this principle several instruments, differing
somewhat in detail, have been constructed. The pendulum with
the recording plate is fixed by a catch which is capable of being
moved certain distances along the arc through which the pen-

-34 PRACTICAL PHYSIOLOGY.

dulum swings. "When the arc is lengthened, the velocity is
altered. In its swing the pendulum knocks over, with a catch
attached to the middle of its lower edge, a trigger key placed in
the primary circuit, and by this means the muscle of a muscle-
nerve preparation gives a contraction. The preparation is
arranged in a manner similar to that described above (p. 229) for
the other kind of recording apparatus ; but the frog apparatus
is fixed in a collar to the upright of a firm stand, capable of
being raised or lowered by means of a screw. The upright is
fitted with a circular movement worked by a tangent screw at
its base. The stand, with the frog apparatus, is placed upon a firm
table close to the myograph : this table can not only be raised
and lowered, but is also capable of a circular horizontal move-
ment.

Method. — Allow the pendulum to hang vertically. Arrange
a nerve-muscle preparation on the table as above described.
Cover the glass plate of the pendulum smoothly with
glazed paper ; having smoked it, replace the j)late. Adjust the
lever so that it barely touches the plate at its edge. Arrange a
pair of electrodes under the nerve, interpose a key in the
secondary current of an induction apparatus, and fix the trigger
key (Fig. 36), at a convenient place in the battery circuit.
Close the key in the secondary circuit (key 2), then raise the
pendulum, and fix it in the catch to the right ; see to the adjust-
ment of the writing lever. Close the trigger key, then open the
second key, and set free the pendulum. As it passes the trigger
key, a stimulus will be sent into the nerve by opening the
battery circuit, and a contraction will be recorded on the plate as
it passes the lever. Underneath this tracing, a tracing of a
vibrating tuning fork must be taken, and the latent period may
be marked by returning the pendulum to its place, closing the
second key, and then by carefully approaching the pendulum
catch to the trigger key, which can be kept closed by the finger ;
by slightly raising the lever and allowing it to make a mark
upon the paper, the exact 'point where the current entered the
nerve is recorded. The effects of heat and cold, and of the
action of poisons, can be demonstrated with this myograph, and
also by taking two pairs of electrodes, and placing one a long

PRACTICAL PHYSIOLOGY. 235

distance from, and the other near, the muscle, connecting them to
the end screws of a reverser (Fig. 34), from which the cross
wires have been removed, and alternately throwing the current
into one or the other pair of electrodes ; the effect of having a
long or a short piece of nerve stimulated may be shown by the
difference in the length of the curve. By having two trigger
keys arranged at small distances, two coils, batteries, and elec-
trodes, the effect of two stimuli acting one after the other may
be shown.

The Spring Myograph. — In this instrument the clockwork is
replaced by the momentum imparted by a strong coiled spring.
The recording surface is, as in the case of the pendulum myo-
graph, a glass plate ; and in this instrument, as in the other, the
glass plate in its course opens a trigger key, and a contraction
similarly ensues, as the apparatus, induction coil, key, and elec-
trodes are arranged in an almost exactly similar way.

Muscle Currents. — An-ange a Thomson's galvanometer in
a dark chamber, and place the scale at about three feet east and
west facing it. Light the paraffin lamp, and, after having set
the mirror free by raising the screw adjustment at the top of
the instrument, adjust the lamp so that the light falls well on
it. By means of the magnet, adjust the mirror until it throws
its light upon the zero of the scale. To the binding screws of
the galvanometer attach the screws from the shunt, and for the
sake of practice allow a weak current to pass from a Daniell :
by attaching wires from the battery to the shunt, with the plug
in the hole marked ^^, the needle will be deflected, as indicated
by the movement of the spot of light on the scale : the light
will probably move in the same direction as the current. Pre-
pare two pairs of non-polarizable^ electrodes, and connect them
with the shunt ; the plugs being in the shunt, place the
electrodes so that their plugs touch : on opening the shunt
there will be no deflection. Now take a pithed frog, and
having dissected out the sciatic nerve, fix the femur in a cork

♦ A non-polarizable electrode is made by half-filling a piece of glass tube
with china clav moistened with saline solution and projecting as a plug from
one end. and then filling up the tube with a saturated solution of zinc sul-
phate, into which dips an electrode of well-amalgamated zinc.

236 PRACTICAL PHYSIOLOGY.

plate by means of a pin, and having attached a ligature to the
tendo-achillis, tie it firmly to a hook, and stretch it slightly ;
remove the remainder of the frog from the gastrocnemius thus
prepared ; place the electrodes near, having inserted the plugs
in the shunt so that one touches the middle, and the other one
end ; now open the plug i, the light will quickly pass off the
scale ; re-insert the plug, and remove another, so as to allow
less of the muscle current to pass through the galvanometer,
say lo^Qo ; then note the amount and direction of the current,
from the direction of the deflection and the degrees of the scale
the light has passed over. Similarly the position of the elect-
rodes may be altered, and the currents noted, and the general
laws demonstrated. For the purpose of showing the negative
variation of the muscle current when the muscle enters into
contraction, arrange under the nerve a pair of electrodes con-
nected with an induction coil ; the rest of the apparatus being
as before.

Electrotonus. — Prepare a nerve-muscle preparation, and re-
move it from the body, taking care that the sciatic nerve is
uninjured, and very long. Place the preparation in a moist
chamber, and arrange the nerve over two pairs of non-polariz-
able electrodes ; connect one pair of electrodes with an induc-
tion coil, and arrange for single shocks ; connect the other pair
with a rheochord, the binding screws of which are also joined
to the end screws of a reverser, to the middle screws of which
the wires of one or more Daniell's elements are connected.
Find out the exact minimal current which will cause a con-
traction, and then move the secondary coil a little farther away
from the primary. On making or on breaking the primary
current there will be no contraction ; but if now a descending
galvanic current, which is called the polarizing current, be sent
into the nerve, an induction current weaker than the normal
minimal will cause a contraction. This will also occur for some
time after the galvanic current has been shut off. Similarly, it
may be shown that if the current be ascending, an induced
current, stronger than the minimal, will be required to produce
a contraction ; in like manner, the effects of a series of shocks
may be shown, plus an ascending or descending continuous cur-

PRACTICAL PHYSIOLOGY. 237

rent. So we see that the irritability of the nerve is increased
during the passage of a constant current in a descending direc-
tion, and diminished if it be in a contrary direction. If the
relative position of the electrodes of the induction and polaris-
ing current be reversed, an exactly opposite effect occurs in all
particulars.

OTHER PROPERTIES OF MUSCLE.

Elasticity. — Prepare the gastrocnemius attached to the femur,
clamp the femur, attach the gastrocnemius tendon to the lever
of the moist chamber, and load the lever with a ten gram weight.
Allow the lever to mark on a recording cylinder, then load with
twenty to thirty grams, and so on. It will be found that the
extensibility gradually diminishes for equal increments of
weight. On removing the weights, the lever will return to the
same point on the paper from which it started.

Reaction. — Remove from a pithed frog a gastrocnemius which
has been i)erfectly freed from blood, cut it across wdth a sharp
knife, and apply blue and red litmus paper to the ends. It will
be found that there will be a bluish mark on the red litmus.

Tramparency. — Take a flat muscle from a pithed frog {e.g. the
mylohyoid ov sartoriiis), and as soon as possible place it on a slide
in saline solution. Examine with a quarter inch, and focus,
through the muscle some vessel underneath the fibres. It will
be found quite clear, and so prove the transparency of livincr
muscle. On entering into rigor mortis this property disappears..

CHAPTER III.

PHYSIOLOGY OF THE FROG'S HEART.

Anatomical considerations. — Open the abdomiual cavity in a
pithed frog by a longitudinal incision, avoiding the large veins
in the middle line ; cut through the middle of the sternum and
pin out the arms on either side ; pass a thick glass rod down
the oesophagus, open the pericardium, and note the following
points : the two auricles, the conus arteriosus on the right side,
dividing into the two aortse. Lift up the apex and the fine
ligament or frsenum connecting the dorsal aspect of the ventricle
with the pericardium comes into view. Tie a fine ligature to
the frsenum, which can then be severed from the pericardium.
When turned up, notice the auricles again and, behind, all the
sinus venosus ; at the junction of the sinus with the right
auricle, just above the auricular ventricular groove, is seen the
ivhite line. The sinus venosus is then formed by the confluence
of the inferior vena cava, the two hepatic veins, and the two
superior venae cavse. Note the sequence of the rhythmic con-
tractions, of the sinus venosus, auricle, and ventricle.

Position of the ganglionic centres. — The vagi run in company
with the superior venae cavse, and, on reaching the sinus venosus
split into a plexus, which is beset with ganglion cells (Remak's
ganglia). Some of the fibres are gathered together and pass on
into the septum auricularum, where there are again many gang-
lion cells {Bidder's ganglia). These fibres pass down in the
septum to the upper part of the ventricle, where there are
secondary ganglionic masses, particularly on the dorsal aspect.

The inhibitory centre. — Turn up the ventricle and apply elect-

PRACTICAL PHYSIOLOGY.

239

rodes to the white line above mentioned, stimulate with the
interrupted current for a second or two, and notice that the
heart stops in diastole, but soon goes on beating again.

Dissection to expose the vagus.— The easiest way to do this is
to dissect away the integument over the scapula, cut through all
the muscles attached to it, keeping close to the bone, divide the
brachial nerves and vessels and so remove the scapula with the
fore-limb attached. Close under the spot where the aorta of that
side gives off the carotid and axillary vessels lies the cornu of the
hyoid bone ; to this is inserted a thin strip of muscle which

S.V.C. Ganglion of Remak

r.v.c. a

Ganglion of Bidden.

Fig. 37. Gauglia of Frog's Heart. (Aj'ter Carpenter's Phijswlogy.)

can be traced upwards and backwards to its origin on the petrous
bone : this is the inferior petrohyoid. On this muscle lie three
nerves ; the most anterior passes down for a short distance, and
turns sharply forwards and upwards, towards the tongue (glosso-
])haryngeal); the middle, a slender nerve, passes down the middle
of the muscle to be distributed to the larynx (the laryngeal
branch of vagus) ; on the posterior border of the muscle lies
the vagus, which passes down over the root of the lung to the
sinus venosus. The nerve generally lies under some pigmented
veins and fascia.

240

PRACTICAL PHYSIOLOGY.

Stimulate the vagus. — (a) With a very weak interrupted cur-
rent, and with the arrangement known as Helmholtz modifica-
tion. Notice that the beat is slow by lengthening the diastole.
(&) With a strong current, the heart stops, after one beat, in
diastole.

IiUrinsic nervous mechanism.— {\) Excise the heart with a
sharp pair of scissors, leaving behind the sinus venosus, place
the excised auricles and ventricle into h per cent, salt solution.
Notice that the sinus goes on beating as before, but the excised

Fig. 38. Dissection showing position of Yagus in the Frog, (a) Glosso-
pharyngeal nerve ; (J) bvpoglossal nerve; ir) vagus; (f?) laryngeal nerve ;
(e) larynx; (/j petrobyoid musde ; {g) aorta; {h)\\mg; (i) auricle; (k)
bulbus arteriosus ; i7) ventricle.

heart stops for a time and then goes on again, especially if
stimulated ; the rhythm, however, is different from that of the
sinus.

(2) Separate the auricles from the ventricle ; iboth go on
beating, but with independent rhythm.

(3) Separate the upper third of the ventricle from the lower
two-thirds. The latter will no longer contract rhythmically,
but will respond to a single stimulus with a single beat, the
latent period being very long.

PRACTICAL PHYSIOLOGY. 241

SUinnius' ej-periment.— Fass a ligature between the auricle and
sinus veuosus below, and between the aorta) and venae cavse
superiores above. Tie it tightly, so that the sinus is separated
by the ligature from the rest of the heart. The heart stops in
diastole, but the sinus beats naturally. On stimulating the
flaccid heart single, and sometimes a succession of, beats may
be obtained. If now the heart be separated by a clean incision
along the line of the ligature, it will resume an independent
rhythm to that of the sinus.

Action of certain jJoixons. — Muscarin or PUocavpbie. — Place
the excised heart in neutral saline solution, with a trace of
muscarin or pilocarpine solution. It will become quite motion-
less in diastole, ^l/ro/»?/?.— Transfer the motionless heart to a
•2 per cent, solution of atropin in neutral saline solution.
Notice the gradual return of the beat. Atropin is the antagonist
of muscarin and pilocarpine.

Further action of J tropin.- Inject under the skin of the back
a few drops of atropin solution, 1 per cent. After about ten
minutes pith the frog and make a vagus preparation. Stimulate
the vagus; no inhibition follows, even with the strongest cur-
rents. Stimulate the inhibitory centre, and again no stoppincr
of the beat results. The vagus nerve-endings are paralysed.
Stannius' experiment, however, succeeds as with the normal
heart.

Frog-Ueart and Rheoscopic Limb. — Prepare the hind leg of a
vigorous frog, together with a long length of uninjured sciatic
nerve, and in the same pithed frog expose the heart and open
the pericardium. Arrange the frog on a glass plate, and also
the limb on another, then allow the nerve to fall upon the
ventricle. Each time the ventricle contracts, a contraction
occurs in the limb.

Graphic Methods. — For this purpose a hollow cylindrical box,
about three inches long and one inch in diameter, is fixed upon
a metal support. The box is provided wiih two metal tubes, by
means of which water at various temperatures may be passed
through it by attaching to the metal tubes gutta-percha tubes,
the one passing from a vessel fixed on a stand at some distance
above the frog box, and the other similarly fixed somewhat

16

242 PRACTICAL PHYSIOLOGY.

below. The lever is thus made : a glass rod, of the thickness
usually employed as a stirrer, is taken, and with a blow- pipe
flame it is softened sufficiently to allow of its being drawn out
at the softened part to great fineness : the fine part is then
broken at a point about five inches from the unaltered glass
tube, which is now similarly drawn out the other side, leaving
a knob of glass between the two thinner parts. On this side all
the thinned part is removed, and the glass now remains with a
thin arm about five inches long. A square piece of cork is now
passed along the thin glass to the knob, and through this a fine
needle is passed. The needle can be adjusted in bearings which
are fixed to the edge of the box. A second piece of cork is
passed along the lever arm, and is adjusted and cut so that its
point, directed downwards, can rest upon the ventricle of the
heart. After these corks have been put in place, the writing
end of the lever may be made by allowing the extremity to be
softened for a few seconds in the flame of a spirit lamp. The
frog heart-box can be adjusted to the recording cylinder, and
for the purpose of recording the contraction the cylinder should
revolve slowly. (1) Having exposed the heart of a pithed frog,
tie a ligature to its fraenum, and, cutting through the vessels, lift
it by the ligature to the heart-box ; having first allowed water
at about 10° C. to pass through it, moisten the heart by placing
a little serum on the top of the box by means of a capillary-
pipette. Adjust the lever so that the cork rests well upon the
ventricle, and the writing lever marks on the recording drum
record-tracings of the contractions at various temperatures,
cooling the water down by means of ice to 1° C, and then
raising it by increments to 20° C, and compare the tracings.
(2) If, instead of a heart beating in the ordinary way, a
Stannius preparation (p. 241) be substituted, the heart may be
stimulated by weak induction shocks, the apparatus being
arranged so that the electrodes just touch the ventricle ; and
with a key in the secondary circuit, and the kick-over key in
the primary circuit, definite regular contractions may be com-
pared, and the effects of temperature in altering the rapidity
and the strength of the contraction seen, 'as well as the latent
period recorded.

PRACTICAL PHYSIOLOGY. 243

Endocardial Pressure. — A large frog (Rana esculenia) is taken,
and the heart is exposed in the usual manner, the pericardium
opened, the fnenum ligatured, and the heart turned over by the
ligature. A cut is made into the bulb, and by this means a
double cannula is passed into the ventricle, a ligature is passed
round the heart, and the cannula is tied in tightly. The vessels
are then divided beyond tho ligature, and the cannula, with the
heart attached, is removed. To one stem of the cannula a tube
is attached, communicating with a reservoir of a solution of
dried blood in -70 saline solution, and filtered, which is capable of
being raised or lowered in temperature by being surrounded by
a metal box which contains hot, cold, or iced water. Attached
to the other end is a similar tube, which communicates by a T
piece with a small mercury manometer, provided with a writing
style, and also with a vessel into which the serum is received. The
apparatus being arranged so that the movements of the mercury
can be recorded by the float and the writing style on the slowly
revolving drum, and after some serum has been allowed to pass
freely through the ventricle, both tubes are clipped, the second
one beyond the J piece, and the alterations in the pressure are
recorded. The effects of fluids at various temperatures should
similarly be recorded in the manner indicated above.

Roy's Tonometer. — By this apparatus the alterations in
volume which a frog's heart undergoes during contraction are
recorded by the following means : A small bell-jar, open above,
but provided with a firmly fitting cork, in which is fixed a double
cannula, is adjustable by a smoothly ground base upon a circular
brass plate, about two to three inches in diameter. The junc-
tion is made complete by greasing the base with lard. In the
plate, which is fixed to a staud adjustable on an upright, are two
holes, one in the centre, a large one about one-third of an inch
in diameter, to which is fixed below a brass grooved collar,
about half an inch deep ; the other hole is the opening into a
pipe provided with a tap (stopcock). The opening provided
with the collar is closed at the lower part with a membrane of
animal tissue, which is loosely tied by means of a ligature
around the groove at the lower edge of the collar. To this
membrane a piece of cork is fastened by sealing-wax, from

Ki— 2

244 PRACTICAL PHYSIOLOGY.

which passes a wire, which can be attached to a lever, fixed on a
stage below the apparatus. "When using the apparatus, fix the
bell- jar by means of cord, drop a little glycerin into the collar
closed by membrane, and fill the jar with olive oil. Now pre-
pare, in the way above described, the heart of a large frog, tie
in the cannula, which is, as before mentioned, fixed in the
cork ; the tubes of the cannula communicating with the reser-
voir of serum on the one hand, and with a vessel to contain the
serum after it has run through on the other. Pass the cannula
with heart attached into the oil, and firmly secure the cork.
Now open the tap, raise the membrane a little, and allow a few
drops of the oil to pass out ; shut the tap, and let go the mem-
brane. By these means the lever will be found to be adju^^ted
to a convenient elevation. Allow the lever to write on a
moving drum, pass serum through at various temperatures, and
compare the tracings. After a short time the heart will stop
beating ; but two wires are arranged, the one in the cannula,
the other projecting from the plate in such a way that the heart
can be moved against them by shifting the position of the bell-
jar a little. The wires act as electrodes, and can be made to-
communicate with an induction apparatus, so that single induc-
tion shocks can be sent into the heart to produce contractions,,
and if need be, by means of the trigger key, at one definite
point in the revolution of the recording cylinder.

CHAPTER lY.

NERVOUS SYSTEM.

Functions of the Medulla Oblongata in the Frog. — Hold
the frog as if about to pith it (see p. 215) : divide the skin,
oc3ipito-atlantoid membrane and medulla by a transverse cut
with a sharp scalpel ; destroy the brain by thrusting a pointed
piece of wood into it. The operation should be almost blood-
less. The frog should be allowed to rest on its belly for a short
time, to enable it to recover from the shock. Before long it
will be found to have assumed a nearly normal attitude. It
does not, however, make any si)ontaneous movement, provided
that it is kept moist and at an equable temperature. If the flank
be gently stroked, the muscles will twitch ; and if the stimulus
be more violent, bilateral movements will occur. This is best
seen in a frog which is freely suspended. If the skin about the
anus be pinched, both legs will be simultaneously drawn up.

Functions of the Roots of the Spinal Xerves. — Divide the skin
along the back of a frog, whose brain alone has been destroyed,
as in the previous experiment. Separate the muscles of the
back, so as to expose the arches of the vertebrae, which should
be carefully cut away with a pair of blunt-pointed scissors.
The roots of the nerves will then be seen within the spinal canal.
Expose the roots of the eighth, ninth, and tenth nerves, taking
the greatest care not to touch them, by completely removing the
surrounding structures. The posterior roots will then be seen
to be the larger and the more superficial ; they conceal the
anterior roots. Select the largest of the roots now visible — it is
thit of the ninth nerve— and pass a fine silk ligature round it

246 PRACTICAL PHYSIOLOGY.

without touching it more than is necessary. Tighten the liga-
ture. At tho nv^vAQ instant movements will be noticed in some
part of the body of the frog. Cut the nerve between the
ligature and the cord ; movement will again take place. Place
the proximal portion of the divided nerve upon a pair of elect-
rodes in connection with a Du Bois Reymond's induction
apparatus ; decided movements will occur on the pass^age of a
current, whilst no such movements are seen when the distal
extremity of the nerve is stimulated in the same manner.

Cut away the posterior root, and repeat the preceding experi-
ments with the anterior root ; movements will occur as soon as
the root is touched, as well as when the ligature is tightened,
and when the nerve is divided. Tetanus ensues upon stimula-
tion of the distal portion of the nerve ; but the passage of an
electric current through the central end produces no result.

Functions of the Spinal Cord. — In a preparation in which the
brain has been destroyed, and the cord divided below the
medulla, (a) the reflex function of the cord may be shown by
irritating the surface of the skin by means of small pieces of
filter paper dipped in acid, and placed in various situations ;
contraction of certain muscles usually follows for the purpose
of getting rid of the irritation. The groups of muscles are as
a rule regularly brought into action Avhen particular parts are
irritated, {h) If the irritation occur in one leg, and that leg
be prevented from moving, the other leg will act ; but after a
time general contraction may occur, [c) If the irritation be
very great, or if the frog be under the influence of strychnia,
general convulsions may occur.

APPENDIX.

ON THE PRESERVATION OF NORMAL AND
PATHOLOGICAL MUSEUM PREPARATIONS.

The only satisfactory method of making permanent preparations of
entire organs or tissues is to obtain the specimen whilst it is quite fresh.
When this can be done it is placed in a zinc or earthenware basin, and a
small stream of fresh water is allowed to flow upon it for twenty-fonr
hours. At the end of this time it is deprived of the blood which it con-
tains by gently squeezing along the course of the main vessels. It is then
dissected in such a manner as to show the especial features for which it
is preserved. It will be found that the dissection is more readily and
cleanly performed at this stage than after the preparation has been
placed in spirit. It is then put back into the basin and the water is
allowed to flow over it until the greater part of its colouring matter has
been washed away. In cold weather this is readily effected, but in
summer it must be carefully watched to prevent decomposition setting
in. The amount of water which flows over the specimen is of less im-
portance than its constant renewal.

As soon as the washing is complete, which will usually be in forty-
eight to seventy-two hours after its commencement, the preparation
should be suspended by means of silken threads in the glass jar in which
it will eventually be preserved. A mixture consisting of half methy-
lated spirit and half water is then poured over it. The bottle should
afterwards be conspicuously labelled and set aside, the spirit being
changed as often as it becomes discoloured. In dealing with large
numbers of specimens it will be found convenient to place in each jar
an ungi'ound microscopic slide or other slip of glass, upon which a re-
ference CO the specimen has been made by means of a w riting diamond,
details being at the tame time entered in a book kept for the purpose.

248 APPENDIX.

As soon as the spirit in which the specimen is immersed remains clear,
the final dissection should be made under weak spirit, all loose pieces of
connective tissue being removed with a pair of curved scissors, the
muscles being carefully cleaned, and the various points of interest being
demonstrated to their greatest advantage. The preparation is then sus-
pended in a mixture of proof spirit and water in the proportion of one
part of spirit to two of water. Proof spirit is employed as the perma-
nant preservative agent because it does not become cloudy with sudden
atmospheric changes, as is so frequently the case with methylated
alcohol. The glass jar must be provided with a ground rim, upon which
a glass cover of moderate thickness fits flush. The cover is luted to the
bottle by a cement prepared according to the follo^\ing formula :

Melt together, in an iron or earthenware vessel, one pound of gutta-
percha and one pound of asphalt, and when they are thoroughly mingled
p>our them on a slate or stone slab which has previously been moistened
with water. Roll the mass into cylinders of convenient length and
thickness, and use the sticks in the same way as sealing-wax. The glass
cover shoiild be gently warmed before it is pressed upon the hot cement
which has been spread along the rim of the glass jar. A weight, vary-
ing from four to twelve pounds according to the size of the jar, is placed
upon the freshly-cemented top for a period of forty-eight hours, after
which the superfluous cement is scraped away and a neatly applied
ring of Brunswick black is painted round the edge and top of the
cover in such a way as to conceal the line of junction.

The method here detailed is not applicable to every specimen. The
hollow \iscera, such as the uterus, bladder, etc., after they have been
washed for twenty-four hours, and after the removal of redundant
tissue, should be stuffed with tow, cotton-wool, or horse-hair, the aper-
ture being subsequently sewn up. They should then be suspended for
seventy-two hours in 64 per cent, methylated spirit, after which the
stuffing should be removed, the preparation being placed in the ordinary
mixture of methylated spirit and water, until they are ready for mount-
ing. These preparations are put into strong spirit to cause them to set
in the form given to them by the stuffing. Structures which deteriorate
in water, such as the brain and other nervous tissues, and those in
which it is desired to preserve, as far as possible, the original colour,
should be placed at once in strong spirit.

Large anatomical and physiological preparations may be embedded in
plaster of Paris, in white earthenware basins with ground rims, the basin
being subsequently filled \\-ith diluted proof spirit, and the cover sealed
down with a cement made by mixing together litharge and gold size to
form a paste. As in the case of the glass jar, a weight must be placed

APPENDIX. 249

upon the cover to keep it firmly adherent to the basin until the cement
begins to dry, which will not usually take place much under a week.

Specimens showini^ colloid degeneration, tuljercular synovitis, villous
tumours, etc., may be preserved in a mixture of equal parts of glycerine
and distilled water, to which has been added a trace of corrosive subli-
mate (1 part in 1500) or of pure carbolic acid.

In a few cases where it is necessary to preserve as far as possible the
colour and translucency of the preparation, as in voluntary muscle
infested with trichina, Wickersheimer's fluid may be employed. This
fluid can be obtained from the importers, Messrs. W. Schacht and Co.,
26, Finsbury Pavement, E.G., at a price of 2s, 6d. per quart bottle, or it
may be manufactured according to the accompanying formula :

Alum ... ... ... 100 grammes.

Caustic potash .. .. 60 ,,

Chloride of soda... ... 25 „

Nitrate of potash ... 12 „

Arsenious acid ... ... 10 „

Dissolve the ingredients in three litres of boiling water. Cool. Filter,
and afterwards add to each litre of fluid iOO c.c. of glycerine and
100 c.c. of proof spirit.

The fresh preparation should be entirely immersed in the fluid for
ten days, and it should then be suspended in a glass jar with a small
quantity of the fluid beneath it. The jar should be sealed in the usual
manner. Neither the glycerine process nor Wickersheimer's fluid, how-
ever, have been found to yield very satisfactory results, and for valuable
specimens it is better to adopt the alcohol method.

Small specimens, such as ovaries, sections of human eyes, etc., may
be conveniently preserved and shown, by means of glycerine jelly, in
the following manner :

After clearing away all redundant tissue, the specimen should be
placed in Miiller's fluid for a month. If its contents are suspected to
be fluid, or semi-fluid, it should be frozen by placing it in a mixture of
powdered ice and salt, and whilst it is hard it should be divided in the
required direction with an ordinary sharp table-knife. The colouring
matter of the Miiller's fluid must then be dissolved out by placing the
specimen in a solution of chloral hydrate (40 grains to the ounce), the
chloral being changed as often as it becomes discoloiired. The prepara-
tion is then placed for two or three days in a mixture consisting of one
part of glycerine and three parts of water, from which it is transferred,
for forty-eight hours, to a stronger mixture of equal parts of glycerine
and water. It is then ready to mount in glycerine jelly, which is made

2 50 APPENDIX.

thus : One part of Coignet's (Paris) gold label gelatin* is soaked in six
parts of distilled water until it is thoroughly swollen. It is then heated,
and six parts of glycerine, to which a trace of pure carbolic acid has
been previously added, is mixed with it, and the whole is filtered whilst
it is hot through white filtering paper or cotton-wool — the heat being
maintained during the filtration by means of a funnel vriih a hot- water
jacket. The resulting jelly is preserved for usp in a st<jppered bottle.

To mount in glycerine jelly, small glass capsules similar to those em-
ployed in histological work are required, but they should be made of
thicker and more brilliant glass. The jelly is liquefied by placing the
bottle which contains it in hot water, and it is then poured over the
preparation, which has been previously placed in the capsule. In the
case of sections of the eye, care must be taken not to pour the jelly over
the cut edge, lest the retina be detached. All bubbles should be care-
fully removed, and whilst the jelly is setting the specimen should, if
necessary, be kept below the surface by gently pressing upon it with a
needle fixed in a handle. When the jelly is quite firm its surface should
be covered with an even coating of zinc paint, over which again a layer
of shellac may be spread.

The glass jars should be round and of clear white glass free from
bubbles. Large oval bottles frequently crack spontaneously, owing, so
far as can be ascertained, to the extreme difficulty which is experienced
in getting them properl}' annealed. The square bottles made in Hol-
land, although very much cheaper and useful as stock bottles, are not
of sufficiently good glass to be of much service in a well-kept museum.
The covers of the basins, as well as of the large glass jars, should be
provided with a hole plugged with a well-ground glass stopper — as,
apart from the convenience of filling them without disturbing the
cement, it will serve to prevent the covers cracking across as a result of
variations in the bulk of the spirit due to alterations in the atmospheric
conditions.

THE ORGAN OF JACOBSON.

Preparation. — By placing the anterior portion of the head of a young
guinea-pig in a ^ per cent, solution of osmic acid for twenty-four hours,
and subsequently in absolute alcohol for 48 hours. A trace of hydro-
chloric acid is added to decalcify the bone, and the sections are made in
a plane parallel to the tip of the nose.

* Price 2s. lOd. per pound. The orduaary gelatins sold in packets in this
country, even Nelson's No. 1 gelatin, used for photographic purposes, will
not give a transparent jelly.

APPENDIX. 251

Stnidia-e. — The organ of Jacobsou is bilateral, and consists of a tube
lying in the lower or osseous part of the septum narium. It is flattened
when seen in transverse section, so as to appear kidney-shaped, and is
surrounded by a special investment of hyaline cartilage which is fre-
quently incomplete. This cartilage is known as Jacobson's cartilage.
The epithelium lining the tube is similar to that covering the mucous
membrane of the nasal cavity, but the epithelium lining the inner or
mesial side of the canal is thicker than that on the outer side. The
thicker epithelium is called the sensory epithelium. Beneath the epi-
thelium is a fibrous layer, and still deeper are glands and a tissue con-
sisting of a cavernous system of bloodvessels. Numerous branches of
the olfactory nerve supply the organ, which also contains a few
unstriated muscle fibres. Nothing is known of its function except that
it is accessory to the sense of smell.

PREPARATION OF UREA.

Concentrate urine to a fourth of its bulk, and add a cold saturated
solution of oxalic acid. This throws down crystals of oxalate ; remove
them and boil with some chalk, and a little water. Oxalate of lime is
precipitated, and can be filtered off together with the excess of chalk.
Carbonic acid is given off, and urea remains in solution, and may be
crystallized out by concentrating (Berzeliub).

Or mix urine with animal charcoal and evaporate gently to dryness.
Extract with alcohol and concentrate till a drop will crystallize, then
set aside to cool.

BACTERIA STAINING.
It may be useful, and not out of place, to devote a few lines to
the subject of bacteria staining.

(1) Stainimj cover-<jlais /iltiis of dried material, e.g., sputum, pus,
blood, or masses of micro-organisms from a cultivation. The material
should be spread out in the thinnest possible film upon a cover-glass,
dried in the flame of a spirit-lamp to coagulate all the albumen, and
placed with the film downwards upon some drops of the staining fluid
in a watch-glass for five minutes. It should then be washed quickly in
dilute spirit, then in distilled water, dried in the flame of a spirit-lamp,
and mounted in Canada balsam. Sometimes the washing has to be
done in acid solutions instead of in water. This is the case with the
bacilli of tubercle. In that case, too, double-staining should be used, as
will be described below.

(2) Staininy .-iectioiis of tissues &\io\Adi be done in much the same way,
but after staining in one dye it is usual to pass them through a second to
double-stain. In this way the bacteria are stained one colour, whilst

252 APPENDIX.

the tissue itself is another. The combinations are very various. After
double-staining, the sections are passed rapidly through the dehydrating
and clearing solutions, and mounted in Canada balsam.
For single stains the following solutions are most useful :
i. An aqueous solution of Spiller's purple, to which a few drops of
spirit have been added.

ii. An aqueous solution of methylen blue.

For double stains, it is only necessary to give the folloMring combina-
tions out of very many :

i. Stain No. 1. Fuclmne, made thus :
Anilin oil, 5 to 25 c. c.
Distilled water, 100 c.c.
Shake well together and filter.
To 100 c.c. of anilin water thus prepared add 11 c.c. of
alcoholic solution of fuchsine.

Stain the sections for at least thirty minutes, slightly warming the
solution. Wash in nitric acid, 1 in 3, then in water, and transfer to

Stain No. 2. Mefhi/Ieu blue, or Vesuvin, the former a saturated
watery solution, the latter a 1 to 2 per cent, solution for a few minutes.
Wash in alcohol, clear in clove oil or oil of cedar, and mount in Canada
balsam.

ii. Weigert's 1st Stain :

2 per cent, watery solution of gentian violet
Saturated watery solution of anilin

Mix.
2nd Stain :

Bismarck brown...

Sp. Vini rect. (sp. gr. "SSO)

Distilled water ...

Fifteen minutes in this.
(E-ecommended by Klein for tubercle, who, however, states that
the colour fades. )

iii. Ehrlich -Weigert's formula for sections :

Saturated watery solution of anilin ... 100 cc.
Saturated alcoholic solution of fuchsin,

or methyl violet, etc. ... ... 11 cc.

After staining in Ehrlich-Weigert's fuchsin, wash in distilled water,
immerse it in alcohol for a moment, and then place in the following
solution :

Distilled water ... ... ... ... 100 cc.

Saturated alcoholic solution of methylen

blue ... ... ... ... ... 20 cc.

et

12

c.c.

100

c.c.

1

grm

10

c.c.

100

c.c.

APPENDIX. 253

Formic acid (pure) | i n •

(Or glacial acetic acid) j lU mm.

Leave the .-ections in this s<jlution for one to two hours, and then
treat in the ordinary manner with water, alcohol, and oil of cloves.

iv. For anthrax, a combination of aqueous mtthjihn blue (saturatt-d
Solution) and toMu (1 percent, in alcohol) i.su.sed. The eosin solution is
added drop by drop to a little of the methylen blue in a watch-glass, as
long as the precipitate which forms is redissolved. Stain for 1 to 3
minutes, wash in .spirit, fix in commercial benzine, clear in a mixture
of benzine and oil of cloves, and mount in Canada balsam.

For staining tubercle bacilli in tissues, we strongly recommend
the following method :

First of all half fill an ordina»y-sized test-tube with a solution of
fuchsin in anilin water, as in the above method iii. , but with somewhat
more fuchsin solution, say 15 per cent, of the alcoholic solution, adding a
few drops of absolute alcohol to keep it in solution ; warm the solution to
about 45'' C, filter into a porcelain dish, and keep the solution at about
the same temperature with a spirit lamp ; place the sections in this
solution and keep them in it for half an hour (or cover-glass specimens
ten minutes) then transfer into the washing fluid thus made :

Of a 10 per cent, solution of nitric acid ... 1 part.
Methylated spirit ... ... ... ... 1 part,

Distilled water ... .. . ... 1 part,

and wash thoroughly for several minutes.

Then transfer to a filtered solution of methylen blue (saturated) for
5-10 minutes. Afterwards wash in a saturated solution of sodium car-
bonate for 15 minutes, then in water. Xext take the specimens out
one by one on a section lifter, dehydrate quickly with absolute alcohol,
and place upon a glass slide. Then allow a few drops of the turpentine
and creasote mixture to fall upon the section, inclining the slide so that
the fluid quickly runs from it as soon as the latter appears transparent.
Remove the clearing fluid with filter paper and mount in Canada
balsam.

Although a long process, by its use the crystals so often present in
the nitric acid washed specimens are avoided, and the methylen blue
forma an excellent background for the red bacilli.

254

APPENDIX.

FORMULAE.

The following formulae may prove useful :
Cohn's Normal Fluid.

•1 grm. Potassium phosphate.
■1 ,, Magnesium sulphate (crystallised).
•01 ,, Calcium triple phosphate.
20 c.c. Distilled water.
And also the above with "2 grm. ammonium tartrate added, for
'cultivation' purposes.

Pasteur's Fluid :

Potassium phosphate ...

20 parts

Calcium ,,

2 ,,

Magnesium sulphate ...

2 ,,

Ammonium tartrate

100 „

Cane sugar

... 1,500 „

Water

... 8,476 „

10,000
Cane sugar to be omitted when Pasteur's fluid without sugar is
required (Huxley and Martin).

Mayer's Solution (with Pepsin) :

15 per cent, sokition sugar-candy

. 20 c.c.

Dihydropotassic phosphate

•1 grm

Calcic phosphate

. -1 ,,

Magnesic sulphate

. -1 M

Pepsin ...

. -23 „

(Huxley and Martin.)

INDEX.

Aberration, chromatic, 6
spVerical, 6
Accessory apparatus, 44
Acid albumin, 151, ISO, 195 ;
differentiation between acid and
alkali, 152
Acid hsematin, 175
Action of reagents on blood, 48
Adenoid tissue, 65, 99, 133
Adipose „ 66
Adjustment of microscope, 2
Adrenals, structure of, 130
Agminated glands. 104
Air cells, 1 1 1

Albumin, acid, 151, 180, 195 ;
differentiation of, 152
alkali, 15-2, 180
casein, 152, 179, 180
classification of, 149
egg, 150 _

examination of, 195
native, 150
nature of, 148
solubility of, 149
serum, 151, 179
tests, 148, 188, 195
Albuminuria, test for, 188, 193
Alcohol, absolute, 18
carmine, 28
hsematoxylin, 27
methylated, 18
Alimentary canal, histology of,
93-107
Large intestine, 104
(Esophagus, 99
Salivary glands, 98
Small intestine, 102
Stomach, 100
Teeth, 95 ; development of, 96

Alimentary canal, tongue, 93
Tonsils, 99

Alkali albumin, 152,195; haematin,
175

Alloxan, 168

Alveoli, 111

Alum caruiine, 28

Ammonium bichromate, 18
chromate, 18
molybdate, 81
urate test for, 191

Amrjeboid movement, study of, 53

Ampullae, structure of, 137

Amyloid substance, 157

Analysis of gall stones, 183

urinary calculi, 191

Angular aperture, 7

Anilin dyes, 29-31

directions for staining
with, 34, 251

Anode, 223

Anthrax staining, 253

Aperture, angular, 7

Apochromatic lens, 9

Apparatus, epitome of, 46, 147

Appendices epiploicse, 104

Aqueous humour, 16

Areolar tissue, 64

Army razor, 22

Arterial schema, 203

Arteries, structure of, 91

Auditory nerve, structure of, 79

organs, histology of, 135-
138

Auerbach's plexus, 88

Axis cylinder, 79

B.

Bacteria staining, 251
Baker, 35, 7iote

256

INDEX.

Baryta solution, 41

Battery for production of electri-
city, 217

Beale's carmine, 28

Beck's microscopes, 13

Bellini, tubes of, 119

Bertini, columns of, IIS

Bichromate, ammonium, 18
potassium, 18

Bile acids, preparation of, 182
test for, ISl

Bile, chemistry of, ISl

in urine, tests for, 181, 188

Bile pigment, test for, 181

Bismaik brov\-n, 30

Bismuth test for sugar, 163

Biuret reaction, 149

Bladder, structure of, 120

Blood, 47-57

Action of reagents on, 48-50
Amphibian, 57
Circulation of, 201-210
Chemistry of, 173-179
Coagulation of, '173, 178
Crystals, 55, 56
Double staining of, 57
Effect of cold on, 173
Estimation of by haemoglobin,

175
Gas chamber for, 48, 55
Histology of, 47-57
Lake condition of, 173
Mammalian, 56
Physiology of, 173, 201
Plasma of, 178
Pressure of, 205-209
Reaction of, 173
Spectrum of , 174, i7o
Serum of, 177
Staining of, 52, 57
Tests for, 56, 173, 174,
Vertebrate, 56
Warm stage for, 54

Blood corpuscles, 47

Method of counting.
Movements of, 55

Blood plates, 48

Blood-vessels, histology of, 91, 92

Bone, 68-72

Chemistry of, 158
Compact, histology of, 69

190

>1

Bone, Development of, 70-72
General characters, 68, 69
Preparation, 68
Spongy, 70

Borax carmine, 28

Bcittchers te.-t for sugar, 163

Bowman's sarcous elements, 75

Boxes for embedding, 21

Brain, histology of, 87

Bronchus, structure of, 111

Briicke's disdiaclasts, 75

Brunner's glands, 103

Brunswick black, 40

Bulb, structiire of, 85

Butter, chemistry of, 179

C.

Cabin'ets, 44

Cacao butter for embedding, 21
Calcifj'ing cartilage, 67
Calculi, analysis of, 191

examination of, 191
Camera lucida, 11
Canada balsam, 39, 40

Directions for mounting in,

38
Preparation of solution, 38
Cannula, method of making, 209
Capillaries, stnacture of, 92, 132
Capillifurm processes, 137
Carbo-hydrates, chemistry of, 160-

164
Carbolic acid, as a mounting solu-
tion, 38
in urine, 191, 193
Carboluria, test for, 191. 193
Cardiac muscle, histology of, 76
Cardiograph, 209
Carmine, 27, 28

directions for staining
with, 34
Cartilage, 66-68

Development of bone in, 70
Elastic, 67
Fibrous, 67
Hyaline, 66
Ossifying, 67
Casein, 152, 179

Castor oil, as an embedding mix-
ture, 21
as a mounting fluid, 38

INDEX.

257

Cathcart's microtome, 26
CeUs,

Cartilage, 67
Connective tissue, 6-3, 141
Central, 102
Ganglion, 83
Muscle, 76
Nerve, 83
Parietal, 101
Peptic, 101
Salivary, 98
Tendon, 62
Cementum, 96
Cementing reagents, 40
Cerebellum, histology of, 86
Cerebrum, histology of, 87
Ceruminous glands, 115
Chemical stimulation of muscle,

227
Chlorides in urine, quantitative
test for, 171
in urine, qualitative
test for, 180
Chloride of gold, 32
Cholesterin, properties of, 182

test for, 183
Chondrin, chemistry of, 159

test for, 197
Choroid, histology of, 145
Chromic acid, 17, 41
Chylous urine, test for, 191
Ciliary movement, 59

muscle of Riolani, 140
processes, 145
Ciliated epithelium, 59
Circulation, arterial schema to

demonstrate, 203
Circulation, microscopical exami-
nation of, 201-203
Classification of anilin dyes, 30
Cleaning slides and cover-glasses,

36
Clearing, 35
Clove oil, 35

Coagulated proteids, 156
Coagulation of blood, 178
milk, 180
Cochineal dye, 29
Cochlea, histology of, 135
Cohnheim's fields, 73, 75

Cohn's normal fluid, formula for,
254

Cold, effects of, on blood, 173

effects of, on excitability of
nuiscle, 219

Columnar epithelium, 59

Commutator, 225

Compartments, muscle, 75

Compound microscope, 5

Condenser, 10

Coni vasculosi, 123

Conjunctiva, histology of, 140

Connective tissues, histology of, 62

Constriction, lianvier's, 77, 79

Continental model of microscope, 2

Contractile discs, 75

Cord, spinal, histology of, 79-85

Corium, structure of, 114

Cornea, histology of, 140

Corneal corpuscles, preparation of,
141

Cornicula laryngis, 67

Corpus cavernosum, 124
luteum, 125
spongiosum, 124

Corpuscles, blood, coloured, 47
colourless, 47, 53, 54
counting, 50
feeding of, 50
preparation of, 52
staining, 52
tendon, 62
of Hassall, 129

Corti, organ of, 136

Cover -glasses, 36

measurement of, 36
cutting of, 36
cleaning of, 36

Cowper's glands, 124

Crouch's microscopes, 13,

Crusta petrosa, 96

Crypts of Lieberklihn, 103

Crystallin, chemistry of, 153

Crystalline lens, structure of 143

Crystals, blood, 56

Curare, effects of, 202, 226

Curdling milk, methods of, 180

Currents, ascending, 223

continuous, electrical ac-
tion of on muscle, 217

17

258

INDEX.

Currents, descending, 223

efifect of variations in

strength of, 22-i
extra, 221
interruption of, 222
regulation of strength
of, 223

Ciitting sections, 19-27

Cylinder-axis, 79

Cystin, test for, 194

D.
Dammar solution, 39, 40
Daniell's cell, arrangement of, 217
Degenerations of spinal cord, 81
Dehydration, 35
Delicate tissues, the preparation

of, 41
Dentine, histology of, 95-97
Derived albumins, 151-153
Descemet, membrane of, 141
Description of microscope, 2-5
Development of Ixjne, 70-72
of teeth, 96-98
Dextrine, 162, 196
Diabetic urine, 189
Dialysis, 151
Diaphragms, 3
Directions for

Choosing microscope, 12

Dissociating, 41

EmVjeddinfr, 21

Hand section -cutting, 20-23

Hardening, 17

Mounting, 35-40
in glycerin, 37
in Canada balsam, 38

Softening, 41

Staining, 27-35

Teasing, 40
Disc, contractile, 75

Hensen's, 75

interstitial, 75

lateral, 75
Discus proligerus, 125
Disdiaclasts, 75
Dissecting microscope, 5
Dissociation of tissues, 41
Double staining, 34
Drawing microscopical objects, 11
Drop bottles, 39

Drum for recording, 206, 227
Du Bois Reymond's key, 218, 223
induction coil,

218
rheochord,223
Ductless glands, histology of, 128-

131
Dyes, anilin, 30

E.

Ear

histology of, 135-138
Ampullae, 137
Cochlea, 135
Semicircular canals, 137
Egg albumin, 150
Ehrlich - "Weigert's method

staining bacteria, 252
Elastic cartilage, 67
stripe, 64
tissue, 64
Elasticity of muscle, 237
Elastin, chemistry of, 159
Electrical stimulation of muscle by
continuous current, 217 ; by in-
duction shocks, 219 ; by unipolar
stimidus, 221
Electricity, action of on blood, 49
Electrodes, negative, 223

preparation of, 216 ;

of

•23:

note

positive, 223
Electrotonus, 236
Embedding box. 21

materials, 20
Embryonal tissue, 65
Enamel, structure of, 95, 97
cap, 97
cuticle, 97
organ, 97
Endocardial pressure in heart of

frog, 243
Endochondral bone, 70
Eudomysium, 75, 76
Endoneurium, 78
Endothelium, 60

preparation of, 60
English model of microscopes, 4
makers of microscopes, 13
Eosin, 29, 79

INDEX.

259

Eosin, directions for staining with,

34
Kpidennis, 11."^
Ei)idi(lyinis, structure of, 123
Epiglottis, lOS
Epineuriuin, 78
Epithelium, 59

Ciliated, 59

Columnar, 59

Glandular, 60

Pigment, GO

Squamous, 58

Transitioual, 59
Ether freezing, methods of, 25, 26
Eustachian tube, structure of, 67
Examination of fresh tissues, 16 ;
of hardened tissues, 17 ; of cir-
culation, 201 ; of organic sub-
stances, 195-200 ; of a solid
substance, 198 ; of urinary cal-
culi and deposits, 191-194
Excitability of muscle, 219
Extra current of Faraday, 221
Eyelids, structure of, 139
Eye pieces, 3, 9

histology of, 139-146

Ciliary processes, 145

Conjunctiva, 140

Choroid, 145

Cornea, 140

Crystalline lens, 143

Eyelids, 139

Iris, 144

Lachrymal gland, 140

Lens, 143

Retina, 142

Sclerotic, 145

F.
Fallopian tubes, structure of, 126
Faraday, extra current of, 221
Faradization of muscle, 232
Farrant's solution, 37
Fasciculi of muscle, 75
Fat, chemistry of, 166, 179

structure of, 66
Fatigue curve, 232
Fehling's test for sugar, 189
Fermentation test for sugar, 163,
190

Ferments, pancreatic, 186
pepsin, 185
ptyalin, 184
rennet, 180, 186
Ferrein, pyramids of, 118
Fibres, nerve, structure of, 78
Fibrin, 155, 178
Fibrinogen, 154, 178
Fibrinoplastic globulin, 154
Fibro-cartilage, 67
Fibrous tissues, 62
Fish, examination of circulation

in, 203
Flogel's granular layer, 75
Forceps, 46

Fresh tissues, examination of, 16
Freezing mixtures, 25
Frog board, 201, 228

examination of circulation

in, 201
experiments with heart of,
240-244
Frog-heart box of Roy, 243
Funiculi of nerve, 78

G.

Galactophorous ducts, 127
Gall bladder, structure of, 107

stones, analysis of, 183
Galvani's experiment, 222
Galvanometer, method of using,

235
Ganglia of heart, 238
Ganglion cells, 83, 89
Gas chamber for experimenting

with blood, 55
Gases, action of, on blood, 48
Gastric juice, chemistry of, 185
Gelatin, properties of, 157

test for, 197
Gelatinous tissue, 65
Genito-urinary organs, histology
of, 117-127

Bladder, 120

Fallopian tubes, 126

Kidney, 117-120

Ovary, 124

Penis, 123

Prostate, 121

Testes. 122

Ureter, 120

17—2

260

INDEX.

Genito-urinary organs, terms, 125

Va.= deferen?, 121

Vesiculse seminales, 12-3
Giannuzzi, crescents of, 98, 99
Giant cells, 68
Glands,

Adrenal, 130

Agminated, 103

Bowman's, 138

Briinner's, 103

Ceruminous, 115

Cowper's, 124

Gastric, 101

Kidney, 117-120

Lachrymal, 110

Lieberkiihn's, 103

Liver, 105-107

Lymphatic, 132

Mammary, 126

Meilxjmian, 139

Mohl's, 139

Mucous salivary, 98

Nuhn's, 9-4

Pancreas, 104 ; action of, 186

Peptic, 101

Peyer's, 103

Pituitary bodv, 129

Pineal, 129

Prostate, 121

Pyloric, 101

Salivary, 98

Sebaceous, 115

Serous, 99

Spleen, 133

Supra -renal, 130

Sweat, 114

Th}-mus, 128

Tliyroid, 128
Glandular epithelium, 60
Glass covers, 36
slides, 35
Glisson, capsule of, 106
Globin, 155
Globulins, 153, 177, 185, 195

Crystallin, 153

Fibrinogen, 154, 178

Myosin, 153

Paraglobulin, 154, 177

Vitellin, 155
Glvcocholic acid, preparation of,
182

Glycerin,

chemistry of, 165
as a mounting fluid, 37
jelly, 3S, 249
Glycogen, chemistry of, 162
preparation of, 161
test for, 196
Glucose, chemistry of, 163

tests for, 163, 189, 190,

197, 199
to distinguish from lac-
tose, 199
GmeHn's test for bile pigments,

ISl
Gold chloride, 19, 32

size, 40
Graafian follicles, 125
Granular layer, Flogel, 75
Grape sugar, properties of, 163.

See Gluco-^e
Graphic method as applied to the
circulation, 203 ; to muscle, 227 ;
to the pulse, 204 ; to the blood
pressure, 205 ; to the heart, 241
Grey matter, spuial cord, struc-
ture of, 82-84
Guaiac test for blood, 174
Gum arable solution, preparation
of, 26

H.

Ha IK, structure of, 115

Hand- cutting, 20, 22

Hardened tissues, examination

of, 17
Hardening reagents, 17-19

Absolute alcohol, 18

Ammonium bichromate, 18
chroraate, 18

Chromic acid, 17

Chromic acid and spirit, 17

Gold chloride, 19

Mercuric chloride, 18

Methylated spirit, 18

Midler's fluid, 18

Nitric acid, 18

Osmic acid, 19

Palladiiun chloride, 19

Picric acid, IS

Potassium bichromate, 18
Hardening, directions for, 1 7, 19

INDEX.

261

Hasaall, concentric corpuscles of,
129

Hitmacytonieter, 51
Hsematin, 175
Hajmatoporphyrin, 175
Hiematoxylin, 27

directions for stain-
ing with, 33
Weigert's, 80
Haemin crystals, 56
Hsemochromagen, 175
Haemoglobin, 55

chemistry of, 174
preparation of cry-
stals of, 55
Hsemoglobinometer, 175
Hartnack's microscopes, 14
Haversian canals, 69

interspaces, 69
systems, 69
Heart, action of drugs on, 241

alteration in volume of

during contraction, 243
endocardial pressure in, 243
experiments with, 239-244
histology of muscle of, 76
influence of vagus on, 240
nervous mechanism of, 239
of frog described, 230
Stannius' experiment, 242
Heat, effects of, on excitability of

muscle, 219, 232
Heidenhain, semilunes of, 98, 99
Heller's test for albumin, 150
Helmholtz's modification of the

magnetic interrupter, 221
Henle, looped tube of, 118
Hensen's disc, 75
Histology, practical, 1-146
Human blood, histology of, 47
Hyaline cartilage, 66, 67
Hydrochloric acid, 41
Hydrocele fluid, experiments with,

178
Hypobromite test for urea, 169

Immersion lenses, 8-10
Indol, test for, 187
Induction coil, 218

Induction shock, passage of through

muscle, 218
Infundibulum of kidney, 118

of lung, 111
Injection of tissues, 42
Ink, staining with, 29, 79
Inosite, chemistry of, 164

test for, 199
Interlobular veins, 106
Interrupter, magnetic, 220
metronome, 222
spring, 222
Interstitial disc, 75
Intestine, histology of, large, 104

small, 102
Intralobular veins, 106
Involuntary muscle, histology of,

76
Iodized serum, 16, 41
Iris, structure of, 144
Irrigation, 48

Jacobson's organ, histology of,

250
Jelly, glycerin, mounting in, 250
preparation of, 38,
249

K.

Kathode, 223

Key, Du Bois Reymond's, 217,223

Morse's, 217

trigger, 229
Kidney, 117-120
Knives, 44

Krause's membrane, 75
Kymograph, 2o6

Lachrymal gland, histology of,

140
Lactiferous ducts, 127
Lactose, 163, 180, 199

to distinguish from glu-
cose, 199
Lacunae of bone, 69
Lake condition of blood, 173
Lamina fusca, 145
vitrea, 145
Lantermann, sections of, 79

262

INDEX.

Lardacein, 157

Large intestine, structure of, 104

Laryngoscope, method of using,

213
Larynx, structure of, 109
Lateral discs, 75
Leitz's microscopes, 14
Lens, apochromatic, 9

crystalline, structure of, 143
dry, 8

immersion, 8-10
Lenses, 6, 8, 9, 10, 12
Leucin, preparation of, 187

test for, 199
Leucocytes, 47

method of feeding, 50
movements of, 53
Lieberkiihn, crypts of, 103
Liebig's test for chlorides, 171

for urea, 171
Lifter, section, 36
Light, sources of, for microscope,

10
Liver, structure of, 105-107
Logwood, 27

directions for staining
with, 33
Lung, structure of, 109-112
Lymphatic cells, 63

glands, 133
Lymphatics, structure of, 132
Lymphoid tissue, 94, 99, 133

M.

Macula germinativa, 125
Magnetic interrupter, 220
Malpighi, corpuscles of, 134

pyramids of, 117, 118
rete of, 113
Mammary gland, structure of, 126
Marine glue, 40
Materials, embedding, 20

injecting, 42
Matrix of cartilage, 67
Mayer's solution, formula for, 254
Measuring of cover glasses, 36
Mechanical stimulation of muscle,

227
Medland, 35, note
Medulla oblongata, functions of

in frog, 245

Medulla oblongata, structure of,

85
Medulla of bone, 70
Medullary sheath, 78
Medullated nerve fibres, structure

of, 78
Meissner's plexus, 89
Membranovis bone, 71
Mercuric chloride, 18
Methsemoglobin, 175
Methylated spirit, 18, 41
Metronome as a current inter-
rupter, 222
Metacasein, 187
Microscope, description ( f , 1-5
di?secting, 2, 5
directions for choosing,

12
lamp, 10
m.akers of, 13-15
Microtomes
Beck's, 24
Boecker's, 24
Cathcart's, 26
Caldwell's, 24
Ether, 25
Jung's, 24
Leitz's, 24
Ranvier's, 23
Reichert's, 24
Rutherford's, 23
Stirling's, 23
Williams', 25
Zeiss's, 24
Milk, chemistry of, 179
sugar, 163, 180
to distinguish from glucose,
199
Millon's reagent, 148, 158, 180

method of preparing, 147,
note
Minimal contraction, 236
Moist chamber, 230
Molybdate, ammonium, 31
Moore's test for sugar, 163
Morse's key, 217
Mounting, 35

fluids, 37
Mouth epithelium, 58
Mucin, chemistry of, 158
test for, 197

INDEX.

263

Muco-salivary glands, 99
Mucous glands, 94, 98, 101

tissue, 65
Miiller, fibres of, 142
Miiller's fluid, 18
Multiple staining, 34, 35
Murexide test, 168
Museum, preparation of specimens

for, 247-250
Muscle, cells, 96

chemical excitability of,

227
currents, 235
effect of heat and cold on,

232
efifect of fatigue on, 232
effect of poisons on, 226,

232
elasticity of, 237
electrical excitability by
continuous current, 217
electrical excitability by
induction shock, 219,
231
electrical excitability by
unipolar stimulation,
221
faradization of, 232
graphic representation of
the contraction of, 227-
230
histology of, 73-76
mechanicalexcitabOityof,

227
properties of, 214-232
reaction of, 237
sugar, chemistry of, 164
thermal excitability of ,227
time measurement of

curve, 233
transparency of, 237
Muscle-nerve preparation, 216,

230
Muscular tissue, structure of

striped, 74 ; unstriped, 76
Myograph, 228

pendulum, 233
spring, 235
Myosin, 153

N.
Nachet's microscopes, 14

Nail, stnicture of, 116
Xaphthalamine, test for, 187
Native albumins, nature of, 150
Natural currents in muscle and

nerve, 236
Needles, 40, 46
Negative variation, 236
Nerve cells, 83,89
Nerve-muscle preparation, 216
Nerves, electrc^tonus in, 236

functions of roots in frog,

245
negative variation in, 236
Nerve plexuses, 88-90
Nerves, structure of, 77
Nervous system :
Cerebellum, 86
Cerebrum, 87
Degeneration of, 81
Medulla, 85

Spinal cord, functions of, 246
structure of, 81-
85
Sympathetic, 88
Tissue, structure of, 77-90
Nervous mechanism of frog's heart,

238
Neurilemma, 78
Neuroglia, 79, 82, 88
Nitrate of silver, 31
Nitric acid, 18

Non-medullated nerves, 79, 88
Non-striped muscle, 76
Normal saline solution, 16, 48,

254
Nose, histology of, 138, 250
Nosepiece, 15
Nuhn, glands of, 94

O.

Object glass, 16
Objectives, 6, 8, 9, 10, 12
Oculars, 2, 9
Odontoblasts, 97
(Esophagus, 99
Oil, castor and spermaceti, 21

clove, 35
Oils and fats, chemistry of, 165
Olfactory cells, 138

organ, 138, 250

nerve, 139

264:

INDEX-

Olive oil and white wax, 20

chemistry of, 165
Ophthalmoscope, 211
Optic nerve, structure of, 79
Organic substances, examination

of, 195-200
Osmic acid, 19, 32, 66
Osseous tissue, chemistry of, 158

histology of, 68-72
Ossifying cartilage, 67
Ost^blasts, 6S, 71
Ost-eoclasTs, 72
Osteodentine, 96
Ovary, 124
Ovula Xabothi, 126
Ovum, structure of, 125
Oxalates in urine, 189, 194
Oxyhsemoglobin, crystals of, 55

spectrum of, 174

P.

Pacciniax bodies, 89
Palladium chloride, 19, 32
Pancreas, digestive action of, 1S6

structure of, 104
Papillae of tongue, structure of, 93
Paraffin and lard, for embedding,

20
Paraglobulin, 154, 177
Parietal cells, 101
Parotid gland, structure of, 99
Parkes' microscopes, 13
Pasteur's fluid, formula for, 254
Pa\Vs test for sugar, 190
Pendulum myograph, 233
Penis, structure of, 123
Pepsin, digestive action of, 185
Peptic cells, 101

glands, 101
Peptones, properties of, 155, 155,

186
Perforating fibres, 69
Perichondrium, 67
Perimysium, 75, 76
Perineurium, 78
Periosteal bone, 71
Periosteum, structure of, 70
Pettenkofer's test for bile salts,

181
Peyer's glands, 103

Phosphates, test for in milk, 180

in urine, 189
quantitative test for,
172
Physiological chemistry, 147-200
Picric acid, 18, 41
Picric acid test for sugar, 163
Picrocamiin, 28
Pigment, 60

Pineal gland, struchire of, 129
Pithing of frogs, 215
Pituitary body, structure of, 129
Plasma of blood, experiments

with, 178
Plain muscles, histology of, 74, 76
Plexuses of sympathetic nerve,

90
Pneumogastric nerve, dissection
of, 239
influence of upon
heart in frog,
240
Poisons, influence of on heart, 241
on muscle,
226, 232
Potassium, acetate, 37

bichromate, 18, 41
sulphocyanate, 185
Powell and Leland's microscopes,

15
Practical histology, 1-146

physiology, 201-246
Primary marrow cavities, 71
Primitive fibrils, 79

nerve sheath, 78
Prostate, structure of, 121
Proteids, 148-159
acid, 151
alkali, 152
casein, 152
classification of, 149
egg, 150

examination of , 195, 196
nature of, 148
serum, 151
solubility of, 149
tests for, 148, 156, 158,
195
Pseudostomata, 111
Ptyalin, action of, 184
Pulp of teeth, structure of, 96

INDEX.

265

Pulse, clenKtnstration of bysphyg-

nidgraph, 20 4
Purkinje's cells, 86
Purpiirin, 29

Pus iu urine, tests for, 190, 194
Pyloric j^lands, 101

Q.

Quantitative estimation of sugar,
189

Quantitative estimation of chlo-
rides in urine, 171

Quantitative estimation of phos-
phates in urine, 172

Quantitative estimation of urea,
169-171

R.
Rabbit, method of demonstrating

the blood pressure in, 205, 209
Rauvier's microtome, 23

constriction, 79
Razor, 22

Reaction of blood, method of test-
ing, 173
Reagents, action of, on blood, 48-

50
Reagents, for fresh tissues, 16
hardening, 17
list of, 46
mounting, 37
softening, 41
staining, 27-35
Recording cylinder, 227
Reichert's microscopes, 14, 15
Reissner, membrane of, 135
Remak, fibres of, 89

ganglion of, 238
Rennet, 180, 186
Respiratory tract, histology of,
108-112

Bronchi, 111
Epiglottis, 108
Larynx, 109
Lung, 111
Trachea, 109,110
Rate Malpighii, 113

testis, 1 22
Retina, histology of, 142
Reverser, 225

Revmond, Du Bols, key of, 223i

228
induction coil, 218. 221, 228
rhecjcliord of, 223
Rheochord, 223
Rheoscopic frog, 226, 241
Rigor mortis, 237
Roberts' test for sugar by fermen-
tation, 190
Riolani's muscle, 140
Roy, frog- heart box of, 243

Tonometer of, 243
Russell and West's test for urea,

169
Rutherford's microtome, 23

S.

Salicylic acid in urine, 191, 193

Saline solution, 16, 48

Saliva, chemistry of, 184

Salivary glands, histology of, 98

Sarcolemma, 75

Sarcous elements, 75

Schema, arterial, 203

Schiflf's test for uric acid, 168

Schreger, lines of, 96

Schultze's, Max, muscle corpus-
cles of, 76

Schwann's nucleated nerve -sheath,
78

Schwann's primitive substance,
78

Scissors, 45

Sclerotic, structure of, 145

Sebaceous glands, 115

Section by hand, 20-23
by microtomes, 23-27
Cutters, 22-27
Cutting, 19
Directions for, 19, 20
Lifters, 36

Sections of Lantermann, 79

Semilunes of Heidenhain, 98, 99

Semicircular canals, structure of,
137

Serous glands, 94, 99

Serum albumin, 151, 177, 179,
195, 196

Serum albumin, method of ob-
taining, 151

•266

INDEX.

Serum of blood, experiments with,

177
Sharper's perforating fibres, 69
Simple microscope, 5
Silver nitrate, 31
Skin, structure of, 113
Slides. 36

Small intestine, structure of, 102
Softening reagent^;, 41
Solubilities of proteids, 149
Solution, gum, 26

Farrant's, 37

saline, 16, 48

Spectrum analysis of blood, 174

of bile, ISl
Spermatozoa, 123
Spermaceti and castor oil for em-
bedding, 21
Sphygmograph, 204
Spinal cord, degeneration of, SI
functions of. 246
nerves, origin of, 85
peculiarities of dif-
ferent regions of,
84
structure of, 79-85
Spleen, structure of, 133
Spongy bone, 70
Spring inteiTupter, 222, 232

myograph, 235
Squamous epithelium, 58
Staining, blood, 52, 57

directions for, 33-35
multiple and double, 34
reagents, 27-35
Stanley's microscopes, 13
microtomes, 26
Stannius' experiment, 241
Sta:*ch, chemistry of, 160

test for, 196
Stephens' ink, staining with, 29,

79
Stethoscope, 210
Stokes' fluid, 174

reduced haematin, 175
Stomach, structure of, 100
Stomata, 61

Strieker's warm stage, 55
Striped muscle, structure of, 73
Submaxillary gland, structure of,
PS

Sugar, grape, 163

in urine, tests for, 189

milk, 163, 180

quantitative estimation of
glucose, 189
Sulphocyanate of potassium, 185
Suprarenal capsule, 130
Sweat glands, 114
Swift's microscopes, 13
Sympathetic nerve system, 88

Tadpole, examination of circula-
tion in, 201
Taste buds, structure of, 94
Taurocholic acid, preparation of,

1S2
Teazing, directions for, 40
Teeth, structure of, 95

development of, 96
Tendon corpuscles, 62

termination of muscle in,
73
Testes, structure of, 122
Tests for

Acid albumin. 151

Albumins, 148-153, 195

Alkali albumin, 152

Alloxan, 168

Amyloid substance, 157

Bile acids, ISl

Bile pigment, 181

Blood, 55, 56, 174, 190

Casein, 152, 180

Calcium, 185

Carbonates, 184

Carbolic acid, 191

Chlorides, 169, 171, 180, 184

Cholesterin, 182

Chondrin, 159, 197

Ch^'luria, 191

Coagulated proteids, 156

Cystin, 194

Crvstallin, 153

De"'xtrin, 162, 196, 199

Diabetic urine, 163, 189

Egg albumin, 150, 196

Elastin, 159

Fat, 165, 166

Fibrin, 155

Fibrinogen, 154, 178

INDEX.

267

Tests for fibrinoplastin, 154. 178

(ielatin, 157, 197

Globin, 155

Globulins, 153, 185, 195

Glucose, 163, .see Grape sugar

Cilycerin, 165

Glycogen, 162, 196, 199

Grape sugar. Bismuth, 163
Br.ttcher's, 163
Fehliug's, 189
Feruienlation,

163
Indigo carmine,

163
Moore's, 163
Pavy's, 190
Picric acid, 163
Tromn.er's, 163,

197, 199
Quantitative
test for, 163,
189

Indol, 1S7

Inosit, 164, 199

Lacto.se, 163, 180, 199

Lardacein, 157

Leucin, 199

Magnesium, 185

Metacasein, 1S7

Milk sugar, 163, ISO, 199

Mucin, 158, 185, 197

Murexide, 168

Myosin, 153

Naphthalamine, 187

Oil, 165

Oxalates, 189, 194

Paraglobulin, 154

Pepsin, 185

Peptones, 155

Phosphates, 168, 172, 180,
184

Potassium, 184

Potassium sulphocyanate, 185

Proteids, 148, 156, 158, 195

Pus, 190, 194

Salicylic acid, 191

Saliva, 184

Schiff's, 168

Seralbumin, 151, 195, 196

Sodium, 185

Starch, 161, 196

Tests for sulphates, 169, 184
Tyrosin, 197
Urates, 189, 191, 198
Urea, 168, 169, 198
Uric acid, 168, 198
Urine, alltuminous, 188
bilious, 181, 188
blood in, 190
carbolic acid in, 191,

193
chloiides in, 169, 171
chylous, 191
cystin in, 192, 193
diabetic, 103, 189
mucus in, 167, 194
morbid, 188, 194
normal, 167-172
oxalates in, 189
phosphates in, 168,

172, 189
pus in, 190
rhubarb in, 192
salicylic acid in, 191,

193
sugar in, 163, 189
sulphates in, 169
Vitellin, 155
Tetanus, 220, 232
Thermal stimulation of muscle,

227, 232
Thymus gland, structure of, 128
Thyroid gland, structure of, 128
Time measurement of muscle

curves, 233
Tongue, histology of, 93
Tonometer, Roy's, 243
Tonsils, structure of, 99
Tooth, structure of, 95

development of, 96
Trachea, structure of, 110
Transitional epithelium, 59
Trigger key, 229
Trommer's test for sugar, 163, 197»

199
Tubercle bacillus, method of stain-
ing, 253
Tunica albuginea, 122
granulosa, 125
Turnover key, 229
Tyros in, preparation of, 187
test for, 197

268

INDEX

U.
UxiPOLAR excitation of muscle,

221
Unstriped muscle, histology of, 74,

76
Urari, effects of, 202, 226
Urates, tests for, 189, 191, 198
Urea, nitrate, 167, 198
oxalate, 168
properties of, 167
quantitative estimation of,

169
preparation of, 166, 251
Ureter, structure of, 120
Urethra, structure of, 123
Uric acid, 16S, 1S8, 198
Urinary calculi, analysis of, 191
Urine, albuminous, 188
bilious, 181, 188
blood in, 190
carboluria, 191, 193
chemistry of, 167-172
chlorides in, 169, 171
chylous, 191
cystin in, 192, 193
diabetic, 163, 189
examination of normal,

167-172
examination of unhealthy,

lSS-194
morbid, 188, 192
mucus in, 167, 194
nonnal, 167-172
oxaluria, 189
phosphatic, 168, 172, 189
pus in, 190
rhubarb in, 192
salicylic acid in, 191, 193
sugar in, 163, 189
sulphates in, 169
urates in, 189, 191
Uterus, structure of, 125
Uvea, 144

Vagus in frog, dissection to ex-
pose, 239

Vagus, influence of upon heart of
frog, 240

Valvulae conniventes, 103

Variations in strength of continu-
ous currents, 224

Vas deferens, 121

Vas efferens, structure of, 123

Veins, structtue of, 91

Verick's microscopes, 14

Vesiculae seminales, 123

Villi, 103

Vitellin, 155

Vitelline membrane, 125

Vitellus, 125

Vitreous humoiu-, 65

W.

Warm stage, 54

Wax masses, for embedding, 20

Weigert's method of staining
nerve-tissue, 80

Weigert's method of staining
bacteria, 252

Wharton's tissue, 65

White blood corpuscles, move-
ments of, 54

White fibrous tissue, 62

Wickersheimer's fluid, preparation
of, 249

Williams' microtome, 25

X.

XaxthbOx boteic test for albumin,

14S

z.

Zeiss's microscopes, 13, 14

microtome, 24
Zona fasciculata, 130

glomerulosa, 130

pellucida, 125

reticularis, 130

1.0ND0X : BAILUEBE, TIXDALL, AND COX. KI2«G WILLIAM a^KEET, STRASU.

V M ,

I I i