IRLF

The Ruling of the Turck Disc. Explanation of the Squares. — In the first place,
we have the square which encloses the entire ruled surface. This is made up of
nine squares, each i mm. square. These are the squares to use in connection with
leukocyte counts with the white pipette. They may be termed the large squares.
The very smallest squares which can be found are those made by the intersection of
the triple ruled lines in the center; they are 1/40 mm. or 25 microns square and are
never used for any purpose, except possibly in connection with the counting of bacteria
in a vaccine. It will be observed that it requires four of these very small squares to
make one of the squares usually designated as the small square.

There are 400 small squares in each large square, consequently, as there are nine
large squares, the entire ruled surface consists of 3600 small squares. There are
4000 small squares, in i cubic mrn.

The unit in estimating the leukocyte or red cell content of blood is the cubic
millimeter. The unit is i/iooo of a cubic centimeter.

In making a leukocyte count we usually take the white pipette, which has the
mark II just above the bulb, and draw up the blood to 0.5 and then with suction we
fill the pipette to the II mark with the diluting fluid for which a 1/2% solution of
glacial acetic acid in water is most satisfactory. This gives a dilution of 1-20.

Counting with the 2/3 inch objective all of the highly refractile dots representing
leukocytes in one of the i mm. squares at either of the four corners we note the num-
ber and mentally multiply by 20 (the number of times the blood was diluted). As
the depth of the diluted blood between the ruled surface of the haemacytometer
slide and the under surface of the cover-glass is only i/io of a millimeter, we multiply
the figure as above obtained by 10 to get the number of cells in a 1-20 dilution of
blood in a space of one cubic millimeter.

Example: Counted 90 leukocytes; 90X20=1800X10=18,000: equals number
of leukocytes in i cubic mm. of blood.

For red counts we use the red count pipette which has the 101 mark just above the
bulb. Taking up blood to 0.5 we draw up the diluting fluid to 101. This gives a
dilution of 1-200. Counting the red cells in five of the aggregations of 16 small
squares (1/20 mm.) thus having counted 80 small squares we have counted 1/50
of the total number of small squares in a cubic mm., there being 4000 small squares
in a cubic mm. Consequently the number of red cells in 80 small squares multiplied
by 50 and then by the dilution of 200 gives the number of red cells in one cubic
mm. of the blood examined.

It is well to make a second preparation and record the average of the two counts.

OR, HENKY HORH

VST & GRANT AVE,

SAA' FRANCISCO, CAL

Principal normal and pathological blood-cells with average size,
percentage in a normal differential count and the diseases in which
certain pathological cells are more or less pathognomonic.

LIBRARY OF

r :FORNIA

The diameter of the bottom of this Petri dish is 3 inches or 7.5 +
centimeters.

The area of a circle is equal to the square of the radius multiplied
by TT or 22/7.

i 1/2 in.=radius. i 1/2X1 1/2 = 2.25. 2.25X22/7 = 7.07 square
inches.

3.75 cm. = radius. 3.75X3.75 = 14.06. 14.06X22/7=44.1 square
centimeters.

Number of bacterial colonies in i sq. in. averages, approximately,
75. Number in 7.07 sq. in. = 530.

Number of bacterial colonies in i sq. cm. averages, approximately,
12. Number in 44.1 sq. cm. = 5 28.

In a microscopic field, if the diameter were 8 small squares (1/20
mm.), the radius would be 4 small squares and the area of such a round
field would be 4X4 = 16X22/7 = 50+. Such a field would contain 50
small squares.

PRACTICAL BACTERIOLOGY, BLOOD WORK
AND ANIMAL PARASITOLOGY

STI TT

BY THE SAME AUTHOR

The Diagnostics and Treatment

OF

Tropical Diseases

ILLUSTRATED PREPARING

PRACTICAL

Bacteriology, Blood Work

AND

Animal Parasitology

INCLUDING

Bacteriological Keys, Zoological Tables
and Explanatory Clinical Notes

BY

E. R. STITT, A. B., Ph. G., M. D.

MEDICAL INSPECTOR, U. S. NAVY; GRADUATE, LONDON SCHOOL OF TROPICAL MEDICINE; HEAD
OF DEPARTMENT OF TROPICAL MEDICINE, U. S. NAVAL MEDICAL SCHOOL; PROFESSOR OF
TROPICAL MEDICINE, GEORGETOWN UNIVERSITY; LECTURER IN TROPICAL MEDI-
CINE, JEFFERSON MEDICAL COLLEGE; FORMERLY ASSOCIATE PROFESSOR
OF MEDICAL ZOOLOGY, UNIVERSITY OF THE PHILIPPINES AND
INSTRUCTOR IN BACTERIOLOGY AND PATHOLOGY,
U. S. NAVAL MEDICAL SCHOOL.

Third Edition, Revised and Enlarged
With 4 Plates and 106 Other Illustrations Containing 513 Figures

PHILADELPHIA

P. BLAKISTON'S SON & CO/

1012 WALNUT STREET
1914

FIRST EDITION, COPYRIGHT, 1909, BY P- BLAKISTON'S SON & Co.

SECOND EDITION, COPYRIGHT, 1910, BY P. BLAKISTON'S SON & Co.

THIRD EDITION, COPYRIGHT, 1913, BY P. BLAKISTON'S SON & Co.

?T,H % . yrAf&Lp .PRESS. YORK. PA

01

PREFACE TO THE THIRD EDITION.

In the preparation of the third edition of this laboratory manual it
soon became evident that the new material to be added would increase
the size of the book beyond that which would permit its being readily
carried in one's pocket. It has, however, been possible to keep the size
of the book within the limits considered desirable by the use of a smaller
type in a considerable proportion of the paragraphs so that in this way
and by increasing the number of lines on each page it has been possible
to add extensively to the subject matter and with only an increase of
about sixty-five pages.

The advantage attaching to more ready reference obtained by the
alternation of different sizes of type would appear to make this plan an
improvement over the old.

While the chapters dealing with bacteriology have been added to
and made to include more recent advances it will be noted that in the
section on animal parasitology the subject matter has been greatly
increased.

In the revision of the chapter on protozoa I am greatly indebted to
Professor Minchin's recent work on the Protozoa and in those relating
to arachnoids and insects to the very practical volume of Colonel
Alcock entitled " Entomology for Medical Officers."

The illustrations have been added to and many which did not seem
to bring out sufficiently details of anatomy have been replaced by others
more satisfactory in that respect.

The three plates of the cestode, trematode and nematode ova were
drawn by Mr. L. Avery under the supervision of P. A. Surgeon Garrison,
U. S. N. and it is believed that they will be found more satisfactory than
similar plates contained in works on animal parasitology.

Several new tables have been added among which special attention
is called to the one on urinary findings in various diseases of the genito-
urinary system and also to the key to the intestinal bacteria attached
to the inside of the board cover.

A chapter on " Disinfectants and Insecticides" giving the practical

VI PREFACE TO THE THIRD EDITION

application of methods of carrying out these important Public Health
questions has been added.

In the chapter on " Immunity" a modification of Emery's technic
for the Wassermann test has been incorporated — the use of Noguchi's
reagents with Emery's technic. The subject matter of the sections on
vaccines and anaphylaxis has been extensively revised.

E. R. S.

PREFACE TO THE SECOND EDITION.

THE fact of the necessity for a second edition of this manual of lab-
oratory and clinical diagnosis in a little more than a year would indicate
that the original arrangement of material should be adhered to.

Each section of the book had been carefully revised and much new
matter added. In particular has that part of the book relating to ani-
mal parasitology been rewritten and almost doubled in extent, and a
chapter on Poisonous Snakes added. In the chapter on " Practical
Methods in Immunity" the most recent advances in the Wassermann
test and practical agglutination methods have been incorporated as well
as a brief discussion of the question of Anaphylaxis.

The section on " Clinical Bacteriology and Animal Parasitology of
the Various Body Fluids and Organs" has been revised to meet the most
recent advances in clinical diagnosis. This section not only answers
as a cross index to the importance of the various bacteria and animal
parasites in practical clinical work, but gives a concise, practical state-
ment as to how to proceed in the examination of various secretions and
excretions. This information is difficult to obtain in the larger works
on clinical diagnosis by reason of its being taken up under many different
headings.

A method is given for the making of differential counts in the same
preparation as that for making the leukocyte count which has the advan-
tages of accuracy and the saving of time.

Several new illustrations have been added — the one of poisonous
snakes has been taken from Stejneger's report.

The plan of making this little volume a practical one has been con-
tinued in the second edition; theoretical considerations have been
brought out only when necessary to a proper understanding of some
recent or difficult laboratory method.

The very elementary considerations and definitions have not been
given because in order to present a compact and at the same time a
practical working guide it has been necessary to eliminate that which

vii

viii PREFACE TO THE SECOND EDITION

seemed least essential. Furthermore, instruction in biological science
is now a part of the requirements of candidates for admission to the
various medical schools.

At the request of many who have found the book of assistance I
have added an outline of those methods in the chemical examination
of urine and gastric contents which have seemed to me to be most essen-
tial in the making of diagnoses. In the tropics I have found the deter-
minations of total nitrogen and nitrogen eliminated as ammonia to be
exceedingly valuable in diagnosis. Methods for such determinations,
as elaborated by Assistant Surgeon E. W. Brown, U. S. Navy, of the
U. S. Naval Medical School, have proven satisfactory and have been
incorporated in this section which is to be found in the Appendix.

Every effort has been made to keep the book within the limits of a
pocket manual.

Owing to my absence from the United States I have to thank Dr.
Charles S-. Butler for correcting the proof. For the revision of the
index I am indebted to Mr. John P. Griest.

E. R. S.

PREFACE TO THE FIRST EDITION.

WHILE a member of the Naval Examining Board and examiner in
bacteriology and clinical microscopy, I have during the past six years
had an opportunity to judge of the qualifications of several hundred
graduates of the various medical schools of the country from the stand-
point of practical application in the laboratory of that which they had
learned as undergraduates.

More particularly I have made it a point to ascertain from the suc-
cessful candidates, while under instruction at the Naval Medical School,
the features of their laboratory courses, which had seemed to them
most practical; such methods being subsequently tested in our own
class work.

As a result I have endeavored to incorporate in this manual methods
which have been submitted to the criticism of postgraduate students
from all the leading medical schools of the country, and which have
been considered by them adapted to the requirements of practical,
speedy, and satisfactory clinical laboratory diagnosis.

For the laboratory worker the most valuable asset is common sense
and he mifst be able to bring to mind the possibilities of the production
of various artefacts and results from trivial errors in technic. It has
been my object to point out where such mistakes may arise, the reasons
for obtaining results differing from those ordinarily obtained and the
means employed to eliminate as far as possible such results.

We are too apt to neglect the trivial details of stains, reaction of
media, and the like, yet it is only when every detail of technic has been
rigidly carried out that we are in a position to judge of the significance
of an object observed in a microscopical preparation.

In bacteriology, candidates were frequently able to give the cul-
tural and morphological characteristics of all the important pathogenic
organisms, yet when it was required of them to outline the procedure
by which they would differentiate members of the typhoid-colon groups
when encountered in a plate made from feces, the problem appeared
to them impossible. They possessed the information, but did not
know how to apply it.

ix

X PREFACE TO THE FIRST EDITION

In practical work, organisms can only be separated culturally by the
use of Keys and for this reason Keys are given at the beginning of each
division of bacteria. These enable one to quickly place the organism
isolated in its respective group. Only methods of differentiation which
are applicable in a physician's private laboratory are given. Practical
methods for making the final identification by agglutination or other
immunity tests are described. Technic for immunizing animals to
furnish such sera is given in detail.

The giving of the cultural characteristics in a systematic tabulated
Key gives space in the notes for presenting the salient points in the
pathological and epidemiological aspects of each organism.

I have endeavored to give a scientific yet practical classification of
the important pathogenic moulds, a subject about which there exists
greater confusion in the minds of students than for any other. In the
nomenclature I have followed Gedoelst's "Les Champignons Parasites."

In the chapter on Media Making, it is believed that anyone after
reading this section and following the instructions will be able to satis-
factorily and without the adjuncts of a large laboratory make any kind
of media. The directions as to titrations are given in detail because
it is beginning to be recognized that reaction of media in bacteriology is
of as great importance as staining is in blood work.

The section on Blood Work is practical and gives a method for mak-
ing a Romanowsky stain which is quick and reliable. The chapter on
Normal and Pathological Blood gives in a few pages the more important
points to be born'e in mind in considering a possible diagnosis.

While there is no difference between the laboratory requirements of
medical work in the tropics and that in temperate climates, unless by
reason of such measures of diagnosis being indispensable in the tropics,
it has, however, been my endeavor to treat every tropical question,
whether in blood work, bacteriology, or animal parasitology, in a more
complete way than is usual in manuals of this character. Therefore it
is believed that his little book will be of great service to the laboratory
worker in the tropics.

It is only from working under Doctor Charles W. Stiles in his course
of laboratory instruction in Animal Parasitology in the United States
Naval Medical School that I feel justified in presenting a concise out-
line of the subjects in medical zoology which appear to me to be most
important for the physician.

The system of arranging tables, showing the families, genera, etc.,

PREFACE TO THE FIRST EDITION XI

in which each species belongs will, it is believed, greatly simplify the
matter of classification for the medical student. The points given
under each parasite are believed to be practical ones. When a parasite
has only been reported for man two or three times, very little space is
given to it.

Part IV summarizes the various infections which may be found in
different organs or excretions of the body and embraces both bacterial
and animal parasites. Practical methods for examining material are
also given.

The chapter on Immunity, in which the theoretical side is immedia-
ately illustrated by the practical application will tend to simplify this
bug-bear of the medical student.

The illustrations have been selected with a view to bringing out
points which are difficult to state briefly in the text, and furthermore
they have been grouped together so that comparison of similar parasites
is possible without turning from page to page.

I have in particular to thank Hospital Steward Ebeling of the Navy
for his care in bringing out such details.

By reason of the authority of Braun, it has been considered sufficient
to give in the tables only the proper zoological name of the parasite as
given in the 1908 German edition. The synonyms have been omitted
for consideration of space.

The works chiefly consulted in addition to that of Braun have been:
Albutt's System of Medicine; Osier's System of Medicine; Muir and
Ritchie's Bacteriology; Mense's Tropenkrankheiten ; Blanchard's Les
Moustiques; Guiart and Grimbert's Diagnostic; Ehrlich's Studies in
Immunity; Stephens and Christopher's Practical Study of Malaria;
Daniel's Laboratory Studies in Tropical Medicine; Manson's Tropical
Diseases; Gedoelst's Les Champignons Parasites; Neveu-Lemaire
Parasitologie Humaine; Chester's Determinative Bacteriology; Leh-
mann and Neumann's Bacteriology.

E. R. S.

CONTENTS.

PART I. BACTERIOLOGY.

CHAPTER I.— APPARATUS.

The Microscope, i; — Apparatus for sterilization, 5; — Cleaning glassware,
8; — Concave slides, fermentation tubes, 10.

CHAPTER II.— CULTURE MEDIA.

Nutrient bouillon, 17; — Standardization of reaction, 19; — Sugar-free
bouillon, 20; — Glycerine bouillon, 21; — Peptone solution, 21; — Nutrient
agar, 21; — Glycerine agar egg medium, 23; — Gelatine, 23; — Litmus milk,
24; — Potato, — 25; — Blood serum, 25; — Blood agar, 26; — Bile and faeces
media, 26; — Culture media for protozoa, 29.

CHAPTER III.— STAINING METHODS.

Loffler's methylene blue, 33; — Carbol fuchsin, 33; — Gram's method, 33; —
Acid-fast staining, 35;— Neisser's stain, 36;— Capsule staining, 37;— Fla-
gella staining, 38; — Spore staining, 39; — Staining of protozoa, 39.

CHAPTER IV.— STUDY AND IDENTIFICATION OF BACTERIA. GENERAL CONSIDERA-
TIONS.
Methods of isolating bacteria, 41; — Classification, 43; — Use of keys, 45.

CHAPTER V. — STUDY AND IDENTIFICATION OF BACTERIA. Cocci.

Key, 49; — Streptococci, 50;— Sarcinae, 53; — Staphylococci, 54; — Pneumo-
coccus, 55; — Gram-negative cocci, 57.

CHAPTER VI. — STUDY AND IDENTIFICATION OF BACTERIA. SPORE-BEARING
BACILLI.

Key, 63; — Anthrax, 64; — Cultivation of anaerobes, 67; — Malignant cedema,
69; — B. botulinus, 70; — B. tetani, 72; — B. aerogenes capsulatus, 75.

CHAPTER VII. — STUDY AND IDENTIFICATION OF BACTERIA. BRANCHING, CURV-
ING BACILLI. MYCOBACTERIA. CORNYEBACTERIA.

Acid-fast bacilli, 76; — Tubercle bacillus, 78; — Leprosy bacillus, 82; —
Non-acid-fast branching bacilli, 84; — B. mallei, 84; — B. diphtheriae, 85;
— Hofman's bacillus, 89; — B. xerosis, 89.

CHAPTER VIII.— STUDY AND IDENTIFICATION OF BACTERIA.

Gram-negative bacilli, Hemophilic bacteria, 91; — Influenza bacillus, 92; —
Friedlander's bacillus, 95; — Plague, 95; — Eberth, Gartner, and Escherich
groups, 99; — Typhoid, 100; — Dysentery, 105; — Chromogenic bacilli, 109.

xiii

XIV CONTENTS

CHAPTER IX. — STUDY AND IDENTIFICATION or BACTERIA.
Spirilla, 112; — Chlolera, 112.

CHAPTER X. — STUDY AND IDENTIFICATION OF MOULDS, 117.

CHAPTER XI. — BACTERIOLOGY OF WATER, AIR, AND MILK.
Water, 127; — Milk, 132; — Air, 135.

CHAPTER XII.— PRACTICAL METHODS IN IMMUNITY.

Methods of obtaining immune sera, 141; — Agglutination tests, 143; —
Deviation of the Complement, 145; — Fixation of the Complement, 146; —
The Wassermann reaction, 147; — Opsonic power and preparation of vac-
cines, 156; — Anaphylaxis, 161.

PART II. STUDY OF THE BLOOD.

CHAPTER XIII.— MlCROMETRY AND BLOOD PREPARATIONS.

Micrometry, 169; — Haemoglobin estimation, 172; — Counting blood, 174; —
Study of fresh blood, 177; — Blood films, 179; — Staining blood films, 179; —
lodophilia, 185; — Occult blood, 186.

CHAPTER XIV.— NORMAL AND PATHOLOGICAL BLOOD.

Color index, 188; — Red cells, 188; — White cells, 190; — Eosinophilia, 196; —
Leukocytosis, 197; — Lymphocytosis, 199; — Diseases with a normal leuko-
cyte count, 199; — The primary anaemias, 199; — Secondary anaemias, 201;
—The leukemias, 202.

PART III. ANIMAL PARASITOLOGY.
CHAPTER XV. — CLASSIFICATION AND METHODS, 211.

CHAPTER XVI.— THE PROTOZOA.

Rhizopoda, 218; — Flagellata, 222; — Infusoria, 231; — Sporozoa, 233; —
The malarial parasite, 235.

CHAPTER XVII.— THE FLAT WORMS.

Flukes, 245; — Liver flukes, 247; — Intestinal flukes, 249; — Lung flukes,
250; — Blood flukes, 251; — Cestodes, 253; — Somatic taeniasis, 259.

CHAPTER XVIIL— THE ROUND WORMS.

Filariidae, 265; — Key to filarial larvae, 269; — Trichinosis, 271; — Hook-
worms, 274; — Ascaridae, 277; — Leeches, 280.

CHAPTER XIX.— THE ARACHNOIDEA.

The mites, 282;— The ticks, 285;— The Linguatulidae, 289.

CHAPTER XX.— THE INSECTS.

The Pediculidae, 292; — The Diptera, 298; — Biting flies, 299.

CHAPTER XXL— THE MOSQUITOES.

Dissection of mosquitoes, 311; — Differentiation of Culicinae and Anophe-
linae, 312; — Classification of Culicidae, 313.

CHAPTER XXII.— THE POISONOUS SNAKES, 317.

CONTENTS XV

PART IV. CLINICAL BACTERIOLOGY AND ANIMAL

PARASITOLOGY OF THE VARIOUS BODY

FLUIDS AND ORGANS.

CHAPTER XXIII. — DIAGNOSIS or INFECTIONS OF THE OCULAR REGION, 325.

CHAPTER XXIV. — DIAGNOSIS OF INFECTIONS OF THE NASAL CAVITIES, 328.

CHAPTER XXV. — EXAMINATION OF BUCCAL AND PHARYNGEAL MATERIAL, 330.

CHAPTER XXVI.— EXAMINATION OF SPUTUM, 333.

CHAPTER XXVII.— THE URINE, 337.

CHAPTER XXVIII.— THE FAECES, 345.

CHAPTER XXIX.— BLOOD CULTURES AND BLOOD PARASITES, 351.

CHAPTER XXX.— THE STOMACH CONTENTS, 354.

CHAPTER XXXI.— EXAMINATION OF Pus, 355.

CHAPTER XXXII.— SKIN INFECTIONS, 357.

CHAPTER XXXIII.— CYTODIAGNOSIS, 359.

CHAPTER XXXIV.— RABIES AND VACCINIA, 362.

APPENDIX.

PREPARATION OF TISSUES FOR EXAMINATION IN MICROSCOPIC SECTIONS, 371.

MOUNTING AND PRESERVATION OF ANIMAL PARASITES, 377.

PREPARATION OF NORMAL SOLUTIONS, 380.

DISEASES OF UNKNOWN ETIOLOGY, 381.

CHEMICAL EXAMINATION OF THE URINE, 383.

CHEMICAL EXAMINATION OF THE GASTRIC CONTENTS, 392.

CHEMICAL TESTS OF F^CES, 393.

DISINFECTANTS AND INSECTICIDES, 394.

BACTERIOLOGY, BLOOD-WORK AND
ANIMAL PARASITOLOGY.

CHAPTER I.
APPARATUS.

THE MICROSCOPE.

THE most important piece of apparatus for the laboratory worker
is the microscope. Very satisfactory microscopes can be purchased in
this country. Instruments of standard German make are in use in
many laboratories and appear to give general satisfaction. It is impos-
sible to do good microscopical work unless the microscope gives and
continues to give good definition and the working parts remain firm.
Folding microscope stands are now made which are perfectly satisfac-
tory, such instruments, however, have only the advantage of occupying,
less space in a case so that unless the question of compactness is involved,
as in an outfit for the military services or for a microscopist who travels

about a great deal, the ordinary rigid horseshoe base is to be preferred.

•
A mechanical stage is almost a necessity in connection with blood- work and its

use is advantageous in bacterial preparations. For the study of tissue sections the
moving of the slide with the ringers is preferable. Therefore, the mechanical
stage should be capable of ready attachment or removal. For the examination of
colonies growing in Petri dishes we also use the stage unencumbered with the me-
chanical stage. A triple or quadruple nose-piece, according to the number of
objectives used, is also indispensable.

Objectives. — To meet the demands of clinical microscopy there
should be three objectives, preferably a i6-mm. (2/3 -in.), a 4-mm.
(i/6-in.) and a 2-mm. (i/i2-in.) homogeneous oil immersion. The
Zeiss AA is a ly-mm. objective, and the Leitz No. 3, an i8-mm. one.
The Zeiss D is about 4. 2-mm. and the Leitz No. 6, a 4. 4-mm. A dust-
proof quadruple nose-piece with four objectives will be found a great
convenience (in addition to the 2/3 -in. and i/i2-in. objectives, a i/4-in.

2 APPARATUS

for urine and blood counting, with a i/8-in. for examining hanging-drop
preparations and for quick examination of blood smears). An apo-
chromatic objective costs about three times as much as an achromatic
one and, except in photographic work, has little if any advantage.

As regards oculars (eye-pieces) a No. 2 and a No. 4 will best meet
the requirements. For high magnification a No. 8 may be of service.
The Zeiss oculars are numbered according to the amount they increase
the magnification given by the objective; thus a No. 2 increases the
magnification, given by the objective alone, twice; a No. 8, eight times.
Some oculars are classified according to the equivalent focal distance,
and are referred to as i/2-in., i-in., and 2-in. oculars.

The oculars in common use are known as negative oculars, by which is meant
an ocular in which the lower lens (collective) assists in forming the real inverted image
which is focused at the level of the diaphragm within the ocular. When using a disc
micrometer, it is supported by this diaphragm, and the outlines of the image are
cut by the rulings on the glass disc, and so we are enabled to measure the size of the
object examined. The measurement of various bacteria, blood-cells, and parasites is
exceedingly simple and assists greatly in the study of bacteria, and is indispensable
in work in animal parasitology. (For details of micrometry see section on blood-
work.) When an ocular is termed positive, it refers to an ocular which acts as a
simple microscope in magnifying the image, the image being formed entirely by the
objective and being located below the ocular.

Objectives are usually designated by their equivalent focal distance. It is im-
portant to remember that the equivalent focal distance does not represent the work-
ing distance of an objective, by which is meant the distance from the upper surface
of the cover-glass to the lower surface of the objective. Thus a i/4-in. objective
may have to be approached to the object so that the distance intervening may be
only 1/6 in. or even less. This explains the frequent inability to locus an object
when a high-power dry objective (i/6-in. or i/8-in.) is used with a rather thick cover-
glass — the objective possibly having a short working distance, so that the thickness
of the cover-glass does not allow of any free working distance.

Instrument makers generally specify the thickness of cover-glass to be used with
a certain tube length, but as a practical matter it will be found convenient to use
No. i (very thin) cover-glasses. The principal objection to these is that they are
more fragile than the No. 2, but with a little practice in cleaning cover-glasses this
is negligible. Immersion lenses are less affected than dry lenses by the question of
a certain thickness of cover-glasses for a certain tube length.

One of the most fruitful causes of the crushing of microscopical
objects and the overlying cover-glass or, what is far more important,
the breaking of the cover-glass of a hanging-drop preparation and
consequent risk of infection is the attempt to focus with the fine adjust-
ment. It should be an invariable rule for the worker to bring his object-

USE OF THE MICROSCOPE 3

ive practically into contact with the upper surface of the cover-glass,
then using the coarse adjustment (rack and pinion) to slowly elevate
the objective, looking through the eye-piece at the same time. In
other words, obtain focus with the coarse adjustment and maintain it
with the fine adjustment (micrometer screw). The fine adjustment
should only be used after the focus is obtained.

In using the oil-immersion objective always dip the lens in the oil and practically
touch the cover-glass — the eye being at a level with the stage — before beginning
to focus. With the coarse adjustment one can feel the contact with the cover-
glass, which is impossible with the fine adjustment. It saves time and disappoint-
ment to make a preliminary examination of a preparation requiring the high dry
or immersion lens with a low power (2/3-in.) before employing the higher power;
in this way we locate or center a suitable field for study.

It will be observed that objectives frequently have their numerical aperture
marked on them. This is expressed by the letters N.A. From a practical stand-
point this gives the relative proportion of the rays which proceeding from an object
can enter the lens of the objective and form the image. Of course, the greater the
number of rays, the greater the N.A. , the better the definition, and consequently
the better the objective. Immersion oil, having the same index of refraction (1.52)
as glass, would not deflect rays coming from the object and so prevent their enter-
ing the objective, as would be the case if we used a dry objective with an interven-
ing air space. In this case a portion of the rays would be turned aside by the dif-
ference in the refractive index of air. As a rule, the higher the numerical aperture,
the better the objective and the less the working distance. In blood counting, the
cover-glass being comparatively thick, it may happen that with a i/6-in. of high
numerical aperture there may not be sufficient working distance to bring the blood-
cells into focus, which could be done with an objective of lower numerical aperture.
Consequently, we must always consider the matter of working distance as well as
that of numerical aperture. The skill of the optician, however, can obviate this
defect in an objective of high numerical aperture so that it may combine the qualities
of perfect definition with sufficient working distance.

Practical Points in the Use of the Microscope. — An important
matter in the use of the microscope is to get all the details possible with
a low power before using a higher power. This, of course, does not
apply to a bacterial preparation where it is necessary to use a i/i2-in.
or a high-power dry lens. It is well, however, in a bacterial or blood
preparation to first examine the smear with the 2/3-in. objective in
order to determine suitable areas for examination with the oil-immer-
sion objective. With tissue sections it is not only advisable to begin the
study with the lowest power, but even an examination with the unaided
eye or with a magnifying glass, before using the microscope, will give a
surprising amount of information.

4 APPARATUS

After using the oil-immersion objective the lens should be wiped clean of oil
with a strip of Japanese lens paper or with a silk handkerchief. If the oil should
dry on the surface of the lens it may be removed with a drop^of xylol on a piece of
lens paper. Immediately afterward the lens should be dried. Dried oil on a lens
often causes the lens to be considered defective. Accidental contact of the dry
objectives with oil is not uncommon and should always be thought of when satis-
factory optical effects are not obtainable.

It is advisable to cultivate the use of both eyes in doing microscopical work.
When using one eye the other should be kept open with accommodation relaxed.
It is this squinting of the unemployed eye which so often fatigues. A strip of card-
board 4 or 5 inches long, with an opening to fit over the tube of the microscope,
leaving the other end to block the vision of the unused eye, will prevent the strain.
This apparatus can be purchased in vulcanite.

A warm stage for the study of living protozoa may be extemporized by taking
a piece of copper about the size of the stage and with a strip projecting out ante-
riorly for 5 or 6 inches. The under surface of the plate is covered with flannel and
a hole about i inch in diameter cut out of the center. The proper amount of heat
is applied by a flame impinging on the tongue-like projection of the copper plate.

Direct sunlight or excessively bright light is to be avoided. If such conditions
must exist a white shade or muslin curtain drawn across the window is a necessity.
Light from the north and from a white cloud is the most desirable. South of the
equator a southern light. In the tropics a piece of plate glass fitted into the lower
part of a wire screen frame gives good lighting, keeps out dust, and does not interfere
greatly with the circulation of the air.

The technic in connection with proper illumination is probably more important
than any other point; unless the light is utilized to the best advantage, the best re-
sults cannot be obtained. In examining fresh blood preparations or hanging drops
the concave mirror should be used and the light almost shut off by the iris dia-
phragm so as to give a contour picture. In examining a stained blood or bacterial
preparation, the Abbe condenser should be properly focused so as to best illuminate
the stained film. In many instruments set-screws are provided which check the
elevation of the Abbe condenser when the proper focus is reached. Inasmuch as
the light from the condenser should come to a focus exactly level with the object
studied, it is evident that a fixed position for the condenser would not answer when
slides of different thickness were used. Always use the plane mirror when examin-
ing stained bacterial or blood films, as a color image is desired. Ordinarily in ex-
amining tissue sections, the Abbe condenser should either be put out of focus by
racking down or by the use of the concave mirror and the narrowing of the aperture
of the iris diaphragm. Swing-out condensers are now made which are very con-
venient. The proper employment of illumination only comes with experience, and
one should continue to manipulate his mirrors, diaphragm, and condenser until
the best result is obtained. Then study the specimen.

For microscopical work in a laboratory not properly supplied with windows or
for night work the frosted incandescent bulb is very satisfactory.

Dark Ground Illumination. — Very valuable information, especially
as regards the detection of treponemata in material from hard chancres

DARK GROUND ILLUMINATION 5

or mucous patches, may be obtained by the use of dark ground illumina-
tion. There are many different types of apparatus for this purpose.

The bacteria or spirochaetes are intensely illuminated and show as
brilliant silvery objects in contrast to the dark background.

When the morphological details of a brightly illuminated object in the dark
field can be distinctly observed it is proper to use the term dark ground illumina-
tion. When only particles, usually surrounded by bright and dark rings, and not
showing any structure, are observed in the dark field the proper designation is ultra-
microscopic. An apparatus using only the short waves of the ultra-violet spectrum
enables one to observe particles no larger than i/io of a micron. For this appa-
ratus it is necessary to employ photographic plates. In using the i/i 2-inch ob-
jective with dark ground illumination a funnel-like base is supplied on which we
screw the nickle plated front mount of the objective. Before using the dark- field
apparatus it must be centered with a low power. This is carried out by getting
concentric rings parallel with the circle of the microscopic field. Immersion con-
tact b.etween the front surface of the Abbe condenser and the under surface of the
slide carrying the preparation must be made before focussing the i/i2th objective.
As a source of illumination we may use a small arc-lamp or a Nernst lamp or an
incandescent gas lamp. In using an arc-lamp one must have a suitable rheostat
according to the electrical current employed. Information as to voltage and nature
of current must be given the one supplying the apparatus.

In making preparations the slides and cover-slips should be scrupulously clean
and the material thinly spread out and free of bubbles.

APPARATUS FOR STERILIZATION.

For the purpose of sterilizing glassware, media, and old cultures
there are three methods ordinarily employed. The hot-air sterilizer,
in which a temperature of about 150° C. is maintained for one hour, is
ordinarily used for the sterilization of Petri dishes, test-tubes, pipettes,
etc. If the temperature is allowed to go too high, there is danger of
charring the cotton plugs and also of causing the development of an
empyreumatic oil which makes the plugs unsightly and causes them to
stick to the glass. Again we must be careful not to open the door until
the temperature has fallen to 60° C., otherwise there is danger of crack-
ing the glassware. Where gas is not obtainable, the hot-air sterilizer is
not a very satisfactory apparatus.

The Arnold sterilizer is to be found everywhere and can be used on
blue-flame kerosene-oil stoves as readily as with gas burners. The
most convenient form, but more expensive, is the Boston Board of
Health pattern. The ordinary pattern, with a telescoping outer por-
tion, answers all purposes, however. In the Arnold, sterilization is

6 APPARATUS

effected by streaming steam at 100° C. It is usual to maintain this
temperature for fifteen to twenty-five minutes each day for three suc-
cessive days. The success of this procedure — fractional sterilization —
is due to the fact that many spores which were not killed at the first
steaming have developed into vegetative forms within twenty-four

FIG. i. — Dressing sterilizer showing cylinder containing water (K) heated either
by gas or Primus kerosene lamps.

hours, and when the steam is then applied such forms are destroyed.
Experience has shown that all the spores have developed by the time
of the third steaming, so that with this final application of heat we
secure perfect sterilization.

It is customary to use the Arnold for sterilizing gelatin and milk

STERILIZATION 7

media, even when the autoclave is at hand, the idea being that the
greater heat of the autoclave may interfere with the quality of such
media. The most convenient autoclave is the horizontal type, such as
is to be found everywhere for the sterilization of surgical dressings.

The source of heat may be either gas, the Primus kerosene-oil lamp or steam
from an adjacent boiler. More recently a method of employing kerosene, gaso-
lene, or alcohol with a gravity system has been perfected. During the past 6 years,
in the laboratory of the U. S. Naval Medical School, we have been using a dressing
sterilizer, made by the American Sterilizer Co. , with which it has been possible to
most satisfactorily carry out all kinds of sterilization, thus doing away with the
use of the Arnold and the hot-air sterilizer. It is impossible to sterilize ordinary
fermentation tubes in the autoclave on account of the boiling up of the media and
wetting of the plugs. This is still done with the Arnold. By use of the Durham
tubes — which are to be preferred, except for gas analysis — sugar media can be thus
sterilized.

Should a small bubble remain in the top of the small inverted inner tube after
removal from the autoclave, one may make a mark with a grease pencil at the line
of the bubble; or, if preferred, the basket of Durham tubes can be heated to boiling
for ten minutes in a pan of water or in the Arnold when, after cooling, the bubSle
will be found to have disappeared.

Glassware will come out from such an autoclave with wrappers as dry and plugs
of the test-tubes as stopper-like as could be effected in a hot-air sterilizer.

The objection which exists in the use of some autoclaves, as regards condensa-
tion on dressings or apparatus, does not exist in this type. The mechanism, by
which the inner and outer chambers are connected and disconnected, and that for
vacuum production, rests in the simple turning of a lever from mark to mark. We
have been able with a gas burner to obtain a pressure of 15 pounds in less than ten
minutes. In sterilizing test-tubes we place them in small rectangular wire baskets,
6X5X4 in. These baskets are to be preferred to round ones, as they pack more
satisfactorily in the refrigerator used for storing media. In sterilizing flasks, test-
tubes, Petri dishes, throat swabs, pipettes, etc., it has been our custom, after ex-
posing to 20 pounds' pressure for twenty minutes, to produce a vacuum for two or
three minutes; then with the steam in the outer jacket for a few minutes to thor-
oughly dry the articles in the disinfecting chamber. The valve to the inner chamber
is then opened to break the vacuum; the door is now opened, and the articles re-
moved in as dry a state as if they had been in the hot-air sterilizer. Articles, how-
ever, can be thoroughly dried without the use of a vacuum, simply allowing the steam
to remain in the outer jacket with the steam cut off from the inner chamber.

PRESSURE AND TEMPERATURE TABLE.

5 pounds' pressure, 107. 7° C., 226° F.

10 pounds' pressure, n5-5° C. , 240° F.

15 pounds' pressure, 121.6° C. , 250° F.

20 pounds' pressure, 126.6° C., 260° F.

25 pounds' pressure, JSQ-S0 C. , 267° F.

30 pounds' pressure, 134-4° C., 274° F.

8 APPARATUS

All such articles as Petri dishes, pipettes, swabs, etc., are wrapped in cheap
quality filter-paper, making a fold and turning in the ends as is done in a druggist's
package. Old newspapers answer well for this purpose. The sterile swab can be
used for many purposes in the laboratory. They are most easily made by taking
a piece of copper wire about 8 inches long, flattening one end with a stroke of a
hammer, then twisting a small pledget of plain absorbent cotton around the flat-
tened end. After wrapping, the swabs are sterilized in bunches. We not only
use them for getting throat cultures, but in addition for culturing faeces, pus, or
other such material. The pus is obtained with a swab, which material is then dis-
tributed in a tube of sterile bouillon or water. With the same swab the surface of
an agar plate is successively stroked. This method is almost as satisfactory as the
German one of using bent glass rods for this purpose. Everyone has encountered
the difficulties attendant upon the bending of platinum wires and also the possi-
bility of destroying your organisms by an insufficiently cooled wire.

CLEANING GLASSWARE.

It is a routine in our laboratory for everything to go through the
sterilizer at 125° C. before anything else is done. This is a safe rule
when dealing with dangerous pathogenic organisms (especially tetanus
and anthrax).

As soon as taken out of the sterilizer the contents are emptied, and the tube or
dishes placed in a i % solution of washing soda and boiled. This thoroughly cleans
them. As the washing soda slightly raises the boiling-point and also makes the
spores more penetrable, it would appear that under ordinary circumstances, it
would be sufficient to place all contaminated articles in a dishpan with the soda
solution, and boil for at least one hour, not using a preliminary sterilization in the
autoclave. The tubes are now cleaned with a test-tube brush, thoroughly rinsed
with tap water and placed in a i% solution of hydrochloric acid for a few minutes;
then rinsed thoroughly in water and placed in test-tube baskets, mouth downward,
and allowed to drain over night. Some laboratory workers boil their test-tubes
and other glassware in water containing soap or soap powder and, after a thorough
rinsing in tap water, drain. Hydrochloric acid should not be used after the soap
as it will cause the formation of an unsightly coating difficult to remove. When
thoroughly dry they may be plugged and sterilized. To plug a test-tube, pick out
a little pledget of plain absorbent cotton about 2 inches in diameter from a roll.
Place it over the center of the tube and with a glass rod push the cotton down the
tube about an inch. The cleaning fluid commonly used in laboratories consists of
one part each of potassium bichromate and commercial sulphuric acid with ten
parts of water. This is an excellent mixture for cleaning old slides, etc. , especially
when grease or balsam is to be gotten rid of. It is very corrosive, however. An
efficient and less corrosive method for cleansing slides and cover-glasses is to leave
them over night in an acetic acid alcohol mixture (two parts of glacial acetic acid
to one hundred parts of alcohol). After drying and polishing out of this mixture,

HANGING DROP 9

it is well to pass the slides and cover-glasses through the flame of a Bunsen burner
or alcohol lamp to remove every vestige of grease. Ordinarily, rubbing between
the thumb and forefinger with soap and water, then drying with an old piece of
linen, and finally flaming will yield a perfect surface for making a bacterial
preparation.

FIG. 2. — i, Inoculation of tubes; 2, plugging of tubes; 3, filling tubes; 4, Smith's
fermentation tube; 5, Durham's fermentation tube.

CONCAVE SLIDES, FERMENTATION TUBES.

The concave slide is ordinarily used for making hanging-drop prep-
arations for the examination of bacteria as to motility, capsules, size
and arrangement. To prepare a hanging-drop preparation for the
study of motility it is best to place a loopful of the young bouillon

FIG. 3. — Hanging drop, over hollow ground slide. (Williams.)

culture or a loopful of salt solution into which is then emulsified a
small amount of growth from an agar slant, in the center of the cover-
glass; now having applied with a brush a ring of vaseline around the

10 APPARATUS

concave depression in the slide we apply the slide as a cover to the
cover-glass which latter adheres to the ring of vaseline. The completed
hanging-drop preparation can now be turned over and placed on the
stage of the microscope.

A substitute which is equally good may be made by spreading a ring or square
of vaseline — smaller than the cover-glass to be used — in the middle of a plain slide.
Then putting a loopful of salt solution in the center of the space, and inoculating
with the culture to be studied, we finally cover it with a cover-glass, gently pressing
the margins down on the vaseline. This gives a preparation for the study of motility
or agglutination which does not dry out for hours, and is easier to focus upon than
the concave slide hanging-drop preparation

In examining a hanging drop first use a low-power objective and, having brought

FIG. 4. — Blood serum coagulating apparatus.

into focus the margin of the drop as a center line, change to a r/6- or i/8-in. objective.
By this procedure a thin layer of fluid is brought under the high dry objective in-
stead of the deeper layer in the center of the drop. It is not advisable to use an
immersion objective with a hanging-drop preparation.

The light should be cut down to a minimum with the iris diaphragm and the
concave mirror used. When we have finished examining the preparation the cover-
glass should be pushed over with the forceps so that a corner projects and we then
seize this with the forceps, lift up the cover-glass and drop it into the disinfecting
solution along with the slide.

The fermentation tube with a bulb and closed arm is expensive,
difficult to clean, and is easily broken. It is, however, convenient in
the determination of the gas formula of an organism. It's use is
described under water analysis. As a substitute in the study of gas
production and in water bacteriology, the Durham tube is to be
recommended.

Into a test-tube, about 1X7 in., we introduce the special sugar media, then
drop down a small test-tube (1/2X3 mO with its open end downward. Insert the

ACID PROOFING FOR DESKS

II

plug of the large tube and sterilize. During sterilization the fluid enters the mouth
of the smaller tube and fills it, and when the medium is subsequently inoculated, if
gas forms, it appears in the upper part of the closed end of the smaller tube.

For inspissating blood-serum slants a regular inspissator is desirable.
This is nothing more than a double-walled vessel, the space between
the walls being filled with water.

As a substitute one may take the common rice cooker (double boiler). Fill the
outer part with water; and in the inner compartment pack the serum tubes properly
slanted on a piece of wood or a wedge-shaped layer of cotton. Place a weight on
the cover of the inner compartment to sink it
into the surrounding water, and allow to boil
for one or two hours. This same apparatus
may be used for their sterilization on two sub-
sequent days, but it is better to sterilize in the
autoclave or Arnold. As regards a working
desk, it will be found convenient to have an
arrangement similar to the ordinary flat-top
desk, with a tier of drawers on each side. A
block of wood with holes bored in it to contain
dropping-bottles may be placed in the upper
lef thand drawer. In this way the stains are as
accessible as if they encumbered the desk. It is advisable to paint the inside of this
drawer black so that the light may not cause the staining reagents to deteriorate.

A very popular method of preparing the surfaces of laboratory
desks, sinks, and tables is the application of the so-called " acid-proofing."
This gives an ebony-like finish- which is not affected by strong acids.

In using it the surface of the wood must be new (free of any varnish, oil, or
paint; if previously so coated the surface must be planed).

FIG. 5. — Rice cooker.

Solution i.

Potassium chlorate,
Cupric sulphate,
Water,

125.0 gms.
125.0 gms.
i ooo.o c.c.

Apply two coats of this solution at least 1 2 hours between applications. When
thoroughly dry apply two coats of solution No. 2.

Solution 2.

Aniline oil,
Hydrochloric acid,
Water,

I2O.O C.C.

180.0 c.c.

IOOO.O C.C.

When the treated surface is thoroughly dry apply one coat of raw linseed oil
with a cloth. After this is dry wash with very hot soapsuds.

1 2 APPARATUS

An aspirating bottle on a shelf elevated two feet, with rubber tubing and glass
tip leading to a small aquarium jar or other desk receptacle, makes a good substitute
for a small sink and faucet. A Hoffman screw clamp on the rubber tube controls
the flow of water.

Ordinary glass salt cellars will be found very useful, where the watch-glass is
employed. They may also be wrapped, sterilized, and used to contain fluids for
inoculating, etc.

A glass-topped fruit jar or a specimen jar containing a disinfecting solution for
contaminated slides, etc., should be on every working desk. A good solution is that
of Harrington (corrosive sublimate, 0.8; commercial HC1, 60.0 c c.; alcohol, 400.0
c.c.; water, to IOOG.O c.c.).

A very simple method of making a disinfectant similar to lysol is
to put one part of cresol or crude carbolic acid and one part of soft soap
in a wide-mouthed bottle over night. The resulting compound makes
a perfect solution with water and a 5% solution of this will be found
at least equal to a 5% phenol solution. In addition to using as a
desk jar disinfectant it is excellent for disinfecting faeces, sputum, etc.

For use in making loops and needles, platinum wire of 26 gauge will be found
most suitable. The handle made of glass rod is preferable to the metal ones. One
end is fused in the flame and, holding the 3- to 4-in. piece of platinum wire, with
forceps, in the same flame, insert the glowing metal into the molten glass.

For making smears from faeces, sputum, and the like, wooden tooth-picks are
very convenient; the kind with the spatulate end is preferable.

When gas is obtainable, the maintaining of a constant temperature for the
body temperature incubator (38° C.) and the paraffin oven (60° C.) is best secured
by the use of some of the various types of thermo-regulators. The Reichert type
is the one in general use, although there are many features about the Dunham and
Roux regulators which are advantageous.

If the pressure of the gas-supply varies from time to time, it is essential to regu-
late this by the use of a gas-pressure regulator (Murrill's is a cheap and satisfactory
one).

Incubators, controlled electrically, can be obtained of certain foreign makers,
and are quoted in catalogues of American dealers. It is probable that the Koch
petroleum lamp incubator is the most satisafctory one where gas is not obtainable.
They should be of all metal construction, and not with a wood casing, on account of
the danger from fire. They cost from twenty-five to fifty dollars.

An incubator may be extemporized by putting the bulb of an incandescent
electric lamp in a vessel of water. The proper temperature may be obtained by
increasing the amount of water or by covering the opening more or less completely
with a towel. The test-tubes to be incubated can be put into a fruit jar or tin can,
which receptacle is placed in the vessel heated by the lamp.

Emery suggests the use of a Thermos bottle as an incubator.

The vacuum bottle should be first warmed by pouring in warm water. After-
ward the bottle should be three-fourths filled with water at 100° F.

CAPILLARY BULB PIPETTES 13

Schrup suspends his cultures and thermometer in the water by threads attached
to pins in the cork of the vacuum bottle. The plug should be paraffined or covered
with a rubber cap. As regards the matter of a low-temperature incubator (for
gelatin work), this is best met by using a small refrigerator. The ice in the upper
part maintains an even cold, and by connecting up an electric lamp in the lower part
of the refrigerator we can easily maintain a temperature which only varies one or
two degrees during the twenty-four hours.

With a i6-candle-power lamp a temperature of about 25° C. is maintained (this
is too high, being about the melting-point of gelatin) ; with an 8-candle-power, one

10.

FIG. 6. — i, 2, 3, Drawing out glass tubing; 4, 5, Wright's rubber bulb capillary
pipettes showing grease pencil mark for making dilutions; 6, 7, Wright's U-tubes;
8, 9, 10, methods of drawing out test-tubes for vaccines in opsonic work; n, bac-
teriological pipette.

about 21° to 23° C. ; and with a 4-candle-power, from 18° to 20° C. ; the box being
about 20X30X36 inches.

When much serum reaction work is done, an electrically run centrifuge is a great
convenience.

A filter pump attached to the water faucet, preferably by screw threads, is
almost indispensable for filtering cultures, etc., and for cleaning small pipettes,
especially the hsemocytometer pipettes. Such a filter or vacuum pump with a
vacuum gauge is more easily controlled.

The niter pump is indispensable when using the various types of porcelain or
Berkefeld niters. The Punkal or Muencke types of filter are the most convenient in

14 APPARATUS

filtering toxins or in the sterilization of certain media when heating would be
unadvisable.

With the possible exception of the platinum loop, there is no piece
of apparatus so applicable to many uses as the capillary pipette made
from a piece of glass tubing.

These may be made in a great variety of shapes. The one with a
hooked end, the Wright tube, is the best apparatus for securing blood

-L-AVERY-

FIG. 7. — i, Apparatus combining various methods for culture of anaerobes; (a)
Hofmann clamp for connecting with vacuum pump; (b) pyrogallic at bottom of
bottle for Buchner's O absorption method; (c) deep glucose agar stab covered with
sterile liquid petrolatum (see anaerobes). 2, One-fourth inch capillary loop U tube
for making two nitric acid albumin tests (see chemical examination of urine). 3,
Piece of tubing bent to hold slide for steaming smears in flame. 4, Schmidt's fer-
mentation apparatus, as modified by using graduated cylinder (see under faeces).
5, One-fourth inch glass tubing, 4 1/2 inches long with corks at each end. For
contrifuging faeces for ova. 6a, Apparatus connected with sterile centrifuge
tube for taking blood from vein of man or a guinea-pig or rabbit's heart. 6b,
Erlenmeyer flask which can be used instead of centrifuge tube. See under sections
Immunity and Blood. 7, A graduated pipette with Hofmann clamp applied to
rubber bulb for precise delivery of measured quantities of liquids.

for serum tests. The crook hangs on the centrifuge guard and by
filing and breaking the thicker part of the tube the serum is accessible
to a capillary rubber bulb pipette or to the tip of a haemocytometer
pipette. In this way dilutions of serum are easily made. The capil-

BACTERIOLOGICAL PIPETTES 15

lary pipette is made by taking a piece of i/4-in. soft German glass tub-
ing, about 6 inches long, and heating in the middle in a Bunsen flame,
revolving the tubing while heating it. When it becomes soft in the
center, remove from the flame and with a steady even pull separate the
two ends. The capillary portion should be from 18 to 20 inches in
length. When cool, file and break off this capillary portion in the
middle. We then have two capillary pipettes. By using a rubber
bulb, such as comes on medicine droppers, we have a means of sucking
up and forcing out fluids by pressure with the thumb and forefinger of
the right hand. The bulb should be pushed on about 1/2 to 3/4 in.;
this gives a firmer surface to control the pressure on the bulb.

A bacteriological pipette is made by drawing out a g-inch piece of tubing about
3 inches at either end, then heating in the middle we draw out and have two pipettes
similar to the one shown in the drawing. A piece of cotton is loosely pushed in
just above the narrow portion. These may be wrapped in paper and sterilized for
future use. They may be made perfectly sterile at the time of drawing out.

Where gas is not at hand, the Barthel alcohol lamp gives a flame similar to that
of the Bunsen lamp and is equally satisfactory for heating glass tubing. By making
a collar with a lateral opening to fit the burner of a Primus lamp a powerful side-
flame is obtained which is almost as suitable for glass blowing as the Bunsen blast
usually employed.

CHAPTER II.
CULTURE MEDIA.

WHILE there are certain advantages in sterilizing the glass test-
tubes prior to filling them with media, yet this may be dispensed with —
the sterilization after the media has been tubed being sufficient. If a
dressing sterilizer is at hand, this is preferable for sterilizing such media
as bouillon, potato, and agar (10 to 15 pounds' pressure for fifteen min-
utes). Milk should be sterilized with the Arnold, subjecting the media
to three steamings for twenty minutes on three successive days. Gelatin
may be sterilized in either way, but preferably in the autoclave at 7
pounds' pressure for fifteen minutes. As soon as taken out of the ster-
ilizer it should be cooled as quickly as possible in cold water. This
procedure tends to prevent the lowering of the melting-point of the fin-
ished gelatin and also preserves its spissitude.

Blood-serum is preferably solidified as slants in a blood-serum inspissator. This
requires one to two hours. The subsequent sterilization in the autoclave or Arnold
should not be done immediately after making the solidified slants, but on the subse-
quent day. If done on the same day, many of the slants are ruined by being dis-
rupted by bubbles. The preparation of blood-serum slants or slants of egg media
can be conveniently carried out in a rice cooker (double boiler). Place the tubes
in the inner compartment of the cooker, obtaining the slant desired B"y manipulating
an empty test-tube, or with a towel or cotton batting on the bottom. Then cover
the tubes with another towel. The outer compartment should contain water alone
(no 25% salt solution). The inner compartment should be weighted down so that
it is surrounded by water — the light tubes not being sufficient to sink it. Allowing
the water in the outer compartment to boil one or two hours will inspissate or solidify
the slants satisfactorily. The sterilization on subsequent days may be carried out
in the same apparatus, although it is more efficient if done in an Arnold or an auto-
clave. (This sterilization in the rice cooker makes the media too dry.)

In making media a rice cooker is almost essential; at any rate, it is so if ease,
expedition, and unfailing success in preparation are to be achieved. As it is neces-
sary to make the contents of the inner compartment boil, the temperature of the
water in the outer compartment must be raised. This is done by using a 25%
solution of common salt or a 20% solution of calcium chloride in the outer compart-
ment instead of plain water. Should CaCla be carried over to media in inner com-
partment (as by thermometer) coagulation of albumin and clearing of medium will
be prevented.

16

NUTRIENT BOUILLON 17

A 15% solution of salt raises the boiling-point 2 1/2° C.; a 20%, 3 1/2° C.,
and a 25%, 4 1/2° C. The raising of the boiling-point by calcium chloride is about
the same for similar strength solutions.

Although the Bacteriological Committee of the A P. H. Association recom-
mends special steps to be taken in the preparation of gelatin and agar, yet for clinical
purposes it will be found satisfactory to keep on hand a stock of bouillon, and when
it is desired to make agar or gelatin to simply prepare such media from the stock
bouillon in the way to be subsequently given.

NUTRIENT BOUILLON.

This may be made either from fresh meat or from meat extract.
Media from fresh meat are usually lighter in color and possibly clearer.
In the Philippines, however, certain measures employed for the preser-
vation of the meat made it very difficult to prepare clear bouillon from
it, so that meat extract was used entirely. There is very little differ-
ence, if any, in the nutritive power of media made in either way. The
chief objections to fresh meat as a base are: i. It takes more time and
trouble. 2. The reaction, due to sarcolactic acid and acid salts, is
quite acid, so that it is necessary to titrate and neutralize the excess of
acidity. 3. The reaction of the finished media tends to change unless
the boiling at the time of making was very prolonged. 4. It is not
infrequent to have a heavy precipitate of phosphates thrown down at
the time of sterilization, thus making it necessary to repeat the process
of filtration and sterilization.

If fresh meat is used, take about 500 grams (one pound), remove fat and cut it
up with a sausage mill or purchase the meat already cut up as for a Hamburg steak.
It makes little difference whether the amount be 100 grams more or less. Place
the chopped-up meat in a receptacle and pour 1000 c.c. of water over it. Keep in
the ice chest over night and the next morning skim off with a piece of absorbent cot-
ton the scum of fat; then squeeze out the infusion with a strong muslin cloth, mak-
ing the amount up to 1000 c.c. This meat infusion contains all the albuminous mate-
rial necessary for the clarification of the bouillon. It is convenient to designate this
meat base as Meat Infusion to distinguish from the base containing meat extract.

Having obtained 1000 c.c. of this 50% meat infusion, we dissolve in it i% of
Witte's peptone and 1/2% of sodium chloride. While there is a sufficiency of the
various salts necessary for bacterial development in the meat juices, yet there is not
enough to give the best results when bouillon cultures of various organisms are used
for agglutination tests; and furthermore, when bouillon is used for blood cultures,
disintegration of the red cells, with clouding of the clear medium, may occur if there
be not sufficient salt present to prevent this.

The salt and the peptone are best put in a mortar, and adding about one ounce
of the meat infusion we make a pasty mass; then we gradually add the remaining
infusion until solution is complete. It is sometimes recommended to use a temper-

1 8 CULTURE MEDIA

ature of 50° C. to facilitate the solution of the peptone. This is not necessary, and
if the temperature is not watched closely it might go up to 65° C. or higher and we
should lose the clearing albuminous material from its coagulation. Of this rather
cloudy solution take up 10 c.c. with a pipette and let it run out into a porcelain
dish. Add 40 c.c. of distilled or rain water and about six drops of a 0.5% phenol-
phthalein solution. (Phenolphthalein, o. 5 ; dilute alcohol, TOO c. c. ) Bring the con-
tents of the porcelain dish to a boil and continue boiling for one or two minutes in
order to expel all CC>2. Now from a burette filled with decinormal sodium hydrate
solution, run in this solution until we have the development of a faint but distinct
pink in the boiling diluted bouillon which is not dissipated on further boiling.

It is more satisfactory to take burner from beneath the porcelain dish just before
running in the N/io solution, again boiling so soon as a pink color is obtained.
Having obtained the light pink coloration we read off the number of c.c. or frac-
tions of a c.c. of N /io sodium hydrate solution added to produce the color. This
number gives the acidity of the bouillon in percentage of N/i acid solution.

Percent acid means that so many c.c. of N/i acid added to 100 c.c. of the medium
at the neutral point would give that percentage reaction. Thus 11/2 c.c. of N/i
HC1 solution added to 100 c c. of medium at o, would give us i 1/2% of acidity
or +1.5.

Percent alkaline means so many c.c. of N/i sodium hydrate solution added to
100 c.c. of the medium at the neutral point. Thus a 1/2% alkaline medium would
be one whose alkalinity would correspond to the addition of 1/2 c.c. of N/i
NaOH to 100 c.c. of the medium at o. It is written —0.5.

If we took 100 c.c. of the medium and put it in a beaker and then ran in N/i
NaOH solution from a burette, it will be readily understood that if we had to add
3 1/2 c.c. of N/i NaOH to obtain the pink color, it would show that the acidity of
the 100 c.c. of medium, being tested, corresponded to 3.5 c.c. of N/i acid solution,
and that its acidity was equal to 3 1/2% of N/i acid solution, or that its reaction
was +3.5.

As N/i NaOH solution is too corrosive for general use in a burette, and as io c.c.
of medium is more convenient to work with than 100 c.c., we use a solution one-
tenth the strength of the N/i NaOH and we take only one-tenth of the 100 c.c. of
medium. In this way it is the same from a standpoint of directly reading off our
percentage reaction as if we had 100 c.-c. of medium and used N/i NaOH solution.
The A. P. H. Association recommends 5 c.c. of the medium and the use of N/2O
NaOH. As the N/io NaOH is always at hand for titrating gastric juice, the N/io
is used instead.

Should it be found difficult to carry on the titration while boiling the end reaction
may be fairly accurately determined in the cold. Deliver into a beaker from a
pipette io c.c. of the bouillon and make up to 50 c.c. with distilled water and add
5 drops of o. 5% phenolphthalein solution. Then run in N/io NaOH from a burette
and continue to add the N/io NaOH solution from the burette, drop
by drop, until the addition of a drop fails to show any intensifying of the purplish
violet color at the spot where it came in contact with the diluted bouillon in the
beaker. This marks the end reaction. A reaction of about +o. 7 in the cold gives a
delicate pink with phenolphthalein as an indicator. Titration in the cold is not very
satisfactory with gelatin and agar.

TITRATION OF MEDIA 1 9

Having determined the percentage acidity 'of the 10 c.c. sample tested, we easily
calculate the number of c.c. of N/i NaOH solution required to be added to the 1000
c.c. of bouillon to obtain a reaction corresponding to the neutral point of phenol-
phthalein. It is more exact to take the average of two titrations.

As 100 c.c. of medium would require 3 1/2 c.c., 1000 c.c. would require 10 times
as much, or 35 c.c. N/i NaOH solution. Having measured out and added 35 c.c
of the N/i NaOH solution to the meat infusion, containing salt and peptone, we
have a solution which is exactly neutral to phenolphthalein, or o. It is usually
considered that a reaction of about i% acid is the optimum reaction for bacterial
growth. Hence we should now add i% of N/i HC1 solution to the medium. This
would be accomplished by adding 10 c.c. of N/i HC1 solution to the 1000 c.c. of
neutralized medium, and we would have a medium with a reaction of +i. If we
desired a reaction of i% alkalinity we would add an additional c.c. of N/i NaOH
solution to every 100 c.c. of the medium at o, or 10 c.c. for the 1000 c.c. of medium.
The reaction would then be — i.

As a matter of convenience, we usually determine the reaction of the medium,
which is always more or less acid, and then add enough N/i NaOH to reduce the
acidity to the percentage we desire to set the medium, instead of neutralizing all
the acidity present and then, in a second operation, restoring the acidity to the
point desired.

Thus finding the acidity of the medium to be 3 1/2% and desiring to give it
an acidity of i%, we would add only 2 1/2 c.c. of N/i NaOH to every 100 c.c. of
medium, or 25 c.c. for the zoooc.c. of medium. The reaction would then be found
to be +i.

The neutral point of litmus is not a sharp one, but it corresponds rather closely
with a reaction of +1.5 to phenolphthalein. The recommendations of the A. P. H.
Association call for making the titration with the medium boiling. If the color of
the end reaction at boiling-point be obtained, it will be found that when cool it
deepens until it corresponds to the rich violet-pink of the end reaction in the cold
or vice versa.

To summarize:

Take Peptone, 10 grams

Sodium chloride, 5 grams

50% meat infusion, 1000 c.c.

Dissolve the peptone and sodium chloride in the meat infusion and
add enough N/i NaOH to make the reaction +i.

Put the solution in the inner compartment of a rice cooker and bring
to the boiling-point and maintain this temperature for twenty minutes.
The calcium chloride or sodium chloride in the outer compartment of
the rice cooker enables us to secure a boiling temperature for the con-
tents of the inner compartment. Do not stir the bouillon that is being
heated, as the pultaceous membranous mass of coagulated albumin
makes nitration easy. Filter. The filter-paper in the funnel should be

20 CULTURE MEDIA

thoroughly wet with water before pouring on the bouillon. This is to
prevent clogging of the pores of the filter-paper. Make up the quantity
of filtrate to 1000 c.c. by adding water.

If greater exactness is demanded than answers for ordinary clinical work, it is
advisable to again titrate and again adjust the reaction or to simply record the exact
reaction. It is more convenient to have a counterpoise to balance the inner compart-
ment and then to add water to the medium until a kilo weight, in addition to the
weight balancing the container, is just balanced. Then titrate, adjust the reaction
(if so desired), and filter. Sterilize in the autoclave at 115° C. for fifteen minutes
or in the Arnold on three successive days. The use of a balance is preferable in the
preparation of bouillon, necessary in making gelatin and imperative in making
agar media.

BOUILLON MADE FROM LIEBIG'S MEAT EXTRACT.

Place in a mortar 3 grams of Liebig's extract, 10 grams of peptone and 5 grams
of sodium chloride. Dissolve the whites of one or two eggs in 1000 c.c. of water.
Then add this egg-white water, little by little, to the extract, peptone, and
salt in the mortar until a brownish solution is obtained. Pour this into the inner
compartment of a rice cooker; apply heat to the outer compartment containing
the salt or calcium chloride solution, allow to come to a boil and to continue
boiling for fifteen to twenty minutes. Do not stir. Place inner compart ment on
the scales and its counterpoise and a one-kilo weight on the other side. Add
water until the two arms balance. Filter and sterilize.

The reaction of media made with Liebig's meat extract rarely exceeds +0.75
(from +0.6 to +0.9). Consequently for growing bacteria it is unnecessary to titrate
and adjust reactions unless precision is demanded.

SUGAR-FREE BOUILLON.

Inoculate nutrient bouillon in a flask with the colon bacillus. Allow to incubate
at 37° C. over night. Pour the contents into a sauce-pan and bring to a boil to kill
the colon bacilli. Put about 15 grams of purified talc (Talcum purificatum, U. S. P. )
in a mortar. Add the dead colon culture, stirring constantly. Then filter through
filter-paper. It may be necessary to again pass the filtrate through the same
filter until the sugar-free bouillon is perfectly clear.

For all ordinary purposes the very small amount of sugar in bouillon made from
Liebig's meat extract may be neglected in determining gas production; so that under
such conditions the various sugars could be added directly to the meat-extract
bouillon.

SUGAR BOUILLONS.

The sugar media ordinarily used for determining fermentation or gas production
are those of glucose and lactose. In special work such carbohydrates as saccharose
and maltose are used. The alcohol mannite is used in differentiating strains of
dysentery bacilli.

PEPTONE SOLUTION 21

To make, simply dissolve i or 2% of the sugar in sugar-free bouillon or that
made from meat extract. Tube in Durham's or the ordinary fermentation tubes and
sterilize in the autoclave at only about 5 pounds' pressure for 15 minutes, or in the
Arnold. Ordinary peptone solution is a good substitute for sugar-free bouillon.

Too high a degree of heat may turn the sugar bouillon brownish. The nature
of the sugar itself ma}7 further be affected by too high a temperature.

CALCIUM CARBONATE BOUILLON.

Where we wish to cultivate such organisms as streptococci and pneumococci in
massive cultures we may add small fragments of marble (calcium carbonate) so
that any inimical excess of acid may be neutralized. North used a glucose bouillon
containing calcium carbonate in the production of massive cultures of B. bulgaricus.

GLYCERINE BOUILLON.

Add 6% of glycerine to ordinary bouillon. It is chiefly used in the cultivation
of tubercle bacilli.

PEPTONE SOLUTION (DUNHAM'S).

Dissolve i% of Witte's peptone and 1/2% of sodium chloride in distilled water.
Filter, tube, and sterilize. Peptone solution may be used as a base for sugar media
instead of bouillon. It is the medium used in testing for indol production. This
test is made by adding from 6 to 8 drops of concentrated H2SO4 to a twenty-four- to
forty-eight-hour-old peptone culture of the organism to be tested. If the organism
produces both indol and a nitroso body, we obtain a violet-pink coloration, "cholera
red. " If no pink color is produced on the addition of the sulphuric acid, add about
i c.c. of an exceedingly dilute solution (i : 10,000) of sodium nitrite.

It is very important in determining the "cholera red" reaction to know that the
peptone used will give the reaction as it is not given by true cholera strains with
certain samples of peptone.

For the Voges-Proskauer Reaction. — Fill fermentation tubes with a 2% glucose
Dunham's peptone solution and sterilize. After inoculation with the organism to
be tested incubate for three days. Then add 2 to 3 c.c. of strong caustic potash
solution. The development of a pink color on exposure to the air is a positive
reaction (the color of a weak eosin solution).

Hiss' SERUM WATER MEDIUM.

Take one part of clear beef serum and add to it about 3 times it's bulk of water.
Heat the mixture in the Arnold for 15 minutes to destroy any diastatic ferment which
might be present. Color to a deep transparent blue with litmus solution and then
add i% of any of the various sugars used in fermentation tests. Sterilize in
the Arnold by the fractional method.

NUTRIENT AGAR.

In making agar medium it is preferable to use powdered agar, as this goes into
solution more readily than the shredded agar. The reaction of agar is slightly

22 CULTURE MEDIA

alkaline, so that if i 1/2 to 2% of agar is added to nutrient bouillon having a reaction
of +i the finished product will be found to be about +0.8.

To make: Weight out 15 to 20 grams of powdered agar and place in a mortar.
Make a paste by adding nutrient bouillon, little by little, and when a smooth even
mixture is made, pour in into the inner compartment of a rice cooker and add the
remainder of the 1000 c.c of bouillon. The use of the balance is preferable.

The outer compartment of the rice cooker should contain the 25% salt solution.
Bring to boil, and the agar will be found to have entirely gone into solution after
five to ten minutes of boiling.

Then, using a funnel which has been heated in boiling water and which contains
a small pledget of absorbent cotton, we filter the agar, tube it, and sterilize it in the
autoclave or Arnold. One and one-half percent agar can be readily filtered through
filter-paper and gives a clearer medium.

By taking of meat extract 3 grams, peptone 10 grams, salt 5 grams, powdered
agar 15 grams, the white of one egg and 1000 c.c. of water, making at first apasteof
all the ingredients in a mortar, then gradually adding the remainder of the 1000 c.c.
of water, putting in the rice cooker, bringing to a boil without stirring, allowing to
boil fifteen minutes and then filtering through absorbent cotton placed between two
layers of gauze in a hot funnel, we obtain a satisfactory medium, the reaction of
which will be from -fo. 7 to +0.9. It is very important not to interfere with the pul-
taceous coagulum which forms on the surface of the boiling agar.

Where very exact adjustment of the reaction of the finished pro-
duct is desirable the method of preparation of the Committee on
Water Analysis of the American Public Health Association is to be
preferred.

Dissolve 15 grams of agar in 500 c.c. of water in the inner compartment of the
rice cooker previously described. After the agar is in solution (after 10 to 15 minutes
boiling) remove the inner compartment, containing the 3% agar solution, and
allow it to cool to about 55° C. Mix in the mortar, as described in the
directions for making nutrient bouillon from Liebig's extract, 3 grams of Liebig's
extract, 10 grams of peptone and 5 grams of sodium chloride in 500 c.c. of water
containing the whites of one or two eggs. Heat this mixture to 50 to 55° C. and pour
it into the agar solution, in the inner compartment, which has been cooled to about
55° C. Now titrate this mixture containing 500 c.c. of double strength agar and 500
c.c. of double strength peptone, meat extract and salt solution. The resulting
1000 c.c. gives i 1/2% agar and i% peptone solution. Having adjusted the
reaction by the addition of the necessary amount of N/i acid or alkali, we place the
inner compartment in the outer one of the rice cooker, bring to a boil and filter
through filter-paper which has been wetted with boiling water. The filtration can
be carried out in the autoclave or in an Arnold sterilizer. Of course the ordinary
filtering through gauze and cotton will answer where clearer media is not an object.

GLUCOSE AGAR.

Add the agar to i or 2% glucose bouillon and proceed as for ordinary agar. If
preferred, the glucose agar can be made by rubbing up meat extract 3 grams, peptone

GELATIN 23

10 grams, salt 5 grams, glucose 10 grams and 15 grams of agar in 1000 c.c. of water
containing the white of egg (one to two eggs), then boiling in the rice cooker and
filtering.

GLYCERINE AGAR.

Add the agar to 6% glycerine bouillon instead of nutrient bouillon, or the gly-
cerine may be added to nutrient agar which has been melted. Glycerine agar with
a reaction of o makes an excellent base for blood and serum media for use in cul-
turing delicate pathogens.

GLYCERINE AGAR EGG MEDIUM.

Take the white and the yolk of one egg and mix thoroughly in a vessel kept
between 45° and 55° C. with an equal amount of glycerine agar. Tube the medium,
inspissate in a rice cooker as for serum tubes, and sterilize as for blood-serum tubes.

This makes an excellent medium for growing tubercle bacilli. As egg medium
has a tendency to be dry, it is well to add i c.c. of glycerine bouillon to each slant
before autoclaving.

NUTRIENT GELATIN.

Place in a mortar 3 grams of Liebig's extract, 10 grams of peptone and 5 grams
of sodium chloride. Dissolve the whites of one or two eggs in 1000 c.c. of water.
Then add this egg-white water, little by little, to the meat extract, peptone and salt,
in the mortar, until a brownish solution is obtained. Pour this into the inner
compartment of the rice cooker and bring the temperature up to 45° C. (This
preliminary elevation of temperature is better carried out in some heated water in
a pan, as the heating by means of the salt solution in the outer compartment of the
rice cooker is difficult to control, so that a temperature approximating 70° C. might
be obtained and the albumin of the white of egg coagulated. The temperature in
the outer compartment might be approaching boiling before the contents of the inner
compartment would show 45° C.) Now take about 120 grams of "gold label" or
other good quality gelatin (12%) and crush it down in the meat extract egg- water
solution in the inner compartment of the rice cooker.

The gelatin quickly goes into solution at 45° C. Gelatin being quite acid it will
probably be found upon titration that the reaction is about +4%. N/i NaOH
solution is added to bring the reaction to about +1%. or 3 c.c. N/i NaOH for
each 100 c.c., provided the reaction were exactly +4%. The procedure is the
same as for bouillon. The color reaction is not quite as distinct with gelatin as with
bouillon.

Having neutralized and allowed to boil for fifteen minutes, we filter through
filter-paper in a hot funnel. As it is very important that gelatin should be perfectly
clear, it is better to filter through filter-paper than through cotton. The filter-paper
should be very thoroughly wetted with very hot water before filtering gelatin or
agar.

24 CULTURE MEDIA

Tube the "medium and sterilize, either in the Arnold on three successive days or
in the autoclave at 8-10 pounds' pressure for ten minutes. The tubes should be
cooled as quickly as possible in cold water after taking out of the sterilizer.

AGAR GELATIN MEDIUM (NORTH).

Lean chopped beef or veal, 500 grams.

Agar, 10 grams.

Gelatin, Gold label, 20 grams.

Peptone, Witte's, 20 grams.

Sodium chloride, 5 grams.

Distilled water, q.s., 1000 c.c.

Extract the chopped beef with 500 c.c. distilled water for 18 hours, strain through
muslin and combine the ingredients in the usual way. Adjust the reaction to the
neutral point, using phenolphthalein as indicator.

North states that this medium is excellent for streptococci, pneumococci and
diphtheria bacilli because it is soft, moist, and can be used at 37° C.

It is claimed to be of special value for carrying stock cultures.

LITMUS MILK.

Milk for media should be as fresh as possible. It should then be put in a 1000
c.c. Erlenmeyer flask, sterilized for fifteen minutes in the Arnold, and set over night
in the refrigerator. The next morning the milk beneath the cream should be
siphoned off. The short arm of the siphon should not reach the bottom of the flask
so as to avoid the sediment. Add sufficient litmus solution to this milk to give a
decided lilac tinge; tube and sterilize in the Arnold on three successive days.

Litmus milk which apparently is as satisfactory as the above as regards nutritive
quality and cultural characteristics can be made from certain canned milks which
have not been condensed or sweetened and which do not contain chemical pre-
servatives. The "Natura" brand of milk is the one I have experimented with.

Litmus Solution. — A simple solution may be made by digesting the powdered
cubes repeatedly with hot water, mixing the extracts, and, after allowing them to
stand all night, decanting the solution from the inert sediment into a clean bottle.

In litmus solution so made, however, a red dye is also present while calcium and
other salts are dissolved out. For bacteriological purposes a pure solution of the
blue dye should be used. This is called "azolitmin." It is freely soluble in water
but insoluble in alcohol.

It can be conveniently prepared as follows: Weigh out 2 ounces of powdered
litmus; digest repeatedly with fresh quantities of hot water until all the coloring
matter is dissolved out; allow to settle, and decant off the fluid from the insoluble
powder. Add together the extracts, which should measure about a liter. Evap-
orate down the solution to a moderate bulk, then add a slight excess of acetic acid,
so as to convert all carbonates present into acetates. Continue the evaporation,
the later stages over a water bath, until the solution becomes pasty. Add 200 c.c.
of alcohol, and mix thoroughly. The alcohol precipitates the blue coloring matter,
while a red coloring matter, together with, the alkaline acetate present, remains in

BLOOD SERUM 25

solution. Transfer to a filter. Wash out the dish with alcohol and add this to
the filter. Wash the precipitate on the filter with alcohol. Dissolve the pure
coloring matter remaining on the filter in warm distilled water and dilute to 500 c.c.
Azolitmin solution prepared in this way is more sensitive than ordinary litmus
solution.

Azolitmin in powder can be purchased from dealers in chemicals.

POTATO SLANTS.

Take Irish potatoes and scrub throughly with a stiff brush. Then pare off
generously all the outer portion. From the white interior cut out cylinders with a
cork borer. These cylinders should be of 1/2 to 3/4 of an inch in diameter. Di-
vide a cylinder by a diagonal cut. This gives a plug with a flat base, the other
extremity being a slant. These potato plugs should be left in running water over
night or washed with frequent changes of water. This prevents the blackening of
the plug. Into a i-in. test-tube drop a pledget of absorbent cotton well moistened
with water. Then drop in the potato plug, base downward. Sterilize in the auto-
clave at 15 pounds for fifteen to twenty minutes, to insure sterility.

For glycerine potato, soak the plugs in 6% glycerine solution for about one hour.
Then drop a pledget of absorbent cotton moistened with the same glycerine
solution into the test-tubes and follow it with the potato plug. Sterilize in the
autoclave.

BLOOD-SERUM.

The blood of cattle should be collected in large pans or pails at the abattoir.
This vessel of blood should then be kept in the cold-storage room and the next
morning the more or less clear serum will have been squeezed out from the clot.
Collect this serum and keep in the ice chest for future use. If to be kept for along
time, it is advisable to add about 2% of chloroform to the serum in tightly corked
flasks. This will not only keep the serum, but will eventually sterilize it.

To make Loffler's serum, take one part of glucose bouillon and three parts of
blood-serum. Mix, tube, and coagulate the albumin in the inspissator or rice cooker,
giving the tubes a proper slant before heating. Sterilize the following day in the
autoclave as previously directed (7 Ibs.) or in the Arnold on three successive days.

A SUBSTITUTE FOR ORDINARY BLOOD-SERUM.

Add from 10 to 15 c.c. of i% glucose bouillon to the white and yolk of one egg,
make a smooth mixture in a mortar and tube. Inspissate and sterilize as for ordin-
ary serum slants. The morphology of the diphtheria bacilli and the luxuriance of
growth is similar to that of cultures on Loffler's serum.

When this medium is to be used for culturing tubercle bacilli add about i c.c.
of glycerine bouillon to each tube before final sterilization in the autoclave. The
cotton plugs should be paraffined to prevent drying of the slants in the incubator.
This medium seems to answer as a substitute for Dorset's egg medium. While
glycerine bouillon favors growth of human tuberculosis, it is not so satisfactory for

26 CULTURE MEDIA

bovine tuberculosis as plain glucose bouillon. Tim is better than the various
white of egg substitutes usually recommended. (Pouring a little alcohol in the mor-
tar and moistening the sides by tilting, then burning off the alcohol, in a measure
sterilizes the mortar. If the egg is cracked open with a sterile knife, a medium can
be prepared which will be sterile as the result of the two-hour inspissation in the rice
cooker.) By covering the tube with a rubber cap or preferably, by heating the plugged
end of the test-tube, quickly withdrawing the cotton plug and dipping the part of
the plug which enters the tube into hot melted paraffin, then quickly reintroducing
the plug, the contents of the tube will be prevented from drying out. This procedure
is essential for growing tubercle bacilli. Dorset's egg medium for the cultivation
of tubercle bacilli consists of the whole egg, which is emulsified as above, and heated
at 70° C. for from four to five hours each day for two days. To provide moisture
about i c.c. of sterile 6% glycerine solution is added to each slant.

HYDROCELE, AND BLOOD AGAR.

To tubes of melted agar at 50° C, add from i to 3 c.c. of hydrocele or ascitic
fluid, observing aseptic precautions. For blood agar the blood from a vein should
be received into a sodium citrate salt solution to prevent coagulation, and added
subsequently as for hydrocele fluid. Allow the agar to solidify as a slant, or as a
poured plate.

BLOOD-STREAKED AGAR.

Sterilize the lobe of the ear and puncture with a sterile needle. Collect the ex-
uding blood on a large platinum loop and smear it over the surface of an agar slant.
It is advisable to incubate over night as a test for sterility. Plates or slants of glycer-
ine agar of neutral reaction smeared with blood give the best results when such
delicate pathogens as pneumococci, streptococci, gonococci or meningococci are to
be cultured.

BILE MEDIA.

Secure ox bile from the abattoir or human bile from cases of gall-bladder drain-
age in hospitals. Put about 10 c.c. in each tube and sterilize. Some prefer to add
i% of peptone. Conradi's medium is ox bile containing 10% of glycerine and 2%
of peptone. This is the medium for blood cultures in typhoid, etc.

The bile lactose medium now used in water analysis is made by adding i% of
lactose to ox bile and tubing in fermentation tubes. As a substitute for fresh bile
one may use a 15 to 20% solution of a good quality of inspissated ox gall (Fel Bovis
Purificatum). A liver bouillon made by using 500 grams of finely divided beef
liver in 1000 c.c. of water with i% peptone, and prepared as for meat infusion broth,
is a good substitute for bile.

RECTOR'S BILE LACTOSE NEUTRAL RED MEDIUM.

This is recommended in the isolation of the colon bacillus as superior to lactose
litmus agar. It consists of 10% of dried ox bile, i% of peptone, and i 1/2% agar.

MEDIA 27

After the medium is filtered and tubed we add i% of lactose and i% of a i-ioo
neutral red solution. Colon colonies have a distinct purplish red zone. Furthermore
the bile inhibits the growth of many organisms which give pink colonies on lactose
litmus agar. MacConkey's bile salt medium contains 1/2% of sodium taurocholate
and is colored with neutral red.

THALMAN'S MEDIUM FOR THE GONOCOCCUS.

Five hundred grams of lean, finely minced beef are placed in 1000 c.c. of distilled
water and allowed to stand over night in an ice box. It is then filtered and the fil-
trate made up to 1000 c.c. with distilled water. To 100 c.c. of the beef juice add
i 1/2 grams of agar, and boil for 15 minutes. Then add 2 grams of glucose, and bring
the reaction to plus 0.6 by addition of N/iNaOH. Tube, sterilize, slant, and in-
cubate over night. No peptone or salt is required.

PLATING MEDIA FOR F^CES WORK.

The media of Endo, Conradi-Drigalski and the lactose litmus agar medium are
probably the most satisfactory of the numerous ones that have been proposed for
plating out faeces. A convenient way of preparing any one or all of these, and which
apparently gives media equal to that prepared according to the original formulae,
is as follows:

Liebig's extract, 5 grams.

Salt, 5 grams.

Pep ton, 10 grams.

Agar, 30 grams.

Water to make 1000 c.c.

Prepare as for ordinary nutrient agar, with the difference that the reaction should
be brought down to o. Some prefer a reaction of +0.2.

A stiff agar (3%) is employed to check the diffusion of acid beyond the colony.

FOR ENDO'S MEDIUM.

Keep this agar base in 100 c.c. quantities in Erlenmeyer flasks instead of test-
tubes. (If more convenient smaller quantities may be put in the flask.) When
needed for plating, melt a flask of this agar, and while liquid add to the 100 c.c.
six drops of a saturated alcoholic solution of basic fuchsin, and then about twenty
drops of a Treshly prepared 10% solution of sodium sulphite. The sulphite solution
decolorizes the intense red of the fuchsin to a light rose pink. This color fades to a
light flesh or pale salmon color when cold. Now add 5 c.c. of a freshly prepared
hot aqueous 20% solution of chemically pure lactose. If only occasionally using
such media, tube in 20 c.c. quantities and add one drop of the basic fuchsin and four
drops of the sodium sulphite solution and i c.c. of the hot freshly prepared lactose
solution to a tube of the melted agar base just before pouring the plate. This medium
contains i% of lactose. Kendall prepares an Endo medium which only contains
i 1/2% of agar and with a reaction just alkaline to litmus (about plus 1.2%).

28 CULTURE MEDIA

Colon bacilli show on this medium as vermilion colonies, which in about forty-
eight hours have a metallic scum on them. Typhoid and dysentery colonies are
grayish. Streptococci a deep red.

FOR LACTOSE LITMUS AGAR.

Color the agar base with litmus solution to a lilac color. Then add 5 c.c. of the
hot freshly prepared lactose solution in distilled water. This may be tubed, putting
10 c.c. in each test-tube, or put in quantities of 50 or 100 c.c. in small Erlenmeyer
flasks. It is then sterilized in the autoclave (10 pounds for fifteen minutes) or in
the Arnold.

FOR CONRADI-DRIGALSKI MEDIUM.

To 100 c.c. of lactose litmus agar add i c.c. of a solution of crystal violet (crystal
violet o.i gram, distilled water 100 c.c.). The medium is then ready to put into
plates. Colon colonies are pink. Typhoid and dysentery colonies, a bluish-gray.

CONRADI'S BRILLIANT GREEN MEDIUM.

Take of Liebig's extract 20 grams (2%), peptone 10 grams (i%), agar 30 grams
(3%)and water to 1000 c.c. This amount of meat extract should give about the
proper acidity, +3. If not, the reaction should be adjusted to that point. Filter
through cotton, tube 150 c.c. amounts into 250 c.c. Erlenmeyer flasks and sterilize.

Then add i c.c. of a i to 1000 aqueous solution of brilliant green (Hochst) and
i c.c. of a i% solution of picric acid to the flasks containing 150 c.c. of the melted
agar. Sterilization after adding the dyes precipitates them and is unnecessary.
Pour the finished medium into large Petri dishes and inoculate the surface with the
faeces.

Brilliant green does not interfere with agglutination as does malachite green.

This medium is considered by some authorities the one of choice in isolating
typhoid bacilli from faeces and urine.

The surface of the poured plates of Endo, Conradi-Drigalski, and the brilliant
green media should be dried in the incubator before smearing with the faeces. For
routine work I prefer Endo's medium followed by Russell's double sugar agar.

SELECTIVE MEDIA FOR CHOLERA.

Dieudonne's medium rests on the ability of cholera to grow when alkali is present
in such amounts as to inhibit the growth of other faecal bacteria.

Take equal parts of defibrinated blood obtained at the slaughter house and
normal NaOH solution. Mix 30 parts of this alkaline blood mixture with 70 parts
of hot 3% nutrient agar. The poured plates should be left half open over night in
the incubator otherwise even cholera will not grow on the plates.

Krumwiede has as a formula for his medium equal parts of whole egg and water,
to which 50% water egg mixture is added an equal amount of 12 1/2% crystal
sodium carbonate solution. This alkaline egg mixture is steamed for 20 minutes.

MEDIA FOR PROTOZOA 2Q

To prepare add 30 parts of this alkaline egg mixture to 70 parts of meat extract free
3% agar. (No meat extract; only peptone and salt.) The cholera colony has a
hazy look, like a little wad of absorbent cotton sticking to the surface with a metallic
luster halo.

RUSSELL'S DOUBLE SUGAR AGAR.

A fairly stiff agar (2 to 3%) with a reaction of about plus 0.7 is colored with litmus
solution to produce a distinct purple violet color. It may be necessary to add more
alkali. To this litmus tinted agar is added i% of lactose and 0.1% of glucose and
the medium as thus prepared is tubed and slanted. Sterilization should be carried
on in the Arnold, on two successive days, as the autoclave temperatures tend to
break up the sugars.

On these slants typhoid shows a delicate growth on the violet slant with a deep
pink in the butt of the tube. The paratyphoids show gas bubbles in a pink butt with
a violet slant.

The colon bacillus turns both slant and butt a deep pink and the butt is filled
with gas bubbles. To inoculate this medium we take material from a suspicious
colony grown on Endo and smear the material on the slant; then with the same
platinum needle we stab into the butt.

Culture Media for Protozoa.

MEDIUM OF MUSGRAVE AND CLEGG.

Dissolve in 1000 c.c. of water 0.3 to 0.5 gram Liebig's extract and 0.3 to 0.5
gram of common salt. If desired for plating add 2 to 3 % of agar.

A very satisfactory substitute is ordinary nutrient bouillon diluted one to ten.

MEDIUM OF SMITH.

Glucose i.o gram; Peptone i.o gram; NaCl 0.2; Aqua destill. 1000.0; Na2CO3 0.3.
Agar q. s. is added for solid medium.

MEDIUM OF CASTELLANI.

This is an aqueous medium containing i% of lactose and 10% of agg albumin.
This may replace water of condensation in an agar slant.

NOVY MACNEAL MEDIUM.

Cover 125 grams of chopped up beef with 1000 c.c. of water and place over
night in the refrigerator. Strain and add 20 grams of peptone, 5 grams salt, 10 c.c.
of normal sodium carbonate solution and 20 to 25 grams agar. Prepare as for
nutrient agar and sterilize. To i part of this one-quarter strength meat infusion
nutrient agar, when melted and cooled down to 60° C., add twice its volume of de-

30 CULTURE MEDIA

fibrinated rabbit's blood. This medium is the standard one for the culture of cer-
tain trypanosomes and other protozoa. Under the designation N.N.N. medium
(Nicolle Novy MacNeal) Nicolle has modified the medium so that there is only salt
and agar in the base to which the blood is added instead of one containing meat ex-
tract and peptone. It is the Hb which seems essential in the culture of various
protozoa. Rogers used citrated salt solution, which was slightly acidified with citric
acid, in his culturing of Leishmania from the splenic blood of cases of kala azar.
Incubation at 22° C.

ROW'S H^MOGLOBINIZED SALINE MEDIUM.

Take 10 c.c. blood from rabbit's heart or arm vein of man, defibrinate the blood
and then add 10 volumes of distilled water to lake the cells (liberation of Hb). One
volume of this laked blood solution is added to two volumes of sterile 1.2% salt
solution.

CULTURE MEDIA FOR TREPONEMATA.

I. NOGUCHI formerly first inoculated material containing treponemata into the
testicle of rabbits, obtaining by this procedure a pure culture, after a few transfers
to the testicles of other rabbits. He now grows the organism directly from serum
from a chancre. Test-tubes 2 by 20 cm. are filled with 15 c.c. of a medium consist-
ing of 2 parts of 2% slightly alkaline agar to which when melted and cooled down
to 50° C. is added i part of ascitic or hydrocele fluid. At the bottom of the medium
in the tube is placed a fragment of fresh sterile tissue, preferably a piece of rabbit's
kidney or testicle. After the medium solidifies a layer of sterile paraffin oil is run
in so that it covers the solid medium to a depth of 3 cm. Tne material is inoculated
at the bottom of the tube with a capillary pipette. Incubation at 37° C. is carried
on for two weeks. The tissue acts by removing any oxygen that may be present in
the depths of the medium. Anaerobiosis is a necessary condition. Many specimens
of ascitic fluid are unsuited.

II. Serum Agar of Muhlens and Hofmaim.— Fill sterile test-tubes one-third
full with horse serum. This is sterilized on three successive days at 55° C. Then
add an equal amount of a 3% agar containing 0.5% glucose which has been melted
down and cooled to 50° C. The mixed serum agar is then kept at 55° C. for two hours.
Such tubes are inoculated as for ascitic agar rabbit tissue media and incubated under
anaerobic conditions, preferably in a flask from which the air has been exhausted and
the remaining oxygen absorbed as shown in the anaerobic bottle described and
illustrated in Fig. 7.

WELLMAN'S PLACENTAL AGAR.

Fresh human placenta is thoroughly ground up in a meat chopper, after first
washing out the blood by running sterile salt solution through the attached vessels.
To each kilo of the macerated placental tissue is added i liter of distilled water.
This mixture is allowed to infuse for forty-eight hours at refrigerator temperature,
after which it is passed through a No. N Berkefeld which has been previously tested

PLACENTAL AGAR 31

and found to hold back ordinary bacteria. The first half-hour's nitrate is usually
found to be perfectly sterile. To facilitate this filtration the cylinder of the filter
is filled with fine, clean, sterile sand until the candle is completely covered. The
filtrate is either tubed or added to 2% sterile, previously melted agar at 40° to 41° C.,
mixed, and slanted. No titration or other preparation is necessary, except that
the medium is placed at a temperature of 40° C. for two days to inactivate the comple-
ment, as suggested by Bass in the use of human blood cultures. Fresh human
placenta contains over 30% of the hydrolytic products of protein digestion, and will
therefore secure growths of strictly parasitic or feebly vegetative bacteria, and possi-
bly protozoa, that are grown with great difficulty or not at all on ordinary media.
For instance, the acid-fast organisms from bits of leprous tissue, either of human
or rat origin, grow on this medium so readily that microscopic growth can be dis-
cerned in from five to seven days. From human tuberculous glands, urine, or cerebro-
spinal fluid the same method will give a growth of B. tuberculosis that can be distin-
guished in from seventy-two hours to a few days.

CHAPTER III.
STAINING METHODS.

IN order to study a bacterial or blood specimen the first essential is
a properly prepared film; the matter of staining is of less importance.
The slide or cover-glass, after cleaning with soap and water or by special
solutions, should be polished with a piece of old linen. If a glass sur-
face is free of grease a loopful of water will smear out evenly and over
the entire surface. The only quick practical way to make the slide
or cover-glass grease free is to burn the surface for a moment in a Bunsen
or alcohol flame. The cover-glass must not be warped. To make a
preparation, apply a small loopful of distilled water on the slide or cover-
glass and, touching a colony with a platinum needle, stir the transferred
culture into the loopful (not drop) of water. The mistake is almost
invariably made of taking up too much bacterial growth. Fluid cul-
tures do not need dilution. Smearing the mixture over a large part of
the cover-glass or over an equal area of a slide, it is allowed to dry. If
very little water is used, the preparation dries readily. Otherwise it can
be dried in the fingers high over a flame. As soon as dry, the cover-glass
should be passed three times through the flame, film side up, to fix the
preparation. Slides may be fixed by passing them five times through
the flame, but the method by burning alcohol recommended for fixing
blood-films gives more satisfactory bacterial fixation. For routine work
the stain recommended is a dilute carbol fuchsin. Drop about 5 to 10
drops of water on the cover-glass, then add one drop of carbol fuchsin.
Allow the dilute stain to act from one to two minutes, then wash in
water, dry between small squares of filter-paper (4X4 inches), and
mount in balsam or the oil used for the i/i 2-inch immersion objective.

By far the best mounting medium is liquid petrolatum. This not only has the
advantage of always being of proper consistence for mounts, as opposed to Canada
balsam, which must frequently be made thinner with xylol, but it is less sticky and
does not develop the acidity which causes balsam mounts of Romanowsky stains
to fade. Furthermore, it has superior optical qualities. It is also applicable for
mounting small insects and sporangia of moulds. For permanent preparations the

32

GRAM'S STAINING METHOD 33

border of the cover-glass should be sealed with gold size or some other cement.
Some prefer to mount directly in water without preliminary drying. It is good
practice to make a rule to always keep the smeared side of the preparations up —
never allowing it to be reversed. By this simple rule, preparations can be carried
through the most complicated staining methods without the necessity of scratching
the cover-glass, etc., to see which is the film side. In grasping a cover-glass with a
Cornet or Stewart forceps, be sure that the tips are well by the margin of the glass,
otherwise the stain will drain off. In staining with slides, the grease pencil and the
glass tubing, as recommednded under Blood Smears, will be found useful. The dilute
carbol fuchsin and Lb'ffler's methylene blue are probably the best routine stains. As
a rule better preparations are obtained with dilute stains than with more concen-
trated ones.

Loffler's Alkaline Methylene Blue. — Saturated alcoholic solution
of methylene blue, 30 c.c. ; one to ten thousand caustic potash solution,
100 c.c. (Two drops of a 10% solution KOH in 100 c.c. of water
makes a i : 10,000 solution.)

Carbol Fuchsin (Ziehl-Neelsen). — Saturated alcoholic solution
basic fuchsin, 10 c.c.; 5% aqueous solution carbolic acid, 100 c.c.

Gram's Method. — The most important staining method in bac-
teriological technic and the one so rarely giving satisfactory results to
the inexperienced is Gram's stain. In using this method, the following
points must be kept in mind:

1. Laboratory cultures (subcultures) which have been carried over for years
frequently lose their Gram characteristics.

2. Cultures which are several days old or dead or degenerated do not stain
characteristically.

3. The aniline gentian violet deteriorates when exposed to light in two or three
days — it should be kept in the dark. It should have a rich, creamy, violet
appearance.

4. The iodine solution deteriorates and becomes light in color. It should be of
a rich port- wine color.

5. The decolorizing with 95% alcohol should stop as soon as no more violet
stain streams out. This is best observed over a white background, washing at
intervals. Do not confuse stain on forceps for that on preparation.

6. The preparation should be thin and evenly spread. Some prefer carbol
gentian violet to aniline gentian violet. (Saturated alcoholic solution of gen-
tian violet, i part; 5% aqueous solution of carbolic acid, 10 parts.) This tends to
overstain.

The formula for aniline gentian violet is i part of saturated alcoholic solution
gentian violet and 3 parts of aniline oil water (made by adding 2 c.c. aniline oil to
100 c.c. distilled water, shaking violently for three to five minutes and then filtering
several times to get rid of the objectionable oil droplets which, in a Gram-stained
preparation, show as confusing black dots).
3

34 STAINING METHODS

The following stock solutions of Weigert are recommended:

No. i. No. 2.

Gentian violet, 2 grams. Gentian violet, 2 grams.

Aniline oil, 9 c.c. Distilled water, 100 c.c.

Alcohol (95%), 33 c.c.

These stock solutions keep indefinitely. Mix i c.c. of No. i with 9 c.c. of No. 2.
Filter. This keeps about two weeks and is the solution to pour on the preparation.
It may be kept on from two to five minutes. Some hasten the staining by steaming
as for tubercle bacilli. Next wash the preparation with water and flood the cover-
glass with Gram's iodine solution. Some bacteriologists simply pour off excess of
aniline gentian violet and immediately drop on the iodine solution. It is well to
repeat the application of the iodine solution a second time. The iodine solution is left
on one minute or until the preparation has a coffee-grounds color.

Gram's Iodine Solution.
Iodine, i gram.

Potassium iodide, 2 grams.

Distilled water, 300 c.c.

After washing off the excess of iodine solution at the tap, drop on 95% alcohol
and decolorize until no more violet color streams out. Now wash again and counter-
stain either with the dilute Carbol fuchsin or with a saturated aqueous solution of
Bismark brown.

The Gram-positive bacteria are stained a deep violet.

In staining smears of pus for gonococci or other Gram-negative bacteria it is
best to first stain with the gentian-violet solution for two to five minutes. Then
wash and examine the preparation mounted in water. The organisms stand out
prominently. After noting the presence of the cocci treat the smear with the
Gram solution and proceed as in the usual Gram staining technic.

Stained by Gram's Method. Not Stained by Gram's Method.

S. pyogenes aureus. Meningococcus.

S. pyogenes albus. M. catarrhalis.

S. pyogenes. M. melitensis.

M. tetragenus. B. typhosus.

Pneumococcus. B. coli communis.

Anthrax bacillus. B. dysenteriae (Shiga).

Tubercle bacillus. Sp. cholerae asiaticae.

Lepra bacillus. B. pyocyaneus.

Tetanus bacillus. B. mallei.

Diphtheria bacillus. B. pneumoniae (Friedlander).

B. aerogenes capsulatus. B. proteus.

Oidium albicans. B. of influenza.

Mycelium of actinomyces. B. of bubonic plague.

Saccharomyces. B. of chancroid.

Hofman's bacillus. B. of Koch- Weeks.

B. xerosis. Gonococcus.

ACID-FAST STAINING 35

Practically all pathogenic cocci are Gram-positive, except the Gono-
coccus, the Meningococcus, the M. catarrhalis, and the M. melitensis.

Practically all pathogenic bacilli are Gram-negative, except the
spore-bearing ones (exception B. malig. cedemat.), the acid-fast ones
and diphtheria and diphtheroid organisms.

The bacillus of glanders is Gram-negative.

Method for Staining Acid -fast Bacilli. — i. Carbol fuchsin, with gen-
tle steaming for three to five minutes or in the cold for fifteen minutes.

2. Wash in water.

3. Decolorize in 95% alcohol containing 3% of hydrochloric acid
(acid alcohol), until only a suggestion of pink remains — almost white.

4. Wash in water.

5. Counterstain in saturated aqueous solution of methylene blue
or with Loffier's methylene blue.

6. Wash, dry, and mount.

The steaming of the slides with carbol fuchsin is most conveniently carried out
by resting the slides on a piece of glass tubing bent into a V or U shape.

A method in which the organisms or granules which stain by the Gram method,
and to which so much importance is attributed by Much, may be stained, as well
as those retaining acid-fast properties, has been proposed by Fontes. The method
is to stain the preparation with carbol fuchsin, decolorize with acid alcohol, then carry
through the various steps of the Gram method, counters taining however, with Bis-
mark brown. Fontes in his method used i part of absolute alcohol and 2 parts of
acetic acid as the decolorizing agent. I have obtained, however, just as satisfactory
results with the acid alcohol. By this method the acid-fast tubercle bacilli show as
red rods dotted with violet granules. Those which do not fully retain acid-fast
properties show as zigzag violet lines.

Herman's Stain for Tubercle Bacilli. — It has been claimed that this stain gives
better satisfaction than the Ziehl-Neelsen. It consists*of two solutions: (i) ammo-
nium carbonate in distilled water, i%; (2) crystal violet (methyl violet 6B) in 95%
ethyl alcohol, 3%. The two solutions are kept in separate bottles and, for staining,
i part of (2) is mixed with 3 parts of (i). The sections are placed on a cover-glass,
the water evaporated, and about seven drops of the staining mixture are placed
on the specimen and allowed to steam for one minute over a water-bath. Place for
a few seconds in 10% nitric acid and then in 95% alcohol to decolorize. Mount
without a counterstain or use eosin i% or a very dilute fuchsin. The organisms
are purple. This staining method may be applied to smears of concentrated or
unconcentrated sputum in the same manner as for sections of tissue.

Smith's formol fuchsin :

Saturated alcoholic solution basic fuchsin, 10 c.c.

Methyl alcohol, 10 c.c.

Formalin, 10 c.c.

Distilled water to make 100 c.c.

36 STAINING METHODS

This gives a very sharp differentiation of bacteria and nuclear structures. It
has a purplish tinge. Fixation by heat gives the best staining. Allow the stain to
act for two to ten minutes. It should not be used until after standing twenty-four
hours, and after standing about two weeks it appears to lose its sharp staining power.

Archibald's Stain. — This is an excellent bacterial stain and has
been highly recommended by Blue and McCoy in plague work.

Solution No. i. Solution No. 2.

Thionin, 0.5 Methylene blue, 0.5

Phenol crys., 2.5 Phenol cry s., 2.5

Formalin, i.o Formalin, i.o

Water, 100.0 Water, 100.0

Dissolve for twenty-four hours. Mix equal parts and filter. Stain smears
fixed by heat or otherwise for ten seconds.

Nicolle's Carbol Thionin.

Sat. sol. thionin in 50% alcohol, 10 c.c.

Carbolic acid solution (2%), 100 c.c.

Pappenheim's Stain. — Take a very small portion of methylene green on the point
of a penknife and shake it into a test-tube. Then take up twice as much pyronin
and deposit it in the same test-tube. Fill the test-tube one-half full with water and
the solution should have a distinct reddish-violet color. A drop on a piece of filter
paper shows a violet center and peripheral green ring. The solution should be fresh.
Stain from two to five minuted. Differentiate with a little resorcin on a penknife
point dissolved in one-quarter of a test-tube full of alcohol. Dehydrate, clear
and mount. Polymorphonuclear nuclei stain greenish; nuclei of mononuclears and
plasma cells from bluish-red to dull violet. Cytoplasm of lymphocytes and plasma
cells purplish-red. Bacteria red.

Romanowsky Stains. — See under section on Blood. For mount-
ing specimens showing chromatin staining, as malarial parasites, try
panosomes, intestinal flagellates etc., liquid petrolatum is to be highly
recommended. The chromatin staining lasts without any fading for
at least two years. The acidity of balsam causes rapid fading of the
chromatin.

Neisser's Stain for Diphtheria Bacilli.

Solution No. i. Solution No. 2.

Methylene blue, o.igram. Bismark brown, 0.2

Alcohol, 2 c.c. Water (boiling), 100 c.c.

Glacial acetic acid, 5 c.c. Dissolve the stain in the boiling

Distilled water, 95 c.c. water and filter.

Dissolve the methylene blue in the
alcohol and add it to the acetic acid
water mixture. Filter.

CAPSULE STAINING 37

To stain: Fix the preparation. Pour on the dilute acetic acid methylene blue
solution and allow to act from thirty to sixty seconds. Wash. Then pour on the
Bismark-brown solution, and after thirty seconds wash off with water. Dry and
mount. The bodies of the bacilli are brown with dark blue dots at either end.

Neisser recommends only five seconds as the time of application of each solution.
He also recommends that the culture be only nine to eighteen hours old and that the
temperature of the incubator shall not exceed 36° C. Incubation at 37° C. gives
satisfactory results.

Ponder's Stain For Diphtheria Bacilli.

Toluidin blue (Grubler), 0.02 gram.

Glacial acetic acid, i c.c.

Absolute alcohol, 2 c.c.

Distilled water to 100 c.c.

The film is made on a cover-glass and fixed in the usual way. A small quantity
of the stain is spread on the film and the cover-glass is turned over and mounted as
a hanging- drop preparation. The metachromatic granules of the diphtheria bacilli
stain with striking intensity. With diphtheroids, the more intense staining sharply
differentiates from ordinary cocci and bacilli, which show in the preparation only
as faint light blue bodies. It is a most excellent stain for bringing out the ascopores
of yeasts. In my opinion the stain is more valuable than the Neisser method.

Capsule Staining. — The best method for studying bacteria, as to
presence of capsules, is in the hanging drop, with the greater part of the
light shut off by the diaphragm.

In material where capsules are well developed, as in pneumonic sputum, the
Gram method of staining brings out the capsule perfectly. This is of diagnostic
value, as the more or less nonpathogenic pneumococci common about the mouth
do not seem to show a capsule when stained in this way. The India ink method
of staining gives good resalts for capsules.

The most beautiful method of staining capsules is the latest one
proposed by Muir.

1. Prepare thin film, dry and stain in carbol fuchsi none-half minute; the prepa-
ration being gently heated (steamed).

2. Wash slightly in 95% alcohol, then wash well afterward in water.

3. Flood preparation in mordant for five to ten seconds.

Mordant. — Sat. aqueous sol. mercuric chloride, 2 parts

Tannic acid (20% aqueous sol.), .2 parts

Sat. aqueous sol. potash alum, 5 parts

4. Wash in water thoroughly.

5. Treat with 95% alcohol for one minute. (The preparation should have a pale
red color.)

6. Wash well in water.

38 STAINING METHODS

7. Counterstain with methylene blue one-half minute.

8. Dehydrate in alcohol. Clear in xylol and mount. (May simply dry speci-
men with filter-paper.)

Rosenow's Capsule Stain. — Make a very thin smear of the pathological material
and when nearly dry cover the preparation for ten to twenty seconds with 10%
tannic acid solution. Wash in water and blot. Stain with aniline gentian violet
by gently steaming for one-half to one minute. Wash in water. Apply Gram's
iodine solution for one-half to one minute. Decolorize in 95% alcohol and then stain
with alcoholic solution of eosin. Wash in water, dry and mount.

Flagella Staining. — Inoculate a tube of sterile water (gently) in
upper part, with just enough of an eighteen to twenty-four-hour-old
agar culture to produce faint turbidity. Incubate for two hours at 37°
C. From the upper part of culture take a loopful and deposit it on a
cover-glass. Dry in thermostat for one to five hours or over night.
Use perfectly clean cover-glasses. To stain by

Muir's Modified Pitfield Method.

i. Flood specimen with mordant. Steam gently one minute.

Mordant. — Tannic acid (10% aqueous solution), 10 c.c.

Sat. aq. sol. mercuric chloride, 5 c.c.

Sat. aq. sol. alum, 5 c.c.

Carbol fuchsin, 5 c.c.

Allow precipitate to settle or centrifuge. Keeps only one week.

2. Wash well in water for two minutes.

3. Dry carefully — preferably in incubator.

4. Pour on stain. Steam gently one minute.

Stain. — Sat. aq. sol. alum, 10 c.c.

Sal. ale. sol. gentian violet, 2 c.c.

(May use carbol fuchsin instead of gentian violet.)
Stain only keeps two days.

5. Wash well in water. Dry and mount.

Zettnow's Flagella Staining Method.

Solution I. — Dissolve 2 grams of tartar emetic in 40 c.c. water.

Solution II. — Dissolve 10 grams tannin in 200 c.c. water. To the 200 c.c.
solution II, warmed to 50 or 60° C., add 30 c.c. of the tartar emetic solution.
The turbidity of the mordant should entirely clear up on heating. The mordant
should keep for months when a small crystal of thymol is added to it.

Next dissolve i gram silver sulphate in 250 c.c. distilled water. Of this solution
take 50 c.c. and add to it drop by drop ethylamine (this comes in a 33% solution)
until the yellowish-brown precipitate which forms at first is entirely dissolved and
the fluid is entirely clear. It requires only a few drops. The bacterial preparations
prepared as described above are floated in a little mordant contained in a Petri dish

STAINING OF PROTOZOA 39

which is heated over a water bath for five to seven minutes. Take the dish contain-
ing the preparation off the water bath and as soon as it becomes slightly opalescent
as the result of cooling remove the cover-glass preparation and wash thoroughly in
water. Then heat a few drops of the ethylamine silver solution upon the mordanted
cover preparation until it just steams and the margin appears black. Next wash
thoroughly in water and mount. This gives the most satisfactory results of any
method I have ever experimented with.

Spore Staining. — The most satisfactory spore staining method is
really the negative staining of the spore obtained when a bac-
terial preparation is stained by dilute carbol f uchsin or Loffler's methy-
lene blue. The spore appears as a highly refractile piece of glass in
a colored frame.

The acid-fast method, as for tubercle bacilli, gives good results.
The decolorizing, however, must be lightly done, otherwise the spore
will lose its red stain.

Holler's Method. — Fix films and then treat with chloroform for one or two
minutes. Wash thoroughly and treat with a 5% solution chromic acid for one
minute. Wash in water and then stain as for acid-fast organisms with carbol
f uchsin. Use a i% sulphuric acid solution instead of the 3% acid alcohol.

Agar Jelly Staining Method of H. C. Ross.

Very clear i 1/2% solution of agar is colored with Unna's polychrome
methylene blue, Giemsa's solution, thionin or Gram's solution of iodine. Very
thin smears of blood, faeces or gastric content sediment are made and either
fixed lightly in the flame or air dried. A drop of the melted colored agar solu-
tion is placed on the smeared cover-glass and this is mounted immediately on a
clean slide. The preparation is ready for examination in about two minutes.

The Staining of Protozoa.

Unless staining albuminous material it is well to add a little blood-
serum or white of egg to the preparation — about one loopful to a smear.
The serum or white of egg is best preserved by the addition of 2 % chloro-
form and kept tightly corked.

Giemsa's Method. — Fix moist smears with a fixative made by
adding i part of 95% alcohol to 2 parts of saturated aqueous solution
of bichloride of mercury. Keep in this solution twelve hours. Now
wash for a few seconds in water and then for about five minutes with a
dilute Lugol's solution (KI, 2 gm.; Lugol's solution, 3 c.c.; Aqua, 100
c.c.). Now wash in water and then in a 0.5% solution of sodium thio-
sulphate to remove the iodine which was used to remove the mercury.

40 STAINING METHODS

Wash in water five minutes, then stain with Giemsa's stain as used in
blood work for one to ten hours. Wash and mount.

Vital Staining of Protozoa with Neutral Red Solution. — As a stock
solution one uses a 0.5% aqueous solution of neutral red.

The drop of salt solution or water on the slide should be tinged a
light violet-rose color with a fraction of a loopful and the faeces or other
material emulsified in this.

Protozoa take a rose-pink color with a distinct differentiation be-
tween endoplasm and ectoplasm.

Should the faeces be quite alkaline the neutral red will be decomposed
with the formation of bilirubin-like crystals.

The Giemsa formalin method described under Blood Work is of
value in certain cases.

Highly to be recommended for the staining of protozoa, whether in smears or in
sections, is the Panoptic method.

1. Wright's or Leishman's stain for one minute.

2. Dilute with water and allow dilute stain to act for three to ten minutes.
Wash in water and then

3. Pour on dilute Giemsa's stain. Allow to stain from thirty minutes to twenty-
four hours. Differentiate with i : 1000 acetic acid solution until blue stain just
shows commencing diffusion into the acetic acid. Then wash in water, 95%
alcohol, absolute alcohol and treat with xylol and mount in liquid petrolatum.

With preparations other than blood smears, as sections, it is better to go from
95% alcohol to oil of origanum, then mount.

Owing to the great value of a sharp nuclear picture in differentiating amoebae
it is of great importance to use some iron haematoxylin method. That of Weigert
is given in the appendix.

Fix moist smears, film surface down, in Zenker's fluid for five to ten minutes.
Wash in water, treat with Gram's solution and wash with 70% alcohol until all the
yellow color is discharged. Wash in water. Then stain with Mallory's phospho-
tungstic hamatoxylin for one-half hour. Wash clear and mount. See appendix.

Mallory's Differential Stain for Amoebae. — Staining in saturated aqueous
solution thionin for from three to five minutes. Next differentiate in 2% aqueous
solution oxalic acid for one-half to one minute. Then wash in water, clear and mount.
Nuclei of amoebae are stained a brownish red.

CHAPTER IV.

STUDY AND IDENTIFICATION OF BACTERIA— GENERAL
CONSIDERATIONS.

IN order to study bacteria it is necessary to isolate them in pure
culture. This may be accomplished by taking one or more loopfuls of
the material and mixing it in a tube of melted agar or gelatin. From
this first tube one or more loopfuls are transferred to a second tube of
melted agar or gelatin, and from this a third transfer is made, thereby
giving us tubes in which the distribution of the bacteria is one or more
hundred times less in the second than in the first tube, and equally
more dilute in the third than in the second. When we pour the con-
tents of the tubes into Petri dishes we would have the bacterial colonies
on the first plate so thick that it would be impossible to pick up a single
colony with a platinum needle without touching an adjacent one. On
the second plate the distribution might be such that we should have
discrete, well separated colonies, material from which could be taken up
on the point of the needle or loop without touching any other colony.
If the second plate did not meet these requirements, the third would.

In clinical bacteriology we work almost entirely with organisms
preferring blood-heat temperature, hence it is necessary to use agar
or blood-serum as standard media for the obtaining of isolated colonies.
Gelatin is of little value for this purpose in medical work. In using
agar it will be remembered that it solidifies at a temperature slightly
below 40° C. and does not melt again until it is subjected to a tempera-
ture practically that of boiling. Again, if the temperature of the media
exceeds 44° C. it may affect injuriously the organisms we wish to study.
Consequently it requires careful attention and quick work to inoculate
the tubes, mix, transfer and pour into plates within the limits of a tem-
perature which injures the organisms, and one which brings about the
solidification of the agar.

Again, we not only have colonies developing from organisms which
have been fixed at the surface as the agar solidified in the plate, but
more numerous ones developing from bacteria caught in the depths

42 STUDY AND IDENTIFICATION OF BACTERIA

of the media. Therefore we have superficial and deep colonies. Ex-
cept to the person of great experience, all deep colonies look alike and
there is at times great difficulty in deciding whether a colony is deep or
superficial. It is in the matter of trying to obtain information from the
differences in deep colonies that the greatest difficulties in the study

FIG. 8. — Petri agar plate. Made by spreading scrapings from the mouth over
sterilized nutrient agar; after forty-eight hours in the thermostat the light "colonies"
develop. Streaked plate. (Delafield and Prudden.}

of bacteriology arise. By using the method of simply stroking plates
along five or six parallel lines from one side of the plate to the other
with a bent glass rod, platinum loop, or a small cotton swab, we obtain
colonies which are well separated and which are entirely superficial.
The material as pus, faeces, throat membrane, etc., should be evenly
distributed in a tube of sterile water or bouillon; the swab which was

KOCH'S SOLID MEDIA 43

originally used for obtaining the material being then pressed against the
sides of the test-tube to express excess of fluid and then stroked gently
over successive lines on one plate. Or, if the organisms be very abun-
dant, over a second plate without recharging it from the inoculated
tube.

According to my experience a very satisfactory method is to take a loopful from
the bouillon tube suspension of the pus or faeces and deposit the fluid in the platinum
loop on the left half of the poured plate then, without recharging the locp. we touch
the right half of the plate. Now taking a bent glass rod from a jar of 95% alcohol
we flame it and to cool the same we press the bent portion into the middle of the
plate. This also divides the surface of the plate into two portions. Then rubbing
the bent rod over the smaller amount of the material on the right side we carry it
over the entire right side. Then go to the loopful deposited on the left side with the
rod and rub it over this side. For urine, deposit one drop on one side and 5 drops
on the other. A smear from pus, sputum, urine or throat culture should always be
made first in order to get an idea as to the degree of dilution which is necessitated
before plating out.

To obtain isolated colonies on blood-serum or blood-streaked agar,
which can be touched and by transfer obtained in pure culture, we
simply smear the material on a slant of either medium. Then, without
sterilizing the loop, we smear it thoroughly over a second slant, and so on
to a third, or possibly a fourth or fifth.

At present the classification of the bacteria is very unsatisfactory
from a scientific standpoint. The nomenclature abounds in instances
where three or four terms are used in naming a single bacterium, in-
stead of the single generic name and single specific one as is used in
zoological nomenclature. This matter of nomenclature is a subordi-
nate factor in the confusion when we begin to investigate and find that
different names have been applied to apparently the same organism.
The slightest variation in morphological, locomotor, or biological
characteristics seems to be considered sufficient by many observers to
justify the description of a new species, and, of course, the giving of a
new name. Many of these names which are now retained were applied
prior to the epoch-making introduction of gelatin media by Koch (1881)
and consequently at a time when the isolation of organisms in pure
culture was a matter of extreme difficulty and uncertainty. One of
the first facts noted by the student in taking up bacteriology is the
difficulty in determining motility; this property should always be
tested on young cultures in bouillon. In Brownian movement there
is a sort of scintillating movement, but the bacterium does not move

44

STUDY AND IDENTIFICATION OF BACTERIA

from that part of the field. In current movement all the bacteria
swarm in the same direction, going very fast at times, and then more
slowly. If in great doubt, the mounting of the organisms in a 2%
solution of carbolic acid will stop movement if it be true functional
motility, while Brownian and current movement are not interfered
with. In true motility bacteria move in opposite and in all directions,

FIG. 9. — Chart in use at the U. S. Naval Medical School.

and move away from the place where first observed unless degenerated
or dead.

At times we judge of motility by the presence of this characteristic in a few of
the organisms seen in the microscopic field, the vast majority of the bacteria not
showing motility. A source of error can be present when the bacteria are emulsified
in a drop of water which might contain motile bacteria.

CHARACTERISTICS OF BACTERIA 45

Reaction of media is of the greatest importance in causing variation
in the functions of bacteria, and is one which has until recently been
almost entirely neglected. In describing an organism at the present
time it is always necessary to note the reaction of the media, the tem-
perature at which cultivation took place, and the age of the culture
when examined.

In the following keys the term bacterium has been used as a general
designation for all schizomycetes. Migula calls motile rod-shaped
organisms bacilli, and nonmotile ones bacteria. Lehmann and Neu-
mann call spore-bearing organisms bacilli, and nonspore-bearing ones
bacteria.

The B. typhosus is very motile and does not possess spores. Accord-
ing to Migula, it would be the Bacillus typhosus; according to Lehmann
and Neumann, the Bacterium typhosum. The B. anthracis has spores
and is nonmotile. Hence it would be Bacterium anthracis, according
to Migula, and Bacillus anthracis, according to Lehmann and Neumann.

In the use of the keys at the head of each group of organisms it will
be observed that the primary separation is on the basis of morphology
—the cocci in one group, the bacilli in three subgroups: one for those
rod-shaped organisms showing branching and curving forms, one for
the spore bearers and one for the simple rods. The spirilla are grouped
by themselves.

An important method of differentiation is the reaction to Gram's
stain. It should be remembered that organisms carried along on arti-
ficial media often lose their Gram staining characteristics; hence it is
desirable to determine this staining reaction in cultures freshly isolated.
Be sure that the stains, especially the aniline gentian violet and the
iodine solution, have not deteriorated. There is no more important
stain than this, and none which requires greater experience. The chief
causes of conflicting results are i. working with old cultures and 2. not
having satisfactory staining solutions.

Motility, as stated above, is at times difficult to determine. For this
purpose young eighteen-hour-old bouillion cultures are preferable, and
the preparation should be made by applying a vaseline ring to the slide,
then putting a drop of the bouillion culture in the center of the ring
(or a drop of water inoculated from an agar slant growth), then putting
on a cover-glass. By this method current movement is done away with
and the preparation keeps for hours. This is a convenient method for
agglutination tests.

46 STUDY AND IDENTIFICATION OF BACTERIA

Liquefaction of gelatin is a very important means of differentiating. When a
room-temperature incubator is not at hand (20° to 22° C.), it is better to put the
inoculated gelatin tube in the body-temperature incubator, and from day to day test
the power of solidifying with ice-water. If the organism digests the gelatin (a
liquefier), the medium will remain fluid when placed in ice- water; if the organism
is a nonliquefier, the medium in the tube becomes solid. Of course we lose the
information to be obtained from the shape of the area of liquefaction.

FIG. 10. — Series of stab cultures in gelatin, showing modes of growth of different
species of bacteria. (Abbott.)

For routine work the only sugar media used are the glucose and the
lactose bouillon. These are of the utmost importance in differentiating
organisms of the typhoid and colon group. Following Ford, these
intestinal bacteria have primarily been separated by their action on
litmus milk — whether turning it pink or only slightly changing or not
changing at all the original color.

Examine the colonies on Petri plate at first with the unaided eye,
then with a hand magnifying glass or low-power objective, using re-

KOCH'S POSTULATES 47

fleeted and transmitted light alternately. Having determined the
presence of two or more different kinds of colonies, make a ring with
wax pencil around one or more of each kind of colony, numbering them.
The slides or culture tubes used in determining the species of organism
present in the plate should bear the same number as that of the colony
from which the material was taken. A convenient procedure is to put
a loopful of water on a clean cover-glass and to emulsify material from
a colony in it. Then invert over a concave slide without vaselining
the circumference of the concavity. After examining for motility,
smear out and dry the bacterial preparation. Then fix in the flame
and stain with aniline gentian violet for two to five minutes. Wash
and mount the preparation in water. Afterward pass through the usual
Gram technic.

After this inoculate the various culture media from similar colonies.
One may inoculate a tube of bouillon from a single colony and later
on inoculate the other culture tubes.

In testing for gas production it is better to use the Durham fermenta-
tion tube as small amounts of gas may not be easily detected with deep
stab cultures into glucose or lactose agar.

If a Durham or Smith tube be not at hand the production of gas may be deter-
mined by observing bubble formation on the surface of the sugar bouillon culture.
As none of the pathogenic cocci produce gas, fermentation tubes are unnecessary
where cocci are to be studied. The litmus milk tube gives data as to acid production.

An important point is to wait at least forty-eight hours (in the case
of M. melitensis, four to seven days) before reporting on the cultural
findings on the agar or blood-serum slant or plate upon which the
material is smeared (pus, exudate, blood, etc.).

Should an organism be encountered in original investigations these
requirements as to etiological relationship should be carried out (Koch's
postulates), i. The organism should be constantly present in that
particular pathological condition. 2. Such bacteria should be isolated
in pure culture from the pathological material. 3. Such pure cultures
when inoculated into suitable animals should reproduce the pathologi-
cal conditions and should be capable of a second isolation in pure culture
from such an experimental animal. For various reasons, such as unsuit-
able animals or artificial media, these requirements are impossible of
execution with several organisms which are generally recognized as the
causes of certain diseases.

The experimental animals most frequently employed in the diagno-

48 STUDY AND IDENTIFICATION OF BACTERIA

sis of bacterial diseases are the guinea pig, the rabbit, the white rat
and the white mouse. In the following diseases the most suitable
animals for inoculation are :

1. Tetanus — mice or guinea-pigs subcutaneously. The spasms begin in the limbs
nearest the site of inoculation.

2. Pneumococci and streptococci — mice intraperitoneally or rabbits intraven-
ously.

3. Staphylococci — rabbits.

4. Diphtheria, tuberculosis, anthrax and malignant oedema — the guinea-pig
subcutaneously.

5. Glanders and cholera — the guinea pig, intraperitoneally.

6. Plague — guinea-pigs, cutaneously or subcutaneously.

In the cutaneous method of infection the material, as from a plague bubo, or
the sputum from pneumonic plague, is thoroughly rubbed with a glass rod upon
the shaven surface of the guinea-pig.

In the subcutaneous method one can use a hypodermic needle (the all glass
syringe with platino-iridium needle is the best) or an opening can be cut with the
scissors, a pocket then opened up with the forceps and a piece of tissue inserted to
the bottom of the pocket with the forceps.

The large ear vein of the rabbit is used for intravenous inoculation. This can be
made to stand out with either hot water or xylol.

In intraperitoneal injections the animal is best held head down so that the
intestines gravitate downward. The shaven skin is pinched up and the needle
inserted in the median line.

CHAPTER V.

STUDY AND IDENTIFICATION OF BACTERIA— COCCI. KEY

AND NOTES.

Streptococcus Forms. — Cells, divide to form chains.

I. Gelatin not liquefied.

1. Haemolytic zone on blood agar.

a. Very slight acidity in lactose litmus bouillon. S. pyogenes. Tends to
produce arthritis in experimental animals. Often a granular sediment in
bouillon.

b. Marked acidity but no gas production in lactose litmus bouillon. S.
acidi lactici. Non pathogenic. Forms diffuse cloudiness in bouillon.

2. Greenish appearance about colonies on blood agar.

a. No tendency to capsule formation. S. viridans. Produces endocarditis
in experimental animals.

b. Distinct capsule formation in pathological material or on favorable media.
S. lanceolatus (Pneumococcus). Gram positive, lance-shaped cocci with
bases apposed within a capsule.

c. Very marked capsule development on all media. S. mucosus. A. strepto-
coccus with extraordinary capsule development, up to io(i in width, S.
mesenterioides, is not pathogenic.

II. Gelatin liquefied.

Streptococcus coli gracilis. (Cocci quite small — 0.2 to 0.4/1. In faeces.)
A tube-like liquefaction, chains rather long; only slight growth on agar.

Constant inhabitant of stools of meat diet.
Sarcina Forms. — Cells divide in three dimensions of space. (Packets).

A. No pigment production on agar.

a. Sarcina alba. (Colonies finely granular.)

b. Sarcina pulmonum.

B. Yellowish pigment.

a. Sarcina lutea. (Colonies coarsely granular.)

b. Sarcina flava. (Colonies finely granular.)

C. Rose-red pigment.

a. Sarcina rosea.

Micrococcus Forms. — Cells divide irregularly in various directions.
I. Gram-positive cocci.
A. Cocci — round.

i. Divide in two planes at right angles. Tetrad formation. Merismopedia.
a. M. tetragenus. Moist white viscid colones. No liquefaction of
gelatin. Capsule.

4 49

50 STUDY AND IDENTIFICATION OF BACTERIA

2. Divide irregularly. Bunch of grapes arrangement. (Staphylococci.)

a. Gelatin not liquefied. M. cereus albus.

b. Gelatin liquefied. / M. (Staphylococcus) pyogenes albus.

\ M. (Staphylococcus) pyogenes aureus.

c. Gelatin very slightly liquefied.

S. epidermidis albus. (Stitch coccus.)
B. Cocci — biscuit-shape.

Diplococcus crassus. (May be mistaken for meningococcus.)
On ordinary agar we have a scanty growth resembling the streptococcus.
Colonies on ascites agar are smaller than those of meningococcus. It produces acid
in glucose, maltose and lactose.
II. Gram-negative cocci.

A. Grow only at about incubator temperature.

1. Grow only on blood or serum media. Gonococcus.

2. Grow on blood serum media, or glycerine agar.

a. Diplococcus intracellularis meningitidis. (Produces acid in glucose
and maltose but not in lactose.)

3. Grows on ordinary media. Micrococcus melitensis.

B. Will grow at room temperature as well as at 37° C.

a. Micrococcus catarrhalis. Does not produce acid in glucose or maltose.

b. M. pharyngis siccus. Colonies dry and tough and adhere to medium.
NOTE. — Other biscuit-shaped Gram negative organisms resembling the meningo-
coccus are (a) Diplococcus flavus. The colonies show yellow pigment and we have
three varieties according to the depth of the yellow color, (b) M. pharyngis siccus
and (c) M. cinereus chiefly have coarse dry colonies on ascitic agar.

STREPTOCOCCUS FORMS.

Those cocci tending to arrange themselves in chains are usually
described as streptococci. (Ogston, 1881; Rosenbach, 1884.)

When we consider that certain bacilli at times assume an arrange-
ment which we term strepto-bacilli, yet have no relationship, it would
suggest that the matter of chain morphology is simply a characteristic
common to many entirely different cocci.

Again old laboratory cultures of streptococci may show alternations of cocci
and rods giving the appearance of the dots and dashes of the Morse code. Further-
more unsuitable media may bring about various involution types in an organism
primarily streptococcal.

It is often difficult to distinguish streptobacilli from streptococci morphologic-
ally and the same is true of diplococci and diplobacilli. These bacillary pairs and
chains however often show bipolar staining and are almost invariably Gram
negative.

While streptococci tend to assume chain formation in pus and tissues they often
appear as diplococci in blood.

STREPTOCOCIC 51

The essential point to bear in mind is that the finding of a strepto-
coccus does not necessarily explain an infection, because normally
streptococci are among the organisms most frequently and abundantly
found in plates made from normal buccal and nasal secretions. It is
well to be very conservative when reporting streptococci as the etiolog-
ical factor from lesions of the throat or nose.

Probably the most practical point in the differentiation of strepto-
cocci, next to that of pathogenicity, is the occurrence of long or short
chains, the virulent ones tending to appear in chains of from ten to
twenty cocci, while the normal inhabitants of the nose, mouth and
faeces generally tend to be in shorter chains.

FIG. ii — Streptococcus pyogenes. (Kolle and Wassermann.)

As regards virulence, this is exceedingly variable — it is soon lost,
but may be restored either by inoculating streptococci along with
various other organisms or by passage through successive rabbits.
The rabbit is the most susceptible animal and should be inoculated in
one of the prominent ear veins. If the needle of the syringe is not in-
serted in the vein it will be difficult to force in the material and a swell-
ing will immediately show itself.

Besides the morphological and pathogenic variations, Schottmuller
has noted differences where these organisms are grown on i part of
blood and 3 to 6 parts of agar. On this medium Strep, erysipelatis
has a hemolytic action, the laking of the red cells bringing about a more
or less clear ring surrounding the colony. The short-chain strepto-
cocci do not have a hemolytic halo. The pneumococcus has a

52 STUDY AND IDENTIFICATION OF BACTERIA

greenish zone. Streptococci which are profoundly toxic and which
have been isolated from milk-borne epidemic sore throats differ from
the ordinary S. pyogenes in being encapsulated, not tending to form
chains and producing only slight haemolysis on blood agar.

Some of the English authorities have introduced biochemical
methods of differentiating: the Strep, pyogenes coagulating milk, re-
ducing neutral red, and producing acid in lactose, saccharose, and
mannite media.

S. pyogenes does not produce acid in inulin media while the pneu-
mococcus does,

A freshly prepared solution of sodium taurocholate, 5%, added to an equal
amount of a twenty-four-hour bouillon culture of S. pyogenes does not disintegrate
the cocci or, at any rate, not within a few minutes. The reverse is true of
the pneumococcus.

When we consider the biochemical variations which a single organism, as the
colon bacillus, may exhibit, the value of such methods of differentiating may well
be questioned. The question of the symbiotic relationship, which, when established
between two or more bacteria, may cause harmless organisms to take on virulence,
would appear to be a more important consideration.

Almost without exception, human streptococci are Gram positive.
Their colonies are quite small, but distinct and discrete. In appear-
ance the colonies of streptococci and pneumococci are practically iden-
tical. In a blood-serum throat culture pneumococcus and streptococcus
colonies are the smallest, diphtheria ones are quite small and discrete,
but slightly flatter. (Always examine the water of condensation for
streptococci.) The sarcina and staphylococcus colonies are much
larger.

Streptococcic colonies on blood agar are much more moist and luxuriant than
on ordinary agar. A very important point, in judging whether a streptococcus
or other organism is pathogenic in a given infection, is to examine smears from the
pus or other material in a Gram-stained specimen for information as to abundance
and, in particular, phagocytosis of any organism, before plating out.

Streptococci are commonly the cause of diffuse phlegmonous inflam-
mations, while the staphylococci cause circumscribed lesions. Strepto-
cocci cause necrosis and do not characteristically produce pus. The
importance of the streptococcus as a secondary infection in diphtheria,
tuberculosis, small-pox, and even in typhoid fever must always be kept
in mind. It is this infection which does not respond to diphtheria
antitoxin, and not the diphtheria one.

SARCINA FORMS 53

When freshly isolated from human lesions streptococci often show only a slight
virulence for animals. Hence massive doses are indicated and intravenous or
intraperitoneal injections. The guinea-pig is not very susceptible to streptococci;
the rabbit and white mouse being the animals of choice.

In nondiphtheritic anginas, puerperal fever, ulcerative endocarditis
and coccal enteritis it is the streptococcus which is usually the cause.
It has been claimed that acute articular rheumatism is due to a short-
chain streptococcus (M. rheumaticus), which is best isolated from
material from an acute joint infection, but may also be isolated
occasionally from the blood. It produces much acid and clots milk in
two days. The growth is described as being more luxuriant than that
of S. pyogenes. It is about 0.5/4 in diameter.

The majority of investigators have reported streptococci from acute joint inflam-
mations and bacilli from chronic infectious joint affections. Goadby has considered
a streptobacillus, somewhat resembling Ducrey's bacillus of chancroid, which
exhibits marked pleomorphism and Gram variations, and grows best on egg albumin
agar of plus 3 reaction as the cause of arthritis deformans and alveolar osteitis.
Inoculation of cultures of this organism into or around the knee-joints of rabbits
has produced lesions similar to those of rheumatoid arthritis.

SARCINA FORMS.

These are best observed in hanging-drop preparations, when they
can be seen as little cubes, like a parcel tied with a string, and by noting
them when turning over, it will be seen that they are different from the
tetrads which only divide in two directions of space. At times the
packet formation is not perfect and it will be difficult to distinguish
such as sarcinae. All sarcinae stain by Gram. If the staining of
sarcinae be too deep it may obscure the lines of cleavage. Sarcinae are
nonmotile.

Various sarcinae have been isolated from the stomach, especially
when there is stagnation of stomach contents. Sarcinae have also been
found in the intestines. In plates the S. lutea is frequently a contami-
nating organism, being rather constantly present in the air. The
demonstration of sarcina morphology should always be made from
liquid media, as bouillon. Urine makes an excellent medium.

MICROCOCCUS FORMS.

This grouping includes all cocci which do not show chain or packet
formation. It will be found convenient to divide them into two classes

54

STUDY AND IDENTIFICATION OF BACTERIA

according to their staining by Gram. The M. tetragenus, S. pyogenes
aureus and the pneumococcus stain by Gram, while the gonococcus,
the meningococcus, the M. catarrhalis and the M. melitensis are Gram
negative.

M. tetragenus. — This organism is frequently found associated with
other organisms in sputum, especially with tubercle and influenza
bacilli. The colonies are white, slightly smaller
than staphylococci and are quite viscid.

It was formerly considered unimportant in
disease, but the idea now prevails that it is re-
sponsible for many abscesses about the mouth,
especially in connection with the teeth. Injected
subcutaneously into mice, it produces a septicaemia
and death in three or four days. The blood shows
great numbers of encapsulated tetrads. It has been
reported twice as a cause of septicaemia in man.

Staphylococci. — To cocci dividing irregularly
and usually forming masses which are likened to
clusters of grapes the term staphylococcus is ap-
plied. While there have been experiments which
show that by selecting pale portions of a yellow
colony, eventually a white colony could be pro-
duced, yet, as a practical consideration, it is con-
venient to consider at least two types of staphy-
lococci: the Staphylococcus pyogenes aureus and
the Staphylococcus pyogenes albus. In culturing
from the pus of an abscess or furuncle we generally
obtain a golden coccus, while in material from the
nose or mouth, the staphylococcus colonies are
almost invariably white. As regards the common
skin coccus, this will be found to produce a white
colony. A coccus which very slowly liquefies gelatin and has been
supposed to cause stitch abscesses is the S. epidermidis albus.

While it is customary to look for a golden colony in the case of organisms show-
ing virulence, yet at times a cream-white colony may develop from cocci of great
virulence.

The S. pyogenes citreus is considered as of very feeble pathogenic power. Cer-
tain cocci whose colonies have presented a waxy appearence have been designated
as S. cereus albus and S. cereus flavus, respectively. They are of very little practi-

FIG. 12. — Gektine
culture Staphylococ-
cus aureus one week
old. (Williams.)

THE PNEUMOCOCCUS 55

cal importance. The Staphylococcus pyogenes aureus grows readily at room tempera-
ture, but better at 37° C. It coagulates milk and renders bouillon uniformly turbid.
It grows on all media, as blood-serum, agar, potato, etc. It has been proposed to
distinguish it from skin staphylococci by its power of producing acid in mannite.
Ordinarily the individual cocci are about i// in diameter, but they vary greatly in
size according to the age of the culture and other conditions. The aureus, as it
is frequently called, is not only often found in circumscribed processes, but it is a
frequent cause of septicaemia, osteomyelitis, endocarditis, etc.

In infection of bone tissue the Staphylococcus is by far the most frequent cause.
It is well to remember that insignificant staphylococcal infection may lead to sep-
ticaemia. In the tropics, where resistance is often lowered and staphylococcal skin
infections common, continued fevers are often septicaemias. It is the organism
most frequently concerned in terminal infections. The lowered resistance of the
patient permits of their passage through barriers ordinarily resistant. Not only
should this be kept in mind when such organisms are isolated at an autopsy, but
as well the fact that their entrance may have been agonal or subsequent to death.

The Pneumococcus of Fraenkel. — (Weichselbaum differentiated
organisms causing pneumonia in 1886.) This is by far the most com-
mon cause of pneumonia, whether it be of the croupous, catarrhal, or
septic type. It is also frequently found in meningitis, empyema, endo-
carditis and otitis media. It should not be confused with the pneumo-
bacillus of Friedlander, which, although possessing a capsule like the
pneumococcus, differs from it by being Gram negative, being a bacillus
and having large viscid colonies. The pneumococcus is the cause of
more than 80% of the cases of pneumonia. It does not grow below
20° C. and is best cultivated on blood-serum, or blood-streaked agar.
On plain agar it grows as a very small dew-drop-like colony, which is
slightly grayish by reflected light. It produces considerable acid, thus
acidifying and usually coagulating litmus milk. It produces acid in
inulin media which the streptococcus fails to do. The colony is smaller
and more transparent than a streptococcus colony. In sputum or
other pathological material it is best recognized by the presence of a
capsule inclosed in which are two lance-shaped cocci with their bases
apposed. In artificial culture we rarely get the capsule. It also some-
times grows in short chains like a streptococcus. The* best medium
for differentiating is the serum of a young rabbit; in this it grows as a
diplococcus, while streptococci show chains. The best method of
isolating it in pure culture is to inject the sputum into the marginal
ear vein of a rabbit or subcutaneously into a mouse. Death results
from septicaemia in about two days and the blood teems with pneu-
mococci. Usually the pneumococcus quickly loses its virulence, and

56 STUDY AND IDENTIFICATION OF BACTERIA

also dies out in a few days unless transferred to fresh media. The
best medium for its preservation is rabbit's blood agar; this also main-
tains the virulence. On this medium the colonies are larger than on
agar and they present a greenish appearance.

The pneumococcus growth emulsifies very readily and evenly so that suspensions
for vaccines are easily made.

It is a well-known fact that the pneumococcus is a frequent inhabitant of the
nasal, pharyngeal, and buccal cavities. The explanation of infection is either on
the ground of lowered resistance of the patient or enhanced virulence of the organism.
Oscar Richardson has reported an organism in cases of lobar pneumonia, cerebro-
spinal meningitis, mastoid disease, etc., bearing resemblance to both pneumococci
and streptococci — the Streptococcus capsulatus. It differs from the pneumococcus

**•£•*« *;.

*f T*k~i

•«* ' ds*

r

• •m.m^*^-'*jf

FIG. 13. — Pneumococcus, showing capsule, from pleuritic fluid of infected rabbit,
stained by second method of Hiss. (Williams.)

in that the colonies on blood-serum are viscid and like irregular flecks of mucus.
The characteristic culture is a glucose agar stab. (Reaction must not exceed +0.5.)
From the line of puncture there are flail-like projections extending outward from
one-fifth to one-fourth of an inch. The capsule persists on ordinary culture media.
This organism resembles the Streptococcus of Bonome of the French.

In a study of blood and sputum cultures from thirty- two cases of lobar
pneumonia Hastings and Boehm found blood and sputum positive bacteriologically
in eleven cases. In nine of these cases the pneumococcus was isolated and in
two a haemolysing streptococcus. In the other twenty-one cases the sputum
cultures were bacteriologically positive in eighteen of the cases and negative in
three. In nine cases the pneumococcus was isolated, in two cases B. coli, in
one case M. catarrhalis, in one case a staphylococcus, in two cases staphylococci
and streptococci, in one case B. influenzas. The percentage of positive blood

GONOCOCCUS 57

cultures was 30.3. Cole obtained 30% of positive blood cultures. The blood was
taken into flasks of bouillon in dilution of 1-50.

Diplococcus crassus. — This is a Gram positive, kidney-shaped
diplococcus, which might be confused with the M. catarrhalis or the
meningococcus by ordinary staining methods. It is larger than the
meningococcus.

In throat cultures I have isolated on several occasions a Gram positive diplococcus
which is at times biscuit-shaped, at times irregularly spherical. It possesses two
or three metachromatic granules, so that in a Neisser stain for diphtheria the appear-
ance of these granules may be confusing.

Using Ponder's toluidin blue stain I have observed granule staining in organisms
of round or oval morphology which were suggestive of the ascospore staining of
yeasts.

Gram Negative Cocci. — It is important to bear in mind that there
are many cocci of varying shapes, which in cultures or in smears from
the throat, nose or faeces are Gram negative. These are not well classi-
fied or described. To distinguish the three important kidney-shaped
diplococci, it can be most easily accomplished by cultural methods,
using hydrocele agar (ascites or blood agar will answer), ordinary blood-
serum and plain agar. The gonococcus will only grow on the hydro-
cele agar; the meningococcus will grow on this, but likewise grows on
ordinary blood-serum. The M. catarrhalis will grow on plain agar as
well as on other media.

Other Gram negative organisms of confusing morphology are M.
pharyngis siccus, the colonies of which show great crinkly dryness, and
M. pharyngis flavus.

Gonococcus (Neisser, 1879). — This organism is characteristically
a diplococcus, the separate cocci being plano-convex with their plane
surfaces apposed. (Biscuit shape, coffee-bean shape.) They are gen-
erally found grouped in masses of several pairs, most strikingly in pus
cells or epithelial cells, but also found extracellularly. Except in the
height of the disease, there is a great tendency for the organisms to show
involution forms, so that instead of biscuit-shaped diplococci we have
round, irregular and uneven cocci. It is therefore advisable in search-
ing smears from chronic gonorrhoea to continue the search of Gram-
stained specimens until some fairly typical diplococci are found. There
is nothing requiring greater discrimination than a diagnosis from such
a smear. At the commencement of a gonorrhoea the epithelial cells are
abundant and gonococci are found adhering to them or lying free.

58 STUDY AND IDENTIFICATION OF BACTERIA

Later on, at the acme of the discharge (the creamy, abundant discharge) ,
it is in the pus cells we find them and they may be so abundant that 10
to 20% of the pus cells may contain them. In the subacute stage the
epithelial cells, which practically disappear when the discharge is so
abundant, begin to reappear, and in the chronic stage the epithelial
cells are the chief ones, and are the ones, on which we find an occasional
gonococcus, often distorted in shape.

The best method of diagnosis in cases of chronic gonorrhoea is to have the patient
drink beer and eat the stimulating food previously interdicted, to take active ex-
ercise and to have a sound passed. To obtain material for examination ihe glans
penis should be washed and the patient who has presented himself with a full
bladder should pass a portion of the contained urine. Next the prostate aj*9 seminal

FIG. 14. — Gonococcus. Film from urethral pus. (Coplin.)

vesicles should be massaged with the patient standing but bent over and the penis
pendant. The drops of discharge from the massage should be received in a small
Petri dish and finally the remaining urine should be passed into a sterile bottle.
Smears and cultures should be made from the sediment of the two urinary specimens
and from the secretions of the massaged prostate and vesicles.

The smears made from the resulting discharge or centrifuged urine will probably
contain gonoccocci if they are present in the urethra. In the female the favorite
sites are the urethra and the cervix uteri. In municipal examinations it is customary
to make two smears: one from the urethral meatus and a second from the cervix.
The vagina is not a suitable soil for their development. In female children it is
most often found in the discharge of the vulvovaginitis.

MENINGOCOCCUS 59

In addition to the genital organs, the gonococcus may at times invade and be
isolated from the eye (gonorrhoeal ophthalmia), the joints, rarely as a cause of
endocarditis and possibly as the factor in septicaemia. Grown upon hydrocele or
ascites agar, or blood-streaked agar, or upon blood agar from man or the rabbit,
the colonies appear as irregular, minute, dew-drop spots. By the second or third
day the involution forms are abundant, and within four to seven days the culture
will probably be found to be dead. Unless frequent transfers are made, it will be
best kept alive on blood agar. The organism grows best at 37° C., and will not
grow below 25° C. It will not grow on plain or glycerine agar or ordinary blood-
serum unless the transfer of considerable pus in inoculating the slants gives it a
suitable culture medium. In material from joints, it is in the fibrin flakes that the
gonococci are most .apt to be found, if found at all.

By heating the blood-streaked agar tubes to 56° C. for twenty minutes (in-
activation-destroying complement and hence bactericidal power of blood on slant)
greater success in primary cultures will be obtained.

In culturing gonococci the transfer of material to culture media
should be made with the least delay possible.

The most satisfactory medium is Thalman's medium upon the
slanting surface of which we have deposited two or three drops of
human serum. Blood may be taken from a vein or the Wright U tube
may be used and after centrifuging the sterile serum is taken off with
a capillary bulb pipette and deposited on and smeared out on the slant.

Diplococcus intracellularis meningitidis (Weichselbaum, 1887). —
This is the organism of epidemic cerebrospinal meningitis, and is
frequently termed the meningococcus. The diplococcus is Gram
negative and biscuit-shaped and is, like the gonococcus, chiefly con-
tained in pus cells. It is also found free in the cerebrospinal fluid
withdrawn from cerebrospinal fever cases. There is a greater tendency
to variation in size and shape than is the case with the gonococcus,
which latter, in fresh material, shows a striking uniformity morpholog-
ically. The meningococcus is at times not abundant— early in the case,
however, the picture may be similar to that of gonorrhoea.

On blood-serum the colonies appear after twenty-four to forty-eight hours as
discrete, very slightly hazy colonies, about one-tenth of an inch in diameter. On
serum agar, as ascites or hydrocele agar, they grow best. Unless considerable
cerebrospinal fluid is transferred with the inoculating loop, they do not grow on
plain agar. They will grow at times on glycerine agar. The organism is very
sensitive to light, cold and drying. It ferments dextrose and only grows at blood
temperature, thus distinguishing it from the M. catarrhalis. It is scarcely patho-
genic for laboratory animals, with the exception of the mouse and guinea-pig,
when intraperitoneal injections but not subcutaneous ones give results. Intradural
injections give results. The cultures die out very rapidly, so that it is necessary to

60 STUDY AND IDENTIFICATION OF BACTERIA

make transfers every one or two days. The meningococcus has been isolated from
the nasal secretions of patients. The possibility of these organisms being the M.
catarrhalis must be considered.

Flexner has shown that in monkeys, which are susceptible to the disease, in-
jections of cultures of M. intracellularis into the spinal canal is followed by migra-
tion of the cocci to the nasal cavity both free and in phagocytic leukocytes.

The meningococcus has a very slight resistance to sun or drying so
that its aerial transmission seems doubtful. It is supposed to effect
an entrance by the nares, thence reaching the cerebral meninges. Inf ec-

FIG. 15. — Diplococcus intracellularis meningitidis and pus cells. (Xiooo.)

(Williams.}

tion is probably by direct contagion. Several cases have been re-
ported where with a high leukocytosis the cocci have been found in the
polymorphonuclears of blood smears and in cultures from the blood.
(In about 25% of blood cultures where from 5 to 10 c.c. are employed.)

By the use of initial injections into horses of killed cultures followed by alternate
injections into horses of living diplococci, then seven days later of an autolysate
made from different strains; seven days later again injecting living diplococci; thus
alternating material every week, an antiserum of value has been obtained by Flexner.
The immunization requires about one year. In using, withdraw about 20 c.c. of
patient's cerebrospinal fluid with a syringe, and then inject, through the same needle,
an equal quantity of the serum. The injection is repeated every day for three or
four days.

For diagnosis, make smears and cultures from cerebrospinal fluid.
The sediment from the centrifuged material gives better results. In
tuberculosis the lymphocytes preponderate; in cerebrospinal meningitis
the polymorphonuclears.

MALTA FEVER 6 1

It has been stated that a point of difference between the phagocytosis with the
gonoccoci and the meningococci is that the meningococci invade and at times destroy
the nucleus of the polymorphonuclear, which is not true of gonococci. The appear-
ance of large phagocytic endothelial cells, often containing polymorphonuclears, in
the centrifuged cerebrospinal fluid is a favorable prognostic sign. At times there
does not appear to be any relation between the number of phagocytic polymorpho-
nuclears and the severity of the case.

Vincent has recommended a precipitin test for epidemic cerebrospinal meningitis
which has the advantages of being simple and more immediate than cultures and of
particular value in those cases when meningococci cannot be found in the smears or
in cultures from the cerebrospinal fluid. It is performed by adding one or two drops
of antimeningococcic serum to a tube of fresh cerebrospinal fluid which has been
cleared by centrifugalization for 10 to 15 minutes. After adding the serum the tube
is placed in the incubator at 52° C. for two to five hours together with a control
tube. The formation of a precipitate (turbidity) shows a postive test.

Micrococcus catarrhalis (Seifert, 1890). — This organism has been
specially studied by Lord. It resembles the meningococcus strikingly
and can only be differentiated by cultural procedures. It grows on plain
agar and at room temperature, and does not produce acid in glucose
media. It not only occurs in the nasal secretions of healthy people,
but appears to be responsible for certain coryzas and bronchial affec-
tions, resembling influenza. It also is responsible for certain epidemics
of conjunctivitis.

The original cultures may show only slight growth whereas the sub-
cultures prove luxuriant.

The colonies are larger, more opaque, and have a more irregular
wavy border than the round colonies of the meningococcus.

Micrococcus melitensis (Bruce, 1887). — This is the organism of
Malta or Mediterranean fever, sometimes called undulant fever, on
account of successive waves 'of pyrexia running over several months.
The disease has a very slight mortality (2%), and the lesions are chiefly
of the spleen, which is large and diffluent. The organisms can best be
isolated from the spleen.

M. melitensis is only about 0.3/1 in diameter. The characteristics are its very
small size and the dew-drop minute colonies on agar, which at incubator temperature
only show themselves about the third to the sixth day. It is nonmotile and Gram
negative. In bouillon there is a slight turbidity.

Many laboratory infections have been recorded.

The organism occurs in peripheral circulation, it having been cultivated from
blood very successfully by Eyre. He takes blood at the height of the fever, and in
the afternoon. Formerly it was customary to isolate by splenic puncture.

Infection is chiefly by means of the milk of infected goats. The organisms are

62 STUDY AND IDENTIFICATION OF BACTERIA

excreted in the urine of patients, and a diagnostic point is to make plates from the
urine. Such urine applied to abraded surfaces causes infection.

The serum of patients shows agglutinating power as early as the fifth day of
the disease, and this may persist for years after recovery. Nicolle has advised using
serum heated to 56° C. for 30 minutes for the agglutination test, nonspecific agglu-
tinins being thereby destroyed. Carriers may be of importance in Malta fever and
are best detected by agglutination tests.

A high mononuclear increase may be found in this disease.

CHAPTER VI.

STUDY AND IDENTIFICATION OF BACTERIA. SPORE-
BEARING BACILLI. KEY AND NOTES.

A. Grow aerobically.

1. Stab culture in gelatin has branches growing out at right angles to line of stab.

a. Has no membrane on bouillon or liquefied gelatin. Projecting branches
from line of stab only at upper part of line of growth. Absolutely nonmotile,
Ends sharply cut across or concave. ANTHRAX GROUP.

b. Has thick whitish membrane on bouillon and surface of liquified gelatin.
Projecting branches all along the line of stab. Sluggishly motile. MY-
COIDES GROUP. (B. mycoides. B. ramosus.)

2. Stab cultures in gelatin do not show projecting branches.

a. Potato cultures do not become wrinkled. At first slightly moist, later
dry and mealy. SUBTILIS GROUP. (Hay bacillus group.) Actively
motile with more or less square ends and a central spore which is of the same
diameter or only slightly larger than the bacillus. The yellow subtilis is at
times found in water. The colonies on potato are of a cheese-yellow color.
The bacilli are very large and show a sluggish, worm-like motion.

B. megatherium often shows a granular or beaded appearance in a Gram
preparation. The narrow spores are never central, usually between center
and end, and rather elongated. It most nearly resembles the sporulating
bacillus of malignant oedema but if the spore is quite terminal and bulging
may resemble B. tetani.
Cultures of B. megatherium are somewhat similar to B. coli colonies.

b. Potato cultures at first even growth but after a few days become wrinkled.
VULGATUS GROUP. (Potato bacillus.)

B. vulgatus shows marked wrinkling, like intestinal coils. B. mesentericus
show slight wrinkling and a network-like appearance.

Two water bacilli belonging to this group are the B. mesentericus fuscus
(brown growth) and B. mesentericus ruber (red growth).

NOTE. — The following cultural characteristics are common to all the above spore
bearers.

1. Liquefaction of gelatin.

2. Milk slowly and incompletely coagulated with very little change in reaction.
Later the coagulum is digested.

3. No gas in either glucose or lactose.

4. No indol.

5. All are Gram positive.

6. All digest blood-serum.

63

64 STUDY AND IDENTIFICATION OF BACTERIA

B. Grow only anaerobically.

1. Rods very little swollen by centrally situated spores.

a. Motile. B. cedematis maligni. (Gram negative.)

b. Non motile. B. aerogenes capsulatus. (Capsule.)

2. Spores tend to be situated between center and end.

a. No liquefaction of gelatin. B. butyricus.

b. Gelatin liquefied slowly.

B. botulinus. Milk not coagulated.

B. anthracis symptomatici.

B. enteritidis sporogenes. Milk coagulated with abundant gas.

c. Gelatin liquefied rapidly. B. cadaveris sporogenes. Very motile.

3. Spores situated at end of rod. Drum-stick sporulation. TETANUS GROUP.

The following table taken from Lehmann and Neumann, based
on pathogenic effects, is of great practical value. After inoculation
of some animal subcutaneously with the suspected material we have:

A. No particular symptoms at site of inoculation.
Absorption of the soluble toxin causing:

(1) General symptoms of tetanus. B. tetani.

(2) Botulism poisoning symptoms. Pupillary symptoms. Paralysis of
tongue and pharynx. Cardiac and respiratory failure.

B. Local symptoms marked at site of inoculation. Hemorrhagic emphysematous
oedema.

(1) Motile.

(a) Gram negative. B. cedematis maligni.

(b) Gram positive. B. anthracis symptomatici.

(2) Nonmotile.

B. aerogenes capsulatus or B. phlegmonis emphysematosse.

SPORE-BEARING AEROBES.

Bacillus anthracis (Pollender discovered 1849. Davaine recog-
nized nature 1863. Koch proved 1876). — Of the aerobic spore-bearing
bacilli this is the only one of particular medical importance.

Anthrax is an important disease in domestic animals, especially
sheep and cattle. The characteristic postmortem change in animals
is the greatly enlarged, friable, mushy spleen. Man is much less suscep-
tible than these animals, but is more so than the goat, horse, or pig.
The Algerian sheep has a high degree of immunity, as has the white rat.
The brown rat is quite susceptible. The disease in man chiefly occurs
among those working with hides, wool, or meat of infected cattle. The
two chief types in man are: i. Malignant pustule and 2. Woolsorter's
disease. An intestinal type is also recognized. Malignant pustule

ANTHRAX 65

results from the inoculation of an abrasion or cut; thus it frequently
shows on the arms and the backs of those unloading hides. It first
appears as a pimple, the center of which becomes vesicular, then
necrotic.

FIG. 1 6. — Anthrax bacilli. Cover-glass has been pressed on a colony and then fixed
and stained. (Kolle and Wasscrmann.)

FIG. 17. — Anthrax bacilli growing in a chain and exhibiting spores.
(Kolle and Wasserman.)

A ring of vesicles surrounds this central eschar and a zone of congestion, the
vesicles. The lymphatics soon become inflamed as well as neighboring glands.
If the^mstule is not excised and death occurs, there is not much enlargement of the
spleen and the bacteria are not abundant in the kidneys, etc., as with animals.
Man seems to die from a toxaemia rather than a septicaemia.

In woolsorter's disease there is great swelling and oedema of the bronchial and
mediastinal glands. The lungs show oedema, which about the bronchi is hemorrhagic.

5

66 STUDY AND IDENTIFICATION OF BACTERIA

The bacillus is 5 to 8/* by i to i i/2//. It has square cut or concave
ends and is often found in chains. It is Gram positive. Colonies, by
interlacing waves of strings of bacteria, show Medusa head appearance.
For cultural characteristics see key. Spores develop best at a tempera-
ture of 30° C. They stain with difficulty.

Stiles thinks that animals are infected by eating the bones of animals which have
died of anthrax, cutting buccal mucous membrane, and so becoming infected.
Spores do not form in an intact animal body, but they do form after a postmortem
or the disintegration of the body by maggots. For this reason it is better not to
open up the body of the animal, but to make the diagnosis by cutting off an ear.
Dried spores will live for years and will withstand boiling temperature for hours.

In vaccinating animals against anthrax, Pasteur used two vaccines. The first
is attenuated fifteen days at 42.5° C. The second, attenuated for only ten days, is
given twelve days later.

FIG. 18. — Bacillus anthracis in blood of rabbit. (Coplin.}

In taking material from a malignant pustule before excision, be
careful not to manipulate it roughly, as bacteria may enter the circula-
tion. Make cover-glass preparations, staining by Gram. Make
culture on agar. Blood cultures are usually only positive later in the
disease. Inoculate a guinea-pig or a mouse subcutaneously.

The guinea-pig dies in about forty-eight hours and shows an cedematous gelatin-
ous exudate at site of inoculation. The blood is black and swarms with anthrax
bacilli. It is the best example of a septicaemia.

An organism with a central spore and morphologically resembling
B. anthracis, but motile, has been reported as occurring in the stools
of pellagrins. Gelatine stabs show a cup-shaped liquefaction in about

SPORE BEARING ANAEROBES

67

five days. No change in milk. The colonies are slimy and opaque.
The organism is said to be agglutinated by the serum of pellagra cases.
The name B. MAYDIS has been given to it.

SPORE-BEARING ANAEROBES.

There are three very important pathogens in this group — that of
malignant oedema; that of botulism, and the organism of tetanus.

The B. enteritidis sporogenes is of importance in connection with
indications of faecal contamination of water. In connection with B.
aerogenes capsulatus, there is some
question as to whether the exten-
sive oedema produced by it may
not usually be from a terminal or
cadaveric infection. At any rate
necrotic material seems necessary.

It should be stated that our
knowledge of the differential cul-
tural characteristics of anaerobes
is unsatisfactory. The exact
methods which are in use for
aerobes have not been applied
to anaerobic organisms.

To Cultivate Anaerobes. —
Probably the apparatus giving
the most perfect anaerobic con-
ditions is the Novy jar, in which
the air has been replaced by
hydrogen. The difficulties attending the method are:

FIG. 19. — Novy jar.

1. Unless a special apparatus (Kipp's) is at hand, there may be difficulty in pre-
venting the sulphuric acid from frothing over when poured on the zinc. It
should, at first, be added in small quantities at a time — well diluted (i to 6).

2. Various wash-bottles are required: one containing silver nitrate solution for
traces of AsHs and one with lead acetate for H^S and another with pyrogallic
acid and caustic soda for any oxygen that may come over.

3. Mixtures of hydrogen and air explode. Consequently, in determining whether
all air has been expelled and in its place an atmosphere of hydrogen exists, it

is necessary to see if the escaping gas burns with a blue flame. Unless this
is collected in a test-tube and examined, we may have an explosion.

4. Except in a large laboratory, where the apparatus is set up and ready for use,
too much time would be required.

5. Simpler methods appear to give as good results.

68

STUDY AND IDENTIFICATION OF BACTERIA

In Tarozzi's method, pieces of fresh sterile organs are added to
bouillon. Pieces of kidney, liver, or spleen are best suited. After
adding the tissue the media may be heated to 80° C. for a few minutes
without interfering with the anaerobic condition producing properties
of the fresh tissues. This method is practically the same as that
recommended by Smith (see Tetanus). This is also a feature of
Noguchi's method of culturing Treponema pallidum,

The Method of Liborius.

In this it is necessary to have a test-tube containing about 4 inches of a i%
glucose agar. Glucose acts as a reducing agent and furnishes energy. It is con-
venient to add about i/io of i% of sulphindigo-
tate of soda; the loss of the blue color at the site
of the colony enabling us to pick them out. The
tube of agar should be boiled just before using to
expel remaining oxygen from the tube. Now
rapidly bring down the temperature to about 42°
C., by placing the tube in cold water, and inocu-
late the material to be examined. A second or
third tube may be inoculated from the first, just
as in ordinary diluting methods for plate cultures.
Having inoculated the tubes, solidify them as
quickly as possible, using tap water or ice-water.
The anaerobic growth develops in the depths of
the medium. Some pour a little sterile vaseline
or paraffin or additional agar on the top of the
medium in the tube as a seal 'from the air.
Others have recommended the inoculation of
some aerobe, as B. prodigiosus, on the surface.
This latter method is not advisable. A deep stab
culture is often sufficient.

The Method of Buchner.

In this method one gram each of pyrogallic
acid and caustic potash or soda for every 100 c.c.
of space in the vessel containing the culture is
used to absorb the oxygen. It is convenient to
drop in the pyrogallic acid; then put in place the
inoculated tubes or plates; then quickly pouring
in the amount of caustic soda, in a 10% aqueous
solution, to immediately close the containing
vessel. A large test-tube in which a smaller one containing the inoculated medium
is placed, and which may be closed by a rubber stopper, is very convenient. A
good rubber-band fruit jar is satisfactory. A desiccator may be used for plates.

FIG. 20. — Arrangement of
tubes for cultivation of anae-
robes by Buchner's method.

(Williams.)

MALIGNANT (EDEMA 69

An excellent method for anaerobic plates, either in a desiccator with the pyrogallic
acid and caustic soda, or less satisfactorily in the open air, is to sterilize the parts
of the Petri dish inverted; that is, the smaller part is put bottom downward in
the inverted cover (as one would set one tumbler in another). Then, in using,
unwrap the Petri dish, lift up the inner part, pour in the inoculated medium
into the upturned cover. Then immediately press down the inner dish, spreading
out a thin film of the medium between the two bottoms.

J. H. Wright's Method.

Make a deep stab culture in glucose agar or gelatin, preferably boiling the media
before inoculating. Then flame the cotton plug and press it down into the tube so
that the top lies about three-fourths of an inch below the mouth of the test-tube.
Next fill in about one-fourth of an inch with pyrogallic acid; then add 2 or 3 c.c. of
a 10% solution of caustic soda, and quickly insert a rubber stopper. This method
is one of the most convenient and practical, and is to be strongly recommended.

Method of Vignal.

In this a section of glass tubing (1/4 in.) is drawn out at either end, as in making a
bacteriological pipette, with a mouth-piece containing a cotton plug. The liquid
agar or gelatin is then inoculated and the medium drawn up into the tube. In a
very small flame the capillary narrowings are sealed off, and we have inside the tube
very satisfactory anaerobic conditions. To get at the colonies, file a place on the
tube and break at this point.

To obtain material for examination and isolation in pure culture from the deep
agar stab-tube, it is best to loosen the medium at the sides of the tube with a heated
platinum spud or a flattened copper wire. Then shake the mass out into a sterile
Petri dish. It is dangerous to break the tubes with a hammer as some do.

A Combination Method.

Recently as shown in the illustration in Fig. 7, I have been combining various
methods so that very satisfactory anaerobic conditions are obtained. First, a
deep agar stab of freshly sterilized glucose agar is made. The surface of this is then
covered with sterile paraffin oil. The proper amount of pyrogallic acid is then
deposited in a salt mouth bottle. The rubber stopper with the glass and rubber
tubing is then firmly pushed in and connection made with a filter pump.

In five to ten minutes almost all the air will be exhausted when the Hofmann
clamp is screwed up tight and the bottle disconnected from the vacuum pump.
The glass tubing end is then inserted into a graduate holding 10% caustic soda
solution, the Hofmann clamp unscrewed, and the necessary amount of caustic soda
having been run in, as noted under Buchner method, we again close the screw clamp
and incubate.

B. oedematis maligni (Pasteur, 1877). — This is the vibrion septique
of Pasteur. It is found in garden soil and in street sweepings. It is

7O STUDY AND IDENTIFICATION OF BACTERIA

the cause of an acute cellular necrosis attended with serous sanguino-
lent exudation and with more or less emphysema. The organism
only becomes generalized in the blood about the time of death and
postmortem. Therefore, it is not a septicaemia, as is anthrax. The
bacillus is an organism about the size of anthrax (y/Jt by 0.8), but is nar-
rower and does not have the same square cut or dimpled ends. Further-
more, it is motile, Gram negative and an anaerobe. The guinea-pig
is very susceptible, and about the time of death and postmortem there
may be seen long flexile motile filaments, 1 5 to 40 /* long, which move
among the blood cells as a serpent in the grass (Pasteur).

In cultures it grows out very slightly from the line of stab, giving a jagged
granular line, differing from tetanus. Spores form best at 37° C. — requiring about
forty-eight hours. It liquefies gelatin. In examining an exudate from a suspected
case, note the presence of spores centrally situated. Inoculate a guinea-pig. Death
occurs in about two days. There is intense hemorrhagic emphysematous oedema at
the site of inoculation, the cedematous fluid however does not show spores. The
bacilli do not appear in the blood until about the time of death and it is an assistance
in diagnosis to put the dead body of the guinea-pig in the incubator for a few hours.
The subcutaneous tissue contains fluid and gas. There is present the foul odor of
an anaerobe. Examine for the long filaments showing flowing motility. Be sure
to stain by Gram. (Negative.) For cultures, heat the material (either from a
wound or from a guinea-pig) which shows spores to a temperature of 80° C. for
from fifteen minutes to one hour. Then inoculate glucose agar stab culture and grow
anaerobically. Courmont differentiates anthrax from malignant oedema by in-
jecting into ear-vein of rabbit. The injection of malignant oedema in this way,
instead of subcutaneously, tends to immunize.

B. botulinus (Van Ermengem, 1896). — This is the organism which
produces botulism, a form of meat poisoning. It is a spore-bearing
anaerobe and must not be confused with another organism associated
with meat poisoning — the B. enteritidis of Gartner. The spores are
at the end and are not very resistant; a temperature of 80° C. often
killing them.

In botulism the meat becomes infected after the animal has been slaughtered;
in Gartner meat poisoning the cow meat was infected at the time of slaughter — it
was from a sick animal. Thorough cooking of the meat protects against botulism
but not certainly against Gartner meat poisoning.

There are dysphagia, paralysis of eye-muscles, and cardiac and respiratory
symptoms (medulla). The symptoms are due to the elaboration of a soluble toxin
of the same nature as that of diphtheria and tetanus. There is no fever and con-
sciousness is preserved.

An antitoxin which it is stated has therapeutic value in botulism has been

BOTULISM 71

prepared in the usual way by Kempner. Without serum treatment death occurs in
about 40% of cases and takes place between twenty-four and forj^eight hours.

The bacillus has been isolated from sausage and ham. It is a large bacillus —
5 to io/iX ij". It is slightly motile and stains by Gram. It produces gas in glucose

Fig. 21. — Bacillus of botulism, (Kolle and Wassermann.}

FIG. 22.— Symptomatic anthrax (Rauschbrand) bacilli showing spores. (Kolle

and Wassermann.)

media. It grows best at 22° and only slightly at 37° — hence it is dangerous only
irom its soluble toxin, the bacilli not developing to any extent in the body.

For this reason botulism patients are not a source of danger, it is the infected
meat alone which causes the disease. On the contrary where the meat poisoning
is due to the Gartner or paratyphoid group infection may take place from the patient's
discharges.

72 STUDY AND IDENTIFICATION OF BACTERIA

When the toxin is introduced, it requires a period of incubation of
twelve to twenty hours. Symptoms of gastrointestinal disorder may
come on shortly after the ingestion of the toxin containing food, these
however are not the specific manifestations, as are the eye symp-
toms, etc.

An important point is that ham may not appear decomposed and yet contain
many bacilli and much toxin. It is a very potent toxin — as little as one-thousandth
of a c.c. may kill a guinea-p'ig. In man the toxin is apparently absorbed from the
alimentary canal. For diagnosis inject an infusion of the ham or sausage which
was eaten of into a guinea-pig, and characteristic pupillary symptoms with death
by cardiac and respiratory failure will result.

Cultures may be made in glucose agar.

The culture is disrupted by gas. Incubation at room temperature and in the
dark is necessary. There is a rancid odor. The characteristic point is the pro-
duction of a powerful soluble toxin which produces symptoms when no bacilli are
present.

B. tetani (Nicolaier, 1885; Kitasato, 1889).— This is the most
important organism of the anaerobic spore bearers. Its characteristics
are the tetanic symptoms produced by the toxin and the strictly termi-
nal drum-stick spores. Spores are difficult to find in material from
wounds infected with tetanus, but readily develop in cultures. Prior to
the formation of spores the organism is a long thin bacillus (4 X 0.4^) . It
is motile and Gram positive. It liquefies gelatin slowly and does not
coagulate milk.

Theobald Smith recommends growing it in fermentation tubes containing
ordinary bouillon, but to which a piece of the liver or spleen of a rabbit or guinea-pig
has been introduced at the junction of the closed arm and the open bulb. By this
method spores develop rapidly in from twenty-four to thirty-six hours. Sporulation
is most rapid at 37° C. As there is always liability to postmortem invasion of
viscera by ordinary saprophytes, Smith recommends that great care be taken not
to handle the animal roughly in chloroforming and in pinching off pieces of the
organ at autopsy. The animal must be healthy, and the tubes to which the piece
of tissue is added must be proven sterile by incubation. Smith calls attention to
the uncertainty of the temperature at which tetanus spores are killed. He shows
that some require temperature only possible with an autoclave. In view of the
danger of tetanus, it is advisable to carefully autoclave all material going into bac-
terial vaccines, such as salt solution, bottles for holding, etc.

Tetanus seems to grow better in symbiosis with aerobes; hence a
lacerated dirty wound with its probable contamination with various
cocci, etc., and its difficulty of sterilization, offers a favorable soil. The
tetanus bacillus gives rise to one of the most powerful poisons known;

TETANUS 73

it is a soluble toxin like diphtheria toxin, and it is estimated that 1/300
of a grain is fatal for man.

Rosenau has established an antitoxin unit for tetanus which has the power of
neutralizing one thousand minimal lethal doses. Practically, it is ten times the
least quantity of antitetanic serum necessary to protect the life of a 350 grams
guinea-pig from a test dose of tetanus toxin furnished by the hygienic laboratory.
(The necessity of some definite unit is apparent when tests have shown that serum
stated to contain six million units per c.c. only had a value of 90 of the official
American units.) Consequently it is a unit ten times as neutralizing as the diph-
theria antitoxin one. The antitoxin of tetanus is less efficient than that of diphtheria
for the following reasons:

i. There is about three times as great affinity in vitro between diphtheria toxin
and antitoxin as is the case with tetanus.

FIG. 23. — Tetanus bacilli showing end spores. (Kolle and Wassermann.)

2. The tetanus toxin has greater affinity for nerve cells than for antitoxin.

3. Treatment with antitoxin is successful after symp.toms of diphtheria appear.
With tetanus it is almost hopeless after the disease shows itself. Hence the impor-
tance of the early bacteriological examination of material from a suspicious wound
(rusty nail).

4. The tetanus toxin ascends by way of the axis cylinder, and the antitoxin
being in the circulating fluids cannot reach it, whereas with diphtheria both toxin
and antitoxin are in the circulation. Diphtheria also selects the cells of paren-
chymatous and lymphatic organs which are more tolerant of injury than the nerve
cells. The dose of tetanus antitoxin as a prophylactic is 1500 units; as a curative
agent 5000 to 20,000 units. Recent experience shows that it should be injected
intravenously when symptoms have manifested themselves.

That the disease is due to toxin is shown not only experimentally, but also if
spores are carefully freed of all toxin by washing, and then introduced they do not
cause tetanus — the polymorphonuclears engulfing them. The importance of the

74 STUDY AND IDENTIFICATION OF BACTERIA

presence of ordinary pus cocci in a tetanus wound may be that the activity of the
leucocytes in phagocytizing them allows the tetanus bacillus to escape phagocytosis.

This would also explain the importance of nec-
rotic tissue in a lacerated wound — the phagocytes
taking this up instead of tetanus bacilli. The
toxin is digested by the alimentary canal juices
and infection by that atrium is improbable.
The infection occurs especially through skin
wounds, and also from those of mucous mem-
brane. While tetanus is like diphtheria, a dis-
ease in which the bacilli are localized and do not
spread, yet recently Richardson has obtained
tetanus bacilli in pure culture from the tributary
lymphatic glands of a "rusty nail" wound of
foot. The cultures inoculated into root of tail
of a white rat caused the rat's death in forty-
eight hours with typical "seal gait" attitude of
tetanus in rats.

The usual period before symptoms occur is
fifteen days. The shorter the period of incuba-
tion, the more probably fatal the disease. The
horse is the most susceptible animal, next the
guinea-pig, then the mouse. Fowls are practi-
cally immune.

In examining for tetanus, scrape out
the material from the suspected wound
with a sterile Volkmann spoon and put
it in a tube containing blood-serum.
Place this in an incubator. We have
here the principle of the septic tank —
the cocci and other aerobes grow lux-
uriantly and enable the tetanus bacillus
to develop. From day to day smell the
culture, and if an odor similar to the
penetrating, sour, foul smell of the stools
of a man who has been on a debauch be
detected, it is suspicious. The nonde-
velopment of a foul odor is against
FIG. 24.— B. aerogenes capsu- tetanus. Also make smears from the
latus agar culture showing gas material and examine for drum-stick
formation. (Williams.) T, ,, f

spores. If these are found, heat the

material to 80° C. for one-half hour, to kill nonsporing aerobes and
facultative anaerobes, and then inoculate a deep glucose agar tube

THE GAS BACILLUS 75

and cultivate by Wright's method. The fusiform lateral outgrowth
about the middle of the stab is characteristic.

A more rapid method is to draw up the material, provided it be pus (tissue
scrapings may be emulsified in sterile salt solution) into a capillary bulb pipette.
Then seal off the end and heat the capillary bulb pipette and its contents in a water
bath at 80° C. for 15 minutes. Next break off the sealed tip and stick the pipette
into a deep tube of glucose agar. When the point reaches the bottom, force out the
material along the line of the stab as the pipette is withdrawn. Cover the surface of
the agar with sterile liquid petrolatum and incubate. Better anaerobic conditions
obtain where the Buchner or Wright method is employed.

Tetanus produces no gas. Material for examination is best obtained with a bulb
pipette (containing a little sterile salt solution) which is plunged into the agar and the
salt solution forced out and drawn in where a proper growth is noted.

Spores form in thirty-six to forty-eight hours. In injecting test animals it is
advisable to divide the material to be injected into two portions; one animal is
injected with the material alone, the second animal with tetanus antitoxin at the
same time the material is injected. Only the first animal dies with tetanic symptoms.

B. aerogenes capsulatus (Welch, 1891).— This bacillus is appar-
ently widely distributed. It is possibly the same organism as Klein's
B. enteritidis sporogenes, which is constantly present in faeces. It is a
large capsulated organism, which does not form chains. Spores are
produced on blood-serum. These are frequently absent on other
media. It is questioned whether its pathogenicity is other than ex-
ceedingly feeble, the presence of the bacillus in emphysematous find-
ings at postmortem being attributed to terminal or cadaveric invasion.

Cases, however, in the Philippines, have been reported following carabao horn
wounds, in which most serious and fatal results attended emphysematous lesions
showing this bacillus. The isolation of a Gram positive bacillus from a lacerated
wound discharge, even in the absence of emphysema, is almost diagnostic.

In milk cultures we have coagulation and from the subsequent development
of gas the disruption of the coagulum into shreds. An odor of butyric acid is
developed.

Cultures in litmus milk show these shreds plastered against the sides of the tube
and showing a pink color.

It is the cause of "foamy organs" occasionally present at autopsy.

The best method of diagnosis is to inoculate the culture or material
into the ear vein of a rabbit, kill it and then incubate the body at 37° C.
Gas is generated in the organs in a few hours.

Achalme isolated a large bacillus from a fatal case of rheumatism which is now
considered as having no relation to acute rheumatism and which was probably B.
aerogenes capsulatus.

Kendall has called attention to the importance of this organism in a certain
proportion of cases of summer diarrhcea of infants. (See under chapter on faeces.)

CHAPTER VII.

STUDY AND IDENTIFICATION OF BACTERIA. MYCO-

BACTERIA AND CORYNEBACTERIA. KEY

AND NOTES.

Key for Bacilli. — Having branching characteristics, as shown by
parallelism, branching, curving forms, V-shapes, clubbing at ends, seg-
mental staining, etc.

/ Cultures more or less wrinkled and dry.
Acid-fast. Mycobactermm. \ ,,

(^ More like moulds.

I. Grow rapidly on ordinary media at room temperature.

Examples: Timothy grass bacillus of Moeller (B. phlei).

Mist bacillus. Butter bacilli as reported by (i) Rabinowitsch
and (2) Petri.

II. Only grow at about incubator temperature. Scanty growth or none at all

on ordinary media. Media of preference are: (a) solidified blood-serum,
(b) glycerine agar, (c) glycerine potato and (d) egg media.

1. Cultures fairly moist, luxuriant, and flat. Op. temp. 43° C.
a. Bacillus of avian tuberculosis.

2. Cultures scanty, wrinkled, and dry. Appear in ten to fourteen days. Op.
temp. 38° C. Bacilli longer, narrower, more regular in outline and staining
than bovine; vacuolation more marked (2.5^). Smear from organs of
inoculated guinea-pig shows few bacilli. Less virulent for rabbits.

a. Bacillus of human tuberculosis.

Cultures as above, but even more scanty. Bacilli shorter, thicker, less
vacuolated (1.5^). Smear from organs of guinea-pig shows many bacilli.

b. Bovine tubercle bacilli.

3. Very difficult to cultivate (Czaplewski).
Smegma bacilli of various animals.

III. Noncultivable by ordinary methods. Cultivable in symbiosis with amoebae.
(Clegg.) Duval cultivated an acid-fast bacillus on N.N.N. medium con-
taining i% glycerine. Bayon cultivated on placental juice glycerine agar a
slightly acid-fast diphtheroid which changed to acid fast in peritoneum of
mouse. Bayon's organism thought to be similar to Kedrowsky's diphtheroid
of leprosy.

i. B. leprae. Found chiefly in nasal mucus and in juice from lepra tubercles.
Less often in nerve leprosy.

ACID FAST BACILLI 77

f Colonies more flat and moist.
Nonacid-fast. Corynebactermm. < _..

( Like other bacteria.

I. Do not stain by Gram's method.

i. B. mallei (Glanders). Characteristic culture is that on potato. Growth
like layer of honey by third day. Becomes darker in color, until on eighth
day is reddish-brown or opaque with greenish-yellow margin.

II. Gram positive.

1. Very luxuriant growth on ordinary media. Colonies often yellow to brown-
ish. B. pseudodiphtheriae. Shorter, thicker and stain uniformly.

2. Moderate growth on ordinary media. B. diphtheriae. Best media are
blood-serum (Loffler's) or glycerine agar. Has metachromatic granules at
poles.

3. Scanty and slow growth on nutrient media. B. xerosis.

THE GROUP or ACID-FAST BRANCHING BACILLI.

There is a large and ever-increasing number of organisms which
have the same staining reactions as the tubercle bacilli, but which differ
in four important essentials of:

1. Growing readily on any media.

2. Showing more or less abundant growth or colonies in twenty-
four hours.

3. Having no pathogenic power for guinea-pigs when inoculated
subcutaneously.

4. Not requiring body temperature for development, but growing
at room temperature.

Many of these organisms, if injected intraperitoneally into guinea-pigs will pro-
duce a peritonitis with false membrane. Some also produce granulation tissue
nodules which may be confused with true tubercles. For this reason it is well to
study the lesions in experimental tuberculosis in the guinea-pig. Injected subcuta-
neously, on either or both sides of the posterior abdomen with the needle pointing
toward the inguinal glands, we may have caseation and ulceration at the site of
inoculation. The glands in relation enlarge and caseate. Smears from these show
T. B. The marked and characteristic change is the enormous enlargement of the
spleen, which is studded with grayish and yellow tubercles. Make smears and cul-
tures from the spleen. The death of the guinea-pig usually occurs in about two
months. The lesions may be looked for at three to five weeks.

These nonpathogenic acid-fast bacilli are of greatest importance by reason of
their possible confusion with the true tubercle bacilli. Their colonies correspond
more or less with different types of tubercle bacilli colonies, being either dry and wrint
kled like human, or moist and irregularly flat as avian. Eventually the mois-
colonies become dry and wrinkled. They have been isolated from:

1. Butter and milk.

2. From grasses, especially in timothy grass infusion.

78

STUDY AND IDENTIFICATION OF BACTERIA

3. In various excretions of animals, as in
dung, urine, etc.

4. Normally in man — from skin, nasal
mucus, cerumen, and tonsillar exudate.

It is important to remember that such organisms
have very rarely been reported from pulmonary
lesions, and when present they have been considered
as probably causative.

The present view is that the finding of tubercle
bacilli in sputum has practically as great value as it
had before we knew of these various acid-fast bacteria.

Tubercle Bacillus (Koch, 1882). — This is
a rather long, narrow rod, 3X0.3;*. In the
human type it tends to show a beaded appear-
ance, this not being due to spores, however.
In the bovine type the staining is more solid,
the organism shorter and thicker, and shows
even a more scanty growth than human T. B.
It has been established that many of the
tuberculous affections of man, especially those
of the skin, bone, and mesenteric glands, are
of the bovine type, while, as a rule, pulmo-
nary and laryngeal lesions are of the human
type. Experiments by various commissions
in different countries have shown that human
and bovine types are very closely related and
that not only may a bovine strain affect man,
but that human T. B. may infect young
calves. As bacilli of the bovine type have
frequently been reported in intestinal and
mesenteric tuberculosis of children it shows
the importance of sterilizing cows' milk.
Koch considers human infection from bovine
sources as of very rare occurrence.

FIG. 25. — Bacillus tuber-
culosis; glycerine agar-agar
culture, several months old.
(Curtis.)

Although Kossel has found only two cases of bovine
T. B. in 709 cases of pulmonary tuberculosis yet for
the other types the findings are different. Leaving
out of consideration the frequency of infections with

bovine T. B. in children, recent statistics have shown that in adults about 4% of
cervical adenitis, 22% of tabes mesenterica and 3.5% of bone and joint tuberculosis
are due to bovine strains of T. B.

TUBERCULOSIS 79

The British Royal Commission in its final report of July, 1911, considered three
types of T. B.

I. The bovine type belonging to the natural tuberculosis of cattle.
II. The human type. The type more generally found in man.
III. The avian type, belonging to natural tuberculosis of fowls.

The bovine type grows slowly on serum and at the end of two to three weeks
shows only a thin grayish uniform growth which is not wrinkled and not pigmented.
The human type grows more rapidly and tends to become wrinkled and pigmented.
Subcutaneous inoculation of 50 mg. of culture into the neck of calves produced
generalized tuberculosis. A similar injection of human T. B. does not cause general-
ized tuberculosis but only an encapsulated local lesion.

Intravenous injection of o.oi to o.i mg. of bovine T. B. into rabbits causes general
miliary tuberculosis and death within five weeks. With human T. B. in doses of
o.i to i.o mg., similarly injected, the majority of rabbits live for three months.

Subcutaneous injection of 10 mg. bovine T. B. causes death in 28 to 101 days.
Similar injection of human T. B. in doses up to 100 mg. did not kill the rabbits after
periods of from 94 to 725 days. The duration of life in injected guinea-pigs is longer
with human than with bovine T. B.

Subcutaneous injections of bovine T. B. into cats produces generalized tubercu-
losis while the cat is resistant to human T. B. thus given.

Recent statistics (Beitzke) show tuberculous lesions in 58% of
adults at autopsy — Naegli's figures were about 90%.

It is a question whether the avian type is absolutely distinct; many
experiments having indicated the impossibility of infecting fowls
with human T. B. Nocard, by inserting collodion sacs containing
bouillon suspensions of human T. B., claims to have changed these to
the avian type. The avian type grows at 43° C. fairly luxuriantly ras a
moist, more or less spreading culture. It grows much better on gly-
cerinated agar than on serum. Morphologically they are like the
human type, but show less tendency to form compact masses. Very
pleomorphic. Have been reported from sputum of man (doubtful).

Fowls become infected by intravenous or subcutaneous injection or as the result
of feeding. After feeding the lesions are chiefly of the alimentary tract; after in-
jections, of spleen, liver and lungs. Avian T. B. is more virulent for rabbits than
human T. B. but less so than bovine T. B. The mouse is the only animal besides the
rabbit in which avian T. B. can cause a generalized tuberculosis. The conclusions
are that there is no danger to man from avian T. B. With the bovine type it is
quite different as nearly one-half of the deaths in young children from abdominal
tuberculosis were due to bovine T. B. and to that type alone. Not only in children,
but in adolescents suffering from cervical gland tuberculosis, a large proportion were
caused by bovine types. The bovine type is also an important factor in lupus.

There is also a fish tuberculosis. This organism grows much more

8o STUDY AND IDENTIFICATION OF BACTERIA

rapidly than the other types (three to four days), and grows best at 24°
C., growth ceasing at 36° C. The colonies are round and moist.

It is certain that many of the symptoms usually noted in the tuberculous are due
to secondary infections. Pettit, by careful blood cultures, obtained the pneumo-
coccus in 24 cases and the streptococcus in 36 cases out of 130 cases studied. He
used from 5 to 20 c.c. of blood from the vein. Positive blood cultures were obtained
in 68% of far-advanced cases, 45% of advanced cases and 16% of incipient cases.

The best culture medium for primary cultures is blood-serum or,
better, a mixture of yolk of egg and glycerine agar. Dorset's egg me-
dium is also used. In subcultures, either glycerine agar, glycerine
potato, or glycerine bouillon make good media. In inoculating media
from tuberculous material, as, say, from a tuberculous gland or, more
practically, from the spleen of a guinea-pig, the material must be
thoroughly disintegrated or rubbed on the surface of the media so that
individual bacilli may rest on the surface of the culture media. In
growing in flasks in glycerine bouillon a surface growth is desired. The
cylindrical flask of Koch gives a better support to the pellicle than an
Erlenmeyer one. In inoculating, a scale of such a surface growth or a
grain from the growth on a slant should be deposited on the surface of
the glycerine bouillon in the flask.

Inasmuch as the filtrate from cultures has little texic effect, the
poison is assumed to be intracellular.

Koch's old tuberculin, which was simply a concentrated glycerine bouillon
culture, is now principally used in veterinary diagnosis. It was prepared as follows:

After four to six weeks the surface growth begins to sink to the bottom of the
flask. This fully developed culture is evaporated over a water bath at 80° C. to
one-tenth the original volume. It is then filtered, the final product containing
about 40% of glycerine.

Koch's tuberculin "R" or new tuberculin was introduced in 1897. In this,
virulent bacilli are dried in vacuo, ground up in water and centrifuged. The first
supernatant fluid (T. O.) is discarded. Subsequent trituration and centrifugaliza-
tion, preserving each time the supernatant suspension, gives the new tuberculin.
It has been found at times to contain virulent T. B.

Koch's bazillen emulsion has been more recently introduced by Koch (1901).

This is simply a suspension of ground up bacilli in 20% glycerine solution.
Another preparation is the bouillon filtrate of Denys. This is the unheated filtrate
of broth cultures of human T. B. It contains 1/4% phenol.

In the use of T. R. and of bazillen emulsion, Sir A. Wright recommends doses
of 1/4000 of a milligram, and he rarely goes beyond i/iooo of a milligram in treatment.
These products come in i c.c. bottles containing 5 mg. of bacillary material. It is
convenient to remove 2/10 of a c.c., containing i mg. Add this to 10 c.c. of glycerine
salt solution with 1/4% of lysol. Each c.c. contains i/io mg. One c.c. of this stock

DIAGNOSIS OF TUBERCULOSIS 8 1

solution added to 99 c.c. of salt solution, with 1/4% of lysol, would give a working
solution, each c.c. of which would contain i/iooo mg. of tuberculin.

For diagnostic reactions we have, besides the method of injecting
tuberculin and noting presence or absence of fever, six more recent
diagnostic tuberculin tests: i. Variations in opsonic index. 2. Instil-
lation into one eye of a drop of 1/2% or i% solution of purified tuber-
culin. Reaction is shown by redness, especially of inner canthus, in
twelve to twenty-four hours (Calmette). A previous instillation may
sensitize a nontuberculous case and a second application of the drop may
give an erroneous diagnosis. 3. The cutaneous inoculation method
(similar to ordinary vaccination methods). Scarify two small areas on
the arm (i/io inch in diameter), about 2 inches apart. Rub in one
a drop of old tuberculin, in the other a drop of 25% tuberculin. As a
control scarify a spot midway and to one side of the others and rub in
one drop of 0.5% carbolic glycerine. The appearance of bright red
papules in twenty-four hours indicates reaction (von Pirquet). This is
the method of preference. 4. Intracutaneous inoculation of one drop
of a i-iooo, i-ioo or i-io dilution of old tuberculin (Mantoux and
Moussu). Webb recommends hypodermic needle points which have
been dipped in old tuberculin and the points allowed to dry. A drop
of water is placed on the skin and the needle points having been mois-
tened in it are plunged through the skin and withdrawn with a twist.
A definite lump shows a positive reaction. 5. Ointment tuberculin
test. Rub in 50% ointment of tuberculin in lanolin. Reaction is
shown by dermatitis with reddened papules in twenty-four to forty-
eight hours (Moro). 6. Inoculation of bovine and human tuberculin
to diagnose type of infection (Detre). Of questionable value.

Ebright injects the suspected material into the subcutaneous tissue of one side
of the abdomen of three guinea-pigs. At the end of one week an injection into the
other side of the abdomen of one of the guinea-pigs of 1/4 c.c. tuberculin is given.
Twenty-four hours later smears are made from the original site of inoculation and
examined for tubercle bacilli. If negative this is repeated with a second guinea-
pig at the end of the second week and finally at the end of the third week with the
third guinea-pig.

Bloch's method is to damage the lymphatic glands in the inguinal region by
squeezing the tissue between the fingers. Injections made there of tuberculosis
material show abundant tubercle bacilli in these damaged glands in ten to twelve
days.

In staining it is better to use the Ziehl-Neelsen method, decolorizing
with 3% hydrochloric acid in 95% alcohol. The alcohol, for all prac-
6

82 STUDY AND IDENTIFICATION OF BACTERIA

tical purposes, enables us to eliminate the smegma and similar bacilli,
these being decolorized by such treatment. There are two objections
to the Gabbett method, where decolorizer and counterstain are combined :
i. We cannot judge of the degree of decolorization — we are working
in the dark; and 2. the matter of elimination of smegma bacilli is
impossible.

Pappenheim's method, in which corallin and methylene blue are dissolved in
alcohol, does not appear to have an advantage over acid alcohol. As a practical
point when the question of tuberculosis of the genito-urinary tract is involved,
inoculate a guinea-pig with urinary sediment.

It must be remembered that in young cultures of tubercle bacilli many of the
rods are nonacid-fast, taking the blue of the counterstain, while older rods are acid-
fast. This frequently causes suspicion of a contaminated culture.

Discussion has arisen as to the granules of Much. These are considered by Much
as resistant forms while others consider them degeneration forms of tubercle
bacilli. At any rate material containing only these Gram positive granules and no
acid-fast rods may when injected into animals give rise to tuberculosis and acid-
fast bacilli.

The combination of the acid-fast and Gram staining methods as recommended
by Fontes is very satisfactory.

Bacillus Leprae (Hansen, 1874). — This is the cause of leprosy. In
nodular leprosy the organism is readily and in the greatest abundance
found in the juice of the tubercles of the skin, and secretions of
ulcerations of nasal and pharyngeal mucosa.

The earliest lesion is probably a nasal ulcer at the junction of the bony and
cartilaginous septum. Scrapings from this ulcer may give an early diagnosis.

In the skin they are chiefly found in the derma packed in the so-called lepra
cells. The process is granulomatous but does not show the caseation of tubercu-
losis or the predominant plasma cells of syphilis. The bacilli are also found engulfed
in the endothelial cells lining the lymphatics.

They are also found in the glands in relation to the superficial lesions. The
bacilli are found in smaller numbers in the liver and spleen. In anaesthetic or nerve
leprosy they are found in small numbers in the granuloma tissue which affects the
interstitial connective tissue of the peripheral nerves. Also, rarely, in the anaesthetic
spots of nerve leprosy.

In morphology and staining reactions they are almost identical
with the tubercle bacillus. The main points of distinction are : i. The
fact of the leprosy bacilli being found in enormous numbers, especially
in large vacuolated cells (lepra cells), and lying in the lymph spaces.
They are frequently beaded and lie in masses which have been likened
to a bundle of cigars tied together, so that smears show the bacilli in

LEPROSY 83

prodigious numbers. It may be necessary to examine for long periods
of time, smears made .from tuberculosis lesions of skin before rinding a
single organism. 2. Leprosy bacilli have not been cultivated with
absolute certainty 3. Injected into guinea-pigs, they do not produce
lesions.

There have been many reports of positive findings with the Wassermann test in cases
of tubercular leprosy but such reports are considered doubtful by many. Butler,
in the Philippines, has found that the lepers gave no higher percentage of positive
Wassermann reactions than did the nonleprous native patients at his clinic.

Recently a leprosy-like disease of rats has been reported in which there are two
types: i. A skin affection and 2. a glandular one. In this disease, acid-fast
bacilli, alike in all respects to leprosy bacilli, have been found. Deanehas obtained
a diphtheroid-like organism in culture, which is nonacid-fast. This same finding
has been obtained in cultures considered positive in human leprosy.

Quite recently it has been claimed that the leprosy bacillus has been
cultivated by excising aseptically the subcutaneous portion of lepromata
and dropping the leprous tissue into salt solution, the resulting growth
being like a streptothrix. This was the basis of the Nastin treatment
which is now more or less discredited. In 1909 Clegg reported that by
smearing plates containing amoebae with spleen pulp of lepers (in which
the bacilli were abundant) he obtained growth of an acid-fast bacillus.
He was able to carry on these organisms in subculture for several genera-
tions. A vaccine made from these bacilli does not seem to have been
successful.

Duval states that he has cultivated the lepra bacillus on Novy-Mac-
Neal media to which i% glycerine had been added. He states that
white mice can be inoculated and a pure culture obtained from the
peritoneal cavity. According to Duval it grows best at 32° to 35° C.
and is not killed by a temperature of 60° C. It is most easily obtained
by injecting white mice intraperitoneally with material from leprous
tissues.

Bayon considers the cultures of Duval and Clegg as not shown to
have characteristics which would separate them from the saprophytic
group of acid-fast organisms. He thinks that Kedrowsky's nonacid-
fast diphtheroid is one stage in the typical acid-fast leprosy bacillus.
He states that sera of lepers showed the complement fixation test with
antigen made from cultures isolated by himself as well as with the Ke-
drowsky culture, which tests were negative with Duval's culture.

For diagnosis we should use both smears from the nasal mucus and

84 STUDY AND IDENTIFICATION OF BACTERIA

from ulcerated lepromata or from the scrapings from intact tubercles.
Some advise centrifuging with salt solution, but this is rarely necessary.
The most practical method is by taking a capillary bulb pipette which has been
drawn out into a fine point. The point is broken off as with a Wright's blood sticker
and inserted deep into the corium. The serum which results is drawn up, smeared
out on a slide and stained. The best method is to excise a small portion of skin
or mucous membrane, fix it in absolute alcohol or Zenker's fluid. Cut thin sections
in paraffin. Stain with carbol fuchsin, decolorize with acid alcohol, and then stain
with haematoxylin. This gives the location of the bacilli. This is also a good
method for tuberculous tissues. It is claimed that the B. leprae stains more easily
and loses its color more rapidly than the tubercle bacillus. Some prefer to stain
the leprosy bacillus by Gram's method, it as well as the tubercle bacillus being
Gram positive.

NONACID-FAST BRANCHING BACILLI.

Bacillus mallei (Loffler and Shutz, 1882). — This is the cause of a
rather common disease of horses. When affecting the superficial
lymphatic glands, it is termed " farcy;" when producing ulceration of
nasal mucous membrane, the term " glanders" is used.

In man there are two types of glanders — chronic and acute. In the
chronic form an abrasion becomes infected from contact with glanders
material and an intractable foul discharging ulceration results. This
may persist for months with lymphatic involvement or may become
acute. The acute form may also develop from the start and the cases
are usually diagnosed as pyaemia. Death invariably results in acute
glanders. The bacillus is a narrow, slightly curved rod, about 3X0.3;*.
It is nonmotile and Gram negative. It at times presents a beaded
appearance. In subculture on agar or blood-serum the growth is
somewhat like typhoid but more translucent. In original cultures from
pus or tissues the colonies may not show themselves for forty-eight
hours.

As the organism does not tend to invade the blood stream, blood cultures are
apt to be negative. The glanders bacillus grows best on an acid glycerine agar

( + 2).

The characteristic culture is that on potato. Grown at 37° C., we
have a light brown mucilaginous growth, which by the end of a week
spreads out and takes a cafe au lait color. The potato assumes a dirty-
brown color. This and the inoculation of a guinea-pig are the chief
diagnostic measures. If the material is injected intraperitoneally into
a male guinea-pig, marked swelling of the testicles is noted within

DIPHTHERIA 85

forty-eight hours, at the earliest, to seven to ten days. Cultures
should be made from this swollen testicle as other organisms than
glanders may bring it about.

Only the B. pyocyaneus and cholera vibrios give a similar coloration of potato.
These organisms, however, are easily differentiated. The glanders bacillus is the
most dangerous of laboratory cultures and should be handled with extreme care.

The best stains are carbol thionin and formol fuchsin. In sections stained with
carbol thionin the bacilli are apt to be decolorized by the subsequent passage of
the section through alcohol and xylol. This may be avoided by blotting carefully
after the thionin, then clearing with xylol or some oil and mounting. Nicolle's
tannin method is a good one.

Mallein is prepared by sterilizing cultures that have grown in glycerine bouillon
for about a month by means of heat (100° C.). The dead culture is then filtered
through a Berkefeld filter and the filtrate constitutes mallein. It is chiefly used as
a means of diagnosing the disease in horses. The reaction consists in rise of tempera-
ture and local oedema. The dose is about i c.c.

Agglutination and complement fixation tests are also used for diagnosing glanders.

Bacillus diphtherias (Klebs discovered, 1883; LofHer cultivated,
1884). — The diphtheria bacillus is found not only in the false mem-
brane which is so characteristic of the disease, but may be found in
abundance in the more or less abundant secretions of nose and pharynx.
In studying the epidemiology of diphtheria, especial attention must be
given to the examination of nasal discharges.

Infection of the larynx and middle ear are not very rare. The
mucous membrane of the vagina or the conjunctiva may also be infected.
The B. diphtherias may be in pure culture lying entangled in the fibrin
meshes or contained within leukocytes in the membrane or be asso-
ciated with staphylococci, pneumococci, or especially streptococci.
These latter complicate unfavorably and cause the suppurative con-
ditions about the neck. In fatal cases the diphtheria bacillus may be
found in the lungs. Ordinarily, however, it remains entirely local and
does not get into the circulation or viscera.

It produces soluble absorbable poisons which are designated toxin
in the case of the one responsible for the acute intoxication, paren-
chymatous degeneration and death; and toxone for the poison which
produces cedema at the site of inoculation and postdiphtheritic palsy.
The injection of the soluble poisons alone without the bacilli produces
the symptoms of the disease.

The bacilli tend to appear as slightly curved rods, showing varying
irregularities in staining, as banding or beading, and in particular the

86 STUDY AND IDENTIFICATION OF BACTERIA

presence at either end of small, deeply staining dots (metachromatic
granules). These granules may be seen in an eighteen-hour culture,
but within thirty-six hours, may be abundant.

These are well seen with Loffler's blue, but better with Neisser's method. In
culture they also show swelling at one or both ends or clubbing. In secretions or in
culture they show V-shapes or false branching and, what is most characteristic,
the parallelism — four or five bacilli lying side by side like palisades. Being a Gram

FIG. 26. — Bacillus of diphtheria. (X 1000.) (Williams.)

positive organism while the majority of the other pathogenic bacilli are Gram
negative, it is of greatest importance to stain smears by this method. It is not so
strongly tenacious of the gentian violet as the cocci, so decolorization should not be
carried too far.

The best medium for growing it is Loffler's blood-serum.

An egg medium, made of the whole egg with glucose bouillon as described pre-
viously, is as suitable as Loffler's serum. Coagulated white of egg answers fairly
well, as will a hard-boiled egg — the shell at one end being cracked and the white cut
with a sterile knife. This smooth side is then inoculated and the egg placed cut
side downward in a sherry glass. If an incubator is not at hand a tube may be carried
next the body in a pocket. The bacillus grows better on glycerine agar than on
plain agar. On such plates they appear as small, coarsely granular colonies with a
central dark area. In size the colonies resemble the streptococcus. On blood-
serum the colonies are larger — 1/12 to 1/8 inch in diameter.

The diphtheria bacillus grows luxuriantly on blood agar and like the streptococcus
pyogenes has a yellowish laked zone around the colony. The Hofman and the
Xerosis bacillus do not seem to have this haemolytic power. In bouillon it tends

DIPHTHERIA ANTITOXIN 87

to form a surface growth. It is at the surface that the toxin function is most marked,
hence in growing diphtheria for toxin formation we use Fernbach flasks which expose
a large surface to the air. It is a marked acid producer — bouillon with a + 1 reaction
becoming +2.5 to +3 in thirty-six hours. The filtrate from a two- or three- weeks-
old broth culture is highly toxic, and is usually referred to as diphtheria toxin. It
is used in injecting horses to produce antitoxin. Ehrlich uses as a standard to meas-
ure the toxicity of toxin the minimal lethal dose (M. L. D.). This is the amount
of toxin which will kill a 35o-gram guinea-pig in just four days. Some toxins have
been produced whose M. L. D. was 1/500 c.c., so that i c.c. of such toxin would kill
500 guinea-pigs. Theoretically, the measure of an antitoxin unit is the capacity
of neutralizing 200 units of a pure toxin. (On exposure to light, etc., toxin loses

FIG. 27. — Diphtheria bacilli involution forms. (Kolle and Wassermann.)

its toxic power and is termed toxoid.) Inasmuch, however, as toxoneand toxoid
are also present, we may practically consider an antitoxin, or immunizing unit
(i.e., Immunitatseinheit) as about capable of making innocuous 100 M. L. D.

In the preparation of antitoxin horses are employed; the method being to inject
the bouillon filtrate or toxin subcutaneously at weekly intervals for a period of three
or four months. When each c.c. of the serum of the horse is found to contain about
250 to 500 antitoxin units the horse is bled from the jugular vein. Some sera contain
as much as 1300 units in a cubic centimeter.

Methods of purifying and concentrating antitoxin are now employed by certain
makers, the principle being that the antitoxin in the horse serum is precipitated
with the globulins which come down on half saturation with ammonium suplhate.
In this way, as the content in horse serum proteids is lessened, the anaphylactic
dangers are lessened.

As a curative measure, from 2500 to 5000 units should be injected.
If the injection is delayed or the case very serious the dose should be
10,000 units. As much as 50,000 units has been given in severe cases.
The prophylactic dose is 500 units.

88 STUDY AND IDENTIFICATION OF BACTERIA

Sudden death after administration of antitoxin has been reported
in cases of status lymphaticus. (See anaphylaxis).

In obtaining material from a throat, be sure that an antiseptic gargle
has not been used just prior to taking the throat swab. The part of the
swab which touched the membrane or suspicious spot should come in
contact with the serum slant. This is best accomplished by revolving
the swab. An immediate diagnosis is possible in probably 35% of cases
by making a smear from a piece of membrane. In doing this Neisser's
stain or the toluidin blue stain are usually considered the most satis-
factory. I prefer the Gram stain, however. The diphtheria bacilli
found in such smears are not apt to be clubbed and stain more
uniformly.

If there is any doubt about the nature of an organism in a throat culture, always
stain: i. with Loffler's alkaline methylene blue for two minutes; 2. with Gram's
method, being careful not to carry the decolorization too far, and 3. by Neisser's
method. With Loffler's you obtain a picture which, after a little experience, is

FIG. 28. — B. diphtherias stained by Neisser's method. (Williams.')

characteristic; at times the polar bodies show as intense blue spots in the lighter
blue bacillus. One is liable to confuse cocci lying side by side for diphtheria bacilli
with segmental or banded staining. This difficulty is not apparent when Gram's
staining is used. This gives us great information, as the diphtheria and the pseudo-
diphtheria are the only small Gram positive bacilli usually found in the mouth.
The cocci are also well brought out. Neisser's stain gives a picture which, when
satisfactory, is almost absolutely characteristic. You have the bright blue dots
lying at either end of the light brownish-yellow rods. When first isolated from a
throat, the diphtheria bacillus is apt to stain characteristically by Neisser. Later

DIPHTHEROID BACILLI 89

on, in subculture, there may be no staining of the polar bodies. Neisscr originally
recommended five seconds' application, with an intermediate washing, for each of
his two solutions. Thirty seconds for each is probably preferable. Some authorities
recommend five to thirty minutes. It is well to bear in mind that about 2% of the
people in apparent health carry diphtheria bacilli of the granular or barred type in
their throats and of these about one in five will prove virulent for the guinea-pig.

It is essential when a question exists as to the nature of a diphtheria-
like organism to test it as to virulence. While there are exceptions,
especially in freshly isolated colonies, yet as a rule a severe infection
yields virulent organisms and vice versa. Pure cultures are best obtained
by streaking material from the throat on glycerine agar plates. From
an isolated colony inoculate a tube of bouillon. From such a twenty-
four-hour-old culture inoculate a guinea-pig with two or three drops
subcutaneously in the shaven abdomen. Escherich considers a fatal
result with 1.5 c.c. of such a bouillon culture a satisfactory test as to
virulence. After death, which occurs in two or three days, the adrenals
are enlarged and haemorrhagic.

Diphtheroid Bacilli. Pseudodiphtheria Bacillus. Hofman's Bacil-
lus.— Under these terms various Gram positive bacilli have been de-
scribed as occurring in nose and in skin diseases.

Their chief importance is in connection with their presence in the
throats of healthy people. Probably approximately 10% of people
harbor such organisms as against i to 2% with granular types. Some
authorities believe it possible for these diphtheroids to be capable of
being transformed into virulent diphtheria bacilli. This seems im-
probable. Such organisms are often found in urethral discharges,
either alone, or with gonococci or other organisms.

1. They very rarely give the blue dot staining at the two ends. Exceptionally
they may give a dot at one end. Neisser attaches importance to the dots
at both ends as showing diphtheria.

2. They tend to stain solidly or at most with only a single unstained segment.
They are shorter, thicker, and do not curve so gracefully as the true diphtheria
bacillus. They are stockier.

3. They produce very little acid in sugar media, not one-half that produced by
true diphtheria.

4. They are nonpathogenic for guinea-pigs.

5. Many of them grow quite luxuriantly and often show chromogenic power.

Xerosis Bacillus. — This organism is frequently found in normal
conjunctival discharges. There is question as to its pathogenesis, and
the finding of this organism should not exclude the previous presence of

QO STUDY AND IDENTIFICATION OF BACTERIA

strictly pathogenic organisms, such as the gonococcus or the Koch-
Weeks. It resembles the diphtheria bacillus in being Gram positive
and showing parallelism, but differs i. in being nonvirulent for guinea-
pigs; 2. in requiring about two days for the appearance of colonies; 3.
in not showing Neisser's granule staining, and 4. in producing very little
acid in sugar media.

CHAPTER VIII.

STUDY AND IDENTIFICATION OF BACTERIA. GRAM
NEGATIVE BACILLI. KEY AND NOTES.

KEY to the recognition of nonspore-bearing, nonchromogenic, non-
Gram-staining, nonbranching bacilli.

(NOTE. — Some books say that the proteus group is Gram positive. It is, how-
ever, usually negative.)

Do not grow on ordinary media. Require blood agar (haemophilia bacteria), serum
agar, or blood-serum.

Minute dew-drop colonies.

1. Influenza bacillus. Requires blood media.

2. Koch- Weeks bacillus (conjunctivitis). Serum agar best medium.

3. Miiller's bacillus of trachoma. Like Koch- Weeks bacillus, but easier to
cultivate.

4. Morax diplobacillus of conjunctivitis. Grows well and produces little pits
of liquefaction in Loffler's blood-serum.

5. Bordet-Gengou bacillus of whooping-cough. Does not grow on Loffler's
serum. Requires blood or ascitic fluid agar.

6. Ducrey's bacillus (soft chancre). Requires almost pure blood. Forms
chains.

Grow well on ordinary media.

I. Cultures in litmus milk. PINK.

A. Nonmotile.

Lactis aerogenes group. B. lactis aerogenes.

Produce gas in glucose, lactose, and saccharose. No liquefaction of gelatin.
Short, stubby bacteria, often showing capsules. Intermediate between
the colon and Friedlander group.

B. Motile.

1. No nliquef action of gelatin.

a. B. coli group. Coagulation of milk. No subsequent peptonization.
Gas in glucose and lactose, none in saccharose. Indol produced.
Neutral red reduced.

2. Liquefaction of gelatin.

a. B. cloacae group. Gas in glucose, slight in lactose. Slow coagulation
of milk. Subsequent peptonization.

II. Cultures in litmus milk. LILAC.
A. Nonmotile bacilli.

i. No gas generated in glucose or lactose bouillon.

a. Haemorrhagic septicaemia group. These are oval bacilli with tendency
to bipolar staining.

91

g 2 STUDY AND IDENTIFICATION OF BACTERIA

Colonies smaller and less opaque than those of B. coli.

Examples: B. pestis, B. suisepticus, B. cholerae gallinarum (chicken

cholera).

B. pseudo tuberculosis rodentium (very similar to plague).

B. pestis is absolutely nonmotile, does not liquefy gelatin, does not

produce indol, produces slight acid in glucose but not in lactose bouillon,
b. Dysentery group. Colonies similar to those of B. coli.

Divided into two classes according as mannite is acted on:

Those not giving acid — nonacid group — (Shiga-Kruse).

Those giving acid — acid group — ( Fl ex ner- Strong).
2. Gas generated in glucose bouillon not in lactose.

a. Friedlander group. Give very viscid, porcelain-like colonies.

Tendency to capsule formation in favorable media.

Examples: B. pneumonise, B. capsulatus mucosus, B rhinoscleromatis.
B. Motile bacilli.

1. Do not liquefy gelatin.

a. Do not produce gas in either glucose or lactose bouillon.

Typhoid, or Eberth group. No indol. No coagulation of milk. No
reduction of neutral red.

b. Gas generated in glucose, not in lactose media. Milk not coagulated.
Neutral red reduced.

Gartner group. This includes:

Pathogenic types for man; as B. enteritidis, B. icteroides, B. paratyphoid
B, B. psittacosis. Nonpathogenic for man; as B. cholerae suum (hog
cholera) .

2. Liquefy gelatin.

a. Proteus group. Colonies at first round later amoeboid, spreading.
Produce gas in glucose, not in lactose. Produces foul odor.
B. zopfii type of Proteus group does not liquefy gelatin; colonies at first
round, later amoeboid, spreading. Foul odor in cultures. Gelatin stab
shows lateral branching.

NOTE. — The Friedlander and the lactis aerogenes group, differing culturally
chiefly in carbohydrate fermentation activities, organisms considered as belonging
to the Friedlander group rather than to the lactis aerogenes group may show acid
in litmus milk. Where an organism having the characteristics of B. coli, but
fermenting saccharose, is found, it is termed B. coli communior. A non-gas
producing colon type organism has been designated B. coli anaerogenes. Cer-
tain organisrns which turn litmus milk lilac and which liquefy gelatin, but do not
produce gas in sugar media, belong to the "Booker" group. Other organisms
which acidify and coagulate litmus milk but do not liquefy gelatin or produce gas
in glucose or lactose media have been placed in the "Bienstock" group. The
proteus or Hauser group is composed of organisms showing various functions;
Proteus vulgaris liquefying gelatin rapidly, P. mirabilis slowly and P. zenkeri
not at all.

GRAM NEGATIVE BACILLI REQUIRING SPECIAL MEDIA.

Bacillus influenzae (Pfeiffer, 1892). — This organism is the type of
the so-called haemophilic bacteria— organisms whose growth is restricted

INFLUENZA 93

to media containing haemoglobin. • The influenza bacillus seems to
grow better on slants freshly streaked with blood than on those which
have been made for some time, and they appear to grow better on this
surface smear of blood than on a mixture of agar and blood.

The influenza bacilli are most likely to be isolated from the sputum of broncho-
pneumonia due to this organism. It has also frequently been found in the nasal
secretions of influenza patients. Exceptionally, it is present in the blood, and has
been isolated in cases of meningitis from cerebrospinal fluid. It also occurs at times
in anginas, but then usually associated with other organisms. Infection probably
only takes place by contact. It is a very small bacillus which in sputum tends to
show itself in aggregations, especially centering about M. tetragenus. It stains
rather faintly when compared with cocci, so that a smear of sputum stained with
formol fuchsin shows a deep violet staining for the M. tetragenus or other cocci,
and scattered around in a clump-like aggregation we see these minute, rather faintly
stained rods. They also tend to stain more deeply at either end, so that they some-
times appear as diplococci. Gram's method, counterstaining with formol fuchsin.
is excellent for their demonstration. The red bacilli and the violet-black cocci are
easily distinguished.

To cultivate them, rub the sputum, or at autopsy the material from
a lung, on a slant smeared with human blood (pigeon's blood is also
satisfactory), and then without sterilizing the loop, inoculate a second
blood slant; then a third, and possibly a fourth. The colonies appear as
very minute dewdrop-like points which seem to run into each other in a
wave-like way. To test such colonies we should transfer a single colony
to plain agar and blood-serum, trying not to carry over any blood. If
the least trace of blood is carried over, they may grow on agar or blood-
serum. Organisms resembling the influenza bacillus have been isolated
from whooping-cough. Such organisms have also been found in the
fauces of well persons. In many epidemics of influenza the bacillus
has not been isolated, or success has obtained in only a small proportion
of the cases. Etiological factors in conditions more or less resembling
influenza may be the Streptococcus, Pneumococcus, or M. catarrhalis.
The influenza bacillus seems to grow best in symbiosis with some other
organism, especially with S. pyogenes aureus.

Koch -Weeks Bacillus (Koch, 1883). — This produces a severe
conjunctivitis. It is very common in Egypt and is also a frequent cause
of conjunctivitis in the Philippines and in temperate climates.

Smears made from conjunctival secretion show large numbers of
small Gram-negative bacilli, especially contained within pus cells, but
also lying free. They are more difficult to cultivate than the influenza
bacillus, but the same general methods hold. The vitality of this

94 STUDY AND IDENTIFICATION OF BACTERIA

organism is very slight so that. almost immediate transference, of
material is necessary. Flies are an important factor in Egypt.
The period of incubation is short, twelve to thirty-six hours. The
best medium is a mixture of glycerine agar and hydrocele or ascites
fluid. At first we rarely obtain pure cultures. The colonies are
dewdrop-like and first show themselves in about thirty-six hours in
incubator cultures.

FIG. 29.— The Koch- Weeks Bacillus. (Hansell and Sweet.)

Diplobacillus of Morax. — This organism causes mild blepharo-con-
junctivitis chiefly at the internal angle of the eye. They are about i or
2/J. long and tend to occur in pairs or short chains. Some claim that
they are Gram positive.

Culturally the formation of little pits of liquefaction in Loffler's serum within
twenty-four hours which later become confluent may be regarded as fairly character-
istic. They do not grow on nutrient agar.

After two or three days on blood-serum rather marked involution
forms occur. While usually causing a more or less chronic conjuncti-
vitis they may at times produce a keratitis.

Bacillus of Chancroid (Ducrey, 1889). — These are short cocco-
bacilli, occurring chiefly in chains. They show bipolar staining. They
grow best in a mixture of blood and bouillon.

Bacillus of Bordet-Gengou. — This bacillus was reported as the cause
of whooping-cough by Bordet and Gengou in 1906. (Czaplewski and
Reyher had previously reported oval bipolar staining organisms, as the
cause of pertussis, and other authors influenza-like organisms.)

PLAGUE 95

The bacillus is oval, Gram negative, shows bipolar staining, somewhat resembles
B. influenzae and grows only on uncoagulated serum media, as blood or ascites agar.
The original cultures are very scanty so that the colonies are difficult to recognize.
In subcultures the growth is more flourishing. The organism is only found in white,
thick, leukocyte abounding sputum, of the beginning of the disease. Hence per-
tussis is probably contagious only at the onset.

Complement binding and agglutination reactions have been obtained. For
diagnosis stain the sputum. Remember that pertussis gives a mononuclear leuko-
cytosis of 15 to 50 thousand.

GRAM NEGATIVE BACILLI GROWING ON ORDINARY MEDIA.

Bacillus pneumonias (Friedlander, 1882). — This organism is
responsible for about 5% of the cases of pneumonia. It is usually
termed the pneumobacillus to distinguish it from the pneumococcus;
at other times Friedlander's bacillus. The name of Fraenkel attaches
to the pneumococcus. Morphologically, it is a short, thick bacillus,
and in pathological material, as sputum, shows a wide capsule. It is
nonmotile and Gram negative. The colonies on agar are of a pearly
whiteness and are markedly viscid. On potato it shows a thick viscid
growth containing gas bubbles. The characteristic culture is the nail
culture of a gelatin stab. The growth at the surface is heaped up like a
round-headed nail, the line of puncture resembling the shaft of the nail.
It does not liquefy gelatin. It does not produce indol, and does not
produce gas in lactose bouillon — differences from the colon bacillus —
with which it may be confused in cultures, as it does not then possess a
capsule. If in doubt, inject a mouse at the root of the tail. Death
from septicaemia occurs in two days. The peritoneum is sticky and
numerous capsulated bacilli are present in the blood and organs. The
organisms which have been isolated from rhinoscleroma and ozcena
are practically identical with the B. pneumonias. This group of organ-
isms is generally referred to as the Friedlander group. Similar organ-
isms have been isolated from the discharges of middle-ear diseases and
in anginas. Cases have been reported where the B. pneumonias was
the cause of septicaemia in man.

Bacillus pestis (Kitasato, Yersin, 1894). — This is the organism of
plague. It is primarily a disease of rats. It is the member of the group
of haemorrhagic septicaemias (Pasteurelloses), from which man suffers.

Other Pasteurelloses are chicken cholera, swine plague, mouse septicaemia and
rabbit septicaemia. This is a widely distributed group and may include saprophytic
organisms as well as those noted for their virulence.

g STUDY AND IDENTIFICATION OF BACTERIA

B. cholerae gallinarum and B. suisepticus are approximately similar in size and
cultural requirements to B. pestis. The oval bacillus with bipolar staining in smears
from tissues is very characteristic for both of them. Another name for swine plague
(B. suisepticus) is infectious pneumonia of swine. The organism is chiefly found in
the lungs.

Where the plague bacilli are found chiefly in the glands, we have
bubonic plague; when in lungs, pneumonic plague; when localized in
the skin and subcutaneous tissue, the cellulo-cutaneous; and when as a
septicaemia, septicaemic plague. An intestinal type is recognized by

FIG. 30. — Colonies of plague bacilli forty-eight hours old. (Kolle and Wassermann.)

some authors. It must be remembered that in all forms of plague the
lymphatic glands show hemorrhagic oedema; it is in bubonic plague,
however, that the areas of necrosis with periglandular oedema are
prominent. Where the symptoms are slight, mainly buboes, the term
pestis minor is sometimes used; the typical disease being termed pestis
major. In pneumonic plague we have a bronchopneumonia.

In smears from material from buboes, from sputum, or in blood smears, as well
as from blood or spleen smears from experimental animals, we obtain the typical
morphology of a coccobacillus (1.5X0.5^) with very characteristic bipolar staining;
there being an intermediate, unstained area. Very characteristic also is the appear-
ance in these smears of degenerate types which stain feebly and show coccoid and
inflated oval types. The presence of these involution forms associated with typical
bacilli is almost diagnostic for one with experience. Inoculating tubes of plain agar
and 3% salt agar with this same material, we obtain in plain agar cultures organisms
which are typically small, fairly slender rods, which do not stain characteristically
at each end and are not oval. The smear obtained from the salt agar presents most
remarkable involution forms — coccoid; root-shaped, sausage-shaped forms, ranging

PLAGUE CULTURES

97

from three to twelve microns in length, more resembling cultures of moulds than
bacteria. Another point is that on the inoculated plain agar we are in doubt at
the end of twenty-four hours whether the dewdrop-like colonies are really bacterial
colonies or only condensation particles. By the second day, however, these colonies
have an opaque grayish appearance, so that now, instead of questioning the presence
of a culture, we consider the possibility of contamination.

Blood cultures in septicsemic plague may show from 5 to 500,000 per c.c. Smears
from the blood in such cases are positive in only about 17%.

FIG. 31. — Pest bacilli from spleen of a rat. (Kolle and Wassermann.}

The plague bacillus grows well at room temperature — -its optimum
temperature being 30° instead of 37° C., as is usual with pathogens.
Next to the salt agar culture, the most characteristic one is the stalactite
growth in bouillon containing oil drops on its surface. The culture
grows downward from the under surface of the oil drops as a powdery
thread. These are very fragile, and as the slightest jar breaks them, it
is difficult to obtain this cultural characteristic.

While Klein states that B. coli, proteus vulgaris and, in particular, B. bristolensis
may be mistaken for plague bacilli, if bipolar staining alone be relied upon, yet it is
B. pseudotuberculosis rodentium which may confuse an experienced worker. While
this latter is only moderately pathogenic for rats yet the fact that rats may be
immunized to B. pestis by inoculation with B. pseudotuberculosis rodentium brings
up the suspicion of identity of the two organisms. In diagnosing always use animal
experimentation. Owing to the difficulty in emulsifying plague bacilli, agglutination
tests are not satisfactory.

Albrecht and Ghon have shown that by smearing material upon the
intact, shaven skin of a guinea-pig, infection occurs. This is the most
crucial test.

g8 STUDY AND IDENTIFICATION OF BACTERIA

A pocket made by cutting the skin of a guinea-pig with scissors and extended
subcutaneously with scissors or forceps, into which a piece of the suspected plague
tissue is thrust with forceps, is more practical than injecting an emulsion with hypo-
dermic syringe.

Mice inoculated at the root of the tail quickly succumb. Rats, this being pri-
marily a disease of rats, are of course susceptible. Other rodents, as squirrels, are
susceptible. It has been suggested that a rodent, the Siberian marmot, or tarabagan
(Arctomys bobac) might be the starting-point of plague outbreaks. In natural
plague of rats, the lesions which establish a diagnosis even without the aid of a
microscope are dark red, subcutaneous injection of .the flaps of the abdominal walls
as they are turned back, fluid in the pleural cavities, cedematous haemorrhagic

FIG. 32. — Pest bacillus involution forms produced by growing on 3% salt agar.

(Kolle and Wassermann.)

periglandular infiltration and swelling of the neck glands, and in particular a creamy,
mottled appearance of the liver. The neck glands are chiefly involved because the
flea prefers to inhabit the skin of the neck. Smears from the spleen will show the
oval bacilli.

A chronic rat plague, which may be a factor in keeping up the disease, is char-
acterized by enlargement of the spleen and the presence within it of nodules contain-
ing plague bacilli. McCoy has noted that the frequency of the cervical bubo in
rats, noted by the Indian Commission (72%), was not found in California. The
glands show periglandular infiltration and injection as well as enlargement.

Recent investigations in India have definitely determined the fact
that the flea (Xenopsylla cheopis) is the intermediary in the trans-
mission of plague from rat to rat and from rat to man. In primary
pneumonic plague the infective nature is very great and appears to be
by the respiratory atrium (From man to man). This was the terri-
fying type of plague in the black death of the fourteenth century.

TYPHOID-COLON GROUP 99

Strong and Teague have shown that of 39 plates exposed before the mouths of
patients with pneumonic plague, with marked dyspnoea and pulmonary oedema, but
without coughing, only one plate showed plague bacilli. In 39 other experimental
plate cultures with coughing on the part of the patients there were 15 plates showing
plague bacilli.

The droplet method of infection is therefore the important one in plague
pneumonia.

As these droplets are expelled to a considerable distance not only should the
respiratory inlets be protected by masks but the conjunctivas with glasses and
abrasions with protective coatings.

For diagnosis make smears and cultures from material drawn from
a bubo by a syringe. (At a later stage, when softening begins, there may
not be any bacilli present.) Also, if pneumonic plague, from the
sputum. Blood cultures and even blood smears may be employed in
septicaemic plague. Formol fuchsin and Archibald's stain make satis-
factory stains. Always inoculate a guinea-pig with the material either
by rubbing it in with a glass spatula on the shaven skin or by sub-
cutaneous injection. For prophylaxis the most important method is
that of Haffkine. Stalactite bouillon cultures of plague are grown for
five to six weeks. These are killed by a temperature of 65° C. for one
hour. Lysol (1/4%) is added to the preparation and from 0.5 to 4 c.c.
injected, according to the age and size of the individual treated. Sus-
ceptibility is reduced about one-fourth, and of those attacked after
previous vaccination, the mortality is only about one-fourth of what it is
among the noninoculated. Strong prepares a prophylactic vaccine
from living plague cultures rendered avirulent. Yersin's serum, made
by injecting horses with dead plague cultures and afterward with
living ones, is of value prophylactically and has possibly considerable
curative power.

The Eberth, Gartner and Escherich Groups. — From a standpoint
of cultures in litmus milk and sugar bouillon we can divide the organ-
isms related to typhoid at one extreme and the colon at the other into
three groups.

i. The Eberth or typhoid group. There are three important patho-
gens in this group: the B. typhosus, the B. dysenteriae, and the 3.
faecalis alkaligenes. The color of litmus milk is practically uhaltereU'
and there is no gas production in either glucoSecr lactose bouillon. Np
coagulation of milk. No reduction' oi ftcutral red. ' The J3. typb'osiis.
and the B. faecalis alkaligenes are actively motile, while' the B. dysen-
teriae is nonmotile or practically so.

100 STUDY AND IDENTIFICATION OF BACTERIA

During the first twenty-four to forty-eight hours there is a moderate
acid production by typhoid, so that the milk culture is less blue, while
with the B. faecalis alkaligenes the alkalinity is intensified from the start,
so that the blue color is deepened.

2. The Gartner or hog cholera group. Besides organisms im-
portant for animals and probably at times for man, such as B. cholerae
suum and B. psittacosis and B. icteroides (interesting historically as
having been reported as the cause of yellow fever by Sanarelli), we have
two pathogens: i. B. enteritidis (Gartner's bacillus) and 2. B. para-
typhoid B. In this connection it may be stated that the present view
is that hog cholera is caused by an ultra-microscopic organism and not
by the B. cholerae suum.

These organisms cannot be separated culturally, but only by im-
munity reactions. They do not turn litmus milk pink. They produce
gas in glucose bouillon, but not in lactose. They very powerfully re-
duce neutral red with the production of a yellowish fluorescence. They
do not coagulate milk. There is a transient acidity in the litmus milk,
but becoming shortly afterward alkaline, the lilac-blue color is intensi-
fied. Both organisms are motile.

3. The Escherich or colon group. These turn litmus milk pink,
coagulate milk, reduce neutral red, and show varying degrees of motility.
The three groups of organisms just described are nonliquefiers of gela-
tin. Two intestinal organisms, the B. cloacae and the Proteus vulgaris,
differ in liquefying gelatin.

Bacillus typhosus (Eberth, 1880; Gaffky, 1884).— This organism
may be isolated from the stools, urine, and the blood of typhoid patients.

At postmortem it can be best isolated from the spleen, but is also present in
Peyer's patches which have not ulcerated. When ulceration has occurred contami-
nation with B. coli. is almost sure. Cultures may be obtained from the liver also.
In sections made from spleen the Gram negative bacilli are apt to be decolorized.
Thionin, then blotting and clearing in oil or xylol, shows the clumps of bacilli lying
between the cells.

Formerly it was supposed that by the differences in the thickness of the film of
a colony or by its varying shades of grayish-blue, we possessed data of importance in
differentiating typhoid from related organisms.
? ;Th£ cj&ipmes look like grapevine leaves.

GrOwth *;en.,pota>x>w,as A!S(? considered as affording information. At present,
the, biochemical reactions give us-uucrroation assisting in differentiation, and the
agghVci nation '• 'aijd bactpriolytic phenomena, the final diagnosis. The various
plating medfe/are corisiderecl Uftder- media for plating out faeces.

Not only do we find ny'pcrplasi'a ef rftfe/endothelial cells in the lymphoid tissue

TYPHOID FEVER

101

of Peyer's patches and the mesenteric glands and the spleen, with subsequent necro-
ses, but focal necroses of the same character are found in the liver.

A striking feature of the pathology of typhoid fever is the long-con-
tinued persistence of the organisms in the gall-bladder and elsewhere.
It is beginning to be believed that a previous typhoid infection, pos-
sibly so mild as to have passed unnoticed, is at the basis of gall-bladder
infections and resulting gall-stones. Various bone infections, especially
osteomyelitis, have shown the typhoid bacilli in pure culture. For-
merly it was supposed that the typhoid bacillus brought about its lesions
by a local infection centered in the ileum. The present view is that
typhoid bacilli effect an entrance into the blood stream through some
lymphoid channel, as by tonsil or other alimentary lymphoid structure.
Of animals, only the chimpanzee seems to be susceptible.

FIG. 33. — Seventy-two-hour-old culture of typhoid bacillus on gelatin. (Kolle and

Wassermann.)

They develop in the general lymphatic system, the spleen in partic-
ular, where they are protected from the bactericidal power of the blood.
After a time, however, approximately the period of incubation, they
become so abundant in these lymphatic organs that they are carried
over into the general circulation. Then as a result of bacteriolysis the
intracellular toxins are liberated and symptoms develop. If bac-
teriolysis takes place other than in the blood we have various suppurative
processes. As a result of the formation of antibodies, the development
in spleen, etc., is checked but should these immunity reactions become
less potent relapses may occur or various local infections manifest
themselves.

102 STUDY AND IDENTIFICATION OF BACTERIA

As the bacilli do not multiply to any extent in the blood itself the disease cannot
be considered as a typical septicaemia but as a bacteriaemia.

Typhoid bacilli can be isolated from the blood during the latter
period of incubation and rarely after the tenth day of the disease.
It is a practical point that the time to isolate the bacteria from the
blood is in the first days of the attack. The diagnosis by agglutina-
tion is only expected after the seventh to tenth day. Agglutination
may not appear until during convalescence, and in about 5% of the
cases it is absent. It, as a rule, disappears within a }tear.

Very little success has b«en obtained with curative sera. Chantamesse, by
treating horses with a nitrate from cultures of typhoid bacilli on splenic pulp and
human defibrinated blood, claimed to have obtained a curative serum possessing
antitoxic power. Wright's method of prophylactic inoculation is now being em-
ployed in the British army with apparent success. In this, twenty-four- to forty-
eight-hour-old cultures are killed at 53° C.; 1/4% of lysol is then added. An injec-
tion of 500 million bacteria is made at the first inoculation, and ten days later an
injection of one billion. The British prefer to inject subcutaneously in the infra-
clavicular region and at the insertion of the deltoid. The Germans consider three
injections as conferring greater immunity.

Russell has obtained splendid results in the U. S. Army with his method of
vaccination. In this three injections are given at intervals of ten days, the dosage
being 500,000,000, for the first and 1,000,000,000 for each of the two succeeding
injections.

Typhoid vaccines sterilized with 0.5% of phenol appear to keep much longer and
to have a higher immunizing power than those prepared by sterilization with heat
and subsequent addition of the antiseptic.

Typhoid bacilli may be found not only in the blood, urine and faeces but as well
in the sputum of cases showing pulmonary involvement. They have also been found
in the cerebrospinal fluid of cases showing meningeal symptoms. At the autopsy
they may be found in the spleen, Pyer's patches, mesenteric glands and liver.

A very important discovery is that certain persons, who may have
had only a slight febrile attack, may eliminate typhoid bacilli for years
in their faeces (typhoid carriers). The bacilli are also eliminated for
considerable periods in the urine. Distinction is now being made
between acute carriers (convalescents) and chronic carriers.

The most satisfactory method of detecting carriers is by examina-
tion of faeces or urine plated out on Endo's medium. While carriers
usually give a Widal reaction this is by no means constant. Typhoid
carriers are said to maintain a high opsonic index.

The urine and faeces of typhoid convalescents should be proven negative by cul-
tural procedure before discharging J;hej3atients.

TYPHOID CARRIERS 103

Vaccination seems to be a very satisfactory measure in bringing about the dis-
appearance of typhoid bacilli in the dejecta of carriers.

For laboratory diagnosis, blood cultures during the first week and
agglutination tests during the second week and onward are the practical
methods.

Along with the, agglutination tests the urine and faeces should be cultured on
Endo's plating medium and later transferred to Russell's medium for cultural
identification. The positive identification, provided the culture so isolated shows the
cultural characteristics of typhoid, is made by testing the bacilli for agglutination
with a known typhoid serum. Instead of the usual blood cultures one may use the
clot in the Wright U-tube for culturing and the serum remaining after centrifugaliza-

K$b

FIG. 34. — Bacillus of typhoid fever, stained by Loffler's method to show flagella.
(X 1000.) (Williams.)

tion for the Widal test (clot culture). B. typhosus appears in the blood in relapses.
Kayser considered that about 27% of cases of typhoid in Strasburg were caused by
raw milk, 17% by contaminated water, 17% by contact with typhoid, and 10% were
due to typhoid carriers. Other cases were due to infected food, and about 13% were
of origin impossible to determine. These latter may have been due to unrecognized
typhoid carriers. He does not attach the same importance to fly dissemination as
do American authors.

Contact infection is the great factor in perpetuating typhoid fever
but this agency shows diminishing cases each year provided water and
milk supplies are safe. The leading European cities as a result of a safe

IO4 STUDY AND IDENTIFICATION OF BACTERIA

water supply rarely show more than about three typhoid deaths
per 100,000 population per year. Edinburgh shows less than one per
200,000 for the year 1910. In American cities rates of twelve to fif-
teen per 100,000 are common.

The Gartner or Meat-poisoning Group. — Under this designation
may be considered the organisms which cause gastrointestinal disorders
of varying degrees, infection with which is usually brought about by the
ingestion of meat obtained from diseased cattle. Unless the meat is
thoroughly cooked the bacilli in the interior may not be killed.

In this group may be placed B. enteritidis, the typical meat-poisoning organism,
B. paratyphoid B, B. Danysz, B. Aertryck, B. typhi muriiim and B. suipestifer.

B. suipestifer or the hog cholera bacillus was formally thought to be the cause of
this important epizootic. It is found in the intestines of quite a percentage of
healthy hogs. The cause is now known to be a filterable virus.

These organisms are alike morphologically and culturally and show quite a
tendency to bipolar staining and reduction of neutral red with fluorescence in
forty-eight hours. B. paratyphoid B, B. Aertryck and B. suipestifer are alike
from an agglutination standpoint, while B. enteritidis and B. Danysz show similar-
ity in this respect. B. paratyphoid A stands by itself.

Paratyphoid Bacilli (Achard and Bensaude, 1896; Schottmuller,
1901). — Cases resembling mild attacks of typhoid occasionally show
agglutination for paratyphoid bacilli. These organisms have also
been isolated from the blood, as with typhoid. Two types have been
recognized: the paratyphoid A and the paratyphoid B. The latter
occurs in 80% of such cases. Culturally, paratyphoid B. cannot be
separated from Gartner's bacillus. In paratyphoid A there is less gas
produced in glucose bouillon than with paratyphoid B, and the primary
acidity of litmus milk is not succeeded by a subsequent alkalinity. It
does not seem practical to draw a fine distinction between these two
strains.

Paratyphoid B. not only gives symptoms resembling a mild typhoid infection,
but may show symptoms more like those of meat poisoning or even cholerine. It
is more pathogenic for laboratory animals than is B. typhosus. The development of
antibodies upon immunizing a man or animal with paratyphoid organism does not
seem to approach that obtained with typhoid.

Bacillus enteritidis (Gartner, 1888).' — This organism has been
frequently isolated from cases of gastroenteritis from ingestion of in-
fected meat.

Meat from healthy animals which has been in contact with that of diseased
animals may become injected. The simple act of placing a piece of infected meat

DYSENTERY 105

on a sound piece may infect the latter. It has been noted that the bacteria, or their
toxins, may be distributed unevenly in the meat eaten, so that one person consuming
the same meat may be made very ill while others eating this meat may escape
infection. Infection of food may occur not only from unclean handling but from the
material carried by flies or even from the faeces of mice or rats deposited on food-
stuffs.

This organism is very pathogenic for laboratory animals, producing a haemor-
rhagic enteritis and at times a septicaemia. Where meat has been contaminated with
Gartner's bacillus toxins may have been produced, and symptoms of poisoning with
acute gastroenteritis would occur shortly after ingestion. This is not a true toxin
as it does not require a period of incubation before manifesting its toxic action. It
is interesting to note that this toxin is not destroyed by the boiling temperature, thus
differing from the toxin of the other important meat-poisoning (botulism) bacillus —
B. botulinus — which is rendered innocuous by a temperature of 65° or 70° C. If
there is only a little toxin introduced with the contaminated meat, the symptoms
will be delayed one or two days. Such organisms have been isolated in pure culture
from cases with high fever, marked intestinal derangement, with considerable blood
in the rather fluid stools. In two cases studied the disease was at first diagnosed
as a severe typhoid infection. Klein thinks the organism of Danysz's virus (to
kill rats during plague epidemics) may be identical with B. enteritidis.

Proteus vulgaris.— This organism is often encountered in plates
made from faeces, or sewage contaminated water.

It is common in decaying meat or cheese, and cases of even fatal poisoning
with marked gastrointestinal symptoms and cardiac failure have been reported.
At times it is the cause of cystitis. The colonies on agar are moist and unevenly
spreading (amoeboid). The bacilli are very motile, long and slender, tend to form
filaments and, as a rule, are Gram negative. It digests blood-serum and is a rapid
liquefier of gelatin. In litmus milk it coagulates with a soft clot and an alkaline
reaction. Subsequently the litmus is reduced and the clot digested giving a dirty
yellowish-brown fluid. Indol is rarely produced. The cultures generally have a
putrefactive odor. In infective jaundice (Weil's disease) this organism has been
reported as the cause. Organisms of this group were formerly designated as B.
termo.

Bacillus dysenteriae (Shiga, 1898). — -Dysentery bacilli produce a
coagulation necrosis of the mucous membrane of the large intestine
and occasionally of the lower part of the ileum. Polymorphonuclears
are contained in the fibrin exudate.

It was formerly thought that these lesions were of local origin, but the present
view is that toxins are produced which, being absorbed, are eliminated by the
large intestine with resulting necrosis. Flexner, by injecting rabbits intravenously
with a toxic autolysate, produced characteristic intestinal lesions. The toxin with-
stands a temperature of 70° C. without being destroyed. The toxin may cause
joint trouble.

106 STUDY AND IDENTIFICATION OF BACTERIA

There are two main types of dysentery bacilli:

1. Those producing acid in mannite media — 'the acid strains (Flex-
ner-Strong types).

2. Those not developing acid in mannite (Shiga-Kruse types).
Ohno finds that fermentative reactions do not correspond to immunity
ones. Thus an acid strain used to immunize a horse may produce a
serum more specific for a nonacid strain. The Shiga type is very toxic
in cultures, while the Flexner type does not seem to possess a soluble
toxin.

The Shiga strains are apt to cause a paresis of the hind extremities of the injected
rabbit which may be followed by paralysis and death. At the Lister Institute
injections of a soluble toxin produced a serum of marked antitoxic power. Such a
dysentery serum, which is probably both antitoxic and antimicrobic, is of curative
value. Shiga immunized horses with polyvalent cultures and obtained a polyvalent
serum which has reduced the death rate about one-third.

The dysentery bacillus is present in the milky white, leukocyte filled
blood flecked mucous stools during the first five or six days of the dis-
ease. By the tenth day it has probably disappeared. Lactose litmus
agar is the most satisfactory plating medium. The stool of the first
two days may give practically a pure culture. The staining of a smear
from the muco-purulent stool is rich in phagocytic cells, many of them
packed with Gram negative bacilli. In all cultural respects the dysen-
tery bacillus resembles the typhoid, and the only practical method of
distinguishing these two organisms, other than by agglutination reac-
tions, is by the nonmotility or exceedingly slight motility of the dysen-
tery bacillus.

The characteristic of nonmotility is of greatest differentiating value and the
reports of slight motility are probably from misinterpretation of molecular movement
as motility. The dysentery bacilli do not form those threads or whip-like filaments
so characteristic of typhoid cultures and are somewhat plumper. The dysentery
bacillus is not found in the blood and hence is not eliminated in the urine. It is
found in mesenteric glands. In dysentery patients agglutination phenomena do
not show themselves until about the twelfth day from the onset. Hence, this
procedure is of no particular value in diagnosis. It is of value, however, to identify
an organism isolated from the stools at the commencement of the attack, using
serum from an immunized animal or a human convalescent for the agglutination
test.

Butler has suggested taking serum from dysentery convalescents, noting the
strain involved, and preserving it by taking up with filter paper as recommended
by Noguchi for the Wassermann haemolytic amboceptor. This I consider very

THE COLON BACILLUS 107

valuable as it is very difficult to immunize rabbits with a Shiga strain on account of
its great toxicity.

There seems to be very little agglutination power in the serum of convalescents
from Shiga strains. Flexner strains give agglutination, but early in convalescence
the serum is not apt to have a titre of more than 1-50.

Morgan has reported as the cause of certain cases of bacillary dysen-
tery a bacillus known as B. Morgan, No. i. It is motile, produce indol,
and in glucose bouillon gives a very slight amount of gas.

It does not change mannite and does not produce a primary acidity in litmus
milk. This organism is a frequent cause of summer diarrhoea of children. Flies
from houses with such cases often show Morgan's bacillus. A dysentery type much
like the Flexner Strong strain is often found in the enteric affections of children in
the United States.

In Japan, dysentery-like epidemics of a very fatal disease, termed
ekiri, occur among young children. The organism is very motile, pro-
ducing gas and acid in glucose but not in lactose media. It is reported
to at times show indol production. Apparently a member of the
Gartner group.

More recently a strain of dysentery bacilli, known as Type F, has
been considered of importance. This organism is very closely related
to the Flexner strain and only differs from it in that it requires about
48 hours to turn mannite litmus media pink and that maltose litmus re-
mains blue. An organism showing similar cultural characteristics has
been recently recovered from faeces of laboratory rabbits by German
workers investigating the problem of whether certain animals might
serve as carriers for dysentery.

B. COLI, B. LACTIS AEROGENES, B. CLOACAE.

While the COLON BACILLUS chiefly inhabits the large intestine, the B.
lactis aerogenes is to be found in the upper part of the small intestine.
While they may be separated on the ground of motility, yet it is by the
greater fermentative activity of the B. lactis aerogenes that they are
best separated. Some consider them as only representing different
strains of the same organism. B. lactis aerogenes is closely related to the
pneumobacillus and at times shows capsules. Some consider that the
B. coli produces a bactericidal substance which inhibits the growth of,
or destroys, pathogenic bacteria which may have passed the destructive
influences of the gastric juice; others that this effect is due to their free

108 STUDY AND IDENTIFICATION OF BACTERIA

growth and the development of phenol and various putrefactive sub-
stances. The probable importance of the colon bacillus in protecting
the organism is shown by the fact that where numerous colonies of patho-
genic organisms may be cultivated from fseces we may find a diminu-
tion in number or absence of the colon bacillus. This condition may be-
observed in infections with the organisms of dysentery, cholera, typhoid,
and paratyphoid. While its normal function is probably protective,
yet the B. coli is an important pathogenic agent, it being frequently the
organism isolated from purulent conditions within the abdominal cav-
ity, especially in appendicitis and lesions about the bile ducts. It is
particularly prone to cause lesions of the bladder and pelvis of the kid-
ney. In the treatment of colon cystitis by vaccines of dead colon
bacilli, the most brilliant results in opsonic therapy have been obtained.

Sir A. Wright thinks that certain cases of mucous colitis may be due
to colon infection and that vaccination may cure them. The colon
bacillus is fully considered under the bacteriology of water.

B. CLOACA was isolated first from sewage by Jordan. It is, as a rule,
a rapid liquefier of gelatin, and in its reactions with sugars and litmus
milk resembles the colon bacillus.

Where the gelatin liquefaction is slow or slight it may be distinguished
from B. coli by its gas formula which is about three times as much CO 2
as H, just the reverse of that of the colon bacillus. B. lactis aerogenes
is often found in sewage. It is one of the causes of the souring of milk.

B. ACIDOPHILUS, B. BlFIDUS, B. BULGARICUS.

These are often termed the long rod group of lactic acid bacteria
in contradistinction to certain other Gram positive bacilli which
are short and oval and which are confused with the so-called milk
streptococci.

The long rod group often forms chains and often shows metachromatic granules
which stain with Neisser's method. They are'readily distinguished from Gram nega-
tive lactic acid producers, of which the type is B. lactis aerogenes, by their Gram
positive staining. B. acidophilus often give the impression of a diphtheroid in a
Gram stained faeces smear. It is nonmotile and often shows polar granules. Grows
only at temperatures above 22° C., op. 37° C. It grows better anaerobically than
aerobically and then shows the clubbed involution characteristics of B. bifidus;
so that some consider these organisms the same, the morphology of B. bifidus being
the result of anaerobiosis. Original cultures are best made in i% glucose and i%
acetic acid bouillon. Some authorities consider B. bifidus the most important

CHROMOGENIC BACILLI IOQ

representative of the large intestine flora. B. lactis acidi is less thermophilic than
B. acidophilus and coagulates milk which B. acidophilus does not do. Certain
polar granule bacteria, as B. granulosum, found in Yoghurt, are similar to B. acido-
philus but coagulate milk; no gas. B. bulgaricus is the type of the group and is
discussed under milk.

Rodella thinks B. acidophilus, B. bifidus, B. gastrophilus and the Boas-Oppler
bacillus identical. B. bulgaricus is said to never show polar granules. B. bulgaricus
and the group of organisms similar to it found in buttermilk, etc., are widely used in
the treatment of various intestinal troubles. North has used cultures of B. bulgari-
cus for extermination of undesirable organisms in other parts of the body than the
alimentary canal (used as applications in nasal, throat or geni to-urinary infections).

CHROMOGENIC BACILLI.

These are identified by the color of their colonies on agar. The
B. pyocyaneus is the most important one of them in medicine, but the
B. prodigiosus is also of interest medically. A violet chromogen, the
B. violaceus, which is motile and liquefies gelatin, has been described
under many names. It has been found in water.

An orange-yellow chromogen, the B. fulvus, is nonmotile and varies
as to its liquefaction of gelatin.

B. pyocyaneus (Gessard, 1882).— This organism is frequently
termed the bacillus of green or blue pus. It is a small (2.5X0.5/0
motile Gram negative bacillus.

It is generally a slender delicate bacillus often showing thread-like
arrangement but at times it may appear as short plump rods. It
grows readily at room or incubator temperature. It liquefies gelatin
rapidly. The green color diffuses through the agar or gelatin on which it
grows, so that we not only have the green-colored colony, but the me-
dium as well is colored. Upon potato the colonies are more of a deep
olive green to dirty brown.

No gas is produced in either glucose or lactose bouillon; blood-serum is digested,
the pitted surface showing a reddish-brown color. The protein ferment pyocyanase
has been used to remove diphtheritic membrane and for treatment of M. catarrhalis
nasal catarrhs. There are two pigments — a green water soluble one and a blue
one soluble in chloroform.

It is widely distributed in water and air, and is frequently isolated
from faeces. The B. fluorescens liquefaciens of water seems to be simply
a strain of B. pyocyaneus. The B. pyocyaneus is frequently associated
with other pus organisms in abdominal abscesses.

110 STUDY AND IDENTIFICATION OF BACTERIA

In addition to having an endotoxin, it produces a soluble toxin similar to diph-
theria toxin. This toxin differs from those of diphtheria and tetanus in that it can
stand a temperature of 100° C., while those of diphtheria and tetanus are destroyed
at about 65° C. The fact that the union between toxin and antitoxin is only of a
binding, neutralizing nature is best shown by taking a mixture of pyocyaneus toxin
and antitoxin which is innocuous and heating it. This destroys the antitoxin, but
does not injure the toxin. We now find that the original toxicity has returned. The
antitoxins of diphtheria and tetanus are more stable than the corresponding toxins;
hence, this experiment would be impossible with them, as upon heating we should
first destroy the toxin.

On account of the frequent association of B. pyocyaneus with other
organisms of better recognized pathogenicity it has until more recently
been considered rather harmless; this view can no longer be entertained
as it is frequently the sole cause of middle-ear inflammations, intestinal
disorders and possibly at times of septicaemia.

FIG. 35. — Bacillus pyocyaneus. (Kolle and Wassermenn.)

B. prodigiosus. — 'This is a very small coccobacillus which shows mo-
tility in young bouillon cultures. It is Gram negative. The colonies
on agar or other solid media show a rich red color. The pigment only de-
velops at room temperature; it is absent in cultures taken out of the
incubator. The B. prodigiosus is frequently found on foodstuffs,
especially bread, where it may simulate blood. It liquefies gelatin rap-
idly and gives a diffuse turbidity to bouillon. It is probable that B.
indicus and B. kilensis are strains of B. prodigiosus.

Coley's fluid, which has been used in cases of inoperable sarcoma and other
malignant growths, is a culture prepared by growing very virulent streptococci in

COLEY'S FLUID in

bouillon for ten days. This streptococcus culture is now inoculated with B. prodi-
giosus, and after another ten days the mixed culture is killed by heat at 60° C. and the
sterile product injected. Coley injected about one-twentieth of a c.c. of this vaccine.
At present he uses nonnltered, heat sterilized bouillon cultures of a streptococcus
obtained either from a case of erysipelas or septicaemia. To this is added material
from agar cultures of B. prodigiosus, grown separately and sterilized before adding
to the sterilized streptococcus bouillon culture.

CHAPTER IX.

STUDY AND IDENTIFICATION OF BACTERIA. SPIRILLA.
KEY AND NOTES.

KEY to recognition of gelatin liquefying, motile and Gram negative
spiral or comma-shaped organisms.

A. Do not give the nitroso-indol reaction with sulphuric acid alone in twenty-four
hours.

1. Produce an abundant moist cream-colored growth on potato at room tempera-
ture.

a. Finkler and Prior's spirillum (Vibrio proteus). Liquefaction of gelatin
very rapid. No air-bubble appearance at top of liquefied area. Cultures
have foul odor. Milk coagulated. Thicker spirillum than cholera. Iso-
lated from cholera nostras.

2. Scanty growth or none at all on potato at room temperature. Only a mod-

erate yellowish growth when incubated about incubator temperature,
a. Spirillum tyrogenum (Deneke's spirillum). Does not liquefy gelatin so
rapidly as Finkler Prior. Thinner and smaller spirillum than cholera.

B. Give the nitroso-indol reaction with sulphuric acid within twenty-four hours.

1. Very pathogenic for pigeons.

a. Spirillum metschnikovi. Liquefies gelatin about twice as rapidly as
cholera. Gives bubble appearance at top of stab.

2. Scarcely pathogenic for pigeons,
a. Spirillum choleras asiaticae.

Nonmotile, nonliquefying and Gram positive spirilla have also been described.
There is also a large group of phosphorescent spirilla.

Spirillum cholerae asiaticse (Koch, 1884).— Typically, the mor-
phology of this organism is that of the comma (Comma bacillus of
Koch). It also frequently shows S shapes, and often appears in long
threads showing turns. When freshly isolated from cholera material
they, as a rule, show a fairly typical morphology, but after subcultures
in the laboratory variations are common, so that rod forms and round
involution shapes give a picture altogether at variance with the comma
shape.

Even in recent cultures of undoubted cholera we may have different types, as
coccoid forms and slender rods. Ohno has noted the fact that the same strain of
cholera will give at one time vibrio forms and again coccoid or rod forms, depending

112

CHOLERA

on the reaction of the media. Inasmuch as the recognition of vibrio shapes is of
importance in diagnosis he recommends that material from a stool be inoculated
into three tubes of peptone solution of reaction +0.3, —0.5 and — i, respectively, one
of which would probably show vibrio morphology.

FIG. 36. — Cholera spirilla. (Kolle and Wassermann.)

The cholera spirillum is very motile (a scintillating motility) and
liquefies gelatin fairly rapidly, although more slowly than any of the
spirilla mentioned in the key. The colony on gelatin was formerly
considered characteristic, but like most cultural characteristics, it is now

FIG. 37. — Involution forms of the spirillum of cholera. (Van Ermengen.}

considered as being only of confirmatory value; it is not specific. These
colonies show in twenty-four hours as small granular white spots which
have a spinose periphery. An encircling ring of liquefaction now
makes its appearance and the highly refractile (as if fragments of spark-

8

STUDY AND IDENTIFICATION OF BACTERIA

ling glass) colony can be separated into a granular center, a striated
periphery, and a clear external ring of liquefaction.

On gelatin stabs the liquefaction produces a turnip-like hollow at the top of the
puncture — the air bubble appearance. It gives the nitroso-indol reaction with sul-
phuric acid alone (cholera red). Kraus attaches importance to the fact that cholera
does not produce a haemolytic ring on blood agar as do the pseudocholera spirilla;
a difficulty is that many pseudospirilla do not haemolize. Furthermore, true cholera
strains may occasionally show haemolysis, especially in laboratory cultures. Quite
a discussion has arisen in connection with a spirillum isolated from cases of diarrhoea
(no symptoms of cholera) in pilgrims at El Tor. This organism gave the immunity
reactions (agglutination) of true cholera but on account of its haemolytic power has
been considered as distinct from cholera. Such a view would seem to be untenable.
Sp. choleras grows very rapidly on peptone solution and this is the medium for the
enrichment test to be later described. On this it may form a pellicle. On agar
the colony is more opalescent (more of a translucent grayish blue) than the typhoid.
It does not grow on potato except at incubator temperature. It
does not coagulate or turn acid litmus milk. The spirilla are
found in myriads in the rice-water discharges, these white
flakes being desquamated epithelial cells. They penetrate the
crypts of Lieberkuhn, but rarely extend to the submucosa.
The symptoms are due to an endotoxin.

Cholera may be transmitted from water supplies,
when the outbreak is apt to be widespread and in
great numbers from the start. Also by indirect con-
tagion, as by flies or on lettuce, etc. A very im-
portant point is that we have well persons whose faeces
contain virulent cholera spirilla (cholera carriers).

Cholera spirilla disappear from the stools of cholera
patients very rapidly, usually in five to ten days.
Cholera carriers are therefore of less importance epi-
riUum of cholera demiologically than typhoid carriers,
stab c u 1 1 u r e in jt js we^ to remember however that cases have been reported
oW.atl CFrTe n kll of Positive findings after a period approximating two months
and Pjeifer.} from the onset of the attack of cholera. Another important

consideration is that the vibrios may be absent at one examina-
tion and be present at a later one. Purgatives seem to influence the reappearance
of the spirilla. An acid reaction of the faeces, such as that induced by lactic acid
bacteria, would apparently be of value in the prophylaxis of cholera carriers.

Greig has found infection of the bile of the gall-bladder or ducts in 80 cases in
271 cholera autopsies. While cholera spirilla are soon crowded out by intestinal
bacteria, thus explaining the short period during which cholera spirilla are excreted
by convalescents, this is not true when the cholera vibrio gets into the bile ducts or
gall-bladder, where ideal conditions prevail for a prolonged life. In fact bile has

FIG. 38. — Spi-

CHOLERA DIAGNOSIS 1 15

recently been recommended as a selective medium for cholera enrichment. Greig
found one cholera convalescent excreting cholera vibrios 44 days after the attack.
Of twenty-seven persons who had been in contact with cholera patients six
were excreting cholera vibrios though apparently well.

To identify such spirilla immunity reactions are necessary:

1. Injected intraperitoneally into guinea-pigs, it produces a perit-
onitis and subnormal temperature. This reaction exists for
spirilla other than the true cholera spirillum.

2. Intramuscular injections into pigeons are only slightly patho-
genic, if at all.

3. The agglutination test is the most practical. In this we use serum
from an immunized animal, in dilution of from 100 to 1000. It is
rare that true cholera vibrios fail to agglutinate in serum of i to
500 and even sera of i to 10,000 dilution give the reaction. Serum
of cholera convalescents may show agglutination as early as the
tenth day; it is usually best shown about the third week. Dunbar's
quick method is very practical. Make two hanging-drop prepa-
rations, using mucus from the stool as the bacillary emulsion. To
one add an equal amount of a i : 50 normal serum; to the other a
i : 500 dilution of immune serum. Cholera spirilla remain motile
in the control, but lose motility and become agglutinated in the
preparation with the immune serum.

4. Pfeiffer's phenomenon. If cholera spirilla are introduced into the
peritoneal cavity of immunized guinea-pigs (or if together with a
i : 1000 dilution of immune serum the mixture is injected intra-
peritoneally into normal guinea-pigs) and at periods of ten to
sixty minutes after injection, material is removed by a pipette from
the peritoneal cavity, the spirilla have lost motility, have become
granular and degenerated. Pseudospirilla are unchanged. This
reaction may be carried on in a pipette, using fresh serum.

Antisera for the treatment of cholera have not proved successful. Prophylac-
tically, there are two prominent methods: i. That of Haffkine, where live cholera
spirilla are injected subcutaneously; and 2. Strong's cholera autolysate. In this
cholera cultures are killed at 60° C. The killed culture is then allowed to digest
itself in the incubator at 37° C. for three or four days (peptonization). The prepara-
tion is then filtered and from 2 to 5 c.c. of the filtrate is injected. Ferran was the
first to use vaccines.

For diagnosis : i . take a fleck of mucus, make a straight smear and
fix; stain with a i : 10 carbol fuchsin. The comma-shaped organisms
appear as fish swimming in a stream.

Il6 STUDY AND IDENTIFICATION OF BACTERIA

2. Inoculate a tube of peptone solution. The cholera spirilla grow
so rapidly, and being strong aerobes, they grow on the surface of
the fluid so that by taking a loopful from the surface, we may in
three to eight hours obtain a pure culture. Should there be a
pellicle present, this should be avoided in the transfer by tilting
the tube slightly, so that the material near the surface be obtained
without touching the pellicle. Inoculate a second tube from the
surface of this first and, if necessary, a third (enrichment method).

3. Test for cholera red reaction. (Simply adding from three to five
drops of concentrated chemically pure sulphuric acid to the first
or second peptone culture after eighteen to twenty-four hours'
growth. Some specimens of peptone do not give the reaction.)
At times we only get the cholera red when we have a pure culture
of cholera.

4. Smear a fleck of mucus or, better, the three hour surface growth
of a peptone culture on a dry agar surface in a Petri dish. From
colonies developing, make agglutination and, if desired, cultural
tests. It is by immunity reactions that we identify cholera spirilla.
The surface moisture of plates is best dried by the filter-paper top.

The cholera colony is easily distinguished from the ordinary faecal bacterial
colonies by its transparent, bluish-gray, delicate character. It emulsifies with the
greatest ease. A practical, quick method is to make smears from suspicious colonies,
stain for one minute with dilute carbol fuchsin and if vibrios are present to make
two vaseline rings on a single slide allowing ample space at one end for handling the
preparation safely. Inside of one ring deposit with a platinum loop a drop of salt
solution and inside the ring nearest the end which is to be held by fingers or forceps,
deposit a loopful of i to 500 or i to 1000 dilution of cholera serum. The emulsion
in the salt solution remains uniformly turbid and under a low power of the microscope
(2/3 in.) shows a scintillating motility. The emulsion made into the drop of serum
quickly shows a curdy agglutination and upon examination with the 2/3-in. objective
shows clumping and absence of motility. Cover-glasses placed over the two
vaseline rings assist in the study of the preparation.

CHAPTER X.

STUDY AND IDENTIFICATION OF MOULDS.

CLASSIFICATION or THE FUNGI.

Order Suborder

Phycomycetes Zygomycetes

Ascomycetcs

Gymnoascus

. Carpoascus

Family Genus

Species

JVtticor • -,

M. corymbifer

^ M. mucedo

Rhizomucor

R. septatus

Rhizopus

R. niger

!S. cerevisiac

Saccharomyces '

S. anginas

S. blanchardi

Saccharo-

mycetes

Endomyces

E. albicans

Cryptococcus

r C. gilchristi
C. hominis

T. sabouraudi

T. tonsurans

r Trichophyton <

T. violaceum

T. mentagro-

Gymno-

phytes

asceae

T. cruris

,

Microsporum

M. audouini

, Achorion

A. schoeleini

Penicillium

P. crustaceum

A. fumigatus

Perisporia- ,

Aspergillus

A. concentricus

ceae

A. pictor

A. niger

Discomyces

D. bo vis
k D. madurae

Madurella

M. mycetomi

JMalassezia

M. furfur

Microsporoides

M. minutissi-

mus

Trichosporum

T. giganteum

Sporotrichum

S. beurmanni

Hyphomycetes

NOTE. — In many of the works on bacteriology considerable space is given to
the so-called Higher Bacteria. The organisms are chiefly considered under the names
Leptothrix or forms in which are found simple nonbranching threads, Cladothrix
or thread-like forms with false branching and Streptothrix or forms showing true
branching. It is not practical to consider any separate group distinct from the so-
called Lower Bacteria on the one hand and the Fungi on the other.

117

Il8 STUDY AND IDENTIFICATION OF MOULDS

• i

THE FUNGI.

The Thallophyta are plants in which there is no differentiation be-
tween root and stem.

The classes of Thallophyta which are of interest medically are i . the
Algae and 2. the Fungi.

Some include Lichenes as a separate class. These are really sym-
biotic organisms — 'Fungi parasitic on Algae.

The Algae contain chlorophyll, with the exception of Cyanophyceae.
To the order Cyanophyceae it is considered that the family of bacteria
belong.

The fungi do not possess chlorophyll. They are in their simplest forms ramifying
filaments called hyphse. The vegetative hyphae which intertwine in tangled threads,
as a support, are termed the mycelium, while those which project upward are called
the aerial hyphae and are the ones which bear the conidia or spores.

The aerial hypha which carries the fruiting organ encasing the conidia (sporan-
gium) is called the sporangiophore and the more or lesg rounded termination of this
hypha, which projects into the sporangium, is called the columella.

The hypha may be composed of one cell or of many cells separated by septa
(septate).

The orders of the class Fungi which are of interest medically are:
i. the Phy corny cetes; 2. the A<comycetes; 3. the Hyphomycetes.

Phycomycetes. — These produce a copious network-like mycelium, which is non-
septate, and reproduce asexually by means of a sporangium, a case-like structure
borne on the clubbed extremity of an erect hypha (columella) and containing numer-
ous spores or, as in the case of the suborder Oomycetes, reproduction is by hetero-
gamy. (Dissimilar sexual cells — a smaller male, antheridium, and a larger female,
oogonium. By fertilization by antherozoids from the antheridium penetrating the
oosphere we have oospores.)

The suborder Zygomycetes reproduces either asexually (a sporangium filled with
spores) or by isogamy (two similar but sexually differentiated cells conjugate and
form on fusion a zygospore).

Belonging to this suborder we have four families, only one of which, the Mucor
family, is of importance medically. In this family we have three genera: Mucor,
without rhizoids; Rhizopus, with rhizoids and unbranched aerial hyphae and,
Rhizomucor, with rhizoids and ramified mycelium.

Two species of Mucor are of pathogenic importance.

i. Mucor mucedo and 2. Mucor corymbifer. These moulds develop especially
in external cavities as nasopharynx and external ear.

Pulmonary and generalized infections have also been reported. The pathogenic
species have smaller spores and grow best at 37° C. The thick, coarse, cotton-like
mould seen on horse manure is a Mucor. The sporangium, the organ of fructifica-

YEASTS IIQ

tion, contains the spores within its interior. The M. mucedo has thick silver-gray
mycelium, with large sporangia, 150 ft in diameter, containing oval spores, 5 X Qj".
The M. corymbifer, which has been reported from a generalized infection, con-
sidered as typhoid, shows a snow-white mycelium. The sporangia are 20 to 4o/x
and the spore about 3jw in diameter.

Rhizopus niger has a columella which becomes distorted into a mushroom shape
after the spores have been discharged from the sporangium. This mould has been
considered as the cause of a mycosis of the tongue.

Ascomycetes. — -In this order are included many of the parasitic
moulds. The most distinctive characteristic is the formation of asco-
spores in an ascus (little sac). It is an enlarged extremity of a hyphal
branch in which a definite number of spores, usually eight, is formed.
The ascus usually ruptures at its tip. Other members of the order are

FIG. 39. — Yeast cells. Saccharomyces cerevisiae. (Coplin.)

formed from hyphae by the separation of cells in succession from the
free cells. The mycelium is septate.

The order is divided into those with naked asci (Gymnoascus) and those having
a perithecium or investing layer about the ascus (Carpoascus).

Belonging to the suborder Gymnoascus we have i. the family of Saccharomycetes,
which reproduce by budding and in which the asci are without any semblance of a
sheath, and 2. a family in which there is an indication of the formation of a peri-
thecium— this may be termed the Gymnoasceae family.

Saccharomycetes. — There are three genera: Saccharomyces, Endomyces, and
Cryptococcus.

Saccharomyces. — These reproduce by budding, have ascopores and no mycelial-
like threads.

S. cerevisiae. — This is the ordinary yeast fungus. Used at times as an antiseptic.
S. anginae. — Found in a case of angina.

120 STUDY AND IDENTIFICATION OF MOULDS

S. blanchardi. — Found in a jelly-like tumor mass of the abdomen. The budding
cells varied from 2 to 20/1. Probably identical with S. tumefaciens, reported
as the cause of a subcutaneous tumor about region of Scarpa's triangle.
Endomyces. — Forms spores in the interior of filaments, or by ascus formation or by
chlamydospores (resistant spore-like structures with a thick membrane which
project from the extremities or sides of the hyphae as bud-like structures).

E. albicans. — The organism of thrush. It produces a false membrane, especially
on buccal surfaces, which is easily detached and beneath which the mucosa is
intact. Grows only in acid media. Hence propriety of alkaline treatment.
Cryptococcus. — Reproduces by budding, but ascospore formation not observed.
Not a well-recognized genus. The diseases caused by it are termed blasto-
mycoses.

C. Gilchristi. — The cells are about i6/* in diameter and have a thick, double
contoured membrane. They reproduce by budding. The skin lesions resemble
various infectious granulomata and diagnosis rests on the finding of budding

FIG. 40. — Thrush fungus. (Kolle and Wassermann.)

or sporulating cells. It may invade internal organs. Original cultures are
obtained with some difficulty and then best with LofHer's serum. Subcultures
grow readily. Potato is a good medium and on it we may have both mycelial
and yeast-like growth. Guinea-pigs can be inoculated subcutaneously. A
mould, somewhat similar, is the Coccidioides immitis of Ophuls. This has a
mycelial growth in tissues, this distinguishing it from the former fungus. The
infection frequently becomes generalized. The small bodies, about 3jw, in the
Molluscum contagiosum cells are thought by some to be yeasts. They are more
probably artefacts. Plimmer's bodies in cancer cells belong in this group.
They also are probably other than parasites.

C. linguae pilosae. — This is a more or less elongated yeast-like organism and sup-
posed to be the cause of black tongue, a benign affection of the lingual papillae.

Gymnoascea. — -Belonging to the family Gymnoasceae we have the
genera Trichophyton, Microsporum and Achorion.

RINGWORM

121

The trichophytons are generally known as the large-spored ring-worms. The
spores are in chains and may be inside the hair or both outside and inside. Many
of them are of animal origin, especially from the horse and the cat. The spores are
from 5 to 7/1.

The mycelium is greatly segmented, shows simple or dichotomous branching,
and produces spores within the mycelium.

T. tonsurans. — Gives a crater-like culture with fine marginal rays. Fungus
wholly inside the hair. Causes most of the large-spored scalp ringworms and
many body cases.
T. sabouraudi. — Has a heaped-up festooned sort of culture. There is a similar

10.

FIG. 41.- — More common fungi, i, Culture of Achorion schoenleini (favus);
2, culture of Trichophyton tonsurans; 3, culture of Trichophyton sabouraudi; 4,
sporangium of Aspergillus; 5, culture of Trichophyton mentagrophytes; 6, culture
of Microsporum audouini; 7, mycelium and spores of Malassezia furfur; 8, Crypto-
coccus gilchristi; 9, A and B, sporangium and mycelium of Mucor corymbifer; 10,
Penicillium; n, Saccharomyces tumefaciens; 12, Discomyces bovis.

fungus with a violet culture. These cause some of the scalp and beard ring-
worms.

T. mentagrophytes. — This is the T. megalsporon endoectothrix of Sabouraud.
The external spores are in chains or in short mycelial threads, not mosaics of
spores, and are of very unequal size (2 to n microns). There are varieties
from horse, cat, and bird. The lesions are more inflammatory than those of
the endothrix class. Most of the beard and body ringworms belong to this
group — very few scalp cases. The cultures are finely rayed.

122

STUDY AND IDENTIFICATION OF MOULDS

Some give yellow cultures, others white and one derived from birds a rose-
colored culture.

Microsporum audouini. — This is the so-called small-spored ring-
worm and is a very common and highly contagious affection of the scalp
in children in England and France; less so in other countries.

It is almost never seen in the tropics. It almost exclusively affects the hairy
scalp. The spores are 2 to 3^ in diameter. The broken stump of the hair is char-
acteristic. The fungus is packed as a mosaic of spores, forming a white sheath,
chiefly on the outside of the hairs. It gives a downy-white culture.

9-08 .

FIG. 42. — Tropical fungi, i, concentric rings of Aspergillus concentricus;
2, sporangium of A. concentricus; 3, Aspergillus pictor; 4, Microsporoides minu-
tissimus; 5, Trichosporum giganteum; 6, black granules of Madurella mycetomi;
7, yellow grains of Discomyces madurae.

Achorion schoenleini is the cause of favus. The cultures are rather
wrinkled. It is characterized by the scutulum or favus cup. This is a
sulphur-yellow pea-sized cup with a central lusterless hair. Affected
hairs may not show a cup. Favus is not so contagious as ringworm.
It chiefly affects the hairy scalp, but may also invade the nails and even
the body.

Microscopical examination shows great irregularity of spores and mycelium, the
latter being irregularly disposed and of varying thickness and length and wavy

ACTINOMYCOSIS 123

instead of straight as in Trichophyton. There is also the greatest irregularity in
the refractile favus spores — they are gnarled and bizarre shaped, in contrast to the
regular ovals or spheres of the ringworm fungus. Cultures show ridges or
convolutions.

In the suborder Carpoascus we have to consider the family Perisporiaceae. In
this family the asci are completely inclosed by the investing membrane, the peri-
thecium. When this rots the spores are set free. There are two genera of interest,
Penicillium and Aspergillus.

In Penicillium we have vertical branches with strings of conidia. In Aspergillus
these conidia arise from a globular termination of the hypha.

Penicillium. — While Penicillium does at times form perithecia, yet they char-
acteristically show chains of spores. The common P. glaucum resembles a hand
with terminal beads, more than the hair pencil, from which the name is derived.
P. crustaceum. — Is the common blue-green mould. It has been deemed patho-
genic in cases of chronic catarrh of the eustachian tube and in gastric hyper-
acidity.

Aspergillus. — These have sterigmata carrying chains of spores, these sterigmata
being little processes projecting out from the knob-like termination of the aerial
hypha (columella). Of the pathogenic Aspergilli we have:

1. A. fumigatus. — This has been considered as the cause of pellagra. A pul-
monary mycosis resembling phthisis may be due to this species.

2. A. repens. — This has been found in the auditory canal and may produce a
false membrane.

3. A. flavus. — This has been found in the discharges of chronic ear diseases.

4. A. nidulans has been reported as one of the causes of mycetoma showing white
granules. It has also been considered a cause of aural mycosis.

5. A. concentricus. — This is the cause of an important tropical ringworm, tinea
imbricata. The scales are dry, like pieces of tissue-paper. There are generally
about four rings which do not heal in the center. General appearance is that
of watered silk. There are no inflammatory lesions. Common in Malay
peninsula. Also found in some parts of the Philippines and in China. Some
authorities consider the fungus to be a Trichophyton.

6. A. pictor. — This is the cause of a skin affection of Central America. In the
affection colored spots appear on the skin, chiefly on face, forearms, and chest.
The disease is attended with a mangy odor. Spots are of various colors; if
the superficial epithelium is affected we have a dark violet color. Deeper
involvement gives red spots.

Hyphomycetes. — In this order are grouped certain genera which
cannot properly be assigned to any of the other orders. They are also
designated Fungi Imperfecti, for the reason that the fruiting bodies
characteristic of the other orders have not been satisfactorily observed.

Discomyces bovis. — This is the well-known ray fungus, the cajise of actinomycosis.
In man it is at times found in chronic suppurative conditions attended with much
granulation tissue. Such pus may show small yellow-gray granules about the
size of a pin's head. When spread out between two slides the central portion

124 STUDY AND IDENTIFICATION OF MOULDS

shows a network of mycelium with bulbous thread-like rays going to the periphery.
The "clubs " at the periphery are degenerate structures and do not stain by Gram.
The central mycelium is Gram positive. This mould is essentially an anaerobe
and should be cultivated in a deep glucose agar stab. It may also be cultivated
in bouillon. In this it grows at bottom. Growth is dry and chalky. In diagnosis
look for the little granules. Curetting of the sinuses may give the "ray fungus"
when they are not found free in the pus.

Discomyces madurae. — This is a ray fungus found in the yellow "fish-roe" granules
of madura foot. It is strictly aerobic in cultures, thus differing from actinomy-
cosis. For diagnosis proceed as for D. bo vis.

Madurella mycetomi. — This is the cause of the black "gunpowder" granules of
madura foot. It is a mycelial mass with rather oval shaped swollen segments.
It is at times cultivable on potato and agar as felted masses of gray growth, which
later become almost black.

Malassezia furfur. — This is the fungus of tinea versicolor. It is common both in
temperate and in tropical climates. It is characterized by dirty yellow spots
about covered parts of the body. Scrapings show a profusion of mycelial threads
and interspersed spores. It is very difficult to cultivate. The organism usually
termed the bottle bacillus is really a fungus having the characteristics of the
genus Malassezia. It is thought to be the cause of pityriasis of the scalp.
Microsporoides minutissimus. — This is generally considered as the cause of Ery-
thrasma or dhobie itch, a very common intertrigo of the tropics. It is character-
jzed by its narrow mycelium and small spores. Various fungi are found in this
affection. Castellani considers the chief cause of dhobie itch to be a trichophyton,
T. cruris.

Clinically this affection shows festooned areas of a bright red color which tend
to clear up in the center becoming fawn color. As a result of the intolerable itching
and scratching the affection tends to spread from its favorite sites — the inner sur-
faces of the thighs and the scrotum. The spores and mycelium are abundant at
the onset but later, one may not find any evidence of the mould. In some of the
rapidly spreading cases I have found a symbiosis of fungus and coccus, the bacterial
elements lying packed in aggregations scattered through the mycelial ground work.

Culturally these cocci were S. pyogenes aureus.

Trichosporum giganteum. — This is the cause of a disease of the hairs, known in
Columbia as "Piedra," so called from the small gritty-like masses along the
length of the hair. These spores are arranged like mosaics about the hair.
Sporotrichum beurmanni. — This fungus has a narrow mycelium (2/0 and branches
in all directions. The spores appear as little grape-like clusters of oval spores
(3 to 5ju) at the end of a filament. It is readily cultivated, showing as a small
white growth about the eighth day.

The fungus of Sporotrichosis develops in tissue by budding, not showing the
mycelial growth seen in artificial cultures. Potato makes a good medium and often
such cultures show pigmentation.

This mould produces indolent, glistening, subcutaneous tumors which are pain-
less. They may ulcerate and give off a brownish discharge. They resemble tuber-
culous or syphilitic lesions.

Certain organisms which resemble both moulds and bacteria, having branching

FUNGI 125

filamentous forms and at the same time having a spore-like method of reproduction,
are known under the names Streptothrix or better Nocardia. It is chiefly in various
pathological processes of the lungs that they have been observed, but in addition
they have been noted in brain, glands, kidney and subcutaneous tissue.

The infections are most likely to be confused with phthisis and glanders. The
organisms are easily cultivated and in staining reactions are midway between T. B
and Actinomycosis.

DIAGNOSIS OF FUNGI.

The most expeditious way to examine for fungi is to treat the scales
or hairs with a 10% solution of caustic potash or soda. Then crush
between two slides; heat moderately over the flame and examine.

Tribondeau's method is to treat the scales with ether, then with alcohol, and
finally with water. Next put the sediment (it is convenient to use a centrifuge)
in a drop of caustic soda solution. Cover with a cover-glass, and after the prepara-
tion has stood about an hour run glycerine under the cover-glass.

A very satisfactory method is to scrape the scales with a small scalpel, and smear
out the material so obtained in a loopful of white of egg or blood-serum on a glass
slide. By scraping vigorously the serum may be obtained from the patient. After
the smear has dried, treat it with alcohol and ether to get rid of the fat. It may then
be stained with Wright's stain or by Gram's method. The ordinary Gram method
may be used or the decolorizing may be done with aniline oil, observing the decolor-
ization under the low power of the microscope.

Yeasts are best examined in hanging drop on the plain slide with vaselined cell,
as given under Blood.

An excellent way to examine moulds is to seize some of the projecting sporangia
from the surface of a plate with forceps and mount in liquid petrolatum. I have
found that moulds in scales from skin or infecting various mites or insects will show
a growth in this medium when mounted on a slide and covered with a cover-glass.

CULTIVATION OF FUNGI.

Moulds grow well on media with an acid reaction, so that by adjust-
ing the reaction to + 2% or even higher, we permit of the growth of the
fungi, but inhibit bacterial development.

Glycerine agar, bread paste, or potato media are all suitable, but the best medium
is that of Sabouraud:

Maltose, 4 grams.

Peptone, i gram.

Agar, i . 5 grams.

Water, 100 c.c.

Make the reaction about +2.

126 STUDY AND IDENTIFICATION OF MOULDS

Before inoculating media with moulds, some recommend placing the material in
60% alcohol for one or two hours to kill the bacteria. The moulds withstand such
treatment.

In cultivating moulds it is best to use small Erlenmeyer flasks, containing about
one-fourth of an inch of media on the bottom, for the development of the colonies.
In order to separate the mould we may take the hair or scales on a sterile slide and
cut them into small fragments with a sterile knife. Then moisten a platinum loop
from the surface of an agar slant, touch a fragment with the loop, and when it adheres
transfer it to the agar slant. Make four or five inoculations on the surface and
from suitable growth, after four to seven days, inoculate the medium in the Erlen-
meyer flask.

Plauth recommends receiving the mould material between two sterile glass slides.
Seal the edge of the slide with wax and place the preparation in a moist chamber
for four to seven days. From developing fungus growth inoculate the medium in
the Erlenmeyer flask. A Petri dish containing several layers of thoroughly mois-
tened filter-paper in top and bottom makes a satisfactory moist chamber.

CHAPTER XI.
BACTERIOLOGY OF WATER, AIR, MILK, ETC.

BACTERIOLOGICAL EXAMINATION OF WATER.

WHILE in a chemical examination as to the character of a water
there are certain relations between the free and albuminoid ammonias,
nitrates, nitrites, chlorides, etc., which indicate the probable animal as
against vegetable nature of the organic matter present, yet it is a more
or less presumptive evidence. In a bacteriological examination of
water the finding of the colon bacillus may from a practical standpoint
be considered as positive evidence of human faecal contamination.
Theoretically, the possibility of organisms being present corresponding
culturally to B. coli and derived from cereals is to be considered. Also
the faeces of animals contain an organism which cannot be differentiated
from the colon bacillus.

In detecting sewage contamination in water to which varying amounts of sewage
had been added, it was found that the bacterial tests were from ten to one hundred
times more delicate than the chemical ones.

As showing sewage contamination of water, the presence of the B. coli has been
generally accepted as the most satisfactory indication. The English authorities
consider sewage streptococci and the spore-bearing B. enteritidis sporogenes as of
value as indicators as well as the B. coli — the presence of sewage streptococci indi-
cating very recent sewage contamination and that of the B. enteritidis sporogenes,
in the absence of streptococci and colon bacilli, as evidence of sewage contamination
at some period more or less remote.

In the United States the colon bacillus alone is considered the indi-
cator of sewage contamination, and all tests, presumptive or positive,
are based on the presence of this organism.

It is not the finding of the colon bacillus but rather the question of
it's relative abundance that is involved in a water analysis. Thus the
finding of one colon bacillus in 50 c.c. of water would not have weight as
showing contamination, but the presence rather constantly of the colon
bacillus in i c.c. or less makes contamination of a water supply
probable.

127

T28 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

In collecting samples of water for bacteriological examination, the following
points should be considered:

1. The bottles, which should have a capacity of from 25 to 100 c.c., should be
sterile. Sterilization may be effected by heat or by rinsing with a little sulphuric
acid and subsequently washing out thoroughly with the suspected water before col-
lection. The utmost care must be exercised that the fingers do not come in contact
with the glass stopper of the neck of the bottle while filling it. If the specimen is
to be sent some distance, it should be packed in ice to prevent bacterial development.
Frankland states that a count -of 1000 became 6000 in six hours and 48,000 in forty-
eight hours. In water packed in ice for a considerable time, however, the bacterial
count may diminish.

2. If collecting from city water supplies, secure the sample direct from the mains
and let the water run from the tap a few minutes before collection. If the water be
taken from a pond, stream, or cistern, be sure that the specimen comes from at least
10 inches below the surface. As sedimentation is the most important method in
self-purification of rivers and ponds, it will be understood that any stirring up of the
mud on the bottom will enormously increase a bacterial count.

Quantitative Bacteriological Examination.

if Deliver definite quantities of the water to be examined into tubes of liquefied
gelatin or agar and plate out the same in a series of Petri dishes.

A more practical method is to deliver the water from the graduated pipette into
the empty sterile dish. The water should be deposited in the center of the plate
and the melted gelatin or agar poured directly on the water and then, carefully
tilting to and fro, mix the water and the media. One set of plates should be of gelatin
and incubated at room temperature; a similar set should be of lactose litmus agar and
incubated at 38° C. If the water is highly contaminated, it is necessary to dilute
it; thus, with river water, which may contain from 2000 to 10,000 bacteria per c.c.,
a dilution of i to 100 would be desirable.

Ordinarily it will be sufficient to deliver from a sterile graduated pipette 0.2, 0.3,
and 0.5 c.c. of the water in each of two sets of plates: one set for gelatin, the other for
agar.

When gelatin is not at hand or convenient to work with, the gelatin plates may
be replaced by others of lactose litmus agar for incubation at room temperature.
After twenty-four hours at 38° C. or forty-eight hours at 20° C., the count should be
made.

Example. — Forty colonies were counted on the gelatin plate containing 0.2 c.c.
(1/5) of the water. The number of organisms would be 200 per c.c. Ten colonies
were counted on the agar plate containing 0.2 c.c. and incubated at 38° C. Number
of bacteria developing at body temperature equals 50 per c.c.

There is no strict standard as to the number of bacteria a water should contain
per c.c. Koch's standard of 100 colonies per c.c. is generally given. It is by the
qualitative rather than the quantitative analysis that one should judge a water.

If there should be very many colonies on a plate, the surface can be marked off
into segments with a blue pencil. If very numerous, cut out of a piece of paper a
space equal to i square centimeter. By counting the number of colonies inclosed

COLON BACILLUS IN WATER

I2Q

in this space at different parts of the plate, we can strike an average for each space of
i square centimeter. To find the number of such spaces contained in the plate,
multiply the square of the radius of the plate by 3.1416. Then multiply this number
by the average per square centimeter, and we have the total number of colonies
on the plate. This is the principle of the Jeffers disc.

The relative proportion between the bacterial count at 20° C. and that at 38° C.
is of great importance from a qualitative standpoint, as will be seen later.

2. Deliver into a series of Durham fermentation tubes containing glucose
bouillon and into another series containing lactose bouillon varying definite amounts
of the water to be examined. In tubes showing the presence of gas in both glucose
and lactose bouillon the evidence is presumptive that the colon bacillus is present.
For the positive demonstration plates must be made from such tubes as show gas.

It is sufficient to deliver from graduated pipettes in each series quantities of
water varying in amount from o.i c.c. to 10 c.c. In our laboratory we inoculate
with o.i c.c., 0.2 c.c., 0.5 c.c., i c.c. and 10 c.c. of the suspected water. If the o.i c.c.
tubes show gas, we have reason to assume that the water contained at least 10 colon
bacilli per c.c. If only the 10 c.c. tubes showed gas — those with less amounts not
having gas — we would be in a position to state that the water contained the colon
bacillus in quantities of 10 c.c., but not in quantities of i c.c. or less. Many authori-
ties regard water as suspicious only when the colon bacillus is present in quantities
of 10 c.c. or less; waters of good quality frequently showing the presence of the colon
bacillus in quantities of ico to 500 c.c.

It is generally accepted that if a water shows the presence of the
colon bacillus in quantities of i c.c. or less, it should be regarded as
suspicious.

At the present time the medium that gives the least source of error in carrying
out the quantitative presumptive tests is the lactose bile. It is made by adding
i% of lactose and i% of peptone to ox bile, and fermentation tubes of the media
showing gas may be considered as very probably containing the colon bacillus. The
percentage of error with this method is reported to be only 11%, while with glucose
fermentation tubes the error is more than 50%. Gas formation is usually shown in
forty-eight hours, but it is advisable to continue the incubation for seventy-two hours.
These presumptive tests are chiefly of value in highly contaminated waters. Even
with this method plates should be made.

3. As the colon and sewage streptococci ferment lactose with the production
of acid and hence produce pink colonies on lactose litmus agar, much information
can be obtained from the proportion existing between the number of pink colonies
and those not having such a color. Waters of fair degree of purity rarely give any
pink colonies.

Qualitative Bacteriological Examination.

General Considerations. — In some countries the proportion of
liquefying to nonliquefying colonies on gelatin plates is considered of
importance. Certain sewage organisms belonging to the proteus and
9

130 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

cloaca groups liquefy gelatin; consequently, if the proportion of liquefy-
ing to nonliquefying be greater than as i to 10, the water is considered
suspicious. The test is not considered by American authorities as of
any particular value.

The American Public Health Association recognizes the importance
of the information obtained from a comparison of the number of organ-
isms developing at 38° C. and those developing at 20° C. Bacteria
whose normal habitat is the intestinal canal naturally develop well at
body temperature, while normal water bacteria prefer the average
temperature of the water in rivers and lakes. Consequently when the
number of organisms developing at 38° C. at all approximates the num-
ber developing at 20° C., there is a strong suspicion that sewage or-
ganisms may be present. Normal waters give proportions of i to 25
or i to 50, while in sewage contaminated waters the proportion may be
as i to 4 or less.

In addition, the appearance of pink colonies on the lactose litmus
agar is of great assistance in judging of a water. Both sewage strepto-
cocci and the colon bacillus give pink colonies — 'those of the streptococci
are smaller and more vermilion in color. Microscopic examination
will differentiate the cocci from the bacilli. It is well to bear in mind
that the pink colonies after twenty-four hours may turn blue in forty-
eight hours from the development of ammonia and amines. Conse-
quently the lactose litmus agar plates should be studied after twenty-
four hours.

A good water supply will rarely show a pink colony, while in a sew-
age-contaminated one the pink colonies will probably predominate.

The diagnostic characteristics considered important by the Ameri-'
can authorities in reporting the colon bacillus (Recently designated
excretal colon bacillus) are:

1. Typical morphology, nonsporing bacillus, relatively small and often quite
thick.

2. Motility in young broth cultures. (This is at times unsatisfactory, as some
strains of the colon bacillus do not show it even in young bouillon cultures).

3. Gas formula in dextrose broth. Of about 50% of gas produced, one- third
should be absorbed by a 2% solution of sodium hydrate (CC^). The remaining gas
is hydrogen. (Later views indicate that the gas formula is exceedingly variable and
should not be depended upon. To carry out this test one fills the bulb of a fermen-
tation tube with the caustic soda solution, holding the thumb over the opening
or witih a rubber stopper, the bouillon culture and the soda solution are mixed
by tilting the fermentation tube to and fro. The total amount of gas is first re-

TYPHOID AND WATER 131

corded and then that remaining after the CO2 has been absorbed is reported as
hydrogen.)

4. Nonliquefaction of gelatin.

5. Fermentation of lactose with gas production.

6. Indol production.

7. Reduction of nitrates to nitrites.

To these may be added the acidifying and coagulation of litmus milk without
subsequent digestion of the casein. The production of gas and fluorescence in glu-
cose neutral red bouillon is also a very constant function of the colon bacillus.
B. coli aerogenes is similar to B. coli with the exception of nonmotility and production
of gas in saccharose media. B. coli anaerogenes is also similar to B. coli but does not
produce gas in glucose and lactose.

NOTE. — The reduction of neutral red with a greenish-yellow fluorescence is very
striking and has been suggested as a test for the colon bacillus. Many other organ-
isms, especially those of the hog cholera group, have this power. It is convenient,
however, to color glucose bouillon with about i% of a 1/2% solution of neutral red.

On the plates made for the detection of colon bacillus may be found
certain organisms which have origin in fecal contamination. The more
important of these are those of the paratyphoid, cloaca and proteus
groups. In addition, the B. fecalis alkaligines has not rarely been
isolated. Among natural water bacteria there may be present either
the liquefying or the nonliquefying B. fluorescens. These colonies
have a yellowish-green fluorescence.

Certain chromogenic cocci and bacilli are found in uncontaminated
waters as B. indicus or B. violaceus. From surface washings we obtain
certain soil bacteria as B. mycoides, B. subtilis, B. megatherium. One
of the higher bacteria which shows long threads, Cladothrix dichotoma,
is common, and is characterized by a brown halo around it's gelatin
plate colony.

Isolation of the Typhoid Bacillus from Water.

This is probably the most discouraging procedure which can be
taken up in a laboratory. Only the most recent reports of such isola-
tion from water supplies, which have been verified by immunity reac-
tions, can be accepted and of these the number of instances is exceed-
ingly small. Owing to the long period of incubation, the typhoid
organisms may have died out before the outbreak of an epidemic
suggests the examination of the water supply.

There have been various methods proposed for the detection of the B. typhosus
in water. A method which would offer about as reasonable a chance of success as
any other would be to pass 2 or 3 liters of the water through a Berkefeld filter; then

132 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

to take up in a small quantity of water all the bacteria held back by the filter. Then
plate out on lactose litmus agar and examine colonies which do not show any pink
coloration. The dysentery bacillus has about the same cultural characteristics as
the typhoid one, so that it is important to note motility. If from such a colony
you obtain an organism giving the cultural characteristics of B. typhosus, carry out
agglutination and preferably bacteriolytic tests as well. Some strains of typhoid,
especially when recently isolated from the body, do not show agglutination.

The Conradi Drigalski, the malachite-green, and various caffeine containing
plating media have been highly recommended.

Isolation of the Cholera Spirillum from Water.

The method proposed by Koch in 1893 does not seem to have been improved
upon by later investigators. To 100 c.c. of the suspected water add i% of peptone
and i% of salt. Incubate at 38° C., and at intervals of eight, twelve, and eighteen
hours examine microscopically loopfuls taken from the surface of the liquid in the
flask. So soon as comma-shape organisms are observed, plate out on agar. The
colonies showing morphologically characteristic organisms should be tested as to
agglutination and bacteriolysis. Inasmuch as the true cholera spirillum shows a
marked cholera-red reaction it is well to inoculate a tube of peptone solution from
such a colony and add a drop of concentrated sulphuric acid after incubating for
eighteen hours. The rose-pink coloration is given by the cholera spirillum with the
acid alone — the nitroso factor in the reaction being produced by the organism.

BACTERIOLOGICAL EXAMINATION OF MILK.

A bacterial milk count is of comparatively little value as showing
whether a milk is dangerous or not. As a matter of fact, a milk which
contains several million of bacteria per c.c. might be less dangerous
than one containing only a few thousand, especially if in the latter
there were numerous liquefiers and gas producers present. There is,
however, one point of importance in connection with the quantitative
estimation of bacteria in milk, and that is the fact that in order to keep
the development of the bacteria within the limits of 10,000 to 50,000
per c.c., it is necessary that the requirements of cleanliness in milking
and the rapid cooling of the milk after obtaining it and the keeping of
the temperature below 50° C. be rigidly observed. If a milk has a
high count it shows some error in the handling of the milk. Anderson
has found that top milk contains from ten to five hundred times as many
bacteria as bottom milk. Centrifugally raised cream contains more
bacteria than that forming by gravity. In making a quantitative bac-
teriological examination, the principle is the same as with water.

MILK I33

Make a known dilution of the milk with sterile water; add definite quantities of
this diluted milk to tubes of melted <. gar or gelatin, and pour into plates. The diluted
milk may also be delivered in the center of the plate and the melted agar or gelatin
poured directly on it, mixing thoroughly. Always shake the bottle well before
taking sample.

Example. — Added i c.c. of milk to 199 c.c. of sterile water in a large flask (500
to 1000 c.c.) After shaking thoroughly, take i c.c. of this i : 200 dilution and add
it to 99 c.c. of sterile water. Shaking thoroughly, we have a dilution of 1:20,000.
Of this we added 0.5 c.c. to a tube of gelatin or agar. After incubation the plate
showed 75 colonies. Therefore the milk continued in each c.c. 75 X 2X 20,000 (dilu-
tion) = 3,000,000 — the number of bacteria in each c.c. of milk.

Lactose litmus gelatin or agar is to be preferred in milk-work, as the normal
lactic acid bacteria produce reddish colonies which are very striking. A standard
easily attained for high-grade, certified milk would be 5000 to 10,000 per c.c.

In the qualitative examination of milk, many dairies employ the fermentation
tube, any organism producing gas being considered undesirable. Again liquefying
organisms, as shown by the presence of such bacteria in the gelatin plates, is evidence
of probable contamination by faecal bacteria. A question which seems difficult to
decide is as to the general nature of the so-called normal lactic acid bacteria of milk.
Some describe them as very short, broad bacilli with very small colonies, fermenting
lactose with the formation of lactic acid. Others consider that the streptococci are
the organisms which are concerned with the normal fermentative changes. In
examining specimens of milk considered the best on the market, I have repeatedly
found the small red colonies on lactose litmus agar to be in chains of either Gram
positive streptococci or streptobacilli. Of the acid-forming bacilli in milk we have
i. the B. lactis acidi group. These are oval cells about 0.9 microns by 0.6 microns,
often in chains. They are Gram positive and nonmotile. They may be the same
as Streptococcus lacticus of Kruse. They curdle milk with a homogeneous clot — this
being due to the fact that they do not produce gas in lactose media. 2. The B.
coli aerogenes group. These are gas producers. (See under water.) 3. The B.
bulgaricus group. In connection with the organisms present in the tablets used
for treating milk to produce lactic acid for the treatment of intestinal disorders, and
considered to be normal lactic acid bacteria, I have found both streptococci and
bacilli. These have all agreed, however, in not producing gas in either lactose or
glucose fermentation tubes.

The organism upon which special stress is laid in these so-called lactic acid pro-
ducers is the B. bulgaricus. This is a large, nonmotile organism with square ends
like anthrax. It often occurs in long chains and does not possess spores. It is Gram
positive and often shows metachromatic granules like those of the diphtheria bacillus.
Colonies show in forty-eight hours which resemble streptococcus ones, but are
more contoured on the surface. It produces a deep vivid pink in litmus milk, while
milk streptococci only cause a light pink. It produces a very large amount of acid
(3%). Little or no growth on ordinary laboratory media or below O° C. (Op. temp.
42° C.).

Heinemann states that it occurs normally in human faeces and various fermented
milks — also in gastric juice when HC1 is absent. To isolate, put milk or faeces into
a broth containing 0.5% acetic acid and 2% glucose. Transfer to litmus milk after

134 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

twenty-four hours and from such tubes plate out on milk serum agar (coagulate
boiling milk with a few drops of acetic acid, filter and add i% peptone, 2% glucose
and 1.5% agar).

As they grow in very acid media the term acidophilous is applied. It was sup-
posed that these bacteria were peculiar to certain fermented milks as matzoon and
yogurt. Hastings has shown the group to be present in milk in the United States
and considers the source to be the alimentary tract of cows.

Another source of information as to the quality of a milk may be
derived from a study of the number of leukocytes or pus cells contained
in i c.c. of the milk. It must be understood that cellular elements
which differ only slightly from true pus cells may be found in the milk
of healthy cows and may be found in great numbers. Statements have
been made that such cells are neither amoeboid nor phagocytic.

The Doane-Buckley method is probably the most accurate. In this you throw
down the cellular contents of 10 c.c. of milk in a centrifuge revolving about 1000
times a minute for ten to twenty minutes. Then remove supernatant milk and add
0.5 c.c. of Toisson's' solution to the sediment. You thus have the leukocytes of
10 c.c. contained in 0.5 c.c. (Concentrated twenty times.) Make a haematocytom-
eter preparation as for blood and find the average munber of cells for each square
millimeter. Then multiply this by 10 to get the number of cells in a cubic millimeter.
As a cubic millimeter is one thousand times smaller than a cubic centimeter, you
multiply the number per cubic millimeter by 1000. Then, as the milk was concen-
trated twenty times, you divide by 20. (If it were diluted twenty times, you would
multiply by 20.)

Example. — Found an average of 50 cells per square millimeter. This would
make 500 per cubic millimeter, and 500,000 per c.c.; then 500,000 divided by 20
would give 25,000.

There is no agreement as to a standard for allowable leukocytes. Even in
apparently healthy animals they may exceed 100,000 per c.c. Doane has suggested
500,000 per c.c. as a preferable limit.

The smear methods for determining the number of leukocytes present do not
compare in accuracy with the volumetric ones.

To summarize, we may state that the bacterial count is an indicator
of the care used in handling the milk while the presence of harmful
bacteria (qualitative examination) or numerous pus cells indicates
disease in the cow. During 1912 severe epidemics of sore throat due
to a streptococcus, S. epidemicus, were traced to milk of cows having
probably suffered from mastitis. In Baltimore the milk had been
pasteurized by the flash method which indicates the unreliability of this
process.

Pasteurization of Milk.— The objections to this method of preserving milk
have been(i) that the lactic acid bacteria which have been by some credited with

AIR 135

antagonism to harmful bacteria, would be destroyed by pasteurization, (2) the more
rapid development of bacteria in milk that has been pasteurized (3) interference with
nutritive qualities and (4) pasteurized milk does not show its deterioration as does
unpasteurized milk, thus failing to give a clue as to the age of the milk.

The United States Bureau of Animal Industry in studying this important phase
of the milk question has grouped the milk bacteria into three classes (a) acid -forming,
(b) putrefactive (liquefying) and (c) inert bacteria. In their investigations it was
found that many acid- forming bacteria withstood temperature as high as 168° F.,
so that pasteurized milk was soured just as is raw milk, but more slowly. They found
that pasteurized milk showed fewer putrefactive bacteria than raw milk, so that
even should it be a fact that injurious toxins were produced by spore-bearing putre-
factive organisms the development of such organisms would be even less in pasteu-
rized milk.

The statement so often advanced that bacteria develop more rapidly in pasteu-
rized milk than in raw milk was proved fallacious.

It was recommended that holding the milk for thirty minutes at 145° F. was a
far better method of pasteurizing than quickly bringing the milk to a temperature of
185° F. (flash method). All admit the great value of the killing of important patho-
gens (typhoid, cholera, streptococci, etc.).

BACTERIOLOGICAL EXAMINATION OF AIR.

In Paris a cubic meter of air was found to contain the following
number of organisms:

Suburbs. — 'Winter, 145 moulds, 170 bacteria.

Summer, 245 moulds, 345 bacteria.

City Hall. — -Winter, 1345 moulds, 4305 bacteria.

Summer, 2500 moulds, 9845 bacteria.

Air of hospitals, especially after sweeping, may contain 50,000
bacteria per cubic meter. There does not seem to be any particular
relation between the amount of carbon dioxide in air and the bacterial
content.

Petri's Rough Method. — Exposure of a lactose litmus agar plate (capacity 100
sq. cm.) for five minutes will give the number of organisms present in ten liters of air.
Multiply by 100 for one cubic meter.

The two groups of organisms usually found in air are i. bacteria and 2. moulds.
Moulds (spores) may be carried by currents of air; bacteria, however, are generally
carried about by particles of dust or finely divided liquids (spray). On the lactose
litmus agar plate staphylococci and streptococci show as bright red colonies.

Sedgwick-Tucker Sterile Granulated Sugar Method. — Sterilize aerobioscope
and introduce granulated sugar on support. Again sterilize (not over 120° C. in
dry-air sterilizer). Allow a given quantity of air to pass through; then shake the
sugar into wide part of aerobioscope. Now pour in 10 or 15 c.c. of melted gelatin

136 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

(40° C.) to dissolve sugar. Roll tubes as for Esmarch roll cultures, and incubate
at room temperature. To draw air through the aerobioscope, connect the small end
with a piece of rubber tubing which is attached to a tube in the stopper of an aspirat-
ing bottle. Having poured a definite quantity of water into the aspirating bottle,
allow the water to run out. The same quantity of air will be drawn through the
sugar of the aerobioscope as the amount of water passing out of the aspirating bottle.
The bacteria and moulds are caught by the sugar.

Example. — Passed ten liters of air through the aerobioscope. The bacteria in
this quantity of air showed 75 colonies when incubated at 20° C. The unit being
one cubic meter or one thousand liters, we have only obtained the bacteria of one
hundredth of the unit. Hence multiplying 75 by 100 gives 7500 bacteria as present
in one cubic meter of the air examined.

FIG. 43. — Sedgwick-Tucker aerobioscope. (Williams.)

In comparing the results with the aerobiscope with those obtained
by exposing a plate as in Petri's method for ten instead of five minutes,
it was found that the latter was sufficiently in accord to make it a satis-
factory approximate quantitative method. The simplicity and ease of
access to the colonies developing on it make it preferable when the air of
operating-rooms or hospital wards is to be examined.

Of the fungi ordinarily obtained in examinations of the air the blue-green mould
and the red yeast are the most common. B. subtilis and sarcina types of cocci are
the most common bacterial colonies found upon exposed plates. Sewer air is as a
rule free from bacteria, due probably to the fact that bacteria tend to adhere to
moist surfaces.' The importance of Fliigge's droplet method of contamination of
the air of a room is brought out in the discussion of infection with pneumonic plague.
This is an important method in the transmission of tuberculosis.

CHAPTER XII.
PRACTICAL METHODS IN IMMUNITY.

THAT which prevents the gaining of a foothold by disease organisms
in the animal body or which neutralizes their harmful products or de-
stroys the parasites is termed immunity. In the main, the question
of immunity hinges on the powers of resistance of the human body
and the aggressiveness or virulence of the invading organism. It must
always be kept in mind that immunity is only relative; thus the fowl,
which is practically immune to tetanus, may be made to succumb by
reducing its resistance by refrigeration or by increasing the amount of
poison introduced. The insusceptibility which the fowl has to tetanus
or which man has to many diseases of animals is best termed inherent
immunity, and is at present only a subject of theoretical interest.
When immunity to a given disease is obtained as a result of an attack
of the disease in question or by laboratory methods of inoculation, this
is termed properly an acquired immunity, and in the former case is a
naturally acquired immunity or "natural immunity" and in the second
is an artificially acquired immunity or " artificial immunity."

Immunity may be divided into that which is inherent and that which is acquired.
Inherent immunity is such as is observed in the resistance of Algerian sheep to
anthrax (ordinary sheep are very susceptible) or the fowl to tetanus and is of interest
theoretically rather than practically. Acquired immunity may be brought about
naturally as by an attack of a disease or artificially by laboratory measures.

As a result of an attack of a disease or in response to the stimulus
of the injection of the organisms or its products, we have developed in
the man so injected certain specific antagonistic properties to that
organism, which are usually demonstrable in the blood-serum or other
body fluids, and to which we apply the terms agglutinating power,
opsonic power, or bacteriolytic power. The term antibody is also
applied. All three powers may be present together in equal or in vary-
ing degree or one or more may be absent. By agglutinating power we
mean that which causes evenly distributed organisms to come together
and form clumps. By opsonic power we mean that which so alters the

137

PRACTICAL METHODS IN IMMUNITY

resistance of bacteria that the phagocytes ingest them. By bacterio-
lytic power we mean that which brings about disintegration or lysis of
the specific organism. The bacterium which causes the disease or
which is used in inoculation for the production of immunity is termed
the specific organism.

Of the different kinds of immunity only artificial immunity will be considered.
This may be obtained in two ways: By injecting the bacteria or their products
into man or animals and as the result of the activity of the cells of the animal invaded,
antibodies are formed which neutralize the toxins (antitoxins) or bring about lysis

of the specific bacteria (bacterioly-
sins). These antibodies which are
supposed to be thrown off (free re-
ceptors) from those body cells which
have suitable fixation powers for the
invading toxin molecule or bacterium
may remain potential for months or
years and so confer a more or less en-
during immunity.

These fixation points are known
as cell receptors and are intended for
the assimilation of various foodstuffs
by the cell. If destroyed by the
toxin or bacterium they are repro-
duced in great excess by nature.

Not only may bacteria act
in this way but foreign cells,
such as red cells or various
parenchymatous cells, when in-
jected, give rise to antagonistic
substances which act as factors in
their destruction — haemolysins
for red cells, cytolysins for dif-
substance which is injected and
produced is called an antigen.

FIG. 44. — Receptors of the first order
uniting with toxin. (Journal of the A merican
Medical Association, 1905, p. 955.) a, Cell
receptor; b, toxin molecule; c, haptophore
of the toxin molecule; d, toxophore of the
toxin molecule; e, haptophore of the cell
receptor.

feient parenchymatous cells. The
in reaction to which antibodies are
This is termed "active immunity."

When we take the serum of a man or animal immunized actively and inject
it with its contained antibodies into a second animal or man, we confer an immunity
on the second animal; but as his cells take no active part in the production of the
immunity, but are only passive, we term this immunity "passive immunity." If
this serum which is introduced in passive immunity only neutralizes the toxic prod-
ucts of the infecting bacteria, we term it antitoxic passive immunity and designate
the immune serum as antitoxic serum. If it- destroys the organism, we call it anti-

TOXINS

139

microbic serum, and the immunity, antimicrobic passive immunity. Some immune
sera are both antitoxic and antimicrobic.

It is well to remember that some organisms produce a toxin which is given off
while the bacterium is alive; and in other instances the toxin is intracellular and is
only given off when the bacterium disintegrates; consequently, an antimicrobic
serum may cause the liberation of toxin. Diphtheria, tetanus, or botulism antisera
are instances of antitoxic sera, while practically all others are antimicrobic. There
is but one factor to consider in an antitoxic serum and that is the protoplasmic
particles which are thrown off from the cell in response to the injury incident to
the attack upon the cell by the
toxin particles. This free particle
in the circulation represents the
entire mechanism of antitoxic-
immunity. It is capable of unit-
ing with the toxin molecule and
neutralizing its toxic power, or
rather so binding its combining
end (haptophore group) that it is
incapable of attaching itself to a
cell, so that the poisonous end of
the toxin (toxophore group) can-
not have access to the cell.

The term toxin, strictly
speaking, is applicable only
to such bacterial poisons as
(i) require a period of in-
cubation before being capa-
ble of manifesting toxic
symptoms and (2) can pro-
duce antitoxins.

FIG. 45. — Receptors of the second order
and of some substance uniting with one of them.
Journal of the American Medical Association,
1905, p. 1113.) c, Cell receptor of the second
order; d, toxophore or zymophore group of
the receptor; e, haptophore of the receptor; /,
food substance or product of bacterial disin-
tegration uniting with the haptophore of the
cell receptor.

(For further discussion of toxins
and antitoxins see under diphthe-
ria, tetanus, botulism, and pyocy-

aneus infections.) In antimicrobic sera we have two factors to consider, the
first is a protoplasmic particle quite similar to the antitoxin molecule, but which
in itself has no power of injuring its specific bacterium. This particle is gen-
erally referred to as the amboceptor or immune body. It is the specific product of
the activity of a specific bacterium or foreign cell against the body cells attacked.
It withstands a temperature above 56° C. and of itself is incapable of injuring the
bacterium in response to whose attack it was produced. The second factor in the
bacteriolysis of the specific bacterium, or the haemolysis of the specific foreign cell,
is something normally present in the serum of every animal, and which is capable
of disintegrating a foreign cell or bacterium, provided it can have access to the cell
or bacterium through an intermediary amboceptor (hence the amboceptor is some-

140

PRACTICAL METHODS IN IMMUNITY

times called an intermediary body). This something is called the "complement."
It is by some called "alexine," by others cytase (Metchnikoff). The complement
cannot act upon and destroy an invading bacterium or cell unless the amboceptor
is present to make the necessary connection. The complement is destroyed by a
temperature of 56° C., so that, if we heat the serum from an immune animal to 56° C.,
the complement it naturally contains is destroyed, and the amboceptor it contains
which is not injured by such a temperature, is incapable of destroying bacteria or
cells, unless we replace the complement which has been destroyed by fresh comple-
ment. This is done experimentally by adding the serum of a nonimmunized animal
which contains the complement, but no specific immune body (amboceptor) to the
heated serum. This is termed "activating," and a serum so treated is said to be
"activated." When an immune serum has been heated to 56° C., it is said to have
been "inactivated."

FIG. 46. — Receptor of third order, and of some substance uniting with one of
them. (Journal of the American Medical Association, 1905, p. 1369.) c, Cell recep-
tor of the third order — an amboceptor; e, one of the haptophores of the amboceptor,
with which some food substance or product of bacterial disintegration (f) may unite;
g, the other haptophore of the amboceptor with which complement may unite;
k, complement; h, the haptophore; z, the zymotoxic group of complements.

Antimicrobic sera are not as efficient in treatment as antitoxic ones.
It might be that if we could use homologous sera for treating man instead
of the usual heterologous ones from the horse better results might
obtain.

It would appear that a more hopeful outlook will obtain by combin-
ing serum therapy with chemo-therapy, thus a combination of anti-
pneumococcic serum with sodium oleate seems capable of producing cura-
tive results which neither alone can bring about.

Again, a combination of vaccination (active immunization) with

SERUM DIAGNOSIS 141

the injection of the antimicrobic serum (passive immunization) has been
thought by some to be of value. When we allow a mixture of bac-
teria or cells to remain in contact with their specific immune serum
which has been inactivated, the amboceptors attach themselves to the
bacteria or cells, so that now, upon adding normal serum (comple-
ment), these bacteria or cells are so prepared that the complement
can disintegrate them. This experiment is termed " sensitizing " and
cells so treated are said to be "sensitized."

METHODS FOR OBTAINING IMMUNE SERA.

While a convalescent from a disease may be utilized to obtain an
antitoxic, agglutinating, opsonic, or bacteriolytic serum against the
specific bacterium, yet this is more conveniently obtained from an
animal which has been immunized against the bacterium or cell in
question. The rabbit is the most convenient animal to employ for the
production of immune sera where the object is to have at hand a serum
for use in diagnosis.

Where sera are used on an extensive scale, as in the production of
curative sera, larger animals are employed. There are two applications
of serum diagnosis: i. Where the bacterium is known and the serum
is to be diagnosed. 2. Where the serum is known and the bacterium
is to be diagnosed.

The first is employed by testing the agglutinating or bacteriolytic power of the
serum taken from a patient upon pure cultures of the organism which is suspected
as the cause of the disease. The Widal test (agglutination) is the best instance of
this procedure. This method is of practical value in the diagnosis only of typhoid,
Maltg, fever, and para-typhoid. In diseases like cholera and bacillary dysentery,
the disease has run its course before agglutinating power becomes apparent in the
serum. This method, however, may be used to prove that a convalescent has suf-
fered from a suspected disease. Thus, by testing the agglutinating power of a serum,
one or two weeks after recovery from a suspicious case of ptomaine poisoning, we
may be able to demonstrate that the case in question was cholera. The second
method has wider application, and is the one in which we use the sera of animals
which have been immunized with known bacteria. Organisms isolated from urine,
faeces, or blood of patients, or those obtained from water or food supplies may be
identified by testing the agglutinating, opsonic, or bacteriolytic power of known
sera against them. This has a wide range of applicability. The testing of the
opsonic power of the sera in man or animals immunized against plague, and possibly
cerebrospinal meningitis, seems to give more definite information than do agglutina-
tion or bacteriolytic tests. With the majority of other organisms, however, the
agglutination test is the one almost always preferred.

142

PRACTICAL METHODS IN IMMUNITY

Even in a small laboratory there are no particular difficulties in the way of hav-
ing on hand rabbits immunized against typhoid, paratyphoid, Malta fever, acid-
producing and nonacid-producing strains of dysentery, cholera, etc. Just as we
inject men with vaccines prepared from various bacteria in opsonic therapy, so we
inject animals to produce sera for diagnosis. We may use either a bouillon culture
or the growth on agar slants taken up with salt solution as the inoculating material.
This is heated for one hour at 60° C. to kill the bacteria. Where we desire to produce
a serum which will disintegrate red blood cells (haemolytic serum), we inject about
5 c.c. of the washed red cells of the animal for which we wish to produce a specific

FIG. 47. — i, Red cells -f- normal serum. No amboceptor. No haemolysis. A,
Complement; B, normal red cell. 2, Red cells + immune serum. Complement and
amboceptor. Haemolysis. C, Complement; D, amboceptor; E, haemolyzed red
cell. 3, Red cells + immune serum heated to 56° C. Inactivated. Complement
destroyed. No haemolysis. F, Destroyed complement; G, amboceptor; H, red
cells. 4, Red cells + heated immune serum + fresh serum. (Activated by con-
tained complement.) Haemolysis. I, Destroyed complement; J, fresh complement;
K, amboceptor; L, haemolysed red cell. 5, Diagram showing antitoxin production.
a, Toxin molecule; b, antitoxin molecule; c, neutralization of toxin by antitoxin.
6, Diagram showing bacteriolysin. d, Complement; e, amboceptor; /, bacillus.

serum. For details see method of preparing haemolytic amboceptor serum under
Noguchi's mcdification of Wassermann test. For preparing a serum for the bio-
logical blood test we inject the rabbit with human serum in quantities of about
5 c.c. every fifth day. About one week after the last injection the antiserum ob-
tained from the injected rabbit should be strong enough for one-tenth of a c.c. to
produce turbidity when added to i c.c. of a i-iooo dilution of human serum in salt
solution. Various controls are necessary when used in medico-legal work.

AGGLUTINATION TESTS 143

For obtaining an agglutinating or bacteriolytic serum for bacteria we inject
about i c.c. of the killed bacterial bouillon culture subcutaneously or into the peri-
toneal cavity of the rabbit. The easiest way to inject the rabbit is to hold the animal
head down and plunge the needle in the median line into the abdominal cavity,
forcing in the contents of the syringe. The intestines gravitate downward and by
entering the needle below the limits of the bladder we avoid injuring any vital part.
It may be more satisfactory to at first inject only about 1/2 c.c., and then if there is
very little reaction, as shown by the appetite and spirits of the rabbit, to inject
about four days later i c.c. About four or five injections at intervals of three to
five days will usually produce an immune serum.

Injection of the antigenic material (blood cells, serum or bacterial emulsion) into
the marginal ear vein may be employed. With this method, however, I have had
several rabbits die in what was considered anaphylactic shock. (For the method
of immunizing rabbits to produce a hsemolytic serum see Wassermann test.) Some
animals do not seem to be capable of producing antibodies, so that it may be necessary
to use one or more rabbits before a satisfactory serum is obtained. The most con-
venient way of obtaining serum for a test is to cut across one of the marginal veins
of the rabbit's ear, and collect the blood in a Wright's U-tube. Centrifugalizing,
we have the serum ready for use.

The vein can be made to stand out prominently by applying a compress dipped
into very hot water. When a large amount of serum is desired it is better to use a
test-tube with two pieces of glass tubing passing through a double perforated rub-
ber stopper. To one of the projecting pieces of glass tubing a stout hypodermic
needle is attached through the medium of 8 inches of rubber tubing and to the
second piece of glass tubing passing through the stopper of the large test-tube
another piece of rubber tubing is attached for suction. To obtain blood from the
rabbit find the ensiform cartilage and insert the needle in the notch to the left and
gently force it upward. Applying suction with the mouth the blood flows into the
test-tube so soon as the needle enters the heart. By placing the tube of blood in
the refrigerator the serum separates out from the clot. The removal of 20 to 30 c.c.
of blood does not seem to affect the animals in the least and they can be used in this
way time and time again. The immune body and agglutinin in serum remain
active for weeks when kept in the refrigerator. The complement and opsonin,
however, begin to deteriorate at once and have disappeared by the fifth day. Con-
sequently, for opsonic and bacteriolytic and haemolytic experiments, fresh serum
— twelve to twenty-four hours — must be used, or it may be activated.

AGGLUTINATION TESTS.

There are two methods of testing the aggultinating power of a
serum — -the microscopical and the macroscopical or sedimentation
method.

i. For the microscopical method draw up serum to the mark 0.5 of the white
pipette. Then draw up salt solution to the mark n. This when mixed gives a
dilution of i to 20. One loopful of the diluted serum and one loopful of a bouillon

144 PRACTICAL METHODS IN IMMUNITY

culture or salt solution suspension of the organism to be tested gives a dilution of
i to 40. One loopful of the 1-20 diluted serum and 3 loopfuls of the bacterial sus-
pension give a dilution of 1-80. These two dilutions answer in ordinary diagnostic
tests. The red pipette with a i-ioo or 1-200 dilution may be used where dilutions
approaching i-iooo are desired. Having mixed the diluted serum and the bacterial
suspension on a cover-glass, we invert it over a vaselined concave slide and examine
with a high power, a dry objective (1/6 in.). It is simpler to make a ring of vaseline
to fit the cover-glass and make the mixture of diluted serum and culture in the center
of this ring or square. Then apply the cover-glass, press it down on the vaseline
ring and examine as with the ordinary hanging drop. In making dilutions it is
preferable to use salt solution, as the phenomenon of agglutination requires the
presence of salts. Ordinarily, thirty minutes is a sufficient time to wait before
reporting the absence of agglutination. Agglutination is more rapid at body
temperature than at room temperature. In reporting agglutination, always give
time and dilution. It is absolutely necessary that a control preparation be prepared
in every instance; that is, one with the bacterial culture alone or with a normal
serum of the same dilution as the lowest used. Some normal sera will agglutinate
in i to 10 dilution, and group agglutinations (as paratyphoid with typhoid serum)
may occur in i to 40 or possibly higher. It is very unusual for sera to agglutinate
any other bacteria than the specific one in dilutions as high as 1-80.

2. For the macroscopical or sedimentation test, take a series of small test-tubes
(3/8X3 in.) and deposit i c.c. of salt solution in each of the series. Now, having
taken an empty test-tube, drop 4 drops of serum in it and then add 12 drops of salt
solution. This approximately gives i c.c. of a 1-4 dilution of the serum. With a
rubber-bulb capillary pipette, which has been graduated to hold 16 drops or i c.c.,
draw up the contents of the tube containing the i to 4 serum and add it to the next
tube containing i c.c. of salt solution. This gives a dilution of i to 8. Now mix
thoroughly by drawing up and forcing out with the bulb pipette, and then withdraw
i c.c. and add to the next tube containing i c.c. of salt solution. This gives a dilu-
tion of i to 1 6. Having mixed as before, again withdraw i c.c. of the mixture and
add it to the i c.c. in the next tube. We now have a dilution of i to 32. Again
withdrawing i c.c. and adding it to the fourth tube containing i c.c. of salt solution
we have a dilution of i to 64. In tube i there is i c.c. of a dilution of the serum of
i to 8; in tube 2, there is i c.c. of a dilution of i to 16; in tube 3 of i to 32. Tube
4 contains 2 c.c. of i to 64. Now adding i c.c. of a culture of typhoid or any other
organism, we have the dilution of the serum in each tube doubled. Tube i now
contains a serum in dilution of i to 16, acting on the bacteria; tube 2 of a i to 32;
tube 3 of a i to 64. Now place these tubes in the incubator and, after two to five
hours or overnight, we examine for the clearing up of the supernatant fluid. If
the serum in a certain dilution agglutinates, the clumps gravitate to the bottom
and the upper part becomes clear. If so desired, these dilutions may be carried on
to i to several hundred in the same way. It is safer to work with dead cultures
instead of living ones. To prepare, take a twenty-four-hour agar slant culture of
typhoid or paratyphoid and emulsify in salt solution (about 6 c.c. to a slant).

By adding o.i of i% of formalin to the typhoid emulsion and placing in the ice-
box the cultures will be found sterile in about three days. The emulsion should be
shrken twice daily while undergoing sterilization in the ice-box. Such cultures

COMPLEMENT DEVIATION 145

are not easily contaminated and appear to retain their agglutinable qualities for
several months. The macroscopic methods are preferable with such dead cultures.

A very convenient method in general use in Germany is the follow-
ing: Make dilutions of serum in ordinary test-tubes (3/4X6 in.) as
described for the samll test-tubes. Then take a loopful (2 mg.) of
culture from an eighteen to twenty-four-hour-old agar culture and
emulsify it thoroughly in the dilution in the first test-tube — repeat the
process in the second tube and so on. This procedure is much safer
than when live cultures are added with a pipette. Again, the dilution
is unchanged by this addition whereas it is doubled when an equal
volume of culture is added to the diluted serum. A control should
always be made in normal salt solution. After incubating, observe
flocculent precipitates (agglutination) by tilting the fluid in the tubes
to form a thin layer and to obtain the most advantageous light and look
for a fine curdy precipitate (aggutination) or a uniformly turbid emul-
sion (negative reaction).

The method of using a slide with two vaselined rings, one containing
an emulsion in the specific serum and the other in salt solution is of
great practical value. This method is described under cholera.

Pfaundler under the designation of a thread reaction showed that organisms tended
to grow in thread forms in a culture medium containing the homologous serum.
Mandelbaum has suggested this as a means of diagnosing typhoid. Take ordinary
bouillon containing 1% of sodium citrate. Inoculate it with a culture of typhoid.
Now with a bulb capillary pipette take up one part (as marked by a wax pencil) of
the patient's blood and fifteen times as much of the citrated bouillon just inoculated
with typhoid. Mix the blood and citrated bouillon on a sterile slide or in a test-tube
and after drawing up into the lower part of the expansion of the capillary pipette,
seal off the capillary end. Now place the sealed-off pipette upright in an incubator
and after four or five hours take out from the expanded end a loopful of the clear
supernatant fluid (the blood cells settle to the bottom) and if the typhoid bacilli
are in chains instead of being single and motile it shows a positive reaction.

DEVIATION OF THE COMPLEMENT.

It has been found that if there is not sufficient immune body in a
mixture of normal serum, containing abundant complement, and bac-
terial emulsion, only a portion of the bacteria will be destroyed. In-
creasing the amount of immune body with a constant quantity of normal
serum, we reach a point where all the bacteria are destroyed. Now,
if we continue to increase beyond this point the addition of immune

10

146 PRACTICAL METHODS IN IMMUNITY

serum, the destruction of the bacteria ceases, and the cultures will again
contain myriads of living bacteria.

To carry out the test, make a series of tubes containing mixtures of bacteria
with the same quantity in each of normal serum. Thus, each tube contains 1/2 c.c.
of bacterial emulsion and 1/2 c.c. of i-io normal serum. Now inactivate a tube of
i -zoo immune serum and to each of the tubes of normal serum and bacterial emulsion
add increasing drops of the inactivated i-ioo immune serum. Thus, i drop to No. i
tube, 2 drops to No. 2 tube, and so on. After incubating for two hours, we take a
pipette and plate out a fraction of a drop in an agar plate. The limit at which bac-
teriolysis is complete is shown by there being an absence of colonies.

Beyond or below that point colonies are more or less abundant. The explanation
of this phenomenon of deviation or deflection of the complement is that where we
have an excess of amboceptors for available receptors on the bacterial cells, only a
portion of the amboceptors can attach themselves to their specific bacteria. The
free amboceptors, not being able to form a union with the bacterial cell receptors
(for which they have a greater affinity), combine with the complement present.
Unless the complement be in excess, there will be no free complement left to join
onto the amboceptors attached to the bacterial cells, and consequently bacteriolysis
does not take place and the plate cultures show an abundance of colonies.

FIXATION OR ABSORPTION OF THE COMPLEMENT.

One of the controversies in connection with the nature of the com-
plement is that regarding the question of the unity of complements
or whether there exist different kinds of complements for different
amboceptors (unity and multiplicity of complement). To prove that
a single complement will act with varying amboceptors, Bordet and
Gengou showed that the same complement would activate both haemo-
lytic and bacteriolytic immune bodies. If to a mixture of typhoid
bacteria and inactivated typhoid immune serum some guinea-pig serum
is added and the mixture be allowed to remain at 37° C. for two hours,
and then sensitized red cells be added and the mixture again be placed
in the incubator for two hours, no haemolysis will be found to have
occurred, because the bacteria have absorbed all the guinea-pig com-
plement through the intervening typhoid amboceptors, and there is no
complement left to haemolyze the red cells through the specific blood-
cell amboceptors. If, instead of immune typhoid serum, the serum of a
.normal person had been used, there would have been no amboceptors
to unite the.complement to the bacterial cells. The complement would
then be at hand to unite with the sensitized red cells subsequently added
and bring about their haemolysis, as shown by the ruby color of the

COMPLEMENT FIXATION 147

supernatant fluid. This phenomenon of Bordet and Gengou has been
utilized by Wassermann for the diagnosis of diseases where cultures are
not applicable. It is in the diagnosis of syphilis that it is best known.
It having until recently been impossible to obtain cultures of Treponema
pallidum, we use an emulsion of the liver of a syphilitic foetus, which
has been filtered so as to be clear, instead of a culture. The syphilitic
liver, as can be observed by staining according to Levaditi's method, is
packed with spirochaetes.

While Noguchi has recently obtained pure cultures of the organism of syphilis
yet the antigen prepared from such cultures was not found as satisfactory by Craig
and Nichols as that from the liver of a syphilitic foetus, cases of syphilis which showed
strongly positive tests with ordinary antigen not giving a positive test with the spe-
cific antigen.

It has now been found that lecithin or, preferably, emulsions of various normal
organs may be substituted as antigen for the syphilitic liver, the antigenic power
being due to lipoids. Aqueous extracts contain in addition to lipoids, substances
which render the antigen unstable — alcoholic extracts are more stable and contain
less anticomplement.

For the immune bodies we take the serum of the patient, or if a case of locomotor
ataxia or general paresis, the cerebrospinal fluid.

EMERY'S TECHNIC FOR THE WASSERMANN TEST.

Owing to technical difficulties with the method of making and
employing the antigen and amboceptor features of the original Emery
test, I have retained the principle of the test but substituted the
reagents prepared in exact accordance with Noguchi's directions.

Briefly stated, the principle of Emery's test consists in the employ-
ment of fresh human serum for supplying complement and the pri-
mary incubating of the hemolytic system (human red cells and rabbit
serum immune to human red cells) at the same time as the incubation
of the antigen and serum but in separate tubes. Then in the second
period of incubation to add these " sensitized" cells to the serum
antigen combination.

In Noguchi's method all reagents are incubated together in the
first period with the exception of the amboceptor paper (dried serum
of rabbit immune to human red cells), which is not added until the
period of incubation for complement binding is completed and the
second incubation commenced. Time is saved in the Emery tech-
nic, inasmuch as the red cells are already sensitized by the hemo-

148 PRACTICAL METHODS IN IMMUNITY

lytic amboceptor when added to the tubes, and hemolysis shows itself
almost immediately in tubes when the complement has not been
absorbed by the antigen through syphilitic antibodies.

Noguchi has called attention to the fact that protein constituents of certain
aqueous or alcoholic extracts may have the power to fix complement through certain
intermediaries existing in fresh serum which, however, does not obtain for inacti-
vated sera (sera heated to 56° C. for 15 minutes).

Pure lipoidal substances as contained in Noguchi's acetone insoluble antigen,
however, do not act in this way.

Consequently by using such an antigen we eliminate the objection to employing
fresh human serum in the test for syphilitic antibodies.

As giving more uniform hemolytic results and as being more stable and easier
of employment, I have made use of Noguchi's directions for taking up the serum of
the rabbits, immunized to human red cells and his method of standardizing this
'' amboceptor " paper. In practice, I measure off the length of paper correspond-
ing to 8 to 10 Noguchi units and dissolve the dried serum in such paper in i c.c. of
salt solution. This makes a satisfactory and uniform substitute for the sterile
immune serum used by Emery. It has been noted that the dried rabbit serum on
the paper may contain a certain amount of complement even when several months
old, consequently, to avoid confusion, I invariably inactivate this serum paper solu-
tion by heating to 56° C. for 5 minutes.

Method : i. Take blood from the finger or ear in a large Wright U tube (1/4 inch
in diameter). Place in 37° C. incubator for 15 minutes (to increase yield of serum)
and then centrifuge.

2. Graduate a capillary pipette for i volume and 4 volumes.

3. Into each of a series of small test-tubes put 4 volumes of normal salt solution.
(These tubes are most conveniently made by breaking off 2 1/2 to 3 inch lengths of
i/4-inch soft glass tubing and then fusing one end in the flame to make a small test-
tube.)

Make a distinguishing mark, e. g., X, on end of tube with blue- wax pencil and use
this tube to hold control. Mark the other tubes I, II, III, and so on. When differ-
ent sera are to be tested they may be distinguished by lines either above or below,
or with circles, also marks with red-wax pencil may be used.

4. Make a i to 10 dilution of stock antigen solution in salt solution.

To Tube I add 4 volumes of i to 10 antigen, thus making 8 volumes of i to 20
antigen in Tube I. Mix thoroughly by manipulating bulb of pipette. Then trans-
fer 4 volumes of the i to 20 from Tube I to Tube II, and so on through the series.
When the dilution in the last tube has been made throw 4 volumes away.

The 4 volumes of dilution of the antigen in the respective tubes will then be:
In Tube I, i to 20; in Tube II, i to 40; in III, i to 80; in IV, i to 160, and so on.

5. Add i volume of serum to be tested to control tube X, and to each of the tubes
I, II, III, etc., in succession. (If the serum be added to the antigen tubes before the
control tube, -antigen might be carried over to the control.)

6. If the serum has been inactivated restore complement by adding i volume
of a 40% fresh guinea-pig serum. Also use 2 volumes of this inactivated human
serum instead of i.

WASSERMANN TECHNIQUE

149

7. Incubate at 38° C. for 30 minutes. This allows syphilitic antibody, if pres-
ent, to bind complement.

8. As soon as the above mixtures have been made and put in the incubator
prepare the "haemolytic system" by adding i volume of 20% emulsion of washed
human red cells to 4 volumes of solution of amboceptor paper (8 to 10 Noguchi
units of amboceptor paper dissolved in i c.c. of salt solution and then heated to 55° C.
for 5 minutes makes a suitable amboceptor solution — thus of a paper of which 4 mm.

CONTROL 1to2° U'40 Ito8° I10|6° 't'320 lt,640
ANTIGEN ANTIGEN ANTIGEN ANTIGEN ANT1OEN ANTIGEN

FIG. 48. — i. Capillary pipette being graduated by drawing up i and 4 drops
from a watch-glass, (a) Blue pencil mark of i drop or i volume, (b) Mark of
volume of 4 drops. 2. Graduated centrifuge tube containing sodium citrate normal
salt solution. 3. Tube with 10 amboceptor units in i c.c. of salt solution. 4. Mix-
ture of i volume 20% emulsion red cells and 4 volumes inactivated amboceptor
solution. 5. Small glass tubes for Emery test. 6. Method of transferring from
tube to tube. 7. Making a Wright U-tube — the end "a" to be used as a capillary
pipette.

was the unit we should cut off about 40 mm., place in test-tube and extract the dried
serum with i c.c. of salt solution), and place this haemolytic system in incubator
alongside the tubes already there. To obtain the washed red cells allow 4 to 10
drops of blood to drop into a graduated centrifuge tube containing salt solution to
which has been added i % of sodium citrate to prevent coagulation. After shaking,
centrifuge. Pour off supernatant fluid, replace with salt solution, again shake and
centrifuge — this sediment of red cells is to be diluted with 4 volumes of salt solution
(20% emulsion). (Incubation hastens sensitization of the red blood cells. Agglutina-
tion of red cells also occurs.)

PRACTICAL METHODS IN IMMUNITY

9. At the expiration of 30 minutes from the commencement of incubation for
complement binding, add i volume of haemolytic system to each of the tubes, I, II,
III, etc., in the order of antigen dilution.

10. Finally, after washing pipette in salt soultion, add i volume of haemolytic
system to control in tube X. (If the haemolytic system should be added to the con-
trol tube before the antigen tubes, complement from the control tube might be
carried over to the antigen tubes.)

FIG. 49. — i. Copper water bath 12X12X8 inches, (a) Thermometer to show
38° C. (b) Tubes containing antigen dilutions, (c) Tube containing hamolytic
system incubating along with the antigen tubes. 2. Ordinary rice cooker with copper
holder for test-tubes.

Shake each tube thoroughly. Allow them to incubate for a few minutes. Then
examine tubes I, II, III, etc., for haemolysis. The control should, of course, show
haemolysis. The antigen tubes should show a white, supernatant fluid over the in-
tact red cell sediment in the tubes with the low dilutions and even in the highest
dilutions, where the serum is strongly positive. In a weakly positive serum, in-
hibition of haemolysis may only show in the first two tubes and haemolysis show in
those tubes having higher dilutions of antigen.

It will be noted that the reagents are made in accordance with
Noguchi's directions. Even in those cases where fresh guinea-pig
serum is employed to replace complement, absent from the human
serum tested, we employ the 40 % solutions used in Noguchi's tech-

ANTIGEN 151

nic. It is possibly better to start with a 1-30 dilution in the first
antigen tube instead of with a 1-20. It is also an advantage to titrate
the human complement.

Preparation of Acetone Insoluble Antigen. — Take about 50 grams of finely
divided beef, dog, or rabbit heart or liver and triturate in a mortar to a paste. Pour
on this paste 500 c.c. of absolute alcohol and keep the mixture in a corked bottle
in the 37° C. incubator for five to seven days. (We use beef heart and 96% alcohol.)
Next filter through paper and collect the filtrate in a large shallow dish and hasten
evaporation with the aid of a current of air from an electric fan directed upon the
uncovered surface.

Within twenty-four hours only a sticky residue should remain. This is taken up
in about 50 c.c. of ether and the turbid ethereal solution kept over night in the refrig-
erator in a corked bottle.

In the morning there will be found about 45 c.c. of clear supernatant fluid which
is decanted off and allowed to evaporate to about 15 c.c.

Now to this 15 c.c. add about 150 c.c. of acetone and a precipitate will form
which collects at the bottom of the measuring cylinder. Now pour off the
supernatant acetone and let the sediment stand until it is of a resinous con-
sistence. Now dissolve 0.3 grams in i c.c. of ether and then add 9 c.c. of methyl
alcohol. This gives the stock antigen solution.

In using the antigen solution for the Emery or Noguchi test we dilute i c.c. with
9 c.c. of salt solution. This opalescent, working, antigen emulsion should be made
up fresh on the day of preparing the tests.

About one-half of these antigens are lacking in power to absorb
complement in the presence of syphilitic sera. More rarely they
may absorb complement with a nonsyphilitic serum (anticomplemen-
tary) or they may have a haemolytic action. Consequently a new stock
antigen should be tested as to its reliability—

1. A mixture of 0.4 c.c. working antigen emulsion, 0.6 c.c. salt solution, and o.i
c.c. of a 10% suspension of washed red cells when incubated at 37° C. for two hours
should not show any haemolysis.

2. A mixture of 0.4 c.c. working antigen emulsion, 0.6 c.c. salt solution, o.i c.c.
of a 40% solution of fresh guinea-pig serum, and 2 units of amboceptor and incubated
at 37° C. for one hour should show haemolysis when we now add o.i c.c. of a 10%
washed red-cell emulsion and the whole then again incubated for two hours at 37° C.
(The antigen did not absorb complement in the absence of syphilitic antibodies.)

3. A mixture of 0.2 c.c. of a i to 10 dilution of working antigen emulsion, 0.8
c.c. of salt solution, i drop of syphilitic serum, o.i c.c. of a 40% dilution of fresh
guinea-pig serum, and 2 units of amboceptor should be incubated at 37° C. for one
hour. When we then add o.i c.c. of a 10% suspension of washed red cells and
again incubate for two hours we should fail to obtain haemolysis. (The antigen can
absorb complement through the intermediation of syphilitic antibodies.)

Preparation of Amboceptor Paper. — In order to secure blood from the vein of a

152 PRACTICAL METHODS IN IMMUNITY

man or the heart of the immunized rabbit, the most convenient method is with the
use of an Erlenmeyer flask with a rubber stopper having two perforations in the stop-
per. To one of the projecting pieces of glass tubing a stout hypodermic needle is
attached through the medium of about 8 inches of rubber tubing, and the second
piece of glass tubing is bent at an angle as it leaves the stopper to provide a suction
tube. With a man, constrict the upper arm sufficiently to stop venous return with
an Esmarck rubber bandage or a towel. Paint tincture of iodine over a prominent
vein at the bend of the elbow. Gentle suction will cause the blood to flow into the
needle tube and thence into the flask.

The blood as it is taken from the arm should be received in about 50 c.c. of normal
salt solution containing i% of sodium citrate. About 28 to 30 c.c. are usually suffi-
cient. Now throw down this red-cell suspension in three or four centrifuge tubes.
The resulting sediment should be washed and rewashed with salt solution. Two
to three washings with salt solution suffice.

Now take a large healthy rabbit, shave the lower abdomen and paint the surface
with tincture of iodine. The easiest way to inject the rabbit is to hold the animal
head down and plunge the needle of a large glass hypodermic syringe containing the
washed red-cell sediment into the abdominal cavity in the median line. The intes-
tines gravitate downward and by entering the needle below the limits of the bladder
we avoid injuring any vital part.

Make the injections at intervals of five days and give increasing amounts at each
successive injection. Thus, first injection, 5 c.c.; second injection, 8 c.c.; third
injection, 10 c.c.; fourth injection 12 c.c.; and at the fifth injection give about 15 to
20 c.c. of washed red-cell sediment. It is well to dilute the cell sediment with an
equal amount of salt solution. About ten days after the last injection, we take some
blood in a Wright's tube from a vein of the ear and dilute the serum to make a i to
100 dilution. To i c.c. of a i% emulsion of red cells we add o.i c.c. of a 20% dilu-
tion of guinea-pig's fresh serum — similar combinations being made in a series of
10 tubes. To each of these tubes we add varying amounts of the i to 100 dilution,
o.i c.c. in the first, 0.2 c.c. in the second, 0.3 c.c. in the third, and so on. If we
obtain haemolysis in the tube containing 0.2 c.c. of i to 100 dilution of serum but not
in that containing o.i c.c. we note that the serum has a titre of about i to 500. If
the o.i c.c. gave haemolysis, the serum would have a titer of i to 1000.

Having ascertained that the haemolytic serum is sufficiently strong we shave the
left side of the thorax of the rabbit and enter the needle of the apparatus similar to
that used for taking the blood from a man's vein in one of the intercostal spaces of the
left side.

Having introduced the needle, feel for the heart beat and then plunge the needle
into the heart. We can withdraw about 30 c.c. of blood without injury to the rabbit.
This blood should be received in a clean empty flask and set, over night, in the re-
frigerator. The following morning pour off the clear serum into a clean Petri dish
and saturate, one by one, squares of filter paper with the serum. Allow the filter
paper to dry on a piece of unbleached muslin. Noguchi recommends Schleich and
SchulPs paper No. 597. When thoroughly dry cut strips 10 mm. wide. This makes
the amboceptor paper. To standardize, take a series of tubes containing i c.c. of
a i% emulsion of red cells and add o.i c.c. of 20% dilution of guinea-pig serum for
complement. Next cut across the amboceptor paper strip pieces of varying width,

WASSERMANN TESTS 153

as i mm., 2 mm., 3 mm., 5 mm., and so on. The narrowest strip which gives hasmo-
lysis in one hour equals one unit. Thus if a piece 5 mm. wide was required to pro-
duce haemolysis, 5 mm. of the paper would have a value of one unit.

NOGUCHI'S METHOD.

For the suspension of red cells use a 1/2% suspension of washed
human red cells.

For complement use fresh guinea-pig serum in a dilution of i part
to i 1/2 parts of salt solution (40%).

Experiment. — 'Take 4 small test-tubes (12 by 125 mm.) label id, ib
and 20, 2b, respectively. Into la and ib each put i drop of the serum
of the patient to be tested and into ia and 2b each put i drop of the
serum of a person known to give a positive test for syphilis. Next
add to each of all four tubes i c.c. of the 1/2% suspension of washed
red cells. Then add to each tube o.i c.c. of the 40% fresh guinea-pig
serum. Now add to tube la and tube ia each o.i c.c. of the i to 10
antigen dilution (opalescent working antigen emulsion). Tubes ib and
2b are controls not containing antigen. Mix contents of tubes thor-
oughly and incubate at 37° C. for one hour or for 1/2 hour in a water
bath. Now add to each of the four tubes 2 units of the immune
haemolytic serum, as measured off on the amboceptor paper strip — thus
with a paper of which 2 mm. equals i unit, drop into each tube 4 mm.
of the strip.

The tubes without antigen (ib and 26) should show good haemolysis.
Tube 20, that of the known syphilitic, with antigen, should not show
haemolysis and that of the person examined (10) should show haemolysis
in case the test is negative for syphilis. Moderately positive cases may
show a slight trace of haemolysis. In case the tubes without antigen are
negative (no haemolysis), repeat the test with smaller amounts of human
serum. It may be advisable to employ the serum of a person known to
be free from syphilis. In this case we should use two additional tubes,
30 and 3#, conducting the test as for the syphilitic control serum.

Many workers prefer to use inactivated serum for the test. In this case we should
add four times as much of the inactivated serum as for the unheated serum.

Inactivation not only destroys complement but likewise diminishes the strength
of the antibody content of the serum. Factors such as character of food and general
condition influence the complement strength of guinea-pig serum so that it is ad-
visable to titrate the guinea-pig serum. To do this take i c.c. of a i% emulsion of
human red cells and drop in one unit of amboceptor paper. The amount of comple-

154 PRACTICAL METHODS IN IMMUNITY

ment which will entirely haemolize the red cells in one-half an hour in water bath
equals one unit of complement. For the test use two units of complement.

THE WASSERMANN TEST.

In the Wassermann reaction the rabbits are injected with sheep
red cells which have been washed twice with salt solution by aid of the
centrifuge. About five injections with, on the average, the quantity of
red cells contained in 5 c.c. of sheep blood given at intervals of five days
gives a strong haemolytic serum if taken about one week after the last
injection. The method is to take in a test-tube 0.2 c.c. inactivated
human serum (heated for one hour at 56° C.), o.i c.c. fresh serum from
guinea-pig for complement, i unit antigen and 3 c.c. normal salt solu-
tion; then to incubate for one hour at 37° C. (An antigen unit is the
amount that will inhibit haemolysis of i c.c. of 5% emulsion of sheep
cells when mixed with 0.2 c.c. luetic serum and o.i c.c. guinea-pig comple-
ment.) Then add 2 units of amboceptor and i c.c. 5% emulsion of
sheep red cells, shake and incubate for one hour. (The amount of
haemolytic serum that will haemolize i c.c. of a 5% emulsion of sheep
red cells to which o.i c.c. guinea-pig serum has been added, in one hour,
is an amboceptor unit.)

The same technic is employed with the control test-tube except that
the antigen unit is not put in.

The Noguchi method gives a positive reaction with nonsyphilitic
sera in about 7% of cases. The Wassermann gives a negative result
in about 9% of syphilitic sera. These figures show the advantage of
checking one against the other.

There seem to be certain sera when with a clinical history of syphilis we
obtain a positive Wassermann with unheated serum and a negative one with
inactivated serum. In order to obtain information with the same serum heated
and unheated I would recommend, when it is inconvenient to carry out the original
Wassermann technic, to employ the Noguchi technic with inactivated serum and
the Emery technic with fresh unheated serum. In any case when serum cannot be
tested within twenty-four hours it should be inactivated, as unheated serum tends
to become anticomplementary.

Cherry thinks anticomplementary bodies are found during chloroform anaes-
thesia. If the antigen should also have anticomplementary action the total might
give a negative result.

By heating the serum for half an hour at 56° C. (inactivation) the positive
results obtained in certain cases of cancer, nephritis, scarlet fever, leprosy and tuber-
culosis may be avoided; the syphilitic antibody alone being thermostable. The

WASSERMANN TESTS 155

thermostability of serum of inherited syphilis is the highest — that of primary
syphilis the least of luetic sera.

McDonagh states that in the primary stage the Wassermann is
positive in 40% of cases. In secondary cases 97% give positive results
when treatment has not been instituted. In tertiary syphilis about
70% are positive.

In 268 cases at the medical clinic of Johns Hopkins Hospital, Clough
failed to obtain a positive reaction in ninety-nine cases which were
negative clinically.

In forty-five cases of syphilis he obtained 73% of positive results.
Excluding cases which had received thorough treatment 82% were
positive. Tabes gave 40% and general paresis 100 %. In five cases
of primary syphilis four gave positive reactions.

Based upon the observation of Bauer, that human serum contains haemolytic
amboceptors for sheep corpuscles, and of Hecht, that the complement normally
present in human serum would suffice without the addition of guinea-pig serum
complement, the following method of Fleming is easy of application.

For the test we use:

1. Alcoholic extract of rabbit's heart, made by washing the recently removed
heart with salt solution to remove all blood. Cut into small pieces and grind in a
mortar with sand and for every gram of heart add 5 c.c. of 95% alcohol. Keep the
mixture at a temperature of 60° C. for two hours and filter. This is the stock
solution. For use dilute it ten times with normal salt solution.

2. A 5% emulsion of washed sheep red cells, prepared as for the Wassermann
test.

3. Suspected and control sera.

With a capillary bulb pipette take up one part of serum and four parts of the
heart antigen, mix on a glass slide, again draw up into the capillary pipette and,
leaving a separating air space, next draw up one part of 5% emulsion of sheep red
cells. Then seal off tip of pipette and incubate at 37° C. for one hour. Now file
off tip and mix the red cells with the serum and antigen and again draw up into the
capillary pipette and incubate a second time for two hours. Haemolysis or the
reverse is shown in the fluid overlying the cell sediment. Various controls should
be made using normal and known syphilitic sera; also with normal salt instead of
serum.

The objections to methods using human serum for complement are
i. the great variation in the complement content of different human
sera; 2. human complement requires about ten times as much ambo-
ceptor as guinea-pig complement and is less sensitive to fixation, and
3, the statement is made by s>me workers that while homologous
complement and amboceptor may be efficient yet the complement of a

156 PRACTICAL METHODS IN IMMUNITY

serum will not act upon its homologous antigen. This is not true
because the complement of human serum invariably haemolyzes the
homologous antigen (human red cells).

The various precipitate tests that have been proposed are unreliable. The
precipitate reactions with bile salts give better results than with lecithin, this latter
showing positive results in almost one-half of non-syphilitic cases.

DETERMINATION or OPSONIC POWER AND THE PREPARATION
or VACCINES.

In that which has been considered in the previous pages only the
theories of Ehrlich have been brought out. In order to understand the
problems involved in the study of opsonins the phagocytic theory of
immunity brought forward by Metchnikoff must be studied. Ehrlich's
views would seem to hold with diseases where there is an increase in
bacteriolytic or antitoxic power of the serum while in such diseases, as
are caused by pathogenic cocci, the phagocytic element is operative as
there is an absence of bacteriolytic power in the serum of the person
with the infection.

There are two kinds of phagocytes, the microphages (represented
by the polymorphonuclears) which on phagolysis or disintegration give
off microcytase, a bactericidal substance. Cytase is the same as
complement or alexine.

The microphages are chiefly bactericidal while the macrophages,
represented by the large mononuclears of the blood and fixed connective-
tissue cells, exert their action on protozoa or animal cells.

Phagocytes may either act by ingesting bacteria and destroying
them intracellulary or they may as a result of phagolysis bring about
bacteriolysis extracellularly. According to Metchnikoff the intra-
cellular bacteriolysis explains why an individual may possess immunity
and yet his serum fail to show any bacteriolytic power.

The following modification of Leishman's method takes very little time and skill
and is applicable in the determination of the organism concerned in an infection, as
in Wright's method. The control of vaccine treatment by taking opsonic indices
from time to time does not seem to have met with much favor in this country — the
sources of error being as great, if not greater, than ordinary variations in the opsonic
index during the negative and positive phases.

Method. — We start with a i% solution of sodium citrate in salt solution. With
this emulsify a twelve to twenty-four-hour agar slant growth of the organism to be
tested using 6 to 8 c.c. of the citrated salt solution. The bacterial emulsion is now

OPSONIC INDEX 157

poured into a bottle or sealed off in a test-tube and shaken thoroughly in a shaker or
by hand. The emulsion is then centrifuged to throw down the bacterial clumps and
the supernatant slightly turbid bacterial suspension poured off. If working with a
dangerous pathogen it is advisable to kill the organisms as in making vaccines.

Now with a capillary bulb pipette so graduated that the one volume mark con-
tains about two drops we draw up one volume of citrated salt solution. Then
having made a break with an air column, we take up one volume of the patient's
blood. Again make an air break and draw up one volume of the citrated salt solu-
tion bacterial emulsion. The three volumes are then immediately forced out into
a small test-tube, made from three inches of 3/16- inch glass tubing, as shown in the
Emery technic for the Wassermann. The citrate prevents coagulation of the blood
and the contents of the tube are well mixed by drawing up and ejecting with the
capillary bulb pipette. Incubate this small test-tube at body temperature for
15 minutes, shaking the contents once or twice during the incubation period. Ex-
actly at the expiration of the period of incubation (usually 15 minutes although at
times 10 minutes or 30 minutes may be desirable) place the tube in a centrifuge and
throw down the cell sediment. Next pipette off the supernatant fluid and then
plunge the pipette to the bottom of the tube and draw off the greater part of the
sediment at the bottom. This consists largely of the red cells the leukocyte layer
on the surface being undisturbed.

Now mix the remaining cell sediment and smear out on a slide or preferably
between two cover-glasses as in Ehrlich's method. The smear is fixed by burning
off a film of alcohol and stained with dilute carbol fuchsin or methylene blue. The
granule staining with Wright's stain makes it slightly confusing.

A second similar preparation but using blood from a normal person as a control
is then made. Counting the phagocytized bacteria in a given number of poly-
morphonuclears, we obtain an average number of bacteria phagocytized per cell.
Repeating the count with the control or normal blood, we likewise have the average
number of bacteria taken up per cell. Dividing the patient's average by the normal
average, we have the opsonic index. If the average for fifty of the patient's cells was
eight and that of the control only four, the patient's index would be two, or twice the
normal. The practical value of this test is that where two or more organisms are in a
body fluid we may ascertain the causative organism by noting marked variation
from the normal in the patient's opsonic index for that particular organism and not
for the other organism. This variation may be of the nature of a high or low opsonic
index.

METHOD or WRIGHT FOR OBTAINING OPSONIC INDEX.

While other observers had previously noted the presence of sub-
stances in immune sera which so acted on the bacteria that phagocytosis
was made possible, yet it was to Wright and Douglas, in 1903, that the
existence of this factor in phagocytosis was brought forward and the
estimation of such substances made practicable.

To this substance the name opsonin was given — 'the Greek word

158 PRACTICAL METHODS IN IMMUNITY

from which it is derived indicating preparation of the food — that is,
the opsonin so alters or sensitizes the bacteria that they can be engulfed
or phagocytized by the polymorphonuclear leukocytes (the microphages
of Metchnikoff). About the same time Neufeld and Rimpau noted
the presence of a substance in immune sera which so acted on bacteria
as to prepare them for phagocytosis. Their designation" bacterio-
tropic substance" is practically synonymous with opsonin.

In 1902 Leishman introduced the method of determining the "phagocytic index."
By taking one part of blood and one part of an emulsion of the bacteria in question
and keeping the mixture in a moist chamber at body temperature for a standard
time, as 15 to 30 minutes, and then spreading the blood-bacteria mixture and
staining the film with Leishman or Wright's stain he counted the number of bacteria
in a certain number of polymorphonuclears, and by dividing obtained the average
number per leukocyte of bacteria phagocytized.

The Wright technic for determining the phagocytic average, and
from this the opsonic index, is as follows:

Blood is taken from the patient and at the same time from a normal individual,
or preferably the blood of several normal individuals is pooled. This blood is best
collected in a Wright's tube, although it may be received in a small test-tube. After
coagulation and separation of the serum, the serum is ready for use.

The next step is to prepare the leukocyte emulsion. For this we fill a centri-
fuge tube with normal salt solution, to which has been added i% sodium citrate
— the latter to prevent coagulation. Then having pricked a finger congested by a
constricting rubber band, from 15 to 20 drops of blood are added to the citrated
salt solution, and the mixture thoroughly shaken. After centrifugalization for about
5 minutes the red corpuscles will be thrown to the bottom of the tube with the leu-
kocytes forming a superimposed layer. In order to free the leukocytes entirely from
serum admixture, the supernatant citrated salt solution is pipetted off, and a fresh
tubeful of salt solution is added to the blood-cell sediment. Again shaking, we then
centrifuge, obtaining for a second time a sediment of blood cells with the leukocytes
in the superimposed layer. In some laboratories the washing in salt solution is
again repeated, but for all practical purposes two washings as described above suffice.

The superimposed layer of white cells may now be pipetted off from the heavier
red cells (of course, containing a large admixture of red cells) to be used as a leuko-
cyte cream — or by slanting the centrifuge tube we can pipette off the proportion
of the leukocyte mixture needed from the bottom, sides or top of the slanted layer
of blood cells.

Having prepared our leukocyte emulsion, and the serum from the normal in-
dividual as well as that from the patient, it only remains to prepare our bacterial
emulsion. For bacteria in general, with the exception of tubercle bacilli, we simply
take up a small loopful of a young agar culture (eighteen hours or less), and emulsify
it uniformly with salt solution, added by degrees until the suspension amounts to
1/2 to i c.c., and giving a faint turbidity. To thoroughly distribute and especially

BACTERIAL VACCINES 159

to break up clumps repeated suction and ejection with a capillary pipette provided
with a rubber nipple is satisfactory.

The presence of clumps in a bacterial emulsion invalidates the estimation of
phagocytosis, for the reason that a leukocyte will take up a clump of twenty or more
bacilli as readily as one separate organism.

Having at hand (i) the suspension of leukocytes, (2) the bacterial emulsion,
and (3) the sera of the patient and the normal individual, we are ready to proceed
with the test.

Using a capillary bulb pipette with a pencil mark to indicate i volume we draw
up to the mark (i) the leukocyte cream. Then wiping off the tip of the pipette we
draw up this volume of leukocyte emulsion about one-half an inch to make an air
break between this and (2) i volume of the bacillary emulsion. Again making an
air space we draw up (3) the serum of the normal individual. This gives 3 columns in
the capillary tube with intervening breaks of air. We next eject the three con-
stituents into a watch-glass and thoroughly mix them by alternate suction and ejec-
tion with the tube and nipple. When mixed we draw the mixture up into the same
capillary tube, seal off the capillary end in the flame and put in an incubator for
exactly 15 minutes.

We next repeat the process identically except that the patient's serum is used in-
stead of that of the normal individual.

These tubes having been kept at the same temperature for the same length of
time are then taken out, the contents blown into a watch glass, mixed thoroughly
a second time, and then a smear is made — a drop of the mixture being deposited
on a very clean slide and the smear made by a second narrower slide (by cutting off
the corner of the slide) which is drawn along in a zigzag way. The smears are then
stained (Leishman's or Wright's blood stain or Ziehl-Neelson's for tubercle bacilli)
and the number of the bacteria in from fifty to one hundred leukocytes counted.
This number divided by the number of cells gives the phagocytic average.

The phagocytic average of the patient's tube divided by that of the normal in-
dividual's tube gives the opsonic index. Thus,, in counting 100 cells we find 500
phagocytized cocci in the patient's tube, giving an average of 5, and in the normal
individuals blood we get 1000, an average of 10. Then the opsonic index would
be 5 -Mo, or 0.5.

PREPARATION OF VACCINES.

It has been found satisfactory to make use of stock vaccines in
gonorrhceal and tuberculous affections. In treatment of tuberculosis
Wright prefers Koch's T. R. or Neu Tuberculin in doses of from 1/5000
to 1/800 of a milligram. Some prefer Koch's more recent bazillen
emulsion. In case of other infections, however, and preferably with
gonorrhceal infections, the causative organism should be isolated from
pus, sputum, urine, blood, or other material (autogenous vaccine).

In the making of vaccines all media and 'apparatus should be sterilized with
scrupulous care to avoid the danger of tetanus infection. Having isolated the organ-

160 PRACTICAL' METHODS IN IMMUNITY

ism, it is inoculated upon one or more agar slants, and after a growth of from five
to seven hours with streptococci and pneumococti, or with eighteen hours for staphy-
lococci and colon, the growth on these inoculated slants is taken up with salt solu-
tion, thoroughly shaken up in the diluting solution and standardized.

The most practical way is to gently rub off the growth on the agar in about i or
2 c.c. of salt solution with a platinum loop. Then pour the bacterial emulsion into
a sterile test-tube and repeat the process with three to five agar slants, until we have
from six to 10 c.c. of the emulsion in the sterile test-tube. By heating to melting-
point in the flame a piece of glass tubing and attaching it to the rim of the test-
tube (also melted), we have a handle with which to draw out the test-tube when
heated about i inch from the mouth in a blowpipe flame. Drawing this out, we let it
cool, and then filing the constricted portion we break it off and seal it in the flame.
By shaking up and down vigorously for five to fifteen minutes, or preferably in a
mechanical shaker the bacteria are distributed evenly in the salt solution. A piece
of platinum wire, twisted into corkscrew shape, and fused in the drawn out end of
the containing test-tube helps in breaking up the bacterial emulsion and is a great
aid in the preparation of streptococcic or diphtheroid vaccines.

The sealed test-tube is then placed in a water-bath at 60° C. and heated at this
temperature for one hour. Again shake. The constricted sealed end is again filed
off and a few drops shaken out in a watch glass for standardization, and at the same
time a few drops are deposited on an agar slant as a test for sterility. (Incubation
for twenty-four hours should not show growth.)

Wright found that by taking a definite quantity of blood and a
similar quantity of bacterial emulsion, mixing the blood and bacterial
emulsion, then making a smear and staining, it was possible to de-
termine the ratio of bacteria to red cells, and from this the number of
bacteria per cubic centimeter could be determined. For example, if
we find three bacteria to each red cell we should have 15,000,000
bacteria to i cubic millimeter. (There being 5,000,000 red cells to the
cubic millimeter.) As i cubic centimeter is 1000 times greater than i
cubic millimeter, there would be 15,000,000,000 bacteria in each cubic
centimeter of such an emulsion, or vaccine, as it is termed.

The standardization made with a haemacytometer is best done by drawing up
the vaccine to 0.5 with either the red or white pipette, according to concentration,
and then sucking up i to 10 dilute carbol fuchsin to n or 101. Allow the bacteria
to settle on the shelf for ten minutes before counting. Count as in making a red
count.

A more satisfactory diluting fluid is that recommended by Callison. It is:
Hydrochloric acid 2 c.c., Bichloride of mercury (1-500 aq. sol.) 100 c.c., and sufficient
i% aqueous solution of acid fuchsin to color the diluting mixture a deep cherry red.
The diluting fluid should then 'be filtered. The bichloride forms an albuminate on
the surface of the bacteria which promotes rapid sedimentation and the fuchsin
stains the bacteria.

BACTERIAL VACCINES l6l

Having determined the strength of the stock vaccine, we should
prepare a dilute vaccine for injection. This is most conveniently
carried out by filling vials with 50 c.c. of salt solution, plugging with
cotton, then sterilizing in the autoclave. A sterile rubber cap is now
drawn over the mouth of the vial. Sterility is insured by plunging the
rubber cap and neck in boiling water. If the stock vaccine showed
5,000,000,000 bacteria per c.c. and we desired to have a vaccine con-
taining 200,000,000 bacteria per c.c., it would be necessary to draw out
2 c.c. of the salt solution by means of a sterile .syringe needle inserted
through the rubber cap and replace it with 2 c.c. of the bacterial
emulsion. Example: In introducing 2 c.c. of a vaccine containing
5,000,000,000 bacteria per c.c., we throw in 10,000,000,000 bacteiia in
a volume equal to 50 c.c. Then each c.c. of the 50 c.c. in the bottle
would contain 10,000,000,000 divided by 50 or 200,000,000 in each c.c.
If we only want a vaccine containing 100,000,000 per c.c. we should only
add i c.c. We now add 1/4% of trikersol to the vaccine in order to
insure sterility. (Introduced with syringe, inserting needle through
rubber cap.) The syringe is best sterilized by drawing up vaseline
or olive oil heated to 150° C., and the neck and rubber cap of the
bottle in boiling water. We now draw up the desired dose of bacteria.
If glass syringes are used, simply boiling in water suffices. The
ordinary doses are: For gonococci, streptococci, pheumococci, and
colon vaccines, 5,000,000 to 50,000,000. For staphylococci 200,000,000
to i, 000,000,000.

Wilson gives the following minimum and maximum doses expressed in millions:

Streptococcus, 6 and 68.

Gonococcus, 45 and 900.

Meningococcus, 300 and 900.

M. melitensis, 700 and 1400.

B. coli, 16 and 240.

B. typhoid (treatment) 100 and 250.

B. typhoid (prophylaxis) 500 and 1000.

B. pyocyaneus, 34 and 1000.

B. pneumoniae, 44.

Staphylococci, 150 and 900.

B. tuberculosis, 1/20000 to 1/200 milligram.

ANAPHYLAXIS.

This is a term which indicates the opposite of prophylaxis. It was
noted that after a period of incubation of at least ten days a second

1 62 PRACTICAL METHODS IN IMMUNITY

injection of horse serum produced symptoms of respiratory embarrass-
ment, convulsions and, at times, death. The primary injection had
during the period of incubation sensitized the cells to this particular
proteid.

This phenomenon of sensitization in the case of rabbits bears the
name of Arthus, and as applied to guinea-pigs sensitized with diphthe-
ria antitoxin sera the name Theobald Smith, and it is stated by Muir
and Ritchie that active research as to anaphylaxis may be said to date
from the discovery of the phenomenon of Theobald Smith.

Rosenau and Anderson working with guinea-pigs showed that small
doses were efficient for sensitization, that the condition was trans-
missible from mother to offspring and that a second animal could be
sensitized by being injected with the serum of a sensitized animal.

This group of symptoms, the so-called anaphylactic shock, which is apt to set
in within a few minutes after the second injection, is often preceded by restlessness
and great excitement and together with the dyspnoeic manifestations there is cardiac
weakness and great fall of blood-pressure. The more serious symptoms and at times
death are more apt to appear after intracerebral injections than after intraperitoneal.
Subcutaneous injections are least apt to produce anaphylactic symptoms. Our
attention to this phenomenon commenced with the study of "serum sickness" or
"serum disease." In this an erythematous rash or urticaria associated with more
or less oedema comes on after eight to twelve days from the time of the first and only
injection of horse serum. It is supposed to be due to the fact that some of the serum
originally injected remains unchanged in the tissues so that when the sensitization
takes place there is present and at hand the same foreign proteid to bring about
anaphylactic symptoms.

Immunization against anaphylaxis is possible by repeating injection of the sen-
sitizing serum or proteid during the period of incubation.

It is important to note that this hypersusceptibility appears to be
very rarely of importance in the matter of the administration of a
second injection of diphtheria antitoxin after the period of anaphylactic
incubation.

As a rule the death or untoward effects of the injection of serum are
in cases of status lymphaticus. Cases in man do occur, however, but
with extreme infrequency, in which within a few minutes after the only
injection of serum the patient becomes restless, shows symptoms of
cardiac and repiratory embarrassment and may be dead in a very short
time.

According to Rosenau and Anderson individuals who have asthmatic tendencies
as well as those who have had serum injections ten to twelve days or longer prior to

ANAPHYLAXIS 163

the second injection should be considered as possible subjects for anaphylactic
shock.

Vaughan recommends that when this is to be feared one should only give about
o.i c.c. of the serum and after an interval of two hours, provided no untoward symp-
toms set in, to give the full amount of the injection. Besredka advises heating the
serum to 56° as a guard against anaphylactic shock.

The condition of hypersusceptibility or anaphylaxis is at times
termed allergy. Thus in a person who has been successfully vacci-
nated a reaction shows at the site of inoculation within twenty-four
hours which does not appear in the nonimmune person for a period two
or three times as long. The diagnostic tests with tuberculin and
luetin are hence often referred to as allergic reactions.

It may here be stated that some investigators are of the opinion that
our views not only as to immunity but as to the essential nature of
infectious diseases may be later on found to rest in production of
anaphylaxis.

The name "Anaphylactine" has been applied to the sensitizing substance pro-
duced during the period of incubation.

It has been proposed to employ this phenomenon as a diagnostic measure. By
taking the serum of a tuberculous patient, which would contain the sensitizing sub-
stance, and injecting it into the peritoneal cavity of a rabbit, the animal would
be sensitized and an injection of tuberculin a few hours later would bring about the
phenomena of anaphylaxis in the rabbit.

This passive anaphylaxis, as it is termed, usually requires approximately twenty-
four hours for sensitization. This passive anaphylactic sensitization seems to dis-
appear in two weeks. It has been advised to passively sensitize guinea-pigs with
the serum of the person about to be injected and then twenty-four hours after inject
the guinea-pigs with the curative serum. If untoward results occur in the guinea-
pigs the patient should not receive the injection.

Recently Hagemann has found the following technic valuable in the diagnosis
of surgical tuberculosis. Guinea-pigs are inoculated intraperitoneally with tuber-
culosis cultures and by the end of the second week such pigs are sensitized. The
suspected material, as serous effusion, is injected intracutaneously and within
twenty-four to forty-eight hours a distinct swelling of the skin with a bluish-red
center, which is surrounded by a porcelain white ring and outside of this a zone
of inflammation, shows a positive test.

NOTES ON BACTERIOLOGY.

NOTES ON BACTERIOLOGY.

NOTES ON BACTERIOLOGY.

NOTES ON BACTERIOLOGY.

NOTES ON BACTERIOLOGY.

PART II.
STUDY OF THE BLOOD.

CHAPTER XIII.
MICROMETRY AND BLOOD PREPARATIONS.

MlCROMETRY.

IN the examination of blood and faeces preparations, especially
when the identification of animal parasites is in question, there is noth-
ing that assists more than a knowledge of the measurements of the object
studied. The making of such measurements microscopically is termed
micrometry.

Micrometry is also indispensable in bacteriology and cytodiagnosis
as well as in animal parasitology.

The most practical way of making these measurements is with an ocular microme-
ter. These can be bought separately, or a glass disc (disc micrometer) with lines
ruled on it can be dropped into the ocular to rest on the diaphragm inside the ocular.
The ruled surface of this glass diaphragm should be placed downward. As was
stated in connection with the microscope, the image of the object is formed at the
level of the diaphragm rim inside the ocular, consequently the lines of the image cut
those of the lines ruled on the glass in the ocular. Once having standardized the
value of the spaces of the ocular micrometer for each different objective, all that is
necessary subsequently in measuring is to count the number of lines or spaces which
the image of the object fills and then, knowing the value of each space for that ob-
jective, to multiply the number of spaces by the value of a single space.

The unit in micrometry is the micron. This is usually written p
and is the i/iooo part of a millimeter. There are 1000 microns in a
millimeter.

To standardize: For this purpose it is necessary to have a scale
of known measurements. The stage micrometers are usually ruled
in spaces of o.i and o.oi mm. The lines which are i/io of a milli-
meter apart are consequently separated by a distance of 100 microns;
those i/ 100 of a millimeter apart are separated by a distance of 10
microns.

169

iyo

MICROMETRY AND BLOOD PREPARATIONS

The ocular micrometer is usually ruled with 50 or 100 lines or spaces,
separated by longer lines into groups of 5 and 10.

Having brought the lines on the stage micrometer to a focus, we
determine the number of spaces on the stage micrometer which the 50
divisions of the ocular micrometer cover. To distinguish the ruling of
the ocular from that of the stage micrometer, revolve the ocular with
the fingers.

FIG. 50. — Micrometry diagrams, i. Ocular micrometer with stage micrometer.
50 spaces of ocular micrometer cover two 100 micron spaces and ten 10 micron spaces;
equal 300 microns. Each division on ocular micrometer equals 6 microns. 2.
Ocular micrometer subtending image of whip worm egg. 9 spaces of ocular mi-
crometer cover Whipworm egg. Each space equals 6 microns. Whipworm egg
equals 54 microns. 3. Ocular micrometer with ruling of haemacytometer. 50
spaces of ocular micrometer cover space equal to width of 6 small squares 50X6 = 300
microns. Each division of ocular micrometer equals 6 microns.

The tube length which is used at the time of standardizing must
always be adhered to in subsequent measurements.

Example : With a 2/3-inch objective, the 50 rulings of the ocular micrometer
fill in fifteen of the i/io millimeter rulings (ioo/<) and three of the i/ioo millimeter
spaces (IQ/X). Consequently the 50 spaces of the ocular cover 1530 microns (15 X 100
= 1500; 3X10 = 30). Then if 50 spaces equal 1530 microns, one space would equal

BLOOD PREPARATIONS 171

30.6 microns. With the i/6-inch objective the 50 ocular spaces would cover about
three of the i/io millimeter (ioo/*) spaces of the stage micrometer. Then the 50
spaces would equal 300 microns and one space would equal 6 microns.

The ruling of the slide of a Thoma-Zeiss haemocytometer will answer as well as
a stage micrometer. The small squares are 1/20 of a millimeter square, consequently
the distance between the lines bordering the small square is 1/20 millimeter or 50
microns.

Now, if with the i /6-inch objective, the 50 lines on the ocular fill in the spaces of
six small squares, the length of such a space would be 50X6 = 300 microns. This
divided by 50 spaces would equal 6/*.

Should there be 100 spaces on the ocular micrometer instead of 50, it would
only be necessary to divide the length in microns of the ruled surface of the stage
micrometer covered by the 100 lines of .the ocular micrometer by 100. The quotient
would give the value in microns of each space of such an ocular micrometer.

The most accurate instrument for measuring is the filar micrometer. These are
expensive. Measurements can also be made with the camera lucida, but it takes
considerable time to make the adjustments necessary, so that it is not convenient.
With an ocular micrometer one can make measurements of blood-cells, amoebae, etc.,
in a few seconds — it only being necessary to slip in the ocular micrometer.

Rule for determining the magnifying power of microscopic lenses: Measure the
diameter of the lens of the objective in inches — the approximate equivalent focal
distance is about twice the diameter. Dividing 10 by the equivalent focal distance
gives the magnifying power of the lens. This should be multiplied by the number
of times the ocular magnifies. Example: The diameter of the lens of the objective
was found to measure 1/2 inch, the focal distance would then be about i inch.
Dividing 10 by i we have 10 as the magnifying power of the lens of the objective.
If we were using a No. 4 ocular, the magnifying power would be approximately forty.

BLOOD PREPARATIONS.

To obtain blood, except for blood cultures, use either a platino-
iridium hypodermic needle which can be sterilized in the flame, a small
lancet, or a surgical needle with cutting edge.

When using such surgical needles it is a good plan to sharpen the cutting edge on
a fine-grained whetstone. Afterward the needle should be sterilized by boiling.
Sterilization of a needle in the flame blunts the cutting edge. A steel pen with one
nib broken off or the glass needle of Wright may also be used. To make a glass
needle, pull straight apart a piece of capillary tubing in a very small flame. Tap
the fine point to break off the very delicate extremity. Scarcely any pain attends
the use of such a needle. In puncturing either the tip of the finger or lobe of the
ear a quick piano-touch-like stroke should be used. The ear is preferable, as it is
less sensitive and there is less danger of infection. Before puncturing, the skin
should be cleaned with 70% alcohol and allowed to dry. It is advisable to sterilize
the needle before using it.

172 MICROMETRY AND BLOOD PREPARATIONS

The first drop of blood which exudes should be taken up on the paper
of the Tallquist hsemoglobinometer, using subsequent ones for the blood
pipettes and smears. If it is necessary to make a complete blood ex-
amination, it is rather difficult to draw up the blood in the pipettes,
dilute it, and then get material for fresh blood preparations and films
without undue squeezing, which is to be avoided. Of course, fresh
punctures can be made. Ordinarily, complete blood examinations are
not called for. It is only a white count or a differential count or
an examination for malaria that is required.

As a practical point it is very rare that a red count is indicated. There is one
point not sufficiently recognized by physicians and that is that a call for a routine
blood examination is not apt to be as carefully conducted as one calling for a specific
feature. Without disparaging the necessity of routine examinations of urine as
well as blood it is a fact that the internist who knows what he wants gets better
results from the laboratory man.

HEMOGLOBIN ESTIMATION.

The most accurate instrument for this purpose is the Miescher
modification of the v. Fleischl haemoglobinometer.

The magenta-stained glass wedge for comparison with the diluted blood is similar
in each instrument, but by the use of a diluting pipette accurate dilutions are possible
in the Miescher. There are two cells provided — one 12 millimeters high, the other
15 millimeters; the idea of this being to enable one to make separate comparisons
and to select the central part of the glass- wedge scale, where comparison is more
accurate than at the ends. As these cells contain columns of diluted blood propor-
tionately as 5 to 4, we should have similar readings when we multiply the reading
on the scale with the 15 mm. cell by 4/5.

The mixing pipette is graduated with the marks 1/2, 2/3 and i/i — the first
giving a dilution of i to 400 (when the diluent, 3,0.1% soda solution, is drawn up to
the mark above the bulb) the second of i to 300 and the last of i to 200.

Artificial light preferably from a candle is necessary. There is a table accom-
panying each instrument which shows the value for that particular instrument in
milligrams per liter of haemoglobin for any reading obtained on the scale.

The apparatus is expensive, requires considerable time and care in the making
of estimations, and is exclusively an instrument for a well-equipped laboratory.

Sahli's Haemometer. — A simple and apparently very scientific
instrument which has been recently introduced is the Sahli modifica-
tion of the Gower haemoglobinometer. Instead of the tinted glass, or
gelatin colored with picrocarmine to resemble a definite blood dilution,
Sahli uses as a standard the same coloring matter as is present in the tube
containing the blood. By acting on blood with ten times its volume of

HEMOGLOBIN ESTIMATION

173

N/io HC1, haematin hydrochlorate is produced, which gives a brownish-
yellow color. In the standard tube, which is sealed, a dilution repre-
senting i% of normal blood is used. To apply this test, pour in N/io
HC1 to the mark 10 on the scale of the graduated tube. Add to this 20
cubic millimeters of the blood to be examined, drawn up by the capillary
pipette provided. So soon as the mixture assumes a clear bright dark
brown color, add water drop by drop until the color of the tubes matches.
The reading of the height of the aqueous dilution on the scale gives
the Hb. reading. The tubes are encased in a
vulcanite frame with rectangular apertures.
This gives the same optical impression as
would planoparallel glass sides.

The most accurate readings are obtained with ar-
tificial light in a dark room but almost as satisfactory
comparisons can be obtained with natural light from
a window. It is advisable to turn the ruled side
around so that one may match colors without being
influenced in his determination by the scale.

The apparatus must be kept in a dark place as
strong light will change the color of the standard
tube. It is recommended that the N/io HC1 be
preserved with chloroform.

Tallquist's Haemoglobin Scale. — This is
a small book of specially prepared filter-pa-
per with a color-scale plate of ten shades of
blood colors. These are so tinted as to match
blood taken up on a piece of the filter-paper
and are graded from 10 to 100. So soon as the
blood on the filter-paper has lost its humid
gloss, the comparison should be made. This
is best done by shifting the blood-stained
piece of filter-paper suddenly from one to the
other of the holes cut in each shade — -the
piece of filter-paper being underneath the color

plate. At least a square centimeter of the filter paper should be stained
by the blood. Daylight coming from a window to the rear or at the side
should be used in making the comparison. The error with this method
is probably not over 10% after a little experience. If the colored plate
is not kept in the dark, the tints tend to fade.

FIG. 51. — Sahli's haemo-
globinometer. (Greene.}

174

MICROMETRY AND BLOOD PREPARATIONS

To' COUNT BLOOD-CORPUSCLES.

The instrument almost universally used is the Thoma-Zeiss haemacy-
tometer. The apparatus consists of two pipettes, one for leukocytes,
graduated to give a dilution of i to 10 or greater; the other for red cells
to give a dilution of a i to i oo or greater. The white pipette has the mark
ii above the bulb and the red pipette the mark 101. In addition, there
is a counting chamber. This consists of a square of glass with a round
hole in the center. Occupying the center of this round hole is a circu-
lar disc of glass of less diameter, SD that an encircling channel is left.

The square and the circle of glass are
cemented to a heavy glass slide. The sur-
faces of each are absolutely level and
highly polished. That of the circular disc
is ruled into squares of varying size and is
exactly i/io of a millimeter below the
level of the surface of the surrounding
glass square.

When a polished piano-parallel cover-
glass rests on the shelf, as the outer square
glass is termed, there is a space left be-
tween its under surface and the ruled disc
of o. i millimeter. The channel around the
disc is termed the moat or ditch. The most
desirable rulings are those of Turck and of
Zappert. In these the entire ruled surface

consists of nine large squares, each i millimeter square. These are sub-
divided, and in the central large square are to be found the small squares
used for averaging the red cells. These small squares are 1/20 of a milli-
meter square and are arranged in nine groups of sixteen small squares
by bordering triple-ruled lines. As the unit in blood counting is the
cubic millimeter, if one counted all the white cells lying within one of
the large squares (i millimeter square), he would have only counted the
cells in a layer i/io of the required depth, so that it would be necessary
to multiply the number obtained by 10. This product, multiplied by
the dilution of the blood, would give the number of white cells in a cubic
millimeter of undiluted blood.

FIG. 5 2. — Thomas- Zeiss blood
counter showing pipette, count-
ing chamber, and ruled field.
(Greene.)

To make a red count: Having a fairly large drop of blood, apply the tip of the
10 1 pipette to it and, holding the pipette horizontally, carefully and slowly draw up

THE COUNTING OF RED CELLS 175

with suction on the rubber tube a column of blood to exactly 0.5 or i. The variation
of 1/25 of an inch from the mark would make a difference of almost 3%. If the
column goes above 0.5, it can be gently tapped down on a piece of filter-paper until
the 0.5 line is cut. Now insert the tip of the pipette into some diluting fluid and,
revolving the pipette on its long axis while filling it by suction, you continue until
the mark 101 is reached. A variation of 1/25 of an inch at this mark would only
give an error of about 1/30 of i%. After mixing thoroughly by shaking for one
or two minutes, the fluid in the pipette below the bulb is expelled (this, of course, is
only diluting fluid). A drop of the diluted blood of a size just sufficient to cover
the disc when the cover-glass is adjusted, is then deposited on the disc and the cover-
glass applied by a sort of sliding movement, best obtained by using a forceps in
one hand assisted by the thumb and index-finger of the other.
Among diluting fluids Toisson's is probably the best:

Sodium chloride, i gram

Sodium sulphate, 8 grams

Glycerine, 30 c.c.

Distilled water, 160 c.c.

Dissolve the sodium chloride and the sodium sulphate in the glycerine water and
add sufficient methyl or gentian violet to give a rich violet tint.

A 2 1/2% solution of potassium bichromate makes a very satisfactory diluting
fluid in the counting of red cells.

A salt solution of about i% strength, tinged with about i drop of a saturated
alcoholic solution of gentian violet to about 50 c.c., is a good substitute, or the salt
solution alone will" answer when no white count is to be made at the same time as the
red one.

It is important to work quickly in adjusting the cover-glass, or there will be cells
settling in the center of the drop from a greater depth than the one which the apposi-
tion of the cover-glass makes (i/io millimeter deep).

A good preparation should show:

1. Presence of Newton's rings.

2. Absence of air bubbles.

3. Entire surface of ruled disc covered.

4. Equal distribution of cells.

Before counting, about five minutes should be allowed for the set-
tling of the cells.

It will be remembered that the small squares are 1/20 millimeter square. The
depth of fluid from upper surface of shelf to lower surface of cover-glass is i/io mm.
Hence each space embraced by the small square and the depth of fluid is 1/4000 of
the unit used in estimating number of corpuscles in blood, or i cubic millimeter
(1/20X1/20X1/10=1/4000). Count 100 of the small squares .(this enables one
to use decimals). There are nine squares between triple-ruled lines, each containing
sixteen small squares. Count the number of corpuscles in the sixteen small squares
contained in upper left-hand triple-ruled square. Put down this count. Next

176 MICROMETRY AND BLOOD PREPARATIONS

count corpuscles in the adjoining sixteen squares. Put down this count. Then in
third sixteen squares. Put down the number. Now move down to next row of
three triple-ruled squares. Count the number of corpuscles in each of the three
sixteen-square spaces and set down the numbers for addition. We have now counted
ninety-six small squares (6X 16). Count at any place four additional small squares
and add number of blood-cells contained therein to those in the ninety-six small
squares already counted. Divide the sum by 100 or simply point off two decimals.
This gives the average for each small square. Multiply this by the dilution and then
(as the small square is only 1/4000 cu. mm.) by 4000. This will give the number of
corpuscles in i cubic millimeter. Example: 100 small squares contained 655 red
cells. Pointing off, 6.55 equals average number of red cells per small square.
Multiply by dilution (200) and then by 4000 (the small square is 4000 times smaller
than the unit: i cu. mm.) — 6.55X200=1310X4000 = 5,240,000.

At least 100 small squares, and preferably 200 should be counted.
If the blood appears normal, one may simply count the number of red
cells in five of the sixteen small square spaces (eighty small squares).
Having added the numbers and multiplying by 10,000, you obtain the
number of cells in i cubic millimeter. (Eighty small squares is 1/50 of
the unit of i cu. mm., or 4000 small squares. The blood dilution being
i to 200, we have 50 X 200 X number of cells in eighty small squares.)

In counting, count corpuscles lying on the lires above and to the right. Do not
count those lying on lines below and to the left.

In the small squares count only corpuscles lying in the space or cutting the upper
line. This prevents counting the same cell twice.

To Count White Cells.— Draw up the fluid in the white pipette to
the mark 0.5. Then, still holding the pipette as near the horizontal as
possible, because the column of blood tends to fall down in the larger
bore, draw up by suction a diluting fluid which will disintegrate the red
cells without injuring the whites. The best fluid is 0.3 % of glacial acetic
acid in water. This makes the white cells stand out as highly refractile
bodies. Some prefer to tinge the fluid with gentian violet. The 0.5
mark is preferred because it takes a very large drop of blood to fill the
tube up to the i mark and if there is much of a leukocytosis a i to 10
dilution is not sufficient. In leukaemic blood it is better to use the red
pipette with the 0.3% acetic acid solution.

The blood having been drawn up to 0.5, we have a dilution of i to 20.
Making a preparation, exactly as was done in the case of the red count,
we count all of the white cells in one of the large squares (i sq. mm.).
The cross ruling greatly facilitates this. Note the number. Then
count a second and a third large square. Strike an average for the large
squares counted and multiply this by 10, as the depth of the fluid gives

THE COUNTING OF WHITE CELLS 177

a content equal to only i/io of a cubic millimeter. Then multiply
by the dilution. Example: First large square 50; second large square
70; third large square 60. Average 60. Then 60X10X20 = 12,000,
the number of leukocytes in i cubic millimeter of blood. The count
may be made with a low power (2/3 -inch objective) as the leukocytes
stand out like pearls. It is better, however, to use a higher power, so
that pieces of foreign material may be recognized and not enumerated
as white cells.

When it is desired to make a white count with the same preparation as is used for
the red one, especially if the ruling is of the old style (only central ruling and not in
nine large squares as with Zappert and Tiirck), it is advisable to make use of the
method of counting by fields. With a Leitz No. 4 ocular and a No. 6 objective, with
a tube length of 120 millimeters, it will be observed that the field so obtained has a
diameter of eight small squares. Now, remembering that the area of a circle equals
the square of the radius multiplied by TT, or 3.1416, we have the following calculation:
The diameter being eight small squares, the radius would be four small squares.
Squaring the radius, we have sixteen. This multiplied by 3.1416 gives us fifty.
This means that every field, with the microscope adjusted as stated, contains fifty
of the small squares, or 1/80 of the unit of one cubic millimeter of the diluted blood.

By keeping a single red cell in view while moving the mechanical stage from right
to left or from above downward, we know that a new field of fifty small squares is
brought into view when the circumference of the field cuts this individual cell.
Example: As 2000 small squares would ordinarily be a sufficient number to count
for a white count, this would require us to count the number of leukocytes in forty
of the designated microscopic fields (this, of course, is only one-half the unit, hence
we should multiply by 2). Counted forty fields and noted fifty white cells. 50X2
= 100X200 (the dilution in red pipette) = 20,000. Consequently 20,000 would
represent the number of leukocytes in one cubic millimeter of the blood examined.

After making a blood count, the hsemacytometer slide should be cleaned with soap
and water and then rubbed dry, preferably with an old piece of linen. As the
accuracy of the counting chamber depends upon the integrity of the cement, any
reagent such as alcohol, xylol, etc., and, in particular, heat, will ruin the instrument.
The pipettes should be cleaned by inserting the ends into the tube from a vacuum
pump, as a Chapman pump. First draw water or i% sod. carbonate solution
through the pipette, then alcohol, then ether, and finally allow air to pass through
to dry the interior. If the interior is stained, use i% HC1 in alcohol. If a vacuum
pump is not at hand, a bicycle pump or suction by mouth will answer.

PREPARATIONS FOR THE STUDY OF FRESH BLOOD.

Many authorities prefer a fresh-blood specimen to a stained dried
smear in the study of parasites of the blood. In malaria in particular
there is so much information as to species to be obtained from a fresh
specimen that the employment of this method should never be neglected.

178 'MICROMETRY AND BLOOD PREPARATIONS

While waiting for the film to stain one has five or six minutes which
could not be better spent then in examining the fresh specimen which
only requires a moment to make.

Manson's Method. — -Have a perfectly clean cover-glass and slide. Touch the
apex of the exuding drop of blood with the cover-glass and drop it on the center of
the slide. The blood flows out in a film which exhibits an "empty zone" in the
center. Surrounding this we have the "zone of scattered corpuscles," next the
"single layer zone" and the "zone of rouleaux" at the periphery. It is well to ring
the preparation with vaseline. When desiring to demonstrate the flagellated bodies
in malaria, it is well to breathe on the cover-glass just prior to touching the drop of
blood.

The Method of Ross is very easy of application and gives most satisfactory
preparations. Take a perfectly clean slide, and make a vaseline ring or square of
the size of the cover-glass. Then, having taken up the blood on the cover-glass,
drop it so that its margin rests on the vaseline ring. Gently pressing down the cover-
glass on the vaseline makes beautiful preparations which keep for a very long time.
If it is desired to study the action of stains on living cells, this method is also appli-
cable. A very practical way to do this is to tinge 0.85% salt solution containing i%
sodium citrate (the same as is used in opsonic work) with methylene azur, gentian
violet, or methyl green. With a Wright bulb pipette, take up one part of blood,
then one part of tinted salt solution. Mix them quickly on a slide and then deposit
a small drop of the mixture in the center of the vaseline ring and immediately apply
a cover-glass and press down the margins as before. This method will be found of
great practical value.

A METHOD FOR MAKING DIFFERENTIAL LEUKOCYTE COUNT IN SAME
PREPARATION AS FOR WHLJE COUNT.

Employ the same technic as in making the ordinary white count
but using as a diluting fluid a 2 % formalin solution to which has been
added one drop of Giemsa's stain for each c.c. just before making the
blood examination.

The best results are obtained when the mixing in the pipette bulb is done im-
mediately after taking up the blood and diluent. Recently I have found it necessary
to add enough N/i NaOH to the commercial formalin to bring it to +i. Of this
I use i 1/2% in a 1/2% glycerine solution instead of water.

The usual technic in making the haemocytometer preparation is employed-
using a Tiirck ruling. Count the leukocytes in the three upper or lower i sq. mm.
squares, divide by 3 to obtain an average per sq. mm., multiply by 10 for the con-
tent of a cubic millimeter and then by 20 for the dilution. (Blood to 0.5; diluent
to IT.) This can be done mentally and requires no calculation on paper. Having
counted the leukocytes, again go over the same portion of the ruled surface and count
the polymorphonuclears and estimate the percentage of these to the total leuko-
cytes. The majority of disrupted cells in a dry-stained preparation are transitionals
hence the percentage of polymorphonuclears by this method is lower.

STAINING OF BLOOD 179

It is unnecessary in such a count to have an assistant; of course, in making a
complete differential count it is preferable to have some one tabulate or labor-
iously to do this one's self.

The red cells are practically diaphanous and not disintegrated as when acetic
acid is used as a diluent, consequently it is easy to make out the particular red cell
as to size, etc., containing a malarial parasite.

The best results are obtained with a i/6-in objective. Higher powers are of
course impracticable by reason of the thickness of the cover-glass of the haemocy-
tometer.

The following are the appearances of the various leukocytes.

Eosinophiles. — In these the bilobed nucleus stains rather faintly and the color
is greenish blue. The eosinophile granules show easily as coarse, brickdust-colored
particles.

Polymorphonuclears. — The nucleus stains a deep, rich, pure violet but less in-
tense than that of the small lymphocyte. The shape of the nucleus is typically
three or four lobed but even when of the horseshoe shape of a transitional nucleus
is easily recognizable by the intensity of the violet staining. That which makes the
polymorphonuclears very easy of differentiation is the distinctness of the cell out-
lines produced by the fine yellowish granulations in the cytoplasm.

Small Lymphocytes. — The nucleus is perfectly round and stains a deep violet.
It is almost impossible to make out any cytoplasmic fringe.

Large Lymphocytes. — The nucleus here is round and of a lighter violet than that
of the small lymphocyte. The cytoplasm is blue, nongranular, and sharply defined
from the nucleus.

Large Mononuclears. — These show a washed-out, slate-colored nucleus which
blends with the gray slate-blue staining of the cytoplasm so that there is an in-
definiteness of outline in the more or less irregularly contoured nucleus.

Transitionals. — These show the same characteristics as the large mononuclears,
but with a more faintly stained and more indented nucleus. The large mono-
nuclears and transitionals stand out as slate-colored cells. When very much degen-
erated these cells have a greenish hue.

Mast Cells. — The granulations show as a rich maroon or reddish-violet color.

The young ring forms of malaria show as violet-blue areas in the red cells. When
half-grown or approaching the merocyte stage, the containing red cell takes on a
faint pink coloration, thereby differentiating it from the noninfected red cells. At
the same time the parasite is extruded and has the appearance of a violet-blue body
projecting from the margin of the red cell. It is as if a blue body were budding
from a pink one.

It is an easy matter with this method to count the number of trypanosomes or
malarial crescents in a cubic millimeter of blood.

PREPARATION AND STAINING OF DRIED FILMS.

When preparations are desired for a differential count, Ehrlich's
method of making films is to be preferred, as the different types of leuko-
cytes are more evenly distributed. In making smears by spreading,

i8o

MICROMETRY AND BLOOD PREPARATIONS

there is a tendency for the polymorphonuclears to be concentrated at
the margin while lymphocytes remain in the central part of the film.

In Ehrlich's method we have perfectly clean dry cover-slips. Take
up a small drop of blood without touching the surface of the ear or finger.
Drop this cover-glass 'immediately on a second one and as soon as the
blood runs out in a film, draw the two cover-slips apart in a plane par-
allel to the cover-glasses. Slide them apart. Ehrlich uses forceps to
hold the cover-glasses to avoid moisture from the fingers.

Of the various methods of spreading films on slides there is none

FIG. 53. — Blood technic. i, 2, 3, Method for making blood smear on slide; 4,
U tube for resting slides while staining; 5, slide showing grease pencil marking,
marking prevents stain from overflowing; 6, method for drawing apart cover-glasses
in making blood smear.

equal to that described by Daniels. In this the drop of blood is drawn
along and not pushed along. The films are even, can be made of any
desired thickness by changing the angle of the drawing slide, and there
is little liability of crushing pathological cells. Take a small drop of
blood on the end of a clean slide. Touch a second slide about 1/2
inch from end with the drop and as soon as the blood runs out along
the line of the slide end, slide it at an angle of 45° to the other end of the

BLOOD SMEARS l8l

horizontal slide. The blood is pulled or drawn behind the advancing
edge of the advancing slide. An angle less than 45° makes a thinner
film; one greater, a thicker film.

Instead of a slide a square cover-glass may be used and if the edge be smooth it
makes a more satisfactory spreader than the slide. Many workers prefer the Ross
thick-film method in examining for malaria. In this about one-half of a drop of
blood is smeared out over a surface about equal to that of a square cover-glass and
allowed to dry. It is then flooded with a 0.1% aqueous solution of eosin for
about 15 minutes. The preparation is then gently washed with water and then
treated with a polychrome methylene-blue solution. After a few seconds this is
carefully washed off and the preparation dried and examined.

Instead of the Daniels method some prefer to take up the drop of blood on the
slide on which the smear is to be made, about 1/2 inch from the end. Then apply
the spreader slide and so soon as the drop runs along the end of the spreader slide
proceed as above described.

Of the various methods of making smears by means of cigarette paper, rubber
tissue, needles, etc., the best seems to be to take a piece of capillary glass tubing and
use this instead of a needle in making the film. There is one advantage about the
strip of cigarette paper touched to the drop of blood and drawn out along the slide
or cover-glass, and that is that it is almost impossible not to make a working prepa-
ration by this method.

In the making of smears the chief points are to make the smears as
soon after taking the blood as possible and to have slides and cover-
glasses scrupulously clean. It is well to flame all slides and cover-
glasses which are to be used for blood-work. This is the best method
of getting rid of grease.

Fixation of Film. — In Wright's, Leishman's, and other similar stains the methyl-
alcohol solvent causes the fixation. In staining with Giemsa's stain, Ehrlich's
tri-acid, hsemotoxylin and eosin, Smith's formol fuchsin, and with thionin, separate
fixation is necessary. For Giemsa and thionin, either absolute alcohol (ten to
fifteen minutes), or methyl alcohol (two to five minutes) answer well.

Formalin vapor, for five to ten seconds, is also used for fixation. For Ehrlich's
tri-acid, haemotoxylin and eosin and formol fuchsin, heat gives the best results.
The best method is to place the films in an oven provided with a thermometer.
Raise the temperature of the oven to 135° C. and then remove the burner. After
the oven has cooled, take out the fixed slides or slips.

Some prefer to place a crystal of urea on the slide, then hold it over the flame
until the urea melts. This shows that a temperature between 130° and 135° C. has
been reached.

One of the handiest methods is to drop a few drops of 95% alcohol on the slide
or cover-glass. Allow this to flow over the entire surface; then get rid of the excess
of alcohol by touching the edge to a piece of filter-paper for a second or two. Then
light the remaining alcohol film from the flame and allow the burning alcohol to
burn itself out. A chemical fixation which gives good fixation for haematoxylin

182 MICROMETRY AND BLOOD PREPARATIONS

and tri-acid stains (not equal to heat) is a modification of Zenker's fluid (Whitney).
To Muller's fluid, which is potassium bichromate 2 grams, sodium sulphate i gram,
and water 100 c.c., add 5 grams of bichloride of mercury and 5 c.c. of nitric acid
(C. P.). Fixation is obtained in five seconds.

When using corrosive sublimate fixation one should after thorough washing in
water treat the film with Gram's iodine solution for about two minutes and then wash
with 70% alcohol until the yellow color of the film disappears. Methyl alcohol
for two minutes is satisfactory.

Staining Blood-films. — As separate staining with eosin and methyl-
ene blue rarely gives good preparations and as the modifications of the
Romanowsky stain recommended are easy to make and employ, and
give much greater information, the separate method of staining is not
recommended. The most satisfactory single stain is thionin.

Rees' Thionin Solution. — Take of thionin 1.5 gram, alcohol 10 c.c., aqueous
solution of carbolic acid (5%) 100 c.c. Keep this as a stock solution. It should
be at least two weeks old before using. For use, filter off 5 c.c. and make up to
20 c.c. with water.

1. Fix films (a) by heat, (b) by alcohol and ether, or (c) preferably by i%
formalin in 95% alcohol for one minute.

2. Stain for from ten to twenty minutes. Wash and mount. Malarial parasites
are stained purplish; nuclei of leukocytes, blue; red cells, faint greenish-blue.

Ehrlich's Triacid or Triple Stain. — There are required:

1. Sat. aq. sol. orange G. (Dissolve 3 grams in 50 c.c. water.)

2. Sat. aq. sol. acid fuchsin. (Dissolve 10 grams in 50 c.c. water.)

3. Sat. aq. sol. methyl green. (Dissolve 10 grams in 50 c.c. water.)

These three solutions may be kept as stock solutions. They keep well in the dark.
To make the stain, add 9 c.c. of No. 2 (acid fuchsin) to 18 c.c. of No. i (orange G.).
After they are mixed thoroughly, add 20 c.c. of No. 3 (methyl green). Then after
the first 3 ingredients are well mixed, add 5 c.c. of glycerin. Mix, then add 15 c.c.
of alcohol; again mix, and finally add 30 c.c. of distilled water. Keep the mixed
stain about one week before using. The best fixatives are heat and Whitneys'
modified Zenker. To use, stain films from two to five minutes; then wash and
mount. The triacid stain is a good tissue stain. The objections to the tri-acid
stain are that it does not stain malarial parasites or mast cells and that failure to
obtain good results is of frequent occurrence.

Wright's Method. — The stain is made by adding i gram of methylene blue (Grubler)
to 100 c.c. of a 1/2% solution of sodium bicarbonate in water. This mixture is
heated for i hour in an Arnold sterilizer. The flask containing the alkaline methyl -
ene-blue solution should be of such size and shape that the depth of the fluid does
not exceed 2 1/2 inches. When cool, add to the methylene-blue solution 500 c.c.
of a i to 1000 eosin solution (yellow eosin, water soluble). Add the eosin solution
slowly, stirring constantly until the blue color is lost and the mixture becomes
purple with a yellow metallic luster on the surface, and there is formed a finely
granular black precipitate. Collect this precipitate on a filter-paper and when
thoroughly dry (dry in the incubator at 38° C.) dissolve 0.3 gram in 100 c.c. of pure

ROMANO WSKY STAINING METHODS 183

methyl alcohol (acetone free). Wright lately has recommended using o. i in 60 c.c.
methyl alcohol. This constitutes the stock solution. For use filter off 20 c.c. and
add to the filtrate 5 c.c. of methyl alcohol.

A modification by Batch is very satisfactory. In this method instead of poly-
chroming the methylene blue with sodium bicarbonate and heat, the method of
Borrel is used. Dissolve i gram of methylene blue in 100 c.c. of distilled water.
Next dissolve 0.5 gram of silver nitrate in 50 c.c. of distilled water. To the silver
solution add a 2 to 5% caustic soda solution until the silver oxide is completely
precipitated. Wash the precipitated silver oxide several times with distilled water.
This is best accomplished by pouring the wash-water on the heavy black precipitate
in the flask, agitating, then decanting and again pouring on water. After removing
all excess of alkali by repeated washings, add the methylene-blue solution to the
precipitated silver oxide in the flask. Allow to stand about ten days, occasionally
shaking until a purplish color develops. The process may be hastened in an incu-
bator. When polychroming is complete, filter off and add to the nitrate the i to
1000 eosin solution and proceed exactly as with Wright's stain.

In Leishman's method the polychroming is accomplished by adding i gram of
methylene blue to 100 c.c. of a 1/2% solution of sodium carbonate. This is kept at
65° C. for twelve hours and allowed to stand at room temperature for ten days
before the eosin solution is added. The succeeding steps are as for Wright's stain.

In all Romanowsky methods distilled water should be used. If not obtainable,
the best substitute is rain-water collected in the open and not from a roof.

Method of staining:

1. Make films and air dry.

2. Cover dry film preparation with the methyl-alcohol stain for one minute
(to fix).

3. Add water to the stain on the cover-glass or slide, drop by drop, until a
yellow metallic scum begins to form. It is advisable to add the drops of water
rapidly in order to eliminate precipitates on the stained film. Practically, we may
add i drop of water for every drop of stain used.

4. Wash thoroughly in water until the film has a pinkish tint.

5. Dry with filter-paper and mount.

Red cells are stained orange to pink; nuclei, shades of violet; eosinophile granules,
red; neutrophile granules, yellow to lilac; blood platelets, purplish; malarial parasites,
blue; chromatin, metallic-red to rose-pink

Giemsa's Modification of the Romanowsky Method. — This is one of
the most perfect of the modifications. The objection is that greater
time in staining films is required than with the Wright or Leishman
method and the stain is very expensive.

Take of Azur II eosin 0.3 gram. Azur II 0.08 gram.

Dissolve this amount of dry powder in 25 c.c. of glycerine at 60° C.
Then add 25 c.c. of methyl -alcohol at the same temperature. Allow
the glycerine methyl-alcohol solution to stand overnight and then filter.
This is the stock stain. To use: Dilute i c.c. with 10 to 15 c.c. of dis-

184 MICROMETRY AND BLOOD PREPARATIONS

tilled water. If i to 1000 potassium carbonate solution is used instead
of water it stains more deeply.

The alkaline diluent is used to obtain the course stippling in malig-
nant tertian (Maurer's clefts). Having fixed the smear with methyl
alcohol for one to five minutes, pour on the diluted stain, and after
fifteen to thirty minutes wash off and continue washing with distilled
water until the film has a slight pink tinge. For Treponema pallidum
stain from two to twelve hours.

While the Romanowsky methods are more satisfactory for differential counts and
for the demonstration of the malarial parasites, and especially for differentiating
species, yet by reason of the liability to deterioration in the tropics of methylene
blue the hsematoxylin methods may be preferable. Many workers in blood-work
and cytodiagnosis prefer the haematoxylin.

1. Fix the film either by heat with methyl alcohol for two minuetes or with Whit-
ney's fixative. Heat is to be preferred.

2. Stain with Meyer's hemalum or Delafield's haematoxylin for from five to
fifteen minutes according to the stain. Frequently three minutes will be
found sufficient. To make the hemalum, dissolve 0.5 gram of hae matin in 25
c.c. of 95% alcohol. Next dissolve 25 grams of ammonia alum in 500 c.c. of
distilled water. Mix the two solutions and allow to ripen for a few days. The
stain should be satisfactory in two or three days.

To make Delafield's haematoxylin, dissolve i gram of haematoxylin crystals in
6 c.c. of 95% alcohol. Add this to 100 c.c. of saturated aqueous solution of
ammonia alum. After exposure to light for a week, the color changes to a
deep blue-purple. Add to this ripened stain 25 c.c. of glycerine and 25 c.c.
of methyl-alcohol and, after it has stood for about two days, filter. The stain
should be filtered from time to time as a sediment forms. This makes a stock
solution which should be diluted 10 to 15 times with water when staining.

Mink's Modification of TJnna's Haematoxylin.

Haematoxylin, i gram.

Alum, 8 grams.

Sulphur (sublimed), i gram.

Glycerine, 30 c. c

Alcohol, 50 c.c..

Water, 100 c.c.

Dissolve the hsematoxylin in the glycerine in a mortar. Dissolve the alum in the
water and add it to the glycerine haematoxylin in the mortar. Then add the sulphur
and the alcohol. The solution ripens in about three to four days. Allow the sedi-
ment to remain in the bottom of the bottle containing the stain and filter off small
quantities as needed.

3. Wash for two to five minutes in tap water to develop the haematoxylin color.

4. Stain either with a i to 1000 aqueous solution of eosin or with a 1/2 of i%

COAGULATION RATE OF BLOOD 185

eosin solution in 70% alcohol. The eosin staining only requires fifteen to
thirty seconds.
5. Wash and examine.

lODOPHILIA.

This reaction is supposed to be due to the presence of glycogen,
especially in the polymorphonuclears, in suppurative conditions.

It has been stated that a differentiation between the joint involvement in gonor-
rhoeal infection and in articular rheumatism may be made from iodophilia being
present in the gonococcus infection.

Make blood-smears on cover-glasses as usual, and after they dry, but without
fixation, mount them in a drop of the following solution:

Iodine, i part.

Potassium iodide, 3 parts.

Gum arabic, 50 parts.

Water, 100 parts.

Small brown masses in the polymorphonuclears indicate a positive iodophilia.

Viscosity of the Blood. — This is estimated by observing the relative height to
which blood rises in capillary tubes as compared with water, and normally varies
from three to five. The higher the haemoglobin content the greater the viscosity.
Viscosity is high in arterio-sclerosis and diabetic coma, low in the anaemias of
nephritis.

Coagulation Rate of Blood. — This determination is of value in con-
nection with operations on jaundiced patients.

Wright's coagulometer is a standard instrument but is cumbersome.

A simple method of determining the rate is to take a- piece of capil-
lary glass tubing and hold it downward from the puncture to let it
fill for 3 or 4 inches. Then at intervals of thirty seconds scratch
with a file the capillary tubing at short distances and break off between
the fingers. When coagulation has taken place a long worm-like co-
agulum is obtained. Normally coagulation occurs in about three to
four ^minutes, when the temperature is that of the hand in which the
tubes are conveniently held. Rudolf recommends placing the tubes in
metal tube containers in a Thermos bottle at 20° C. He gives the normal
coagulation rate for this temperature as 8 minutes, while at a tempera-
ture below this the period is lengthened. Age and sex do not influence
the rate. Sabrazes, the originator of this method found no appreciable
variation in tubes from 0.8 to 1.2 mm. diameter.

1 86 MICROMETRY AND BLOOD PREPARATIONS

In Biirker's test you mix a drop of blood and a drop of distilled water on a slide
and with a capillary tube sealed off at the end stir the mixture every half minute.
So soon as fibrin threads appear you have coagulation.

SPECIFIC GRAVITY or THE BLOOD.

Hammerschlag has a method for the determination of the Hb.
percentage based upon the specific gravity of the blood.

In this method a mixture of benzol and chloroform is made of a specific gravity
of about 1050. A medium size drop of blood is then taken up with a pipette and
dropped into the mixture. If it sinks add more chloroform from a dropping bottle,
if it tends to rise, more benzol. The mixture in which the drop of blood tends to
remain stationary, near the top of the mixed benzol and chloroform, has the same
specific gravity as that of the blood. This is determined by an accurately graduated
hydrometer. The normal average specific gravity for men is 1059, for women 1056.
A table, giving the Hb. percentage corresponding to the specific gravity accom-
panies the outfit.

To determine the necessity for intravenous infusion in cholera Rogers has re-
cently recommended the employment of small bottles containing aqueous solution
of glycerine with specific gravities varying from 1048 to 1070, increasing the specific
gravity in each successive bottle by 2°.

An accurate urinometer will suffice to determine the specific gravity. Drops
of blood from the cholera patient are deposited at the center of the surface of the
fluid in the bottles from a capillary pipette. If the specific gravity of the blood is
1062 at least a liter of saline or sodium bicarbonate solution is needed. If 1066 at
least two liters. Formerly he estimated the indications by blood pressure con-
sidering a pressure of 80 in Europeans 01 of 70 in natives as indicating intravenous
injections.

OCCULT BLOOD.

When the presence of blood cannot be recognized by macroscopical
or microscopical methods (occult blood) we must resort to spectro-
scopic or chemical tests. It is in connection with blood in the faeces
that these tests for occult blood are chiefly called for. Before making
such tests on faeces it is advisable to have the patient on a meat-free
and green-vegetable-free diet for two or three days. It is chiefly in
carcinoma or ulcerations of the gastro-intestinal tract that such exami-
nations of the faeces are required.

Haemin Crystal Test (Teichman).— Prepare a solution of o.i gram each of KI,
KBr, and KCL in 100 c.c. of acetic acid. This is a stable solution. Mix some of
the material with a few drops of the solution on a slide, apply a cover-glass and
warm the material until bubbles begin to appear (gentle steaming), then examine
for dark-brown crystals.

OCCULT BLOOD 187

Blood in the Urine. — The most rapid method of detection is by using the micro-
spectroscope. An ordinary hand spectroscope will answer however.

Donogany's test is very satisfactory. To 10 c.c. of urine add i c.c. ammonium
sulphide solution and i c.c. of pyridin. The urine will assume a more or less deep
orange color according to its blood content. The spectrum of alkaline methaemo-
globin or hgemochromogen will be obtained. See illustrations under urine.

In making the guaiac or other tests it is a good plan to repeatedly filter the blood-
containing urine through the filter. Then touch a spot on the moist filter with the
guaiac or benzidin solution and then finally drop on this so treated spot a drop or
two of hydrogen peroxide solution.

Blood in Faeces or Gastric Contents. — Take 5 grams of faeces and rub it up
thoroughly in a mortar with 15 c.c. of a mixture of equal parts of alcohol, glacial
acetic acid and ether. Filter through an unmoistened pleated filter paper re-
peatedly until only 3 to 4 c.c. remain of the filtrate. The faeces filtrate can be
first tested chemically by depositing a few drops in the center of 3 or 4 circles of
white filter-paper placed in a Petri dish or upon an ordinary white plate.

The moistened spot is then treated with a few drops of a freshly prepared alco-
holic solution of guaiac resin (about \ gram of guaiac resin is broken up into
small fragments and shaken up in about 3 c.c. of alcohol) and finally there is drop-
ped upon the spot a few drops of a solution of hydrogen peroxide. Waves of blue
color extending out into the moistened filter-paper show a positive test for blood.

For the benzidin test pour on this fasces filtrate-moistened filter-paper a few
drops of the following solution: 2 c.c. of a saturated alcoholic solution of benzidin
2 c.c. of solution of peroxide of hydrogen and two drops of glacial acetic acid. (Blue.)

If the aloin test is preferred we treat the filtrate-moistened filter-paper with a
few drops of a 3% solution of aloin in 70% alcohol and then treating the spot
with hydrogen peroxide solution. Brick red colour.

More reliable is the spectroscopic test. For this we take about 3 c.c.
of the concentrated ether, acetic acid, alcohol faecal nitrate and add to it
2 c.c. of pyridin. Then add not more than 2 to 3 drops of ammonium
sulphide solution. (The ammonium sulphide solution should be kept
in an amber-coloied, glass-stoppered bottle. The solution should be
freshly prepared every 10 days.) Examine the solution, contained in a
small test-tube, with the spectroscope and the two absorption bands of
methaemoglobin-alkaline (haemochromogen), between D and E, show
a positive blood test. Comparison should be made with fresh blood,
in which the absorption band in the yellow is nearer line D
(oxyhaemoglobin spectrum).

CHAPTER XIV.
NORMAL AND PATHOLOGICAL BLOOD.

IN considering what may be termed normal blood, it must be borne
in mind that the normal varies for men, women, and children:

Hb. Red cells. Leukocytes.

Men, 90 to 110%, 5 to 5 1/2 million, 7500.
Women, 80 to 100%, 4 1/2 to 5 million, 7500.
Children, 70 to 80%, 4 1/2 to 5 million, 9000.

COLOR INDEX.

This is obtained by dividing the percentage of the haemoglobin by
the percentage of red cells, five million red cells being considered as
100%. To obtain the percentage of red cells it is only necessary to
multiply the two extreme figures to the left by two. Thus if a count
showed the presence of 1,700,000 red cells, the percentage would be
34 (17X2=34). If the Hb. percentage in this case were 50; then the
color index would be 50-^34, or 1.4.

In normal blood the color index is, approximately, i.

In anaemias we have three types of color index: i. The pernicious
anaemia type, which is above i. Here we have a greater reduction in
red cells than we have of the haemoglobin content of each cell. 2. The
normal type, when both red cells and haemoglobin are proportionally
decreased, as in anaemia following haemorrhage. 3. The chlorotic type.
Here there is a great decrease in haemoglobin percentage, but only a
moderate decrease in the number of red cells. Hence the color index
is only a fraction of i. For example, in a case of chlorosis we have 40%
of haemoglobin and 90% of red cells, 40^90 = 0.4.

RED CELLS.

In considering the corpuscular richness of a specimen of blood, it
must be remembered that this does not necessarily bear any relation to
the quantity of blood in the body. Thus, a more or less bloodless-

PATHOLOGICAL RED CELLS 189

looking individual, the total quantity of whose blood is greatly reduced,
may, notwithstanding, give a normal red count. In examining a speci-
men of peripheral blood we get a qualitative, not a quantitative result.

Normally, we have an increase in red cells in those living at high altitudes.
An altitude of two thousand feet may increase the red count about one million, and
a height of six thousand feet about two million. Profuse sweats and diarrhoeas also
increase the red count. Pathologically, in chronic polycythemia with cyanosis
and splenic enlargement, we have a red count of about ten million. In cyanosis
from heart disease, etc., and in Addison's disease there is also an increase in red
cells.

The normal red cell or ery throcyte measures about 7.5^ in diameter. It is non-
nucleated and normally stains with acid dyes, taking the pink of eosin or the orange
of orange G. If larger, 10 to 20;*, it is called a macrocyte; if smaller, 3 to 6/*, a
microcyte.

Anisocytosis is a term applied to a condition where marked varia-
tion in size of the red cells occurs.

Macrocytes are rather indicative of severe forms of anaemia, the
microcytes, of less grave types. When the red cell is distorted in shape,
it is called a poikilocyte. Care must be exercised that distorted shapes
are not due to faulty technic. Crenation and vacuolation of red cells
are marked in poorly prepared specimens.

In addition to variation in size and shape, we also have pathological
variation in staining affinities.

Polychromatophilia.— This shows itself by red cells taking a brown-
ish to a dirty blue tint, as is frequently seen in immature red cells,
especially nucleated ones.

Granular basophilic degeneration (also termed punctate baso-
philia and stippling) refers to the presence of blue dots in the pink back-
ground of stained red cells. It is found in many severe anaemias, as
pernicious anaemia, the leukaemias, malarial cachexia, etc. It is very
characteristic of lead poisoning.

The nucleated red cell, while normal for the marrow, is always
pathological for the blood of the peripheral circulation. Normoblasts
have the diameter of a normal red cell. The nucleus is round and stains
intensely with basic dyes, often appearing almost black. Another
characteristic is that it frequently appears as does the setting in a ring.
Some give the term microblast to smaller nucleated forms. In normo-
blasts the red cell proper stains normally. The megaloblasts not only
have a greater diameter than the normoblast, but the nucleus is poor
in chromatin, stains less intensely and is less distinctly outlined. In-

I QO NORMAL AND PATHOLOGICAL BLOOD

stead of being round, the nucleus is irregular and may be trefoil in shape.
The cytoplasm surrounding the nucleus shows polychromatophilia.
This contrasted with the pure blue of the lymphocytes should differen-
tiate. Nornioblasts are found in secondary anaemias, and especially in
myelogenous leukaemia. Megaloblasts are peculiarly characteristic of
pernicious anaemia. Enormous megaloblasts are sometimes termed
gigantoblasts.

In aplastic anaemia (a severe type of pernicious anaemia), in contrast to ordinary
pernicious anaemia, nucleated reds are very rarely found. There is also very little
poikilocytosis, and the color index is about normal. It is a rare, rapidly fatal
anaemia, particularly of young women.

It does not show remissions, runs a rapid course, and is attended with a marked
increase of lymphocytes. The bone marrow of the femur is pinkish yellow and
homogeneous.

The term leukanaemia has been employed to describe conditions
which partake of the characteristics of pernicious anaemia and leukaemia.

WHITE CELLS.

Owing to the conflicting views as to origin, nature, and functions of
the various leukocytes, their classification is in a state of confusion.
As regards the appearance of the cells, this of course varies as the stain
used, and it requires considerable experience for a single individual to be
able to positively recognize the difference between a lymphocyte and
a large mononuclear when one specimen is stained with a Romanowsky
stain, another with Ehrlich's triacid, and a third with haematoxylin and
eosin. This, of course, is intensified when different persons adhere to
the method of staining which they prefer and are at a loss to appreciate
differences which are brought out by some other stain used by some
other person. Even with the same stain used with different specimens
of blood we find the staining characteristics of various leukocytes imper-
ceptibly merging the one into the other, so that at times it is impossible
for one, even with his own standard of differentiation, to be sure whether
he is dealing with a lymphocyte or a laige mononurlear. The difficulty
is even greater when we deal with Turck's irritation forms and with
myelocytes.

Without going into the various granule stainings so thoroughly
brough out by Ehrlich, we shall immediately take up the question of
a practical classification for use in making a differential count. As the
Romanowsky method of staining (Wright, Leishman, or Giemsa) gives

THE LEUCOCYTES IQI

us information not yielded by either haematoxylin and eosin or the tri-
acid, the points of differentiation to be referred to in that which follows
is with blood so stained.

In considering the staining affinities of different parts of the leuko-
cytes1, it is convenient to divide such into basic ones, acid ones, and thos-e
which may be said to be on the border line between these — the so-called
neutrophilic affinities.

With Wright's stain we have the eosinophile or oxyphile affinity of
the granules of eDsinophiles for acid dyes, in this case eosin. The
nuclei and basophile granules have affinities in greater or less degree for
basic stains (the blue and the violet shading resulting from methylene
blue as modified by poly chroming) . With the granules in the cyto-
plasm of the poly morphonucl ears and neutrophilic myelocytes, and to a
less extent in the transitional, we have a staining which merges into a
yellowish-red on the one extreme and into a lilac on the other. As a
standard, neutrophilic granules should be a mean of these extremes.

Not only by reason of the authority of Ehrlich, but because such a
division gives all variations, which can then be combined by one pre-
ferring a simpler classification, it would seem proper to divide the nor-
mal leukocytes into :

1. Small Lymphocytes. — These are small round cells about the size
of a red corpuscle with a large centrally placed, deeply violet staining
nucleus and a narrow zone of cytoplasm. This cytoplasm may not be
more than a mere crescentic fringe. This is the type of lymphocyte
which makes up the greater proportion of the leukocytes in chronic
lymphatic leukaemia. At times these cells seem to be composed of
nucleus alone.

2. Large Lymphocytes. — These are of the same type as small
lymphocytes, but possessing more cytoplasm. The nucleus, while
round and taking a fairly deep rich violet stain, does not stain so deeply
as the nucleus of the small lymphocytes. The cytoplasm is a clear,
translucent, pure blue. It may contain pinkish granules known as azur
granules, but these are of rathei large size and do not mar the glass-like
appearance. They are from 9 to 15^ in diameter and are common in
children. In the acute lymphatic leukaemias they at times predominate.

3. Large Mononuclears. — -These are large round or oval cells with
a nucleus which has lost the richness of violet staining of the lymphocyte
nucleus. The nucleus is furthermore frequently irregular in outline
or may show the commencing indentation of the transitional nucleus.

1 92 NORMAL AND PATHOLOGICAL BLOOD

There is not that sharp distinction between nucleus and cytoplasm
that exists in the lymphocytes. The cytoplasm of the large mononu-
clear gives the impression of opacity, as if it were frosted glass instead
of clear glass. The neutrophile mottling which begins to appear causes
a disappearance of the pure blue character of the cytoplasm of the
lymphocyte. It is principally by the washed-out staining of the nucleus
and the opaque lilac of the cytoplasm that we differentiate them from
the lymphocytes. They greatly resemble Tiirck's irritation forms or
plasma cells and may be confused with myelocytes.

4. Transitionals. — These appear as but a later stage in the decay
of the large mononuclears; the nucleus is more indented, frequently
horseshoe-shaped, and has a washed-out violet shade of less intensity
than that of the large mononuclears. These are the cells so often dis-
rupted in smears.

These four kinds of cells are frequently referred to as the lymphocyte
series, and although many authorities consider that the small lympho-
cyte represents a more mature cell than the others of this class, yet it
is thought by others that the age of the cell increases as we go from small
lymphocytes to large lymphocytes, thence to the large mononu clear;
and then in the transitional we have the decrepit stage which precedes
dissolution. The old view that the transitional was the precursor of
the polymorphonuclear has few advocates at the present time.

While it is convenient to consider these hyaline cells as representing
different stages in development, yet from a standpoint of immunity this
is untenable. The large mononuclears and transitionals are the cells
in which we find certain animal cells and pigment phagocytized, as is
the case in malaria. These cells are the macrophages of Metchnikoff
and are probably derived from the bone marrow.

The lymphocytes take origin from the lymphoid tissue, and very
probably the large lymphocyte is a younger, more immature cell than
the small lymphocyte.

Ehrlich and Naegeli regard the large mononuclears as of myeloid
origin while Pappenheim considers them to belong to the group of
lymphocytes.

A normal percentage of large mononuclears and transitionals com-
bined should not exceed about 4%.

In addition to the series of leukocytes just considered we have pres-
ent normally in the blood three types of granular cells distinguished
according to the staining affinity of their granules. These are:

ARNETH INDEX 1 93

1. Polymorphonuclear Leukocytes. — -This cell normally constitutes
the greater proportion of the leukocytes. It is an amoeboid, actively
phagocytic cell, about 10 or 12 /* in diameter, and is the microphage of
Metchnikoff. Bacteria are actively phagocytized by this cell, and it is
the cell concerned in determining the opsonic power of blood to various
bacteria. It has fine lilac granules which are termed neutrophilic (ep-
silon granules). The single nucleus is rich in chroma tin and is lobose
like the kernel of an English walnut; frequently it resembles the letter
z. These cells are derived from the neutrophilic myelocytes of the bone
marrow. It is in these cells that the glycogen, or iodophil granules,
appear in certain suppurative conditions.

A great deal of interest has been aroused in the so-called Arneth index, espe-
cially in connection with prognosis in tuberculosis and various pyogenic infections.
The basis of the test is that polymorphonuclears showing only one or two nuclear
nodes are considered immature while those having three, four or five nuclear nodes
possess greater phagocytic power.

A normal distribution is as follows:

Class I. Class II. Class III. Class IV. Class V.

6% 35% 42% 16% i%

To obtain the Arneth index add to the sum of the polymorphonuclear percent-
ages of cells containing one and two nodes one-half of the percentage of those having
three nodes. In the above we have as the normal Arneth index 62.

In an advanced case of tuberculosis we might have an index of 79, obtained as
follows :

Class I. Class II. Class III. Class IV. Class V.

20% 45% 28% 6% i%

2. Eosinophile Leukocytes.— These are very striking cells with
coarse granules staining brilliantly pink, the eosinophile, oxyphile, or
acidophile granules (alpha granules of Ehrlich). The cells are a little
larger than the polymorphonuclears. The normal eosinophile is to be
distinguished from the eosinophilic myelocyte by its possessing two
distinct lobes in the nucleus. At times we find three nuclei. The
nucleus of the myelocyte is round. The eosinophile is the cell so
frequently increased in infections by intestinal animal parasites.

3. Mast Cells.— These also have coarse granules, but they stain a
deep violet blue. Hence they are basophile granules (gamma granules).
In fresh blood these granules do not show up very well, thus they can be
distinguished from the highly refractile granules of the eosinophile.

13

194 NORMAL AND PATHOLOGICAL BLOOD

The trilobed nucleus stains less intensely than the granules. As a rule,
the mast cell is about the size of a polymorphonuclear.

In a differential count of normal blood we find about the following percentages.

Polymorphonuclears, 65 to 70%, about 5000 per c. mm.

Small lymphocytes, 20 to 25%, about 1500 per c. mm.

Large lymphocytes, 5 to 10%, about 500 per c. mm.

Large mononuclears, i to 2%, about 100 per c. mm.

Transitionals, 2 to 4%, about 200 per c. mm.

Eosinophiles, i to 2%, about TOO per c. mm.

Mast cells, 1/4 to 1/2%, about 25 per c. mm.

The leukocytes which are found in the peripheral circulation only
in pathological additions are:

1. Neutrophilic Myelocytes.— The common type is a large cell
with a large centrally placed, feebly staining nucleus. This may be
recognized by the difficulty of distinguishing the nucleus from the cyto-
plasm, there being no sharp line separating these parts of the cell. They
imperceptibly merge into one another. They differ from a large mono-
nuclear in that the cytoplasm is distinctly dotted with neutrophile
granules and that we cannot make out a distinct line of separation of a
slightly irregular or indented nucleus from the surrounding slightly
neutrophilic cytoplasm. Cornil has described a very large myelo.cyte
with eccentrically placed nucleus and neutrophilic granules.

Myelocytes are at times found with both basophilic and neutrophilic
granules, and may rarely be seen to have all three kinds of granules on a
single myelocyte, acidophile, basophile, and neutrophile.

2. Eosinophilic Myelocytes. — These can be distinguished from
normal eosinophiles by their possessing a single round nucleus, not
bilobed. These myelocytes may be as large as a normal eosinophile,
but frequently are no larger than a red cell.

The neutrophile myelocyte is characteristic of spleno-myelogenous
leukaemia, the eosinophile one ofmyelogenic leukaemia. The occurrence
of an occasional myelocyte is frequently noted in conditions having a
leukocytosis. In diphtheria their presence in numbers is of bad prog-
nostic import. Myelocytes are of diagnostic importance in metastases
of malignant tumors.

3. The Irritation Cell of Turck, or Plasma Cell,— This cell has a
faintly staining, eccentrically placed nucleus, and a dark opaque blue,
frequently vacuolated, cytoplasm. They are usually recorded as large

BLOOD PLATELETS I 95

mononuclears. Turck supposed them to appear in the circulation as
the result of bone-marrow irritation.

4. Myeloblasts. — These cells are found in myeloid leukaemia and
though often mistaken for lymphocytes or large mononuclears they are of
marrow origin. The nucleus stains more intensely than that of the
large mononuclear and the cytoplasm is more deeply blue stained than
that of the large lymphocyte. They also contain three or four nucleoli.

Pyronin methyl-green staining is best for demonstrating the nuclei.

5 . Pathological Large Lymphocytes. — These are as a rule much larger
than normal large lymphocytes and show poorer staining of both
nucleus and cytoplasm. The nuclei often show the appearance of
division into two or more lobes, thus showing the characteristics of
Rieder cells. They may be confused with large mononuclears but
are considered to be derived from the germinal centers of various lym-
phoid tissues. They are found in leukaemic and pseudo-leukaemic
conditions.

BLOOD PLATELETS.

These are normally present in blood in the number of about 350,000 per cubic
millimeter. They disintegrate very quickly after the blood is withdrawn. Wright
has demonstrated that they are pinched-off projections of giant cells of the bone
marrow. They consist only of protoplasm, no nuclear material. They do not
contain haemoglobin. In conditions where giant cells are less abundant, as in per-
nicious anaemia, the blood platelets are less abundant. In myelogenous leukaemia
they are very abundant. They vary in size from 2 to 5/f according as a larger or
smaller pseudopod of a giant cell has been broken off. Stained with Wright's stain,
they are more purplish than blue and show thread-like projections. They are often
mistaken for the protozoal causes of various diseases. Especially are they confused
with malarial parasites when lying on a red cell. The blood plate has no brick-
red chromatic material; it is purplish rather than blue, and has no pigment grains.
It is advisable to compare these isolated blood plates with the larger or smaller ag-
gregations scattered about the smears. In this way their true character is apparent.
In addition to blood platelets, which in fres.h blood can only be observed when a
fixative is used, we have other confusing bodies.

The haemokonia of Muller are small, highly refractile bodies showing active
oscillatory movement. They are supposed to be cast-off granules of eosinophiles
or other leukocytes, or possibly derived from nuclei. As this blood dust or haemo-
konia is found in a marked degree in lipaemia it may be that the particles are fat.
It is interesting that this lipaemia is absent after the taking of large quantities of
fat in cases with serious pancreatic trouble. The serum of a normal individual is
rather turbid after slight indulgence in butter. Pinched-off fragments of red cells
may also appear as possible protozoal bodies.

196 NORMAL AND PATHOLOGICAL BLOOD

LEUKOPENIA.

This is a term used to designate a reduction in the normal number
of leukocytes. A leukocyte count of 5000 would represent a slight
leukopenia; one of 2000, a marked leukopenia. In the later stages
of typhoid, and in acute miliary tuberculosis, we expect a moderate
leukopenia.

The leukopenia of typhoid is moderate and is often preceded in the first few
days by a moderate neutrophile leukocytosis. Later on we have a decided increase
in the lymphocytes. A marked diminution or absence of eosinophiles is so character-
istic that any increase in eosinophilic percentage negatives a diagnosis of typhoid.

Paratyphoid gives a similar blood picture.

Chronic alcoholism and chronic arsenic poisoning cause a reduction
in the number of the white cells. Pernicious anaemia shows a marked
leukopenia, as is also the case with Band's disease. Two tropical dis-
eases, kala-azar and dengue, show a marked leukopenia, the counts
often being below 2500. During the apyrexial period of malaria we
may have a white count of 5000, ^

It has recently been claimed that a leukopenia with a. coincident
marked reduction in the lymphocytes is characteristic of ifceasles and
that this occurs several days before the Koplik spots appear?*v

Kocher notes that in exophthalmic goiter the leukocyte count is considerably
diminished and that the polymorphonuclears are not much more than one-half the
usual percentage while the percentage of the lymphocytes is almost double the
normal.

X-ray treatment tends to destroy leukocytes in the exposed region, especially
polymorphonuclears. The small lymphocytes are least affected.

EOSINOPHILIA.

Where the eosinophiles are increased to 5%, we have a moderate
eosinophilia. In some cases of infection with intestinal parasites,
especially hook-worms, but also from other parasites, as round and
whip-worms, we may have an eosinophilia of 30 to 50%. In Guam,
among the natives, it is difficult to find an eosinophile count under 15%.
The eosinophilia tends to disappear when the anaemia becomes very
severe.

The eosinophilia of trichinosis is best known, and a combination of
this blood finding with fever and marked pains of mucles, would justify
the excision of a piece of muscle for examination for encysted embryos.

LEUCOCYTOSIS 197

In true asthma eosinophilia is marked, and its absence is of value in indi-
cating other causes for the condition. Certain skin diseases, especially
pemphigus, show eosinophilia.

Eczema and psoriasis are not apt to give more than 3 or 4% eosinophiles. A
rather high degree of eosinophilia is found in mycosis fungoides.
Scabies also gives an eosinophilia.

The proportion of eosinophiles in the blood of children is greater
than in that of adults.

Increase of both eosinophiles and mast cells is found in myelogenous
leukaemia.

LEUKOCYTOSIS.

It is to an increase in the polymorphonuclears that this term is
usually applied, the 'term lymphocytosis or eosinophilia being employed
where ^vvhite cells of eosinophile or lymphocyte nature are increased.
We have physiological leukocytosis in the latter weeks of pregnancy,
also in the new-born, and in connection with digestion.

Pathological Leukocytosis.— Pneumonia. In this disease we have a
leukocytosis of 20,000 to 30,000 or higher. The eosinophiles are almost
absent. A normal leukocyte count in pneumonia makes a prognosis
unfavorable.

The leukocyte count drops about the time of the crisis, and with
the reappearance of eosinophiles is a favorable sign. A moderate
leukocytosis occurs in carcinoma and sarcoma.

Septic processes. The leukocyte count is of great value, especially
when we obtain a leukocytosis with 80 to 90% of polymorphonuclears,
as in appendicitis, cholecystitis, or other suppurative conditions.

According to Cabot, leukocytosis varies in infections as follows:

1. Severe infection — good resistance; early, marked and persistent leuko-
cytosis.

2. Slight infection — slight resistance; leukocytosis present, but not
marked.

3. In fulminating infections we may have no increase in whites, but a
higher percentage of polymorphonuclears.

4. Slight infection and good resistance may not be productive of leuko-
cytosis.

It is in connection with the question of operation in appendicitis
or similar conditions that the matter of a leukocyte count is of prime
importance. If there be a leukocytosis but with less than 75% of

198 NORMAL AND PATHOLOGICAL BLOOD

polymorphonuclears it indicates an infection of little virulence or a
walled-off process with an exacerbation. It is difficult to form an opin-
ion when the polymorphonuclears are under 80%. Leukocytosis with
polymorphonuclear percentage of 85 to 90 indicates immediate opera-
tion; percentages over 90 point to peritonitis and if with such per-
centages of polymorphonuclears there is absence of leukocytosis the
prognosis is grave.

Spirochaeta fevers, as relapsing fever, may give a leukocytosis of
from 25,000 to 50,000.

Smallpox, especially at time of pustulation, plague, scarlet fever,
and liver abscess give a leukocytosis of from 12,000 to 15,000.

Smallpox often shows a very large percentage of very characteristic large
mononuclears.

FIG. 54. — Leukocytosis (40,000); sixteen polymorphonuclears in field. (Cabot.}

The leukopenia and lymphocyte increase in measles are important points in
differentiating it from scarlatina.

With meningitis counts of 25,000 are not unusual, in abscess of the
brain the white count rarely exceeds 15,000.

Poliomyelitis and polioencephalitis give a slight leukocytosis during
the febrile accession.

Erysipelas and epidemic cerebrospinal meningitis also give a leukocytosis of
from 15,000 to 20,000. In malignant diseases we sometimes have a moderate
leukocytosis. Rogers states that in liver abscess, with a leukocytosis of 15,000 to
20,000, we have only about 75 to 77% of polymorphonuclears — there being also
a moderate increase in the percentage of large mononuclears.

Drugs such as antipyrin may give a leukocytosis. The leukocyte increase of
pilocarpine is rather a lymphocytosis.

THE PRIMARY ANAEMIAS 199

LYMPHOCYTOSIS.

Of course, the disease in which we have the most marked lymphocy-
tosis is lymphatic leukaemia.

The lymphocytosis of typhoid fever has been taken up under leuko-
penia.

Whooping-cough may give a lymphocytosis of 20,000 to 30,000.

Young children have normally an excessive proportion of lymphocytes. This
is apt to be particularly marked in hereditary syphilis. Enlarged tonsils may
give rise to a lymphocytosis of 10,000 to 15,000, when more than 50% of the white
cells will be lymphocytes. Rickets and scurvy give a lymphocytosis.

Varicella and mumps may also give a a increase in the percentage of
lymphocytes.

Malta fever is a disease which may show quite a mononuclear increase.

DISEASES IN WHICH THERE is A NORMAL LEUKOCYTE COUNT.

Uncomplicated tuberculosis, influenza, Malta fever, measles, try-
panosomiasis, malaria, syphilis, and chlorosis. In malaria we have
a leukocytosis at the time of the rigor, while during the apyrexial period
there is a moderate leukopenia. In malaria we have a marked increase
in the percentage of the large mononuclears and transitionals. These
may form from 25 % to 35 % of the leukocytes. When beaiing particles
of pigment they are known as melaniferous leukocytes — macrophages
which have ingested malarial material. In dengue, at the time of the
terminal rash, we may have as great a percentage of large mononuclears.
In this disease, however, we have a great diminution of polymorphonu-
clears from the start (25 to 40%). Instead of a large mononuclear we
have at the onset a lymphocytic increase. There is an increase of large
mononuclears in trypanosomiasis.

The white count is about normal in uncinariasis (Ashford's average
was 7800). Some have reported a leukopenia in severe cases.

While eosinophilia is the most marked feature in hook-worm disease
yet in very severe cases it may be absent.

THE PRIMARY ANAEMIAS.

Chlorosis. — In chlorosis it is the reduction of haemoglobin with the
slight numerical variation from normal of the red cells that makes for
a diagnosis. The color index is very low. There is nothing abnormal

200 NORMAL AND PATHOLOGICAL BLOOD

about the leukocytes. Microcytes may be present, and very occasion-
ally a normoblast. Macrocytes and megaloblasts are always absent.
Blood of chlorotics is very pale and very fluid and coagulates rapidly,
hence frequency of thrombosis.

Spleen, liver, and lymph glands as a rule normal.

Simple Primary Anaemia. — This condition is not recognized by many authors,
but is a convenient term under which to group anaemias which are neither chlorosis
nor pernicious anaemia and for which no assignable cause can be designated. It
is a secondary anaemia without a cause. In it color index is about normal, there
is no change in the leukocytes and cases go on to recovery.

Pernicious Anaemia. — In pernicious anaemia we obtain a very
fluid, but normally colored drop of blood upon puncture. The yellow

FIG. 55. — Pernicious anaemia. M.m, Megaloblasts; n, normoblast; s, stippling
(punctate basophilia). (Cabot.)

marrow of the long bones is transformed into a soft, bright red lymphoid
tissue, smears from which show great numbers of megaloblasts. Areas
of fatty degeneration are characteristic, especially the tiger-lily spots
in the heart muscle. Iron-containing pigment (hemosiderin) is found
in the liver, spleen, and kidneys. Areas of degeneration in the spinal
cord may account for nervous symptoms. The red cells frequently
fall below 2,000,000 with patients going about. Cases have been re-
ported with counts under 200,000. The color index is high. Megalo-
blasts are the most characteristic qualitative change in the red cells.
Megaloblastic crises may at certain times show enormous numbers
of megaloblasts. Cases often present remissions in which no megalo-
blasts can be found. In such cases the presence of many macrocytes

SECONDARY ANAEMIAS 2OI

should prevent an examiner's reporting against a pernicious anaemia
previously diagnosed.

Poikilocytosis, polychromatophilia, and stippling are also features
of the disease. Normoblasts are far less frequent than megaloblasts
and there is usually a moderate lymphocytosis. Myelocytes may be
present, but their precursors, the myeloblasts, are probably more fre-
quently met with.

Cases of pernicious anaemia show remissions during which the patient is ap-
parently on the road to recovery. Such improvements are only temporary. The
remissions may last from two months to possibly three or four years. Especially
in the anaemia of Dibothriocephalus latus do we have a picture of pernicious anae-
mia. It is supposed to be due to a toxin present in the heads of these tape-worms.

Blood changes more or less like those of pernicious anaemia have at times been
noted in children with tuberculosis of bovine nature. The human strain of T.B.
does not seem to produce such changes.

An acute disease showing a rapidly developing anaemia of the pernicious anaemia
type is verruga peruana in which the bone, marrow seems especially involved.

SECONDARY ANAEMIAS.

These are the anaemias which can be definitely traced to some dis-
ease not of the haemopoietic system.

There are two main groups — those following haemorrhage and those
secondary to various diseases. If the haemorrhage is sudden and great,
the resulting condition is one of oligochromaemia — chlorotic in type.
Normoblasts are usually found after the third day.

The low Hb. percentage is apt to continue for several weeks. There is also an
increase in the percentage of polymorphonuclears.

It is a question whether prolonged operation or those requiring narcosis are
justified where the reduction in Hb. is under 40%. (According to Miculicz, 30%
is the minimum).

Where the loss of blood is gradual, as in gastric cancer or severe haemorrhoids
the picture may more nearly approach that of pernicious anaemia. Secondary
anaemias usually show a moderate leukocytosis. In chronic nephritis and prolonged
suppurative conditions normoblasts and macrocytes are rare — moderate poikilo-
cytosis with the presence of many microcytes being the rule.

In fatal anaemia from chronic acetanilide poisoning high color index, macrocytes
and megaloblasts have been noted.

In some secondary anaemias, as in syphilis, carcinoma, and tuber-
culosis, we have a chlorotic color index (chloro-anaemias).

In secondary anaemias polychromatophilia, poikilocytosis, and punc-
tate basophilia (stippling )may be present. This latter is very marked

202 NORMAL AND PATHOLOGICAL BLOOD

in lead poisoning, but in certain cases of malarial cachexia it may be
equally prominent. The only form of nucleated red cell seen is the
normoblast, in very small numbers, or it may not be present.

Megaloblasts are practically never seen, except in some of the very severe para-
sitic anaemias, as the broad Russian tape-worm infection. The red cells generally
number between 2,000,000 and 4,000,000, thus differentiating chlorosis. The
leukocytes are frequently increased to 15,000. In the anaemia of splenic anaemia
there is a marked leukopenia. In anaemias from malignant tumors the color index
is usually of the chlorotic type — the haemoglobin content of the red cells being
more affected than the number. Normoblasts are usually present, and this find-
ing may differentiate gastric cancer from ulcer. In bone marrow metastases
megaloblasts may be expected. Myelocytes and so-called tumor cells (large cells
with faintly-staining vacuolated nuclei and frut little cytoplasm) may also be found.
As a rule, there is a moderate leukocytosis in malignant disease. Eosinophiles
may be largely increased in sarcoma.

THE LEUKAEMIAS.

It is in the leukaemias that we have the greatest increase in the num-
ber of white cells. These cases show more or less anaemia, but we may
have cases of myelogenous leukaemia showing 250,000 leukocytes per
cubic millimeter without particular change in the red cells. The 'more
marked the red-cell change the more severe the condition.

There are two well-defined types of leukaemia, the lymphatic and the
splenomyelogenous. It must be borne in mind, however, that while
a greater change in the lymphatic glands may produce the lymphatic
type, yet even in such cases we expect to find alteration in bone marrow
and spleen; that is, there is a general involvement of the haemopoietic
system in all leukaemias, the activity being most marked in spleen and
bone marrow in certain cases and in lymphatic glands in others.

Myelogenous leukaemia is a very rare disease, about five times as
rare as pernicious anaemia. Lymphoid leukaemia is still more rare.

Splenomyelogenous Leukaemia (myeloid leukaemia). — The differen-
tiation of the blood picture of this disease from leukocytosis does not
depend on the number of leukocytes, but on the presence and large
proportion of myelocytes. We expect both neutrophilic and eosino-
philic myelocytes in myeloid leukaemia — the proportion of these varies,
but, as a rule, the neutrophilic one is the common one. The blood in
advanced cases is milky and shows a most marked buffy coat. The
marrow is largely replaced by a yellow pyoid material. The spleen
may weigh 10 pounds.

THE LEUKAEMIAS

203

The leukocyte count is on the average from 200,000 to 500,000. Cases are
reported of more than 1,000,000 white cells. The neutrophilic myelocytes make
up about 30 to 40% of these and, about equal in number, are found the polymor-
phonuclears, while the percentage of the lymphocytes is decreased (2 to 5%) and
normal eosinophiles, eosinophilic myelocytes, and large mononuclears make up the
remaining percentages. We usually have great numbers of normoblasts. Megal-
oblasts may be rarely found. The red count is usually about 2,500,000 and the
color index low.

Lymphatic Leukaemia. — -In this we have glandular enlargements,
but not such large masses as in Hodgkin's disease. The red cells are
usually reduced about one-half and the color index is a little below
normal. Normoblasts are rarely found. Myelocytes, as a rule, are

FIG. 56. — Myelogenous leukaemia, m, Myelocyte; p, polymorphonuclear; b, mast
cell; n, normoblast. (Cabot.)

absent, but may amount to 5% of the leukocytes. The predominating
leukocyte (75 to 98%) is the small lymphocyte. In acute lymphatic
leukaemia the large lymphocytes predominate.

These however are pathological and differ from the large lymphocyte in not
having azur granules and the nucleus stains poorly and is often indented. The
leukocyte count is never so great as in myeloid leukaemia, rarely exceeding 125,000.

Pseudoleukaemia.— Hodgkin's disease is usually considered as a
disease with marked glandular enlargements, but with a negative blood
picture, or at any rate only a moderate leukocytosis with a relative
increase of lymphocytes.

The red cells are usually above 3,000,000. It has been considered
that an increased percentage of transitionals (10 to 15%), should a
leukopenia coexist, is^characteristic.

204 NORMAL AND PATHOLOGICAL BLOOD

Undoubtedly the view that so-called lymphosarcomata, lymphatic
leukaemia, and Hodgkin's disease merge into one another and that they
represent a malignant cell formation in the haemopoietic system is the
conservative one to take.

A certain proportion of cases of Hodgkin's disease, however, show
endothelial proliferation and a chronic fibroid change.

In Kundrat's lymphosarcoma we have a neutrophile leukocytosis
and a diminution of the lymphocytes. The spleen and liver are rarely
involved.

Another condition with swelling of the lymphatic glands, which do not how-
ever fuse, is the so-called granulomatosis.

In this we have a polymorphonuclear leukocytosis of from 20,000 to 50,000 with

>•

FIG. 57. — Lymphatic leukaemia, p, polymorphonuclear; m, megaloblast; e, eosino-
phile. Twenty-one lymphocytes in this field. (Cabot.)

an increase in the percentage of eosinophiles. The lymphocytes are absolutely
and relatively decreased. In granulomatosis there is no tendency to haemorrhage.

Splenomegaly.— The best known anaemia associated with splenic
enlargement is Banti's disease.

Banti's disease also has a very low color index and leukopenia. In
this the primary affection is of the spleen which becomes greatly en-
larged. The accompanying cirrhosis of the liver with its symptoms of
ascites, etc., differentiate it. Splenectomy often cures the disease.
The leukopenia is one showing not only a diminution of polymorphonu-
clear percentage but of cells of the lymphocyte type as well.

There is a considerable increase in the large mononuclear percentage. Nu-
cleated reds and myelocytes are invariably absent. It must be remembered that

SPLENIC ANAEMIA 205

we have a group of cases showing splenomegaly which are syphilitic in origin and
which as a rule give a positive Wassermann. Clinically or haematologically they
resemble true Band's disease but pathologically the spleen shows a fibrosis instead
of the marked increase in lymphatic tissue characteristic of Band's disease.

In the tropical splenomegaly or kala azar we have a marked leukopenia with
a marked reduction in the percentage of poly morphonu clears. The Gaucher type
of splenic anaemia does not show as pronounced and early an anaemia as in Band's
type.

Certain conditions which partly resemble myelogenous leukaemia
and partly pernicious anaemia are designated leukanaemia. Some con-
sider this to belong to the group of diseases in which the multiple mye-
loma is placed.

In splenomegalic polycythaemia we have a red count of from 9 to
10 millions. The Hb. percentage may be 200. There is also a
leukocytosis up to 50,000. Patients are cyanosed and have a very large
spleen.

Splenic anaemia of infancy usually occurs between the ages of twelve
and twenty-four months. The spleen is notably enlarged and in many
cases the liver is equally so. The red cells are not greatly diminished
in number, two and one-half to three millions being usual findings.
Nucleated reds are abundant. While a leukocytosis of 30,000 to 50,000
is often present it is markedly less than that of splenomyelogenous
leukaemia and the increase in white cells is more of those of lymphocyte
type.

The color index is very low.

Another splenomegaly of children, clinically resembling kala azar, is caused
by Leishmania infantum.

NOTES ON BLOOD WORK.

NOTES ON BLOOD WORK.

NOTES ON BLOOD WORK.

NOTES ON BLOOD WORK,

NOTES ON BLOOD WORK,

PART 111.
ANIMAL PARASITOLOGY.

CHAPTER XV.

GENERAL CONSIDERATIONS OF CLASSIFICATION AND
METHODS.

ANIMALS that are in all respects alike we term a Species. Of course
the male and female of a species may be very unlike, but as a result of
mating they produce young having characteristics similar to the parents.
Now, if, as in the case of the mosquitoes causing yellow fever, we find
some with straight silvery lines and others uniformly showing crescentic
silveiy bands about thorax, yet resembling each other closely in the
respect of being dark, brilliantly marked mosquitoes, we should con-
sider them as being separate species with a certain relationship to which
the term Genus is applied.

The term "genus" is of wider application than the word " species."
Thus animals which agree in the main characteristics of size, proportion
of parts, and general structure are placed in the same genus.

In naming a species we always first write the name of the genus
which has a Greek or Latin name, commencing with a capital, and follow
with the specific term, which latter commences with a small letter.
Thus we designate the dark silver-marked mosquitoes as belonging to
the genus Stegomyia; those showing the characteristics of curved silver
bands and two central parallel lines (lyre pattern) on dorsal surface of
thorax we designate as Stegomyia calopus; the species with only the
straight silver lines we call Stegomyia scutellaris.

If the specific name is a modern patronymic we add i in the case of a man or
ae for a woman to the exact and complete name of the person.

Again, certain genera show resemblances which enable us to make broader
groupings to which we apply the term Subfamily. Thus the genus Stegomyia
and the genus Culex have the similar characteristics of palpi in the female being
shorter than the straight proboscis; we therefore classify all species of Stegomyia

211

212 CONSIDERATIONS OF CLASSIFICATION AND METHODS

and all species of Culex under the designation Culicinae. The name of a subfamily
ends in "inse." Now, again, certain insects are different from others in having
scales on the wings. We find that not only do the Culicinae have such character-
istics; but the same is observed with the Anophelinae and other similar scale-wing
insects. All of these we term a Family and we speak of the Culicidae, meaning
the family of mosquitoes. The name of a family ends in "idae." Many families
are not subdivided into subfamilies, but are directly separated into genera. Again,
a genus may have only a single species.

At times a family may be raised to superfamily rank — the subfamilies then
becoming families. Thus the families Ixodidae and Argasidae belong to the super-
family Ixodoidea. The termination for a superfamily is oidea.

When there are a number of families agreeing closely in some striking character-
istic, we group them together into an Order; thus, the family of mosquitoes closely
resembling many other families of insects in possessing a pair of well-developed
wings are grouped in the order Diptera; all of which resemble certain other animals
in the possession of a distinct head, thorax and abdomen with three pairs of legs
projecting from the thorax. This collection of animals we call a Class; thus, we
speak of the class Insecta. It will be observed that the insects have no internal
skeleton, but instead a chitinous cuticle, the exoskeleton. Spiders, ticks, etc.,
resemble them in this respect, and we now apply to all such animals the wider
designation, Branch or Phylum Arthropoda.

Inasmuch as the animal kingdom is divided into the branches Protozoa, Pori-
fera, Ccelenterata, Echinodermata, Vermes, Arthropoda, Mollusca and Chordata,
we see that the branch is the largest grouping we employ. To descend in the scale
we have belonging to the branch, the classes; to the class, the orders; to the order,
the families; to the family, the subfamilies; to the subfamily, the genera; to the
genus, the species. Occasionally a species is further divided into subspecies.

By a type species we understand the species of a genus always re-
ferred to as representing the genus.

While other species of a genus may for good reason be transferred to another
genus the type species is permanently in the genus. Many favor alliteration for
type species, as Heterophyes heterophyes. When a species is transferred to a new
genus the specific name goes with it.

The male animal is designated by the sign of Mars (cf ), the female by that of
Venus (9).

There are certain terms employed in animal parasitology which it is necessary
to understand. Among these we shall refer to the following:

1. True Parasitism. — By this is understood the condition where the parasite
does harm to the host, deriving all the benefit of the association. A good example
of this would be the hookworm infecting man or animals.

2. Mutualism. — In such an association there is mutual benefit to each party
of the association. An instance of this would be the presence of colon bacilli in
the intestines. The bacillus is furnished a suitable habitat and in return protects
its hDst against strictly pathogenic bacteria.

Another example would be the oyster crab found inside the oyster shell.

ZOOLOGICAL NOMENCLATURE 213

3. Commensalism. — Here there is benefit to the parasite, but no injury to the
host. An example of this kind would be furnished in the case of the Trichomonas
vaginalis which lives in the vaginal mucus, but so far as known, does no injury to
the host.

If the Entamceba coli be nonpathogenic this would be another example.

4. Nomenclature.— When the thousands of different species, genera,
etc., of animals is considered, it will be readily perceived that, unless
some system existed for theii designation, indescribable confvsion
would prevail. To avoid this, the International Code, based on the
rules of Linnaeus (tenth edition of Systema naturae, 1758, is basis of
binary zoological nomenclature), requires Latin or Latinized names.

In printed matter the zoological name should be in italics, that of the family
in Roman type. The name of the author of a specific name is written immediately
after the name without punctuation and may be followed by the year of publica-
tion set off by a comma, thus: Ascaris lumbricoides Linnaeus, 1758. Should the
name of the author appear in parentheses it indicates that he proposed the specific
name but placed the species in another genus than that in which it now appears,
and the name of the author responsible for placing the species in the present genus
may be written after the name of the original author of the species; for example,
Davainea madagascariensis (Davaine, 1869) Blanchard, 1891, tells us that Davaine
proposed the specific name madagascariensis in 1869 but placed it in some other
genus and that Blanchard in 1891 transferred it to the genus Davainea. There
are certain rules governing the naming of animals. Of these, the law of priority
provides that the oldest published name, under the code, of any genus or species
is its proper zoological name. The history of the naming of the organism of syphilis
illustrates this well. Schaudinn gave this organism in 1905 the name of Spiro-
chaeta pallida. Ehrenburg, in 1838, had used the name Spirochaeta for animals
of a different character, so that this designation of the genus was not permissible
under the code. Villemin, a little later, proposed the generic name Spironema.
This term, however, was found to have been used in 1864 by Meek for a genus of
molluscs and by Klebs in 1892 for a genus of flagellates. Consequently, being a
homonym, it was not available.

(A generic name can be applied to only one animal genus and if a similar name
is subsequently given another genus it is a homonym and is to be rejected.)

On December 2, 1905 Stiles and Pfender then proposed the name Microspiro-
nema, but as Schaudinn published on Oct. 26, 1905 the designation Treponema,
the name Treponema pallidum had to be accepted as the proper zoological name
for the organism of syphilis.

Of unusual interest is the question of the name of the old-world hookworm.
Dubini, in 1843, named a nematode found by him in man Agchylostoma. By the
law of priority this spelling would have been the correct one had he not stated in
a footnote that the generic name was derived from two Greek words a-f-yvkoo and
orofia. Having indicated the origin of the name it became subject to the rules
for correct transliteration, which is Ancylostoma.

214 CONSIDERATIONS OF CLASSIFICATION AND METHODS

In case of larva and adult or male and female, formerly considered different
animals but subsequently found to be the same, the oldest available name becomes
the name of the species.

Another point is that names are not definitions, consequently the fact of lack
of appropriateness of any name is no objection to its continuation. This will ap-
peal to anyone as a wise provision, because if a different name were substituted
each time a designation more descriptive or applicable was invented it would be
utterly destructive to system. When it is considered that some of our parasites
have approximately fifty different designations, for the most part given by med-
ical observers, it will be appreciated how much the zoologist has aided us in trying
to eliminate all but the single proper zoological name.

The objections so frequently heard among physicians in connection with adopt-
ing new names for old ones are not well founded. Wherever confusion has reigned,
the establishment of order always results in temporary greater confusion. There
is no doubt that the student taking up this subject a few years hence will have the
satisfaction, thanks to the zoologist, of only having to burden his mind with one
name for one parasite.

There is only one correct name for an animal and all other names
are synonyms.

The principal cause of changes of names is that our conception of the relation-
ships of animals changes.

5. Terminology. — This applies to appropriate designations for different organs,
symptoms, etc., and is not subject to any rule other than that of good usage.

Thus the terms cirrus in the case of the male copulatory organ of flukes, spicule
for the same in nematodes and penis in connection with insects would be instances
of terminology.

6. Pseudoparasitism. — Where organisms enter the body accidentally and when
such sojourn in the body of man plays no part in the life history of the organism
we employ the term pseudoparasitism. For example: Fly larvae swallowed by
man and passed out in the faeces. We also use the terms temporary parasites
(bedbug) and permanent parasites (liver fluke).

7. Hosts. — The animal in which a parasite undergoes its sexual life is called the
definitive or final host, that in which it passes its larval existence the intermediary
host. For example: Man is the intermediary host of the malarial parasite, the
mosquito the definitive host. A single animal may, however, be both definitive
and intermediary host; thus Trichinella may pass its larval existence in the muscles
of man and its sexual life in his intestines.

8. Heredity, Congenitalism. — Hereditary characteristics are those which were
present in the ovum or spermatozoon before fertilization; congenital ones those
which occur after fertilization. South African tick fever is probably an instance
of heredity, the spirochaetes having been found in the ovary and ova of the female
tick.

9. Heterogenesis, Parthenogenesis. — Offspring differs from parent, but after
one or more generations there is reversion to the parent form.

Strictly speaking the term heterogony applies to reproduction when a sexual

PARTHENOGENESIS 215

generation alternates with a parthenogenetic one. Where a nonsexual genera-
tion, as by division or budding, alternates with a sexual one the process is called
metagenesis. In parthenogenesis reproduction eggs develop without the occurrence
of fertilization by spermatozoa.

In coccidiosis we have a sexual cycle (sporogony) alternating with a nonsexual
one (schizogony). In the infection with Strongyloides we have a sexual cycle
alternating with a parthenogenetic one. In malaria we have a sexual generation,
a nonsexual one and according to Schaudinn, a parthenogenetic one, which latter
accounts for malarial relapses.

CHAPTER XVI.

THE PROTOZOA.

CLASSIFICATION OF PROTOZOA.

Class

Order

Rhizopoda Gymnamoeba

(Sarcodina)

These throw out protoplas-
mic projections called pseudo-
podia.

Flagellata

(Mastigophora)
These move by means of
undulating membranes or
flagella.

Infusoria Heterotricha

(Ciliata)

These have contractile vacu-
oles and numerous fine cilia
which are shorter than flagella
and have a sweeping stroke.

Sporozoa

These have no motile organs.
They live parasitically in the
cells or tissues of otlrer animals.
Reproduction by spores.

Genus

Entamoeba

Leydenia

Species
E. coli

E. histolytica
E. tetragena
E. buccalis

L. gemmipara

(S. recurrentis

S. vincenti

S. duttoni

S. carteri

S. refringens

Schizotrypanum S. cruzi

Treponema

' T. pallidum
T. pertenue

Trypanosoma

T. gambiense
T. rhodesiense

Trichomonas

T. vaginalis
T. intestinalis

Lamblia L- intestinalis

Babesia B. bigemina

{L. donovani

L. tropica

L. infantum

Balantidium B. coli

Coccidiaria

Eimeria
Isospora

Haemosporidia Plasmodium

E. stiedae
I. bigemina

P. vivax
P. malarise
P. falciparum

2l6

BINUCLEATA 217

NOTE. — Hartmann and others have grouped the Haemosporozoa and the Haemo-
flagellata in an order BINUCLEATA. The main characteristic is the possession of
two differentiated nuclei, the kinetonucleus and the trophonucleus, at some develop-
mental or transitional stage. While trypanosomes plainly show these characteris-
tics certain others, as the malarial parasites and the leishman-donovan bodies, hav-
ing been modified as the result of cell parasitism, do not do so. This grouping to-
gether of the blood flagellates and sporozoa under the name Binucleata has been con-
sidered by many protozoologists as possibly convenient but not resting on sufficient
ground to cause organisms with similar life histories as Plasmodium and Coccidium
to be separated and the former to be placed with the blood flagellates in a new
grouping.

THE PROTOZOA.

By the term protozoa we understand a branch of animals in which a
single cell is morphologically and functionally complete; it is not one of
a number of cells going to make up a complex individual and dependent
on such a combination as is the case with the metazoa (there is no
differentiation into tissues in protozoa).

Recognizing the fact that certain protozoa have characteristics which make
it impossible to draw a distinction between them and plants Haeckel has proposed
the name Protista as a designation for all simple and primitive living organisms
whether they be plants or animals. In such a classification we would have the
kingdom of Protista as well as the animal and vegetable kingdoms. In such a
grouping the bacteria would be the lower types and the fungi and protozoal organ-
isms the higher ones.

The protozoal cells are made up of protoplasm which is divided into nucleus
and cytoplasm. The cytoplasm is at times separated into an external, hyaline
portion, the ectoplasm or ectosarc and an internal granular portion, the endo-
plasm or endosarc. The functions of the ectosarc are protective, locomotor, ex-
cretory and sensory; those of the endosarc trophic and reproductive. Protozoa
may be holozoic (animal like) or holophytic (plant like), saprophytic (fungus
like), or parasitic (living at the expense of some other animal or plant).

The nucleus is characterized by concentration of the so-called chromatin sub-
stance of the cell. This chromatin however is usually combined with achromatin.
The usually accepted test for chromatin is the staining affinity for basic aniline
dyes. This test is now known to be unsatisfactory as other substances than* chro-
matin may stain even more intensely. When chromatin is scattered through the
cytoplasm, as extranuclear aggregations, such chromatin granules are called chro-
midia. There are cells where the chromidia take the place of the nucleus and from
which a nucleus may be formed. Chromidia may arise from nuclei and nuclei
from chromidia. The nucleus is made up of a network of linin in which achro-
matic reticulum is contained the nuclear sap or karyolymph. As a rule an achro-
matic nuclear membrane, continuous with the reticulum, separates the nucleus
from the cytoplasm. In addition we have a substance which is achromatic (plas-
tin) and which is the imbedding substance for chromatin grains. These plastin
chromatin combinations are called karyosomes. The nucleoli are probably pure
plastin. Plastin is to be regarded as a secretion or modification of chromatin

2l8 THE PROTOZOA

made to serve as a matrix for the chromatin. Chromatin may be concentrated
in a single mass so that the nuclear space looks like a vesicle with a central chroma-
tin mass (vesicular nucleus) or numerous chromatin grains may be scattered through
the nuclear space (granular nucleus). The centrosome, which presides over cell
division, is usually located just outside the nucleus. In some protozoa however
the centrosome is within the nucleus and is often seen inside of a karyosome and is
then called a centriole. The centrosome may also function over kinetic activities
(flagellar motion) and is then termed blepharoplast.

Certain protozoa, as trypanosomes, show a differentiation of nuclei, the larger
trophonucleus governing the functions of general metabolism and the smaller kine-
tonucleus directing the motor activities. Infusoria have a larger macronucleus
which contains vegetative chromatin and a smaller micronucleus which contains
reserve reproductive chromatin.

Reproduction of protozoa may be by fission, when the nucleus and cytoplasm
divide into two by simple division.

When the nuclei divide into a number of daughter nuclei, which is followed by
multiple division of the cytoplasm, we have sporulation.

Instead of fission we may have sexual reproduction or conjugation (zygosis).
Here the nuclei of the separate sexual individuals (gametes) are termed pronuclei
and the product of their fusion a synkaryon.

Where a single cell has division of its nucleus with subsequent fusion of these
daughter nuclei to form a synkaryon the process is termed autogamy.

If two similar cells conjugate the term is isogamy; if dissimilar as the macro-
gametes and microgametes of malaria, anisogamy.

The process of sexual union is termed syngamy and is of two kinds (i) when
the two gametes fuse completely or copulation and (2) when they remain separate
and only exchange nuclear material or conjugation.

The structures of protozoa concerned in movement, metabolism, etc., are termed
organelles. Of the former, pseudopodia, flagella, cilia and myonemes (contractile
fibrils which give support to the body cell of certain protozoa) may be given and
food vacuoles and contractile vacuoles of the latter. The contractile vacuole
which is probably an excretory organelle is absent in almost all parasitic protozoa.
It is however present in ciliates.

RHIZOPODA (SARCODINA).

In this class of protozoa the pseudopodia serve the double purpose
of nutrition and locomotion. These protoplasmic extensions may be
quite broad or very narrow — the lobose and the reticulose.

As a rule, the thicker the pseudopod the more rapid the movement.
Some rhizopods have hard shell-like coverings which are secreted in or
on the ectosarc. These skeletons have openings through which the
pseudopods project. The pseudopodia may be made up only of ecto-
plasm or both ectoplasm and endoplasm may take part. Amoeboid
movement always starts in the ectoplasm. In addition to the nucleus,

THE AMCEB^E OF MAN

2IQ

which the so-called chromatin-staining methods bring out as reddish
areas, we frequently observe smaller aggregations of chromatin-staining
material in the cytoplasm. This extranuclear chromatin is supposed
to play a part in .the more intricate divisions which such protozoa un-
dergo. Food vacuoles and contractile vacuoles are present in many
rhizopods.

Entamoeba coli (Amoeba coli). — This is considered by Schaudinn to
be a harmless inhabitant of the intestines and its presence in the faeces
is not considered of importance.

FIG. 58. — Various protozoa, i, Entamoeba coli; 2, Entamceba histolytica; 3,
Leydenia gemmipara; 4, Trichomonas vaginalis; 5, Trichomonas intestinalis; 6,
Lamblia intestinalis; 7, flagellated Leishmania donovani; 8, Leishmania donovani
in phagocyte; 9, Eimeria stiedae; 10, Isospora bigemina; n, Trypanosoma gambiense;
12, Balantidium coli.

It is now recognized that amoebae of man are not cultivable. When we obtain
cultures on the various nutrient poor agar plates, formerly so much used, we find
that the amoebae belong chiefly to water amoebae, in particular a Limax.

The only safe way in recognizing amoebae in stools is to note amoeboid movement.
The encysted amcebae, except by the experienced, can scarcely be differentiated
from many vegetable cells and especially from large phagocytic cells, of probable
endothelial origin. By the use of neutral red in very dilute solution the granular
endoplasm will be observed to take up the brick-red stain.

220 THE PROTOZOA

A method for bringing out the nuclear features is as follows: take a loopful of
2% acetic acid and a loopful of 2% formalin. Tinge the mixture to a rose color
with neutral red and then stir in a little saturated aqueous solution methyl green,
using a tooth pick which has been dipped into the methyl green.

In staining with iron haematoxylin or better with phosphotungstic haematoxylin
proper fixation is very important. Fix in 100 parts of sat. aq. sol. bichloride to
which is added 50 c.c. absolute alcohol and 5 drops glacial acetic acid. The stain
should be poured on the moist smear of faeces. The fixative should be heated to
60° C. and should only act for 10 to 20 seconds. Then place in cold sublimate
alcohol for 10 minutes wash in 70% alcohol colored to a rich port wine color with
iodine, then in 70% alcohol, then in water and then stain as preferred. Some like
a carmine stain.

E. coli varies greatly in size (8 to 40^). There is no well-marked distinction
between a granular interior and a more compact, hyaline exterior. The nucleus is
centrally situated, is distinct, and on staining with Wright's stain shows the chro-
matin coloration. The nucleus is rich in chromatin and with iron haematoxylin it
shows four chromatin aggregations lining the nuclear membrane. It is sluggishly
motile and is of a grayish-white color. When stained it does not show a distinction
between endoplasm and ectosarc. The infecting stage is an encysted form with
eight nuclei or spores.

Entamceba histolytica (Amoeba dysenteriae).— This is considered
the pathogenic amoeba. Schaudinn considered that it was by the
possession of its tough, tenacious glassy, and highly refractile ectoplasm
that it was able to bore its way into the submucosa of the large intestine
and bring about those gelatinous-like necroses, which, by undermining,
eventually result in dysenteric ulcerations.

It was also thought to be the species found in tropical liver abscess.
As described by Schaudinn, it has a marked differentiation between the
glassy ectoplasm and the granular endoplasm. The nucleus is indis-
tinct, eccentric, or even peripherally situated, and stains feebly.

The movement is more active and the color more greenish-yellow than E. coli.
Craig notes the characteristic staining of the E. histolytica, this being a dark blue
ectoplasm encircling a lighter blue endoplasm. In dividing, there is a process of
budding. These little spore-like bodies form at the periphery of the encysted amoeba
and are the infecting stage. Faeces should be examined as soon as possible after
the stool is passed in order that one may have the best opportunity to observe
movement. A particle of mucus pressed down with a cover-glass makes a satis-
factory preparation. If necessary to dilute, use blood-warm salt solution — not
plain water.

Amoebae were first described by Lambl in 1859. Found by Loesch in dysenteric
stools in 1875. Councilman and Lafleur in 1891 separated amoebae into pathogenic
and nonpathogenic strains.

Kartutis produced dysentery in cats by introducing dysenteric stools into the
rectum. Kruse and Pasquale produced dysentery with liver abscess pus which

AMCEB.E

221

was bacteriologically sterile. Shiga in 1898 separated the bacillary type of dysen-
tery from the amoebic one. Schaudinn, in 1903, stated that E. histolytica was the
pathogenic amoeba of man. Viereck found encysted amoebae in dysenteric stools
containing four nuclei. This amoeba is now believed to be the common pathogenic
amoeba of man and is named E. tetragena.

Entamceba tetragena. — This amoeba has a homogenous and highly
refractile ectoplasm with a nucleus richer in chromatin than E. his-
tolytica. It has a central karyosome which varies in size.

FIG. 59. — Human amoebae showing vegetative and encysted stages. Water
amoebae for comparison. (ia) Entamceba coli; (16) E. coli (encysted); (2) E.
japonica; (30) E. histolytica; (36) E. histolytica (encysted); (30) E. histolytica per-
ipheral buds; (40) E. tetragena; (46) E. tetragena (encysted); (50) water amoeba,
vegetative; (56) water amoeba, encysted.

In an iron haematoxylin preparation this karyosome shows a central spot or
centriole which niay fill up most ol the nuclear space but in such case is surrounded
by a clear zone with the karyosome ring outside. Hartmann found that some of
Schaudinn's specimens were E. tetragena and the belief is now growing that the life
history of a nucleus resolving into chromidia which collected at the periphery and
formed the peripheral infecting spores was an error in observation on the part of
Schaudinn and that the true life history of the pathogenic human amoeba is that of
E. tetragena. In such case E. tetragena and E. histolytica applying to the same

222 THE PROTOZOA

amoeba we must drop the name E. tetragena by reason of priority of E. histolytica.
Craig now takes this view.

Wenyon has recently produced dysentery in kittens by infecting them with
material containing the four spores of the encysted E. tetragena. He also produced
liver abscesses in one of the kittens experimentally infected with dysentery.

Entamoeba buccalis. — This has an ectoplasm similar to E. histolytica, but has a
centrally situated nucleus, the nucleus, however, is poor in chromatin.

Obtained from the mouths of persons with dental caries. It does not appear
to have pathogenic characteristics.

Castellani has reported an intestinal amoeba with an undulatory membrane.
He has given it the name of E. undulans.

Leydenia gemmipara. — It is a question whether these bodies were animal
parasites or simply body cells showing amoeboid movement. They were found
in the ascitic fluid of two cases of carcinomatosis. They varied in size from 3 to 36^.

FLAGELLATA (MASTIGOPHORA).

In this class of protozoa the adults have flagella for the purposes of
locomotion and the obtaining of food.

Some flagellates more or less resemble rhizopods in being amoeboid and in having
an ectoplasm and an endoplasm. The body is frequently covered by a cuticle
(periplast). Some flagellates have a definite mouth part, the cytostome, which leads
to a blind oesophagus; others absorb food directly through the body wall. In addi-
tion to flagella, some flagellates possess an undulating membrane. All flagellates
possess a nucleus and some have contractile vacuoles. The flagellum may arise
directly from the nucleus or from a small kinetic nucleus, the blepharoplast (micro-
nucleus or basal granule).

The most important flagellates of man are the haemoflagellates. Among these
we may include the blood spirochaetes and the organism of syphilis, which have
many resemblances to the spiral forms of bacteria, together with the three genera in
which protozoal characteristics are marked, namely, Leishmania, Trypanosoma
and Trypanoplasma. In addition we have flagellates in the intestinal canal and in
the vaginal secretion. Some authors place the genus Piroplasma with the flagellates
and there has been controversy concerning the nature of certain projections from
these bodies. It would seem preferable, however, to consider them under the
Sporozoa.

Spirochaeta.

The generic term Spirochseta is applied to flagellates having a spiral
shape, an undulating membrane, and no flagella. This genus is one
about which there are two views : one, that the members belong to the
bacteria; the other, that they are protozoa. The absence of demonstra-
ble nucleus and blepharoplast makes them apparently vegetable in nature
while the variations in thickness, the fact of transmission by an arthro-

RELAPSING FEVER 223

pod, and indications of a longitudinal, rather than a transverse division,
would indicate protozoal affinities.

It would seem from recent investigations that both methods occur — longitudinal
division occurring when there are few organisms in the blood and transverse at the
height of the infection.

Minchin has adopted the name Spiroschaudinnia, proposed by Sambon, for the
parasitic blood spirochaetes

S. recurrentis. — This is the organism of relapsing fever. It was formerly con-
sidered a bacterium and was termed the Spirochaeta obermeieri (discovered by
Obermeier in 1873).

It is present in the blood of persons suffering from the disease during the pyrexia.
During the apyrexia they are not found in the peripheral circulation. At this time
they are present in great numbers in the spleen where they are actively phagocytized.

FIG. 60. — Spirochaetae of relapsing fever from blood of a man. (Kolle and

Wassermann.}

The disease is supposed to be transmitted by bedbugs or lice. Monkeys are sus-
ceptible and, after passage of the organism through monkeys, rats can be infected.

S. duttoni.— This is the cause of South African tick fever or " tete-fever. " The
disease is similar to relapsing fever, but there are generally four or five febrile par-
oxysms with apyrexial intervals. The disease is readily transmitted to ordinary
laboratory animals, especially the rat.

A certain degree of immunity is conferred by an injection with a certain spiro-
chaete, but this does not hold for other species; thus, rats which have recovered
from S. recurrentis can be infected by S. duttoni and vice versa. The disease is
transmitted by the bite either of the adult or larval Ornithodoros moubata. Koch
found spirochaetes in the eggs of the ovaries of ticks which had fed on persons with
the disease. It is thus an instance of hereditary transmission.

Leishman, who believes in the protozoal nature of these organisms, has observed
clumps of chromatin granules in the Malpighian tubes and in the ovaries of infected
ticks, which granules he considers developmental stages. Material showing such

224 THE PROTOZOA

granules but no spirochaetes has brought about spirochaete infection in mice. He
considers that infection probably occurs through material voided from the Mal-
pighian tubes rather than through the medium of veneno-salivary secretions.

Other spirochaetes that have been considered as pathogenic for the type of re-
lapsing fever in India and that of America are the S. carteri and the S. novyi.

Nicolle has shown with relapsing fever of Algiers that the body louse can trans-
mit the infection by spirochaete containing material from the crushed louse being
rubbed into the wound made by the louse in biting. Eggs from an infected louse
hatch out infected young lice, thus showing the hereditary transmission. It is
now also considered that infection with South African relapsing fever by O. moubata
occurs by the rubbing in of spirochaete containing faeces into the wound made by
the bite of the tick. These, as with plague infection from the contaminated faeces
of the rat flea, are instances of the contamination mode of infection. Noguchi has
recently cultivated the various species of pathogenic human spirochaetes by employ-
ing a method similar to that used in cultivating the organism of syphilis. He noted
longitudinal division in his cultures.

S. vincenti. — This is a very delicate spiral-shaped organism which has been found
in conjunction with a fusiform bacillus in a throat inflammation, usually termed
Vincent's angina. ,

S. refringens. — This Spirochaeta is frequently associated with the Treponema
pallidum and is common in genital ulcerations. It is thicker, has less regular and
more flattened curves and stains more readily. By "dark ground illumination" it
is thicker, of a yellow tint instead of pure white, and moves in its entire length.

Treponema.

The genus Treponema has no undulating membrane and has a
flagellum at each end.

Treponema pallidum (Spirochaeta pallida). — -This is the cause of
syphilis. It is characterized by the very geometric regularity of the
spirals, which are deeply cut, and in focusing up and down continue in
focus (like a corkscrew). They require about thirty minutes to stain
distinctly with Giemsa's stain and the attenuated ends or flagella should
always be noted before reporting their presence.

Treponemata are found in the cellular areas surrounding the thickened blood-
vessels and in the coats of the larger arteries. To stain them in section Levaditi's
method is the best.

The India-ink method of Burri is highly recommended. Take one loopful of
secretion from a chancre and deposit it on one end of a slide. Surround this drop
with five loopfuls of distilled water and five loopfuls of Giinther and Wagner's ink.
Mix and make a smear as for blood. When dry examine with the oil immersion
objective and the treponemata will be found to stand out as white spirals against
a dark background. Treponemata often appear as if bent in the middle.

Harrison prefers collargol to India-ink. One part of collargol is put in a bottle
with 19 parts of water and well shaken. This shaking is repeated. One loopful

SYPHILIS

225

of the suspected serum and one loopful of the collargol suspension are mixed and
smeared out and examined as for the India-ink method.

T. pallidum has been cultivated anaerobically in horse serum by Schereschewsky.
The cultures contained other organisms. Muhlens, by growing anaerobically on
horse-serum agar (i to 3), claims to have obtained pure cultures. Animal inocu-
lations with this material were negative, however.

-AVERY-

FIG. 6 1. — Binucleata, (Haemoflagellata and Haemosporozoa). i. Schizo-
trypanum cruzi; (a) Merozoite just entering r.b.c.; (£) fully developed trypanosome
form in blood; (c) form found in intestine Conorhinus; (d) form in salivary
gland of Conorhinus; (e) merocyte from the schizogenous cycle in lungs. 2. Leish-
mania donovani; (a) Parasites from spleen smear, free and packed in phagocytic
cell; (b) and (c) flagellate forms from cultures. 3. Trypanosoma gambiense. 4.
Plasmodium vivax; (a) young schizont; (b) uninfected red cell; (c) red cell, punctate
basophilia; (d) merocyte; (e) macrogamete; (/) adult schizont. 5. Plasmodium
malariae; (a) half-grown schizont showing equatorial band; (b) macrogamete; (c)
merocyte; (d) young schizonts. 6. Plasmodium falciparum; (a) red cell showing
multiple infection; (b) young ring form; (c) crescent; (d) young schizont on per-
iphery of r.b.c. 7. (a) Treponema pallidum; (b) Spirochaeta refringens. 8. Trepon-
ema pertenue.

Noguchi has cultivated T. pallidum under strict anaerobic conditions in a medium
of ascitic fluid containing a piece of fresh sterile tissue, preferably placenta. The
growth is faintly hazy and does not have an offensive odor. Spirochaeta micro-
dentium shows similar morphology but the cultures have a foul odor. Sp. macro-
dentium is similar culturally but differs morphologically.

When cultures of T. pallidum, grown for one or more weeks in ascitic fluid agar
IS

226 THE PROTOZOA

and ascitic fluid are ground in a mortar, heated to 60° C. for one hour then, with the
final addition of i% trikresol, we have an emulsion called "luetin." This extract
produces an allergic reaction on the skin of certain syphilitics (Luetin reaction).
To carry out the test luetin is introduced intradermally at the insertion of the left
deltoid and a control emulsion of agar media injected in the right arm. A negative
result shows as an erythema without pain or papule formation. Positive reactions
show as papules vesicles or even pustules giving rise to discomfort for several days.
While the control side usually becomes normal in forty-eight hours yet in latent
and tertiary syphilis the control may show almost as marked a reaction. The term
" Umstimmung " is applied to this susceptibility to trauma of the skin of those having
tertiary syphilis. Some cases of parasyphilitic infections which are negative to the
Wassermann test give a positive luetin reaction.

Noguchi has recently demonstrated T. pallidum in all layers of the cerebral
cortex except the outermost one in 12 cases out of 70 cases of general paresis examined.

In diagnosis either use the dark ground illuminator or make a thin smear from
the sanious oozing after vigorous friction of the chancre with gauze, taking up this
blood-stained serum on the end of a slide and smearing the surface of a second slide
with the adhering material. It is in most cases more satisfactory to curet the lesion,
in this way obtaining material from the areas of the thickened arteries.

In the diagnosis of cerebrospinal syphilis we use, in addition to the Wassermann
test of the blood, (i) the Nonne-Apelt reaction in which about i c.c. of a saturated
aqueous solution of ammon. sulphate is added to an equal amount of cerebrospinal
fluid. If turbidity or rather opalescence appear immediately, or within three minutes,
the test is positive. (2) The counting of the lymphocytes in the cerebrospinal
fluid. A lymphocytosis occurs in cerebrospinal syphilis, tabes and general paresis.
(3) The Wassermann test, using the cerebrospinal fluid instead of blood-serum.

T. pertenue. — An organism of similar morphology was first reported
by Castellani as present in yaws. It is found in smears and sections as
with T. pallidum.

A point of distinction between these spirochaetes is that the T. pallidum is found
in abundance in sections from a chancre about the thickened arteries in the corium,
while in sections from a yaws nodule the T. pertenue is found chiefly in the region
of the interpapillary pegs of the Malpighian layer of the epidermis where they bound
the papillary layer of the corium.

T. pertenue has been cultivated in the same way as T. pallidum and Nichols has
infected rabbits by intratesticular injection. A disease of Guam known as gangosa
is possibly connected with a tertiary form of yaws. In persons who have had yaws
a positive Wassermann reaction seems to be given in a higher percentage than is
true for syphilis. Salvarsan is also more specific for yaws than for syphilis.

Trypanosoma.

The genus Trypanosoma has a more or less spindle-shaped body,
along one border of which runs an undulating membrane. There is one
flagellum bordering the membrane and projecting like a whip posteriorly.

SLEEPING SICKNESS 227

There is a nucleus (macronucleus) and a blepharoplast (micronucleus — •
centrosome), the latter being located anteriorly as a chromatin staining
dot or rod. From this blepharoplast the flagellum proceeds posteriorly
bordering the undulating membrane and projecting freely beyond the
posterior end. The nucleus is larger, nearer the posterior end, and does
not stain so intensely as the blepharoplast.

Some consider that the trypanosome developed from types with a single anterior
flagellum proceeding from a blepharoplast. The moving of the blepharoplast with
the flagellum to the other end would make the flagellar end the anterior end. This
controversy as to which is the anterior end is the cause of confusion.

T. gambiense. — This is the trypanosome causing human trypano-
somiasis, the latter stage of which is known as sleeping sickness. It is
from 17 to 2%/J. long, and from 1.5 to 2/* wide. Blepharoplast oval.

It was first discovered in smears from blood by Ford in 1901, and recognized as
a trypanosome by Dutton in 1902, and observed in 1903 by Castellani in the cere-
brospinal fluid of patients with sleeping sickness. It is now proposed to consider
cases where trypanosomes are not present in the cerebrospinal fluid as in the first
stage; when present, as in the second stage.

It is very difficult to distinguish the human trypanosome from some of the other
pathogenic ones by staining methods. The immunity test is the most reliable.
An animal recovered from an infection by a certain trypanosome does not possess
immunity for other pathogenic ones. Novy and McNeal cultivated T. lewisi in
water of condensation on blood agar at room temperature and Thomson and Sinton
have recently cultivated both T. gambiense and T. rhodesiense by using rat's blood
instead of rabbit's blood in the N.N.N. medium. It is present in the blood, usually
in exceedingly small numbers, and in the lymphatic glands of patients. It is by
puncture of the glands that we have the best means of finding the parasites. It is
also found in the cerebrospinal fluid in sleeping sickness. The parasite stains readily
with Wright's stain. The transmitting agent is the Glossina palpalis.

The life history of T. gambiense is not so well understood as that of certain other
organisms. There seem to be certain periods when even with trypanosomes in the
peripheral circulation tsetse flies do not become infected. From about 2 to 5% of
flies seem to become infective in experiments. When blood containing the so-called
short form of trypanosomes is ingested by G. palpalis they reach the gut and remain
there unattached. From the fifth to the seventh day they seem to become scarce
in the digestive tract but later they reappear in quantity. About the eighth to the
eighteenth day long slender forms pass into the proventriculus and later reach the
salivary glands as long slender forms. They multiply in the glands and develop into
short crithidial forms which later become similar to those found in the peripheral
circulation. Robertson considers that the important development takes place in
the salivary glands and not in the intestine while Kleine thinks the mature forms
the first to appear in the gut. It requires eighteen to twenty days or longer for the
complete development and flies so infective remain so for the remainder of life.

228 THE PROTOZOA

Some authors consider types representing male, female and indifferent forms to be
noted during the developmental cycle.

Other authorities think it possible that trypanosomes may encyst in the digestive
tract, and so the flies transmit the disease along with their faeces. This does not
seem to be possible in connection with human infections. Koch found several cases
where infection had taken place by coitus. This is the method of infection in T.
equiperdum, a trypanosome disease of horses.

The various trypanocidal remedies, atoxyl, arsacetine, etc. , have not proven very
satisfactory. One of Ehrlich's latest products, arsenophenyl-glycine, however, has
given encouraging results; horses affected with surra having been cured by its use.
In man it has been given in doses of i gram without ill effects.

T. rhodesiense. — -This is a trypanosome reported for man by
Stephens and Fantham. The nucleus, instead of being in the center
as in T. gambiense, is quite near the blepharoplast. It is much more
virulent for laboratory animals than T. gambiense. It is transmitted
by G. morsitans and the developmental cycle is similar to that of T.
gambiense except that it seems that the important developmental cycle
occurs in the gut of the fly.

Schizotrypanum cruzi (Trypanosoma cruzi) Chagas, 1909.— A
human trypanosomiasis found in the state of Minas Geraes, in Brazil, is
caused by this protozoon. Cruz states that the specific protozoon is
transmitted by a bug of the genus Conorhinus (Reduviidae).

This trypanosome is remarkable for the large size of its blepharoplast. In length
it is only a little longer than the diameter of a red cell. It is cultivable on blood
agar and can be transmitted to various laboratory animals, as guinea-pig, white
mice, and monkeys.

Cruz thinks that a non-sexual cycle occurs in general tissues of man but that a
special sexual cycle occurs in the lung capillaries. In the lungs the parasite loses its
flagellum and becomes oval in shape. Subsequently eight daughter spores develop.
These spores or merozoites are liberated into the general circulation and each one
penetrates a red cell and develops into an adult trypanosome. When ingested by
Conorhinus they lose the flagellum and assume an oval Leishmania form, which
multiply by fission. Eventually there are produced trypanosome types which get
into the salivary glands and thence into man. Chiefly a disease of children with
swelling of neck, axillary and groin glands, anaemia, enlarged spleen, oedema of
eyelids and irregular fever. Usually fatal in children but less so in adult. In adults
apt to have goiter.

Of the more important trypanosome diseases of animals may be mentioned:

1. Nagana. Pathogenic for domesticated animals in South Africa. T. brucei.

2. Surra. Pathogenic for horses in India and Philippines. T. evansi.

3. Dourine. Transmitted by coitus in horses. T. equiperdum.

4. Mai de caderas. Affects horses in South America. T. equinum.

A harmless infection, especially in sewer rats, is due to T. lewisi. Transmission of

KALA AZAR. 22Q

this rat trypanosomiasis can apparently be brought about through the agency of
both fleas and lice. In the flea there is apparently a developmental cycle of a dura-
tion of one week.

There are many trypanosomes in birds, fish, frogs, etc.

Trypanoplasma.

The genus Trypanoplasma has a rather large blepharoplast, from which arise
two flagella. One extends forward as a free anterior flagellum, while the other
projects posteriorly, running along the border of the undulating membrane. This
genus is not known for man.

Leishmania.

The genus Leishmania includes three species: L. donovani, the para-
site of kala azar, L. tropica,the parasite of oriental sore andL. infantum
the cause of a leishmaniasis among children in northern Africa.

The disease known as ponos, which exists in the Grecian islands Spezzia and
Hydra has been found by Galle to be a leishmaniasis. Nicolle has found a disease
of very young children (as a rule in the second year of life) in Tunis due to L.
infantum. This protozoon morphologically resembles L. tropica but is smaller.
It is found chiefly in the spleen, liver and bone marrow. The symptoms are extreme
anaemia, splenic, and to a less degree, hepatic enlargement. Irregular temperature,
rapid pulse and a mononuclear leukocytosis and transient oedema are also noted.
It can be inoculated into the dog and monkey; other animals are practically immune.

A similar disease has been noted in Italy, Malta, and Portugal. L. infantum
grows rapidly in Novy MacNeal medium, in which medium L. donovani will not grow.
Furthermore inoculation of L. donovani into dogs and monkeys has been unsuccess-
ful. These are undoubtedly different species, inasmuch as in sections of India,
where tropical ulcer was common, there was no kala azar, and in Assam where kala
azar prevailed there were no Leishman-Donovan bodies to be found in smears from
the tropical ulcerations there present, except rarely in cases of general infection.
L. tropica has been cultivated by Nicolle.

It is interesting that the parasite of kala azar cannot be cultivated except in
sterile media while that of oriental sore will grow in media contaminated with cocci.

These parasites are typically intracellular, being within either polymorpho-
nuclears, which contain only one or two of the bodies, or in large mononuclears, in
which there may be as many as six. They may be packed, however, in phagocytic
endothelial cells.

In kala azar smears taken during life we may find the bodies imbedded in a faintly
blue staining matrix; after death and in sections of tissue such an appearance is
not seen. In the spleen they are not found in the Malpighian bodies, but in the
phagocytic cells lining the lymph spaces. The parasites occur in the peripheral
circulation in about 80% of the cases. They abound in the liver and spleen. The
parasite is oval and about 2 X 3/£. There are two distinct chromatin staining masses.
The larger nucleus is more or less spherical, peripherally situated, and stains faintly,

230 THE PROTOZOA

while the smaller chromatin mass is generally rod-shaped and stains intensely. It
has been recently recommended that instead of liver or splenic puncture for the
demonstration of these bodies, a blister be raised and a smear from that containing
many polymorphonuclears might show these bodies. The affection is characterized
by a leukopaenia so that it is very difficult to demonstrate the parasites in ordinary
blood smears.

By cultivating the parasites obtained from splenic puncture in acidified sodium
citrate solution at room temperature, Rogers succeeded in obtaining flagellated forms
similar to Herpetomonas. An anterior flagellum proceeds directly from the blephar-
oplast. The bedbug is supposed to be the intermediary host.

Patton has recently noted that when bedbugs feed on kala-azar patients who have
the L. D. bodies in their peripheral circulation that the parasites develop into the
flagellate stage in the bedbug and are present in great numbers from the fifth to the
eighth day. These flagellate forms change into postflagellate ones by the twelfth
day and are then found in the stomach. If, however, he allowed the bedbugs to
have a second feeding of human blood after the infecting feeding the flagellates dis-
appeared within twelve hours. This is apparently an important point in epidemi-
ology. Patton succeeded in infecting a white rat with intraperitoneal injection of
splenic emulsion from a kala-azar patient.

L. infantum is transmitted from dog to dog by the dog flea, P. serraticeps and
the same agent probably transfers the parasite from dog to man. Experiments
would indicate that the Indian form of kala azar is not a disease which can be trans-
mitted to dogs.

The genera Herpetomonas and Crithidia are frequently found in the alimentary
tract of insects and have caused confusion in the search for developmental forms of
various pathogenic flagellates in transmitting insects. In Herpetomonas, of which
the type species is H. muscae domesticae, the body is spindle-shaped with a rather
blunt flagellar end and an attenuated anterior end. In Crithidia both extremities
are pointed and the blepharoplast is situated toward the center quite near the tropho-
nucleus. In Herpetomonas the blepharoplast is near the rather blunt flagellar
extremity at some distance from the nucleus.

There is no undulating membrane in either of these genera, this differentiating
them from Trypanosoma.

Darling has reported from Panama a protozoon somewhat like Leishmania in
which the cells of lungs, liver, spleen, and lymphatic glands contained numerous
parasites about 3 to 4/4 in diameter, slightly oval in outline, and containing a large
and small chromatin staining mass. He has given it the name Histoplasma
capsulata.

Trichomonas.

Trichomonas vaginalis. — This parasite has a fusiform body and is about
It has three flagella arising from the anterior end and an undulating membrane. It
lives in vaginal mucus which has an acid reaction A change of reaction, as at men-
struation, causes them to disappear. Forms similar to the T. vaginalis have been
found in the intestine and in sputum from putrid bronchitis.

INFUSORIA. 231

These flagellates are generally considered harmless, although doubt as to this is
expressed by some authors.

Lamblia.

Lamblia intestinalis. — These parasites are aboutioX i5/* and have a pear-shaped
body with a depression at the blunt anterior end. This depression enables the
flagellate to attach itself to the summit of an epithelial cell. Around the depression
are three pairs of flagella which are constantly in motion. Another pair of flagella
project from either side of the blunt little tail-like projection. When stained, the
parasites have a pyriform shape with two chromatin staining areas on either side
of the anterior end. When encysted, they assume an oval shape. This parasite
is generally considered as of little importance, but inasmuch as, when in great num-
bers in the caecum and appendix, they may give rise to symptoms resembling appen-
dicitis and as they are responsible for a chronic and intractable diarrhoea associated
with mental and physical depression, this is undoubtedly an affection only minor in
importance to amoebic infection. It is a common infection in the tropics.

INFUSORIA (CILIATA).

The Infusoria are the most highly developed of the Protozoa.

The bodies of Infusoria are oval and may be free or attached to a stalk-like
contractile pedicle, as with Vorticella, or they may be sessile. The cilia, which are
characteristic, may be markedly developed around the cytostome (mouth) and serve
the purpose of directing food into the interior, while others act as locomotor organs.
The body is enveloped by a cuticle which may only have one opening or slit, to serve
as mouth; or it may have a second one, a cytopyge or anus. Usually the faecal
matter is ejected through a pore which may be visible only when in use. They usu-
ally have a large nucleus and a small one. Infusoria tend to encyst when conditions
are unfavorable (as when water dries up in a pond). When the cilia are evenly
distributed over the entire body of the ciliates we have the order Holotricha; when
ciliated all over, but with more prominent cilia surrounding the peristome, we call
the order Heterotricha. It is to this order that the Infusoria of man belong.

Balantidium coli. — -This is the only ciliate of importance in man.
It is a common parasite of hogs. It is from 60 to ioo/i long by 50 to
'jofJ. broad, and has a peristome at its anterior end which becomes narrow
as it passes backward. It has an anus. The ectosarc and the endosarc
are distinctly marked. The cuticle is longitudinally striated.

These parasites cause an affection similar to dysentery and may bring about a
fatal termination. It is almost impossible to escape noticing the actively moving
bodies if a faecal examination is made. When encysted they are round.

Another ciliate, the B. minimum, 25 x 15^, has also been reported for man.

Nyctotherus faba has a kidney-shaped body and is about 25 by 15;*. It has a
large contractile vacuole at the posterior end. It has a large nucleus in the center
with a small fusiform micronucleus lying close to it. It has only been reported
once for man.

DESCRIPTION OF PLATE I.

(Kolle and Wassermann. )

Malarial Parasites.

4

1. Two tertian parasites about thirty-six hours old, attacked blood-corpuscles
swollen.

2. Tertian parasite about thirty-six hours old ; stained by Romanowsky's method.
The black granule in the parasite is not pigment but chromatin. Next to it and to
the left is a large lymphocyte, and under it the black spot is a blood plate.

3. Tertian parasite, division form nearby is a polynuclear leukocyte.

4. Quartan parasite, ribbon form.

5. Quartan parasite, undergoing division.

6 Tropical fever parasite, (^stivo-autumnal.) In one blood-corpuscle may
b2 seen a smaller, medium, and large tropical fever-ring parasite.

7. Tropical fever parasite. Gametes half-moon spherical form. Smear from
bone marrow.

8. Tropical fever parasite which is preparing for division heaped up in the blood
capillaries of the brain.

Asexual Forms.

9. Smaller tertian ring about twelve hours old.

10. Tertian parasite about thirty-six hours old, so-called amoeboid form.

1 1. Tertian parasite still showing ring form forty-two hours old.

12: Tertian parasite, two hours before febrile attack. The pigment is beginning
to arrange itself in streaks or lines.

13. Tertian parasite further advanced in division. Pigment collected in large
quantities.

14. Further advanced in the division. (Tertian parasite.)

232

PLATE I.

COCCIDIA. 233

SPOROZOA.

This class of Protozoa gets its name from the method of reproduction
— sporulation. These parasites rarely show binary fission. While
the sporozoa are found within cells, in the tissues and in internal cav-
ities, as intestine and bile ducts, yet it is as inhabitants of the blood that
they have their greatest importance for man — -these are known as
Haemosporidia. A sporozoon may be either naked or amceboid or be
covered with a distinct cuticle.

NOTE. — Sporozoa are divided into two subclasses — the Telosporidia and the Neo-
sporidia. In the former the vegetative activity of the protozoon goes on to full growth
at which time the reproductive activity commences. With the Neosporidia,
however, the growth and reproduction go on at the same time.

Among the Telosporidia we have the orders Gregarinaria, Coccidiaria, and
Haemosporidia

Gregarines are chiefly parasites of arthropods and worms and are not known
for man or the higher vertebrates.

The subclass Neosporidia is practically of no importance in human parasitology,
only the order Sarcosporidia having been reported for man. From an economic
standpoint, however, the order Myxosporidia is of great importance — Nosema being
the cause of pebrine, a disease destructive to the silkworm. In this the eggs of an
infected N. bombycis may be infected.

Coccidiaria.

The parasites of the order Coccidiaria are almost exclusively found in the intes-
tines and in the organs connected with it. In the vegetative stage it lives within an
epithelial cell, which it destroys. Afterward it falls into the lumen lined by this
epithelial cell and sporulates, either by the method of schizogony or sporogony.

Owing to their egg-like shape, coccidia have often been considered as the ova of
intestinal parasites, and vice versa. Upon swallowing an oocyst with its contained
sporozoites the membrane of the oocyst is digested in the duodenum and the sporo-
zoites liberated. They enter epithelial cells, as of intestine, and reproduce by
schizogony. After a varying number of nonsexual cycles sporogony commences,
sporonts being produced instead of schizonts. The female sporont is fertilized by
the microgamete which is an elongated body provided with two flagella. These
microgametes are formed from the male sporont and when thrown off from the
periphery they enter (usually a single one) the macrogamete. After fertilization a
resistant membrane is formed and the term oocyst is used. Within the oocyst
are found smaller cysts, the sporocysts, in which the sporozoites are formed.

The cycle is very similar to that of malaria except that no arthropod host is
required for the sexual cycle. The spores which are formed in schizogony are known
as merozoites.

Merozoites may best be distinguished from sporozoites by the presence of a
nuclear karyosome, this being absent in sporozoites. In Eimeria we have the
oocyst containing four sporocysts with two sporozoites in each sporocyst while in
Isospora we have an oocyst containing two sporocysts with four sporozoites in each.

Eimeria stiedae. — This sporozoon is usually known as the Coccidium cuniculi

DESCRIPTION OF PLATE II.

(Kolle and Wassermann. )

Malarial Parasites.

15. Complete division of the parasite. Typical mulberry form.

1 6. To the left is the completed division form, an almost developed gamete, which
is to be recognized by its dispersed pigment.

1 7. A tertian ring parasite, small size broken up.

1 8. Three-fold infection with tertian parasite. The oval black granules are the
chromatin granules.

19. To the left, tertian parasite with large, sharply demarked, and deeply colored
chromatin granules. To the right, tertian parasite. Both thirty-six hours old.
Both probably gametes.

20. Tertian parasite thirty-six hours old, ring form.

21. Tertian parasite with beginning chromatin division, with eight chromatin
segments.

22. Tertian parasite chromatin division farther advanced with twelve chromatin
granules, in part triangular in form.

23. Completed division figure of a tertian parasite. Twenty-two chromatin
granules.

24. The young tertian parasites separating themselves from each other. The
pigment remains behind in the middle.

25. Quartan ring parasite, which is hard to differentiate from large tropical ring
or small tertian ring.

26. Quartan ring lengthening itself.

27. Small quartan ribbon form.

28. The quartan ribbon increases in width. The dark places consist almost
entirely of pigment.

234

PLATE II.

MALARIA 235

or C. oviforme. It is most frequently found in the epithelium of the bile ducts.
It has very rarely been reported for man. In these cases (about five) cysts of the
liver have been found containing coccidia. The parasite is about 40 X 20^, and is
oval in shape with a double outlined integument. The sporozoites, which form
inside, are falciform in shape. These escape and enter fresh epithelial cells, and
thus the process of schizogony goes on. The parasites of the liver are larger than
those found in the intestines, these latter being only about 3oX is/*. In the faeces
the form most often found is the oocyst, about 40 X 2O//. Infection takes place by
ingestion of the oocyst.

Isospora bigemina. — This parasite, formerly called the Coccidium bigeminum,
lives in the intestinal villi of dogs and cats. It is about 12 X 8ft. and shows a highly
refractile envelope (oocyst) containing two biscuit-shaped sporocysts within each of
which are four sporozoites. It has been reported for man three times.

Haemosporidia.

Of the Sporozoa found in the blood (Haemosporidia) , the malarial
parasites are the only ones connected with disease in man.

In addition to man, infections with parasites of a similar nature are found in
monkeys (Plasmodium kochi; the sexual forms alone seem to be present), in birds
(Hsemamceba relicta; this organism is usually designated Proteosoma) . An infection
of crows and pigeons of like nature is Halteridium. Numerous haemosporidia
have been reported for bats, various other mammals, tortoises, lizards, etc.

The life history of the malarial parasite is one of the most interesting chapters
in medicine. Laveran discovered the parasite in 1880. In 1885, Golgi noted that
sporulation occurred simultaneously at time of malarial paroxysm. Koch, Golgi,
and Celli demonstrated existence of different species for different types of fever.
King and Laveran (1884) considered possibility of mosquito transmission. Manson
(1894) formulated hypothesis that gametes were destined to undergo development
in the mosquito from observing that flagellated bodies only appeared some time
after the blood was withdrawn.

Ross (1895) demonstrated that flagellation takes place in the stomach of the
mosquito. McCallum (1897) saw fertilization of macrogametes by microgametes
of Halteridium. Opie recognized differences in sexual characteristics.

Ross (1898) demonstrated life cycle of bird malaria (Proteosoma), showing for-
mation of zygotes and presence of sporozoites in salivary glands. Grassi and Big-
nami proved the cycle for Anophelinae for human malaria. In 1900 (Sambon and
Low), infected mosquitoes from Italy were sent to London, where, by biting, they
infected two persons.

Life History. — When man is at first infected by sporozoites we have
starting up a nonsexual cycle which is completed in from forty-eight to
seventy-two hours, according to the species of parasite. The falciform
sporozoite bores into a red cell, assumes a round shape and continues
to enlarge (schizont). Approaching maturity, it shows division into a
varying number of spore-like bodies. At this stage the parasite is

DESCRIPTION OF PLATE III.

(Kolle and Wassermann.)

Malarial Parasite.

29> 3°> 31- The quartan ribbon increases in width. The dark places consist
almost entirely of pigment.

32. Beginning division of the quartan parasite and the black spot in the middle
is the collected pigment.

33. Quartan ring.

34. Double infection with quartan parasites.

35. Wide quartan band. The fine black stippling in the upper half of the para-
site is pigment.

36. Beginning division of the quartan parasite. The chromatin (black fleck)
is split into four parts.

37. Division advanced, quartan parasites.

38. Typical division figure of the quartan parasite.

39. Finished division of the quartan parasite. Ten young parasites, pigment in
the middle.

40. Young parasites separated from one another.

41. Small and medium tropical ring, the latter in a transition stage to a large
tropical ring.

42. Small, medium and large tropical ring, together in one corpuscle.

236

PLATE III.

16

MALARIA

237

termed a merocyte. When the merocyte ruptures, these spore-like
bodies or merozoites enter a fresh cell and develop as before.

At the time that the merocyte ruptures it is supposed that a toxin is
given off which causes the malarial paroxysm. The cycle goes on by
geometric progression from the first introduction of the sporozoite, but
it is usually about two weeks before a sufficient number of merocytes
rupture simultaneously to produce sufficient toxin for symptoms (period
of incubation). This cycle is termed schizogony.

9.

FIG. 62. — Sexual and nonsexual cycle of malaria, i, Schizonts; 2, merocyte;
3, merozoites; 4, macrogamete; 5, microgametocyte; 6, and 7, gametes in stomach of
mosquito; 8, microgametocyte throwing off microgametes; 9, microgamete fertilizing
macrogamete; 10, vermiculus or zygote; n and 12, zygotes; 13, zygote distended
with sporozoites; 14, sporozoites.

After a varying time, whether by reason of necessity for renewal of
vigor of the parasite by a respite from sporulation, or whether from a
standpoint of survival of the species, sexual forms (gametes) develop.
Some think that sporozoites of sexual and nonsexual characteristics are
injected at the same time. It is usually considered, however, that
sexual forms develop from pre-existing nonsexual parasites.

These gametes show two types: the one which contains more pig-
ment, has less chromatin, and stains more deeply blue is the female — -a
macrogamete; the other with more chromatin, less pigment, and staining

DESCRIPTION OF PLATE IV.

(Kolle and Wassermann. )

Malarial Parasite.

43. To the left a young (spore) tropical parasite. To the right a medium and
large tropical parasite.

44. An almost fully developed tropical parasite. The black granules are pig-
ment heaps.

45. Young parasites separated from one another. Broken up division forms
twenty-one new parasites.

46. To the left a red blood-corpuscle with basophilic, karyochromatophilic gran-
ules. Prototype of malarial parasite. On the right a red blood-corpuscle with re-
mains of nucleus.

Sexual Forms or Gametes.

47. An earlier quartan gamete (macrogamete in sphere form), female.

48. An earlier quartan gamete (microgametocyte), male.

49. Tertian gamete, male form (microgametocyte).

50. Tertian gamete, female (macrogamete).

51. Tertian gamete (microgametocyte) still within a red blood-corpuscle.

52. Macrogamete tertian within a red blood-corpuscle.

53. Tropical fever. (^Estivo-autumnal) gamete, half-moon (crescent) still
lying in a red blood-corpuscle. In the middle is the pigment. The concave side
of the crescent is spanned by the border of the red blood-corpuscle.

54. Gamete, tropical fever parasite.

55. Gamete of tropical fever parasite heavily pigmented.

56. Gamete of the tropical fever parasite (flagellated form), microgametocyte
sending out microgametes (flagella or spermatozoa).

238

PLATE IV.

MALARIA 239

grayish-green rather than blue is the male— a microgametocyte. When
the gametes are taken into the stomach of the Anophelinae, the male cell
throws off spermatozoa-like projections, which have an active lashing
movement and break off from the now useless cell carrier and are there-
after termed microgametes. These fertilize the macrogametes and this
body now becomes a zygote.

By a boring-like movement the zygote goes through the walls of the
mosquito's stomach, stopping just under the outer epithelial layer of
the stomach or mid-gut. It continues to enlarge until about the end
of one week it has grown to be about 6o/* in diameter and has become
packed with hundieds of delicate falciform bodies.

Zygotes of benign tertian show little rod-like particles of yellowish pigment —
those of malignant tertian black clumps, which, however, are not so coarse as those
of quartan.

The matuie zygote now ruptures and the sporozoites are thrown off
into the body cavity. They make their way to the salivary glands and
thence, by way of the veneno-salivary duct in the hypopharynx, they
are introduced into the circulation of the person bitten by the mosquito,
and start a nonsexual cycle. As the sexual life takes place in the mos
quito, this insect is the definitive host — man is only the intermediary host.

It must be remembered that only certain genera and species of Anophelinae are
known malaria transmitters; thus Stephens and Christophers, in dissecting 496
mosquitoes of the species M. rossi, did not- find a single gland infected with sporo-
zoites. With M. culicifacies, however, twelve in 259 showed infection.

This is one of the methods of determining the endemicity of malaria or the
malarial index. There are two other methods: i. by noting the prevalence of
enlarged spleen, and 2. by determining the number of inhabitants showing malarial
parasites in the blood. This index is best determined from children between two
and ten years of age, as children under two years show too high a proportion of para-
sites in the peripheral blood while those over ten years of age show too great an in-
cidence of enlarged spleens.

There are three species of malarial parasites: i. the Plasmodium
vivax, that of benign tertian — cycle, forty-eight hours; 2. the Plasmo-
dium malariae, that of quartan — cycle, seventy- two hours; and 3. the
Plasmodium falciparum, that of aestivo-autumnal or malignant tertian
— cycle of forty-eight houis.

Variations in cycles may be produced by infected mosquitoes biting on successive
nights, so that one crop will mature and sporulate twenty-four hours before the
second. This would give a quotidian type of fever. In aestivo-autumnal infections
anticipation and retardation in the sporulation cause a very protracted paroxysm,
lasting eighteen to thirty-six hours; this tends to give a continued or remittent fever
instead of the characteristic type.

240

THE PROTOZOA

UNSTAINED SPECIMEN (FRESH BLOOD).

P. vivax.
'(Benign tertian.)

P. malarias.
(Quartan.)

P. falciparum.
(Malignant tertian)
Costive-autumnal. )

Character of the

Swollen and light

About the size

Tendency to distortion

infected red cell.

in color after

and color of a

of red cell rather than

eighteen hours.

normal red cell.

crenation. Shriveled

appearance. (Brassy

color. )

Character of young

Amoeboid outline.

Frosted glass

Small, distinctly round,

schizont.

Hyaline. Rare-

disc. Very

crater-like dots not

ly more than slight amoeboid

more than one-sixth di-

one in r.c. Ac-

motion.

ameter of red cell.

tive amoeboid

Two to four parasites

movement.

in one red cell common.

One third diam.

of r. c.

Character of ma-

Amoeboid outline.

Rather oval in

Only seen in overwhelm-

ture schizont.

No amoeboid

shape. Slug-

ing infection. Have

movement.

gish movement

scanty fine black pig-

of peripherally

ment clumped together.

placed coarse

black pigment.

Pigment.

Fine yellow

Coarse almost

Pigmented schizonts

brown granules

black. Shows

very rare in periph.

which show ac-

movement only

circulation except in

tive motion in

in young to

overwhelming infec-

one-half grown

half-grown

tions. Tend to clump

schizont. Mo-

schizont.

as excentric pigment

tion ceases in

mass blocks.

full-grown

schizont.

STAINED SPECIMEN.

P. vivax.
(Benign tertian.)

P. malariae.
(Quartan. )

P. falciparum.
(Malignant tertian)
C^Estivo-autumnal. )

Character of in-
fected red cell.

Larger and light-
er pink than
normal red cell.
Shows "Schiiff-
ner's dots."

About normal
size and stain-
ing.

Shows distortion and
some polychromato-
philia and stippling.
Rarely we have coarse
cleft-like reddish dots
— Maurer's spots.

MALARIA

STAINED SPECIMEN. — (Continued.)

241

P. vivax.
(Benign tertian)

P. malariae
(Quartan.)

P. falciparum
(Malignant tertian)
(^stivo-autumnal.)

Character of young

Chromatin mass

Rather thick

Very small sharp hair-

schizont.

usually single

round rings

like rings, with a

and situated in

which soon tend

chromatin mass pro-

line with the

to show as equa-

truding from the ring.

ring of the ir-

torial bands.

Often appears on per-

regularly out-

iphery of red cell as a

lined blue para-

curved blue line with

site.

prominent chromatin

dot. Frequently two

chromatin dots.

Character of half-

Vacuolated loop-

More marked

tNot often found in per-

grown schizont.

ed-like body

band forms

ipheral circulation.

with single chro-

stretching

Chromatin still com-

matin aggrega-

across r. b. c.

pact.

tion. Schiiff-

ners dots.

Character of ma-

Fine pigment

Coarse pigment

Very rarely seen in per-

ture schizont.

rather evenly

rather peripher-

ipheral circulation in

distributed in

ally arranged in

ordinary infection.

irregularly out-

an oval para-

Pigment clumps early.

lined parasite.

site.

Character of me-

Irregular division

Rather regular Sporulation occurs in

rocyte.

into fifteen or

division into spleen, brain, etc.

more spore-like

eight or ten

Rarely in peripheral

chromatin dot

merozoites —

circulation. Eight to

segments.

Daisy.

ten chromatin staining

merozoites.

Character of mac-

Round deep blue.

Round, similar

Crescentic, deep blue,

rogamete.

Abundant,

to P. vivax but

pigment clumped at

rather coarse

smaller.

center, chromatin '

pigment, chro-

scanty and in center.

matin at per-

iphery.

Character of mi-

Round, light

Round like P.

More sausage-shaped

crogametocyte.

green-blue, pig-

vivax.

than crescent. Light

ment less abun-

blue. Pigment scat-

dant, chroma-

tered throughout.

tin abundant

Chromatin scattered.

and located

centrally or in

a band.

242 THE PROTOZOA

In full grown schizonts we find the chromatin in separate aggregations through-
out the parasite while the pigment is clumped. In gametes the pigment is
scattered and the chromatin is in a single mass.

If many young ring forms are present during pyrexia it is probable that the
infection is E.A.

In parthenogenesis, as observed in P. vivax, the nonsexual forms and the males
die off leaving only the female forms. The nucleus divides into a dense and light
portion. The latter degenerates and the former goes on to merozoite formation.

This is Schaudinn's explanation of relapses. Another explanation of latent
malaria is by conjugation of two ring forms.

In the diagnosis of malaria one should always examine both a fresh
specimen and a stained one, as each method gives valuable information
in differentiating species. When time will not permit the examination
by both methods, always use the smear stained by Wright's stain, as
the small peripherally situated rings of aestivo-autumnal fever may
escape notice in a fresh specimen.

For the cultivation of malarial parasites (Bass) the blood in 10 to 20 c.c. quan-
tities is taken from the patient's vein and received in a centrifuge tube which con-
tains i/io c.c. of 50% glucose solution. A glass rod, or piece of tubing, extending
to the bottom of the centrifuge tube is used to defibrinate the blood. After centrif u-
galizing there should be at least i inch of serum above the cell sediment. The
parasites develop in the upper cell layer about 1/50 to 1/20 inch from the top. All
of the parasites contained in deeper lying red cells die. To observe the development,
red cells from this upper i/20-inch portion are drawn up with a capillary bulb pipette.

Should the cultivation of more than one generation be desired, the leukocyte
upper layer must be carefully pipetted off, as the leukocytes immediately destroy
the merozoites. Only the parasites within red cells escape phagocytosis. Sexual
parasites are much more resistant, and the authors think they observed partheno-
genesis. The temperature should be from 40 to 41° C. and strict anaerobic condi-
tions observed, ^stivo-autumnal organisms are more resistant than benign tertian
ones. Dextrose seems to be an essential for the development of the parasites.

Bass considers that P. vivax has a flat amoeboid like structure which enables it
to squeeze through the brain capillaries while adult schizonts of P. falciparum have
a solid oval form which causes them to be caught in the capillaries.

Belonging like the malarial parasite to the Haemosporidia we have a group of
parasites known as the PIROPLASMS. The correct name for these parasites is Babesia
but they are better known under the name Piroplasma. They are minute organisms,
usually pear or rod shape, which invade the red corpuscles. They produce no
pigment but destroy the corpuscle and set free the Hb. which is excreted in enor-
mous amounts by the kidneys. It is this which gives the name redwater to the better
known Texas fever of cattle. Organisms of this kind have been thought of in con-
nection with blackwater fever of man. Seidelin has claimed that a parasite of
similar nature, P ar a plasma flavigemim, was the cause of yellow fever.

At one time spotted fever of the Rocky Mountains was supposed to be due to
a parasite named Babesia hominis.

CHLAMYDOZOA

243

SARCOSPORIDIA.

Sarcosporidia are sporozoa found in the striped muscles of various
mammals and birds. They are common in the pig and mouse and
have been reported for man in three well-authenticated cases. In the
last, Darling found these protozoa in the biceps muscle of a negro patient
in Panama. In Baraban's case the laryngeal muscles at autopsy were
found to show cysts about 1/15 inch long which contained sickle-shape
sporozoites about g/* long.

They are known also as Miescher's tubes when in mus-
cle fibers. They are divided into three genera: Miescheria and
Sarcocystis when parasitic in muscle fiber; Balbiania, when
parasitic in the in tervening connective tissue of the muscles.
The method of transmission is unknown. In some places more
than 50% of the sheep and pigs may show infection.

Miescheria has a thin membrane surrounding the cyst while
that of Sarcocystis is thickened and radially striated by small
canaliculi.

As the young trophozoite grows nuclei increase and a definite
membrane forms which the sporoblasts eventually fill. Ac-
cording to Minchin the Sarcosporidia contain only one genus,
Sarcocystis. It is never parasitic for invertebrate hosts and
while occasionally found in birds and reptiles it is pre-emin-
ently a parasite of the higher vertebrates. As a rule, they are
harmless parasites but the Sarcocystis muris is very pathogenic
for the mouse. Closely related to the order Sarcosporidia is the
parasite Rhinos poridium kinealyi.

Rhinosporidium kinealyi. — It causes pedunculated tumors of
nasal cavity. The pansporoblasts enlarge in the center of the
connective tissue of the nasal polyp and contain about 12
sporoblasts. When mature the cystic-like polyp bursts and the
sporoblasts are liberated to extend the infection.

FIG. 63. — Mie-
scher's sac from
the musculature
of a hog. X30
diameters. (After
Ostertag.)

CHLAMYDOZOA.

These organisms are generally considered as being protozoal in
nature and as a rule belong to the filterable viruses, which is the desig-
nation for the infectious principles of those diseases, in which filtration
of defibrinated blood or serum through a Berkefeld filter capable of
holding back so small an organism as the M. melitensis, does not pre-
vent the infection being transmitted when introduced by the proper
atrium of infection. The Chlamydozoa are also characterized by the
occurrence of "cell inclusions."

244 THE PROTOZOA

The best known infections of this group of diseases in man are smallpox, vaccinia,
rabies, trachoma, molluscum contagiosum, and foot and mouth disease. There are
many such infections in other animals. The cell inclusions are regarded as prod-
ucts of cellular reaction to a virus which is more or less impossible of demonstra-
tion. The discovery of exceedingly minute granules in some of these diseases, as
in variola and trachoma, has suggested that, as a reaction to the invasion by such
a granule, the cell throws an enveloping mantle about the invading particle. To
designate this we use the name Chlamydozoa.

The generic name Cytorrhyctes has been applied to certain of these viruses,
thus C. vaccinias develops within the epithelial cells of stratified epithelium. In
vaccinia, Councilman and his colleagues consider that the development only takes
place in the cytoplasm of the cell. In variola, however, the developmental cycle
affects the nucleus.

Cytorrhyctes luis, reported as the cause of syphilis, sporulates in the blood-
vessels and in the connective tissue, not in epithelial cells.

Cytorrhyctes scarlatinas was reported by Mallory to have been found in the skin
in four cases of scarlet fever.

CHAPTER XVII.

FLAT WORMS.

CLASSIFICATION OF THE PLATYHELMINTHES (FLAT WORMS).

Class

Family

Trematoda

Fasciolidae

Paramphistomidse

Schistosomidse

Genus

Fasciola

Fascioletta

Fasciolopsis

Dicrocoelium

Paragonimus

Opisthorchis

Clonorchis

Heterophyes

Cladorchis

Gastrodiscus

Schistosomum

Cestoda

,., f Dibothriocephalus
Dibothnocephahdae I

Tseniidae

Diplogonoporus
Dipylidium

Hymenolepis

Taenia
Davainea

Species

F. hepatica
F. ilocana

F. buski

D. lanceatum
P. westermanii
O. felineus
C. sinensis
C. endemicus
H. heterophyes

C. watsoni

G. hominis

S. haematobium
S. japonicum
S. mansoni

D. latus
D. grandis
D. caninum
H. nana

H. diminuta

T. solium

T. saginata

D. madagascariensis

NOTE. — Two larval Taeniidaeare found in man (Cysticercus cellulosae and Echino-
coccus polymorphus).

Also two larval Dibothriocephalidae (Sparganum mansoni and Sparganum
prolifer).

Two parasites often referred to as ophthalmic flukes have been reported lying
between the crystalline lens and its membrane. They have been considered as
possibly trematode larvae. Distomum ophthalmobium was found in 1850 in the eye
of a child and Monostoma lentis in the eye of an old woman.

TREMATODES OR FLUKES.

Flukes are generally leaf-like in outline, rarely cylindrical, and exhibit
marked variation in size and shape. They are nonsegmented and do

245

246 FLAT WORMS

not have cilia on ectoderm. Very characteristic of them is the posses-
sion of suckers by which they hold on to the skin or alimentary system of
their host.

They are divided into two orders: i. the Monogenea in which the egg gives rise
to a larva which later becomes the adult and 2. the Digenea. It is to this latter that
the flukes parasitic in man belong. This order is characterized by the fact that the
larva becomes parasitic in some second animal and then gives rise to a second gen-
eration of larvae which latter develop into adults.

The largest human fluke, Fasciolopsis buski, is from two to three inches (50 to
75 mm.) in length, while the Heterophyes heterophyes is less than 1/12 of an inch
(2 mm.) in length. The most important fluke, the liver fluke, Clonorchis endemicus,
is flat and almost transparent, while the almost equally important lung fluke, the
Paragonimus westermanii, is oval, almost round and reddish-brown in color. With
the exception of the Schistosomidse, all flukes are hermaphrodites, and, with the
exception of this family, all flukes have operculated eggs. The only other opercu-
lated (with a lid) eggs we meet with in man are those of the Dibothriocephalidae.

The three important families of flukes parasitic for man are: i.
Paramphistomidas — flukes with two suckers situated at either extremity.
2. Fasciolidae — -flukes with two suckers, one terminal, the other adjacent
to it and situated ventrally. This family includes the important geneia
Fasciola, Opisthorchis, Dicroccelium, Fasciolopsis, and Paragonimus.
In Paragonimus and Heterophyes the genital pore is posterior to the
acetabulum, in the other genera it is anterior. Fasciola has a den-
dritic intestinal canal which is not the case with Clonorchis, Fascio-
lopsis, Fascioletta, Opisthorchis and Dicroccelium. In Dicroccelium
the testicles are anterior to the uterus, in Opisthorchis, Clonorchis,
Fasciolopsis and Fascioletta they are posterior. Fasciolopsis and
Clonorchis have branched testicles (the former a very large fluke-
Clonorchis of medium size) while those of Opisthorchis are lobed.

3. Schistosomidae : In this family we have a leaf-like male which by
a folding in of its sides makes a channel for the thread-like female. The
sexes are separate, not hermaphroditic as with the Fasciolidae and
Paramaphistomidae.

Flukes have two suckers which, except in the Paramphistomidae, are quite near
each other — one is termed the oral sucker and the other the ventral sucker or acetabu-
lum. The intestinal tract consists of a pharynx, proceeding from the oral sucker,
which bifurcates and terminates in blind intestinal casca.

At the posterior extremity is an excretory pore which is at the termination of
a duct which divides into ramifying branches. This is the water-vascular system.
The testes, of various shapes and relations to the uterus, are more or less centrally
situated and have vasa deferentia. In some flukes the receptaculum seminis is a

FLUKES 247

conspicuous organ. The vitellaria are bilateral branching glands which pour
nutrient material into the ootype. It is in the ootype that the eggs are formed,
and opening into it we have the adjacent ovary. The shell gland is near the ovary.

A canal, known as Laurer's canal, leads from the ootype to the exterior, the
function of which is in question. It is probable that as trematodes have no sperma-
theca, the spermatozoa from other flukes enter by way of this canal. The life his-
tory of the important human flukes is unknown. It is supposed that this, in a meas-
ure, may resemble that of the common liver-fluke of sheep (sheep rot). In this the
eggs containing a ciliated embryo (miracidium) pass out in the faeces. This embryo
is hatched out and, gaining the water, swims about actively until it reaches some
suitable mollusk (Limnaea truncatula). By means of a pointed end, it bores its
way into the body of the gasteropod and in the pulmonary chamber becomes a
bag-like structure (the sporocyst) from the germinal cells of which develop a creature
with an alimentary canal (redia). The rediae tend to break out of the sporocyst and
wander to the liver of the snail. These rediae may give rise to a second generation
of rediae.

From the rediae minute little worms resembling adult flukes in possessing suckers,
but differing in the possession of a tail, develop (cercaria). Having reached maturity,
these cercariae leave the rediae, and, as in case of Fasciola hepatica, lose the tail, be-
come encysted on blades of grass, to be eaten by sheep and again commence the cycle.
The encysted cercariae develop into adult liver flukes. It is probable that with many
flukes the cercarise enter some host, as mollusk, insect, or fish, and that it is by eat-
ing such animals as food that man becomes infected. Looss thinks it possible that
the miracidium of Schistosomum haematobium may bore its way directly into man,
as do the larvae of the hookworm. Manson also suggests that the reporting by Mus-
grave of 100 mature lung flukes in a psoas abscess makes it very probable that these
parasites entered the body as miracidia. The idea in China is that the infection
with the common liver fluke of man is brought about by eating fish. Fluke disease
is generally known as distomatosis or distomiasis.

LIVER FLUKES.

Fasciola hepatica (Distomum hepaticum). — This fluke, while of
enormous economic importance by reason of destruction of sheep, has
only been reported twenty-three times in man, and in these instances
does not seem to have occasioned marked symptoms.

It has a cone-shaped anterior projection and is about 11/4 inch (30 mm.) long.
The intestinal canal, as well as the testicles, is branched. There is, however, a possible
importance of F. hepatica in connection with a peculiar affection known as "halzoun. "
This results from the eating of raw goat-liver, and it is supposed that the flukes
crawl up from the stomach and, entering the larynx or attaching themselves about
the glottis, produce the asphyxia characteristic of the disease.

Dicrocoslium lanceatum. — This has only been reported seven times in man.
The symptoms are unimportant. The fluke is about 1/3 of an inch (8 mm.) long,
with testicles anterior to the uterus.

248

FLAT WORMS

Clonorchis endemicus (Opisthorchis sinensis).— This fluke and the
C. sinensis are the most important of the human liver flukes. Until
recently these flukes were known as Opisthorchis sinensis.

Looss has separated this genus from Opisthorchis principally by the character-
istic of branching testicles — those of Opisthorchis being lobed. This fluke is very
common in China and Japan — in certain sections of Japan 20% of the population
being infected. This fluke is about 1/4 to 1/2 inch (8 mm.) long and C. sinensis
about 3/4 of an inch long and 1/6 of an inch broad (16 X 4 mm.) When squeezed

FIG. 64. — Trematodes of man, natural size, i, Clonorchis endemicus (Opisthor-
chis sinensis); 2, Gastrodiscus hominis; 3, Dicrocoelium lanceatum; 4, Heterophyes
heterophyes; 5, Schistosomum haematobium; 6, Fasciola hepatica; 7, Paragonimus
westermanii; 8, Fasciolopsis buski; 9, Opisthorchis felineus; 10, anatomy of C. en-
demicus (enlarged). G. P., genital pore; V. S., ventral sucker; V. G., vitelline glands;
R. S., receptaculum seminis; T., branched testicles.

out of the thickened bile ducts it is so transparent and glairy as almost to resemble
glairy mucus. As many as 4000 of these parasites have been found in a case, chiefly
in the liver, but at times in the pancreas. This fluke is supposed to produce most
serious symptoms, as indigestion, swelling and tenderness of liver, ascites, oedema,
and a fatal cachexia. As a matter of fact, many physicians in China attribute very
little pathogenic importance to it. The disease is diagnosed by the presence of the
ova in the stools. The source of infection is probably through the eating of uncooked
fish.

Kobayashi has examined various mollusks and fish for trematode larvae. He

FLUKES 249

succeeded in infecting nine kittens and two cats by feeding them with certain fresh-
water fishes whose flesh contained trematode larvae. These fish were found in
districts where human distomiasis was common. The view is taken that the two
species of Clonorchis are identical.

Opisthorchis felineus. — This fluke is smaller than the C. endemicus, and is a
common parasite of the gall bladder and bile ducts of cats. There are two lobed
testicles in this species instead of dendritic ones as in C. endemicus. In certain
parts of Siberia the parasite is found in more than 6% of the human autopsies. The
symptoms are similar to those caused by C. endemicus.

Other liver flukes of less importance which have been reported for man are:
i. Opisthorchis noverca. This was found in bile ducts of two natives of Calcutta.
It was lancet-shaped and covered with spines.

2. Metorchis truncatus: This is a small fluke, 1/12 inch (2 mm.) long, squarely
cut across at its posterior end and covered with spines. This was possibly found
once in man.

Intestinal Flukes.

Cladorchis watsoni (Amphistomum watsoni). — This fluke is about 1/3 of an
inch (8 mm. ) long, of oval outline but broader at posterior end and has an indistinct
oral sucker and a large sucker at the other end. This parasite has only been reported
once. Eggs, 125 X 75/*-

Gastrodiscus hominis (Amphistomum hominis). — This fluke is about 1/4 of an
inch (6 mm.) long and has a disc-like acetabulum about 1/6 of an inch in diameter
from which proceeds a teat-like projection, bearing an oral sucker. While it has
only been reported twice for man, indications are that it is probably fairly common
in India and Assam. Eggs, 150 X 72fi.

Fasciolopsis buski (Distomum crassum). — This is probably a rather common
parasite in India, as Dobson found the eggs in i% of the stools of more than 1000
coolies. The fluke is from 2 to 3 inches (40 to 70 mm.) in length and about 1/2 of an
inch (12 mm.) in breadth. It is thick, brown in color, and has a very large acetabu-
lum, three times the size of the oral sucker and located almost adjacent to it. The
branched ovary and shell gland lie in the center with the branched testicles posterior.
The coiled uterus is anterior to the testicles. Eggs, 125 X 75j". These parasites
cause dyspeptic symptoms and an irregular diarrhoea. It is also called Distomum
crassum. F. rathouisi is now considered to have been a shrunken F. buski, as it
seems to be anatomically similar to F. buski. Kwan's fluke reported from Hong
Kong, was possibly F. buski.

Heterophyes heterophyes (Cotylogonimus heterophyes). — This exceedingly
small fluke (2 X 0.5 mm.), which can be recognized by its small size (less than 1/12
of an inch long) and large, prominent acetabulum, was formerly supposed to be rare.
The oral sucker is much smaller than the acetabulum. The elliptical testicles lie
at the extreme posterior end. Cuticle has scale-like spines. The eggs are 30X1 7/*.
Very characteristic of this genus is the large sucker-like genital pore just below and
to one side of the acetah^ulum. Looss has shown that it is quite common in Egypt,
he having found it twice in Alexandria in nine autopsies. The parasites occupy the
ileum. It is common in dogs.

250

FLAT WORMS

Fascioletta ilocana. — This is a small fluke, about 1/4 inch (6 mm.) long. There
are two massive testicles in the posterior part of body. The acetabulum is promi.
nent. The egg of this small fluke is quite large (ioc/0 and has an operculum.
These trematodes were found by Garrison in five natives of Luzon, P. I., after treat-
ment with male fern.

FIG. 65. — Anatomy of a tape-worm, Tasnia solium (A., longitudinal, B., cross
section); a fluke, Paragonimus westermanii (C)., male and female nematode, Oxyuris
vermicularis (D.). A. i, Testes; 2, yolk glands; 3, shell glands; 4, ovary; 5, vagina;

6, vas deferens; 7, uterus before branching; 8, water-vascular system. B. i, Cuticle;
2, circular muscle; 3, ovary; 4, testes; 5, uterus; 6, excretory canal; 7, nerve cord.
C. i, Oral sucker; 2, acetabulum; 3, uterus; 4, testes; 5, excretory canal; 6, ovary;

7, yolk glands. I), (a) Female, i, Vulva; 2, uterus; 3, bulb of oesophagus; 4, anus;
(b) Male, i, Bulbous mouth end; 2, testes; 3, spicule; 4, alimentary canal. E.
Egg of P. westermanii.

LUNG FLUKES.

Paragonimus westermanii (Distoma ringeri).— In certain parts of
Japan and Formosa it is estimated that as many as 10% of the inhab-
itants may harbor this parasite.

It is also common in China, and recently many cases have been reported in the
Philippines. Dr. Stiles states that around Cincinnati, Ohio, there was at one time
quite a heavy infection among the hogs, so that it may be that certain cases diagnosed
in man as pulmonary tuberculosis are paragonimiasis.

BILHARZIASIS 251

It is popularly known as endemic haemoptysis on account of the accompanying
symptoms of chronic cough and expectoration of a rusty-brown sputum. After
violent exertion, and at times without manifest reason, attacks of haemoptysis of
varying degrees of severity come on. The characteristic ova are constant in the
sputum and establish the diagnosis. The fluke itself is a little more than 1/3 of an
inch (8 mm.) long and is almost round on transverse section, there being, however,
some flattening of the ventral surface. The acetabulum is conspicuous and opens
just anterior to the middle of the ventral surface. Eggs about 90 x 65 /*.

The branched testicles are posterior to the laterally placed uterus and the
genital pore opens below the acetabulum. The branched ovary is opposite the uterus
on the other side.

It is rather flesh-like in appearance and is covered with scale-like spines. The
flukes are usually found in tunnels in the lungs, the walls of which are of thickened
connective tissue. There may be also cysts formed from the breaking down of
adjacent tunnel walls. In addition to lung infection with this fluke, brain, liver,
and intestinal infections may be found. Musgrave was the first one to call attention
to the frequency of general infection with this parasite (paragonimiasis) in the
Philippines. He found it in seventeen cases in one year. The life history, beyond
the stage of miracidium, is unknown.

Another fluke which has been reported from the lung is Fasciola gigantea (very
similar to F. hepatica). This was coughed up by a French officer who had been in
Africa.

BLOOD FLUKES.

Schistosomum haematobiuin. — Flukes of the circulatory system
are of great importance in Egypt, South Africa, Japan, and the West
Indies. The disease is named bilharziasis after Bilharz who in 1851
first associated the parasite and the disease.

It seems probable that there are at least three human species, differentiated
principally by the appearance of the egg. In the blood-fluke disease of Egypt,
(S. haematobium), the parasite chiefly infects the bladder and the egg has a terminal
spine. The terminal-spined ovum is also found in the rectum and in the faeces. In
the West Indies, as shown by the reports of Surgeon Holcomb from Porto Rico,
rectal bilharziasis is rather common. In these cases the egg is practically always
lateral-spined. Looss thinks that the lateral-spined egg is the product of an unfer-
tilized female S. haematobium. These flukes differ from other human flukes in
possessing nonoperculated eggs as well as in having the sexes separate. The adults
of this species, the S. mansoni, are scarcely, if at all, to be distinguished from the
S. haematobium. Leiper has recently noted a difference in that the male of S.
mansoni has 7 testicles as against 4 for S. haematobium. With S. japonicum, the
name of the Eastern species, there is not only the difference that the eggs are without
spines, but, in addition, the skin of the adult parasite is not tuberculated, as is the
case with the other two species. It is slightly smaller, the acetabulum projects more
prominently, and the lower part of the male infolds more markedly than in S.

FLAT WORMS

haematobium. Catto considers that the S. japonicum may live in both arteries and
veins. The other two species only live in branches of the portal vein. The blood
flukes are about 1/2 inch (13 mm.) long. All of these flukes live separately until
maturity. At this time the female enters what is known as the gynaecophoric canal
of the male; this canal is formed by the infolding of the sides of the flat male fluke,
thus giving a rounded appearance to the male. The female is longer than the male
(about 5/6 of an inch long), and is thread-like and of a darker color. Her two
extremities project from the canal of the male in which she lives.

The oral sucker of the male is infundibuliform and is smaller than the peduncu-
lated acetabulum. In the female the oral sucker is larger than the acetabulum.
The eggs are fusiform, yellowish in color, have a thin shell and a terminal spine.

The most prominent symptoms of the Bilharz disease are haemat-
uria and bladder irritation; later on calculus formation. In rectal
bilharziasis the symptoms are more those of bleeding piles or of a mild
dysentery.

There may also be involvement of the appendix. In the Japanese infection
the symptoms point more to liver and spleen, there being ascites, cachexia, and a
bloody diarrhoea.

The eggs of the S. japonicum are readily found in the fasces; they are about
100 X 70/4. They are oval, transparent, and with a smooth shell, within which can
be made out the outlines of an embryo. Upon adding water the ciliated embryo
begins to show movement in about ten minutes and shortly afterward bursts out
of the shell and swims about actively. It is more melon-shaped than the miracidium
of S. haematobium.

The life history is not known of any of these flukes. Looss conjectures that it
is probable that the miracidium enters the skin, not requiring an intermediary host.
Frequent experiments have failed to show any mollusk, etc., which attracted the
embryo. Evidence seems to show that those who are constantly wading about in the
water of the pools or the mud of the fields are the ones most subjected to infection.

Katsurada, by experiments with a cat and dog, has proved that infection will
take place through the shaved skin of an animal held in infected water — none of
the water being allowed to enter by mouth. Fully developed miracidia and male
and female flukes were found in the portal vein. It is thought that further develop-
ment of the miracidia in the body may account for the heavy infection.

Turner has recently noted the frequency of bilharzial affections of the lungs in
South Africa (50% in natives) and he thinks this may be an important factor in
prevalence of lung diseases in the natives of this region. He considers bathing in
contaminated waters of prime importance in the causation of the infection which
he thinks is probably by way of the skin.

A recent view is that the miracidium enters while bathing by the preputial
channel, hence the value of circumcision.

If urine containing eggs is diluted with water the miracidium breaks out of the
shell and swims about as if in search of some desired object.

The view is also entertained that the miracidium may gain access to the body
through the drinking water; there is much evidence against this. However access

TAPE WORMS

253

to the body is gained, it is known that the larval forms make their way to the liver
where they develop. Arriving at maturity, the males and females become united
and proceed to the terminal branches of the portal vein, where the irritating eggs,
given off by the female, give rise to the symptoms.

Ova or the Parasitic Worms or Man •
TREMATODA

N TO SCALE X ICOO

Heterophyes
heterophyes

(after Loo*S.IQO,'5) ,«y

Di
coe

l&ncetxtum

Opisthorchis
felineus^ne

Clqnorcliis Clonorchis
sihensis endemicus

(Nodi:i.-il fiui:. LOOM.IW)

Fasciola Fasciolopsis

heptxtica buskii (*^^

{n.f\crtoo«s.l905)

FIG. 66. — Trematode ova.

CESTODE OR TAPE-WORM INFECTIONS.

The cestodes and trematodes constitute the two great divisions of
the flat worms. Anatomically, a tape-worm may be considered as a
series of individual flukes united in one ribbon-like colony. The
cestode segments, or proglottides are covered by an elastic cuticle and
in their interior usually contain striated elliptical bodies composed of
calcium carbonate about 5 to 25/1 according to the species in which they
are found.

These calcareous bodies are characteristic of cestode tissue. They have been
mistaken for coccidia. There is no mouth or alimentary canal in tape-worms, the
segments absorbing their nourishment through the general surface.

254

FLAT WORMS

A tape-worm is divided into the segment-producing controlling head and the
series of segments or proglottides together known as the strobila. The head and neck
together form the scolex. Tape-worm heads are provided with suctorial or hook-
like organs, or both, to enable them to hold on to the intestinal mucosa.

The hooks when present on the anterior extremity of the head are carried by a
protrusible structure called the rostellum.

The importance of the head is generally recognized by the well-
known fact that the permanent evacuation of one of these parasites is
only arrived at when the head as well as the segments is expelled.
Otherwise, additional segments will be produced.

10

7-0%

FIG. 67. — Tape-worms. A. i, 2 and 3, Scolex, proglottides and ovum of Taenia
solium; B. 4, 5, 6 and 7, Scolex, prologlottides and ovum of Dibothriocephalus latus;
C. 8, 9, and 10, Scolex, proglottides and ovum of Taenia saginata. The onchosphere
in 10 is shown within the outer yelk coating (frequently seen in stools). In 3 only
the onchosphere within the embryonal shell is shown.

Even in tape-worms twenty-five to thirty feet in length, the head is no larger
than a small shot. It carries the suckers or hooklets which best enable us to differ-
entiate the different species. The segments adjacent to the head are immature —
the sexually-mature ones being found from the middle of the body onward. The
sexually-mature segment possesses a varying number of testicles: three in Hymeno-
lepis nana and as many as 2000 in Taenia saginata. As with the flukes, they also
have vasa deferentia, cirrus, ovaries, yolk glands, uterus, genital pore, etc. The

TAPE WORMS 255

location of the genital pore and the character of the branching of the uterus are of
the greatest importance in differentiation. The sexually-mature proglottides may
either expel their ova, when these would be found in the faeces or, as is common,
they break off and pass out themselves in the faeces. Then they either expel the
eggs or may be eaten by some animal and in this way effect an entrance for their ova.
It is an important practical point that the fasces of a patient with T. solium or T.
saginatamay not show any ova, these passing out in the intact segments. The oval
operculated eggs of Dibothriocephalus latus, however, are constantly in the faeces.
The "hexacanth" or six-hooked embryo,-also called the onchosphere, is the essen-
tial part of the egg. The embryonic envelope is dissolved off in the alimentary
canal of the animal ingesting it, and the onchosphere bores its way through the gut
to later become encysted in various tissues. In some tape-worms a ciliated embryo
is liberated from the egg shell and swims about actively to enter some fish or other
animal. When the six-hooked embryo reaches its proper tissue, the hooklets are
discarded and a scolex similar to the parent one is developed. At this time we have
a bladder-like structure with the scolex inverted in it. This is termed the proscolex
stage. This little cyst with its scolex when ingested by another animal is digested,
and the scolex, establishing itself in the intestine, develops a series of segments.
The ciliated embryo of the D. latus does not form a cyst, but instead a worm-like
creature similar to the adult. This is termed a Plerocercoid.

If the larval stage shows a single cyst and a single head, it is termed
Cysticercus; if multiple cysts but only one head to each cyst, Ccenurus;
while with multiple cysts and multiple heads in each cyst the term
Echinococcus is used.

Where there is very little fluid in the cyst and the larva is of minute
size, as with the Hymenolepis, the term Cercocystis is employed.

KEY TO CESTODE GENERA.

I. Head with two elongated slit-like suckers — Genital pores ventral — Rosette
uterus. Dibothriocephalidcz.

(A) Single set of genital organs in each segment. Dibothriocephalus.

(B) Double set of genital organs in each segment. Diplogonoporus.

(C) Immature fofms showing characteristics of Dibothriocephalidse— (collective
group). Sparganum.

II. Head with four cup-like suckers; genital pores lateral. T&niida.

(A ) Uterus with median stem and a varying number of lateral branches. Tania.

(B) Uterus without median stem and lateral branches.

(1) Genital pores single. Rostellum with not more than two rows of hooks.

(a) Suckers armed with numerous small hooklets. Fifteen to twenty
testicles in each segment. Davainea.

(b) Suckers not armed. Three testicles in each segment. Hymenolepsis.

(2) Genital pores double. Rostellum with four or five rows of hooks.
Dipylidium.

256 FLAT WORMS

INFECTIONS.

Taenia saginata (Tsenia mediocanellata).— This very widely dis-
tributed tape-worm is often termed the unarmed tape-worm, to dis-
tinguish it from the T. solium or armed tape-worm.

It is from 10 to 25 feet long and has several hundred proglottides. The small
pear-shaped head has four pigmented elliptical suckers and no hooklets. The seg-
ments are plumper than those of T. solium, hence the name saginata. The single
lateral genital pore projects markedly and in a series of segments presents, as a rule,
first on one side, and then on the opposite side of the next segment (alternating).
The best way to distinguish a segment of the T. saginata from the T. solium is by
counting the number of lateral uterine branches; these number fifteen to thirty, are
quite delicate and branch dichotomously. The lateral divisions of the uterus of
the T. solium are tree-like in their branching and only number five to twelve on each
side.

T. solium has three ovaries while T. saginata has only two. The ox is the inter-
mediate host. The eggs of Taenia have an oval outer shell which is filled with
rather translucent, refractile yolk, often in globules. Within the oval shell is the more
rounded cell of the six-hooked embryo with its thick striated membrane. The outer
shell is often absent in the eggs found in the faeces, only the shell of the six-hooked
embryo being found. The six-hooked embryo, having worked its way from the
alimentary canal to the muscles or liver of the ox, becomes encysted (Cysticercus
bovis). This little bladder-like structure is about 1/4 by 1/3 inches, and contains
but a small amount of fluid. Being ingested by man's eating raw or imperfectly
cooked meat, the adult stage becomes established in his alimentary canal.

It is probable that the various raw-meat cures have made the infection more com-
mon. In Abyssinia the infection is said to be universal, and a man without a tape-
worm to be a freak. An important point is the fact that the larval stage almost
never appears in man. It is this fact which makes it a so much less dangerous para-
site than the T. solium, which readily establishes a larval existence in man if the ova
are introduced into the human stomach. Cooking meat always destroys the
cysticercus. A period of about two months elapses after the ingestion of the cysti-
cercus before the mature segments pass out of the rectum. These not only make
their exit with the faeces, but are also capable of wandering out at other times. In
this they differ from the segments of T. solium. T. saginata next to Hymenolepis
nana is the common tape-worm of the United States. Dr. Stiles has examined
several hundred tape-worms in the United States during the past few years and
has found only one T. solium.

Abnormalities of the scolex and proglottides are not uncommon with T. saginata.
This is less frequently the case with T. solium.

Tsenia solium. — -The measly-pork tape-worm is smaller than the
T. saginata and differs from it in having a globular head, with a rostellum
which is crowned by twenty-six to twenty-eight hooklets.

In T. saginata a depression takes the place of the armed rostellum; the suckers

DWARF TAPE WORM 257

of T. saginata are, however, much more powerful than those of T. solium. The
segments have only five to ten coarse branches and are expelled only at the time of
defecation. The segments or the ova having been ingested by a hog, the six-hooked
embryo is liberated and becomes encysted chiefly in the tongue, neck, and shoulder
muscles of the hog, as an invaginated scolex. Pork containing this cysticercus
(Cysticercus cellulosae) is known as measly pork. This cysticercus contains much
more fluid than that of the ox and is from 1/4 to 4/5 of an inch long. If one by
chance should carry the egg on his fingers to his mouth, as the result of examining
mature segments, the larval stage may be established in man. If this infection is
not heavy, very few symptoms may be observed. The cysticercus, however, tends
to invade the brain, next in frequency the eye, and so causes convulsions, death or
blindness. Instead of only being the size of a pea, these cysts, when forming in the
brain, may be the size of a walnut or larger. T. solium is comparatively common in
North Germany, but is exceedingly rare in England and the United States.

Taenia africana. — This is an unarmed tape-worm, only about 5 feet long. It
was found in a native soldier in German East Africa. -

Garrison has reported from the Philippines a tape-worm with an unarmed
rostellum, V-shape and spiral formation of the uterine stem with compact structure
of the gravid uterus under the name of Taenia philippina. Another tape-worm,
T. confusa of which only segments were found was reported by Ward from Nebraska.

Hymenolepis nana (Taenia nana).— This is generally known as the
dwarf tape-worm — -it is the smallest of the human tape-worms. It is
from 1/4 of an inch to 1/2 inch in length, and is less than 1/25 of an
inch in breadth. (zoX i m.m.)

The genus Hymenolepis has lateral genital pores, all of which are on the same
side. These lateral genital pores cannot be made out in specimens as ordinarily
examined. The head has four suckers and a rostellum, which is usually invaginated.
The rostellum has a single row of twenty-four to thirty hooklets encircling it. Of
the 150 to 200 narrow segments the terminal ones are packed with eggs which in the
last two or three seem to fill entirely the disintegrating segments. It would seem
that the fully mature segments disintegrate and in this way the eggs are set free in
the surrounding intestinal contents.

The worms as found in fresh faeces after taeniacide treatment are frequently in
an advanced state of disintegration so that it is impossible to make out the head or
hooklets.

The eggs of this species are quite characteristic, there being two distinct mem-
branes. The inner one has two distinct knobs, from which thread-like filaments
proceed. The eggs of the H. diminuta have a thicker, striated, outer membrane
and there are no filaments. The eggs of the Dipylidium caninum are similar, but are
found in the faeces in aggregations — several eggs in a packet.

The dwarf tape-worm has been found to be the most common tape-
worm in the United States. Dr. Stiles found it in about 5% of children
in a Washington orphanage.
17

258 FLAT WORMS

It has been estimated that in certain parts of Italy 10% of the children may be
infected. The symptoms, expecially nervous ones, may be marked in this infection.
It has been incriminated as a cause of chyluria. Although very small, yet the num-
ber of parasites may be very great, even more than 1000. In a case that I treated
with thymol there were 1500 worms expelled. A form found in rats, which may be
identical with H. nana, does not require an intermediate host. The six-hooked
embryo bores into the intestinal villus and there develops a Cercocystis (larva of
small dimensions with but little fluid). When fully developed, it drops into the
lumen of the gut, and a new parasite is added to the already existing number of
parasites. This explains the heavy infection. H. diminuta and H. lanceolata have
also been reported for man a few times.

H. diminuta is much larger than H. nana, being about 10 inches long. The
suckers are small and the rostellum insignificant and unarmed. The intermediate
host is some insect, as a moth; the definitive, the rat. As man is not liable to eat
the insect hosts the infection is rare in man. Twelve cases have been reported for
man of which 5 were from the U. S.

H. lanceolata is common in geese and ducks.

Dipylidium caninum (Taenia cucumerina) (T. flavopunctata) . — This is a com-
mon parasite of dogs and cats. The larval stage is passed in lice .and fleas. The
cases of human infection have been principally in children, probably from getting
dog lice or fleas in their mouths. The number of infections reported for man is
about 40 and of these about 30 in children. The head has four suckers and a
rostellum, which has three or four rows of encircling hooklets. The segments have
the shape of melon seeds and have bilateral genital pores.

Davainea madagascariensis. — This tape-worm has been found in Siam and
Mauritius. It is about 10 inches long. The head has four suckers and a rostellum
with ninety hooklets. The suckers have rings of hooklets. The genital pores are
unilateral. The cockroach is supposed to be the intermediate host.

There have been about 10 cases reported (Madagascar, Siam and British
Guiana). There has also been reported a D. asiatica, the single specimen, however,
lacking a head so that the exact genus is doubtful. It has been reported twice in
children in Breslau. The intermediate host is thought to be a cyclops. Garrison
reported cases from the Philippines.

DlBOTHRIOCEPHALID^E INFECTIONS.

Dibothriocephalus latus (Bothriocephalus latus). — This is fre-
quently termed the broad Russian tape-worm. It has a small olive-
shaped head with two deep winding suctorial grooves on each side ; it has
neither rostellum nor hooklets.

The segments are quite broad., being about 1/2 by 1/5 inch. At the end of the
strobila they are more nearly square. The segments are very numerous, 3000 or
more. The fully developed worm is about 30 feet long. The uterus in each segment
is rosette-shaped and the genital pore is ventrally situated. The eggs of this species
have an operculum and a ciliated embryo. This ciliated embryo swims around and
either enters some fish, especially pike, directly or through an as yet unknown inter-

HYDATID CYSTS 259

mediary. This parasite produces an intense anaemia similar to pernicious anaemia.
It is a frequent parasite in Switzerland, Bavaria, Japan, Scandinavia, and Russia.
Recently several cases have been reported from our Northwest, and some of the fish
of the waters of that region are said to be infected. The larva is a pleroceroid and
is about i inch long. It is said that salting, smoking, or other ordinary methods of
preserving fish will not kill it.

A tape-worm, Diplogonopoms grandis has been reported from Japan. In this
there are two complete sets of genital organs to each segment.

SOMATIC T^ENIASIS.

While rarely we may have the larval stage of T. solium present in
man, and while certain bothriocephalid larvae (Sparganum mansoni
and Sparganum proliferum) infect man, yet they are unimportant as

FIG. 68. — Daughter cyst from FIG. 69. — A group of daughter

hydatid cyst, considerably en- cysts from hydatid cysts,

larged. (Coplin.} (Coplin.}

compared with the larval stage of the Taenia echinococcus. The
adult stage of this parasite is passed in dogs. It is one of the smallest
tape-worms known, being only about 1/6 inch long. It has a head with
four suckers and a rostellum encircled with hooks. There are only three
to four segments. The larval stage, on the contrary, gives one of the
largest of larval cestodes. In man it may reach the size of a child's
head. The larval stage is also found in hogs and sheep, and it is prob-
able that by reason of the dog's eating the echinococcus cyst of such
animals at the abattoir we owe the increase in this serious infection.

Man contracts the infection from association with dogs. The disease is peculiarly
prevalent in Iceland. As stated above, the adult stage is passed in the intestine of
the dog. Should the egg-bearing segments passed by the dog contaminate the hands

260

FLAT WORMS

of man and a single egg be ingested, we may have hundreds of Taenia larvae produced.
The six-hooked embryo, leaving its shell, bores its way through the walls of the
alimentary tract and especially seeks the liver, just as the embryo of T. solium seeks
the brain and eye.

Griffith notes that in Australia from 10 to 15% of hydatid cysts
occur in the lungs. The cyst wall is quite thin and the hydatid cachexia
seems to appear earlier in the lung than in the liver cases.

In the development of the cyst, after the embryo has come to rest at some point
in the liver, we have formed at first an indistinctly laminated external envelope

s.

FIG. 70. — Tape-worms, i, 2, and 3, Head, melon-shaped segments and egg
packet of Dipylidium caninum; 4, 5, 6 and 10, entire worm magnified, head, larval
stage in intestinal villus and ovum of Hymenolepis nana; 7, echinococcus cyst; A,
mother cyst; D, daughter cyst; E, granddaughter cyst; C, scolex in brood capsule;
B, brood capsule; G, parenchymatous layer; F, laminated layer; 8 and 9, Taenia
echinococcus; 9, natural size.

with coarsely granular fluid contents. Later on the contents become transparent,
and two distinct layers can be observed: i. The external, markedly laminated one,
and 2. the internal one, made up of small cells externally and large cells and cal-
careous corpuscles internally. This internal lining membrane is known as the paren-
chymatous or germinal layer. When the external layer is incised it curls up by
reason of its elasticity. This is characteristic of such a cyst. In addition, we have
an enveloping connective-tissue capsule formed by the surrounding liver substance.
From the germinal layer arise the brood capsules and the scolices. In these brood

CESTODES

261

capsules we have the cellular layer external — just the reverse of the mother cyst.
Scolices may develop either on the outside or inside of these brood capsules. It is
interesting to note that one onchosphere may develop hundreds of scolices. From
the parenchymatous layer of the mother cyst, daughter cysts are formed; these have
an external stratified layer and an internal parenchymatous one; within them a vary-
ing number of scolices may develop. From these daughter cysts, granddaughter
cysts may arise — all within the mother cyst — and hence are termed endogenous.

At times the daughter cysts work their way external to the mother cyst and pro-
ceed to develop in a manner similar to the endogenous formation. The exogenous

Ova or the Parasitic Worms or Man
CESTODA

DRAWN TO SCALE X . • I <S O O

Tivniix
solium

n Diplo^a^ Dibothrio^

cfonoporus cephalus nymenolepis ntxna.

^m^ 3 f*r>cJ*^r*ln 1*>\tlI«S/ \ te.rtcrRon8Qm,fro«BSt.tlc»!9(O)

/?%v dr&nais. iuius(*rten,o<m.«>«)

If 'l)\m O (after Ioo»».l$a6) . _,=-— ¥-1 t • '•

s^^\nymenolepi

iw^«^%^r ^x \ diminuta

TeemaXgp^ f%\ \^^^fn>.^^^

sadinata .

OM'f'i-^'iow) T\- i- i

Uipylidium

num^mrs.^iooo)

Cestode sepments

DRAWN TO SCALt X IO W

ggjj^^jgj ^^MauMtTciy,

Decvtvinea

Eni njL, SSL- *"!fc

solium eaninum Dibothriocephalus latus — s

m*™/"" (/.S.A'tu-atMedicaJ School.

FIG. 71. — Cestode ova.

development is rare in man, but common in hogs. Hydatids containing no scolices
are called sterile. These cysts may be as large as a child's head, but are usually
smaller. The fluid of these cysts contains about i% of NaCl, also a trace of sugar;
in addition there is a toxin which produces urticaria and acts as a cardiac depressant.
If any quantity should escape into the peritoneal cavity at operation, it may cause
death. Hydatids develop very slowly, and the duration of the disease is usually
from two to eight years.

Echinococcus multilocularis is possibly due to a species different from T. echino-
coccus. In this we have a honeycomb arrangement with cavities filled with a gela-

262 FLAT WORMS

tinous material. The majority of these cysts are without scolices. This form of
hydatid is very fatal.

Sparganum mansoni (Bothriocephalus liguloides) . — This is a larval bothrio-
cephalid which is about 5 to 10 inches long and has been reported ten times in Japan.
It has been found in various parts of the body, as in pleural cavity, tissues about
kidney, and in abscess of the thigh. They have been found in the urethra and under
the conjunctiva. They resemble ribbon-like strings of fat.

Sparganum prolifer (Plerocercoides prolifer). — This has been reported from
Japan as a larval form in the subcutaneous tissue. Stiles has found these larval
forms in skin lesions in Florida. They show themselves as bizarre grub-like forms.
They reproduce by budding.

CHAPTER XVIII.

THE ROUND WORMS.

CLASSIFICATION or THE NEMATHELMINTHES (ROUND WORMS ) .

Class.

Nematoda

Family.
Angiostomidae

Filariidae

Genus.

Species.

Trichotrachelidae

Strongylidae

Ascaridae

Strongyloides

S.

stercoralis.

Dracunculus

D.

medinensis

F.

bancrofti

F.

loa

F.

perstans

Filaria

F.

demarquayi

F.

ozzardi

F.

philippinensis

IF.

volvulus

Trichuris

T.

trichiura

L Trichinella

T.

spiralis

Eustrongylus

E.

gigas

Strongylus

S.

apri

Trichostrongylus

T.

instabilis

Triodontophorus

T.

deminutus

CEsophagostoma

0.

brumpti

Phy salop tera

P.

caucasica

Ancylostoma

A

duodenale

Necator

N. americanus

Ascaris <

f A.

A

lumbricoides

t A.

canis

[ Oxyuris

O.

vermicularis

Gigantorhynchus

G. gigas

{Hirudo

H

medicinalis

Limnatis

L. nilotica

Hasmadipsa

H. ceylonica

Acanthocephala
Hirudinea

NOTE. — The Strongyloides stercoralis was formerly described under two desig-
nations: (i) Anguillula intestinalis, a parasitic generation and (2) Anguillula ster-
coralis, a free living generation.

ROUND WORMS OR NEMATODES.

All nematodes are covered by a cuticle which varies in thickness,
and is frequently ringed. The cuticle is moulted three or four times.
The cuticle is formed by the underlying ectoderm which is, as a rule,

263

264 THE ROUND WORM

markedly developed in four ridges which divide the body into quadrants.
Within the ectoderm is the body cavity, a space in which the reproduc-
tive organs lie in a clear fluid. The excretory system usually consists
of two tubes which discharge near the head.

While the alimentary canal is more or less tube like in appearance it shows near
the mouth a muscular oesophagus with a bulb-like expansion at the commencement
of the remainder of the intestinal tract.

The testis and ovary are generally tube like. The sexes are, as a rule, separate.
The male can usually be recognized by its smaller size, its curved or curled posterior
end, and at times exhibiting an umbrella-like expansion — the copulatory bursa.
The spicules, chitinous copulatory structures, may be observed drawn up in the worm
or projected out of the cloaca. The genital opening of the female is ventral and
usually about the mid-point; that of the male is close to the anus.

Certain papillae in the region of the anus are valuable in differentiation. As a
rule nematodes develop in damp earth from the eggs as rhabditiform larvae. Very
few nematodes are viviparous (Filaria, Trichinella).

The families Gnathostomidae and Anguillulidae are of very little
importance in human parasitology. Gnathostoma siamense was once
found in a breast tumor and Rhabditis pellio once in the urine.

Anguillula aceti, the vinegar eel, has been reported from the genito-urinary tract
several times. Such cases can be explained by the prior contamination of the urine
bottle or by the use on the part of the patient of a vinegar vaginal douche. The
genera Rhabditis and Anguillula belong to the family Anguillulidae.

A case of infection with a small nematode found in the papules of a skin infection,
in a French boy is recorded as due to Rhabditis niellyi. The present view is that
the parasites were embryos of A. duodenale, boring into the skin.

ANGIOSTOMHXE.

In this family we have heterogenesis.

Strongyloides stercoralis. — This parasite was formerly thought
to be the cause of Cochin-China diarrhoea. It presents two genera-
tions: i. Parasitical or intestinal form. 2. The free living or faecal
form.

i. The intestinal form (also known as Anguillula intestinalis) is represented only
by females. These are about 1/12 of an inch (2 mm.) long and reproduce partheno-
genetically. They have a pointed, four-lipped mouth, and a filariform oesophagus
which extends along the anterior fourth of the body. The anus is situated near the
sharpened posterior end, the vulva about the lower third of the body. The uterus
contains a row of 8 to 10 elliptical eggs which stand out prominently in the posterior
part of the body by reason of being almost as wide as the parent worm. They
usually live deep in the mucosa and the embryos emerge from the ova laid in the

FILARIASIS 265

mucosa. The embryos escape from the eggs while still in the intestine, so that in
the faeces we only find actively motile embryos. The eggs, which are strung out
in a chain, never appear in the faeces except during purgation. As they greatly
resemble hookworm eggs, this is a point of great practical importance. In fresh
faeces we find hookworm eggs and Strongyloides embryos. The embryos are rather
common in stools in the tropics. These embryos have pointed tails and are about
250 X 13/4. They have a double cesophageal bulb. They are about 250^ when they
first emerge but may grow until they will approximate 500/4 in the faeces. If the
temperature is low, these rhabditiform embryos develop into filariform embryos,
which being ingested form the infecting stage. It has been demonstrated that
infection of man may also take place through the skin. If the temperature is warm,
25° to 35° C., these embryos develop into:

2. The free living form, Anguillula stercoralis. In this we have males and
females, with double oesophageal bulbs, the male about 1/30 of an inch (3/4 mm.) long
with an incurved tail and 2 spicules and the female about 1/25 inch (i mm.) long
with an attenuated tail; these copulate and we have produced rhabditiform larvae,
which later change to filariform ones. At this time the length is about 550 microns.
These, being ingested, start up the parasitical generation. If these do not reach the
intestine they die out.

FILARIID.E.

This family is of the greatest importance to man. It is also one
about which much confusion exists as to the adult type; hence anyone
finding adult filariae should fix them in hot 5% glycerine alcohol
(alcohol 70%), and subsequently mount in glycerine gelatin. Formalin
is not to be used, other than for a very brief period (2 to 6 hours) and
then followed by the lacto-phenol method.

These worms are most likely to be seen as writhing thread-like worms, especially
in the lymphatic glands and connective tissue, and about body cavities. They have
a lipped or simple mouth and a filariform oesophagus. The male has an incurved
tail with-preanal and postanal papillae which may be even corkscrew-like as in F.
immitis. The spicules are unequal or there may be but one. The female is ovovivi-
parous, the vulva is at the anterior end and the uterus usually double.

Dracunculus medinensis (Filaria medinensis).— The Guinea or
Medina worm, of which until recently only the female was known, is
of great importance in parts of India, Africa, and Arabia. The female
is a thread-like worm, about 20 to 30 inches long. The habitat is the
subcutaneous and intermuscular connective tissue, especially of the
lower extremity. It develops without symptoms. Finally a blister-
like area appears on the surface of the leg, particularly about ankle-
joint, which soon forms a painful ulcer. From this opening the

266

THE ROUND WORMS

anterior end of the worm projects to pour forth its striated embryos
upon contact with water.

The mouth is terminal and the body uniformly cylindrical. The uterus is a con-
tinuous tube filled with sharp-tailed, transversely striated embryos, 650X17^, and
constitutes the greater part of the body, the alimentary canal being pressed to one
side. The genital organs probably discharge through the oesophagus. The body
when being extracted is rather transparent. The tip of the tail is bent, forming a
sort of anchoring hook. Recently Leiper fed monkeys on bananas containing in-

7-08.

FIG. 72. — Round worms (Filariidae). i. Hooked posterior extremity and ante-
rior extremity of Dracunculus medinensis; 2, cross section of uterus filled with embryos,
D. medinensis; 3 and 4, free embryo and embryos of D. medinensis in intermediate
host (Cyclops); 5, natural size of female Filaria bancrofti; 6, embryo of F. bancrofti
in blood; 1 7, tail of male F. bancrofti; 8, male and female of F. loa (natural size);
9, tuberculated integument and posterior end of male F. loa; 10, posterior end of male
F. perstans; n, male of F. bancrofti (natural size); 12, blunt-tailed embryo of F. per-
stans; 13, sharp-tailed embryo of F. demarquayi.

fected Cyclops, and at the autopsy six months later obtained both male and female
forms.

As regards the life history, Fedschenko, in 1870, showed that the embryos when
liberated swam around in water and finally entered the bodies of species of the genus
Cyclops. The female tends to come to the surface in the lower extremities, and
experiments show that if on the blister-like points of emergence some water be
squeezed out from a sponge, the uterus will eject a milky-looking fluid containing

FILARIASIS 267

myriads of embryos. This would indicate that the worm selects the lower extremity
so that the embryos may gain access to the Cyclops when the host is wading through
the water.

Leiper showed that a strength of HC1 equal to that of gastric juice killed the
Cyclops, but made the Dracunculus embryos very active. From this he judged that
infection must probably take place from drinking water containing infected Cyclops.
The suggestion of Leiper that wells harboring Cyclops be treated with steam, intro-
duced by a pipe, seems to be valuable. The disease is known as "Dracontiasis."

Filaria loa (Filaria oculi). — This is a thread-like worm of West
Africa about i to 2 inches long. The cuticle is characterized by
distinct wart-like structures.

The anterior extremity is like a truncated cone with two papillae at the base of
the cone. The wart-like cuticular protuberances or bosses are about 12 to 15
microns in height. The females are 2 to 3 inches (50 to 70 mm.) long and about 1/2
mm. broad.

The males are smaller than the females and have three preanal papillae and two
postanal ones. There are two short unequal spicules. The life history is not satis-
factorily established. The young are born ovoviviparously, and it has been sug-
gested that the localized oedemas, known as Calabar swelling, may be due to the
irritation produced by these eggs. These swellings are of hen's egg size, painless,
do not pit on pressure and last about three days. They occur especially on the hands
and arms. The embryos almost exactly resemble those of F. bancrofti. They have
a diurnal periodicity, however, appearing in the blood about 8 A. M., increasing to
noon and disappearing about 9 p. M. The adult worms have a tendency to
wander about in the subcutaneous connective tissue, especially about the region of the
orbit or even under the conjunctiva.

Adult worms of F. loa have been found and extracted, with an absence of the
filarial embryos in the peripheral circulation of the patient. Leiper has just noted
two species of Chrysops as intermediate hosts, the embryos developing in the
salivary glands.

Filaria bancrofti (Filaria sanguinis hominis). — -This is the most
important of the filarial worms. It is a common infection in South
China, India, the West Indies, and in the Pacific Islands, especially
Samoa.

In medical books the embryos have been designated Filaria sanguinis hominis.
This species is the cause of the common manifestations of filariasis, such as elephanti-
asis, varicose groin glands, chyluria, lymph scrotum, etc.

Filarial diseases are prone to lymphangitis attacks. Thus in lymph scrotum an
erysipelatoid condition of the scrotum with high fever and chills may result. This
condition is at times mistaken for malaria. Varicose groin glands may be mistaken
for hernia. In the Philippines very few symptoms are noted in those affected with
filariasis. Occasionally chylocele or chyluria is reported.

F. bancrofti lives in lymphatics of trunk and extremities. At times the fine
white thread-like worjms may be seen as writhing coils in lymphatic glands.

268 THE ROUND WORMS

The sexes are usually found together. The females are about 3 inches long
and the males less than 2 inches. The tails of both sexes are incurved, but that
of the male is more so. The head is club-shaped. The vulva opens 1.2 mm. from
the anterior end. There are 2 uterine tubules. The sheathed embryos are supposed
to be born viviparously and Manson supposes that as a result of injury to the parent
worm and resulting extrusion of eggs, the blocking of lymph channels occurs.

A very interesting fact is that people with elephantiasis fail to show larvae in the
peripheral circulation. Manson considers that it is due to the blocking of the lymph
channels.

These embryos show a nocturnal periodicity. During the day they
remain in the lungs, and larger arteries.

If the patient sleeps in the day time and is active at night the nocturnal perio-
dicity or presence of embryos in peripheral circulation is inverted. In the case of
F. loa, however, a change of habits does not change the periodicity of the filarial
embryos, they continue to appear in the peripheral circulation by day even if the
patient sleeps at that time.

The disease is transmitted especially by Culex fatigans. The sheathed embryos,
getting into stomach of mosquito, wriggle out of the sheath, they then bore their
way through walls of stomach and enter into a sort of passive stage, during which
further development takes place. They finally become distributed in the muscles
of the thorax and make their way along the fleshy labium, to enter the wound in
a person bitten by a mosquito, by way of Button's membrane. This takes about
twenty days at which time the larvae are about 1/16 inch long and have an alimentary
canal.

Filaria perstans. — The adults are found in connective tissue and deeper fat,
especially about the mesentery and abdominal aorta.

The female is about 3 inches (75 mm.) long; the male is rarely found and is less
than 2 inches long. These worms are characterized by incurved tails, the extremity
of which has two triangular appendages giving a bifid appearance. The embryos
do not possess a sheath and have a blunt tail. The life history is unknown. Both
mosquito and tick have been incriminated. The embryos are always present in
the peripheral circulation — hence perstans. There does not seem to be any symp-
tomatology.

It is of historical interest that F. perstans was once considered the cause of
sleeping sickness.

Filaria volvulus. — This is a rather common parasite of Central Africa. The
male is about 11/2 inches (35 mm.) and the female about 5 inches long. The
females are so interlaced in the fibro-cystic swellings that it is difficult to determine
their length. The tumors start from the presence of a worm in a lymphatic. The
tumors are easily enucleated. Adults are striated. They are found in cystic tumors,
especially about the axilla and popliteal space. The cystic contents contain abundant
sheathless larvae about 300/4 long; they are not found in the peripheral circulation.
Life history unknown, although it has been suggested that a species of Glossina
may be concerned.

Filaria demarquayi. — The habitat of this filarial worm is the West Indies.
The embryo has no sheath and has a sharp tail. Other filarial species which have

WHIP WORMS 269

been reported are F. magalhaesi, F. ozzardi, F. volvulus, F. powelli, and F. philip-
pinensis. A species called F. gigas is now considered to have been only the hair
of the leg of a fly. The embryos have usually been given such names as F. nocturna,
F. diurna, etc. Of course the embryos and the parent should have the same name.
It has been proposed to designate these embryos the same as the parent, but with
the use of the term Microfilaria instead of Filaria.

The points usually noted in the description of filarial embryos are:

1 . Presence or absence of periodicity of embryos in peripheral
circulation.

2. Presence or absence of a sac sheath around the embryo.

3. Accurate measurements.

4. Shape and description of head and tail ends.

5. Character of movement.

6. Location of V spot and break in cell column in stained
specimens.

KEY TO FILARIAL LARVAE.

A . Sheath present.

1. No periodicity.

F. philippinensis. Tightly-fitting sheath; not flattened out beyond extremi-
ties. Tail is pointed and abruptly attenuated. Lashing progression move-
ment. 320X6.5^.

2. Periodicity exhibited.

a. Nocturnal periodicity.

F. bancrofti (F. nocturna). Pointed tail; loose sheath; lashing movement.
300X7.5,". V spot QOjW from head; break in cells 50^ from head.

b. Diurnal periodicity.

F. loa (F. diurna). Pointed tail; loose sheath; 245 by 7 microns. V spot
60 to 70 microns from head, break in cells 40 microns from head.

B. Absence of sheath. None of these exhibit a periodicity, being continuously present.

1. Blunt tail — F. perstans. 200X4.5^-

2. Sharply-pointed tail:

a. F. demarquayi. 2ioX5/*.

b. F. ozzardi. 215X5,".

NOTE. — A filarial embryo, F. powelli, reported once. It has a sheath, nocturnal
periodicity, and is about i^oXs^1-

TRICHOTRACHELID^E.

These have a long thin neck and a thicker terminal portion. The
oesophagus is of the single row of cells type. The anus is terminal;
there is only one ovary.

Trichuris trichiura (Trichocephalus dispar). — This is usually called

270

THE ROUND WORMS

the whip-worm — 'the thickened body representing the handle and the
narrow neck the lash. It is one of the most common parasites in both
temperate and tropical climates.

The egg is very characteristic in having an oval shape with knobs at either extrem-
ity. It resembles a platter with handles. The male is almost 2 inches long, and
has the terminal portion curled up in a spiral. It has a single terminal spicule.

The female is a little longer than the male, and has the terminal part in the shape
of a comma instead of being coiled. The neck only contains the oesophagus which

FIG. 73. — Round worms, i, Encysted embryo of Trichinella spiralis; 2, male
and female of T. spiralis; 3, male and female of Trichocephalus trichiurus; 4, egg
of T. trichiurus; 5 and 6, head and male and female of Ascaris canis; 7, 8, and 9, head,
egg and male and female of Oxyuris vermicularis; 10, n and 12, head, egg and tail of
Ascaris lumbricoides; 13 and 14, head and egg of Echinorhynchus gigas; 15, 16 and
17, parthenogenetic female and rhabditif orm and mariform embryos of Strongyloides
stercoralis.

is contained in a groove in large cells which form a single row like a string of pearls.
These cells play a digestion role. The vulva opens at the upper end of the thickened
terminal end which contains an intestine lying between the ovary and uterus.
The great powers of resistance of the ova may account for their general distribution;
they may live for months under conditions of freezing and so forth. There is no
intermediate host. The worm arrives at sexual maturity in about one month after
ingestion. The whip-worm prefers the caecum, but also lives in the lower end of the
ileum and the appendix.

TRICHINOSIS

271

The neck burrows into the mucosa, and much importance has been attributed
by the French to the possibility of this paving a way for the entrance of pathogenic
bacteria. They do not seem to produce serious symptoms.

Trichinella spiralis (Trichina spiralis). — -The cause of trichinosis is
usually termed Trichina spiralis in medical works.

The adults live in the duodenum and jejunum; the males are about 1/16 of an
inch (1.5 mm.) long with two tongue-like caudal appendages and without a spicule.
These two lateral projections enable the male to hold the female in copulation —
the cloaca being evaginated to act as a penis.

The females are about 1/7 of an inch (0.3 to 0.4 mm.) long. The female gives
off embryos from the vulva which is near the mouth end (viviparous).

These parasites can be seen with an ordinary magnifying glass. With higher

FIG. 74. — Trichina spiralis (Ziegler).

powers the oesophagus has the appearance of a serrated line instead of an cesophageal
bulb. The male is about 40^ broad and has a prominent testicular enlargement
filling the posterior extremity. The female is about 6o/x broad and has a rounded
posterior extremity with a prominent slit-like cloaca. It is in this posterior extremity
that the female increases in size as she becomes filled with eggs. The vulva is in the
anterior third. After fertilization of the females the males die, and the females
bore into the intestinal mucosa and begin to produce embryos to the number of
more than 1000 each. These gain access to the lymph channels and are distributed
by the blood stream to the striated muscles. Embryos reaching other tissues fail to
develop.

It is about ten days before they reach the muscle. In the muscle they become
encysted as the oval lemon-shaped areas containing coiled-up embryos that every-
one is familiar with. These oval areas are about 450X250;* and have a chitinous
capsule.

The encysted trichinae are found chiefly in the muscle fibers of the tongue and

272 THE ROUND WORMS

diaphragm and may remain alive as long as ten to twenty years; finally, however,
the cyst undergoes calcareous infiltration and the embryo dies.

When uncoiled the embryo is about i mm. long with the mouth at the attenuated
end. Among cannibals it would be easy to keep the cycle going by eating improperly
cooked or raw human meat, the parasite being thus transmitted.

As this would not explain the transmission among civilized men, the
following is the life history: Man obtains his infection from eating raw
pork, the embryos encysted in the muscle of the hog being liberated in
the stomach, and the males and females developing in the intestine as
above described. The hog may gain his infection by eating the meat
of other hogs or rats. These rats eat scraps of pork at slaughter houses
and become infected. Being cannibals, rats when once infected, con-
tinue to propagate the infection. In man, during the first two or three
days while the adults are breeding in the intestine, we have gastroin-
testinal symptoms.

It is during this period or at any rate before the fifth day that
purging may be of benefit. About ten to twenty days after infection
the embryos begin to wander and we have the acute muscle pains.
In the diagnosis we should try to obtain specimens of the pork which
has caused the trouble in order to examine for encysted trichinae, or
to feed to white rats or rabbits, subsequently examining the diaphragm
of these animals for encysted trichinae or the intestine for adult trichinae.
Excision of a small piece of the deltoid of man may confirm the
diagnosis. The best method is to take blood in 3% acetic acid,
centrifuge, and examine for larvae.

During the diarrhceal stage we may examine the stools for adult worms, in
particular dead males or possibly actively motile embryos — these latter are about
90X6^.

Always examine the blood for eosinophilia.

It is well to remember that the parts of meat which trichinae prefer (muscle of
diaphragm, of neck, etc.) are often used in sausage. Unfortunately it is almost
impossible to detect the embryos in sausage meat.

STRONGYLID.E.

In this family the male has a caudal bursa, a prehensile sort of ex-
pansion at the posterior end for copulatory purposes.

The mouth is usually provided with six papillae and at times with a
chitinous armature. Those without the chitinous armature are in-
cluded in the subfamily Strongylinae (Strongylus, Trichostrongylus)

STRONG YLIDJ2 273

while those having an armed mouth are in the subfamily Sclerosto-
minae (Ancylostoma, Necator, Triodontophorus, (Esophagostoma,
Physaloptera).

Eustrongylus gigas (Strongylus renalis). — -This is the largest round
worm infecting man; it is usually found in the pelvis of the kidney
(giant strongyle).

Two or more worms may so distend the kidney as to convert it into a mere shell.
Pain, haematuria and other symptoms of pyuria, together with the finding of the eggs,
make the diagnosis. There seem to be seven authentic and eight doubtful cases
of infection in man.

The females are about 40 inches (i m.) long and about 1/3 of an inch (8 mm.)
in breadth while the male is about 10 inches (25 cm.) long.

The collar-like copulatory bursa of the male distinguishes it from Ascaris as
does also the dark red color. The source of infection is unknown but it has been
suggested that the larval stage may exist in fish.

Many of the reported cases were simply fibrinous clots from ureters or wandering
round worms.

The very characteristic ova, with gouged-out oval depressions, may be found
in the urine, and are diagnostically confirmatory.

Strongylus apri. — This nematode is common in the lungs of hogs, producing
a bronchitis in young animals but apparently harmless for adult ones. It has been
reported once from the lungs of a six-year-old boy. The male is about i inch (25
mm.) long with two long spicules. The female is about 2 inches long and has a
sharply hooked posterior extremity with the vulva just beyond the bend. The mouth
has six lips. The eggs contain embryos when laid.

Trichostrongylus instabilis. — This is a small strongyle formerly known as Stron-
gylus subtilis. The male is about 1/6 of an inch (4 mm.) long, and the female about
1/4 of an inch (6 mm.). Anteriorly it tapers to a pointed head end which is only
about one-tenth the thickness of the posterior extremity. The male has a bursa
and two prominent equal spicules. It has been found in the upper part of the small
intestine of inhabitants of Egypt and Japan. It does not appear to produce symp-
toms. Ova like hookworm ones (63 X 4 1 ft) •

Triodontophorus deminutus. — This is a small round worm with three forked
teeth taking origin from the pharyngeal lobes. The collar-like mouth orifice is
made up of 22 rounded plates just inside the round mouth opening. They are less
than 1/2 inch long and have once been found in the intestinal canal.

(Esophagostoma brumpti. — Six young females were found in a cyst of the colon
in an African negro. They were about 1/3 inch (8 mm.) long. The anterior end
presents an ovoid protuberance with a second cuticular inflation just below it. The
buccal capsule is very shallow and surrounded by about a dozen chitinous plates.
The mouth has six papillae.

This species has recently been reported by Thomas in a native of Brazil.

Physaloptera caucasica. — Mouth with two equal laterally placed lips, each hav-
ing three papillae and three teeth. The male has a lancet-shaped posterior extremity
and is about 1/2 inch long (14 mm. by 0.71 mm.). Female is about i inch long
18

274 THE ROUND WORMS

(27 mm.) with a rounded tail end. Found only once in the alimentary canal of a
native in the Caucasus. Leiper has recently reported a species P. mordens from
Uganda, one case.

Ancylostoma duodenale (Dochmius duodenalis.) — The hookworm,
so called from the hook-like appearance of the ribs of the copulatory
bursa or from the hook-like projection of the head dorsally, is probably
the most important of the parasitic worms. This species in Europe
and Africa and the Necator americanus in the New World cause an
immense amount of invaliding. The Egyptian anaemia and the Porto
Rican anaemia are caused by this parasite.

Goeze found a hookworm in a badger in 1782. He named the parasite Ascaris
criniformis. Froelich, in 1789, found hookworms in the fox and called them hook-
worms from the hook-like ribs of the copulatory bursa. He proposed the generic
name Uncinaria. Therefore Uncinaria belongs to the hookworms of the fox and
is not valid for any human species.

In 1838, Dubini found a hookworm as a human parasite. On account of the
four ventral teeth projecting from the mouth he gave it the name Agchylostoma or
correctly Ancylostoma.

Bilharz and Griesinger noted the connection of the parasite with Egyptian
chlorosis, but it was not until the time of the St. Gothard tunnel (1880), that the
importance of the parasite was recognized. Grassi noted the diagnostic value of
the ova in faeces in 1878. In 1902, Stiles noted and described the hookworm found
in the United States as different and proposed the name Uncinaria americana, later
changed to Necator americanus. A. J. Smith had also recognized the morphological
differences.

Hookworms may be found in the small intestine (jejunum) of man
in enormous numbers. They either produce their effects by feeding on
the mucosa or by causing loss of blood.

The males are little more than 1/3 of an inch (9 mm.) long and the females little
more than 1/2 inch (13 mm.) in length. The male can readily be distinguished
by his umbrella-like expansion or copulatory bursa. The tail of the female is pointed.
The vulva of A. duodenale is located in lower half of the ventral surface; that of
N. americanus in upper half. The large oval mouth of the Old World hookworm
has four claw-like teeth on the ventral side of the buccal cavity and two on the dorsal
aspect. In N. americanus the buccal capsule is round, smaller and the ventral
teeth are replaced by chitinous plates. Dorsally there are two similar but only
slightly developed lips or plates. A very prominent conical dorsal median tooth
projects into the buccal cavity. Through it passes the duct of the dorsal
cesophageal gland. The copulatory bursa of the N. americanus is also different,
being terminally bipartite and deeply cleft in the division of dorsal ray rather than
tripartite and shallow as with the A. duodenale.

The delicate-shelled eggs pass out in the faeces, and in one or two days a rhabditi-
form embryo (200X14/0 is produced.

THE HOOK WORM

275

The mouth cavity of the embryo is about as deep as the diameter
of the embryo at the posterior end of the mouth cavity; that of Strongy-
loides is only about one-half as deep as the diameter.

A temperature of i° C. kills the eggs in twenty- four to forty-eight hours. After
moulting twice, it remains rather quiescent but still lying inside the discarded skin.
It reaches this stage in from four to fourteen days according to the temperature.

The soil in the area of the hookworm-egg-laden stool becomes infested with these

FIG. 75. — i a, Copulatory bursa of Necator americanus, showing the deep cleft
dividing the branches of the dorsal ray and the bipartite tips of the branches; also
showing the fusion of the spicules to terminate in a single barb. Scale i/io mm.
ib, Branches of dorsal ray magnified. 2a, The buccal capsule of N. americanus.
2b, The same magnified. 3a, Copulatory bursa of Ancylostoma duodenalc, showing
shallow clefts between branches of the dorsal ray and the tridigitate terminations.
Spicules hair-like. 3b, The dorsal ray magnified. 4a, The buccal capsule of A.
dnodcnale, showing the much larger mouth opening and the prominent hook-like
ventral teeth. 4b, the same magnified. $a, Egg of N. americanus. 5b, Egg of
A. duodenale. 6a, Rhabditiform larva of Strongyloides as seen in fresh faces.
6b, Rhabditiform larva of hookworm in faeces eight to twelve hours after passage
of stool.

larvae which will even climb up blades of grass. It is for this reason that children with
their bare feet are so liable to infection. (If the larvae get into water they sink to the
bottom.) It is at this stage that it burrows into the skin of man, producing the so-
called "ground-itch " at the site of entrance. Having gained access to the lymphatics
and veins, they eventually reach the lungs. Here they get into the bronchioles and

276 THE ROUND WORMS

undergo a third moulting. They then work their way up the trachea to the glottis
and are swallowed to then become adults in the intestine. Dr. Stiles, while accept-
ing this theory of the life history, thinks it probable that infection is also brought
about by swallowing directly some infecting stage.

Very young dogs can be infected with human hookworm larvae, but infection of
man with the dog hookworm (A. caninum) has not been reported.

The infecting stage is not a young larva but one in which the cuticle
of a former larval stage instead of being cast off remains and acts as a
protecting sheath for the more mature larva within. In this stage
larvae may remain alive for six to twelve months and have greater
powers of resistance than younger larvae. Introduction, either by skin
or mouth, of these cuticle-covered larvae is followed by finding of eggs in
the faeces in about fifty days.

It has been claimed that where ordinary microscopical examination for ova will
show 40% of infections and methods involving concentration 55% that cultural
methods will show 99%. A convenient method of culturing is to make a pile of
filter-paper circles of 2 inches diameter and about 1/4 inch high and place in the center
of a 4-inch Petri dish. Fill the dish with water about to the height of the filter-paper
and spread a thick layer of faeces on the top of the filter-paper island. The larvae
hatch out in about six days and swim out into the clear surrounding water. They
are best found by centrifuging the fluid containing them.

Of the three standard drug treatments that of thymol seems to be preferable
to betanaphthol and vastly so to eucalyptus oil. In giving thymol it is imperative
that neither alcohol in any form nor fats in any form be given on the day of treatment.
Stiles prefers to divide his thymol into three doses, 1/3 at 6 A.M., 1/3 at 7 and 1/3
at 8 followed by epsom salts at 10 A. M. The patient should be on a restricted
diet and be given two doses of salts on the two days preceding the administration
of thymol.

Necator americanus. — This is the species of hookworm found in
the southern states of the United States and the West Indies.

It is very prevalent in Guam, L. I. It was found by Looss in pigmies from
Central Africa, so that this parasite was undoubtedly brought to America by slaves.
It is not rare in Ceylon, India and the Philippine Islands.

The copulatory bursa of this parasite has double spicules which fuse terminally
in a barb, while A. duodenale has two fine hair-like spicules. The head of Necator
has a more marked dorsal bend than Ancylostoma.

To sum up the differences between this species and A. duodenale
we have with Necator: i. Smaller oral cavity which shows rib-like
projections leading to the two ventral plates instead of the four promi-
nent projecting teeth. 2. The dorso-median ray of the caudal bursa is
deeply cleft and shows bipartite divisions terminally instead of having

ASCARID.E

277

a shallow cleft and tripartite division. 3. The vulva of the female is
placed in the anterior third.

The eggs of N. americanus are larger than those of A. duodenale. In hook-
worm disease we have ground itch, tibial ulcer, anaemia, interference with physical
and mental development and, in bad cases, dirt eating.

Shade, moisture and sandy soil seem essential factors for the development of
hookworm. Prophylaxis is essentially one connected with soil pollution. The

Ova or the Parasitic Worms or Man
NEMATODA

SCALE. x ieoo

A A

ORAWN

CrVMthout outer
envelope (Modt

/roin Stiles. \<t<£. an

ArShowind /
sides syifi-f,
metrically H
convex [{

(after Loos 5.,»«3t

over to
I show one
laid? flatten

edloriginoi)

Oxyuris

vermiculorisA.B.

Strongylus
subtilis

s, 1995)

Asccxris" Atfchvlostoma

lumbricoides ^^^^ d^SSSSS/g..,

A R f^ T\ Tr-i/^V-ii »t-i t_* Looss'ws')" in fresh stool

rrichuris Strongvloides

trichium ste'rcomlis

(OriginAl» x^bJ^LnjJiSUS

w: Embryo
M in stool
after 12 to46
, -- . hours.

Agchylostoma
duodenale

.MKHjt,

IOOS)

FIG- 76. — ^Nematode ova.

subjection of the faeces to some septic tank process is more reliable than chemical
disinfection or burying the faeces 8 to 10 inches under ground.

These have three papillae around oral cavity, one dorsal and two
ventral. The male has two equal-length spicules. An intermediary
host is not needed in the life history of this family.

Ascaris Lumbricoides. — The male round or eel worm is from 5 to

278 THE ROUND WORMS

8 inches (18 cm.) long and the female from 7 to 15 inches (30 cm.) in
length. They are from 1/7 to 1/4 of an inch (5 mm.) in diameter.

It is probably the most common parasite of man, especially in
children and as it does not require an intermediate host infection takes
place through food or drink or by ringers of children who have been
playing where soil pollution exists.

The normal habitat is the upper part of the small intestine, hence the ease with
which they are vomited up. The three papillae-like lips with a constriction just
behind are easily studied with a hand glass. The very long, whitish, convoluted,
thread-like tubes of the uterus lead to the- opening of the vulva anteriorly and ven-
trally. The male has two large lance-like spicules.

The body of the worm is transversely striated and resembles the ordinary earth-
worm, but is more grayish than red. The ova are very characteristic with a rough
mammillated exterior. This at times is shelled off and we have a smooth egg which
may be mistaken for eggs of other parasites. The eggs leave the body in the faeces

FIG. 77. — Anterior extremity of Ascaris lumbricoides ; A, seen from front; B, seen
from dorsal surface. (Tyson after Railliet.)

and after a long time — a few weeks to several months, according to temperature —
develop an embryo which remains in the shell until swallowed by man. It is stated
that they will remain alive for years. On being swallowed, the embryo leaves the
egg and we have males and females developing in the small intestine. In countries
where such parasites abound, as in Guam and the Philippines, the possibility of their
getting into the peritoneal cavity through operative measures on the intestine must
always be thought of.

Guiart considers it probable that Ascaris may suck blood, produce intestinal
ulcerations and bacterial infections, and perforate intestine. Their entrance into
bile ducts or into larynx (vomited) must be considered.

At autopsy they may be found perforating the appendix or even filling up the
pancreatic duct.

Some think that the symptoms of itching of nose and anus, vertigo, or convul-
sions and anaemia may be due to a toxin secreted by the worm.

Ascaris Canis. — This is a parasite of the dog and cat, but is occasionally found
in children. It is much smaller than the A. lumbricoides — male is 2 to 3 inches long,

THE PIN WORM 279

female 4 to 5 inches in length. The parasites are characterized by the presence of
wing-like projections from the anterior end (arrow-like head).

Oxyuris vermicularis. — This parasite is also known as the pin-worm
or seat-worm and is more frequent in children than in adults.

The male is about 1/6 of an inch long and the female a little less than 1/2 inch
in length. The male has an incurved tail with a single spicule and the female a long
tapering tail. The vulva is in the upper third.

These worms have a clear slightly bulbous, pipe mouth-piece-like projection
surrounding the three-lipped anterior extremity. There is a well-marked bulb
cesophagus.

The eggs are thin-shelled plano-convex, and show a coiled-up embryo. After
ingestion of eggs, the adults develop in the small intestine where copulation takes
place; the males then die. The fertilized females go to the caecum and colon where
they remain until they reach maturity. At this time the females wander to the rec-
tum where they either expel their ova or themselves work their way out of the anus.
This usually occurs at night, and the scratching induced by the itching causes the
eggs to be widely spread about the region of the anus. The worms may also wander
into the vagina, urethra, or under prepuce. It will be seen that as a result of the
scratching, the fingers become contaminated with o^a which may be carried to the
mouth and so cause a fresh infection, no intermediate host being required. The
examination of the material under the finger nails of children harboring this parasite
may show eggs under the microscope. A knowledge of the life history — the early
location in the small intestine, and later on in the large — shows that treatment should
be dual in its direction — enemata for the gravid female in the rectum and santonin
and calomel for the young adults in the small intestine.

The diagnosis is preferably made by examining the stools for the
white, thread-like females which are expelled after a diagnostic dose of
calomel and salts, rather than by searching for the eggs.

These females, which are packed with embryo containing eggs, may be seen
wriggling on the surface of the freshly passed fasces. In handling these worms
one should be careful as they are apt to cause infection should the eggs get on the
fingers.

ACANTHOCEPHALA.

These are called thorn-headed worms on account of a proboscis which projects
anteriorly like a little peg.

There are several rows of hooks surrounding this projection which are directed
backward to enable the parasite to attach itself to the intestinal wall. The worm
absorbs nourishment through the general body wall, there being no alimentary
canal or mouth. These worms are common in hogs. The three-shelled eggs are very
striking and the intermediate stage is in June bugs.

The Echinorhynchus or Gigantorhynchus gigas. — This parasite is about 6 inches
(15 cm.) long for the male and 10 to 12 inches (25 cm.) for the female. It has

280 THE ROUND WORMS

transverse rings and resembles Ascaris but is more white in color. It is said to be
not uncommon in southern Russia.

The Echinorhynchus or Gigantorhynchus moniliformis might be contracted
by persons eating death-watch beetles as is sometimes done for the improvement of
the complexion.

HIRUDINEA (LEECHES).

Hirudo medicinalis. — This is the leech used medicinally for the abstraction of
blood. They have a secretion which prevents coagulation of the blood so that when
they are removed the wound still continues to bleed.

Limnatis nilotica. — This species has been found in many parts of Northern
Africa and, gaining access to the stomach through drinking-water, it wanders to
the pharynx, nares, and even trachea. Manson refers to a case of obstinate epis-
taxis and headache caused by a leech in the nostril.

This leech is about 4 inches long (8 to 10 cm.) and about 1/2 inch (1.2 cm.)
broad. The dorsal surface is greenish brown with narrow orange brown borders.
The young leeches are only about 1/8 inch ( 3 mm.) long and taken in with the drink-
ing water may attach themselves to the surface of some mucous membrane and after
some weeks reach adult size.

Haemadipsa ceylonica. — Whese are land leeches found in India, Philippines,
Australia, and South America. They are only about i inch (25 mm.) long and are
slender. They leave the damp earth to climb shrubs and from there to drop on
animals or man passing through the forest. The bites are painless, but may be fol-
lowed by ulcers. They may get into the nostrils.

They will even penetrate thick clothing in order to reach the skin.

CHAPTER XIX.
THE ARACHNOIDS.

CLASSIFICATION OF THE ARACIINOIDEA.

Order.

Acarina

Family.

Trombidiidae
Gamasidae

Tyroglyphidae
Sarcoptidas
DemodicidcTg
Tarsonemidae

Subfamily.

Ixodidae

Argasinae

Ixodinae

Genus.

Species.

Trombidium

T. holosericeum

Dermanyssus

D. galling

Tyroglyphus

/ T. farinas
\ T. longior

Sarcoptes

S. scabiei

Demodex

D. folliculorum

Pediculoides

P. ventricosus

[Argas

/ A. persicus
\ A. miniatus

Ornithodoros

O. savignyi

Ixodes

I. ricinus

Hyalomma

H. aegyptium

Rhipicephalus

R. bursa

Dermacentor

{D. reticulatus
D. andersoni

Margaropus

M. annulatus

Amblyomma

A. hebrasum

Hsemaphysalis

H. leachi

SLinguatula

L. rhinaria

Porocephalus

P. constrictus

Linguatulida

The class Arachnoidea and the class Insecta belong to the phylum
Arthropoda. This phylum contains a greater number of species than
does any other phylum.

While the lobsters, crabs and water fleas, which belong to the class
Crustacea, are important zoologically, they are of very slight impor-
tance medically. Besides the Crustacea we have the thousand-legged
worms or Myriapoda.

The different classes of Arthropoda resemble the segmented worms
but have as distinction the possession of jointed appendages which
proceed from the somites in pairs. Some of the pairs of limbs are for
locomotion; at times, certain ones may be specialized for food taking.

The somites or divisions of the body have a chitinous exoskeleton.

281

282 THE ARACHNOIDS

Respiration takes place through the medium of gills in the Crustacea
and by tracheal tubes in the Myriapoda, Arachnoidea, and Insecta.

The Arachnoidea have no antennae while the Myriapoda and Insecta
have a single pair of antennae, the former having numerous pairs of
legs or jointed appendages while the latter have only three pairs of legs.
The Arthropoda have segmented bodies, but they differ from the worms
in having jointed appendages for the purpose of taking in food and
moving from place to place. They also have an exoskeleton which is
more or less unyielding from the deposit of chitin in the cuticle. This
cuticle is not a true skin but only a secretion of the epidermis. .

Within this external skeleton we have a dorsal digestive system and
a ventral nervous system.

THE ARACHNOIDEA.

The Arachnoidea differ from the Insecta in having the head and
thorax fused together. They also have four pairs of ambulatory appen-
dages, while the insects only have three pairs. The Arachnoidea never
have compound eyes — 'these when present being simple. Of the two
orders of Arachnoidea of interest medically the Acarina is far more
important than the Linguatulida.

ACARINA.

Of the acarines we are chiefly interested in the mites and the ticks.
The acarines do not show any separation of the abdomen from the
cephalo-thorax. A hexapod larva develops from the egg; this is suc-
ceeded by an octopod nymph which differs from the adult in not having
sexual organs.

In addition to the four pairs of legs in the fully developed acarine there are two
other paired appendages, the chelicerae. in front of the mouth, and the pedipalps
on either side of the mouth.

Trombidiidae.

These generally have a soft, more or less hairy integument and are often brightly
colored. The two eyes are often pedunculated and the cheliceras are lancet shaped
and the palps project beyond the rostrum as claw-like appendages. A tip-like
appendage on the apical segment of the palps is characteristic. A very common
and annoying member of this family is the hexapod larva of the Trombidium
holosericeum. It is usually designated Leptus autumnalis. Popularly it is termed
"harvest mite," "red bug" or "jigger." They are found in the fields in the autumn

ACARINA 283

and attack both man and animals. The condition (itching and redness) produced
is at times called autumnal erythema. There is a Trombidium in Mexico which
has a predilection for the skin of the eyelids, prepuce, and navel. The Kedani mite,
an orange-red larval mite about 250 by 125 microns is believed by the Japanese
authorities to bring about infection with Japanese river fever or Tsutsugamushi,
as the result of transmitting either a bacterium or protozoon by its bite. The
disease somewhat resembles typhus, although an eschar at the site of the bite and
lymphatic involvement is present.

Gamasidae.

Of the Gamasidae. which generally have a hard leathery body and1 styliform
piercing chelicerae, delicate five jointed palps and styliform hypostome, only the
Dermanyssus gallinse is of interest. This coleopterous mite infests chicken-houses
and sucks the blood of the inmates. They will also attack man. Poultrymen may
be troubled with a sort of eczema on the backs of the hands and forearms, similar
to scabies, resulting from bites by these mites. They measure 350X650^. They
have no eyes.

Tyroglyphidae.

Mites of this family live on cheese, flour, dried fruits, etc. They are small,
without eyes, and have a smooth skin and a cone-like appearance of the mouth
parts which are largely formed by the chelate chelicerae. They are chiefly of
importance because of their being occasionally found in urine, faeces, etc., and being
striking objects, the question of pathogenicity arises. The T. longior has been
associated with intestinal trouble (probably a coincidence, patient having eaten
cheese containing these mites).

Glyciphagi are found in sugar and are the cause of what is known as "grocers'
itch." Rhizoglyphus parasiticus is reported to be the cause of an itch-like affection
of the feet of coolies on tea plantations. To distinguish: the dorsum of Glyciphagus
is hairy or plumose; Tyroglyphus has both claws and suckers on tarsi, while Rhizo-
glyphus has only claws.

Sarcoptidae.

These are small eyeless mites with a transversely striated cuticle. They live
on the epidermis of man and various animals. The rostrum is chiefly made up of
chelate chelicerae with quite short three jointed, rather adherent palpi. It is the female
that makes the tunnels in the skin between the fingers, on penis, flexor surface of
forearm, etc. The male dies off after copulation. The female passes through four
stages: i. larva; 2. nymph; resembles adult, but has no sexual organs; 3. the pubes-
cent female; 4. the egg-bearing female. A pair of itch mites may produce 1,500,000
descendants in three months. Transference of eggs, larvae or pubescent females
does not seem to transmit scabies. It is the egg-laden female only. The human
itch mite, Sarcoptes scabiei, is an oval mite, the male is 250X150^; the female is
about 400X300^. Besides the difference in size, the male may be distinguished from
the female by the fact that the third and fourth pairs of legs in the female have

284

THE ARACHNOIDS

bristles, but in the male, the fourth pair has suckers. The tunnels made by the
female have the egg-bearing female at the blind end; scattered all along are fasces,
eggs, larvae; the eggs being next the mother and the more mature young at the en-
trance to the gallery. A diagnosis can be made from the finding of either eggs or
larvae. The eggs are 140^ long and hatch out in four to five days. A female
becomes mature in about two weeks.

In treating itch with sulphur preparations the adult females and immature itch
mites are killed; the eggs, however, are not affected. Hence a second treatment
about ten days after the first is necessary to kill the young mites, which have devel-

- l_. AV E.R V —

FIG. 78. — Arachnoidea exclusive of ticks. (ia) Sarcoptes scabiei, female;
(ib) S. scabiei, male; (2) Demodex folliculorum; (3) Trombidium akamushi, hexapod
larva (Kedani mite); (4) Trombidium holosericeum larva (Leptus); (5) Dermanyssus
gallinae; (6) Tyroglyphus longior; (ja) Pudiculoides ventricosus, male; (76) P. ventri-
cosus, young female; (jc) P. ventricosus impregnated female; (8) Porocephalus
armillatus; (go) Linguatula serrata, female; (96) L. serrata, larva.

oped subsequent to the first treatment,
of itch mites.

Different animals have different species

Demodicidae (Hair Follicle Mites).

Demodex folliculorum. — This is a vermiform acarine about 400^ long; the eggs
are about 75^ long; they chiefly live in the sebaceous glands of nose and forehead.

Tarsonemidae.

This acarine family shows a complete sexual dimorphism. The Pediculoides
ventricosus is oval and about 125X75," for the male which has claws at the extremi-

DCODID^E 285

ties of the anterior and posterior pairs of legs; the two other pairs have hooklets
and a sucking disc. The female is about twice as long but of the same breadth as
the male, and has claws only on the anterior legs.

The chelicerae are needle like with inconspicuous palps and the front and rear
pairs of legs are widely separated. The gravid female is like a ball and is about
IQOO/J. in diameter.

They live on wheat and may be found in wheat straw, which, if handled, may be
followed by a severe skin eruption with an irregular fever.

Ixodidae.

This family of the Arachnoidea is one of great medical interest and
of growing importance. It has recently been proposed to raise the
ticks to a superfamily, Ixodoidea and to divide it into the families
Argasidae and Ixodidae.

While only proven the intermediary hosts in the case of the organism of African
tick fever and the as yet undiscovered cause of spotted fever of the Rocky Moun-
tains, there is considerable speculation as to the possibility of blackwater fever being
due to a Babesia (Piroplasma). Piroplasmata of animals seem to be invariably
transmitted by ticks.

Very important diseases due to these small pear-shaped organisms within red
cells are .known for various animals, the best known being that of cattle in Texas
and known as Texas fever. Other piroplasmata diseases are Rhodesian fever
(cattle), heart water (sheep), and malignant jaundice of dogs. In these diseases
there are pathological features which resemble blackwater fever of man.

It is of interest to note that it was with the transmission of Texas
fever through an intermediate host (the tick) that Smith and Kilborne
(1889-1893) established the zoological principle of transmission of
disease through arthropod intermediary hosts. This led up to the
work on malaria, yellow fever, etc.

Ticks differ from insects in having four pairs of legs, only two pairs of mouth
parts, and no antennae. They differ from other acarines in having a median probe-
shaped puncturing organ, the hypostome, which is beset with numerous teeth
projecting backward, and in possessing stigmal plates. The head, or capitulum,
or rostrum, is the part which projects anteriorly from the body. This carries the
piercing parts which are the single hypostome or dart and a pair of piercing chitinous
structures, the chelicerae which lie above the hypostome. As a sheath for these
delicate biting parts we have a segmented pair of palpi or pedipalps. The mouth
is a slit between the chelicerae and hypostome.

Two depressed pitted areas on the dorsal surface of the capitulum in the adult
female are known as porose areas. Very important structures are the stigmal
plates. These are striking mosaic-like areas which are located just posterior to
each hind leg in the Ixodinae and between the third and fourth legs in the Argasinae.

286 THE ARACHNOIDS

As the greatest confusion exists as to the classification of ticks, Dr. Charles W.
Stiles has now in hand a system of classifying ticks according to the appearance
of these plates as seen under the high power of a microscope. There is great varia-
tion in the outline and general picture of these stigmal plates in the different species.
The stigmal orifice, the opening of the tracheal system, is in the center. The
Ixodinse have a scutum or shield-like chitinous structure on the dorsal surface. It
covers almost the entire back of the tick in the male and only a small portion anteri-
orly in the female. The genital opening is toward the anterior part of the ventral
surface. The anus, with anterior or posterior anal grooves, is near the posterior
third of the venter. The legs have six segments, the coxa being flattened out on the
surface of the body and the terminal tarsus ending with a pair of hooks and at times
with a pulvillus. The nymph has stigmal plates but has no genital opening while
the larva has neither genital apertures nor stigmal orifice.

Life History of Ticks. — -This varies greatly according to the sub-
family, genus, and species. The female Ornithodoros savignyi lays
about 140 eggs. The larva does not leave the egg, but moults inside,
and finally emerges as an eight-legged nymph. It lives in the dust in
the cracks of the native huts and comes out at night to feed on the sleep-
ing natives. As the possibilities for destruction are not so great as with
many Ixodinae the necessity for thousands of eggs is not imperative for
the continuation of the species as with the Ixodinae. With some of
the Ixodinae the females lay from 5000 to 20,000 eggs during several
days or weeks and then die. The eggs are preferably deposited near
grass. The egg stage lasts from two to six months, when the six-legged
larva (" seed tick ") emerges. It crawls up a blade of grass and gets on a
passing animal. After feeding, or at times without taking nourishment,
the larva drops to the ground, and changes to the pupal stage which has
four pairs of legs. The pupa crawls up a blade of grass and gets on a
passing animal (the second host). Feeding, it falls to the ground where
it remains eight to ten weeks. It moults and develops into an adult
tick. These males and females gain access to a third animal host — •
the males fecundate the females, after which the female gorges herself
with blood; afterward dropping off the animal and laying eggs. With
some ticks fewer hosts suffice.

Cleland has noted reports of serious symptoms, chiefly cardiac and visual, from
the bite of ticks in Australia (Ixodes holocyclus). This is exceptional, however, as
the symptoms following the bites of such ticks are only those of skin irritation.

Classification of Ixodidae.

Subfamily Argasinae. — Head concealed by body when viewed
dorsally. No scutum. Stigmal plates between third and fourth legs.

287

Adults have no suckers beneath claws. Slight sexual dimorphism.
Anus near middle of venter. Skin rough.

Genus Argas. — Body narrow in front. Margins thin and acute. No eyes.
The A. persicus (Miana bug) of Persia has been supposed to be concerned in the
transmission of a serious disease. Rostrum some distance from anterior margin.
It is also called the fowl tick and transmits fowl spirillosis.

Genus OrnUhodoros. — Margins of body rounded. Skin has many irregular
tubercles. Rostrum even with anterior margin so that ends of palpi slightly project.
It is the intermediate host of Spirochaeta duttoni. (South African tick fever.)

O. moubata is very common in Africa living in cracks in mud floors and bites
severely the sleeping natives. The larva makes its first moult inside the egg so
that it shows 4 pairs of legs when it emerges. Christy thinks it may transmit
Filaria perstans.

O. savignyi has two pairs of eyes near base of mouth parts.

<f . X^A^

EIG. 79. — OrnUhodoros moubata. (Murray from Doflein.)

Subfamily Ixodinse. — 'Mouth parts project in front of body when
viewed dorsally. Scutum present. Stigmal plates posterior to fourth
pair of legs. Adults have suckers beneath claws. Skin finely striated.

Anus behind middle of venter.

Sexual dimorphism marked. Male has well-developed scutum;
female has porose areas.

Section Ixodce. — Transverse recurved preanal groove in female. Male has ventral
surface covered with chitinous plates. No eyes. Genus Ixodes.

Ixodes has long rostrum with slender palpi — palpi narrow at base, leaving gap
between them and hypostome.

Section Rhipicephalus. — No preanal, but postanal groove in female. Ventral

288

THE ARACHNOIDS

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LINGUATULIDA 289

surface of male without adanal plates in Dermacentor, Haemaphysalis, Aponomma
and Amblyomma, but with one or two pairs in Hyalomma, Rhipicephalus and
Margaropus.

In the genera Hyalomma, Aponomma and Amblyomma the palpi are long and
slender and of about uniform width of segments.

In Hyalomma the segments of palpi are of about equal length. In Aponomma
and Amblyomma the second palpal segment is much longer than the others. Ambly-
omma differs from Aponomma in being very ornate and in having eyes.

In the genera Haemaphysalis, Dermacentor, Rhipicephalus, and Margaropus
the palpi are short.

Haemaphysalis has very broad rostrum, triangular palpi, and no eyes. Derma-
centor has a square rostrum with short thick palpi, the second and third joints being
as broad as long. Dermacentor andersoni transmits spotted fever of the Rocky
Mountains — not D. reticulatus.

Rhipicephalus has palpi without transverse ridges and comma-shaped stigmal
plates. The stigmal plates of Margaropus are nearly circular and the palpi have
acute transverse ridges externally. Margaropus annulatus transmits Texas fever
of cattle. This tick is also called Boophilus bovis or B. annulatus. Some authors
term it Rhipicephalus annulatus. Larvae developing from eggs of female ticks
which have fed on cattle infected with Texas fever transmit the disease which is due
to a protozoon Babesia bigemina.

LINGUATULIDA (TONGUE WORMS).

These are vermiform acarines more or less distinctly annulated. They have
retractile hooks at either side of the elliptical mouth.

If the hooks are to be considered not as degenerated legs but antennae and palpi,
then there is no vestige of legs in the adult. The sexes are separate.

Linguatula rhinaria. — This has been observed in man both in larval and adult
stages.

The male is white and about 3/4 inch long while the female is about 4 inches
long, tadpole shape, yellowish in color, and has about ninety segments, lives in "the
nasal cavity and frontal sinus of dogs, rarely in horses and sheep, and very rarely
in man.

The female lays embryo-containing eggs which, gaining freedom through the
nasal mucus, are swallowed by various animals. A larva develops which bores its
way through the gut and encysts in the liver or mesenteric glands. After several
moultings, they work their way again to the intestines and so get out of the body
of their host; or they may wander to lungs and trachea and either escape or take up
their position in the nostrils to become adults and produce eggs. Consequently,
one animal may act as intermediate and definitive host or these cycles may take
place in distinct animal hosts.

The larval form (1/5 in.) is far more common in man than the adult. Symptoms
are referred to liver in both larval and adult stage, and epistaxis and nasal symptoms
for adult stage only.

Porocephalus constrictus. — The adult form P. moniliformis lives in the lungs of
snakes and the eggs are probably ingested by drinking water. These eggs develop

2QO THE ARACHNOIDS

into a curled-up, ringed larva, about 1/2 inch long with twenty-three rings, which
is encysted especially in the liver or lungs. These escape and are swallowed by the
snakes, their definitive hosts.

While in the liver or lungs of man the patient may have signs of bronchitis,
hepatitis or peritonitis. Cases usually only discovered at postmortem. Parasites,
however, might possibly be found in sputum or faeces.

CHAPTER XX.
THE INSECTS.

CLASSIFICATION or THE CLASS INSECTA.

Order. Family. Subfamily. Genus.

Species.

Siphuncu-

{Pediculus

P. capitis

lata

Pediculidae

Phthirius

P. vestimenti

P. pubis

Rhynchota

Acanthiidae

Acanthia

A. lectularia

(Hemip-

Conorhinus \

C. megistus

tera) { Reduviidae

C. sanguisuga

f

{ Pulex

P. irritans

Xenopsylla

X. cheopis

Siphonap-

Pulicin

ae Ceratophyllus

C. fasciatus

tera

Pulicidae

Ctenocephalus

C. serraticeps

[ Ctenopsylla

C. musculi

Sarcops

>yl- Sarcopsylla

S. penetrans

linae

Simulidae

Simulium

S. reptans

(buffalo gnats)

Psychodidag

Phlebotomus

P. papatasii

(moth midges)

Chironomidae

Ceratopogon

C. pulicaris

(midges) f Culicinae Culex

C. fatigans

Culicidae \ Anophe- Anopheles

A. maculipennis

[ linae

(Tabanus

T. bovinus

Tabanidas

Haematopota

H. pluvialis

(horseflies)

1 Pangonia

P. beckeri

( Chrysops

C. dispar

Diptera

f
Glossina

G. palpalis
G. morsitans

Stomoxys

S. calcitrans

Muscidae

Musca

M. domestica

Auchmeromyia

A. luteola

Calliphora

C. vomitoria

Lucilia

L. caesar

Chrysomia

C. macellaria

(screw-worm)

Sarcophagidae

iSarcophaga
Ochromyia

S. carnaria
O. anthropophaga

GEstridae

Dermatobia
Hypoderma

D. cyaniventris
H. diana

2QI

2Q2 THE INSECTS

INSECTA.

The class Insecta has one pair of antennas, three pairs of mouth parts
(the fused labium being considered as one pair), and three pairs of legs.
They have three divisions of the body — head, thorax, and abdomen.

The head carries the antennas and mouth parts; the thorax, which is divided
into the pro-meso and meta thorax, carries upon the ventral surface of each thoracic
segment a pair of legs and on the dorsal surfaces of the two posterior segments a
pair of wings. The abdomen does not support appendages. The air is supplied
by means of tracheae — branching breathing tubes which have external openings or
stigmata. The tracheae are stiffened by spiral chitinous bands. The Malpighian
tubules are excretory organs of the alimentary system and excrete nitrogenous
waste material. Insects have two pairs of wings, the second pair of which is fre-
quently rudimentary and shows simply as knob-like projections. These are termed
halteres or balancers. In some insects both pairs of wings are rudimentary, as in
Siphonaptera.

Where insects show metamorphosis we have voracious worm-like larvas coming
out of eggs; these larvae are succeeded by a quiescent nonfeeding encased pupa
which finally develops into an imago or fully developed insect. An insect which
does not present this developmental cycle shows incomplete matamorphosis. Of
the class Insecta only the Siphunculata Rhynchota, Siphonaptera, and Diptera
are of special importance.

SIPHUNCULATA.
These are small flat wingless insects not showing metamorphosis.

The Pediculidse.

In this family there are no wings and there is no metamorphosis.
The acorn-shaped eggs (nits) are deposited on hairs of the host.

Pediculus capitis. — The female is about 1/12 of an inch long; the male smaller-
They vary in color according to the color of the hair of the host. The eggs are de-
posited on the hairs of the head in number of 6c which hatch out in about six days.
The thorax is as broad as the abdomen. The male louse is rounded off posteriorly
and shows a dorsal aperture for a pointed penis, while the female is recognized by a
deep notch at the apex of the last abdominal segment. There seems to be a marked
preference exhibited by lice for their own peculiar racial host. It has recently been
suggested that this might account for certain peculiarities in infection where different
races were living together and under similar conditions as to food and environment,
and yet only one race contracts the disease (beriberi). The head louse has been
found to harbor leprosy bacilli when living on a leper.

Pediculus vestimenti. — This louse lives about the neck and trunk and deposits
its eggs in the clothing. They number about 75 and hatch out in three or four days
and become mature in about two weeks. Unlike the fleas there is no grub stage.

LICE

293

It is almost twice the size of the P. capitis and the abdominal seg-
ment is broader than the thorax. The abdomen is less markedly
festooned than that of P. capitis; is less hairy and contains 8 segments
as against 6 for P. capitis.

It has recently been shown to transmit typhus fever and more recently Nicolle
has demonstrated it as a carrier of relapsing fever, the spirochastes being introduced
by the material from the crushed louse being rubbed into the wound by the scratch-
ing of the victim (just as with the flea in plague) and not by the bite itself.

FIG. 81. — Siphunculata and Rhynchota. i. Pediculus capitis. 2, Pediculus
vestimenti. 2a. Protruded rostrum of Pediculus. 3. Phthirius pubis. 4. Acan-
thia lectularia. 5. A. rotundata. 6. Conorhinus megistus.

Phthirius pubis. — This louse is popularly known as the crab louse. The female
is little more than 1/25 of an inch in length, and the male a trifle less. They are
almost square. The second and third pair of legs are supplied with formidable
hooks. They have a preference for the white race and live about the pubic region.
The female lays about a dozen eggs, which hatch out in about a week.

RHYNCHOTA.

The Rhynchota are insects possessing a sucking beak in which the
lower lip forms a long thin tube or rostrum which can be bent under the

294 THE INSECTS

head or thorax. Inside this tube are biting parts — mandibles and max-
illae. The metamorphosis in this order is not marked.

They have no palpi. The lower lip or labium or beak has it's
edges curved to form the tube and it is only covered by the labrum at
it's base. With the Diptera the labrum goes into the formation of the
sucking tube. The mandibles and maxillae are bristle-like structures
serrated at the tip. The mandibles are grooved internally and form
when apposed a tube for blood.

The Acanthiidae.

These have a flattened body, a three-jointed rostrum, and four-
jointed antennae. Their wings are atrophied.

Acanthia lectularia (Cimex lectularius). — This is the cosmopolitan bedbug.
It measures about 1/5 by 1/8 of an inch (5 by 3 mm.). It is of a brownish-red color.
The most conspicuous feature of the bedbug is the long proboscis continuous with
the dorsal integument of the head and tucked under the ventral surface. There
are two prominent eyes and two four-jointed antennae. There are eight abdominal
segments. The bedbug lives in cracks and crevices, especially about beds. It is
said thay can migrate from house to house. At any rate, they are frequently trans-
ferred with wash clothes. They have a penetrating odor when crushed. The
female deposits about fifty eggs at a time in cracks and in ten days they hatch out
into larvae which pass insensibly into adults by a series of five moultings; this deposit-
ing of eggs occurs about four times a year.

The bedbug is very probably the intermediate host in kala azar and it has been
incriminated in connection with typhus fever and relapsing fever.

In India the A. rotundata is the one encountered. It is of a dark mahogany
color, has a smaller head, narrower abdomen, thick rounded prothoracic borders
and is more densely covered with hairs than A. lectularia. The prothorax of A.
lectularia is flattened at the side.

*

Reduviidae.

These bugs have a long narrow head and a distinct neck. The
antennae are long and slender. The antennae in the genus Conorhinus
are inserted about midway between the eyes and point of the head.

Conorhinus sanguisuga. — This is known as the Texas or Mexican bedbug,-
and was formerly the foe of the common bedbug, but having gotten a taste for
human blood through the Cimex or Acanthia, it now prefers man. It is extending
toward the North. It has wings. The bites are much more severe than those
of the common bedbug. It is of a dark brown color, nearly an inch in length, with
a long, flat, narrow head and a short thick rostrum. They can run as well as fly.
They bite at night.

FLEAS 2Q5

CONORHINUS MEGISTUS. — This is called "Barbeiro" in Brazil on account of its
preference for biting the face. The Schizotrypanum cruzi undergoes a develop-
mental cycle in this bug which transmits the disease.

SlPHONAPTERA.

These are laterally flattened wingless insects and undergo a complete
metamorphosis.

Pulicidae.

This family is divided into two subfamilies — the Pulicinae and the
Sarcopsyllinae. In the former the female remains practically unchanged
after fecundation, in the latter the abdomen becomes enormously dis-
tended with eggs, and the female remains stationary after her impreg-
nation in the burrow which she has made under the skin.

Pulicinae. — Formerly, with the exception of infection with Dipylidium caninum,
the fleas were only under suspicion as carriers of disease; ideas having been enter-
tained as to their being possible transmitters of relapsing fever, typhus fever and
kala azar. Trypanosoma lewisi is transmitted by fleas, either Pulex irritans or
C. canis. The trypanosome undergoes development in the flea and the infecting
material is in the faeces of the flea and transmission occurs by the licking on the part
of the rat of faeces from an infected flea. The infection has no connection with the
puncture wound of the flea as is the case with plague. As a result of the convincing
experiments of the British Plague Commission, their role in the transmission of
plague has been absolutely established. It is by the bite of the Xenopsylla cheopis
that plague is chiefly transmitted from rat to rat, and in bubonic and septicaemic
plague it is apparently the intermediary .in human infection.

The average capacity of a flea's stomach is about 0.5 cmm. so that with a rat
dying with septicaemic plague and with possibly 100 million bacilli to one c.c. of
blood the flea would take in about 5000 bacilli. Furthermore these multiply in
the alimentary canal so that the digested blood teems with bacilli when reaching
the anus of the flea. The plague bacilli are passed out with the faeces and these
being rubbed into the puncture of the flea bite bring about infection. The puncturing
apparatus of the flea consists of a pointed epipharynx and two distally serrated
mandibles. These chitinous biting parts are contained in the labium which divides
distally into two labial palps. The maxillae are conspicuous triangular structures
and, projecting farthest anteriorly, are the conspicuous four-jointed maxillary palps,
often mistaken for antennae. By the apposition of the internally grooved mandibles
to the epipharynx a tube is formed through which the blood is sucked up. The
antennae are inconspicuous and are in close apposition to the sides of the head,
behind the eyes, and can only be well made out with a lens. Fleas have three pairs
of legs, and the male can be distinguished from the female by its smaller size and the
conspicuous coiled-up penis within the abdomen. The female has a conspicuous
gourd-like spermatheca which varies in shape in different species. The body of

296 THE INSECTS

the flea is flattened laterally. They may or may not have eyes, and certain con-
spicuous structures called combs are of importance in classification. In the meta-
morphosis of the flea the eggs are hatched out in dust of crevices, etc., into bristled
larvae in about one week. The larva forms a cocoon and develops into a nymph
which has three pairs of legs. The nymphs emerge from the cocoon as adult fleas
in about three weeks after the larva forms it.

KEY TO THE FLEAS.

A. With combs.

1. Eyes present.

a. Combs along inferior border of head and on prothorax.
Ctenocephalus serraticeps.

b. Combs only on prothorax. Ceratophyllus fasciatus.

2. Eyes absent.

a. Collar of combs on prothorax and four short ones along
inferior border of head. Ctenopsylla musculi.

B. Without combs.

a. Ocular bristle arises near upper anterior margin of eye. A
line between this and the oral bristle approximately vertical.
Two bristles posterior to antennae. Xenopsylla cheopis.
Lxmopsylla cheopis. Formerly Pulex cheopis.

b. Ocular bristle arises near lower anterior margin of eye. A line
between this and the oral bristle approximately horizontal.
One bristle posterior to antennae. Pulex irritans.

The common human flea of Europe is the Pulex irritans; that of the United States
the Ctenocephalus serraticeps or dog flea. The flea that is implicated with plague
is the Xenopsylla cheopis. It resembles P. irritans, but is more yellow than brown
in color. It also has a greater number of bristles on the head. The ocular bristle
runs above and in front of the eye; that of P. irritans below. It is principally the
flea of Mus decumanus, the sewer rat; but the house rat, M. rattus, becomes infected
from coming in contact with the sewer rat in the basement.

Ceratophyllus fasciatus is the common rat flea of Europe and the U. S. In the
tropics X. cheopis is the common rat flea (98% in India). Ctenocephalus serrati-
ceps, Ctenopsylla musculi and Pulex irritans have also been frequently found on
both Mus norvegicus and M. rattus. To distinguish M. norvegicus from M. rattus
we have in the former (i) ears which barely reach the eyes when laid forward and
(2) tail rather shorter that length of head and body together (only 89% of length
of head and body together). With M. rattus the tail is longer than the head and
body together (25% longer) and the extended ear covers or reaches beyond the
middle of the eye. M. rattus has a sharper nose, longer and more delicate tail
and thinner ears than M. norvegicus (formerly M. decumanus).

M. alexandrinus is a variety of M. rattus. Rats and mice belong to the family

SARCOPSYLLA

Muridae and the common mouse is M. musculus.
Rodentia of the class Mammalia.

297
They belong to the order of

Sarcopsyllinae.

Belonging to the subfamily Sarcopsyllinae, the Sarcopsylla penetrans (Derma-
tophilus penetrans) is of great importance in tropical countries. It is known as
the chigoe, nigua, or jigger. The male and virgin female are unimportant
as they do not penetrate^ the skin but act as ordinary fleas. The female, which
when unimpregnated is only about 1/24 of an inch long, when' impregnated
bores its way into the skin of man, especially about the toes," soles of the

FIG. 82. — Fleas, bedbugs and ticks. A, Lcemopsylla cheopis; B, P. irritans;
C, Ctenopsylla musculi; D, bedbug; E, cross section of rostrum of Ornithodorus;
F. longitudinal section of Ornithodorus.

feet or finger-nails, and in the chosen site develops enormously, becoming
as large as a small pea. This enlargement takes place in the second and
third abdominal segments and is packed with eggs measuring about 400 microns
long and numbering about 100. A small black spot in the center of a tense rather
pale area is characteristic. The metamorphosis is similar to that of the flea. Sar-
copsylla can be differentiated from the flea by the proportionately larger head to
the body, and especially by the fact that the head is the shape of the head of a fish,
distinctly pointed. With the fleas the lower border of the head comes out in a
straight line to join the curve of the upper part. In the Sarcopsylla lower and upper
border of head are both curved.

298

THE INSECTS

DlPTERA.

The insects of the order Diptera are of great importance medically
in a variety of ways, either by the direct irritation of their bites, by their
transmitting disease directly, as does the common house fly typhoid
fever, or by acting as intermediate hosts for various parasites. They
are characterized by mouth parts formed for puncturing, sucking, or
licking. They present a complete metamorphosis, larva, pupa, and
imago. As a rule, the Diptera have a distinct pair of wings, the second
pair being rudimentary. With the Aphaniptera or Siphonaptera the

FIG. 83. — i and 2, male and female Xenopsylla cheopis. 3, Head of Cerato-
phyllus. 4 and 5, male and egg distended female of Sarcopsylla penetrans.

wings are practically absent. Under the Aphaniptera, we have to
consider the Pulicidae or flea family.

The order Diptera is usually divided into the following suborders: i. Orthorrha-
pha: Diptera with larvae having a differentiated head. The imago breaks through
the larval or pupal case by a T-shaped break and has no frontal lunule (an oval space
just above the root of the antennae). The Orthorrhapha are divided into: a. Nemo-
cera (with long, many jointed antennae) and b. Brachycera (with short antennae)
2. Cydorrhapha: larvae without differentiated head. The imago escapes through
an anterior opening and has a lunule and ptilinum (an inflatable projecting organ

BITING FLIES 2 99

just above the root of the antennae). If the halteres are covered by a scale (squama)
we have calyptrate Cyclorrhapha; if not, acalyptrate. These squamae are large
enough in the calyptrate species to even conceal the halteres when the fly is looked
at from above. 3. Pupipara: the larvae are extruded ready to begin the pupal state.

The males of flies where the two compound eyes come together above the antennae
are referred to as holoptic, if more or less widely separated as dichoptic. Ocelli are
three single eyes usually, when present, situated in the triangular space between the
compound eyes in the front (the space separating the compound eyes).

In studying the biting flies it is very important to recognize the anterior, small,
or mid-cross vein. This short transverse rib or vein is the key to wing venation.
Beneath it is the discal cell and it bounds the first posterior cell internally or basally.
It is also of great value in differentiating Culicidae. The character of the antennae
should also be noted carefully. The study of the bristles about head, thorax, and
abdomen (chaetotaxy) is more difficult. Anyone taking up the study of flies should
carefully note the wings, etc., of Musca domestica. By putting a few house flies
on moist horse manure in a gauze-covered bottle the entire metamorphosis may be
observed.

Tabanidae.

This is the family of horseflies, gadflies, breeze flies or green-headed flies. It
is the most numerous family of the Diptera — there being more than 1000 species.
The females are blood suckers; the males live on flowers and plant juices. The eyes
are usually very brilliant in color, and in the male make up the greater part of the
head.

They belong to the suborder Orthorrhapha and in the group of short antennae
flies (Brachycera). Five posterior cells are always present.

The antennae consist of three segments. No arista. The epipharynx is tube
like, the hypopharynx has a groove and both are awl shaped. The pair of maxillae
are serrated and the mandibles lancet like. They have rather coarse maxillary
palps. The labellae are prominent at the extremity of the fleshy labium. They are
thick set flies and rarely show color. The body of the larva has eleven segments
with a small but distinct head. The eggs are deposited in masses on the leaves or
stems of plants about marshy places. The larva is carnivorous.

Tabanus autiunnalis. — Is about 3/4 of an inch long; it is dark in color, and has
four longitudinal bands on the thorax. The last joint of the antennae has a crescentic
notch. The wings do not overlap.

Hsematopota pluvialis. — In the Haematopota there is no crescentic antennal
notch, and the wings overlap. The abdomen is narrower than in Tabanus. The
brimp, one of the Haematopota, bites man severely.

Pangonia beckeri. — The genus Pangonia is characterized by a very long, slender,
and more or less horizontal proboscis.

Chrysops dispar. — Chrysops has three ocelli, in this respect differing from the
genera Tabanus and Haematopota. The wings are widely separated and spotted.
The antennae of Chrysops are especially long and slender. Chrysops and Haemato-
pota produce the greatest amount of pain from their bites. The Tabanidae are not
implicated as intermediate hosts in the transmission of disease. By their bites,

300

THE INSECTS

however, they may transmit disease directly, as with anthrax. Two species of
Chrysops have been found to transmit Filaria loa.

Muscidae.

The Muscidae, Sarcophagidae, and CEstridae are calyptrate Cyclorrhapha.

The common housefly, M. domestica, is the best example of this family.

The arista is feathered both dorsally and ventrally with straight hairs. The
fourth longitudinal vein bends down in a rather sharp angle as compared with
Stomoxys, which gives the first posterior cell rather a fusiform appearance. The eyes
are close together in the male, far apart in the female. The female lays about 125

FIG. 84. — Wing venation of A, Tabanus; B, Stomoxys; C, Glossina.

eggs in a heap preferably in fermenting horse manure. The larva comes out in about
thirty-six hours. Very characteristic are the stigmata decorating the blunt posterior
ends. (See illustration.)

The larval stage lasts seven to ten days and then the barrel- shaped pupal stage
is entered upon. This lasts about three days when the adult fly emerges. This
fly is incapable of biting, the piercing organs being fused with the labium, but may
transmit disease directly, carrying infectious material from the source, as in faeces,
to the food about to be ingested. Their r61e in typhoid fever is one of immense
importance. By reason of its hairy sticky legs, habits of frequent defecation and
constant regurgitation the housefly is an important agent in the spread of cholera,
dysentery, infantile diarrhoeas and tropical ophthalmias as well as typhoid.

BITING FLIES

3OI

In the Muscidae the antennae hang down in front of the head in three segments
and have an arista plumose to the tip. The first posterior cell is narrowed. There
are no bristles on abdomen except at tip.

(I) Stomoxys, Haematobia and Glossina have a more or less
elongated proboscis adapted for biting. Stomoxys has delicate palpi,
shorter than the proboscis, and arista feathered only on the dorsal side
with straight hairs. Haematobia has club-like palpi about as long as
proboscis and arista with hairs dorsally and ventrally. Glossina has
thick set but not clubbed palpi and an arista feathered on the dorsal
side with branching hairs.

(II) Musca, Calliphora, Chrysomyia, Lucilia, and Cordylobia do
not have a proboscis adapted for biting.

FIG. 85. — Common housefly (Musca domestica): Puparium at left; adult next,
larva and enlarged parts at right. All enlarged. From circular 71 (by L. O.
Howard), Bureau of Entomology, U. S. Department of Agriculture.

Stomoxys calcitrans. — These greatly resemble the common housefly in size
and shape. They can be easily distinguished by the black, piercing proboscis extend-
ing beyond the head. There are longitudinal stripes on the thorax and spots on the
abdomen. The proboscis on examination will be seen to be bent at an angle near its
base. The palps are short and slender. The wings diverge widely.

The female lays about 60 banana-shaped eggs in horse manure. These hatch
out in three days as larvae which turn into pupae in two or three weeks. After about
ten days the fly emerges. The genus Stomoxys includes vicious biters. This is
the fly which comes into houses before a rain, and which has given the common house-
fly the reputation of biting before a rain. Stomoxys may be implicated in trans-
mitting surra (Trypanosoma evansi).

It has now assumed great importance as a transmitter of poliomye-
litis and possibly of pellagra.

302

THE INSECTS

The horsefly (Haematobia irritans) rarely bites man. In these the palpi are much
longer than in Stomoxys, being as long as proboscis. These palps are also thick
and spatulate.

Glossina palpalis. — This is the tsetse fly that is responsible for the
transmission of human trypanosomiasis (sleeping sickness).

The tsetse fly is a small brownish fly about 1/3 of an inch long. The pro-
boscis extends vertically and has a bulb at its base. The arista is plumose
only on the upper side and the individual hairs are themselves feathered.
The wings are carried flat, closed over one another like the blades of a pair of scissors

FIG. 86. — Insects in which the adult stage is important, (i) Stomoxys cal-
citrans; (2) S. calcitrans, larva; (3) Tabanus bovinus; (4) Tabanus larva; (5) Glossina
palpalis; (6) G. palpalis, side view; (7) G. palpalis pupa; (8) Glossina palps and arista.

and project beyond the abdomen. The most characteristic feature of the tsetse
fly is the way the fourth longitudinal vein bends up abruptly to meet the mid cross
vein and then curves downward to run parallel with the third longitudinal vein.
In Stomoxys, the wings separate; in Haematopota they just meet, and in Glossina
they cross. Glossinae bite chiefly in the daytime.

The tsetse fly does not lay eggs, but gives birth to a single full-grown larva
almost as large as the mother which immediately bores its way into the soil and
becomes a pupa.

The pupal stage is about a month and the larval stage in the mother about two
weeks. G. palpalis bites in the day time. Both males and females bite. Glossina

BLOW FLIES

303

morsitans transmits the cattle trypanosome disease, nagana and the human infection
due to Trypanosoma rhodesiense.

Auchmeromyia luteola. — This is an African fly, the larva of which is known as
the " Congo floor maggot,' ' and is a blood sucker. The larva is of a dirty- white color
and about 2/3 of an inch long. It crawls out at night and feeds on the sleeping
natives. This is the only known instance of a blood-sucking larva.

Calliphora vomitoria and Lucilia caesar. — These are flies with brilliant metallic-
colored abdomens, commonly called blow flies in the case of Calliphora and blue-
bottle flies for Lucilia. They deposit their eggs on tainted meat and in wounds.

11

FIG. 87. — Insects in which the larval stage is important, (i) Chrysomyia
macellaria; (2) C. larva; (3) Dermatobia cyaniventris larva, early stage (ver ma-
caque); (4) D. cyaniventris larva, later stage (torcel or berne); (5) D. cyaniventris;
(6) Auchmeromyia luteola; (7) A. luteola, larva; (8) Sarcophaga magnifica; (9)
S. magnifica larva; (10) Anthomyia pluvialis; (n) A. pluvialis larva.

«

Many cases of obscure abdominal trouble are probably due to the larvae of these
flies. Intestinal myiasis is undoubtedly of greater importance than has been
thought. The larvae, with hook-like projections anteriorly and a ringed body, can
easily be recognized in the faeces. They have been mistaken for flukes. They
also have a tendency to be attracted by those with ozena and the larvae may develop
in the nostrils.

Chrysomyia macellaria. — This is known as the screw-worm when in the larval
stage. The adult fly resembles the blue-bottle flies. It is distinguished from them,
however, by the presence of black stripes on thorax. These flies are very common
over nearly all North and South America. The thorax is striped. The eggs, which

304 THE INSECTS

number 250 or more, when deposited in the nostrils or in wounds, develop into the
screw-worm larva, which may, by going up into the frontal sinus, cause death.
These larvae have twelve segments with rings of minute spines.

Ochromyia anthropophaga (Cordylobia anthropophaga or Tumbu Fly). — This
is an African fly whose larvae develop under the skin of man and animals. It is
known as the Ver de Cayor. The larva resembles the Ver Macaque, is rather
barrel shaped and beset with small spines. It bores its way into the skin and makes
a lesion like a boil which has a central opening through which the larva breathes.

Sarcophagidae.

These are known as "flesh flies." The most important characteristic is the fact
that the arista is plumose up to the mid-point, beyond which it is bare. They are
usually thick set and moderately large flies.

Sarcophaga carnaria. — This is a grayish fly with three stripes on thorax and
black spots on each segment of the abdomen. It is viviparous. The larvae gain
access to nasal and other cavities and there develop. Cases of death have been
reported. Naturally, the fly deposits its larvae on decaying flesh. . In times of war
all of these flies become important by reason of "maggots" in the wound. These
larvae are the most common ones in intestinal myiases. The mouth booklets are
strongly curved and separate. Each abdominal segment has a girdle of spines.
The anterior end is somewhat pointed. The hind stigmal plate is in a deep cavity.

(Estridae.

The flies of this family are usually called botflies. The mouth parts are almost
vestigial. They have a large head with a somewhat bloated-looking lower portion.
They are often rather hairy. The larvae which develop from the eggs are parasitic
either in the alimentary canal or the subcutaneous tissues.

Dermatobia cyaniventris. — These are large, thick-set flies about 3/5 inch long,
with prominent head and eyes, small antennae, and a marked narrowing at the
junction of thorax and abdomen. The thorax -is grayish and the abdomen a metallic
blue. The larvae are deposited under the skin in various parts of the body. When
the larvae move they cause considerable pain. At first the larva is club-shaped,
but later on it becomes oval. The former is called Ver Macaque, the latter Torcel.

Hypoderma diana. — The larval form of this fly has been reported three times
for man. It forms tumors under the skin which it is thought may reach this location
by proceeding in some way from the alimentary canal.

In Hypoderma the arista is bare while in Dermatobia the upper border is plumose.

CHAPTER XXI.
THE MOSQUITOES.

MOSQUITOES (Culicidae) are of the greatest importance medically,
not only from their influence upon health in general by reason of inter-
ference with sleep and possibly from direct transmission of disease, but,
more specifically, they are the only means by which it at present appears
possible to bring about infection with such diseases as yellow fever,
malaria, filariasis, and possibly dengue. In addition, many diseases
of animals are transmitted by mosquitoes.

The Culicidae differ from all other Diptera in having scales on their
wings and generally on head, thorax, or abdomen.

To identify a mosquito, examine a wing and note the scales; also note the presence
of two distinct fork cells and, in addition, that the costal vein passes completely
around the border of the wing, making a sort of fringe with its scales. Mosquitoes
undergo a complete metamorphosis, there developing from the egg a voracious,
rapidly-growing larva; next, a nongrowing, nonfeeding stage — the pupa or nymph.
There the head and thorax are combined in an oval body, from the back of which
projects the siphon tubes; and tucked in ventrally is a small tail- like appendage.

The fully developed insect emerges from the pupa.

The Culicidae belong to the suborder Nematocera. These have long articulated
antennae and include four families: Culicidae Chironomidae, Simulidae, and Psycho-
didae.

The principal mosquito-like, blood-sucking Diptera which are
frequently mistaken for mosquitoes — 'none of which have scales on their
wings — are the following:

1. Chironomidae or Midges. — The blood-sucking species of Chironomidae, which
are found in most parts of the world, belong chiefly to the genus " Ceratopogon."
These midges are of very small size, about 1/12 of an inch long, are able to
get through netting and, usually being in swarms, they are exceedingly trouble-
some. The antennas have thirteen joints and the wings are shorter than the
abdomen and have only longitudinal veins. One of the. midges, the "jejen"
of Cuba, is a great scourge, its small size enabling it to enter eyes and nostrils.
The larva of Chironomus is a red worm-like creature; the pupa has a tufted
head.

2. Simulidae or Buffalo Gnats. — These are small blood-thirsty insects only about
1/8 of an inch in length. The thorax is humped, the legs are short and the

20 305

3°6

THE MOSQUITOES

proboscis short and inconspicuous. The antennas have eleven joints but
are rather short. One species, the S. damnosum, known by the natives of
Uganda as "Mbwa," is greatly dreaded; its bites causing swellings and sores.
Sambon has considered Simulium reptans as the transmitting agent of pellagra.
Psychodidse or Moth Flies. — These are small, hairy, slender midges, with
long legs and a short proboscis. The antennae are long, hairy and consist
of 12 to 1 6 joints. Palpi 4 jointed. They are only about 1/12 of an inch in
length. The hairy wings have numerous longitudinal veins. Some, as

FIG. 88. — Mosquito-like insects belonging to families Chironomidas, Simulidae
and Psychodidae. (i)Phlebotomus papatasii; (2) P. papatasii (natural size); (3)
P. papatasii (larva); (4) P. papatasii larva (natural size); (5) Ceratopogon pulicaris;
(6) C. pulicaris (natural size); (7) Chironomus larva; (8) Attitude of a Simulium;
(9) Simulium reptans; (10) Larvae of Simulium.

Phlebotomus, have an enlongated proboscis and are vicious blood suckers.
It has been suggested that they may be of importance in the transmission of
tropical ulcer. A fever of about three days' duration found in Bosnia, char-
acterized by leukopenia and similar to dengue and known as Phlebotomus
or Pappataci fever, has been thought to be caused by the bite of infected P.
papatasii.

Phlebotomus is common in the tropics and may transmit surra. The pro-
boscis is much shorter than that of mosquitoes.

Mosquitoes have three main parts of the body — 'the head, the
thorax, and the abdomen. On the head, the space behind the two

MOSQUITOES

307

compound eyes is called the frons, in front, and the occiput posteriorly.
The nape is back of the occiput. The bulbous prolongation of the frons which
projects over the attachment of the proboscis is the clypeus. The clypeus is hairy
in the Culex; scaly in Stegomyia. The proboscis is straight in all mosquitoes of
importance medically. It consists of a fleshy, scaled, gutter-shaped portion be-
neath, known as the labium, which terminates in two hinge-joint processes — the
labella. At the end of the labium is a thin membrane (Button's membrane). It
is through this that filarial embryos are supposed to pass on their way from the
interior of the labium to enter the person bitten. The labium may be considered
as the sheath of a knife, holding and protecting the slender, blade-like penetrating

FIG. 89. — Anatomy of mosquito, i, Dorsal view of mosquito; 2, wing of mos-
quito; A, costal vein; B, mid cross vein; C, posterior cross vein; D, first fork-cell;
E, second fork-cell; 3, various types of scales; a, flat head scales; b and c, Mansonia
wing scales; d, upright forked head scales; e, f, g and h, various shapes of thoracic
scales.

organs. Lying in this groove we have, from above downward, the horseshoe-
shaped labrum — epipharynx, the under surface of which is open. This when closed
by the underlying hypopharynx forms a tube through which the blood is sucked
up by the mosquito. In the hypopharynx, which somewhat resembles a hypoder-
mic needle, is a channel, the veneno-salivary duct. It is down this channel that
the malarial sporozoite passes. There are two pairs of mandibles and two pairs
of maxillae on either side of the hypopharynx — the mandibles above and the maxillae
below. The serrations of the maxillae are coarser than those of the mandibles.
The sensory organs, the palps, lie on either side of and slightly above the proboscis.

3o8

THE MOSQUITOES

These are of the utmost importance in differentiating mosquitoes and must not
be confused with the antennae, which are attached above the palpi and at the sides
of the clypeus. These antennae are of importance in distinguishing the sex of the
mosquito.

The thorax is largely made up of the mesothorax, at the posterior margin of
which is a small, sharply-defined piece, the scutellum; this may be smooth or trilo-
bed. Underneath and posterior to the scutellum is the metanotum; the metano-
tum is bare in Culicinae, has hairs in Dendromyinae and scales in Joblotinae.

There is a pair of wings attached to the posterior part of the mesothorax and,

FIG. 90. — Distinguishing characteristics of mosquito larvae and fly antennae.
Siphon tubes of i, Stegomyia, 2, Culex, 3,Tasniorhynchus; mental plates of 4, Taenio-
rhynchus, 5, Stegomyia, 6, Culex; larval antenna- of 7, Culex, 8, Stegomyia, 9, Anoph-
eles; antennae of 10, Muscidae, n, Tabanidae, 12, Simulidae, 13, Sarcophagidae.

more posteriorly still, a pair of rudimentary wings (halteres) attached to the
metanotum. The three pairs of legs are attached to the thorax.

There are nine segments in the abdomen. The genitalia arise from the termi-
nal segments as bilobed processes. In the male there is a pair of hook-like appen-
dages or claspers, between which, and ventrally situated, are the harpes, also a pair
of chitinous processes.

In considering the question of the possible danger which might arise from the
introduction of a case of yellow fever, malaria, or filariasis, it would give the greatest
information if mosquito ova were at hand so that we could by watching the develop-
ment from egg to larva, pupa, and insect, have all the points from which to decide
as to the genera developing in the given locality. It is generally a very easy matter

MOSQUITO

309

to dip out large numbers of larvae from the pools and having noted the character-
istics of the larvae, to do the same when the pupae develop; so that we have only to
verify our identification when the insect emerges from the pupa.

THE OVA.

The egg raft of Culex, containing about 250 ova, is quite perceptible on the
surface of the water as a black, scooped-out mass, about 1/5 of an inch in length.
The eggs are set vertically in the raft. The eggs of the Stegomyia are laid singly
and have a pearl-necklace-like fringe around them.

ATE H/MRS

~-- " UATERAL. ABDOM. HAIRS

ANTENNA

MOUTH BRUSH

FIG. 91. — i. Asiphonate larva. Anopheles. 2. Siphonate larva. Stegomyia.

The Anophelinae eggs are oval in shape with air-cell projections from either side.
They are laid in triangle and ribbon patterns. The markings of these air cells
vary and have been used for differentiation. The length of time of the egg stage
varies according to temperature and other conditions — one to three days for Stego-
myia and two to four days for Anophelinae. The Anophelinae are more difficult
to raise than Culex or Stegomyia.

LARV.E.

The

There are two great classes of larvae — the siphonate and the asiphonate.
latter are always Anophelinae.

The Culicinae larvae have a projecting breathing tube at the posterior extremity
which is called a respiratory siphon. This projects off at an angle from the axis

3io

THE MOSQUITOES

of the body, the true end of which terminates in four flap-like paddles. If you di-
vide the length of the siphon by the breadth, you get what is known as the siphon
index. In Culex the siphon is long and slender, in Stegomyia it is short and barrel-
shaped. When at the surface the Culex larva has its siphon almost vertical and the
body at an angle of about 45°.

The Stegomyia larva hangs more vertically. As a rule, the hairs proceeding
from the sides of Culex larvae are straight and the head relatively large. There
are also no palmate hairs along the sides.

The Anophelinae larvae have a small head which is capable of being twisted
around with lightning-like rapidity. They are darker in color and have no siphon;

^''°"

Fig. 92. — Metamorphosis of mosquitoes, i, 2, 3, 4, and 5, Eggs, larva, pupa,
and heads of male and female Culex; 6, 7, 8, 9, and 10, eggs, larva, pupa, and heads
of male and female Anopheles; n, 12, 13, 14, and 15, eggs, larva, pupa, and heads
of male and female Stegomyia.

float parallel to the surface of the water; have long lateral branching hairs, and on
the sides of each of the five or six middle abdominal segments they have a pair of
palmate hairs. These palmate hairs are supposed to aid them in keeping their
position on the surface of the water. The larvae are usually called "wigglers."
The duration of the larval stage is from i to 2 weeks, according to the temperature.

THE PUP.E.

These have a bloated-looking cephalo-thorax and a shrimp-like tail — the latter
the abdomen. Very important in examining them with a lens is to note the char-
acteristics of the siphon tubes which project from the dorsal surface. These siphons

DISSECTION OF THE MOSQUITO

311

are long and slender in Culex and project from the posterior portion of the head
end. In Anophelina? they are broadly funnel-shaped and arise from the middle
of the head end. The siphon of the Stegomyia is triangular.

The bulbous end of the Culex nymph is more vertical than the horizontally-
placed cephalo-thorax of Anopheles. The duration of pupal life is short — only
one to three days. At the end of this time the pupa comes to the surface and
straightens out. The integument then splits dorsally and the perfect insect emerges.
It dries its wings for a time on its raft-like pupal skin and then flies away.

From the above it will be seen that the stages in the metamorphosis of the mos-
quito take about two weeks: one to three days for egg stage; seven to ten days for
larval stage, and two to three days for pupal stage.

DISSECTION or THE MOSQUITO.

The easiest way to secure a mosquito for dissection is to use an ordinary plugged
test-tube. Slipping the open end of the test-tube over the resting mosquito,

FIG. 93. — Anatomy of mosquito, i, Cross section of proboscis of mosquito; 2,
anatomy of mosquito, longitudinal section; 3, tip of proboscis of mosquito; a, labrum-
epipharynx; b, hypopharynx; c, manidible; d, maxilla.

by a slight movement, the insect will fly toward the bottom. Then quickly insert
the plug. If it is not desired to study the scales, the best way to kill the mosquito
is by striking the tube sharply against the thigh. If it is also desired to study the
scale characteristics it is better to put a drop or so of chloroform on the lower
part of the cotton plug. The vapor falls to the bottom of the tube and kills the
mosquito. Take the mosquito out, pull off legs and wings, and then place the
body in a drop of salt solution on a slide. Bile has been recommended. Then
hold the anterior end of the thorax by pressure of a needle. With a needle

312 THE MOSQUITOES

in the other hand, gently crush the chitinous connection between the sixth
and seventh segments of the abdomen. Then holding the thorax firm, steadily
and gently pull the last segments in the opposite direction. If this is done
properly, a delicate gelatinous white mass will slowly float out in the salt
solution. One should be able to secure the alimentary canal as far up as
the proventriculus, which is just anterior to the stomach, the part in which the
malarial zygotes develop. Proceeding from before backward, we have the
proventriculus, which is a sort of muscular ring at the opening of the stomach
or mid-gut. Leading from the stomach we have the hind-gut, which ends in the
rectum. Taking origin at the posterior end of the stomach and festooning the hind-
gut are five longitudinal tubes — the Malpighian tubules. These are characterized
by large granular-like cells with a prominent refractile nucleus. They are re-
garded as the renal structures. It is in these tubules that the embryo of the Filaria
immitis of the dog develops. In the female mosquito, the parts withdrawn may
seem to be largely made up of the white oval ovaries. These are connected with
the spermathecae, in which the spermatozoa are stored after fecundation by the
male. In the male the testicles are quite distinct. Next to the examination of the
stomach for zygotes, which appear as wart-like excrescences on its outer sur-
face, the most important structures are the salivary glands, where the malarial
sporozoites are found. The easiest way to dissect out the salivary glands is to
press down firmly, but gently, on the anterior part of the thorax, and then with the
shaft of a second needle, pressing on the head to gently draw the head away from
the thorax, so that by this expression and traction movement you extract them
with the head segment. They are very minute and are to be told by their ex-
ceedingly highly refractile appearance. To stain for sporozoites, pick up the head
end, and with forceps draw the severed neck along a clean dry slide, trying at the
same time to smear out the adherent salivary glands. After drying, stain
with Wright's stain. The sporozoites are narrow falciform bodies about I2/J. in
length, with a central chromatin dot.

A matter about which there is dispute is as to whether the salivary glands
communicate with the alimentary canal. Theobald states that there is no con-
nection between them.

DIFFERENTIATION OF CULICIN^E AND ANOPHELIN^E.

It is impossible even for an entomologist to differentiate mosquitoes
without recourse to elaborate keys and tables. It is a comparatively
easy matter, however, to decide as to whether the mosquito is a probable
malaria transmitter or not.

While certain characteristics of the male are used to separate the
^Edinae from other subfamilies, yet it is only with the female that we
concern ourselves in differentiating the Culicinae from the Anophelinae.
Therefore, it is first necessary to distinguish the male from the female.
If the antennae have not been torn off, this can be decided by the highly
adorned plumose antennae of the male, those of the female being sparsely

CLASSIFCATION OF MOSQUITOES

decorated with short hairs. The palpi of the Anophelinae tend to be
clubbed, while those of the Culex are straight. If the antennae have
been broken off, look for the claspers at the end of the abdomen.
Having determined that the insect is a female, we then proceed to place
it either in the subfamily Culicinae or Anophelinae by a study of the
relative length of the palpi to the proboscis. If the palpi are shorter
than the proboscis, it belongs to the Culicinae; if as long or longer, to
the Anophelinae. The palpi of the female Megarhininae are also long,
but the proboscis is curved.

Having settled on the subfamily, we separate the genera by con-
sidering such points as character and distribution of scales on back of
head, wings, thorax, and abdomen; banding of proboscis, legs, abdomen,
and thorax, shape of scales on wings, and location of cross-veins.

FIG. 94. — Anopheles. FIG. 95. — Culex.

Resting positions of anopheles and culex insects. (Drawn by C. 0. Waterhouse.}

In the resting position Culex allows the abdomen to droop, so that it is parallel
to the wall. The angle formed by the abdomen with head and proboscis gives a
hunchback appearance.

Anopheles when resting on a wall goes out in a straight line at an angle of about
45°. It resembles a bradawl.

Classification.

There are four subfamilies of Culicidae, differentiated according to
the palpi:

i. Palpi as long or longer than i. Palpi as long as proboscis in females; proboscis
proboscis in male. straight. Anophelincs.

2. Palpi as long or shorter than proboscis in females;
proboscis curved. Megarrhinina.

3. Palpi shorter than proboscis in females. Culicina.

>. Palpi shorter than proboscis in male and female.

THE MOSQUITOES

The important ones from a medical standpoint are the Anophelinae
and Culicinae.

Anophelinae.

i.' Scales on head only; hairs
on thorax and abdomen.

2. Scales on head and thorax
(narrow curved scales).
Abdomen with hairs.

Scales on head and thorax
and abdomen. Palpi
covered with thick scales.

1. Scales on wings, large and lanceolate. Anopheles,
Palpi only slightly scaled.

2. Wing scales small and narrow and lanceolate.
Myzomyia. Only a few scales on palpi.

3. Large inflated wing scales. Cydoleppteron.

i. Wing scales small and lanceolate. Pyrelophorus.

i. Abdominal scales only on ventral surface.
Thoracic scales like hairs. Myzorhynchus.
Palpi rather heavily scaled.

2. Abdominal scales narrow, curved or spindle-
shaped. Abdominal scales as tufts and dorsal
patches. Nyssorhynchus.

Abdomen almost completely covered with scales
and also having lateral tufts. Cellia.

4. Abdomen completely scaled. Aldrichia.

NOTE. — Of the above genera only Cydoleppteron and Aldrichia are unproven
malarial transmitters.

The Megarhininae are of no importance medically.
The genus Megarhinus has the following characteristics:

1. Large mosquitoes with brilliant metallic coloring. (Elephant mos-
quitoes.)

2. Long, curved proboscis.

3. Caudal tufts of hairs on each side of abdomen.

The ^Edinae are not known to play any role in transmission of diseases. This
subfamily is characterized by having the maxillary palpi much shorter in both
males and females than the proboscis.

One genus Sabethes is very characteristic, owing to dense paddle-like scale
structures on two or more legs.

STEGOMYIA

315

Differentiation of Culicinae Genera.

i. Posterior cross-vein nearer
the base of the wing than
the midcross-vein.

1. Proboscis curved in female. Psorophora.

2. Proboscis straight in female.

A. Palps with three segments in the female.

a. Third segment somewhat longer than
the first two. Culex,

b. The three segments equal in length.
Stegomyia.

B. Palps with four segments in the female.

a. Palps shorter than the third of the pro-
boscis. Spotted wings. Theobaldia.

b. Palps longer than the third of the pro-
boscis. Irregular scales on w i n g s .
Mansonia,

C. Palps with five segments in the female.
Taniorhynchus.

2. Posterior cross- vein in line with midcross-vein. Joblotina.

3. Posterior cross- vein further from base of wing than midcross-vein.

Mucidus.

Of the Culicinae the genus Stegomyia is of importance on account
of yellow fever. The totally efficient hosts for filariasis (filarial embryos
found in the thorax and proboscis) are chiefly among the genus Culex.
The genera Mansonia and Taeniorhynchus may also transmit filariasis.
Some think the Anophelinae genera "Cellia" and "Myzomyia" may
transmit filariasis as well as malaria.

The genus Culex is implicated in dengue.

Stegomyia. — This is the most important culicine genus. These are mosquitoes
with silver markings. The head, entirely covered with flat scales, has also some
upright forked scales. Scutellum has dense flat scales. S. calopus is deep blackish-
brown with two thoracic parallel lines with curved silver-white lines outside (lyre
marking). Banding of thorax, abdomen, and legs.

S. calopus bites only at night after the first feeding. The first meal of blood
however may be taken in the day time. To become infected it must take blood
from a yellow-fever patient in the first two or three days of the disease. After
sucking the blood of a yellow-fever patient the mosquitoes cannot transmit the
disease by biting a nonimmune to yellow fever for a period of eleven days. After
this time the mosquito remains infective for its life — in one instance 57 days.

S. scutellaris has a single silver stripe down the center of thorax. Mosquitoes
of this genus are often called "Tiger mosquitoes." The larvae have short, barrel-
shaped siphons. They breed particularly in receptacles about the house.

S. pseudoscutellaris, which resembles S. scutellaris, but has white bands only,
at the sides of the abdominal segments, is thought to transmit filariasis in Fiji.

Cidex. — Male palpi long and acuminate. Head has narrow curved and up-

316 THE MOSQUITOES

right forked scales. Laterally, flat scales. C. fatigans supposed to carry dengue
as well as Filaria bancrofti. It also transmits Proteosoma of birds, the life history
of which in this mosquito paved the way to the epochal discoveries in connection
with malarial transmission by anophelines. This is a brown mosquito with pale
yellow banding of each abdominal segment. The legs are brown except for the
coxae and femora.

Theobaldia. — These Culicinas have spotted wings resembling Anophelmas.
These spots are due to aggregations of scales, not to dark scales. Male palps are
clubbed (like Anopheles).

Mucidus. — This genus has a mouldy look from long twisted gray scales. The
legs are densely scaled.

Mansonia. — This genus is characterized by broad flat asymmetrical wing scales.
As the wing scales are brown and yellow the wings are mottled.

Grabhamia. — Wings have pepper-and-salt appearance with short fork cells.

Taniorhynchus. — This genus is characterized by dense wing scales, which are
broadly elongated with truncated apex.

Acartomyia. — Much like Grabhamia, but scales of head give ragged appearance.
Male palpi clubbed.

A. zammittii was supposed to be concerned in Malta fever, but it is now known
that transmission is by medium of milk of infected goats.

CHAPTER XXII.
POISONOUS SNAKES.

SNAKES belong to the class Reptilia and the order Ophidia. They
are divided into colubrine snakes (Colubridae) and viperine snakes
(Viperidae).

Of the Colubridae the Hydrophinas or sea-snakes with rudder-like compressed
tail and the Elapinae with round tails are most important.

Many of our harmless snakes such as the garter-snake and blacksnake belong
to the Colubridae.

The cobras belong to the subfamily Elapinae and are best known by a neck-like
expansion or hood. The only poisonous colubrine snakes in the United States are
the beadsnake (Elaps fulvius) often called the Florida coral snake, and the sonoran
coral (Elaps euryxanthus).

The beadsnake is black with about seventeen broad crimson bands, which
bands are bordered with yellow.

Although small, they are very venomous. The upper jaw has anteriorly grooved
fangs, which appendages are not present in the nonpoisonous coral snakes, these
latter having teeth in the upper jaw so that the wound shows four rows of punc-
tures instead of two rows and one larger puncture on each side to mark the entrance
of the fangs.

In Asia there are many important poisonous colubrine snakes; the cobra (Naja
tripudians), the King cobra (Naja bungarus) and the Kraits (Bungarus fasciatus).

All of the Australian poisonous snakes are colubrines.

The Viperidae which are characterized by a triangular head and tubular poison
fangs are the most important poisonous snakes in America. The rattlesnake
(Crotalus), the copperhead (Agkistrodon), and the water moccasin being widely
distributed in the United States.

There are many harmless snakes which more or less resemble these "Pit Vipers"
as the rattlers, moccasins, and copperheads are called. This term refers to a deep
hole or pit found on the side of the head between the nostril and the eye. It is a
blind sac.

Some divide the Viperidae into the Crotalinse, which possess the pit and the
Viperinte which do not have this structure.

The poison fangs are grooved or perforated and connected with the poison
glands which resemble salivary glands and may be almost an inch in length in large
snakes. The tongue is slender and forked and is a tactile organ.

The jaws are remarkable for their great extensibility, not only vertically, but
laterally, by the ligamentous connections of the two halves of the mandible or
lower jaw.

317

318 POISONOUS SNAKES

As the fangs are directed backward it is necessary for the snake when striking
to open widely the jaws and bend back the neck. The fangs are then brought
forward and erected by the spheno-pterygoid muscles. The snake bite is a com-
bination of bite and blow. The functional fangs of colubrine snakes however are
not mobile.

In addition to the possession of the pit, these vipers have a more or less trian-
gular head and in particular a single row of large scales on the under surface posterior
to the vent (anus) , while the harmless snakes show an elongated oval head and two
rows of large ventral scales posterior to the vent.

FIG. 96. — i, Single row of scales posterior to vent (poisonous snakes — water
moccasin); 2, double row scales of harmless snake (Natrix); 3 and 5, side and dorsal
view of head of pit viper; 4 and 6, side and dorsal view of head of harmless snake
(Natrix) ; 7 and 9, bite puncture and skull of Elaps; 8 and 10, bite puncture and skull
of harmless snake.

In examining the wound made by a snake the two punctures of the
fangs indicate the bite of a poisonous snake. If these fang puncture
points are far apart it shows that a large snake, and probably one
capable of injecting a greater amount of venom has given the bite.

When a snake strikes the fangs move from the horizontal to the erect position,
the mouth being widely open. When the fangs enter the jaws close and pressure
is exerted on the poison glands so that the venom pours out.

The amount of venom varies with the size and condition of the snake, an adult
cobra yielding about i c.c.

SNAKE VENOM 319

The cobra, after having bitten, remains attached for a short time while the
daboia strikes with the greatest rapidity and immediately releases itself.

Cobra and krait bites (colubrine snakes) produce more or less
similar symptoms such as paralysis of articulation with nausea and
vomiting and later paralysis of the respiratory apparatus. There is
only an insignificant reaction at the point of bite.

The venom is mainly neurotoxic, causing death by paralysis of cardiac and re-
spiratory centers. Cobra venom is also very haemolytic. This haemolysin is acti-
vated by the normal complement of the serum of the animal poisoned, the haemolysin
as contained in the venom not being toxic when alone. Lecithin also has the
property of activating the hemolytic amboceptor of venom.

In rattlesnake bites (viperine snakes) there is marked pain at the
site of the wound with much swelling and haemorrhagic infiltration.
The swelling and petechial mottling spread up the limb from the point
of entrance of the venom. Cold sweats, nausea, weak heart, and syn-
cope are common.

Rattlesnake venom is active chiefly on account of it's haemorahagin or rather
endotheliolysin, which destroys the endothelial lining of blood-vessels.

Venoms may also contain proteolytic ferments which may account for the
softening of muscles in snake bite cases. The toxic effect of the venom takes place
without an appreciable incubation period, hence different from true toxins.

The most venomous snakes seem to be the sea-snakes (Enhydrina). This
venom is almost entirely neurotoxic.

The tiger snake of Australia is almost equally venomous and the krait (B.
cceruleus) next. The rattlesnake is about one-fifth as venomous as the krait.

Certain venoms greatly increase the coagulability of the blood so that intravas-
cular thromboses may occur. It is chiefly with the venoms of Daboia and Bun-
garus that such thromboses are likely to occur and this accounts for the almost
instantaneous death which at times results from bites of such snakes.

The nonspecific treatment of snake-bite poisoning is i. by applying a tight
ligature above the site of the bite. The ligature, which should preferably be a
rubber band, is to be applied about a single bone extremity, not about one with
two supporting bones. 2. The making of deep incisions about the fang punctures
and thorough irrigation with a strong solution of potassium permanganate. Rogers
has recommended that the punctures be enlarged with a lancet and the resulting
wound packed with crystals of permanganate.

Recently Bannerman has shown that a dog bitten by a cobra cannot be saved
by free incision and the rubbing in of permanganate crystals. It may however be
saved by the immediate injection of 10 c.c. of a 5% solution of permanganate, but
not if two minutes has elapsed. Bites from the daboia are fatal, however the per-'
manganate be applied.

He therefore does not consider the permanganate treatment of any practical
value. Rogers thinks that Bannerman's experiments with dogs do not give a true

320 POISONOUS SNAKES

idea of the value of permanganate because he has had success in experimenting with
cats and because it has saved human lives. Chromic acid injections (i%) have
also been recommended.

Internally alcohol does not seem to be of any value, in fact many of the deaths
have been attributed to excessive ingestion of whiskey. Strychnine in large, almost
poisonous doses, was highly recommended in Australia but the statistics seem to
make the value of this remedy doubtful.

Antivenins. — The active agents of snake venoms may be either of the nature
of haemorrhagins, neurotoxins, or fibrin ferments. In colubrine snakes the neuro-
toxin vastly predominates while with the viperines it is the haemorrhagin. Certain
Australian snakes contain all three bodies in about equal proportion while with
the rattlesnakes of America it is almost entirely the haemorrhagin which causes
the poisoning. The Elaps of Florida is a colubrine snake and its venom is neuro-
toxic in nature.

The cause of death in colubrine snake bites is chiefly from paralysis of the respira-
tory centers while with the Pit Vipers it is chiefly from haemorrhages in the vital
organs. Antitoxins have been prepared against both viperine and colubrine venoms
and these are specific, a colubrine antivenin will not be of value against a viperine bite.
Antivenins should be administered either intravenously or intramuscularly. The
amounts recommended for injections to neutralize a fatal dose of snake poison vary
from 100 to 300 c.c. of the antivenin serum. There is no accurate standardization.

NOTES ON ANIMAL PARASITOLOGY.

NOTES ON ANIMAL PARASITOLOGY.

NOTES ON ANIMAL PARASITOLOGY.

NOTES ON ANIMAL PARASITOLOGY.

PART IV.

CLINICAL BACTERIOLOGY AND ANIMAL PARA-

SITOLOGY OF THE VARIOUS BODY

FLUIDS AND ORGANS

CHAPTER XXIII.
DIAGNOSIS OF INFECTIONS OF THE OCULAR REGION.

IT is advisable before taking material for cultures or smears to
cleanse the nasal area of the eye-lids, and especially about the caruncles,
with sterile salt solution. Then, by gently pressing on the lids, we may
be able to get pure cultures of the organism causing the infection.
Normally, we may find in the region of the caruncles various skin
organisms, especially staphylococci, giving white colonies.

The xerosis bacillus and white staphylococci may be considered normal findings
in the conjunctival sac. Streptococci and pneumococci have also been reported
from apparently normal conjunctival secretions.

A small particle of sterile cotton, wound on a toothpick, with the aid of a sterile
forceps, makes an excellent swab for obtaining material for smears; the same may
first be drawn over an agar surface in a Petri dish in a series of parallel lines of in-
oculation before making the smears on slide or cover-glass.

When there is considerable discharge, a capillary pipette, with a rubber bulb,
may be used to draw up sufficient material for cultures and smears. Be sure to
round off the end of the pipette in the flame and not to use a very fine capillary
tube.

In conjunctival cultures, plates of glycerine agar or agar plates smeared with
blood are to be preferred, as the gonococcus and Koch- Weeks bacillus will only
grow on blood or hydrocele agar. The diphtheria and xerosis bacilli grow well on
glycerine agar.

In addition to the white staphylococcus, the streptococcus may be present
when inflammation of the nasal duct exists.

The Streptococcus is at times responsible for a pseudo-membranous conjunctivi-
tis. The Staphylococcus is as a rule the cause of phlyctenular conjunctivitis.

325

326 INFECTIONS OF THE OCULAR REGION

The pneumococcus is a fairly common cause of serpiginous corneal ulcerations.
Active treatment is necessary.

It is now recognized as advisable to make an examination for the
pneumococcus before perfoiming operations on the eye as serious
results may follow if the pneumococcus be present. It is the organism
frequently found in dacryocystitis and, in the case of traumatism, may
bring about panophthalmitis. .

Corneal ulcerations are not apt to appear even with a pneumococcal conjunc-
tivitis unless there be an injury of the epithelium.

The B. xerosis is possibly a harmless organism and must not be accepted as ex-
plaining an infection unless other factors have been eliminated. The true diph-
theria bacillus, which the xerosis so much resembles, may cause a pseudomembran-
ous inflammation.

The B. pyocyaneus may cause severe purulent keratitis as well as conjunctivitis.
The pyocyaneus toxin appears to be a factor.

The gonococcus and the Koch-Weeks bacillus are usually responsible for the
very acute cases of conjunctivitis. Both these organisms are characteristically
intracellular and are Gram negative.

Conjunctivitis in the course of epidemic cerebrospinal meningitis has been
found to be due to the meningococcus.

The diplobacillus of Morax and Axenfeld is more common in chronic, rather
dry affections of the conjunctiva, chiefly involving the internal angle and showing
a morning accumulation of the secretion. The bacilli are found in twos, more
rarely in short chains. They are generally free but may be found in phagocytic
cells. They resemble Friedlander's bacillus morphologically but do not have
capsules.

In cases of ozena with involvement of the nasal ducts Friedlander's bacillus
may be found.

Even in cases without ozena, capsulated, Gram negative bacilli of the Fried-
lander group have been frequently reported in conjunctival inflammation and in
dacryocystitis as well.

The nodules of the eye-brows give the most convenient area to take
material from in the diagnosis of leprosyf either the fluid expressed
after scraping or a piece of tissue cut into sections. Conjunctival ul-
ceration in leprosy may show abundant bacilli as is also true of corneal
ulceration.

Ordinarily it is impossible to find tubercle bacilli in tuberculous conjunctival
discharges.

The discharge from a tuberculous dacryocystitis may show them satisfactorily.
Animal inoculation is preferable in the diagnosis of ocular T. B. The pneumo-
coccus is, however, the most important organism in dacryocystitis — rarely the B.
coli.

OCULAR INFECTIONS 327

In a gonorrhceal ophthalmia the secretion is much more abundant and there is
an absence of contaminating organisms, the reverse of infection with the confusing
M. catarrhalis. As a matter of fact, large numbers of M. catarrhalis may be present
in the conjunctival secretion with only slight irritation being observable.

In keratomycosis the cause has been ascribed to Aspergillus fumigatus.

Certain fungi of the genus Microsporum have been thought to be the cause of
trachoma, as have also certain bacillary forms. One should be very conservative
about reporting fungi in smears or cultures of external surfaces.

The larval stage of Taenia solium (Cysticercus cellulosae) has a predilection for
eye as well as brain. It is usually situated beneath the retina.

The question as to the nature of the so-called ophthalmic flukes is taken up under
trematodes. Echinococcus cysts have been reported in the orbit.

The adult Filaria loa tends at times to appear under the conjunctiva or in the
subcutaneous tissue of the eye-lids.

Fly larvae have been reported from the conjunctival sacs in the helpless sick.

Demodex may cause an obstinate blepharitis.

Prowazek has thought that certain fine dots within the cytoplasm of epithelial
cells, which stain best by Giemsa's method and which he considered protozoal in
nature, were the cause of trachoma.

CHAPTER XXIV.

DIAGNOSIS OF INFECTIONS OF THE NASAL AND AURAL

CAVITIES.

IN taking material from the nasal cavities, for bacteriological
examination, it is well to wash about the alae with sterile water and
then have the patient blow his nose on a piece of sterile gauze and take
the material for culture or smear from this. If the material is purulent
and located at some ulcerating spot, it is best to use a speculum, and
either touch the spot with a sterile swab or use a capillary bulb pipette
with a slight bend at the end.

Normally, we find only white staphylococcus colonies and colonies of short-chain
streptococci. The M. tetragenus, B. xerosis, and Hoffman's bacillus are also
occasionally found.

In some cases of ozena we may find an organism of the Friedlander type in pure
culture.

Biscuit-shaped diplococci, both Gram negative and positive, are to be found
either normally or in cases of coryza. M. catarrhalis has probably been frequently
reported as the meningococcus. Still, the meningococcus has been found in the
nasal secretions of patients with cerebrospinal meningitis. B. influenzas and the
pneumococcus have also been frequently found in cultures from the nasal secretions.

Diphtheria involving the nasal cavity must always be kept in mind, and in
quarantine investigations the examination of the nasal secretions culturally should
be a part of the routine.

The tubercle bacillus may be found in nasal ulcerations; it is, however, only
present in exceedingly small numbers. On the other hand, one of the best diagnostic
procedures in leprosy is to examine smears from nasal mucous membrane for the
B. leprae. In such ulcerations the bacilli are found in the greatest profusion. Rarely
glanders may cause ulcerations.

B. proteus is frequently responsible for the production of foul odors in nasal
discharges but does not seem to produce inflammatory conditions of the nasal
mucosa. It simply decomposes the discharges. Various fungi have been reported
from the nose, but in such a region the strictest conservatism in reporting should be
observed.

Recently sporozoa have been reported in a case of nasal polyp. (Rhinosporidium.)

So many degenerative changes in epithelial cells resemble protozoal forms that
such findings require ample confirmation.

The larval form of Linguatula rhinaria is a rare parasite of the nasal cavities;
it is not infrequent, however, in the nostrils of dogs.

328

EAR INFECTIONS 329

Various fly larvae are far more common, and the " screw- worm,"
the larva of the Chrysomyia macellaria, is common in certain parts
of tropical America, and may by its burrowing effects cause fatal results.

The larvae of Sarcophaga have in particular been found in the nasal cavities of
children. Myriapods, while of very little importance elsewhere, have been reported
more than thirty times from the nasal fossae.

In a study of the bacteriology of otitis media, in 277 cases, Libman
and Celler found streptococci present alone in 81%, streptococcus
mucosus in 10% and the pneumococcus in 8%; staphyococci, B. pyo-
cyaneus and B. proteus have also been found. Mixed infections are
common.

Streptococci are the organisms which most often cause sinus thrombosis and brain
abscess. The influenza bacillus has been reported as a cause of acute otitis media.

Nonvirulent diphtheroid bacilli are not infrequently obtained in cultures from
ear discharges.

Other organisms which have been isolated from middle ear or mastoid discharges
are B. coli, M. catarrhalis, M. tetragenus and Friedlander's bacillus.

B. typhosus may be found in middle-ear discharges of persons who have had an
attack of typhoid fever.

The middle ear is normally free of bacteria, but in affections of the throat, as
with streptococci, pneumococci, and diphtheria bacilli, these organisms may infect
it by way of the Eustachian tube.

The moulds are of greater importance in affections of the external auditory canal
than the bacteria. The cerumen seems to make a good culture medium so that
various species of Aspergillus, Mucor, etc., develop and close the canal. These
infections are often introduced by the patient's finger. Various mites and fly larvae
have been reported from the ear.

CHAPTER XXV.
EXAMINATION OF BUCCAL AND PHARYNGEAL MATERIAL.

IN a preparation made from material taken by a sterile swab from
the region of the normal buccal and pharyngeal cavities and stained by
Gram's method we are struck by the variety of organisms present.

Gram positive and Gram negative staphylococci are present, as are also strepto-
cocci, pneumococci, leptothrix forms, and very probably yeasts and sarcinae types
with many Gram negative bacilli. If pseudo-diphtheria organisms are present,
we have these showing a Gram positive reaction. If this material is smeared on
agar plates and cultured at 37° C., we are struck by the fact that the colonies on
the plates may be exclusively staphylococcal and streptococcal.

FIG. 97. — Vincent's angina. Spirochaeta vincenti. (Coplin.}

•*

It is very difficult, if not impossible, to distinguish a pneumococcus colony from
•a streptococcus one on a plate culture. The presence or absence, however, of the
pneumococcus is distinctly shown in the Gram-stained smear, either by its lance-
shaped morphology or the presence of a capsule. It has been my experience that
smears from about 15% of normal individuals show capsulated pneumococci.

In diphtheria examinations we rely chiefly on the cultural findings on Lb'ffler's
serum. Where the process is streptococcal or due to the organisms associated with
Vincent's angina, the immediate examination of a smear from the suspected spot
or area gives greater diagnostic information. The streptococcus being so abundant
in cultures from normal throats, it is difficult to determine its significance in a cul-

330

THROAT INFECTIONS 331

ture; abundance of streptococci in a smear from an ulceration or bit of membrane,
however, is of etiological import.

By staining with Neisser's method it is possible to make an imme-
diate diagnosis of diphtheria from a smear from a piece of membrane
in about 25 % of cases. It is well, however, to always culture such
material. The toluidin blue stain of Ponder is the best stain for
diphtheria.

Material from the throat is ordinarily best obtained with a sterile copper-wire
cotton-pledget swab. The platinum loop usually bends too easily. A sterile for-
ceps may be more convenient for obtaining particles of membrane. It is believed
that ulcerative conditions of the throat, associated with the presence of the large
fusiform bacillus and delicate spirillum, which make the picture of Vincent's angina,
are more common than is usually so considered.

In Giemsa stained smears from the dirty membrane covering the ulcerated area
of Vincent's angina there are usually two types of the fusiform bacillus to be seen;
one rather slender, pale blue with maroon dots at either end, the other rather thicker
and of a uniform maroon staining. The spirilla are from 10 to 18 microns long and
the fusiform bacilli from 5 to 7 microns.

As a rule, only cultures on serum are made and very rarely direct smears. If a
smear were always made and stained by Gram's method (with a contrast stain of
dilute carbol fuchsin) at the same time the culture was made, it is probable that
much information of value would be obtained.

The B. fusiformis is an anaerobe which gives a fetid odor but culturally has no
distinct characteristics. The spirillum has not been cultivated. It has been
thought that the bacillus and spirillum are different stages of the same organism.
At times aggregations of the fusiform bacillus give the appearance of branching so
characteristic of diphtheria organisms. Being Gram negative, however, the differ-
entiation is easily made — the B. diphtherias being Gram positive. Again the
attenuated ends of the fusiform bacillus are diagnostic.

Direct smears are the procedure of choice in streptococcal and
pneumococcal anginas as well as in Vincent's angina.

Unless very familiar with the morphology of Treponema pallidum and using
Giemsa's staining procedure, we should be very conservative in reporting such an
organism from suspected syphilitic ulcerations of the throat.

The thrush fungus (Endomyces albicans) may be easily demonstrated in a Gram-
stained specimen as violet mycelial structures.

Yeasts due to food particles are not infrequently observed in smears and cultures
from the mouth.

Actinomycosis may develop about a carious tooth and the finding of the ray
fungus in the granules from the pus may give the diagnosis.

Amoebae and flagellates have been reported from the mouth. Also in the re-
markable disease "halzoun," flukes have been found to be the cause of the asphyxia.

332 EXAMINATION OF BUCCAL AND PHARYNGEAL MATERIAL

In the tropics, round worms may be vomited up and, lodging in the pharynx,
may have to be extracted.

During the campaign of Napoleon in Egypt many cases of leech involvement of
the nasal and buccal cavities were noted. The parasite was the Limnatis nilotica
which gained access to the upper pharynx through drinking water from springs and
pools. Many such cases continue to be reported from the Mediterranean basin.

CHAPTER XXVI.
EXAMINATION OF SPUTUM.

FREQUENTLY the material submitted for examination as sputum is
simply buccal or pharyngeal secretion, or more probably secretion
from the nasopharynx, which has been secured by hawking. It should
always be insisted upon that the sputum be raised by a true pulmonary
coughing act, and not expelled with the hacking cough so frequently
associated with an elongated uvula. When there is an effort to deceive,
some information may be obtained from the watery, stringy, mucoid
character of the buccopharyngeal material and also fiom the presence
of mosaic-like groups of flat epithelial cells (often packed with bacteria).
The pulmonary secretion is either frothy mucus or mucopurulent mate-
rial, and if the cells are alveolar they greatly resemble the plasma cells.
At times these cells may contain blood pigment granules (heart-disease
cells).

In the microscopic examination a small, cheesy particle, the size of a pin head,
should be selected. This should be flattened out in a thin layer between
the slide and cover-glass and should be examined for elastic tissue, heart-disease
cells, eggs of animal parasites, amoebae, and fungi. Echinococcus booklets, Cursch-
man spirals besprinkled with Charcot-Leyden crystals, and haematoidin and fatty
acid crystals may also be observed.

Curschman spirals indicate bronchial as against cardiac or uremic asthma.
Charcot-Leyden crystals have no special significance, except in certain tropical
diseases when these crystals often are present in paragonomiasis sputum and in
the pus of amoebic liver abscesses discharging by way of the lungs.

It may facilitate the examination of the sputum for elastic tissue and actinomy-
cosis and other fungi to add 10% sodium hydrate to the preparation.

To make smears for staining, the sputum should be poured on a flat surface,
preferably a Petri dish, and a bit of mucopurulent material selected with forceps. A
dark back-ground facilitates picking out the particle. A toothpick is well adapted
to smearing out such material on a slide. After using the toothpick it can be burned.
When dry, the smear is best fixed by pouring a few drops of alcohol on the slide,
allowing this to run over the surface, and then, after dashing off the excess of alcohol,
to ignite that remaining on the film in the flame and allow to burn out.

A mark with a grease pencil, about 1/2 inch from the end, gives a convenient
surface to hold with the forceps and also prevents the stain subsequently used from
running over the entire surface. A piece of glass tubing about 12 inches long bent

333

334 EXAMINATION OF SPUTUM

into a narrow V shape makes a very satisfactory rest for the slide in staining and is
convenient for the steaming of staining solution over the flame.

Sputum should as a routine measure be stained by the Ziehl-Neelson method and
by Gram's method.

In examining for tubercle bacilli it may be necessary to employ some method for
concentrating the bacterial content of the sputum prior to making the smear. A very
satisfactory method is that of Miihlhauser-Czaplewski. Shake up the sputum with
four to eight times its volume of 1/4% solution of sodium hydrate in a stoppered
bottle. When the mixture has become a smooth, mucilaginous-looking fluid, add a
few drops of phenolphthalein solution and bring the pink mixture to a boil.

Then add drop by drop a 2% solution of acetic acid, stirring constantly, until
the pink color is just discharged. If the least excess of acid is added over that just
sufficient to cause the pink color to disappear, mucin will be precipitated. Now pour
this mixture into a centrifuge tube and smear the sediment on a slide and stain for
tubercle bacilli.

Tubercle bacilli usually occur nested in clumps of sputum. There-
fore, when few in number it is only by chance that they may be found.
Concentration methods aim to dissolve these clumps of sputum and
collect, free from mucus, whatever bacilli may be present. There are
many concentration methods for sputum. One of these has been
given above. Uhlenhuth's method has some advantages over others
in the solvent used: i. It breaks up the sputum very rapidly; 2. it
immediately dissolves all organisms except acid-fast ones; 3. applied
in not too concentrated form and for not too long a time, tubercle
bacilli are not killed, so that by washing the sediment carefully by
several dilutions and centrifugings we have in the sediment viable
tubercle bacilli which we may attempt to cultivate upon Dorset's or
other suitable media with the reasonable hope that contaminations will
not choke them out or prematurely kill the inoculated guinea-pig; 4.
it has less effect upon the staining properties of tubercle bacilli than
any other material used in concentration methods.

To make this solvent (antiformin) take double the quantity of chlorinated lime
and sodium carbonate required by the U. S. Pharmacopoeia and prepare according
to U. S. P. directions. To the finished liquor sodae chlorinatae (Labarraque's solu-
tion) add 7 1/2% of sodium hydrate.

The Liquor sodae chlorinatae of the Br. P. is slightly stronger and some English
authorities recommend a mixture of equal parts of this Labarraque's solution and
r5% sodium hydrate solution. As a rule one part of antiformin to five parts of
sputum is sufficient. Very tenacious sputum may require one part to four parts
of sputum. If more antiformin is used the specific gravity is too much increased
and the bacilli are damaged. The fluidification is hastened at incubator
temperature.

To five parts of sputum add one part of antiformin, shake well and place in

TUBERCULOUS SPUTUM 335

incubator for one hour. To 10 c.c. of the homogeneous mixture add 1.5 c.c. of a
solution made up of one part chloroform and nine parts alcohol. Shake violently
and centrifuge for 15 minutes. Mix the sediment wit)/ egg albumin, smear out and
stain.

When it is desired to culture the tubercle bacilli mix 20 c.c. of sputum with 65 c.c.
sterile water and add 15 c.c. antiformin. Stir the mixture with a glass rod. After
30 minutes to two hours we should have a homogeneous mixture. Centrifuge for
15 minutes or longer, wash the sediment twice with sterile salt solution and smear out
the well-washed sediment over serum or glycerine egg slants. The tubes should be
covered with black paper and the plugs paraffined. It must be remembered that for
culturing tubercle bacilli we must protect the growth from sunlight as this will kill
the organism. If fluid culture media are inoculated the transferred material should
be deposited on the surface. Should the particle sink growth will not occur.

Sputum smears stained by some Romanowsky method or by the haematoxylin-
eosin stain are best adapted for the study of various cells, and in particular of the
eosinophile cells so characteristic of bronchial asthma. In sputum from cancer of
the lungs the large vacuolated cells may be found.

When examining the sputum of the bronchopneumonia of influenza the formol
fuchsin gives the best results. The influenza bacilli are found in little masses, fre-
quently grouped about small collections of M. tetragenus. The cocci stain a rich
purplish-red, while the small influenza bacilli take on a light pink color.

T. B. sputum showing a mixed infection with streptococci or pneumococci or with
the influenza bacillus makes for a bad prognosis. M. tetragenus, which often is
present when cavities exist, does not seem to be so unfavorable prognostically.

Red cells show up well in specimens stained by the Romanowsky method; if
rouleaux formation is marked, it may indicate pulmonary infarction.

In culturing sputum a mucopurulent mass should be washed in
sterile water and should then be dropped into a tube of sterile bouillon.
With a sterile swab it should be emulsified and successive streaks made
along the surface of an agar or glycerine agar plate. In obtaining cul-
tures from influenza sputum, first smear the material thoroughly over
a blood-serum slant; then inoculate, by thorough smearing over the
surface of successive blood-stieaked agar slants, the material on the
surface of the blood-serum slant. The platinum loop should be trans-
ferred from one slant to another without recharging. The influenza
bacillus seems to grow better if the blood-streaked agar slants are
prepared just before inoculating with the sputum. All that is necessary
is to sterilize an ear, puncture and take up the exuding blood with a
large loop. Cultures for tubercule bacilli are impracticable except with
antiformin. A guinea-pig should be inoculated.

The blood-stained watery sputum of plague pneumonia should be cultured on

336 EXAMINATION OF SPUTUM

plates of plain agar and 3 % salt agar at the same time. An ordinary smear stained
with carbol thionin, however, practically makes a diagnosis.

Pneumococci, M. catarrhalis, and Friedlander's bacillus in sputum are best
demonstrated by Gram's method of staining.

The distinct capsule staining of the pneumococci in a Gram preparation of
sputum from a suspected case of pneumonia is of value in diagnosis.

The finding of the ray fungus (D. bovis) in sputum gives the diagnosis of actino-
mycosis. Streptothrix infections of lungs have been confused with tuberculosis.

Moulds, especially Aspergilli, may be found in sputum. Species of Mucor,
Cryptococcus, and Endomyces have also been reported.

Amoebae from liver abscess rupturing into the lung may be found. Very impor-
tant pulmonary infections are those with Paragonimus westermanii. This is recog-
nized by the presence of operculated eggs in the sputum.

A fluke, F. gigantea, was once found in sputum.

Hydatid cysts, either of the lung or of the liver, rupturing into the lung, may be
recognized by the presence of echinococcus hooklets. The material is bile-stained
if from the liver.

Strongylus apri has been reported once from the lungs and embryos might be
found in the sputum. In pulmonary bilharziosis Schistosoma eggs may be found in
the sputum.

The test for ALBUMEN IN THE SPUTUM is of value in the diagnosis of pulmonary
tuberculosis.

About 10 c.c. of fresh sputum as pure as possible from saliva is mixed with an
equal quantity of water and 2 c.c. of a 3% solution of acetic acid to remove mucin.
After filtering the filtrate is tested for albumin. The test is obtained also in
pneumonia and pleurisy with effusion.

CHAPTER XXVII.

THE URINE.

MATERIAL for staining is best obtained by centifuging the urine,
then pouring off the supernatant urine, then draining the mouth of
the centrifuge tube against a piece of filter-paper so that we have only
the pus sediment to finally remove with a capillary bulb pipette and
make smears.

The addition of a loopful of egg albumen or blood serum to about twice that
amount of urinary sediment gives better results. (See under Staining Methods.)

Orange 'Yellow Green

B C D c

Oxy haemoglobin

MeJkaemo<globin - alkaline

Mefhaemoglobin — -faintly acid

Corborv monoxide haemoqlobm

haemoglobin

FIG. 98. — i, Various absorption bands of spectrum; 2, crystals of glucosazone
(Phenylhydrazine sugar test); 3, Cammidge crystals (interacinar type of pancreati-
tis); 4, Cammidge crystals (interlobular type of pancreatitis).

The smear may be stained directly by Wright's method or after fixing by heat
with Gram's stain, T. B. stain, or haematoxylin and eosin. The latter is the best for
the staining of epithelial cells and animal parasites; the Gram method for bacteria.

It is frequently difficult to distinguish the spores of moulds from red blood-cells
except by measurement and staining reactions. Spores of moulds rarely exceed
five microns.

22

337

338

THE URINE

It is difficult to determine the presence of blood in urine in higher dilution than
i to 300 with the spectroscope. The ordinary occult blood test will show it in much
higher dilution.

To secure urine for bacteriological examination catheterization is rarely necessary
in men — in the case of women it is the proper method.

The glans penis and meatus should be thoroughly washed with soap and water,
after which dilute alcohol (50%) should be used. The greater part of the urine
first passed should be rejected and only the last portion passed should be caught in
a sterile receptacle. A drop of this urine may be either streaked over the surface
of an agar or a lactose litmus agar plate, or so treated after being first diluted in a
tube of sterile bouillon.

The lactose litmus agar medium is very useful in distinguishing typhoid or para-

O o

On*

oW |.

XJcwx

FIG. 99. — Starches and fibers found in urine.

typhoid colonies (blue) from colon, and streptococcus or staphylococcus colonies
(pink). The urine may be added to tubes of melted agar and then poured.

The most satisfactory procedure is to deposit one drop on a poured plate and five
drops on a second plate. The surface is smeared over with a bent glass rod first
smearing out the single drop and then going to the second plate without a second
sterilization. Neutral glycerine agar or blood agar is desirable for such organisms
as pneumococci or streptococci and, for the gonococcus, Thalman's medium smeared
over with a few drops of human serum.

Cystitis from a colon infection gives an acid urine; that caused by Proteus
vulgaris an alkaline urine.

BACTERIAL INFECTIONS OF URINE 339

The old designation B. termo so often employed in connection with the bacteri-
ology of the urine in older works applied to the proteus group and M. ureae to ordi-
nary staphylococci.

The bacillus of typhoid and the micrococcus of Malta fever are also
found in the urine. This elimination in urine of bacilli by typhoid
carriers is of great importance in the spread of the disease.

While the smegma bacillus in urine may be differentiated from the tubercle
bacillus by the former losing its red color, by prolonged decolorization with acid
alcohol, yet it is chiefly by the subcutaneous inoculation of the guinea-pig that we
should diagnose genito-urinary tuberculosis. Inject the sediment after centrifuging.

The method recommended by Gasis which depends on the alkali fast properties
of the T. B. has not given me satisfactory results.

Gonococci are reported from Gram-stained smears.

To culture gonococcus material the transfer to culture media should be made
almost immediately after obtaining the material from the patient. M. catarrhalis
is a rare finding.

Staphylococcus and Streptococcus infections about the mouth as well as such
infections in heart or joint may show the presence of the causative organisms in
the urine. At times bacterial infections of the kidney may give symptoms of renal
stone.

As it is much easier to culture urine than blood a bacteriological examination of
the urine may give us the desired information and the organism for the autogenous
vaccine. Salt mouth bottles with cotton plugs, when sterilized, make cheap and
satisfactory containers. The urine should be plated out as soon as possible after
its passage. As a rule when organisms are present in the urine they are in such
numbers that the question of contamination rarely arises.

Yeasts and moulds frequently contaminate urine, especially diabetic urine,
after it has been passed. Amoebae and flagellates (Trichomonas vaginalis in females)
may be found in urine.

Eggs of Schistosomum haematobium (bilharziosis) are important diagnostic
findings; these are terminal-spined. Those of rectal bilharziosis are, as a rule,
lateral-spined.

In chylous urine the filarial embryos may be found. This examination is facili-
tated by centrifugalization.

The eggs of the E. gigas may be recognized in urinary sediment by their pitted
appearance.

The vinegar eel may be found in the urine of females who have used vaginal
douches of vinegar.

Echinococcus hooklets, scolices, or laminated membrane have been found in the
urine.

The larval dibothriocephalid, Sparganum mansoni, has been reported three times
in urine (urethra).

Oxyuris from the vagina may be found in urine.

Various mites may be found in urinary sediment as the result of lack of care in
the washing of the receptacle and are entirely accidental.

340

THE URINE

Unless having the characteristics of the itch mite and in a person showing scabies
lesions about the genital organs the diagnosis of the mite as A. Scabiei should
not be made.

Crystals of biliverdin may be found in the urinary sediment in marked jaundice.
They somewhat resemble crystals of tyrosin but are brownish in color while those
of tyrosin are black. Furthermore, it is excessively rare to find crystals of leucin
and tyrosin in the urinary sediments, and in such diseases as acute yellow atrophy of
the liver, the urine should be concentrated to one-tenth its volume and the residue
treated with alcohol. The tyrosin crystalline sheaves and the leucin striated
globules crystallize out from the alcohol.

I

s

FIG. IOQ. — Epithelium from different areas of the urinary tract, a, Leukocyte
(for comparison); b, renal cells; c, superficial pelvic cells; d, deep pelvic cells; e,
cells from calices; /, cells from ureter; g, g, g, g, g, squamous epithelium from the
bladder; h, h, neck-of-bladder cells; i, epithelium from prostatic urethra; k, urethra!
cells; I, I, scaly epithelium; m, m' ', cells from seminal passages; n, compound granule
cells; o, fatty renal cell. (Ogden.}

URINARY SEDIMENTS.

Turbidity of the urine is most often due either to bacterial contamination,
amorphous urates (sedimentum lateritium) or phosphates.

Urates go into solution upon heating and phosphates upon the addition of a few
drops of acetic acid.

In turbidity due to bacteria contaminating the urine subsequent to its passage
it is best to call for another sample.

To preserve urinary sediments formalin is the best for casts and epithelial cells
while for general use one may employ a piece of camphor or the addition of one
volume of saturated borax solution to four volumes of urine.

URINARY CRYSTALS

341

Chloroform does not answer for sediments as it does for urine to be examined
chemically. To take up a sediment insert a pipette to the bottom of the tube with
the opposite opening closed by a finger, then tease the sediment into the pipette
opening in the centrifuge tube, by manipulating the fingers.

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FIG. 101. — Deposit in acid fermentation, a, Fungus; b, amorphous sodium urate;
c, uric acid; d, calcium oxalate.

In a urine of acid reaction we may find the following unorganized sediment:
I. Amorphous sodium or potassium urates. Usually yellowish red. Heat and
alkali bring about solution.

-i,

FIG. 102. — Deposit in ammoniacal fermentation, a, Acid ammonium urate; b,
ammonium magnesium phosphate; c, bacteria.

II. Uric acid. Whetstone crystals of yellowish-red color. Soluble in alkalis
but not by heat. Abundant sediment of uric acid crystals may be due to too great
concentration or too great acidity of the urine rather than to the so-called uric acid
diathesis.

342 THE URINE

III. Calcium oxalate. Octahedral crystals or dumb-bell shapes which are
highly refractile. Often due to diet (asparagus, tomatoes, spinach, rhubarb, etc.).

IV. Cystin occurs in six-sided crystals which are soluble in ammonia.
In a urine of alkaline reaction we may expect:

I. Triple phosphates (NH4 MgPO4). Usually in coffin-lid or fern-like form.
Easily soluble in acetic acid.

II. Calcium phosphate and calcium carbonate which effervesce on the addition
of acid.

III. Ammonium urate. These show as the thorn-apple structures.

The presence of ammonium urate, particularly if with triple phosphates, denotes
bacterial decomposition within the genito-urinary tract provided the urine is just

i

FIG. 103. — Fatty and waxy casts, a, Fatty casts; b, waxy casts.

(Greene.)

passed. Pus cells derived from the site of inflammation should be present also.
While certain bacteria might possibly bring on chemical changes without giving rise
to inflammation yet such a possibility is so rare as to be negligible. In the presence
of amorphous phosphates one should always think of exogenous sources as vegetable
diet or withdrawal of proteid food before thinking of disordered metabolism.

Organized Sediment. — An occasional leukocyte may be found in the urine of
healthy people. Any abundance of leukocytes indicates inflammation of genito-
urinary tract. Some workers count the pus cells in urine by the same technic used
for the leukocyte count of the blood. A urine having 100,000 pus cells per c.mm.
will give as a result about 0.1% albumin.

Leukocytes are found in abundance at times in the urine of women without

URINARY CASTS 343

pathological significance. Red blood cells usually show as pale doubly ringed
bodies. They appear in inflammations, particularly stone or schistosome infection.
They may be found in conditions where toxins are being eliminated through the
kidneys, as in tuberculosis. The menstrual period of women must be kept in mind
in the examination of urine sediments.

Epithelial Cells. — For morphology of cells from different locations see illustration.
It is almost impossible to state positively the origin in the genito-urinary tract of
certain cells. Very trustworthy evidence however is finding of epithelial cells on
casts or the so-called compound granine cells (fatty degenerated renal epithelium).
Sheets of more or less small round or caudate epithelial cells are rather significant of
pyelitis. Vaginal epithelium resembles that gotten from scraping the buccal
mucosa.

Of casts we have (i) hyaline, narrow and homogenous. They do not prove
nephritis. (2) Epithelial casts. Usually indicative of nephritis but very slight
inflammatory processes can cause them. (3) Blood casts. (4) Granular casts.
If coarse granules rather significant of chronic nephritis. Finely granular casts do
not seem to have any more significance than hyaline ones. As a matter of fact
under dark ground illumination hyaline casts show a granular structure. (5) Waxy
casts are highly refractile, show fissuring of margins and are of serious prognostic
import (chronic nephiitis).

Cylindroids are drawn out bodies showing tapering ends, irregularity of diameter
and longitudinal striations.

It will be found that a 2/3 in. objective gives almost all the information
required as to casts. It is quicker and gives more positive information.

Mounting a sediment in Gram's solution or tinging it with the merest trace of
neutral red is of much assistance.

344

THE URINE

Special features

Sudden onset. CEdema
often marked, especially
of face. Mild or even
severe uraemic symptoms.
Pulse tension increased
but heart not hyper-
trophied.

Marked oedema. Uraemia
common. Hypertrophied
left ventricle. Blood pres-
sure increased.

No oedema until later.
High blood pressure (200
to 250). Cardiac hyper-
trophy; often uraemia and
albuminuric retinitis.

No uraemia. Symptoms
attributable to heart.

Reaction of urine acid.
No tenesmus.

Reaction of urine alkaline
or very faintly acid.
Tenesmus.

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CHAPTER XXVIII.
THE FJECES.

IT is advisable to examine a stool macroscopically befoie taking up
the miscroscopical examination. The mucus shreds or casts of the
bowel in mucous colitis or membranous enteritis may give the
diagnosis of obscure abdominal pain. Pus in stools may often be noted
without the aid of the miscroscope.

The normal stool is sausage shaped and soft. Neither the special form of scybal-
ous masses called sheep pellets nor the pencil-like nor the tape-like excrement prove
the existence of stricture of the intestinal lumen although suggestive of such a con-
dition. The mucus of bacillary dysentery is opaque and grayish from the great
number of pus and phagocytic cells. It is well to remember that Charcot Leyden
crystals, which are practically always absent from bacillary dysentery stools, are
not infrequent findings in the amoebae containing stools; of course, these crystals
appear in other intestinal parasite infections.

In obstruction of the common bile duct we have acholic, whitish, foul-smelling
stools. If the putty color be due to bacterial change exposure to the air will restore
the brownish tinge.

Sprue stools are white-wash to putty colored, pultaceous, and filled with air
bubbles. The amount is excessive.

Fatty stools are best examined microscopically.

As so many solid masses resemble gall-stones it is well to dissolve the suspected
mass in hot alcohol and examine for cholesterin crystals upon evaporation of the
alcohol.

If the faecal examination is to be made for the diagnosis of amoebae,
in a case where the characteristic mucus stools are not present, or to
verify the existence of flagellates, it is best to give a dose of salts early
in the morning and examine the liquid stools which follow such treat-
ment. This treatment is satisfactory for examination for intestinal
parasites or ova.

A very practical way of obtaining amoebae is to pass a rectal tube or a piece of
drainage tube with fenestrations into the bowel, and amoebae may be found in the
mucus filling the perforations in the tube.

If the purpose of the examination is to determine the digestive power
of the alimentary tract for proteids, carbohydrates, or fats, it is best
to use a test diet, as that of Schmidt and Strasburger.

345

346

THE FAECES

Prior to using this test diet, one should familiarize himself with the macroscopic
and microscopic appearances resulting from such a diet in a normal person; informa-
tion is then at hand to judge of variations from the normal. The examination of
the faeces of persons, on ordinary and specifically undetermined articles of diet, is
very unsatisfactory when the state of digestion of muscle fibers and the question
of fat digestion are at issue.

In examining the faeces of the normal person and likewise with the patient, wait
until the second or third day so that the faeces of previous diets may have passed out.
A charcoal powder taken before commencing the diet serves as an indicator.

Diet: breakfast, 7 A. M., bowl of oatmeal gruel (40 grams oatmeal, 10 grams
butter, 200 c.c. milk, 300 c.c. water). Also one very soft-boiled egg (i min.) and 50
grams zwieback. In the forenoon, 500 c.c. of milk.

For dinner, 2 o'clock, chopped beef broiled very rare (125 grams with 20 grams
butter poured over it). Also a potato puree (200 grams mashed potato, 50 grams
milk, 10 grams butter). Also 1/2 liter of milk and 50 grams zwieback.

FIG. 104. — Microscopical constituents of faeces, (v. Jaksch.} a, Muscle fibers;
b, connective tissue; c, epithelium; d, leukocytes; e, spiral cells; /, g, k, i, various
vegetable cells; k, "triple phosphate" crystals; /, woody vegetable cells; the whole
interspersed with innumerable microorganisms of various kinds.

For supper, 7 o'clock, the same articles as for breakfast.

This detailed diet may be varied to suit circumstances as regards interchanging
meals. Furthermore, the milk may be taken in the form of tea or cocoa or cooked
with the other food. Even a small amount of wine may be permitted. The diet
taken, however, should absolutely conform to the following requirements: i. the
taking of 1/4 pound chopped beef, a portion of which should be half raw; 2. the milk
taken should amount to about a quart; 3. about 4 ounces of bread or toast and from
4 to 8 ounces of potato puree should be eaten daily.

The detailed diet contains about no grams albumin, 105 grams fat and 200
grams carbohydrates with a fuel value of 2247 calories.

The stool is best collected in quart fruit jars and examined as soon after evacua-
tion as possible. The wooden spatula like tongue depressors are well adapted to
handling the specimen.

FAT IN THE F^CES 347

Having familiarized one's self with the degree of digestion of muscle, starch,
and fat in a normal person, we are in a position to judge of the state of assimilation
in a patient.

The first part of the test is the macroscopical one. For this grind up a faecal
mass of 1/2 to i inch diameter in a mortar, gradually adding water until it has the
consistence of a broth. About 1/2 c.c. of this emulsion should now be squeezed
out between two slides and studied against a dark surface and then when held up
to the light. The normal stool gives a rather uniform brownish homogeneous layer.
Connective-tissue remnants (indicative of gastric derangement) show as whitish
fibers. Undigested muscle tissue remnants as reddish-brown splotches. Fat
particles as whitish-yellow clumps. Potato remnants appear like sago grains and
mash out easily like mucus. Mucus is best noted in the fecal mass before making
the emulsion. In the microscopical test of this emulsion.

We judge of muscle digestion by the intactness of the striations.
If a muscle remnant is only a homogeneous yellowish particle, it shows
satisfactory digestion. If it is rectangular, with well-defined cross
striations, it shows poor digestion for meat (Azotorrhcea). A loopful
of faeces should be smeared into a drop of Gram's solution for starch-
digestion determination. Normally there should be no blue-staining
starch granules.

Soaps are gnarled bodies everted like the pinna of an ear, while soap crystals
are comparatively coarse and do not melt on application of gentle heat as do the more
delicate fatty acid crystals. Neutral fat is in round or irregular globules. The best
stain for fat is Sudan III (saturated solution of Sudan III in equal parts of 70%
alcohol and acetone).

Mix up the fasces with dilute alcohol (50 to 70%) and then add a drop of the above
solution and apply a cover-glass quickly. The fat globules show as orange or
golden-yellow bodies.

By rubbing up a small portion of the faeces in 36% acetic acid, applying a cover-
glass and heating over a flame until the preparation shows bubbles, we convert the
soaps and other fat combinations into free fatty acids which show as more or less
numerous highly refractile bodies which show a crystalline structure as the prepara-
tion cools. By practice one learns the amount of such globules to expect with differ-
ent fat contents in stools.

Steatorrhoea, or the presence of fat in abnormal quantities in the
faeces, is shown by the pale, bulky, greasy stools as well as in the micro-
scropical examination.

Average for normals in i gm. dried fasces:

Total fat,
Total fatty acid,
Total soap,
Total neutral fat,

225 mg.
86 mg.

74-7 mg-
64.4 mg.

(22.5%)
(37. 9% of all fat)
(33. 4% of all fat)
(28. 5% of all fat)

348 THE FAECES

In normal cases the only fat elements recognizable are yellow calcium or color-
less soaps.

As quantity of fat increases (as say 500 to 600 mg.) droplets of neutral fat appear
with or without increase in number of soap masses. Also needles and splinters
of fatty acid and soaps appear. Much connective-tissue debris shows defect in
gastric digestion, as only the stomach digests connective tissue.

A test for activity of fermentation should be made by using a Schmidt
apparatus.

A distinct evolution of gas in twelve hours shows starch digestion defect. Such
fasces are acid. A delayed production of gas (after twenty-four hours) shows albu-
min decomposition. Such faeces show an alkaline reaction. The apparatus is
shown in Fig. 7. Into a stocky salt mouth bottle we put approximately 5 grams
of faeces which have been rubbed up into an emulsion with water and fill the bottle
with water. The remaining portion of the apparatus consists of a test-tube or a
graduated cylinder fitted with a doubly perforated rubber stopper. One U-shaped
glass tube passing through this stopper connects with a second test-tube. This tube
serves as a receptacle for any water which may come over from the water-filled tube
or graduated cylinder and has an opening punched out of the bottom of the test-
tube. The other opening in the twice perforated cork admits a straight tube which
connects with a large rubber stopper which fits into the bottle for the fasces. To
prepare, fill the graduated cylinder, then push in the doubly perforated cork which
is connected with the side receiving tube and the large rubber cork. This latter is
then pushed down to fit tightly into the bottle filled full with the faeces emulsion.

In addition to the faeces examination we should check the results from the test
diet with indican and nitrogen partition determinations of the twenty-four hour
urine specimen — the ratio of ammonia nitrogen to total nitrogen indicating the func-
tional power of liver and the indican the question of stasis in lower part of small
intestine.

The most satisfactory test for bile in the faeces is to emulsify a small
particle of faeces in a saturated aqueous solution of bichloride of mercury,
preferably with a wooden tooth pick, on a concave glass slide. After
one or more hours hydrobilirubin-containing faeces show a salmon pink
color and bilirubin ones a green color. One should familiarize himself
with these reactions in normal cases.

In examining a liquid stool after salts, it is well to color the drop of faeces, which
is to be covered with the cover-glass, with a small loopful of 1/2% solution of neutral
red. If diluting fluid is used, it should be salt solution, and not water. The neutral
red tinges the granules of the endoplasm of amoebae and flagellates a very striking
rose pink color, thus differentiating them from vegetable cells or body cells.

Whether examining the thin faeces or the mucus particle, it is well to reserve
report on amoebae or flagellates until motion is observed. Encysted protozoa are
difficult to diagnose.

When a smear preparation is desired, we may smear out a fragment of mucus
and stain by Romanowsky's or Gram's method. The character of the bacteria

BACTERIAL EXAMINATION OF FAECES 349

present appears to be of diagnostic value — especially in the case of infants and young
children. Beautiful preparations may be made by mixing the faeces with water, then
centrif uging for one minute. This throws down vegetable debris and crystals. Now
decant the supernatant fluid, which holds the bacteria in suspension, and add an
equal amount of alcohol. Again centrifuge, decant, and smear out and examine
the bacterial sediment.

Simply taking a small mass of faeces and emulsifying it with a
wooden toothpick on a concave slide in 70% alcohol — then, after the
sediment settles, taking up a loopful with platinum loop from the sur-
face and smearing out, gives a very satisfactory smear. Gram's method,
with dilute carbol fuchsin counterstaining, gives the best picture.

The Boas-Oppler bacillus may be found in the stools in this way. Normally,
a Gram-stained stool shows a great preponderance of Gram negative bacilli and such
a finding in a measure excludes cancer of the stomach. Organisms which are Gram
positive as well as the Boas-Oppler bacillus are, i. Lactic acid bacilli — these show
Gram negative areas in the slender bacilli. 2. A type of bacillus similar in size to the
colon bacillus but Gram positive and noncultivable (found in acid stools).
3. Bacilli of the B. subtilis type.

It is very important to examine the fasces for T. B. With children a diagnosis
of tuberculosis may be made in this way when the sputum cannot be obtained,
the pulmonary secretion being swallowed. The preparation on the concave slide
as described above should be stained for T. B.

To culture for typhoid, dysentery, cholera, or other bacteria, take up the material
in a tube of sterile bouillon and smear it out with a swab over a lactose litmus agar
plate or an Endo or Conradi-Drigalski plate. Before streaking the plates they should
be very dry on the surface. This can be best done by pouring into a plate with a
circular piece of filter-paper in the lid and placing in the incubator for one-half hour
to dry. The filter-paper absorbs the moisture. Then inoculate the surface of the
plate with the faecal material.

In summer complaints of infants and children the organisms con-
cerned are as a rule related to various dysentery strains of bacilli.
Kendall in 293 stool examinations found the gas bacillus (B. aerog^
capsul.) in 22 cases. The gas bacillus produces intestinal disorders
which are not benefited by lactose but by buttermilk (lactic acid bac-
teria). For diagnosis, a loopful of the faeces is emulsified in a tube of
sterile milk or litmus milk. The emulsion is heated to 80° C. and held at
this temperature for 20 minutes. After incubation for 1 8 to 24 hours,
preferably anaerobically, we get (i) a shreddy disruption of the casein,
(2) the smell of rancid butter and (3) fully 80% of the casein is dissolved.
Smears show short thick Gram positive rods with slightly rounded ends.
B. Subtilis is sometimes found but does not give a rancid odor nor the
strong disruption of the clot.

350 THE FAECES

It was until recently thought that Cammidge's reaction (urine) when associated
with azotorrhoea and steatorrhcea made for a diagnosis of chronic pancreatitis.
At present very little importance is attached to the Cammidge reaction.

Loss of weight, anaemia, diarrhoea and pains in the upper abdomen are important
indications of pancreatic trouble. As chronic pancreatitis is often associated with
cholelithiasis jaundice is frequently present. Glycosuria is not often present.
While functional tests are important they do not make for a sure diagnosis.

Miiller's method for pancreatic functioning determination is to give a calomel
purge two hours after a meal. A little of the liquid stool is smeared on the surface
of blood-serum and the tube incubated at 60° C. (paraffin oven). If the surface is
smooth, no trypsin was present; if dotted with spots of digestion liquefaction, it
shows that the pancreatic secretion is present.

In Schmidt's nucleus test small cubes of beef are hardened in absolute alcohol
and then tied up in tiny silk bags. These are recovered from the faeces and sec-
tioned. Complete preservation of nuclei indicates a total absence of pancreatic
functioning provided the passage of the tissue be not too rapid as by diarrhoea.

Epithelial cells are generally more or less disintegrated. In the mucus of
•bacillary dysenteric stools, however, large intact phagocytic cells are frequent,
which may be mistaken for encysted amoebae.

Triple phosphate crystals are frequently observed in faeces, as may also be crys-
tals of various calcium salts. Charcot-Leyden crystals are rather indicative of
helminthiases.

Various flagellates, and in particular Lamblia, may be responsible for diarrhceal
conditions which may cause rather serious symptoms.

Balantidium coli has been reported several times as the cause of dysenteric
conditions. Coccidiadea are found in the fasces.

It is in the faeces we examine either for the parasites or for their ova
in connection with practically all the flukes, except the lung fluke and
the bladder fluke; for intestinal taeniases and for practically all the
round worms, except the filarial ones.

Bass has recommended that faeces which have been made fluid be centrifuged
and the supernatant fluid containing vegetable debris be poured off. The sediment
contains hookworm eggs. Then pour on sediment a calcium chloride solution of
sp. gr. 1050. Again centrifuge and decant. Next add calcium chloride solution
of a sp. gr. of 1250 and centrifuge. This brings to the surface the hookworm eggs
which may be pipetted off. As a rule, the finding of hookworm eggs is very easy
without such a technic.

In the tropics, the examination of the faeces vastly exceeds in value
that of urine and is possibly more important than blood examinations.

The larvae of various insects may at times be detected in the stools, as well as
certain acarines (cheese mites, etc.).

The test for occult blood is indicated in helminthiases as well as in the conditions
for which it is usually tested.

CHAPTER XXIX.
BLOOD CULTURES AND BLOOD PARASITES.

CLINICALLY, the most important examinations of the blood for
parasites is for the presence of various bacterial infections and for
certain blood protozoa and also filarial embryos.

The modern method of culturing blood, especially for the detection of typhoid
or paratyphoid bacilli, is by the use of the bile media of Conradi. Test-tubes are
filled with 7 to 10 c.c. of i% peptone ox bile, or ox bile alone, and the medium is
sterilized in the autoclave. It is good practice to place the syringe in a plugged
test-tube containing salt solution, with the needle unscrewed. After autoclaving,
the sterile syringe can be taken to the bedside in the test-tube. Using a wide test-
tube, a forceps can be sterilized at the same time and used to attach the needle to the
barrel of the syringe.

By using a piece of glass tubing into which the needle is inserted we may sterilize
the syringe easily in the test-tube. The glass tubing prevents the steel needle from
coming in contact with the glass of the test-tube and so prevents cracking the test-
tube. Instead of a syringe a better apparatus is a test-tube or an Erlenmeyer flask
\yith a double perforation stopper for insertion of two pieces of glass tubing, one
joined to a piece of rubber tubing carrying the needle and the other attached to a
rubber tube for suction by the operator's mouth. See Fig. 7.

The skin should be scrubbed gently with green-soap solution and water for about
three minutes. The skin of the area to be punctured should then be sterilized by
the gentle application of Harrington's solution (not scrubbed) for one-half minute,
and should then be washed with sterile water. It appears to be safe to simply
scrub the area with 70% alcohol for one or two minutes. Applications of pure
carbolic acid on a gauze wad for a few seconds followed by neutralization with 70%
alcohol gives satisfactory sterilization. The present method of sterilizing skin
for taking blood or inoculating vaccines is simply to smear the site of entrance for
the needle rather heavily with tincture of iodine. A tourniquet is now applied to
distend the vein, and the needle is inserted in the direction of the venous flow.
Withdrawing 5 to 10 c.c. of blood, we loosen the tourniquet, (otherwise the blood
may flow from the puncture) then withdraw the needle, and force out abo'ut 1/2 c.c.
into the first bile tube, about i c.c. into the second, and 2 or 3 c.c. into the third.
It is well to reserve some of the blood for Widal tests.

The bile tubes are now incubated for ten to twelve hours and then transfers
are made to bouillon tubes. These bouillon tubes can be used in six to eight hours
for testing the organism against known typhoid or paratyphoid sera. Test-tubes
containing 10 c.c. of ordinary bouillon with i% of sodium citrate are as satisfactory
as bile media.

351

352 BLOOD CULTURES AND BLOOD PARASITES

Some prefer a 2% sodium glycocholate in bouillon while others use a 2% solution
of ammonium oxalate in bouillon for blood cultures.

Some prefer to streak plates of lactose litmus agar with material from the bile
tubes instead of inoculating the bouillon tubes. Contamination with staphylococci
or the presence of staphylococci, streptococci, or plague bacilli in septicagrmc con-
ditions show easily accessible colonies.

Schotmuller adds i to 3 c.c. of blood to liquefied agar at 45° C., and after mixing
pours into plates. The standard method formerly was to add the blood to an
excess of bouillon (i to 5 c.c. of blood to 100 c.c. or more of bouillon).

A very useful procedure in the isolation of streptococci, pneu-
mococci, plague and anthrax bacilli is to inject i to 2 c.c. of blood
into suitable animals. When injecting mice use only about 0.2 c.c.

By using the bile media, we can take the blood from the ear in typhoid cases,
if preferred. Then if chance staphylococcic contamination occurs, such colonies
are readily differentiated from typhoid ones by the pink color on lactose litmus
agar. For culturing blood in septicaemic conditions, the blood should always be
drawn from the vein and cultured either by mixing i to 2 c.c. with melted agar
and then pouring plates or by transferring to bouillon in excess (at least ten times
as much bouillon as blood) and after eighteen to twenty-four hours' incubation
plating out. For streptococcus and pneumococcus blood agar plates are to be pre-
ferred, the pneumococcus giving green colonies with only a suggestion of haemolysis
while the streptococcus gives an opaque colony with a distinct haemolytic zone
surrounding it.

Typhoid cultures are best obtained in the first week of the disease,
after that time the Widal is the test of preference.

If a paratyphoid serum is not at hand for testing, it may suffice to inoculate a
glucose bouillon tube or a Russell lactose glucose litmus slant; gas production
indicates paratyphoid. This test should be applied when a very motile organism
does not show agglutination with a known typhoid serum. Anthrax and glanders
should be considered in blood cultures.

In Malta fever it must be remembered that colonies do not show themselves
for several days. Addition of blood to melted agar is a good procedure.

Blood for culturing typhoid or the paratyphoids may be taken
with a Wright's tube from the ear or finger. Dipping the hand in hot
water assists the flow of blood. The supernatant serum after centrifu-
galization should be pipetted off with a sterile pipette and reserved
for agglutination tests while the clot is dropped into a bile tube.
(Clot culture.)

Rosenberger was the first to insist upon the importance of examination of blood
for T. B. Brem considered that many cases of finding of acid-fast bacilli were not
of T. B. The Kurashigi-Schnitter method for tubercle bacilli in blood is to take

BLOOD PARASITES 353

about i c.c. blood and put in a centrifuge tube containing 5 c.c. of 3% acetic acid.
After the red cells are thoroughly laked centrifuge, pipette off supernatant fluid and
dissolve the sediment in 5 c.c. antiformin. When dissolved add 5 c.c. absolute
alcohol and centrifugalize for twenty minutes. Smear out the sediment and stain.

The examination of the blood for the parasites of malaria, filariases, kala-azar
and spirillum fevers has been discussed under their respective headings.

With trypanosomes from human trypanosomiasis, smears from gland juice or
cerebrospinal fluid seem more satisfactory to examine than blood smears unless the
blood is taken in 5 to 10 c.c. quantities and centrifuged in sodium citrate salt solution.

The latest method in the diagnosis of trichinosis is to take 5 to 10 c.c. of blood
from a vein at the time of the migration of the embryos to the muscles (10 to 20
days). This is forced out into a centrifuge tube containing 3% acetic acid, and
the sediment examined for trichina larvae.

CHAPTER XXX.
THE STOMACH CONTENTS.

FROM a microscopical standpoint there is comparatively little that
is of value in the examination of the gastric contents; there is nothing
very specific about the findings.

A test meal is not a necessity as in the chemical examination, but
either vomitus or material withdrawn with a stomach-tube two or more
hours after an ordinary meal suffice.

The most satisfactory specimen is one taken before the giving of
the test meal.

The washings from the stomach are allowed to stand until the
sediment has fallen to the bottom and an examination of this is made.

The microscopical diagnostic points in connection with distinguishing cancer of
the stomach from nonmalignant dilatation are: i. Fragments of cancer tissue.
These are very rarely found and are most difficult to diagnose. 2. The presence of
flagellates in the early stages of cancer (the so-called anacid stage preceding the
development of lactic acid) . As flagellates prefer an alkaline medium, they disappear
after the acidity due to lactic acid comes on. 3. The presence of the Boas-Oppler
bacillus. There* are probably several organisms so designated. They are lactic acid
producers and are characterized by being very large bacilli (7X1/0 and arranged in
long chains which stretch across the field of the microscope. They are Gram posi-
tive and do not form spores. They can be cultivated on media rich in blood and
are aerobic. They should only be reported when present in great abundance and in
long chains. Heinemann thinks it probable that the Boas-Oppler bacillus, Lepto-
thrix buccalis, and B. bifidus may be identical with B. bulgaricus (see under Milk).
4. The absence of sarcinae and yeasts. The presence of these sarcinae and fungi in
vomitus is indicative of a simple dilatation.

In chronic gastritis the picture of mucus entangling large numbers of epithelial
cells is characteristic.

In examining the sediment from the filter-paper after filtering off the stomach
contents always use a dilute Gram solution (about i to 4) for mounting the sediment.
Muscle fibers, yeast cells, red blood-cells, and epithelial cells are stained a golden
yellow. Starch granules are stained blue while fats are unstained and show as glo-
bules of varying sizes.

354

CHAPTER XXXI.
EXAMINATION OF PUS.

Pus may be collected for examination either i. with a platinum loop,
2. with a sterile swab, 3. with a bacteriological pipette or 4. with a
hypodermic syringe.

It is always well to make a smear and stain it by Gram's method at
the same time that cultures are made. The Gram stain gives informa-
tion as to the abundance of organisms in the pus and as to the probable
findings in the culture. Pneumococci and streptococci are differenti-
ated from the staphylococci in this way without the necessity of more or
less extended cultural methods.

Smears from material examined for gonococci may show Gram negative diplo-
cocci which, however, do not generally have the morphology of the gonococcus.
They are furthermore extracellular.

The M. catarrhalis has been reported from urethral smears though very rarely.
Diphtheroid organisms are not uncommon. Gram positive cocci are rather common
in smears from discharges of chronic gonorrhoea.

When autogenous vaccines are to be made, the isolation of the exciting organism
is necessary. This is best done by streaking the pus, taken up with a sterile swab
and emulsified in a tube of bouillon, over the surface of an agar plate. Practically
as convenient and providing a more nutritious medium is to smear the material on
a loop or swab over the surface of a blood-serum slant, then to inoculate a second
tube from the first without recharging the loop or swab, and so on until three or four
tubes are inoculated. Isolated colonies should be obtained in a third or fourth tube.

In examining blood-serum slants inoculated with purulent material, always
examine the water of condensation for streptococci.

A bacteriological pipette is very useful when pus is to be sent to a laboratory;
the tip can be sealed in a flame and the cotton plug at the other end insures the
noncontamination of the contents. The material may be drawn up either with the
mouth or with a rubber bulb.

The hypodermic syringe is very useful in puncturing buboes, etc.,
especially in plague. A small pledget of cotton on a toothpick dipped
into pure carbolic acid and touched to a spot over the bubo, which after
about thirty seconds is soaked with alcohol, makes a sterile anaesthetic
spot at which to introduce the needle of the syringe. It must be remem-

355

356 EXAMINATION OF PUS

bered that when plague buboes begin to soften, the plague bacilli may
be replaced by ordinary pus organisms.

It is remarkable how frequently we get pure cultures from abscess material. In
purulent material from abdominal abscesses we are apt to obtain mixed cultures,
especially the colon bacillus and B. pyocyaneus, in addition to ordinary pus
organisms.

When it is a question between streptococci and pneumococci, it is well to inocu-
late a mouse; the capsulated pneumococci at the autopsy make the diagnosis.

Animal inoculation is also necessary in plague and glanders, and possibly anthrax.
When tetanus is suspected, it should be examined for as described under Tetanus.
Tuberculosis should also be identified by inoculating a guinea-pig, as well as by acid-
fast staining and culture, if there is any doubt as to the nature of the material.

The black or yellow granules of madura foot, as well as those of actinomycosis,
should be examined as recommended in the section on fungi.

Amcebae, coccidia, and larval echinococci may be found in purulent material,
as may also various other animal parasites, as fly larvae, sarcopsyllae, etc.

The pus from an amoebic abscess of the livei is as a rule sterile when
cultured.

The examination at the time of operation or exploration frequently
shows an absence of amoebae as well as of bacteria. Two or three days
later amoebae may be found in the pus draining from the abscess cavity.

Flukes, round- worms, and whip- worms may as a result of their wandering from
the intestinal lumen cause abscesses.

Serious ulcerations may follow infection with the Guinea-worm.

CHAPTER XXXII.
SKIN INFECTIONS.

CULTURAL methods are as a rule to be preferred in the bacterio-
logical examination of the skin.

This is best done by washing the surface to be examined with soap and water, in
order to eliminate chance organisms which may have settled on the surface of the
skin in dust or as a result of contact with material containing them. Scrapings are
then made with a sterile dull scalpel, and this material is emulsified in a drop of
sterile water in the center of a Petri dish. A tube of melted agar at 42° C. is then
poured on the inoculated drop and, by mixing, the bacterial flora is distributed over
the entire surface of the plate. Of the colonies developing on such plates probably
80% will be found to be staphylococci, and of these the greater proportion will
be staphylococci showing white colonies.

Occasionally the aureus or citreus may be isolated.

Streptococci and colon bacilli are rarely found.

The Staphylococcus pyogenes aureus is the organism usually isolated from
furuncles, circumscribed abscesses, and carbuncles.

Streptococci are the organisms to be expected in phlegmonous infections.

Cold abscesses, which are frequently due to tuberculous infection, are, as a rule,
sterile.

Acne pustules may show staphylococci or the microbacillus of acne may be
present.

The Bacillus acnes is a short broad bacillus often showing a beaded
appearance when stained by Gram's method. It is Gram positive
According to Hartwell it grows readily on glucose agar when cultivated
anaerobically (Wright's method). Colonies appear in four to five days.

Sabouraud's medium for its culture is: Peptone 20 grams, glycerine 20 grams,
glacial acetic acid 5 drops, agar 15 grams and water 1000 c.c. The bottle bacillus,
which morphologically resembles a yeast, is considered to be the cause of dry pityri-
asis capitis. It may also be found in the comedones of children.

In the tropics, an organism which at times produces lesions similar to impetigo
and again pemphigoid eruptions and at other times wide-spreading erysipelatous
conditions gives cultural characteristics similar to S. pyogenes aureus. It is probably
only a virulent aureus. It has been described under the name of Diplococcus pem-
phigi contagiosi.

The Staphylococcus epidermidis albus, or stitch abscess coccus, is considered by
Sabouraud to be the cause of eczema seborrhoicum.

357

358 SKIN INFECTIONS

It is in scrapings from the skin of lepromata that we find acid-fast
organisms in the greatest profusion. In tuberculosis of the skin the
tubercle bacilli are exceedingly scarce. Inoculation of a guinea-pig
will piobably give positive results with the tubercle bacillus. The
leprosy bacillus is noninoculable for experimental animals.

Anthrax. and glanders cause skin lesions which can only be surely diagnosed
culturally or by animal inoculation.

Plague bacilli may be isolated from the primary vesicles appearing at the site of
the flea bite.

Tropical phagedaena is thought by some to be due to a sort of diphtheroid or-
ganism. The organisms of Vincent's angina may cause tropical ulcer.

The skin diseases due to fungi are discussed under that section. Of the skin
affections caused by animal parasites, ground itch is the most important. This is a
form of dermatitis due to the irritation set up by the hook-worm larvae penetrating
the skin of the foot and leg.

The Sarcopsylla penetrans or jigger (sand flea) is an important agent in ulcera-
tions about the foot.

Certain acarines cause skin lesions, as is also the case with the larvae of certain
flies.

The itch mite (Sarcoptes scabiei) is an important animal parasite of the skin.

The various lice, fleas and bed bugs are well understood as causes of skin irrita-
tion.

Filarial infections are also important especially the ulcers of the
Guinea-worm, Calabar swellings of F. loa, the cystic tumors of F.
volvulus and the varicose groin glands and elephantiasis of F. bancrofti.

Leeches, as H. ceylonica, may cause serious ulceration.

Oxyuris may cause a severe irritation about the region of the groin and inner
surfaces of the thigh, and especially about the vulvar region of female children.

Gnathostomum siamense, a nematode with two lip-like structures and spine-
like structures covering its anterior one-third, has been found once in a tumefaction
of the breast.

Plerocercoid larvae of Dibothriocephalidae have been found in the subcutaneous
tissues.

Certain skin diseases, as Oriental sore and yaws, are protozoal in origin.

CHAPTER XXXIII.
CYTODIAGNOSIS.

THIS method of diagnosis is chiefly employed in the examination of
cellular sediments of pleural, ascitic, and ceiebrospinal fluid.

The fluids which pathologically collect in the serous cavities are
divided into two classes, i. the transudates, which form as the result
of some circulatory inadequacy and 2. the exudates, which result from
inflammatory processes.

Transudates have little or no fibrin and very few cellular elements and do not
contain nucleo-albumin. Exudates contain nucleo-albumin and usually have a
specific gravity above 1018, while that of the transudates is lower than 1018.

There are two simple methods for differentiating transudates and exudates.
Moritz adds 2 drops of a 5% solution of acetic acid to the fluid to be tested. A
heavy, cloud-like precipitate shows the fluid to be of inflammatory origin (an
exudate). A transudate may produce a slight opalescence. Rivalta's test consists
in dropping a drop of the fluid to be tested into a cylinder containing 2 drops of
glacial acetic acid in 100 c.c. distilled water. A nebulous cloud as the drop of fluid
sinks shows an exudate.

For pleural fluids we should receive the material in centrifuge tubes about one-
fourth filled with 2% sodium citrate salt solution. This prevents clotting. Having
thrown down the sediment, the supernatant fluid is poured off, and in its place a i %
aqueous solution of formalin is added. After mixing and allowing to stand for about
five minutes, centrifugalization is again repeated and, pouring off the supernatant
formalin solution, we make smears from the sediment. This is either stained by a
Romano wsky method or, after fixing with heat (burning alcohol), the smear is
stained with haematoxylin and eosin.

With ascitic fluid it is usually sufficient to centrifuge the fluid, then decant off the
supernatant fluid and drain by means of a piece of filter-paper held at the
mouth of the upturned tube. The sediment adheres to the bottom of the tube and
is best emulsified with the small amount of fluid remaining by means of a bulb
pipette. The material is sucked up, smeared out on a slide with a second slide as
for blood and stained preferably with Giemsa after fixation. H. E. staining brings
out mitotic figures best. If the fluid has coagulated it is best to take a little of the
coagulum and stain it with neutral red as for vital staining. It is difficult to disso-
ciate the cells from the clot. The wet Giemsa method described for blood gives good
results with puncture fluid sediments.

At the time of securing fluid for cytodiagnosis, cultures should be made on blood-

359

360 CYTODIAGNOSIS

serum for various pyogenic bacteria and, if tuberculosis is suspected, inoculation
of a guinea-pig is indicated.

The interpretation of cellular sediments is more difficult than many books would
indicate, there being many factors which tend to complicate the findings.

The polymorphonuclears in purulent fluids often show fatty degeneration,
swollen and faintly staining nucleus or a breaking up of the nucleus into small deeply
staining masses (nuclear fragmentation). Such fragments in the smear may be con-
fusing. The endothelial cells often show fatty degeneration in the cytoplasm and
we often note bacteria and other cells which have been phagocytized by them.
Where proliferation of endothelial cells is going on actively the cells show a rather
deeply staining cytoplasm as compared with the light staining cytoplasm of the cells
in transudates.

The following are the leading differentiations:

1. A smear showing almost entirely lymphocytes with a few red
cells and very rarely a polymorphonuclear indicates a tuberculous
process.

2. Where a pyogenic process is engrafted on a tuberculous one, we
have still the red cells, some degenerated lymphocytes, and in particular
polymorphonuclears showing fragmentation of their nuclei.

3. When a hydro thorax results from chronic heart or kidney disease,
the characteristic cell is the endothelial cell, which greatly resembles a
large mononuclear. These cells often are arranged in plaques.

4. Some authorities consider that the cancer cell can be recognized
by its occurring in masses and having a markedly vacuolated cytoplasm.
It has been claimed that they contain glycogen by which means we can
distinguish them from endothelial cells which they so much resemble.
If such cells should show mitosis the finding would be suggestive.
For mitotic figures wet fixation with some bichloride fixative, with H. E.
staining, is best.

Jousset introduced inoscopy as a means of diagnosing tuberculosis. The fluid
was allowed to coagulate and was then digested with an artificial gastric juice. The
digested material was then centrifuged and the sediment examined for tubercle
bacilli. This process does not seem to have met with much favor in this country.
(Using sodium citrate obviates the necessity for digesting the coagulum.)

The same points will hold for ascitic fluid as for pleural fluid.

In taking cerebrospinal fluid for culture and cytodiagnosis we use a stout anti-
toxin needle without attaching a syringe. Aspiration is responsible for many of
the ill effects of lumbar puncture. The needle should be about 4 inches long for an
adult. Sterilize the skin and needle as described for blood cultures from a vein.
To make a lumbar puncture, place patient on left side with knees drawn up. A line
at the level of the iliac crests passes between the third and fourth lumbar vertebrae.
Select a point midway between the spinous processes of these lumbar vertebrae and

CYTODIAGNOSIS AND PARASYPHILITIC DISEASE 361

enter the needle 2/5 of an inch to the right of this point, pushing the needle inward
and upward. Collect the material in a sterile test-tube. Make cultures on blood-
serum and then centrifugalize and examine the sediment as for pleural fluids.

In general terms it may be stated that:

1. A lymphocytosis indicates a tuberculous process.

2. An abundance of polymorphonaclear and eosinophilic leukocytes
indicates a meningococcic, streptococcic, influenza or pneumococcic
infection.

When the case is one of meningism there are very few. cells. In poliomyelitis
there is a cell increase of which 90% may be lymphocytes.

A method of examination considered by neurologists as of differential diagnostic
value is to count the number of cells in a cubic millimeter of the cerebrospinal fluid.
The technic is to use a gentian- violet- tinged 3% solution of acetic acid. This is
drawn up to the mark 0.5, and the cerebrospinal fluid is then sucked up to n. After
mixing, the cell count is made with the haemocytometer. Normally we have only
one or two cells per cubic millimeter, but in tabes or general paresis this is increased
to 50 or 100 cells (greatest at onset of disease).

The test for globulins as showing parasyphilitic disease is taken up under Tre-
ponema pallidum. The technic of the Wassermann test with cerebrospinal fluid
is discussed under that test. Any excess of urea in the cerebrospinal fluid is a sure
sign of renal inadequacy.

Trypanosomiasis gives a cellular increase very similar to syphilis.

In the work of the French Sleeping Sickness Commission five cells per cubic
millimeter was taken as normal.

CHAPTER XXXIV.
RABIES, VACCINIA AND THE FILTERABLE VIRUSES.

RABIES is a disease of dogs and wolves, but is communicable to man
and domesticated animals. The virus, whatever it may be, resides in
the saliva and nervous structures. It is destroyed by a temperature of
50° C. In man the period of incubation is usually from three weeks to
three months, but may be shorter or may extend over one year.

Bites about the face and those with marked lacerations are particularly serious.
Bites of rabid wolves give about four times as great a mortality as those of dogs.
In the dog there are two types of the disease — dumb rabies and furious rabies.

By inoculating rabbits subdurally with an emulsion of the brain or spinal cord
of a rabid animal, and successively the medulla of this rabbit subdurally into other
rabbits, we finally so increase the virulence of the infection that rabbits die in six days.
Beyond this it is impossible to increase the virulence and it is termed "fixed virus."
The pathogenic power of this virus is also changed so that it is not apt to cause rabies
if injected subcutaneously. To attenuate this virus the spinal cord of the rabbit
is removed and is dried over caustic potash at a temperature of 23° C. The cord is
divided into segments about i inch in length. Drying for about fifteen days seems
to entirely destroy the virus.

To prepare the material for prophylactic injections a small portion of the cord is
emulsified with normal salt solution and injected subcutaneously. The German
method is to commence with a cord that has been desiccated only eight days. At
first injections are given daily, and it is possible to inject three days' cords by the
sixth day. The immunity is "active" and the immunizing agent is a "vaccine."
Like vaccine virus the product can be preserved (for probably a month) by the use
of glycerine so that it is now possible to send the material for inoculation from the
laboratory preparing it.

The treatment lasts for about twenty days. In the diagnosis of rabies in dogs
it is preferable to preserve the animal so that the development of the symptoms
may be observed.

In case the dog has been killed, it may be possible to make a diagno-
sis by means of the Negri bodies. These are round or oval bodies from
i to 2on in diameter, which may be found in the nerve-cells, especially
those of the cotnu ammonis (Hippocampus major).

These bodies were first described by Negri in 1903. In street rabies large amoe-
boid forms from 18 to 23;* may be found, while in the nerve tissues of animals with
"fixed" virus only minute forms, 0.5/4 or less, may be detected. The fact that the

362

RABIES

363

virus will pass through a Berkefeld filter is no argument against its protozoal
nature. Calkins considers it to be of rhizopod affinity. The term Neuroryctes
hydrophobias has been given it. The bodies are present four to seven days
before the onset of symptoms. They may be demonstrated by staining smears of
gray brain substance by some Romanowsky method, especially by the Giemsa stain.
The smears should be made by mashing the thin slice of gray matter taken from i.
Cornu ammonis, 2. Region of fissure of Rolando — in dog crucial sulcus — or 3.
Cerebellum, with a cover-glass against the slide. Afterward the cover-glass is gently
drawn along the slide.

The smear on the slide is then fixed in methyl alcohol for two to three minutes,
washed with water and covered with a stain made by adding 3 drops of Sat. ale.
sol. of basic fuchsin to 10 c.c. of distilled water and then adding 2 c.c. of Loffler's
methylene blue solution. The stain on the slide is then steamed gently and after-
ward washed with water and dried.

FIG. 105. — Two nerve cells of hippocampus major (smear preparation) showing
Negri bodies. A, Negri bodies; B, inner bodies within the Negri bodies. (After
Reichel, American Veterinary Review.)

As their relation to the nerve-cell is more or less disturbed by such a method
it is preferable to fix brain tissue from the region of the cornu ammonis for five to
seven hours in Zenker's fluid, then to imbed in paraffin and make sections. These are
stained with Giemsa's stain and the Negri bodies are brought out as iliac-red bodies
in the blue cytoplasm of the nerve-cells. It is necessary to differentiate in 95%
alcohol.

In the Lentz method the 3;* sections, after removal of the paraffin, are flooded
with absolute alcohol. They are then stained with a 1/2% solution of eosin in 60%
alcohol for one minute. Wash in water and next stain for one minute in Loffler's
methylene blue. Again wash in water. Apply Lugol's solution to the section for
one minute and then differentiate alternately in methyl alcohol and water until the
section is pink. After washing in water, again stain with Loffler's blue for one-half
a minute, then wash in water and dry carefully with filter-paper. Now differentiate
in alkaline alcohol (i drop of a 5% solution NaOH in 30 c.c. absolute alcohol) until

364 RABIES, VACCINIA AND THE FILTERABLE VIRUSES

the section is pink, then quickly differentiate in acid alcohol (i drop 50% acetic
acid in 30 c.c. absolute alcohol) until a slight blue outline to the ganglion cells is
obtained. Treat rapidly with absolute alcohol and xylol and mount in balsam.
The Negri bodies show as light carmine pink bodies on the light blue ground of the
gangh'on cells. In the interior of the pink bodies dark blue dots or rings may be
observed.

This method can also be used for brain smears.

In addition to examining for the Negri bodies, a rabbit may be inoculated
subdurally with a sterile salt-solution emulsion of the medulla of the dead dog.

If the brain and medulla of the dog are to be sent to a laboratory
for examination they should be packed in ice or placed in glycerine.
Take of glycerine one part and one part water. Sterilize the diluted
glycerine by boiling, allow to cool, and drop the pieces of brain tissue
into this. This does not kill the virus.

When from advanced putrefaction, or otherwise, the Negri bodies cannot be
found the changes in the Gasserian ganglia may give a diagnosis. In typical lesions
the ganglion cells are more or less completely destroyed and replaced by cells of
other types.

When a person is bitten by a dog suspected of being rabid the following
simple measures should be instituted. The dog should be kept under observation
in a safe quiet place and will show clinical evidence of rabies within five days and
will die shortly afterward in case rabies exists. When the animal dies the head
and several inches of the neck should be removed and packed in ice and sent to the
nearest laboratory.

Antirabic serum has been prepared by injecting sheep with emulsions of rabbits'
cord and brain — at first intravenously, then subcutaneously.

The thorough cauterization of the dog-bite wound with pure nitric
acid, as soon as possible after the bite, is imperative even when the Pas-
teur treatment can be given later.

VACCINIA is a disease produced artifically by the injection of vaccine virus ob-
tained from the calf. The material for vaccine is taken from vesicles about
one week after the inoculation. The most potent material is in the pulp at the base
of the vesicle and not in the lymph which exudes from the vesicle. The pulp is
ground up and mixed with an equal amount of glycerine, which acts not only as a
preservative but as a mild antiseptic for nonsporing bacteria. The calves are autop-
sied after the pulp has been curetted from the inoculated skin of the abdomen to be
sure that no disease exists in the calves. The virus is afterward tested for pus organ-
' isms, tetanus, and foot and mouth disease.

Guarnieri in 1892 first observed small bodies near the nucleus of infected epi-
thelial cells, He called them Cytoryctes vacciniae. Calkins regards these bodies
as well as the Negri bodies as being rhizopods and the distributed chromatin as
idiochromidia (granules of nuclear chromatin within the cytoplasm).

RABIES 365

THE FILTERABLE VIRUSES.

The first disease of which the virus was found to be capable of pass-
ing through the finest porcelain filter was that of foot and mouth disease
(LofBer and Frosch, 1898).

The filter which is ordinarily used for testing for the passage of such
disease agents is the Berkefeld filter, one made of diatomaceous earth.
Of the infections belonging to man in which such a passage of blood or
serum through the pores of a porcelain filter, capable of keeping back
even such a small bacterial organism as that of Malta fever, but which
does not hold back their virus, we have the following: foot and mouth
disease, trachoma, molluscum contagiosum, vaccinia, variola, rabies,
typhus fever, measles, scarlet fever, yellow fever, dengue, Papataci
fever and poliomyelitis.

There are many diseases of this nature which are important among the domes-
ticated animals, such as pleuropneumonia of cattle, African horse sickness and hog
cholera. The viruses of pleuropneumonia of cattle and poliomyelitis have been
obtained in artificial cultures. Some of these viruses seem related to bacterial
infections and others to protozoal ones. These viruses differ as to method of trans-
mission, pleuropneumonia of cattle being transmitted by inhalation, rabies and
vaccinia by the cutaneous atrium, hog cholera by ingestion and many of those sup-
posed to have protozoal affinities, as yellow fever, Papataci fever and horse sickness
by mosquitoes.

As a rule these viruses are destroyed by a temperature of 55° C. in a few minutes.

MISCELLANEOUS NOTES.

MISCELLANEOUS NOTES.

MISCELLANEOUS NOTES.

MISCELLANEOUS NOTES.

MISCELLANEOUS NOTES.

APPENDIX.

A— PREPARATION OF TISSUES FOR EXAMINATION IN
MICROSCOPIC SECTIONS.

i. Fixation:

a. It is most important that the tissues to be examined be placed in the fixing
fluid as soon after death or operation as possible. Degenerative changes are in this
way avoided.

b. The piece of tissue to be fixed must not be too large. Using a sharp scalpel,
or preferably a razor, a slab of tissue about one-half an inch square and not more
than one-fifth of an inch thick should be dropped into the bottle containing the fixa-
tive. The bottom of this bottle should have a thin layer of cotton with a piece of
filter-paper covering it. There should be at least twenty times as great a volume
of fixing fluid as of tissue to be fixed. Delicate tissues, as pieces of gut, should be
attached to pieces of glass, wood, cardboard, or blotting paper before being placed in
the fixative.

c. The most convenient fixative for the average medical man is a 10% solution
of ordinary commercial formalin (4% of formic aldehyde gas), either in water or,
preferably, in normal salt solution. Fixation is complete in from twelve to twenty-
four hours. By placing in the incubator, at 37° C., two to twelve hours in the forma-
lin solution suffices. If fixed in the paraffin oven (56° C.), fixation is accomplished
in about one-half hour.

Formalin once used for fixation must be thrown away.

The fixative which probably gives the best histological pictures and with which
we obtain the most satisfactory haematoxylin staining is Zenker's fluid. This is
Miiller's fluid containing 5% of corrosive sublimate. It also contains 5% of glacial
acetic acid, which latter is only added just before we are ready to fix the piece of
tissue. Miiller's fluid is:

Pot. bichromate, 2 . 5 grams.

Sod. sulphate, i . o grams.

Water, 100.0 c.c.

Zenker's fluid fixes in about twenty-four hours. After all corrosive sublimate
fixatives we should wash the tissues in running water for twelve to twenty-four
hours. The precipitate of mercury in the tissues is best gotten rid of by treating the
section on the slide with Lugol's solution, rather than the tissue in bulk with iodine
alcohol.

In Orth's fluid we add 10% of formalin to Miiller's fluid (recommended for nerve
tissue).

371

372 APPENDIX

A saturated corrosive sublimate solution in salt solution with the addition of 5%
of glacial acetic acid may be used as a substitute for Zenker's fluid.

2. Dehydration. — After washing for twelve to twenty-four hours in running
water, following corrosive sublimate fixation, or simply washing for a few minutes
after formalin, the tissues should be placed in 70% alcohol. They may be kept in
this indefinitely. If they are to be sent to a laboratory for sectioning, it is advisable
to moisten a pledget of cotton in 70% alcohol and fill in the bottom of the bottle with
it. Then drop in the tissues and pack in gently over them sufficient 70% alcohol-
saturated cotton to fill up the bottle. All the alcohol should be absorbed by the
cotton so that if the bottle should break in transit there would be no damage from the
alcohol. The stopper of the bottle should be paraffined or sealed with wax.

Tissues may be left in the 70% alcohol twelve to twenty-four hours and should
then be transferred to 95% alcohol for an equal time. They are then transferred to
absolute alcohol, where they remain from two to twelve hours and are then placed in
xylol. The time in xylol should be as short as possible. So soon as the tissue looks
clear it should be removed — thirty minutes to two hours.

3. Imbedding. — The tissue is now transferred to melted paraffin. Paraffin melt-
ing at 48° C. for winter work, and that melting at 54° C. for summer is to be recom-
mended. The time in the paraffin should not be prolonged. Two hours will
ordinarily suffice. Some leave in the paraffin for twelve to twenty-four hours.

Next take a paper box (made of stiff writing-paper folded over a square of wood)
and fill with the melted paraffin. As quickly as possible drop in the piece of tissue
taken out of the paraffin bath with heated forceps and, so soon as the paraffin begins
to solidify on the surface, place the paper box in ice water. When paraffin is rapidly
cooled, crystallization is less.

The Acetone Method. — Take the tissues out of the 70% alcohol and place in ace-
tone. After remaining in acetone for one to two hours, the tissues should be trans-
ferred to fresh acetone for an equal length of time. Dry calcium chloride in the bot-
tom of the acetone bottles keeps it dehydrated. They should then be placed in
xylol for about one-half hour and then embedded in paraffin as directed above.

The Chloroform Method. — The procedure may be the same as in the method of
passing through alcohols to xylol, substituting chloroform for xylol and then trans-
ferring to paraffin.

Where absolute alcohol is not obtainable, very satisfactory results may be
obtained by transferring to a mixture of 95% alcohol and chloroform after immer-
sion in 95% alcohol. Then going from the alcohol-chloroform mixture to pure
chloroform, thence to paraffin.

Rapid paraffin imbedding methods.

When a piece of tissue is not more than one-fourth inch square and one-eighth
inch thick, it is very easy to run it through in three to six hours. Thus:

10% Formalin (in 37° C. incubator), i hour.

70% Alcohol (in 37° C. incubator), i hour.

95% Alcohol (in 37° C. incubator), i hour.

Absolute Alcohol (in 37° C. incubator), 1/2 hour.

Xylol (in 37° C. incubator), 1/2 hour.

Paraffin (in 55° C. incubator), 1/2 to 2 hours.

APPENDIX 373

Method of Lubarsch. — In this excellent method small pieces of tissue not more
than 1/5 inch thick are placed in a wide test-tube containing 10% formalin for 10 to
15 minutes, changing the fluid twice. Transfer to 95% alcohol 10 minutes changing
alcohol once. Absolute alcohol, for 10 minutes changing twice. Pure aniline oil
until tissues are transparent, 15 to 30 minutes. Xylol, changing two to three times
or until the xylol is no longer yellow, 10 to 20 minutes. Imbed in paraffin for 20
minutes to i hour. During the entire process keep the test-tube in a water bath
or incubator at 50° C. It is preferable to have a good microtome. The best is that
of Minot. Very satisfactory sections can be cut with the various types of student
microtomes, costing from $12 to $20.

(In using a hand microtome, a razor with a flat edge is necessary. After expe-
rience, sections thin enough for histological but not for bacteriological examination
can be made.)

If the piece of tissue is properly dehydrated and imbedded, thin sections (3 to
io/0 should be easily obtained, provided the knife be sharp. One advantage about
the paraffin method is that it is only necessary to have a small part of the blade in
proper condition. With celloidin the entire cutting edge must be perfect. Having
cut the sections, they should be dropped on the surface of a bowl of warm water
(45° C.). This causes the section to flatten out evenly.

Decalcification. — This is best accomplished by fixing in 10% formalin for twenty-
four hours, then placing a small piece of the bone (not exceeding one-half inch square
and one-fifth of an inch thick) in concentrated sulphurous acid.

This decalcifies in about 2 to 7 days. Wash thoroughly in alkaline water
and then in tap water. Pass through alcohols and xylol and imbed and section as
before described.

To Stain Sections. — It is first necessary to affix the section to a slide or cover-glass.
To attach the section firmly to the slide, so that it will not become detached in
subsequent treatment, pick up a section on a strip of cigarette paper.

A sheet of cigarette paper is cut into about five pieces (1/2X1 1/2 inches).
Inserting the strip of cigarette paper under the section, it is easily lifted up out of
the water. Then apply the slip of cigarette paper, section downward, to a perfectly
clean slide. Blot with a piece of filter-paper, then strip off the piece of filter-paper
leaving the section smoothly applied to the slide. Next place in the 37° C. incubator
for twelve to twenty-four hours and the section will be found to be so firmly attached
that it will not be dislodged by subsequent treatment.

For Immediate Diagnosis. — Take a loopful of albumin fixative (white of fresh
egg, 50 c.c.; glycerin, 50 c.c.; sodium salicylate, i gram) and deposit it on a cover-
glass. Now take up a loopful of 30% alcohol (i drop of 95% alcohol and two drops
of water) and applying it over the albumin fixative, smear out the mixture uniformly
over the cover-glass.

2. Pick up a section on a strip of cigarette paper and apply it to the prepared sur-
face on the cover-glass. Blot with gentle pressure with a piece of filter-paper over
the strip of cigarette paper, and strip off this latter, leaving the section attached to
the cover-glass.

3. Now, turning the flame of the Bunsen burner down very low or with a small
alcohol flame, we hold the cover-glass in a Stewart's forceps, section side up, over
the flame and slowly lower it until the paraffin is observed to melt. This shows a

374 APPENDIX

temperature of about 50° C. The section is fixed by the coagulation of the albumin
at about 70° C. To obtain this temperature lower the cover-glass still more, and the
moment vapor is seen to rise from the section it indicates the attachment of the sec-
tion to the cover-glass.

4. Flood section on cover-glass or slide with xylol; this dissolves out the paraffin.
It is better to pour off the first xylol and drop on fresh xylol (one minute).

5. Remove xylol with two applications of absolute alcohol (one minute).

6. Treat specimen with two or three applications of 95% alcohol (one to two
minutes).

7. Next wash in water (one to two minutes).

8. Flood specimen with hasmalum or Delafield's haematoxylin (three to seven
miautes).

9. Wash in tap water for about two to five minutes until a purplish tinge is devel-
oped in the section. The alkali in ordinary tap water develops this color.

10. Apply i to 1000 eosin for thirty seconds to one minute.

11. Wash in water; then in 95% alcohol; then in absolute alcohol.

12. Apply a few drops of xylol and as soon as the section is perfectly transparent
mount in balsam.

The staining by haematoxylin and eosin is the best for the study of the histology
of a section. It only requires about ten minutes to run a preparation through for
diagnosis by this method.

The reagents are best kept in dropping-bottles.

The staining of sections on slides is exactly as for those on cover-glasses. Cop-
lin's staining jars are very convenient for use in staining slides.

Where the cover-glass method is used, staining by Gram's method, acid-fast stain-
ing, capsule staining, etc., may he carried out as for bacterial preparations.

For staining Gram positive bacteria in sections, the Gram method as for bacterial
preparations, using dilute carbol fuchsin as a counterstain, gives good results.

For Gram negative bacteria stain with thionin as for blood preparations (ten to
twenty minutes). Then differentiate in i to 500 acetic acid solution for ten to twenty
seconds, wash with water, then with 95% alcohol, and quickly through absolute
alcohol and xylol.

Nicolle's Method. — i. Stain with Lo frier's methylene blue ten to fifteen minutes.

2. Differentiate in i to 500 acetic acid ten to twenty seconds.

3. Place in i% solution of tannin for a few seconds (fixes color).

4. Wash in water, then into 95% alcohol, absolute alcohol, xylol, and balsam.

WEIGERT'S IRON H^MATOXYLIN.

Solution I.

Haematoxylin, i gram.

Alcohol (95%), 100 c.c.

This must be allowed to ripen for some days and does not keep over six months.

Solution II.

Liq. Ferri sesquichlor. sp. gr. 1.124 (about 10%) 4 c.c.

HC1, i c.c.

Water, 100 c.c.

APPENDIX 375

Mix equal parts of number one and number two. The mixture only keeps about
three days. The HC1 prevents overstaining.

This stain followed by Van Giesen's stain gives more perfect results than any
common method of staining. The iron haematoxylin intensifies the sharpness of
the Van Geisen differentiation.

Van Giesen's Stain. — Take of i% aqueous solution acid fuchsin from 5 to 15
c.c. Saturated aqueous solution picric acid 100 c.c. The method of using is to first
stain with haematoxylin in the usual way. Then pour on the picric-acid fuchsin
solution and allow to stain for one to five minutes. Wash, pass through alcohols
and xylol and mount in balsam.

Connective-tissue fibers, axis cylinders, and ganglion cells are stained a bright
garnet red. Myelin, muscle fibers, and cells generally are stained yellow. Nuclear
staining is that of hasmatoxylin. The stronger stain is used for nerve tissue; the
weaker, for demonstrating connective tissue in tumors.

Levaditi's Method. — Take small pieces of tissue, about 2 mm. in thickness, and
harden in 10% formalin for twenty-four hours and then in alcohol for the same period;
then wash in water for a short period. They are stained in a freshly made solution
of silver nitrate 1.5% for three successive days, changing the solution each day,
maintaining the blood temperature, and excluding light. The tissue is then placed
in a 2% solution of pyrogallic acid, with the addition of 5% formalin. After remain-
ing in this for twenty-four hours, light being excluded, they are passed through
85%, 95%, and absolute alcohol, respectively; embedded in paraffin; and cut in
about 5 micron sections. Equally good results may be obtained by allowing the
silver nitrate to act at room temperature and embedding in celloidin.

Romanowsky. — Staining sections with Romanowsky stains is not very satis-
factory. The differential staining seems to fade out in passing through the alcohols.
This may be avoided by blotting the section after staining and differentiation and
then applying the xylol to the blotted section. After staining with Giemsa's stain
for ten to fifteen minutes, differentiate with i to 500 acetic acid. When the section
has a pinkish tinge, wash in water, dry, clear in xylol, and mount.

Good tissue staining may be gotten with Wright's stain. After removing the
paraffin with xylol and the rylol with absolute alcohol, pour on a sufficient number of
drops of stain and immediately dilute with an equal number of drops of water.
Allow the diluted stain to remain for three to five minutes. Next wash in water,
differentiate, until the tissue has a pinkish tinge, in i to 500 acetic acid. This
differentiation is best done in a tumbler of the dilute acetic acid.

After washing in water, quickly pass through 95% and absolute alcohol, clear in
xylol, and mount.

Skin Sectioning. — Of all tissues that of skin offers the greatest difficulty in pre-
paring sections. The best results can probably be obtained by fixation in picro-
sublimate (saturated aqueous solution picric acid i part; saturated aqueous solu-
tion bichloride of mercury one part); to this stock mixture add 5% glacial acetic
acid just before using. Fix small pieces of skin six to eighteen hours. Transfer
direct to 70% alcohol in which the tissue may be kept indefinitely.

For sectioning run through alcohols to absolute and then to a mixture of absolute
alcohol and carbon bisulphide (equal parts). Leave until tissue sinks, then transfer
to pure carbon bisulphide until tissue sinks. Then transfer to a saturated

376 APPENDIX

solution of paraffin in carbon bisulphide and thence to paraffin. Bisulphide of
carbon has the disadvantage of foul odor and inflammability but does not seem to
render tissues brittle and difficult to section as does xylol.

NEUROLOGICAL STAINING METHODS.

Neuropathology practically dates from the introduction of Marchi's method of
staining in 1885.

Ordinary osmic acid stains both normal and pathological fat. With Marchi's
method only the oleic acid of fatty degeneration is stained.

The method is not useful until three or four days have elapsed from the onset
of the condition causing the degeneration and it is applicable for only three or four
months because by that time phagocytes have taken up the pathological fat which
is stained in the Marchi method. The Weigert method is the one to use after a
period of three or four months. In Weigert's stain only the normal myelin sheath
is stained and the lack of staining of myelin sheaths in degenerated areas is the basis
of the stain. For demonstrating axonal reactions or other degenerative changes
in nerve cells, as shown by bulging of the concave sides of the cells, eccentric nucleus
and granular appearance of the tigroid bodies, Nissl's method is the best.

For neuroglia fiber staining Mallory's phosphotungstic acid haematoxylin is to
be recommended.

I. For Marchi's Method. — Small pieces of nerve tissue are hardened in Miiller's
fluid for seven to ten days and are then transferred to a mixture of two parts Miiller's
fluid and one part of a i% osmic acid solution and should remain in this mixture
for about seven days. The tissue thus treated is run through alcohols and imbedded
in paraffin in the usual way.

II. For Weigert-Pal Method. — Thin slices of tissue are fixed in 10% formalin
in about four days. The tissue should then be transferred to 5% potassium bichro-
mate for about twelve days. The tissue is then imbedded and sections cut. If
only recently mordanted these sections may be at once stained with Weigert's
haematoxylin for twelve to twenty-four hours (10 c.c. ripened 10% solution haema-
toxylin in absolute alcohol and 90 c.c. water). Wash in water to which about 2%
of a saturated solution of lithium carbonate has been added. Now differentiate
from one-half to five minutes in 1/4% solution of potassium permanganate until
the gray matter looks a brownish-yellow. Next treat sections with oxalic acid
i gram, potassium sulphate i gram and water 200 c.c. until the gray matter is
almost colorless. This takes only a few seconds. Wash in water, pass through
alcohols and xylol and mount in balsam.

III. For Nissl staining either thionin or Giemsa staining is satisfactory.

IV. For neuroglia fiber staining use Mallory's Phosphotungstic acid haematoxylin.

Take of haematein ammonium, o.i gram.

Water, 100.0 c.c.

Phosphotungstic acid crystals (Merck) 2.0 grams.

Dissolve the haematein in a little water with the aid of heat, and add it after it is
cool to the rest of the solution; no preservative is required. If the solution stains
weakly at first, it may be ripened by the addition of 5 c.c. of a 1/4% aqueous

APPENDIX 377

solution of potassium permanganate, or it may be allowed to stand for a few weeks
until it ripens spontaneously.

MAKING AND STAINING OF FROZEN SECTIONS.

The various types of ether freezing microtomes are not very satisfactory when
only used occasionally. With the general introduction of cylinders containing
compressed carbon dioxide, which is used for aerating waters, we have at hand a
practical and convenient method of making frozen sections.

The instrument makers furnish a freezing microtome of the Bardeen type which
can be attached directly to the cylinder by a revolving clamp nut.

It is necessary to have a stand to support the iron cylinder in a horizontal posi-
tion The tissue, which may be taken at operation for immediate diagnosis, or
which preferably has been fixed in formalin for twelve to eighteen hours is immedi-
ately placed in water. If the tissues have been in alcohol it will require hours of
washing before they can be frozen. The piece of tissue which is to be frozen should
not be more than one-fifth of an inch thick. Having placed the piece of tissue on
the freezing box of the microtome we turn the valve of the cylinder to allow the
gradual escape of gas. When frozen solid, we elevate the freezing box holding the
frozen tissue, by revolving a graduated disc with the left hand. In the right hand
we firmly grasp a well-sharpened blade of a carpenter's plane mounted in a wooden
handle. This is held at an angle of 45 degrees to the polished ways of the micro-
tome. By alternate shoving and withdrawing of the blade, held rigidly, we accumu-
late on the blade a number of sections. Then dip the blade in a vessel of water to
detach the sections which float in the water. Keep repeating the process until
numerous satisfactory sections are obtained. Handles for holding the Gillette
razor blades are good substitutes for the carpenter's plane.

These sections may be picked up with a strip of cigarette paper and applied to a
clean slide upon which a very small loopful of albumin fixative has been smeared
out with 30% alcohol. The piece of cigarette paper with the section underneath
is then firmly smoothed out upon the slide with filter-paper. The piece of cigarette
paper is then carefully stripped off and the section remains attached to the slide.
By careful heating over a very small flame, until the vapor just arises, the section
is fixed to the slide and we can then stain the section in any way that may be desired.

NOTE. — The procedures for carrying the tissues through celloidin are not given
as it requires perfect condition of the entire cutting surface of the microtome knife
and a considerable time for the passage through regents and celloidin. It is more
suitable as a method when sections for class work are to be prepared.

B— MOUNTING AND PRESERVATION OF PATHOLOGICAL SPECIMENS
AND ANIMAL PARASITES.

To Mount Small Round Worms. — Wash the hook, whip, or filarial worm in salt
solution, then drop in 70% alcohol containing 5% of glycerine; the glycerine-alcohol
mixture being at a temperature of 60° C. When cool, pour into Petri dishes and
allow the alcohol to evaporate in the 37° C. incubator.

Mount in glycerine jelly, preferably in a concave slide, and ring the preparation

378 APPENDIX

with gold size. The following is the formula for Kaiser's glycerine jelly: Soak one
part of gelatin in six parts of distilled water for two hours. Then add seven parts
of glycerine. To the mixture add i % of carbolic acid, warm for fifteen minutes,
with constant stirring, and then filter through cotton.

To Prepare Tape -worms. — Wash in salt solution. Wrap around a piece of glass
as a glass slide and fix in salt solution containing 2 to 5% of formalin. Then keep
the preparation permanently in 70% alcohol. If preferred, the specimen may be
run through alcohols and xylol and mounted in balsam.

Larvae. — Mosquito larvae may either be prepared as for small round worms or
they may be dropped into 70% alcohol at 60° C. and then passed through alcohols
and cleared in xylol and mounted in balsam. Flukes and insects may require treat-
ment with hot (60° to 70° C.) solution of 10 to 20% sodium hydrate solution. Then
wash thoroughly in water and subsequently pass through alcohols to xylol and mount
in balsam. Clove oil or cedar oil clears more slowly, but makes specimens less
brittle than does xylol. Another satisfactory method is to drop insects or larvae
into acetone at 60° C. and after being in this from one to twelve hours to clear in
xylol or clove oil and mount in balsam.

^ Nematodes. — Looss has a method of first washing a small nematode or delicate
fluke in salt solution. Then pouring this first salt solution out of the test-tube in which
the washing was carried out, to add fresh salt solution, and then an equal amount
of saturated aqueous solution of bichloride of mercury. The shaking is easily
carried on in the test-tube. After washing in water the worm is passed through
alcohols, one strength of which should contain iodine. Clear in xylol and mount in
balsam.

An excellent method is that of Langeron.

After washing in salt solution fix for a few hours in 5% formalin. Then transfer
to lactophenol which has been diluted with an equal amount of water. Allow to
remain in this solution for several hours and then transfer to pure lactophenol in
which fluid the specimens are to be mounted. Ring with paraffin or with gold size.
(To make lactophenol take two parts of glycerine and one part each of distilled
water, crystallized carbolic acid and lactic acid.)

A quick method of preparing small nematodes for examination is to fix them for
from two to twelve hours in 5 to 10% formalin, this being heated to 60° C. at the
time the worms are dropped into it. Then transfer to the following solution:

Glucose syrup (glucose, 48; water, 52), 100 c.c.

Methyl alcohol, 20 c.c.

Glycerine, 10 c.c.
Camphor, q.s. (a small lump for preservation).

They may be mounted directly in this and the cover-slip ringed with about 60° C.
paraffin, followed with gold size.

Preparations so cleared and mounted in glycerine jelly should also be ringed with
paraffin or °ome cement.

Flukes, cestodes, and nematodes are best stained with carmine. The following
is a good formula.

Dissolve, by boiling, 4 grm. carmine in thirty drops HC1 and 15 c.c. water. Then

APPENDIX 379

add 95 c.c. of 85% alcohol and filter while hot. Neutralize with ammonia until
precipitate begins to form. Then filter cold.

i. Stain parasites taken from 70% alcohol for five to twenty minutes. 2. Dif-
ferentiate in 3% hydrochloric acid. 3. Pass through alcohols to xylol and mount in
balsam.

Mites, Fleas and Various Small Insects. — By simply taking one or two drops
of liquid petrolatum and mounting the specimen in it then covering with a cover-
glass one is able to study the details of these objects almost as well as if they were
passed through acetone and xylol into balsam. Liquid petrolatum is also most
excellent for mounting the aerial hyphas of fungi with their sporangia as well as for
Romanowsky stained blood smears.

Pathological tissues which are to be sent to a laboratory for sectioning or to be
kept for future study should be fixed by one of the methods given in Section A of
the appendix.

Formalin fixation is the more convenient — that with Zenker's fluid the more
perfect. After fixation with Zenker's fluid the pieces of tissue must be washed in
running water over night.

After fixation the pieces of tissue are transferred to 70% alcohol in which they may
be kept indefinitely.

For preservation of gross specimens the method KAISERLING is generally used.

Fix for from one to five days in Solution I.

Solution I.

Formaldehyde, 200 c.c.

Water, 1000 c.c.

Nitrate of potassium, 15 grams.

Acetate of potassium, 30 grams.

The position of the specimen should be changed from day to day. There must
be at least five times as much fluid as specimen. Drain and transfer to 80% alcohol
for a few hours, then into 95% alcohol until the natural color is just restored.

Finally preserve in

Acetate of potassium, 200 grams.

Glycerine, 400 c.c.

Water, 2000 c.c.

It is advisable to keep these specimens in the dark as light destroys the natural
color.

To Prepare Flies or Mosquitoes for Transmission Through the Mails. — Wrap
the insect carefully in a piece of tissue paper (toilet-paper answers). Impregnate
sawdust with 5% carbolic acid solution and fill around the tissue paper in the
box containing them. (Barely moisten.)

It is very satisfactory to take a tube form vial with a cork from the inner sur-
face of which two small shallow holes have been bored, one containing parafor-
maldehyd, the other camphor. The insect is mounted upon a pin stuck in the
cork, which latter is inserted and parafined externally.

380 APPENDIX

C— PREPARATION OF NORMAL SOLUTIONS.

A normal solution is one which contains the hydrogen equivalent of an element,
expressed in grams, dissolved in sufficient distilled water to make 1000 c.c. The
hydrogen equivalent is the atomic weight of any element divided by its valence. In
a base, salt or acid, we use the molecular weight in grams divided by valence.

What may be considered as the valence of a base is shown by the number of
hydroxyls combined with it; that of an acid by the number of replaceable hydrogen
atoms which it contains.

To make a normal solution, dissolve in distilled water a weight in grams equal
to the sum of the atomic weights of the substance divided by its valence, and make
up the volume to exactly 1000 c.c.

NaOH is univalent. Na = 23. O = i6. H = i. Dissolve 40 grams NaOH
in water and make up to exactly 1000 c.c.

Oxalic acid is COOH — COOH+2H2O which gives it a molecular weight of 126.
As it contains two carboxyl groups it is dibasic, and it is necessary to divide the
molecular weight by 2, so that for a normal solution of oxalic acid we dissolve 63
grams in a volume of distilled water made up to 1000 c.c.

If a chemical laboratory is not accessible one may prepare normal solutions with
an error so slight as to be unimportant in clinical work in the following way:

Sodium hydrate being very hygroscopic, it is impossible to accurately prepare a
normal solution by directly weighing out the substance. Instead, select perfect
crystals of oxalic acid, such as can be obtained in a drug store, and weigh out on the
most accurate apothecary scales obtainable exactly 6.3 grams of the most perfect
crystals in the bottle. Put these preferably in a volumetric flask and make up with
distilled water to 1000 c.c. Less accurate is the use of a measuring cylinder. If
care is used this should give N/io solution of oxalic acid in which the error is less
than i%.

Having N/io acid at hand, we may prepare N/io NaOH in the following way:
Weigh out an excess of sodium hydrate (5 grams of stick caustic soda) and dissolve
in 1 100 c.c. of distilled water. Take up 10 c.c. of this solution with a pipette and let
it run into a beaker. Add six drops of phenolphthalein solution. This gives a violet-
pink color. Fill the burette with the N/io oxalic acid solution and let it run into
the sodium hydrate solution in the beaker until the pink is just discharged. Reading
off the number of c.c. of the N/io acid used, we know the strength of the sodium
hydrate solution. It is well to repeat the titration and take an average.

If 10.5 c.c. of the oxalic acid solution were required it would show that the sodium
hydrate solution was stronger than N/io, as only 10 c.c. would have been necessary
if the NaOH solution had been N/io. It is therefore necessary to dilute the sodium-
hydrate solution in the proportion of 10 to 10.5. Measure exactly 1000 c.c. of the
too concentrated sodium-hydrate solution and add to it 50 c.c. of distilled water, mix
thoroughly, and we have 1050 c.c. of N/io solution of NaOH. ioooX 10.5 = 10,500.
10,500-7-10= 1050.

. As Acidum hydrochloricum U. S. P. is about two-thirds water (68.1%) to make
N/io HC1, which would require 3.65 in 1000 c.c., it would be necessary to take about
three times this amount of U. S. P. acid. Take 12 c.c. of the acid and add distilled
water to make uoo c.c. Put 10 c.c. of this dilute solution in a beaker. Add phenol-

APPENDIX 381

phthalein solution and titrate. If n c.c. of N/io NaOH were required it would be
necessary to add 100 c.c. of water to a volume of 1000 c.c. of the diluted hydrochloric
acid. 1000X11 = nooo-f-io= noo.

Other acid and alkali solutions can be made as for N/io HC1 and N/io NaOH.

D— DISEASES OF UNKNOWN OR NOT DEFINITELY DETERMINED

ETIOLOGY.

OF TEMPERATE CLIMATES.

Acute Articular Rheumatism. — Various bacteria have been reported as cause.

Epidemic Poliomyelitis. — Material from the cord of child with the disease
when injected subdurally, intravascularly, or into the peritoneal cavity of monkeys
produced the disease in the animals inoculated. The virus has been passed through
three generations of monkeys (Flexner).

The virus has been found in the brain, spinal cord, mesenteric and salivary glands
of monkeys and may remain in the nasal muscosa of monkeys as long as five months.
This would indicate the existence of human chronic carriers. With the possible
exception of the rabbit only man and the monkey are susceptible. This would
indicate that the virus is directly transferred from man to man. The virus is
highly resistant to drying and light. It will remain alive for months in dust. It is
not sterilized by pure glycerine during many months of contact. It is possibly
transmitted by a biting fly, Stomoxys calcitrans.

Flexner and Noguchi have recently cultivated the virus of poliomyelitis by
employing ascitic fluid to which had been added a fragment of sterile rabbit kidney
and nutrient agar, this culture medium being covered with a layer of paraffin oil.
The growth is obtained under anaerobic conditions. The minute colonies are
composed of globular or globoid bodies from .15 to .3 mikron in diameter. These
bodies may be single or in chains or in masses. In older cultures bizarre forms are
obtained. Monkeys bave been inoculated with the cultures.

Foot-and-mouth Disease. — Probably due to an ultramicroscopic organism.

Measles. — Cause entirely unknown. Hektoen has shown that blood contains
the virus.

Anderson has found that the virus of measles can pass through a Berkefeld filter
and loses its infectivity after heating for 15 minutes at 55° C. In infecting monkeys
it was found that the blood of patients with measles was infective only just before
and for about twenty-four hours after the appearance of the eruption. Mixed nasal
and buccal secretions were infective for monkeys for about forty-eight hours from the
time of the eruption. The scales from desquamating cases were not capable of
infecting monkeys hence it was thought that measles was not contagious during the
period of desquamation.

Mumps. — Herb has implicated a diplococcus. Inoculations into Steno's duct of
monkeys successful.

Rabies. — Probably the Negri bodies.

Roetheln (German Measles).— Nothing known.

Scarlet Fever. — Streptococci seem most probable cause (S. anginosus). Mallory
has implicated epithelial protozoa.

382 APPENDIX

Recently cell inclusions in polymorphonuclears have been supposed to be diag-
nostic; other diseases however seem to give them as measles, diphtheria, etc.

Small-pox and Vaccinia. — Guarnieri and Councilman have implicated epithelial
protozoa.

Spotted Fever of the Rocky Mountains. — Supposed to be due to an unknown
protozoon transmitted by a tick, D. andersoni.

Typhus Fever. — It has been suggested that the cause may be a protozoon trans-
mitted by vermin.

Recent work by Anderson and Ricketts has shown that the blood of human cases
is infective for monkeys. The virus does not seem to pass through a Berkefeld
filter and the epidemiology points to the body louse as the transmitting agent.

Varicella. — Entirely unknown.

Whooping Cough. — Influenza-like bacilli have been implicated. Bordet-
Gengou bacillus.

Or TROPICAL CLIMATES.

Ainhum. — A disease characterized by a constricting fibrous ring, especially of
little toe, often leading to spontaneous amputation.

Beriberi. — Various microorganisms and food factors suggested. A form of
multiple neuritis, occurring chiefly in countries where rice is the staple food, char-
acterized by oedema and marked cardiac and respiratory embarrassment. The
vagal involvement produces grave symptoms. Rice from which the pericarp has
been largely removed, polished rice, implicated.

Blackwater Fever. — Considered as a malarial disease, but thought by some to
be possibly caused by a protozoon — a Babesia (Piroplasma). A disease usually
occurring in malarial patients characterized by rapid febrile onset, early jaundice,
asthenia, pain in loins and the pathognomonic haemoglobinuria.

Dengue. — Supposed to be due to a protozoon transmitted by Culex fatigans.
A disease characterized by sudden onset, high fever for three or four days, pains
in the postorbital regions, back and about joints. A remission occurs on the third
to fifth day followed by a secondary rise of temperature and a measles-like eruption.
Leukopenia and reduction in the percentage of polymorphonuclears. Virus exists
in the blood and is filterable.

Goundou. — Symmetrical bony tumors of nasal processes of superior maxillary
bones.

Pellagra. — A disease about which two etiological views exist (i) that it is con-
nected with the ingestion of spoiled maize, the other that it is of protozoal nature
and transmitted either by Simulium reptans or Stomoxys calcitrans. It is char-
acterized by (i) a sprue-like stomatitis and disorders of alimentary canal (2) an
erythema usually limited to parts exposed to the sun and characterized by marked
symmetry and striking delimitation from the sound skin and (3) various neuro-
logical manifestations and a toxic psychosis which may go on to confusional insanity.
The disease is characterized by annual recurrences in the spring with improvement
in the winter.

Rat-bite Disease. — A disease caused by the bite of rats. Rather common in
Japan. Five weeks after bite when wound has healed, high fever sets in, cicatrix

APPENDIX 383

becomes inflamed with lymphangitis and swollen glands. The fever falls in a few
days to be succeeded by other febrile paroxysms. An erythematous eruption accom-
panies the second paroxysm. Supposed to be due to a protozoon.

Sprue. — A form of chronic diarrhoea characterized by diaphanous thinning
of gut and ulcerations of buccal cavity.

Tsutsugamushi. — A disease of Japan somewhat resembling typhus fever.
Supposed to be due to a protozoon transmitted by the Kedani mite.

Verruga Peruana. — A disease with a fever characterized by a profound involve-
ment of the bone marrow producing very rapidly an anaemia resembling that of
pernicious*anaemia. Pains of bones and joints marked. Death may occur before
the eruption, which appears as patients improve, either as red spots which enlarge
to the size of pea (miliary) or as pedunculated lesions as large as a pigeon's egg
(nodular). The lesions are very haemorrhagic and may appear in crops. The
nodular form is located chiefly about the knees and elbows but the miliary form
may cover entire skin surface and mucous membranes.

Yellow Fever. — Supposed to be due to a protozoon transmitted by the Stego-
myia calopus. A disease characterized by sudden onset, rachialgia, albuminuria
and jaundice about the third day. Pulse becomes slow even with rising temperature.
Black vomit often precedes fatal termination. Virus exists in the blood and is
filterable.

E— CHEMICAL EXAMINATION OF URINE.

For the prevention of decomposition when a urine is not examined shortly after
voiding, chloroform (10 to 20 drops added to a tightly corked bottle) or formalin
(4 or 5 drops to a pint of urine) are ordinarily employed. Formalin is better for
microscopical material, but, owing to its reducing power, should be substituted by
boric acid in urine to be examined for sugar. For clearing urine, turbid by reason
of bacteria, rubbing up with Talcum purificat. U. S. P. and filtering is recommended.

A twenty-four-hour specimen is necessary for accurate work. The urine should
be collected in clean separate bottles. Where pus comes from the bladder the
proportion of pus in each bottle will be practically the same; if from the kidneys
the amount will vary in the different bottles.

The amount of urine varies in different individuals (water or beer habit). It is
usually given as from 1000 to 1500 c.c.

Long proposes to substitute 2.6 for Haeser's coefficient which, if multiplied by
the two final figures of the specific gravity taken at 25° C., gives the weight of urin-
ary solids in 1000 c.c.

Albumin. — Practically serum albumin alone is clinically important.

The two usual tests are i. Heat test and 2. Heller's nitric acid test. For the
former, add 3 to 10 drops of 5% acetic acid to the perfectly clear urine in a test-
tube and bring to a boil. By boiling the upper portion a turbidity in contrast with
the clear lower portion may be obtained.

A more delicate test for albumin is the following: Add to a test-tube half filled
with filtered urine one-fifth it's volume of a saturated aqueous solution of sodium
chloride; heat to the boiling point; add two to five drops of fifty per cent, acetic
acid and heat again. This test may serve to distinguish nucleo-albumin, as most

384 APPENDIX

forms of nucleo-proteid found in urine do not react to the test, while serum albumin
does. Thus where a positive nitric acid test is present, and no precipitate occurs
with this test, the proteid present is usually nucleo-proteid.

For Heller's test, pour a small amount of nitric acid into a narrow test-tube
and, while holding the tube at an angle of about 45°, superimpose a layer of the urine
to be tested, which is delivered drop by drop from a pipette and allowed to flow
down the side of the tube.

This test can be converted into a quantitative one which is sufficiently accurate
for clinical purposes. It is based on the fact that a specimen of urine containing
0.003% °f albumin will give a perceptible ring at the layering of the urine and acid
in two minutes. If the ring appears at once or in a few seconds the albumin con-
tent is greater. From the qualitative test an idea can be formed as to the amount
of albumin which the urine contains, a heavy ring forming immediately showing a
considerable albumin content. Probably the highest elimination of albumin is
found in chronic parenchymatous nephritis where it may run from i to 3%. In
an ordinary case of acute nephritis 0.5% would be an average content.

Recently I have been using for both qualitative and quantitative albumin tests
the apparatus shown in Fig. 7. This is simply a five-inch piece of one- fourth-inch
soft glass tubing heated at a point 2 inches from one end, drawn out for about two
inches and bent to form a U tube with one end shorter than the other. This form
of tube enables one to perform two tests with the same column of nitric acid and is
easily cleaned and dried. They may be kept suspended around a glass tumbler's
rim. Taking up a small amount of nitric acid with a capillary bulb pipette it is
deposited in the capillary curve of the bent tube. This acid pipette should be
kept attached to the acid bottle. With a second pipette the urine is deposited in
the short arm of the U tube and the presence of albumin shows by a distinct ring
at the junction of urine and acid in the clear capillary tubing. The long arm will
serve for the introduction of a second specimen of urine for the albumin test.

For quantitative test we dilute the filtered urine with one or more parts of nor-
mal salt solution according to the intensity of the albumin ring. A very convenient
way of making the dilution is with a graduated centrifuge tube. Make a one to
ten dilution of the urine, mix and draw up with a bulb pipette and deposit in the
short arm of the U tube. A distinct ring forms in 2 or 3 seconds. Pour off one-
half of the diluted urine and make up with an equal amount of saline. Deposit
this 1-20 dilution in the long arm. The ring forms in about a minute. With
further testing it is found that a one to forty dilution shows a perceptible ring in
just 2 minutes. This final and successful dilution multiplied by 0.0033 gives the
percentage of albumin in the urine (40X0.0033=0.13%).

Should it be desired to determine the nature of the proteids present either in
urine or in exudates or transudates the following method is applicable. Determine
the percentage of total proteid by the method employed above. Then throw down
the globulins by the addition of an equal amount of a saturated solution of ammo-
nium sulphate, filter and estimate the proteid content of the filtrate. The differ-
ence between that and the total gives the percentage of globulin/ The filtrate is
now treated with 5% acetic acid until a precipitate of nucleo-proteid ceases to form;
the fluid is filtered and the clear filtrate (which should not show any turbidity with
a drop of 5 % acetic acid) is tested for its proteid content, which represents the

APPENDIX 385

serum albumin. When the combined percentage of globulins and serum albumin
is subtracted from the total proteid percentage we have the percentage of
nucleo-proteid.

Bence-Jones Body. — (Albumose.) Perform the heat test for albumin. The
appearance of a heavy precipitate which partially clears on boiling suggests albu-
mose. If albumose is present a cloud will appear in the nitrate on cooling. The
precipitate formed with nitric acid, if due to albumose, disappears with heat, that
of serum albumin does not.

As another test for the Bence-Jones body, usually present in multiple myelomata,
that of Boston is of value. Mix 15 c.c. urine in a test-tube with an equal amount of
saturated NaCl solution. Add 2 c.c. of 40% NaOH solution and shake the contents
of the tube thoroughly. Heat the upper contents of the tube to boiling and add lead
acetate solution (10%) drop by drop continuing the heating. A brown to black
precipitate (sulphur) shows this form of albumin.

In tests requiring the removal of albumin boil the urine and add dilute acetic
acid until the precipitate is flocculent, then filter.

SUGAR.

Fehling. — Pour equal parts of Fehling's copper solution (34.639 grams of copper
sulphate in 500 c.c. of water) and Fehling's alkali solution (173 grams sodium potas-
sium tartrate and 50 grams sodium hydrate in 500 c.c. water) into a test-tube. Mix
and dilute the deep Wire solution with two parts of water. Heat the upper portion
of the diluted Fehling's solution in the flame to boiling and drop in from a pipette
the urine to be examined. A yellowish to red precipitate shows the presence of
sugar.

Fehling's test will show the presence of i/ioo of i % of glucose in an
aqueous solution but is vastly less delicate for sugar in urine. This is due to the
power of the creatinin in urine of holding the reduced suboxide of copper in solu-
tion. An important point is that the creatinin is broken up by prolonged boil-
ing hence the puzzling precipitates one gets at times after a long period of boiling
are explained in this way. Glycuronic acid may cause a doubtful reaction. If the
precipitated cuprous oxide is in very fine granules the color is greenish, if less fine,
greenish-yellow and if quite coarse, reddish.

Creatinin holds in solution the copper suboxide formed by uric acid as well as
that resulting from very small glucose content of urine.

As a test for doubtful glycosuria it is well to give 100 grams of pure glucose. A
normal person should deal with such an amount without showing sugar reaction of
the urine.

Phenylhydrazin (Kowarsky). — Mix five drops of pure phenylhydrazin in a test-
tube with ten drops of glacial acetic acid. Shake lightly and add 15 drops of satu-
rated solution of NaCl. This makes a pasty mixture. Now add 10 c.c. of the urine
and bring carefully to a boil over a small flame and continue to boil gently for two
minutes. Upon cooling a yellowish crystalline precipitate falls more or less rapidly
according to the sugar content of the urine. If the urine contains 0.2% or more of
sugar the precipitate appears in a few minutes. The test is sensitive for 0.03% of
sugar.

25

386 APPENDIX

Fermentation Test. — This is the surest test for sugar in the urine. It will show
the presence of 0.05% of glucose. Instead of the Einhorn apparatus one may be
extemporized by taking a 50 c.c. cylinder, filling it to overflowing with the urine
which has previously been rubbed up with a piece of compressed yeast the size of a
hazel-nut. The urine should be made acid with tartaric acid to prevent ammoniacal
decomposition with the formation of CO2. A small 3-in. test-tube is filled with the
yeast-treated urine and dropped mouth downward into the 50 c.c. cylinder. The
apparatus is incubated for twenty-four hours and the presence of gas in the closed
end of the test-tube shows that sugar was present. A control to determine that the
yeast does not contain sugar is advisable. To utilize this test as a quantitive one,
first accurately take the specific gravity of the urine; then add the yeast and
fill the test-tube and cylinder as directed above. Next pour off or pipette off the
urine exactly to the 50 c.c. mark. Incubate for twenty-four to forty-eight hours
and make up the loss by evaporation, with distilled water. After the urine has
cooled down to room temperature the contents of tube and cylinder are thoroughly
mixed (the small tube having been withdrawn with a pair of forceps), then filtered
to remove the sediment of yeast and then brought to the exact original volume of
50 c.c. with distilled water to make up the loss by evaporation. (If there should be
doubt as to the completion of the fermentation of the glucose a qualitative test for
sugar can be made.) The specific gravity is again taken and the difference between
this and the first reading multiplied by 0.23. Example: Specific gravity of unfer-
mented urine, 1.030, that of urine after incubation, 1.022. Difference, 8X0.23 =
1.84%.

It is advisable to have two good urinometers, one to register from 1000 to 1025,
a second to register from 1025 to 1050.

Benedict's New Method for Quantitative Determination of Sugar in Urine.

The solution for quantitative work has the following composition:

Copper sulphate (pure crystallized) 18.0 c.c.

Sodium carbonate — crystallized (100 grams of anhydrous

salt will answer) 200 . o gm.

Sodium or potassium citrate 200 . o gm.

Potassium sulphocyanate 125.0 gm.

5 % potassium ferrocyanid solution 5.0 c.c.

Distilled water to make total volume of 1000.0 c.c.

With the aid of heat dissolve the carbonate, citrate and sulphocyanate in enough
water to make about 800 c.c. of the mixture, and filter if necessary. Dissolve the
copper sulphate separately in about 100 c.c. of water and pour the solution slowly
into the other liquid, with constant stirring. Add the ferrocyanid solution, cool and
dilute to exactly one liter. Of the various constituents, the copper salt only need be
weighed with exactness. Twenty five c.c. of the reagent are reduced by 50 mg. of
glucose.

Sugar estimations are conducted as follows: The urine, 10 c.c. of which should
be diluted with water to 100 c.c. (unless the sugar content is believed to be low), is
poured into a 50 c.c. burette up to the zero mark. Twenty-five c.c. of the reagent
are measured with a pipette into a porcelain evaporating dish (25-30 cm. in diameter),

APPENDIX 387

10 to 20 gm. of crystallized sodium carbonate (or one-half the weight of the anhy-
drous salt) are added, together with a small quantity of powdered pumice-stone or
talcum, and the mixture heated to boiling over a free flame until the carbonate has
entirely dissolved. The diluted urine is now run in from the burette, rather rapidly
until a chalk white precipitate forms, and the blue color of the mixture begins to
lessen perceptibly, after which the solution from the burette must be run in a few
drops at a time, until the disappearance of the last trace of blue color, which marks
the end point. The solution must be kept vigorously boiling throughout the entire
titration. If the mixture becomes too concentrated during the process, water may
be added from time to time to replace the volume lost by evaporation. The cal-
culation of the percentage of sugar in the original sample of urine is very simple.
The 25 c.c. of copper solution are reduced by exactly 50 mg. of glucose. Therefore
the volume run out of the burette to effect the reduction contained 50 mg. of the
sugar. When the urine is diluted 1:10, as in the usual titration of diabetic urines,
the formula for calculating the per cent, of sugar is the following:

'„ times 1000 equals per cent, in original sample, wherein X is the number of

cubic centimeters of the diluted urine required to reduce 25 c.c. of the copper
solution.

In the use of this method chloroform must not be present during the titration.
If used as a preservative in the urine it may be removed by boiling a sample for a
few minutes, and then diluting to its original volume.

This solution will keep indefinitely and it is claimed by Benedict, that compari-
son with the polariscope and by Allihn's gravimetric process will show it to be more
accurate than any of the ordinarily used methods.

APPROXIMATE QUANTITATIVE ESTIMATION with Fehling's solution. (One c.c. of
Fehling's solution is reduced by 5 mg. glucose.)

Measure off 2 c.c. of Fehling's solution in a pipette and put in a test-tube or
small beaker and dilute with 20 c.c. of water.

Bring the diluted Fehling's to boiling and drop in drop by drop the urine from
a dropping-bottle for which the number of drops per c.c. has been noted. Esti-
mating 20 drops to the c.c. if 2 drops of urine are required to reduce the copper it
would show a sugar percentage of the urine of 10. Four drops 5%, 8 drops
2-5%> 16 drops 1.25%, 32 drops 0.6%, 64 drops 0.3%, 100 drops 0.2%.

The Pancreatic Reaction of Cammidge in the Urine.

Cammidge claims that there is a definite and important relationship between
his pancreatic reaction in the urine and disease of the pancreas. The results of
some workers go to support this view, particularly when considered in connection
with the examination of the faeces for neutral fat.

The principle of the reaction depends upon the formation in the urine of a sub-
stance having the characters of an unfermentable pentose sugar after boiling with
hydrochloric acid. It is not present in the original urine as such, and forms an
osazone on treatment with phenylhydrazine, easily distinguished from the corre-
sponding compound of glucose. As the presence of glucose would seriously interfere
with the success of the reaction, all specimens of the urine examined must be care-

388 APPENDIX

fully tested for glucose, and, if present, it must be removed by fermentation with
yeast cake. Glucose is rarely present.

The technic of the reaction requires considerable time, but is easy of manipula-
tion, and should be readily carried out in any hospital. The urine, if alkaline,
must be made acid in reaction, and any albumin or sugar present must be removed
and the urine made up to its original bulk with distilled water. To 40 c.c. of the
clear filtered urine are added 2 c.c. of concentrated hydrochloric acid, and the mix-
ture gently boiled for ten minutes in a small flask, using a funnel in the neck as a
condenser. It is now cooled and distilled water added to again make up the con-
tents to 40 c.c., owing to the loss by evaporation. Eight grams of lead carbonate are
now slowly added to neutralize the excess of acid. After standing for a few minutes
the flask is again thoroughly cooled and the contents filtered until perfectly clear.
The filtrate is then well shaken with 8 grams of powdered tribasic lead acetate, and
the resulting precipitate removed by filtration, which is repeated until perfectly
clear.

The excess of lead in solution must now be removed by treating with 4 grams of
powdered sodium sulphate; the mixture is heated to boiling, then thoroughly cooled
and filtered. From the filtrate are measured 17 c.c.; this is transferred to a small
flask with funnel condenser and there are added 2 grams of sodium acetate, 0.8
grams phenylhydrazine hydrochloride and i c.c. of 50% acetic acid. The mixture
is then boiled gently for ten minutes, filtered into a test-tube with a mark showing
15 c.c., and made up, if necessary, to that point with hot distilled water. The fil-
trate is carefully stirred and left to stand over night.

The quantity and time of deposit of the crystals will depend upon the degree
of extension of the inflammatory process in the pancreas. Thus, in well-marked
cases, a light-yellow flocculent precipitate should appear in a few hours, but in less
characteristic cases it may be necessary to leave the preparation over night before
a deposit occurs. Under the microscope the precipitate is seen to consist of long,
light-yellow, flexible, hair-like crystals of pentosazon, arranged in delicate sheaves.

Urinary Tests in Connection with Acidosis.

The determination of the ammonia quotient, which is the ratio of N eliminated
as ammonia to total nitrogen elimination, has assumed great importance by reason
of its connection with various forms of acid intoxication, as in diabetes, pernicious
vomiting of pregnancy, and various hepatic diseases.

The degree of acidosis is better determined by the quantitative estimation of
nitrogen elimination as ammonia than by estimating quantitatively the amount of
diacetic and /?-oxybutyric acid in the urine. Normally we have about 0.7 gram of
ammonia eliminated daily. In acidosis this may rise to 5 or 10 grams and instead
of being from 3 to 5% of the total N, it may amount to 30 to 50%.

Formalin Method for the Estimation of Ammonia.

Free ammonia reacts with formalin to form hexamethylenetetramine. If
sodium hydrate is added to neutralized urine in the presence of formalin free am-
monia is liberated and reacts with the formalin. So soon as all the ammonia has
been liberated, the end reaction occurs.

APPENDIX 389

Ronchese first utilized this principle and Mathison found that pot. oxalate
made the end reaction sharper. Brown found that preliminary clearing with lead
subacetate made the end reaction still sharper and removed certain nitrogenous
substances which reacted with formalin making the result only about 5% higher
than with Schaffer's method. The technic is as follows: About 60 c.c. of filtered
urine are treated with 3 grams of basic lead acetate, well stirred, allowed to stand
a few minutes and filtered. The filtrate is treated with 2 grams of neutral potas-
sium oxalate well stirred and filtered; 10 c.c. -of the clear filtrate are diluted to 50
c.c. with distilled water; a few drops of i% phenolphthalein solution are added.
The mixture will be slightly alkaline or acid. Five grams potassium oxalate
are added and stirred. It is exactly neutralized with decinormal NaOH or H2SO4.
Twenty c.c. of 20% commercial formalin, previously made neutral, are added, and
the solution again titrated with decinormal NaOH to neutralization. Every c.c.
of decinormal NaOH corresponds to 0.0017 gram NHs. The quantity of ammonia
is then calculated on the basis of the twenty-four-hour volume. Example: The
10 c.c. of urine required 4 c.c. N/io NaOH to give a pink color. 4X0.0017 =0.0068.
Then 100 c.c. urine would contain 0.068 and 1000 c.c. (twenty-four-hour urine am-
ount) 0.68 gram of ammonia.

ESTIMATION OF TOTAL NITROGEN.

Principle. — The nitrogenous material of the urine is converted into ammonium
sulphate on boiling with H2SO4. The ammonia is then estimated as described
under estimation of ammonia by the formalin method.

Technic. — Solutions required :

1. Twenty per cent, commercial formalin previously made neutral with NaOH.

2. N/io NaOH.

3. Forty per cent. NaOH.

Ten c.c. of filtered urine are pipetted into a Kjeldahl or Koch flask; 10 c.c. of
concentrated H2SO4 and 10 grams K2SO4 are added. The mixture is heated over
a free flame, gently at first to avoid foaming, and is finally brought to a boil, which
is continued until the mixture is perfectly clear, usually requiring forty-five minutes
to an hour. The contents are cooled and quantitatively transferred to a 200 c.c.
volumetric flask and i c.c. of phenolphthalein solution added. The greater part of
the acidity is now neutralized by adding about 30 c.c. of the 40% NaOH. It is
cooled under a water tap and made up to the 200 c.c. mark; 10 c.c. are taken, diluted
to 50 c.c. with distilled water and exactly neutralized with N/io NaOH. Twenty
c.c. of the formalin solution are now added and the titration again performed. The
pink end reaction is beautifully clear and sharp. The second reading multiplied
by the factor 0.0014 gives the amount of nitrogen in grams in 10 c.c. of the fluid.
It is then computed for the twenty-four-hour volume as for N, eliminated as ammonia.
Example: It required 5 c.c. N/io NaOH — 5X0.0014 = 0.007. As original 10 c.c.
were diluted to 200, the 10 c.c. taken for titration would only be 1/20; hence 0.007 X
20 = 0.14 gram for 10 c.c or 1.4 for 100 c.c. or 14 grams for 1000 c.c.

The amount of urea, which represents from 85 to 90% of the total nitrogen, is
usually determined instead of the total N. The hypobromite and hypochlorite
methods are, however, lacking in accuracy, and more exact methods of urea estima-
tion are more time-consuming than the one just given for total N.

390

APPENDIX

Probably the most convenient test for urea is the hypobromite method, using
the Doremus ureometer with a side tube connected to the closed arm of the fermenta-
tion tube by a glass stop cock.

The reagent is prepared by taking 70 c.c. of a 30% stock solution of NaOH,
diluting it with 180 c.c. water and then adding 5 c.c. of bromine, stirring until the

bromine is dissolved. This solution if stored
in a cool dark place will keep about one week,
The urine to be tested must be free from
sugar and albumin and contain less than i %
of urea. Ordinarily the urine must be di-
luted two to four times to obtain a speci-
men containing less than i%. In using
this improved Doremus ureometer the closed
portion of the U tube is filled with the hy-
pobromite solution, and the urine introduced
by allowing it to run in from the side tube
by opening the glass cock arranged for that
purpose. After the gas has risen and the
instrument has stood for a short time the
readings may be made in grams to the liter,
or in percentage.

This urea determination is only a rough
clinical one.

Gerhardt's Test for Diacetic Acid.

Add a few drops of ferric chloride solu-
tion to 10 to 50 c.c. of urine so long as a
precipitate continues to form. Then filter
and to the filtrate add more ferric chloride
solution. A bordeaux red color shows dia-
cetic acid. The test is sensitive. As a con-
trol to show that the color is not due to drug
elimination (antipyrine, salicylates, etc.)
boil a specimen which gave the test for
three to five minutes. If the color was due

to drugs it will be obtained with a boiled sample while such treatment drives off the
diacetic acid. In Hurtley's test add 2.5 c.c. HC1 and i c.c. of i% sol. of sod.
nitrate to 10 c.c. urine. Shake and allow to stand 2 minutes. Now add 15 c.c.
strong ammonia followed by 5 c.c. of 10% sol. ferrous sulphate. The slow produc-
tion of a violet colour shows positive test (2 hours). Shows i part aceto-acetic
acid in 50,000.

If the urine shows a well-marked Gerhardt reaction it is well to test for /?-oxy-
butyric acid.

The following modification of Lange's test by Hart is a satisfactory one. The
principle involved is the removal of acetone and diacetic acid by heat, then oxidizing
/?-oxybutyric acid to acetone with hydrogen peroxide and then testing for acetone.

FIG. 106.

-Doremus-Hinds Ure-
ometer.

APPENDIX 3QI

Method: Take 20 c.c. of urine, dilute with an equal amount of water and add a
few drops of acetic acid. Next boil in a beaker until the original amount of diluted
urine is reduced to 10 c.c. (originally 40 c.c.). Dilute this evaporated urine with an
equal amount of water, giving us 20 c.c. In each of two test-tubes put 10 c.c. of
this 20 c.c. To one tube add i c.c. of hydrogen peroxide and warm gently, without
boiling, for one minute; then cool. The other tube is left untreated. Next, to each
test-tube add 10 drops of glacial acetic acid and 5 to 10 drops of a freshly prepared
sodium nitroprusside solution and mix. Next carefully overlay each tube with
about 2 c.c. of concentrated ammonia. If /2-oxybutyric acid were present in the
tube treated with the hydrogen peroxide and thereby oxidized to acetone a violet-
red ring will develop at the point of contact while in the untreated tube there will
be no such color ring.

A yellowish-brown ring from the presence of creatinin may show in the untreated
tube. It is well to allow the tubes to stand for three to four hours before finally
reporting the absence of /3-oxybutyric acid. It will probably show 0.2%.

Acetone. — To one-sixth of a test-tube of urine add a crystal of sodium nitro-
prusside. Make strongly alkaline with NaOH. Shake. The addition of a few
drops of glacial acetic gives a purple color to the foam, if acetone is present.

Diazo Reaction. — To 5 c.c. sulphanilic acid solution (sulphanilic ac. i pt., HC1
50 pts., aq. 1000 pts.) add two drops of a 0.5% solution of sodium nitrite. Add an
equal quantity (5 c.c.) of urine. Shake and add quickly 2 or 3 c.c. of ammonium
hydrate. A carmine color, especially in the foam, shows a diazo reaction. If the
reaction is positive, and the mixture is allowed to stand for 24 hours, a precipitate
forms, the upper margin of which exhibits a green, greenish-black or violet zone.

Indican. — Take 10 c.c. urine and treat it with i c.c. of sol. of lead subacetate.
Filter. Of this nitrate take 6 c.c. and treat with an equal amount of Obermayer's
reagent; allow to stand for 5 minutes then shake gently with 2 c.c. of chloroform.
Obermayer's reagent is strong HC1 containing 2 parts of ferric chloride to the liter
— o.i gram to 50 c.c. of HC1.

A more exact method is to pour off the supernatant acid urine. Wash the
chloroform with water, then pour off as much of the supernatant water as possible
and add 10 c.c. of alcohol. A clear blue fluid results.

Urobilin. — Urobilin appears in considerable quantity in urine when there is
much destruction of red cells, as in pernicious anaemia, internal haemorrhage, and in
malaria cachexia. The best test is that of Schlesinger. To the unfiltered urine
add an equal amount of a saturated solution of zinc acetate in absolute alcohol.
Shake, add a few drops of Lugol's solution and filter. Fluorescence in the nitrate
shows the presence of urobilin. The degree of blood destruction is indicated by the
intensity of the fluorescence.

Bile Pigments. — A satisfactory test is that of Rosin (Trousseau). Overlay 10
c.c. urine with about 5 c.c. of dilute tincture of iodine (i to 10 of 95% alcohol).
An emerald green ring at the point of contact shows the presence of bile coloring
matter.

Phenolsulphonephthalein Test for Renal Efficiency.

Geraghty has recently stated that in 35 cases where an autopsy made it possible
to verify the accuracy of this test that the lesions as revealed at autopsy corre-

392 APPENDIX

sponded closely with the results of the test. Again in 30 nephrectomies the condi-
tions found were in accordance with the results of the test. The general opinion of
those who ;have used the test is that it is more reliable than cryoscopy and far easier
of application. The technic is as follows: One c.c. of the phthalein solution
containing 6 mg. is injected intramuscularly or subcutaneously. The drug can be
bought in ampules ready for use. About twenty minutes before injecting the drug
the patient is given from 200 to 400 c.c. of water to drink. After the injection the
bladder is emptied with a catheter and the time is accurately noted when the urine
which subsequent to the emptying of the bladder and being allowed to drop into a
test-tube containing one drop of a 25% sodium hydrate solution first shows a pink-
ish tinge. This is recorded as the time of appearance of the drug in the urine and
normally is about 10 minutes. The catheter is then withdrawn and the urine that
is passed in the first hour collected and subsequently that passed in the second hour.
To each hour's specimen sufficient 25% sodium hydrate is added to give a purple-
red color and the entire amount is then poured into a liter flask and made up to
1000 c.c. A similar treatment is employed for the urine of the second hour. The
amount of drug eliminated in each hour is then determined by a colorimeter.

Cabot has proposed the use of a series of ten test-tubes containing solutions of
the drug representing from 5% to 50% of the drug dose, each tube containing 5%
more than the preceding one. These comparison solutions may be made up with
the patient's urine obtained at the time of emptying the bladder so that the con-
fusion which may obtain when water is used is avoided. It has recently been pro-
posed to make the standards with water and use a piece of yellow glass for match-
ing. The urine to be tested made up to 1000 c.c. as previously described is then
poured into a test-tube of similar size and matched.

In normal cases Cabot got 46% of the drug eliminated in the first hour, the
average for the second hour being 17%. The quantity of urine secreted in either
hour has no relation to the test, which is the percentage of drug eliminated. In
cases with serious kidney disease the amount of drug eliminated in the first hour
may range from 5 to 12%.

When the question of the kidney involved arises, the urine must be taken by
ureteral catheterization or by a separator.

F— CHEMICAL EXAMINATION OF GASTRIC CONTENTS.

The test breakfast ordinarily used is that of Ewald (one shredded wheat biscuit
or two small pieces of toast with 400 c.c. of water is what is usually given). This
Ewald breakfast is a low-grade stimulant to acid production. It is given in the
morning on an empty stomach. If at supper, the night before, the patient partake
of raspberry jam the finding of the characteristic seeds in the stomach contents
the next morning would be evidence of lack of motor activity. The Fischer meal
which contains a 4-ounce Hamburg steak in addition to the water and toast of the
Ewald is withdrawn after three hours.

The stomach tube is more easily passed if it be thoroughly chilled in ice water
without the use of any lubricant.

The stomach tube should be passed one hour after the Ewald breakfast and if
more than 50 c.c. of fluid be obtained it indicates stasis or hypersecretion.

APPENDIX 393

Filter the gastric contents and test first for free HC1. The most reliable and
sensitive test is that of Gunsberg. The reagent, which should be freshly prepared,
consists of phloroglucin 3 grams, vanillin i gram, and absolute alcohol 30 c.c. By
mixing 2 drops of gastric juice and an equal quantity of Gunsberg reagent in a small
porcelain dish and carefully heating above a flame we obtain a carmine red color
if free HC1 be present. A water bath is preferable.

For lactic acid a modification of Strauss' method is quite satisfactory. Shake,
in a test-tube, 5 c.c. of gastric contents with 20 c.c. of ether, allow to settle and
pour off 5 c.c. of the supernatant ether into another test-tube. To this ether add
20 c.c. of water and 2 drops of a i to 9 solution of ferric chloride and shake well.
The presence of i% of lactic acid will give an intense greenish color.

Having determined the presence or absence of free hydrochloric or lactic acid,
we should make a quantitative test of the various factors producing the acidity of
gastric juice (a modified Topfer test). These are: i. Free HC1. 2. Combined HC1.
3. Acid salts, and 4. Total acidity.

To 10 c.c. of filtered gastric contents, in a beaker, add 3 drops of dimethyl-
amido-azo-benzol solution (a 1/2% solution in 95% alcohol). In the presence of
free HC1 the fluid becomes a rich carmine pink.

After reading the burette run in N/io NaOH solution until the pink color is
discharged and a light yellow color is obtained. This reading multiplied by 10
gives the amount of free HC1 in degrees, a degree corresponding to i c.c. N/io
NaOH. Next add 6 drops of a 1/2% alcoholic solution of phenolphthalein to the
light yellow fluid in the beaker. Again titrating the same preparations we add
N/io NaOH until a faint but distinct pink color is produced. The number of c.c.
added for the free HC1 plus the number to give the pink color when multiplied by
10 gives the total acidity in degrees. (For example: 2.5 c.c. N/io NaOH used to
obtain yellow color — 2.5X10 = 25 or acidity due to free HC1. After adding the
phenolphthalein, 4 c.c. N/io NaOH required to produce pink color — 4+2. 5 X
10 = 65 or total acidity in terms of acidity. This means that it would require 65
c.c. N/io NaOH to neutralize 100 c.c. of gastric juice. A total acidity of 60
is about normal. To obtain percentage in HC1 multiply by 0.00365; thus. 65 X
0.00365 = 0.23 HC1.)

Having determined the total acidity add 3 c.c. of 10% neutral calcium chloride
solution to the gastric contents already in the beaker. As a result of the formation
of acid calcium phosphate the pink color is discharged. Again add N/io NaOH
from the burette until the pink color is restored. The number of c.c. used gives the
amount of acid salts present.

From the figures for the total acidity subtract the sum of that for free HC1 and
for acid salts and the remainder will give the acidity due to combined HC1.

G— CHEMICAL TESTS OF FAECES.

To test for acidity Kaplan rubs up 5 grams faeces in 30 c.c. distilled water. Put
2 c.c. of the emulsion in a test-tube and add a few drops of phenolphthalein solution.
Titrate with N/io NaOH to a pink. A normal stool from a Schmidt test-diet re-
quires about 1.5 c.c. N/io NaOH. After fermentation the stool may be quite acid
or more alkaline than before the fermentation test.

394 APPENDIX

Test for Pancreatic Ferments. — To obtain a stool for ferment examination,
calomel 2 to 3 grains, or phenolphthalein 5 grains is to be preferred to salts. The
Fuld-Gross-Goldschmidt test uses for trypsin testing a solution of casein, i gram,
sodium carbonate, i gram chloroform i c.c. to i liter of water. If the stool is not
very liquid 5 grams of faeces are rubbed up with 20 c.c. salt solution and filtered.
Dilutions of i to 10, i to 100 and i to 1000 are made and 0.5 and i c.c. of these dilu-
tions added to 6 test-tubes each containing 5 c.c. of the casein solution. The tubes
are incubated for 24 hours at 38° C. and completion of the digestion tested by add-
ing 5% acetic acid which should not cause a precipitate in tubes in which digestion
is complete.

The estimation is made by units, one unit being the digestive power of one c.c.
of faeces filtrate to digest i c.c. of casein solution. If i c.c. of the i to 1000 faeces
dilution digested 5 c.c. of casein solution it would represent 5000 units. If i c.c. of
i to 10 dilution it would be 50. As there are 5 c.c. of the casein solution we multi-
ply the dilution of faeces by 5 for i c.c. or by 10 if we had only 0.5 c.c. of faeces dilution
in the tube tested.

For amylopsin a similar technic is followed using a i % solution of soluble starch
instead of the 0.1%. casein solution. The end reaction is tested by adding i
drop of N/io iodine solution to each of the starch tubes and fasces dilution after
24 hours of incubation. The absence of a blue color shows completion of starch
digestion.

The normal ferment content of the fasces rarely falls below 200 units and
may be as high as 10,000. Cases showing a ferment value of only 25 to 50 units
are very suspicious as regards pancreatic disease.

H— DISINFECTANTS AND INSECTICIDES.

By disinfection is meant the destruction of injurious bacteria.

Sterilization is where all living things are destroyed.

Germicides are substances which kill bacteria while antiseptics are those which
are inimical to the growth of bacteria.

Formalin is antiseptic in 1-50,000 dilution but germicidal only in 1-20.

Deodorants may or may not be antiseptic or germicidal. An insecticide may
or may not be a germicide and vice versa.

In disinfection we must consider

(1) Strength of solution.

(2) Time of application.

(3) Nature of medium in which disinfectant acts.

By Coefficient of Inhibition we mean time and concentration necessary to prevent
development of bacteria.

By Inferior Lethal Coefficient we mean time and concentration necessary to kill
nonspore-bearing bacteria.

By Superior Lethal Coefficient we mean time and concentration necessary to
kill spore-bearing bacteria.

Disinfectants may be (A) physical (B) gaseous (C) chemical.

(A) Of the physical disinfectants we have

(i) Sunlight. The red and yellow rays practically inert. The violet and

APPENDIX 395

ultra violet most active. Direct sunlight kills plague bacilli in less than one hour
— typhoid bacilli in six.

(2) Burning. Very efficient but expensive.

(3) Boiling. Especially m carbonate of soda solution for about one hour is a
very efficient disinfectant. Nonspore-bearing bacteria are killed almost instantly
by a boiling temperature. One must remember that the boiling temperature is
lower at mountainous elevations.

(4) Steam. Extremely efficient. The condensation of the steam on the object
to be sterilized gives off latent heat and produces a vacuum.

(B) Of the gaseous disinfectants we have the very efficient germicide formal-
dehyde gas and the weakly germicidal, but potent insecticide, sulphur dioxide.

Formaldehyde gas is practically valueless as an insecticide.

Bromine, chlorine and hydrocyanic acid gas have a certain degree of efficiency
but are not of practical application. Hydrocyanic acid gas is especially dangerous
on account of its extreme toxicity.

(i) Formalin. — This is a 40 % solution of formaldehyde gas, but is as a rule
of less strength from evaporation or otherwise. Formaldehyde is efficient as a
surface disinfectant when the temperature is above 50° F. and the air contains at
least 60 % of moisture. It is not efficient in cold dry rooms. Owing to its lack
of penetrating power it is not efficient for the disinfection of mattresses, or similar
articles. To prepare a room for disinfection we must measure the cubic space
to ascertain the necessary amount of formalin to use and stuff up or better paste
up with newspaper all cracks and openings.

In the production of formaldehyde gas the more expensive autoclaves and lamps
have largely been replaced by the simple formalin permanganate method. In this
one pours 500 c.c. of formalin on 250 grams of potassium permanganate for each
1000 cubic feet with six to twelve hours' exposure.

In employing this method, take a pan partly filled with water. Place in this
a second metal or glass receptacle containing the permanganate. Then pour the
formalin on the permanganate crystals.. The gas is generated in great amount in
a few seconds. The receptacle containing the permanganate and formalin should
be large enough to contain ten times the volume of formalin, as there is a tendency
for the mixture to foam over the sides of the dish.

Another practical method is the formalin-sheet-spraying one. The formalin
(40%) should be sprayed on sheets suspended in the room in such a manner that
the solution remains in small drops on the sheet. Spray not less than 10 ounces
of formalin (40%) for each 1000 cubic feet. Used in this way a sheet will hold
about 5 ounces without dripping or the drops running together. The room must
be very tightly sealed in disinfecting with this process and kept closed not less than
twelve hours. The method is limited to rooms or apartments not exceeding 2000
cubic feet. The formalin may also be sprayed upon the walls, floors, and objects
in the rooms.

Paraform Lamps. — For single rooms the use of the paraform lamp is quite con-
venient. Special lamps can be obtained to burn the paraform tablets or a pint
tincup will suffice for the heating of i ounce of paraform. The lamp or alcohol
flame under the receptacle must not be high enough to ignite the paraform which
burns readily and in so doing does not give off formaldehyde gas. One ounce of

396 APPENDIX

paraform is sufficient for a space of 500 cubic feet. One can dissolve 2 ounces
of paraform in 8 ounces of boiling water and then pour this over 4 ounces of
potassium permanganate in a two gallon pail.

N. Y. Health Department Method.— After a prolonged series of tests the N. Y.
Department of Health gave preference to the following formula.

Paraformaldehyde 30 grams, potassium permanganate 75 grams, water 90
grams. The chemicals are mixed in a deep quart pan and the water is added and
the mixture stirred. The evolution of gas is slow in starting but is complete in
five to ten minutes.

It was found that 87% of the gas was evolved and the quantities given above
suffice to disinfect 1000 cubic feet in four hours. It is well to put the small pan
containing the chemicals in a larger one to prevent danger of fire and soiling of the
floor by the frothing of the mixture.

Sulphur Dioxide. — Sulphur dioxide is fairly efficient, but requires the presence
of moisture. It is only a surface disinfectant and is lacking in penetrating proper-
ties. An atmosphere containing 4.5%. can be obtained by burning 5 pounds
of sulphur per 1000 cubic feet of space. This amount requires the evaporation or
volatilization of about i pint of water. Under these conditions the time of ex-
posure should be not less than twenty-four hours for bacterial infections. A shorter
time will suffice for fumigation necessary to kill mosquitoes and other vermin. Dry
sulphur dioxide produced by burning 2 pounds of sulphur for each 1000 cubic feet
of space will answer for this purpose. An exposure of from two to three hours is
sufficient.

The sulphur may be burned in shallow iron pots (Dutch ovens), containing not
more than 30 pounds of sulphur for each pot, and the pots should stand in vessels
of water. The sulphur pots should be elevated from the bottom of the compart-
ment to be disinfected in order to obtain the maximum possible percentage of com-
bustion of sulphur. The sulphur should be in a state of fine division, and ignition
is best accomplished with alcohol (special care being taken with this method to
prevent damage to cargo or vessel by fire), or the sulphur may be burned in a special
furnace, the sulphur dioxide being distributed by a power fan. This method is
peculiarly applicable to cargo vessels.

Liquefied sulphur dioxide may be used for disinfection in place of sulphur dioxide
generated as above, it being borne in mind that this process will require 2 pounds
of the liquefied gas for each pound of sulphur, as indicated in the above
paragraphs.

Sulphur dioxide is especially applicable to the holds of vessels or to apartments
that may be tightly closed and that do not contain objects that would be injured
by gas. Sulphur dioxide bleaches fabrics or materials dyed with vegetable or ani-
line dyes. It destroys linen or cotton goods by rotting the fiber through the agency
of the acids formed. It injures most metals. It is promptly destructive of all
forms of animal life. This property renders it a valuable agent for the extermina-
tion of rats, insects, and other vermin. Sulphur dioxide is a germicide only in the
presence of moisture, and even then will not kill spore-bearing organisms. If cloth-
ing is washed immediately after sulphur disinfection the rotting effect will be greatly
lessened. If used in spaces containing machinery all metal parts should be coated
with vaseline.

APPENDIX 397

CHEMICAL SOLUTIONS.

Bichloride of mercury is usually sold in the form of antiseptic tablets. As a
disinfectant for the infectious diseases it is usually used in a strength of i-iooo.
The solution should be made in a wooden or earthenware vessel. As bichloride
forms inert albuminates it should not be used in the disinfection of sputum, faeces
or any albuminous excreta. It must be remembered that bichloride is a mordant
so that any stains in soiled clothing will remain permanent. For disinfection of
clothing the material should be left in i-iooo bichloride for one hour. Dishes
for food should never be disinfected in bichloride on account of the danger from
poisoning. Floors and walls may be disinfected with i-iooo bichloride applied
with a mop. Allow the solution to dry on the floor or walls.

Formalin. — A 5% solution of commercial formalin in water (50 c.c. formalin
950 c.c. water) makes a satisfactory disinfectant for soiled clothing. It is also
valuable for albuminous material. The disinfectant must act in a strength of 5%
so that if one pint of faeces is to be disinfected we should add one pint of a 10% for-
malin solution and allow it to act for one hour.

Carbolic Acid. — It is soluble in water to the extent of about 5% and in such
strength it is an efficient disinfectant. The solution should be made with hot water.

In standardizing disinfectants carbolic acid is used as the standard. It how-
ever is expensive and there is often difficulty in making up satisfactory solutions.
More efficient and more convenient is the Liquor cresolis comp. U. S. P. This may
be prepared by mixing up equal parts of cresol and soft soap as noted on page 12.
This has a value according to tests made in the Hygenic Laboratory of 3, making
it in tests without organic matter three times as efficient as carbolic acid. Under
similar conditions lysol had a value of 2.12 creolin 3.25 and trikresol of 2.62.

Equal parts of a 5% solution of Liq. Cresol. Comp. and the faeces, urine
or sputum to be disinfected is satisfactory for disinfection provided the mixture
is allowed to stand for one hour. Liq. Cresol. Comp. (5%) is an excellent
disinfectant for contaminated bedclothing, etc. It is also most suitable for the
disinfection of floors and walls.

Lime. — It must be remembered that air-slaked lime is inert as a disinfectant.
For disinfecting faeces freshly prepared milk of lime is excellent. It is made by
mixing unslaked lime with four times its volume of water. An equal quantity
should be added to the faeces to be disinfected.

Chlorinated Lime. — This can be purchased in air-tight containers and when the
package is opened it should give off a powerful odor of chlorine.

For a working disinfectant solution add i pound to 4 gallons of water.
This is satisfactory for mopping floors and for disinfecting faeces, sputum and urine,
equal parts of the excreta and disinfecting solution being mixed and allowed to stand
for one hour. For disinfection of drinking water one teaspoonful of Jchlorinated
lime to i pint of water makes a stock disinfectant. For use one teaspoonful of
this stock solution is added to 2 gallons of the drinking water to be disinfected.
Let stand at least 1/2 hour.

INSECTICIDES.

The following notes are taken chiefly from the U. S. P. H. Service directions.
SULPHUR DIOXIDE — obtained as described above — destroys all animal life.

398 APPENDIX

In the case of vessels, when treated for yellow fever infection, the process shall
be a simultaneous fumigation with sulphur dioxide, 2 % volume gas, and two
hours' exposure, in order to insure the destruction of mosquitoes.

In the case of vessels when treated for plague the process with sulphur dioxide
shall be as follows:

Without cargo: The simultaneous fumigation with sulphur dioxide gas not less
than 2% for six hours' exposure.

With cargo: Fumigation with sulphur dioxide gas, 4 %, six to twelve
hours' exposure, according to stowing.

Infected vessels may require partial or complete discharge of cargo, and frac-
tional fumigation for efficient deratization.

Pyrethrum. The fumes of burning pyrethrum may be used to destroy mos-
quitoes in places where there are articles liable to be injured by the use of sulphur.

Four pounds per 1000 cubic feet space for two hours' exposure will kill, all or
practically all of the mosquitoes but precautions should be taken to sweep up and
destroy any that may have escaped.
Pyrethrum stains walls, paper, etc.

The oxides of carbon, as used at Hamburg, are efficient to destroy rats but do
not kill fleas or other insects. They are obtained by burning carbon, coke, or char-
coal, in special apparatus, and the gas as produced consists of about 5 %
carbon monoxide, 18 % carbon dioxide, and 77 % nitrogen.

Twenty kilos of carbon, coke, or charcoal are used for every 1000 meters of
space. The gas is allowed to remain in the ship for two hours and from seven to
eight hours are allowed for it to leave it. This is about equivalent to i 1/3 pounds
of carbon (coke) to 1000 cubic feet of air space. As this gas is very fatal to man
and gives no warning of its presence, being odorless, a small amount of sulphur
dioxide should be added to give warning of its presence. As it does not kill fleas
it cannot be depended on for complete work, where there is evidence of plague
among rats on the vessel, as the infected fleas would infect the rats coming aboard
after the deratization.

The articles named as disinfectants which can obviously destroy animal life can
be used for that purpose when applicable, as steam for bedding, fabrics, etc. For-
maldehyde is not applicable for this purpose.

For fleas the best insecticides are (i) crude petroleum (fuel oil) which is at times
called Pesterine, (2) an emulsion of kerosene oil made as follows: kerosene 20 parts,
soft soap i part and water 5 parts. The soap is dissolved in the water by aid of
heat and the kerosene oil gradually stirred into the hot mixture.

For cockroaches there is nothing so good as sodium fluoride. By sprinkling
the powder about the haunts of the cockroaches they are gotten rid of in a few days.

For exterminating rats and in this way secondarily the rat-fleas besides the
ordinary poisons such as As., P., etc. Rucker has recommended a poison composed
of plaster of Paris, 6 parts, pulverized sugar i part and flour 2 parts. This mixture
should be exposed in a dry place in open dishes. To attract the rats the edge of
the dish may be smeared with the oil in which sardines have been packed.

Wise and Minett report good results from the use of crude carbolic acid as a
larvicide for mosquitoes. They added about i teaspoonful for each 2 cubic feet
of water in the pool. Of course the ordinary method for destroying mosquito larvae
is by covering the surface of the water in the cistern or pool with a layer of petroleum.

INDEX

Abbe condenser, 4
Abscess, bacteria in, 356
Acanthia lectularia, 291, 294

rotundata, 294
Acarina, 282
Acidosis, 388
Acartomyia, 316
Acetone, for sections (see tissue), 372

in urine, 391
Acid-fast bacteria, 76, 77

siaining, 35
Acid proofing, u
Actinomycosis (see Discomyces), 123,

336, 356
.Kdina?, 314
Agar, egg, 23

gelatin, North, 24

glucose, 22

glycerine, 23

nutrient, 21

placenta], 30

plating, 41
Ancylostoma duoderiale, 263, 274, 350,

358
Agglutination, macroscopical, 144

microscopical, 143
Ainhum, 382

Air, bacteriological examination of, 135
Albumin in urine, 383
Albumin in sputum, 336
Albumose in urine, 385
Aldridr'a, 314
Alexin, 140
Amboceptor, 139, 151
Ammonia in urine, 388
Amoebae, 219, 336, 348, 356
Anaemia, aplastic, 190

infantum, 205, 229

pernicious, 190, 200

primary, 199

secondary, 201
Ana-robes, 64, 67

Buchner method, 68

combination method, 69

cultivation of, 67

Liborius method, 68

Tiroz/i's method, 68

Yignal method, 69

Wright method, 69

Anaphylaxis, 161
Anaphylactic shock, 162
Anginas, 331
Anguillula, 264

Animal inoculations, 48, 143, 152
Animal parasites, general classification,
211

mounting of, 377

nomenclature in, 213

preservation of, 378
Anisocytosis, 189
Anophelinae, 291, 314
Anthrax, 63, 64

vaccination, 64
Antiformin, 334
Antigen, 138, 147, 151
Antitoxin, 138

botulism, 70

diphtheria, 87

pyocyaneus, no

tetanus, 73
Antivenins, 320
Appendicitis blood count, 197
Arachnoidea, 281
Argas, 281, 287
Arneth index, 193
Ascaris, canis, 263, 278

lumbricoides, 263, 277, 332
Ascitic fluid (cytodiagnosis in), 360
Aspergillus, concentricus, 117, 123

flavus, 123

fumigatus, 123

nidulans, 123

pictor, 123

repens, 123

Auchmeromyia luteola, 303
Azolitmin, 24

Babesia, 242

Bacillus, acidi lactici, 133

acidophilus, 108

acnes, 357

aerogenes capsulat., 64, 75, 349

Aertyrck, 104

anthracis, 64

anthracis symptomat., 64

bifidus, 108

botulinus, 70

bulgaricus, 108

399

4OO

INDEX

Bacillus, cloacae, 108

coli, 107, 130, 338, 356

diphtherias, 77, 85, 328, 330

dysenteriae 105, 349

enteritidis (Gartner), 70, 104

enteritidis sporogenes, 127

fecalis alkaligines, 99

fusiformis, 331

icteroides, 100

influenzas, 92

lactis agrogenes, 107

leprae, 82, 326, 328, 358

mallei, 84, 328, 358

mycoides, 63

of avian tuberculosis, 79

of Bordet-Gengou, 91, 94

of bovine tuberculosis, 79

of chancroid, 94

of chicken cholera, 96

of Hofmann, 89

of hog cholera, 104

of Koch-Weeks, 93, 326

of malignant cedema, 69

of mouse septicaemia, 95

of Morax, 94, 326

of smegma, 76, 82

of timothy grass, 77

of trachoma (Muller), 91

paratyphosus (A. and B.), 104, 352

pestis, 95, 295, 335, 352, 355

pneumoniae (Friedlander), 95

prodigiosus, no

proteus, 105

pseudotuberculosis rodentium, 92

psittacosis, 92

pyocyaneus, 109

subtilis, 63

suipestifer, 104

tetani, 72, 356

termo, 339

tuberculosis, 78, 326, 328, 334, 349

typhosus, 100, 131, 196, 349, 352

violaceus, 109

vulgatus, 63

xerosis, 89, 325

zopfii, 92

Bactera, identification of, 41
Balantidium coli, 231
Band's disease, 204
Bed bug, in Kala azar, 230
Bence-Jones albumin, 385
Benedict sugar test, 386
Beriberi, 382
Bienstock group, 92
Bile media, 26, 351
Bile pigments, 391
Bilharziasis, 251

infection in, 339

Binucleata, 217
Black water fever, 382
Blood, coagulation rate, 185

color index of, 188

counting red cells, 174

counting white cells, 176

counting with microscopic field, 177

cultures of, 351

differential count (normal), 194

differential count (in haemacytom-
eter), 178

dried films, 179

fixation of, 181

fresh preparations, 177

making preparations, 171

normal count, 199

occult, 1 86

red cells of, 188

specific gravity of, 186

spectroscopic test, 187

staining of, 1 79

tubercle bacilli in, 352 ^

viscosity of, 185

white cells of, 190
Blood platelets, 195
Blood serum, coagulating apparatus, 10

preparation of, 25
Boas-Oppler bacillus, 354
Booker group, 92

Bordet and Gengou phenomenon, 146
Bordet amd Gengou bacillus, 94
Bothriocephalus, 258
Bottle bacillus, 357
Botulism, 70
Bouillon, glycerine, 21

calcium carbonate, 21

Liebig's extract in, 20

nutrient, 17

standardizing reaction of, 18

sterilization of, 20

sugar, 20

sugar-free, 20
Broth media, 17
Buccal secretions, 330

Calliphora vomitoria, 303

Cammidge reaction, 350, 387

Capsule staining, 37

Carbol-fuchsin stain, 33, 35, 160

Casts in urine, 343

Cellia, 314

Cells, in blood, 188, 190

in cytodiagnosis, 360
Cerebrospinal fluid, 60, 147, 360

puncture for. 360
Cestoda, 245, 253

key to genera, 255
Charcot-Leyden crystals, 333, 350

INDEX

401

Chlorinated lime, 397
Chlorosis, 199
Cholera, 112

carriers in, 114

diagnosis, 115

in water, 132

media for, 28
Cholera red, 21, 116
Chironomidae, 305
Chlamydozoa, 243
Chromatin stains, 40, 183
Chromidia, 217
Chromogens, 109
Chrysomyia macellaria, 303
Chrysops, 299
Chyluria, 267
Cladorchis watsoni, 249
Cladothrix, 117
Classification, animal kingdom, 211

arachnoidea, 281

bacilli, branching, 76

bacilli, gram negative, 91

bacilli, spore bearing, 63

bacteria, 43

cocci, 49

flat worms, 245

fungi, 117

insects, 291

mosquitoes, 291, 313

protozoa, 216

round worms, 263

spirilla, 112
Clonorchis endemicus, 248

sinensis, 248
Coccidiaria, 233
Coccidium (see Eimeria and Isospora),

233

Coley's fluid, in
Colon bacillus, 107

in water, 130
Colonies, isolation of, 43
Color index, 188
Colubrine snakes, 319
Commensalism, 213
Complement, 140

absorption of, 146

deviation of, 145
Conjunctival infections, 325
Conorhinus, 294
Conradi-Drigalski medium, 28
Conradi-brilliant green medium, 28
Corrosive sublimate, 397
Cover-glasses, 2
Cover-glass preparations, 32
Crithidia, 230
Cryptococcus gilchristi, 120

linguae pilosae, 120
Ctenopsylla musculi, 296
26

Culicinae, 315

Culture media, agar, 21

bile media, 26

blood agar, 26

blood serum, 25

bouillon, 17

cholera media, 28

egg media, 23, 25

faeces media, 27

gelatin, 23

gelatin agar (North), 24

Hiss' serum water, 21

litmus milk, 24

peptone solution, 21

potato, 25

protozoal, 29

Russell's double sugar, 29

sterilization of, "5, 16

sugar bouillon, 20

titration of, 18
Cycloleppteron, 314
Cysticercus, 255
Cystitis, 344
Cytodiagnosis, 359
Cytorrhyctes luis, 244

scarlatinae, 244

vaccinae, 244, 364

Dark ground illumination, 4, 224

Davainea madagascariensis, 258

Demodex folliculorum, 284

Deneke's spirillum, 112

Dengue, 382

Dermacentor andersoni, 289

Dermatpbia cyaniventris, 304

Desk-microscopic, n

Dhobies itch, 124

Diazo reaction, 391

Dibothriocephalus latus, 258

Dicroccelium lanceatum, 247

Dieudonne's cholera medium, 28

Differential leukocyte count, 194

Diphtheria, 85

diagnosis of, 88, 331
diphtheria-like bacilli, 89
media for growing, 25
Neisser's stain, 36, 88
toxin of, 87

Diplococcus, crassus, 57

intracellular. meningitidis, 59
lanceolat., 55

Diplognoporus grandis, 259

Diptera, 298

Dipylidium caninum, 215, 228

Disinfecting solution, 12, 394

Disinfectants, 394

Distomiasis, 247

Dorset's egg medium, 26

402

INDEX

Double boiler, n
Dracunculus, 265
Dum dum fever, 229
Dunham's solution, 21
Dysentery, amoebae in, 220, 348

bacilli, 105

bacilli in fasces, 349

Ear affections, 328
Eberth group, 99
Echinococcus cysts, 259
Echinorhynchus gigas, 279
Ehrlich, blood film method, 180

granule staining, 190

tri-acid stain, 182
Eimeria stiedae, 233
Emery's test 147
Ekiri, 107
Endo medium, 27
Endomyces albicans, 120
Endothelial cell in cytodiagnosis, 360
Entamceba, buccalis, 222

coli, 219

histolytica, 220, 348

tetragena, 221
Eosinophiles, 193
Eosinophilia, 196
Escherich group, 99
Eustrongylus gigas, 273, 339
Exudates, 359
Eye-piece (see Ocular), 2
Eye-strain, 4
Eye infections, 325

Faeces, 345

amoebae in, 348

bile in, 348

culturing, 349

diet for examination of, 346

fats in, 347

fermentation test, 348

pancreatic test, 394

plating media, 27

soaps in, 347
Fasciola gigantea, 251

hepatica, 247
Fascioletta ilocana, 250
Fasciolopsis buski, 249
Fat in faeces, 347
Fauces, 330
Favus, 122

Fehling sugar test, 385
Fermentation tubes, 10
Films (blood), 179
Filter pump, 13
Filterable viruses, 365
Filaria, bancrofti, 267, 339, 358

demarquayi, 268

Filaria embryos, key to, 269

loa, 267, 327, 358

medinensis, 265

ozzardi, 269

perstans, 268

philippinensis, 269

powelli, 269

volvulus, 268
Fixation, blood films, 181

tissues, 371
Flagella staining, 38
Flagellata, 222
Flat worms, 245
Fleas, 295

key to, 296

Flugge's droplet infection, 99, 136
Flukes, 245

of blood, 251

of intestines, 249

of liver, 247

of lungs, 250
Focus, microscopical, 3
Foot and mouth disease, 381
Formalin, 395
Friedlander group, 91, 95
Frozen sections, 377
Fungi, Achorion, 122

Ascomycetes, 119

Aspergillus, 123, 327

classification of, 117

Cryptococcus, 120

cultivation of, 125

diagnosis of, 125

Discomyces bovis, 123

Discomyces madurae, 124

Hyphomycetes, 123

Imperfecti, 123

Madurella mycetomi, 124

Malassezia furfur, 124

Microsporoides, 124

Microsporum audouini, 122, 327

Mucor, 118

Penicillium, 123

Rhizopus, 118

Saccharomycetes, 119

Trichophyton, 121

Trichosporum giganteum, 124

Gall stones, 345
Gartner group, 100
Gas production, 47
Gastric contents, 354

chemical examination of, 392
Gastrodiscus hominis, 249
Gelatin, 2, 3

liquefaction of, 46

General paralysis (spinal fluid in), 361
Gentian violet stains, 33

INDEX

403

Giemsa's stain, 183
Glanders, 84, 358
Glassware, cleaning of, 8
Glossina palpalis, 302
Gnathostoma siamense, 264, 358
Gonococcus, 27, 57, 326, 339
Gonorrhoea, 57
Goundou, 382
Grabhamia, 316
Gram method, 33

negative bacteria, 34

positive bacteria, 34

solution, 34

Granular degeneration (red cells), 189
Granules (white cells), 191
Guinea worm, 265

Haemacytometer, 174
Haemadipsa ceylonica, 280
Haematopota, 299
Haematoxylin stain, 184
Haemin crystals, 186
Haemoglobin estimation, 172
Haemoglobinometers, Miescher's, 172

Sahli's, 172

Tallquist, 173
Haemosporidia, 235
Haffkine, cholera vaccine, 115

plague prophylactic, 99
Halzoun, 247
Hanging drop, 9
Hemokonia, 195
Heredity, 214
Herpetomonas, 230
Heterogenesis, 214, 264
Heterophyes heterophyes, 249
Hirudo, medicinalis, 280

nilotica, 280
Hiss' serum- water, 21
Histoplasma, 230
Hodgkin's disease, 203
Hook worms, 274, 350
Hosts, 214

Hydatid disease, 259, 356
Hydrocele agar, 26
Hymenolepis, nana, 257

diminuta, 258
Hyphomycetes, 123
Hypoderma diana, 304

Illumination, dark ground, 4
Immersion objectives, 3
Immune sera, antimicrobic, 139

antitoxic, 139

diphtheria, 87

in diagnosis, 141

preparation, 141

tetanus, 73

Immunity, active, 138

natural, 137

passive, 138
Incubators, body temperature, 12

electrical, 12

petroleum lamp, 12

room temperature, 13
Indican in urine, 391
Indol, test for, 21
Influenza, 92
Infusoria, 231
Inoculation animals (tuberculosis), 48,

77

animals (plague), 99

of media, 47
Insecticides, 397
lodophilia, 185
Insecta, 292
Isospora bigemina, 235
Itch mite, 283, 358
Ixodidae, 285

Japanese river fever, 283, 383
Joints, gonococcus in, 59

Kaiserling solution, 379

Kala azar, 229

Kedani mite, 283, 383

Key to branching, curving bacilli, 76

to cocci, 49

to filarial embryos, 269

to fleas, 296

to Gram-negative bacilli, 91

to spirilla, 112

to spore-bearing bacilli, 63
Kidney diseases, table, 344
Koch's postulates, 47
Kundrats lymphosarcoma, 204

Laboratory desks, n
Lactic-acid bacteria, 108
Lactophenol, 378
Lamblia intestinalis, 231
Lamp, primus, 15
Larvae, fly, 303

mosquito, 309

mounting, 278
Leeches, 280
Leishmania, 229

media for, 30

donovani, 229

infantum, 229

tropica, 229
Leprosy, 82,^326, 358

diagnosis of, 83

in rats, 83
Leptothrix, 117
Leukaemia, 202

404

INDEX

Leukaemia, lymphatic, 203

splenomyelogenous, 202
Leukocytosis, 197
Leukopenia, 196
Levaditi stain, 375
Leydenia gemmipara, 222
Light in microscopical work, 4
Linguatula rhinaria, 289
Liquefaction of gelatine, 46
Litmus, 24
Liver abscess, 198
Loeffler serum, 25
Loemopsylla cheopis, 296
Luetin, 226
Lumbar puncture, 360
Lymphocytosis, 199
Lymphocytes, large, 191

small, 191
Lymphosarcoma, 204

Madura foot, 124
Macrogamete, 237
Magnifying power, 171

of oculars, 2
Malaria, 235

cultivation, 242

diagnosis of, 242

differential tables, 240, 241

life cycle, 235

life history, 235

index, 239

Romanowsky stain in, 183
Mallein, 85

Mallory's amoeba stain, 40
Malta fever, 61
Mansonia, 316
Marchi method, 376
Mast cells, 193
Measles, 381
Megarhininae, 314
Meat poisoning, 70, 104

group of bacteria, 104

toxin of, 105
Malignant pustule, 64
Mechanical stage, i
Media (see culture media), 16
Megaloblast, 189
Melaniferous leukocytes, 199
Meningococcus, 59, 331
Metorchis truncatus, 249
Micrococcus, 53

catarrhalis, 61

cinereus, 50

melitensis, 61, 339

pharyngis siccus, 50

rheumaticus, 53

tetragenus, 54
Microgametocyte 237

Micrometer disk, 2, 169

standardization of, 1 70

screw, 3

Micrometry, 2, 169
Microscope, i
Microscopical sections (see tissue), 373

quick diagnostic method, 373
Milk, bacteriological examination of,
132

B. bulgaricus in, 133

lactic-acid bacteria in, 133

leukocytes in, 134
Mites, 282

Mononuclear leukocytes, 172
Mosquitoes, anatomy of, 307

classification of, 313

dissection of, 311

larvae of, 309

ova of, 309

pupae of, 310
Motility 43, 45

Brownian, 43

current, 43

Moulds (see Fungi), 117
Mounting parasites, 377
Much's granules, 35, 82
Mucidus, 316
Mumps, 381
Mus norvegicus, 296
Musca domestica, 300
Muscidae, 300
Mutualism, 212
Myeloblasts, 195
Myelocytes, 174
Myzomyia, 314
Myzorhynchus, 314

Nasal infections, diphtheria in, 328

leprosy in, 328
Nastin in leprosy, 83
Necator americanus, 276
Negri bodies, 362
Neisser's stain, 36, 331
Nematocera, 298, 305
Nematoda, 263
Neosporidia, 233
Nervous tissue, 376
Nissl method, 376
Nitrogen determination, 388, 389
Nocardia, 125
Noguchi test, 153

media for treponemata, 30
Normal solutions, 380
Nomenclature, in animal parasitology,
213

law of priority in, 213
Normoblasts, 189
North's gelatin agar, 24

INDEX

405

Notes, blank, bacteriology, 165
blood work, 206
parasitology, animal, 321

Novy MacNeal (N.N.N.) medium, 29

Numerical aperture, 3

Nyssorhynchus, 314

Occult blood, 1 86
Ocular infections, 325

animal parasites in, 327

bacilli in, 325

gonococcus in, 326

M. catarrhalis in, 61

pneumococcus in, 326
Objectives, i
Oculars, 2

CEsophagostoma brumpti, 273
(Estridse, 304
Opisthorchis, felineus, 249

noverca, 249

sinensis, 248
Opsonic power, 156

apparatus in, 13

determination of, 157
Ornithodoros, 287
Orthorrhapha, 298
Otitis, 329

Ova in faeces, 253, 261, 277
Oxyuris vermicularis, 279

Pancreatic tests, 350, 394
Pangonia, 299
Panoptic staining, 40
Paragonimus westermani, 250
Parasitism, 242
Parthenogenesis, 214
Pasteurized milk, 134
Pasteurelloses, 95
Pebrine, 233

Pediculoides ventricosus, 284
Pediculus capitis, 292

vestimenti, 292
Pellagra, 67, 382
Penicillium crustaceum, 123
Petri dishes, 41, 42
Phagocytosis, 156
Pleiffer's phenomenon, 115
Pharyngeal secretions, 330
Phenolsulphonephthalein test, 391
Phenylhydrazin test, 328
Phlebotomus, 306
Phthirius pubis, 293
Physaloptera, 273
Piedra, 124
Pinta, 123
Pipettes, bacteriological, 14

capillary bulb, 15
Piroplasmata, 242

Plague, 95

diagnosis of, 99

flea in, 98, 295

pneumonia, 98

prophylaxis, 99
Platinum wire, 12
Pleural fluids (cytodiagnosis), 359
Pneumococcus, 55, 326
Poikilocytes, 189
Poliomyelitis, 381

Polymorphonuclear leukocytes, 193
Porocephalus constrictus, 289
Protista, 217
Protozoa, 216

culture of, 29

discussion of, 217

staining of, 39
Pseudoleukaemia, 203
Psychodidae, 306
Pulex, cheopis, 296

irritans, 296
Pulicidae, 295
Pupipara, 299
Pus, cultures from, 355

tetanus in, 74
Pyretophorus, 314

Rabies, 362

preservation of dog in, 364
Rats, 296

Rat-bite disease, 382
Reaction of media, 18, 45

standardization of, 18
Red blood-cells, counting of, 1 74

fformal, 189

nucleated red cells, 189

polychromatophilia, 189

punctate basophilia, 189
Relapsing fever, 223
Rhabditis pellio, 264
Rheumatism (acute), 381
Rhinosporidium, 243
Rhizoglyphus parasiticus, 383
Rhizopoda, 218
Rhizopus, 119
Rhynchota, 293
Rice cooker, n, 16
Ring- worms, 121

Rocky Mountain spotted fever, 289, 382
Roetheln, 381
Romanowsky stains, 183
RosS thick film, 181
Round worms, 263
Row's haemoglobin medium, 30
Russell's double sugar medium, 29

Sabouraud's medium for moulds, 125
Saccharomyces, anginosae, 119

406

INDEX

Saccharomyces, blanchardi, 120

cerevisiae, 119
Sarcina lutea, 53
Sarcoptes scabiei, 283
Sarcophaga carnaria, 304
Sarcopsylla penetrans, 297
Sarcosporidia, 243
Scarlet fever, 381
Schistosomum hsematobium, 251

japonicum, 252

mansoni, 251
Schizotrypanum, 228
Screw worm, 303

Sections, making and staining, 3 73
Septicaemia, 55

Serum (see immune serum), 141
Sewage, iri water, 127
Shiga's bacillus, 105
Simulidae, 305
Siphonaptera, 295
Siphunculata, 291, 292
Skin infections, 357

itch mite, 358

leprosy in, 358

pus cocci in, 357

sarcopsylla, in, 358
Sleeping sickness, 227, 302
Slides, cleaning, 8

concave, 9
Smallpox, 244, 365
Snakes, 317
Sparganum, mansoni, 262

prolifer, 262
Spectroscope, 187
Spirillum choleras asiaticae, 112

metschnikovi, 112

of Finkler, Prior, 112

tyrogenum, 112
Spirochaeta, 222

duttoni, 223, 287

recurrentis, 223, 293

refringens, 224

vincenti, 224
Spiroschaudinnia, 223
Splenic anaemia, 205
Splenomegaly, 204
Spores, spore-bearing bacilli, 63

staining, 39
Sporotrichosis, 124
Sporotrichum f eurmanni, 1 24
Sporozoa, 233
Sprue, 383
Sputum, 333

albumin test in, 336

amoebae in, 336

antiformin for, 334

centrifugalization for T.B., 334

culturing, 335

Sputum, fixing smears, 333

Paragonimus eggs in, 336

plague pneumonia, 335
Stage, warm, 4
Staining methods, 32
Stains, acid fast, 35

agar jelly, 39

Archibald's, 36

Balch's, 183

capsule, 37

carmine for worms, 278

carbol fuchsin, 33

flagella, 38

for Negri bodies, 363

Giemsa's, 183

Gram's method, 33

haematoxylin, 184, 376

Herman's, 35

Leishman's, 183

Levaditi's, 375

Loffler's methylene blue, 33

Neisser's, 36

Nicolle's, 36

Panoptic, 40

Pappenheim's, 36

Ponder's diphtheria, 37

protozoal, 40

Romanowsky, 36, 183, 375

Smith's formal fuchsin, 35

spore, 39

tri-acid, 182

Van Giesen's, 375

Wright's, 182
Staphylococcus, 50, 54

epidermidis albus, 54

pyogenes albus, 54

pyogenes aureus, 54
Stegomyia, 315
Sterilization, Arnold, 5, 16

autoclave, 5, 16

glass ware, 5

hot air, 5

pathogenic bacteria, 8
Stomach contents, 354, 392

Boas-Oppler bacillus, 354

cancer cells in, 354
Stomoxys, 301
Stool examination, 345
Streptococcus, 49, 50

capsulatus, 56

coli gracilis, 49

fecalis, 51

pyogenes, 52
Streptothrix, 125
Strong, cholera prophylactic, 115

plague vaccine, 99
Strongylidae, 272
Strongyloides stercoralis, 264

Remarks

Found in faeces and sewage -contami-
nated water. Differs from B. typhos.
by marked alkali production.

Blood cultures first week — 'agglutina-
tion afterward.

Nonacid strain, highly toxic.

Acid mannite strain, moderate toxici*

Much like Flexner strain. No ac id
maltose.

Found in summer diarrhoea of childn

Little gas. No fluorescence n. rt
Litmus milk acid in third day.

Much gas. Marked reduction n.
with yellow fluorescence. Litr.
milk alkaline third day. 2.

B. choleras suis, B. icteroides, 1.
Danysz virus and B. paratypL
closely related (Gaertner group

There is also a B. coli anaerogenes which
is like B. coli but does not form gas

Very nearly related to Friedlander's bac-
illus as well as to B. coli. 3.

Differs from B. coli in liquefaction of
gelatin and shows slow production
of gas in lactose.

Three types — Proteus vulgaris rapid
gelatin liq.; P. mirabilis, slow gelatin
liq.; P. zenkeri, no gelatin liq. Spread-
ing growths characteristic. 2.

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