Main Lib.
«rio. Dept.

BIOLOGY

LIBRARY

G

THE AUTHOH AT WORK.

LABORATORY METHODS

OF

HISTOLOGY AND BACTERIOLOGY.

BY

J. H. HOLMAN, M. D.,

Professor of Histology and Bacteriology, Meharry Medical College;
Analyst Mercy Hospital, Nashville, Tenn.

NASHVItLE, TENN.:
NATIONAL BAPTIST PUBLISHING BOARD.

1903.

THE LIBRARY OF
CONGRESS,

Two Copies Received

MAY 18 1903

Copyright Entry

CLASS

>T

COPY A.

XXc, No.

5

BIOLOGY

V.'-'

0

COPYRIGHT, 1903,

BY
JOHN H. HODMAN.

THIS VOLUME
IS RESPECTFULLY DEDICATED

TO

THE ALUMNI OF

MEHARRY MEDICAL COLLEGE,

NASHVILLE,

TENN.

268470

PREFACE.

This manual of Histology and Bacteriology has been
more particularly prepared to facilitate the study of
these important branches of medicine by the author's
classes. While the author makes no pretension to orig-
inal research in this book, he has endeavored to include
the useful and practical points of the more exhaustive
works on the subject. In every case the shortest and
best methods are used. He has avoided technical terms
where practicable and has sought to express the truth
in simple language. He has provided blank sheets for
drawing and extra notes before and after each part
of this volume.

The author wishes to express his gratitude to friends
for encouragement, especially to Dr. G. W. Hubbard,
M. D., Dean of Meharry Medical College, and Prof.
W. Patterson, A. M., Assistant Professor of Chemistry
in the same institution. He wishes to thank his as-
sistant, Mr. A. Lyman Paey, for the illustrations,
most of which being original, having been sketched
from actual specimens prepared by the students in the
laboratory of Meharry Medical College. He also

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6 PREFACE.

wishes to express his gratitude to the National Baptist
Publishing Board for the excellent and artistic manner
in reproducing the illustrations and getting out the
work.

The author embraces this opportunity to acknowl-
edge his obligations to the following authorities for
information, viz.: Sobotta's Human Histology, Bohm
and DavidofFs Histology, Leroy's Essentials of His-
tology, and McFarland. Eyre and Sternberg's Bacteri-
ologies. J. H. HOLMAN.

Meharry Medical College, Nashville, Tenn., May,
1903.

CONTENTS.

PART I.

Page.
CHAPTER I.

THE MICROSCOPE AND ITS ACCESSORIES n

CHAPTER II.

MANIPULATION AND CARE OF THE MICROSCOPE. . . 14
CHAPTER III.

GENERAL HISTOLOGY 26

CHAPTER IV.

THE DEVELOPMENT OF ANIMAL TISSUE 33

CHAPTER V.

EPITHELIAL TISSUE 35

CHAPTER VI.

CONNECTIVE TISSUE 38

CHAPTER VII.

BLOOD TISSUE 49

CHAPTER VIII.

MUSCLE TISSUE 56

CHAPTER IX.

THE VASCULAR SYSTEM 61

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8 CONTENTS.

Pa^e.

CHAPTER X.
THE LYMPH VASCULAR SYSTEM 65

CHAPTER XL
THE NERVES 72

CHAPTER XII.
THE SKIN AND ITS APPENDAGES 85

CHAPTER XIII.
THE DIGESTIVE TRACT 95

CHAPTER XIV.
THE URINARY TRACT 109

CHAPTER XV.
THE REPRODUCTIVE ORGAN 114

CHAPTER XVI.
THE RESPIRATORY SYSTEM 120

CHAPTER XVII.

THE SPECIAL SENSES 123

PART IL

URINALYSIS 133

PART IIL
BACTERIOLOGY 159

TECHNIC, REAGENTS AND STAINS 195

PARTI.

HISTOLOGY

LABORATORY METHODS OF HIS-
TOLOGY AND BACTERIOLOGY.

CHAPTER L

THE MICROSCOPE AND ITS ACCESSORIES.

The microscope is an instrument which magnifies an
object by means of its optic apparatus. There are two
kinds of microscopes, the simple and compound. The
compound is used in Histology and Bacteriology. The
parts and use of the same will be taught from time to
time in the Laboratory. The base is the part that
rests upon the table and supports all the rest of the
instrument. The pillar is the perpendicular part ex-
tending from the base to the back part of the stage.
The arm is the part that is attached to the pillar and
works on a hinge- joint. The mirror is attached to
the arm in front and works by means of a universal
joint; its function is to give light to tn,e observer
while examining the object. The stage is the plat-
form upon which the object rests while it is being ex-

(i-i)

12 LABORATORY METHODS OF

amined. The aperture is the circular opening in the
stage; its diameter can be varied according to the
amount of light required to see the object distinctly by
the use of the diaphragm, which is the circular disk
placed under and attached to the platform. The body is
the cylindrical attachment attached to the arm in front
and supporting the coarse adjustment, draw tube and
objectives. The eyepiece and the objectives are the
main parts of the instrument. Since the eyepiece and
objectives are the essential parts of the instru-
ment, it is very important that we know enough
about them to use them intelligently. The eye-
piece consists of a field glass and an eyeglass,
which are placed in the positions indicated by
their names. The coarse adjustment is placed on
the rear of the arm, at the point where the arm and
body meet. Its use is to obtain a focus. The fine ad-
justment is placed on the top of the arm and is used to
complete the focus obtained by the use of the coarse
adjustment. The draw tube is placed in the body of
the instrument and is used to increase its magnifying:
power.

Exercise No. i. — Study of the Microscope.

Make a study of the microscope at your locker and
learn to use all the parts. Take the material given you
and find a focus of the same. Your focus is the point

HISTOLOGY. 13

where you can see the object best. Since you under-
stand the principles of the microscope, we will not
spend the time to explain them, but will use the instru-
ment.

CHAPTER IL

MANIPULATION AND CARE OF THE MICRO-
SCOPE.

1. In taking an instrument from the case, grasp it
by the base and pillar.

2. Free it from dust, with the paper supplied.

3. Place the instrument on the worktable, with the
opening of the base from you.

4. Place your eye over the eyepiece, and at the same
time manipulate the reflector until you get a good
light.

5. Place the object to be examined on the stage and
lower the objective to a point just above the slide and
cover-glass, with your head on a level with the stage
while doing so.

6. Now regulate the light by means of the dia-
phragm.

7. Place your eye over the eyepiece and rack up-
wards until you reach the point where you can see the
object.

8. Note what you see in your notebook.

9. Make drawings of what you see.

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HISTOLOGY. 15

Handle the instrument with more than ordinary
care; never let anything touch the lenses. The in-
strument must be kept clean. Be careful and learn
to use the instrument with both eyes open. Do not
vise it so long that an aching sensation will be pro-
duced in the eyes.

The accessories to the microscope are many, but we
will not use all of them ; since we want a working
knowledge of the following we will learn their use:

The Substage, which is placed under the stage, is
to support the Abbe condenser.

The Iris diaphragm is used to regulate the light
which passes through the condenser.

The Abbe is a lens of a very short focal distance and
gives a very strong light.

The Mechanical Stage is an instrument used to
manipulate the object while upon the stage.

The Camera Lucida is an instrument used to assist
the student in making the drawings.

The Polariscope is used to examine objects by means
of polarized light.

The Micrometer is an instrument used to determine
the magnifying power of the microscope. There are
two kinds of micrometers, viz. : an eyepiece and a stage
micrometer. Determine the magnifying power of the
instrument at your desk.

16 LABORATORY METHODS OP

1. Take the stage micrometer and place it upon
the stage of the microscope.

2. Build a platform as high as the stage of the in-
strument, using books for the purpose.

3. Place upon your platform a piece of white paper.

4. Now focus the lines on the stage micrometer,
keeping both eyes open.

5. The lines on the micrometer will be seen upon
the paper.

6. Now make a pinhole in the paper between any
two lines.

7. Multiply the distance between the pinholes in
the paper by the denominator of the fraction on the
scale of the micrometer. The product will be the mag-
nifying power of the instrument.

The Microtome is an instrument that is used to cut
tissue into very thin sections.

The Paraffine Bath is an apparatus that is used to
infiltrate the tissue with paraffine.

The Cornet Forceps is an apparatus used to hold the
cover glass.

The Centrifuge is an apparatus used to precipitate
the solid from a fluid.

Slides and Cover-Glasses are accessories used to re-
tain or protect the tissue (or object).

HISTOLOGY. 17

Exercise No. 2 — Use the Polariscope.

1. Place the microscope upon the table and take off
the objective.

2. Attach the analyzer.

3. Reattach the objective to the lower end of the
analyzer.

4. Place upon the stage the specimen.

5. Now place upon the specimen the selenite plate.

6. Now attach the polarizer and proceed to examine
the preparation.

Use the Abbe :

1. Place the object upon the stage and get a good
light.

2. Screw the Abbe around in place and regulate the
light by the use of the iris diaphragm.

3. Examine, note and draw.
Exercise No. 3. — Use the Microtome.

1. Place the tissue in the" clamps of the instrument
and fasten it.

2. Set your knife so that it will be at an angle with
the tissue.

3. Have the block of tissue of the right size, and
gently but firmly draw the knife across the tissue.

18 LABORATORY METHODS OF

4. Regulate the thickness of the tissue by the use of
the milled head on the under surface of the microtome.
(B. & L. is the best for students' use.)

Exercise No. 4. — Use the Centrifuge.

1. Attach the instrument to the table and place the
tubes in place.

2. Revolve the tubes moderately for three minutes.

3. Take the sediment out and examine with the mi-
croscope.

The Haematokrit is used in the same way, only re-
volved more rapidly.

Exercise No. 5. — Make a spread of yeast-plant.

1. Obtain some yeast and place it in a little water in
a test tube ; set aside in a warm place for a short while.

2. Take a pipette and compress the bulb so as to get
some of the yeast and water in the pipette.

3. Now place a small drop of this material on the
slide and examine.

4. Examine with H. P. (1-6 in. Obj.)

5. Observe the morphology, grouping, budding and
search for a chain.

6. Make drawings and note all you see.

This lesson is to illustrate the mode of reproduction
of cells, morphology (or shape), the contents of cells,

HISTOLOGY. 19

the function, and the spores (or seed of cells). W;e
use the plant cells because they are very large and are
easy to obtain and possess all the properties of the ani-

CBLLS OP BREWERS' YEAST.

1, A group of two cells; 2, dark spot represents a vacu-
ole; cell forming a bud.

mal cells. The size of the yeast cells is 1-4000 to i-
2500 of an inch in diarneter. Their form is ovoidal.
They have the power to produce fermentation. In
studying the plant under the microscope you will do
well to note whether there is any chlorophyll in the
plant or not. In this way you will be able to make your

20 LABORATORY METHODS OF

experiments of very great value to you. This work is to
introduce the student to the study of Histology. The
word Technic means the various manipulations neces-
sary to get the tissue ready for the microscope.

The term Histology means the microscopic study of
tissues. The unit of tissues is a cell. It is our work
to investigate the relation that the cell and intercellular
substance bear to the tissues and organs of the animal
or plant. Since we are studying the human body we
will confine ourselves to the study of the tissues and
organs of animals, i. e., cat and dog, mostly.

In the beginning we will study the cells of the yeast-
plant, spirogyra and protococcus in the plant kingdom,
and the green euglena and slipper animalcule in the
animal kingdom — each one for a special purpose: the
euglena, for its property of containing chlorophyll in
its body and for its possessing the power of motion;
the slipper animalcule, for its mode of reproduction
and its mode of locomotion.

We will begin with the yeast plant and go upwards
and see if there are any changes in the vegetable king-
dom that resemble those in the animal kingdom.

Exercise No. 6. — Make a spread of Protococcus.

I. From the green growth on a tree or fence, take a
small bit of the growth and place it in a petri-dish,
moisten and put in a warm place for twelve hours,

HISTOLOGY.

21

PROTOCOCCUS.

Showing morphology, grouping and fission.

2. When the cells have begun to vegetate place
some of them on the slide and irrigate with water.

3. Cover with cover-glass and examine.

4. Use H. P. and observe grouping, cell contents,
and reproduction.

5. Irrigate with acetic acid and search for a nucleus.
The lesson here is to find some reagent that will find

a nucleus without staining the preparation. Acetic
acid is the one. This is used in all of our work when
we look for a nucleus.

22 LABORATORY METHODS Otf

Exercise No. 7. — Make a spread of scrapings of some
organ and examine.

1. Take a knife and scrape the surface of the organ
and place the material on a slide; cover and examine.

2. Note all you see, and make drawings.

Exercise No. 8. — Make a normal Salt Soiution. (NaCl.
7 grams to 1,000 c.c. of uwter.)

This fluid is to be used in all cases when you want
to dilute the scrapings.

Exercise No. 9. — Make a spread of Spirogyra.

SPIROGYRA.
1, Showing mode of reproduction; 2, nucleus.

HISTOLOGY. 23

1. Obtain from a pond or brook a small quantity of
the growth and place it in a glass vessel with some
water.

2. Examine with H. P.

3. Search for nucleus, note the shape of the cells,
note the arrangement o>f the cells in the filament, ob-
serve the arrangement of the chlorophyll in the cells,
irrigate with acetic and make drawings.

Exercise No. 10. — Irrigation.

1. Place the cover glass on the spread.

2. Place a drop of reagent on one side of the prepa-
ration and a piece of blotting paper on the other.

3. Look through the instrument.

4. Note what you see and draw.

Any reagent may be used for irrigating. Irriga-
tion is only used on fresh spreads.

E.rcrcise No. n. — The Slipper Animalcule.

1. Take some of the scum from the top of the hay
infusion.

2. Examine with H. P.

3. Make out all the structures of the animal, note
and draw.

24;

LABORATORY METHODS OF

SLIPPER ANIMALCULE.
1, Nucleus; 2, cilia.

Exercise No. 12.— Dissociate some tendon of an ox.

i. Place some of the tendon in enough weak soda so-
lution to cover it and let it remain for two days.

Exercise No. 13.— Tease some of the tendon which
has been dissociated in the soda solution.

1. Fix a sewing needle in a pen-holder.

2. Use as a teasing instrument.

3. Tear the tissue into small bits and place them on
the slide.

HISTOLOGY. 25

4. Examine with H. P. and L. P. When using the
L. P. you may dispense with the use of the cover-glass.

CHAPTER IIL

GENERAL HISTOLOGY.

The Cell : — A cell is the unit of all the living organ-
isms. All cells originate from a pre-existing cell. A
complete cell consists of the following: a cell wall,
protoplasm, nucleus, nucleolus and centrosome. All
cells are not complete : some may have a nucleus, and
some may not; some may have a cell wall, and some
may not. A complete cell is called a typical cell, and
one that is incomplete is called an atypical cell. The
cell body is a mass of protoplasm, having in it a vary-
ing amount of delicate fibers called "spongioplasm."
Along the course of these we see several little nodes.
These nodes are called microsomes. That part of the
protoplasm near the nucleus is called the nucleoplasm,
and that part near the cell wall is called the exoplasm.
The power of motion is located in the protoplasm.
This is illustrated in the Amoeba, a small animal which

moves by means of pseudopods, i. e., a part of the pro-
(26)

HISTOLOGY.

: ( '" AMOEBA.

1, Pseudopods.

toplasm flows to one side of the body of the animal,
and then the rest of the protoplasm flows in the same
direction, until the whole animal changes its position.

Exercise No. 14.

1. From the scum of the hay infusion pick out a
submerged leaf.

2. Apply the cover-glass to the leaf and place on the
slide.

28 LABORATORY METHODS otf

3. Examine, note the manner of motion and sketch
the animal in several positions.

THE CEXL WAU,.

The cell wall is a delicate membrane surrounding
the cell-body, and is derived from the cell-body.

Exercise No. 15. — Make a microscopic preparation of
Protococcus and observe the cell wall.

The Nucleus: — This is the highly refractive body
found in the cell-body, and is composed of the nuclear
membrane, nuclear network and nucleolus, seen only
under very H. P. This can be seen in the protococ-
cus preparation.

Centrosomes : — These are highly refractive bodies
seen in the nucleus, and they preside over the repro-
duction of cells, while the nucleus transmits the hered-
itary traits of the parent cell to the new one. — Leroy.

The properties of a cell are motion, reproduction,
metabolism, growth, irritability and function. Metab-
olism is that property of a cell by which it takes in
nutriment and assimilates the same, and the property
of excreting the waste matter, which is the result of
metabolism. Irritability is the property of a cell
to respond to nervous stimulation. The function of
a cell is the work the cell will do. Reproduction of a

HISTOLOGY. 29

cell is the power to reproduce a like cell. This is ac-
complished in two general modes : Direct and Indirect.
The Direct is observed in the protococcus and yeast
plants. The Indirect is studied in the higher animals.
For convenience the stages of changes are noted sepa-
rately. They are as follows : ( I ) Resting nucleus ;
(2) the Skein; (3) the Rosette; (4) the Aster; (5)
the Diaster; (6) the Daughter Rosette; (7) the'
Daughter Skeins; (8) the Daughter Nuclei. Each
one of these will be observed in the experiment with
the growing tip of the onion.

Exercise No. 16. —

1. Grow some onions in a glass jar in some water.

2. Cut off some of the tips of the growing rootlets.

3. Fix them in formalin solution.

4. Harden in alcohol for I hour.

5. Dehydrate with absolute alcohol.

6. Place the tissue in equal parts of ether and abso-
lute alcohol for twelve hours ; then place it in thin eel-
loidin for twelve hours.

7. Now place it in thick celloidin for twelve hours.

8. Embed, section, mount and stain.

9. Examine with H. P. and the oil immersion.

Technic for making the solution. The formalin is
made as follows ;

30 LABORATORY METHODS OF

1. Take a 40 per cent solution of formaldehyd in
water or alcohol. Make a 10 per cent solution. The
celloidin solution is made by taking 5 grams of
celloidin and 100 grams, i. e., c.c., of the mixture of
equal parts of ether and absolute "alcohol. Mix.

2. The thick celloidin is made by taking the same
mixture and adding celloidin until the mixture is as
thick as a sirup.

By sectioning we mean cutting the tissue into very
thin sections, so that they can be easily made trans-
parent, by the next steps which are to follow. By
clearing a tissue we mean that we make the tissue so
transparent that it is of the same transparency as that
of the glass slide on which the tissue is mounted. Sev-
eral chemicals are used for this purpose. They all
depend on their indices of refraction for their value.
They are called clearing agents.

A piece of tissue to be carefully and properly
mounted must go through the following steps :

1. The cells must be killed.

2. The cells must be fixed.

3. The tissue must be hardened.

4. The tissue must be dehydrated.

5. The tissue must be dried.

6. The tissue must be infiltrated.

7. The tissue must be embedded, : 4

HISTOLOGY. 31

8. Sectioned ; embeding material taken out ; then the
tissue fixed on the slide, stained, mounted and labeled.
After the student has carefully done this he will not
have any trouble.

Exercise No. 17. —

1. Applr to the slide a section of the outer skin of
an onion -bulb. (Apply the section and add the stain.)

2. Wash in water to remove the excess of stain.

3. Dry with blotting paper; add a dehydrating me-
dium.

4. Wipe off the dehydrating medium, which is usu-
ally glycerine.

5. Add xylol.

6. Apply balsam1 to the section and place cover-glass
on the section balsam downward.

7. Label and keep in a horizontal position until bal-
sam hardens (or study at once). Make out all you
can about cells in this preparation; /. e., shape, size,
contents and motility.

Exercise No. 18. —

Make a preparation similar to that above and apply
a drop of iodine solution arid examine. Now remove
the cover and apply a drop of sulphuric acid. The

32 LABORATORY METHODS OF

stain will take reddish-brown ; then the acid will attack
the cellulose and convert it into dextrine; then the
whole will be reddish-brown. At first all the starch will
be blue, due to the presence of the iodine solution.
Look for Brownian movement; this is an independent
movement which is not due to vitality.

Exercise No. 19. — Make a section of a potato and
search for starch granules.

1. Place the potato in the microtome and cut sec-
tions 20 microns thick.

2. Place a section on the slide and search 'for a

field in which to make observations.

3. Note the morphology of the cells and the con-
tents of the same.

4. Place a cover on the section and irrigate with
iodine solution.

5. Make notes.

This is the mode of making a micro-chemical ex-
amination. Note that the morphology in this case is
not the same as in the onion cell,

CHAPTER IV.

THE DEVELOPMENT OF ANIMAL TISSUE.

1. The cell or ovum of the female, which is gener-
ated in the ovary, passes down the fallopian tube and
meets the spermatozoon, the male element, provided
conception has occurred.

2. The ovum there multiplies by indirect division
into a mass that resembles a mulberry ; hence the mul-
berry stage.

3. The cells do not all grow with the same rapidity,
so that there are soon formed three distinct layers of

, the original mass, i. e., the cpiblast, the hypoblast and
the mesoblast. The epiblast is the uppermost layer,
the hypoblast is the under layer, and the mesoblast is
the middle layer.

Now is the time when tissue differentiation begins.
All the tissues of the body are formed from these three
layers; hence the terms: mesoblastic, hypoblastic and
epiblastic, so often used in medical literature. A tis-
sue is composed of cells and intercellular substance,

working in common to accomplish some purpose.
3 (33)

34 HISTOLOGY.

In this manual you will find the tissue origin under
each tissue, as well as on this page. From the epiblast
or ectoderm are derived the following tissues : nerv-
ous tissue, the epithelium, covering the surfaces of
the body, the enamel, the nails, the hair, the organs of
special sense, and all glands, except those which open
into the alimentary tract, from the oesophagus down-
ward ; from the mesoblast or mesoderm are derived
blood, blood vessels, all the connective tissues, mus-
cles, dentine and cementum ; from the hypoblast or
hypoderm are derived the epithelial lining of the ali-
mentary canal and the glands opening into it. Tissues
may be placed into four groups : epithelial, muscular,
ncri'ous wid connective.

CHAPTER V.

EPITHELIAL TISSUES.

In this manual Epithelial Tissue is considered
first.

Exercise No. 20. —

EPITHELIAL CELLS.

a, Squamous; b, columnar; 1, goblet cells; a, ciliated
epithelial; 1, cilia; 2, cell wall; 3, nucleus located in the
protoplasm.

(35)

36 LABORATORY METHODS OF

Collect upon the tongue some saliva and place it
on the slide; cover and examine with H. P. Do you
see any blood vessels? Are the cells all of the same
shape? What figure would be the nearest to their
shape? What have you concluded as to the manner
in which they get their nourishment? Do they re-
semble scales? If so, they are of the squamous va-
riety. There are four varieties and it is our purpose to
study them in detail. These cells are reproduced by
mitosis. Give the function of this variety of cells.

Exercise No. 21. —

1. Place upon the slide some scrapings from the
upper part of the pharynx of a frog.

2. Examine with H. P. Note the following: shape,
cilia, contents and size of nucleus. The functions of
the cilia is to give motion to substances passing over
them. This variety is columnar in shape, at the same
time ciliated.

Exercise No. 22. —

Examine the scraping from the stomach of some
animal and note the shape of the cells there. This is
done in1 the same manner as the others. You will find
that the cells are of a columnar variety.

HISTOLOGY. 37

Exercise No. 23. — Make a preparation of liver cells

Do the same as Exercise No. 21.

P. S. — A scalpel is used in scraping.

Under the head of Epithelial there are several modi-
fied cells. Some of them contain mucin and others a
pigment. Sometimes their shape takes on the nature
of all the four varieties. They are then called transi-
tional epithelial cells. Again they are modified accord-
ing to function. They are then called specialised epi-
thelial cells; i. e., glandular and neuro epithelial.

CHAPTER VI,

CONNECTIVE TISSUE.

Connective Tissue is of mesodermic origin. It
serves to hold the different parts of the body tog-ether,
and contains more intercellular substance than the
epithelial tissue. It serves to support the organs
of the body as well. There are ten varieties of this tis-
sue:

WHITE FIBROUS AND YELLOW ELASTIC TISSUES.

1, White fibrous; 2, yellow elastic,
(38)

HISTOLOGY. 39

i. — WHITE FIBROUS TISSUE.

Study the white fibrous tissue.

1. Tease a piece of tendon of an animal and place
it on the slide.

2. Stain with carmine.

3. Observe the fibers running in a wavy line across
the field. This tissue is found in the following- places :
tendon, omentum, subcutaneous tissue and a few other
places.

II. — YELLOW ELASTIC TISSUE.

Take the ligament of an ox's neck and prepare, stain,
mount and study.

To differentiate yellow elastic tissue from white fi-
brous : take a scalpel and cut each one. The white
fibrous will remain unaltered, while the yellow will
curl up. Under the microscope the white will appear
to be in a straight or wavy line, while the yellow will
appear to be in branched processes. If you boil them
the white will yield gelatin, and the yellow yields elas-
tin.

III. AREOLA TISSUE.

It is composed of white, fibrous, yellow elastic and
adipose tissues, in varying amounts.

LABORATORY METHODS OF

AREOLA TISSUE.

1, Yellow elastic tissue; 2, white fibrous tissue,
dark spots represent wandering cells.

The

IV. — MUCOUS TISSUE.

This tissue is composed of large branching cells
surrounded by a soft gelatinous substance (mucin).
In embryo it is found in the umbilical cord. Study
this tissue.

Technic: Make a spread of some fluid that exudes
from the cut end of an umbilical cord and observe the
shape, size and contents of the cells.

HISTOLOGY. 41

V. — ADIPOSE TISSUE.

This tissue is composed of large, poorly stained cells,
and is found in a large number of places in the body.
These cells are bound together by areola tissue. The
fat cells are derived from the fixed connective cells.

ADIPOSE TISSUE.
1, Nucleus; 2, protoplasm; 3, fat drop; 4, cell wall.

The protoplasm is converted into fat, and the nucleus
is pushed to one -side and finally leaves the cell-body.
Fat surrounds every tissue of the body except the brain
and spinal cord. This tissue is used as a cushion for
the eye.

42 LABORATORY METHODS OF

VI. — RETlFORM TISSUE.

This variety of tissue is composed of a number of
white fibers and a varying amount of connective tissue
cells.

RETIPORM TISSUE.
Showing only the fibrous tissue.

Exercise No. 24. —

Take a section of a fresh spleen and shake it in an
indifferent fluid for several minutes; then place it on
the slide and examine. An indifferent fluid is a fluid
that has no effect on the tissue which is placed in it,

HISTOLOGY.

43

i VII. — ADENOID TISSUE.

This tissue is simply retiform tissue having a vary-
ing amount of lymphoid cells entangled in its meshes.

VIII. — CARTILAGE TISSUE.

This is a variety of connective tissue that is widely
distributed. There are three main varieties of carti-
lage ; viz. :

WHITE FIBRO-CARTILAGE.

1, White fibers; 2, cartilage cells; 1, 2, 3, 4, cartilage
cells in different stages of reproduction.

44 LABORATORY METHODS OF

White Fibro-Cartilage. — This variety is composed
of the following material: matrix, capsule, peri-
chondrium, cells and lacunae. The matrix has em-
bedded in it a few white fibers : hence the name. The
matrix is a homogeneous material, semisolid in con-
sistency, and will yield chondrin on boiling. The oval
shaped cells lie embedded all in the matrix, in groups
of two, four and eight, etc. The cells are those oval
bodies scattered all in the matrix. The lacunae are
those hardened areas found near the cells on all sides,
those white threads that run all in between the cells
and give the fibrous nature to the cartilage. The peri-
chondrium is the membrane that is found on the out-
side of the cartilage, and is composed of two layers:
one to protect the cartilage; the other to furnish new
cells in the growing cartilage.

Hyaline Cartilage is composed of. a matrix, peri-
chondrium, cells and lacunae.

Exercise No. 25. — Mount a T. $. of trachea of a cat;
stain unth carmine.

1. Cut the section with the microtome 20 microns
thick.

2. Place the section on the slide and stain.

3. Wash off excess O'f stain with acid alcohol.

4. Wash in 70 per cent, alcohol; then 80 per cent,
and 90 per cent,

HISTOLOGY.

5. Wash in 100 per cent.

6. Clear up with some of the clearing agents.

7. Dry and mount in balsam.

T. S. is the abbreviation for transverse section.

45

FIBRO ELASTIC CARTILAGE.

The Fitiro Elastic Cartilage. — This variety of tissue
is composed of yellow elastic tissue in addition to the
material in the white fibrous cartilage.

IX —BONE TISSUE. **w'

This variety of tissue gives shape to the body and is
therefore very hard. It is composed of the following

46 LABORATORY METHODS OF

T. S. BONE.
1, Haversian canal; 2, lacunae; 3, canaliculi.

parts : H aversion system, Haversian canals, latmellae,
lacunae, canaliculi and bone cells. The Haversian sys-
tem is composed of Haversian canals, a number of
canaliculi, a number of lamellae and a number of bone
cells. The cells lie in the bone matrix in much the
same way as they do in the cartilage matrix. The
lacunae are also very much the same. The bone cells
send out processes for the purpose of conveying- nutri-
ment to all parts of the bone ; these processes are called
canaliculi. The Haversian canals cafry the small

HISTOLOGY. 47

blood vessels into the bones. The lamellae, arranged
around the Haversian canal, are called the Haversian
lamellae. The lamellae running parallel with the outer
surface of the bone are called the ground lamellae, and
those running between the Haversian system, are called
the interstitial lamellae. There are little canals run-
ning from the periosteum to the nearest Haversian la-
mellae. These canals are called Volkmann's canals.
Occasionally uncalcified lines are seen passing ob-
liquely or transversely through the lamellae. These,
are called the lines of Sharpey. The periosteum
consists of fibrous connective tissue, and is divided
into two layers. One layer is the bone-forming
layer and the other is the bone-protecting layer. Bone-
marrow is composed of two kinds of cells, the red and
yellow. The yellow is mostly of the fat variety, and
the red is mostly of red blood cells. The blood vessels
of the bone tissue are distributed through the Haver-
sian system and the canals of Volkmann. The perios-
teum is richly supplied with blood vessels.

Bone develops in two ways : centers of ossification
and perichondrial. The perichondrial is as follows:
the cells of the immature bone pass down the lines of
Volkmann and locate themselves in a center called a
center of ossification. Ossification means the depo-

48 ' HISTOLOGY.

sition of salts of lime in the tissues. These salts are
usually deposited in cartilage, and then it is called
bone. Dentine is a variety of bone, and .will be dis-
cussed more fully when we come to the subject of the
teeth.

No. 26. — Make a preparation of cartilage
from the end of bone in the fresh state.

1. Take a sharp knife and cut a piece of cartilage
from the end of a long bone and section.

2. Mount in salt solution.

3. Stain.

4. Study, draw and note.

Look for the following: matrix, cells, capsule, lacu-
nae and perichondrium.

Exercise No. 27. — Examine the L. $. of bone given
you.

Look for the following: Haversian system, Haver-
sian canal, lacunae, bone cell and canaliculi.

Exercise No. 28. — Examine the T. S. section of the
bone given you.

Look for the same as you did in the previous exer-
cise. Make drawings in both cases ; note and study.

Exercise No. 29. — Examine the cuts in your text and
make a. draining of all you see.

CHAPTER VIL

BLOOD TISSUE.

[The tenth variety of Connective Tissue mentioned in
this work.]

Blood is a tissue of mesodermic origin, and has n
fluid intercellular substance, known as plasma. The
cellular elements are many, viz. : white corpuscles, red
cells, and the platelets. The blood is distributed to
all parts of the body by means of tubes called arteries,
and is returned to the heart by means of the veins.
The capillaries are the terminals of the arteries and the
beginning of the veins. Their walls are very thin, and
admit a change of gases between the blood and the
tissues.

Exercise No, 30. — Obtain a drop of blood from your
finger.

1. Sterilize the lance and prick the part selected.

2. Wipe off the first drop of blood with a clean cloth
wet in alcohol.

4 (49)

50 LABORATORY METHODS OF

3. Constrict the part above the puncture and obtain
another drop.

4. Place this drop on the slide and cover, or touch
the edge of two clean cover-glasses to the drop, so that
a very thin spread of blood can be secured between
them; place on the slide and examine.

5. Note the shape of the red cells; the manner of
grouping on the slide ; search for nuclei ; look for stain
in the cells ; note the central part of the cell, and meas-
ure the cell with the micrometer.

6. In the same preparation examine the white cor-
puscles which are found adhering to the slide.

Exercise No. 31. — Use the Haemacytometer, count the
cells of both varieties, in a c.m.

1. Take 995 c.m. of sodium sulphate solution; spe-
cific gravity, 1028; temperature, 15 C.

2. Take 5 c.m. of the blood.

3. Mix in the mixing jar with the spud.

4. Take the instrument and fill the cell with the
mixed blood and solution.

5. Place the instrument under the microscope and
let the whole stand a few minutes ; then count the num-
ber of cells in ten squares; strike the average; then
multiply the average by 10,000, since the blood has

HISTOLOGJ. 51

been diluted and since the squares are only one-tenth
of a m.m. square. The average number of red cells
is about 5,000,000 in each c.m. of blood in men, and
4,500,000 in women.

Exercise No. 32. — Use the Haemoglobinometer.

1. Take the instrument and set it up in front of a
piece of white paper.

2. Obtain 20 c.m. of blood and place it in the tube
provided for the reception of the blood.

3. Dilute the blood with water until the blood mix-
ture and the standard mixture in the tube coincide in
color.

If the blood has the required amount of haemoglobin
in it, when you have added the water up to the gradu-
ate 100, the two colors will coincide. If it reaches the
color before the required amount of w&ter has been
added, there is not enough haemoglobin in the speci-
men; and if it requires more water than the 100, there
is too much haemoglobin.

Exercise No. 33. — Use the Haematokrit.

I. Set up the centrifuge and place the Haematokrit
in place; revolve the centrifuge very rapidly for three
minutes.

52 LABORATORY METHODS OF

2. Observe the position of the heavier materials in
the blood.

3. Hold the instrument between you and the light;
see how far the red cells register on the graduate.
This is a tube of small bore and is arbitrarily gradu-
ated. It registers per cent.

Exercise No. 34.— Make a spread of frog's blood.

i. Snip the nose of the animal with a sharp instru-
ment and obtain a small drop of blood.

FROG'S BLOOD.
1, Leukocytes; 2, oval red cells.

HISTOLOGY. 53

2. Collect it in the same way that you did the blood
of man.

3. Examine the shape of the corpuscles ; search for
a nucleus in the red corpuscles; measure the cells in
both diameters, and examine the white corpuscles for
the amoeboid movement.

Exercise No. 35. — Make a spread of human blood and

apply the reagents given you, viz.: salt solution,

acetic acid, and water.

Irrigate in the usual way. In the salt preparation
r - 1 e what you see ; give the cause. Do the same with
all the others. Do red cells have a cell-wall? (Leroy
and Sobotta say they do>.

The colorless corpuscles are of five varieties: [mono-
nuclear lymphocytes, poly nuclear lymphocytes, large
lymphocytes, eosinophiles and basophiles. These are
all larger than the red blood cells. They all originate
from the lymph glands. They are separated into their
classes by the use of different stains, and the use of the
eye-piece micrometer. A large per cent of them has
the power oi moving like the ameba; hence the term
ameboid movement, as applied to the white blood cells.
Blood shadows are red blood cells that have been sub-
jected to the action of water, and have the haemoglo-
bin dissolved out, leaving the shadow of the cell. Blood
platelets are small red cells. The crystals found in

54

LABORATORY METHODS

blood are haemoglobin, haematin, haematoidin. The
most important are the hemin, which can be demon-
strated by the following method :

HUMAN BLOOD.

1, Biconcave red blood cells; 2, red blood cells on edge;
3, spherical red blood cells; 4, red Hood cells after the ac-
tion of water; 5, leukocytes; 6, rouleaux. Crenated red
cells to left of the rouleaux.

Obtain a drop of blood in the usual way, and apply
to it some salt solution and some acetic aicid; let the
whole dry for a few minutes. Examine with H. P.

The red blood cells are derived from the mesoblast,
and are nucleated and colorless when first formed.

HISTOLOGY. 55

These multiply and lose their nuclei and acquire hemo-
globin. Later in life the spleen and red marrow of the
bone contribute to the supply of red cells. The white
blood cells are derived mostly from the lymphoid tis-
sue and spleen. The lymphoid corpuscles become dis-
lodged and carried along the blood current or lymph
current, as the case may be. "In these places they un-
dergo a mature change." — Lcroy.

Exercise No. 36. — Make a stained preparation of
blood.

1. Make a spread in the usual way.

2. Fix high over the flame.

3. Apply eosin 30 minutes.

4. Haematoxylin 3 minutes.

5. Dry, mount and label.

6. Study and preserve,

Search for the four varieties of lymphocytes and
observe the formation of rouleaux in the red cells.

CHAPTER VIII

MUSCLE TISSUE.

This variety of tissue is of mesoblastic origin, and is
divided into three classes : voluntary, involuntary and
heart muscle. All muscle tissue is rich in blood vessels,
lymphatics and nerves. The blood vessels break up
into capillaries, which form a net work around each
individual fiber. The nerves terminate in special end-
organs under the sarcolemma.

The voluntary or skeletal muscle is the most wide-
ly distributed. It forms all the skeletal muscles, and
is under the control of the will. It is composed of
long, cylindrical fibers, by some authors called cells.
These are the largest cells in the body — so large that
one cannot be placed in the field of the microscope at
once. Sometimes they are il/2 inches long. They
are all surrounded by a delicate membrane, or cell-wall,
called sarcolemma, just under which are placed the
nuclei of the cell. The transverse striations that are
seen all along the course, are said to be due to the pres-
ence rnd make-up of the sarcous elements. There are
seen running along the long diameter of the cell deli-
(56)

HISTOLOGY.

57

cate lines, which are called sarcostyles and can be
demonstrated by taking a piece of the tissue and disso-
ciating the same. This will separate the lines into in-
dividual fibrils. Each fibril is called a sarcostyle. The
space between the sarcous elements is filled with sub-
stance, which is called sarcoplasm, and is analogous
to the protoplasm in other cells. This protoplasm is
endowed with the special power of motion, or con-
tractility. If we use a very H. P. we will be able

SECTION OF VOLUNTARY MUSCLE.

A. Fiber or Cell. 1. Sarcolemma; 2, fibrillae.

B. Two fibers, one showing torn sarcolemma as a clear

space.

C. A sarcostyle; 1, Fusiform body; 2, a ball; 3, lighc

spaces.

58 LABORATORY METHODS OP

to observe the lines of Hensen and the membrane of
Krause. These lines are located in the sarcous ele-
ments. The sarcous elements are composed of the fol-
lowing parts, viz. : a spindle-shaped body, light space,
and a bill. Placed end to end they make a fiber or cell.
The lines of Hensen are those lines seen dividing the
spindle-shaped bodies, and the membrane of Krause is
the membrane or line drawn across the light bands.
The following wrappings are found enveloping the
muscle: endomysium, the membrane around the fiber.
A number of fibers (wrapped in the endomysium) is
put together and forms a bundle; this bundle is
wrapped in another membrane, called a perimysium. A
number of these bundles is put together and enveloped
in another membrane, called epimysium.'

Involuntary Muscle. — This variety of tissue is net
under the control of the will. The cells are fusiform
in shape, and have their nuclei centrally located. They
are joined together by overlapping each other; ait the
point where the cells meet a cement substance is seen.
They are found in the tissues that are not under the
control of the will, i. e., the intestines, uterus, and rec-
tum.

Heart Muscle. — This variety of muscle is found only
in the heart, and is not the same as the involuntary
muscle, in shape or function. The cells are short, rec-

HISTOLOGY. 59

tangular shape, with branching interlacings, as well
as a cement substance between the cells. With a H. P.
can be seen a lairge number of fine transverse striations

L. S. HEART MUSCLE.

in each cell; also a number of longitudinal lines are
seen in them. The cell has a single nucleus centrally
located. Between each one is found the capillaries
and fibrous tissue, which serves to hold the tissue in
place.

Exercise No. 37. — Mount some fresh muscle tissue.

Take some teased muscle, place it on the slide and
examine. Search for the following parts, and make

£0 HlSTOLOGt.

drawing: cells, nuclei, and the enveloping membranes.
Proceed in like manner with all three varieties.

Exercise No. 38. — Mount the prepared tissue supplied,
and study, cover, label and preserve.

CHAPTER IX.

THE VASCULAR SYSTEM.

The vascular system is composed of the heart, ar-
teries, veins and capillaries. The heart is a muscular
organ, lined by an endocardium, which is the continua-
tion of the tunica intima of the blood vessels. Next
to the endocardium is the myocardium from within
out. The myocardium is the heart muscle. The outer
surface of the heart is covered by the pericardium:
this is a serous membrane, of white fibrous tissue, and
covered by endothelial cells. Numerous ganglia are
found in the tissues of the heart, along the course of
the nerves. The nerve supply of the heart is derived
from the pneumogastric and sympathetic system. The
fibers running from these centers are both medullated
and non-medullated. The first is from the pneumo-
gastric, and the second is from the sympathetic sys-
tem. The valves of the heart are simply broad folds
of the endocardium, between* which are a small amount

(61)

62 LABORATORY METHODS OP

of fibrous tissue. The lymphatics of the heart are
many and are found in two places, viz. : around the
coronary arteries and between the muscle fibers The
capillaries are minute ramified arteries, which have lost
their outer coat and hence become very thin. This
thin space is where the white blood cells leave the ves-
sels in inflammation; the spaces are called stigmata.
The blood vessels are richly supplied with lymphatics
which are seen ramifying in the outer coat of the
larger arteries and surrounding the smaller capillaries.

T. S. BLOOD VESSEL.
Showing bands of yellow elastic tissue.
Tunica intima; 2, Tunica media; 3, Tunica

HISTOLOGY. 63

The veins are different from the arteries in that they
have thinner coats and less muscle tissue; their walls
are thinner than the arteries and their lumen is larger.
They have more fibrous tissue than the arteries. Ar-
teries are composed of three coats. The tunica intiw*,
the tunica media, and the tunica adventitia. The in-
ternal coat is lined with endothelial cells resting upon
a basement membrane of white fibrous tissue. Just
under this coat is located a band of yellow elastic
tissue, called the fenestrated membrane of Henle.
The middle coat is composed of the smooth muscle
running around the artery, and having a few fibers of
fibrous tissue interspersed with the muscle cells, es-
pecially the large arteries. The outer coat is com-
posed of white and yellow elastic tissue in abundance.
The nerve supply of the artery is derived from the
vasomotor system, and the blood supply of a part is
regulated by the contraction or expansion of the mus-
cle fibers in the middle coat of the artery. The outer
coat is for strength ; the inner one for smoothness ; the
middle one for contraction and expansion. All the
blood vessels are of mesoblastic origin. The vessels
are closed tubes.

Exercise No. 39. — Make a section of a blood vessel^
mount, stain and preserve.

54 HISTOLOGY.

Technic: Use the alcohol hardening method. Ob-
serve the coats, and make out the yellow elastic tissue.

Exercise No. 40. — Make a stained preparation of an
artery and vein.

CHAPTER X.

THE LYMPH VASCULAR SYSTEM.

The lymphatics are in all particulars like the blood
vessels in structure, .except that even the largest
lympatics have only a small amount of muscle tissue.
In all the tissues and organs there are small spaces that
are called pericellular spaces. These spaces are the
beginning of lymphatic vessels. The small vessels
continue to join until they reach the thoraic duct, which
pours its contents into the sub-clavian vein : at this
point the inner court of the duct is folded over on it-
self and forms a valve to prevent blood from flowing
into the duct. All along the course of the vessels are
found similar valves, which prevent the lymph from
flowing but one way. While all the tissues of the body-
are rich in lymph vessels, the ones supplied most are
the white fibrous, areola, blood vessels and nerves. In
the last two locations they are called perivascular and
perineurial lymphatics, respectively. The lymph is a
clear fluid, having floating in it a large number of
cells and fat granules. The cells are very much like
5 (65)

66 LABORATORY METHODS OF

the small lymphocytes of the blood. The fat is al-
ways present in varying amounts, but is increased after
a meal. The cells of the lymph eventually become
the white blood cells. — Osbnrn.

DIFFUSE ADENOID TISSUE.

In many positions throughout the body there are
found small nodes oi adenoid tissue located in the
tunica propria of mucous membranes, as in the lungs
and digestive tract. This variety of adenoid tissue is
called diffused. In some places this tissue is present
in small amounts, and fades away into the surrounding
tissue without presenting any line of demarcation. In
other locations this tissue is surrounded by a band of
white fibrous tissue, having dispersed it in a few
smooth muscle fibers. This band is called a capsule.
The node and band is called a lymph-follicle. When
these nodes occur in the course of a lymph vessel, they
are called lymph glands. These glands are usually
bean-shaped and the capsule sends down little bands of
white fibrous tissue, separating the gland into divisions.
These bands are called trabecula, and the divisions,
lobules. The trabeculae subdivide, forming the reti-
form tissue of the gland, in which are found the Mal-
pighi?n corpuscles of the lymph glands. The blood
vessels enter the capsules. The nerves of these glands

HISTOLOGY.

67

are of both varieties, viz. : medullated and non-medul-
lated. That portion of the gland which is located near
the capsule is called the cortical portion, and that por-
tion near the center of the gland is called the medul-
lary portion.

THE SPLEEN.

The spleen is a lymphoid gland, and differs from
the other lymphoid glands in that it has no medullary
portion and the blood supply is differently arranged.

SECTION OF SPLEEN.

1, -Capsule; 2, Malpighian corpuscles; 3, intralobular
vein; 4, splenic pulp. The large bands of fibrous tissue
represent the trabeculae.

68 LABORATORY METHODS OF

The splenic artery enters the spleen at the hilum
and passes up the trabeculae for a certain distance,
and then leaves them and passes into the splenic
pulp ; here the artery loses its outer coat and con-
tinues as the lymphoid tissue of the spleen. In this
place the adenoid tissue collects into a dense mass and
the mass is called the Malpighian corpuscle of the
spleen. Here the germ center is found. The veins
begin where the artery ends and pass to the trabeculae,
where they pass out of the organ by following the tra-
beculae to the hilum. A few lymphatics are found in
the capsule and trabeculae. The nerves of the glands
are few in number, and are usually distributed to the
blood vessels.

Exercise No. 41. — Harden the spleen; mount, stain
and examine. Follow the formalin method; stain
with eosin and haematoxylin.

Exercise No. 42. — Do the same with a lymph nodule.
THE THYMUS.

The thymus is a gland. In early life it is mostly
ephithelial in nature, and in later life it becomes a
lymphoid gland, presenting all the characteristics of
the spleen. The blood vessels and nerves are found
in the trabeculae and send other branches into the

HISTOLOGY. 69

lymphoid tissue. The thymus is the only lymphoid
gland that has no germ center, but cell division can be
demonstrated in the gland. Hassal's corpuscles are
the dense bodies found in the thymus. They are re-
garded as epithelial remains of the gland in early life.

THE TONSILS.

The tonsils are situated in the mouth at the base of
the tongue. They are surrounded by a dense mem-
brane of white fibrous tissue on the under side, and by
the mucous membrane, which is made up of pavement
epithelial cells of the buccal cavity, on the outer side.
The membrane dips down sometimes into the sub-
stance of the tonsil, and is called the crypts. The ton-
sils are supplied with blood vessels, lymphatics . and
nerves, which enter the organ through the capsule.
They are lymphoid bodies.

SEROUS CAVITIES AND MEMBRANES.

The serous membranes may be considered as ex-
panded lymphatics. These membranes are composed
of a thin sheet of fibrous tissue, supporting a layer of
thin, irregular, polyhedral cells, placed edge to edge
and united by a cement substance. At very short dis-
tances there are little openings in the mucous mem-
brane, called stomata, which are the mouths of lym-

70

LABORATORY METHODS OP

phafic g-lands. Sometimes the cells do not adjust
themselves completely, and a space is left, called pseu-
do-stomata. The pleura, pericardium and peritoneum
are serous membranes. The serous membranes are
not in contact with the air.

THE MUCOUS MEMBRANES.

This variety of tissue is in such a position that it
may come in contact with the air. It is composed of
the following structures : epithelial cells, basement

DIAGRAM OF THE DIFFERENT TYPES OF GLANDS.

1, Simple tube; 2, compound tube; 3, simple coiled tube;

4, compound saccular; 5, simple saccular,

HISTOLOGY. 71

membrane, and beneath the basement membrane there
is a layer of smooth muscle, called muscularis mucosie.
Along the course of the membrane there are several
openings which represent the months of glands.
These glands are simply an infolding of the mucous
membrane. Several types of glands are recognized.
All of them are some type of a tube. See cut for
types of glands. The different glands will be dis-
cussed as they are studied.

CHAPTER XL

THE NERVES.

The nerves are highly specialized fibers, beginning in
a cell and ending in a special end-organ. Their func-
tion is to carry impulses to and from the cell. The
combination oi a cell, end-organ, and nerve fiber, is
called a neuron. A nerve cell is one of the largest cells
in the body and possesses all the essentials of any
cell, viz. : nucleus, nucleolus and protoplasm. They
have no cell wall. The cells vary greatly in shape, and
each point of the cell is called a pole. One of the points
is called an axis cylinder. The points arising from the
cell at other points than the one called axis-cylinder
are called dendrites. The nerves are named according
to the number of processes they have. The first type
and second type cells are those cells that have either a
long or short axis cylinder. The first is a first type
cell, and the second is a second type cell. Neurogha
cells are small cells that give off several short proc-
esses, which act as a connective tissue. The cell is
called a glia cell, and the fibrils are called neuroglia.
(72)

HISTOLOGY. 73

GANGLIA.

These bodies are nerve centers, consisting of nerve
cells and nerve fibers. Some of the fibers end in' the
ganglion, while others pass on to more distant points.
The brain and spinal cord may be termed a group of
large ganglia. The whole ganglion is covered with a
fibrous tissue, called a sheath.

NERVE

The nerve fiber is composed of the axis cylinder of a
nerve cell, and three protecting membranes: the axi-
lemma, the medullary sheath and the neurilemma.

In the medullary nerves, at regular intervals, there
appear constrictions, called the nodes of Ranvier.
These nodes are the result of an absence of the medul-
lary sheath at these points. The space between any
two nodes is called internode. In the center of an in-
ternode is found the nerve corpuscle, located directly
under the neurilemma. The non-medullated nerve
fibers are found in the sympathetic system. Each fiber
consists of an axis cylinder, neurilemma and an oval
nucleus upon the surface of the fiber. There are two
theories concerning the formation of the axis cylinder :
one is that the nerve cells fuse themselves together,
end to end, ancj form a long thread; the other is that

74 LABORATORY METHODS OF

the nerve cells have a large number of processes, and
one of these functions as an axis cylinder. In both
cases the neurilemma corresponds to a cell membrane.
A medullated fiber is a fiber with a medullary sheath,
and a non-medullated fiber is a fiber without a medul-

T. S. SPINAL CORD.

Showing investing membranes and distribution of blood
vessels in the white and gray substances.

lary sheath. The nerves end in the muscle tissue by
expanding into a small plate of nerve fiber. There are
two main divisions of nerves, viz. : central and sympa-
thetic nerves. We will study each of these in detail,

HISTOLOGY. 75

The central nerves are studied by taking a section
of the spinal cord and "dissociating the tissue, then
scraping the anterior cornua and spreading the ma-
terial on a slide and examining it in the fresh state.
The best method is to study a stained section and com-
pare results with the cuts in all good books on the
subject.

Exercise No. 43. — Take a T. $. of the spinal cord of
a dog and scrape the anterior cornua, and examine
the scrapings.

Use the saline rnount; search for the following: A
large nerve cell having its processes. Locate the axis-
cylinder of the cell.

Exercise No. 44. — Examine the nerve of a dog or ox
in salt solution.

Technic same as above.

Exercise No. 45 — Make a T. S. of spinal cord from
tissue that has been hardened. Stain, mount and
preserve.

Search for the following: the gray matter, consist-
ing of ganglion cells, neuroglia and fibers ; the white
matter, consisting of meduHated fibers and connective
tissue. 'The white matter surrounds the gray. The gray

76 LABORATORY METHODS OF

T. S. SPINAL, CORD— CAT.

1, Anterior median fissure; 2, posterior median fissure;
3, anterior roots; 4, posterior roots; 5, central canal; 6,
anterior gray commissure; 7, posterior gray commissure;
8, anterior white commissure; 9, posterior white commis-
sure; 10, anterior columns; 11, posterior lateral column;
12, posterior column; 13, substantia gelatinosa; 14, 15, 16,
17, 18, groups of motor cells; 19, column of Burdoch; 20,
column of Goll.

matter is in the shape of the letter H. The surface of
the cord is divided into four areas: anterior, pos-
terior and two lateral. On dividing the cord into
halves, from before backwarc)s? and then dividing

HISTOLOGY. 77

each half into three columns, we have the anterior,
posterior and lateral columns of each half, making
six columns in the cord. In the lower cervical and up-
per thoracic region two more divisions appear in the
posterior column, the column of Goll (placed near the
posterior fissure) and the column of Burdoch. The
white matter is connective tissue and nerve fibers, hav-
ing a large number of glia cells interspersed in them.
The nerve fibers have a medullary sheath, but no neit-
rilemma. "The nerve fibers run in the same direction
as the long axis of the spinal cord ; a few may be seen
which run across the cord." — Leroy. Around the en-
tire cord are found three protecting membranes: the
dura mater, arachnoid and pia mater. The space be-
tween the dura mater and arachnoid is called the sub-
dural space. The space between the arachnoid and
pia mater is called the sub-arachnoidaj space. All
these membranes are composed of white fibrous tissue
and endothelial cells. They are all more or less con-
nected by fine fibers of fibrous tissue, which run trans-
versely to the long axis of the cord. The Pacchion-
ian bodies are large papillae projecting upward into the
subdural space. The blood vessels are found in the
dura mater and pia mater; but none are found in the
arachnoid. They are most numerous in the pia mater.
The gray matter of the cord is arranged in the shape
of the letter H. The long arms of the H are directed

78 LABORATORY METHODS OF

from before backwards, and the short arm is directed
from side to side, joining the long arms at their mid-
dle points. The long arms are comma-shaped, with
the big end of the comma directed anteriorly, i. e., to-
wards the front of the cord. This end does not ap-
proach the surface as near as the small end of the
comma. The small end is called the posterior horn,
while the large end is called the anterior horn of the
cord. In the center of the transverse arm of the H. is
seen a small opening, called central canal, lined with
ciliated epithelial cells, and containing a fluid called cer-
ebro spinal fluid. The transverse arm of the H is called
the gray commissure, and the white matter imme-
diately in front and behind it is called respectively an-
terior and posterior white commissure. Some histolo-
gists divide the gray commissure into anterior and
posterior, with reference to central canal. There is a
narrow cleft in the white substance, beginning at the
anterior median periphery, and extending to within a
short distance of the gray commissure, called anterior
median fissure. A narrower but similar fissure is
seen on the posterior side of the gray commissure,
called posterior median fissure. On each limb of
the letter H of the cord there are three horns; an-
terior, posterior and lateral horns, located as their
names indicate. From the anterior horn emerge the
anterior roots, and the posterior horns receive the pos-

HISTOLOGY. 79

terior roots. From the lateral horn emerge the motor
nerve axis-cylinders, the same as in the anterior horn.
The nerve cells of the anterior horn resemble a -straight-
ened comma, with a very long tail. The reticular
processes, column of Clark, and the substantia gelatin-
osa, enter the structure of the posterior horn. The
substantia gelatinosa covers the horn and surrounds
the central canal. The nerves of the anterior horn and
the column o>f Clark are very large and stellate. The
blood vessels of the cord enter it mostly from the pia
mater, and branch repeatedly to form capillaries, which
run parallel with the fibers in the wihite matter, but
form a net work in the gray matter. In both cases,
they are surrounded by neuroglia tissue. There are
thirty-one pairs of nerves springing from the spinal
cord. The motor nerves are anterior, while the sen-
sory nerves are posterior. They are distributed to the
skin and other parts of the body, and terminate in a
special nerve-ending.

Exercise No. 46. — Make a T. $. of the spinal cord.

Tcchnic: Mullers fluid, paraffine, haematoxylon and
eosin.

Locate the following: (L. P.) Enclosing mem-
branes, median fissures, gray matter, white matter,
axis-cylinders in the white matter, T. S. with their en-

80 LABORATORY METHODS OF

closing membranes, the horns of the cord in the gray
matter, the different .shaped and sized nerve cells in the
anterior horn, central canal, anterior and posterior
roots, the different commissures, and the entrance of
the blood vessels in the pia mater.

CEREBRUM.

The cerebrum is composed of five layers of nerve
cells, which merge imperceptibly into each other.
They are :

1. The molecular layer. This is the first layer, and
is composed of a number of finely granular cells, with
medullated fibers and neuroglia. The cells of Cajal
are found in this layer.

2. l^he layer of small pyramidal cells. This is located
under the first layer and the processes that leave the
cells form a net work. The axis-cylinders leave the
basal ends of the cells and send collaterals in the me-
dulla. The dendrites enter the molecular layer.

3. The layer of large pyramidal cells. This differs
from the second only in size of the cells, and in this
layer there is no network sent to the layer above.

4. The layer of irregular cells. This layer is com-
posed of oval or polygonal cells, which have no special
dendrites. Each axis-cylinder sends out collaterals,
and enters the medulla to become one or two nerve
fibers.

HISTOLOGY.

81

SECTION OF CEREBRUM.
Showing morphology and arrangement of the cells.

5. Spindle-shaped cells. This layer lies next to the
medulla, and is composed of spindle-shaped cells, with
nerve fibers between them. The cells are arranged
parallel with the fibers. These five layers constitute
the cortex of the cerebrum. In these, that part of the
nervous force dedicated to the cerebrum, is carried on.
The fibers extending from the cortex, with the connect-
ive tissue structure holding the nerve fibers together,
constitute the medulla of the cerebrum.
6

82 LABORATORY METHODS OF

The five layers of the cerebrum are the molecular
layer, the small pyramidal layer, the large pyramidal
layer, the layer of irregular cells and the layer of spin-
dle-shaped cells.
Exercise N~o. 47. — Make a T. S. of the cerebrum.

Technic : same as the spinal cord.

1. Search for the layer of the granular cells, large
and small pyramidal cells, and the layer of irregular
and oval cells.

2. Examine the medulla of the cerebrum ; note the
presence of connective tissue cells, or glia cells.

3. Search for spider and Cajal cells. In the first
layer will be found the cells of Cajal; in the second
layer will be found the small pyramidal cells; in the
third layer will be found the large pyramidal cells;
in the fourth layer will be found the irregular cells;
in the fifth layer will be found the oval cells.

CEREBELLUM.

The cerebellum is composed of three layers: The
outer molecular, the middle granular or rust colored
layer, and the inner medullary tract. The outer layer
consists of three varieties of nerve cells: the cells of
Purkinje, the polypo-lar or multipolar, and the stellate
cells. The middle layer contains two varieties of cells :
the granular cells and the short stellate cells. The
inner layer contains two varieties of cells : the centrifu-

HISTOLOGY. 83

gal neuraxis of the Purkinje cell and the two types of
centripetal cells, one type sending their neuraxis into
the granular layer. These fibers are called mossy fibers
and do not extend through the granular layer. The
other type of cells sends the collaterals of the cells into
the layer where the Purkinje cells are found and seem
to ascend the neuraxis of the Purkinje cells, and are
called climbing fibers of the centripetal cells. The
central gray nucleus are found in the medullary por-
tion of the cerebellum, and are a number of multipolar
cells with many dendrites. The medullary or white
substance of the cerebellum is arranged in a layer of
branching sheets, and when cut, presents in the cut
section a tree-like arrangement, called arbor vitae. For
more extensive work on this subject see Leroy, So-
botta, and Bohm and Davidoff.

THE MEDULLA OBLONGATA.

This structure is the same as that of the spinal cord,
the chief difference being in the arrangement of the

structures.

SYSTEM OF SYMPATHETIC NERVES.

This system is composed of a large number of gan-
glia and nerve fibers, connected by small fibers. The
fibers are non-medullated. They are arranged into

84 HISTOLOGY.

three large groups, viz.: the cardiac ganglia, the epi-
gastric ganglia and the hypogastric ganglia. These
ganglia are located in front of the spinal column.

NERVE ENDINGS.

There are three varieties of nerve endings : free
nerve endings occurring in the skin, mouth, spinal
cord, etc., terminal corpuscles occurring in the skin,
and neuro-epithelial, occurring in the perceptive or-
gans, viz. : mouth, eye, nose and ear.

Exercise No. 48. — Examine the skin T. S.

Technic : Harden in alcohol, embed in celloidin, and
stain with haematoxylin and eosin.

I. Search for the four layers of the cutis-notha, hair
follicle, hair in place, hair muscle, sweat gland, se-
baceous gland, nerve endings and pigment of the skin.

In what layers are all these objects found?

A sweat gland is a simple tubular gland.

What lesson do you get from the skin bearing on the
nervous system?

CHAPTER XIL

THE SKIN AND ITS APPENDAGES.

The cutaneous system comprises the skin, hair and
glands. The teeth are sometimes considered as be-
longing to this system. The skin is composed of two
distinct structures, viz. : the cuticle and cutis. The
cuticle is derived from the ectoderm and the cutis is
derived from the mesoderm. The cuticle consists of
four layers ; the stratum corneum, the stratum lucidum,
the stratum granulosum and the stratum Malpighii.
The first layer is composed of cells that have lost the
appearance of ever having been nucleated, called the
cornified layer. The second layer is composed of cells,
wilier! are polyhedral in shape and have almost lost
their nuclei. The third layer is composed of cells ir-
regularly polyhedral in shape and are joined together
by little spines. These cells are called prickle cells,
and outside of these are seen other cells, which have a
granular protoplasm. These two layers constitute the
stratum granulosum. The fourth layer is composed
of cells, which are columnar in shape and are in a
stage of active growth, as shown by the property they

(85)

86 LABORATORY METHODS OF

L. S. SKIN.— HUMAN.

1, stratum corneum; 2, stratum lucidum; 3, stratum
granulosum; 4, stratum •Malpighii; 5, corium; 6 and 7,
vessels of the skin.

have of straining so easily. This layer is the one that
contains the pigment that gives color to the skin in the
colored races and, in some locations, in the white race.
This layer rests upon the true skin. The true skin is
composed of two layers of fibro-elastic tissue, called
papillae of the true skin. The nerves end in the true
skin, in end corpuscles, and in the cuticle in an arboriz-
tioti manner.

HISTOLOGY. 87

TH£ SEBACEOUS GLANDS.

These glands are of the compound saccular type and
are located in the papillae of the corium. They consist
of acini, secreting cells and a duct. The sudoriferous
glands belong to the tubular type, and consist of a
basement membrane and gland cells and a duct. The
gland is located deep down in the tissue beneath the
skin. The deep end is coiled upon itself and the su-
perficial end is straight nearly all the way from the
coil to the cuticle. Just at the cuticle the duct becomes
tortuous and makes its way out as a little depression on
the surface of the skin. All these structures will be
seen when you study the skin.

NAILS AND HAIR.

The nails are derived from the epidermis, and are
composed of horny cells. The parts of a nail are
nail-body, nail-bed and nail-matrix. The nail-bed is
composed of the stratum Malpighii. The nail, there-
fore, grows in length and thickness by new cells from
the matrix. The body of the nail is composed of the
other layers of the cuticle. The stratum lucidum takes
the greater part in the formation of the nail as the
transparency indicates.

The hair, like the nail, is derived from the skin, and
is found almost all over the body. There are no hairs

88 LABORATORY METHODS OF

in the palms of the hands and soles of the feet. The
hair consists of three divisions: the shaft, that part
above the skin; the root, that part in the skin; the
bulb, that part from which the hair grows — in fact,
it is a part of the root. The hole in which the hair
rests is called the follicle. The shaft consists of three
layers : the corticle, cortical and medulla. The cuti-
cle is the outermost covering and consists of epithelial

L. S. HUMAN HAIR.

1, cuticle; 2, inner root sheath; 3, Henle's layer; 4, me-
dulla of hair; 5, papillae of hair; 6, hair bulb; 7, vessels of
hair.

HISTOLOGY. 89

cells overlapping each other. The cortical is the thick-
est and contains the coloring- matter of the hair. The
medulla is not always present, but when it is, it con-
tains air and gives an appearance according to tta
light used to examine the specimen.

At the bottom of the hair follicle is the fibrous tissue
that contains the blood vessels of the hair. The sheaths
of the hair are named according to their location. The
arrectores muscles of the hair are composed of a few
fibers of smooth muscle-tissue arising in the fibrous
of the corium, and cause the hair to stand on ends in
case of fright. A longitudinal section of the hair pre-
sents for examination : shaft, composed of three lay-
ers of modified epithelial cells ; root, that part of the
hair within the skin; the bulb, a division of the root;
the papillae, an elevation of the true skin into the root
of the hair; the sheaths, surrounding the root and
bulb, called root sheaths (the opening and the sheaths
are called the hair follicle) ; sebaceous glands, com-
posed of a saccular variety of glands, located about
midway the root of the hair.

The theories as to the growth of hair are two : first,
that the hair grows from an infolding of the epidermis
of the skin ; second, that the hair grows from the papil-
lae at the bottom of the root. The nerves of the hair
are few and extend only to the duct of the sebaceous

90 LABORATORY METHODS OP

gland. There is only one fiber to each hair. The
shedding -of the hair is a difficult subject to under-
stand, but the theory is that the cells in the papillae
harden and therefore the hair loosens and falls out.
These are called bulb hairs. The papillary hairs are
shed in this way from the external root sheath there
arises a bud, which grows downward and develops
into a new hair, which gradually pushes the old hair
out. The condition called goose flesh is caused by a
contraction of the muscles of the hair. The nerves
supplying the muscles of the hair are derived from the
sympathetic nerves and are called pilomotor nerves.

TH£ MAMMARY GLANDS.

These glands are included in the cutaneous glands.
They are a variety of compound saccular glands, and
are histologically considered as a tube being lined with
a basement membrane and glandular epithelial cells
with large nuclei. The opening in the gland is called
acinus. The end of the tube is called the duct. A
large number of these glands is put together and forms
a lobule having a small amount of white fibrous and
connective tissue placed around it. A large num-
ber of these lobules is put together and forms a lobe.
The gland, as we see it, is a number of glands — i. e.,
just as the liver is composed of separate livers, so is

HISTOLOGY.

91

T. S. MAMMARY GLAND.

Showing lobules, connective tissue, and acini lined with
columnar epithelial cells.

this gland. The mammary is richly supplied with
blood vessels and lymphatics. The lymphatices of
this gland communicate with those of the axilla. The
secretion of it is called milk. Milk is a mixture of
fat, leukocytes and colostrum corpuscles.

Exercise No. 49. — Make a T. S. of the mammary
gland.

Technic: Formalin, celloidin and H. & E.
Look for the lobe, lobule, acinus and connective tis-
sue elements.

CHAPTER XIIL

THE DIGESTIVE TRACT.

THE; MOUTH.

The mouth is lined with a mucous membrane con-
sisting of stratified epithelial cells. Upon the surface
of the mouth are seen the ducts of numerous glands.
Between this layer of epithelial cells and the submu-
cous tissue are found the papillse of the mouth. They
are composed of fibrous tissue. The gums have no
glands in them. The blood supply is derived from the
mucous membrane blood supply. The lymphatics are
from the same source.

THE:

The teeth are said by some to be a part of the tegu-
mentary system. The structure of a grown tooth is as
follows : every tooth has a neck, crown and root. The
crown is composed of a modified epithelial cell tissue,
called enamel. The enamel is the hardest substance
in the body, and is covered by a membrane of epithelial
(92)

HISTOLOGY. 93

cells, the cuticula dentis. The enamel substance is ar-
ranged in columns or prisms called enamel prisms.
They are stuck together with a substance more dense
than themselves, called enamel-cement. The lines of
Retzius are parallel lines running from the dentine to
the cuticula dentis in the enamel. They represent the
periodic deposition of the salts of lime in the enamel.

THE DENTINE.

The dentine is the tissue between the enamel and the
pulp-cavity. It is composed of little tubes about two
and one-half microns in diameter. They are surround-
ed by the membrane of Newmann. In their course they
take an S-shaped direction, giving rise to the lines of
Schrager. Peculiarly, irregularly branched spaces are
seen in the dentine, called interglobular spaces. They
represent the uncalcified spaces in the dentine.

The cementum is located on the tooth from the
crown downward, and is composed of bone plates hav-
ing no Haversian canals as a rule. The peculiarity
of the cementum is, it has a large number of Sharpey's
fibers, which are abundant in those arears where noe
bone corpuscles are found. They are found where the
bone has not calcified. — Bohm and Davidoff.

The pulp cavity is the cavity found in the central
portion of the tooth, and contains the pulp. It is very

94-

LABORATORY METHODS OF

large. The pulp is composed of fibrous tissue, nerve
fibers, a semifluid substance and an artery. The ar-
tery enters the cavity and breaks up into* capillaries.
The nerves supply the tooth substance by the branch-
ing method.

The epiblast and hypoblast, both enter into the com-
position of the tooth. The epithelial of the primitive
jaw becomes thickened and grows down into the sub-
jacent tissue and forms the dental ridge. From the
lower outer border of this flask-shaped mass of cells,

L. S. TOOTH.

1, enamel; 2, dentine; 3, pulp cavity; 4, neck of the
tooth; 5, cementum; 6, pericementum ; 7, apical foramen.

HISTOLOGY. 95

dental bulbs grow (one for each milk tooth), and sub-
sequently become hollow on the under surface and cov-
er a conoid upgrowth of connective tissue cells (the
papillae). The dental bulbs continue to expand one
for each tooth, assuming the form of the crown of the
tooth, and the inner layer of the cells become columnar.
Each of these columnar cells secretes an enamel prism,
and the bulb is now called the enamel organ. "While
this has been in progress the outer cells of the papillae
have elongated to form the odontoblasts which deposit
the dentin. From what has been said, it will be seen
that the enamel is deposited outwards and the dentin
inwards. The remains of the papillae become the pulp
of the tooth. The permanent teeth are formed simi-
larly. The cementum is deposited later by the alveolar
periosteum." — Lcroy.

Exercise No. 50 — Examine the L. $. of the tooth
supplied.

Make out the structure enamel.
Exercise No. 51.— Examine the T. S. of the tooth.

ESOPHAGUS.

The esophagus is composed of four coats, viz. : mu-
cous, sub mucous, muscularis mucosae, muscular and
fibrous coat.

96 ' LABORATORY METHODS OF

The mucous coat is composed of stratified epithelial
cells resting upon a basement membrane.

The muscularis mucosse is composed of a .band of
smooth muscle located just under the mucous coat.

The submucous coat consists of fibrous tissue, lym-
phatics, nerves, blood vessels and glands, which empty
their contents into the esophagus.

The muscular coat is composed of two layers of
muscle tissue, the inner layer being placed circularly
and the outer layer placed longitudinally. The upper
third is composed of the involuntary variety.

The fibrous coat is composed of a thin layer of white
fibrous tissue and a few fibers of yellow elastic tissue.

Exercise No. 52. — Make a T. $. of the esophagus.

Technic: Formalin, celloidin and H. & E.
Make out all the coats and describe the tissue in
each.

STOMACH.

The stomach consists of the usual four coats : mu-
cous, submucous, muscular and fibrous. The muscu-
laris mucosae belongs to the mucous coat.

The mucous coat consists of a basement membrane
resting upon a stroma of connective tissue, and sup-
porting a layer of columnar epithelial cells. In this

HISTOLOGY. 97

layer of cells are found a few goblet cells. The glands
of the stomach are of two varieties; the peptic and
pyloric. The peptic are of the tubular variety, while
the pyloric are of the compound tubular variety, and
are located in the pyloric end of the stomach. Under
the microscope the type of glands will tell what end
of the stomach is under consideration. The peptic
glands secrete pepsin, and the gastric glands secrete
the acid of the stomach.

The submucous coat consists of loose fibrous tissue,
lymphatics, blood vessels, nerves and occasionally a
little diffuse adenoid tissue is found. This layer is
thrown up into folds, forming the rugae and depres-
sions of the stomach.

The muscular coat consists of three coats of mus-
cle tissue ; an inner circular, a middle longitudinal and
an outer oblique layer.

The fibrous coat is made up of white fibrous tissue
derived from the peritoneum, and is covered on the
outside with endothelial cells. The outer coat of mus-
cle tissue is usually absent. The lymph-follicles are
not numerous in the stomach.

SMALL INTESTINES.

The small intestines consist of the usual four coats,
arranged in the usual manner. The folds in the in-
testines are called villi, and are composed of the same

7

98 LABORATORY METHODS OF

T. S. SMALL INTESTINES.

1, mucosa, covered with columnar epithelial cells; 2, sub-
mucosa; 3, muscular layers; 4, fibrous layer.

tissues as the rugae of the stomach. The goblet cells
are abundant in this tissue. In this coat there are a
few smooth muscle fibers which give rise to the mus-
cularis mucosae. The submucous coat is the same as
that of the stomach.

The muscular coat is made up of two layers : an in-
ner circular layer and an outer longitudinal layer,
separated by a small amount of connective tissue.

HISTOLOGY. 99

The serous coat is the same as that of the stomach.

SMALL INTESTINE.— CAT.

1, villus; 2, submucosa; 3, muscularis mucosae; 4, Bnm-
ner's gland located in the submucosa.

The glands of the small intestines are of four vari-
eties : Lieberkuhn's glands are found in the mucosa
of the entire intestinal tract, between the villi. They
are of the simple tubular type. The glands of Brunner
are of the same type as the ones above, but divide into
many branches; therefore they look like a racemose
gland. They are serous and not of mucous type.
These are the duodenal glands. The solitary glands
are found throughout the intestinal tract and are lymph

\
100 LABORATORY METHODS OF

THREE VILLI, SHOWING THE ARRANGEMENT OP
THE VESSELS.

follicles. "Foyer's patches are elongated structures
consisting of from ten to sixty .adjacent solitary
follicles connected by their cortical zones." — Sobotta.
They occur in the mucosa and submucosa. The nerve
supply of the intestinal tract is derived from the sym-
pathetic and cranial nerves.

Exercise No. 53. — Examine the fallowing: esopha-
gus, stomach, s\mal! intestines and large intestines.

Technic: Corrosive sublimate and celloidin. Stain
with H. Car. and H. & E.

HISTOLOGY. 101

THE LARGE INTESTINES.

SECTION OP DOG'S LARGE INTESTINE.— PREPARED

BY A. L. PAEY.

1, lumen of gland; 2, columnar epithelial cells; 3, gob-
let cells; 4, mucosa; 5, submucosa; 6; muscular layers;
7, fibrous layer.

THE LARGE INTESTINES.

The large intestines differ only from the small in
that they have a fewer number of glands in them. The
lymph follicles and the glands of Lieberkuhn are the

102

LABORATORY METHODS OP

T. S. LARGE INTESTINES SHOWING LYMPH NOD-
ULES.

ones found. The rectum is lined with stratified epithe-
lial. Its mucous membrane is thrown into folds called
rectal valves.

THE UVER.

The liver is composed of lobes and lobules, having
varying amounts o>f connective tissue between each lob-
ule and between each lobe. This fibrous tissue is
called the capsule of Glisson. A lobule of the liver is
composed of the following: beginning, in the center,
you find the intralobttlar vein; surrounding this (with

HISTOLOGY.

103

H. P.), you see the liver cells; following these to the
outer edge of the lobule, you see the interlobular vein
enclosed in a few fibers of Glisson's capsule. At the
same point you see the bile capillaries leaving the lob-
ule.

The blood of the interlobular branch of the hepatic
artery and the blood of the interlobular branch of the
portal vein mix and pass into the lobule between two
lobules, called the interlobular vein, and then pass to
the center and out of the lobule through the intralobu-

A— T. S. LIVER— CAT.
1, lobules; 2, interlobular connective tissue.
B. — 1, liver cells.

104 LABORATORY METHODS OP

lar vein. A number of these intralobular veins unite
to form the sublobular vein, and these again unite to
form the hepatic veins, which carry the blood to the
inferior vena cava. Between each liver cell are seen
the beginning of the bile capillaries, which pass out to
the edge of the lobule and empty into the bile ducts in
that place. The bile ducts unite to form the large bile
duct. The large duct passes on to the duodenum.'
When there is more bile than wanted for the digestive
tract, the bile is backed into the bile cyst. This cyst
is called gall bladder, and is placed on the under side
of the liver. The bladder and ducts are composed of
white fibrous tissue lined with columnar epithelial cells.
Under this is the muscle tissue, and under the muscle
tissue is some loose areola tissue — then comes the layer
of fibrous tissue, lined with endothelial cells. The lym-
phatics are in the form of perivascular lymphatics.
The nerves are of both varieties : medullated and non-
medullated. The liver cells form the chief study of
the liver.

Exercise No. 54. — Study all the tissue from the in-
jected tissue furnished. And make T. $. of the
tissue furnished.

Stain, mount, study and preserve.

HISTOLOGY. 105

TH£ SALIVARY GLANDS.

The salivary glands are the parotid, submaxillary,
sublingual and the pancreas. They are all of a true
glandular nature, having a duct, which divides and
subdivides until it reaches the alveolus of the gland.
The little alveolus is in reality the lumen of the duct
lined with glandular epithelial cells. Kach individual
alveolus and its basement membrane is called a lobule.
Each lobule has an enclosing membrane of white fi-
brous tissue separating it from the next lobule. As
these duct branches grow larger a second band of
white fibrous tissue is thrown around a number of
them, forming a lobe. A band of white fibrous tissue
is thrown around the whole mass. This band is called
a capsule. The whole mass is called a gland. The
duct, after leaving the alveolus, is lined with columnar
epithelial cells. In some glands it is lined with two
layers of cells. The salivary glands are named ac-
cording to their secretion: mucous, serous and mixed
glands. The glands are richly supplied with blood ves-
sels. The artery enters the gland at the point where
the duct leaves it, and when it reaches the acini it
breaks up into a network of capillaries that reunite to
form the veins, which leave the gland, accompanied by
the artery. The lymphatics are found with the artery
as far as the 'acini. The nerves are of both varieties and

106 LABORATORY METHODS OF

reach as far as the acini. There are found small
ganglia in the glands.

THE PAROTID GLAND.

This gland is a serous gland. It presents for an ex-
amination a capsule, septa of connective tissue, lobes

T. S. SUBMAXILLARY GLAND.
1, fibrous connective tissue between the lobules.

and lobules. The lobe is composed of lobules, connec-
tive tissue between the lobules and a duct. The lobule
contains acini and ductules. The acini is the terminal
of the tube of which the gland is composed. The com-

HISTOLOGY. 107

position of acini : glandular cells, resting on a base-
ment membrane and the lumen of the tube, or gland.
The ductules discharge their contents into the ducts.

Exercise No. 55. — Study, mount and preserve the sec-
tion suppliea.

Note the method of stain used in your book.

l'..rercise No. 56. — M\a\ke a study of the pancreas in the
same way you did the parotid.

Note the difference, if there is any. The ducts of
all glands are lined with columnar epithelial cells.
Sometimes the cells are arranged in two layers. (See
the scheme ot the salivary-glands in Bohm and David-
off, page 227.)

Exercise No. 57. — Make a study of the gland tissue
supplied and make out all the parts named as be-
longing to a gland.

Mount and preserve.

Exercise No. 58. — Make a T. $. of all the glands of
the month and note the difference, if any, in them.

Make drawings of all of them. There are numerous
small glands in the lips and oral cavity generally, and
are named according to their location.

108 HISTOLOGY.

Exercise No. 59. — Make T. $. of the lip.

Study, mount and preserve.

THE PANCREAS.
The pancreas is the abdominal salivary gland and is

SECTION OF PANCREAS.

in structure the same as the other glands of the same
name.

Exercise No. 60. — Make a T. $. of the pancreas.

Study, mount and preserve.
Note the technic in all cases.

CHAPTER XIV.

THE URINARY TRACT.

THE KIDNEY.

This organ is a compound tubular gland, consisting
of about twenty lobules, surrounded by a dense layer
of white fibrous tissue called a capsule. In this cap-
sule is found some smooth muscle tissue. The kid-
ney is of a bean shape. And the hilum is directed to-
wards the median line of. the, abdominal cavity. At
the hilum the artery enters, and the vein and ureters
leave. The nerves are of both kinds : the medullated
and non-medullated.

The outer investment is called a capsule, having in
it a few smooth muscle fibers. The cortex of the kid-
ney is located just under the capsule and constitutes
one-third of the kidney substance. It contains the
labyrinth, Malpighian bodies and uriniferous tubules.
The labyrinth is . the space found between the upper
end of the uriniferous tubules, and extends down-
wards to the point where they all unite to empty into

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HO LABORATORY METHODS OF

the calyces of the kidney. It will be remembered that
the uriniferous tubules are gathered into bundles and
then proceed downwards until they reach a point just
above the calyces, where they join several other bun-
dles forming a mass of tubules which empty into the
calyces. There are several such masses in one kidney.
These masses are called the medullary substance of
the kidney. The beginning of the medullary substance
is nearly like the fingers of your hand. Hold your
hand up before you and both extend and separate your
fingers. You will then have a fair example of the
medullary rays, medullary substance, labyrinth and
tube of Bellini, the fingers representing the medullary
rays ; the spaces between them representing the laby-
rinth, the palms representing the medullary substance
and the wrist the tube of Bellini. The tube of Bellini
is the excretory duct of the uriniferous tubules.

The blood vessels enter the kidney at the hilum and
pass upwards and give off branches to form the glorri-
erulus. The glomerulus is a coil of an artery, which
enters the capsule of Bowman at the same point where
the vein leaves the capsule. In this body the urine is
separated, in part, from the blood. (For the theory of
the secretion of urine, see the text on Urine.) The uri-
niferous tubule is the tube which carries the urine from
the glomerulus to the calyces of the kidney, and it

HISTOLOGY.

Ill

SCHEME OF URINIFEROUS TUBULES AND GLOM-
ERULI.

], afferent artery; 2, efferent vein; 3, glomerulus; 4,
neck of the uriniferous tubule; 5, proximal convoluted tu-
bule; 6, spiral tubule; 7, descending limb of Henle's loop;
8, Henle's loop; 9, ascending limb of Henle's loop;
10, spiral portion of the ascending limb; 11, zigzag tubule;
12, distal convoluted tubule; 13, straight collecting tubule.

leaves the glomerulus at the opposite pole from the
artery and vein. The uriniferous tubule takes a tortu-
ous course and has received names accordingly: I, the
neck, lined with low cuboidal epithelial cells; 2, the
convoluted tubule, lined . with low columnar cells ; 3,
the spiral tubule, lined with the same as No. 2; 4, the
descending limb of Henle, lined with squamous cells;

112

LABORATORY METHODS OF

DIAGRAM OP KIDNEY.

1, intralobular artery; 2, glomerulus; 3, afferent vessel
(vein) 5, intralobular vein; 6, venous capillaries of the
cortex; straight uriniferous tubule.

5, Henle's loop, lined with squamous cells ; 6, the as-
cending limb of Henle, lined with the same sort of cells
as the loop; 7, the irregular tubule, lined with striated
columnar epithelial cells; 8, the distal convoluted tu-
bule, lined with granular epithelial cells; 9 the arched
collecting tubule, lined with low cuboidal cells; 10, the
straight collecting tubule, lined with columnar cells;
n, the excretory duct, lined with tall columnar cells.

HISTOLOGY. 113

The sinus of the kidney is formed by the union of the
excretory duct and is lined with white fibrous tissue of
the tubules, and is continuous with the ureters.

Hirer cise No. 61. — Make sections of the tissue given
you and study, mount and preserve.

URETERS.

The ureters are the two tubes that convey the urine
from the kidneys to the bladder and are composed of
three coats ; viz : mucous, muscular and fibrous. They
are lined with squamous cells.

THE BLADDER.

The bladder is composed of the same number of
coats as the ureters. It is lined with squamous cells.

THE URETHRA.

The urethra consists of two coats lined in different
parts with different cells — in the prostatic portion with
transitional cells; the middle portion with stratified
columnar; and in the penile portion with columnar
cells.

CHAPTER XV.

THE REPRODUCTIVE ORGANS.

The reproductive organs of the male are the testes,
prostate glands, Cowper's gland and the penis.

THE

The testes are composed of tubular glands, and pro-
duce the spermatozoa. They are covered with a cap-
sule of fibrous tissue. The -coverings of the testes are
tunica vaginalis, tunica albuginea and tunica vascu-
losa. These coats dip down into the organ and divide
it into lobes. The lobes are composed of convoluted
tubules, straight tubes and a net work of tubes. The
convoluted tubules are lined with four layers of epithe-
lial cells, beginning on the basement membrane, and
coming outwards. You see the parietal cells, then the
mother cells, then the spermatoblasts and finally the
spermatozoa — all these layers are found in the lumen
of the convoluted tubule. And the spermatozoa are the

(114)

HISTOLOGY.

115

T. S. TESTICLE,

Showing capsule, septa of connective tissue and lobules,
generating cells. The straight tube and the network of
tubes are a continuation of the same tube. These tubes
all unite before they leave the epididymis and form the
duct of the testes, called vas deferens. It consists of
three coats, (a very thick muscular coat). The vasa
efferentia makes up the epididymis. The globus major
is the head of the epididymis and the globus minor is
the tail of the same. The vas deferens constitutes the
spermatic cord. The hard mass of fibrous tissue upon
which rests the net work of tubules is called the medi-

116 LABORATORY METHODS OF

astinum. The spermatozoon is a long, comma shaped
body. It is divided into three parts: head, middle
piece and tail. The vesicitla seminal is is the expanded
portion of the vas deferens in addition to having a few
mucous glands of tubular type in its mucous mem-
brane. "The blood supply enters the tunica vasculosa
and mediastinum." — Leroy. Lymphatics are found in
two locations : in the tunica albuginea and in the space
between the tubules. The nerves are of both varities
and are found in the spaces between the seminiferous
tubules.

THK PSNIS.

The penis is composed of two parts, the corpora
cavernosa and the corpus spongiosum, having a large
amount of connective tissue between them. The blood
supply is derived from the dorsal and the corpora cau-
ernosa arteries. The lymphatics empty into the deep
lymphatics of the pelvis. The nerves are of both
varieties. They are derived from the pudic and hypo-
gastric plexus of the sympathetic. The nerves end in
nerve corpuscles or end bulbs. The glands of the gen-
erative organs are of the tubular type.

THK OVARIES.

The ovaries are the female organs of generation,
placed one on each side in the pelvic region. They are

HISTOLOGY. 117

covered with two capsules : the germ epithelial layer of
cells, derived from the peritoneum and a tough band
of fibrous tissue, called tunica albuginea. The sub-
stance of the ovary is divided into two parts, the corti-
cal and medulla, which merge into each other. In the
young ovary one of the cells of the germ epithelial
layer becomes cut off and drops down into the medulla.

L. S. OVARY.— CAT.

1, germinal epithelium; 2, immature Graafian follicles;
S, stroma; 4, blood vessels in stroma; 5, a Graafian follicle
from which the ovum has escaped; 6, 7, ova in different
stages of development; 8, a fully developed ovum; 9, cor-
pus -luteum.

118 LABORATORY METHODS OP

A band of fibrous tissue from the stroma encloses this
cell. This band is called theca follicnli, and is divided
in two parts, an inner and an outer. The first cell is
called primordial ovum. Just inside of the theca fol-
liculi is seen a granular zone, called membrana granu-
losa. Inside of this stratum is the discus proligerus.
Now, inside of all this is the ovum, a large circular
body, enclosed in two cell walls ; viz : the zona pellucida,
and the vitclline membrane. In this space between the
ovum and the vitelline membrane is found the vitellus.
Situated to one side of the litellus is located the ger-
minal vesicle. In the center of the germinal vesical
is the germinal spot. A Graafian follicle that has lost
its ovum is called a corpus luteum. The blood vessels
enter the ovary at the attachment of the broad liga-
ments and pass into the theca folliculi. The lymphat-
ics are most numerous . in the medulla. The nerves
are of both varieties, and supply the follicles.
THE: FAi,u)piAN TUBES.

The fallopian tubes, as all other tubes, consist of
three coats. The mucous coat in these tubes is lined
with ciliated epithelial cells. The blood vessels are
the same as in the other cases.

THE UTERUS.

The uterus is the same in structure, only the muscu-
lar tissue predominates in this organ. The Nabothian

HISTOLOGY. 119

glands are in the mucous coat of the uterus. They are
called the uterine glands.

THE VAGINA.

The vagina is lined with stratified, pavement epithe-
lial cells. The muscle tissue is arranged in two layers,
an inner (circular) and outer (longitudinal), and is
composed of the smooth variety. The region of the
clitoris and the labia minora contains many sebaceous
glands. In the region of the urethra, mucous glands
are found called Bartholin's glands.

CHAPTER XVL

THE RESPIRATORY SYSTEM.

This system comprises the nose, epiglottis, trachea,
larynx, bronchi, lungs and pleura. These tissues are
derived from the mesoderm and the epiderm. The
nerve supply is from the cerebro spinal and sympathetic
system. The epiglottis is chiefly yellow cartilage. Its
mucous surface is covered sparingly with taste buds.
The larynx is cartilagenous. The trachea is composed
of three coats : mucous, submucous and fibrous. The
mucous coat consists of ciliated epithelial cells, base-
ment membrane and elastic fibers. The submucous
coat consists of cartilage, glands, elastic tissue and non-
striated muscle. The cartilage is of the hyaline vari-
ety and is arranged in the shape of a horseshoe. "The
large bronchi are like the trachea in structure, only the
cartilage is not arranged in a loop, but is arranged in
plates." — Sobotta. A bronchus having a diameter not
over I micron is called a bronchiole. They still have a
layer of muscle and a layer of cubic epithelial cells.
These bronchioles lead into the respiratory bronchioles,
(120)

HISTOLOGY.

121

LUNGS.

and these in turn lead into the alveolar ducts. The
alveolus is divided into separate chambers, all
communicating with one opening, called an in-
fundibular space. The alveolus is lined with a
single layer of epithelial cells. The blood vessels fol-
low the course of the bronchus and break up into cap-
illaries around the alveolus, and there the blood under-
goes the gaseous change. The blood changes its
name from venous to arterial. The lymphatics and
nerves are scattered all in the substance of the lung
tissue. The nerves are of both varieties, and they fol
low the course of the blood vessels.

122 HISTOLOGY.

Exercise No. 62. — Make T. S. of the lungs.

Note the technic.

Observe all the points in the tissue.

CHAPTER XVII.

THE SPECIAL SENSES.

The special senses are located in the nose, eye,
tongue, skin and ear.

THE NOSE.

The mucous membrane of the nose is divided into
two tracts, the respiratory and the olfactory. The ol-
factory is the tract that furnishes the cell in which the
nerves end that carry the sensation of odor to the cen-
ter of olfaction or smell. It consists of two layers of
epithelial cells. The cells that receive the sensation
are very long and nucleated. The cells in which the
sensation is generated are placed beneath a layer of
tall, columnar cells. The nerves are of both kinds.
The special nerves communicate with the olfactory
center. The sensory nerves do not come in contact
with the olfactory cells. They are derived from the
trifacial, while the others are derived from the ol-
factory nerve. The blood vessels are found in the
sub-epithelial layer ending in capillaries. The lym-
phatics are numerous.

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124

LABORATORY METHODS OF

THE EYE.

The eye is the organ of vision and consists of a hol-
low globe divided into two chambers : the anterior and
posterior, separated by the iris and lens. In front of
the anterior chamber is the cornea and behind is the
lens. The posterior chamber is composed of the
sclera, choroid and retina. The sclera is the white of
the eye. The choroid is the coat between the retina
and the sclera. The retina is the vision-producing
membrane. The cornea is composed of five layers.

SECTION OF RETINA.
1, pigment layer; 2, layer of rods and cones.

HISTOLOGY. 125

The lens is a biconvex body, placed between the an-
terior and posterior chambers of the eye, and held in
place by the suspensory ligaments. The anterior cham-
ber is filled with a clear fluid called aqueous humor,
and the posterior, with a fluid called vitrous humor.
The lymphatics are found around the nerves of the
eye and in a few other places. The nerves are derived
from the ciliary nerves. The retina contains ten layers.

1. The layer called internal limiting membrane.

2. The layer of nerve fibers.

3. The layer of ganglion cells.

4. The inner molecular layer.

5. The nuclear layer.

6. The outer molecular layer.

7. The outer nuclear layer.

8. The external limiting membrane.

9. The layer of rods and cones.
10. The layer of pigment cells.

Make sections of tissue supplied, according to direc-
tions.

THE EAR.

The special organ of the sense of hearing is lo-
cated in the ear. The ear is of rnesodermic origin.

126

LABORATORY METHODS OF

TONGUE:.

The tongue is the special organ of the sense of taste,
and is placed in the oral cavity. It is covered with a
layer of stratified epithelial cells. The mucous mem-
brane studded with the little projections called, papilke,
upon which you find little taste buds. The m.uscle
layers run in every direction. It is well supplied
with blood vessels and lymphatics. The nerves are of

SECTION OP THE TONGUE.

1, mucosa, showing three papillas, covered with squamous
epithelial cells; 2, stratified epithelium; 3, taste buds; 4
muscular layer; 5, adipose tissue.

HISTOLOGY. 127

the sensory and taste variety. The papillae are of three
varieties, filiform, fungiform and circumvalate. They
all have a few taste buds connected with them. The
tongue is divided into halves by the septum linguae.
In the tongue will be found several tissues of the
body, viz : fat, muscle, epithelial cells and fibrous
tissue.

Exercise No. 63. — Make T. S. of the tongue and ex-
amine.

PART II.

URINALYSIS

INTRODUCTION.

It always gives me pleasure to give my experience
in our chosen profession, especially when it is in any
way connected with the analysis of urine, a subject
which I think is of vital importance in the diagnosis of
diseases, since by it we may arrive at a definite conclu-
sion as to the nature and location of the pathological
state. The older physicians for many years depended
entirely on a sugar and albumin test of urine. But
that no longer satisfies the investigating mind of the
scientific physician, since sugar and albumin are only
two of the many pathological elements found in the
urine. The methods of discovering the constituents
are few and simple, and most of the methods require
only a short time to complete them; so, you see, for
time spent and money expended on one hand and
knowledge gained and scientific treatment established
on the other, there is no sort of comparison. Now,
gentlemen, the value of these analyses will depend on
our knowledge of pathology.

Pathology is only perverted Physiology. Since we
know that the urine is the sewage of the body and that

(131;

132 INTRODUCTION.

it is the principal way by which the soluble material
of the blood and tissues leave the economy, we naturally
look to the urine to find any pathologic changes that
might be taking place in the body. These changes
are of two varieties : i . An increase in the normal
constituents of the urine. 2. An introduction into the
urine of some foreign matter that may be acting as a
poison ; in fact, anything introduced into urine that is
not normally found in it is a poison, illustrated in the
case of poisons, as treated in Toxicology. This variety
is of most interest to the specialist. The first variety
is oi most interest to the physician, since in ordi-
nary diseases it is either an increase or decrease of the
normal constituents of the urine. An analysis of the
urine not only gives us an idea of the changes going
on in the urinary tract, but in other places. A chem-
ical examination is essential, since it gives an idea of
the changes going on in the body a distance from the
urinary tract, while the microscope only detects the
solids in the specimen. How . are these changes de-
termined ? We are now going to talk about the most
importiant constituent of the urine, since by it the nitro-
genous material leaves the body.

URINALYSIS.

The analysis of the urine is very important to the
physician and surgeon. It consists of three depart-
ments, viz. : chemical, physical and microscopical. In
order to gain any information from an analysis, it is
necessary to understand the secretion of the urine.
There are two theories as to the secretion of it, viz. :
the capsule of Bowman, acting as a filter, separates
the urine from the blood ; the epithelial cells lining the
uriniferous tubules secrete a part of the urine. In
fact, both processes go on at the same time. The first
is Ludwig's theory, and the second is Bowman's. The
experiment of Heidenhain settled the question of the
epithelial cells having a part in the secretion of the
urine. He injected a coloring matter into the veins of
an animal. After a suitable time he made sections of
the kidneys of the animal, and found the coloring mat-
ter in the cells of the uriniferous tubules, but none in
the capsule of Bowman. Other experiments prove the
theory good, for when a patient has uremia, the urinif-
erous tubules have lost their epithelial lining, as shown
by the microscope on analyzing the urine. The theory

of Lurwig is proven by the change in the amount of

(133)

134 LABORATORY METHODS OF

urine after drinking a large amount of water. The
urine is increased in quantity but not in quality. (For
the histology of the kidney, see the illustration of that
organ.) The urine is the sewage of the system,
and, therefore, contains the results of all the tissue me-
tabolism. The changes the tissues undergo in the
body you understand from the knowledge of physiol-
ogy. The urine being a filtrate of the blood, it would
be natural to suppose that it was of the same reaction
as the blood ; but not so. It is acid, while the blood is
alkaline. The cause of this change was proven by
C. H. Ralf to be the result of vital phenomena. The
solutions necessary for an analysis of the urine are
hydrochloric acid, sulphuric acid, acetic acid, nitric
acid, liquor potassii, liquor sodii, liquor sodium aqua
carbonate, liquor baric chloride, ammoniae and liquor
magnesium sulphate, Hain's solution, silver nitrate
solution, liquor plumbic acetate, alcohol, water and
hypobromite solution.

The apporatus. A notebook, two dozen test-tubes
and a test-tube rack, two conical glasses, spirit lamp,
beakers and watch glasses, funnels, 2,000 c.c. vessel,
graduates, filter paper, water bath, tripod, swab for
cleaning the test tubes, a microscope and several pi-
pettes. Other things will be added as we go on with
the work.

URINALYSIS. 135

The following solution should be made by the phy-
sician: hypobromite solution and Haines' solution.

The others can be purchased at a drug store. The
first is prepared as follows : 25 c.c. of water to 100
grams of sodium hydrate ; the bromine and sodium hy-
drate must be kept "in separate vessels. When ready
to make the test, take 10 c.c. of the soda solution and
i c.c. of the bromine, add equal amount of water, mix,
then put the whole into the ureometer, then add the
urine.
Technic.

1. Put the solution in the ureometer, filling the arm
and one-half of the bulb.

2. Add i c.c. of urine to the solution ; use a pipette.

3. Read the amount of urea in the specimen by
means of the graduate on the arm of the instrument.
This reading represents the amount in 24 hours in a
liter of urine. The normal amount ranges from 20 to
33 grams. The urea is decreased or increased in many
pathological conditions of the system. (For details
on the subject, see Practice, and for source of urea, see
Physiology; for composition and for reaction between
the solution and the urea, see Medical Chemistry.)

In urinalysis all vessels must be clean. For chem-
ical analysis, the specimen must be that of the urine
representing the 24 hours urine, if not the whole.

136 LABORATORY METHODS OF

For microscopic examination it is best to have the
urine as fresh as possible. In the summer time it is
best to add to each four ounces of urine 10 grains of
sodium salicylate to prevent decomposition.

The physical properties of the urine are too well
known to require special mention here. (See Physi-
ology oh the following points: color, odor, transpar-
ency, reaction and amount. ) The color is straw ; the
reaction is acid; the odor is peculiar to itself; the
amount ranges from 1000 c.c. to 1500 c.c. ; normal
urine is transparent. In disease all of this is changed,
each change representing a pathological process.
Sometimes several properties are changed in one case.
(For diseases represented by the changes, see Prac-
tice.) The method of making the physical analysis is
simple. Use the senses given you. For reaction
use litmus paper. Specific gravity of the urine is de-
termined by using the urinometer. The normal grav-
ity is 1 020 to 1025.
Method :

1. Fill the cylinder of the urinometer two-thirds.

2. Set it on a level table, and introduce the bulb of
the instrument in the center of the cylinder; when the
bulb comes to rest, read off the graduate on the stem of
the bulb. In case you have not enough urine to find
the gravity, you must add water enough to float the
bulb ; then multiply the gravity of the mixture by the

URINALYSIS. 137

sum of the water and urine together. This is not prac-
ticable only in case the urine is of high gravity.

The specific gravity of the urine depends upon the
solids in it. The most abundant solid is the urea; the
other solids are in the shape of salts— therefore, the
acids and metals are in the combined state. The tests
ior the metals are the same as the ones used in analytical
chemistry; this is also true with the acids. The de-
composition of urine is accompanied with a peculiar
odor, caused by the urea breaking up into its constit-
uents. The elimination of the urea represents the tis--
sue metabolism, a process which is explained under the
head of Metabolism in this text. The urea represents
about two per cent, of the solids, and the solids repre-
sent about four per cent, of the urine. "Schroder's ex-
periment is as follows : he injected into the liver by the
portal vein a mixture of ammonium carbonate and
blood, and on examining the blood in the hepatic vein,
it was found to contain urea in abundance. This does
not occur when the same experiment is performed with
any other organ of the body, so that his experiment
proves the great importance of the liver in urea for-
mation."— Kirk.

The accumulation of urea in the body is called urae-
mia, and the same may be said of the accumulation of
uric acid ; only the term is uricacidaemia. There is

138 LABORATORY METHODS OF

some discussion as to the relation between urea and
uric acid. The ratio between them is as one to forty-
five. (For further information see Medical Chemistry.)
At certain times of the day the reaction of the urine
differs widely. The time when it is acid is called the
acid tide, and when it is alkaline it is called the alkcb-
line tide.

What are the most abundant constituents of the
urine ?

What is said of the acids and metals in the urine?

What is the cause of the odor of stale urine?

Examine the spread of urine after the most im-
proved method.

How do you make a microchemic-analysis ?

UREA.

With the ureometer make a test for urea.

What is the normal per cent, of urea?

What does urea represent in the body ?

Give Schroder's theory in reference to the formation
of urea, as taught by an experiment.

Where is urea formed in the body ?

What is urea accumulation in the blood called?

Is there any connection between urea and uric acid?

What is an accumulation of uric acid in the blood
called?

URINALYSIS. 1 39

How is uric acid formed for experimental purposes?

Take 10 c.c. of urine and 2 c.c. of hydrochloric acid ;
put the acid in the urine and set aside for two hours.
Examine under the microscope.

Note the shape of the crystals.

What is meant by the acid tide and the alkaline tide?

MICROCHEMIC ANALYSIS.

Take a drop of urine to be examined and put it on
the slide ; also a drop of the reagent to be used. Place
it on the slide near the urine, put a cover-glass on the
drops, and watch the reaction.

Set a method by which you will always work in your
own office or laboratory.

ABNORMAL, CONSTITUENTS IN THE URINE.

Albumin in the urine is the first to consider. Albu-
min is coagulated by heat and certain chemicals, so we
make use of these principles in our analysis.

Exercise No. i. — Secure your key and clean out your
locker. Keep your locker locked. Note the quan-
tity of a liter as the demonstrator measures it out.

Each student should collect the urine for 24 hours
from a patient and bring it to the laboratory to analyze.

140 LABORATORY METHODS OF

Keep the urine in a tightly stoppered bottle to prevent
evaporation. From the total quantity for the whole
twenty-four hours samples should be taken to make
tests.

SPECIFIC GRAVITY.

With the urinometer take the gravity of the urine
and note the same. Dilute the urine one-half and take
the gravity again. Note the result. Give the range of
gravity in normal urine. Name what conditions will
increase the gravity ; also what conditions will decrease
the same. (See Physiology.)

NORMAL COI/DR.

What is the normal color of urine? Give the cause
of the same. Note the color of ten specimens of urine.
(Read Physiology on the urine.)

REACTION OF URINE.

The reaction of urine is due to what? Under what
circumstances is it alkaline? How do you test the
reaction of the urine?

COMPOSITION OF URINE.

Total grams 1,500.000

Water 1,440.000

URINALYSIS. 141

Solids 60.000

Urea 35-°°o

Uric acid -75°

Sodium chloride 16.500

Phosphoric acid 3-5°°

Sulphuric acid 2.000

Ammonia .625

Creatinin .900

Chlorine I i.ooo

Potassium ' 2.500

Sodium 5-5°o

Calcium .260

Magnesium .210

TEST FOR CHLORIDES IN URINE.

Take 10 c.c. of urine, add to it I drop of potassium
chr ornate,; add to this silver nitrate until the color
changes to red, noting the amount of silver added. Now
calculate the amount of chlorine present. I c.c. of sil-
ver nitrate corresponds to .00586 grams of sodium chlo-
ride. A more practical method is to take a small
amount of urine and add to it I drop of I in 8 silver
nitrate solution. Note results. If a curdy precipitate
occurs, which is not divided on shaking the urine, the

142 LABORATORY METHODS OF

quantity is normal, or if it is divided easily, the quan-
tity is diminished.

The presence of albumin in small quantity does not
make a change in the reaction of the chemicals. (For
clinical significance, see Tyson on Urine.)

PHOSPHATES IN URINE.

To 10 c.c. of urine add a small amount of ammonia.
Warm gently until the earthy phosphates begin to
separate. Set aside 10 or 15 minutes until they are
completely settled ; then measure the height of the pre-
cipitate, and if it is i mm. high, the quantity is normal.
(For clinical see Tyson on Urine.) This is the test
for the earthy phosphates.

THE AUCAUNE PHOSPHATES

are determined by adding to the urine a small quantity
of magnesium fluid, which precipitates the phosphates
into a cloudy precipitate. This cloud is normal. The
more the cloud, the more the phosphates.

SULPHATES.

To> a small amount of urine add a drop of barium-
chloride, and if a cloudiness is produced it indicates
that a normal quantity of sulphates are present. If a

TTRINALYSIS. 1 43

curdy precipitate is produced, it indicates an increased
quantity. (For clinical, see Tyson on Urine.)

1. Make a standard solution of sodium phosphate
(10.885 grains of well crystallized sodium phosphate)
in distilled water and dilute to a liter. (50 c.c. then
contains .01 centigram of phosphoric acid.

2. Make a saturated solution of potassium-ferrocy-
anide.

3. Make a solution of sodium acetate by dissolving
10 grams of sodium acetate in 100 c.c .of acetic acid
c. p. and diluting the 1,00 c.c. with water.

4. Make a solution of uranium acetate, such that I
c.c. will correspond to .005 milligrams of phosphoric
acid made as follows : Dissolve the uranium acetate in
water until the proper strength is obtained.

Step i. Take 50 c.c. of standard solution of sodium
phosphate in a beaker with 5 c.c of the solution of so-
dium acetate. Heat to 90 degrees C.

Step 2. Let the uranium solution run in until the
warm mixture ceases to precipitate. This is done from
a burette.

Step 3. Take a small amount of the potassium so-
lution and place it on a clean white dish ; then transfer
a drop of the warm mixture to the dish containing the
potassium solution. If the reddish brown precipitate

144 LABORATORY METHODS OF

does not appear, continue the adding of the uranium
solution until it does. Then read off the amount used.
This is the amount that will precipitate o.i decigram
of phosphoric acid. Now make a quantity of the
uranium solution, say a liter.

SULPHATES.

Make a solution so that i c.c. of the solution of bari-
um chloride will precipitate 12.50 milligrams of sul-
phuric acid prepared as follows :

1. Solution.. Dissolve 30.50 grams of barium chlo-
ride in a liter of water.

2. Solution the same strength as the one above, but
a different solution. Dissolve 27.57 grams of potas-
sium sulphate in a liter of water.

CHLORIDES.

1. Make a saturated solution of potassium chro-
mate.

2. Make a solution of silver nitrate such as I
c.c. will precipitate 10 milligrams of NaCl, as fol-
lows : Dissolve 29 grams of stick silver nitrate in a
liter of water.

ABNORMAL URINE.

Sugar solution. 30 grains of copper sulphate dis-
solved in one-half ounce of water; then add one-half

URINALYSIS. 145

ounce of glycerine. Thoroughly mix. To this mix-
ture add 5 ounces of liquor potassse. Make i liter of
this.

ALBUMIN.

Albumin is one of the abnormal constituents of the
urine. The presence of the same is indicative of
some lesion in the kidneys, especially in the uriniferous
tubules, according to Bowman's theory. This may be
a nephritis. The method of finding the presence of
the same is simple. It depends upon the fact that
albumin is coagulated under certain conditions. These
we must now study. Heat the urine at the top for a
short while and, if albumin is present, there will be a
white ring at the junction of the cold and hot urine,
which is the test for albumin.

Make an acid test for albumin. Take a small quan-
tity of nitric acid, and place it in a test-tube and al-
low a small amount of urine to flow down the side of
the tube. If albumin is present, there will be a
white ring or zone at the point of contact. This is
said to detect i part in 100,000 parts of urine. But
this is not as delicate as the heat test.

GRAVIMETRIC METHODS.

Gravimetric methods are of extreme importance to
the physician in cases of Bright' s Disease. But the
10

146 LABORATORY METHODS OF

method is so long that a busy physician has not time
to follow it ; so we must depend on some approximate
methods, the best of which are to precipitate the albu-
min by means of boiling the urine and adding a few
drops of nitric acid and setting it aside for twelve
hours. Shake the specimen once or twice in order to
get a uniform mixture of the materials in the urine.
A more accurate way is to take an Esbach's albumi-
nometer. (See your own drowings for the same.)

Method : Take the specimen of urine and put it
into the apparatus to the graduation U, then put in the
reagent to the letter R ; put in the stopper ; shake well ;
set aside until next period; read off the amount of
albumin on the albuminometer.

Each graduation denotes i gfam of dried albumin
in a liter of the urine.

The next method is to use the centrifuge, which is
very simple to use, and is very accurate in results.
Learn to use the centrifuge.

The amount of albumin found in a specimen is very
variable, but not over two per cent, in any case. This
is a very large amount.

Sugar in the urine is the next important abnormal
ingredient. The detection of the same depends on the
power that sugar has of reducing the copper salts into
cuprous hydrate, which is yellow or reddish yellow.

URINALYSIS. 147

Hain's Solution is the best to use in the test. The mere
change is not conclusive of the presence of sugar,
since a reddish brown is the test for sugar — that is, the
color desired, but we may judge from the color to a
certain extent, whether sugar is present or not.

FERMENTATION TEST.

The fermentation test is performed as follows:
Take a saccharometer and place in it a small
part of a cake of yeast and fill it up to the bulb with
the urine to be tested; set aside until the next period,
and read off the amount of sugar in twelve hours in
a liter of the urine. The graduations on one side of
the apparatus represent grams to the liter; on the
other, grains to the ounce.

PRACTICAL QUESTIONS IN NORMAL URINALYSIS.

Give the histology of a urine-bearing tube.

Give the theory of Ludwig in regard to the secretion
of the urine.

Give C. H. Ralf's experiment on the cause of the
change in reaction between the urine and the blood.

Give the histology of a glomerulus of the kidney.

Give the theory of Bowman in regards to the secre-
tion of the urine.

Give Heidenhain's theory of secretion of urine as
shown by an experiment.

148 LABORATORY METHODS OF

Give the conclusions of the same experimenter.
How did you determine the amount of water in the
urine ?

How much urine voided in twenty- four hours?
How much urea in a sample of normal urine for
twenty- four hours?

How did you make the urea test? Describe in de-
tail the process.

What solution did you use?

What are the most abundant constituents of the
urine?

Give the formula for urea.

Give the test for chlorides in urine, phosphates,
lution.

Give the properties of urea.

Give the test for chlorides in urine, phosphates,
urates, sulphates, sugar, albumin and bile.

What is the color, odor, reaction and specific grav-
ity of normal urine?

1 Name and describe the apparatus used in determin-
ing the above.

Give cause of each property.

What is the consistence of normal urine?

Give five causes why the urine may be cloudy.

URINALYSIS. 149

Give the test for nucleo-albumin.

Give the source of peptones in the urine; also test.

Give the test for bile in the urine.

Where is bile made in the body?

Give the indican test.

What is the presence of indican in the urine called?

MICROSCOPIC URINALYSIS.

Take a spread of the urine that you used when you
precipitated the earthy phosphates and place it on the
slide and examine.

Method: With a long pipette, take some of the
urine from the bottom of the sedimentation glass and
place it on the slide, cover with a cover-glass, using
just enough urine to fill the space between the cover
glass and the slide. Focus and make drawings of
what you see. (See the Atlas.) Wipe off the slide,
cover, and receive the spread that the assistant will
give you ; examine. (See Atlas.) Take a small quan-
tity of urine and set aside for a while to settle and use
the pipette on the same.

What method of making a spread is this?

Take the same amount of urine £nd do the same with
it. Only use the centrifugal apparatus.

Why did you use the centrifuge?

Find the reaction of the urine you used in finding
the earthy phosphates.

LABORATORY METHODS OF

Find the specific gravity of the urine; note the dif-
ference between the normal gravity and this one.

What is the cause of the decrease in gravity?

Dilute the same urine to one-third and find gravity
again.

Find the reaction of the urine you brought the first
day, also the gravity.

What has taken place in the sample since you have
been studying it-?

In case you have not enough urine to find the grav-
ity, how will you proceed to find it?

What is the gravity?

Test the sample of urine for urea after the hypo-
bromite method.

What is the percentage of urea?

What ureometer do you use, and what advantage
has it over other kinds?

What is the formula for urea?

Give the reaction between the solution and the urea.

EXAMINATION OF SEDIMENT.

Make a test of the sediment in the urine that has
become cloudy from standing twenty-four hours, and
determine the nature of the sentiment.

URINALYSIS. 151

Method : Pour in the tube some of the urine and heat
it, and if a cloudy precipitate appears, the sediment is
due to earthy phosphates. If the cloud remains after
the addition of an acid, it may be due to albumin ; if it
disappears, it may be due to the presence of phos-
phates; if it disappears on the addition of heat alone
it is due to urates; if on the addition of an acid the
piecipitate disappears with the evolution of gas, the
precipitate is due to carbonates.

What is the cause of ammoniacal odor in urine that
has been standing for a while at a moderate tempera-
ture?

What causes the white precipitate that forms at the
bottom of the vessel?

How can you tell when the alkalinity is due to a
fixed alkali or to a volatile alkali?

Method : Test a sample of urine with litmus paper,
then dry the paper, and if the blue remains, the
alkalinity is due to a fixed alkali; if the blue disap-
pears, it is due to a volatile alkali.

NUCI<£0- ALBUM IN.

Test for nucleo-albumin. This is a very simple test.
It is performed as follows : Take a small quantity of
the urine to be tested and dilute the sample to reduce
the salts that hold the albumin or mucin, which can

152 LABORATORY METHODS OP

be differentiated by simply boiling it with a mineral
acid. If a substance is produced that is reduced by
cupric oxid the substance is mucin; if not reduced, it
is nucleo^albumin.

PEPTONES.

Test for peptones in the urine: Place a drachm
of Fehling's Solution in a test-tube, and put a small
amount of urine in the tube, and if peptones are pres-
ent a halo of a rose-colored hue will float, forming the
test for peptones. .(For the pathology for peptonura
see Practice of Medicine.)

SUGAR TEST.

Moore's Test for sugar in the uf ine : Take a small
quantity of urine in a test-tube and one-half as much
strong alkali and boil, and if a reddish-brown or yel-
lowish-brown precipitate appears, sugar is present.
This precipitate intensifies as the boiling is continued.

HYDROBILIRUBIN.

Test for hydrobilirubin or urobilin : Add a small
quantity of ammonia to the urine to be tested and fil-
ter the same. To the filtrate add a little chloride of
zinc, and if a green color appears, urobilin is present.

URINALYSIS. 153

UROERYTHRIN.

To a small amount of urine to be examined, add a
small amount of neutral acetate of lead, and if a yel-
low color appears, uroerythrin is present. (See Ty-
son for clinical significance or the Practice of Medi-
cine.)

URATES.

What is the brick-dust deposit in the urine?

The murexide test for urates is a practical test.
Place a little urates in a tube and evaporate the same
after adding a little nitric acid. A yellow residue is
left. Add a little ammonia and the residue turns vio-
let, which forms the test for the urates or uric acid.
On the addition of potassium solution the color be-
comes blue.

Give the formula for ammonium carbonate, also
ammonium carbamate. (See Medical Chemistry.)

Test for bile in the urine : Take a small amount of
urine and put it in a test-tube. Now let a small
amount of commercial nitric acid run down the side
of the tube on the urine. If bile coloring matter is
present, a play of colors will be seen at the point of

154 LABORATORY METHODS OF

contact of the urine and the acid (green, blue, violet,
red and yellow). This is Gmelin's Test for Bile.—
Tyson.

BLOOD.

This is a common occurrence. The presence of the
same is detected by the color and the use of the mi-
croscope. The cause of the blood in the urine is a
common question asked the physician. It is caused
as follows: i. Local or renal congestion. 2. Trau-
matism of the ureters, bladder, calculi, acute cystitis,
and urethritis, simple and specific. 3. General dis-
eases, such as malarial-fever. The color of the urine
is the chief index to the beginning of the analysis.
The microscope comes in next. It is the surest method
to find the corpuscles in the urine before you say you
are dealing with a case of Haematuria. Since there
are so many other elements that resemble blood in color
it is not safe to go by the color alone. There are
many tests for blood, chemically, but these are not ab-
solute, so we will not spend the time on them. (See
Practice.)

PYURIA.

The presence of pus in the urine is called pyuria.
There are two tests for pus in the urine. 1st. Any

URINALYSIS. 155

strong alkali added to the urine will give a ropy pre-
cipitate. 2. Any good microscope will give you a fair
outline of pus corpuscles, which is the best test for
a busy physician.

What causes pus in the urine? Any lesion of the
urinary tract, where pus is being formed, will result
in a deposition of pus in the urinary tract. (These
conditions are many.)

PART HI.

BACTERIOLOGY

BACTERIOLOGY.

Bacteriology is the science of the minute organisms
that inhabit the sea, land and atmosphere — in fact,
every place that can be inhabited. Their morphology
is simple. They are of three general classes, viz.:
rods, spheres and curves.

Classify the spheres into cocci : Monococci, *. e.,
one sphere; diplococci, two spheres; streptococci,
spheres arranged in a chain; staphylococci, spheres ar-
ranged in a bunch. Staphylococci are divided into
several names, according to the color of the growth
on the medium.

GENERAL BIOLOGY OF BACTERIA.

There are six points to consider in the cultivation
of bacteria: food supply, reaction, moisture, gaseous
surroundings, temperature and light.

We will first consider the food supply, which -is the
medium on which they grow. It is, in nature, the
different substances that they accidently fall on and
grow ; but, in the laboratory, it is the different media

that we make for their sustenance or growth. Bac-

(159)

160 LABORATORY METHODS OF

teria serve a useful purpose in nature by breaking up
the complex substances that we find in nature into
simpler ones.

The reaction of the medium is of importance, since
many bacteria will not grow on any but an acid me-
dium, and all but a very few require an alkalin me-
dium.

Moisture is the next to consider. All bacteria re-
quire moisture; but some can live with less than oth-
ers; some can stand drying for several days and then
remain vital when placed in a suitable place for
growth (for an example, take the anthrax).

The relation of bacteria to the gases of the atmos-
phere is another important consideration. Some can
live without the oxygen, while others cannot live even
a short while without it; still another variety can live
with or without the presence of air.

Temperature is the next to be considered. The
point where the bacteria will grow best is called the
optimum temperature. This is usually the same as
that of summer weather. The temperature in which
they grow with the greatest difficulty is called the
minimum temperature.

MEDIA.

1. Take beef extract 3 grams

2. Peptone 10 grams

BACTERIOLOGY. 161

3. Sodium chloride , , . > 5 grams

4. Water 1000 c.c.

Boil until ingredients are dissolved (fifteen to

twenty minutes) ; add water from time to time to
keep up the 1 ,000 c.c.; allow to cool slightly; meas-
ure out 1,000 c.c., and place them in a large flask.

Determine the Reaction. — Process: Take phenolph-
thalein as an indicator and normal 20 sodium hydrox-
ide as an alkali ; take 5 c.c. of the medium and 40
c.c. of distilled water; put 12 drops of the indicator
into the mixture and proceed to add the alkali from the
burette until the indicator shows sufficient quantity of
the alkali, which can be observed by watching the color
of the fluid as it changes from a colorless to a red.
Calculate from this how much sodium hydroxide will
be required to alkalinize the whole liter of medium;
then add and boil forty-five minutes. Keep up the
level by adding water; filter and distribute in the ves-
sels you are going to use. Sterilize fifteen minutes each
day for three successive days in the Arnold sterilizer.

PEPTONE SOLUTION.

Take sodium chloride, 0.5 grams; take peptone, i.oo
gram; water, 100 c.c.

We will make other media from time to time as they

are needed. Everything is sterilized.
31

162 LABORATORY METHODS OF

Agar and gelatine are simply bouillon hardened, or
stiffened by them.

Everything must be labelled in the laboratory ac-
cording to the model shown you.

Examine an inoculating needle, draw and learn the
use of the same in more than one method.

Notice the demonstrator, as he inoculates one tube.
Describe in full the process in your notebook.

Inoculate several agar and gelatin tubes, some on a
slant surface and some on a square surface.

It is necessary that the glassware be thoroughly
cleaned before it is used. Several methods have been
suggested, but the best is the one which follows:
First, take cleaning fluid and wash the vessels in if.
The fluid is made as follows : dissolve 80 grams of
potassium bichromate in 300 c.c. of warm water;
when cooled, slowly mix the solution by constant stir-
ring with 450 grams of sulphuric acid ; store the liquid
in a glass bottle with glass stopper.

CLEANING TEST-TUBES.

Wash them in warm soapsuds; then place them in
the cleaning fluid, twenty minutes; then pour the solu-
tion back into the bottle ; wipe the tubes dry and drain

BACTERIOLOGY. 163

in the sink on the drain-board; wash them until the
color of the solution disappears.

FLASKS AND PETRI DISHES.

Wash them with soap and water ; then fill them with
the fluid and allow it to remain ten minutes. Proceed
as in the other case.

COVER GLASSES AND SLIDES.

Drop the cover glasses in the fluid one by one and
let remain ten minutes. Take them out and wash
them in alcohol; then place them in the sterilizer at a
temperature of 160 degrees C for one hour. If a drop
of bouillon is spread on a cover glass, the drop will
spread out evenly if the cover-glass is clean.

PLUGGING TUBES.

-This is a simple process, but much depends on the
way you do it. The plug should be neatly made and
firmly rolled just large enough to fit the test-tube or
flask. They are usually made of cotton-batting, which
should be sterilized before being used. The plug
must not be too large or too small. It is best to have
them just large enough to fit the tube or flask and no
larger. The whole must be sterilized.

164 LABORATORY METHODS OF

METHOD OF STERILIZING.

Place the vessels in the sterilizer and light the gas;
watch the thermometer; keep the temperature up to
135 degrees C for one hour; then shut off the gas and
let the temperature go down to 45 degrees C, and then
open the door and take the vessels out. Store in the
locker till wanted.

Light is another factor in the growth of bacteria.
It is a very decided germicide to certain bacteria.
It is probably the ultra violet and green rays that do
the killing of the germs. But light and heat both are
more certain to kill them than either heat or light
alone. — Muir and Ritchie.

Movement of Bacteria. — This is variable. Some
will move wlien they are in the act of reproducing.
In what is commonly called the spore stage some
move more rapidly than at other times ; some less.
Some substances will cause bacteria to move towards
them, such as salts of potassium and sodium. This
property is called CHemiotaxsis, positive and negative.

The Part Played by Bacteria in Nature. — As has
been said, the function of bacteria is to break up the
complex molecule into more simpler ones, as shown
by the souring of milk, the curdling of cheese and
the fermentation of sugar solution.

BACTERIOLOGY. 165

Saprophytic and Parasitic Bacteria. — The bacteria
that grow on dead organic matter is called sapro-
phytic, the variety that grow on living organic matter
is called parasitic.

The variability of bacteria is of much importance,
since the line of demarcation is not yet definitely de-
termined.

The death of bacteria is usually determined by the
fact that in a medium in which they were growing
luxuriantly, on inoculating a new supply of the same
medium they fail to grow.

Those agents which kill the greatest number of bac-
teria in the shortest time are carbolic acid and bi-
chloride of mercury (the first in a solution of I to
1,000; the second in a solution of I to 20.)

These are called germicides. Those agents that so
alter the medium that bacteria will not grow on it are
called antiseptics.

The best germicides are heat, acids and the heavy
metals in solution.— Muir and Ritchie.

CULTIVATION OF BACTERIA.

In order to study bacteria, it is necessary to have
them growing apart from other bacteria. In nature
this is not so since they are mixed up in general in the
medium; it is only in the blood and tissues that we

166 LABORATORY METHODS OF

find a few in the isolated state. When we have suc-
ceeded in obtaining a pure culture, we have only taken
the germ from its mixed state and have it growing on
a medium that is suited to its growth and development.
These pure cultures are absolutely necessary to a suc-
cessful study of the germ in question.

The Study of the Methods of Sterilization.— Heat
is the best, whether in the form of dry or moist heat.
This is the manner in which we do it : take a platinum
wire and heat it to a bright red, then let it cool to a
red heat; it is now ready to be inserted into the cul-
ture tube.

All instruments are sterilized in the bunsen before
they are used. The glassware is sterilized by a dry
heat. This is done by the use of the hot-air oven.
Watch the demonstrator use the oven.

The method of using the oven is simple and will
be used a great deal before we are through this work.
This method is not used in sterilizing the media as it
will evaporate the fluid media and scorch the solid be-
fore it will reach the temperature 135 degrees C.

Sterilizing by Moist Heat. — Boil the apparatus for
five minutes in a covered vessel, and that will insure
sterilizing for almost any variety of bacteria, but will
not kill spores; so we boil the material about twenty
minutes for three successive days. Steam sterilis-

BACTERIOLOGY. 167

ers are used in the following manner : in using the Ar-
nold and the Koch Sterilisers, put the media in the
steriliser at the same time you put in the water, and
that will insure the media getting the same amount of
heat that the water gets. Boil the whole fifteen min-
utes for three successive days. This is Tyndall's in-
termittent method. Each of these will be demonstrated
in the class.

Sterilisation by steam at high pressure is a quick
method but is not adapted to media work. It is car-
ried on by means of the autoclave. Sterilisation by
low pressure is adapted to one kind of media — that is,
blood-serum, on Tyndall's principle.

Plating of Bacteria. — Take a sterilised platinum
wire ; let it cool slightly ; then introduce it into the col-
ony of bacteria and then into tube No. I ; shake
well; then sterilise again; take a loop full from
tube No. i and inoculate tube No. 2 with the same;
sterilise; take a loop from tube No. 2 and inoculate
tube No. 3 ; pour the contents into the same number of
Petri dishes and number them according as the tubes
were numbered; set aside until next day and note the
change if any. This will be explained as we go along
in our work. Always sterilise your needle before and
after use. (The media must be melted.)

Hanging-Drop Culture. — This is made by simply
taking the drop of media containing the growth and

168 LABORATORY METHODS OF

placing it on the top side of a cover-slip and turning
the cover-slip down 'on the slide and watching the mo-
tion or non-motion of the germ.

COIyONY COUNTING.

1. Take a Petri dish and divide it off into equal seg-
ments and place it on the table, with a black surface
under the dish.

2. Take I c.c. of the fluid to be used and carefully
mix it with a tube of gelatine.

3. Pour the contents of the tube into the dish.

4. Incubate twenty-four hours at the room temper-
ature.

5. With the aid of a lens count the colonies that ap-
pear in the dish. (Each colony represents a bacterium
to begin with.)

BACTERIOLOGICAL EXAMINATION OF THE BLOOD.

1. Sterilise the part carefully with 1-1,000 perchlo-
ride of mercury ; then wash off the mercury with alcu-
hol; let the alcohol evaporate; wipe the part with a
dry sterile cloth or absorbent cotton.

2. Prick the part with a needle prepared for the
purpose by sterilising with dull heat.

3. Take a loop of the blood from the part and put
it in the media, and if you get a growth, make the
whole test for bacteria as directed further on.

BACTERIOLOGY. 169

Urine and water are examined in the same way (in
general the vessels are cleaned).

Filtration of bacteria is an important process in the
study of the organisms. It is done by means of a por-
celain tube which is the only perfect filter known.
Watch the demonstrator use the filter.

Examine water for Bacteria. — Take the water and
place it in the plating dish and after a time count the
colonies.

Method: Take a definite quantity of water and
put it in a dish of agar-agar; put the same in an
incubator for twenty- four hours; take out and count
the colonies with the aid of a lens. Each colony re-
presents a single germ.

Practical Use of the Analysis. — First, we find the
B. Coli and B. Typhosus in water. The earth is an-
other place where we find a great number of bacteria,
i. e., the B. tetani.

DETERMINING THE THERMAL DEATH POINT OF BAC-
TERIA.

This is a simple procedure. Take some bouillon
and inoculate it with some living bacteria; place it in
a water bath and introduce a thermometer; turn on
the gas and note the temperature; when the tempera-
ture reaches the body temperature take out one of

170 LABORATORY METHODS OF

the tubes and inoculate a new tube with the contents
of the hot tube ; wait a while and take out another tube
and proceed as in the above case, and so on, until you
have taken out several tubes, all at a different tem-
perature. Set the whole aside until the next period
and note which one has the growth on it. It will usual-
ly be the one that stays in the bath the shortest time
which will give the growth. The ones on which there
is no growth are dead and the temperature at which
they died is known by the reading on your tube, since
you noted the temperature when you took out each
eye of bouillon to inoculate the new tube.

DETERMINE THE STRENGTH OF ANTISEPTICS AND GER-
MICIDES.

Take some of the culture and subject it to the action
of different strengths of the germicides in a Petri dish
Any germ will serve for the test, or if the agent is a
gas, we will place some pieces of blotting paper, which
have been soaked in some good medium, and scatter
them in a closed chamber and set the gas generator
going. After a while take some of the paper and
smear the surface of a sterile medium and the growth
will not take place if the germicide is strong enough.

INOCULATING ANIMALS.

The mouse, guinea pig, rabbit, and white rat are the
ones that are used most. Take the animal and sterilise

BACTERIOLOGY. 171

the parts to be inoculated; then take an eye of the
growth on a needle after having made the incision at
the root of the tail of the animal ; inoculate the animal
and keep him in a cage to prevent him from spreading
the disease, if it is pathogenic. There are other meth-
ods. Fill a sterile hypodermic and inoculate the animal
in the dorsal veins of the ear and observe the same pre-
cautions as in other cases. This determines the
pathogen etic or non-pathogenetic properties of the bac-
teria.

Staining bacteria is a very important step in the
study of bacteria. We will take that up when we
come to stain the several kinds of bacteria.

Diagnosing bacteria is very difficult and is not free
from error. But we do it this way: we observe the
color or growth of the medium on which they grow
best. Their action in the hanging-drop preparation,
their morphology, their mode of bunching, their
source, their relation to air, their chemical production,
their action on an animal (pathogenetic property), their
reaction with Gram's stain and analine dyes.
These are the points by which we make a diagnosis of
a colony of bacteria. A few others we will discuss as
we come to them.

COVSR-GLASS PREPARATIONS.

These are simple to make, but we must be careful
not to contaminate the growth when we take the eye

172 LABORATORY METHODS OP

from the tube to make the spread on the cover-glass.
This is prevented by taking the needle and heating
it to a bright red in the flame of the bunsen, and let-
ting it cool a little before placing it in the culture or
growth. The spreading of the material on the cover
and drying in the air, and then passing it three times
times through the flame, then applying the stain, ac-
cording to the special process that we will learn as we
go along, constitute the technic.

METHOD OF EXAMINING THE MATERIAL THAT A PHY-
SICIAN MAY BRING TO YOU TO EXAMINE.

Several cover preparations must be made; one to
be stained with Gram's method, another to be stained
with carbolfuchsin, one with gentian violet, and an-
other with a mordant to stain the spores, if any are
present.

Gelatin plates should be made and kept at room
temperature. Now, if no growth has appeared on the
plates in twenty-four hours, it is well to try some oth-
er medium. But, if a growth does occur, you must
take a tube for your research that has only about 200
colonies on it. Now the first question to decide is,
Are all the colonies of one or of many species? The
question is answered by noting the characteristics of
the different species. This is not always known. Note

BACTERIOLOGY. 173

all yoii can from the plate, and all you can from the
hahging-drorj culture, and all you can from the mi-
crtfecdpe arid the reaction oh certain media, also re-
action on certain animals. The technic will be dis-
cussed in this chapter.

MICROSCOPIC EXAMINATION OF AN UNKNOWN.

Make the spread of the twenty-four hour culture;
note the form, the size and the appearance of the pro-
toplasmic contents regarding its reaction to a stain ; the
method of. grouping.

Has it a capsule?

Do the bacteria stain with a watery stain?

Do they require a mordant to stain them or not?

How do they behave toward Gram's stain?

Are they motile ?

Do they form spores?

Do they possess flagella?

What is the best temperature for their growth?

Growth on Media :— What are the characteristics of
the growth? What is its relation to oxygen? What is
the best temperature for its growth? Observe its
growth on gelatine stab-culture, rate of growth, form

174 LABORATORY METHODS OP

of growth, both on and in the substance, presence or
absence of liquefaction, color, presence or absence of
gas formation, odor and relation to reaction of media,
streak culture, shake culture, plate culture and the
condition of the colonies. Note the reaction on agar
in the same way, also bouillon and special media.
Note reaction on animals when inoculated.

Inoculating Animals. — The animal is either a rat,
mouse, guinea-pig, or rabbit. Method : Take the ani-
mal, and sterilize the part selected for the operation,
and inoculate the same and watch results. If the ani-
mal dies in twenty-eight days, the germ is pathogenic.
But it usually dies in less time. The best location for
the inoculation is in the dorsal veins of the ear or the
root of the tail.

Autopsies on Dead Animals. — Method : Take a
strong cord, a trough and the dead animal ; tie the ani-
mal in the trough, soak the animal in carbolic acid
solution to prevent the dry hairs from flying in the
room during the autopsy, hence contaminating the air
of the room; now open the body and examine. The
animal should be burned after the operation.

INOCULATION AND INCUBATION.

To inoculate a tube of medium, take a culture and
place it between the thumb and index-finger; take a
tube of the medium that you are going to inoculate

BACTERIOLOGY. 175

and place it between the index and second finger ; take
an inoculating needle and sterilize it in the flame; let
it cool a little; then place it in the culture; after hav-
ing taken out the plugs as directed in the class room,
insert the needle in the culture tube, then transfer
an eye to the tube of medium .and replug both tubes.
Pass the culture on to the next. After having ster-
ilized the needle, pass it also. Incubate the medium
tube for twenty-four hours at room temperature after
having' labeled the tube as follows: name, date and
medium. ,

Hanging-drop Preparation. — Take the hanging-
drop slide and make a preparation in the usual way
and examine the same. Note the following: shape,
size, motility and spore-formation.

Dry Preparation. — Make a dry preparation after the
usual manner, viz : Take the cover slip and clean the
same by the use of alcohol ; pass it three times through
the flame ; make a thin spread from the culture ; dry
in the air; pass over the flame until it is fixed, then
place the whole under the misroscope and examine.

Watery-Stain Preparation. — Make a spread in the
usual way and then apply the stain a few seconds ; and
wash in a glass of water; place under the microscope
and examine.

176 LABORATORY METHODS OF

Spore Stain. — Make a spread in the usual way and
stain for spores.

Flagella Stain. — Make the spread in the usual way
and stain for flagella.

Gram's Stain. — Place the spread on the cover in
the usual way ; apply the stain and note results.

This is what the reagents give us in regards to the
diagnosis of bacteria. It is in order to take up
the appearance of cultures of a studied germ. Inocu-
late all the media that you have and note the results
on the same. Inoculate them in all the known ways;
i. e., stick, stab, smear and slant inoculations.

Note all the changes that will occur in twenty-four
hours. This will help you very much in the diagnosis
of the germ in hand.

Distribute the Media: — Take three tubes of agar-
agar, and three tubes of gelatine and three of bouillon.
Inoculate one bouillon and two gelatine, one slant and
one stick in -the same way as the gelatine. Incubate
these for twenty-four hours. Note all the reactions
that occur in that time.

QUIZ.

What are bacteria?
Give their morphology.

BACTERIOLOGY. 177

Where are they found?

What are the media on which they grow best?

How do we get rid of them?

What is the best means of getting rid of spores ?

How hot must the medium be to kill all known
spores ?

What is the temperature to be obtained before you
can take out the glassware when you are sterilizing
it?

How do you use the hot-air sterilizer?

How do you lower the temperature in the hot-air
sterilizer ?

Are media sterilized in the hot-air sterilizer?

What are spores?

What are chromatic granules ?

What do you learn from the use of the hanging-
drop culture?

How did you make the cell for the culture ?
Did you find any bacilli, any cocci, any spirilla?
How did you use the moist-sterilizer?
Why do you use the discontinuous method?

What is meant by sterilizing?
12

178 LABORATORY METHODS OP

What is meant by pathogenic, saprophytic, chromo-
genic and pyogenic bacteria?

Are bacteria easily seen with the ordinary instru-
ment?

At what temperature do they grow best?
Are all bacteria disease producers?
Are all bacteria chromogenic?
How did you fold the filter for nitration?
What is the advantage in this over the old way?
What medium did you make?
Are the principles the same?

Take a large tube of water and sterilize over the
flame.

How can you tell when the tube is sterilized?
What is the temperature of boiling water ?
Are the spores destroyed in the water — if not, why
not?

If we use a steam boiler can we destroy the spores?
If so, why can we?

What is the name of the steam boiler that we use
in sterilizing cultures?

Name all the agents used in sterilizing, as a class.
Name two methods of using the dry heat.

BACTERIOLOGY. 179

What is the name of the rod and wire that you use
in inoculating the medium in your hanging-drop
culture ?

How hot do you make the wire?

What kind of heat did you use in sterilizing the
media?

What kind of heat do you use in sterilizing instru-
ments ?

Inoculation of Tubes. — Watch the demonstrator in-
oculate a tube of bouillon, then do the same. Incu-
bate it for twenty-four hours.

Make a stained spread of the material given you and
examine the same.

Determine the class to which the germs belong,
whether cocci or bacilli.

Consult the atlas for the answer to the query.

Review the lesson you had the last period ; note the
likeness or unlikeness to this lesson.

Are all bacteria pathogenic or not?

Note their color in a growth?

Make a stained preparation of the germs at your
table and examine.

Note in your notebook the points observed.

Determine the thermal death-point of streptococci
in bouillon.

180 LABORATORY METHODS OP

Method: Put some water in the water-bath; put
the tube of culture in it, and heat to 56 degrees C. ; let
it remain for fifteen minutes.

Inoculate a second tube from this one, and store in
your locker. Note the tube next day and see if there is
any growth in the tube. If not, the thermal death-point
is 56 degrees C. This method is the one we follow
at all times.

Learn the theory of reproduction of bacteria. Note
the color on the board which represents the stained
germ and the stained spore.

tfOOD SUPPLY.

The medium on which bacteria grows is termed
food. We are to determine the best food on which
they grow. The best ones are bouillon, agar-agar and
gelatin. In order to determine the reactions of the
medium after the germs have been growing on the
medium, it must be of a composition the same as that
of the tissues and juices of the body, or the same as
that of the dead organic bodies. This we attempt to
do when we make the different media. The properties
of media which must be observed are clearness 'and so-
lidity. The characteristic growth of the culture is ob-
served on solid media.

BACTERIOLOGY. 181

Name the processes you went through when you
made the stained preparation of the germs.

What is the best method to be followed when you
make a preparation of bacteria — the stain or the other
methods ?

What are the three best stains for ordinary use?

You may use the oil-immersion lens when you make
a good preparation.

Give the preparation of some of the stains in daily
use. . ,

Make a drawing to illustrate the morphology of
bacteria.

Describe in detail the method of making a dry
spread.

Watch the demonstrator make a plate of the air
germs.

Note the date and medium.

Note the temperature of the room when the plate is
made.

Give Tyndall's principle of intermittent sterilizing.

TYPHOID

Typhoid fever is an infectious disease caused by the
presence of the B. Typhosus. The B. was discovered

182 LABORATORY METHODS OP

by Ebert and Koch, in 1880, and was first secured in
pure culture from the spleen and affected lymphatic
glands by Gaffky, in 1884. — McFarland.

The organism is a short, small bacterium, I to 20
.microns in length and .5 to .8 microns in thickness.

The ends are rounded and sometimes they unite in
chains, especially in the potato growth.

The organisms are actively motile, the motility be-
ing due to the presence of a long flagellum. They
stain very well by Loffler's method, and they are used
to demonstrate the presence of flagella in the study of
bacteria in the laboratory. The organisms stain well
with the ordinary stains. They lose their stain with
Gram's stain.

The bacillus has no spore; the dark spot seen at
the end of the germ is a chromatic granule.

The bacterium is both a parasite and a saprophyte.
The bacterium is sometimes present in green vegeta-
bles which have been sprinkled with water contami-
nated with the organisms. This brings to mind a few
years ago when the city ordered that certain springs be
closed on account of the prevalence of the bacillus ty-
phosus, and at the same time prevented the marketmen
from sprinkling their vegetables with water from the
brooks on their way to town. Their reason for such
action is evident when we know that the organisms

BACTERIOLOGY. 183

thrive well in water. Their resistance to all germicides
has been spoken of already. They grow well at
room temperature, and boiling in water kills them,
since they are killed at the temperature of 60 degrees C.
This brings to mind another well-known fact, that the
people in certain large cities are requested to boil all
the drinking water before it is used. We know the rea-
son why such a course is taken when we know the
death-point of the germ. Cold has no effect on the
germ, and certain chemicals must be very strong be-
fore they will kill them. The best chemicals are bi-
chloride of mercury and carbolic acid. The bacilli are
killed in a short time by drying. So a hot-air steril-
izer will be all right for sterilizing the vessels used
in the work on this kind of bacteria.

The best method of obtaining the typhoid germs is
to take the animal after it is dead and get the germs
from the spleen and lymphatic gland; but you can get
them from the dejecta of the patient with difficulty.

The bacilli are sure to be present in the patient after
the second or third week. Right here is the place to
try Widal's Test.

The method of making a pure-culture from a pa-
tient's f eces is as follows : Take several tubes of gela-
tin and melt the gelatin ; have 10 c.c. of the medium in
each tube; now add to each tube i-io c.c. of carboi-

184 LABORATORY METHODS OF

ic acid. In the first tube put a loop full of the broken
up feces and take a loop full from this tube and trans-
fer to tube number two and so on. Next plate all the
tubes and number them. The carbolic acid kills all
the saprophytes and leaves only the bacillus typhosus
and its near congener, the bacillus coli. The gelatin
will not be liquefied.

For Widal's reaction read McFarland.

Serum diagnosis has been attempted. This is on
the principle of attenuation of germs by the presence
of another species.

The Bacterium Pyocyanus was the antitoxic growth
used. This is on the principle of attenuation of germ
by the presence of another species. This as yet has
not come into use.

Make a spread of bacillus anthracis.

What is the morphology of the same?

Are they often found in man?

Give the approximate diameter of the germ and the
length in microns.

Do you observe the morphology of the germs and
spores in them,

Biology.— The bacilli grow well on all the ordi-
nary media— best grown at 35 degrees C. ; death-point,
60 degrees C. They withstand dry heat well, and

BACTERIOLOGY. 185

must be heated up to a very high degree before they
are destroyed. Spores are of the usual variety. The
action of the gastric juice kills them. They effect ani-
mals lower than man ; therefore, they are a disease for
the veterinary surgeon to study. The best method of
getting rid of the dead animal is to burn it or bury it
under a pile of slacked lime several inches deep.

Examine the tap water for bacteria by making the
plate cultures in the usual way, as follows: Take the
gelatin and melt it and pour it into the Petri dish;
then take i c.c. of water from the hydrant and pour
it into the dish on the medium, and note results in a
few days.

Plate the pus in the vessel, and examine the same
on the slide after staining.

Make a stained preparation of the urine on the
table.

Tell to what class the bacteria belong, i. e., if they
are cocci or bacilli.

Some stain with blue and others with red.
Make a study of the bacillus of hog-plague.
Make the usual cover preparation; stain with fuch-
sin; make drawings of the same.

Examine bacillus prodigiosus; note the color of the
colonies.

186 LABORATORY METHODS OP

Use the colony counter and make calculation on the

results.

Make a note of the method you would follow in
making a bacteriologic diagnosis, stating the points
that you would observe in each step.

Make a spread of bacillus tuberculosis and stain
with fuchsin and methyline blue — the first, thirty min-
utes ; the second, one-half minute. Examine under the
oil lens.

Make a spread of spirillum cholera and stain with
fuchsin.

Tell how you would determine the following points
in the study of a certain germ.
First, the pathogenesis or non-pathogenesis.
Second, the chromogenesis.
Third, the motility.
Fourth, the morphology.

Fifth, the reaction to stains, and Gram's method.
Sixth, the reaction on gelatin.
Seventh, the reaction on all media.
Eighth, the source.

Ninth, the germicide for the germs.
Tenth, the thermal death-point for the germ.
Eleventh, the best temperature for its growth.

BACTERIOLOGY. 1 87

How do you prevent your mind from letting slip

the points observed?

Make stained spread from the scrapings from your
mouth; note the morphology.

Determine the death-point of streptococci as a
class, after the following manner : Put a number oi;
t. t. in an incubator and heat the water to 56 degrees
C. and let remain fifteen minutes. Inoculate a tube of
agar and place it in your locker until next period, and
note if there is a growth or not on the medium. If
there is, the germ or organism is not killed at the tem-
perature of 56 degrees C.

Study Streptococcus' and Sarcina. Note the effects
on bouillon, and on agar-agar, on gelatin, and on pota-
to. (See Sternberg on Streptococcus.)

See McFarland on biology of bacteria and note the
points of interest to you as a bacteriologist. See what
he says about the appearance of the colonies on the
different media; note the similarity between his de-
scription and your notes. Note the appearance of the
colonies on a gelatin plate.

Note the color of the growth of bacillus prodigio-
sus, sarcina luteus, staphylococcus-pyogenes.

Make a plate of gelatin and inoculate it with a drop
of water to see if there are any bacteria in the water.

Study spore formation of bacteria.

188 LABORATORY METHODS OF

Study suppurative diseases, their causes and cure.

Study the cause of gonorrhoea; the method of mak-
ing the spread for the discovery of the cocci.

Study mixed infection.

Make a cover spread of the pus on the table and
classify the germs in it.

INFLUENZA.

The disease Influenza is a widespread disease and
is more prevalent in winter than in any other time.
It is caused by the presence of a certain bacillus called
Bacillus Influenzae. They are short rods 1.5 microns in
length and .3 microns in breadth. The bacilli sometimes
occur in chains and sometimes in bunches. They lose
the stain by Gram's method. They are non-motile
and do not form spores. They are best stained with
carbol fuchsin. They grow best on blood agar and
can be grown best on the agar by smearing the me-
dium with the sputum of the patient. The lower an-
imals' blood is as good as that of the human subject.
The bacilli grow well at room temperature ; their opti-
mum temperature is that of the body. The power of
resistance of this organism is of a low order. The
mode of distribution of this bacillus is by direct con-
tact with the mucus and pus of the person affected
with influenza. The location in the body is unusally

BACTERIOLOGY. 189

in the respiratory organs. They are always mixed
with other organisms. The purest cultures can be se-
cured from the greenish yellow-pus that may be col-
lected from the person so affected. As the disease
advances, we may be able to find the germs in the
leucocytes of the blood. It is a peculiar fact that the
bacilli remain in the sputum for a while after the dis-
ease is cured.

Method of Making the Examination. — Take some
of the greenish yellow pus and make the usual cover-
glass preparation, and stain the same with carbol-
fuchsin.

Give the use of the agglutinating test of Widal.

What is the thermal death-point of bacillus typho-
sus?

Watch the demonstrator use the anaerobic culture
tube.

Widal's Test is as follows: Take one drop of the
blood of the patient and mix it with nine drops of
water; place one of these drops on a cover-glass;
make ready a hanging-drop slide; now transfer one
drop of a culture of bacillus typhosus and examine
at once. If an agglutination occurs within fifteen
minutes the patient has typhoid fever.

Make a spread of bacillus anthracis and examine
it, using the oil lens. Study spore- formation from this
bacillus.

190 LABORATORY METHODS OF

What is the theory of immunity ?

Express your opinion on the subject.

Give the theory of phagocytosis.

Demonstrate the anaerobic culture tube.

Experiments on Animals. — These are for the pur-
pose of demonstrating the pathogenesis of the germ.
Take the animal and put it in Voge's Holder, head
downward. Take an antiseptic solution, sterilize the
part selected for the injection, and at the same time
sterilize your syringe in hot water. Now remove the lid
of your Petri dish and draw the required amount of
growth in the syringe. (The syringe is graduated in
c.c. and i-io of c.c., so that you can get a definite
amount.) Inject the material in the animal and put
the animal in the cage until next period. Examine the
animal and see if it shows signs of disease.

Taking the Temperature of an Animal. — Take the
animal and place it in the Vogue, head downward ; put
the thermometer in the rectum,, and note degree.

Make a spread of the material on the table and stain
with fuchsin.

Bacillus Pyocyaneus is a short rod. It is three mi-
crons in length and has one flagellum; does not form
spores; frequently forms chains of four or six;
can exist with or without oxygen. It -stains well with

BACTERIOLOGY. 191

the ordinary stains and will not retain Gram's stain.
It is the cause of green pus in a wound. It produces
the green color to the pus of old sores.

Stain a spread of bacillus Flouresence Liq., and
note the likeness of the two.

Stain the dejecta at your desk and make out the
morphology of the species, and try to differentiate the
colon bacilli from the typhoid bacilli, if such are pres-
ent. Consult the atlas and find the germs which are
most like 'the ones on the slide.

What lesson of practical value can you get from this
experiment ?

How do you determine the power of any germicide ?
Note the process in your notebook.

How would you get the material to the city bacteri-
ologists ?

Name five diseases which you must report to the
city authorities.

How would you make an examination of milk in
case you were called on to do so?

What bacteria are most likely to be in the milk?

REAGENTS
AND STAINS

From OSBURN'S MANUAL.

13

TECHNIQ REAGENTS AND STAINS.

VEGETABLE SECTION.

NO. I. METHOD FOR STAINING FRESH VEGETABLE SEC-
TIONS.

(1) Apply section to slide and add rosanilin violet,
one to five minutes.

(2) Wash in water to remove excess of stain.

(3) Dry with blotting paper and add glycerine to
dehydrate.

(4) Remove excess of glycerine and add glycerine
again to thoroughly dehydrate.

(5) Wipe off excess of glycerine and add xylol
twice.

(6) Apply to cover-glass a drop or two of xylol-
balsam, and, having wiped off the excess of xylol from
the slide, drop it gently (balsam down) upon the sec-
tion. Then apply general pressure with dissecting
needle to spread out the balsam.

(7) Label and keep in a horizontal position until
the balsam is hardened.

(195)

196 TECHNIC,

Vegetable Sections. — To stain paraffin or celloidin
sections of plant structures, the methods are practically
the same as those given below for animal sections.

ANIMAL SECTIONS.

NO. 2. CARMINE METHOD FOR FREE SECTIONS.

1 I ) Apply section to slide and wash with thirty-five
per cent alcohol.

(2) Add lithium carmine sufficient to cover section,
one to five minutes.

(3) If necessary, remove excess of stain with acid
alcohol, five to ten seconds.

(4) Dehydrate with increasing strength of alcohol
— thirty-five per cent, seventy-five per cent, and ninety-
five per cent, and absolute.

(5) Wipe off excess of alcohol, and when section is
partly dry add creosote to clear up, five to ten minutes.

(6) Wipe off excess of creosote and mount with
balsam.

(7) Centre cover-glass and apply pressure to spread
out the balsam.

(8) Label and lay aside in horizontal position,
cover-glass up, until the balsam hardens.

REAGENTS AND STAINS. 197

NO. 3. CARMINE) METHOD WITH AFFIXED PARAFFIN
SECTIONS.

1 I ) Apply to the center of the slide a thin layer of
collodion-clove-oil mixture.

(2) Center and attach the section, applying the heat
of a spirit or Bunsen flame.

(3) Immerse in xylol two minutes and in turpen-
tine ten minutes to remove paraffin. Sections im-
mersed in turpentine alone should remain twenty min-
utes.

(4) Wash with alcohol, decreasing strengths, using
thirty-five per cent alcohol last.

(5) Apply lithium carmine, one to ten minutes.

(6) Remove excess of stain with acid alcohol.

(7) Dehydrate with alcohol, increasing strengths.

(8) Dry and clear up with creosote, five to ten
minutes.

(9) Wipe of excess of creosote and mount in bal-
sam.

(10) Center cover-glass.

( 1 1 ) Label and lay aside in horizontal position until
balsam hardens.

198 TECHNIC,

NO. 4. CARMINE METHOD FOR AFFIXED CEUXDIDIN SEC-
TIONS.

(1) Center section and affix with collodion mix-
ture.

(2) Stain with lithium carmine, one to five minutes.

(3) Remove excess of stain with acid alcohol, five
or ten seconds.

(4) Apply seventy per cent alcohol.

(5) Apply eighty per cent alcohol.

(6) Apply ninety-five per cent alcohol a few sec-
onds.

(7) Clear up with creosote.

(8) Remove excess of creosote with blotting paper.

(9) Mount with balsam and center cover-glass.

(10) Label and lay aside in a horizontal position.

NO. 5. HAEMATOXYUN METHOD -FOR FREE SECTIONS.

1 I ) With section on slide, apply alcohol of the same
strength as staining solution.

(2) Stain with diluted hsematoxylin, one to ten
minutes.

REAGENTS AND STAINS. 199

(3) Remove excess of stain with thirty-five per
cent alcohol.

(4) Dehydrate with alcohols, increasing strengths.

(5) Clear up with creosote or cedar oil.

(6) Center section and apply balsam and cover-
glass.

(7) Center cover-glass.

(8) Label and lay aside in horizontal position until
balsam hardens.

NO. 6. HAEMATOXYUN METHOD FOR AFFIXED SECTIONS.

(1) Apply to slide a thin layer of egg-albumen and

glycerine.

(2) Center section and flatten it by gently touching
with end of finger.

(3) Apply heat of flame until paraffin melts (sec-
tions that have been flattened upon water should be
heated much longer than others).

(4) Remove paraffin with xylol or turpentine.

(5) Remove xylol, etc., with alcohol, decreasing
strengths.

(6) Stain with diluted hsematoxylin, one to ten
minutes.

200 TECHNIC,

(7) Remove excess of stain with thirty-five per cent
alcohol.

(8) Dehydrate with alcohol.

(9) Clear up with creosote, five to ten minutes.

(10) Wipe off excess of creosote and mount with
balsam.

(u) Center cover-glass.

(12) Label and keep in horizontal position until
balsam hardens.

NO. 7. HAEMATOXYLIN METHOD FOR AFFIXED
DIN SECTIONS.

( I ) Center section and affix with collodion mixture.

(2) Stain with diluted hsematoxylin, one to ten min-
utes.

(3) Remove excess of stain with acid alcohol.

(4) Apply seventy per cent alcohol.

(5) Apply eighty per cent alcohol.

(6) Apply ninety-five per cent alcohol a few sec-
onds.

(7) Clear up with creosote, five to ten minutes.

(8) Remove excess of creosote with blotting paper.

(9) Mount with balsam and center cover-glass.

REAGENTS AND STAINS. 201

(10) Label and lay aside in a horizontal position

until balsam is hardened.

/

NO. 8. HA£MATOXYUN-£OSIN METHOD.

(1) If desired, affix section to slide with egg-albu-
men and glycerine or collodion and clove-oil.

(2) If a paraffin -section, remove paraffin with xylol
or turpentine or both ; remove xylol, etc., with alcohol.

(3) Apply thirty-five per cent alcohol.

(4) Stain with diluted hsematoxylin, one to five
minutes.

(5) Apply thirty-five per cent alcohol to remove ex-
cess of stain.

(6) Stain with alcoholic eosin about five minutes.

(7) Apply ninety-five per cent alcohol to remove ex-
cess of stain and dehydrate.

(8) Clear up with cresote.

(9) Remove excess of creosote and mount with bal-
sam.

(10) Center cover-glass and label.

(n) Lay aside in horizontal position until balsam
hardens.

Staining Unicellular Organisms.
It is often desirable to examine materials \\ithout
staining. This is accomplished by placing upon the

202 TECHNIC,

glass slip a drop of the material tc be examined and
applying cover-glass. A hair placed under the cover
glass will prevent the object from being crushed and
allow of free motion in the case of living organisms.
Should it be desired to stain such preparations, two
methods may be pursued, irrigation and cover-glass
staining.

NO. 9. IRRIGATION AND STAINING MICRO-ORGANISMS.

(1) Place upon the slide a drop of material to be
studied.

(2) Apply cover-glass.

(3) At the edge of the cover-glass, by means of a
pipette, place a drop or two of the reagents or stain.

(4) By means of a triangular piece of blotting paper
applied at the opposite edge of the cover-glass, absorb
the moisture from the preparation, thus drawing under
the stain.

NO. 10. COV£R-GI,ASS PREPARATIONS.

(1) Make a thin spread of the substance to be ex-
amined upon a sterilized cover-glass.

(2) Using a Cornet forceps, dry the preparation by
holding it between the fingers above the flame.

REAGENTS AND STAINS. 203

(3) When dry pass the cover-glass three times
through a flame, keeping the preparation up.

(4) Apply stain.

(5) Wash in distilled water by dipping the covier-

glass in the water two or three times.

(6) Examine as a water mount or, if desired, dry
and mount in balsam.

(7) Label and lay aside in a horizontal position until
balsam hardens.

Note. — The above method may be used for all sim-
ple staining. Special methods, however, are often used*
and they will be given as required.

LABORATORY EXERCISE;. — Centering and labeling.
Upon the under side of your box-cover make an
outline of a slide. The pencil should have a needle
point. Connect opposite angles and place over the in-
tersection of the lines a cover-glass. Be sure that the
centre of the cover-glass coincides with the center of
the diagram. Now, carefully make an outline of the
cover-glass. This outline may be used for centering
both the sections and the cover-glass. Make a draw-
ing of this outline and also a drawing of a
slide with labels and cover-glass in situ. Fill in the
forms of labels in second diagram, using the following
data : A transverse section of the muscle of a normal

204 TECHNIC,

cat was stained with lithium carmine and mounted in
balsam in October i, 1891, by John Smith.

Drawings. For this work the student should pro-
vide himself with a No. 5 or a No. 6 H Faber pencil,
a small rule or triangle and a sheet of thin blotting
paper. The pencil should be kept sharpened to a
needle point. The majority of students will say: I
cannot draw. An honest and faithful effort will often
produce gratifying results. Let every line mean some-
thing. Be scrupulously neat in all your work. Re-
member that this work will furnish a better exhibit of
character and ability than any other task of the labora-
tory.

Abbreviations. The following abbreviations are em-
ployed in this text:

Transverse section — T. S.
Longitudinal section — L. S.
Vertical section— V. S.
Low power — L. P.
High power — H. P.
Cubic centimeter — c. c.
Micro-millimeter — u.
Millimeter — mm.
Grami — g.

REAGENTS AND STAINS. 205

REAGENTS AND STAINS.

In preparing the following regeants it is well to re-
member that the weight of a cubic centimeter of water
is one gram, and that a liter contains 1,000 cubic centi-
meters. The formulae that are given are those most
commonly used and are briefly stated :

NORMAL, FLUIDS.

Distilled imter.- — A supply of distilled water should
be constantly at hand for the preparation of the re-
agents and stains.

Normal saline. — This is prepared by dissolving one
part, by weight, of sodium chloride in 150 parts of dis-
tilled water.

MACERATING FLUIDS.

Dilute alcohol. — This may be prepared by mixing
one part of ninety-five per cent alcohol with two parts
of distilled water. Other fluids used for this purpose
are solutions of potassium bi-chromate, two per cent,
and caustic potash, twenty-five per cent.

DECALCIFYING FLUIDS.

Picric acid. — Make a saturated aqueous solution of
picric acid. This is an excellent fluid for decalcifying

206 TECHNIC,

bones, serving at the same time as a staining reagent.
Crystals should be added from time to time, so that
some undissolved crystals will always remain in the
bottom of the vessels.

Nitric acid. — Use a ten per cent volumetric solution
in water. Decalcifkation occurs in five to ten days.

FIXING REAGENTS.

Absolute alcohol. — Specimens should remain in this
reagent from one to six hours, according to size.

Perenyi's fluid —

Nitric acid (ten per cent) 40 cc.

Chromic acid (0.5 per cent) 30 cc.

Alcohol 30 cc.

A good reagent for embryos and adult tissues. Time,
three to twelve hours ; dehydrate with alcohol.

Erlicki's fluid- —

Potassium bi-chromate 2.5 grams.

Cupric sulphate 0.5 grams.

Water 100 cc.

Aqueous Solution —

Corrosive Sublimate —

Corrosive sublimate I gram.

Water 95 cc.

REAGENTS AND STAINS. 207

Alcoholic Solution —

Corrosive sublimate I gram.

Alcohol (95 per cent) 99 cc.

Used for special purposes and specially foi alimen-
tary tract. Time, twenty-four hours, hardening in al-
cohol, to which a few crystals of iodine have been
added.

Chromic acid. — Use a 0.5 per cent solution, dehy-
drating with alcohol in the dark.

Muller's fluid—

Potassium bi-chromate .... 25 grams.

Sodium sulphate 10 grams.

Water 1,000 cc.

Pulverize the solids before adding water, and use a
piece of camphor in the solution to prevent the forma-
tion of fungi. Good for general use and especially val-
uable for central nervous system. Requires from two
to six weeks. Wash in water for several days and de-
hydrate with alcohol.

Plemming's fluid —

Chromic acid (one per cent solution).
46 cc.

208 TECHNIC,

Osmic acid (two per cent solution)

12 CC.

Glacial acetic acid 3 cc.

Especially valuable for delicate tissues. Time,
twenty-four hours ; dehydrate with alcohol.

HARDENING REAGENTS.

Midler's fluid, corrosive sublimate solution, chro-
mic acid, and others of the reagents named above may
be used for hardening purposes. For general use, iai/-
cohol will be found invaluable. The tissue should be
passed through increasing strengths of alcohol, seventy
per cent, eighty per cent, ninety per cent ninety-five
per cent, and absolute. It should be allowed to re-
main twenty- four hours in each, except that one to six
hours will suffice for absolute alcohol. Ethyl alcohol
should be used, or, in lieu of this, methyl alcohol
makes a good substitute. To prepare absolute alcohol,
dehydrated copper sulphate may be added to ethyl or
methyl alcohol. This will absorb the water present.

EMBEDDING MEDIA.

Paraffin and celloidin are extensively used for em-
bedding tissue.

REAGENTS AND STAINS. 209

FIXATIVES.

Collodion and clove oil mixture. — Mix one part of
collodion with three parts clove-oil.

Egg-ulbumen and glycerine. — Filter the whites of
several eggs and add to the filtrate an equal volume of
glycerine. To the mixture add a few drops of carbolic
acid or a small piece of thymol to prevent putrefaction.

PARAFFIN SOLVENTS.

Xylol, turpentine, chloroform, and benzole are com-
monly used to remove paraffin from sections. A gooci
plan is to immerse the slide containing the section for a
few moments in xylol, and then transfer to turpen-
tine for ten minutes.

STAINING SOLUTIONS.

The following staining preparations are those most
frequently used, and will be found adequate to the
work required in this text. Should others be needed,
the formulae can be obtained from more advanced
works.

Hanstein's rosanilin violet —

Methyl violet I gram.

Fuchsin I gram.

Distilled water 100 cc.

14

210 TECHNIC,

Note. — This is a valuable stain for vegetable sec-
tions. It should be diluted for use as desired.

Lithium carmine —

Carmine 2.5 grams.

Lithium carbonate (saturated

solution) 100 cc.

The carmine should be dissolved in cold solution.
Sections stain rapidly, and. should be decolorized with
acid alcohol.

D ala-field's haeinatoxylin —

1. Hsematoxylin I gram.

2. Absolute alcohol 6 cc.

3. Amonium alum (saturated

sol.) 6 cc.

4. Glycerine 25 cc.

Methyl alcohol . . ; 25 cc.

Process. — Dissolve (i) in (2); add this solution to

(3) ; expose to air and light for a week; filter and add

(4) and (5) ; allow it to stand for a long time exposed
to air and light.

Alcoholic Eosin for Sections. — Make a saturated al-
coholic solution. This is used as a ground stain in
connection with hsematoxylin ; also as a blood stain.

REAGENTS AND STAINS. 211

Magenta for blood, etc. —

Magenta i gram

•Alcohol (eighty-five per cent). 50 cc.

Water 150 cc.

Glycerine 200 cc.

Meythelene blue for blood —
Make a saturated aqueous solution.
Carter's carmine mass for injecting —

Carmine 3 grams

Strong ammonia 6 cc.

Glacial acetic acid 6 cc.

Coignet's French gelatin ... 7 grams
Water 80 cc.

Process. — "Place the finely cut gelatine in 50 cc. of
water for five hours ; dissolve the carmine in mortar
with a little water and add the ammonia; let stand for
two hours and then pour into a bottle, rinsing the mor-
tar with the remainder of water ; place the gelatin and
water on a water-bath until the gelatin melts. To the
carmine fluid add the acetic acid, a few drops at a
time (rinsing mortar thoroughly) until the fluid be-
comes crimson. To the melted gelatin add the crim-

212 REAGENTS AND STAINS.

son carmine, little by little, with continual stirring.
Keep in a cool place with surface covered with meth-
ylated spirit. When wanted for use, dissolve on water-
bath and filter through flannel wrung1 out of hot
water." (Fearnley's Method.)

CLEARING AGENTS.

Those commonly used are cedar oil, creosote, clove-
oil, xylol, and aniline oil. Clove oil cannot be used
with celloidin sections.

MOUNTING MEDIA.

Glycerine jelly and Canada balsam, are commonly
used for mounting purposes. For the laboratory, bal-
sam will be found a satisfactory medium. Should
xylol be used for clearing, the balsam should be dis-
solved in xylol. Chloroform balsam may be used in
in xylol. Chloroform balsam may be used in sections

For the formulae of reagents and stains required for
work in bacteriology and urinalysis, the reader is re-
ferred to the chapters in which is discussed the micro-
technique of these subjects.

MICROSCOPIC TECHNIQUE.

I. REAGENTS AND STAINS.

(l) DECOLORIZING SOLUTIONS.

Twenty-five per cent aqueous solutions of hydro-
chloric, nitric, and sulphuric acids may be used for de-
colorizing.

(2) ACID ALCOHOL.

Hydrochloric acid I part

Alcohol (seventy per cent) 100 parts

(3) IODINE SOLUTION.

Iodine I gram

Potassium iodide 2 grams

Water 90 cc.

(4) CARBOL tfUCHSIN.

Fuchsin I cc.

Alcohol 10 cc.

Dissolve and add 100 c.c. of five per cent solution of
carbolic acid. Filter.

(213)

214 TECHNIC,

(5) ACID METHYLENE BLUE.

Sulphuric acid 16 cc.

Water 90 cc.

Methylene blue 2 grams

This stain should be prepared fresh from time to
time. The carbol fuchsin improves with age.

(6) LOFFLER'S ALKALINE METHYLENE BLUE.

Concentrated alcoholic solution of
methylene blue 30 c.

Potassium hydrate (aqueous solu-
tion i-ioooo) : 100 cc.

This is especially useful in staining the bacillus of
diptheria.

(7) ANILINE-WATER GENTIAN VIOLET.

Aniline oil 5 cc.

Water 100 cc.

Mix, shake vigorously, filter; the fluid after filtra-
tion should be perfectly clear; add

Alcohol 10 cc.

Alcoholic solution of gentian violet, n cc.

This solution should be freshly prepared about every
two weeks.

REAGENTS AND STAINS. 215

(8) LQFFLER'S MORDANT FOR FLAGELLA.

Tannic acid 2 grams.

Water 8 cc.

Saturated solution of ferrous sul-
phate 5 cc.

Saturated alcoholic solution of
fuchsin I cc.

(9) ANILINE-WATER DYE FOR STAINING SPORES.

Saturated alcoholic solution of
fuchsin or gentian violet .... 1 1 parts

Aniline oil water 100 parts

Abs. alcohol 10 parts

Keeps well for ten days.

(lO) AQUEOUS STAINS.

Saturated aqueous solutions of fuchsin, gentian vio-
let, and methyline blue will be found useful for all
simple staining.

(ll) ALCOHOLIC SOLUTIONS.

Saturated alcoholic solutions of fuchsin, gentian-
violet, and methyline blue should be kept on hand to

216 TECHNIC,

be used in simple staining and in connection with
other stains.

II. STAINING METHODS.

(1) SIMPLE STAINING.

This consists in using a single stain.

(2) DOUBLE STAINING.

This consists in using two stains, one to stain spores,
protoplasm, etc., and the other as a ground stain. The
following methods will illustrate double staining:

STAINING OF SPORES.

(a) Make a cover-glass spread, dry and pass three
times through the flame.

(b) Add aniline- water gentian -violet.

(c) Heat until the preparation begins to boil; re-
move for a minute. Repeat this process six times.

(d) Wash in three per cent hydrochloric acid-alcohol
one minute.

(e) Wash in water.

REAGENTS AND STAINS. 217

(f) Counter- stain with aqueous methylene blue half
a minute.

(g), Wash in water, .

(h) Dry and clear up with xylol.

(i) Mount in balsam.

STAINING, OF

(a) Mix upon the cover-glass a portion of the cul-
ture with a drop of water, using care not to break off
the delicate flagella.

(b) Dry and pass three times through a flame.

(c) Apply Loffler's mordant one minute, warming
gently.

(d) Wash in water.

(e) Stain with aniline-water fuchsin.

(f ) Wash- in water. ,

(g) Dry and mount in balsam.

GRAM'S METHOD FOR BACTERIA.

(a) Make a cover-glass preparation by the usual
method.

(b) Stain with aniline- water gentian-violet solu-
tion, two to five minutes, warming slightly.

(c) Add Gram's iodine solution one and one-half
minutes.

218 TECHNIC,

(d) Apply alcohol, repeatedly, long as stain contin-
ues to come away from the preparation.

(e) Wash in water and examine as a water mount.

(f) If desired, dry and mount in balsam.

GABBETT'S METHOD FOR TUBERCULOSIS, ETC.

(a) Make a cover-glass smear of the sputum, pus,
blood, or urine to be examined. After the preparation
is dry, affix by passing three times through the flame.

(b) Using a Cornet forceps, apply carbol-fuchsin
five to ten minutes, heating until steam appears.

(c) Wash in water.

(d) Apply acid methylene blue for one minute.

(e) Wash in water.

(f) Dry and mount in balsam.

Staining Tissues for Bacteria.

Tissues may be stained in Gram's method by the fol-
lowing process :

METHOD FOR STAINING BACTERIA IN SECTIONS.
IN SECTIONS.

(a) Using an aqueous solution of fuchsin, gentian-
violet or methylene blue; apply stain for about five
minutes.

(b) Wash in water.

REAGENTS AND STAINS. 219

(c) Apply an aqueous solution of acetic acid, one
per cent, for one minute.

(d) Apply alcohol two minutes.

(e) Clear up with xylol.

(f) Mount with balsam.

INDEX.

Abbe's condenser, 15, 17
Accessories to the micro-
scope, 15
Acetic acid action on cells,

21, 53, 54
Acids stains, see technic and

stains

Adenoid tissue, 66
Adipose tissue, 41
Alcohol as a hardening agent,

29

Alveolar ducts, 121
Alveolus, 121
Amoeba, 26

Amoeboid movement, 53
Analyzer, 17
Animalcule, 23
Anterior chamber of the eye,

124
Anterior column of cord,

77

Anterior cornua, 75
Apical foramen, 94
Aqueous humor, 125
Arachnoid, 77
Areas of the cord, 76
Arrectores muscle of the

hair, 89
Arteries, 61

coats of, 63
Axis cylinder, 73
Axilemma, 73
Animal autopsies on, 174

inoculation, 170, 175
Anthrax, 160
Antiseptics, 170

Attenuation of germs,

Bacilli, 182
Bacteria, 159, 177

agglutination test, 183, 189

anaerobes, 190

biology of, 184

classification of 159

cultivation of, 165

death of 169

food supply of, 159

microscopic examination of,
173

movement of, 164

parasitic, 165

saprophytic bacteria, 165

spores of, 176

temperature of growth, 160,

162

Bacteriological diagnosis. 186
Bacteriology, 159
Bartholin's glands, 119
Basement membrane, 96
Basophiles, 53
Bellin's tube, 110
Bile capillaries, 104

duct, 104
Bladder, 113
Blood tissue, 49

platelets, 49

corpuscles, 49

plasma, 49

shadows, 53

origin of, 53, 55

crystals, 53
Bone, 45

(221)

222

INDEX.

canaliculi, 46

cells, 46

development of, 47

blood vessels, 47
Bronchi, 120
Bronchiole, 120
Brownian movement, 32
Brunner's glands, 99
Budding, 18
Bulb hairs, 90
Bacteria, 159
Bouillon, 179

Camera lucida, 15
Capillaries, 61

bile, 104

lymph, 65
Capsule of Glisson, 102

of glands, 105

of lymph glands, 66
Cardiac muscle-cells, 58
Carmine stain, see chaptei

on stains and reagents
Cartilage, 43

capsule of, 44

fibro-elastic, 45

hyaline, 44

lacunae of, 44

matrix of, 44
Cells, 18, 19

blood cells, 49

bone, 46

cartilage, 44

of stomach, 96

ciliated, 36

connective-tissue

containing pigment, 86

of cerebellum, 82

double staining of blood,
55

eosinophile, 53

epithelial, 35

fat, 41

fixing of, 30

ganglion, 73

germinal, 117

gland, 70

goblet, 98, 101

muscle, 56

nerve, 72

neuroglia, 72

of Purkinje, 83

olfactory, 123

polynuclear, 53

pyramidal large, 82

small, 82

spindle, 81
Cell division direct, 29

indirect, 29
Cellodin, see the chapter on

technic

Cementum, 93
Centrosomes, 28
Chondrin, 44
Choroid, 124
Ciliated cells, 36
Circulatory system, 61
Circumvallate papillae, 127
Clitoris, 119

Coil-glands of the skin, 87
Commissures, 76, 78
Compound microscope, 11
Condenser, 15
Connective tissue, 38
Convoluted tubule, 114
Corium, 89
Cornea, 124
Corpus luteum, 118
Cortical layer of hair, 88

nodule of lymphatic gland,
100

substance of kidney, 10
Cover-glass, 16
Cowper's gland, 114
Crystalline lens, 125
Cutical, 85
Cutis, 85

Carbol-fuchsin, 188
Cholera, 186
Cocci, 159

Colony counting, 168
Culture media, 160

INDEX.

223

hanging-drop, 167, 175
incubation of,
plate, 174
shake, 174

Decalcifying fluid, see technic
Delafield's haematoxylin,
Dendrites, 72
Dentine, 93
Dentinal bulbs, 95
Diaster, 29

Direct cell-division, 29
Discus proligerous, 118
Dura mater, 77

Ear, 125
Ectoderm, 34
Elastic fibers, 45
Embedding media,
Enamel, 93
Endocardium, 61
Endomysium, 58
Emd-organ, 56
Epimysium, 58
Epithelium, 35

cut of, 35
Esophagus, 95
Exoplasm, 36
Eye, 124

Fallopian tubes, 118

Fat cells, 41

Female generative organs, 16,

17, 18, 19

Fenestrated membrane 63
Fibro-cartilage, 45
Filiform papillae, 127
Flagella, see cilia
Fungiform papillae, 127
Flagella, 176, 182

Ganglia, 73

Genital organs of male, 114
Germ center of lymphoid tis-
sue, 68
Germinal cells of ovary, 118

spot, 118

vesicle, 118
Gonococcus, 188
Gram's method, 172, 176
Glands, type of, 70

structure of, 71

submaxillary, 106
Glisson's capsule, 102
Glomerulus, 110
Goll's column, 77
Gower's haemacytometer, 50
Graafian follicle, 118
Gray matter, 77

Hair, 87

shedding of, 90
Hassal's corpuscles, 69
Haversian canal, 46
Heart, 61
Haematin, 54
Haematoidin, 54
Haemin, 54
Haemoglobin, 54
Hilum of lymph glands, 68
Histology, general, 20, 26
Hyaline cartilage, 44

Immersion lens,

Incubate, 168

Immunity, 190

Indifferent fluid,
salt solution, 22

Indirect cell-division, 29

Infiltration,

Infiltrated, 30

Influenza, 188

Injection fluids
gelatin-carmin

Inoculation of animals, 174

Intermoleciilar layer of ret-
ina, 125

Interglobular spaces, 93

Interglobular artery, 103
vein, 103

Intestines, 97
large, 101

224

INDEX.

mucous membrane of, see

the stomach
nerve supply, 100
small, 97
technic, 100
Intralobular artery of kidney

112

vein of, 112
Iris diaphragm, 15

Kidney, 109

arched collecting portion

of tubule, 112
blood vessels of, 110
cortex of, 109
distal convoluted tubule of,

112

medullary substance of, 110
straight collecting tubule,

112

Knife of microtome, 77
Krause's membrane, 58

Lacunae of bone, 46
Lamellae of, 46
Large intestines, 101
Lateral columns of the cord,

77

Layer of Henle, 88
Leucocytes, see lymphocytes
Lens, 15

Abbe, 15

of the eye, 125
Lieberkuhn's glands, 99
Ligaments of the eye, 125
Lingual mucous membrane,
126

papillae, 127
Liquor folliculi,
Liver, 102

vascular system of, 103
Lobules of liver, 102
Loop of Henle, 112
Lungs, 120

lymphatics of, 121
Lymph glands, 67

capillaries, 65

follicles, 66

of the tongue, 126

solitary glands, 100
Lymphocytes, 53
Lymphoid tissue
Lymph-vessels, 65

Male generative organs, 114
Malpighian corpuscles, 68
Mammary gland, 90
Matrix; 44

of bone, 46

of nail, 87

Mediastinum testis, 115
Medullary sheath, 77
Medullated nerve fibers, 74
Membrana propria,
Mesoderm, 34

Microscope and its accesso-
ries, 11

coarse adjustment of, 12

compound, 11

description of, 11

fine adjustment, 12

parts of, 11

simple, 11

study of, 12
Microtome, 16

student's 18
Milk, 91

Mitosis, see indirect cell-di-
vision

Mixed glands, 105
Molecular movement of cells,

see Brownian movement
Mononuclear cells, 53
Morula, 33
i Mount,

| Mouth glands of, 107
! Mucous glands, 105
Muscle cell, 56

cardiac, 58

heart, 58

smooth, see involuntary, 58

striped, see voluntary, 56

INDEX.

225

fiber, 56
tissue, 56

Muscularis mucosae of the in-
testines, 98

Myocardium, 61

Nails, 87

bed, 87

body, 87

matrix, 87
Nerves, 72

end-organs, 56

cells of, 72

fibers, 73

medullated, 74

non-medullated, 74
Neurilemma, 73
Neuroglia cells, 72
Neurone, 72
Nitric acid,
Nodes of Ranvler, 73
Nodules,

lymph, 66
Nuclear membrane, 28

stain

Neucleolus, 28
Nucleus, 28

Objectives, 12
Odontoblasts, 95
Olfactory tract, 123
Ossification, 47
Ovary, 117

germinal epithelial layer,
117

medullar and cortical sub-
stance, 117

blood vessels of, 118
Ovum, 118

Pacchionian bodies, 77
Pancreas, 108
Papillae, 127

circumvallate, 127

filiform, 127

fungiform, 127
Paraffin,
15

Parotid gland, 106

Penis, 116

Pericardium, 61

Perichondrium, 44

Perimysium, 58

Periosteum, 47

Pia mater, 77

Picro carmin as a stain, see
technic

Pigment layer of the eye, 12b

Plural staining, see double in
the technic

Portal vein, 103

Primitive jaw, 94

Prostatic portion of the ure-
thra, 113

Protococcus, 20

Protoplasm, 26

Pseudopod, 26

Pulp of spleen, 68

Purkinje's cell, 82

Pyramidal cells, 80

Rectum, 102

Red blood corpuscle, 51
estimation of, 50

Respiratory system, 120

Retina, 124

Respiratory bronchiole,
layers of, 125

Retzius, 93

Rouleaux of red blood corpus-
cles

Salivary glands, 105

blood supply, 105

lymphatics of, 105

nerve supply, 105

scheme of, 70
Saprophyte, 182
Sarcolemma, 56
Sarcous elements, 56
Sclera, 124
Sebaceous glands, 87
Section staining, see technic
Sectioning, see technic

226

INDEX.

Sense organs, 123
Sensory nerve endings, 123
Sharpey's fibers, 47
Sinus of kidneys, 113
Skin and its appendages, 85

glands of the skin, 87

nerve endings of, 86

pigment of, 113

structure of, 85
Slides, 16

Slipper animalcule, 23
Solitary lymph-follicle, 99
Specimens drawings of, 5
Spermatoblasts, 114
Spermatozoa, 114
Spermatozoon, 116
Spinal cord, 75

anterior median fissure, 78

gray matter, 77

horns of, 78

posterior median fissure, 78

white substance of, 75

ganglion cells of, 73
Spindle cells, 81
Spirogyra, 22
Spleen, 67
Splenic pulp, 68
Spongioplasm, 26
Stains, see technic
Sterilization, 114, 161
Sterilizers, 165'
Stomach, 96
Stomata, 69

Stratified epithelium, 126
Streptococcus, 187
Subarachnoid space, 77
Subdural space, 77,
Submaxillary gland, 105
Sudoriparous glands, 87
Suspensory ligament, 125
Sweat-glands, 87
Sympathetic gangla

Taste-buds, 126

Tease, 39

Technic, reagents and stains

Teeth, 92
structure of, 92

Tendon, 39

Test-tubes, 162

Theca folliculi, 118

Thymus gland, 68

Tongue, 126

Tooth pulp, 95

Tubule

uriniferous, 109
straight collecting, 112
schematic drawing of, 111
tunica albuginea, 114

Theory of immunity,
phagocytosis, 190

Tubercle bacillus, 186

Ureters, 113
Urethra, 113
Urinalysis, 129

albumin, 14

albuminometer Esbach's,
146

alkaline phosphate, 142

apparatus required for
urine examination, 134

bile, 153

Gmelin's test for, 154

blood in, 154

cherniqal reaction of, 140

chlorides in, 141

color, 140

composition of, 140

creatin, 141

creatmin, 141

Fehling's solution, 152

fermentation test, 147

haematuria, 154

Heidenhain's theory as to
the secretion of the urine,
133

hydrobilirubin, 152

introduction. 131

kidney, 133

Ludwig's theory, 133

mucin, 151

INDEX.

227

nitric acid test, 145
nucleoalbumin, 151
odor of urine,
peptones, 152
phosphates, 142
pus in the urine, 154
quantity of, 140
reaction of urine, 134
reagents required in the

analysis of the urine, 135
secretion of urine, 133
sediment in urine, 150
selecting a specimen of

urine, 135
specific gravity of the urine, |

140

sugar in the urine, 146
sulphates in the urine, 146,
urates, 135
urea, 135
uroerythrin, 153
Uterus, 118
glands of, 119

Vacuole, 19
Vagina, 119

glands of, 119
Vasa afferentia

efferentia

vas deferens, 115
Veins,

intralobular, 102

portal, 103
Valve, 65

rectal, 102

Villi of small intestines, 97
Volkmann's canal, 47

Widal's serum diagnosis, 183

Wandering cells, 40

White blood corpuscles, 53

Xylol, see technic
Yeast, 18

Zona pellucida, 118
tissue of, 120

1903

.QPYOELTOCAT.DW
MAY 18

-I 'y3

UNIVERSITY OF CALIFORNIA LIBRARY

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