MEMCAL

LUIBMAl^Y

IN HEMORIAM
O.W. JONES, SR.

BOOKS

JOSEPH McFARLAND, M. D.

Pathogenic Bacteria and Protozoa
Octavo of 878 pages, illustrated. Cloth,

$3.50 net. Seventh Edition

Pathology

Octavo of 856 pages, with 437 illustra-
tions. Cloth, $5.00 net. Second Edition

Biology: General and Medical

i2mo of 440 pages, with 160 illustra-
tions. Cloth, $1-75 net.

A TEXT-BOOK

UPON THE

PATHOGENIC BACTERIA
AND PROTOZOA

FOR

STUDENTS OF MEDICINE AND
PHYSICIANS

BY

JOSEPH MCFARLAND, M.D.

Professor of Pathology and Bacteriology in the Medico-Chirurgical College,

Philadelphia; Professor of Pathology in the Woman's Medical College

of Pennsylvania ; Pathologist to the Philadelphia General Hospital

and to the Medico-Chirurgical Hospital, Philadelphia; Director

of the Laboratories of the Henry Phipps Institute ; Fellow

of the College of Physicians .of Philadelphia, etc.

TOtb 293 flllustrations, a number of tbem in Colors

Seventh Edition, Thoroughly Revised

PHILADELPHIA AND LONDON

W. B. SAUNDERS COMPANY
1912

Copyright, 1896. by W. B. Saunders. Reprinted September, 1896. Re-
vised, reprinted, and recopyrighted August, 1898. Reprinted November,
1898. Revised, reprinted, and recopyrighted August, 1900. Reprinted
June, 1901. Revised, entirely reset, reprinted, and recopyrighted May,
1903. Reprinted August, 1904. Revised, reprinted, and recopyrighted
May, 1906. Reprinted August, 1907. and May, 1908. Revised, reprinted,
and recopyrighted August, 1909. Revised, reprinted, and recopyrighted
September, 1912.

Copyright, 1912, by W. B. SAUNDERS COMPANY.

PRINTED IN AMERICA

PRESS OF

W. B. SAUNDERS COMPANY
PHILADELPHIA

TO

MY HONORED AND BELOVED GRANDFATHER

jflfcr, 5acob (Brim

WHOSE PARENTAL LOVE AND LIBERALITY ENABLED ME TO PURSUE
MY MEDICAL EDUCATION

THIS BOOK IS AFFECTIONATELY DEDICATED

PREFACE TO THE SEVENTH EDITION.

To his scientific friends, whose continued appreciation
and patronage have made necessary the preparation of this
seventh edition of the PATHOGENIC BACTERIA, the author
desires to extend his sincere thanks.

In so far as his pages have been found a useful and reli-
able guide, he is elated; in so far as they may have failed,
he feels humiliated, but 'is stimulated to renewed and more
earnest endeavors on their behalf.

The flight of time has brought with it many changes, but
perhaps in no department of learning have they come in
greater number or with more startling rapidity than in
Microbiology.

When, some eighteen years ago, the author was appointed
to give the first systematic course of lectures upon Bacteri-
ology, in the Medical Department of the University of Penn-
sylvania, there were few text-books suitable for the use of
students, and the preparation of the "Pathogenic Bacteria"
seemed to be a justified, though a doubtful venture. To-day
our shelves groan beneath the weight of many excellent vol-
umes.

When the " Pathogenic Bacteria" appeared, all of the
existing books were general in character. The title adopted
by the author seemed to be of doubtful expediency, lest it
should limit the success of the work by contracting the
sphere of its usefulness. But it was fortunate in meeting
with a cordial reception, and, in spite of its title, soon
came to be looked upon and used as a general text-book.
All of the early revisions were directed toward increasing
its general usefulness and making it serviceable in all the
fields in which Bacteriology was taught or practised.

But now, times have changed, and it can no longer be said
that anything short of a many volume encyclopedia can be
regarded as an adequate "general" work upon Bacteriology.
There are now excellent books devoted to microbiology; to

7

8 Preface

the systematic classification and identification of bacteria;
to the laboratory methods used in studying them; to the
chemistry and toxicology of their metabolic products ; to the
problems of infection and immunity; to the individual the-
ories of immunity; to the blood-serum therapy; to bacterio-
vaccination and the opsonic index; to complement-fixation;
to the bacteriology of water; to the bacteriology of foods;
to the bacteriology of the dairy ; to the bacteriology of sew-
age and the methods of its disposal ; to the relation of bacte-
riology to agriculture ; to the relation of bacteriology to the
public health; to veterinary bacteriology, and so on and on,
almost without limit. A dozen great international journals
in English, German, and French are devoted to the subject,
and weigh down our shelves with hundreds of ponderous
volumes of innumerable monographs and experimental re-
searches, and one becomes bewildered in his efforts to
"keep up" with the ever-expanding information.

In the meantime the "Pathogenic Bacteria," diverted
from one after another of the fields it had pre-empted, but
for which it was not definitely intended, found and held its
own place as a medical book.

As it became more and more clear that the original inten-
tion of the author was to be realized and the destiny of his
book was to be purely medical, it became equally clear that
the present revision must meet the requirements of that field
as completely and as perfectly as possible.

In the past the "Pathogenic Bacteria" has been devoted
to the consideration of bacteria only When the author was
criticized because it had nothing to say about the higher
fungi and the Protozoa, he pointed out that the title de-
clared the contents of the work, and a change would be
inconsistent with the original purpose.

There was always the feeling that the development of
Protozoology would soon make it necessary for the student
to have a text-book upon the Pathogenic Protozoa, and that
it would then become necessary to divorce the two subjects
again. As, however, knowledge of the protozoa engaged in
human pathology has not so expanded as to make this either
necessary or desirable, and as the future purpose of the
"Pathogenic Bacteria" is to meet the needs of students of
human medicine and pathology, it has become both desir-
able and practicable to change the original plan, depart from
the unwholesome consistency, and, without any important

Preface 9

change in the title, offer to old friends and future patrons a
work that shall endeavor to meet all modern requirements :

By describing all the pathogenic micro-organisms of
importance in human medicine, whether they be bac-
teria or protozoa;

By teaching the laboratory technic with reference to
the needs of medical students and practitioners;

By bringing each micro-organism under consideration
into a historic, geographic, biologic, and pathologic
setting ;

By dwelling upon the anatomic and physiologic disturb-
ances referable to the various micro-organisms;

By describing the lesions occasioned by the different
micro-organisms; and,

By explaining such methods of diagnosis and treatment
as grow out of the knowledge of microbiology in gen-
eral and of the micro-organisms in particular.

JOSEPH McFARLAND.
PHILADELPHIA, September, 1912.

CONTENTS.

PART L— GENERAL.

PAGE

HISTORICAL, INTRODUCTION 17

CHAPTER I.
STRUCTURE AND CLASSIFICATION OF THE MICRO-ORGANISMS 29

CHAPTER II.
BIOLOGY OF MICRO-ORGANISMS 58

CHAPTER III.
INFECTION 78

CHAPTER IV.
IMMUNITY 105

CHAPTER V.
METHODS OF OBSERVING MICRO-ORGANISMS 172

CHAPTER VI.
STERILIZATION AND DISINFECTION 201

CHAPTER VII.
CULTURE-MEDIA AND THE CULTIVATION OF MICRO-ORGANISMS... 224

CHAPTER VIII.
CULTURES AND THEIR STUDY 242

CHAPTER IX.

THE CULTIVATION OF ANAEROBIC MICRO-ORGANISMS 260

11

12 Contents

CHAPTER X. PAGE

EXPERIMENTATION UPON ANIMALS 268

CHAPTER XI.
THE DETERMINATION OF BACTERIA 278

CHAPTER XII.
BACTERIOLOGY OF THE AIR 283

CHAPTER XIII.
BACTERIOLOGY OF WATER 287

CHAPTER XIV.
BACTERIOLOGY OF THE SOIL 294

CHAPTER XV.
BACTERIOLOGY OF FOODS 296

CHAPTER XVI.
DETERMINATION OF THE THERMAL DEATH-POINT OF BACTERIA. . 300

CHAPTER XVII.

DETERMINATION OF THE VALUE OF ANTISEPTICS, GERMICIDES,

AND DISINFECTANTS 302

CHAPTER XVIII.

THE PHAGOCYTIC POWER OF THE BLOOD AND THE OPSONIC

INDEX 307

CHAPTER XIX.
THE WASSERMANN REACTION FOR THE DIAGNOSIS OF SYPHILIS. . . 318

PART IL— THE INFECTIOUS DISEASES AND
THE SPECIFIC MICRO-ORGANISMS.

CHAPTER I.
SUPPURATION 339

Contents 13

CHAPTER II. PAGE

MALIGNANT EDEMA 374

CHAPTER III.
TETANUS 385

CHAPTER IV.
ANTHRAX 4°°

CHAPTER V.
HYDROPHOBIA, LYSSA, OR RABIES 412

CHAPTER VI.
CEREBROSPINAL MENINGITIS 423

CHAPTER VII.
GONORRHEA 43 1

CHAPTER VIII.
CATARRHAL INFLAMMATION 439

CHAPTER IX.
CHANCROID 442

CHAPTER X.
ACUTE CONTAGIOUS CONJUNCTIVITIS 46

CHAPTER XI.
DIPHTHERIA 45 1

CHAPTER XII.
VINCENT'S ANGINA 478

CHAPTER XIII.
THRUSH 484

CHAPTER XIV.
WHOOPING-COUGH.. . 488

CHAPTER XV.
PNEUMONIA 492

14 Contents

CHAPTER XVI. PAGE

INFLUENZA 514

CHAPTER XVII.
MALTA OR MEDITERRANEAN FEVER 520

CHAPTER XVIII.
MALARIA 524

CHAPTER XIX.
RELAPSING FEVER 546

CHAPTER XX.
SLEEPING SICKNESS 554

CHAPTER XXI.
KALA-AZAR (BLACK FEVER) 566

CHAPTER XXII.

YELLOW FEVER 576

CHAPTER XXIII.
PLAGUE 581

CHAPTER XXIV.
ASIATIC CHOLERA 604

CHAPTER XXV.
TYPHOID FEVER 632

CHAPTER XXVI.
DYSENTERY 687

CHAPTER XXVII.
TUBERCULOSIS '. 710

CHAPTER XXVIII.
LEPROSY 762

CHAPTER XXIX.

GLANDERS 775

Contents 15

CHAPTER XXX. PAGE

RHINOSCLEROMA 785

CHAPTER XXXI.

SYPHILIS 787

CHAPTER XXXII.
FRAMBESIA TROPICA (YAWS) 800

CHAPTER XXXIII.

ACTINOMYCOSIS 803

CHAPTER XXXIV.
MYCETOMA, OR MADURA-FOOT 812

CHAPTER XXXV.
BLASTOMYCOSIS 818

CHAPTER XXXVI.

RINGWORM 824

CHAPTER XXXVII.

FAVUS.. . 828

BIBLIOGRAPHIC INDEX 833

INDEX. . 849

PART I. GENERAL

HISTORICAL INTRODUCTION.

BIOLOGY, chemistry, medicine, and surgery, in their evolu-
tion, contributed to a new branch of knowledge, Bacteri-
ology, whose subsequent development has become of in-
estimable importance to each. Indeed, bacteriology illus-
trates the old adage, " The child is father of the man,"
for while it is in part the offspring of the medicine of the
past, it has established itself as the dictator of the medicine
of the present and future, especially so far as concerns the
infectious diseases.

THE EVOLUTION OF BACTERIOLOGY.

I. BIOLOGIC CONTRIBUTIONS j THE DOCTRINE OF SPONTANEOUS
GENERATION.

Among the early Greeks we find that Anaximander
(43d Olympiad, 610 B. C.) of Miletus held the theory that
animals were formed from moisture. Empedocles of
Agrigentum (450 B. C.) attributed to spontaneous genera-
tion all the living beings which he found peopling the
earth. Aristotle (384 B. C.) is not so general in his view
of the subject, but asserts that "sometimes animals are
formed in putrefying soil, sometimes in plants, and some-
times in the fluids of other animals."

Three centuries later, in his disquisition upon the Pytha-
gorean philosophy, we find Ovid defending the same doc-
trine of spontaneous generation, while in the Georgics Virgil
gives directions for the artificial production of bees.

The doctrine of spontaneous generation of life was not
only current among the ancients, but we find it persisting
through the Middle Ages, and descending to our own genera-
tion. In 1542, in his treatise called " De Subtilitate," we

2 I7

1 8 introduction

find Cardan asserting that water engenders fishes, and that
many animals spring from fermentation. Van Helmont
gives special instructions for the artificial production of
mice, and Kircher in his " Mundus Subterraneus " (chapter
"De Panspermia Rerum") describes and actually figures
certain animals which were produced under his own eyes
by the transforming influence of water on fragments of
stems from different plants.*

About 1671, Francesco Redi seems to have been the first
to doubt that the maggots familiar in putrid meat arose de
novo: "Watching meat in its passage from freshness to de-
cay, prior to the appearance of maggots, he invariably ob-
served flies buzzing around the meat and frequently alight-
ing on it. The maggots, he thought, might be the half-
developed progeny of these flies. Placing fresh meat in a jar
covered with paper, he found that although the meat putre-
fied in the ordinary way, it never bred maggots, while meat
in open jars soon swarmed with them. For the paper he
substituted fine wire gauze, through which the odor of the
meat could rise. Over it the flies buzzed, and on it they
laid their eggs, but the meshes being too small to permit
the eggs to fall through, no maggots generated in the meat ;
they were, on the contrary, hatched on the gauze. By a
series of such experiments Redi destroyed the belief in the
spontaneous generation of maggots in meat, and with it
many related beliefs."

In 1683 Anthony van Leeuwenhoek, justly called the
"Father of microscopy," demonstrated the continuity of
arteries and veins through intervening capillaries, thus
affording ocular proof of Harvey's discovery of the circula-
tion of the blood; discovered bacteria, seeing them first in
saliva, discovered the rotifers, and first saw the little glob-
ules in yeast which Latour and Schwann subsequently
proved to be plants.

Leeuwenhoek involuntarily reopened the old contro-
versy about spontaneous generation by bringing forward a
new world, peopled by creatures of such extreme minuteness
as to suggest not only a close relationship to the ultimate
molecules of matter, but an easy transition from them.

In succeeding years the development of the compound
microscope showed that putrescent infusions, both animal
and vegetable, teemed with minute living organisms.
* See Tyndall: " Floating Matter in the Air."

The History of the Subject 19

Abbe" Lazzaro Spallanzani (1777) filled flasks with organic
infusions, sealed their necks, and, after subjecting their con-
tents to the temperature of boiling water, placed them under
conditions favorable for the development of life, without,
however, being able to produce it. Spallanzani's critics,
however, objected to his experiment on the ground that air
is essential to life, and that in his flasks the air was excluded
by the hermetically sealed necks.

Schulze (1836) set this objection aside by filling a flask
only half full of distilled water, to which animal and vege-
table matters were added, boiling the contents to destroy
the vitality of any organisms which might already exist in
them, then sucking daily into the flask a certain amount of
air which was passed through a series of bulbs containing
concentrated sulphuric acid, in which it was supposed that
whatever germs of life the air might contain would be de-
stroyed. This flask was kept from May to August ; air was
passed through it daily, yet without the development of any
infusorial life.

It must have been a remarkably germ-free atmosphere in
which Schulze worked, for, as was shown by those who re-
peated his experiment, under the conditions that he regarded
as certainly excluding all life, germs can readily enter with
the air.

In 1838 Ehrenberg devised a system of classifying the
minute forms of life, a part of which, at least, is still recog-
nized at the present time.

The term "infusorial life" having been used, it is well to
remark that during all the early part of their recognized
existence the bacteria were regarded as animal organisms
and classed among the infusoria.

Tyndall, stimulated by the work of Pasteur, conclusive-
ly proved that the micro-organismal germs were in the
dust suspended in the atmosphere, and not ubiquitous in
distribution. His experiments were very ingenious and
are of much interest. First preparing light wooden cham-
bers, with a large glass window in the front and a smaller
window in each side, he arranged a series of test-tubes in
the bottom, half in and half out of the chamber, and a
pipet, working through a rubber diaphragm, in the top, so
that when desired the tubes, one by one, could be filled
through it. Such chambers were allowed to stand until all
the contained dust had settled, and then submitted to an

20 Introduction

optical test to determine the purity of the contained atmos-
phere by passing a powerful ray of light through the side
windows. When viewed through the front, this ray was vis-
ible only so long as there were particles suspended in the at-
mosphere to reflect it. When the dust had completely
settled and the light ray had become invisible because of
the purity of the contained atmosphere, the tubes were
cautiously filled with urine, beef-broth, and a variety of
animal and vegetable broths, great care being taken that
in the manipulation the pipet should not disturb the dust.
Their contents were then boiled by submergence in a pan of
hot brine placed beneath the chamber, in contact with the
projecting ends of the tubes, and subsequently allowed to
remain undisturbed for days, weeks, or months. In nearly
every case life failed to develop in the infusions after the
purity of the atmosphere was established.

II. CHEMIC CONTRIBUTIONS? FERMENTATION AND PUTREFACTION.

As in the world of biology the generation of life was an
all-absorbing problem, so in the world of chemistry the
phenomena of fermentation and putrefaction were inex-
plicable so long as the nature of the ferments was not
understood.

In the year 1837 Latour and Schwann succeeded in
demonstrating that the minute oval bodies which had been
observed in yeast since the time of Leeuwenhoek were
living organisms — vegetable forms — capable of growth.

So long as yeast was looked upon as an inert substance
it was impossible to understand how it could impart fer-
mentation to other substances; but when it was shown
by Latour that the essential element of yeast was a growing
plant, the phenomenon became a perfectly natural conse-
quence of life. Not only the alcoholic, but also the acetic,
lactic, and butyric fermentations have been shown to re-
sult from the energy of low forms of vegetable life, chiefly
bacterial in nature. Prejudice, however, prevented many
chemists from accepting this view of the subject, and
Liebig strenuously adhered to his theory that fermenta-
tion was the result of the internal molecular movements
which a body in the course of decomposition communicates
to other matter whose elements are connected by a very
feeble affinity.

Pasteur was the first to prove that fermentation is an

The History of the Subject 21

ordinary chemic transformation of certain substances, tak-
ing place as the result of the action of living cells, and
that the capacity to produce it resides in all animal and
vegetable cells, though in varying degree.

In 1862 he published a paper "On the Organized Cor-
puscles Existing in the Atmosphere," in which he showed
that many of the floating particles collected from the atmos-
phere of his laboratory were organized bodies. If these
were planted in sterile infusions, abundant crops of micro-
organisms were obtained. By the use of more refined
methods he repeated the experiments of others, and showed
clearly that ' ' the cause which communicated life to his in-
fusions came from the air, but was not evenly distributed
through it."

Three years later he showed that the organized cor-
puscles which he had found in the air were the spores or
seeds of minute plants, and that many of them possessed
the property of withstanding the temperature of boiling
water — a property which explained the peculiar results
of many previous experimenters, who failed to prevent
the development of life in boiled liquids inclosed in her-
metically sealed flasks.

Chevreul and Pasteur, by having proved that animal
solids do not putrefy or decompose if kept free from the
access of germs, suggested to surgeons that putrefaction in
wounds is due rather to the entrance of something from
without than to changes within. The deadly nature of the
discharges from putrescent wounds had been shown in a
rough manner by Gaspard as early as 1822 by injecting
some of the material into the veins of animals.

III. MEDICAL AND SURGICAL CONTRIBUTIONS, THE STUDY OF THE
INFECTIOUS DISEASES.

Probably the first writing in which a direct relationship
between micro-organisms and disease is suggested is by
Varro, who says: "It is also to be noticed, if there be any
marshy places, that certain minute animals breed [there]
which are invisible to the eye, and yet, getting into the
system through mouth and nostrils, cause serious disorders
(diseases which are difficult to treat)."

Surgical methods of treatment depending for their suc-
cess upon exclusion of the air, and of course, incidentally
if unknowingly, exclusion of bacteria, seem to have been

22 Introduction

practised quite early. Theodoric, of Bologne, about 1260
taught that the action of the air upon wounds induced a
pathologic condition predisposing to suppuration. He also
treated wounds with hot wine fomentations. The wine
was feebly antiseptic, kept the surface free from bacteria,
and the treatment was, in consequence, a modification of
what in later centuries formed antiseptic surgery.

Henri de Monde ville in 1306 went even further than
Theodoric, whom he followed, and taught the necessity
of bringing the edges of a wound together, covered it with
an exclusive plaster compounded of turpentine, resin, and
wax, and then applied the hot wine fomentation.

In 1546 Geronimo Fracastorius published at Venice a
work " De contagione et contagiosis morbis et curatione," in
which he divided infectious diseases into —

1. Those infecting by immediate contact (true contagions).

2. Those infecting through intermediate agents, such as
fomites.

3. Those infecting at a distance or through the air; he
mentions as belonging to this class phthisis, the pestilential
fevers, and a certain kind of ophthalmia (conjunctivitis).

" In his account of the true nature of disease germs, or
seminaria contagionum, ... he describes them as particles
too small to be apprehended by our senses, but as capable in
appropriate media of reproduction, and in this way of in-
fecting surrounding tissues.

" These pathogenic units Fracastorius supposed to be of
the nature of colloidal systems, for if they were not viscous
or glutinous by nature they could not be transmitted by fo-
mites. Germs transmitting disease at a distance must be able
to live in the air a certain length of time, and this condition
lie holds is possible only when the germs are gelatinous or
colloidal systems, for only hard, inert, discrete particles
could endure longer.

" Fracastorius conceived that the germs became pathogenic
through the action of animal heat, and in order to produce
disease it is not necessary that they should undergo dissolu-
tion, but only metabolic change."*

In 1671 Kircher wrote a book in which he expressed the

opinion that puerperal fever, purpura, measles, and various

other fevers were the result of a putrefaction caused by

worms or animalcules. His opinions were thought by his

*"Brit. Med. Jour.," May 7, 1910, p. 1122.

The History of the Subject 23

contemporaries to be founded upon too little evidence, and
were not received.

Plencig, of Vienna, became convinced that there was an
undoubted connection between the microscopic animal-
cules exhibited by the microscope and the origin of dis-
ease, and advanced this opinion as early as 1762. Unfor-
tunately, his opinions seem not to have been accepted by
others, and were soon forgotten.

In 1704 John Colbach described "a new and secret
method of treating wounds by which healing took place
quickly, without inflammation or suppuration."

Boehm succeeded in 1838 in demonstrating the occur-
rence of yeast plants in the stools of cholera, and con-
jectured that the process of fermentation was concerned
in the causation of that disease.

In 1840 Henle considered all the evidence that had
been collected, and concluded that the cause of the infec-
tious diseases was to be sought for in minute living organ-
isms or fungi. He may be looked upon as the real pro-
pounder of the GERM THEORY OF DISEASE, for he not only
collected facts and expressed opinions, but also investi-
gated the subject ably. The requirements which he formu-
lated in order that the theory might be proved were so
severe that he was never able to attain to them with the
crude methods at his disposal. They were so ably elabo-
rated, however, that in after years they were again postulated
by Koch, and it is only by strict conformity with them
that the definite relationship between bacteria and disease
has been determined.

Briefly summarized, these requirements are as follows:

1. A specific micro-organism must be constantly asso-
ciated with the disease.

2. It must be isolated and studied apart from the disease.

3. When introduced into healthy animals it must pro-
duce the disease, and in the animal in which the disease
has been experimentally ' produced the organism must be
found under the original conditions.

In 1843 Dr. Oliver Wendell Holmes wrote a paper upon
the '' Contagiousness of Puerperal Fever."

In 1847 Semmelweiss, of Vienna, struck by the similarity
between fatal wound infection with pyemia and puerperal
fever, cast aside the popular theory that the latter affection
was caused by the absorption into the blood of milk from

24 Introduction

the breasts, and announced his belief that the disease
depended upon poisons carried by the ringers of physicians
and students from the dissecting room to the woman in
child-bed, and recommended washing the hands of the
accoucheur with chlorin or chlorid of lime, in addition to
the use of soap and water. He was laughed to scorn for
his pains.

In 1849 J. K. Mitchell, in a brief work upon the "Crypto-
gamous Origin of Malarious and Epidemic Fevers," fore-
shadowed the germ theory of disease by collecting a large
amount of evidence to show that malarial fevers were due
to infection by fungi.

Pollender (1849) and Davaine (1850) succeeded in
demonstrating the presence of the anthrax bacillus in
the blood of animals suffering from and dead of that dis-
ease. Several years later (1863) Davaine, having made
numerous inoculation experiments, demonstrated that this
bacillus was the materies morbi of the disease. The bacillus
of anthrax was probably the first bacterium shown to be
specific for a disease. Being a very large bacillus and a
strongly vegetative organism, its growth was easily observed,
while the disease was one readily communicated to animals.

Klebs, who was one of the pioneers of the germ theory,
published, in 1872, a work upon septicemia and pyemia,
in which he expressed himself convinced that the causes
of these diseases must come from without the body. Bill-
roth, however, strongly opposed such an idea, asserting
that fungi had no especial importance either in the processes
of disease or in those of decomposition, but that, existing
everywhere in the air, they rapidly developed in the body
as soon as through putrefaction a " Faulnisszymoid " (putre-
factive ferment), or through inflammation a "Phlogisti-
schezymoid" (inflammatory ferment), supplying the neces-
sary feeding-grounds, was produced.

In 1873 Obermeier observed that actively motile, flexible
spiral organisms were present in large numbers in the
blood of patients in the febrile stages of relapsing fever. .

In 1875 the number of scientific men who had entirely
abandoned the doctrine of spontaneous generation and
embraced the germ theory of disease was small, and most
of those who accepted it were experimenters. A great
majority of medical men either believed, like Billroth, that
the presence of fungi where decomposition was in progress

The History of the Subject 25

was an accidental result of their universal distribution, or,
being still more conservative, adhered to the old notion
that the bacteria, whose presence in putrescent wounds as
well as in artificially prepared media was unquestionable,
were spontaneously generated there.

Before many of the important bacteria had been dis-
covered, and while ideas upon the relation of micro-organ-
isms to disease were most crude, some practical measures
were suggested that produced greater agitation and incited
more observation and experimentation than anything sug-
gested in surgery since the introduction of anesthetics —
namely, antisepsis.

"It is to one of old Scotia's sons, Sir Joseph Lister,
that the everlasting gratitude of the world is due for the
knowledge we possess in regard to the relation existing
between micro-organisms and inflammation and suppura-
tion, and the power to render wounds aseptic through
the action of germicidal substances." *

Lister, convinced that inflammation and suppuration
were due to the entrance of germs from the air, instru-
ments, fingers, etc., into wounds, suggested the employ-
ment of carbolic acid for the purpose of keeping sterile
the hands of the operator, the skin of the patient, the
surface of the wound, and the instruments used. He
finally concluded every operation by a protective dressing
to exclude the entrance of germs at a subsequent period.

Listerism, or "antisepsis," originated in 1875, and when
Koch published his famous work on the "Wundinfections-
krankheiten" (traumatic infectious diseases), in 1878, it
spread slowly at first, but surely in the end, to all depart-
ments of surgery and obstetrics.

From time to time, as the need for them was realized, the
genius of investigators provided new devices which materially
aided in their work, and have made possible many discoveries
that must otherwise have failed. Among them may be men-
tioned the improvement of the compound microscope, the use
of sterilized culture fluids by Pasteur, the introduction of
solid culture media and the isolation methods by Koch,
the use of the cotton plug by Schroeder and van Dusch,
and the introduction of the anilin dyes by Weigert.

It is interesting to note that after the discovery of the
anthrax bacillus by Pollender and Davaine, in 1849, there
*Agnew's "Surgery," vol. i, chap. 11.

26 Introduction

was a period of nearly twenty-five years during which no
important pathogenic organisms were discovered, but during
which technical methods were being elaborated, making
possible a rapid succession of subsequent important dis-
coveries.

Thus, in 1873, Obermeier discovered Spirillum ober-
meieri of relapsing fever.

In 1879 Hansen announced the discovery of bacilli in
the cells of leprous nodules, and Neisser discovered the
gonococcus.

In 1880 the bacillus of typhoid fever was observed by
Hberth and independently by Koch, Pasteur published his
work upon "Chicken-cholera," and Sternberg described the
pneumococcus, calling it Micrococcus pasteuri.

In 1882 Koch made himself immortal by his discovery
of and work upon the tubercle bacillus, and in the same year
Pasteur published a work upon "Rouget du Pore," and
Loffler and Shiitz discovered the bacillus of glanders.

In 1884 Koch reported the discovery of the "comma
bacillus," the cause of cholera, and in the same year Loffler
isolated the diphtheria bacillus, and Nicolaier the tetanus
bacillus.

In 1892 Canon and Pfeiffer discovered the bacillus of
influenza.

In 1894 Yersin and Kitasato independently isolated the
bacillus causing the bubonic plague, then prevalent at
Hong-Kong.

A new era in bacteriology, and probably the most tri-
umphant achievement of scientific medicine, was inau-
gurated in 1890, when Behring discovered the principles of
the " blood-serum therapy." Since that time investigations
have been largely along the lines of immunity, immunization,
and the therapeutic serums, the names of Behring, Kitasato,
Wernicke, Roux, Khrlich, Metschnikoff, Bordet, Wasser-
mann, Shiga, Madsen, and Arrhenius taking front rank.

The discovery of the Treponema pallidum, the specific
organism of syphilis, was made in 1905 by Schaudinn and
Hoffmann, long after clinical study of the disease had antici-
pated it to such an extent that when the discovery was finally
made it was unnecessary to modify our ideas of the disease in
any essential.

In the same year, 1905, Castellani discovered the Trepo-
nema pertenue, the cause of frambesia or yaws.

The History of the Subject 27

In 1911 Noguchi succeeded in obtaining pure cultures of
the treponema.

During the time that so much investigation of the prob-
lems of infection was in progress the discoveries were by no
means restricted to the- bacteria and their products, as the
reader might infer from the perusal of a chapter whose pur-
pose is to explain the development of the department of
science now known as Bacteriology. Other organisms of
different — i. e., animal — nature were also found in large
numbers.

In 1875 Losch discovered the Amoeba coli; in 1878 Rivolta
described the Coccidium cuniculi of the rabbit; in 1879
Lewis first saw Trypanosoma lewisi in the blood of the rat;
in 1 88 1 Laveran discovered Plasmodium malariae in the blood
of cases of human paludism; in 1885 Blanchard described
the sarcocystis in muscle-fibers; in 1893 Councilman and
Lafleur studied Amoeba dysenteriae in the stools and tissues
of human dysentery; in 1903 Leishman and Donovan found
the little body Leishmania donovani in the splenic juice of
cases of kala-azar, and in 1903 Dutton and Forde, working
independently, observed trypanosomes — the Trypanosoma
gambienseof African lethargy — in the blood of human beings.

Each of these was followed by new discoveries and addi-
tions in its own sphere, and the systematic consideration of
these protozoan organisms can only be undertaken in a text-
book devoted to animal parasites.

Many of these organisms, however, can be cultivated and
studied by the methods of bacteriology, and, indeed, it is the
progress of bacteriology that has made our ever-increasing
knowledge of them possible.

That the specific micro-organisms of many of the infectious
diseases remained undiscovered was a source of perplexity
so long as it was supposed that all living things must be visible
to the eye aided by the microscope. To-day, thanks to the
invention of the ultramicroscope, that shows the existence
of things too small to be defined, and still more to the adap-
tation of the method of filtration to the study of the diseases in
question, we realize that the " viruses " of disease may be vis-
ible or invisible and that they have no limitations of size.
Just as bacteria readily find their way through paper filters,
so the invisible and hence undescribed viruses — i. e., micro-
organisms— of rabies, poliomyelitis, yellow fever, pleuro-
pneumonia of cattle, foot-and-mouth disease, rinderpest,

28 Introduction

hog-cholera, African horse-fever, infectious anemia or swamp
sickness of horses, fowl plague, small-pox, cow-pox, sheep-
pox, horse-pox, swine-pox, and goat-pox are at some or all
stages able to pass through the Berkefeld or diatomaceous
earth filters, and some of them through the much less porous
unglazed porcelain or Chamberland filters. Thus there is
opened a new world that is ultramicroscopic, but still teems
with invisible living organisms.

Such organisms, interesting as they must be to the biolo-
gist and pathologist, cannot yet be known or described, and,
therefore, seem to be, for the most part, beyond the scope of
the present writing.

CHAPTER I.

STRUCTURE AND CLASSIFICATION OF THE
MICRO-ORGANISMS.

BACTERIA.

WHEN Leeuwenhoek with his improved microscope dis-
covered the new world of micro-organisms, he supposed them,
on account of the active movements they manifested, to be
small animals, and described them as animalculae. The
early systematic writers, Ehrenberg and Dujardin, fell into
the same error, and it was many years before biologists had
arrived at even approximate accuracy in arranging them.
Indeed, for a long time a great number baffled systematic
writers, and no less an authority than Haeckel, in 1878, sug-
gested that they form a group by themselves to be known as
Protista. Such a grouping, however, was unsatisfactory
alike to botanists and zoologists, and, therefore, was used by
few.

It was evident that structure could not be looked upon as
a satisfactory differential character, for between the protozoa,
or most simple animals, and the protophyta, or most simple
plants, the structural differences were too minute to prevent
overlapping. Motion and locomotion had to be abandoned,
since it was common to both groups. Reproduction was
likewise an unreliable means when taken by itself, for much
the same means of multiplication were found to obtain in
both groups. One great physiologic and metabolic differ-
ence was, however, noted: plants possess the power of
nourishing themselves upon purely inorganic compounds,
while animals are unable to do so and cannot live except
upon complex molecular combinations synthesized by the
plants. In this metabolic difference we find the present
criterion for the separation of the living organisms into the
two main groups! But this does not dispose of all of the
difficulties, for there are certain small groups to which it

29

30 Structure and Classification of Micro-organisms

does not apply. Thus, for example, the fungi which, when
judged by other criteria, are undoubted plants, lack the power
of inorganic synthesis, and so resemble animals.

Fortunately, the question is a purely academic one. Though
seemingly at first sight a most fundamental one, it is, in
reality, of trifling importance, for after a limited experience
the student unhesitatingly assigns most of the known or-
ganisms to one or the other groups, and that occasional mis-
takes may be made, and organisms, like the spirochaeta,
appear sometimes in the group of plants among the bacteria,
and in other writings among the protozoa, is a matter of small
consequence so long as the knowledge of the organisms them-
selves is in no particular diminished by the method of classi-
fication.

In discussing the matter Delage says, ' ' The question is not
so important as it appears. From one point of view and on
purely theoretic grounds it does not exist, while from an-
other standpoint it is insoluble. If one be asked to divide
living things into two distinct groups, of which one contains
only animals and the other only plants, the question is mean-
ingless, for plants and animals are concepts which have no
objective reality, and in nature they are only individuals.
If in considering those forms which we regard as true ani-
mals and plants we look for their phylogenetic history and
decide to place all of their allies in one or the other group,
we are sure to reach no result; such attempts have always
been fruitless."

" Huxley pointed out as early as 1876 the extremely close
relationship between the lowest algae and some of the flagel-
lates, and it is the general opinion that no one feature sep-
arates the lowest plants from the lowest animals, and the
difficulty — in many cases the impossibility — of distinguishing
between them is clearly recognized.

' The point of view which demands a strict separation of
animals and plants has, however, little utility save, perhaps,
to determine the limits of a text-book or a monograph."*

The relative position of the pathogenic vegetable micro-
organisms to the other vegetable organisms can be deter-
mined by reference to the following table. The wide sepa-
ration of the bacteria in Group II. and all of the others,
which appear in Group X., should be noted.

* Calkin's, "The Protozoa," p. 23.

Bacteria 31

TABLE I.

THE PLANT KINGDOM.

'"d These primary divisions, like the cor-

3 If responding primary division of animals

.£5 ^ into vertebrata and invertebrata, are

g-2 now falling into disuse

s.
«fr

IF

!]

X

W W £ W3QON03 Jf

iv ii iirfiii Qg,f

~ T o

!

06 E-d-g-FE:

"Ilfp

The various genera to which the pathogenic fungi belong
are by no means closely related to one another, as can at
once be seen by the following amplification of group X.
Eumycetes :

32 Structure and Classification of Micro-organisms

TABLE II.

X. Eumycetes (ev good, MW"?TOS fungus). The true fungi: plants without chlorophyl.
Class i. Phycomycetes (<t>vKos seaweed), alga-like fungi.
Order i. Zygomycetes.

Sub-order — Mucorineae.
Family — Mucoraceae.
Genus — Mucor.
Order 2. Oomycetes.

Class 2. Hemiascomycetes.
Order i. Hemiascales.

Family — Saccharomycetaceae.
Genus — Saccharomyces .
" — Blastomyces (?).

Class 3. Euascomycetes.

Order i. Euascales (contains 45 families).
Family — Aspergilloceae.
Genus— Aspergillus.
" — Penicillium.

Fungi imperfecti.
This is a large supplementary
group, of imperfectly known
fungi not included in the
tabulation.
In it we find Oidium.

Class 4. Laboulbeniomycetes.
Order i. Laboulbeniales.

Class 5. Basidiomycetes.
Sub-class — Hemibasidii.

Order i. Hemibasidiales.

Family — Ustilaginaceae (smuts).
Sub-class — Eubasidii.

Order i. Protobasidiomycetes.

Family— Uredineineae (rusts).
Order 2. Autobasidiomycetes (mushrooms, toad-stools, etc.).

No entirely satisfactory grouping of the bacteria themselves
has yet been achieved, the best characters to be used as
the basis of classification being undecided. The best system
for their provisional arrangement is probably that of Migula,*
or the modification of it suggested by F. D. Chester, f in
which the morphology, sporulation, and appendages of the
bacteria all enter as important features.

CLASSIFICATION OF THE BACTERIA.
I. ORDER: EUBACTERIA (True Bacteria).

A. SUB-ORDER: Haplobacteria (Lower Bacteria).
I. Family COCCACE;E. Cells globular, becoming slightly elongate
before division. Division in one, two, or three directions of
space. Formation of endospores very rare.
(A) Without flagella.

1. Streptococcus. Division in one direction of space, producing

chains like strings of beads.

2. Micrococcus. Division in two directions of space, so that

tetrads are often formed.

3. Sarcina. Division in three directions of space, leading to the

formation of bale-like packages.

* "System der Bakterien," Jena, 1897-1900 (vols. I and II appearing
at different times).

f "Preliminary Arrangement of the Species of the Genus Bacterium,"
"Ninth Annual Report of the Delaware College Agricultural Experi-
ment Station," 1897, Newark, Delaware, U. S. A.

Bacteria 33

(B) Withflagella.

1. Planococcus. Division in two directions of space, like micro-

coccus.

2. Planosarcina. Division in three directions, like sarcina.

II. Family BACTERIACE^E. Cells more or less elongate, cylindric, and
straight. They never form spiral windings. Division in one
direction of space only, transverse to the long axis of the cell.

(A) Without flagella.

1. Bacterium. Occasional endospores.

(B) Withflagella.

2. Bacillus. Flagella arising from any part of the surface.

Endospore-formation common.

3. Pseudomonas. Flagella attached only at the ends of the cell.

Endospores very rare.

III. Family SPIRILLACE^E. Cells twisted spirally like a corkscrew, or
representing sections of the spiral. Division only transverse
to the long diameter.

1. Spirosoma. Rigid; without flagella.

2. Microspira. Rigid; having one, two, or three undulating

flagella at the ends.

3. Spirillum. Rigid; having from five to twenty curved or un-

dulating flagella at the ends.

4. SpirochfBta* Serpentine and flexible. Flagella not observed ;

probably swim by means of an undulating membrane.

B. SUB-ORDER: Trichobacteria (Higher Bacteria).

IV. Family MYCOBACTERIACE^S. Cells forming long or short cylindric
filaments, often clavate-cuneate or irregular in form, and at
times showing true or false branchings. No endospores, but
formation of gonidia-like bodies due to segmentation of the cells.
No flagella. Division at right angles to the axis of rod in fila-
ment. Filaments not surrounded by a sheath as in Chlamydo-
bacteriaceae.

1. Mycobacterivm. Cells in their ordinary form, short cylindric

rods often bent and irregularly cuneate. At times Y-
shaped forms or longer filaments with true branchings
may produce short coccoid elements, perhaps gonidia.
(This genus includes the Corynebacterium of Lehmann-
Neumann.) No flagella.

2. Actinomyces. Cells in their ordinary form as long branched

filaments; growth coherent, dry or crumpled. Produce
gonidia-like bodies. Cultures generally have a moldy
appearance, due to the development of aerial hyphae. No
flagella.

V. Family CHLAMYDOBACTERIACE^. Forms that vary in different
stages of their development, but all characterized by a sur-
rounding sheath about both branched and unbranched threads.
Division transverse to the length of the filaments,
i. Cladothrix. Characterized by pseudo-dichotomous branch-
ings. Division only transverse. Multiplication by the
separation of whole branches. Transplantation by means
of polar flagellated swarm-spores.

* The spirochaeta and some closely related forms are now thought to
be more properly classified among the protozoa than among the bac-
teria. They will, therefore, appear again in the tabulation of the
protozoan organisms.

3

34 Structure and Classification of Micro-organisms

2. Crenothrix. Cells united to form unbranched threads which

in the beginning divide transversely. Later the cells
divide in all three directions of space. The products of
final division become spheric, and serve as reproductive
elements.

3. Phragmidiothrix. Cells at first united into unbranched

threads. Divide in three directions of space. Late in the
development, by the growth of certain of the cells through
the delicate, closely approximated sheath, branched forms
are produced.

4. Thiothrix. Unbranched cells inclosed in a delicate sheath.

Non-motile. Division in one direction of space. Cells
contain sulphur grains.

II. ORDER: THIOBACTERIA (Sulphur Bacteria).

I. Family BEGGIATOACE.£. Cells united to form threads which are
not surrounded by an inclosing sheath. The septa are scarcely
visible. Divide in one direction of space only. Motility ac-
complished through the presence of an undulating membrane.
Cells contain sulphur grains.

There are two families, numerous subfamilies, and thirteen genera in
this order. They are all micro-organisms of the water and soil, and
have no interest for the medical student.

Structure. — Nucleus. — When subjected to the action of
nuclear stains, large vague nuclear formations are usually
observed in the bacterial cells.

Cytoplasm. — The cytoplasm, of which very little exists
between the large nucleus and cell- wall, is sometimes granu-
lar, as in Bacillus megatherium, and sometimes contains fine
granules of chlorophyl, sulphur, fat, or pigment.

Capsule. — Each cell is surrounded by a distinct cell- wall,
which in some species shows the cellulose reaction with
iodin.

The cell-walls of certain bacteria at times undergo a
peculiar gelatinous change or permit the exudation of
gelatinous material from the cytoplasm, and appear sur-
rounded by a halo or capsule. Such capsules are seen about
the pneumococcus as found in blood or sputum, Friedlander's
bacillus, as seen in sputum, Bacillus aero genes capsulatus in
blood or tissue, and many other organisms. Friedlander
pointed out that the capsule of his pneumonia bacillus, as
found in the lung tissue or in the "prune-juice" sputum,
was very distinct, though it could not be demonstrated at
all when the organisms grew in gelatin.

Polar Granules. — By carefully staining an appropriate
organism certain peculiarities of structure can sometimes
be shown. Thus, some bacilli contain distinct " polar gran-
ules" (metachromatic or Babes-Ernst granules) — rounded or

Bacteria 35

oval bodies — situated at the ends of the cell. Their sig-
nificance is unknown. They have been supposed to bear
some relationship to the biologic activity of the organ-
ism, especially its pathogenesis, but this is uncertain, and
a recent work by Gauss* and Schumburgf shows that
they vary with the reaction of the culture-media upon which
the bacteria grow and have nothing to do with their viru-
lence. Bacillus megatherium is peculiar in having its
cytoplasm filled with small granules which stain deeply.
The diphtheria bacillus and the cholera spirillum stain very
irregularly in fresh cultures, as if the tingeable substance
were not uniformly distributed throughout the cytoplasm.
Vacuolated bacteria and bacteria that will not stain, or stain
very irregularly, may usually be regarded as degenerated
organisms (involution forms) which, because of plasmolysis,
or solution, can no longer stain homogeneously.

Flagella. — Many bacteria possess delicate straight or wavy
filaments, called flagella, which appear to be organs of loco-
motion.

Messeat has suggested that the bacteria be classified, ac-
cording to the arrangement of the flagella, into :

I. Gymnobacteria (forms without flagella).
II. Trichobacteria (forms with flagella).

1. Monotricha (with a single flagellum at one end).

2. Lophotrocha (with a bundle of flagella at one end).

3. Amphitricha (with a flagellum at each end).

4. Peritricha (flagella around the body, springing from all

parts of its surface).

This arrangement is-, however, less satisfactory than that
of Migula already given.

Motility. — The greater number of the bacteria supplied
with flagella are actively motile, the locomotory power no
doubt being the lashing flagella. The rod and spiral micro-
organisms are most plentifully supplied with flagella; only
a few of the spheric forms have them.

The presence of flagella, however, does not invariably im-
ply motility, as they may also serve to stimulate the passage
of currents of nutrient fluid past the organism, and so favor
its nutrition. The flagellate bacteria are more numerous
among the saprophytic than the pathogenic forms.

* "Centralbl. f. Bakt.," etc., xxxi, No. 3, Feb. 5, 1902, p. 106.

f Ibid., xxxi, No. 14, p. 694, June 3, 1902.

J "Rivista d'igiene e sanata publica," 1890, n.

36 Structure and Classification of Micro-organisms

Bacillus megatherium has a distinct but limited ameboid
movement.

The dancing movement of some of the spheric bacteria seems to be
the well-known Brownian movement, which is a physical phenomenon.
It is sometimes difficult to determine whether an organism viewed under
the microscope is really motile or whether it is only vibrating. One
can usually determine by observing that in the latter case it does not
change its relative position to surrounding objects.

In some cases the colonies of actively motile bacteria,
such as the proteus bacilli, show definite migratory tenden-
cies upon 5 per cent, gelatin. The active movement of
the bacteria composing the colony causes its shape con-
stantly to change, so that it bears a faint resemblance to an
ameba, and moves about from place to place upon the sur-
face of the gelatin.

Size. — Bacteria are so minute that a special unit has
been adopted for their measurement. This is the micro-
millimeter (//), or one-thousandth part of a millimeter,
equivalent to the one-twenty-five-thousandth (-jTriinr) of an
inch.

The size of bacteria varies from a fraction of a micro-
millimeter to 20 or even 40 micromillimeters.

Reproduction. — Fission. — Bacteria multiply by binary
division (fission). A bacterium about to divide appears
larger than normal, and, if a spheric organism, more or less
ovoid. By appropriate staining karyokinetic changes may
be observed in the nuclei. When the conditions of nutri-
tion are good, fission progresses with astonishing rapidity.
Buchner and others have determined the length of a gener-
ation to be from fifteen to forty minutes.

The results of binary division, if rapidly repeated, are
almost appalling. "Cohn calculated that a single germ
could produce by simple fission two of its kind in an hour ;
in the second hour these would be multiplied to four;
and in three days they would, if their surroundings were
ideally favorable, form a mass which can scarcely be reck-
oned in numbers." " Fortunately for us," says Woodhead,
" they can seldom get food enough to carry on this appalling
rate of development, and a great number die both for want
of food and because of the presence of other conditions
unfavorable to their existence."

Sporulation. — When the conditions for rapid multiplica-
tion by fission are no longer good, many of the organisms
guard against extinction by the formation of spores (Fig. i).

Bacteria 37

Endospores, or spores developed within the cells, are gen-
erally formed in the elongate bacteria, — bacillus and spiril-
lum, — but Zopf has observed similar bodies in micrococci.
Escherich also claims to have found undoubted spores in a
sarcina.

Spores may be either round or oval. As a rule, each

a b c d e }

O o (3^=>

Fig. 1. — Diagram illustrating sporulation: a, Bacillus inclosing a
small oval spore; b, drumstick bacillus, with the spore at the end; ct
clostridium; d, free spores; e and /, bacilli escaping from spores.

organism produces a single spore, which is situated either
at its center or at its end. When, as sometimes happens,
the diameter of the spore is greater than that of the
bacillus, it causes a peculiar barrel shape bulging of the
organism, described as clostridium. When the distending
spore is at the end, a "Trommelschlager," or "drum-
stick," is formed. End-spores are almost characteristic
of anaerobic bacilli. When the formation of a spore is
about to commence, a small bright point appears in the
cytoplasm, and increases in size until its diameter is nearly
or quite as great as that of the bacterium. A dark, highly
refracting capsule is finally formed about it. As soon as
the spore arrives at perfection the bacterium seems to die,
as if its vitality were exhausted.

The spores differ from the bacteria in that their capsules
prevent evaporation and enable them to withstand drying
and the application of a considerable degree of heat. Very
few adult bacteria are able to resist temperatures above
70° C. Spores are, however, uninjured by such temper-
atures, and can even successfully resist the temperature of
boiling water (100° C.) for a short time. The extreme
desiccation caused by a protracted exposure to a dry tem-
perature of 150° C. will invariably destroy them, as will also
steam under pressure. Not only can the spores successfully
resist a considerable degree of heat, but they are also un-
affected by cold of almost any intensity. Von Szekely* found
anthrax spores capable of germination after eighteen years

* "Zeitschr. fur Hygiene," 1903, xuv, 3.

38 Structure and Classification of Micro-organisms

and six months in some dried-up old gelatin cultures found
in his laboratory.

Arthrospores. — The formation of arthrospores is less clear,
and seems to be the conversion of the entire organism into a
spore or permanent form. Arthrospores have been observed
particularly among the micrococci, where certain individuals
become enlarged beyond the normal, and surrounded by a
capsule.

Though the cell-wall of the adult bacterium is easily pen-
etrated by solutions of the anilin dyes, it is difficult to stain
spores, which are distinctly more resistant to the action of
chemic agents than the bacteria themselves.

Germination of Spores. — When a spore is about to germi-
nate, the contents, which have been clear and transparent,
become granular, the body increases slightly in size, the
capsule becomes less distinct, and in the course of time
splits open to allow the escape of a young organism. The
direction in which the capsule ruptures varies in different
species. Bacillus subtilis escapes from the side of the spore;
Bacillus anthracis from the end. This difference can be
made use of as an aid in differentiating otherwise similar
organisms.

So soon as the young bacillus escapes it begins to in-
crease in size, develops a characteristic capsule, and presently
begins the propagation of its species by fission.

Morphology. — The three principal forms of bacteria are
spheres (cocci), rods (bacilli), and screws (spirilla).

Cocci. — The spheric bacteria, from a fancied resemblance to
little berries, are called cocci or micrococci. When they divide,
and the resulting organisms remain attached to one another,
a diplococcus is produced. Diplococci may consist of two at-
tached spheres, though each half commonly shows flattening
of the contiguous surfaces. In a few cases, as the gonococcus,
the approximated surfaces may be slightly concave, causing
the organism to resemble the German biscuit called a "Sem-
mel." When a second binary division occurs, and four result-
ing individuals remain attached to one another, without dis-
turbing the arrangement of the first two, a tetrad, or tetracoc-
cus, is formed. To the entire groups of cocci dividing in two
directions of space so as to produce fours, eights, twelves,
etc., on the same plane, the name merismopedia has been
given. Migula uses the term micrococcus for the unflagel-
lated tetrads, and planococcus for the flagellated forms.

Bacteria 39

If division take place in three directions of space, so as
to produce a cubic " package" of cocci, the resulting aggre-
gation is described as a sarcina. This form resembles a
dice or a miniature bale of cotton. Few sarcinae have
flagella, similar flagellated organisms being called by Migula
planosarcina.

If division always take place in the same direction, so
that the cocci remain attached to one another like a string
of beads, the organism is described as a streptococcus.

Cocci commonly occur in irregular groups having a fan-
cied resemblance to bunches of grapes. Such are called*

/

<D ©

© O

Fig. 2. — Diagram illustrating the morphology of the cocci : a, Coccus
or micrococcus; b, diplococcus; c, d, streptococci; e, f, tetracocci or
merismopedia ; g, h, modes of division of cocci; i, sarcina; /, coccus
with flagella ; k, staphylococci.

staphylococci, and most organisms not finding a place in the
varieties already described are so classed.

Cocci associated in globular or lobulated clusters incased
in a resisting gelatinous, homogeneous mass, have been
described by Billroth as ascococcus, Cocci, solitary or in
chains, surrounded by an incasement of almost cartilaginous
consistence, have been called leuconostoc.

Bacilli. — Better known, if not more important, bacteria
consist of elongate or " rod-shaped forms," and bear the
name bacillus (a rod) . These present considerable variation
of form. Some are ellipsoid, some long and slender. Some
have rounded ends, as Bacillus subtilis; others have square
ends, as B. anthracis. Some are large, some exceedingly
small. Some always occur singly, never uniting to form
threads or chains; others are nearly always so conjoined.

The bacilli divide by transverse fission only, so that the
only peculiarity of arrangement is the formation of threads
or chains. In the older writings, short, stout bacilli were

40 Structure and Classification of Micro-organisms

described under the generic term bacterium. Migula now
employs the term to include only bacillary forms without
flagella. A pseudomonas is a bacillary form with polar
flagella. Some of the flexile bacilli have sinuous move-
ments resembling the swimming of a snake or an eel, and

Fig. 3. — Diagram illustrating the morphology of the bacilli: a, 6,
c, Various forms of bacilli ; d, e, bacilli with flagella ; /, chain of bacilli,
individuals distinct ; g, chain of bacilli, individuals not separated.

are sometimes described as vibrio; but this name also has
passed into disuse, except in France, where spiral organisms
are so called.

Spirilla. — If a rod-shaped bacterium is spirally twisted
and resembles a corkscrew, it is called spirillum. The rigid
forms without flagella are known as spirosoma; rigid forms
with flagella, spirilla and microspira.

A spiral organism of ribbon shape is called spiromonas,
while a similar organism of spindle shape is called a spiru-
lina. One species of spiral bacteria in whose cytoplasm

Fig. 4. — Diagram illustrating the morphology of the spirilla: a, b, c,

Spirilla.

sulphur granules have been detected has been called ophi-
diomonas.

Spiral organisms with undulating membranes are known as
spiroch&ta, but these and the similar genus treponema are
now regarded as more correctly placed among the protozoan
organisms.

The Higher Bacteria

THE HIGHER BACTERIA.

The Higher Bacteria form a group intermediate between
the Schizomycetes, or true bacteria, and the Hyphomycetes,
or molds. In the classification of Migula and Chester
they include the Mycobacteriaceae and the Chlamydobac-
teriaceae. Some, like Petruschky, believe them to be more
closely related to the true molds than to the bacteria.
They are characterized by filamentous forms with real or
apparent branchings. The filaments are usually regularly
divided transversely, so as to appear as if composed of bacilli.

Fig. 5. — Cladothrix, showing false branching. (From Hiss and Zinsser,
" Text-Book of Bacteriology," D. Appleton & Co., publishers.)

The free ends only seem to be endowed with reproductive
functions, and develop peculiar elements that differentiate
the higher from the other bacteria whose cells are all equally
free and independent.

Leptothrix. — These comprise long threads which do not
branch. They are not always easily separated from chains
of bacilli. They rarely appear to play a pathogenic role,
though those inhabiting the mouth occasionally secure a
foothold upon the edges of the tonsillar crypts, where they
grow, with the formation of persistent white patches. This
form of leptothrix mycosis is chronic and difficult to treat.

42 Structure and Classification of Micro-organisms

The leptothrix is a very difficult organism to secure in culture.
The attempts of Vignal* and of Arustamoff f were successful,
but upon the usual culture-media the organisms grew very
sparingly.

Cladothrix. — These also produce long thread-like filaments,
but they occasionally show what is described as false branch-
ing; that is, branches seem to originate from the threads,
but no distinct connection between the thread and the ap-
parent branch obtains. None of the cladothrices is known

Fig. 6. — Streptothrix enteola. Film preparation from peptone-beef-
broth culture, fourteen days at 37° C. X 1000. (Foulerton.)

to be pathogenic. They are frequently organisms of the at-
mospheric dust, and not infrequently appear as "weeds"
in culture-media The colonies grow to about a centimeter in
diameter, are usually white in color, irregularly rounded,
sharp at the edges, more or less concentric, dry and pow-
dery (not velvety) or scaly on the surface. They commonly
liquefy gelatin and blood-serum.

Streptothrix. — These organisms certainly branch. They
also form endospores. Many of them can be cultivated.
Not a few are found under circumstances suggesting patho-

* "Annales de physiologic," 1886.

* Kolle and Wassermann, "Handbuch der Pathogenen Mikro-
organismen," 1903, n, p. 851; Wratsch,i889.

The Yeasts, or Blastomycetes 43

genie action. For a long time there has been a disposition
to regard Bacillus tuberculosis as a form of strep to thrix, since
old cultures show branching involution forms. The old
genus actinomyces is also included by a number of writers
among the streptothrices, so that the Actinomyces bovis of
Bollinger is called Strep to thrix actinomyces, the Actinomyces
madurae, Streptothrix madurae, and the organism found by
Nocard in the disease known as " farcin du bauf," Strepto-
thrix farcinica. There seems, however, no adequate ground
for this arrangement, and the old genus Actinomyces should
be kept. Bppinger, found a streptothrix in the pus of a
cerebral abscess, and Petruschky, Berestneff, Flexner,
Norris, and Larkin have found streptothrices in cases of
pulmonary disease simulating tuberculosis. The organisms
described by these writers were not identical, so that there are
probably several different species. They usually grow well
upon ordinary media and upon solid media form whitish,
glistening, well-circumscribed colonies attaining a diameter
of several millimeters. As they grow old they turn yellowish
or brownish. They liquefy gelatin. Some of the cultures
were not harmful to the laboratory animals, others caused
suppuration.

Actinomyces. — The chief characterization of the organisms
of this group is a clavate expansion of the terminal ends of
radiating filaments. These are seen in sections of diseased
tissues containing the organisms, but rarely are well shown in
the artificial cultures. For further particulars of these or-
ganisms see Actinomyces bovis, etc.

THE YEASTS, OR BLASTOMYCETES.

The organisms of this group are sharply separated from the
bacteria by their larger size, elliptic form, and by multipli-
cation by gemmation or budding.

Each organism is surrounded by a sharply defined,
doubly contoured, highly refracting, transparent cellulose
envelope. Commonly each cell contains one or more distinct
vacuoles. When multiplication is in progress, smaller and
larger buds are formed.

The yeasts, of which Saccharomyces cerevisiae may be
taken as the type, are active fermentative organisms, quickly
splitting the sugars into CO2 and alcohol, and are largely cul-

44 Structure and Classification of Micro-organisms

tivated and used in the manufacture of fermented liquors
and bread. They grow well in fermentable culture-media
and most of them also grow upon the ordinary laboratory
culture-media. Many varieties, some of which produce red
or black pigment, some non-chromogenic, are known. They

Fig. 7. — Blastomycetes dermatitidis. Budding forms and mycelial
growth from glucose agar (Irons and Graham, in "Journal of Infectious
Diseases").

play very little part in the pathogenic processes. Burse has
observed a case of generalized fatal infection caused by an
yeast that he calls Saccharomyces hominis. Gilchrist,
Curtis, Ophuls, and others have seen localized human infec-
tions by blastomycetes. (See Blastomycetic dermatitis.)

THE OIDIA.

These organisms seem to occupy a place intermediate be-
tween the yeasts and the molds — the blastomycetes and the
hyphomycetes. In certain stages they appear as oval cells
which multiply by gemmation, but instead of becoming sep-
arated, hang together. At a later stage of development they
grow into long filamentous formations suggesting the mycelia
of molds, but being less regular. Certain cells also develop
as reproductive organs.

The Oidia

45

They are common micro-organisms of the air and appear as
frequent causes of contamination in culture-media, upon all

Fig. 8. — Oidium, showing the various vegetative and reproductive ele-
ments. X 350 (Grawitz).

forms of which they grow readily, producing liquefaction
where possible. They engage in but few pathogenic processes,
the most familiar being that brought about by Oidium albi-

Fig. 9. — Oidium (Kolle and Wassermann).

cans, which causes the common disease of childhood known as
thrush (q. v.).

46 Structure and Classification of Micro-organisms

THE MOLDS.

In this group it is customary to place a miscellaneous
collection of organisms having in common the formation of
a well-marked mycelium, but being so diversified in other re-
spects as to place them in widely separated groups in the sys-
tematic arrangement of the fungi. Some are correctly
placed among the " Imperfect fungi," some among the Asco-
mycetes, and some among the Phy corny cetes. They are all
active enzymic agents and produce fermentative and putre-
factive changes.

Fig. 10. — Mucor mucedo: i, A sporangium in optical longitudinal
section; c, columella; m, wall of sporangium; sp, spores; 2, a ruptured
sporangium with only the columella (c) and a small portion of the wall
(m) remaining; 3, two smaller sporangia with only a few spores and no
columella; 4, germinating spores; 5, ruptured sporangium of Mucor
mucilaginus with deliquescing wall (m) and swollen interstitial substance
(z); sp, spores (after Brefeld).

1. Ackorion. — The organisms of this genus are character-
ized by a more or less branched hypha, 3 to 5 ^ in diameter,
which break up after a time into rounded or cuboidal spores.
The Achorion schonleini is highly pathogenic and will be
described in the section upon Favus.

2. Tricophyton and Microsporon. — These names are applied
somewhat loosely to organisms affecting skin and hair fol-
licles of men and animals. They form tangled slender my-
celia with many spores of varying size. They occasion

The Molds

47

"ringworm," barber's itch, pityriasis, and tinea. Further
description of the organisms will be found in the section upon
Ringworm.

3. Mucor. — The mucors, or "black molds," belong to the
phy corny cetes. They form a thick, tangled mycelium, in
and above which the rounded black sporongia can be seen
with the naked eye. The mycelium becomes divided at the
time of reproduction. Multiplication takes place asexually
through conidia-spores which develop within sporangia, and
sexually by the conjugation of specialized terminal septate

Fig. 1 1 . — Mucor mucedo. Single-celled mycelium with three hyphae and
one developed sporangium (after Kny, from Tavel).

branches of the mycelium, which conjugate with similar
cells, belonging to other colonies, to form zygospores.

The sporangia form upon the ends of aerial hypha and con-
sist of a smooth spherical capsule within which the spores
develop, to become liberated only when the membrane rup-
tures. The colonies, each of which is unisexual, may be
described as + and — . Colonies of the + type will not
conjugate; colonies of the — type will not conjugate, but
when terminal filaments of + and — come together, conjuga-
tion occurs and zygospore formation takes place.

Mucors are not infrequent organisms of the atmosphere
and occasionally appear as contaminations upon solid culture-
media. About 130 species are known. Of these, Mucor
corymbifer, Mucor rhizopodiformis, Mucor ramosus, Mucor

48 Structure and Classification of Micro-organisms

pusillus, Mucor septatus, and Mucor conoides are said by
Plaut* to be pathogenic when introduced into laboratory
animals. Mucor corymbifer has been known to produce
inflammation of the external auditory meatus in man.f
General mucor mycosis in man has also been observed by
Paltauf J to result from the presence of the same organism.

Fig. 12. — Mucor mucedo. Different stages in the formation and
germination of the zygospore: i, Two conjugating branches in contact;
2, septation of the conjugating cells (a) from the suspensors (&) ; 3, more
advanced stage in the development of the conjugating cells (a) ; 4, ripe
zygospore (6) between the suspensors (a); 5, germinating zygospore
with a germ-tube bearing a sporangium (after Brefeld).

4. Aspergillus and Eurotium. — The organisms of this genus
are included among the Ascomycetes. They are common
organisms of the air and frequent contaminations of the solid
culture-media. To secure them an agar-agar plate can be
exposed to the atmosphere of the laboratory for a short time,
then covered and stood aside for a day or two, when tangled
mycelial growths with rapidly spreading hyphae will usually
be discovered. The recognition is easily made when the
sporangia appear. These are well shown in the accompany-
ing illustration. The mycelium is divided into many cells.

* Kolle and Wassermann, "Die Pathogenen Mikroorganismen,"
1903, i, 552.

t Htickel-Losch in Fliigge, "Die Mikroorganismen." | Ibid.

The Molds

49

Reproduction is asexual and takes place through conidia
spores. The fruit hyphae, which are aerial, terminate in
rounded extremities which are known as columella, from
which many radiating sterigmata arise, each terminating in a
series of rounded spores. A sexual form of reproduction also
takes place through the production of ascospores. Many
species are known, only a few of which are pathogenic.

Fig. 13. — Aspergillus glaucus: A, A portion of the mycelium m, with
a conidiaphore c, and a young perithrecium F, magnified 190 diameters;
B and B', conidiaphore with conidia; B, individual sterigma greatly
magnified; C, early stage of the development of the fructifying organ;
D young perithrecium in longitudinal section; w, the future wall of
the contents; as, the screw, magnified 250 diameters; E, an ascus with
spores from a perithrecium, magnified 600 diameters (duBary).

Aspergillus malignum has been found by von Lindt in the
auditory meatus of man.

Aspergillus nidulans occasionally infects cattle. It is
pathogenic for laboratory animals, usually causing death in
sixty hours. The kidneys are found enlarged to twice their
normal size, and show small whitish dots and stripes of cell
infiltration containing the fungi. The heart muscle, dia-
phragm, and spleen may also be involved. The liver usually
escapes. It takes a large number of spores to infect.

Aspergillus fumigatus. — This is a widespread and not in-
frequently pathogenic form. Its most common lesion is a

50 Structure and Classification of Micro-organisms

pneumomycosis, in which the lung is riddled with small
inflammatory necrotic and cavernous areas containing the
molds. The same condition has occasionally been observed
in human beings, Sticker having collected 39 cases.*

Leber and others have observed keratitis following corneal
infection by this organism.

Aspergillus flavus is also pathogenic.

Aspergillus subfuscus is also pathogenic and highly virulent.

Aspergillus niger. — Pathogenic and found at times in
inflammation of the external auditory meatus.

5. Penicillium. — These are common green molds, widely dis-
seminated throughout the atmosphere and frequent sources
of contamination of the culture-media in the laboratory.

Fig. 14 — Penicillium (Eyrej.

Moist bread exposed to the atmosphere soon becomes covered
with them. They are included in the group of fungi imper-
fecti, and are characterized by a luxuriant tangled septate
mycelium, with aerial fruit hyphae, ending in conidiophores,
each of which divides into two or three sterigmata, the tip of
which forms a chain of rounded spores. The whole germinal
organ thus comes to resemble a whisk-broom or, as Hiss de-
scribes it, a skeleton hand, in which the conidiophore corre-
sponds to the wrist; the sterigmata, to the metacarpal bones;
the chains of spores, to the phalanges.

None of the penicillia is known to be pathogenic either for
man or animals.

Penicillium crustaceum (glaucum) is the most common
source of contamination of the laboratory media.

Penicillium minimum, which may be identical with the
preceding, was once found in the human ear by Sievenmann.

* Nothnagel's Spezielle Path. u. Therap., xiv, 1900.

The Protozoa 51

THE PROTOZOA.

The protozoa are unicellular animal organisms as differen-
tiated from the metazoa which are multicellular animal or-
ganisms. The restriction, implied by the term unicellular is,
however, too narrow, for there are colonial protozoa that con-
sist of many cells, yet share other protozoan characters.

For the purposes of this work, however, all protozoa are
to be regarded as unicellular and the individuals independent
of one another.

Classification. — Many schemes have been devised for
systematically arranging the protozoa, that which follows
being an abbreviation of the standard classification, made to
correspond with the requirements of this work that deals
only with the pathogenic forms.

CLASSIFICATION OF THE PATHOGENIC PROTOZOA.

Phylum PROTOZOA (Trpwrof first, Cwov animal). Unicellular ani-

mal organisms.

Class Rhizopoda (pifa root, 7rw(5of foot). Having soft plasmic
bodies with or without external protecting shells. The con-
tour subject to change through the formation of extensions
known as pseudopods. These may be blunt, rounded, or
lobose, filamentous, or anastomosing. The nutrition is
holozoic or holophytic.
Order GYMNAMCEBA (yvfj.v6c naked). Rhizopoda without ex-

ternal shells or coverings.
Genus Amoeba (a^oifta to change).
Entamoeba.
Chlamydophrys.
" Leydenia.

Class Mastigophora (udanyos whips, <t>6poc to bear). Organ-
isms of well-defined form, naked or surrounded by a well-
defined membrane. Nutrition is holozoic, holophytic, para-
sitic, or saprophytic. Mouth, contractile vesicle, and nucleus
usually present.

Order FLAGEXLATA (Latin, flagellare, to beat). Small organisms
with a well-defined mononucleate body, at the anterior end
or both ends of which are one or more flagella. Actively
motile. May become encysted. Nutrition is holozoic, holo-
phytic, parasitic, or saprophytic.
Family CercomonidcB. Body pyriform, with several anterior

flagella and an undulating membrane.
Genus Cercomonas.
" Trichomonas. .
Monas.
Plagiomonas.

Family Lambliadce. Body pyriform, very much attenuated
behind. Ventral surface shows a reniform depression,
about the posterior part of which there are six flagella.
There are also two flagella at the posterior extremity.
Genus Lamblia (Megastomum).

52 Structure and Classification of Micro-organisms

Family Trypanosomida. Body delicately fusiform. Con-
tains a nucleus, a blepharoplast or centrosome, and an
undulating membrane. A single wavy flagellum arises
in the posterior part of the body close to the centrosome,
passes along the edge of the undulating membrane to the
anterior extremity, where it continues free for some dis-
tance. Nutrition parasitic. Reproduces by division.
Genus Trypanosoma.
" Leishmania.

Babesia.

Family Spiroch<ztid(B. Organisms very long and spirally

twisted. Nucleus indistinct. Multiplication probably

by longitudinal division only. Nutrition is parasitic or

saprophytic.

Genus Spirochaeta. Body flattened, with a very narrow

undulating membrane.

Genus Treponema. Body not flattened. No undulating
membrane. Extremities sharp pointed and terminating
in short flagella.

Class Sporozoa (cxopof a spore, faov an animal). Organisms
unprovided with cilia or flagella in the adult stage. Always
endoparasites in the cells, tissues, or cavities of other animals.
Nutrition is parasitic and osmotic. Reproduction always by
spore-formation, the germs or sporozoites either being produced
by the parent or indirectly from spores, into which the parent
divides.
Subclass Telosporidia. Spore-formation ends the individual life,

the entire organism being transformed to spores.
Order GREGARINIDA. Possess distinct membrane with myo-
nemes during adult life; locomotion mainly by contraction.
Young stages alone (cephalonts) are intracellular parasites,
the adults (sporonts) being found in the digestive tract or the
body cavities. Sporulation takes place after or without
conjugation, but within a cyst that is never formed, while
the parasite is intracellular.

Order COCCIDIIDA. Spherical or ovoid in form, without a
free and motile adult stage. Never ameboid. Sporula-
tion takes place within cysts formed while the organism is
an intracellular parasite.
Genus Coccidium.

" Eimeria.

Order H^MOSPORIDIIDA. Sporozoa of small size living in the
blood-corpuscles or plasma of vertebrates. The adult
form is mobile and in some cases provided with myonemes.
Reproduction by endogenous or asexual sporulation, while
in the host or by exogenous sporulation after conjugation.
Genus Plasmodium.

Subclass Neosporidia. Organisms that form sporocysts through-
out life, the entire cell not being used up in the formation of
the spores.

Order SARCOSPORIDIA. The initial stage of the life history is
passed in the muscle cells of vertebrates. Form is
elongate, tubular, oval, or even spherical. Cysts have a
double membrane, in which reniform or falciform sporo-
zoites are formed.
Genus Sarcocystis.
" Miescheria.
" Balbiania.

The Protozoa 53

Subclass Haplosporidia. Spores provided with large round

nuclei. No polar capsules.
Genus Rhinosporidium.

Class Infusoria (Latin, injusus, to pour into. The organisms were
given this name because they were first found in infusions
exposed to the air). Protozoa in which the motor apparatus
is in the form of cilia, either simple or united into membranes,
membranelles, or cirri. The cilia may be permanent or
limited to the embryonic stages. There are two kinds of
nuclei, macronucleus and micronucleus. Reproduction is
effected by simple transverse division or by budding. Nutri-
tion is holozoic or parasitic.

Subclass Ciliata. Mouth and anus usually present. The
contractile vacuole often connected with a complicated sys-
tem of canals.

Order HOLOTRICHIDA. The cilia are similar and distributed
all over the body, with a tendency to lengthen at the
mouth. Trichocysts are always present, either over the
whole body or in special regions.
Genus Colpoda.
" Chilodon.

Order HETEROTRICHIDA. Organisms possessing a uniform
covering of cilia over the entire body, and an adoral zone
consisting of short cilia fused together into membranelles.
Suborder Polytrichina. Uniform covering of cilia.

Family Bursarida. The body is usually short and pocket-
like, but may be elongated. The chief characteristic
is the peristome, which is not a furrow, but a broad
triangular area deeply insunk, and ending in a point
at the mouth. The adoral zone is usually confined
to the left peristome edge or it may cross over to the
right anterior edge.
Genus Balantidium.

Structure. — From the table it will at once be evident that
the protozoa form an extremely varied group, and that no
kind of descriptive treatment can be looked upon as adequate
that does not consider individuals.

Cytoplasm. — In some of the smaller protozoa, and in certain
stages of others, the cytoplasm appears almost hyaline and
structureless. In most cases, however, it appears granular,
and in the larger organisms, such as ameba, it presents the
appearance which some described as granular, others, as
frothy. The accepted theory of structure teaches that the
protoplasm is honeycombed or frothy, and that it is filled
with endless chambers in which its enzymes and other
active substances, etc., are stored up and its functions car-
ried on.

In addition to these chambers, which are minute and of
uniform size, there are larger spaces called vacuoles, some
of which are the result of temporary conditions — accumu-
lations of digested but not yet assimilated food, etc.; but

54 Structure and Classification of Micro-organisms

others, seen in ameba and in the ciliata, are large, per-
manent, and characterized by rhythmical contractions
through which they disappear from one part of the body sub-
stance to appear in another. These are known as " con-
tractile vacuoles," and are supposed to subserve the useful
purpose of assisting in maintaining cytoplasttiic currents and
so distributing the nourishing juices.

The cytoplasm also contains remnants of undigested or
indigestible foods which constitute the paraplasm or deutero-
plasm. In a few cases granules of chlorophyl are also to

Fig. 15. — Internal parasites: A, Amoeba coli, Losch; B, Monocystis
agilis, I^euck., a gregarine; C, Megastoma entericum, Grassi, a" flagel-
late; D, Balantidium coli, Ehr., a ciliate.

be found in organisms otherwise resembling animals too
closely to be confused with plants.

The cytoplasm may be soft and uniform in quality, or
there may be a surface differentiation into ectosarc, or body
covering, and endosarc, body substance. In the rhizopoda
there is little difference between the two, though certain
fresh-water ameba cover themselves with minute grains of
mineral substance, but in most of the mastigophora and in-
fusoria corticata the ectosarc is characterized by a peculiar
rigidity that gives the animal a definite and permanent form.
From the surface covering or ectosarc coarse threads or fine
hair-like appendages — flagella and cilia — often project. In
many of the infusoria the ectosarc contains trichocysts from
which nettling or stinging threads are thrown out when the
organisms are irritated.

The Protozoa 55

The body substance may show no morphologic differen-
tiation in rhizopoda, but in the corticata there may not only
be a permanent form, but there may be adaptations, such as an
oral aperture, sometimes infundibular in shape and communi-
cating with the soft endosarc through a blind tube. An
anal aperture may also be present.

In the higher infusoria the ectosarc may also be continued
posteriorly to form a stalk, by which the organism attaches
itself ( Vorticella) . Such stalks are contractile.

Nucleus. — In certain protozoa of very simple and indefinite
structure — spirochaeta and treponema — no distinct well-
contoured nucleus can be observed.

In the rhizopoda the nucleus is a distinct organ surrounded
by a nuclear membrane and containing the usual chromatin
and linin.

The greater number of mastigophora possess two distinct
bodies, either a nucleus and a centrosome or a major and
minor nucleus. This is well shown in trypanosoma.

The infusoria vary greatly in the character of the nuclei.
As a rule, there are two indefinite nuclei, the macronucleus
and the micronucleus. Both seem to be essential organs, and
in the phenomena supervening upon conjugation both par-
ticipate. The nuclei of the protozoa are, therefore, extremely
diversified, and vary from the most simple collections of
granules of nuclear substance to large well-formed fantastic-
ally shaped composite organs.

Movement. — Some kind of movement is to be observed at
some period in the life of almost every protozoan.

In rhizopoda with the soft ectosarc the movement consists
of flowing currents by which lobose projections of the body
substance appear now here, now there, in the form of pseudo-
podia, or else a continuous flowing, by which the upper surface
continually coming forward in a thin layer coincides with the
progress of the animal, which continually rolls over and over
as it were.

In mastigophora the movement of the more rigid bodies
is effected through presence of longer or shorter flexile or
rigid coarse threads or " whips." These usually project an-
teriorly— trypanosoma — and by means of a spiral move-
ment draw the cell along with a propeller-like action; sym-
metrically arranged flagella may operate more like oars.

The sporozoa usually manifest very little movement, yet
their sporozoites are motile, and the spermatozoits are also
motile and commonly flagellated.

56 Structure and Classification of Micro-organisms

The infusoria are actively motile through abundant fine
hair-like formations known as cilia. These, multitudinous
as they are, vibrate synchronously with an oar-like move-
ment, propelling the organisms forward or backward or mak-
ing them revolve with great rapidity. Independent cilia
not infrequently encircle the oral aperture, causing a vortex,
in which the minute structures upon which the creatures
feed are caught and carried into the body.

Size. — The protozoa show very great variation in size.
Some of the sporozoa form minute parasites of the red blood-
corpuscles or other cells of the vertebrates. The treponema
is so small that it can slowly find its way through the pores of
a Berkefeld filter.

On the other hand, the sarcoporidium is so large that one
of its cysts, composed of a single organism, can be seen with
the naked eye. Certain protozoa that play no part in morbid
processes — myxosporidia — and so do not come within the
scope of this work, may be several centimeters in diameter.

Reproduction. — The reproduction of the protozoa takes
place both asexually and sexually. It may be that there are
no strictly asexual protozoa, nearly all forms having been
shown upon intimate acquaintance to be subject to occasional
conjugation. Conjugation may result in the loss of individual
identity or the conjugated individuals may again separate.

Whether the reproduction takes place asexually without
conjugation or sexually after conjugation, it always occurs by
division, 'which may be simple and binary or complex and
multiple.

Wherever a distinct nucleus can be found, the multipli-
cation of the protozoa is preceded by some kind of mitotic
change. The more complex the structure of the nucleus, the
more complicated and perfect the mitosis.

The elongate protozoa divide lengthwise, which is some-
times contrary to expectation, as in the cases of treponema
and spirochaeta.

The multitudinous sporozoi'tes into which the zygotes of
the sporozoa divide are commonly the result of anterior divi-
sion into intermediate bodies known as oocysts, ookinetes,
sporocysts, etc. The nuclear substance is first divided so as
to be uniformly distributed among these, then further divided
so that some of it reaches each sporozoite.

In the process of sporulation the entire parent may be
used up, as in the coccidium and plasmodium, or the parent

The Protozoa 57

may continue to live and later form additional sporozoites,
as in sarcocystis.

Encystment. — Nearly all of the protozoa are capable at
times of encysting themselves, i. e., surrounding themselves
with dense capsules by which life may be preserved for some
time amid such unfavorable surroundings as excessive cold,
excessive dry ness, and absence of food. Sometimes the
encysted stage is the spore stage (coccidium), sometimes it
is the adult stage (ameba). Under these circumstances we
find an analogy with the sporulation of the bacteria which is
not for purposes of multiplication, but for self-preservation.
The encysted protozoa are less hardy, however, than the
bacterial and other plant spores, and succumb to compara-
tively slight elevations of temperature.

CHAPTER II.

BIOLOGY OF MICRO-ORGANISMS.

THE distribution of micro-organisms is well-nigh universal.
They and their spores pervade the atmosphere we breathe, the
water we drink, the food we eat, and luxuriate in the soil
beneath our feet.

They are not, however, ubiquitous, but correspond in dis-
tribution with that of the matter upon which they live
and the conditions they can endure. Tyndall* found the
atmosphere of high Alpine altitudes free from them, and
likewise that the glacier ice contained none; but wherever
man, animals, or even plants live, die, and decompose, they
are sure to be present.

Their presence in the air generally depends upon their
previous existence in the soil, its pulverization, and dis-
tribution by currents of the atmosphere. Koch has shown
that the upper stratum of the soil is exceedingly rich in bac-
teria, but that their numbers decrease as the soil is pene-
trated, until below a depth of one meter there are very few.
Remembering that micro-organisms live chiefly upon organic
matter, this is readily understandable, as most of the organic
matter is upon the surface of the soil. Where, as in the case
of porous soil or the presence of cesspools and dung-heaps, the
decomposing materials are allowed to penetrate to a consider-
able depth, micro-organisms may occur much farther below
the surface; yet they are rarely found at any great depth,
because the majority of them require free oxygen for suc-
cessful existence.

The water of stagnant pools always teems with micro-
organisms; that of deep wells rarely contains many unless it
is polluted from the surface of the earth.

It has been suggested by Soyka that currents of air
passing over the surface of liquids might take up organisms,
but, although he seemed to show it experimentally, it is
not generally believed. Where bacteria are growing in
colonies they seem to remain undisturbed by currents of

* "Floating Matter in the Air."

58

Conditions Prejudicial to Growth of Bacteria 59

air unless the surface of the colony becomes roughened or
broken.

Most of the organisms carried about by the air are what
are called saprophytes, and are harmless.

Oxygen. — As all micro-organisms must have oxygen
in order to live, the greater number of them grow best
when freely exposed to the air. Some will not grow at all
where uncombined oxygen is present, but secure all they
need by severing it from its chemic combinations. These
peculiarities divide bacteria into the

Aerobes, which grow in the presence of uncombined
oxygen, and the

Anaerobes, which do not grow in the presence of uncom-
bined oxygen.

As, however, some of the aerobic forms grow almost as
well without oxygen as with it, they are known as optional
(facultative) anaerobes.

As examples of strictly aerobic bacteria Bacillus subtilis,
Bacillus aerophilus, Bacillus tuberculosis, and Bacillus
diphtherias may be given. These will not grow if oxygen
is denied them. The cocci of suppuration, the bacillus of
typhoid fever, and the spirillum of cholera grow almost
equally well with or without oxygen, and hence belong to
the optional anaerobes. The bacilli of tetanus and of
malignant edema, and the non-pathogenic Bacillus butyricus,
Bacillus muscoides, and Bacillus polypiformis, will not
develop at all where any free oxygen is present, and hence
are strictly anaerobic.

The higher bacteria, oidia, molds and protozoa, are for the
most part aerobic and optional anaerobes. Treponema
pallidum seems to be a strictly anaerobic protozoan.

Food. — The bacteria grow best where diffusible albu-
mins are present, the ammonium salts being less fitted
to support them than their organic compounds. Pros-
kauer and Beck* have succeeded in growing the tubercle
bacillus in a mixture containing ammonium carbonate
0.35 per cent., potassium phosphate 0.15 per cent.,
magnesium sulphate 0.25 per cent., and glycerin 1.5
per cent. Some of the water microbes can live in dis-
tilled water to which the smallest amount of organic
matter has been added; others require so concentrated
a medium that only blood-serum can be used for their

* "Zeitschrift fiir Hygiene," etc., Aug. 10, 1894, vol. xvm, No. i.

60 Biology of Micro-organisms

cultivation. The statement that certain forms of bac-
teria can flourish in clean distilled water seems to be
untrue, as in this medium the organisms soon die and
disintegrate. If, however, in making the transfer, a drop
of culture material is carried into the water with the bac-
teria, the distilled water ceases to be such, and becomes a
dilute bouillon fitted to support bacterial life for a time.
Sometimes a species with a preference for a particular
culture medium can gradually be accustomed to another,
though immediate transplantation causes the death of the
organism. Sometimes the addition of such substances
as glucose and glycerin has a peculiarly favorable influ-
ence, the latter, for example, enabling the tubercle bacillus
to grow upon agar-agar.

The yeasts grow best upon media containing sugars, but
can also be cultivated upon media containing diffusible
protein and non-fermentable carbohydrates and glycerin.

The molds flourish upon almost all kinds of organic matter,
but perhaps attain their most rapid development upon media
containing fermentable carbohydrates.

The saprophytic and parasitic protozoa live by osmosis and
absorb through the ectosarc such substances as are capable
of assimilation and nutrition. These forms are cultivable
only upon media containing the same or approximately
the same proteins as those to which they have been accus-
tomed. Thus, to cultivate trypanosoma, blood-serum
must be added to the media.

The larger protozoa live upon smaller animal and vegetable
organisms, which they ingest entire. Such can only be arti-
ficially cultivated provided the attempt be made under con-
ditions of symbiosis with some other and smaller organism
that may constitute the food. Amoeba coli can thus be cul-
tivated in symbiosis with Bacillus typhosus.

Moisture. — A certain amount of water is indispensable
to the growth of bacteria. The amount can be exceedingly
small, however, Bacillus prodigiosus being able to develop
successfully upon crackers and dried bread. Artificial
culture-media should not be too concentrated; at least
80 per cent, of water should be present.

The molds and oidia grow well upon bread that contains
very little moisture. Protozoa usually require fluid media.
Pond-water protozoa can only grow in water, not in con-
centrated culture-media.

Conditions Prejudicial to Growth of Bacteria 61

Reaction. — Should the pabulum supplied contain an
excess of either alkali or acid, the growth of the micro-
organisms is inhibited. Most true bacteria grow best in a
neutral or feebly alkaline medium. There are exceptions
to this rule, however, for Bacillus butyricus and Sarcina
ventriculi can grow well in strong acids, and Micrococcus
urea can tolerate excessive alkalinity. Acid media are
excellent for the cultivation of molds. Neutral or feebly
alkaline media serve best for the cultivable protozoa.

Light. — Most organisms are not influenced by the presence
or absence of ordinary diffused daylight. The direct
rays of the sun, and to a less degree the rays of the electric
arc-light, retard and in numerous instances kill bacteria. In
a recent careful study of this subject Weinzirl* found that
when bacteria were placed upon glass or paper, and exposed
to the direct rays of the sun, without any covering, most
spore-bearing bacteria, including Bacillus tuberculosis, B,
diphtheriae, B. typhosus, s. cholerae asiaticae, B. coli, B. pro-
digiosus, and others are killed in from two to ten minutes.
Certain colors are distinctly inhibitory to the growth, blue
being especially prejudicial,

Treskinskajaf found that sunlight had a marked destruc-
tive effect upon the tubercle bacillus, and varied according
to altitude. By direct sunlight at the sea-level they were
destroyed in five hours: at an altitude of 1560 meters, in
three hours. In winter the time of destruction was about
two hours longer than in summer. In diffused daylight the
time required for destruction was about twice as long as in
direct sunlight. His experiments were performed with
pure cultures dried in a thin layer upon glass.

Certain chromogenic bacteria produce colors only when
exposed to the ordinary light of the room. Bacillus mycoides
roseus produces its red pigment only in the dark. The
virulence of many pathogenic bacteria is gradually attenuated
if they are kept in the light.

Molds and yeasts grow best in the dark, so that in general
it can be said that the vegetable micro-organisms, belonging
to the fungi and having no chlorophyl, need no light and are
injured rather than benefited by it.

The pathogenic protozoa have not been particularly studied

* " Centralbl. f. Bakt. u. Parasitenk. Ref.," xi/ra, Nos. 22-24, P- 68l«
f " Jour. Infectious Diseases," vol. iv, 1907, Supplement, No. 3,
p. 128.

62 Biology of Micro-organisms

with reference to light. Non-pathogenic water protozoa
love the light and die in the dark.

Electricity, X-rays, etc. — Powerful currents of electricity
passed through cultures have been found to kill the organ-
isms and change the reaction of the culture-medium; rapidly
reversed currents of high intensity, to destroy the patho-
genesis of the bacteria and transform their toxic products
into neutralizing bodies (antitoxin?). Attention has been
called to this subject by Smirnow, d'Arsonval and Charin,
Bolton and Pease, Bonome and Viola, and others.

An interesting contribution upon the " Effect of Direct,
Alternating, Tesla Currents and X-rays on Bacteria " was
made by Zeit,* whose conclusions are as follows:

1. A continuous current of 260 to 320 milliamperes passed through
bouillon cultures kills bacteria of low thermal death-points in ten min-
utes by the production of heat (98.5° C.). The antiseptics produced
by electrolysis during this time are not sufficient to prevent the growth
of even non-spore-bearing bacteria. The effect is a purely physical one.

2. A continuous current of 48 milliamperes passed through bouillon
cultures for from two to three hours does not kill even non-resistant
forms of bacteria. The temperature produced by such a current does
not rise above 37° C., and the electrolytic products are antiseptic, but
not germicidal.

3. A continuous current of 100 milliamperes passed through bouillon
cultures for seventy-five minutes kills all non-resistant forms of bacteria
even if the temperature is artificially kept below 37° C. The effect is
due to the formation of germicidal electrolytic products in the culture.
Anthrax spores are killed in two hours. Subtilis spores were still alive
after the current was passed for three hours.

4. A continuous current passed through bouillon cultures of bac-
teria produces a strongly acid reaction at the positive pole, due to the
liberation of chlorin which combines with oxygen to form hypochlorous
acid. The strongly alkaline reaction of the bouillon culture at the
negative pole is due to the formation of sodium hydroxid and the libera-
tion of hydrogen in gas bubbles. With a current of 100 milliamperes
for two hours it required 8.82 milligrams of H2SO4 to neutralize i c.c. of
the culture fluid at the negative pole, and all the most resistant forms
of bacteria were destroyed at the positive pole, including anthrax and
subtilis spores. At the negative pole anthrax spores were killed also,
but subtilis spores remained alive for four hours.

5. The continuous current alone, by means of Du Bois-Reymond's
method of non-polarizing electrodes, and exclusion of chemic effects by
ions in Kruger's sense, is neither bactericidal nor antiseptic. The
apparent antiseptic effect on suspension of bacteria is due to electric
osmosis. The continuous electric current has no bactericidal nor anti-
septic properties, but can destroy bacteria only by its physical effects
(heat) or chemic effects (the production of bactericidal substances by
electrolysis).

6. A magnetic field, either within a helix of wire or between the
poles of a powerful electromagnet, has no antiseptic or bactericidal
effects whatever.

* "Jour Amer. Med. Assoc.," Nov. 30, 1901.

Conditions Prejudicial to Growth of Bacteria 63

7. Alternating currents of a 3-inch Ruhmkorff coil passed through
bouillon cultures for ten hours favor growth and pigment production.

8. High-frequency, high potential currents — Tesla currents — have
neither antiseptic nor bactericidal properties when passed around a bac-
terial suspension within a solenoid. When exposed to the brush dis-
charges, ozone is produced and kills' the bacteria.

9. Bouillon and hydrocele-fluid cultures in test-tubes of non-resistant
forms of bacteria could not be killed by Rontgen rays after forty-eight
hours' exposure at a distance of 20 mm. from the tube.

10. Suspensions of bacteria in agar plates and exposed for four hours
to the rays, according to Rieder's plan, were not killed.

1 1 . Tubercular sputum exposed to the Rontgen rays for six hours,
at a distance of 20 mm. from the tube, caused acute miliary tubercu-
losis of all the guinea-pigs inoculated with it.

12. Rontgen rays have no direct bactericidal properties. The clin-
ical results must be explained by other factors, possibly the production
of ozone, hypochlorous acid, extensive necrosis of the deeper layers of
the skin, and phagocytosis. The action of the x-rays upon bacteria
has been investigated by Bonome and Gros,* Pott,f and others. When
the cultures are exposed to their action for prolonged periods, their
vitality and virulence seem to be slightly diminished. They are not
killed by the x-rays.

Movement. — Rest seems to be the condition best adapted
for micro-organismal development. Slow-flowing movements
do not have much inhibitory action, but violent agitation,
as by shaking a culture in a machine, may hinder or prevent
it. This explains why rapidly flowing streams, whose cur-
rents are interrupted by falls and rapids, should, other
things being equal, furnish a better drinking-water than a
deep, still-flowing river.

Galli- Valerio t has shown, however, that agitation does
not inhibit the growth of the anthrax, typhoid or colon
bacilli or the pneumococcus, but sometimes facilitates it.

Association. — Symbiosis is the vital association of different
species of micro-organisms by which mutual benefit to one
or the other is brought about. Antibiosis is an association
detrimental to one of the associated organisms. Bacterial
growth is greatly modified by the association of different
species. Coley found the streptococcus more active when
combined with Bacillus prodigiosus; Pawlowski, that mixed
cultures of Bacillus anthracis and Bacillus prodigiosus were
less virulent than pure cultures of anthrax; Meunier,§ that

* "Giornal. med. del Regis Esercito," an 45, u. 6.

f "Lancet," vol. n, No. 21, 1897.

t " Centralbl. f. Bakt.," etc., I Orig. 4, xxxvn, Sept. 23, 1904,
p. 151.

§ Societe de Biologic, Seance du n Juin, 1898, "La Semaine medi-
cale," June 15, 1898.

64 Biology of Micro-organisms

when the influenza bacillus of Pfeiffer is inoculated upon
blood agar together with Staphylococcus aureus its growth
is favored by a change which the staphylococci bring about
in the hemoglobin.

A similar advantageous association has been pointed out
by Sanarelli, who found that Bacillus icteroides grows best
and retains its vitality longest when grown in company with
certain of the molds.

Rarely, the presence of one species of micro-organism
entirely eradicates another. Hankin* found that Micro-
coccus ghadialli destroyed the typhoid and colon bacilli,
and suggested the use of this coccus to purify waters pol-
luted with typhoid.

An interesting experimental study of the bacterial an-
tagonisms with special reference to the Bacillus typhosus,
that the student should read, is by W. D. Frost, and appears
in the " Journal of Infectious Diseases," 1904, i, p. 599.

Temperature. — According to Frankel, bacteria will rarely
grow below 16° and above 40° C., but Fliigge has shown that
Bacillus subtilis will grow very slowly at 6° C.; at 12.5° C.
fission does not take place oftener than every four or five
hours; at 25° C. fission occurs every three-quarters of an hour,
and at 30° C. about every half-hour.

The temperature at which micro-organisms grow best is
known as the optimum, the lowest temperature at which
they continue active as the minimum, the highest that can
be endured the maximum.

A few forms of bacteria grow at very high temperatures
(6o°-7o° C.), and are described as thermophilic . They are
found in manure piles and in hot springs. Tsiklinskyf has
described two varieties of actinomyces and a mold that he
cultivated from earth and found able to grow well at 48° to
68° C., though not at all at the temperature of the room.

Most bacteria are killed by temperatures above 60° to
75° C., but their spores can resist boiling water for some
minutes, though killed by dry heat if exposed to 150° C. for
an hour or to 175° C. for from five to ten minutes.

The resistance of low forms of life to low temperatures is
most astonishing. Some adult bacteria and most spores
seem capable of resisting almost any degree of cold. Ravenel %

* "Brit. Med. Jour.," Aug. 14, 1897, p. 418.

t "Russ. Archiv f. Path.," etc., Bd. v, June, 1898.

t "The Medical News," June 10, 1899.

Conditions Prejudicial to Growth of Bacteria 65

exposed anthrax spores to the action of liquid air for three
hours; diphtheria bacilli, for thirty minutes ; typhoid bacilli,
for sixty minutes ; and Bacillus prodigiosus, for sixty minutes,
the\emperature of the cultures being reduced to about — 140°
C., yet in no case was the vegetative capability of all of the
bacteria destroyed, and when transferred to fresh culture
bouillon they grew normally. His researches corroborate
those of Pictet and Yung and others.

To say that bacteria are not injured by cold is a mistake,
as Sedgwick and Winslow* have found that when typhoid
bacilli are frozen, the greater number of them are destroyed,
and that subsequent development of the frozen cultures
takes place from the few surviving organisms.

Bacteria usually grow best at the temperature of a com-
fortably heated room (17° C.), and are not affected by its
occasional slight variations. Some, chiefly the pathogenic
forms, are not cultivable except at the temperature of the
body (37° C.); others, like the tubercle bacillus, grow best
at a temperature a little above that of the normal body.

The temperature endurance of the molds resembles that
of the bacteria. The mycelia are killed at temperatures of
60° C. and over, but their spores endure 100° C. The
yeasts and oi'dia, that have no resisting spores, are killed at
about 60° C. The protozoa are still more sensitive to heat
variations than the plant organisms and are killed by less
extreme variations. Here again, however, the encysted pro-
tozoa endure greater variations than the active organisms.

Effect of Chemic Agents. — The presence of chemic agents,
especially certain of the mineral salts, in an otherwise per-
fectly suitable medium may completely inhibit the develop-
ment of bacteria, and if added to grown cultures in greater
concentration, destroy them. Such substances are spoken
of as antiseptics in the former, germicides in the latter case.
Bichlorid of mercury and carbolic acid are the most familiar
examples of germicides.

Though these agents are supposed to operate in definite
concentrations with almost unvarying result, Trambustif
found it possible to produce a tolerance to a certain amount
of bichlorid of mercury by cultivating Friedlander's bacillus
upon culture-media containing gradually increasing amounts

:"Centralbl. f. Bakt. u. Parasitenk.," etc., May 26, 190x3, Bd.
xxvu, Nos. 1 8, 19, p. 684.

f"Lo Sperimentale," 1893-94.
5

66 Biology of Micro-organisms

of the salt, until from 1-15,000, which inhibit ordinary cul-
tures, it could accommodate itself to 1-2000.

The various chemic agents act in different ways upon the
micro-organisms. Thus, they may combine with the pr'oto-
plasm to make a new and no longer vital cornpound ; or, they
may coagulate or dissolve or dehydrate or oxidize the proto-
plasm to a destructive extent.

The addition of chemic agents to solutions containing
micro-organisms also changes the osmotic pressure. When
an active organism is living in its normal environment, it
contains within its plasm a greater concentration of solutes
than are to be found in the surrounding fluid. Under these
circumstances the pressure on the inside of the ectosarc
or other cell membrane is greater than that on the outer
side, and the cell is in a state of turgor. If now salts are added
so that the solutes on the outside exceed those on the inside,
water is drawn out and the protoplasm is made to shrink or
condense. According to the degree of this change the
organism will be embarrassed, made impotent, or destroyed.
On the other hand, when micro-organisms have enjoyed
a concentrated medium like blood-serum and are suddenly
transferred to distilled water, so much water may be sud-
denly drawn into their protoplasm that they swell up and
may burst and go to pieces. This is particularly true of the
delicate protozoa like the trypanosoma.

Metabolism. — According to their activities, micro-
organisms are classed as —

Zymogens, when they cause fermentation.

Saprogens, when they cause putrefaction.

Chromogens, when they produce colors.

Photogens, when they phosphoresce.

Aerogens, when they evolve gas.

Pathogens, when they cause disease.

The metabolic activities of micro-organisms occasion many
well-known changes in nature. Thus, it is through their
energies that by fermentative and putrefactive changes
organic matter is gradually transformed from complex to
simple compounds. It is by the energy of bacteria that foul
waters are gradually purified, and while it is true that the
presence of large numbers of bacteria in water detracts from
its potability, the very bacteria that cause its condemnation
ultimately effect its purification by exhausting the organic
matter it contains in their own nutrition. In the modern

Fermentation 67

treatment of sewage by the "septic tank " method, the
organic matter contained in the water is consumed through
the agency of anaerobic and aerobic bacteria, until its con-
sumption leaves the water once more clear and pure, the no
longer useful bacteria dying out as the nutrition becomes ex-
hausted.

The promptness with which bacteria attack organic
matter is seen in the changes brought about in foods, some
of which are ruined in flavor or quality, though others
are thought to be improved. Thus, the flavor of butter,
sausage, and cheese, the aroma of wines, and many other
important gustatory characteristics of our foods depend
solely upon the activity of bacteria or other micro-organisms.

Many of these activities are harmless, and, indeed, ad-
vantageous, though the fact that they are not infrequently
accompanied by chemic changes, some of which are poison-
ous, makes it necessary to watch and time their operations
lest acridity, acidity, insipidity, or toxicity of the food re-
place the desired effect.

Briefly considered, the best-known phenomena resulting
from micro-organismal energy are as follows:

Fermentation. — Fermentation is a chemic transforma-
tion of carbohydrates resulting from the activity of micro-
organismal enzymes. The alcoholic fermentation, which is
a familiar phenomenon to the layman as well as to the brewer
and chemist, depends upon the activity of an yeast-plant, one
of the saccharomyces fungi by which the sugar is broken up
into alcohol and carbon dioxid, with some glycerin and other
by-products. The following equation shows the chief changes
produced :

C6H1206 2C2H5OH -f 2C02

Sugar. Alcohol. Carbon dioxid.

There are also several bacteria which produce the acetic
fermentation, though it is generally attributed to Bacillus
aceticus. There are two equations to express this fermen-
tation :

I. CH2CH2OH + O = CH3CHO + H2O
Alcohol. Oxygen. Aldehyd. Water.

II. CH,CHO + O = CH3COOH

Aldehyd. Oxygen. Acetic acid.

A number of different bacilli seem capable of converting
milk-sugar into lactic acid, though Bacillus acidi lactici is

68 Biology of Micro-organisms

the best known and most active acid producer. The butyric
fermentation generally due to Bacillus butyricus may also
be caused by other bacilli. (For an exact description of the
chemistry of the fermentations reference must be made to
special text-books.*) The lactic acid and butyric acid fer-
mentation, have the following equations:

I. C»HMQu + H2Q = C0H1202 + C,Hi,Ofl
Lactose or milk sugar. Galactose. Dextrose.

II. CoHiA = 2CzU,O3
Galactose. Lactic acid.

III. C6H,206 = C4H80^ + C02 + 2H2
Galactose. Butyric acid.

Putrefaction. — Putrefaction is a chemic disintegration
of nitrogenous compounds resulting from the activity of
micro-organisms. The first step in the process seems to
be the transformation of the albumins into peptones, then
the splitting up of the peptones into gases, acids, bases,
and salts. Both fermentative and putrefactive processes
apparently take place through the agency of enzymes.
In the process the innocuous albumins are frequently changed
to toxalbumins, and sometimes to peculiar putrefactive al-
kaloids known as ptomains.

Ptomains. — Vaughan and Novy define a ptomain as " a
chemical compound, basic in character, formed by the action
of bacteria on organic matter." The chemistry of these
bodies is very complex, and for a satisfactory description
of them Vaughan and Novy's bookf is excellent.

Ptomains probably play but a small part in pathologic
conditions. They are formed almost exclusively outside of
the living body, and only become a source of danger when
ingested with the food. It is supposed that cases of ice-
cream and cheese-poisoning are usually due to tyrotoxicon,
a ptomain produced by the putrefaction of the protein sub-
stances of the milk before it is frozen into ice-cream or
made into cheese. The safeguard is to freeze the milk only

*See "Enzymes and Their Applications," by Jean Effront, trans-
lated by S. C. Prescott, New York, 1902; "Micro-organisms and Fer-
mentation," by Alfred Jorgensen, translated by A. K. Miller and
A. E. Lennholm, London, 1900; and the many writings of Christian
Hansen.

f "Ptomaines and Leucomaines," 1888; "Cellular Toxins," 1902.

Production of Gases

when perfectly fresh and avoid mixing the milk, cream,
sugar, and flavoring substances, and allowing the mixture
to stand for some time beforehand.

The occasional cases of " Fleischver-giftung," "meat-
poisoning," or " Botulismus," are due to the development of
toxic ptomains in consequence of the
growth of certain bacteria (Bacillus
botulinus) in the meat. Kaensche*
carefully investigated the subject, and
gives a synoptic table containing all the
described bacteria of this class. His
researches show that there are at least
three different bacilli whose growth
causes the development of poisonous
ptomains in meat. In general these or-
ganisms resemble Bacillus coli.

With the increase of knowledge upon
the toxic character of the bacteria them-
selves, the importance of the toxic pto-
mains has diminished, until at present
we have come to regard them as very
rare causes of disease.

Production of Gases. — Various gases
are given off during decomposition and
fermentation, among them being CO2,
H2S, NH4, H, CH4, and others. Gases
produced by aerobic bacteria usually fly off from the sur-
face of the culture unnoticed, but if the bacterium be
anaerobic and develop at the lower part of a tube of
culture media, a visible bubble of gas is usually formed
about the colonies. Such gas bubbles are almost invariably
present in cultures of the bacilli of tetanus and malignant
edema.

To quantitatively determine the gas-production, some
form of the Smith fermentation-tube is most convenient.
The tube is filled with bouillon containing some sugar,
sterilized as usual, inoculated, and stood aside to grow.
As the gases form, the bubbles ascend and accumulate in
the closed arm. In estimating quantitatively, one must
be careful that the tube is not so constructed as to allow
the gas to escape as well as to ascend into the main
reservoir.

* "Zeitschrift fiir Hygiene," etc., Bd. xxn, Heft i, June 25, 1896.

Fig. 1 6. — Smith's fer-
mentation-tube.

70 Biology of Micro-organisms

For the determination of the nature of the gases produced,
Theobald Smith has recommended the following method :

"The bulb is completely filled with a 2 per cent, solution
of sodium hydroxid (NaOH) and tightly closed with the
thumb. The fluid is shaken thoroughly with the gas and
allowed to flow back and forth from the bulb to the closed
branch, and the reverse several times to insure intimate
contact of the CO2 with the alkali. Lastly, before removing
the thumb all the gas is allowed to collect in the closed
branch so that none may escape when the thumb is removed.
If CO2 be present, a partial vacuum in the closed branch
causes the fluid to rise suddenly when the thumb is removed.
After allowing the layer of foam to subside somewhat the
space occupied by gas is again measured, and the difference
between this amount and that measured before shaking
with the sodium hydroxid solution gives the proportion of
CO2 absorbed. The explosive character of the residue is
determined as follows: The cotton plug is replaced and
the gas from the closed branch is allowed to flow into the
bulb and mix with the air there present. The plug is then
removed and a lighted match inserted into the mouth of
the bulb. The intensity of the explosion varies with the
amount of air present in the bulb. The relative propor-
tion of gases resulting from the fermentation is frequently
of importance for the differential diagnosis of related bac-

TT

teria. Smith has designated this relation of ^o- as the 'gas
formula.' The colon bacillus has a gas formula corre-

TT

spending to ^ — f . Other aerogenic bacilli sometimes
show a formula ^j = ^."

Liquefaction of Gelatin. — As certain organisms grow in
gelatin, the medium becomes partly or entirely liquefied.
This peculiarity is apparently independent of any other
property of the organisms, and is manifested alike by patho-
genic and non-pathogenic forms. The liquefaction is sup-
posed to be dependent upon a form of peptonization. Bit-
ter* and Sternbergf have shown that if from a culture in
which liquefaction has taken place the bacteria be removed
by filtration, the filtrate will retain the power of liquefying
gelatin, showing the property is not resident in the bacteria,
but in some substance in solution in their excreted products.

* " Archiv Fur Hygiene," 1886, Heft 2.
t" Medical News," 1887, No. 14.

Chromogenesis 71

These products were described as " tryptic enzymes " by
Fermi,* who found that heat destroyed them. Mineral
acids seem to check their power to act upon gelatin. For-
malin renders the gelatin insoluble. Some of the bacteria
liquefy the gelatin in such a peculiar and characteristic
manner as to make the appearance a valuable guide for the
differentiation of species.

Production of Acids and Alkalies — Under the head
of "Fermentation" the formation of acetic, lactic, and
butyric acids has been discussed. Formic, propionic, baldri-
anic, palmitic, and margaric acids also result from microbic
metabolism. As the acidity progresses, it impedes, and
ultimately completely inhibits, the activity of the organisms.
The cultivation of the bacteria in milk to which litmus or
lacmoid has been added is a convenient method for detecting
changes of reaction. Rosolic acid solutions may also be
used, the acid converting the red into an orange color.
Neutral red is also much employed for this purpose, the
acids turning it yellow.

The quantitative estimation of changes in reaction can
be best made by titration, and the fermentation-tube culture
can be employed for the purpose. The contents of the bulb
and branch should be shaken together, a measured quan-
tity withdrawn, and titration with ^ sodium hydroxid, or
- hydrochloric acid, performed.

The alkali most frequently formed by bacterial growth
is ammonium, which is set free from its combinations,
and either flies off as a gas or forms new combinations
with acids simultaneously formed. Some bacteria produce
acids only, some alkalies only, others both acids and alka-
lies. Both acids and the alkalies, when in excess, serve to
check the further activity of the micro-organisms.

Chromogenesis. — Bacteria that produce colored colo-
nies or impart color to the medium in which they grow are
called chromogenic; those producing no color, non-chromo-
genic. Most chromogenic bacteria are saprophytic and non-
pathogenic. Some of the pathogenic forms, as Staphylo-
coccus pyogenes aureus, are, however, color producers. It
seems more likely that certain chromogenetic substances
unite with constituents of the culture medium to pro-
duce the colors than that the bacteria form the actual

*"Centralbl. f. Bakt.," etc., 1891, Bd. x, p. 401.

72 Biology of Micro-organisms

pigments; but, as Galeotti * has shown, there are two kinds
of pigment, one being soluble, readily saturating the cul-
ture medium, as the pyocyanin and fluorescin of Bacillus
pyocyaneus, the other insoluble, not tingeing the solid cul-
ture media, but retained in the colonies, like the pigment
of Bacillus prodigiosus. The pigments are found in great-
est intensity near the surface of a bacterial mass. The
coloring matter never occupies the cytoplasm of the bac-
teria (except Bacillus prodigiosus, in whose cells occasional
pigment-granules may be seen), but occurs as an inter-
cellular deposit.

Almost all known colors are formed by different bacteria.
One bacterium will sometimes elaborate two or more colors ;
thus, Bacillus pyocyaneus produces pyocyanin and fluores-
cin, both being soluble pigments — one blue, the other green.
Gessard f has shown that when Bacillus pyocyaneus is culti-
vated upon white of egg, it produces only the green fluor-
escent pigment, but if cultivated in pure peptone solution
it produces only the blue pyocyanin. His experiments prove
the very interesting fact that for the production of fluor-
escin it is necessary that the culture medium contain a defi-
nite amount of a phosphatic salt. Sometimes, when an
organism produces two pigments, one is soluble, the
other insoluble, so that the colony will appear one
color, the medium upon which it grows another. I once
found an interesting coccus, | with this peculiarity, upon
the conjunctiva. It formed a brilliant yellow colony
upon the surface of agar-agar, but colored the agar-agar
itself a beautiful violet. In this case the yellow pig-
ment was insoluble, the violet pigment very soluble and
diffusible through the jelly. Some organisms will only pro-
duce pigments in the -light; others, as Bacillus mycoides
roseus, only in the dark. Some produce them only at the
room temperature, but, though growing luxuriantly in the
incubator, refuse to produce pigments at so high a tem-
perature. Thus, Bacillus prodigiosus produces a brilliant
red color when growing at the temperature of the room,
but is colorless when grown in the incubator. The reaction
of the culture medium is also of much importance in this

* "Lo Sperimentale," 1892, XLVI, Fasc. m, p. 261.
f "Ann. de 1'Inst. Pasteur," 1892, pp. 810-823.
t See Norris and Oliver, "System of Diseases of the Eye," vol. n, p.
489, and "University Medical Magazine," Sept., 1895.

Production of Aromatics 73

connection. Thus, Bacillus prodigiosus produces an intense
scarlet-red color upon alkaline and neutral media, but is
colorless or pinkish upon slightly acid media. Some of the
pigments — perhaps most of them — are formed only in the
presence of oxygen.

Production of Odors. — Gases, such as H2S and NH4, and
acids, butyric and acetic acids, have sufficiently character-
istic odors. There are, however, a considerable number of
pungent odors which seem to arise from independent odor-
iferous principles. Many of them are extremely un-
pleasant, as that of the tetanus bacillus. The odors seem
to be peculiar individual characteristics of the organisms.

Production of Phosphorescence. — Cultures of Bacillus
phosphorescens and numerous other organisms are dis-
tinctly phosphorescent. So much light is sometimes given
out by gelatin cultures of these bacteria as to enable one to
see the face of a watch in a dark room. Gorham found
the photogenesis most marked when the organisms are grown
in alkaline media at room temperature. Most of the phos-
phorescent bacteria are found in sea-water, and are best
cultivated in sea-water gelatin. Some are familiar to
butchers through the phosphorescence they cause on the
surface of fresh meats.

Production of Aromatics. — Phenol, kresol, hydrochinone,
hydro paracumaric acid, and paroxypheny lie-ace tic acid
are by no means uncommon products of bacteria. The
most important is indol, which was at one time thought to
be peculiar to the cholera spirillum, but is now known to
be produced by many other bacteria. The best method of
testing for it is that of Salkowski,* known as the nitroso-
indol reaction. To perform it, 10 c.c. of the fluid to be
tested receive an addition of 10 drops of concentrated sul-
phuric acid. The mixture is shaken in a test-tube. A
few cubic centimeters of a 0.02 per cent, solution of potas-
sium nitrite are then allowed to flow down the side of the
tube. If indol is present, a purple-red color develops at
the junction of the two fluids, f McFarland and Small J
have found that the intensity of this color corresponds to
the quantity of indol present, and that quantitative tests
can be made by means of a comparative color test series.

t "Zeitschrift f. physiol. Chemie," vm, p. 417.

t See Grubs and Francis, "Bull, of the Hyg. Laboratory," No. 7, 1902.

t "Trans, of the American Public Health Association," 1905.

74 Biology of Micro-organisms

The Formation of Nitrates. — A process of fundamen-
tal importance is carried on by certain lowly bacteria of
the soil. Since plants are unable to assimilate the free
nitrogen of the air, but must obtain this element from
the soil in the form of some soluble compound, and since
there is a relatively limited amount of combined nitrogen
in the world, it becomes of the last importance that the
supplies which are continually withdrawn from the soil
should be replaced by the nitrogen liberated in the decay
of organic material. This nitrogen, after a series of putre-
factive changes have occurred, appears as ammonia. The
odor of this gas is often plainly perceptible about manure
heaps. In this form nitrogen is poorly adapted for use by
plants, and moreover may be easily dissipated. An ex-
tensive further process of oxidation is carried on by the
nitrifying bacteria, whereby nitrates are ultimately formed.
These are eminently adapted for use by plants, and so the
soil is rendered continuously capable of supporting vege-
tation.

Nitrosomonas and Nitrosococcus convert ammonia into
nitrous acid, and Nitrobacter oxidizes the latter to form
nitric acid.

These genera are well nigh universal in the soil. They do
not grow on the ordinary culture media, but require special
solutions, free from the diffusive albumins, — free, indeed,
from organic compounds of any sort. Their supplies of
carbon are obtained by the dissociation of carbon dioxid.
It is highly noteworthy that they are thus able to flourish
without food more complex than ammonia, a fact which is
without parallel among organisms devoid of chlorophyl.

Reduction of Nitrates. — A considerable number of bac-
teria are able to reduce nitrogen compounds in the soil
or in culture media, prepared for them, into ammonia.
To the horticulturist this matter is of much interest.
Winogradsky * has described specific nitrifying bacilli which
he found in soil, and asserts that the presence of ordinary
bacteria in the soil causes no formation of nitrites so long
as the special bacilli are withheld.

Reduction of nitrates can be determined experimentally by
the use of a nitrate broth, made by dissolving in 1000 c.c. of
water i gram of peptone and 0.2 gram of potassium nitrate.
The ingredients are dissolved, filtered, then filled into tubes,

* "Ann. de 1'Inst. Pasteur," 1891; "La Semaine medicale," 1892.

Combination of Nitrogen 75

and sterilized. The tubes are inoculated and the results
noted. As nitrites and ammonia are, however, commonly
present in the air and are taken up by fluids, it is always
well to control the test by an uninoculated tube tested
with the reagents in the same manner as the culture.
Two solutions are employed * for testing the culture :

I. Naphthylamin, 0.1 gram, Boi1' cofol> **?• *nd*d* j5.6
Distilled water, 20.0 grams, dilute (1 : 16) hydnc

II. Sulphanilic acid, 0.5 gram.

Hydric acetate, diluted, 150.0 c.c.

Keep the solutions in glass-stoppered bottles and mix equal
parts for use at the time of employment.

About 3 c.c. of the culture and an equal quantity of the
uninoculated culture fluid are placed in test-tubes and about
2 c.c. of the test fluid slowly added to each. The develop-
ment of a red color indicates the presence of nitrites, the
intensity of the color being in proportion to the quantity
of nitrites present. If a very slight pinkish or reddish
color in the uninoculated culture fluid and a deeper red in
the culture develop, it shows that a small amount of
nitrites was already present, but that more have been pro-
duced by the growth of the bacteria.

The presence of ammonia in either fluid is easily deter-
mined by the immediate development of a yellow color or
precipitate when a few drops of Nessler's solution f are
added.

Failure to determine either ammonia or nitrites may
not mean that the nitrates were not reduced, but that they
were reduced to N. It is, therefore, necessary to test the
solutions for nitrates, which is done by the use of phenol-
sulphonic acid and sodium hydroxid, which in the presence
of nitrates give a yellow color.

Combination of Nitrogen. — Not only do bacteria de-
stroy or reduce nitrogen compounds, but some of them
are also able to assimilate nitrogen from the air and so com-
bine it as to be useful for the nourishment of vegetable
and animal life. The most interesting organisms of this

* "Journal of the American Public Health Association," 1888, p. 92.

t Nessler's solution consists of potassium iodid, 5 grams, dissolved
in hot water, 5 c.c. Add mercuric chlorid, 2.5 grams, dissolved in 10
cc. of water, then to the mixture add potassium hydrate, 16 grams,
dissolved in water, 40 c.c., and dilute the whole to 1000 c.c.

76 Biology of Micro-organisms

kind are found upon the roots of the leguminous plants,
peas, clover, etc., and have been studied by Beyerinck.*
It seems to be by the entrance of these bacteria into their
roots that the plants are able to assimilate nitrogen from
the atmosphere and enrich sterile ground. Every agri-
culturist knows how sterile soil is improved by turning
under one or two crops of clover with the plough.

Peptonization of Milk. — Numerous bacteria possess the
power of digesting — peptonizing — the casein of milk. The
process varies with different bacteria, some digesting the
casein without any apparent change in the milk, some pro-
ducing coagulation, some gelatinization of the fluid. In
some cases the digestion of the casein is so complete as to
transform the milk into a transparent watery fluid.

Milk invariably contains large numbers of bacteria, that
enter it from the dust of the dairy, many of them pos-
sessing this power and ultimately spoiling the milk. In
the process of peptonization the milk may become bitter,
but need not change its original reaction.

The phenomena of coagulation and digestion of milk can
be made practical use of to aid in the separation of simi-
lar species of bacteria. Thus, the colon bacillus coagulates
milk, but the typhoid bacillus does not.

Production of Disease. — Micro-organisms that produce
disease are known as pathogenic; those that do not, as non-
pathogenic. Between the two groups there is no sharp
line of separation, for true pathogens may be cultivated
under such adverse conditions that their virulence may be
entirely lost, while those ordinarily harmless may be made
virulent by certain manipulations. In order to deter-
mine that a micro-organism is possessed of pathogenic
powers, the committee of bacteriologists of the American
Public Health Association f recommends that: (i) When a
given form grows only at or below 18° to 20° C., inoculation
of about i per cent, of the body-weight with a liquid culture
seven days old should be made into the dorsal lymph-sac
of a frog. (2) When a species grows at 25° C. and upward,
an inoculation should be made into the peritoneal cavity
of the most susceptible (in general) of warm-blooded animals
— i. e., the mouse, either the white or the ordinary house
mouse. The inoculation should consist of about i per cent.

* See "Centralbl. f. Bakt.," etc., Bd. vii, p. 338.
t "Jour. Amer. Public Health Assoc.," Jan., 1898.

Production of Disease 77

of the body- weight of the mouse of a four- to eight -hour
standard bouillon culture, or a broth or water suspension
of one platinum loop from solid cultures. When such
intraperitoneal injection fails, it is unlikely that other
methods of inoculation will be successful in causing the
death of the mouse. If the inoculations of the frog and
mouse both prove negative, the committee think it un-
necessary to insist upon any further tests of pathogenesis
as being requisite for work in species differentiation.

Production of Enzymes. — Some of these have already been
mentioned as causing fermentation and putrefaction, coagu-
lating milk, dissolving gelatin, etc. There are, however,
others which have interesting and important actions upon
both animal and vegetable substances.

Knowledge upon the subject is just becoming systema-
tized, one of the best writings being by Emmerich and
Low,* who observed that in old cultures of Bacillus pyo-
cyaneus the bacteria become transformed into a gelatinous
mass, and were led to experiment with old and degener-
ating cultures condensed to ^ volume in a vacuum appa-
ratus. The bacteriolytic powers were then found to be
much increased, and they were subsequently able to precipi-
tate from the concentrated culture an enzyme, which they
called pyocyanase. The authors reach the rather hasty con-
clusions that the cessation of growth of bacteria in cultures
depends upon the generation of enzymes; that the enzymes
destroy the dead bacteria; that the enzymes will kill and
dissolve living bacteria and destroy toxins, and, therefore,
are useful for the treatment of infectious diseases; and
that antitoxins are simply accumulated enzymes which the
immunized animals have received during treatment, and
which, appearing in the serum, produce the effects so well
known.

It is probable that many of the toxic effects of bacteria
and their cultures depend upon enzymic substances, the
nature of which we do not yet understand.

* " Zeitschrift fur Hygiene," 1899.

CHAPTER III.
INFECTION*

INFECTION is the successful invasion of an organism by
microparasites. Unfortunately custom has sanctioned the
use of the word in other and sometimes confusing senses,
thus, a table or knife upon which micro-organisms are known
to be or are even supposed to be; the mouth and intestine,
which naturally harbor bacteria of various forms, or a
splinter penetrating the skin and carrying harmless bacteria
into the deeper tissues, are all said by the surgeon to be
" infected," when, in fact, it would be more correct to de-
scribe them as infective.

The term infection should imply an abnormal state result-
ing from the deleterious action of the parasite upon the host.
The colon bacillus is a harmless commensal of the intestine
of every human being, and of most of the lower animals.
The intestine is not " infected," but infested with it, and it is
only when abnormal or unnatural conditions arise that
infection can take place. This form of association of certain
bacteria with certain parts of the body to which they do no
harm, but into which they may rapidly invade when appro-
priate conditions arise, is described by Adami as sub-
infection. The possibility of infection is always there,
though it is but rarely that conditions arise under which it
can be accomplished.

There are two inseparable factors to be considered in all
infections : the organism infecting and the organism infected.
The first is the parasite, the second, the host. Infectivity
and infectability may depend upon peculiarities of either
parasite or host. Organisms that have lived together as
commensals, that is, in a state of neutral relationship for
an almost indefinite period, may suddenly cease their cus-
tomary association, because of newly acquired power of in-
vasion on the one hand, or diminished vital resistance on the

78

Sources of Infection 79

other, and infection take place where it had previously been
impossible.

Bacteria are commonly called saprophytic when they
live in nature apart from other living organisms, and para-
sitic when they live in or upon them. Saprophytic bacteria
when accidentally transplanted from their natural environ-
ment to the body of some animal, for example, may or may
not be capable of continuing life under the new conditions.
In the greater number of cases they die, but sometimes the
new environment seems better than the old, and they mul-
tiply rapidly, invade the tissues in all directions, eliminate
their metabolic products into the juices, and occasion vary-
ing morbid conditions.

The parasitic bacteria live in habitual association with
higher organisms. Sometimes, and indeed most commonly,
it is a harmless association, like that of certain cocci upon
the skin, but occasionally it results in the destruction of
the tissues and the death of the host, as in tuberculosis,
leprosy, etc.

The group of pathogenic organisms has no well-defined
limits, for it is frequently observed that micro-organisms well
known under other conditions, and not known to have been
engaged in pathogenic processes, turn up unexpectedly as
the cause of some morbid condition. Indeed, although we
are acquainted with -u large number of organisms that have
never been observed in connection with disease, we are scarcely
justified in concluding that they are incapable of producing
injury should proper conditions arise.

SOURCES OF INFECTION,

The sources of infection may be exogenous or endogenous;
that is, they may arise through the admission to the tissues
of micro-organisms from sources entirely apart from the
individual infected, or through the admission of some of
those parasitic and usually harmless organisms constantly
associated with him.

Exogenous infections arise through accidental contact
with infective objects belonging to the external world.

A polluted atmosphere may carry into the respiratory
passages micro-organisms capable of colonizing there.
Polluted water or food may carry into the intestine micro-
organisms whose temporary residence may entirely change
the functional and structural integrity of the parts.

8o Infection

Wounds inflicted by the teeth of animals, by weapons, by
implements, or by objects of various kinds, carry into the
tissues micro-organisms whose operations, local or general,
may variously affect the organism to its detriment.

Contact with unclean objects of various kinds — spoons,
knives, cups, blow-pipes, catheters, syringes, dental instru-
ments, etc. — may serve to transfer disease-producing organ-
isms from one person to another who might otherwise never
come in contact with them.

Suctorial insects seem occasionally to act as the medium
by which micro-organisms withdrawn in blood from one
person may be introduced into other persons so that they
become infected. The flea thus brings about the spread of
plague; the mosquito, of malaria; the tsetse fly, of trypano-
somiasis; the tick, of relapsing fever.

Fomites, or objects made infective through contact with
individuals suffering from smallpox, scarlatina, and other
contagious or actively infectious diseases, become the means
through which the specific micro-organisms may be con-
veyed to the well with resulting infection.

Endogenous infections arise through the activity of
micro-organisms habitual to the body. It indicates a
morbid condition of the body by which the defensive mechan-
isms are disturbed, so that organisms harmless under normal
conditions become invasive.

All normal animals are born free of parasitic micro-organ-
isms, but it is impossible for them to remain so because of
the universal distribution of micro-organismal life. The
air, the water, the soil, and the food, as well as the associates
of the young animal, all act as means by which micro-organ-
isms, and especially bacteria, are brought to the surface and
cavities of its body, and but a short time elapses after birth
before it harbors the customary commensal and parasitic
forms.

BACTERIAL TENANTS OF THE NORMAL HUMAN BODY.

The skin and adjacent mucous membranes. The

slightly moist warm surface of the skin is well adapted to
bacterial life, and its unavoidable contact with surrounding
objects determines that a variety of organisms shall
adhere to it. Of these, we can differentiate between forms
whose presence is unexpected and temporary ; others whose

Bacterial Tenants of the Normal Human Body 81

presence may be expected; and still others whose presence
is invariable.

Elaborate investigations upon the bacterial flora of the
skin have been made by Unna*; Mittman,f who studied the
finger-nails, under which he found no less than seventy-
eight different species ; Maggiora, J who isolated twenty-nine
forms from the skin of the foot; and Preindelsberger, § who
found eighty species of bacteria on the hands. Undoubtedly
many of these organisms were accidentally present, and were
at least only semi-parasitic. Not a few were met but once
and were in no sense bacteria of the skin. The skin may
also be temporarily contaminated with bacteria from other
portions of the patient's body, as, for instance, from his
intestine ; thus Winslow || has found the colon bacillus upon
the hands of ten out of one hundred and eleven persons
examined. Wigura** also examined the hands of forty
persons in hospitals, finding tubercle bacilli in two out of
ten persons from phthisical wards in hospitals, colon bacilli
six times and typhoid bacilli once on the hands of nine
attendants in the typhoid wards. He found streptococci
and staphylococci many times. Welchff and Robb and
GhriskeyJ J seem to have been the first to make a clear dif-
ferentiation between the accidentally present bacteria and
the permanently parasitic organisms of the skin, and to show
that certain cocci, producing white and yellow colonies upon
agar-agar, were invariable in occurrence and penetrated to
the lowest epidermal layers.

These cocci, of which Welch describes the most common
as Staphylococcus epidermidis albus, are universally and

* " Monatshefte fiir prakt. Dermatol.," vii, 1888, p. 817 ;vm, 1889,
pp. 293, 562; ix, 1889, p. 49; x, 1890, p. 485; xi, 1890, p. 471; xn,
1891, p. 249.

f'Archiv. f. path. Anat. u. Phys. u. f. klin. Med.," cxm, 1888, p.
203.

f'Giornale della R. Societd d'Igiena," 1889, Fasc. 5, p. 335.
§" Samml. Medic. Schriften," herausg. von der " Wiener klin. Woch-
enschrift," xxii, Wien, 1891; "Rev. Jahresbericht iiber die Fort-
schritten in der Lehre von den pathogenen Mikroorganismen," vii,
1891, p. 619.

|| " Jour. Med. Research," vol. x, p. 463.
** "Wratsch," 1895, No. 14.

ft "Transactions of the Congress of American Physicians and Sur-
geons," n, 1891, p. 1.

tt " Bulletin of the Johns Hopkins Hospital," m, 1892, p. 37,
6

82 Infection

invariably present upon the human skin, and must be re-
garded as habitual parasites.

Where the skin is peculiar in its moisture and greasiness,
however, additional forms are found. Thus, in preputial
smegma, in the axillae, and sometimes about the lips and
nostrils, a bacillary organism, Bacillus smegmatis, is invari-
able, and the recent work by Schaudinn and Hoffman* has
shown that the skin of the genitalia harbors a spiral organism
which they call Spirochseta refringens.

In the external auditory meatus a coccus, Micrococcus
cereus flavus, is almost always to be found in the waxy
secretion.

Upon the conjunctiva as many accidental organisms may
be found as shall have been caught by its moist surface,
though the researches of Hildebrand and Bernheim and
others seem to show that the tears have some antiseptic
power and prevent the organisms from growing, so that in
health there are very few permanent residents of the sac,
certain cocci seeming to be the only constant forms.

The mouth has been carefully studied bacteriologically
by Miller, f who found six organisms — Leptothrix innomi-
nata, Bacillus buccalis maximus, Leptothrix buccalis
maxima, lodococcus vaginatus, Spirillum sputigenum and
Spirochaeta dentinum (denticola) — in every mouth. Prac-
tically the same conclusions were reached by Vincentini. J
These organisms are peculiar in that they will not grow in
artificial culture. In addition to this permanent flora,
Miller cultivated fifty- two other species, some of which were
harmless, some well-known pathogens.

From the mouth these organisms may be traced into the
pharynx and esophagus.

In studying the micro-organisms of dental caries Goodby§
found a large number of organisms which he divided into
three groups: A. Those that produce acids, including
Streptococcus brevis, Bacillus necrodentalis (Goodby),
Sarcina alba, Sarcina lutea, Sarcina aurantiaca, Staphylo-
coccus pyogenes aureus, and Staphylococcus pyogenes sali-
varius (Biondi). B. Those that liquefy blood-serum: Ba-

* "Deutsche med. Woch.," May 4, 1905.
t "Micro-organisms of the Human Mouth," Phila., 1890.
% "Bacteria of the Sputa and Cryptogamic Flora of the Mouth,"
London, 1897.

§ Transactions of the Odontological Society, June, 1899.

Bacteria of the Digestive Apparatus 83

cillus mesentericus rubra, B. mesentericus vulgatus, B.
mesentericus fuscus, Bacillus fuscus, a yellow bacillus,
probably B. gingival pyogenes (Miller), and Bacillus lique-
facium motilis. C. Those that produce pigment, including
the same organisms as group B. In carious dentine two
organisms, Streptococcus brevis and Bacillus necrodentalis,
were invariably present.

The extinction of the great number of bacteria entering
the mouth is referred by most bacteriologists to a bac-
tericidal action of the saliva.

The stomach seems to retain very few of the many
bacteria that must enter it, its persistently acid contents
being inimical to their development. Certain sarcina,
especially Sarcina ventriculi, may be found without any
considerable departure from the normal state. In carcinoma
and other forms of pyloric obstruction with dilatation, the
bacterial flora increases, and in achlorhydria micro-organ-
isms of fermentation make their appearance. They are,
however, accidental and not permanent tenants of the organ.

In carcinoma of the stomach a bacillus, probably one of
the lactic acid groups, early makes its appearance and is of
some diagnostic importance. It is called after its discoverer
the Oppler-Boas bacillus,* also on account of angulations
found in its threads, Bacillus geniculatus. It is a large
bacillus, tending to form long threads easily seen without an
oil-immersion lens. It is probably non-motile, does not
form spores, stains by Gram's method, and is said by Emory f
to divide longitudinally as well as transversely. This, as
he says, will, if proved to be correct, be a most important
means of identifying the species. Cultures are easily made
in media acidified with lactic acid.

The intestine receives such micro-organisms as have
survived whatever destructive influences the gastric juices
may have exerted, and its alkaline contents, rich in proteins
and carbohydrates in solution, are eminently appropriate for
bacterial life. The flora of the intestine is, therefore, in-
creased in number and variety of organisms as we descend
from its beginning to its end. In the small intestine there
may be no bacteria in the upper part of the jejunum, but
in most cases Bacillus lactic aerogenes and bacilli of the
colon groups are found. These increase in number as the

*" Deutsche tned. Wochenschrift," 1905, No. 5.
t "Bacteriology and Hematology," p. 114.

84 Infection

iliocecal valve is reached. The cecum shows large num-
bers of colon bacilli. The rectum contains, in addition,
many putrefactive organisms, such as Bacillus putrificus,
Bacillus proteus vulgaris, members of the Bacillus subtilis
group, and acid-producing organisms, such as Bacillus
acidophilus.

An interesting and thorough study of these organisms
of the bowel and their distribution has been made by Kohl-

Fig. 17.— Sarcina ventriculi (Migula).

brugge.* The total number of permanent residents is not
known. During infancy the predominating organism seems
to be Bacillus lactis aerogenes; during adult life, Bacillus
coli. Streptococci, especially Streptococcus coli gracilis,
are also very common, if not invariable, inhabitants of the
intestine. The total bacteria that finally appear in the
feces, according to the studies of Strasburgerf and Steele,J
may reach the enormous figure of 38 per cent, of the total
bulk.

MacNeal, Latzer, and Kerr,§ in an elaborate work upon the
" Fecal Bacteria of Healthy Men," found that they furnished
46.3 per cent, of the total fecal nitrogen.

: "Centralbl. f. Bakt.," etc., Bd. xxx, 1901, pp. 10 and 70.
f "Zeitschrift fur klin. med.," 1902, xuv, 5 and 6; 1903, XLVIII,
5 and 6.

J "Jour. Amer. Med. Assoc.," Aug. 24, 1907, p. 647.

$ "Journal of Infectious Diseases," 1909, vi, pp. 132, 571.

Bacteria of the Digestive Apparatus 85

Rettger * found the Bacillus enteritidis sporogenes regu-
larly present in the human feces and believes it to be respon-
sible for some of the putrefactive processes that occur there.

The vagina, on account of its acid secretions, harbors
but few bacteria. In a study of the vaginal secretions of
40 pregnant women who had not been subjected to digital
examinations, douches, or baths, Bergholm t found but
few organisms of limited variety.

The uterus harbors no bacteria in health, and but few in
disease. The intervening acidity of the vagina makes it
difficult for bacteria from the surface to penetrate so deeply,
and tha tenacious alkaline mucus of the cervix is an addi-
tional barrier to their progress. Careful studies of the
bacteriology of the uterine secretions have been made by
Gottschalk and Immerwahr J and Doderlein and Win-
terintz. §

The urethra harbors a few cocci which enter the meatus
from the surface and remain local in distribution.

The normal bladder is free from bacteria.

The nose constantly receives enormous numbers of bac-
teria in the dust of the inspired atmosphere. These organ-
isms are too numerous and too various to enumerate, and
might, indeed, comprehend the entire bacterial flora. But
in spite of the large numbers of organisms received, the nose
retains scarcely any, its mucous membranes seeming to be
provided with means of disposing of the organisms. Among
those best able to withstand the destructive influences, and,
therefore, most apt to be found in the deeper passages, are
the pseudodiphtheria bacillus, streptococci, pneumococci,
staphylococci, Bacillus pneumoniae (Friedlander) , Bacillus
subtilis and sarcina. A complete review of the subject with
references to the literature has been made by Hasslauer.||

The larynx and trachea contain very few bacteria and
probably have no permanent parasitic flora.

The lungs harbor no bacteria. A few micro-organisms
doubtless reach them in the inspired air, but the defensive
mechanisms soon dispose of them.

* "Jour, of Biological Chemistry," n, i and 2, Aug., 1906, p. 71.

t "Archiv f. Gynak.," Bd. LXIV, Heft. 3.

J Ibid., 1896, Bd. L, Heft 3.

§ Hegar's " Beitrage fur Geburtshiilfe und Gynakologie," Bd. in,
Heft. 2.

|| "Centralbl. f. Bakt. u. Parasitenk. I. abt. Referata.," Bd. xxxvn,
Nos. 1-3, p. i, and Nos. 4-6, p. 97.

86 Infection

AVENUES OF INFECTION.

The skin seems to form an effectual barrier against the
entrance of bacteria into the deeper tissues. A few higher
fungi — Trycophyton, Microsporon, Achorion, etc. — seem
able to establish themselves in the superficial layers of the
cells, invade the hair-follicles, and so reach the deeper
layers, where morbid changes are produced. The minute
size of the bacteria makes it possible for them to enter
through lesions too small to be noticed. Garre applied a
pure culture of Staphylococcus pyogenes aureus to the skin
of his forearm, and found that furuncles developed in four
days, though the skin was supposed to be uninjured. Bock-
hart moistened his skin with a suspension of the same organ-
ism, gently scratched it with his finger-nail, and suffered
from a furuncle some days later.

The greater number of surgical infections result from the
entrance of bacteria through lesions of the skin. It makes
but little difference to what depth the lesion extends,—
abrasions, punctures, lacerations, incisions, — the protective
covering is gone and the infecting organisms find themselves
in the tissues, surrounded by the tissue lymph, under con-
ditions appropriate for growth and multiplication, provided
no inhibiting or destructive mechanism be called into action.

The digestive apparatus is the portal through which
many infections take place. The Bacillus diphtherise,
finding its way to the pharynx, speedily establishes itself
upon the surface, producing pseudomembranous inflam-
mation there. Typhoid bacilli, dysentery bacilli, cholera
spirilla and related organisms, finding their way to the
intestine, where the vital conditions are appropriate, take
up temporary residence there, to the inconvenience of the
host, who suffers from the respective infections.

Various organisms pass from the pharynx to the tonsils
and so to the lymph-nodes and deeper tissues of the neck,
where their first operations may be observed.

It is supposed by some pathologists that the digestive
tract is a constant menace to health in that it regularly
admits bacteria, through the lacteals, and perhaps through
its capillaries, to the blood, where under slightly abnormal
conditions they might do harm. According to Adami,*

* "Jour, of the American Medical Association," Dec. 16 and 23,
1899, vol. xxxm, Nov. 25 and 26.

The Avenues of Infection 87

the intestine is responsible - for a condition of sub-infection
depending upon the constant entrance of colon bacilla into
the blood. He finds the colon bacillus in the blood, and
traces it to the liver, where its final dissolution takes place
in the fine dumbbell-like granules enclosed in the cells,.
Nicholls* confirms Adami by finding similar dumbbell or
diplococcoid bodies in the epithelial denuded tissues of the
mesentery of normal animals.

Nicholas and Descosf and RavenelJ fed fasting dogs upon
a soup containing quantities of tubercle bacilli, killed them
three hours later, and examined the contents of the thoracic
duct, where tubercle bacilli, some alive and some dead, were
found in large numbers, van Steenberghe and Grysez§
found that carbon particles readily passed through the
intestinal mucosa, entered the lymphatics, were thrown
into the venous circulation, and so carried to the lung, where
anthracosis was produced.

In a subsequent paper || they believe that they have demon-
strated that the tubercle bacillus like the carbon particles
may also pass through the normal intestinal wall, and follow
the same course to the lungs. They believe that pulmonary
tuberculosis thus depends upon ingested and not inhaled
micro-organisms. At my suggestion Montgomery** en-
deavored to repeat the work of van Steenberghe and Grysez
at the Henry Phipps Institute, Philadelphia, but though
many attempts were made by various methods, no carbon
particles were transported from the alimentary to the pul-
monary tissues.

But there are enough experiments recorded to make it
probable that the wall of the intestine is permeable to bacteria,
and that in small numbers they constantly enter the blood of
healthy animals, to be disposed of by mechanisms yet to be
described.

Many of the bacteria penetrating the intestine must be
retained in the lymph nodes; others, as in the experiment
with the tubercle bacilli, meet destruction before they reach
the blood; the remainder must reach the blood alive.

* "Jour. Med. Research.," vol. xi, No. 2.
t "Jour, de Phys. et Path, gen.," 1902, iv, 910-912.
I "Jour. Med. Research." x, p. 460, 1904.

§ "Ann. de 1'Inst. Pasteur," Tome xix, No. 12, p. 787, Dec. 25, 1905.
|| Ibid., 1910, xxiv, 316.
** "Jour, of Med. Research," Aug., 1910, vol. xxm, No. i.

88 Infection

The presence of colon bacilli in the greater number of the
organs shortly after death has led some pathologists to
assume that they readily pass through the intestinal walls
during the death agony, but although experiments have been
made to prove and to disprove it, the matter is still con-
troversial. Undoubtedly in the final dissolution some
change takes place in the constitution of the individual by
which general invasion by bacteria is made more easy than
under normal conditions.

The respiratory apparatus affords admission to a few
micro-organisms whose activities seem more easily carried
on there than elsewhere. Although it is still controversial
whether the inhalation of tubercle bacilli is as frequent a
mode of conveying that organism into the body as was once
supposed, it cannot be denied that its inhalation will account
for the far greater frequency with which tuberculosis affects
the lungs than other organs of the body.

Pneumonia, caused in an immense majority of cases by
the pneumococcus of Fraenkel and Weichselbaum, probably
results from the entrance of the organism into the respira-
tory tissues directly.

The entrance of the unknown infectious agents causing
measles, German measles, smallpox, and scarlatina, can best
be accounted for by supposing that they are inhaled into
the lungs and thus enter the blood.

The genital apparatus is the portal of entry of micro-
organisms whose early or chief operations are local. Among
these are the gonococcus, which causes urethritis, vaginitis,
balanitis, posthitis, endometritis, orchitis, salpingitis, vesicu-
litis, cystitis, oophoritis, sometimes peritonitis, and rarely
endocarditis; the bacillus of Ducrey, that causes the chan-
croid or soft sore; and the treponema of syphilis. In more
rare cases other organisms, such as the common cocci of
suppuration and the tubercle bacillus, may also be trans-
mitted from individual to individual by sexual contact.

The placenta usually forms a barrier through which
infectious agents find their way with difficulty. A study
of this subject by Neelow* shows that the non-pathogenic
organisms do not pass from the mother through the placenta
to the fetus. Some pathogenic micro-organisms, however,
readily pass through, and a few diseases, such as syphilis,
are well known in the congenital form. Pregnant women
* "Centralbl. f. Bakt.," etc., I. Abt. Bd. xxxi, Orig., Aug., 1902, p. 691.

Pathogenesis 89

suffering from smallpox may be delivered of infants with
marks indicative of prenatal disease. Some common
infectious agents, such as the tubercle bacillus, seem to infect
unborn animals with difficulty. The frequency of antenatal
tuberculous infection is, however, somewhat controversial
at present, Baumgarten having reached the opinion, exactly
the opposite of what is commonly believed, that most children
are subject to antenatal infection, though the bacilli sub-
sequently develop and cause disease in only a few of them.

PATHOGENESIS.

This subject can be understood only through a broad
knowledge of the metabolic products of micro-organisms.
In general it may be said that the ability of micro-organisms
to do harm depends upon the injurious nature of their
products. This alone, however, will not explain the phe-
nomena of infection, for in many cases the intoxication is
subsidiary in importance to the invasive power of the micro-
organisms. Some bacteria having but limited toxic powers
possess extraordinary powers of invasion, as Bacillus an-
thracis, and the intoxication becomes important only after the
organisms have penetrated to all the tissues of the body.
Others, with more active toxic properties, have but limited
invasive powers, and a few organisms, growing with difficulty
in some insignificant focus, excite actively destructive
reactions in the tissues with which they come into contact.
Still others, with limited invasive powers, eliminate active
toxic substances, soluble in nature, that enter the circulation
and act upon cells remote from the bacteria themselves, as
in diphtheria and tetanus.

The invasive power, of the organisms depends upon their
ability to overcome the body defenses. This may indicate
activity of the infecting organism, or weakness of the defen-
sive mechanism. The relation of these factors is exceedingly
complex, only partly understood, and will be fully discussed
in the chapter upon Immunity.

For convenience toxins may be described as intracellular
or insoluble, and extracellular or soluble.

The intracellular toxins. These products are but little
known and have only recently begun to attract attention.
Their insoluble nature makes it difficult to isolate them, and
determines the limitations of their activity. Until the in-

go Infection

vestigations of Vaughan, Cooley and Gelston,* and later
Vaughan and his associates, Detweiler,f Wheeler, J Leach, §
Marshall and Gelston, || Gelston,** J. V. Vaughan,ft
Wheeler, ft Leach, §§ Mclntyre,|| || and others, it seemed re-
markable that micro-organisms whose filtered cultures con-
tained little demonstrable toxic substance are sometimes
able to produce active pathogenic effects. By means of
special apparatus in which the micro-organisms could be
cultivated in enormous quantities, and the disintegration of
the micro-organismal masses secured by subjecting them
to high temperatures, to the action of mineral acids or
autolysis, it was discovered that the colon bacilli, typhoid
bacilli, and many supposedly harmless bacteria, contain
intensely active toxic substances. In all probability some
of the toxic substances produced by such means are artefacts,
but enough work has been done to prove that insoluble toxic
substances are present in such organisms, and the toxic
substances obtained by the comminution of culture masses
made solid and brittle by exposure to liquid air, as suggested
by Macfadyen and Rowland; the autolytic digestion of bac-
teria washed free of their culture fluids and suspended in
physiological salt solution, and the dissolution of bacteria
by bacteriolytic animal juices clearly prove that endotoxins
exist.

It seems probable that there is considerable difference in
the readiness with which these intracellular toxic substances
are given up by the bacteria. From some they seem never
to be set free in the bodies of animals into which the bacteria
are injected; thus, Bacillus prodigiosus is harmless for
animals, no matter what quantity is injected, yet active
toxic substances can be extracted from the bodies of these
organisms by appropriate chemical means. From others
they are given off in small quantities either during the life
of the organism, or at the moment of death and dissolution,
as in the case of the typhoid bacillus and streptococci, whose
filtered cultures are almost harmless, though both organisms
are pathogenic.

* "Journal of the American Medical Association," Feb. 23, 1901;
"Trans. Assoc. Amer. Phys.," 1901, "American Medicine," May,
1901.

t "Trans. Assoc. Amer. Phys.," 1902. J Ibid.

§ Ibid. || Ibid. ** Ibid. ft Ibid.

|J "Jour. Amer. Med. Assoc.," 1904, xui, p. 1000.
§§ Ibid., p. 1003. llll Ibid., p. 1073

Pathogenesis 91

The intracellular toxins are limited in action by the
distribution of the bacteria producing them. When these
organisms are but slightly invasive, more or less local reaction
is produced ; when they are actively invasive, general reac-
tions of varying intensity result.

The extracellular toxins, of which those of Bacillus
tetani and Bacillus diphtheriae can be taken as types, have
been known since the early work of Brieger and Fraenkel
and Roux and Yersin. They seem to be excretions of the
bacteria, not retained in the cells, but eliminated from them
as rapidly as they are formed. Thus, in appropriate bouillon
cultures of the diphtheria bacillus, the toxin is present in
large quantity and is highly virulent, but if the fluid be
removed from the bacteria by porcelain filtration and the
remaining bacilli carefully washed, their bodies are found to
be devoid of toxic powers. The poison is most concentrated
where its diffusion is most restricted, thus, agar-agar cultures
of the tetanus bacillus are much more toxic than bouillon
cultures because the soluble principle readily diffuses through
the fluid, but is held by the less diffusible agar-agar.

The soluble toxin is but one of numerous metabolic prod-
ucts of the bacteria. Thus in culture filtrates of the
tetanus bacillus there are at least two very different active
substances, the tetano-spasmin that acts upon the nervous
system with convulsive effect, and the tetano-lysin that is
solvent for erythrocytes.

In all probability all of the culture filtrates of bacteria
are highly complex because of the addition of the various
metabolic products — toxins, lysins, enzymes, pigments, acids,
etc. — of the bacteria, as well as because of changes produced
in the medium by the abstraction of those molecular con-
stituents upon wh'ich the bacteria have fed. This com-
plexity makes it difficult to accurately study the toxins,
which we scarcely know apart from their associated products.

The chemic nature of the toxins differs. Undoubtedly
some are tox-albumins, but others are of different composi-
tion and fail to give the reactions belonging to the compounds
of this group.

The variations observed in toxicogenesis under experi-
mental conditions in the test-tube indicate that similar
variations occur in the bodies of animals, and a few experi-
ments conducted with slight variations in the composition
and reaction of the media in which the bacteria grow will

92 Infection

suffice to show that the exact effect of toxicogenic bacteria
in the bodies of different animals cannot always be accurately
prejudged.

The physiologic and pathogenic action of the extracellular
soluble toxins differs from that of the intracellular and dif-
ficultly soluble toxins in that it is more easily diffused
throughout the animal juices, and that its diffusion is inde-
pendent of the invasiveness of the bacteria, so that a few
organisms growing at some focus of unimportant magnitude,
and causing but little local manifestation, may be able to
produce a profound impression upon remote organs. This
is best exemplified in the case of the Bacillus tetani, which,
finding its way into the tissues under proper conditions,
produces scarcely any local reaction, — indeed, the lesion may
be undiscoverable, — yet may cause the death of the animal
through the intensity of its action upon the central nervous
system.

SPECIFIC ACTION OF TOXINS.

The metabolic products of the greater number of injurious
bacteria are characterized by irritative action upon those
body cells with which they come into contact. If through
the intracellular nature of the poisons and the mildly invasive
character of the micro-organisms this action is restricted
to the seat of original infection, a local manifestation will
result. Its exact nature will, however, be modified to some
extent by other qualities of the bacterial products. Thus,
when in addition to their irritative action which, when mild,
occasions multiplication of the cells of the connective and
lymphoid tissues, and, when extreme, effects the death of
the cells, the products are strongly chemotactic, suppuration
will occur.

Fever and suppuration are, therefore, non-specific actions,
because numerous micro-organisms share the qualities pro-
ductive of these conditions in common.

If the bacteria are rapidly invasive, but still have injurious
products of the intracellular variety, they are apt to share
certain qualities, such as the swelling of the lymph-nodes,
etc., in common, so that such lesions cannot be considered as
specific. So soon as any one of the products is discovered to
give some single lesion peculiar to that organism by which
it is produced, or so soon as the total effect of the activity
of the various products of any micro-organism produces a

Specific Affinity of the Cells for the Toxins 93

typical effect, differing from the total effect of the operation
of other micro-organisms, so that a recognized type of dis-
ease results, it becomes possible to say that the micro-organ-
ism in question is specific.

The most striking examples of the specific action of
bacterio-toxins is, however, seen in those cases where soluble
extracellular metabolic products of bacterial energy are
liberated into the body juices so as to be conveyed by the
circulatory system to all parts of the body. Those cells
most susceptible to its action are then first or most pro-
foundly impressed by it, and definite responses brought
about. Thus, the soluble toxin of tetanus causes no visible
reaction in the cells with which it first comes into contact
at the seat of primary infection, because these cells are
either less susceptible to its influence, or are less well able
to show its effects, than the cells of the nervous system to
which it is secondarily carried by the blood.

SPECIFIC AFFINITY OF THE CELLS FOR THE TOXINS.

The cells of the connective tissue in which the tetanus
bacillus is living show little reaction, but the motor cells of
the central nervous system, having a greater affinity for it,
are profoundly impressed, so that convulsions of the con-
trolled muscular system are brought about. This special
excitation of the nerve cells is specific because no other
bacterio-toxin is known to produce it and it is attributed
to special selective affinities of the nerve cells for the poison.
This affinity has its analogue among the poisons of higher
plants, thus, strychnin has a similar selective affinity and is
also said to be specific in action upon the motor cells.

The venoms of various serpents, especially the cobra, also
have specific reactions, the cells of the respiratory centers
seeming to be most profoundly affected by them.

The diphtheria bacillus, when observed in ordinary throat
infections, is seen to produce a pseudomembranous angina
which results in part from an irritative local action of the
organism, which it shares in common with many others,
and in part from some coagulating product which it shares
in common with a few — pneumococcus, streptocococus, etc.
Neither of these reactions is specific, but subsequent to these
early manifestations comes depressant action on the nervous
cells with palsy, peculiar to the products of the diphtheria
bacillus, and therefore specific.

94 Infection

It is upon the peculiar specific reactions of the bacterio-
toxins and the peculiar susceptibility of certain cells to this
action that the production of distinct clinical manifestations
depend.

THE INVASION OF THE BODY BY MICRO-ORGANISMS.

Some bacteria whose invasiveness is insufficient to enable
them successfully to maintain life in healthy tissues, occa-
sionally get a foothold in diseased tissues and assist in morbid
changes. This is sometimes seen in what is described by
the surgeons as sapremia, in which various saprophytic
bacteria, possessing no invasive powers, by growing in the
putrefying tissues of a gangrenous part, give rise to poison-
ous substances which when absorbed by the adjacent
healthy tissues produce constitutional disturbances, such
as depression, fever, and the like.

Bacteria with limited invasive powers and intracellular
toxins can at best occasion local inflammatory effects.
Such organisms not infrequently vary, however, and when
of unusual vitality may survive entrance into the blood and
lymph circulations and occasion bacteremia, or, as it is more
frequently called, septicemia, a morbid condition charac-
terized by the presence of bacteria in the circulating blood.
When bacteria entering into the circulation are unable to
pervade the entire organisms and continue in the circulation,
they may collect in the capillaries of the less resisting tissues,
producing local metastatic lesions, usually purulent in
character. This form of invasion results in what is surgically
known as pyemia.

The mode by which the entrance of bacteria into the
circulation is effected differs in different cases. Kruse *
believes that they sometimes are passively forced through
the stomata of the vessels when the pressure of the inflam-
matory exudate is greater than that of the blood within
them; that they may sometimes enter in the bodies of
leukocytes that have incorporated them; that they may
actually grow through the capillary walls, or that they reach
the blood circulation indirectly by first following the course
of the lymphatics.

Toxemia results from the absorption of the poisonous
bacterial products from non-invasive bacteria, as in tetanus.

* Fliigge, "Die Mikroorganismen, " vol. i, p. 271.

The Cardinal Conditions of Infection 95

THE CARDINAL CONDITIONS OF INFECTION.

Infection can take place only when the micro-organisms
are sufficiently virulent, when they enter in sufficient num-
ber, when they enter by appropriate avenues, and when the
host is susceptible to their action.

Virulence. — Virulence may be defined as the disease-
producing power of micro-organisms. It is a variable
quality, and depends upon the invasiveness of the bacteria,
or the toxicity of their products, or both.

A few bacteria are almost constant in virulence and can
be kept under artificial conditions for years with very little
change. Other bacteria begin to diminish in virulence so
soon as they are introduced to the artificial conditions of
life in the test-tube. Still others, and perhaps the greater
number, can be modified, and their virulence increased or
diminished according to the experimental manipulations
to which they are subjected.

Variation in virulence is not always a peculiarity of the
species, for the greatest differences may be observed among
individuals of the same kind. Thus, the streptococcus
usually attenuates rapidly when kept in artificial media,
so that special precautions have to be taken to maintain it,
but Hoist observed a culture whose virulence was unaltered
after eight years' continuous cultivation in the laboratory
without any particular attention having been devoted to it.
What is true of different cultures of the same organisms, is
equally true of the individuals in the same culture. To
determine such individual differences is quite easy among
chromogenic bacteria. If these are plated in the ordinary
way it will be found that some colonies are paler and some
darker than others. Conn found that by repeating the
plating a number of times and always selecting the palest
and darkest colonies he was eventually able to produce two
cultures, one brilliant yellow, the other colorless, from the
same original stock.

Decrease of virulence under artificial conditions prob-
ably depends upon artificial selection of the organisms in
transplantation from culture to culture. When planted
upon artificial media, the vegetative members of the bacte-
rial family proceed to grow actively and soon exceed in
number their more pathogenic fellows. Each time the
culture is transplanted, more of the vegetative and fewer

96 Infection

of the pathogenic forms are carried over, until after the
organism is accustomed to its new environment, and grows
readily upon the artificial media, it is found that the patho-
genic organisms have been largely or entirely eliminated and
the vegetative forms alone retained.

Increase of virulence can be achieved by artificial
selection so planned as to preserve the more virulent or
pathogenic organisms at the same time that the less virulent
and more vegetative organisms are eliminated. In cases
in which no virulence remains, the experimental manipula-
tion of the culture is directed toward gradual immunization
of the micro-organisms to the defensive mechanisms of the
body of the animal for which the organism is to be made
virulent. A number of methods are made use of for this
purpose.

Passage Through Animals. — Except in cases where the
virulence of the micro-organism is invariable, it is usually
observed that the transplantation of the organism from
animal to animal without intermediate culture in vitro
greatly augments its pathogenic power. Of course, this
artificially selects those members of the bacterial family best
qualified for development in the animal body, eliminating
the others, and the virulence correspondingly increases.

The increase in virulence thus brought about is, however,
not so much an increase in the general pathogenic power of
the organism for all animals, as toward the particular animal
or kind of animal used in the experiments. Thus, in general,
the passage of bacteria through mice increases their virulence
for mice, but not necessarily for cats or horses; passage
through rabbits, the virulence for rabbits, but not necessarily
for dogs or pigeons, etc.

This specific character of the virulence is well accounted
for by the " lateral-chain theory of immunity," where it will
again be considered.

The Use of Collodion Sacs. — When cultures of bacteria
are enclosed in collodion sacs and placed in the abdominal
or other body cavities of animals, and kept in this manner
through successive generations, the virulence is usually con-
siderably increased. This is one of the favorite methods
used by the French investigators. It keeps the bacteria in
constant contact with the slightly modified body juices of
the animal, which transfuse through the collodion, and thus
impedes the development of such organisms as are not able

The Cardinal Conditions of Infection 97

to endure their injurious influences. Thus it becomes only
another way of carrying on an artificial selection of those
members of the bacterial family that' can endure, and elimi-
nating those that cannot endure the defensive agencies of
those juices with which the organisms come in contact.*

The addition of animal fluids to the culture-media some-
times enables the investigator to increase, and usually
enables him to maintain, the virulence of bacteria. The
cultivation of the organism should embrace a series of genera-
tions in gradually increasing concentrations of the body
fluid employed, until the organism becomes thoroughly
accustomed to it.

In some cases it may be sufficient to use a single standard
mixture, thus: Shaw f found that he could exalt the viru-
lence of anthrax bacilli by cultivating them upon blood-
serum agar for fourteen generations, after which they were
three times as active as cultures similarly transferred upon
ordinary agar-agar.

The increase under such conditions as these probably
depends upon the immunization of the bacteria to the body
juices of the animals, and this whole matter will be under-
stood after the subject "Immunity" has been considered.

Number. — The number of bacteria entering the infected
animal has a very important bearing upon infection, and
may itself determine whether it shall occur or not.

The entrance of a single micro-organism of any kind is
scarcely ever able to effect infection because of the uncer-
tainty of its being able to withstand the defensive mechanisms
of the animal into which it is introduced. In most cases
a considerable number of organisms is necessary in order
that some may survive the new environment. Park points
out that when bacteria are transplanted from culture to
culture, under conditions supposed to be favorable, many
of them die. It seems not improbable, therefore, that when
they are transplanted to an environment in which are present
certain mechanisms for defending the organism against them,
many more must inevitably die. The more virulent an
organism is, the fewer will be the number required to infect.
Marmorek, in his experiments with antistreptococcic serum,
used a streptococcus whose virulence was exalted by passage

* Directions for making and using the capsules are given in the
chapter upon Animal Experimentation,
f "Brit. Med. Jour.," May 9, 1903.

7

98 Infection

through rabbits and intermediate cultivation upon agar-
agar containing ascitic fluid, until one hundred thousand
millionth of a cubic centimeter (un cent milliardieme) was
fatal for a rabbit. In this quantity it is scarcely probable
that more than a single coccus could have been present.
Single anthrax or glanders bacilli may infect rabbits and
guinea-pigs. Roger found that 820 tubercle bacilli from the
culture with which he experimented were required to infect
a guinea-pig, when introduced beneath the skin. Herman
found that it required 4 or 5 c.c. of a culture of Staphylococ-
cus pyogenes to produce suppuration in the peritoneal cavity
of an animal; 0.75 c.c. to produce it beneath the skin; 0.25
c.c. in the pleura; 0.05 c.c. in the veins and o.oooi c.c. in the
anterior chamber of the eye.

In experimenting with Bacillus proteus vulgaris, Watson
Cheyne found that 5,000,000 to 6,000,000 organisms injected
beneath the skin did not produce any lesion ; 8,000,000 caused
the formation of an abscess; 56,000,000 produced a phleg-
mon from which the animal died in five or six weeks and
225,000,000 were required to cause the death of the animal
in twenty-four hours. In studying Staphylococcus aureus
upon rabbits he found that 25,000,000 would cause an
abscess, but 1,000,000,000 were necessary to cause death.

The Avenue of Infection. — The successful invasion of
the body by certain bacteria can be achieved only when
they enter it through appropriate avenues. Even when
invasion is possible through several channels, the parasite
most commonly invades through one that may, therefore,
be regarded as most appropriate, invasion through which
furnishes the typical picture of the infection.

Thus, gonococci usually reach the body through the uro-
genital mucous membranes, where they set up the various
inflammatory reactions collectively known as gonorrhea —
i. e., urethritis, vaginitis, prostatitis, orchitis, cystitis, etc.
These constitute the typical picture of infection. The
organism may also successfully invade the conjunctiva,
producing blennorrhea, but there is no evidence that gono-
cocci can successfully invade the body through the skin,
the respiratory, or alimentary mucous membrane.

Typhoid and cholera infections seem to take place through
the alimentary mucous membrane, and the evidence that
infection takes place by inhalation is slight. It is not known

The Cardinal Conditions of Infection 99

to take place through the urogenital system, the conjunctiva, '
or the skin.

The avenue of entrance not only determines infection,
but may also determine the form that it takes. Thus,
tubercle bacilli rubbed into the deeper layer of the skin
produce a chronic inflammatory disease, called lupus,
that lasts for years and rarely results in generalized tubercu-
losis. Bacilli reaching the cervical or other lymph-nodes by
entrance through the tonsils ,may remain localized, produc-
ing enlargement and softening of the nodes, or passing
through them reach the circulation, in which they may be
carried to the bones and joints and occasion chronic inflam-
mation with necrosis and ultimate evacuation or exfolia-
tion of the diseased mass, after which the patient may
recover. Bacilli entering the intestine in many cases pro-
duce implantation lesions in the intestinal walls; bacilli
inhaled into the lung, or conveyed to it from the intestine
by the thoracic duct and veins, produce the ordinary pul-
monary tuberculosis known as phthisis or consumption.

Inhaled pneumococci colonizing in the pharynx have been
known to produce pseudomembranous angina; in the lungs,
pneumonia ; implanted upon the conjunctiva, conjunctivitis.
Jn these cases we can look upon the type of infection as
depending upon the portal through which the invading
organism found its way into the tissues.

The avenue of entrance is, for obvious reasons, less
important when the micro-organism is of the rapidly invasive
form, whose chief operation is in the streaming blood or in
the lymphatics. Anthrax in most animals is characterized
by a bacteremia regardless of the point of primary infection.
Bubonic plague rapidly becomes a bacteremia regardless of
the entrance of the Bacillus pestis by inhalation into the
lungs, or by way of the lymphatics through superficial
lesions. The failure of the micro-organisms to colonize
successfully when introduced through inappropriate avenues
may be explained by a consideration of the local conditions
to which they are subjected.

When they are introduced beneath the skin, bacteria are,
in most cases, delayed in reaching the circulation, and are
in the meantime subjected to the germicidal action of the
lymph and exposed to the attacks of phagocytes. Many
succumb to these and never penetrate more deeply into the
body. Should any survive, they may be transported to

100

Infection

'the lymph-nodes and there destroyed, or, passing through
these barriers without destruction, and reaching the venous
channels, they have next to pass through the pulmonary cap-
illaries, where they are apt to be caught and destroyed. Fi-
nally, should any escape all these defenses and reach the
general circulation, it is to find the endothelium of the
capillaries prone to collect and detain them until destruction
is finally effected. The systemic circulation is also defended
against such micro-organisms^ as might reach the veins
through lesions or accidents of the abdominal viscera, by
the interposition of the portal capillary network of the liver,
where the bacteria are caught and many of them destroyed,
or passing which, the pulmonary capillary system acts as a
second barrier against them. The deeper the penetration, the
more active the defense becomes, the blood itself furnishing
agglutinins, bacterio-lysins, and phagocytes for the destruc-
tion of the micro-organisms and the protection of the host.

These defenses, however, are of no avail against actively
invasive organisms provided with the means of overcoming
them all through aggressins that destroy the germicidal
humors or toxins that kill or paralyze the cells. When
these are injected directly into the streaming blood they
produce their effects more rapidly than when injected
beneath the skin or elsewhere, because the field of operation
is immediately reached instead of through a roundabout
course in which so many defenses have to be overcome.
Taking anthrax bacilli, whose invasiveness has already been
dwelt upon, as an example, Roger * found that when the
organisms were injected into the aorta, animals died more
quickly than when they were injected into the veins and
obliged to find their way through the pulmonary capillaries
to the general circulation. If the injections were made into
the portal vein, the animals stood a good chance of recovery,
the liver possessing the power of destroying sixty-four
times as many anthrax bacilli as would prove fatal if intro-
duced through other channels.

The conditions differ, however, in different infections,
for when Roger experimented with streptococci instead of
anthrax bacilli, he found that if the bacilli were inoculated
into the portal vein the animals died more quickly than
when they were injected into the aorta, and that when
the bacilli were injected into the peripheral veins the ani-
*" Introduction to the Study of Medicine," p. 151.

The Cardinal Conditions of Infection 101

mals lived longest, the liver seeming to be far less destruc-
tive to streptococci than the lungs.

The Susceptibility of the Host.— Susceptibility is lia-
bility to infection. It is a conditon in which the host is
unable to defend itself against invading micro-organisms.
Unusual or unnatural susceptibility is also spoken of as
predisposition or dyscrasia.

Many animals and plants are naturally without any means
of overcoming the invasiveness of certain parasitic micro-
organisms, and are, therefore, naturally susceptible; others
naturally resist their inroads, but through various temporary
or permanent physiologic changes may lose the defensive
power.

In general, it is true that any condition that depresses or
diminishes the general physiological activity of an animal
diminishes its ability to defend itself against the pathogenic
action of bacteria, and so predisposes to infection. These
changes are often so subtile that they escape detection,
though at times they can be partly understood.

The inhalation of noxious -vapors. It has long been
supposed that sewer gas was responsible for the occurrence
of certain infectious diseases, and when the nature of these
diseases was made clear by a knowledge of their bacterial
causes, the old belief still remained and many sanitarians
continued to believe that defective sewage is in some way
connected with their occurrence. It is difficult to prove
or disprove the matter experimentally. Men who work
in sewers and plumbers who breathe much sewer gas are
not apparently affected by it. Alessi* found that rats,
rabbits, and guinea-pigs kept in cages some of which were
placed over the opening of a privy, while in others the
excreta of the animals were allowed to accumulate, suffered
from a pronounced diminution of the resisting powers.
This would seem to be inconsistent with the habits of rats,
many of which live in sewers. Abbott f caused rabbits to
breathe air forced through sewage and putrid meat infusions
for one hundred and twenty-nine days, and found that the
products of decomposition inhaled by the animals played no
part in producing disease, or in inducing susceptibility to it.

Fatigue is a well-recognized clinical cause of suscep-
tibility to disease, and experimental evidence of its

*"Centralbl. f. Bakt.," etc., 1894, xv, p. 228.
t" Trans. Assoc. Amer. Phys.," 1895.

102 Infection

correctness is not wanting. Charrin and Roger* found that
white rats, which naturally resist infection with anthrax,
succumbed to the infection if compelled to turn a revolving
wheel until exhausted before inoculation.

Exposure to cold seriously influences the resisting power
of the warm-blooded animals. It is an everyday expe-
rience that chilling the body predisposes to " cold " and
may be the starting-point of pneumonia. Pasteur found
that fowls, which resist anthrax under normal conditions,
succumbed to infection if kept for some time in a cold bath
before inoculation.

The reverse seems to be true of the cold-blooded animals,
for Gibierf found that frogs, naturally resistant to the
anthrax bacillus, would succumb to infection if kept at 37° C.
after inoculation.

Diet produces some variation in the resisting powers.
The tendency of scorbutics to suffer from infectious dis-
orders of the mouth, the frequency with which epidemics
of infectious disease follow famines, and the enterocolitis of
marasmatic infants, illustrate the effects of insufficient food
in predisposing to disease. We also find that the infectious
diseases of carnivorous animals are not the same as those of
herbivorous animals, and that the former are exempt from
many disorders to which the latter quickly succumb.
Hankin was able to show experimentally that meat-fed rats
resisted anthrax infection far better than rats fed upon
bread.

Intoxication of all kinds predisposes to infection. Plata-
niaj found that such animals as frogs, pigeons, and dogs
became susceptible to anthrax when under the influence
of curare, chloral, and alcohol. Leo§ found that white rats
fed upon phloridzin became susceptible to anthrax. Wag-
ner || found that pigeons become susceptible to anthrax when
under the influence of chloral. Abbott** found the resisting
powers of rabbits against Streptococcus pyogenes and

* " Compte rendu Soc. de Biol de Paris," Jan. 24, 1890.

f'Compte rendu Acad. des Sciences de Paris," 1882, t. xcix, p.
1605.

JSee Sternberg's " Immunity and Serum Therapy," p. 10; " Cen-
tralbl. f. Bakt.," etc., Bd. vn, p. 405.

§ " Zeitschrift fiir Hyg.," Bd. vn, p. 505, 1889.

|| " Wratsch," 1890, 39, 40.
** " Jour, of Exp. Med.," vol. i, No. 3, 1896.

Mixed Infections 103

Bacillus coli diminished by daily intoxication with 5 to
15 c.c. of alcohol introduced into the stomach through a tube.
Salant* found that alcohol was disadvantageous in com-
batting the infectious diseases because it diminished the
glycogen content of the liver which Collaf had found an
important adjunct in supporting the resisting power.

It is a common clinical observation that excessive indul-
gence in alcohol predisposes to certain infections, notably
pneumonia, and every surgeon knows the danger of pneu-
monia after anesthetization with ether.

Traumatic injury and mutilation of the body are not
without effect upon infection. The more extensive the
damage done to the tissues, the greater the danger of infec-
tion, and the more serious the consequences of infection when
it takes place.

The mutilation of the body by the removal of certain
organs is of disputed importance. There is much literature
upon the effect of the spleen in overcoming infectious agents,
but the experimental evidence seems about equally divided
as to whether an animal is more or less susceptible after
the removal of this organ than it was before.

Morbid conditions in general predispose to infection.
The frequency with which diabetics suffer from furuncles,
carbuncles, and local gangrenous lesions of the skin; the
increased susceptibility of phthisics to bronchopneumonia
of other than tuberculous origin ; the apparent predisposition
of injured joints and pneumonic lungs to tuberculosis; the
extensive streptococcus invasions accompanying scarlatina
and variola ; the presence of Bacillus icteroides and various
other organisms in the blood and tissues of yellow fever
patients, and the presence of Bacillus sui pestifer in the
bodies of hogs suffering with hog cholera, all show the
diminution in the general resisting power of an individual
already diseased.

MIXED INFECTIONS.

The general prevalence of bacteria determines that few
can enter and infect the body of a host without the associa-
tion of other kinds. Therefore their operation in the body
is subject to modifications produced in them or in the host
by these associated organisms.

* "Jour. Amer. Med. Assoc.," XLVII, 18, Nov. 3, 1906, p. 1467.
t " Archiv. Ital. de Biologic," xxvi.

104 Infection

In experimental investigations this fact is not infrequently
forgotten, and it is often remarked with surprise that the
results of inoculation with pure cultures of a micro-organism
may be clinically different from those observed under natural
conditions.

The tetanus bacillus, which endures with difficulty the
effects of uncombined oxygen, flourishes in association with
saprophytic organisms by which the oxygen is absorbed.
The same thing is probably true of other obligatory anaerobic
organisms.

The metabolic products of one species may intensify or
accelerate the action of those of an associated species, or
the reverse may be true, and the products of different organ-
isms, having different chemical composition, may neutralize
one another, or combine to form some entirely new sub-
stance which is entirely different from its antecedents.
Such conditions cannot fail to influence the type and course
of infection.

CHAPTER IV.
IMMUNITY.

IMMUNITY is ability to resist infection. It is the ability
of an organism successfully to antagonize the invasive
powers of parasites, or to annul the injurious properties of
their products. The mechanism of immunity is compli-
cated or otherwise according to circumstances. When the
invasive action of non-toxicogenic bacteria is to be over-
come, certain reactions, mostly on the part of the phagocytic
cells, are called into action; when the toxic products of
bacteria are to be deprived of injurious effects, the reaction
seems to take place between the toxin and certain com-
bining and neutralizing substances contained in the body
juices; when bacterial invasion and intoxication are both
to be" antagonized, both mechanisms are engaged in the
defenses, comparatively simple or exceedingly complex,
according to the conditions involved. The more involved the
conditions of infection become, the more complicated the
defensive reactions become, until it may no longer be pos-
sible accurately to analyze them.

Some have endeavored to refer all of the phenomena of
immunity to the ability of the animal to endure the bacterio-
toxins, and have sought to relegate the reactions against
invasion to a subsidiary place. This is undoubtedly an
error, as the mechanisms are different and the prompt action
of one may make the action of the other unnecessary.
Metschnikoff* found that frogs injected with 0.5 c.c. of
cholera toxin died promptly, but that frogs injected with
cultures of the cholera spirillum recovered without illness.
This would suggest that the recovery of the infected frog
depended upon some defensive mechanism combating the
invasiveness of the bacteria and so preventing the produc-
tion of the toxin to which the frog was susceptible.

Immunity must not be conceived as something insepar-

* " Immunite dans les Maladies Infectieuses," Paris, 1901, p. 150.

105

io6 Immunity

ably associated with infection. The reactions of the body
toward bacteria in the infectious diseases are identical with
those toward other minute irritative bodies, and the reactions
toward bacterio-toxins are identical with those toward other
active substances, so that the only way by which a sat-
isfactory understanding of the phenomena can be reached
is by carefully comparing the reactions produced by bacteria
and their products with those produced by other active
bodies.

Immunity is called active when the animal protects itself
through its own activities, passive when its protection
depends upon defensive substances prepared by some other
animal and forced upon it. Thus, if a frog be injected with
anthrax bacilli, the leukocytes devour the bacteria, destroy
them, and so protect the frog from infection, the immunity
is active because it depends upon the activity of the frog's
phagocytes. But if a guinea-pig previously given anti-
tetanic serum be injected with tetanus toxin, and so recovers
from the toxin, the resisting power, conferred by the anti-
toxin previously injected, does not depend upon any activity
of the animal, which remains entirely passive.

Immunity is largely relative. Fowls are immune against
tetanus, that is, they can endure, without injury, as much
toxin as tetanus bacilli can produce in their bodies, and
suffer no ill effects from inoculation. If, however, a large
quantity of tetanotoxin produced in a test-tube be intro-
duced into their bodies, they succumb to it. Mongooses
and hedgehogs are sufficiently immune against the venoms
of serpents to resist as much poison as is ordinarily injected
by the serpents, but by collecting the venom from several
serpents and injecting considerable quantities of it, both
animals can be killed. Rats cannot be killed by injection
with Bacillus diphtheriae, and Cobbett* found that they
could endure from 1500 to 1800 times as much diphtheria
toxin as guinea-pigs, though more than this would kill them.

Carl Fraenkel has expressed the whole matter very
forcibly when he says: "A white rat is immune against
anthrax in doses sufficiently large to kill a rabbit, but not
necessarily against a dose sufficiently large to kill an ele-
phant."

* "Brit. Med. Jour.," April 15, 1899.

Natural Immunity 107

NATURAL IMMUNITY.

Natural immunity is the natural, inherited resistance
against infection or intoxication, peculiar to certain groups
of animals, and common to all the individuals of those
groups.

Few micro-organisms are capable of affecting all varieties
of animals; indeed, it is doubtful whether any known
organism possesses such universally invasive powers.

The micro-organisms of suppuration seem able to infect
animals of many different kinds, sometimes producing local
lesions, sometimes invading rapidly with resulting bactere-
mia. The tubercle bacillus is known to be pathogenic for
mammals, birds, reptiles, batrachians, and fishes, though it
is still uncertain whether the infecting organisms in these
cases are identical or slightly differing species.

As a rule, however, the infectivity of bacteria and other
micro-organisms is restricted to certain groups of animals
which usually have more or less resemblance to one another ;
thus, anthrax is essentially a disease of warm-blooded ani-
mals, though certain exceptions are observed, and Metschni-
koff has found that hippocampi (sea-horses), perch, crickets,
and certain mussels are susceptible. Among the warm-
blooded animals anthrax is most frequent among the her-
bivora, though some carnivora may also be infected.

Close relationship is not, however, a guarantee that
animals will behave similarly toward infection. The rabbit,
guinea-pig, and the rat are rodents, but though the rabbit
and guinea-pig are susceptible to anthrax, the rat is immune.
This is still better exemplified in the susceptibility of mice
to glanders. The field-mouse seems to be the most suscep-
tible of all animals to infection with Bacillus mallei; the
house mouse is much less susceptible, and the white mouse
is immune. Mosquitos, though closely related, are differ-
ent in their immunity to the malarial parasite. The culex
does not harbor the parasite at all, and of the anopheles,
two very similar species seem to behave very differently,
Anopheles maculipennis being the common definitive host
of the parasite, while Anopheles punctipennis is not known
to be susceptible to it. The same differences may exist among
the members of the human species. It has been asserted
that Mongolians, and especially Japanese, are immune

io8 Immunity

against scarlatina, and that negroes are immune against
yellow fever, but increasing information is to the contraryc

Human beings suffer from typhoid, cholera, measles,
scarlatina, yellow fever, varicella, and numerous other
diseases unknown among the lower animals, even those
domestic animals with which they come in close contact.
They also suffer from Malta fever, anthrax, rabies, glanders,
bubonic plague, and tuberculosis, which are common among
the lower animals. Animals, in turn, suffer from distemper,
hog cholera, Texas fever, swine-plague, chicken cholera,
mouse septicemia, etc., the respective micro-organisms of
which are not known to infect man.

It has already been pointed out that mongooses and
hedgehogs are immune against the venom of serpents from
which other animals quickly die. The tobacco-worm lives
solely upon tobacco-leaves, the juice of which is intensely
poisonous to higher animals, and is also a good insecticide.
Boxed cigars and baled tobacco are often ruined by the
larvae of a small beetle that feeds upon them, and a glance
over the poisonous vegetables will show that few of them
escape the attacks of insects immune against their juices.

These facts are sufficient to show that many animals
are by nature immune against the invasion of microparasites
of certain kinds, and that they are also at times immune
against poisons. Immunity against one kind of infection
or intoxication is, however, entirely independent of all other
infections and intoxications. Immunity against infection
usually guarantees exemption from the toxic products of
that particular micro-organism, though experiment may
show the animal to be susceptible to it. Immunity against
any form of bacterio-toxin usually, though not necessarily,
determines that the micro-organism, though it may be able
to invade the body, can do very little harm.

ACQUIRED IMMUNITY*

Acquired immunity is resistance against infection or in-
toxication possessed by certain animals, of a naturally sus-
ceptible kind, in consequence of conditions peculiar to them
as individuals. It is a peculiarity of the individual, not of
his kind, and signifies a subtile change in physiology by
which latent defensive powers are stimulated to action.
The reactions in general correspond with those of natural
immunity, and comprise mechanisms for overcoming the

Acquired Immunity 109

invasion of pathogenic organisms, for neutralizing or destroy-
ing their toxins or both. As an acquired character and an
individual peculiarity it is not transmitted to the offspring,
though these sometimes also acquire immunity through the
parents. Thus in studying immunity of mice against ricin,
Ehrlich found that the newly born offspring of an immune
mother were not immune, though they subsequently became
so through the mother's milk.

Acquired immunity differs from natural immunity in
being less certain and of variable duration. The animal
may be immune to-day, but lose all power of defending
itself a month hence.

Natural immunity is always active, but certain forms
of acquired immunity are passive.

Immunity may be acquired through infection or intoxica-
tion, and either of these may be accidental or experimental.

(A) Active Acquired Immunity. — i. Immunity Ac-
quired through Infection. — (a) Accidental Infection.—
The most familiar form of acquired immunity follows an
attack of an infectious disease. Every one knows that an
attack of measles, scarlatina, varicella, variola, yellow fever,
typhoid fever, and other common infectious maladies, is a
fairly good guarantee of future exemption from the respec-
tive disease. Immunity thus acquired is not transmissible
to the offspring. Almost everybody has had measles, yet
almost all children are born susceptible to it. It is not
necessarily permanent, as is shown by the not infrequent
cases in which second attacks of measles occur. In some
cases, as after typhoid fever, the immunity is not at first ob-
servable and the patient may suffer from relapses. Later it
becomes well-established and no repetition of the disease is
possible for years.

Sometimes the infection, by which immunity is acquired,
is not exactly similar to the disease against which it affords
protection, as in the case of vaccinia, which protects against
variola. It is still controversial, however, whether cow-pox
is variola of the cow or an entirely different disease. Cow-
pox was, however, common in the days when smallpox
was frequent, and has now become an extremely rare
disease.

(b) Experimental Infection. — i. Inoculation: The oldest
experiments in immunity date from unknown antiquity and
were practised in China and other Oriental countries for the

no Immunity

purpose of preventing smallpox. The Chinese method of
experimentally producing variolous infection was very crude
and consisted in introducing crusts from cases of variola
into the nose, and tying them upon the skin. The Turkish
method was much more neat, in that a small quantity of
the variolous pus was 'introduced into a scarification upon
the skin of the individual to be protected. Lady Montague,
wife of the British Ambassador, brought the so-called
"inoculation" method of preventing smallpox from Turkey
in the early part of the eightheenth century (1718). By both
methods the very disease, variola, against which protection
was desired, was occasioned, the only advantage of the
experimental over the accidental infection being that by
selecting the infective virus from a mild case of variola, by
performing the operation at a time when no epidemic of the
disease was raging, and by doing it at a time when the person
infected was in the most perfect physical condition, the dan-
gers of the malady might be mitigated.

There was always danger, however, that the induced
disease being true variola might prove unexpectedly severe,
and that each inoculated individual, suffering from the con-
tagious disease, might start an epidemic.

2. Jennenan vaccination: In 1791 a country schoolmaster
named Plett, living in the town of Starkendorf near Kiel in
Germany, seems to have made the first endeavor to subject
the oft-repeated observation that persons who had acquired
cow-pox did not subsequently become infected with small-
pox to experimental demonstration by inserting cow-pox
virus into three children, all of whom escaped smallpox.

The father of vaccination, and the man to whom the world
owes one of its greatest debts, was Edward Jenner, who
performed his first experiment on May 14, 1796, when he
transferred some of the contents of a cow-pox pustule on
the arm of a milkmaid named Sarah Nelmess to the arm of
a boy named John Phips. After the lad had recovered from
the experimental cow-pox thus produced, he subsequently
introduced smallpox pus into his arm and found him fully
immunized and rendered insusceptible to the disease. This
led Jenner to perform many other experiments, and record
his observations in numerous scientific memoirs. The
success of his work immediately attracted the attention of
both scientific investigators and sanitarians, and its out-
come has been the establishment of compulsory vaccination

Vaccination 1 1 1

by legal enactment in nearly all civilized countries, with
the result that smallpox, instead of being one of the most
prevalent and most dreaded diseases, has become one of the
most rare and least feared.

The immunity acquired through vaccination is active and
usually of prolonged duration. It is subject to the same
variations observed in other experimentally acquired im-
munities, these variations explaining the occasional failures
which constitute the "stock in trade" of those who still
remain unconvinced of the scientific basis and efficacy of
the procedure.

Though a thorough analysis of the irregularities and ex-
ceptions of vaccination would be of much interest, a brief
mention of the most important must suffice for the present
argument.

The first controversial point is the nature of the "vaccine,"
or virus used in the operation. It is obtained from calves
or heifers suffering from experimental cow-pox, and is a virus
descended from various spontaneous cases of cow-pox
observed in places remote from one another. Experts are
undecided whether cow-pox is variola* modified by passage
through the cow so that the transplanted micro-organisms
are only capable of inducing a local instead of a general
disease, or whether it is an independent affection natural to
the cow.

In reality the matter in unimportant, so long as the desired
effect is accomplished, and the true lineage of the virus is
only a matter of scientific curiosity. As immunity is almost
invariably a specific effect resulting from infection, it would
seem most likely that cow-pox and smallpox were originally
identical.

The advantage of "vaccination" over "inoculation" is
that the induced disease is local and not dangerous except in
rare cases, and that it is not contagious. The natural varia-
tions in the susceptibility of different vaccinated individuals
determine that a few persons cannot be successfully vacci-
nated, being immune to the mildly invasive organisms of
vaccinia, though perhaps susceptible to the actively in-
vasive organisms of variola; that a few individuals shall
prove abnormally susceptible to vaccinia so that the disease
departs from its usual local type and generalizes, but that
in nearly all cases the disease will follow the well-known type
of a local lesion characterized by definite periods of incu-
bation, vesiculation, pustulation, and cicatrization.

ii2 Immunity

The occasional variations in immunity of different in-
dividuals also determine that having been vaccinated once
an individual may not again become susceptible to vaccina-
tion, though he may become susceptible to the more actively
invasive organisms of variola, or that he may soon become
again susceptible to both diseases, or that in very rare cases
no immunity against variola will result from vaccination.
In most cases successful vaccination can be repeated once
or twice at intervals of seven or ten years, and experience
shows that the immunity against smallpox conferred by
vaccination is of longer duration and usually becomes per-
manent after vaccination has been repeated once or twice.

Sanitarians are accustomed to speak of efficient and
inefficient vaccination. These are vague terms and do not
seem to be understood by the laity. Efficient vaccination
is vaccination repeated as often as is necessary. It has
already been shown that individual variations determine
that a few individuals never become immune, hence never
can be efficiently vaccinated. Other persons are efficiently
vaccinated by a single operation. The term is usually inter-
preted to indicate that which experience has shown to be
efficient in average cases.

Failures not uncommonly result from causes having
nothing to do with the problems of immunity. That an
operation of scarification has been performed upon a child,
and that a scar has remained thereafter may mean nothing.
It is not the operation but the disease that achieves the
result, and if the operation be improperly done, poor — i. e.,
old or inert — matter introduced, or if after introduction it
be destroyed by the application of antiseptics, no effect can
be expected. Hence all persons that have been vaccinated
may not have had vaccinia, the essential condition leading
to immunity. Nor does the occurrence of a local lesion act
as a guarantee that vaccinia has been induced. Careful
examination of the resulting lesions should always be made,
that the type of the infection may be studied. It is the
disease, vaccinia, that must occur — three days incubation,
three days vesiculation, three days pustulation, and subse-
quent cicatrization with the formation of a punctate scar.

An arm may be never so sore, may suppurate or even
become gangrenous, without vaccinia having occurred or
the desired benefit attained.

The accidents of vaccination were formerly numerous and

Vaccination 113

sometimes disastrous because of the general inattention to
the quality of the materials used, the mode of inserting them,
the condition of the patient's skin, and the careless treat-
ment of the resulting lesions. When human virus was used,
the transmission of human diseases, such as syphilis and
erysipelas, occasionally took place ; now these are rare acci-
dents indeed. When no attention was paid to the quality of
the bovine virus, and no governmental inspection of labor-
atories required, the accidental contamination of the virus
occasioned a small number of accidental infections of the
patients' arms, but these evils are becoming less and less as
greater attention is given to the details of the process.
Some accidents and some few deaths there will probably
always be, just as there are occasional accidents and occa-
sional fatal results following all kinds of trivial injuries,
though care will eliminate them as the sources of accident
are better understood.

3. Pasteurian vaccination or bacterination: Although the
word vaccination is derived from the Latin vacca, " a cow,"
and was first employed in connection with Jenner's method of
introducing virus modified by passage through a cow, Pasteur,
in honor of Jenner, applied it to every kind of protective
inoculation, and the word bacterination is only introduced for
the purpose of indicating certain differences in the method.

In 1880 Pasteur* observed that some hens inoculated
with a culture of the bacillus of chicken cholera that had
been on hand for some time did not die as was expected.
Later, securing a fresh and virulent culture, these and
other chickens were inoculated. The former hens did
not die, the new hens did. Quick to observe and study
phenomena of this kind, he investigated and found that
when chickens we're inoculated with old and non-virulent
cultures they acquired immunity against virulent cultures.
This led him to the recommendation of the employment of
attenuated cultures as vaccines against the disease, and to the
achievement of great success in preventing epidemics by
which great numbers of the barnyard fowls of France were
being destroyed.

In 1 88 1 Pasteur,! m experimenting with Bacillus anthracis,
observed that if the organism were cultivated at unusually
high temperatures it lost the power of producing spores, and

* "Compte rendu de la Soc. de Biol., 1880, 239; 315 et seq.

t " Compte rendu de la Soc. de Biol. de Paris," 1881, xcn, pp. 662-665.

ii4 Immunity

diminished in virulence. He also found that when the organ-
isms had been so attenuated, they could not regain virulence
without artificial manipulation. It occurred to him that
such organisms, possessing feeble virulence, might be able
to confer immunity upon animals into which they were
inoculated, and he continued to investigate the subject until
he found that by using three " vaccines" or modified cultures
of increasing virulence, it was possible to render animals
immune against the unmodified organisms. This method
was put to practical test with great success, and has since
been extensively practised in different parts of the world.

Arloing, Cornevin and Thomas,* and Kittf found that
exposure of the Bacillus anthracis symptomatici to a high
temperature in the dry state modified its virulence and
devised a practical method of protecting cattle against
symptomatic anthrax by inoculating them with powdered
muscle tissue containing the bacilli attenuated by drying
and exposure to 85 ° C. This method has since been in use
in many countries, and has given excellent satisfaction.

In 1889 Pasteur, { continuing his researches upon the
experimental modification of the germs of disease and their
use as prophylactics, published his famous work upon rabies,
and showed that, although the micro-organism of that
disease had so far eluded discovery, it was contained in the
central -nervous system of diseased animals, where it could
be modified in virulence by drying. By placing spinal cords
removed from rabid rabbits in a glass jar containing cal-
cium chlorid, he was able to diminish the virulence of the
contained micro-organisms according to the duration of the
exposure. The introduction of the attenuated virus was fol-
lowed by the development of a certain degree of immunity.
By repeated inoculation of more and more active viruses
animals acquired complete immunity against street virus.
These experiments formed the basis of the " Pasteur method"
of treating rabies, which is nothing more than immunization
with the modified germs of the disease during the long incuba-
tion period of the disease.

Haffkine§ found that the introduction of killed cultures
of virulent cholera spirilla produced immunity against the
living micro-organisms, and used the method with consider-

"Le Charbon Symptomatique du Boeuf," Paris, 1887.
t "Centralbl. f. Bakt.," etc., i, p. 684.

t "Compte rendu de la Soc. de Biol. de Paris," 1881, cvm, p. 1228.
§"Brit. Med. Jour.," 1891, n, p. 1278.

Immunity Acquired by Intoxication 115

able success for preventing the disease. Later* he applied
the same method, also with considerable success, for the pre-
vention of bubonic plague, and A. E. Wright f followed pretty
much the same method for the prevention of typhoid fever.

In all these cases the immunity induced by the'experimen-
tal manipulations is specific in nature, and variable in inten-
sity, according to the method of treatment adopted and the
thoroughness with which it is carried out. This variability
in the results attained will be much better understood after
the subject of immunization against toxins has been discussed.

2. Immunity Acquired by Intoxication. — Bacterio-toxins
form a miscellaneous group of active bodies of entirely
different chemical composition and physiologic activity.
Some are toxalbumins, some are enzymes, some are bac-
terio-proteins. The true nature of the greater number
of these bodies is unknown, but study of their physio-
logic action has brought forth the important fact that
their behavior toward the body cells is in no way different
from the behavior of the same cells toward other chemical
compounds of similar constitution, and that nearly all
physiologically active bodies introduced into living organisms
produce definite, though not necessarily visible, reactions.

Such reactions are now known as antigenic, and the sub-
stances by which they are induced have been called by
Deutsch antigens. I Since its introduction the precise
meaning given the word by Deutsch has been slightly
changed. As now defined, an antigen is any protein sub-
stance which when injected into the body of a living organism
is capable of producing a chemicophysiologic reaction re-
sulting in the appearance of a self-neutralizing, self-precipi-
tating, self-agglutinating, self-dissolving, or otherwise self-
antagonizing substance known as an antibody.

The antigens are, so far as known, all colloidal substances.
They may be harmful or harmless, active or inert, living or
dead, organized or unorganized. The reactions are specific
and the antibody has specific affinity for that antigen alone
by which its formation has been excited.

All poisonous substances are not antigens, even though a
certain immunity — in the sense of habituation or tolerance —
may follow their repeated administration. One may become

* "Brit. Med. Jour.," 1895, n, p. 1541.
t Ibid., Jan. 30, 1897, i, p. 256.

J Deutsch and Feistmantel, "Die Impfstoffe und Sera," 1903, Leip-
zig, Thieme.

n6 Immunity

habituated or tolerant to a certain quantity of mercury or ar-
senic, and to certain alkaloids, such as morphin, caffein, nico-
tin, cocain, etc., but he does not react as to antigens and no
antibodies are formed. To these various substances he
acquires only a slight degree of tolerance; to the injurious
effects of antigens he may acquire an almost unlimited degree
of immunity through the formation of the antibodies.

From remote antiquity it has been known that those who
regularly consumed small quantities of poisons become ir-
responsive to their action, and it is well known that Mithrid-
ates adopted this mode of defending himself from his enemies.

Chauveau* believed that the immunity conferred by in-
oculations of bacteria was due to the presence of their soluble
products, but the first direct demonstration was given by
Salmon and Smith,! who, as early as 1886, showed that it
was possible to immunize pigeons against the hog-cholera
bacillus by means of repeated injection with cultures exposed
to 60° C., and containing no living organisms. CharrinJ
found it possible to immunize rabbits against Bacillus pyo-
cyaneus by injecting them with the filtered products of cul-
tures of that organism, and Bonome§ similarly to immunize
animals against Bacillus proteus, B. cholera gallinarum and
the pneumococcus. Roux and Chamberland|| and Roux**
were able by the use of boiled cultures of the bacilli of malig-
nant edema, and of quarter evil, similarly to immunize
animals against these respective infections.

The subject was much further elaborated by Roux and
Yersinff in their experiments with diphtheria toxin, by
Behringit in his early studies of diphtheria, and by Kita-
sato§§ in his experiments with tetanus.

These early experiments opened a wide field, through the
investigation of which we now know that the products as well
as the living or dead bacteria of most of the infectious diseases,
when properly introduced into animals, can induce immunity.

(B) Passive Acquired Immunity. — Passive immunity is
always acquired, never natural. It depends upon defensive

*"Ann. de 1'Inst. Pasteur," 1888, 2.

t "Centralbl. f. Bakt.," etc., 1887, n, No. 18, p. 543.

t"Compte rendu," cv, p. 756.

§ "Zeitschrift f. Hyg.," v, p. 415.

|| "Ann. de 1'Inst. Pasteur," 1887, 12.
** Ibid., 1888, 2. ft Ibid., n, 1888, p. 629.

It "Deutsche med. Wochenschrift," 1890, No. 50.
§§ "Zeitschrift fur Hygiene," x, 1891, p. 267.

Passive Acquired Immunity 117

factors not originating in the animal protected, but arti-
ficially or experimentally supplied to it. The fundamental
principle is simple and has become the basis of serum thera-
peutics. If the immunized animal generates factors by
which the infecting bacteria can be destroyed or the activity
of their products overcome in its body, cannot these fac-
tors be removed and the benefit they confer transferred to
another animal?

The first experiments in this direction seem to have
been made by Babes and Lepp,* who found that the
blood-serum of animals immunized to rabies showed a
defensive power when injected into other animals. Ogata
and Jasuharaf found that the subcutaneous injection of
blood-serum from an animal immunized against anthrax
enabled the injected animals successfully to resist infection.
Behring and KitasatoJ found that the blood-serums of
animals immunized against diphtheria and tetanus, when
mixed with cultures of these respective bacilli, neutralized
their power to produce disease. Kitasato § found that if
mice were inoculated with tetanus bacilli, they could be
saved from the fatal infection by the infra-abdominal in-
jection of some blood-serum from a mouse immunized
against tetanus, even after symptoms of the disease had
appeared. Ehrlich|| showed that the blood-serums of animals
immunized against abrin and ricin could save other animals
from the fatal effects of these respective toxalbumins ; Phis-
alix and Bertrand,** and, later, Calmetteft found the blood-
serum of animals, immunized against the venoms of serpents,
similarly possessed the power of neutralizing the poisonous
effects of the venoms. KosselJJ found that the blood-serum
of animals, immunized against the poisonous blood-serum of
eels, contained a body which destroyed or neutralized the
effects of the eels' serum.

Thus, it is shown that in each case in which defensive reac-
tions are stimulated in experiment animals, these reactions are
accompanied by the appearance in the blood-serum of those

* "Annales de 1'Inst. Pasteur," 1889, vol. m.

t "Centralbl. f. Bakt.," etc., ix, p. 25, 1890.

| "Deutsche med. Woch.," 1890, No. 49.

§ "Zeitschrift fur Hygiene," 1892, xu, p. 256.

)| "Deutsche med. Wochenschrift," 1891, Nos. 32 and 44.
** "Compte rendu Acad. des Sciences de Paris," cxvm, p. 556.
ft "Ann. de 1'Inst. Pasteur," 1894, vm, p. 275.
}J "Berliner klin. Woch.," 1898, p. 152.

n8 Immunity

animals of factors that can be utilized to defend other ani-
mals in whose bodies no similar reactions have been produced.

Passive immunity may also be brought about in a few
cases by the injection into the intoxicated animal of sub-
stances, other than immunity products, that have a specific
affinity for the poison. Thus Wassermann and Takaki*
found that when the crushed spinal cord of a rabbit was
mixed in -vitro with tetanus toxin, the poison was quickly
absorbed by the nerve-cells, so that the mixture became
inert and could be injected into animals without harm.
Wassermann also found that the same effects could be pro-
duced in the bodies of animals, and that when the crushed
spinal cord was injected into an animal twenty-four hours,
or a few hours previously, or a few hours after a fatal dose of
tetanus toxin, enough of the combining elements remained
in the blood to fix the toxin before it anchored itself to the
central nervous system of the intoxicated animal. Myersf
found that the ground-up tissue of the adrenal bodies was
able to fix and thus annul the poisonous effects of cobra
venom in vitro.

In all these cases the neutralizing effects are either accom-
plished or initiated by factors prepared experimentally, and
forced upon the animal in whose body their activities are
manifested.

SYNOPSIS OF THE EXPERIMENTAL STUDIES OF
IMMUNITY.

Very important contributions were made by Ehrlich,t in
his work upon the vegetable toxalbumins, ricin, abrin, and
robin. Kossel§ investigated the reactions produced with
toxic eels' blood and found that immunity could be estab-
lished against their hemolytic action. Phisalix and Ber-
trand|| showed that immunity could also be produced in
guinea-pigs against the action of viper venom.

The investigations upon other active bodies were soon
begun. In 1893 Hildebrand ** studied emulsin and found
that it produced a definite reaction with the formation, in

* "Berliner klin. Wochenschrift," Jan. 3, 1898.
t "Lancet," July 2, 1898.

t "Deutsche med. Woch.," 1891, Nos. 32 and 44.
§ "Berliner klin Wochenschrift," 1898.

j| Atti d XI Congr. med. internaz. Roma, 1894, u, 200-202.
** "Virchow's Archives," Bd. cxxxi.

Synopsis of Experimental Studies of Immunity 119

animals injected, of an anti-emulsin. v. Dungern * studied
proteolytic enzymes of various bacteria, and showed that
when gelatin-dissolving enzymes were repeatedly injected
into animals, definite reactions took place, and in the serum
a body appeared that inhibited the action of the ferment in
a test-tube. Gheorghiewskif immunized animals to cul-
tures of Bacillus pyocyaneus, and found that the reaction
provoked caused the appearance in the serum of some body
that prevented the formation of the blue pigment so char-
acteristic of the organism. MorgenrothJ applied the same
principle to rennet, finding that it produced definite reac-
tions, with the formation of an anti-body inhibiting the
coagulation of milk. Bordet and Gengoul found that the
fibrin ferment of the blood of one animal was active in the
body of another animal, producing an inhibiting substance
by which the coagulation of the blood of the first animal
could be delayed.

The studies of Krausll showed a new fact, that when
filtered cultures of the cholera spirillum were introduced
into animals, the serum of these animals, added to the filtered
culture in a test-tube, caused the appearance of a delicate
flocculent precipitate.

Wassermann and Schiitze** found that when cows' milk
was repeatedly injected into rabbits, their serum acquired
the property of occasioning a precipitate when added to
cows' milk, but not when added to goats' or any other
milk. If, however, the rabbit had been repeatedly injected
with goats' milk or human milk, its serum would precipitate
with those milks respectively, and not with cows' milk.
The reaction was thus shown to be specific.

Meyers ft found that the repeated intraperitoneal injec-
tion of egg-albumen into rabbits caused their serum to give
a dense precipitate when added to solutions of egg-albumen.

Tchistowitch ** found that eels' serum injected into ani-
mals produced a reaction in which immunity to its poison-
ous action was associated with the ability of their serum to
produce a precipitate when added to the eels' serum.

* "Miinchener med. Woch.," Aug. 15, 1898.

t "Ann. de 1'Inst. Pasteur," 1899.

t "Centralbl. f. Bakt.," etc., 1899, xxvi, p. 349.

§ "Ann. de 1'Inst. Pasteur," 1903, xvn, p. 822.

|| "Wien. klin. Woch.," 1897.
** "Deutsche med. Woch.," 1900.
ft "Lancet," n, 1900. JJ "Ann. de 1'Inst. Pasteur," vol. 13.

120 Immunity

Closely connected with these various reactions are certain
others variously spoken of as cytotoxic, cytolytic, hemolytic,
bacteriolytic, etc. The first observation bearing upon these
was made by R. Pfeiffer,* who found that when guinea-pigs
received frequent intraperitoneal injections of cholera spirilla
and became thoroughly immunized, their serum behaved
very peculiarly toward the bacteria in the peritoneal cavity
of freshly infected animals, in that it caused them to become
aggregated into granular masses and subsequently to dis-
appear. This became known as " Pfeiffer 's phenomenon."
The serum of the immunized animal was devoid of action
by itself, the serum of the infected animal was inactive, but
the combination of the two brought about dissolution of
the micro-organisms. Later it was shown by Metschnikofff
that the living animal was not a factor in the process, but
that what was seen in the peritoneal cavity could be re-
produced in a test-tube, though not quite as well.

BordetJ made frequent injections of defibrinated rabbits'
blood into guinea-pigs, and obtained a serum that had a
solvent action upon the rabbit's corpuscles in -vitro, and
showed that the induced hemolysis resembled in all points
the bacteriolysis.

Ehrlich and Morgenroth§ studied the hemolytic action
of the serum of goats that had been frequently injected with
the defibrinated blood of sheep and goats, and were able to
point out the mechanism of the corpuscle solution or hemo-
lysis. It was found to depend upon two associated factors,
one of which, the lysin or solvent, was present in normal
blood, and was called "addiment" or "complement," and
another present only in the serum of the reactive animals,
called the "immune body" or "intermediate body."
The former was labile and easily destroyed by heat, the
latter stabile and not affected by heat up to the point of
coagulation. The experiments were confirmed by von
Diingern and many others. It is to be observed in passing
that this reaction differs from the direct solution of the cor-
puscles in vitro by cobralysin, which was studied by Myers, 1 1
and tetanolysin, studied by Madsen,** in that it is inter-
mediate, and only brought about by the cooperation of two

* "Deutsche med. Wochenschrift," 1896, No. 7.
f "Ann. de 1'Inst. Pasteur," 1895. % Ibid., XH, 1898.

§ "Berliner klin. Wochenschrift," 1899.
|| "Trans. Path. Soc. of London," u.
** " Zeitschr. f. Hyg.," 1899, xxxm, p. 239.

Cytotoxic Serums 121

factors, while the action of the lysins of venom, the tetanus
bacillus, the streptococcus, Bacillus pyocyaneus, and other
micro-organisms, is direct and immediate.

Myers found, however, that the hemolytic substance of
venom, and Madsen that the hemolytic products of Bacillus
tetani, also produce reactions in animals, and that when suc-
cessful immunization against them was accomplished, the
serums of the experiment animals became antidotal or in-
hibiting to the action of the respective lysins.

Von Diingern* found that by injecting dissociated epithe-
lial cells from the trachea of oxen into the peritoneal cavity
of guinea-pigs, it was possible to produce epitheliolysins ;
Lindemann,f that emulsions of kidney substance injected
into animals caused them to form nephro-lysins or nephro-
toxins ; Landsteiner J and Metschnikoff § in the same manner
successfully prepared spermatoxin by injecting the sperma-
tozoa of one animal into the peritoneal cavity of another.
Metalnikoffli found that if he introduced the spermatozoa of
a guinea-pig into the peritoneum of another, the spermo-
toxic serum produced was solvent for the spermatozoa of
both. Both Metschnikoff and Metalnikoff also found that
the spermotoxin when introduced into animals was active
in producing anti-spermotoxin by which the destructive
action of the serum upon spermatozoa could be inhibited.

Metschnikoff ** and Funck ff found that animals treated
with emulsions of the spleen, and mesenteric lymph-nodes
of one kind of animal, produced sera whose action was
agglutinative and solvent for leukocytes and lymph-cells.
Delezene { J found that dissociated liver cells injected into
animals similarly caused the formation of a cytotoxic serum
whose specific destructive action was upon them.

All of these reactions are indirect and intermediate, and
take place under appropriate conditions both in the bodies of
animals and in the test-tube.

But the number of antigenic reactions that can be brought
about in the bodies of animals seems to be limitless, and,
strange as it may seem, the antibodies produced in the body

* " Miinchener med. Wochenschrift," 1899.

t "Ann. de 1'Inst. Pasteur," 1900.

t "Centralbl. f. Bakt.," etc., 1899, xxv.

§ "Ann. de 1'Inst. Pasteur," 1899.

i| Ibid., 1900. ** Ibid., 1899-

ft "Centralbl. f. Bakt.," etc., xxvn, 1900.
it " Compte rendu de 1'Acad. des Sciences," 1900, cxxx, pp. 938, 1488.

122 Immunity

of one animal may act as antigens when introduced into
another. Thus, Ehrlich and Morgenroth in their studies of
hemolysis found that serums rich in immune bodies produced
reactions yielding anti-immune bodies, which inhibited the
activities of the respective immune bodies by whose stimula-
tion they were produced.

The reactions which when repeated may lead to immunity
and to the formation of antibodies seem to be followed by
constitutional disturbances much more profound than
would be supposed from the apparent freedom from symp-
toms manifested by the animal. As early as 1839 Magendie
observed that if a rabbit was given an injection of albumin,
and then, some days later, a second injection, it was made
very ill and might die. About 1900 Mattson called the
author's attention in private conversation to the fact that
when guinea-pigs used for testing antitoxic serums were sub-
sequently injected with another dose of serum, they com-
monly died. Not being understood, the matter was not
thought worthy of publication. Otto* speaks of this fatal
action of serums as the "Theobald-Smith phenomenon," the
fact having first been pointed out to him by Smith.

The first to realize the importance of the condition seem
to have been Portier and Richet,| who studied the effect
of extracts of the poisonous tentacles of actiniens upon
dogs which were found to die more quickly and from smaller
doses given at a second injection than at the first. To
this increase of sensitivity to the poison brought about by
the initial dose they gave the name anaphylaxis (av nega-
tive, yoXaZts protection, destroying protection or breaking
down the defenses).

The therapeutic employment of diphtheria antitoxic serum
was scarcely popularized before the medical profession was
shocked by the sudden death of the healthy child of a
noted German professor after a prophylactic injection, and
in 1896 GottsteinJ was able to collect 8 deaths following
the use of the serum, 4 of them being persons not ill with
diphtheria. von Pirquet and Schick§ also pointed out
that in a certain proportion of cases the injection of horse-
serum in man is followed by urticarial eruptions, joint-pains,
fever, swelling of the lymph-bodes, edema and albuminuria,
* von Lenthold, "Gedenkschrift," Bd. i, pp. 9, 16, 18.
t "Compte rendu de la Soc. de Biol. de Paris," 1902.
J "Therap. Monatschrift," 1896.
§ "Die Serumkrankheit," Leipzig and Wien, 1905.

Allergia or Anaphylaxis 123

these symptoms usually appearing after an incubation
period of eight to thirteen days, and constituting what they
call the "serum disease," or allergia. Sometimes these re-
actions are immediate; sometimes death appears imminent,
and, as has been observed, death sometimes occurs.

The investigation of the subject was taken up in 1905
by Rosenau and Anderson*, who pursued it with great in-
terest and industry, by Gay,f Gay and Southard,! and
others.

Experimental study shows that when an animal is injected
with an alien protein of almost any kind, a reaction takes
piace that usually is not completed under six days. If a
second injection is given before the reaction is perfected, the
mechanism of immunity is set in action, and the animal pro-
ceeds to defend itself through the various means described.
If the second administration be deferred, however, until the
first reaction is completed, it seems to find the animal in a
state of disturbed biologic equilibrium, the nature of which
is not understood, but which is characterized by a profound
disturbance that may terminate in death. The reaction is
quite specific; the sensitization, once effected, may continue
throughout the remainder of the life of the animal and be
transmitted from the mother to her offspring through her
blood. The reaction can be brought about by feeding the
protein or by injecting it. It has an important bearing
upon infection and immunity, the chief example being seen
in the tuberculin reaction.

The symptomatology of anaphylaxis is interesting and
characteristic. When it is desirable to study it, a guinea-pig
is first given a sensitizing dose of horse-serum. This may
be very small. Rosenau and Anderson found one guinea-pig
to be sensitized by one-millionth of a cubic centimeter. In
most of their work they used less than ^1^ c.c. It is neces-
sary to wait until the effects of this first injection are com-
pletely over before giving the poisoning dose. This period
of incubation lasts about twelve days. After the lapse of
this time, the second dose, usually about y1^ c.c., is given.
Both doses are given by injection into the peritoneal cavity.

* "Journal of Medical Research," 1906, xv, p. 207; "Bull. No. 29
of the Hygienic Laboratory," Washington, D. C., 1906; "Bull. No.
36," 1907, Ibid.; "Jour. Med. Research," xvi, No. 3, p. 381; "Jour.
Infectious Diseases," iv, No. i, p. i, 1907; "Jour. Infectious Diseases,"
vol. iv, p. 552, 1907.

f "Jour. Med. Research," May, 1907, xvi, No. 2, p. 143.

} Ibid., June, 1908, xvm, No. 3, p. 385.

124 Immunity

The symptoms come on almost immediately. The
animal is profoundly depressed, extremely uneasy, pants
for breath, and suffers from intense itching of the face. It
soon falls, continues to gasp for breath, and dies within an
hour. The disturbances in the body of the animal are
sufficient to account for the symptoms. Extensive lesions
exist, the first to be described by Rosenau* affecting
the mucous membrane of the stomach, which appeared
ecchymotic and ulcerated. Gay and Southard f found
hemorrhages in most of the organs, and believe anaphy-
laxis to depend upon the presence, in the blood of the
sensitized animal, of a substance to which they have given
the name anaphylactin. It seems difficult, however, to
imagine how such a substance could remain in the blood
throughout the entire subsequent life of the animal. Bes-
redka and Steinhardt J found that by the repeated injection
of horse-serum into guinea-pigs, the intervals being too short
to permit anaphylaxis, antianapkylactin could be prepared.

Anaphylaxis is not a disturbance of the cells of the body,
as some have thought, but is at least in part a disturbance
of the composition of the blood, as can be shown by the oc-
currence of what is known as passive anaphylaxis. If the
blood-serum of a sensitized animal be withdrawn and in-
jected into a normal animal of the same kind, it carries the
sensitization with it. The new animal, however, does not
become sensitized at once, but only after some days, hence
it is equally true that the disturbance is not solely in the
blood, else why should not the sensitization be immediately
present upon the injection of the serum?

Anaphylaxis may, furthermore, be local. Thus, when
certain substances like tuberculin are dropped in the eye
there is no effect, but when a second application is made,
after some weeks, the eye may be reddened.

Vaughan has endeavored to explain anaphylaxis by assum-
ing that when the strange protein in the blood reaches the
cells it is slowly broken down by enzymic action, but that
the cells, having once acquired the property of destruction,
seize eagerly upon the protein the next time it is offered, dis-
integrate it rapidly, and so disseminate throughout the body

*"Bull. No. 32 of the Hygienic Laboratory," Washington, D. C.,
October, 1906.

t "Jour. Med. Reserach," July, 1908, xix, No. i, pp. i, 5, 17.

J "Ann. de 1'Inst. Pasteur," xxi, No. 2, February 25, 1907, pp.
117-127.

Explanation of Immunity 125

the degradation products, some of which may be toxic and
account for the reaction.

Anaphylaxis'may play a role in infection. In cases where
an attack of an infectious disease leaves no immunity, the
body may be left hypersensitive to subsequent attacks.

EXPLANATION OF IMMUNITY,

Before the facts now at our disposal had been gathered
together, and before the phenomena of immunity against
infection had been compared with those of intoxication,
Pasteur* and Klebsf endeavored to explain acquired im-
munity by supposing that micro-organisms living in the
infected animal used up some substance essential to their
existence, and so died out, leaving the soil unfit for further
occupation. This was known as the "exhaustion theory."
WernichJ and Chauveau§ thought it more probable that
the micro-organisms after having lived in the body left
behind them some substance inimical to their further
existence. This was known as the "retention theory."
These hypotheses are of historic interest only, and deserve
no more than passing mention, as they both fail to explain
natural immunity or immunity against intoxication.

Karl Roser|| observed that the leukocytes of the bodies of
higher animals sometimes enclosed bacteria in their cyto-
plasm. Koch, Sternberg, and others confirmed the obser-
vation, but no attention was paid to it until Metschnikoff**
correlated it with other known facts and original observa-
tions, and came to the conclusion that the enclosed bacteria
had been eaten by the leukocytes in which they were killed
and digested, and that the behavior of the cells toward the
bacteria afforded an explanation of the mechanism by which
recovery from the infectious diseases takes place. The
original conception upon which this "theory of phagocyto-
sis" was founded, refers recovery in many, if not all of the
infectious diseases, to the successful destruction of the
invading bacteria by the body cells, especially the leukocytes.

* "Compte rendu de la Soc. de Biol de Paris," xci.

t "Arch. f. experimentele Path. u. Pharmak.," xm.

I "Virchow's Archives," Bd. LXXVIII.

§ "Compte rendu de la Soc. de Biol. de Paris," xc and xci.

|| "Beitrage zur Biologic niederster Organismen," Inaugural Dis-
sertation, Marburg, 1881.

** "Virchow's Archives," Bd. xcvi, p. 177; "Ann. de 1'Inst. Pas-
teur," t. i, p. 321, 1887.

1 26 Immunity

These devouring cells Metschnikoff called phagocytes, and
of them he recognized two classes, the microphages , which
are white blood-corpuscles, and the macrop hages, which are
larger cells derived from the endothelial and other tissues.
Metschnikoff, his associates, and his pupils soon collected
evidence sufficient to show that phagocytosis, if not the
chief factor in defending the body from infectious organisms,
is at least an important one. Many of the most interesting
facts are described in Metschnikoff 's books, "Etudes sur Tin

Fig. 1 8. — Phagocytosis; the omentum immediately after injection of
typhoid bacilli into a rabbit. Meshwork showing a macrophage, inter-
mediate forms and a trailer, all containing intact bacilli (Buxton and
Torry).

flammation" and "Immunite dans les Maladies Infectieuses,"
which every interested student of the subject should read.

These studies show that in nearly all cases in which
animals are naturally immune against infection, the leu-
kocytes are active in their phagocytic behavior toward
them; that in acquired immunity, the leukocytes pre-
viously inactive, become active toward them; that the
enclosure of bacteria within the cells sometimes results in
the death of the cells, sometimes in the death of the bacteria ;
that phagocytosis is much more active in diseases in which
the bacteria have limited toxicogenic powers, and in which
they probably exert a positively chemotactic influence upon
the cells, than in cases in which the bacteria are strongly
toxicogenic and probably exert an injurious and negatively
chemotactic influence upon them, and that when the toxico-
genic power of the bacteria is great, many of the phago-
cytes are killed and dissolved — phagolysis. Study of the
primitive forms of animal life shows that amebae constantly

Phagocytosis — Opsonins 127

feed upon smaller organisms, some almost exclusively upon
bacteria, which they are able to kill and digest through an
intracellular enzyme demonstrated by Mouton,* and called
amebadiastase, and regarded as a form of trypsin. The
intracellular digestion of ccelenterate animals is accomplished
by means of actinodiastase, an enzyme discovered by
Fredericq, and studied by Mesml. It seems to be related
to papine and digests albuminoids. The digestion of ery-
throcytes and tissue fragments is accomplished through an
enzyme of the macrophages, which Metschnikoff calls macro-
cytase, that of bacteria through an enzyme of the microphages,
which he calls microcytase. In phagolysis these respective
ferments are liberated into the plasma, imparting to it a
bactericidal and bacteriolytic action similar to that normally
peculiar to the cytoplasm of the cells. The dissemination
of the enzymes in phagolysis, with resulting bacteriolytic
power of the blood plasma and serum, is a later modification
of the original conception of Metschnikoff, that the invading
parasites were eaten up by the phagocytes, and was made
necessary by the investigation of the bactericidal property of
the body juices. The experiments of Wright and Douglasf
indicate that the action of the phagocytes upon the bacteria
is not immediate, but only subsequent to a preparative
action upon the organisms by substances contained in
serum, to which they have given the name " Opsonins" (Lat.
opsono, "I prepare a meal for").

Long before Metschnikoff began his studies of the pha-
gocytes Traube and Gscheidel I observed that the blood-
plasma possessed the power of destroying the vitality of
bacteria. Grohman§ next observed that not only the
intravascular, but also the extra vascular blood possessed
this property. The first exact investigations of the subject
were made by von Fodor. || The systematic investigation of
the bactericidal activity of blood-serum in vitro was next
taken up by Fliigge,** and more particularly by Nuttall,ft
who found that different blood-serums possessed the power

* "Compte rendu de 1'Acad. des Sciences de Paris," 1901, cxxxm,
p. 244.

< t " Proc. Royal Society of London," utxxii, p. 357, 1904-
t " Jahresberichte der Schles. Ges. f. vaterl. Kultur," 1874.
§ " Untersuchungen aus dem physiol. Institut zu Dorpat," Dorpat,
1884; Kriiger.

|| "Centralbl. f. Bakt.," etc., 1890, vn, p. 753-
** "Zeitschrift fur Hygiene," Bd. iv, S. 208.
ft Ibid., Bd. IV, S. 353-

128 Immunity

of killing bacteria in large numbers, but that the bactericidal
power of the serum soon disappeared, after which the serum
became a good culture-medium for the very bacteria it had
formerly destroyed. Metschnikoff objected to the observa-
tions, declaring that all the phenomena were ultimately
referable to the leukocytes, so Nuttall investigated pen-
cardial fluid arid the aqueous humor of the eye, which were
also found to possess bactericidal powers.

The matter was next taken up by Buchner and his as-
sociates,* who showed that the blood-plasma and blood-
serum possessed exactly the same bactericidal effects as the
total blood. Buchner and Nuttall both showed that the
exposure of the bactericidal fluids to a temperature of 56° C.
for a few hours entirely destroyed their activity, though low
temperatures were without effect upon them. Buchner
found that the exposure of the serum to sunlight and
oxygen also destroyed the bactericidal power. Neutraliza-
tion of alkaline serum did not destroy its activity, but
when the serum was dialyzed and the NaCl removed from
it, the germicidal power was lost, to return again when it was
restored. Buchner called the bactericidal principle alexin.

Many interesting facts were collected bearing upon the
bactericidal substance or alexin. Thus Morof showed that
it was proportionally more active in sucking infants than in
adults, and Ehrlich and BriegerJ found that it passed from
mother to offspring in the milk.

At first Buchner regarded alexin as an albumin, but later§
he came to look upon it as a proteolytic enzyme, this view
no doubt resulting from an endeavor to explain the relation
of alexin to immunity against intoxication, in which it was
necessary to show that alexin not only killed bacteria, but
also destroyed toxins.

Hankin|| endeavored to show that there were differences
between the substances destroying the bacteria and those act-
ing upon their toxic products. To the whole group he applied
the term defensive proteids. Those present in natural im-
munity he called sozins, those found in acquired. immunity
phylaxins. Sozins with bactericidal activity he further de-

* " Centralbl. f Bakt .," etc , 1889, Bd v,S. 817; vi, S. 1; "Archivfur
Hygiene," 1891, x, S 727; "Centralbl. f. Bakt ," etc., 1890, vn, S. 76.
f'Jahresb. f. Kinderheilkunde," v, S 396
t "Zeitschrift fur Hyg.," 1893, xm, S. 336.
§ "Munch, med. Woch.." 1899
II "Centralbl. f. Bakt.," etc., xn, Nos. 22, 23: xiv. No. 25.

Defensive Proteins, etc. 129

scribed as mycosozins, those with toxin-destroying activities
as toxosozins. Phylaxins with bactericidal action were called
mycophylaxins; those with toxin-destroying properties toxo-
phylaxins.

Metschnikoff found it unnecessary to change his ideas, and
persisted in referring all the phenomena to the phagocytes
or to enzymes derived from them.

At this point it will be evident to the reader that the
phagocytic theory and the humoral theory contain indubit-
able evidence to show that they are important factors in
defending the body against invading organisms, and that in
each we see mechanisms operative in certain cases. But
we have seen that both Metschnikoff and Buchner are
obliged to strain a point in order to meet the requirements
of increasing knowledge of the subject of immunity.

Thus, when we come to analyze Buchner 's theory of
alexins, we find that if natural immunity depends upon the
ability of the alexins to destroy bacteria, that which takes
place in vitro should correspond with that which takes place
in vivo, and that the invasion of the animal's body by
bacteria should be accompanied by diminution of the bac-
tericidal substance in its blood, which should be used up
before the bacteria can be successful in their invasion.
Experimental evidence is, however, at hand to show that
this is not always true.

Behring and Nissen* found that there was a definite
relation between the bactericidal power of the blood in vitro
and the resisting powers of a large number of animals
studied, but Lubarschf showed the remarkable exceptions of
the rabbit, which is highly susceptible to anthrax, though its
blood is highly bactericidal to the anthrax bacillus, and the
dog, which is scarcely susceptible to anthrax, though its
blood is scarcely bactericidal to the bacillus.

FluggeJ found the bactericidal power of the blood greatly
lessened in thirty-six hours after anthrax infection, and
Nissen that a definite number of bacteria could be killed
by a bactericidal serum, after which the alexin became
inactive. The diminution of the bactericidal power was
shown to occur both in the animal and in the test-tube. He
also showed that the reactions of the bactericidal serums

* "Zeitschrift fur Hygiene," 1890, vm, S. 412.
f "Centralbl. f. Bakt.," etc., 1889, vi, S. 481.
J "Zeitschrift fur Hygiene," iv, S. 208.
9

130 Immunity

were specific, and that when a culture of one kind of bacteria
was injected into an animal, the immediate effect was to
diminish the activity of the serum for that species, though
not necessarily for other species. The diminution of bac-
tericidal energy was shown by him to depend upon the
presence of the bacteria, as the injection of filtrates of bac-
terial cultures did not affect the bactericidal properties of
the serum. This was a very important observation.

There is a correspondence between the behavior of the
phagocytes and the body juices. When the activity of the
phagocytes toward the bacteria is increased, the bactericidal
activity of the serum is usually intensified. But immunity
is only partly explained by alexins and bacteriolysis, for
it embraces the ability of the organism to endure the effects
of toxins some of which are in no way connected with
bacteria.

Tolerance to certain toxins is, of course, natural to many
animals, and tolerance to usually destructive toxins natural
to a few. This toxin-neutralizing or annulling factor
cannot be identical with the bacteria-destroying mechanism.
Cobbett,* Roux and Martin, f and BoltonJ have shown that
horses that cannot be supposed ever to have come into
contact with diphtheria bacilli, vary considerably in their
resistance to diphtheria toxin, and that the serum of the
resisting horses contains something that destroys or neu-
tralizes the toxin in vitro, as well as exerts a protective
influence upon animals into which it is injected. This
substance exerts no inimical action upon the diphtheria
bacilli, beyond what a normal serum would do, therefore
cannot be alexin, but must be antitoxin. Abel§ found
that the blood of healthy men occasionally contained some
substance capable of neutralizing diphtheria toxin; Stern
found one normal serum capable of protecting against typhoid
infection and Metschnikoff one that protected against cholera
infection. Fischel and Wunschheim|| found newly born
babies immune against diphtheria, presumably because of
the presence of a small quantity of demonstrable protective
substance in the blood. These are, however, peculiar and
exceptional cases.

* "Lancet," Aug. 5, 1899, n, p. 532.

t "Ann. de 1'Inst. Pasteur," vin, p. 615, 1894.

t "Jour, of Experimental Medicine," July, 1896, I, No. 5.

§ "Centralbl. f. Bakt.," etc., xvn, pp. 36, 1895.

|| "Zeitschr. fur Heilkunde," 1895, xvi, p. 429-482.

The ''Lateral-chain Theory" of Immunity 131

The most suggestive and fascinating theory of immunity
is that of Ehrlich, and is known as the " Seitenkettentheorie"
or the "Lateral-Chain Theory."*

He began his studies by an investigation into the nature of
toxins and their mode of action. The discovery that there
was no constant relation between the • intoxicating and
antitoxin combining powers of diphtheria toxic bouillon led
him to the conclusion that the toxin molecules possessed two
different affinities, which he described as haptophorous or
combining, and toxophorous or poisoning. The former were
constant, the latter variable. The deterioration in the
strength of the toxic filtrates of bouillon cultures of diph-
theria bacilli was shown to depend upon the transformation
of the toxin into toxoids which were not poisonous, and
was shown to be quite independent of the antitoxin com-
bining affinity of the filtrate which remained unaltered.
The inevitable interpretation seemed to be the existence
in the bouillon of the haptophorous and toxophorous
groups described. Similar toxophorous and haptophorous
groups were shown to exist in other toxins — tetanolysin,
by Madsen, venoms, by Myers, and milk-curdling fer-
ments by Morgenroth. The neutralizing action of the
antibodies produced in the blood of animals immunized to
these various substances depends upon the immediate and
direct combination or union of haptophorous groups in the
antibodies with corresponding haptophorous groups of the
respective toxins or active bodies.

The physiological activities of toxins differ from those of
alkaloids and other poisons differ in three fundamentals:

* The writings of Ehrlich and his associates are so numerous and
scattered, and often so fragmentary, that instead of referring to the
literature according to the method adopted in other parts of this
work, the reader who desires to refer to the original articles can best
do so by making use of the following: Ehrlich, "Die Werthbemessung
des Diphtheric Heilserums," Klinisches Jahrbuch, 1897; Ehrlich, "Die
Konstitution des Diphtheriegiftes," Deutsche med. Woch., 1898 ; "Gesam-
melte Arbeiten zur Irnmunitatsforschung," August Hirschwald, Berlin,
1904 — this work contains the collected papers of Ehrlich and his associ-
ates, Aschoff, " Ehrlich's Seitenkettentheorie und ihre Anwendung auf
die Kiinst.lichen Immunusirungs-prozesse," Jena, 1902, and the chap-
ter upon "Wirkung und Entstehung der Aktiven Stoffe im Serum
noch der Seitenkettentheorie," by Ehrlich and Morgenroth in Kolle
and Wassermann's "Handbuch der Pathogene Mikroorganismen,"
Jena, 1904, Gustav Fischer. Readers unacquainted with the German
language may find the essential facts in Ehrlich's Croonian Lecture,
Proceedings of the Royal Society of London, LXVI, 1900, p. 424, and
in Welch's ''Huxley Lecture," Medical News, LXXXI, 1902, 2, p. 721.

132 Immunity

first in their ability to produce antibodies in the bodies of
animals into which they are injected; second, the mani-
festation of poisonous action only after a definite incubation
period, and, third, an extremely labile composition, by which
the toxin becomes quickly transformed to toxoids.

A study of the physiological action of toxins upon the cells
resulted in showing that certain definite specific affinities
existed, and that the union of the toxin with the cell ante-
dated the production of symptoms. In some cases it was
even found possible to disconnect the anchored toxin by
bringing to the cells haptophorous groups for which the
haptophorous elements of the toxin molecule were known
to have an active affinity. Donitz determined the quantity
of tetanus antitoxin which, injected into the circulating
blood immediately after the toxin, absolutely neutralized

tlsptophiLe Toxofrhlle Uaptofrhore Toxofihore

group. group. group, g™up.t

\
\

Ce

Fig. 19. — Diagram to represent the combining groups of the cell and
of the toxin respectively (after Ehrlich) (Hewlett).

it and rendered all of the circulating toxin innocuous. If the
same quantity of antitoxin was given seven or eight minutes
after the injection of the toxin, death occurred from tetanus,
exactly as if no antitoxin had been given. Evidently the
toxin had anchored itself to the nerve-cells too quickly for
the antitoxin to reach and combine with it. Heymans found
that if an animal was injected with tetanus toxin, and its
entire blood withdrawn immediately afterward and replaced
by transfusion, it died of typical tetanus because in the brief
interval between the toxin injection and the transfusion, the
toxin molecules became anchored to the cell.

The ability of the cells thus to anchor the toxin is supposed
by Ehrlich to depend upon the existence of haptophorous
combining affinities, which he describes as receptors. He
views the mode of toxin reception as depending upon a

The "Lateral-chain Theory" of Immunity 133

mechanism either identical with or analogous to that by
which cellular nutrition is maintained, and points out that
in the case of methylene-blue and other colored substances,
which afford an opportunity to make ocular observations
upon the absorption of the pigment by the cells, only certain
cells absorb the colors.

Cell nutrition is therefore probably carried on through the
agency of receptors by which appropriate nutrient hap-
tophorous groups are apprehended and utilized.

"We now come to the important question of the signifi-
cance of the toxophile groups in organs. That these are in
function especially designed to seize on toxins cannot be for
one moment entertained. It would not be reasonable to
suppose that there were present in the organism many
hundreds of atomic groups, destined to unite with toxins,
when the latter appeared, but in function really playing no
part in the processes of normal life, and only arbitrarily
brought into relation with them by the will of the investi-
gator. It would, indeed, be highly superfluous, for example,
for all our native animals to possess in their tissues atomic
groups deliberately adapted to unite with abrin, ricin, and
crotin, substances coming from far-distant tropics."

"One may, therefore, rightly assume that these toxophile
protoplasmic groups in reality serve normal functions in the
animal organism, and that they only incidentally and by
pure chance possess the capacity to anchor themselves to
this or that toxin."

' ' The first thought suggested by this assumption was that
the atom group referred to must be concerned in tissue
change ; and it may be well here to sketch roughly the laws
of cell metabolism. Here we must, in the first place, draw
a clear line of distinction between those substances which
are able to enter into the composition of the protoplasm, and
so are really assimilated, and those which have no such
capacity. To the first class belong a portion of the food-
stuffs, par excellence; to the second almost all our pharma-
cological agents, alkaloids, antipyretics, antiseptics, etc."

"How is it possible to determine whether any given
substance will be assimilated in the body or not ? There can
be no doubt that assimilation is in a special sense a synthetic
process — that is to say, the molecule of the food-stuff con-
cerned enters into combination with the protoplasm by a
process of condensation involving loss of a portion of its

1 34 Immunity

water. To take the example of sugar, in the union with
protoplasm, not sugar itself as such, but a portion of it,
comes into play, the sugar losing in the union some of its
characteristic reactions. The sugar behaves here as it
does, e. g., in the glucosids, from which it can only be
obtained through the agency of actual chemical cleavage.
The glucosid shows no traces of sugar when extracted in
indifferent solvents. In a quite analogous manner the sugar
entering into the composition of albuminous bodies (gly-
coproteids) cannot be obtained by any method of extrac-
tion; at least not until chemical composition has previously
taken place. It is, therefore, generally easy by means of
extraction experiments, to decide whether any given com-
bination in which the cells take part is, or is not, a synthetic
one. If alkaloids, aromatic amines, antipyretics, or anilin
dyes be introduced into the animal body, it is an easy
matter, by means of water, alcohol, or acetone, according to
the nature of the body, to remove all these substances quickly
and easily from the tissues."

"This is most simply and convincingly demonstrated in
the case of the anilin dyes. The nervous system stained with
methylene-blue or the granules of the cells stained with
neutral red at once yield up the dye in the presence of alco-
hol. We are, therefore, obliged to conclude that none of
the foreign bodies just mentioned enter synthetically into
the cell complex; but are merely contained in the cells in
their free state." .... "Hence with regard to the
pharmacologically active bodies in general, it is not allow-
able to assume that they possess definite atom groups,
which enter into combination with corresponding groups
of the protoplasm. This corresponds, as I may remark
beforehand, with the incapacity of all these substances to
produce antitoxins in the animal body. We must, therefore,
conclude that only certain substances, food-stuffs, par
excellence, are endowed with properties admitting of their
being, in the previously defined sense, chemically bound by
the cells of the organism. We are obliged to adopt the view
that the protoplasm is equipped with certain atomic groups,
whose function especially consists in fixing to themselves
certain food- stuffs of importance to the cell-life." We may
assume that the protoplasm consists of a special executive
center, in connection with which are nutritive side-chains,
which possess a certain degree of independence and which

The "Lateral-chain Theory" of Immunity 135

may differ from one another according to the requirements
of the different cells. And as these side-chains have the
office of attaching to themselves certain food-stuffs, we must
also assume an atom-grouping in these food-stuffs them-
selves, every group uniting with a corresponding combining
group of a side-chain. The relationship of the correspond-
ing groups, i. c., those of the food-stuff, and those of the cell,
must be specific. They must be adapted to one another,
as, e. g., male and female screw (Pasteur), or as lock and
key (E. Fischer). From this point of view, we must con-
template the relation of the toxin to the cell."

"We have already shown that the toxins possess for the
antitoxins an attaching haptophore group, which accords
entirely in its nature with the conditions we have ascribed
to the relation existing between the food-stuffs and the cell
side-chains. And the relation between toxin and cell ceases
to be shrouded in mystery if we adopt the view that the
haptophore groups of the toxins are molecular groups fitted
to unite not only with the antitoxins, but also with the side-
chains of the cells, and that it is by their agency that the
toxin becomes anchored to the cells."

' ' We do not, however, require to suppose that the side-
chains, which fit the haptophore group of the toxins, that
is, the side-chains which are toxophile, represent some-
thing having no function in the normal cell economy. On
the contrary, there is sufficient evidence that the toxophile
side-chains are the same as those which have to do with the
taking up of the food-stuffs by the protoplasm. The
toxins are, in opposition to other poisons, of extremely
complex structure, standing in their origin and chemical
constitution in very close relationship to the proteids and
their nearest derivatives. It is, therefore, not surprising
that they possess a haptophore group corresponding with
that of a food-stuff. Alongside of the binding haptophore
group, which conditions their union to the protoplasm, the
toxins are possessed of a second group, which in regard to
the cell is not only useless, but actually injurious. And we
remember that in the case of the diphtheria toxin there was
reason to believe that there existed alongside of the hapto-
phore group another and absolutely independent toxophore
group." . . . . "As has been said, the possession of a
toxophile group by the cell is the necessary preliminary
and cause of the poisonous action of the toxin." ....

136 Immunity

"If the cells of these organs [organs essential to life] lack
side-chains fitted to unite with them, the toxophore group
cannot become fixed to the cell, which therefore suffers no
injury, i. e., the organism is naturally immune. One of the
most important forms of natural immunity is based upon
the circumstance that in certain animals the organs essen-
tial to life are lacking in those haptophore groups which
seize upon definite toxins. If, for example, the ptomaine
occurring in sausages, which for man, monkeys, and rabbits
is toxic in excessively minute doses, is for the dog harm-
less in quite large quantities, this is because the binding
haptophore groups being wanting, the ptomaine cannot,
in the dog, enter into direct relation with organs essential
to life." . . . "The haptophore group exercises its
activity immediately after injection into the organism, while

Fig. 20. — Shows how the haptophores having united, the toxophores
find a secondary adaptation to the cell, and so can poison it (after
Ehrlich) (Hewlett).

in all toxins — with the perhaps solitary exception of snake-
venom — the toxophore group comes into activity after the
lapse of a longer or shorter incubation period which may,
e. <?., in the case of diphtheria toxin, extend to several weeks."
"The theory above developed allows of an easy and natural
explanation of the origin of antitoxins. In keeping with
what has already been said, the first stage in the toxin
action must be regarded as the union of the toxin by means
of its haptophore group to certain 'side-chains' of the cell
protoplasm. This union is, as animal experiments with a
great number of toxins show, a firm and enduring one. The
side-chain involved, so long as the union lasts, cannot exer-
cise its normal nutritive physiological function — the taking
up of food-stuffs. It is, as it were, shut out from participat-
ing, in the physiological sense, in the life of the cell. We

The "Lateral-chain Theory" of Immunity 137

are, therefore, now concerned with a defect which, according
to the principles so ably worked out by Professor Carl
Weigert, is repaired by regeneration. These principles, in
fact, constitute the leading conception of my theory. If
after union has taken place, new quantities of toxin are
administered at suitable intervals and in suitable quantities,
the side-chains, which have been reproduced by the regen-
erative process, are taken up anew into union with .the
toxin, and so again the process of regeneration gives rise to
the formation of fresh side-chains. In the course of the
progress of typical systematic immunization, as this is
practised in the case of diphtheria and tetanus toxin espe-
cially, the cells become, so to say, educated or trained to
reproduce the necessary side-chains in ever-increasing

Fig. 2 1 . — Cells with various receptors or haptophorous groups of the
first order (a), adapted to combination with the haptophorous groups (6)
of various chemical compounds brought to them. It will be noted that
there is no mechanism by which the toxDphorous elements of the mole-
cules (c) can be brought to the cell.

quantity. As Weigert has confirmed by many examples,
this, however, does not take place by the simple replacement
of the defect; the compensation proceeds far beyond the
necessary limit; indeed, overcompensation is the rule.
Thus the lasting and ever-increasing regeneration must
finally reach a stage at which such an excess of side-chains
is produced that, to use a trivial expression, the side-chains
are present in too great a quantity for the cell to carry and
are, after the manner of a secretion, handed over as needless
ballast to the blood. Regarded in accordance with this
conception, the antitoxins represent nothing more than side-
chains reproduced in excess during regeneration and therefore
pushed off from the protoplasm and so coming to exist in the
free state,"

138

Immunity

The essence of Ehrlich's theory is tersely expressed by
Behring: "The same substance which when incorporated
in the cells of the living body, is the prerequisite and con-
dition for an intoxication, becomes the means of cure when
it exists in the circulating blood."

Figs. 22 and 23. — Show the regeneration of the cell-haptophores or
receptors to compensate for the loss of those thrown out of service.

Fig. 24. — Shows the number
of haptophores regenerated by
the cell becoming excessive, they
are thrown off into the tissue
juice.

Fig. 25. — Explains what anti-
toxins are and how they are
formed. The liberated receptors
in the tissue juice and in the
blood, possess identical combin-
ing affinities with those upon the
cell, and meeting the adapted
haptophorous elements in the
blood, combine with them, thus
keeping them from the cells.

Continuing the quotations from Ehrlich's Croonian Lec-
ture, we find: "In the first place, our theory affords an
explanation of the specific nature of the antitoxins, that
tetanus antitoxin is only caused to be produced by tetanus
toxin, and diphtheria antitoxin through diphtheria toxin.

The "Lateral-chain Theory" of Immunity 139

This very specific nature of the affinity between toxin and
cell is the necessary preliminary and cause of the toxicity
itself. Further, our theory makes it easy to understand the
long-lasting character of the immunity produced by one or
several administrations of toxin, and also the fact that the
organism reacts to relatively small quantities of toxin by
the production of very much greater quantities of anti-
toxin." By the act of immunization, certain cells of the
organism become converted into cells secreting antitoxin at
the same rate as this is excreted. New quantities of anti-
toxin are constantly produced and so throughout a long
period the antitoxin content of the serum remains nearly
constant. The secretory nature of the formation of anti-
toxins has been very strikingly illustrated by the beautiful
experiments of Salmonson and Madsen, who have shown
that pilocarpine, which augments the secretion of most
glands, also occasions in immunized animals a rapid increase
in the antitoxin content of the serum."

"The production of antitoxins must, in keeping with our
theory, be regarded as a function of the haptophore group of
the toxin, and it is easy therefore to understand why, out of
the great number of alkaloids, none are in a position to
cause the production of antitoxins. Conversely, indeed,
I recognize in this incapacity of the alkaloids, in opposition
to the toxins, to produce antitoxins, a further and salient
proof of the truth of the deduction I have previously based
on chemical grounds, that the alkaloids possess no haptophore
group which anchors them to the cells of organs. To formu-
late a general statement, the capacity of a body to" cause the
production of antitoxin stands in inseparable connection
with the presence of a haptophore atomic group. In the
formation of antitoxin the toxophore group of the toxin
molecule is, on the contrary, of absolutely no moment. But
the toxoid modification of the toxins, in which the hapto-
phore group of the toxin is retained, while the toxophore
group has ceased to be active, possesses the property of pro-
ducing antitoxins. Indeed, in some cases of extremely
susceptible animals, immunity can only be attained by
means of the toxoid s, and not by the too strongly acting
toxins." .... "The symptoms of illness due to the
action of the toxophore group, therefore, play no part in the
production of antitoxin." The effect of enzymes upon the
organism with the production of an ti- bodies, and the "specific

140 Immunity

precipitins" caused by the injection of milk, albumin, and
peptones into animals may be looked upon as "having their
origin in the most widely diverse organs, and representing
nothing more than nutritive side-chains, which, in the course
of the normal nutritive processes have been developed in
excess and pushed off into the blood."

' ' Much more complex than in the cases hitherto discussed
are the conditions when, instead of the relatively simple
metabolic products of microbes, the living micro-organisms
themselves come to be considered, as in immunization against
cholera, typhoid, anthrax, swine-fever, and many other in-
fectious diseases. Thus there come into existence alongside
of the antitoxins produced as a result of the action of the
toxins, manifold other reaction products. This is because
the bacterium is a highly complicated living cell of which
the solution in the organism yields a great number of bodies
of different nature, in consequence of which a multitude of
'antikorper' are called into existence. Thus we see, as a
result of the injection of bacterial cultures, that there arise
alongside of the specific bacteriolysins, which dissolve the
bacteria, other products, as, for example, the 'coagulins'
(Kraus, Bordet), i. £., substances which are able to cause
the precipitation of certain albuminous bodies contained in
the culture fluid injected; also the much-discussed agglu-
tinins (Durham, Gruber, Pfeiffer), the antiferments (von
Dungern), and no doubt many other bodies which have not
yet been recognized. It is by no means unlikely that each
of these reaction products finds its origin in special cells of
the body; on the other hand, it is quite likely that the for-
mation of any single one of these bodies is not of itself
sufficient to confer immunity. Thus, in the case of the in-
troduction of bacteria into the body we have to do with a
many-sided production of different forms of 'antikorper,'
each of which is directed only against one definite quality
or metabolic product of the bacterial cell. Accordingly,
in recent times, the practice of using for the production of
immunization definite toxic bodies isolated from the bacterial
cells has been more and more given up, and for this purpose
it is now regarded as important to employ the bacterial cells
as intact as possible." .... "The most interesting
and important substances arising during such an immuniz-
ing process are without doubt the bacteriolysins." ....
"Belfanti and Carbone first discovered the remarkable fact

The " Lateral-chain Theory" of Immunity 141

that horses which had been treated with the blood-corpuscles
of rabbits contain in their serum constituents which are
poisonous for the rabbit, and for the rabbit only." . . .
"Bordet showed shortly thereafter that in the case quoted
there was present in the serum a specific hemolysin which
dissolved the corpuscles of the rabbit. He also proved that
these hemolysins — as had already been shown by Buchner
and Daremberg in the case of similarly acting bodies which
are present in normal blood — lost their solvent property on
being maintained during half an hour at a temperature of
55° C. Bordet added, further, a new
fact, that the blood-solvent property
of those sera which had been deprived
of solvent power by heat, the solvent
action could be restored if certain nor-
mal sera were added to them. By this
important observation an exact analogy
was established with the facts of bacteri-
olysis as elicited by the work of Pfeiffer,
Metschnikoff, and Bordet." ....

"In collaboration with Dr.
Morgenroth, I have sought in regard to
this question, for which hemolysis offered
prospects favorable to experimentation,
to make clear the mechanism concerned
in the action of these two compounds —
the stable, which may be designated
'immune body/ and the unstable, which
may be designated 'complement' — which
acting together effect the solution of the
red blood-corpuscles. For this purpose,
in the first place, solutions containing
either only the 'immune body ' or only the 'complement'
were brought in contact with suitable blood-corpuscles, and
after separation of the fluid and the corpuscles by centrifu-
galization, we investigated whether these substances had
been taken up by the red corpuscles or remained behind in
the fluid. The proof of its location in the one position or in
the other was readily forthcoming, since to restore the
hemolysin to its former activity, it was only necessary to
add to the 'immune body' a fresh supply of 'complement,'
or to the 'complement' a fresh supply of 'immune body'
in order that the presence of the hemolysin in its integrity

Fig. 26. — Com-
bination of cell (a),
amboceptor (6),
and complement
(c) . The ambo-
ceptor may unite
with the cell, but
cannot affect it
alone. The com-
plement cannot
unite with the cell
except through the
amboceptor, hav-
ing no adaptation
to the cell directly.

142 Immunity

might be shown by the occurrence of solution of the red
cells. The experiments proved that, after centrifugalizing,
the 'immune body' is quantitatively bound to the red blood-
corpuscles, and that the 'complement/ on the contrary,
remains entirely behind in the fluid. The presence of the
two components in contact with blood-corpuscles only
occasions the solution of these at higher temperatures, and
not at o° C. And an active hemolytic serum (with 'immune
body' and 'complement' both present) having been placed
in contact with red blood-corpuscles and maintained for a
while at o° C., it was found after centrifugalizing that, under
these circumstances also the 'immune body' had united with
the red blood-corpuscles, but that the 'complement' re-
mained in the serum. This experiment showed that both
components must, at a temperature of o° C., have existed
alongside of one another in a free condition." ....

"But when analogous experiments were undertaken at a
higher temperature it was found that both components were
retained in the sediment.

"These facts can only be explained by making certain
assumptions regarding the constitution of the two com-
ponents, i. <?., of the 'immune body' and the 'complement.'
In the first place, two haptophore groups must be ascribed
to the 'immune body,' one having affinity for a correspond-
ing haptophore group of the red blood-corpuscles and with
which at a lower temperature it quickly unites, and another
haptophore group of a lesser chemical affinity, which at a
higher temperature becomes united with the 'complement'
present in the serum. Therefore at the higher temperature
the red blood-corpuscles will draw to themselves those
molecules of the 'immune body' which in the fluid have
previously become united to the 'complement.' In this
case the 'immune body' represents in a measure the connect-
ing chain which binds the complement to the red blood-
corpuscles and so brings them under its deleterious influence.
Since under the influence of the 'complement' — at least, in
the case of the bacteria — appearances are to be observed
(for example, in the PfeifTer phenomenon) which must be
.regarded as analogous to digestion, we shall not seriously
err if we ascribe to this 'complement' a ferment-like char-
acter." . . . . "Having obtained a precise conception
of the method of action of the lysins of the serum — of the
hemolysins, and thereby also of the bacteriolysins — it be-

The "Lateral-chain Theory" of Immunity 143

comes possible for us to attempt to solve the mystery of the
origin of these bodies. I have in the beginning of this
lecture* fully developed the 'side-chain theory,' according
to which the antitoxins are merely certain of the protoplasm
4 side-chain' which have been produced in excess and pushed
off into the blood.

"The toxins as secretion products of the cells are in all
likelihood still relatively uncomplicated bodies; at least by
comparison with the primary and complex albumins of which
the living cell is composed.

"If we now recognize that the different lysins arise only
through absorption of highly com-
plex cell material — such as red blood-
corpuscles or bacteria — then the ex-
planation, in accordance with what
I have said, is that there are present
in the organism 'side-chains' of a
special nature, so constituted that
they are endowed not only with an
atomic group by virtue of the affini-
ties of which they are enabled to pick
up material, but also with a second
atomic group, which, being ferment-
loving in its nature, brings about the
digestion of the material taken up.
Should the pushing off of these 'side-
chains ' be forced, as it were, by im-
munization, then the 'side-chains'
thus set free must possess both groups,
and will, therefore, in their character-
istics entirely correspond with what

we have placed beyond doubt as regards the 'immune
body' of the hemolysin."

An analysis of this theory shows complete natural im-
munity to depend upon the absence of haptophore groups
(receptors) by which the toxins can be united to the cells.
Extreme sensitivity or susceptibility probably depends
upon the adapted haptophores being present or at least
most numerous upon the cells of highly vital organs ; com-
parative insensitivity or insusceptibility upon the fact that

* These lengthy extracts, through which I have endeavored to
enable Ehrlich to explain his theory to the reader, are taken from his
" Croonian Lecture," delivered before the Royal Society of London,
March 22, 1900.

Fig. 27. — Cell with
receptors of the second
order (a) by which the
cells fix useful mole-
cules, of albumins, etc.,
on one hand (6), and zy-
mogen molecules (c) on
the other hand, and
make use of the one sub-
stance through the ac-
tion of the other.

144 Immunity

the greater number of haptophore groups are attached to
comparatively unimportant cells whose combining affinities
have to be satisfied before combination with more vital cells
can be accomplished. In some cases natural immunity is
increased by the presence of free haptophore groups (anti-
toxin) in the blood.

Acquired immunity against toxins depends upon the
regeneration of the cellular haptophores or receptors which,
being liberated into the body juices, fix the haptophores of
the toxin molecules before they are able to reach the cells
themselves. Antitoxins and other anti-bodies, including the
lysins, consist of liberated cellular haptophores or receptors,
the former having a single combining affinity, the latter a
double combining affinity, by which they unite, on the one
hand, with the cell to be dissolved, on the other with the
complement by which it is to be dissolved. Anti-bodies
having this double combining affinity have been called
' ' amboceptors" by Ehrlich. They are variously known in
different writings as "immune bodies," amboceptors, sub-
stance sensibilisatrice, desmon, and fixateur. The "comple-
ment" or "addiment" of Ehrlich is also called alexin and
cytase. Khrlich conceives every amboceptor and every
complement to be specific, but Bordet and others, while
admitting that the amboceptor is specific, hold that there is
but one complement or cytase.

It has already been shown that MetschnikofFs primitive
conception of phagocytosis, that of the body being defended
against the invasive power of the microparasites by the
incorporation and digestion of the microparasites, had to
be modified to conform to the increasing information upon
the immunity reactions. He has persistently clung to the
idea that the phagocytes are the essential factors, but has
changed the conception of "phagocytosis" to make it
applicable to the condition shown by advancing knowledge.
When invasive organisms enter the body, positive chemotac-
tic influences determine that they are met by a sufficient num-
ber of phagocytes to devour and destroy them. If the invad-
ing organisms are too powerful and the phagocytes are killed
by them and their toxins, the phagolysis or dissolution of the
phagocytes liberates their enzymes into the blood. These
liberated enzymes now act deleteriously upon the invaders,
tending to agglutinate — aggregate them in clumps — and
sensitize them to the future action of the phagocytes by
which they may be taken up. Thus, the blood-serum ac-

The "Lateral-chain Theory" of Immunity 145

quires a "fixing" or "sensitizing" quality from the presence
of the "fixateur" or "substance sensibilisatrice." The
actual solution of the bacteria is accomplished by the
"microcytase" of the leukocytic phagocytes. Thus, we
find that Metschnikoff is prepared to account for the "am-
boceptor" or "immune body" of Ehrlich, which is the
fixateur, and the "complement," which is the " microcytase."
When the phagolysis is excessive, either from infection or
intoxication, both the fixateur and microcytase are free in
the serum. In cases where the bacteria exert a negatively
chemotactic influence upon the leukocytes, no immunity
exists. The reactive phenomena occasioned by the intro-
duction of tissue elements and blood-corpuscles, depend upon
enzymes derived from the macrophages, the "fixateur" as
before, and "macrocytase."

The antitoxins are similarly accounted for: the cellular
digestive enzymes exert their action not only upon the
organized complex molecules of the microparasites, but also
upon their more simple toxic products, fixing or otherwise
altering them until they can be finally destroyed.

It will thus be seen that the two chief theories, though they
appear discordant v/hen explained independently of one
another, are fairly well coordinated. Ehrlich believes the
active bodies to be the products of those cells of the body
for whose haptophorous combining groups the haptophorous
groups of the active bodies engaged, and does not attribute
the function to any particular group of cells ; Metschnikoff
attributes all the activities to the phagocytes, and especially
the leukocytes. Ehrlich looks upon the phenomena as
chemical and pictures them as taking places independently
of the cells; Metschnikoff looks upon them as vital and
brought about by the agency of living cells.

The fundamental ideas embodied in the "lateral-chain theory" of
immunity may, by reversing the hypothesis and considering the bacte-
rial instead of the' body cells to be upon the defensive, be made to ex-
plain other phenomena of immunity. Walker* seems to have been
the pioneer in this field, and his researches show that it is possible to
immunize bacteria against "immune serums" by cultivating them in
media containing increasing proportions of the immune serums. The
bacteria thus cultivated were of increased virulence. The idea was
further amplified by Welch in his Huxley Lecture. f The micro-
organismal cells must be regarded as endowed with receptors of their
own, fitted for combination with adapted haptophorous elements in the
juices reaching them, and therefore capable of reacting toward such

*"Jour. of Path, and Bact," vm, No. 1, p. 34, March, 1902.
f" British Medical Journal," Oct. 11, 1902, p. 1105; "Medical
News," Oct. 18, 1902.

146 Immunity

substances exactly as do the cells of the host. As the host reacts
toward the active products of the bacteria, so the bacteria react toward
the defensive products of the host, and as the cells of the former are
stimulated to the production of immune bodies that shall facilitate
bacteriolysis, so the latter are stimulated to antagonize their action by
producing neutralizing bodies. These neutralizing bodies by which
the defenses of the host are broken down are among those described
by Bail* as " aggressins."

Thus, as the cells of the host invaded are constantly reacting to the
active bodies produced by the invading parasites, so the latter are re-
acting toward the defensive products of the former. If the reactive
processes of the host predominate, immunity and the destruction of
the parasites result; if those of the bacteria predominate, increased
virulence, facilitated invasion, and death of the host may result. This
hypothesis also serves to make clear why micro-organisms entering the
body not infrequently show a marked tendency to colonize in certain
organs and tissues in preference to others.

Supposing accident to determine the tissue in which the primary in-
fection has taken place, a longer or shorter residence in that tissue,
with the resulting more or less marked acquired immunity against the
defensive activities of that tissue, endow the organism with a higher
degree of virulence for it than for other tissues, so that if at some
future time the organism entering the circulation of a new host were
able to colonize in any tissue of the body, its activities could be
more easily and more successfully manifested in that to which it had
already become accustomed, and to which it had acquired a peculiar
adaptability. This adaptability has been made the subject of inter-
esting experimental demonstration by Forssnerf in his work upon the
intravenous injection of streptococci.

SPECIAL PHENOMENA OF INFECTION AND IMMUNITY.

Certain phenomena which present themselves in the course
of infection and immunity, to which reference has already
been made, must now be considered in detail. These are
Specific Precipitation, Agglutination, Antibody Formation,
and Cytolysis.

I. Specific Precipitation. — In 1897 Krausi in studying-
the "specific reactions produced by homologous serums with
germ-free filtrates of bouillon cultures, of cholera, typhoid and
plague bacteria," observed that immune serum brought into
contact with the respective culture filtrate occasioned a
precipitate specific in nature, to which he gave the name
"specific precipitate." It thus became known that in ad-
dition to antitoxins, bacteriolysins, and agglutinins, other
specific bodies were formed.

Bordet§ and Tchist o witch || showed that the phenome-

*" Wiener klin. Woch.," 1905, Nos. 9, 14, 16, 17; " Berl. klin.
Woch.," 1905, No. 15; "Zeitschr. f. Hyg.," 1905, Bd. I, No. 3.
f'Nordiskt Medicinskt Archiv," Bd. xxxv, p. 1, 1902.
J "Wiener klin. Woch.," 1897, No. 32.
§ "Ann. de 1'Inst. Pasteur,'.' 1899, p. 173.
|] " Ann. de 1'Inst. Pasteur," 1899, p. 406.

The Specific Precipitins

147

non was of wide occurrence and had a broad significance, for
they discovered that when the serum of
one animal was injected into another ani-
mal some reaction took place by which the
two serums being subsequently brought
together precipitation took place. The
same was found true of milk. Myers,*
Jacoby,f Nolf,| and others showed that
the faculty of provoking specific precipi-
tins was common to many albuminous
bodies — albumen, globulin, albumose,
peptone, ricin, etc. Kraus in his original
communication dwelt upon the specific
nature of the precipitation, and was
corroborated by Fish, § Wassermann, ||
Morgenroth, and others, and it was shown
that the reaction was sufficiently accurate
to make possible the differentiation of
human and goat's milk. The most im-
portant practical application, however,
came through Uhlenhuth** and Wasser-
man,tt who made use of it for the dif-
ferentiation of bloods for forensic pur-
poses.

Uhlenhuth gave rabbits intraperitoneal
injections of loc.c. of defibrinated blood
at intervals of from six to eight days and
found the blood-serum strongly precipit- complements*
ant after the fifth. He used such serum
for testing the reaction with the bloods of oxen, horses, don-
keys, pigs, sheep, dogs, cats, deer, hares, guinea-pigs, rats,
mice, rabbits, chickens, geese, turkeys, pigeons, and men. •

The method of making the test is important, as careless-
ness of detail will interfere with the accuracy of the result.
The blood to be tested is diluted about i : 100, or until
it has a feeble red color, with tap water, and then freed from

*"Centralbl. f. Bakt.," etc., 1900, Bd. xxxm, and "The Lancet,"
1900, n, p. 98.

f'Archiv fur exper. Path. u. Pharmak.," 1900.
t "Ann. de 1'Inst. Pasteur," 1900, p. 297.
§ "Courier of Medicine," St. Louis, Feb., 1900.
|| " Verhandl. d. Kong. f. innere Med.," 1900, S. 501, Wiesbaden.
**" Deutsche med. Woch.," 1900 and 1901.

ft "Samml. klin. Vortr. v. Volkman," Leipzig, Verlag von Breitkopf
and Hartel, 1902.

Fig. 28— Poly-
ceptor (E h r 1 i c h
and Marshall) such
as can be conceived
to occur in hemo-
lysis and bacterio-
lysis where various
complements are
engaged. a, Re-
ceptor of bacterial
cell ; b, cytophil
group of the ambo-
ceptor; c, dominat-
ing complement ; d,
subordinate com-
plement ; a, /?,
com plementophil
groups of the am-
boceptor, a for the
dominating, /? for
the subordinate

148 Immunity

corpuscular stroma by filtration or decantation. Two cubic
centimeters of it are placed in a small test-tube, and further
diluted with an equal quantity of physiological salt solution
(if more water be added a precipitate of globulin might
take place and spoil the experiment). To such a prepared
blood preparation, from six to eight drops of the immune
serum are added. If the diluted blood come from the same
kind of an animal as that used to immunize the animal
furnishing the test serum, immediate clouding takes place,
and a flocculent precipitate forms. The precipitate never
occurs with any other blood. For those who desire to
acquaint themselves with the best technic for the per-
formance of experiments with the specific precipitates, we
refer the reader to the book of Nuttall mentioned below,
and to a subsequent paper by Nuttall and Inchley,* in which
an apparatus for the accurate quantitative estimation of
the precipitate is described.

Wassermann and Schutzef prepared a test serum by in-
jecting rabbits with human blood, and tested its precipi-
tating powers upon twenty-three other kinds of blood and
found no reaction except with the blood of a baboon, but
the reaction in that case was not nearly so marked as with
human blood.

The most interesting and one of the most important bio-
logical applications of this phenomenon is by Nuttall, whose
work, "Blood Immunity and Blood Relationship" (Cam-
bridge, 1904), should be read by all who wish to study the
subject for its scientific interest as a means of determining
the blood relationship of animals, or its practical medico-
legal importance in recognizing blood-stains. Nuttall
conies to the following conclusions:

" (i) The investigations we have made confirm and extend the ob-
servations of others with regard to the formation of specific precipitins
in the blood-serum of animals treated with various sera. (2) These
precipitins are specific, although they may produce a slight reaction
with the sera of allied animals. (3) The substance in serum which
brings about the formation of a precipitin, as also the precipitin itself,
are remarkably stable bodies. (4) The new test can be successfully
applied to a blood which has been mixed with those of several other
animals. (5) We have in this test the most delicate means hitherto
discovered of detecting and testing bloods, and consequently we may
hope that it will be put to forensic use."

* "Journal of Hygiene," 1904, iv, p. 201.

f "Deutsche med. Wochenschrift," 1900, No. 30.

The Agglutinins 149

The injection of the precipitinogenic serum into animals
results in the formation of anti-precipitins by which their
activity is neutralized.

II. Agglutination. — This phenomenon was first observed
by Charrin and Roger* in the course of some experiments
with Bacillus pyocyaneus. They found that the bacilli
when introduced into serum of animals infected with or
immunized against cultures of Bacillus pyocyaneus ceased
their active movements, became aggregated in clusters and
settled to the bottom, leaving the fluid clear. Observations
confirming and enlarging upon the observation were made
by Metschnikoff, f Issaeff J and others. Gruber and Durham §
made an elaborate and now classic study of the subject, first
employing the term "agglutination" to the phenomenon,
and "agglutinins" to the substances in the serum by which
v it might be brought about. They found that when cholera
or typhoid bacilli are mixed with their respective immune
serums, the organisms lose motility and become aggregated
in clusters, masses or "clumps." They further showed
the reaction to be specific within certain limitations, i. e.,
typhoid immune serum agglutinated typhoid-like bacilli
but no others, etc., and they saw in the phenomenon a
practical means for the differentiation of different, closely
related bacteria, an application that has, indeed, become a
useful one.

It remained for Widal|| to show that it had a much more
important application, in that the micro-organism being
known, the effect produced by a serum upon it would be an
indication of the past infection of the animal from which
the serum was secured. The first practical application was
made in connection with typhoid fever, and the brilliant
success attending it had led to the test being known as the
"Widal reaction" (q.v.).

The agglutinins are stable substances that resist drying
and can be kept dry and active for years. Widal and Sicard
found that they pass with difficulty through a porcelain
filter and do not dialyze. They are precipitated in part by
15 per cent, of sodium chloride that throws down fibrinogen

* "Compte rendu de la Soc. de Biol.," 1899, p. 667.

f'Ann. de 1'Inst. Pasteur," v. 1891.

J Ibid., vii, 1893.

§ " Munchener med. Woch," 1896, No. 9.

|| "Societe Medicale des Hopitaux," June 26, 1896.

1 50 Immunity

and further precipitated with magnesium sulphate, which
throws down globulins. They therefore think they are inti-
mately related to the globulins and to fibrinogen. A tem-
perature of 60° C. diminishes their activity, but they are not
destroyed below 80° C. Sunlight has no effect upon them.

Metschnikoff looks upon agglutination as preliminary to
phagocytosis and to bacteriolysis, and thinks it the effect
of enzymes in the serum preparing and clustering the bacteria
to be taken up by the phagocytes. Ehrlich finds in the
agglutinins nothing more than receptors of what he denomi-
nates the II order, each of which possesses a zymophore and
an agglutinophore group.*

Malvozf found that the addition of chemical substances,
such as safranin, vesuvin, and corrosive sublimate, to cul-
tures of the typhoid bacilli would cause their agglutination.
Typhoid bacilli retained on the Chamberland filter and
washed for a long time, could no longer be agglutinated, and
were found to have lost their flagella and to be without mo-
tion, and led Dineur,| who made additional experiments, to
conclude that agglutination depended upon the flagella.
Malvoz§ found that bacteria were sometimes agglutinated
by their own metabolic products. He prepared a fresh
culture of the first vaccine of the anthrax bacillus by thor-
oughly distributing it through £ c.c. of distilled water, and
then added a loopful of a six-day-old culture. After stand-
ing for a few hours typical agglutinations were observed
under the microscope.

H. C. Ernst and Robey|| think that flagella have nothing
to do with agglutination, which subsequent experiment has
shown to be correct, as many non-flagellated bacteria can
be agglutinated by their respective serums.

Bail,** Joos,tt Eisenberg and VollJt have shown that all
of the agglutinins possess haptophore and agglutinophore
groups, either of which may be destroyed without the other.
Thus typhoid agglutinative serum when exposed to a tem-

* See Nothnagel's "Specielle Pathologic und Therapie," vm, 1901.

f "Ann. de 1'Inst. Pasteur," 1897, No. 6.

t " Bull, de 1'Acad. de Med. de Belgique," 1898, iv, p. 705.

§ " Ann. de 1'Inst. Pasteur," Aug. 25, 1899,

|| " Trans. Cong. Amer. Phys. and Surg.," 1900, p. 26.
** " Archiv f. Hyg.," 1902, XLII, Heft 4.
tf "Zeitschr. f. Hyg.," xxxvi, 1901. p. 422.
tt Ibid., XL, 1902, p. 155.

The Agglutinins 151

perature of 65° C. loses the agglutinophores, and no longer
clumps the bacteria, though it retains the haptophores, and
when brought into contact with the bacteria combines with
them, producing no agglutination, but preventing the action
of any other agglutinogenic serum.

Buxton and Vaughan* find that bacteria differ both in
their agglutinogenic powers and their agglutinability, both
of which must be taken into account in studying the subject.

Theobald Smith f has shown that there are two kinds of
agglutinins, one of which acts upon the bacteria directly, the
other through the flagella. The occurrence of these two
bodies explains some of the incompatible results of previous
experiments.

The Technic of Agglutination Tests. — The subject was
at first investigated with reference to typhoid fever, and a
large literature soon made its appearance. From typhoid
fever the method was extended to one after another of
the infectious diseases, in many of which the agglutina-
tion of the specific bacteria of the disease by the pa-
tient's serum was found to form a valuable adjunct in
diagnosis. While this clinical application was extending,
the method was finding added usefulness in the laboratory,
for it was at once evident that if the reaction was specific,
as it was soon proved to be, the agglutination of the bac-
teria by the serum not only proved the serum to come from
a patient or animal infected by a given bacterium, but proved
as well that the given bacterium was the one infecting. Thus,
the phenomena of agglutination came to be a recognized
laboratory method for the differentiation of bacteria of
similar species.

The reaction is one of the most accurate known to us. It
is so specific that in the case of many organisms it is possible
to tell from what original source they may have come, and
always to tell to what variety they belong by quantitative
estimation. It is, moreover, a simple method for employ-
ment in large laboratories where animals may always be
kept on hand immunized against various cultures, so that
the specific serums may always be at hand. When these
animals are periodically bled, the serums can be sealed
in small tubes and kept an almost unlimited length of time
ready for use when opened and diluted.

* "Jour. Med. Research," July, 1904.
f Ibid., 1904, vol. x, p. 89.

152 Immunity

There is no uniform technic by which to apply the test.
Scarcely any two laboratories employ the same method,
but the results are uniform and the method to be employed,
provided it is free from error, is that found most convenient
to the individual operator.

Certain precautions must be observed, though continued
study of the subject and the employment of newer methods
have shown these to be less numerous.

For clinical application for typhoid fever diagnosis, com-
mercial firms now prepare small cases containing the neces-
sary apparatus and " Kicker's solution" of dead typhoid
bacilli, so that by following the directions the diagnosis can
be made with ease by one inexpert in laboratory manipu-
lations.

In the laboratory the agglutination test may be applied
for the diagnosis of any infectious bacterial disease, pro-
vided the infecting organism be at hand, or for the recogni-
tion of any micro-organism, provided specific serums are
at hand, in the following manner:

If possible, a culture of the microorganism grown upon agar-agar
is to be selected for the purpose. A good-sized platinum loopful of
the culture is taken up and distributed as uniformly as possible through-
out a few cubic centimeters of distilled water. This is best done by
placing the water in a test-tube and then rubbing the culture upon the
glass just above the level of the fluid, until it is thoroughly emulsified,
permitting it to enter the water little by little and, finally, washing it
all down into the fluid. This gives a distinctly cloudy fluid, too con-
centrated to use. Of this one adds enough to each of a series of watch-
glasses or test-tubes, each containing an equal volume of distilled
water (say 2 c.c.), to make the fluid opalescent by reflected light though
transparent by transmitted light. The same quantity should be added
to each, so that they form a uniform series. The serum is next diluted
or otherwise added to the tubes, so that they receive different concen-
trations in a series from the blood of a patient running, say, i : 10, i : 20,
i : 30, i : 40, i : 50, i : 60, i : 80, i : TOO, i : 150, i : 200, i : 300, or a labora-
tory series with artificially prepared serums of high value, running,
perhaps, i: 1000, i: 2000, 1:5000, i: 10,000, 1:50,000, and i: 100,000,
or many times higher dilutions.

If watch-glasses are used, they are stood upon a black surface,
covered, and examined in fifteen, thirty, and sixty minutes by simply
looking at the dark surface through the fluid. If agglutination occur,
the original opalescence gives place to a slightly curdy appearance,
as the uniformly suspended bacteria aggregate in clumps.

If test-tubes are employed, they are best observed by tilting them
and looking through a thin layer of the contained fluid at a dark surface
or at the sky. In either case the flocculent collections of agglutinated
bacteria can be seen.

The test can also be made and observed under the microscope by
the hanging-drop method, but in working with such small quantities
much of the accuracy of the technic is apt to be lost.

Some knowledge is required in order to form correct deductions from

The Antitoxins 153

the experiments. Thus, with typhoid bloods, the agglutination of the
typhoid bacillus usually occurs within an hour in dilutions of i : 50,
but the agglutinability of the culture employed should be known before
the experiment is undertaken.

Similarly, when the method is employed for the differential determi-
nation of bacteria, the value of the serum should be known at least
approximately.

The agglutinins when injected into animals effect definite
chemico-physiological reactions with the formation of anti-
bodies inhibiting their own activity.

HI. The Antitoxins.— In the synopsis of immunity ex-
periments already given the history of the discovery and
development of the anti-bodies has been outlined, together
with references to the original contributions in which they
were made public.

In the section upon the "Explanation of Immunity" we
have seen that the best mode of accounting for the occur-
rence of antitoxins is afforded by Ehrlich in the lateral-chain
theory. He regards them as cell haptophores — receptors —
that are found in excess of the requirements, by cells fre-
quently stimulated by the presence of bacteria-products
possessing adapted haptophores. The receptors are under
normal conditions engaged in maintaining the proper nutri-
tion of the cell; under abnormal conditions (as when pre-
empted by the inert or injurious haptophores of the bacterio-
products) are obliged to increase in number to compensate
for the damage done the cell. The substances by which
anti-body formation can be stimulated must bear a resem-
blance to the normal nutrient substances absorbed by the
cells in that they are provided with haptophore groups
corresponding with the haptophore groups of the cells and
so adapted for union with them. Mineral substances and
alkaloidal substances have no such adaptations, but bacte-
rial products, the toxalbumins of various higher plants,
venoms, enzymes, and other proteid combinations have.
The possession of the haptophore groups determines whether
or not the cell can stimulate anti-body formation, and the
ability to produce anti-bodies shows the existence of the
haptophore groups.

The attachment of the haptophore groups to the cells is
usually shown by morbid action of the cells in cases where
there are associated toxophore groups, as in the case of the
bacterio-toxins, but may not be discovered if there are no
toxophore groups. The combination of the toxin-hap-

1 54 Immunity

tophores with the cell-haptophores can be demonstrated in
the test-tube by crushing the cerebral substance of a rabbit,
and adding tetanus toxin. The toxin becomes fixed by
combination with the cell haptophores or receptors, loses its
further combining powers and fails to affect animals into
which it is subsequently injected. The increased formation
of receptors in consequence of repeated stimulation has been
shown by the effect of abrin upon the conjunctiva. If
dropped into one eye until the conjunctiva is thoroughly
immune against its action, the cells of this eye develop a
greatly increased capacity for absorbing — i. e., fixing — the
abriri as compared with those of the other eye. Thus if the
two conjunctival membranes be dissected out and a certain
quantity of abrin triturated with each, the haptophores of
the cells of the immunized membrane fix the poison so that
it is no longer able deleteriously to affect animals, while
no such effect takes place with the other membrane.

The ability to stimulate the formation of anti-bodies is
entirely independent of any toxic action and is entirely the
work of the haptophores. This is best shown in the fact that
diphtheria toxin that has been heated or otherwise manip-
ulated until its toxic action is lost, still retains the power
of combining with antitoxin, or of producing anti-bodies.

The cells furnishing the haptophore groups or receptors
whose presence in the blood gives it its antitoxic quality
vary in number or quality in different animals. Thus, in
the warm-blooded animals the rapidity with which tetanus
toxin is anchored to the cells of the central nervous system
seems to indicate that those cells, if not the only cells in the
body passing the adapted receptors by which it is anchored,
are the chief cells by which it is absorbed. In the alligator,
however, other cells seem to fix the toxin before it reaches
or connects with those of the nervous system, so that the
alligator, though immune against the action of the toxin,
is able to make antitoxin as well as susceptible animals.

Each introduction of appropriate anti-body forming sub-
stance is followed by an outpouring of the anti-body far in
excess of what would neutralize it, so that after a systematic
treatment has been carried out for some time, the neutral-
izing value of the blood may be a thousand times what would
be necessary to neutralize, the total quantity of active sub-
stance introduced into the animal.

Each anti-body is specific in action, as must be evident

The Antitoxins 155

from its mode of formation. Should it be found, however,
that several active bodies possessed haptophore groups of
identical structure, the anti-body formed by any of them
might be found to possess common neutralizing powers for all.

The animal whose blood contains anti-bodies enjoys
immunity from the active body by which they were formed
only so long as they are present. In some cases, however,
animals that have been long subjected to the immunization
treatment, and whose blood contains large quantities of
free antitoxin, unexpectedly become abnormally sensitive
(hypersensitivity) to the toxin, and may die after receiving a
very small dose. This may be attributed to a difference in
the combining activity of the receptors attached to the cells,
and those separated and free in the serum. If the former
developed a greater affinity for the toxin than the latter,
it would unite with them by preference and intoxication
ensue. If the treatment by which the antitoxins are pro-
duced is interrupted, they immediately begin to lessen in
quantity, and eventually disappear. Their occurrence in
the blood determines that they shall be found in all the body
juices.

Their chemical composition, which experiment shows to
be of proteid nature, determines that when practical use
is to be made of them, they must not be administered by
the stomach, as digestion is usually followed by their
destruction. In infants, the proteid digestion being feeble,
antitoxins pass from the mother's milk to the sucking off-
spring without digestion, but the administration of anti-
toxins by this method at later periods of life is followed
by effects too uncertain to be depended upon. For practical
therapeutic purposes, therefore, the administration must
always be made hypodermically or intravenously.

i. Diphtheria Antitoxin. — This was first utilized for prac-
tical therapeutic purposes by Behring.* As usually pre-
pared by the administration of the toxin, it is essentially
an antitoxin and has no destructive action upon the diph-
theria bacilli. In therapeutics it is employed to neutralize
or "fix" the toxin circulating in the blood, not to destroy
the bacilli, or to effect the regeneration of the tissues injuri-
ously acted upon by the toxin. Martin is of the opinion

*" Deutsche med. Wochenschrift," 1890, Nos. 49 and 50; "Zeit-
schrift fur Hygiene," etc., xn, p. i, 1892; "Die Blutserumtherapie,"
Berlin, 1902.

156 Immunity

that such purely antitoxic serums are inferior to those con-
taining other immunity products, such as bacteriolysins,
and recommends that the whole culture instead of the
filtered culture be used in the immunization of the animal.
If this is done, the bacteriolytic effect is added to the anti-
toxic effects of the serum.

The serum may be used to prevent or to cure diphtheria.

The antitoxin is commercially manufactured at present
by immunizing horses against increasing quantities of diph-
theria toxin until the proper degree of immunity has been
attained, then withdrawing the antitoxic blood. The details
are as follows:

I. The Preparation of the Toxin. — The toxic metabolic products
of the Bacillus diphtheriae are for the most part freely soluble, and
are therefore best prepared in cultures grown in fluid media. The
medium best adapted to the purpose is that recommended by Theobald
Smith.*

To make it, the usual meat infusion receives the addition of a
culture of Bacillus coli, and is stood in a warm place overnight. The
colon bacilli ferment and remove the muscle and other sugars. The
infusion is then made into bouillon, titrated so that the reaction
equals + 1.1 when tested with phenolphthalein. It then receives an
addition of 0.2 per cent, of dextrose, and is sterilized in the autoclave.
To secure the b.est toxic product, the bacilli at hand must be carefully
studied and that naturally possessing the strongest toxicogenic power
employed for the cultures. The greatest toxicity seems to develop
between the fifth and seventh days. If the culture is permitted to
remain in the incubating oven beyond this period, the toxin gradually
is transformed to toxoid and its activity declines. The fatal dose for
a 250-300 gram guinea-pig should be about 0.001 c.c. given hypo-
dermically.

II. The Immunization of the Animals. — All commercial manu-
facturers of diphtheria antitoxic serums now use horses, as recom-
mended by Roux, instead of the sheep, dogs, and goats with which
the earlier investigators worked. The horse is readily immunized,
gives an abundant supply of blood which clots readily and yields a
beautiful clear amber serum.

The horse selected should be in perfect health, and should be tested
with mallein and tuberculin to avoid obscure glanders and tuberculosis.

A small dose of the toxic bouillon — say 0.1 c.c.— should be given
in the beginning, as one occasionally finds exceptionally susceptible
animals that will succumb to larger doses. If a marked local and
general reaction follows, it may be better to try another animal. If
no reaction is brought about, the immunization is carried on as rapidly
as possible. The toxin is injected hypodermatically into the tissues
of the neck, the skin being thoroughly cleaned and disinfected before
each injection. The doses are cautiously increased and may often be
doubled each day. If any unfavorable symptoms arise, treatment
must be interrupted for a day or two. The animal yields good anti-
toxic serum when it can endure several doses of 500 c.c. of the strong
toxin mentioned above.

HI. Bleeding. — When the withdrawal of a small quantity of
blood by a hypodermic needle introduced into the jugular vein shows

* " Journal of Experimental Medicine," May and July, 1899, p. 373.

The Antitoxins 157

that the serum contains a maximum antitoxic strength (300 to 1000
units per cubic centimeter), the horse is ready to bleed. Some horses
can be bled without resistance, but most of them require to be fastened
in appropriate stocks. The blood is taken from the jugular vein,
which is superficial, of large size, and easily accessible. The skin is
carefully shaved over an area about 9 square inches in extent, thor-
oughly disinfected. A small incision is made over the center of the
vein, which is made prominent by pressure at the base of the neck,
and the point of a small sterile trocar being inserted in the incision
through the skin, it is directed obliquely upward into the vein. The
blood is allowed to flow through a sterile tube attached to the cannula
into sterile bottles prepared to receive it. A large horse may furnish
7 to 9 liters ; small horses 5 to 7 liters.

IV. Preparation of the Serum. — The blood is stood away in a
cool place until the clot retracts after coagulation and the clear serum
separates. The serum is then withdrawn under strict aseptic pre-
cautions. It is variously prepared for the market. Some manufac-
turers bottle it without any added preservative; some add a crystal
of thymol; some Pasteurize it; some add carbolic acid; some add
trikresol.

The plain serum would be ideal, but the danger of subsequent con-
tamination through careless treatment makes it rather better to have
an antiseptic added. Trikresol is probably the most satisfactory of
these, though it throws down a precipitate that necessitates the fil-
tration of the product, and leaves the serum slightly opalescent.

V. Determining the Potency of the Serum. — The potency of the
serum is expressed as so many "immunizing units." Only one method
of testing is produced at the present time, though to understand it,
it seems wise to mention the original method from which it was derived.

(A) Behring's Method. — Behring's unit was an arbitrary standard
chosen in consequence of certain conditions existing at the time it
was devised. It is difficult to understand apart from the circum-
stances governing its creation, but may be defined as "Ten times the
least quantity of antitoxin serum that will protect a standard (300 gram)
guinea-pig against ten times the least certainly fatal dose of toxic bouillon."

The method of determining it is not difficult to those skilled in
laboratory technic, and is as follows :

1. Determine accurately the least certainly fatal dose of a sterile
diphtheria toxic bouillon for a standard guinea-pig.

2. Determine accurately the least quantity of the serum that will
protect the guinea-pig against ten times the above determined least
fatal dose of toxin.

3. Express the required dose of antitoxic serum as a fraction of a
cubic centimeter and multiply by 10 ; the result is one unit.

Example: It is found that 0.01 c.c. of a toxic bouillon kills at least
9 out of 10 guinea-pigs, and is therefore the least certainly fatal dose.
Guinea-pigs receive ten times this dose of the toxic bouillon plus
varying quantities of the serum to be tested, measured by dilution —
?ay 2oW c-c-» 7 oW c-c-» JoW c-c- The first two live- The fraction ^V<y
is now multiplied by 10; ^ylnr X 10 = ^ = 1 unit. So we find
that each cubic centimeter of the serum contains 250 units.

This method would be satisfactory were it not for certain variations
in the toxic bouillon by which the strength is worked out. Ehrlich,*
in an elaborate investigation of these changes, has clearly proved that
an ever-changing toxin cannot be a satisfactory standard, because
it does not possess uniform combining affinity for the antitoxin. He
shows by a labored scheme that the toxicity of the bouillon is no

* " Klinisches Jahrbuch," 1897.

158 Immunity

index to its antitoxin-combining power, which, of course, must be
the foundation of the test. The toxin, under natural conditions, is
changed with varying rapidity into toxoids, of which he demonstrates
three groups — prototoxoids, syntoxoids, and epitoxoids. The epi-
toxoids have a greater antitoxin-combining power than the toxin
itself, yet have no toxic action upon the guinea-pigs, hence cause
confusion in the results.

To secure a satisfactory measure of the antitoxic strength of a
serum, it is therefore more important to first determine the antitoxin-
combining power of the toxin or toxic bouillon to be used, than to
determine its guinea-pig fatality, and this is what Ehrlich endeavors
to do.

(B) Ehrlich's Method. — In this method the unit is the same as in
Behring's method, but its determination is arrived at by a very im-
portant modification of the method, by which the standard of measure-
ment is a special antitoxin of known strength, by which the antitoxin-
combining power of the test toxic bouillon is first determined. Ehrlich
began by determining the antitoxic value of a serum as accurately as
possible by the old method and then used that serum as the standard
for all further determinations. The serum was dried in a vacuum, and
two grams of the dry powder were placed in each of a large number
of small vacuum tubes, connecting with a small bulb of phosphoric
anhydride. In this way the standard powder was protected from
oxygen, water, and other injurious agents by which variations in its
strength could be initiated. Periodically one of these tubes was
opened and the contained powder dissolved in 200 c.c. of a mixture
of 10 per cent, aqueous solution of sodium chloride and glycerin.
The subsequent calculations are all based upon the strength of the
antitoxin powder. In Ehrlich's first test serum 1 gram of the dry
powder represented 1700 units. Of the solution mentioned, 1 c.c.
represented 17 units; ^ c.c., one unit.

Having by dilution — 1 c.c. of the first dilution in 17 of water —
secured the standard unit of antitoxin in a convenient bulk for the
subsequent manipulations, it is mixed with varying quantities of the
toxic bouillon to be used for testing the new serums, until the least
quantity is determined that will cause the death of a 250-gram guinea-
pig in exactly four days, when carefully injected beneath the skin
of the animal's abdomen. This quantity of toxin is the test dose.
If the toxic bouillon was "normal" in constitution, it should represent
100 of the least certainly fatal doses that formed the basis of the old
method of testing, but as toxic bouillons contain varying quantities
of toxoids it may equal anywhere from fifty to one hundred and fifty
times that dose.

The test dose of toxic bouillon, having been determined, remains
invariable throughout the test as before, the serum to be tested for
comparison with the standard being modified. The calculation is,
however, different because the guinea-pig is receiving, not ten times,
but more nearly one hundred times the least fatal dose, and the quan-
tity of the antitoxic serum that preserves life beyond the fourth day
is itself the unit.

Example: The sample of serum issued as the standard contains 17
units per cubic centimeter. Serum 1 c.c. + water 16 c.c. = 1 c.c.
is the unit. 1 c.c. of the dilution containing one antitoxic unit is
mixed with 0.01, 0.025, 0.05, 0.075, 0.1 c.c. of the toxic bouillon. All
the animals receiving less than 0.1 c.c. live. A new series is started,
and the guinea-pigs all weighing exactly 250 grams receive 1 unit of
the antitoxin plus toxic bouillon 0.08, 0.09, 0.095, 0.097, 0.1, 0.11,
0.12, etc. It is found that all receiving more than 0.097 die in four

The Antitoxins 159

days, but that the animal receiving that dose, though very ill, lives
longer. The test dose may then be assumed to be 0.1, or it may
be calculated more closely if desired.

To test the serum itself, guinea-pigs weighing exactly 250 grams
are now all given toxic bouillon 0.1 c.c. plus varying quantities of the
serum — ^, ^fa, ^fa, etc. All live except those receiving less than
T£7, which die about or on the fourth day. The serum can then be
assumed to have 400 units per cubic centimeter unless it be desired
to test more closely.

Standard test serums for making tests of antitoxic serums
by the Ehrlich method were first shipped at small expense
from the Kaiserliches Institut fur Serum-Therapie at Hochst-
on-the-Main. At present the Hygienic Laboratory of the
United States Public Health and Marine Hospital Service
has legal control of the manufacture of therapeutic serums
and kindred products in the United States, issuing licenses
to those engaged in legitimate manufacture, and furnishing
a standrad test serum, similar to that of Ehrlich, to those
entitled to receive it.

A full description of " The Immunity Unit for Standard-
izing Diphtheria Antitoxin," by M. J. Rosenau, Director of
the Hygienic Laboratory, can be found in Bulletin No. 21
of the U. S. Public Health and Marine Hospital Service,
Washington, 1905.

As the quantity to be injected at each dose diminishes
according to the number of units per cubic centimeter the
serum contains, it is of the highest importance that therapeu-
tic serums be as strong as possible. Various methods of
concentration have been suggested. Bujwid * and H. C.
Ernst f found that when an antitoxic serum is frozen and
then thawed, it separates into two layers, the upper stratum
watery, the lower yellowish, the antitoxic value of the
yellowish layer being about three times that of the original
serum, the upper layer consisting chiefly of water.

The most satisfactory method of securing a useful concen-
tration is by the employment of the globulin precipitation
as recommended by Gibson, J which is briefly as follows:
The diluted citrated plasma is precipitated with an equal
volume of saturated ammonium sulphate solution and the
antitoxic proteins separated by extracting the precipitate

*"Centralbl. f. Bakt. u. Parasitenk.," Sept. 1897, Bd. xxn, Nos.
10 and 1 1, p. 287.

f "Jour. Boston Soc. of Med. Sci.," May, 1898, vol. n, No. 8, p.
i37-

t "Jour. Biol. Chem.," i, p. 161; m, p. 253.

1 60 Immunity

with saturated sodium chloride solution. The soluble
antitoxic proteins are then reprecipitated from the saturated
sodium chloride solution with acetic acid. This filtered
precipitate is then partially dried between filter-papers and
dialyzed in running water. This yields a final product
which when dried in vacuo is readily soluble in salt solution
and is free from many of the offensive substances in the horse
serum. Steinhardt and Bauzhaf* found that the therapeutic
value of the plasma was not appreciably impaired through
the process of eliminating the albumins and other non-
antitoxic proteins by the salting out methods employed,
and the final dialyzation of the concentrated product,
thus disproving the objection of Cruveilhierf on this point.

2. Tetanus antitoxin was first prepared by Behring and
Kitasato. J It can be employed for the prevention or cure of
tetanus. For the former purpose, hypodermic injections of the
serum may be given in cases with suspicious wounds, or the
wounds may be dusted with a powder made by pulverizing
the dried serum. For treatment the serum must be admin-
istered in frequently repeated large doses by hypodermic or
intravenous injection. The results are less brilliant than
those attained with diphtheria antitoxin because of the avid-
ity with which the cells of the central nervous system take
up the tetanus toxin, and the firmness of the union formed.
An analysis of a great number of cases has, however,
shown that the recoveries following the free administra-
tion of the serum exceed the recoveries effected by other
methods of treatment by about 40 per cent.

By the gradual introduction of tetanus toxin Behring
and Kitasato§ have been able to produce a powerful anti-
toxic substance in the blood of animals.

The method of obtaining tetanus antitoxic serum is like
that employed for securing diphtheria antitoxic serum
(q. v.).

Madsen|| found that for each of the specific poisons,
tetanolysin and tetanospasmin, a specific antitoxin is pro-
duced, the one annulling the convulsive, the other the
hemolytic, properties of the toxin. The usual therapeutic
serums contain both of these.

* "Jour. Infectious Diseases," vol. n, pp. 202 and 264, March, 1908.

f "Ann. de 1'Inst. Pasteur," 1904, xvm, p. 249.

J " Deutsche med. Wochenschrift," 1890, No. 49.

§ Ibid.

|| "Zeitschrift fur Hygiene," 1899, xxxm, p. 239

The Antitoxins 161

Different standards for measuring the strength of the
tetanus toxin and different definitions of the unit of measure-
ment are given in different countries, so that great confusion
and dissatisfaction were experienced until a special committee
of the Society of American Bacteriologists met in New York,
Dec. 27 and 28, 1906, and in collaboration with the United
States Public Health and Marine Hospital Service, Hygienic
Laboratory, formulated a standard unit which has become
the legal unit of measurment for the United States. It is
thus defined :

" The immunity unit for measuring the strength of tetanus
antitoxin shall be ten times the least quantity of antitetanic
serum necessary to save the life of a 35o-grain guinea-pig
for ninety-six hours against the official test dose of a standard
toxin furnished by the Hygienic Laboratory of the Public
Health and Marine Hospital Service." The unit is thus
officially denned, Oct. 25, 1907, in Treasury Circular, No. 61.

Testing tetanus antitoxic serums immediately became
a matter of great simplicity. The governmental laboratory
furnishes the ''test toxin" whose strength is guaranteed,
and what follows is a simple matter of dilution, admixture
with the serum to be tested, and injection animals that are
carefully observed for a few days.

The entire subject, historical, theoretical, and practical,
is treated in Bulletin, No. 43, 1908, of the Hygienic Labora-
tory upon "The Standardization of Tetanus Antitoxin,"
by Rosenau and Anderson.

3. Antivenene or Anti-venomous Serum. — This was
discovered by Phisalix and Bertrand* and made practical
for therapeutic purposes by Calmette.t Calmette found
that cobra venom contained two principles, one of which,
labile in nature and readily destroyed fey heat, was destruc-
tive in action upon the tissues with which it came into direct
contact, the other, stable in nature, was death-dealing
through its action upon the respiratory centers. By heat-
ing the venoms and thus destroying the irritative principle,
he was able to immunize animals against the other, which
he looked upon as the important element of the venom.
The immunized animals furnished an anti-serum, which

* "Compt. rendu de 1'Acad. des Sciences de Paris," Feb. 5, 1894,
Tome cxvm, p. 356.

t "Compt. rendu de la Soc. de Biol. de Paris," 10 Series, Tome i,
p. 1 20, Feb. 10, 1894.
ii

1 62 Immunity

entirely annulled the effect of the toxin (modified venom)
used in treating them. This serum was found to protect
rabbits and other animals against both modified and un-
modified cobra venom, and was used successfully in the
treatment of a number of human beings that had been bitten
by cobras. Calmette, however, erroneously concluded that
because in most venoms studied he was able to find a
larger or smaller proportion of the respiratory poison, it
constituted the essential element of the venom to be an-
tagonized. Arguing from this standpoint, he recommended
his antivenene in all cases of snake-bite, regardless of the
variety of serpent. C. J. Martin* and others showed that
Calmette was wrong, and that his antivenene was useless
in the treatment of the bites of the Australian serpents,
and my own experiments have shown it to be useless
in the treatment of the bites of the American snakes. In
the venoms of our snakes — the rattlesnake, copper-head,
and moccasin — the poison is essentially locally destructive
in action, the fatal influence upon the respiratory centers
being of secondary importance, fatalities from snake-bite
being rare in the United States. Although I made many
attempts to immunize horses against this locally destructive
substance, which has since been carefully studied by Flexner
and Noguchi,f I was unable to do so. Noguchi { and
Madsen and Noguchi,§ however, applied Ehrlich's principle
to the investigation, destroyed the toxophorous group of
the venom molecules, and succeeded in producing an anti-
serum useful in antagonizing the active principle — hem-
orrhagin — of the Crotalus venom.

Antivenene is useful in the treatment of cobra invenoma-
tion, as Calmette has shown by cases treated in his own
laboratory. The serums of Noguchi and others are equally
useful in their respective invenomations, but the opportunity
for successfully employing antivenenes is very small. Few
persons are bitten in places and at times where the remedy
is at hand, and the effects of venom of all kinds are so rapid
that immediate treatment is required. In India and a few
other reptile infected countries, as well as in zoological
gardens where venomous serpents are kept, and in labora-
tories where the snakes are kept for experimental purposes,

* "Intercolonial Medical Journal of Australia," 1897, n, p. 537.
t "Journal of Experimental Medicine," vi, p. 277, 1901-1905.
J Ibid., vm, p. 614, 1906. § Ibid., ix, p. 18, 1907.

The Cytotoxins 163

it is well to be provided with a supply of the serum, but it
has no wide sphere of usefulness.

4. Miscellaneous anti-bodies of many kinds have been
experimentally produced, — anti-enzymes, etc., — but have
no practical application. A knowledge of them is, however,
essential to a thorough understanding of the reactions of
immunity. *In the synopsis of the experiments upon im-
munity reference is made to these bodies and to the litera-
ture bearing upon them.

IV. Cytotoxins.— The reaction takes place through the
combination of the "amboceptor, " " immune body , " "sub-
stance sensibilisatrice," "fixateur," or "desmon" with the
"addiment," "complement," "alexin," or "cytase, "

Hemolysins. — The phenomena of hemolysis caused by
heterologous serums were first studied by Creite* and Lan-
dois,f who studied hemoglobinuria following transfusion.
Subsequent observations were made upon corpuscular
agglutination and solution by venoms by Mitchell and
StewartJ and by Flexner and Noguchi,§ and upon the effects
upon corpuscles of warm-blooded animals, of the poisonous
serum of certain eels by Mosso||, Camus and Gley,** and
Kossel. ft The serious consideration of the subject was,
however, deferred until Belfanti and Carbo ne JJ showed that
if horses were injected with red corpuscles of rabbits, the
serum thereafter obtained from the horses would be toxic
for rabbits; Bordet §§ had shown that the serum of guinea-
pigs injected several times with 3 to 5 c.c. of the defibrinated
blood of rabbits acquired the property of rapidly dissolving
the red corpuscles of the rabbit in a test-tube, and Ehrlich and
MorgenrothHII had shown the mechanism of the hemolytic
action. From this time on the literature of hemolysis rapidly
grew and the subject assumed a more and more important
place in the domain of chemico-physiological research.

* "Zeitschrift f. ration. Med.," Bd. xxxvi, 1869 — quoted by Nuttall
in his "Blood Immunity and Relationships."

t "Zur Lehre von der Bluttransfusion," Leipzig, 1875.

t "Transactions of the College of Physicians of Philadelphia," 1897,
p. 105.

§ "Journal of Exp. Med.," 1901-1905, vi, p. 277.

|| "Archiv. f. Exp. Path, and Pharmak.," xxv, pp. in and 135.
** "Compt. rendu de la Soc. de Biol. de Paris," 1898, p. 129.
ft "Berliner klin. Wochenschrift," 1898.
it "Jour, de la R. Acad. d. Med. de Torino," 1898, No. 8.
§§ "Ann. de 1'Inst. Pasteur," 1898, xn, 688.
HIl " Berliner klin. Wochenschrift," 1899.

1 64 Immunity

The technic of hemolytic studies is comparatively simple,
and it is intended in this chapter to do no more than offer
the student a simple method of performing experiments
which he can modify to suit his own purposes.

The Preparation of Blood-corpuscles. — For the study of hemo-
lysis and hemo-agglutination it is necesary to prepare, a 5 per cent,
suspension of the blood-corpuscles in an isotonic salt (NaCl) solution.
To do this the blood of the animal is permitted to flow into a sterile
tube and is immediately stirred with a small stick or a platinum wire
until completely defibrinated. Some salt solution (0.85-0.9 per cent.)
is then added and the mixture shaken. It is then placed in a sterile
centrifuge tube and rotated until the corpuscles are packed in a mass
at the bottom. The supernatant fluid is poured off, replaced by an
equal volume of salt solution, and shaken until the corpuscles are again
thoroughly distributed. It is then again centrifugated and the fluid
again poured off, after which 95 parts (by volume as compared with
the corpuscular mass) of the salt solution are added and the fluid thor-
oughly shaken to distribute the corpuscles. This slightly greenish-red
fluid is the 5 per cent, solution of corpuscles. It is, of course, not per-
manent, and easily spoils if bacteria enter. It also gradually deterior-
ates through changes in the corpuscles, so that it is not usually useful
after the third day, even when kept on ice.

The hemolytic substance to be investigated must be isotonic with
the corpuscles and therefore must be dissolved in, or diluted with, the
same salt solution as that used for making the corpuscular suspension.
Neglect to observe this requirement may lead to error by diminishing
the tonicity of the solution and inducing spontaneous or hypotonic
disintegration of the corpuscles.

To secure a specifically hemolytic serum one injects an animal —
say a rabbit or guinea-pig — with lo-c.c. doses of defibrinated blood of
the animal for whose corpuscles the serum is to be made hemolytic,
the doses being given intraperitoneally or intravenously, six times, at
intervals of a week. The animal is then bled, the blood permitted to
coagulate, the serum separated and filtered, if necessary.

The contact of the corpuscles and the hemolytic substance is best
conducted in small test-tubes holding about 2 c.c. of the mixed fluids.
It is usually best to work with a constant volume of the blood-corpuscle
suspension and varying quantities or concentrations of the hemolytic
substances. Two observations are to be made, one after thirty minutes
sojourn in the thermostat at 37° C., the other after twenty-four hours
in the ice-box, both observations being made on the same series of tubes.
Hemolysis is shown by the appearance of a beautiful clear red color
of the formerly cloudy greenish suspension. One must notice the dif-
ference between partial and complete hemolysis, different additions
of the hemolytic substance being required for these results.

Cytolysins. — It was soon found that the phenomena of
hemolysis corresponded to those by which many other cells,
vegetable and animal, were destroyed and dissolved through
the activity of immunity products. To such as were applic-
able to bacteria, the term bacteriolysis was applied ; to such
as were applicable to animal cells, the term cytolysis was
applied; epitheliolysis, endotheliolysis, hemolysis, spermato-

The Cytotoxins 365

lysis, etc., being used as specific terms. Delezene* first
produced a leukolytic or leukocyte-destroying serum by
treating animals with the leukocytes of a heterogeneous
species; Metalnikoff , f by injecting the spermatozoa of one
animal into another of another species, produced a sper-
motoxic or spermolytic serum; von DiingernJ produced a
serum capable of dissolving the ciliated epithelium scraped
from the trachea of an ox by injecting the dissociated epi-
thelial cells into an animal; Delezene § found that by
injecting an animal with the dissociated liver cells of a
heterogeneous animal, a hepatolytic serum could be pro-
duced, and so grew up a large literature upon cytotoxins.

The technic of these investigators is not difficult. It is, however,
first necessary to prepare a homogeneous tissue pulp for injection into
the animal that is to furnish immune serum. For this purpose one of
the best forms of apparatus is that of Latapie.|| After the pulp is made,

Fig. 29. — Latapie's instrument for preparing tissue pulp.

it is introduced into the animal in the same manner as the blood for
making the hemolytic serum, the animal bled appropriately, the serum
separated and filtered. The remaining steps do not differ essentially.
The tissue suspension, having about the same concentration as the 5
per cent. NaCl suspensions of the corpuscles, is used as the constant
quantity and the immune serum used as the variable quantity. The
two are brought into contact in small test-tubes, kept for twenty-four

* "Compt. rendu de 1'Acad. de Sciences de Paris," 1900.
t "Ann. de 1'Inst. Pasteur," 1899.
£ "Munchener med. Wochenschrift," 1899.

§ "Compt. rendu de 1'Acad. de Sciences de Paris," 1900, cxxx, pp.
938 and 1488.

II "Ann. de 1'Inst. Pasteur," 1902, xvi, p. 947.

1 66 Immunity

hours in the refrigerator, and the amount of solution gauged by the
naked eye supplemented by microscopical examination of the tissue
elements.

Bacteriolysins. — The first observations upon bacterioly-
sis was made in 1874 by Traube and Gescheidel,* who found
that freshly drawn blood was destructive to bacteria. The
matter was pursued by numerous subsequent investigators
and was explained by Buchner as depending upon alexines.
Pfeifferf described the peculiar reaction known as "Pfeiffer's
phenomenon." Ehrlich and Morgenrothf and Bordet§ de-
scribed the mechanism of cytolysis, explaining the "Pfeiffer
phenomenon" and paving the way for future experiments.

Direct destruction of bacteria by blood-serum and body
juices is rare, and occurs only when the serum contains
appropriate quantities of both factors involved — i. e., ambo-
ceptor and complement. For the usual bacteriolytic
investigations it is, therefore, necessary to consider three
factors: i, The bacteria to be destroyed; 2, the serum fur-
nishing the complement; and 3, the serum furnishing the
immune body.

1. The bacteria to be destroyed should be prepared in the form of a
homogeneous suspension similar to that employed for making the ag-
glutination tests. It is best to use the surface growths from agar-agar,
rubbed between glasses or ground with sodium chlorid solution, added
drop by drop, in a mortar, or well rubbed upon the side of a test-tube
containing the fluid, which is permitted to contact with the mass from
time to time by inclining the tube so that the fluid is able to carry
away the bacteria as they are distributed.

If quantitative estimations are to be made, the number of bacteria
in the suspension must be known or at least a standard quantity must
be employed, as the destructive process is a chemical one, in which the
destructive agents are themselves used up.

2. The serum furnishing the complement is a normal serum-— that is,
the serum from a healthy animal that has undergone no manipulation.

3. The serum containing the amboceptor or the immune body is
obtained from an animal that has been given a high degree of immuni-
zation against the bacterium to be destroyed or dissolved. If it is
desirable, any complement contained in this serum can be destroyed
by heating for a short time to a point just short of coagulation.

These three having been prepared, an appropriate quantity of the
bacterial suspension is placed in a small test-tube, and an appropriate
quantity of the normal serum added. To this mixture of two constants,
varying quantities of the immune serum is added and the tube stood
away for twenty-four hours on ice. In most every case it will be found
that the immune serum contains a great quantity of agglutinating

* " Jahresb. der Schles. Ges. f. vaterl. Kultur," 1874.
f "Deutsche med. Wochenschrift," 1896, No. 7.
J "Berliner klin. Wochenschrift," 1899
§ "Ann. de 1'Inst. Pasteur," xn, 1898.

The Cytotoxins 167

substance, so that the bacteria all fall to the bottom in a short time.
This is independent of bacteriolysis. The bacterial destruction is
gauged by the disappearance of the bacteria which have been dissolved,
or by their failure to grow when transplanted to appropriate culture
media, showing that they have been killed.

By making the bacterial suspension and complementary serum
constant quantities (taking care that not too many bacteria be present),
one is able to estimate the value of the immune serum. By using the
bacterial suspension and a heated immune serum (containing no com-
plement) as constants and varying the addition of complementary
serum, one can estimate the respective values of several complementary
serums. By using both serums as constant factors and varying the
number of bacteria, one can determine the exact bacteriolytic value of
the mixture. By taking out and planting drops from time to time the
rapidity of bacteriolysis can be determined, and by plating out the drops
and counting the colonies one may arrive at percentages of destruction
and express the bacteriolytic process in the form of a curve.

A peculiar phenomenon is sometimes observed and has
been studied by Neisser and Wechsberg,* and is commonly
called by their names. It is the result of "deviation of the
complement," the condition being as follows: When an
animal whose blood-serum is normally possessed of a high
degree of germicidal power is immunized by repeated injec-
tions of a bacterial endotoxin, its serum when examined by
the usual methods fails to show the usual increase in the
specific bactericidal action toward that particular organism,
though it retains its general bacteria-destroying power.
If, however, the serum be greatly diluted, its action is
changed, so that it loses its general bacteria-destroying power
and develops marked increase in the specific destructive
action upon the particular bacteria used in the experiment.
Neisser and Wechsberg attribute the peculiar reaction to the
fact that there being more amboceptors than complements
in the serum, some of the former satisfy their combining
affinities by attaching themselves to the bacteria, some by
attaching themselves to the complement, instead of forming
combinations of all three. If under these circumstances the
serum containing the amboceptors is diluted until their
number becomes approximately equal to the number of com-
plements introduced, any deviation resulting from inequality
of the combining affinities becomes improbable. Bordet
and Gay,f however, have performed experiments tending
to show that these elements do not really unite, thus seem-
ing to controvert the theory of Neisser and Wechsberg, and

*" Munch, med. Wochenschrift," XLVIII, No. 13, p. 697, April 30,
1901.

t "Ann. de 1'Inst. Pasteur," xx, June 25, 1906, No. 6, pp. 267-498.

1 68

Immunity

Bolton * has shown that normal serum may kill relatively
more bacteria when diluted than when undiluted.

A1

II*

Iff

Iff

Fig. 30. — Diagram illustrating the Neisser-Wechsberg phenomenon
of "deviation of complement." In A1 the three black units (c) repre-
sent the quantity of complement necessary for the dissolution of a bac-
terium, and the three white units (/>) the intermediate bodies or am-
boceptors through which they may act. A2 shows these properly
proportioned units properly combined and anchored to the bacterial
cell which will be destroyed. If an excess of amboceptor units be
present, as is suggested in Bl, the resulting combinations and the conse-
quent results may vary according to the differing combining affinities.
Thus, B2 shows an unchanged affinity, i. e., only those amboceptors
unite with bacterial cells that are charged with complement. C2 shows
equal affinity of the amboceptors for complement and for the bacterial
cell, so that charged or uncharged units attach themselves to the cell,
diminishing the complementary action. D2 shows the possible result
when the affinity of the amboceptor for the bacterial cell is diminished
after charging with complement, so that though the complement and
amboceptor combine, there can be no destruction of the bacterium.
Thus, excess of the amboceptor units may "deviate the complement "
and prevent its action.

It was at first hoped that some of these serums and
especially the bacteriolytic serums would have a wide thera-

* "The Bacteriolytic Power of the Blood-serum of Hogs," Bull. No.
95 of the Bureau of Animal Industry, U. S. Dept. of Agriculture.

The Cytotoxins 169

peutic application in cases in which non-toxicogenic bacteria
were invading the body, but experiment and experience have
shown that the laws governing their action greatly limit
their application, and that their effects, when not beneficial,
are bound to be harmful. The difficulty lies in the fact that
when we manufacture such serums we prepare only the
immune body, there being no increase of the complement.

Fig. 31. — Schematic representation of the interfering action of an-
amboceptors and anti-complements. A, Anti-amboceptor action: c,
Complement ; am, amboceptor ; aa, antiamboceptor preventing the am-
boceptor from connecting with the cell. B : c, Complement ; ac, anti-
complement preventing the complement from connecting with the am-
boceptor, am.

To introduce this by itself does the patient no good,
because in most cases the existing infection has brought
about the formation of as much or more ''immune body"
than can be utilized by the complement. To give injec-
tions of active bodies that cannot be utilized is shown by
Comus and Gley* and Kosself to be followed by the forma-
tion of anti-bodies — in this case " anti-immune bodies" — by
which their effect is neutralized. Should anti-immune bodies
be found by this meddlesome medication, the state of the
infected animal would be worse than before, because it would
now be preparing that which by neutralizing the combining
affinities of its own immune bodies, would prevent them from
combining with the elements to be destroyed and so activat-
ing the complements.

No satisfactory method of experimentally increasing the
complement has been devised. If, as Metschnikoff supposes,
the complement is microcytase derived from disintegrated
leukocytes, aseptic suppurations with active phagolysis

* " Compte rendu de 1'Acad. de Sciences de Paris," Jan. i, 1898, 126.

f "Berl. klin. Woch.," 1898, S. 152.

1 70 Immunity

should result in marked increase of the complement. As a
matter of fact, this does take place, but the increase is so
slight that the serum is not practically valuable.

Therapeutic serums whose practical application is based
upon their cytolytic activity must, of necessity, contain
both the essential factors involved in cytolysis, and should
contain them in such proportions that, regardless of other
elements in the blood, they can exercise their combining and
dissolving functions.

We are unable experimentally to accomplish these pre-
requisites, therefore are not in the position to accurately
apply bacteriolytic serums in practice.

V. Complement Fixation.— In 1901 Bordet, while in-
vestigating the nature of the complementary substance,
made a discovery that has now become of great importance,
that is, the " Bordet-Gengou phenomenon," or, as it is now
known, the " fixation of the complement." His method of
procedure was as follows: Blood-corpuscles were sensitized
with appropriate amboceptors and then treated with freshly
drawn normal serum. Hemolysis resulted. If now he added
to the mixture some sensitized blood-corpuscles of a different
species, they did not hemolyze. Clearly, the complement had
been used up in the first hemolysis.

He next found that if, instead of employing blood-corpuscles
for the first test, he used sensitized bacteria — i. e., bacteria
treated with an immune serum containing the amboceptors
appropriate for effecting their solution — the complement would
similarly be used up, " fixed," so that when he subsequently
added sensitized red blood-corpuscles there was no hemo-
lysis.

This reaction was naturally quantitative, the result as de-
scribed depending upon the fact that no more complement
(normal serum) was used in the original hemolysis or bac-
teriolysis than was necessary, and so none left "unfixed"
to effect the lysis or solution of the second factor introduced.

Bordet interpreted his results as indicating that there was
only one complementary or solvent substance, and though
Khrlich subsequently published what he looked upon as
proofs to the contrary, the opinion of Bordet prevails.

In addition, however, Bordet's experiments have been of
practical use. As affording a means of quantitative experi-
mentation they have enabled investigators to measure the
quantity of complement in normal bloods and in immunized

Complement Fixation 171

bloods, and so led to the discovery that for each kind of
animal and for each individual animal the complement is
subject to very little variation. In the course of some three
years they were followed by the investigations of Neisser
and Sachs upon antigens, and made to subserve the useful
purpose of recognizing and differentiating antigenic sub-
stances. Thus, when a certain antibody and its comple-
ment are combined they can only attach themselves to the
particular specific antigen by which the antibody has been
developed. But, what is still more important, they have led
to the invention of methods by which the presence of specific
amboceptors may be determined where they are suspected,
and so have made possible means of arriving at a correct
diagnosis in certain obscure cases of disease in man.

The most important of these measures is the Wassermann
reaction for the diagnosis of syphilis (q. v.). By careful
perusal of the chapter upon the method of performing the
Wassermann reaction the student will learn the general de-
tails of the technic, and can modify them to correspond to
the requirements of other cases in which complement fixa-
tion is to be studied.

CHAPTER V.

METHODS OF OBSERVING MICRO-ORGANISMS.

IT is of the utmost importance to examine micro-organisms
alive, and as nearly as possible in their normal environment,
then to supplement this examination by the study of dead and
stained specimens.

The study of the living organism has the advantage of
showing its true shape, size, grouping, motility, reproduc-
tion, and natural history. It has the disadvantage of being
somewhat difficult because of its small size and trans-
parency.

So long as bacteria were observed only in the natural
condition, however, it was impossible to find them in the
tissues of diseased animals, and it was not until Weigert
suggested the use of the anilin dyes for coloring them that
their demonstration was made easy and their relationship
to pathologic conditions established.

The beauty and clearness of stained specimens, and the
ease with which they can be observed, have led to some serious
errors on the part of students, who often fail to realize the
unnatural condition of the stained bacteria they observe.
It only needs a moment's consideration to show how dis-
turbed must be the structure of an organism after it has
been dried, fixed, boiled, or steamed, passed through several
chemic reagents, dehydrated and impregnated with stains,
etc., to suggest how totally unnatural its appearance may
become.

It is, therefore, necessary to examine every organism,
under study, in the living condition, and to control all
the appearances of the stained specimen by comparison.

L THE STUDY OF LIVING BACTERIA.

The simplest method of observing live bacteria is to
take a drop of liquid containing them, place it upon a slide,
put on a cover, and examine.

172

The Study of Living Bacteria 173

While this method is simple, it cannot be recommended,
as evaporation at the edges causes currents of liquid to flow
to and fro beneath the cover, carrying the bacteria with
them and making it almost impossible to determine whether
the organisms under examination are motile or not. Should
it be desirable that such a specimen be kept for a time,
so much evaporation takes place that in the course of
an hour or two it has changed too much to be of further
use.

The best way to examine living micro-organisms is in
what is called the hanging drop (Fig. 32). A hollow-ground
slide is used, and with the aid of a small camel's-hair pencil
a ring of vaselin is drawn on the slide about, not in, the
concavity at its center. A drop of the material to be ex-
amined is placed in the center of a large clean cover-glass

Fig. 32. — The " hanging drop " seen from above and in profile.

and then placed upon the slide so that the drop hangs in,
but does not touch, the concavity. The micro-organisms
are thus hermetically sealed in an air chamber, and appear
under almost the same conditions as in the culture. Such a
specimen may be kept and examined from day to day, the
bacteria continuing to live until the oxygen or nutriment
is exhausted. By means of a special apparatus (Fig. 33),
in which the microscope is placed, the growing bacteria may
be watched at any temperature, and very exact observa
tions made.

The hanging drop should always be examined at the
edge, as the center is too thick.

In such a specimen it is possible to determine the shape,
size, grouping, division, sporulation, and motility of the
organism under observation.

174 Methods of Observing Micro-organisms

Care should be exercised to use a rather small drop,
especially for the detection of motility, as a large one vi-
brates and masks the motility of the sluggish forms.

When the bacteria to be observed are in solid or semi-
solid culture, a small quantity of the culture should be mixed
in a drop of sterile bouillon or other fluid.'

For observing the growth of bacteria where it is desirable
to prevent movement, Hill * has invented an ingenious
device which he calls the " hanging block.'' His directions
for preparing it are as follows: +

"Pour melted nutrient agar into a Petri dish to the depth of about
one-eighth or one-quarter inch. Cool this agar, and cut from it a
block about one-quarter inch to one-third inch square and of the thick-
ness of the agar layer in the dish. This block has a smooth upper and
under surface. Place it, under side down, on a slide and protect it
from dust. Prepare an emulsion, in sterile water, of the organism
to be examined if it has been grown on a solid medium, or use a broth
culture; spread the emulsion or broth upon the upper surface of the
block as if making an ordinary cover-slip preparation. Place the
slide and block in a 37° C. incub'ator for five to ten minutes to dry
slightly. Then lay a clean sterile cover-slip on the inoculated surface
of the block in close contact with it, usually avoiding air-bubbles.
Remove the slide from the lower surface of the block and invert the
cover-slip so that the agar block is uppermost. With a platinum
loop run a drop or two of melted agar along each side of the agar
block, to fill the angles between the sides of the block and the cover-
slip. This seal hardens at once, preventing slipping of the block.
Place the preparation in the incubator again for five or ten minutes
to dry the agar-agar seal. Invert this preparation over a moist cham-
ber and seal the cover-slip in place with white wax or paraffin. Vaselin
softens too readily at 37° C., allowing shifting of the cover-slip. The
preparation may then be examined at leisure."

With this means of examining the growing cultures, Hill
has acquired interesting knowledge of the fission and budding
of Bacillus diphtherias

If the specimens to be examined must be kept for some
time at an elevated temperature, some such apparatus as
that of Nuttall will be found useful though expensive.

II. STAINING BACTERIA.

In the early days of bacteriology efforts were made to facili-
tate the observation of bacteria by the use of nuclear dyes.
Both carmin and hematoxylin tinge the nuclei of the bac-
teria a little, but so unsatisfactorily that since Weigert in-
troduced the anilin dyes for the purpose, all other stains

* "Journal of Medical Research," vol. vn, No. 2; new series, vol. n,
March, 1902.

Staining Bacteria

175

'have been abandoned. The affinity between the bacteria
and the anilin dyes is peculiar, and in certain cases can be
used for the differentiation of species.

The best anilin dyes made at the present time, and those
which have become
the standard for all
bacteriologic work,
are made in Ger-
many by Dr. Grii-
bler, and in or-
dering stains the
name of this man-
ufacturer should be
specified.

Readers interest-
ed in the biochemis-
try of the subject
will do well to refer
to the excellent
papers by Arnold
Grimme,* upon
"The Important
Methods of Stain-
ing Bacteria, etc.,"
and Marx,f upon
" The Metachro-
matic and Babes-
Ernst Granules."

In this work
special methods for
staining such bac-
teria as have pecu-
liar reactions will
be given together
with the descrip-
tion of the particular organisms, general methods
being discussed in this chapter.

Preparations for General Examination. — For bacterio-
logic purposes thin covers (No. i) are required, because
thicker glasses may interfere with the focussing of the oil-
immersion lenses. The cover-glasses must be perfectly

* "Centralbl. f. Bakt.," etc., Bd. xxxn, Nos. 2, 3, 4, and 5, 1902.

f Ibid., xxxii, Nos. 10 and u, p. 108, 1902.

Fig. 33. — Apparatus for keeping objects
under microscopic examination at constant
temperatures (Nuttall).

only

176 Methods of Observing Micro-organisms

dean. It is therefore best to clean a large quantity in ad-
vance of use by immersing them first in a strong mineral
acid, then washing them in water, then in alcohol, then in
ether, and finally keeping them in ether until they are to
be used. Except that it sometimes cracks, bends, or
fuses the edge of the glass, a more convenient method is
to wipe the glasses as clean as possible with a soft cotton
cloth, seize them with fine-pointed forceps, and pass them
repeatedly through a small Bunsen flame until it becomes
greenish-yellow. The hot glass must then be slowly elevated
above the flame, so as to allow it to anneal. This manceuver
removes the organic matter by combustion. It is not
expedient to use covers twice for bacteriologic work, though
if well cleansed by immersion in acid and washing, they
may subsequently be employed for ordinary microscopic
objects.

The fragility of the covers and their likelihood to be
broken or dropped at the critical moment, make most
workers prefer to stain directly upon the slide. The slide
should be thoroughly cleaned, and if the material to be
examined is spread near one end, the other may serve as a
convenient handle. The slide is also to be preferred if
a number of examinations are to be made simultaneously
or for comparison, as it is large enough to contain a number
of ''smears."

Simple Method of Staining. — The material to be ex-
amined must be spread in the thinnest possible layer upon
the surface of the perfectly clean cover-glass or slide and dried.
The most convenient method of spreading is to place a
minute drop on the glass with a platinum loop, and then
spread it evenly over the glass with the flat wire. Should
it be stained at once it would all wash off, so it must next
be fixed to the glass by being passed three times through a
flame, experience having shown that when drawn through
the flame three times the desired effect is usually accom-
plished. The Germans recommend that a Bunsen burner
or a large alcohol lamp be used, that the arm describe a
circle a foot in diameter, each revolution occupying a second
of time, and the glass being made to pass through the flame
from apex to base three times. This is supposed to be exactly
the requisite amount of heating. The rule is a good one for
the inexperienced.

Inequality in the size of various flames may make it de-

Simple Method of Staining 177

sirable to have a more accurate rule. Novy* suggests that
as soon as it is found that the glass is so hot that it can no
longer be held against the finger it is sufficiently heated for
fixing.

After fixing, the preparation is ready for the stain. Every
laboratory should be provided with " stock solutions," which
are saturated solutions of the ordinary dyes. For pre-
paring them Wood* gives the following parts per 100 as
being sufficiently accurate:

Alcoholic solutions (96 per cent, alcohol). Aqueous solutions (distilled water).

Fuchsin. .3.0 grams.

Gentian violet 4.8 Gentian violet 1.5 grams.

Methylene-blue 7.0 Methylene-blue ..... .6.7

(70 per cent, alcohol).

Scharlach R 3.2 "

Soudan III 0.2 "

(50 per cent, alcohol).
Thionin . . ..0.6 " Thionin. . ..1.2 "

Of these it is well to have fuchsin, gentian violet, and meth-
ylene-blue always made up. The stock solutions will not
stain, but form the basis of the staining solutions. For
ordinary staining an aqueous solution is employed. A small
bottle is nearly filled with distilled water, and the stock
solution added, drop by drop, until the color becomes just
sufficiently intense to prevent the ready recognition of ob-
jects through it. For exact work it is probably best to give
these stains a standard composition, using 5 c.c of the
saturated alcoholic solution to 95 c.c. of water. Such a
watery solution possesses the power of readily penetrating
the dried cytoplasm of the bacterium.

Cover-glasses are apt to slip from the fingers and spill the
stain, so when using them it is well to be provided with
special forceps (Fig. 34), which hold the glass in a firm grip
and allow of all manipulations without danger of soiling
the fingers or clothes. The ordinary sharp-pointed forceps
are unfit for the purpose, as capillary attraction draws
the stain between the blades and makes certain the soiling
of the fingers. In using the special forceps the glass should
not be caught at the edge, but a short distance from it, as

* "Laboratory Work in Bacteriology," 1899.

t " Chemical and Microscopical Diagnosis," N. Y., 1905, D. Apple-
ton & Co., p. 683

178 Methods of Observing Micro-organisms

shown in the cut. This altogether prevents capillary at-
traction between the blades. When the material is spread
upon the slide no forceps are needed, and the method corre-
spondingly simplified. Sufficient stain is allowed to run
from a pipet upon the smear to flood it, but not overflow,
and is allowed to remain for a moment or two, after which it
is thoroughly washed off with water. The smear upon a
slide is then dried and examined at once, a drop of oil of
cedar being placed directly upon the smear, and no cover-
glass used. If the staining has been done upon a cover-glass,
it can be mounted upon a slide with a drop of water between,
and then examined, though this is less satisfactory than ex-
amination after mounting in Canada balsam.

Fig. 34. — Stewart's cover-glass forceps

Sometimes the material to be examined is solid or too
thick to spread upon the glass conveniently. Under such
circumstances a drop of distilled water or bouillon can be
added and a minute portion of the material mixed in it and
spread upon the glass.

When the bacteria are contained in urine or other non-
albuminous fluid, so that the heat used for fixing has nothing
to coagulate and fix the organisms to the glass, a drop of
Meyer's glycerin-albumen can be added with advantage,
though the precaution must be taken to see that this mix-
ture contains no bacteria to cause confusion with those
in the material to be studied.

The entire process is, in brief: (i) Spread the material
upon the glass; (2) dry — do not heat; (3) pass three
times through the flame; (4) stain — one minute; (5) wash
thoroughly in water; (6) dry; (7) mount in Canada
balsam.

To Observe Bacteria in Sections of Tissue. — Har-
dening.— It not infrequently happens that the bacteria to
be examined are scattered among or inclosed in the cells
of tissues. Their demonstration then becomes a matter

Staining Bacteria in Tissues 179

of difficulty, and the method employed must be modified
according to the particular kind of organism. The success
of the method will depend upon the good preservation of
the tissue to be studied. As bacteria disintegrate rapidly
in dead tissue, the specimen for examination should be
secured as fresh as possible, cut into small fragments, and
immersed in absolute alcohol from six to twenty-four hours,
to kill and fix the cells and bacteria. The blocks are then
removed from the absolute alcohol and kept in 80 to 90 per
cent, alcohol, which does not shrink the tissue. Solutions
of bichlorid of mercury* may also be used and are par-
ticularly useful when the bacteria are to be studied in
relation to the cells of the tissues.

Tissues preserved in 95 per cent, alcohol, Muller's fluid,
4 per cent, formaldehyd, and other ordinary solutions
rarely show the bacteria well.

Embedding. — The ordinary methods of embedding suffice.
The simpler of these are as follows :

/. Celloidin (Schering). — The solutions of celloidin are
made in equal parts of absolute alcohol and ether and
should have the thickness of oil or molasses. From the
hardening reagent (if other than absolute alcohol) pass the
blocks of tissue through:

Ninety-five per cent, alcohol, twelve to twenty-four
hours ;

Absolute alcohol, six to twelve hours;

Thin celloidin (consistence of oil), twelve to twenty-
four hours;

Thick celloidin (consistence of molasses), six to twelve
hours.

Place upon a block of vulcanite or hard paraffin, allow the
ether to evaporate until the block can be overturned without
dislodging the specimen; then place in 80 per cent, alcohol

* Zenker's fluid:

Bichromate of potassium 2.5 grams

Sulphate of sodium 1.0 gram

Bichlorid of mercury . . . < 5.0 grams

Water 100.0 "

At the time of using add 5 grams of glacial acetic acid. Permit
the specimens to remain in the solution for a few hours only, then wash
for twenty-four hours in running water and transfer to 80 per cent,
alcohol.

180 Methods of Observing Micro-organisms

until ready to cut. The knife must be kept flooded with
alcohol while cutting.

Celloidin is soluble in absolute alcohol, ether, and oil of
cloves, so that the staining of the sections must be accom-
plished without the use of these reagents if possible.

Celloidin sections can be fastened to the slide, if desired,
by firmly pressing filter paper upon them and rubbing hard,
then allowing a little vapor of ether to run upon them.

//. Paraffin. — Pure paraffin having a melting-point of
about 55° C. is used. The hardened blocks of tissue are
passed through:

Ninety-five per cent, alcohol, twelve to twenty-four

hours ;

Absolute alcohol, six to twelve hours;
Chloroform, benzole, or xylol, four hours;
A saturated solution of paraffin in one of the above
reagents, four to eight hours.

The block is then placed in melted paraffin in an oven
or paraffin water-bath, at 5o°-6o° C., until the volatile
reagent is all evaporated, and the tissue impregnated with
paraffin (four to twelve hours), and finally embedded in
freshly melted paraffin in any convenient mold. In cutting,
the knife must be perfectly dry.

The cut paraffin sections can be placed upon the surface
of slightly warmed water to flatten out the wrinkles, and
then floated upon a clean slide upon which a film of Meyer's
glycerin-albumen (equal parts of glycerin and white of egg
thoroughly beaten up and filtered, and preserved with a
crystal of thymol) has been spread. After drying, the slides
are placed in the paraffin oven for an hour at 60° C., so
that the albumen coagulates and fixes the sections to the
glass.

When sections so spread and fixed upon the slide
are to be stained, the paraffin must first be dissolved in
chloroform, benzole, xylol, oil of turpentine, etc., which
in turn must be removed with 95 per cent, alcohol. The
further staining, by whatever method desired, is accom-
plished by dropping the reagents upon the slide.

///. Glycerin-gelatin. — As the penetration of the tissue
by celloidin is attended with deterioration in the staining
qualities of the tubercle bacillus, it has been recommended

Staining

181

by Kolle * that the tissue be saturated with a mixture of
glycerin, i part ; gelatin, 2 parts ; and water, 3 parts ; cemented
to a cork or block of wood, hardened in absolute alcohol,
and cut as usual for celloidin with a knife wet with alcohol.

Staining. — Simple Method. — For ordinary work the
following simple method can be recommended: After the
sections are cut and cemented to the slide, the paraffin
and celloidin should be removed by appropriate solvents.
The sections are immersed in the ordinary aqueous solution
of the anilin stain and allowed to remain about five minutes,
next washed in water for several minutes, then decolorized
in 0.5 to i per cent, acetic acid solution. The acid removes the
stain from the tissues, but ultimately from the bacteria as
well, so that one must watch carefully, and so soon as the
color has almost disappeared from the sections, they must
be removed and transferred to absolute alcohol. At this
point the process may be interrupted to allow the tissue
elements to be countercolored with alum-carmin or any
stain not requiring acid for differentiation, after which the
sections are dehydrated in absolute alcohol, cleared in
xylol, and mounted in Canada balsam.

The greater number of applications can be made by
simply dropping the reagents upon the slide while held in
the fingers. Where expos-
ure to the reagents is to be
prolonged, the Coplin jar
(Fig- 35) or some more
capacious device must be
employed.

Pfeiffer's Method.— The
sections are stained for one-
half hour in diluted Ziehl's
carbol-fuchsin (pp. 187 and
716), then transferred to
absolute alcohol made feeb-
ly acid with acetic acid.
The sections must be care-

fully watched, and so soon 35._Coplin's staining jar.

as the original, almost

black-red color gives place to a red-violet color they are
removed to xylol, to be cleared preparatory to mounting
in balsam.

* Flugge's " Die Mikroorganismen." vol. i, page 534.

CROSS-SECTION
SHOWING SLIDES
M POSITION,

1 82 Methods of Observing Micro-organisms

Loffler's Method. — Certain bacteria that do not permit
ready penetration by the dye require some more intense
stain. One of the best of these is Loffler's alkaline methy-
lene-blue :

Saturated alcoholic solution of methylene-blue . . 30
1 : 10,000 aqueous solution of caustic potash . . 100

The cut sections of tissue are stained for a few minutes
and then differentiated in a i per cent, solution of hydro-
chloric acid for a few seconds, after which they are dehy-
drated in alcohol, cleared in xylol, and mounted in balsam.

Bacteria, such as the typhoid fever bacillus, which de-
colorize rapidly, do not require the use of acid for the
differentiation, washing in water or alcohol being sufficient.

Gram's Method of Staining Bacteria in Tissue. — Gram
was the fortunate discoverer of a method of impregnating
bacteria with an insoluble color. It will be seen at a
glance that this is a marked improvement on the methods
given above, as the stained tissue can be washed thoroughly
in either water or alcohol until its cells are colorless, without
fear that the bacteria will be decolorized. The details of
the method are as follows: The section is stained from five
to ten minutes in a solution of a basic anilin dye, pure anilin
(anilin oil) and water. This solution, first devised by
Bhrlich, is known as Bhrlich's solution. The ordinary
method of preparing it is to mix the following:

Pure anilin : 4

Saturated alcoholic solution of gentian violet. . 11
Water 100

Instead of gentian violet, methyl violet, Victoria blue, or
any pararosanilin dye will answer. The rosanilin dyes,
such as fuchsin, methylene-blue, vesuvin, etc., will not react
with iodin, and so cannot be used for the purpose. The
anilin-oil solutions do not keep well; in fact, seldom longer
than six to eight weeks, sometimes not more than two or
three; therefore it is best to prepare but a small quantity
by pouring about i c.c. of pure anilin into a test-tube,
filling the tube about one-half with distilled water, shaking
well, then filtering as much as is desired into a small dish.
To this the saturated alcoholic solution of the dye is added
until the surface becomes distinctly metallic in appear-
ance.

Staining 183

Friedlander recommends that the section remain from
fifteen to thirty minutes in warm stain, and in many cases
the prolonged process gives better results.

From the stain the section is given a rather hasty washing
in water, and then immersed from two to three minutes in
Gram's solution (a dilute Lugol's solution) :

lodin crystals 1

Potassium iodid 2

Water ,300

The specimen while in the Gram solution turns a dark
blackish-brown color, but when removed and carefully
washed in 95 per cent, alcohol again becomes blue. The
washing in 95 per cent, alcohol is continued until no more
color is given off and the tissue assumes its original color.
If it is simply desired to find the bacteria, the section can
be dehydrated in absolute alcohol for a moment, cleared
in xylol, and mounted in Canada balsam. If it is necessary
to study the relation of the bacteria to the tissue elements,
a nuclear stain, such as alum-carmin or Bismarck brown,
may be previously or subsequently used. Should a nuclear
stain requiring acid for its differentiation be desirable, the
process of staining must precede the Gram stain, so that
the acid shall not act upon the stained bacteria.

Gram's method rests upon the fact that the combination
of mycoprotein, anilin dye, and the iodids forms a compound
insoluble in alcohol.

The process described may be summed up as follows:

Stain in Ehrlich's anilin- water gentian violet five to

thirty minutes ;
Wash in water;

Immerse two to three minutes in Gram's solution;
Wash in 95 per cent, alcohol until no more color comes

out;

Dehydrate in absolute alcohol;
Clear in xylol;
Mount in Canada balsam.

This method stains the majority of bacteria, but not all,
hence can be used to aid in the differentiation of similar
species:

184 Methods of Observing Micro-organisms

Gram-negative. Gram -positive.

Bacillus anthracis symptomatici ; Bacillus aerogenes capsulatus;
Bacillus coli (whole group); Bacillus anthracis;

Bacillus ducreyi; Bacillus botulinus;

Bacillus dysenteriae; Bacillus diphtherias;

Bacillus icteroides; Bacillus subtilis (whole group);

Bacillus influenzae; Bacillus tetani;

Bacillus mallei; Bacillus tuberculosis (whole acid-

Bacillus cedematis maligni; fast group);

Bacillus pestis bubonica; Diplococcus pneumonias;

Bacillus pneumonias (Friedlander) ; Micrococcus tetragenus;
Bacillus proteus vulgaris; Staphylococcus pyogenes albus;

Bacillus pyocyaneus; Staphylococcus pyogenes aureus;

Bacillus rhinoscleromatis; Streptococcus pyogenes.

Bacillus suipestifer;
Bacillus suisepticus;
Bacillus typhosus (whole group) ;
Diplococcus intracellularis meningitidis;
M icrococcus cat arrh al is ;
Micrococcus gonorrhreae (Neisser) ;
Micrococcus melitensis;
Spirillum cholerae asiaticae;
Spirillum cholerae gallinarum;
Spirillum cholerae nostras;
Spirillum metschnikovi;
Spirillum tyrogenum;
Spirochaete duttoni;
Spirochaete obermeieri;
Spirochaete refringens;
Treponema pallidum;
Treponema pertenue.

No matter how carefully the method is performed, an
unsightly precipitate is sometimes deposited upon the
tissue, obscuring both its cells and contained bacteria.
Muir and Ritchie obviate this (i) by making the staining
solution with i : 20 aqueous solution of carbolic acid instead
of the saturated anilin solution, and (2) by clearing the
tissue with oil of cloves after dehydration with alcohol.
The oil of cloves, however, is itself a powerful decolorant
and must be washed out in xylol before the section is
mounted in Canada balsam.

Gram's method is also employed to aid in differentiat-
ing similar species of bacteria in culture. A thin layer of
a suspension of the bacteria to be examined is spread upon
a slide or cover-glass, dried, and fixed; then flooded with
the anilin-oil gentian violet or other staining solution.
The solution is kept warm by holding the glass flooded
with the stain over a small flame. The process of staining
is continued from two to five minutes. If the heating
causes the stain to evaporate, more of it must be added
so that it does not dry and incrust the glass.

Staining 185

The stain is poured off, and replaced by Gram's solution,
which is allowed to remain from one-half to two minutes,
and gently agitated.

The smear is next washed in 95 per cent, alcohol until
the blue color is wholly or almost lost, after which it can
be counterstained with eosin, Bismarck brown, vesuvin,
etc., washed, dried, and mounted in Canada balsam. Given
briefly, the method is :

Stain with Ehrlich's solution two to five minutes;

Gram's solution for one-half to two minutes;

Wash in 95 per cent, alcohol until decolorized;

Counterstain if desired; wash off the counterstain with water;

Dry;

Mount in Canada balsam.

Nicolle* suggests the following modification of the technic :

a. For Cover-glass Specimens:

1 . Stain for one to five minutes in a warm solution made as fol-

lows: 10 c.c. of saturated alcoholic solution of gentian violet,
100 c.c. of a i per cent, aqueous solution of carbolic acid.

2. Immerse from four to six seconds in the iodine-iodide of potas-

sium solution.

3. Decolorize in a mixture of 3 parts of absolute alcohol and i

part of acetone.

4. Counter stain if desired.

b. For Sections:

1. Stain the nuclear elements of the tissue with carmine. For

this Nicolle prefers Orth's carmine solution (5 parts of Orth's
carmine with i part of 95 per cent, alcohol).

2. Stain in the carbol-gentian violet, as indicated above.

3. Immerse for four to six seconds in the iodine-iodide of potas-

sium solution.

4. Differentiate with absolute alcohol containing 0.33 per cent.

(by volume) of acetone.

5. Treat with 95 per cent, alcohol containing some picric acid

until the tissue is greenish yellow (one to five seconds).

6. Dehydrate with absolute alcohol.

7. Clear with xylol or other appropriate reagent.

8. Mount in balsam.

The Gram-Weigert Stain can be employed with beautiful
results for staining many micro-organisms. It differs from
the Gram method in that anilin oil instead of alcohol is
used for decolorizing. To secure the most brilliant results
it is best first to stain the tissue with alum, borax, or
lithium carmin, and then —

* "Ann. del'Inst. Pasteur," 1895, ix.

1 86 Methods of Observing Micro-organisms

1. Stain in Ehrlich's anilin-oil-water gentian violet,

five to twenty minutes;

2. Wash off excess with normal salt solution;

3. Immerse in dilute iodin solution (iodin i, iodid of

potassium 2, water 100) for one minute;

4. Drain off the fluid and blot the section spread out

upon the slide, with absorbent paper;

5. Decolorize with a mixture of equal parts of anilin

and xylol;

6. Wash out the anilin with pure xylol;

7. Mount in xylol balsam.

Eosin and Methylene-blue (Mallory) make a beautiful
contrast tissue stain for routine work, and also demonstrates
the presence of most bacteria. The success of the method
seems to depend largely upon the quality of the reagents
used and a careful study of their effects. Hardening in
Zenker's fluid is highly recommended as a preliminary. The
details as given by Mallory are as follows:

1. Stain paraffin sections in a 5 to 10 per cent, aqueous

solution of eosin for five to twenty minutes or
longer;

2. Wash in water to get rid of the excess of eosin;

3. Stain in Unna's alkaline methylene-blue solution

(methylene-blue i, carbonate of potassium i, water
100), diluted i : 10 with water, for one-half to one
hour, or use a stronger solution and stain for a few
minutes only;

4. Wash in water.

5. Differentiate and dehydrate in 95 per cent, alcohol,

followed by absolute alcohol until the pink color
returns in the section;

6. Clear with xylol;

7. Mount in xylol balsam.

The nuclei and micro-organisms will be colored blue, the
cytoplasm, etc., red.

Zieler* recommends for the staining of the typhoid,
glanders and other difficultly stainable bacteria, the follow-
ing method of demonstration in the tissues :-

* "Centralbl. f. allg. Path. u. path! Anat." Bd. xiv, No. 14, p. 561.

Staining 187

1. Fix and harden in Miiller-formol solution.
Paraffin imbedding.

2. Staining overnight in Orcein D. (Griibler), 0.1

Officinal "schwefelsaiire" (sulphuric acid), 2.0

70 per cent, alcohol, lOo'o

3. Washing in 70 per cent, alcohol for a short time to remove

the excess of orcein.

4. Washing in water.

5. Staining in polychrome methylene-blue ten minutes to two

hours.

6. Washing in distilled water.

7. Thorough differentiation in glycerin -ether 1 : 2-5 water until

the tissues become pale blue.

8. Washing in distilled water.

9. Seventy per cent, alcohol.

10. Absolute alcohol.

11. Xylol.

12. Balsam.

Glanders bacilli appear dark violet on a colorless back-
ground; typhoid bacilli intense dark red violet.

Method of Staining Spores. — It has already been pointed
out that the peculiar quality of the spore capsules pro-
tects them to a certain extent from the influence of stains
and disinfectants. On this account they are much more
difficult to color than the adult bacteria. Several methods
are recommended, the one generally employed being as
follows : Spread the thinnest possible layer of material upon
a cover-glass, dry, and fix. Have ready a watch-crystalful
of Ehrlich's solution, preferably made of fuchsin, and drop
the cover-glass, prepared side down, upon the surface, where
it should float. Heat the stain until it begins to steam,
and allow the specimen to remain in the hot stain for from
five to fifteen minutes. The cover is then transferred to a
3 per cent, solution of hydrochloric acid in absolute alcohol
for about one minute. Abbott recommends that the cover-
glass be submerged, prepared side up, in a dish of this solu-
tion and gently agitated for exactly one minute, removed,
washed in water, and counterstained with an aqueous solu-
tion of methyl or methylene-blue.

In such a specimen the spores should appear red, and the
adult organisms blue.

I have not found that spores usually color so easily,
and for many species the best method seems to be to place
the prepared cover-glass in a test-tube half full of carbol-
f uchsin :

Fuchsin !

Alcohol 10

Five per cent aqueous solution of phenol crys-
tals . 100

1 88 Methods of Observing Micro-organisms

and boil it for at least fifteen minutes, after which it is
decolorized, either with 3 per cent, hydrochloric or 2-5 per
cent, acetic acid, washed in water, and counterstained blue.

Muir and Ritchie * recommend that cover-films be pre-
pared and stained as for tubercle bacilli (q. V.), decolor-
ized with a i per cent, sulphuric acid solution in water or
methyl alcohol, then washed in water and counterstained
with a saturated aqueous methylene-blue solution for half a
minute, washed again with water, dried, and mounted in
Canada balsam.

Abbott's method of staining spores is as follows:

1. Stain deeply with methylene-blue, heating repeatedly until the

stain reaches the boiling-point — one minute.

2. Wash in water.

3. Wash in 95 per cent, alcohol containing 0.2 to 0.3 per cent, of

hydrochloric acid.

4. Wash in water.

5. Stain for eight to ten seconds in anilin-fuchsin solution.

6. Wash in water.
7- Dry.

8. Mount in balsam.

The spores are blue; the bacteria, red.

Mollerf finds it advantageous to prepare the films, before
staining, by immersion in chloroform for two minutes, fol-
lowing this by immersion in 5 per cent, chromic acid solu-
tion for one-half to two minutes.

The exact technic is as follows:

1. Treat the spread with chlororoform for two minutes.

2. Wash with water.

3. Treat with 5 per cent, solution of chromic acid for one-half to

two minutes.

4. Wash in water.

5. Stain with carbol-fuchsin, slowly heating until the fluid boils.

6. Decolorize in 5 per cent, aqueous sulphuric acid.

7. Wash well with water.

8. Stain in a i : 100 aqueous solution of methylene-blue for thirty

seconds.
The spores should be red and the bacilli blue.

Anjeszky { recommends the following method of staining
spores, which is said always to give good results even with
anthrax bacilli: A cover-glass is thinly spread with the
spore-containing fluid and dried. While it is drying, some
0.5 per cent, hydrochloric acid is warmed in a porcelain

* "Manual of Bacteriology," London, 1897.

f "Centralbl. f. Bakt. u. Parasitenk.," Bd. x, p. 273.

i Ibid., Feb. 27, 1898, xxm, No. 8, p. 329.

Staining 189

dish over a Bunsen flame until it steams well and bubbles
begin to form. When the solution is hot and the smear
dry, the cover-glass is dropped upon the fluid, which is
allowed to act upon the unfixed smear for three or four
minutes. The cover is removed, washed with water, dried,
and fixed for the first time, then stained with Ziehl's carbol-
fuchsin solution, which is warmed twice until fumes arise.
The preparation is allowed to cool, decolorized with a 4-5
per cent, sulphuric acid solution, and counterstained for a
minute or two with malachite green or methylene-blue.
The whole procedure should not take longer than eight to
ten minutes.

Fiocca* suggests the following rapid method: "About
20 c.c. of a 10 per cent, aqueous solution of ammonium are
poured into a watch-glass, and 10 to 20 drops of a saturated
solution of gentian violet, fuchsin, methyl blue, or safranin
added. The solution is warmed until vapor begins to rise,
then is ready for use. A very thinly spread cover-glass,
carefully dried and fixed, is immersed for three to five
minutes (sometimes ten to twenty minutes), washed in
water, washed momentarily in a 20 per cent, solution of
nitric or sulphuric acid, washed again in water, then counter-
stained with an aqueous solution of vesuvin, chrysoidin,
methyl blue, malachite green, or safranin, according to the
color of the preceding stain. This whole process is said to
take only from eight to ten minutes, and to give remarkably
clear and beautiful pictures."

Method of Staining Flagella. — This is somewhat more
difficult than the staining of the bacteria or their spores.

Loffler's Method.} — This is the original and best method,
though somewhat cumbersome, and hence rarely employed
at the present time. Three solutions are used :

(A)-

Twenty per cent, aqueous solution of tannic
acid3.. 10

Cold saturated aqueous solution of ferrous sul-
phate 5

Alcoholic solution of fuchsin or methyl violet

(B) One per cent, aqueous solution of caustic soda.

(C) An aqueous solution of sulphuric acid of such strength
that i c.c. will exactly neutralize an equal quantity of
solution B.

* "Centralbl. f. Bakt. u. Parasitenk.," July i, 1893, xiv, No. i.
f Ibid., 1890, Bd. vii, p. 625.

i go Methods of Observing Micro-organisms

Some of the culture to be stained is mixed upon a cover-
glass with a drop of distilled water making a first dilution,
which is still too rich in bacteria to permit the flagella to
show well, so that it is recommended to prepare a second
by placing a small drop of distilled water, upon a cover
and taking a loopful from the first dilution to make the
second, and spreading it over the entire surface without
much rubbing or stirring. The film is allowed to dry, and
is then fixed by passing it three times through the flame.
When this is done with forceps there is some danger of the
preparation becoming too hot, so Loffler recommends that
the glass be held in the fingers while the passes through the
flame are made.

The cover-glass is now held in forceps, and the mordant,
solution A, dropped upon it until it is well covered, when
it is warmed until it begins to steam. The mordant must
be replaced as it evaporates. It must not be heated too
strongly; above all things, must 'not boil. This solution
is allowed to act from one-half to one minute, is then washed
off with distilled water, and then with absolute alcohol
until all traces of the solution have been removed. The
real stain, — Loftier recommends an anilin- water fuchsin
(Ehrlich's solution), — which should have a neutral reaction,
is next dropped on so as to qover the film, and heated for
a minute until vapor begins to arise, after which it is washed
off carefully, dried, and mounted in Canada balsam. To
obtain the neutral reaction of the stain, enough of the i
per cent, sodium hydrate solution is added to an amount
of the anilin-water-fuchsin solution having a thickness of
several centimeters to begin to change the transparent
into an opaque solution.

A specimen thus treated may or may not show the flagella.
If not, before proceeding further it is necessary to study
the chemic products of the micro-organism in culture
media. If by its growth the organism elaborates alkalies,
from i drop to i c.c. of solution C in 16 c.c. must be added
to the mordant A, and the staining repeated. It may be
necessary to stain again and again until the proper amount
is determined by the successful demonstration of the fla-
gella. On the other hand, if the organism by its growth
produces acid, solution B must be added, drop by drop,
and numerous stained specimens examined to see with
what addition of alkali the flagella will appear. Loffler

Staining 191

fortunately worked out the amounts required for some
species, and of the more important ones the following
solutions of B and C must be added to 16 c.c. of solution
A to attain the desired effect:

Cholera spirillum £-1 drop of solution C •$

Typhoid fever 1 c.c. of solution B

Bacillus subtilis 28-30 drops of solution B

Bacillus of malignant edema .36 or 37 drops of solution B

Part of the success of the staining depends upon having
the bacteria thinly spread upon the glass, and as free from
albuminous and gelatinous materials as possible. The
cover-glass must be cleaned most painstakingly; too much
heating in fixing must be avoided. After using and washing
off the mordant, the preparation should be dried before the
application of the anilin-water-fuchsin solution.

Pitfield's Method. — Pitfield* has devised a single solution, at
once mordant and stain. It is made in two parts, which are
filtered and mixed:

(A)-

Saturated aqueous solution of alum 10 c.c.

Saturated alcoholic solution of gentian violet . . 1 "

(B)-

Tannic acid " 1 gram

Distilled water 10 c.c.

The solutions should be made with cold water, and im-
mediately after mixing the stain is ready for use. The
cover-slip is carefully cleaned, the grease being burned off
in a flame. After it has cooled, the bacteria are spread
upon it, well diluted with water. After drying thoroughly
in the air, the stain is gradually poured on and by gentle
heating brought almost to a boil; the slip covered with the
hot stain is laid aside for a minute, then washed in water
and mounted.

Smith's Modification of Pitfield' s Method.* — A boiling saturated
solution of bichlorid of mercury is poured into a bottle in which
crystals of alum have been placed in quantity more than sufficient
to saturate the fluid. The bottle is shaken and allowed to cool;
10 c.c. of this solution are added to the same volume of freshly
prepared tannic acid solution and 5 c.c. of carbol fuchsin added.
Mix and filter. The filtrate, which is the mordant, is caught

* "Medical News," Sept. 7, 1895.

t ''British Medical Journal," 1901, i, p. 205.

192 Methods of Observing Micro-organisms

directly upon the spread (the liquid must always be filtered at the
time of use) and heated gently for three minutes, but not permitted
to boil. Wash with water and then stain in the following:

Saturated alcoholic solution of gentian violet i c.c.

Saturated solution of ammonium alum 10

Filter the stain also directly upon the slide at the time of
using, and heat it for three to four minutes. Wash thoroughly
in water, dry, and mount in balsam.

Van Ermengem's Method. — Van Ermengem * has devised
a somewhat complicated method of staining flagella, which
has given great satisfaction. Three solutions, which he
describes as the bain fixateur, bain sensibilisateur, and bain
reducteur et reinforcateur, are to be used as follows:

1. Bain fixateur:

2 per cent, solution of osmic acid 1 part

10-25 per cent, solution of tannin 2 parts

The cover-glasses, which are very thinly spread, dried,
and fixed, are placed in this bath for one hour at the room
temperature, warmed until steam arises, and then kept hot
for five minutes. They are next washed with distilled
water, then with absolute alcohol, then again with distilled
water. All three washings must be very thorough.

2. Rain sensibilisateur:

5 per cent, solution of nitrate of silver in distilled water.

The films are allowed to remain in this for a few seconds,
and are then immediately transferred to the third bath.

3. Bain reducteur et reinforcateur:

Gallic acid 5 grams

Tannin 3 "

Fused potassium acetate 10 "

Distilled water 350 c.c.

The preparations are kept in this solution for a few
seconds, then returned to the nitrate of silver solution until
they begin to turn black. They are then washed, dried,
and mounted.

Mervyn Gorden modifies the method by allowing the
preparations to remain in the second bath for two minutes,
transferring to the third bath for one and a half to two

* "Travaux du Lab. d'hygiene et des bact. de Gand.," t. I, p. 3.
Abstracted in the " Central bl. f. Bakt. u. Parasitenk.," 1894, Bd. xv,
p. 969.

Staining 193

minutes, and then washing, drying, and mounting without
returning to the second bath.

Muir and Ritchie find it advantageous to use a fresh
supply of the third solution for each specimen.

Rossi* gives the following directions for staining flagella :

The culture to be examined should be a young culture, not more
than ten, eighteen, or twenty-four hours old. It should be made upon
freshly prepared agar-agar, or upon the reagent after it has been melted
and then congealed, as it is of the utmost importance that the surface
be moist. The culture should be examined by the hanging-drop method
to see that the organisms are actively motile before the staining is at-
tempted.

The staining should be done only after the greatest care has been
taken to see that all the conditions are favorable. For this reason the
cover-glasses employed in making the spreads must be carefully cleaned
with alcohol, then immersed in steaming sulphuric acid for ten to fifteen
minutes. They are then washed in water, then placed in a mixture of
alcohol and benzine (equal parts), wiped with a clean soft cloth, and
passed through the colorless Bunsen flame forty to fifty times, and
then that side of the glass utilized for the "spread" that has been in
direct contact with the flame.

A platinum loopful of the appropriate culture is placed in a drop of
distilled water upon a clean slide and slightly stirred. If conditions
are favorable, it forms a homogeneous emulsion. If clumps appear,
the cultural conditions are not favorable.

If favorable, a loopful of this dilution is added to i c.c. of distilled
water in a clean cover-glass and thoroughly stirred. From the center
of the surface of this fluid a platinum loopful is next taken and placed
upon each of the prepared cover-glasses and, without spreading or stir-
ring, allowed to dry in the air or in an exsiccator.

The staining solutions are made as follows:

(A) A solution of 50 grams of pure crystalline carbolic acid in

looo c.c. of distilled water, to which 40 grams of pure tannin
are added, the whole being warmed on a water-bath until
solution is complete.

(B) Basic fuchsin (rosanilinchlorhydrate) 2.5 grams

Absolute alcohol 100.0 c.c.

(C) Potassium hydrate i .o gram

Distilled water 100.0 grams

Mix solutions A and B and preserve in a well-closed bottle. Place
solution C in a bottle with a pipette stopper. When the staining is to
be done, one pours 15 to 20 c.c. of the A B mixture into a glass-stop-
pered test-tube and adds 2 or- 3 drops of solution C. A precipitate
forms, but quickly dissolves on shaking. More of solution C is added,
and the tube shaken until the solution becomes brown and clouded and
one can see a fine precipitate in a thin layer of the fluid. The fluid is
next filtered several times through the same filter and caught in the
same glass until it will remain clear for several minutes. Then it is
poured on the filter a last time and 4 or 5 drops allowed to fall upon each
of the prepared cover-glasses. In a short time a sheen is observed upon
the surface of the fluid on the cover-glasses, showing that a fine precipi-
tate has formed. When this has occurred, a little experience will show
when the proper moment arrives to throw off the fluid and wash the

* "Centralbl. f. Bakt. u. Parasitenk.," xxxm, Orig., 1903, p. 572.
13

194 Methods of Observing Micro-organisms

cover in distilled water. It is the precipitate that clings to the flagella
and renders them distinctly visible. If no precipitate occurs, the
flagella will not be seen.

L. Smith * offers the following modification of Newman's
method f as being a simple and excellent method of staining
flagella:

The material and cover-glasses are prepared with care as
for the foregoing methods, after which one proceeds as follows :

1. Transfer a loopful of the bacillary emulsion to the clean slide

or cover-glass and allow it to dry in the air.

2. Expose to a mild degree of heat, holding the glass in the fingers —

this is rather drying than actual heating.

3. Allow the stain to drop from a filter upon the film and remain

in contact five to ten minutes.
The formula for the stain is

I. Tannic acid i gram

Potassium alum i

Distilled water 40 c.c.

Dissolve by shaking or allow to stand overnight in the in-
cubator.

II. Dissolve "night blue" J 0.5 gram

95 per cent, or absolute alcohol 20.0 c.c.

Mix I and II thoroughly and remove the heavy precipitate
by filtration. If not used at once, drop from a filter upon
the film. The stain does not keep more than a few days.

4. Wash carefully but thoroughly in water.

5. Apply a saturated aqueous solution of gentian violet for about

two minutes to stain the bodies of the bacteria.

6. Wash thoroughly in water, dry with smooth blotting-paper,

and mount in balsam.

To secure a perfectly clean background for photomicrography, it is
best to stain on a slide. The stain is then poured into a Petri dish, the
slide inverted, the end of the slide used to push aside the film on the
surface of the stain, and the film then immersed downward, one end of
the slide supported, during staining on a match-stick or bit of glass rod.
In this way the adherence of the precipitate to the slide can be avoided.

THE OBSERVATION OF LIVING PROTOZOA.

When protozoa are to be examined in transparent fluids,
such as pond-water or culture fluids in which they have been
artificially nourished, use can be made of a " live-box " or
of the " hanging drop." Ordinarily, however, the organisms
to be examined are contained in blood, in pus, in sputum,
in feces, or in some other more or less opaque fluid, of which
an extremely thin layer must be prepared in order that the

* "Jour. Med. Research," vi, 1901, p. 341.

t" Bacteria," John Murray, London, 2d edition.

t James Strong & Son, Glasgow and Manchester.

Staining Protozoa 195

formed elements may be separated sufficiently for the indi-
vidual cells and organisms to be seen.

Such a thin layer is usually easily obtained by the use of
a slide and cover-glass, and the careful preparation of a good
film.

The slide and the cover-glass should be thoroughly cleansed
and freed from fat and grit and well polished. A compar-
atively small drop of blood — let us say, for example — is
placed upon the center of the slide and immediately covered
with the cover-glass. If the drop is not too large and the
glasses are clean, the weight of the cover-glass causes the
drop to spread, and capillary attraction completes the forma-
tion of a very thin film. The quantity of blood used should
not be sufficient to reach the edges of the cover-glass, else
sometimes the glass is pressed up instead of being drawn down
and moves about too freely. If the examination is to take
enough time to cause the drop to dry, a match-stick dipped in
thin vaselin and drawn about the edge of the cover will
prevent it.

Such a film is usually best examined at or near the center,
where the formed elements are most widely separatedo

The living protozoa in preparations of this kind may be
examined by ordinary illumination by transmitted light, or
with lateral illumination by means of the " dark-field il-
luminator." The latter serves better for the discovery
of the very small transparent organisms — spirochaeta and
treponema — and for the observation of the cilia and flagella.

STAINING PROTOZOA.

It is through the study of stained protozoa that we arrive
at most of our knowledge of their structural details. They
can be stained in blood or fluids upon a slide or in sections of
tissue.

As in the case of the bacteria, it is first necessary to prepare
satisfactory spreads for the purpose. In order that the
description shall be as practical as possible, we will suppose
that the micro-organisms to be stained are in blood — spiro-
chaeta, plasmodium, etc.

As was pointed out above, the protozoa, under such circum-
stances, are distributed among or in cellular elements that
interfere with satisfactory observation unless precautions are
taken to separate them as widely as may be required.

196 Methods of Observing Micro-organisms

i. Cover-glasses. — The glasses should be perfectly clean and freed
from fat, either by washing in alcohol and ether and wiping with
a clean soft cotton cloth or Chinese rice paper, or by flaming.
The drop of blood should be small and should be placed
upon the center of one glass and immediately covered by
another, so held that the corners do not coincide. As soon
as the drop is fairly well distributed the glasses are gently
slid apart.

Fig.

36. — Method of making dry film with two cover-glasses (from
Daniels' " Laboratory Studies in Tropical Medicine").

Slides. — The slides, like the cover-glasses, must be perfectly
clean. The drop of blood is placed upon one slide at about
one-fourth the length of the slide from its end, touched with
the end (it must have ground edges) of the second slide, and
then gently pushed along until the fluid is exhausted.

Fig. 37- — Method of making dry films with two slides (from Daniels'
" Laboratory Studies in Tropical Medicine ").

Staining Protozoa 197

If the covers are to be stained, they can most conveniently
be held in the Stewart forceps. If the slides are used, they
can be held in the fingers.

The stain most useful is that of Romanowsky. It has
many modifications, of which the most used and best known
are Giemsa's, Jenner's, Leishmann's, Wright's, and Marino's.
These stains can be bought either in solution or in tablet
form ready for solution.

Those most highly to be recommended are Wright's and
Marino's.

Wright's Blood-stain. — This is a modification of Leishmann's stain,
to which it is to be preferred because it can be made in a few
hours instead of eleven days. It combines the methylene-blue-
eosin combination of Romanowsky with the methyl -alcohol
fixation of Jenner.

It is prepared as follows:*

''To a 0.5 per cent, aqueous solution of sodium bicarbonate add
methylene-blue (B. X. or "medicinally pure") in the proportion
of i gm. of the dye to 100 c.c. of the solution. Heat the mixture in
a steam sterilizer at 100° C. for one full hour, counting the time
after the sterilizer has become thoroughly heated. The mixture
is to be contained in a flask of such size and shape that it forms
a layer not more than 6 cm. deep. After heating, the mixture is
allowed to cool, placing the flask in cold water if desired, and is
then filtered, to remove the precipitate which has formed in it.
It should, when cold, have a deep purple-red color when viewed,
in a thin layer, by transmitted yellowish artificial light. It
does not show this color while it is warm. To each 100 c.c.
of the filtered mixture add 500 c.c. of a o.i per cent, aqueous
solution of "yellowish, water-soluble" eosin and mix thoroughly.
Collect the abundant precipitate which immediately appears
on a filter. When the precipitate is dry, dissolve it in methylic
alcohol (Merck's "reagent") in the proportion of o.i gr. to
60 c.c. of the alcohol. In order to facilitate the solution the
precipitate is to be rubbed up with the alcohol in a porcelain
dish or mortar with a spatula or pestle.

"This alcoholic solution of the precipitate is the staining fluid. It
should be kept in a well-stoppered bottle because of the volatility
of the alcohol. If it becomes too concentrated by evaporation,
and thus stains too deeply or forms a precipitate on the blood-
smear, the addition of a suitable quantity of methylic alcohol will
quickly correct such fault. It does not undergo any other spon-
taneous change than that of concentration by evaporation."

Method of Staining. — The blood-films are permitted to dry in the air
(not heated) :

1. Cover the film with a noted quantity of the staining fluid by

means of a medicine dropper.

2. After one minute add to the staining fluid the same quantity of

distilled water by means of the medicine dropper, and allow it

*Mallory and Wright, "Pathological Technique," 1911, p. 364.

198 Methods of Observing Micro-organisms

to remain for two or three minutes, according to the intensity
of the staining desired. A longer period of staining may pro-
duce a precipitate.

3. Wash the preparation in water for thirty seconds or until the

thinner portions of the preparation become yellow or pink in
color.

4. Dry and mount in balsam.

Films more than an hour old do not stain so well as fresh ones. Old
films show bluish instead of pink erythrocytes.

Marino's stain* is extremely delicate and gives still more beautiful
results where parasites are present. It is an azur-eosin combination,
prepared as follows:

Solution I:

Methylene-blue (medicinal) 0.5 gram.

Azur II 0.5 "

Water (distilled) 100.0 grams.

Solution II:

Sodium carbonate 0.5 gram.

Water 100.0 c.c.

Pour the two solutions together and stand the mixture in the
thermostat for forty-eight hours at 37° C.; then add 0.2 per
cent, aqueous solution of eosin ("yellowish aqueous eosin").
The quantity of this solution must be varied according to the
blue dyes employed, so as to secure the maximum precipitation.
The exact quantity can only be determined by titration. A pre-
cipitate now forms in the course of twenty-four hours. This is
caught upon a filter-paper and dried.

The precipitate, dissolved in methylic alcohol, in the proportion of
0.04 gm. of the powder to 20 c.c. of the methylic alcohol, forms
the stain.

Method. — The stain is dropped upon the spread so as to cover it,
the number of drops being counted. It is permitted to act for
exactly three minutes for purposes of fixation, then, without pour-
ing off the stain, twice the number of drops of a i : 100,000 aqueous
eosin solution are added. The two fluids gradually mix, trans-
fusion currents are formed, and the specimen is allowed to stand
for exactly two minutes longer. It is during this time that the
staining takes place. A precipitate usually forms upon the
surface of the fluid, so that it must not be poured off, but splashed
off by dropping distilled water upon it from a height. The dis-
tilled water is added until it no longer shows any color, when the
specimen is drained, dried, and mounted in balsam.

The student may also try staining with hematoxylon and
eosin, thionin and eosin, methylene-blue and eosin, or any
other dyes, some of which sometimes bring out special de-
tails of structure. The protozoa do not show the same re-
action to Gram's stain that makes it so useful for differ-
entiating the bacteria.

* "Ann. de 1'Inst. Pasteur," 1904, xvm, 761.

Staining Protozoa in Tissue 199

STAINING PROTOZOA IN TISSUE.

For this purpose the sections should be embedded in paraf-
fin, cut very thin, and cemented to the slides.

Ordinary staining with hematoxylin and eosin is rarely of
much use. Methylene-blue and eosin is better, but still
more useful are the Romanowsky methods, and both the
Wright stain and the Marino stain can, with some modifica-
tion of the time of staining and washing, be employed with
good results.

Still better and more satisfactory for certain protozoa are
the iron-hematoxylin and the Biondi stain.

Heidenhain's Iron-hematoxylin.* — Fix the tissue, by preference, in
Zenker's solution, though alcohol fixation will do. Embed in
paraffin, cut very thin, and fix to the slide.

1. Stain from three to twelve hours in 2.5 per cent, solution of
violet iron-alum (sulphate of iron and ammonium). The sec-
tions should be stood vertically in the solution, so that no pre-
cipitate may form upon them.

2. Wash quickly in water.

3. Stain in a 0.5 per cent, ripened alcoholic solution of hematoxylin
for from twelve to thirty-six hours.

4. Wash in water.

5. Differentiate in the iron-alum solution, controlling the results
under the microscope. The section should be well washed in a
large dish of tap-water before each examination to stop decoloriza-
tion.

6. Wash in running water for a quarter of an hour.

7. Pass through alcohol, xylol, and mount in xylol balsam.

A counterstain with Bordeau R. before or with rubin S. after the
iron stain is sometimes useful.

Biondi-Heidenhain Stain* — The tissues must be fixed in Zenker's
or corrosive sublimate solutions. Embed in paraffin, cut very
thin, fix to the slide.

Stain I. Orange G 8 grams.

Water 100

II. Acid fuchsin \

or Rubin S. J

Water 100

III. Methyl-green 8 grams.

Water 100

Let the solutions stand for several days, occasionally shaking
the bottles to make sure that a saturated solution of each is
secured. At the end of the time set, mix the solutions in the
following preparations :

1 100 parts.

II 20

III 50

* Mallory and Wright, "Pathological Technique," 1911, p. 309-
f Modified from Mallory and Wright, " Pathological Technique,"
1911, p. 289.

200 Methods of Observing Micro-organisms

At the time of staining dilute the mixture i : 60 or i : 100 with
water. To test the solution : ( i ) Acetic acid makes it redder.
(2) A drop of the solution on filter-paper should make a blue
spot with a green center and an orange border. If a red
zone appears outside of the orange, too much acid fuchsin
is present.

1 . Stain the sections from six to twenty-four hours.

2. Wash out a little in 90 per cent, alcohol.

3. Dehydrate in absolute alcohol.

4. Xylol.

5. Xylol balsam.

It is important to place the sections directly from the staining fluid
into the alcohol, because water washes out the methyl-green
instantly.

Ross' Thick Blood-spreads. — In case the number of parasites in the
blood is very small, so that they would be scattered sparingly
over a large area of the ordinary blood spread, Ross* has suggested
a modification of the technic by which they can be more readily
found. To do this a very thick spread is prepared and dried.
As soon as it is dry, and without fixing, the slide is stood vertically
in a vessel filled with distilled water. The red corpuscles at
once begin to hemolyze and the process is carried on to com-
pletion. When all of the hemoglobin has been removed, the
slide is taken out, dried, and then fixed and stained. There
now being no red corpuscles to distract the attention or obscure
the vision, the stained parasites can quickly be found.

Measurement of Micro-organisms. — They can best be
measured by an eyepiece micrometer. As these instruments
vary somewhat in construction, the unit of measurement
for each objective magnification and the method of manipu-
lating the instruments must be learned from dealers' cata-
logues.

Photographing Microorganisms.— This requires special
apparatus and methods, for which it is necessary to refer
to special text-books. f

* "Lancet," Jan. 10, 1903.

f See the excellent chapter upon Photomicrography in Aschoff and
Gaylord's "Pathological Histology," Philadelphia, 1900.

CHAPTER VI.

STERILIZATION AND DISINFECTION*

BEFORE beginning the consideration of the methods em-
ployed for the artificial cultivation of individual micro-
organisms and the preparation of media for that purpose, it
is necessary to have a thorough knowledge of the principles
of sterilization and disinfection in order intelligently to ap-
ply the methods employed for the elimination or destruction
of others whose accidental presence might ruin our experi-
ments.

The dust of the atmosphere, almost invariable in its micro-
organismal contamination, constantly settles upon our glass-
ware, pots, kettles, funnels, etc., and would certainly ruin
every culture-medium with which we experiment did we not
take appropriate measures for its purification and protection.

To get rid of these undesirable " weeds " we make use of
our knowledge of the conditions destructive to bacterial life,
and subject the articles contaminated by them to the action
of heat beyond their known enduring power, or to the action
of chemic agents known to destroy them, or remove them
from fluids into which they have entered by passage through
unglazed porcelain. By all of these methods the articles
are made sterile. Anything is sterile when it contains no
germs of life.

Sterilization is the act of making sterile by destroying or
removing all micro-organismal life, whether infectious or
non-infectious. Disinfection signifies the destruction of the
infectious agents, taking no account of those that are non-
infectious. A germicide is any substance that will kill
germs. It may be used for disinfection and for sterilization.
An antiseptic is a substance that will inhibit the growth of
micro-organisms. It does not necessarily kill them.

The table on page 202 will serve to outline the methods
used for effecting sterilization or the complete destruction
or removal of living organisms:

201

2O2

Sterilization and Disinfection

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is «

151

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PQ PQ

Methods of Sterilization

203

I. The Sterilization and Protection of Instruments
and Glassware. — Sterilization may be accomplished by
either moist or dry heat. For the perfect sterilization of
objects capable of withstanding it dry heat is always to be
preferred, because of its more certain action. If we knew
just what organisms we had to deal with, we might be able
in many cases to save time and gas ; but though some non-
spore-producing forms are killed at a temperature of 60° C.,

pig 3g — Hot-air sterilizer. The gas jets are inclosed within the
space between the outer and middle walls, C, and can be seen at F.
The heat ascends, warming the air between the two Inner walls, which
ascends between the walls, K, then descends over the contents, /, and
escapes through perforations in the bottom, B, to supply the draft at
F, and eventually escapes again at S; R, gas regulator; T, thermometer.

spore-bearers may withstand 100° C for an hour; it is,
therefore, best to employ a temperature high enough to kill
all with certainty. The sterilizing apparatus, or "hot-air
sterilizer," is shown in Fig. 38.

Platinum wires used for inoculation are sterilized by being
held in the direct flame until they become incandescent.

204 Sterilization and Disinfection

In sterilizing the wires attention must be bestowed upon
the glass handle, which should be flamed for at least half
its length for a few moments when used for the first time
each day. Carelessness in this respect may result in the
contamination of the cultures.

Knives, scissors, and forceps may be exposed for a very
brief time to the direct flame, but as this affects the temper
of the steel when continued too long, they are better boiled,
steamed, or carbolized.

All articles of glassware are to be sterilized by an exposure
of one-half to one hour to a sufficiently high temperature —
150° C. or 302° F. — in an appropriate hot-air oven. This
temperature is fatal to all forms of microscopic life.

Rubber stoppers, corks, wooden apparatus, and other
objects which are warped, cracked, charred, or melted
by so high a temperature must be sterilized by exposure to
streaming steam or steam under pressure, in the steam
sterilizer or autoclave, before they can be pronounced
sterile.

It must always be borne in mind that after sterilization
has been accomplished the original sources of contamination
are still present, so that it is necessary to protect the sterilized
objects and media from them.

To Schroder and Van Dusch belongs the credit of having
first shown that when the mouths of flasks and tubes are
closed with plugs of sterile cotton no germs can filter through.
This discovery has been of inestimable value, and has been
one of the chief means permitting the advance of bacteri-
ology. If, before sterilizing, flasks and tubes are carefully
plugged with ordinary (non-absorbent) cotton-wool, they and
their contents will remain free from the access of germs until
opened. Instruments may be sterilized wrapped in cotton,
to be opened only when ready for use ; or instruments and
rubber goods sterilized by steam can subsequently be
wrapped in sterile cotton and kept for use. It is of the
utmost importance to carefully protect every sterilized
object, in order that the object of the sterilization be not
defeated. As the spores of molds falling upon cotton some-
times grow and allow their mycelia to work their way
through and drop into the culture-medium. Roux has em-
ployed paper caps, with which the cotton stoppers can be
protected from the dust. These are easily made by curling a
small square of paper into a " cornucopia," and fastening by

Methods of Sterilization

205

turning up the edge or putting in a pin. The paper is placed
over the stopper before the sterilization, after which no
contamination of the cotton can occur.

II. Sterilization and Protection of Culture=media. —
As almost all of the culture-media contain about 80 per
cent, of water, which would evaporate in the hot-air closet,
and so destroy the material, hot-air sterilization is inap-
propriate for them, sterilization by streaming steam being
the only satisfactory method. The prepared media are

Fig- 39- — Arnold's steam sterilizer (Boston Board of Health form).

placed in previously sterilized flasks or tubes, carefully
plugged with cotton- wool, and then sterilized in an Arnold's
steam sterilizer.

The temperature of boiling water, 100° C., does not kill
the spores, so that one exposure of the culture-media to
streaming steam is of little use. The sterilization must be
applied in a systematic manner — intermittent sterilization —
based upon a knowledge of sporulation.

In carrying out intermittent sterilization the culture-
medium is exposed for fifteen minutes to the passage of

206

Sterilization and Disinfection

streaming steam or to some temperature judged to be
sufficiently high, so that the adult micro-organisms con-
tained in it are killed. As the spores remain uninjured,
the medium is stood aside in a cool place for twenty -four
hours, and the spores allowed slowly to develop into adult
organisms.

When the twenty-four hours have passed, the medium is
again exposed to the same temperature until these newly
developed bacteria are also killed. Eventually, the process

is repeated a third time, lest a few
spores remain alive. When prop-
erly sterilized in this way culture-
media will remain free from con-
tamination indefinitely.

In popular parlance, the inter-
mittent exposure of the culture-
media to steam is spoken of as
sterilization.

A prolonged single exposure to
lower temperatures (6o0-yo0 C.),
known as pasteurization, is em-
ployed for the destruction of bac-
teria in milk and other fluids that
are injured or coagulated by ex-
posure to 100° C. It is appro-
priate only when the organisms to
be killed are without spores and
without marked resisting powers.

Sterilization in the Autoclave. —
If it should be desirable to sterilize
a medium at once, not waiting the
three days required by the inter-
mittent method, it may be done
by superheated steam under a
pressure of two or three atmo-
spheres, sufficient heat being generated to immediately de-
stroy the spores.

Because of its convenience many laboratory workers
habitually use the autoclave for the sterilization of all
media not injured by the high temperature. The steriliza-
tion, to be complete, requires that the exposure shall be
for fifteen minutes at 110° C. (six pounds' pressure).

Fig. 40. — Modern auto-
clave.

Sterilization in the Autoclave

207

The media to be sterilized should be placed in the autoclave, the top
firmly screwed down, but the escape-valve allowed to remain open
until steam is freely generated within and replaces the hot air. The
valve is then closed, and the temperature maintained for fifteen min-
utes or longer if the media be in bulk in flasks. The apparatus should

Fig. 41. — Pasteur-Chamberland filter arranged to filter under
pressure.

be permitted to cool before the valve is opened, and the vacuum be
slowly relieved If the valve be opened suddenly the fluids boil rap-
idly and the cotton plugs may be forced into the tubes or flasks by
the air pressure. The chief objection to the use of the autoclave is that
the high temperature sometimes brings about chemic changes in the
media by which the reaction is altered.

208

Sterilization and Disinfection

Sterilization by Filtration. — Liquids that cannot be
subjected to heat without the loss of their most important
qualities may be sterilized by nitration — i. e., by passing
them through unglazed porcelain or some other material
whose interstices are sufficiently fine to resist the passage
of bacteria. This method is largely employed for the
sterilization of the unstable bacterial toxins that are de-
stroyed by heat. Various substances have been used for
nitration, as diatomaceous earth (Berkefeld filters), stone,

Fig. 42. — Different types of bacteriologic filters : a, Kitasato ; b,
Berkefeld; c, Chamberland ; d, Reichel.

sand, powdered glass, etc., but experimentation has shown
unglazed porcelain to be the only reliable filtering material
by which to remove bacteria. Even this material, whose
interstices are so small as to allow the liquid to pass through
with great slowness, is only certain in its action for a time,
for after it has been repeatedly used the bacteria seem
able to work their way through. To be certain of the efficacy
of any filter, the fluid first passed through must be tested
by cultivation methods to prove that all the bacteria have
been removed. The complicated Pasteur-Chamberland and
the simple Kitasato and Reichel filters are shown in figures
41 and 42.

Disinfection of the Hands 209

The porcelain bougies as well as their attachments must
be thoroughly sterilized before use.

After having been used, a porcelain filter must be dis-
infected, scrubbed, dried thoroughly, and then heated in
a Bunsen burner or blowpipe flame until all the organic
matter is consumed. In this firing process the filter first
turns black as the organic matter chars, then becomes
white again as it is consumed. The porcelain must be dry
before entering the fire, or it is apt to crack.

It should not be forgotten that the filtrate must be received
in sterile receivers and handled with care to prevent sub
sequent contamination.

The filtration of water, peptone solution, and bouillon
is comparatively easy, but gelatin and blood-serum pass
through with great difficulty, and speedily gum the filter.

III. The Disinfection of Instruments, Ligatures,
Sutures, the Hands, etc. — There are certain objects used
by the surgeon that cannot well be rendered incandescent,
exposed to dry heat at 150° C., or steamed continuously, or
intermittently heated without injury. For these objects dis-
infection must be practised. Ever since Sir Joseph Lister
introduced antisepsis, or disinfection, into surgery there has
been a struggle for the supremacy of this or that highly rec-
ommended germicidal substance, with two results — viz., that
a great number of feeble germicides have been discovered,
and that belief in the efficacy of all germicides has been
somewhat shaken; hence the aseptic surgery of the present
day, which strives to prevent the entrance of germs into the
wound rather than their destruction after admission to it.

For a complete discussion of the subject of antiseptics
in relation to surgery the reader must be referred to text-
books of surgery.

The Disinfection of the Hands, etc. — The disinfection of
the skin — both the hands of the surgeon and the part about
to be incised — is a matter of the utmost importance. Wash-
ing the hands with soap, which has marked germicidal
properties, will in many cases suffice to destroy or remove
bacteria from smooth skins. This method, which is regarded
by some surgeons as adequate, is not, however, commonly
regarded as sufficient protection to the patient who might
be infected by any remaining micro-organisms. To over-
come this, many surgeons prefer the use of sterilized gloves

14

210 Sterilization and Disinfection

of thin rubber to all other means of preventing manual in-
fections. Others prefer to use detergent and disinfectant
measures. The method at present generally employed, and
recommended by Welch and Hunter Robb, is as follows:
The nails must be trimmed short and perfectly cleansed.
The hands are washed thoroughly for ten minutes in water
of as high a temperature as can comfortably be borne, soap
and a previously sterilized brush being freely used, and
afterward the excess of soap washed off in clean hot water.
The hands are then immersed for from one to two minutes
in a warm saturated solution of permanganate of potassium,
then in a warm saturated solution of oxalic acid, until
complete decolorization of the permanganate occurs, after
which they are washed free from the acid in clean warm
water or salt solution. Finally, they are soaked for two
minutes in a i : 500 solution of bichlorid of mercury.

Lockwood,* of St. Bartholomew's Hospital, recommends,
after the use of the scissors and penknife, scrubbing the
hands and arms for three minutes in hot water and soap
to remove all grease and dirt. The scrubbing brush ought
to be steamed or boiled before use, and kept in i : 1000
biniodid of mercury solution. When the soapsuds have
been thoroughly washed away with plenty of clean water,
the hands and arms are thoroughly washed and soaked
for not less than two minutes in a solution of biniodid of
mercury in methylated spirit; i part of the biniodid in 500
of the spirit. Hands that cannot bear i : 1000 bichlorid
and 5 per cent, carbolic solutions bear frequent treatment
with the biniodid. After the spirit and biniodid have been
used for not less than two minutes, the solution is washed
off in i : 2000 or i : 4000 biniodid of mercury solution.

It is a mistake to insist upon the employment of disinfect-
ing solutions of a strength injurious to the skin. It must be
obvious to every one that rough skins with numerous hang-
nails and fissures offer greater difficulties to be overcome in
disinfection, and more readily convey micro-organisms into
the wound than smooth, soft skins.

Sterilization of Ligatures, etc.— Catgut cannot be steril-
ized by boiling without deterioration. The present method
of treatment is to dry it in a hot-air chamber and then boil it
in cumol, which is afterward evaporated and the skeins pre-
* " Brit. Med. Jour.," July u, 1896.

Disinfection of Sick-chambers, etc. 211

served in sterile test-tubes or special receptacles plugged with
sterile cotton. Cumol was first introduced for this purpose
by Kronig, as its boiling-point is i68°-i78° C., and thus
sufficiently high to kill spores. The use of cumol for the
sterilization of catgut has been carefully investigated by
Clarke and Miller.*

Catgut may also and equally well be sterilized by the use
of chemical agents. This subject has been carefully reviewed
by Bertarelli and Bocchia,f who regard the method of
Claudius and the modification of it by Rogone as the best.
The method of Claudius is to roll the catgut into skeins and,
without taking any precautions to remove any fat it may con-
tain, place it in a mixture of iodin i, iodid of potassium i, and
distilled water 100. After immersion for eight days the cat-
gut is removed, under aseptic precautions, to alcohol or to
3 per cent, carbolic solution, in which it is indefinitely pre-
served for use.

Ligatures of silk and silkworm gut are boiled in water
immediately before using, or are steamed with the dressings,
or placed in test-tubes plugged with cotton and steamed in
the sterilizer.

Sterilization of Surgical Instruments, etc. — In most
hospitals instruments are boiled, before using, in a i to 2 per
cent, soda (sodium carbonate, sodium bicarbonate, or sodium
biborate) solution, as plain water has the disadvantage of
rusting them. During the operation they are either kept in
the boiled water or in a carbolic solution, or are dried with a
sterile towel. Andrews makes special mention of the fact
that the instruments must be completely immersed to pre-
vent rusting.

Disinfection of the Wound. — Cleansing solutions (normal
salt solution) and disinfecting solutions (such as i : 10,000 to
i : 1000 bichlorid of mercury) are only applied to septic
wounds.

IV. The Disinfection of Sick=chambers, Dejecta, etc.
—The Air of the Sick-room. — It is impossible to sterilize
or disinfect the atmosphere of a room during its occupancy
by the patient. It is entirely useless to place beneath the bed
or in the corner of a room small receptacles filled with car-
bolic acid or chlorinated lime. These can serve no purpose

* "Bull, of the Johns Hopkins Hospital," Feb. and March, 1896.

t "Centralbl. fur Bakt. u. Parasitenk.," Orig. i,, 620.

212 Sterilization and Disinfection

for good, and may do harm by obscuring odors emanating
from harmful materials that should be removed from the
room. The practice is only comparable to the old faith in
the virtue of asafetida tied in a corner of the handkerchief
as a preventive of cholera and smallpox.

Before one is able to make a scientific application of any
germicidal substance it is necessary to become acquainted with
its micro-organism-destroying powers. This may seem at
first thought to be a simple matter, but is, in reality, one of
great complexity and difficulty, for the various micro-organ-
isms show marked variations in their powers of endurance;
different stages in the development of the micro-organisms
show different degrees of resisting power, and the conditions
under which the germicide meets the micro-organism effect
marked variations in action. These factors make it neces-
sary to vary the process of disinfection according to the exact
purpose to be achieved.

Let two examples serve to illustrate these requirements:
Bichlorid of mercury is one of the most powerful, reliable, and
generally useful germicides, but the strength of its solutions
must vary according to the purpose for which they are
intended. It kills cocci and non-sporogenic bacilli in dilu-
tions of i : 10,000 in from five minutes to twenty-four hours,
but to kill anthrax spores requires twenty-four hours' immer-
sion in i : 2000 solution. If albuminous substances are
present in the medium containing the micro-organisms they
precipitate the salt immediately, diminishing the strength of
the solution and so retarding or perhaps preventing the
germicidal action. Again, certain micro-organisms are de-
fended from the action of destructive agents, and among them
the germicides, through the presence of waxy matter in their
substance. Such is the case with the acid-fast organisms,
and notably the tubercle bacillus. Antiformin, a combina-
tion composed of equal parts of liquor sodae chlorinatae and
a 15 per cent, solution of caustic soda, immediately dis-
solves the great majority of micro-organisms, but has no
destructive action whatever upon the tubercle bacillus.

The most useful germicidal substances act destructively
upon the micro-organisms by forming chemical compounds
with their cytoplasm. Thus, the salts of mercury unite with
the protoplasm to form an albuminate of mercury. Other
germicidal agents dissolve or coagulate the protoplasm ; still
others oxidize and so completely destroy the cells. In the

Inorganic Disinfectants 213

process of germicidal action many and varied activities are
at work, and, as all are not understood, the subject is a
difficult one to handle in a limited amount of space. With
the salts, acids, and bases it appears from the researches of
Kronig and Paul* that ionization in solution plays an import-
ant part in the destruction of micro-organisms. They found
that double metallic salts, in which the metal is a constituent
of a complex ion in which the concentration of the dissociated
metal ions is consequently very low, have very little germicidal
power, but that simple salts, in which the condition is reversed,
have correspondingly higher germicidal power. Dissocia-
tion, therefore, seems to have much to do with the matter.

Inorganic Disinfectants.

ACIDS. — These agents are seldom employed, since the concentration

required makes them objectionable.

ALKALIS. — The same holds good with regard to these agents.
SALTS. — In this group we find some of the most powerful and most

useful germicidal substances.

Copper Sulfate. — It is curious and interesting that while this
salt is highly destructive to algae and other low forms of
vegetable life, it is not of much value for the destruction of
bacteria. Its chief use is for the destruction of the green algae
that sometimes render the water of reservoirs dirty and offen-
sive. Some of the salt contained in a gunny-sack and per-
mitted to drag to and fro over the surface of the water behind
a slowly rowed boat usually accomplishes the end, the actual
quantity dissolving in the water being almost infinitesimal.
Mercuric Chlorid (HgCl2). — This is probably the most generally

useful as well as one of the strongest germicides.

A study of its activity under varying conditions is instructive as ex-
emplifying the varying behavior of germicides under the varying con-
ditions under which they may be employed.

First, it makes great difference whether the mercuric chlorid is added
to the substratum containing the bacteria, or whether the bacteria are
added to solutions of the germicide.

Thus, when the salt is dissolved in gelatin in a concentration of
i: i,ooo..ooo, anthrax bacilli cannot grow. If it is dissolved in blood-
serum, the concentration must be increased to i : 10,000 to prevent their
growth.

When the anthrax spores are dropped in solutions of the salt, Kronig
and Paul found that they were killed in twelve to fourteen minutes by
i : 65 solutions; in eighty minutes by i : 500 solutions, and in two hours
by i : looo solutions. When the reaction takes place in albuminous
media Behring and Nochtf found that much more time was required.
Thus, the destruction of the spores by a i : 100 solution required eighty
minutes, and a i : 1000 solution twenty-four hours to completely kill all
of the spores.

LaplaceJ and Panfili§ found that the addition of 5 per cent, of tar-
taric or hydrochloric acid facilitated the germicidal action through the

* "Zeitschrift fur Hygiene," 1897, xxv, i. t Ibid., ix, 432.

J "Deutsche med. Wochenschrift," 1887, 866; 1888, 121.
§ "Ann. Ig. Roma," 1893, m, 527.

214 Sterilization and Disinfection

prevention of albuminate of mercury formation. Liibbert and Schneider
and Behring have used sodium chlorid and ammonium chlorid. Both
of these salts diminish the germicidal action of the mercuric salt about
one-half. Notwithstanding this, however, the "antiseptic tablets" in
common use for surgical and household purposes contain one or both
of these salts, added for the purpose of preventing the precipitation of
the mercuric compounds formed in the presence of alkaline albuminous
materials, such as blood, pus, sputum, feces, etc.

The addition of about 25 per cent, of alcohol to the solution of the
mercuric salt greatly enhances its value. Strong alcoholic solutions
are, however, less useful than aqueous solutions, for the 95 or 100 per
cent, alcohol dehydrates the micro-organisms and prevents the diffusion
currents by which the mercury is carried into their substance.

For most purposes a i : 2000 solution of the mercuric chlorid is to be
recommended.

Silver Nitrate (AgNO3). — The solutions of this salt are probably
more useful than the frequency of their employment might sug-
gest. They have, however, the disadvantages of decomposing
when kept in the light and of making black stains when applied
in concentrated form to the skin or dressings.

The germicidal power of the salt in aqueous solution is less than
that of the mercuric chlorid, but the power in albuminous fluids
is greater. Anthrax spores in blood-serum are killed in seventy
hours in a i: 12,000 solution. The addition of other salts, as
ammonium salts, interferes with the germicidal activity by in-
hibiting ionization.

Combinations of the silver nitrate with albuminous compounds,
and variously known as argonin, argentum casein, argyrol, pro-
targol, etc., have been used where the disinfecting power of the
silver is sought for with the least amount of irritation and the
deepest degree of penetration, as in the treatment of gonorrhea.
Potassium Permanganate (KMnOJ. — Solutions of this salt seem to
act by virtue of a strong oxidizing power. In 2 per cent, solu-
tions anthrax spores are killed in forty minutes; in 4 per cent,
solutions, within fifteen minutes. Koch's experiments showed
less activity of the germicidal power against anthrax spores.
In his hands a 5 per cent, solution seemed to require about a day
to effect complete distraction. A i per cent, solution kills the
pus cocci in ten minutes; a i : 10,000 solution kills plague bacilli
in five minutes.

The chief difficulty in the way of successfully employing this
salt is that it is quickly reduced and its strength destroyed by the
organic substrata in which the bacteria are contained.
HALOGENS AND COMPOUNDS. — Those with the lowest atomic weight

have the greatest disinfecting power.

Chlorin. — This is usually employed in the form of chlorinated
lime. It seems to be a mixture of calcium hypochlorite, Ca(ClO2),
and calcium chlorid, CaOCl2. The addition of any acid, in-
cluding the atmospheric CO2, causes the evolution of Cl. The
powder is readily soluble and solutions of i : 500 kill vegetative
forms of most bacteria in a few minutes (not, however, resisting
spores).

A proprietary compound known as "electrozone," made by
electrolyzing sea-water in such a manner that magnesia and
chlorin are liberated and magnesium hypochlorite and magne-
sium chlorid formed, is a cheap and useful chlorin disinfectant.
Nissen found that 1.5 per cent, of it killed typhoid bacilli in a
few minutes; Rideal, that i : 400 to 500 dilutions of it disinfected
sewage in fifteen minutes; and Delepine, that i : 50 (equal to

Organic Disinfectants 215

0.66 per cent, of chlorin) rapidly killed the tubercle bacillus
and i : 10 (equal to 3.3 per cent, chlorin) killed anthrax spores.
lodin Terchlorid (IC13). — This compound, which is so unstable
that it only keeps in an atmosphere of Cl-gas, has great germi-
cidal action, that probably depends upon the readiness with which
it decomposes. In solutions of i : 1000 it kills vegetative bac-
teria in a few minutes, and in i : 100 it kills anthrax spores with
equal rapidity. The presence of organic and albuminous mate-
rials does not interfere with the germicidal action.

Organic Disinfectants.

Carbolic acid (C6H5OH) is the most important and generally useful
of these. It has the advantage of being cheap and easily kept
and handled. In the pure state it consists of colorless acicular
crystals. When exposed to the atmosphere it takes up water
and gradually becomes a brownish-yellow oily fluid. The
crystals and deliquesced crystals have powerful escharotic proper-
ties and cannot be touched without destruction of the skin.
In 2 to 3 or 5 per cent, solutions carbolic acid destroys most
bacteria within a few minutes. Anthrax and other powerfully
resisting spores, however, require prolonged exposure. Tetanus
spores are said not to be killed in less than fifteen hours. There
is no ionization ; the reagent seems to act by coagulating the bac-
terial protoplasm.

Though carbolic acid has been for a quarter of a century a
favorite surgical disinfectant, the application of 5 per cent,
solution to the skin has so frequently caused gangrene that it is
at present in some merited disfavor.

Closely related to carbolic acid and other products of coal-tar
distillation are orthocresol, metacresol, and paracresol. " Tri-
kresol," a much used antiseptic, is a commercial product con-
sisting of a mixture of all three of the cresols. It is more strongly
germicidal than carbolic acid, but is less soluble in water. It is
or has been largely used for addition to therapeutic serums in
the proportion of 0.4 per cent, as an antiseptic. Such addition
causes the formation of an albuminous precipitate in which,
doubtless, much of the antiseptic is lost, for upon its removal or
even upon its sedimentation resisting forms of bacteria may
grow in the serum. It cannot, therefore, be looked upon as a
reliable preservative.

"Lysol" is said to be a solution of coal-tar cresol in potassium
soap. It has the advantage of forming a lather-like soap, so
that it can be employed both as a cleanser and disinfectant. In
i per cent, solutions it is capable of destroying cocci, typhoid
bacilli, and other micro-organisms of low resisting power.

"Creolin" is also a combination of cresols with potassium
soap. When added to water it immediately forms an emulsion.
It has been much used in obstetric practice, where it has earned
more reputation than it deserves.

''Formalin." — This is Schering's commercial denomination of a
30 to 40 per cent, aqueous solution of formaldehyd gas (H — COH)
or formic aldehyd. The solution is highly germicidal so
long as it is fresh. When exposed for long to the atmosphere
it polymerizes into trioxymethylene and paraformaldehyde
and greatly loses its power. A 10 per cent, solution of formalin
kills pus cocci in half an hour. A 5 per cent, solution kills cholera
spirilli in three minutes; anthrax bacilli, in fifteen minutes;
anthrax spores, in five hours. Pure formalin kills anthrax spores

2l6

Sterilization and Disinfection

in ten to thirty minutes. Strong solutions are extremely irri-
tating and so not applicable in surgery. They are, however, of
great use for household disinfection. Formalin and formaldehyd
gas find their chief usefulness for the aerial disinfection of sick
chambers and domiciles, where they are either used as a spray
or the gas evolved by chemical means or by heat, as will be shown
below.

Peroxid of hydrogen (H2O2) is germicidal through its power to
liberate the nascent O. It quickly decomposes when brought
into contact with organic matter, and, therefore, has a very limited
sphere of usefulness.

The following tables, compiled by Hiss from Fliigge, will
show the comparative values of the commonly employed
antiseptics and germicides:

INHIBITION STRENGTHS OF VARIOUS ANTISEPTICS.
(Adapted from Fliigge, Leipzig, 1902.)

Anthrax
Bacilli.

Other Bacteria.

Putrefactive
Bacteria in
Bouillon.

ACIDS
Sulphuric
Hydrochloric

i : 3000
i : 3000

Choi. spir. i : 6000

Sulphurous

B. mallei i : 700
B. typh. i : 500

i : 6000

Arsenous

i : 200

Boric

i : 800

i : 100

ALKALIES
Potass, hydrox

i : 700

B. diph. i : 600

Ammon hydrox

i : 700

Choi, spir i : 400
B. typh. i : 400
Choi, spir i • 500

Calcium hydrox.

B. typh. i : 500
Choi spir i * i ico

SALTS
Copper sulphate.

B, typh. i : 1 100

Ferric sulphate

Mercuric chlorid . .

B. typh. i : 60,000

Silver nitrate

i 6o,coo

Choi, spir.,

Potass, perman.

I IOOO

B. typhosus i : 50,000

HALOGENS AND COMPOUNDS
Chlorin

i 1500

Bromin

lodin

i 5000

Potass, iodid

Sodium chlor
ORGANIC COMPOUNDS
Ethyl alcohol

i 60

I 12

I IO

Acetic and oxalic acids

B. diph. i : 500

Carbolic acid

I 8oO

B typh. i • 400

Benzoic acid

I IOOO

Choi. spir. i : 600

Salicylic acid
Formalin (40% lormaldehyd)

I 1500

Choi. spir. i : 20,000

Camphor

i 1000

Staphylo. i : 5000

Thymol

Oil mentha pip

I 3000

Oil of terebinth

I 8000

Peroxid of hydrogen

Comparison of Disinfectants

217

BACTERICIDAL STRENGTH OF COMMON DISINFECTANTS.
(Adapted from Fliigge, Leipzig, 1902.)

Streptococci
and Sta-
phylococci.

Anthrax and Typhoid Bacilli,
Cholera Spirillum.

Anthrax spores.

5 minutes.

5 minutes.

2 to 24 hours.

ACIDS
Sulphuric

i : 10
i : 10

i : 100
i : 100

i : 1500
i : 1500
Typhoid i : 700
i : 300 (Gas 10
vol. %)
i =3°

i : 50 in 10 days
i : 50 in 10 days

Cone. sol. in com-
plete disinfection

i : 20 (5 days)
i : 2000 (26 hrs.)

i : 20 (i day)
i : 20 (i hour)

2% (in i hour)
i : iooo(in 12 hrs.)

Alcol. 50% for 4
months with-
out killing1
spores (Koch*)
i : 20 (4 to 45
days) (at 40°
in 3 hours)

(10% in 5 hours)

i : 20 (in 6 hrs.)
i : 100 (in i hr.)
3 : loo (in i hr.)

Hydrochloric

Sulphurous

Sulphurous . ...

Boric

ALKALIES
Potass, hydrox.

i : 5

i :3oo
i : 300
i : looo

Ammon hydrox

Calcium

SALTS
Copper sulphate

Mercuric chlor. . .

i : 10,000 to

IOOO

i : 2coo

i : 10,000
i : 4000

Potass, permang'

i : 200

"Calc. chlorid."

i : 500

i%
i : looo

70% — 10
minutes

Cholera i : 200
Typh. i : 50

i : 300
i : 100

i : 20
i : 200

HALOGENS AND COMPOUNDS

i%
i : 200

70% — 15
minutes

i :6o

i : 300

Trichloridof iodin
ORGANIC COMPOUNDS
Ethyl alcohol

i : 200 to 300
i -.300

i : 3000

i : looo
i : 500

Acetic and oxalic acids.
Carbolic acid

Lysol

Creolin

Salicylic acid

i : 1000
i : TO
Cone.

Formalin (40% formaldehyd)
Peroxid of hydrogen

Certain fundamental principles govern the rationale of
disinfection, and must be kept in mind : (i) the reagent em-
ployed should be known to act destructively upon bacteria ;
(2) it must be applied to the bacteria to be killed ; (3) it must
be applied in sufficiently concentrated form, and (4) it must
be left in contact with the bacteria long enough to accom-
plish the effect desired.

During the period of illness the chamber in which the
patient is confined should be freely ventilated. An abun-
dance of fresh, pure air is a comfort to the patient and a
protection to the doctor and nurse.

After recovery or death one should rely less upon fumi-
gation than upon disinfection of the walls and floor, the

* Koch, Arb. a. d. kais. Gesundheitsamt, i, 1881.

218 Sterilization and Disinfection

similar disinfection of the wooden part of the furniture, and
the sterilization of all else. The fumes of sulphur do some
good, especially when combined with steam, but are greatly
overestimated in action and are very destructive to furnishings,
so that they are rapidly giving way to the more satisfactory,
less destructive, and equally germicidal formaldehyd vapor.

Formaldehyd is probably the best germicide that has yet
been recommended. Its use for the disinfection of rooms
and hospital wards was first suggested by Trillat* in 1892, but
it did not make much stir in the medical world until a year
or more had passed and a 40 per cent, solution of the gas,
under the name of " Formalin," had been placed upon the
market. Care must be exercised in handling the fluid,
that the hands do not become wet with it, as it hardens the
skin and deadens sensation. The vapor is exceedingly irri-
tating to the mucous membrane of the eyes and nose.

The solution can be employed to spray the walls and floors
of rooms, though Rosenauf finds that unless the spray dis-
charged from a large atomizer be very fine, its action is un-
certain.

The original method of disinfection, suggested by Robin-
son,! consisted of the evolution of the gas by volatilizing
methyl alcohol, and passing the vapor over heated asbestos.
Shortly many efficient forms of apparatus were placed upon
the market, for the evolution of the gas or for discharging
it from the solution.

It is not necessary to use a special apparatus in order
to disinfect with formaldehyd; one can, in an emergency,
hang up a number of sheets, saturated with the 40 per cent,
solution, in the room to be disinfected.

The number of sheets must vary with the size of the
room, as each is able to evolve but a certain amount of the
gas, and the quantity necessary for disinfection varies with
the size of the room.

A better method of evolving the gas for purposes of dis-
infection devised by Evans and Russell § is to combine the
solution with permanganate of potassium, when an almost
explosive liberation of the gas takes place.

* "Compte rendu de 1'Acad. des Sciences," Paris, 1892.
t "Disinfection and Disinfectants," P. Blakiston's Son & Co., Phila-
delphia, 1902.

} "Ninth Report of the State Board of Health of Maine," 1896.
§ "Report of the State Board of Health of Maine," 1904.

Disinfection of Sick-chambers, etc. 219

Frankforter* found that a good method of escaping the
undesirable features of the gaseous evolution was to mix the
powder of permanganate of potassium with an equal volume
of sand, so that- the formaldehyd solution is brought more
slowly into contact with the permanganate, under condi-
tions unfavorable to the formation of oxids of manganese,
such as otherwise tend to coat the grains of permanganate
and prevent further reaction between the formaldehyd solu-
tion and the permanganate.

The employment of calcium carbide for the same purpose
is suggested by Evans. f The best results were obtained
when the calcium carbide was in lumps about the size of a
pea; when the formaldehyd solution was diluted with an
equal volume of water, and when the diluted formaldehyde
was added to the carbide in the proportion of 5 c.c. of the
former to 3 grams of the latter. In the permanganate
method the quantity of formalin (or 37-40 per cent, for-
maldehyd in water) should equal 200 c.c. to 1000 cubic feet
of space, but in the carbide method 500 c.c. must be used to
achieve the same result. Evans, therefore, prefers the
permanganate method.

To disinfect with formaldehyd or any gaseous disinfectant,
the room must be carefully closed, the cracks of the windows
and doors being sealed by pasting strips of paper over them.
If an apparatus is used, it is set in action, the discharged
vapor entering the room through the keyhole or some other
convenient aperture, the gas being allowed to act undis-
turbed for some hours, after which the windows and doors
are all thrown open to fresh air and sunlight.

If sheets are hung up, or the permanganate method
employed, the windows and doors, other than that by means
of which the operator is to escape, are closed and sealed.
A dish-pan or wash-tub is placed in the center of the room,
and in it the can containing the permanganate and sand.
The formaldehyd solution is poured on, the operator making
his escape, closing and sealing the door behind him. Any
closets in the room must be left open so that they and their
contents may be disinfected with the room.

So far as is known at present, superficial disinfection by
formaldehyd leaves little to be desired. Care must, how-

* ''Reports and Papers of the American Public Health Association,"
vol. xxxii, part n, p. 114, 1906.
t Ibid., p. 108.

220 Sterilization and Disinfection

ever, be exercised to see that the required volume of gas
is generated to disinfect the apartment. A sufficient con-
centration of the gas is absolutely necessary, and the method
selected should be one capable of discharging the gas in a
short time, so that it immediately pervades the atmosphere.

Disinfection with formaldehyd is, however, only super-
ficial, its penetrating powers being limited. The discharge
of gas into the room should only be preliminary to other and
more thorough disinfection and sterilization of the contents
by the application of solutions of disinfectants to the wood-
work, and to the boiling of the linen, etc.

The Dejecta.— In diphtheria the expectoration and nasal
discharges are highly infectious and should be received in old
rags or in Japanese paper napkins — not handkerchiefs or

Fig. 43. — Pasteboard cup for receiving infectious sputum. When used
the pasteboard can be removed from the iron frame and burned.

towels — and should be burned. The sputum of tuberculous
patients should either be collected in a glazed earthen vessel
which can be subjected to boiling and disinfection, or, as is
an excellent plan, should be received in Japanese rice-paper
napkins, which can at once be burned. These napkins are
not quite so good as the small pasteboard boxes (Fig. 43)
recommended by some city boards of health, because,
being highly absorbent, the sputum is apt to soak through
and soil the ringers. For the fastidious patients in cer-
tain sanatoria, cut-glass bottles with tightly fitting lids
are used to collect the sputum, and as these are not un-
sightly, the patients make no objection to carrying them
about with them. Tuberculous patients should be provided
with rice-paper instead of handkerchiefs, and should have
their towels, knives, forks, spoons, plates, etc., kept strictly
apart from the others of the household and carefully sterilized
after using. Patients whose mental acuity makes their

Disinfection of the Clothing 221

sensibilities very pronounced need never be told of these
arrangements.

The excreta from cases of typhoid fever and cholera re-
quire particular attention. These, and indeed all alvine
matter the possible source of infection or contagion, should
be received in glazed earthen vessels and immediately and
intimately mixed with a 5 per cent, solution of chlorinated
lime (containing 25 per cent, of chlorin) if semi-solid, or with
the powder if liquid, and allowed to stand for an hour before
being thrown into the drain.

Thoughtful consideration should always be given the
germicides used to disinfect the discharges, lest combination
of the chemical with ingredients of the discharge produce
inert compounds. Thus, bichlorid of mercury cannot be
used because it forms an inert compound with albumin.

The Clothing, etc. — The bed-clothing, towels, napkins,
handkerchiefs, night-robes, underclothes, etc., used by a
patient suffering from an infectious disease, as well as the
towels, napkins, handkerchiefs, caps, aprons, and outside
dresses worn by the nurse, should be regarded as infective
and carefully sterilized. The only satisfactory method of
doing this is by prolonged subjection to steam in a special
apparatus; but, as this is only possible in hospitals, the
next best thing is boiling for some time in the ordinary
wash-boiler. In drying, the wash should hang longer than
usual in the sun and wind. Woolen underwear can be
treated exactly as if made of cotton. The woolen outer
clothing of the patient, if infective, requires special treat-
ment. Fortunately, the infection of the outer garments
is unusual. The only reliable method for their sterilization
is prolonged exposure to hot air at 1 10° C. In private prac-
tice it often becomes a grave question what shall be done
with these articles. Prolonged exposure to fresh air and
sunlight will, however, aid in rendering them harmless;
and can be practised when it is not certain that they are
actually infective. Infective articles of wool may be sent to
the city hospital or to one of the moth-destroying and fumi-
gating establishments which can be found in all large cities,
and baked.

The doctor visiting a case of dangerous infection or a hos-
pital for infectious diseases should cover his clothing with a
linen or cotton gown, and protect his hair with a cap, these
articles being disinfected after the visit. By such precau-

222 Sterilization and Disinfection

tions he will avoid spreading infection among his patients or
carrying it to his own family.

The Furniture, etc. — The destruction of infective fur-
niture is unnecessary. The doctor treating a case of infec-
tious disease, if he properly perform his' functions, will save
much trouble and money for his patient by ordering his
immediate isolation in an uncarpeted, scantily, and simply
furnished room the moment an infectious disease is sus-
pected. However, if before his removal the patient has
occupied another bed, its clothing should be promptly dis-
infected.

After the recovery or death of the patient the walls and
ceiling of the room should be sprayed with a formaldehyd
solution, or the room sealed and filled with the vapor.
If they are hung with paper, they should be dampened with
i : 1000 bichlorid of mercury solution before new paper is
hung.

Strehl has demonstrated that when 10 per cent, formalin
solution is sponged upon artificially infected curtains, etc.,
the bacteria are killed by the action of the disinfectant.
This is an important adjunct to our means of disinfecting
the furniture of the sick-chamber.

The floor should be scoured with 40 per cent, formaldehyd
solution, 5 per cent, carbolic acid solution, or i : 1000 bi-
chlorid of mercury solution (no soap being used, as it
destroys the bichlorid of mercury and prevents its action),
and all the wooden articles wiped off two or three times
with one of the same solutions. If a straw mattress was used
it should be burned and the cover boiled. If a hair mattress
was used, it can be steamed or baked by the manufacturers,
who usually have ovens for the purpose of destroying moths,
but which answer for sterilizing closets. Curtains, shades,
etc., should receive proper attention; but, of course, the
greater the precautions exercised in the beginning, the fewer
the articles that will need attention in the end.

The Patient, whether he live or die, may be a means
of spreading the disease unless specially cared for. After
convalescence the body should be scoured with biniodide of
mercury soap, bathed with a weak bichlorid of mercury
solution or with a 2 per cent, carbolic acid solution, or with
25-50 per cent, alcohol, before the patient is allowed to
mingle with society, and the hair should either be cut off
or carefully washed with the disinfecting solution or an

Disinfection of the Patient 223

antiseptic soap. In desquamative diseases it seems best
to have the entire body anointed with cosmolin once daily,
beginning before desquamation begins and having the
unguent well rubbed in, in order to prevent the particles of
epidermis, in which the specific contagium probably occurs,
being distributed through the atmosphere. Carbolated
may be better than plain cosmolin, not because of the very
slight antiseptic value it possesses, but because it helps to
allay the itching which may accompany the desquamative
process.

After the patient is about the room again, common sense
will prohibit the admission of visitors until the suggested
disinfective measures have been adopted, and after this,
touching, and especially kissing him, should be avoided for
some time.

The bodies of those that die of infectious diseases should be
washed in a strong disinfectant solution, and should be given
a strictly private funeral. If this be impossible, the body
should be sealed in the coffin and only the face viewed
through a plate of glass. In my judgment, the body is
best disposed of by cremation.

A dead body cannot remain a source of infection for an
indefinite period. Esmarch,* who made a series of labora-
tory experiments to determine the fate of pathogenic bac-
teria in the dead body, found that in septicemia, cholera,
anthrax, malignant edema, tuberculosis, tetanus, and typhoid
fever the pathogenic bacteria all die sooner or later, more
rapidly during active decomposition than during preservation
of the tissues.

* "Zeitschrift fur Hygiene," 1893.

CHAPTER VII.

CULTURE-MEDIA AND THE CULTIVATION OF
MICRO-ORGANISMS.

IN order to observe them accurately the organisms must be
separated from their natural surroundings and artificially
cultivated upon certain prepared media of standard compo-
sition, in such a manner that only organisms of the same
kind are together. The effects of one organism upon the
growth of another, by neutralizing its metabolic products,
by changing the reaction of the medium in which it grows so
as to inhibit further multiplication, by dissolving the other
species through its enzymes, etc., suffice to show how im-
possible it is to determine the natural history of any organ-
ism unless it be kept strictly away from other species.

Fortunately the same general principles apply equally for
the cultivation of all forms of micro-organismal life, and much
the same media apply in all cases. What is said, therefore,
about the bacteria may be regarded as appropriate for all.

Various organic and inorganic mixtures have been sug-
gested for the cultivation of bacteria, but few have met with
particular favor and become standards. At the present
time certain standard media are used in every laboratory
in the world; all systematic study of the organisms depends
upon the behavior of bacteria upon them, and no study
of micro-organisms can be regarded as complete unless the
behavior of the bacteria upon them has been carefully
considered.

Our studies of the biology of the bacteria have shown that
they grow best in mixtures containing at least 80 per cent, of
water, of neutral or feebly alkaline reaction, and of a com-
position which, for the pathogenic forms at least, should
approximate the juices of the animal body. It might be
added that transparency is a very desirable quality, and that
the most generally useful culture media are those that can
be liquefied and solidified at will.

224

Cultivation of Micro-organisms 225

All accurate bacteriologic culture experiments require
that an exact knowledge of the chemistry of the media used
shall be at hand. The importance of this and the necessity
for having exact information regarding the reaction of the
media are well brought out in the following excerpts from

Fig. 44. — Buret for titrating media. (From Hiss and Zinsser, "Text-
Book of Bacteriology," D. Appleton & Co,, Publishers.)

the Report of the Committee of Bacteriologists of the Amer-
ican Public Health Association :*

"The first thing to obtain is a standard 'indicator' which will give
uniform results. These requirements are best fulfilled by phenol-
phthalein."

" The question of the proper reaction of media for the cultivation
of bacteria and the method of obtaining this reaction have been dis-
cussed in a valuable paper by Mr. George W. Fuller, published in the

* "Jour. Amer. Public Health Assoc.," Jan., 1898, p. 72.
15

226 Cultivation of Micro-organisms

'Journal of the American Public Health Association,' Oct., 1895,
vol. xx, p. 321."

"Method of determining the degree of reaction of culture media :
For this most important part in the preparation of culture media,
burets graduated into one-tenth c.c. and three solutions are required —

"1. A 0.5 per cent, solution of commercial phenolphthalein, in 50
per cent, alcohol.

"2. A — solution of sodium hydroxid.

"3. A — solution of hydric chlorid.

"Solutions 2 and 3 must be accurately made and must correspond
with the normal solutions soon to be referred to.

"Solutions of sodium hydroxid are prone to deterioration from the
absorption of carbon dioxid and the consequent formation of sodium
carbonate. To prevent as much as possible this change, it is well to
place in the bottle containing the stock solution a small amount of
calcium hydroxid, while the air entering the burets or the supply
bottles should be made to pass through a U-tube containing caustic
soda, to extract from it the carbon dioxid."

" The medium to be tested, all ingredients being dissolved, is brought
to the prescribed volume by the addition of distilled water to replace
that lost by boiling, and after being thoroughly stirred, 5 c.c. are
transferred to a 6-inch porcelain evaporating-dish. To this 45 c.c.
of distilled water are added and the 50 c.c. of fluid are boiled for three
minutes over a flame. One cubic centimeter of the solution of phenol-
phthalein (No. 1) is then added, and by titration with the required
reagent (No. 2 or No. 3) the reaction is determined. In the majority of

instances the reaction will be found to be acid, so that the — sodium

hydroxid is the reagent most frequently required. This determination
should be made not less than three times and the average of the results
obtained taken as the degree of the reaction.

"One of the most difficult things to determine in this process is
exactly when the neutral point is reached as shown by the color de-
veloped, and to be able in every instance to obtain the same shade
of color. To aid in this regard, it may be remarked that in bright
daylight the first change that can be seen on the addition of alkali is
a very faint darkening of the fluid, which, on the addition of more
alkali, becomes a more evident color and develops into what might
be described as an Italian pink. A still further addition of alkali
suddenly develops a clear and bright pink color, and this is the reac-
tion always to be obtained. All titrations should be made quickly
and in the hot solutions to avoid complications arising from the presence
of carbon dioxid.

"The next step in the process is to add to the bulk of the medium
the calculated amount of the reagent, either alkali or acid, as may be
determined. For the purpose of neutralization a normal solution of
sodium hydroxid or of hydric chlorid is used, and after being thor-
oughly stirred the fluid thus neutralized is again tested in the same
manner as at first, to insure the proper reaction of the medium being
attained. When neutralization is to be effected by the addition of
an alkali, it not infrequently happens that after the calculated amount
of normal solution of sodium hydroxid has been added, the second
test will show that the medium is acid to phenolphthalein, to the
extent sometimes of 0.5 to 1 per cent. This discrepancy is perhaps
due to side reactions which are not understood. The reaction of the
medium, however, must be brought to the desired point by the further

Bouillon 227

addition of sodium hydroxid, and the titrations and additions of
alkali must be repeated until the medium has the desired reaction
(i. e., 0.0 per cent, to 0.005 per cent. ; see below).

"After the prescribed period of heating, it is frequently found that
the medium is again slightly acid, usually about 0.5 per cent. Without
correcting this, the fluid is to be filtered and the calculated amount of
acid or alkali is to be added to change the reaction to the one desired.
A still further change in reaction is not infrequently to be observed
after sterilization, the degree of acidity varying apparently with the
composition of the media and the degree and continuance of the heat."

"Manner of expressing the reaction: Since at the time the reaction
is first determined culture media are more often acid than alkaline, it is
proposed that acid media be designated by the plus sign and alkaline
media by the minus sign, and that the degree of acidity or alkalinity
be noted in parts per hundred. Thus, a medium marked +1.5
would indicate that the medium was acid, and that 1.5 per cent, of

— sodium hydroxid is required to make it neutral to phenolphthalein ;
while — 1.5 would indicate that the medium was alkaline and that
1.5 per cent, of — acid must be added to make it neutral to the indicator."

''Standard reaction of media (provisional):

"Experience seems to vary somewhat as to the optimum degree
of reaction which shall be uniformly adopted in the preparation of
standard culture media. To what extent this is due to variation in
natural conditions as compared with variations of laboratory procedure
it seems impossible to state. Somewhat different degrees of reaction
for optimum growth are required, not only in or upon the media of
different composition and by bacteria of different species, but also
by bacteria of the same species when in different stages of vitality.
The bulk of available evidence from both Europe and America points
to a reaction of -f-1.5 as the optimum degree of reaction for bacterial
development in inoculated culture media. While this experience is
at variance with that in several of our own laboratories, it has been
deemed wisest to adopt -f-1.5 as the provisional standard reaction of
media, but with the recommendation that the optimum growth reaction
be always recorded with the species."

Many bacteriologists regard a reaction of +1.0 as a
more desirable standard and use it exclusively.

BOUILLON.

This is one of the most useful and most simple media.
It can be prepared from meat or from meat extract, and is
the basis of most of the culture media. The addition of 10
per cent, of gelatin makes it " gelatin" ; that of i per cent, of
agar-agar makes it "agar-agar." The preparation of these
media, however, requires special directions, which will be
given below.

I. To Prepare Bouillon from Fresh Meat. — To 500 grams
of finely chopped lean, boneless beef, 1000 c.c. of clean water
are added and allowed to stand for about twelve hours on

228 Cultivation of Micro-organisms

ice. At the end of this time the liquor is decanted, that
remaining on the meat expressed through a cloth, and then,
as the entire quantity is seldom regained, enough water
added to bring the total amount up to 1000 c.c. This
liquid is called the meat-infusion. To it 10 grams of Witte's
or Fairchild's dried beef-peptone and 5 grams of sodium
chlorid are added, and the whole boiled until the albumins
of the meat -infusion coagulate, titrated or otherwise cor-
rected for acidity, boiled again for a short time, and then
filtered through a fine filter-paper. It should be slightly
yellow and perfectly clear and limpid. Smith,* referring to
bouillon intended for the culture of diphtheria bacilli for
toxin, says that when the peptones are added before boiling
most of them are lost, and therefore recommends that the
meat-infusion be boiled and filtered and the solid ingredients
added and dissolved subsequently. The reaction, which is
strongly acid, is then carefully corrected by titration accord-
ing to the directions already given.

For rough work in students' classes litmus paper may
be used as an indicator for determining and correcting the
acidity resulting from the sarcolactic and other acids in the
meat-infusion, the alkaline solution being added drop by
drop until a faint blue appears on the red paper; or the
method of using phenolphthalein can be employed, the
addition of the alkaline solution being continued until a drop
of the bouillon produces a red spot upon phenolphthalein
paper, made, as suggested by Timpe, by saturating bibulous
paper cut into strips with a solution of 5 grams of phenol-
phthalein to i liter of 50 per cent, alcohol. Acids do not
change the appearance of the paper, but small traces of alkali
turn it red.

If the bouillon is to be employed for exact work, these
crude methods should not be adopted, but chemical titration
according to the method already given should be performed,
After titration the bouillon must again be boiled for a few
minutes, in order to precipitate the acid albumins, as much
water added as has been lost by evaporation, and the fluid
filtered through a pharmaceutic filter.

The filtered fluid is dispensed in previously sterilized tubes
with cotton plugs — about 10 c.c. to each — or in flasks, and
is then sterilized by steam three successive days for fifteen

* "Trans. Assoc. Amer. Phys.," 1896.

To Prepare Bouillon from Meat Extract 229

to twenty minutes each, according to the directions already
given for intermittent sterilization, or superheated in the
autoclave.

The loss of water during boiling is an important matter
to bear in mind, as unless properly replaced it is the cause
of disproportion between the fluids and solids of the media.
The quantity must therefore be measured before filtration
and enough water added to replace what has been lost.
Measuring before filtration is comparatively easy with
bouillon, but difficult with heavy liquids, like the gelatin
and agar-agar solutions. To overcome this difficulty it is
best to make the entire preparation by weight and not by
volume. A pair of platform scales with sliding indicators
will first balance the empty kettle and then show the correct
quantity of each added ingredient. After boiling, the kettle
can be returned to the scale and the exact quantity of water
to be added determined.

II. To Prepare Bouillon from Meat Extract. — When
desirable, the bouillon may also be prepared from beef-
extract, the method being very simple: To 1000 c.c. of
clean water 10 grams of Witte's dried beef-peptone, 5
grams of sodium chlorid, and about 2 grams of beef -extract
are added. The solution is boiled until the constituents
are dissolved, titrated, and filtered when cold. If it be
filtered while hot, there is always a subsequent precipitation
of meat-salts, which clouds it.

Bouillon and other liquid culture media are best dis-
pensed and kept in small receptacles — test-tubes or flasks
— in order that a single contaminating organism, should it
enter, may not spoil the entire quantity. A very convenient,
simple apparatus used by bacteriologists for filling tubes
with liquid media is shown in figure 45. It consists of a
funnel to which a short glass pipet is attached by a bit of
rubber tubing. A pinch-cock, at 6, controls the outflow of
the liquid. The apparatus need not be sterilized before
using, as the culture medium must subsequently be sterilized
either by the intermittent method or in the autoclave after
the tubes are filled. The test-tubes and flasks into which
the culture medium is filled must, however, be previously
sterilized by dry heat, unless the subsequent sterilization
is to be performed in the autoclave, when it may be un-
necessary.

230

Cultivation of Micro-organisms

Sugar bouillon is bouillon containing in solution known
percentages of such sugars as glucose, lactose, saccharose, etc.
As Smith* has pointed out, if the quantity of sugar in the
bouillon is to be accurately known, it is necessary to first
destroy the muscle sugars in the meat-infusion by adding a

Fig. 45. — Funnel for filling tubes with culture media (Warren) : a,
Funnel containing the culture media in liquid condition ; b, pinch-cock
by which the flow of fluid into the test-tube is regulated ; c, rubbei
tubing.

culture of the colon bacillus to the meat-infusion and per-
mitting fermentation to continue overnight before finishing
the bouillon and adding the known quantity of whatever,
sugar is desired. About i per cent, of dextrose, lactose,
saccharose or galactose is all that is required. More may be
injurious. If the bouillon be made from meat extract, fer-
mentation may not be necessary.

The sugar bouillons should not be sterilized in the auto-
clave, as the high temperatures chemically alter the sugars.
* "Jour, of Exp. Med.," n, No. 5, p. 546.

Gelatin 231

GELATIN.

The culture-medium known as gelatin is bouillon to which
10 per cent, of gelatin is added. It has the decided ad-
vantage over bouillon that it is not only an excellent food
for bacteria, and, like the bouillon, transparent, but also is
solid at the room temperature. Nor is this all: it is a
transparent solid that can be made liquid or solid at will.
Leffmann and La Wall have examined commercial gelatins
and found that many of them contain sulfur dioxid in quanti-
ties as great as 835 parts per million. As the varying quan-
tity of this impurity may modify the growth of the culture,
pure gelatin should be demanded, and all gelatin should be
washed for some hours in cold running water after being
weighed and before being added to the bouillon. It is pre-
pared as follows:

To 1000 c.c. of meat-infusion or to 1000 c.c. of water
containing 2 grams of beef-extract in solution, 10 grams
of peptone, 5 grams of salt, and 100 grams of gelatin ("Gold
label" is the best commercial article) are added, and heated
until the ingredients are dissolved. The solution reacts
strongly acid and must be corrected by titration, as already
described. It must then be returned to the fire and boiled
for about an hour. As gelatin is apt to burn when boiled
over the direct flame, double boilers have been suggested,
but unless the outer kettle is filled with brine or saturated
calcium chlorid solution, they are very slow, and when
proper care is exercised there is really no great danger of
the gelatin burning. It must be stirred occasionally, and
the flame should be so distributed by wire gauze or by placing
a sheet of asbestos between it and the kettle as not to act
upon a single point. At the end of the hour the albumins
of the meat-infusion will be coagulated and the gelatin
thoroughly dissolved. Giinther has shown that the gelatin
congeals better if allowed to dissolve slowly in warm water
before boiling. As much water as has been lost by vaporiza-
tion during the process of boiling should be replaced. It
is well to cool the liquid to about 60° C., add the water mixed
with the white of an egg to clear the liquid, boil again for
half an hour, and filter.

If the filter paper be of good quality, properly folded

232 Cultivation of Micro-organisms

(pharmaceutic filter), wet with boiling water, and if the
gelatin be properly dissolved, the whole quantity should
pass through before cooling too much. Should only half
go through before cooling, the remainder must be returned
to the pot, heated to boiling once more, and then passed
through a new filter paper. As a matter of fact, gelatin
usually filters readily. A wise precaution is to catch the
first few centimeters in a test-tube and boil them, so that
if cloudiness show the presence of uncoagulated albumin,
the whole mass can be boiled again. The finished gelatin,
which is perfectly transparent and of an amber color, is at
once distributed into sterilized tubes and sterilized like the
bouillon by the intermittent method. The sterilization can
also be satisfactorily performed by the use of the autoclave
at i io°-i 15° C. for fifteen minutes, but this method is prob-
ably less well adapted to the sterilization of gelatin than of
the other media, as the high degree of heat injures its sub-
sequent solidifying power.

Sterilized gelatin or other culture medium can be kept
en masse indefinitely, but should a contaminating micro-
organism accidentally enter, the whole quantity will be
spoiled ; if, on the other hand, it be dispensed and kept in
tubes, several of them may be contaminated without serious
loss. When properly sterilized and protected, it should keep
indefinitely.

AGAR-AGAR.

Agar-agar is the commercial name of a preparation
made from a Ceylonese sea-weed. It reaches the market
in the form of long shreds of semi-transparent, isinglass-
like material, less commonly in long bars of compressed
flakes, rarely in the form of powder. It dissolves slowly
in boiling water with a resulting thick jelly when cold.
The jelly, which solidifies between 40° and 50° C., cannot
again be melted except by the elevation of its temperature
to the boiling-point. The culture medium made from agar-
agar is nearly transparent, and is almost as useful as gelatin,
as in addition to its ability to liquefy and solidify, it has
the decided advantage of remaining solid at comparatively
high temperatures so as to permit keeping the cultures grown
upon it at the incubation temperature, — i. e., 37° C., — at
which temperature gelatin is always liquid.

The preparation of agar-agar is commonly described in

Agar-agar 233

the text-books as one "requiring considerable patience and
much waste of filter paper." In reality, it is not difficult
if a good heavy filter paper be obtained and no attempt
made to filter the solution until the agar-agar is perfectly
dissolved.

It is prepared as follows: To 1000 c.c. of bouillon made as
described above, preferably of meat instead of beef -extract,
10 to 1 5 grams of agar-agar are added. The mixture is boiled
vigorously for an hour in an open pot over the direct gas
flame or in the double boiler with saturated calcium chlorid
solution in the outside pot. After being cooled to about
60° C., and after the correction of the reaction by titration,
an egg beaten up in water is added, and the liquid again
boiled until the egg-albumen is entirely coagulated.

After the second boiling and the replacement of the
volatilized water, the agar-agar is filtered through a care-
fully folded pharmaceutic filter wet with boiling water.
It may expedite matters to pour in about one-half of the
solution, keep the remainder hot, and subsequently add
it when necessary.

The formerly much employed hot-water and gas-jet
filters are unnecessary. If properly prepared, the whole
quantity will filter in from fifteen to thirty minutes.

Ravenel * prepares agar-agar by making two solutions,
one representing the meat-infusion, but twice the usual
strength, the other the agar-agar dissolved in one-half the
usual quantity of water. The agar-agar is dissolved by
exposure to superheated steam in the autoclave, after which
the two solutions are poured together and boiled until all
of the albumins are precipitated. The coagulation of the
albumins of the meat-infusion serves to clarify the agar-agar.

If agar-agar is to be made with beef -extract, the bouillon
should be made first and filtered when cold, to exclude the
uratic salts which otherwise precipitate in the agar-agar
when cold and form an unsightly cloud.

The finished agar-agar should be a colorless, nearly trans-
parent, firm jelly. It is dispensed in tubes like the gelatin
and bouillon, sterilized by steam, either by the intermittent
process or in the autoclave, and after the last sterilization,
before cooling, each tube is inclined against a slight eleva-
tion, so as to permit the jelly to solidify obliquely and afford
an extensive flat surface for the culture.

* " Journal of Applied Microscopy, "June, 1898, vol. I, No. 6, p. 106.

234 Cultivation of Micro-organisms

After the agar-agar jelly solidifies it retracts so that a little
water collects at the lower part of the tube. This should not
be removed, as it keeps the jelly moist, and also distinctly
influences the character of the growth of the bacteria.

Glycerin Agar=agar. — For an unknown reason certain
bacteria that will not grow upon agar-agar prepared as de-
scribed will do so if 3 to 7 per cent, of glycerin be added after
filtration. Among these is the tubercle bacillus, which, } not
growing at all upon plain agar-agar, will grow well when
glycerin is added — a fact discovered by Roux and Nocard.
The glycerin added to bouillon or any other medium has the
same advantageous influence.

Blood Agar=agar was recommended by R. Pfeiffer for
the cultivation of the influenza bacillus. It is ordinary
agar-agar whose surface is coated with a little blood secured
under antiseptic precautions from the finger-tip, ear-lobule,
etc., of man, or from the vein of one of the lower animals.
Some bacteriologists prepare a hemoglobin agar-agar by
spreading a little powdered hemoglobin upon the surface of
the agar-agar. This has the disadvantage that powdered
hemoglobin is not sterile, and the medium must be again
sterilized after its addition.

The blood agar-agar should be kept in the incubator a day
or two before use so as to insure perfect sterility.

BLOOD-SERUM.

The great advantage possessed by this medium is that it
is itself a constituent of the body, and hence offers oppor-
tunities for the development of the parasitic forms of bac-
teria. If the blood-serum is to be employed fresh, it must
either be heated or kept sufficiently long to lose its natural
germicidal properties. The statement that serum represents
the normal body-juice is erroneous, as it is minus the fibrin
factors and some of the salts, and contains new bodies liber-
ated from the destroyed leukocytes. Solidified blood-serum,
exposed to the heat of the sterilizing apparatus, in no sense
resembles the body- juices.

It is one of the most difficult media to prepare. The
blood must be obtained either by bleeding some good-sized
animal or from a slaughter-house in appropriate receptacles,
the best things for the purpose being i -quart fruit jars with
tightly fitting lids. The jars are sterilized by heat, closed,

Blood-serum 235

and carried to the slaughter-house, where the blood is
permitted to flow into them from the severed vessels of
the animal. It seems advisable to allow the first blood
to escape, as it is likely to become contaminated from
the hair. By waiting until a coagulum forms upon the hair
the danger of contamination is obviated. The jars, when
full, are allowed to stand undisturbed until firm coagula
form within them, after which they are carried to the
laboratory and stood upon ice for forty:eight hours, by
which time the clots will have retracted considerably, and
a moderate amount of clear serum can be removed by sterile
pipets and placed in sterile tubes. If the serum obtained
be red and clouded from the presence of corpuscles, it may
be pipetted into sterile cylinders and allowed to sediment
for twelve hours, then repipetted into tubes. It is evident
that such frequent manipulations afford numerous chances
of infection; hence the sterilization of , the serum becomes
of the greatest importance.

As the demand for serum has been considerable during the
last few years, commercial houses dealing in biologic pro-
ducts now market fresh horse serum, preserved with chloro-
form, in liter bottles. This can be employed with great
satisfaction, the chloroform being driven off during coagu-
lation and sterilization.

If it be desirable to use the serum as a liquid medium, it is
exposed to a temperature of 60° C. for one hour upon each
of five consecutive days. To coagulate the serum and make
a solid culture medium, it may be exposed twice, for an
hour each time — or three times if there be reason to think it
badly contaminated — to a temperature just short of the
boiling-point. During the process of coagulation the tubes
should be inclined, so as to offer an oblique surface for the
growth of the organisms. . The serum thus prepared should
be white, but may have a reddish-gray color if many red
corpuscles be present. It is always opaque and cannot be
melted ; once solid, it remains so.

Koch devised a special apparatus (Fig. 46) for coagulating
blood-serum. The bottom should be covered with cotton, a
single layer of tubes placed upon it, the glass lid closed and
covered with a layer of felt, and the temperature elevated
until coagulation occurs. The repeated sterilizations may
be conducted in this same apparatus, or may be done equally
well in a steam apparatus, the cover of which is not com-
pletely closed, for if the temperature of the serum be raised

236 Cultivation of Micro-organisms

too rapidly it is certain to bubble, so that the desirable
smooth surface upon which the culture is to be made is
ruined.

Like other culture media, blood-serum and its combina-
tions may be sterilized in the autoclave and much time
thus saved. The serum should, however, first be coagu-
lated, else bubbling is apt to occur and ruin its surface.
The autoclave temperature unfortunately makes the prep-
aration very firm and hard, considerable fluid being pressed
out of it.

It is said that considerable advantage is secured from the
addition of neutrose to blood-serum, which prevents its coag-

Fig. 46. — Koch's apparatus for coagulating and sterilizing blood-
serum.

ulating when heated. It can then be sterilized like bouillon
and can subsequently be solidified, when desired, by the
addition of some agar-agar.

Fresh blood-serum can be kept on hand in the laboratory,
in sterile bottles, by adding an excess of chloroform. In the
process of coagulation and sterilization the chloroform is
evaporated ; the serum is unchanged by its presence.

Loffler's Blood-serum Mixture, which seems rather
better for the cultivation of some species than the blood-
serum itself, consists of i part of a beef-infusion bouillon con-
taining i per cent, of glucose and 3 parts of liquid blood-
serum. After being well mixed the fluid is distributed in

Potatoes 237

tubes, and sterilized and coagulated like the blood-serum
itself. As prepared by Loffler it was soft, semi-gelatinous
and semi-transparent, not firm and white ; therefore should
be sterilized at low temperatures. Many organisms grow
more luxuriantly upon it than upon either plain blood-serum
or other culture media. Its especial usefulness is for the
cultivation of Bacillus diphtheriae, which grows upon it
rapidly and with a characteristic appearance.

Alkaline Blood=serum. — According to Lorrain Smith, a
very useful culture medium can be prepared as follows : To
each loo c.c. of blood-serum add 1-1.5 c-c- °f a IO per cent,
solution of sodium hydrate and shake it gently. Put suffi-
cient of the mixture into each of a series of test-tubes, and,
laying them upon their sides, sterilize like blood-serum,
taking care that their contents are not heated too quickly, as
then bubbles are apt to form. The result should be a clear,
solid medium consisting chiefly of alkali-albumins. It is
especially useful for Bacillus diphtheriae.

Deycke's Alkali=albuminate. — One thousand grams of
meat are macerated for twenty-four hours with 1200 c.c. of a
3 per cent, solution of potassium hydrate. The clear brown
fluid is filtered off and pure hydrochloric acid carefully added
while a precipitate forms. The precipitated albuminate is
collected upon a cloth filter, mixed with a small quantity of
liquid, and made distinctly alkaline. To make solutions of
definite strength it can be dried, pulverized, and redissolved.

The most useful formula used by Deycke was a 2.5 per
cent, solution of the alkali-albuminate with the addition of
i per cent, of peptone, i per cent, of NaCl, and gelatin or
agar-agar enough to make it solid.

Potatoes. — Without taking time to review the old method
of boiling potatoes, opening them with sterile knives, and
protecting them in the moist chamber, or the much more
easily conducted method of Esmarch in which the slices
of potato are sterilized in the small dishes in which they
are afterward kept and used, we will at once pass to what
seems the most simple and satisfactory method — that of
Bolton and Globig.*

With the aid of a cork -borer or Ravenel potato cutter
(Fig. 47) a little smaller in diameter than the test-tube ordi-
narily used, a number of cylinders are cut from potatoes.
* "The Medical News," vol. i,, 1887, p. 138.

238 Cultivation of Micro-organisms

Rather large potatoes should be used, the cylinders being
cut transversely, so that a number, each about an inch and a
half in length, can be cut from one potato. The skin is re-
moved from the cylinders by cutting off the ends, after which
each cylinder is cut in two by an oblique incision, so as to
leave a broad, flat surface. The half-cylinders are placed
each in a test-tube previously sterilized, and are exposed
three times, for half an hour each, to the streaming steam of
the sterilizer. This steaming cooks the potato and also
sterilizes it. Such potato cylinders are apt to deteriorate
rapidly, first by turning very dark, second by drying so as to
be useless. Abbott has shown that if the cut cylinders be
allowed to stand for twelve hours in running water before
being dispensed in the tubes, they are not so apt to turn dark.
Drying may also be prevented by adding a few drops of clean
water to each tube before sterilizing.
Some workers insert a bit of glass or a
pledget of glass wool into the bottom of
the tube so as to support the potato
and keep it up out of the water. It is
not necessary to have a special small
chamber blown in the tube to contain
this water, only a small quantity of
which need be added. The special
reservoir increases the trouble of clean-
Fig. 47,-Ravend's mg the tubes.

potato cutter. If the work to be done with potatoes

must be accurate, it may be necessary
to correct their variable reaction, especially if the acids
have not been sufficiently removed by the washing in run-
ning water already described.

To do this the cut cylinders are placed in a measured quan-
tity of distilled water and steamed for about an hour. The
reaction of the water is then determined by titration and
the desired amount of sodium hydroxid added to correct the
reaction, after which the potatoes are steamed in the cor-
rected solution for about thirty minutes before being placed
in the tubes.

A potato-juice has also been suggested, and is of some
value. It is made thus: To 300 c.c. of water 100 grams of
grated potato are added, and allowed to stand on ice over
night. Of the pulp, 300 c.c. are expressed through a cloth
and cooked for an hour on a water-bath. After cooking, the

Litmus Milk 239

liquid is filtered, titrated if desired, and receives an addition
of 4 per cent, of glycerin. Upon this medium the tubercle
bacillus grows well, especially when the reaction of the
medium is acid.

Milk. — Milk is a useful culture medium. As the cream
which rises to the top is a source of inconvenience, it is best
to secure fresh milk from which the cream has been removed
by a centrifugal machine. It is given the desired degree of
alkalinity by titration, dispensed in sterile tubes, and ster-
ilized by steam by the intermittent method or in the auto-
clave. The opaque nature of this culture medium often
permits the undetected development of contaminating or-
ganisms. A careful watch should therefore be kept lest it
spoil.

Litmus Milk. — This is milk to which just enough of a
saturated watery solution of pulverized litmus is added to
give a distinct blue color after titration. Litmus milk is
probably the best reagent for determining acid and alkali
production by bacteria.

The watery solution of litmus, being a vegetable infusion,
is likely to be spoiled by micro-organismal growth, hence
must be treated like the culture media and sterilized by
steam every time the receptacle in which it is kept is
opened.

An excellent method of preparing litmus is given by
Prescott and Winslow* and is as follows:

To one-half pound of litmus cubes add enough water to
more than cover, boil, decant off the solution. Repeat this
operation with successive small quantities of water until
3 to 4 liters of water have been used and the cubes are well
exhausted of coloring matter. Pour the decantations
together and allow them to settle over night. Siphon off
the clear solution. Concentrate to about i liter and make
the solution decidedly acid with glacial acetic acid. Boil
down to about ^ liter and make exactly neutral with caustic
soda or potash. To test for the neutral point, place one
drop of the solution in a test-tube, while one drop of ^r HC1
should turn it red, one drop of ^ NaOHO should turn it
blue. Filter the solution and sterilize at no°C. This
solution should be added to the media just before use in
the proportion of about \ c.c. to 5 c.c. of medium.

* "Elements of Water Bacteriology," John Wiley & Sons, New York,
1904, p. 126.

240 Cultivation of Micro-organisms

If litmus be added to the milk before sterilization, it is apt
to be browned or decolorized, so that it is better to sterilize
the two separately and pour them together subsequently.
It is said that lacmoid is never thus changed, and many
workers prefer it to litmus on that account.

Petruschky's Whey. — In order to differentiate between
acid and alkali producers among the bacteria, Petruschky
has recommended a neutral whey colored with litmus. It is
made as follows:

To a liter of fresh skimmed milk i liter of water is added.
The mixture is violently shaken. About 10 c.c. are taken
out as a sample to determine how much hydrochloric acid
must be added to produce coagulation of the milk, and,
having determined the least quantity required for the whole
bulk, it is added. After coagulation the whey is filtered off,
exactly neutralized, and boiled. After boiling it is found
clouded and acid in reaction. It is therefore filtered again,
and again neutralized. Litmus is finally added to the neu-
tral liquid, so that it has a violet color, changed to blue or
red by alkalies or acids.

The medium is a very useful aid in differentiating the
typhoid and colon bacilli, showing well the alkali formation
of the former and acid of the latter.

Peptone Solution, or Dunham's solution, is useful for the
detection of certain faint colors. It is a perfectly clear,
colorless solution, made as follows:

Sodium chlorid 0.5

Witte's dried peptone 1.0

Water 100.0

Boil until the ingredients dissolve ; filter, fill into tubes and sterilize.

It was for a long time used for the detection of indol.
Garini * found that many of the peptones upon the market
were impure, and on this account failed to show the indol
reaction in cultures of bacteria known to produce it. He
recommends testing the peptone to be employed by the use
of the biuret reaction. The reagent employed is Fehling's
copper solution, with which pure peptone strikes a violet
color not destroyed upon boiling, while impure peptone
gives a red or reddish- yellow precipitate. Both the peptone
and copper solutions should be in a dilute form to make

* "Centralbl. f. Bakt. u. Parasitenk.," xm, p. 790.

Peptone Solution 241

successful tests. The addition of 4 c.c. of the following
solution —

Rosolic acid 0.5

Eighty per cent, alcohol . . . , 100.0

makes the peptone solution a reagent for the detection of
acids and alkalies. The solution is of a pale rose color.
If the organisms cultivated produce acids, the color fades ;
if alkalies, it intensifies. As the color of rosolic acid is de-
stroyed by glucose, it cannot be used in culture media con-
taining it.

Theobald Smith* has called attention to the fact that
many bacteria fail to grow in Dunham's solution, and
recommends that, for the detection of indol, bouillon free of
dextrose be used instead. All bacteria grow well in it, and
the indol reaction is pronounced in sixteen-hour-old cultures.
His method of preparation is as follows: Beef -infusion, pre-
pared either by extracting in the cold or at 60° C., is inocu-
lated in the evening with a rich fluid culture of some acid-
producing bacterium (Bacillus coli) and placed in the ther-
mostat. Early next morning the infusion, covered with a
thin layer of froth, is boiled, filtered, peptone and salt added,
and the neutralization and sterilization carried on as usual.

This method is subject to error caused by the presence in
the medium of indol produced by the colon bacillus. This
can be demonstrated if the tests for indol be sensitive.
Selterf finds that the method of Smith gives inferior results
to a simple culture-medium consisting of water, 90 parts;
Witte's peptone, 10 parts; sodium phosphate, 0.5 part, and
magnesium sulphate, o.i part.

Other culture-media employed for special purposes will be
mentioned as occasion arises.

* "Journal of Kxp. Medicine," Sept. 5, 1897, vi, p. 546.
f'Centralbl. f. Bakt. u. Parasitenk.," Orig. u, p. 465-

16

CHAPTER VIII.
CULTURES, AND THEIR STUDY.

THE purposes for which culture media are prepared are
numerous. Through their aid it is possible to isolate the
micro-organisms, to keep them in healthy growth for con-
siderable lengths of time, during which their biologic peculiar-
ities can be observed and their metabolic products collected,
and to introduce them free from contamination into the
bodies of experiment animals.

The isolation of bacteria was next to impossible until the
fluid media of the early observers were replaced by the solid
culture media introduced by Koch, and exceedingly diffi-
cult until he devised the well-known "plate cultures."

A growth of artificially planted micro-organisms is called
a culture. If such a growth contains but one kind of organ-
ism, it is known as a pure culture.

It has at present become the custom to use the term "cul-
ture" rather loosely, so that it does not always signify an
artificially planted growth of micro-organisms, but may
signify a growth taking place under natural conditions ; thus,
typhoid bacilli are said to occur in " pure culture" in the
spleens of patients dead of that disease, because no other
bacteria are associated with them ; and sometimes, when the
tubercle bacilli are very numerous and unmixed with other
bacteria, in the expectorated fragments of cheesy matter
from tuberculosis pulmonalis, they are said to occur in
"pure culture."

The culture manipulations are performed either with a
sterilized platinum wire or with a capillary pipet of glass.

The platinum wire is so limber that it is scarcely to be
recommended, and a wire composed of platinum and iridium,
which is elastic in quality, is to be preferred. The wires
are about 5 cm. in length, of various thicknesses according to
the use for which they are employed, and are usually fused
into a thin glass rod about 17 cm. in length (Fig. 48). The
wires may be straight or provided with a small loop at the
end so as to conveniently take up small drops of fluid.

242

Technic of Culture Manipulation 243

Heavy wires used for securing diseased tissue from animals
may be flattened at the ends by hammering, and may thus
be fashioned into miniature knives, scrapers, harpoons, etc.,
as desired.

Ravenel has invented a convenient form for carrying in
the pocket. It consists of the platinum wire fastened in a
heavier aluminium wire which in turn fits into a piece of
glass tubing. When carried in the pocket, the position of

Fig. 48. — Platinum needles for transferring bacteria; made from
No. 27 platinum wire inserted in glass rods.

the platinum wire is reversed in the glass tubing and pro-
tected by it (Fig. 49).

Immediately before and immediately after use, the platinum
wire is to be sterilized by heating to incandescence in a flame,
in order that it convey nothing undesirable into the culture,
and in order that it scatter no micro-organisms about the
laboratory.

Capillary glass tubes are employed by the French for
many of the manipulations. They are made of J- or f-inch
glass tubing cut into 25 cm. lengths, heated at the center,

Fig. 49. — Platinum wires for bacteriologic use.

and drawn out to capillary ends about 5 cm. long. They
are sealed at one end and plugged with cotton at the other,
and a number of them, prepared at the same time, sterilized
(Fig. 50). They can be used for all the purposes for which
the platinum wire is employed, and in addition can be used
as containers for small quantities of fluids sealed in them.
When about to use such a tube, its sealed capillary end
should be broken off with forceps, and the tube sterilized
by flaming.

Technic of Culture Manipulation. — In order that ac-
curate results may accrue from the employment of culture

244 Cultures, and their Study

media, and that cultures planted in them may not be con-
taminated through improper technic, it is important habit-
ually to practise certain manipulations by which as much
latitude can be given the operator as is consistent with
thorough defense against contaminating organisms. To this
end the containers of stored culture media should be kept in
an upright position, that the cotton stoppers are not mois-
tened or soiled. If moistened with the culture media, molds
whose spores fall upon the surface of the stoppers may
gradually work their mycelial threads between the fibers
until they appear upon the inner surface and drop newly
formed spores into the contained media, or the cotton stop-
pers may be glued fast and further successful manipulations
prevented.

In handling tubes care must be taken to stand them up in
tumblers, racks, or other contrivances, and not lay them
upon the table so that the contents touch the stoppers.

o

Fig. 50. — Capillary glass tubes.

When the cotton plugs are removed in order that the
contents of the tubes or flasks may be inoculated or other-
wise manipulated the removal and replacement should be
done as quickly as convenient, and the mouth of the tube
should be flamed before removal. They must be held
between the fingers, by that part which projects above the
glass, not laid upon the table, from which dust, and inci-
dentally bacteria, may be taken up and subsequently
dropped into the medium; nor must they be touched with
the fingers at that part which enters the neck of the container
lest they take up micro-organisms from the skin. The
stoppers thus require careful consideration lest they become
the source of future contamination.

So soon as the cotton stopper is removed, the medium is
left without protection from whatever micro-organisms
happen to be in the air, so that it should be replaced as
soon as possible, and every manipulation requiring its
removal performed expeditiously. During the time the

Technic of Culture Manipulation 245

stopper is withdrawn it is wise to hold the tubes or other
containers in an oblique or horizontal position that will aid
in excluding the micro-organisms of the air. Thus, a tube
held vertically can probably more easily receive such organ-
isms than one held horizontally or reversed. Some bacteri-
ologists make inoculations with the tubes reversed in all cases
in which solid media are employed, but it is not at all neces-
sary. If the tubes are held obliquely, the danger of con-
tamination is reduced to a minimum. It is well to adopt
some method of handling the tubes that has given satis-
faction to others and is found convenient to one's self and
habitually practise it until it becomes second nature and
can be done without thought.

The usual method of making a transplantation of bacteria
from culture-tube to cul-
ture-tube is, in detail, as
follows :

In order that any bac-
teria loosely scattered
over the surface of the
cotton stopper, and upon
the glass near the mouth
of the tube, may be de-
stroyed and prevented
from entering the med-
ium as the stopper is with-
drawn, both the tube con-
taining the culture and
the fresh tube to which it
is to be transferred should
be held for a moment in
a flame and rolled from side to side so that all parts are flamed.
The cotton ignites and blazes actively, but the flame can
be extinguished by forcibly blowing upon it and any smolder-
ing remains extinguished by pinching with the fingers. The
tubes are now placed side by side between the thumb and
upward-directed palm of the left hand, the stoppers toward
the operator. The position of the tubes should be such as
to permit one to see the contained media without the fingers
being in the way. The stopper of the tube toward the
left is removed by a gentle twist and placed between the
index and middle fingers of the left hand; the stopper of
the next tube similarly removed and placed between the

Fig. 51. — Method of holding tubes
during inoculation.

246 Cultures, and their Study

middle and ring fingers of the same hand (Fig. 51). If three
or four tubes are to be held, the third stopper can be placed
between the ring and little fingers of the left hand and the
fourth retained in the right hand. The part of each stopper
that enters the tube must not be touched.

The necessary manipulation is usually made with the
platinum wire, which is sterilized by heating to incandes-
cence before using. The wire must not be used while hot,
but cools in a moment or two. The culture is touched, the
wire entering and exiting without touching the tube, and
the bacteria adhering to the wire are applied to the medium
in the other tube, the same care being exerted not to have
the platinum wire touch the glass. After the transfer is
made, the wire is made incandescent in the flame before
being returned to the table or stand made to hold it, and
the stoppers returned one after the other, each to its own
tube, that part entering the tube riot being touched. Each
stopper is given a twist as it -enters the mouth of the tube.

Modifications of tfiese directions can be made to suit the
different forms of containers used, but the essential features
must be maintained.

When any manipulation requires that a tube or flask be
permitted to remain open an unusual length of time, its
contamination from the air can be prevented for some
minutes by heating its neck quite hot. The air about it,
being heated by the hot glass, ascends, forming a current
that carries the bacteria away from, rather than into, the
receptacle.

Isolation of Bacteria. — Three principal methods are, at
present, employed for securing pure cultures of bacteria.
Before beginning a description of them it is well to observe
that the peculiarities of certain pathogenic micro-organisms
enable us to use special means for their isolation, and that
these general methods are chiefly useful for the isolation of
non-pathogenic organisms.

Plate Cultures. — All the methods depend upon the obser-
vation of Koch, that when bacteria are equally distributed
throughout some liquefied nutrient medium that is subse-
quently solidified in a thin layer, they grow in scattered
groups or families, called colonies, distinctly isolated from
one another and susceptible of transplantation.

The plate cultures, as originally made by Koch, require
considerable apparatus, and of late years have given place
to the more ready methods of Petri and von Ksmarch.

Isolation of Bacteria 247

So great is their historic interest, however, that it would be a
great omission not to describe the original method in detail.
Apparatus.— Half a dozen glass plates, measuring about
6 by 4 inches/free from bubbles and scratches and ground at
the edges, are carefully cleaned, placed in a sheet-iron box
made to receive them, and sterilized in the hot-air closet.
The box is kept tightly closed, and in it the sterilized plates
can be kept indefinitely before use.

A moist chamber, or double dish, about 10 inches in diam-
eter and 3 inches deep, the upper half being just enough
larger than the lower to allow it to close over it, is carefully
washed. A sheet of bibulous paper is placed in the bottom,
so that some moisture can be retained, and a i : 1000 bichlorid
of mercury solution poured in and brought in contact with
the sides, top, and bottom
by turning the dish in all
directions. The solution is
emptied out, and the dish,
which is kept closed, is
ready for use.

A leveling apparatus is
required (Fig. 52). It con-
sists of a wooden tripod with
adjustable screws, and a
glass dish covered by a flat
plate of glass upon which a Fig- 52. — Complete leveling ap-
low bell- jar stands. The ESS£b?K™8 Pla* <"*•»».
glass dish is filled with

broken ice and water, covered with the glass plate, and then
exactly leveled by adjusting the screws under the legs of
the tripod. When level, the cover is placed upon it, and it
is ready for use.

Method. — A sterile platinum loop is dipped into the mate-
rial to be examined, a small quantity secured, and stirred
about so as to distribute it evenly throughout the con-
tents of a tube of melted gelatin. If the material under
examination be very rich in bacteria, one loopful may con-
tain a million individuals, which, if spread out in a thin
layer, would develop so many colonies that it would be
impossible to see any one clearly; hence further dilution
becomes necessary. From the first tube, therefore, a loop-
ful of gelatin is carried to a second and stirred well, so as
to distribute the organisms evenly throughout its contents.

248 Cultures, and their Study

In this tube we may have no more than ten thousand organ-
isms, and if the same method of dilution be used again, the
third tube may have only a few hundreds, and a fourth only
a few dozen colonies.

After the tubes are thus inoculated, one of the sterile glass
plates is caught by its edges, removed from the iron box, and
placed beneath the bell-glass upon the cold plate covering
the ice- water of the leveling apparatus. The plug of cotton
closing the mouth of tube No. i is removed, and to prevent
contamination during the outflow of the gelatin the mouth of
the tube is held in the flame of a Bunsen burner for a moment
or two. The gelatin is then cautiously poured out upon the
plate, the mouth of the tube, as well as the plate, being
covered by the bell-glass to prevent contamination by germs
in the air. The apparatus being level, the gelatin spreads
out in an even, thin layer, and, the plate being cooled by the
ice beneath, it immediately solidifies, and in a few moments
can be removed to the moist chamber prepared to receive it*

As soon as plate No. i. is pre-
pared, the contents of tube No.
2 are poured upon plate No.
2, allowed to spread out and

Fig. 53.-Glass bend^ solidify, and then superimposed

on plate No. i in the moist

chamber, being separated from the plate already in the
chamber by small glass benches (Fig. 53) made for the purpose
and previously sterilized. After the contents of all the tubes
are thus distributed, the moist chamber and its contents are
stood away to permit the bacteria to grow. Where each
organism falls a colony develops, and the success of the
whole method depends upon the isolation of a colony and its
transfer to a tube of new sterile culture media, where it can
grow unmixed and undisturbed.

From the description it must be evident that only those
culture media that can be melted and solidified at will can be
used for plate cultures — viz., gelatin, agar-agar, and glycerin
agar-agar. Blood-serum and Loffler's mixture are entirely
inappropriate.

The chief drawbacks to this excellent method are the
cumbersome apparatus required and the comparative im-
possibility of making plate cultures, as is often desirable, in
the clinic, at the bedside, or elsewhere than in the labora-
tory. The method therefore soon underwent modifications,.

Petri's Dishes

249

the most important being that of Petri, who invented special
dishes to be used instead of plates.

Petri's Dishes.— These are glass dishes, about 4 inches
in diameter and J inch deep, with accurately fitting lids.
They were first recommended by Petri* and greatly sim-
plify bacteriologic technic by dispensing with the plates and
plate-boxes, the moist chambers and benches, and usually

Fig. 54. — Petri dish for making plate cultures.

with the levelling apparatus of Koch, though this is still em-
ployed in some laboratories, and must always be employed
when an even distribution of the colonies is necessary in
order that they can be accurately counted.

The method of using the Petri dishes is very simple. They
are carefully cleaned, polished, and sterilized by hot air, care
being taken that they are placed in the hot-air closet right
side up, and after sterilization are kept covered and in that

Fig- 55- — Petri dish forceps.

position. They should be sterilized immediately before
using, or if they must be kept for a time should be wrapped
in tissue paper and then sterilized. The tissue paper pro-
tects the interior from the accidental entrance of dust be-
tween dish and lid, keeps the dish closed, and need not be
removed until the last moment before using.

Time can be saved by sterilizing the dish and cover in
the direct flame, instead of in the hot-air closet, special
forceps (Fig. 55) adapted to holding them having been
devised by Rosenberger.*

* "Centralbl. f. Bakt. u. Parasitenk.," i, No. i, 1887, p. 279.

t "Phila. Med. Jour.," Oct. 20, 1900, vol. vi, No. 16, p. 760.

25°

Cultures, and their Study

The dilution of the material under examination is made
with gelatin or agar-agar tubes in the manner above de-
scribed, the plug is removed, the mouth of the tube cau-
tiously held for a moment in the flame, and the contents
poured into one of the sterile dishes, whose lid is just suffi-
ciently elevated to permit the mouth of the tube to enter.
The gelatin is spread over the bottom of the dish in an
even layer, allowed to solidify, labeled, inverted, so that the
water of condensation may not drop from the lid upon the
culture film and spoil the cultures, and stood away for the
colonies to develop.

To overcome the difficulty of excessive water of conden-
sation Hill has introduced lids made of porous clay, by which
the moisture is absorbed. These can be obtained from
most laboratory purveyors.

Among the other advantages of the Petri dish is the
convenience with which colonies can be studied with a low-
power lens. To do this with the Koch plates meant to
remove them from the sterile chamber to the stage of a
microscope and so expose them to the air, and to con-
tamination, but to examine colonies in the Petri dish, one
simply examines through the thin glass of the bottom dish
without any exposure to contaminating organisms.

Es march's Tubes. — This method, devised by Esmarch,
converts the wall of the test-tube into the plate and dispenses

Fig. 56. — Esmarch tube on block of ice (redrawn after Abbott).

with all other apparatus. The tubes, which are inoculated
and in which the dilutions are made, should contain less than
half the usual amount of gelatin or agar-agar. After inocu-

Colonies

251

lation the cotton plugs are pushed into the tubes until even
with their mouths, and then covered with a rubber cap, which
protects them from wetting. A groove is next cut in a block
of ice, and the tube, held almost horizontally, is rolled in this
until the entire surface of the glass is covered with a thin
layer of the solidified medium (Fig. 56). Thus the tube itself
becomes the plate upon which the colonies develop.

In carrying out Esmarch's method, the tube must not con-
tain too much of the culture medium, or it cannot be rolled
into an even layer; the contents should not touch the cotton
plug, lest it be glued to the glass and its subsequent useful-
ness injured, and no water must be admitted from the melted
ice.

Colonies. — The progeny of each bacterium form a mass

57- — Types of colonies: a, Cochleate (B. coli, abnormal form);
6, conglomerate (B. zopfii) ; c, ameboid (B. vulgatus) ; d, filamentous
(Frost).

Fig. 58. — Surface elevations of growths: a, Flat; b, raised; c, convex;
d, pulvinate; e, capitate; /, umbilicate; g, umbonate (Frost),

which is known as a colony. When these are separated from
one another, each is spoken of as a single colony, and dif-
ferent characteristics belonging to different micro-organisms
enable us at times to recognize by macroscopic and micro-
scopic study of the colony the particular kind of micro-
organism from which it has grown. The foregoing illustra-
tions show the various types of colonies and the legends
the terms used in describing them (Fig. 57).

Growing colonies should be observed from day to day, as
it not infrequently happens that unexpected changes, such
as pigmentation and liquefaction, develop after the colony

252

Cultures, and their Study

is several days old, and indeed sometimes not until much
later. Again, many colonies make their first appearance as
minute, sharply circumscribed points, and later spread upon
the surface of the culture medium, either in the form of a
thin, homogeneous layer or a filamentous cluster. It is
particularly important that in describing new species of
bacteria an account of the appearance of the colonies from
day to day, comparing all of their variations for at least
two weeks, should be included.

Pure Cultures. — Single colonies also subserve a second
very important purpose, that of enabling us to secure pure
cultures of bacteria from a mixture. The usual method of
doing this is by the use of plates, Petri dishes, or Esmarch
tubes according to the methods already described. For

Fig- 59- — Microscopic structure of colonies: 1, Areolate ; 2, grumosej
3, moruloid; 4, clouded; 5, gyrose; 6, marmorated ; 7, reticulate,
8, repand; 9, lobate; 10, erose; 11, auriculate; 12, lacerate; 13, fim-
bricate; 14, ciliate (Frost).

this purpose an isolated colony is selected and carefully
examined to see that it is single and not a mixture of two
closely approximated colonies of different kinds, and then
transplanted to a tube of an appropriate culture medium.
If the colonies are few and of good size, each is transplanted
to the medium directly and without instrumental assistance.
If, however, the colonies are numerous, of small size, and
close together, it may be necessary to do it under a dis-
secting microscope or even a low power of the ordinary
bacteriologic microscope. This operation of transplantation
is familiarly known as fishing.

Fishing. — It requires considerable practice and skill to
fish successfully, and the student should early begin to
practise it. The colony to be transplanted, selected because
of its isolation, its typical appearance, and convenient posi-

Fishing for Colonies 253

tion on the 'plate, is brought to the center of the field and the
plate firmly held in position with the left hand. A sterile
platinum wire is held in the right hand, the little finger, com-
fortably fixed upon the stage of the microscope, being used to
support the hand. As the operator looks into the micro-
scope the point of the platinum wire is carefully brought
into the field of vision without touching either the lens of
the microscope or any part of the plate beneath. Of course,
the wire and the colony cannot be simultaneously looked
upon. When the colony is distinctly seen the platinum
wire appears as a shadow, but the endeavor should be to
make the end of the shadow which corresponds to the point
of the wire appear exactly over the colony. It is then
gradually depressed until it touches the colony and can be
seen to break up and remove some of its substance; or
should the colony be tough and coherent, to tear it away
from the culture medium. It requires almost as much
skill to withdraw the wire from the colony without touching
anything as to successfully approach the colony in the first
place. The bacterial mass adhering to the wire is now
spread upon the surface of agar-agar or stabbed in gelatin
or stirred in fluid medium, as the case may be. The
higher the magnification under which this operation is
done, the more difficult it is. Therefore only low-power
lenses should be employed. The transplantation should be
made immediately after the wire has touched the colony,
the wire not being permitted in the meantime to come into
contact with any other object. Immediately after receiving
the inoculation, the tube containing the culture medium,
held in the left hand, should be passed with a twisting
motion through the flame of a Bunsen burner, so that its
mouth and the cotton plug shall be heated. The cotton
plug of course takes fire, but by vigorously blowing once or
twice the fire can be extinguished. The cotton is then re-
moved with a twisting motion of the plug and placed
between two fingers of the left hand in such a manner that
only the extra-tubular portion of the cotton is touched by
the fingers. During the time that the plug is being with-
drawn the tube should be held as nearly as possible in a
horizontal position, in order that nothing may fall into it
from the atmosphere. The inoculation having been made,
the plug is returned and the platinum wire heated to redness
in order that any remaining bacteria may be killed by the
heat.

254

Cultures, and their Study

B

The Gelatin Puncture Culture. — To make satisfactory
puncture cultures, the gelatin must be firm and not old and
dry. Should the gelatin be soft and semi-fluid at the time
the puncture is made, the bacteria diffuse themselves through-
out and the typical appearance of the growth may be
masked. On the other hand, if the gelatin is old, dry, or
retracted, it is very apt to crack after the culture has been

Fig. 60. — Types of growth in stab cultures. A, Non-liquefying:
1, Filiform (B. coli) ; 2, beaded (Str. pyogenes) ; 3, echinate (Bact.
acidi-lactici) ; 4, villous (Bact. murisepticum) ; 5, arborescent (B.
mycoides). B, Liquefying: 6, Crateriform (B. vulgare, 24 hours);
7, napiform (B. subtilis, 48 hours) ; 8, infundibuliform (B. prodigiosus) ;
9, saccate, (Msp. Finkleri) ; 10, stratiform (Ps. fluorescens) (Frost).

made and thus entirely destroy the characteristics of the
growth. The wire used in the operation should be perfectly
straight, and the puncture should be made from the center
of the surface directly down to the bottom of the tube and
then withdrawn, so that a simple puncture is made. The
resulting appearances as the growth progresses are subject
to striking variations according to the liquefying or non-

The Agar-agar Culture

255

liquefying tendency of the micro-organisms. Various types
of gelatin cultures are shown in the accompanying diagrams,
and it is rather important that the student should familiarize
himself with the terms by which these different growths are
described, in order that uniformity of description may be
maintained. Gelatin cultures may not be kept in the incu-
bating oven, as the medium liquefies at such temperatures.
On the other hand, it must not be kept where the tempera-
ture is too low, else the bacterial growth may be retarded.
The temperature of a comfortably heated room, not subject
to excessive variations, such as are caused by steam heat
and the burning of gas, etc., is about the most appropriate.
Like the colonies, the cultures must be carefully examined
from day to day, as it not infrequently happens that a growth

Fig. 61.— Types of streak cultures : 1, Filiform (B. coli) ; 2, echinulate
(Bact. acidi-lactici) ; 3, beaded (Str. pyogenes) ; 4, effuse (B. vulgaris) ;
5, arborescent (B. mycoides) (Frost).

which shows no signs of liquefaction to-day may begin to
liquefy to-morrow or a week hence, or even as late as two
weeks hence.

The Agar=agar Culture. — Different operations vary in
the exact method adopted in transplanting to agar-agar. In
most cases, a simple stroke is made from the bottom of the
tube in which the agar-agar has been obliquely solidified, and
where it is fresh and moist, to the upper part, where it is
thin and dry. In addition to this, it is advisable to make
a puncture from the center of the oblique surface to the bot-
tom of the tube. This enables us to tell whether the bacteria
can grow as readily below the surface as above. Some
workers always make a zigzag stroke upon the surface of the
agar-agar. This does not seem to have any particular

256 Cultures, and their Study

advantage except in cases where it is desired to scatter the
transplanted organisms as much as possible, in order that
the bacteria will grow, or in order that a large bacterial mass
may be secured.

Cultures upon Potato. — These are made by simply
stroking the surface of the culture medium, the opacity of
the potato making it impracticable to puncture it.

Cultures in Fluid Media. — Here, as has already been
stated, transplantation consists in simply stirring in the
bacteria so as to distribute them fairly well throughout the
medium.

Adhesion Preparations. — Sometimes it is desirable to
preserve an entire colony as a permanent microscopic speci-
men. To do this a perfectly clean cover-glass, not too large
in size, is momentarily warmed, then carefully laid upon the
surface of the gelatin or agar-agar containing the colonies.
Sufficient pressure is applied to the surface of the glass to
exclude bubbles, but not to destroy the integrity of the
colony. The cover is gently raised by one edge, and if suc-
cessful the whole colony or a number of colonies, as the case
may be, will be found adhering to it. It is treated exactly
as any other cover-glass preparation — dried, fixed, stained,
mounted, and kept as a permanent specimen. It is called
an adhesion preparation — " Klatschprdparat."

Special Methods of Securing Pure Cultures.— Pure
cultures from single colonies may also be secured by a very
simple manipulation suggested by Banti.* The inoculation
is made into the water of condensation at the bottom of an
agar-agar tube, without touching the surface. The tube is
then inclined so that the water flows over the agar, after
which it is stood away in the vertical position. Colonies will
grow where bacteria have been floated upon the agar-agar,
and may be picked up later in the same manner as from a
plate.

When the bacterium to be isolated (gonococcus, etc.)
will not grow upon the media capable of alternate solidifica-
tion and liquefaction, the blood-serum, potato, or other
medium may be repeatedly stroked with the platinum wire
dipped in the material to be investigated. Where the first
strokes were made, confluent impure cultures occur; but as
the wire became freer of organisms by repeated contact with
the medium, the colonies become scattered and can be
studied and transplanted.

* " Centralbl. f. Bakt. u. Parasitenk.," 1895, xvii, No. 16.

Special Methods of Securing Pure Cultures 257

In some cases pure cultures may be most satisfactorily
secured by animal inoculation. For example, when the
tubercle bacillus is to be isolated from milk or urine which
contains bacteria that would outgrow the slow-developing
tubercle bacillus, it is necessary to inject the fluid into the
abdominal cavity of a guinea-pig, await the development of
tuberculosis in the animal, and then seek to secure pure cul-
tures of the bacillus from the unmixed infectious material in
the softened lymphatic glands.

In many cases, when it is desired to isolate Micrococcus
tetragenus, the pneumococcus, and others, it is easier to
inoculate the animal most susceptible to the infection and
recover it from the blood or organs, than to plate it out and
search for the colony among many others similar to it.

The growth upon agar-agar is in many ways less charac-
teristic than in gelatin, but as the medium does not liquefy
except at a high temperature (100° C.), it has that advan-
tage. The colorless or almost colorless condition of the
preparation also aids in the detection of chromogenesis.

Sometimes the growth is colored ; at times the production
of soluble pigment colors the agar-agar as well as the growth ;
sometimes the bacterial mass has one color and the agar-agar
another. The growth may be filamentous, or simply a
smooth, shining band. Occasionally the bacterium does not
grow upon agar-agar unless glycerin be added (tubercle
bacillus); sometimes it will not grow even then (gono-
coccus).

Still less characteristic are the growths upon potato.
Most bacteria produce smooth, shining, irregularly extending
growths, that may show characteristic colors.

In milk and litmus milk one should observe change in
color from the occurrence of acid or alkali production,
coagulation, gelatinization, and digestion of the coagulum.

Blood-serum is liquefied by some bacteria, but the major-
ity of organisms have no characteristic reaction upon it. A
few, as the bacillus of diphtheria, are, however, characterized
by rapid development at given temperatures.

While most of the saprophytic bacteria grow well at the
temperature of *a well-wTarmed room, the important patho-
genic forms must be kept at the temperature of the body
either to permit growth or to secure typical development.
To do this satisfactorily an incubating oven or thermostat be-
comes a necessity. Various forms, of wood and metal, are in
the market, one being shown in the illustration (Fig. 62).
17

258

Cultures, and their Study

The growth in gelatin is generally so far removed from
the walls of the tube (a central puncture nearly always being

Fig. 62. — Modern incubating oven.

made in the culture-medium, in order that the growth be
symmetric) that it is impossible to make a microscopic ex-

Microscopic Study of Cultures 259

amination of it with any power beyond that given by a
hand -lens.

MICROSCOPIC STUDY OF CULTURES.

Some attention has been given to the preparation of micro-
tome sections of the gelatin growth, which can be done if the
glass be warmed just sufficiently to permit the gelatin con-
taining the growth to be removed and placed in Miiller's
fluid (bichromate of potassium 2-2.5, sulphate of sodium i,
water 100), where it is hardened. When quite firm it is
washed in water, passed through alcohols ascending in
strength from 50 to 100 per cent., embedded in celloidin, cut
wet, and stained like a section of tissue.

A ready method of doing this has been suggested by
Winkler,* who bores a hole in a block of paraffin with the
smallest size cork-borer, soaks the block in bichlorid solution
for an hour, pours liquid gelatin into the cavity, allows it to
solidify, inoculates it by the customary puncture of the
platinum wire, allows it to develop sufficiently, and when
ready cuts the sections under alcohol, subsequently staining
them with much diluted carbol-fuchsin.

Neat museum specimens of plate and puncture cultures
in gelatin can be made by simultaneously killing the micro-
organisms and permanently fixing the gelatin with formal-
dehyd, which can either be sprayed upon the gelatin or ap-
plied in dilute solution. As gelatin fixed in formaldehyd
cannot subsequently be liquefied, such preparations will last
indefinitely.

Standardizing Freshly Isolated Cultures. — This is a
matter of some importance, as in bringing bacteria into
the new environment of artificial cultivation their bio-
logic peculiarities are temporarily altered, and it takes
some time for them to recover themselves. While the
appearances of the freshly isolated organism should be
carefully noted, too much stress should not be laid upon
them, and before beginning the systematic study of any
new organism it should be made to grow for several suc-
cessive generations upon two or three of the most impor-
tant culture media. Its saprophytic existence being thus
established, the characteristics manifested become the per-
manent peculiarities of the species.

* " Fortschritte der Medicin," Bd. xi, 1893, No. 22.

CHAPTER IX.

THE CULTIVATION OF ANAEROBIC ORGANISMS.

THE presence of uncombined oxygen in ordinary cul-
tures inhibits the development of anaerobic bacteria.
When such are to be cultivated, it therefore becomes
necessary to utilize special apparatus or adopt physical
or chemic methods for the exclusion of the air. Many
methods have been suggested for the purpose, an excel-
lent review of which has recently been published by Hun-
ziker,* who divides them as follows, according to the
principle by which the anaerobiosis is brought about :

1. By the formation of a vacuum.

2. By the displacement of the air by inert gases.

3. By the absorption of the oxygen.

4. By the reduction of the oxygen.

5. By the exclusion of atmospheric air by means of

various physical principles and mechanical devices.

6. By the combined application of any two or more of

the above principles.

This classification makes such an excellent foundation for
the description of the methods that it has been unhesitatingly
adopted.

1. Withdrawal of the Air and the Formation of a
Vacuum. — This method was first suggested by Pasteur and
was later modified by Roux, Gruber, Zupinski, Novy, and
others. It is now very rarely employed. The appropriate
container, whether a tube, flask, or some special device such
as the Novy jar (Fig. 63), receives the culture, and then has
the air removed by a vacuum pump, the tube either being
sealed in a flame or closed by a stop-cock to prevent the re-
entrance of the air

2. Displacement of the Air by Inert Gases. — This
method is decidedly preferable to the preceding, as it

* "Journal of Applied Microscopy and Laboratory Methods," March,
April, and May, 1902; vol. v, Nos. 3, 4, and 5.

260

Displacement of the Air by Inert Gases 261

leaves no vacuum. It is easier to displace the oxygen than
to withdraw it, and any apparatus permitting a combina-
tion of both features, as that designed by Ravenel,* from
which the air can be sucked by a pump, to be later replaced
by hydrogen, can be viewed with favor.

The most simple apparatus of the kind was suggested by
Frankel (Fig. 64), who inoculated a culture-tube of melted
gelatin or agar-agar, solidified it upon the wall of the tube, as
suggested by Esmarch, substituted for the cotton stopper a
sterile rubber cork containing a long entrance and short
exit tube of glass, passed hydrogen through the tube until
the oxygen had been entirely removed, then sealed the
ends in a flame. In this tube the growth of superficial and
deep colonies can be observed. Hansen and Liborius con-

Fig. 63. — Novy's jars for anaerobic cultures.

structed special tubes (Fig. 65) by fusing a small glass tube
into the wall of a culture-tube, and narrowing the upper part
of the tube in a flame. After inoculation, hydrogen is passed
into the small tube and permitted to escape through the
mouth of the large tube until the air is entirely replaced,
after which both tubes are sealed in a flame.

Instead of having a special apparatus for each culture, it
is far better to adapt the principle to some larger piece of
apparatus that can contain a number of tubes or Petri dishes
at a time. For this purpose the jar invented by Novy or the
apparatus of Botkin can be used.

The Novy jar receives as many inoculated tubes as it will

*" Bacteria of the Soil," "Memoirs of the National Academy of
Sciences," First Memoir, 1896.

262 The Cultivation of Anaerobic Organisms

contain and has its stopper so replaced that the openings in
the neck and stopper correspond. Hydrogen gas is passed
through until the air is displaced. This usually takes sev-
eral hours, as the cotton stoppers retain the air in the test-
tubes and prevent rapid diffusion. When the air is all dis-
placed, the stopper is turned so that the tubes are closed.
If it be desired to expedite matters a pump can be used to
withdraw the air, after which the hydrogen is permitted to
enter.

Botkin's apparatus is intended for cultures in Petri dishes.

Cotton jtLvf

Fig. 64.— Frankel's method of
making anaerobic cultures.

Fig. 65. — Liborius' tube for
anaerobic cultures.

It consists of three parts — a deep dish of glass (6), a stand
to support the Petri dishes to be exposed (c), and a bell-
glass (a) to cover the stand and fit inside of the dish. It
can easily be understood by reference to figure 66. The
prepared dishes are stood uncovered in the rack, which is
then placed in the dish forming the bottom of the appara-
tus, and into which liquid paraffin is poured to a depth of
about two inches. The bell-glass cover is now stood in
place and hydrogen gas is conducted through previously
arranged rubber tubes ( d, e) . As soon as the air is displaced

The Absorption of the Atmospheric Oxygen 263

through tube d, both tubes are withdrawn. It is well to
place one Petri dish containing alkaline pyrogallic acid in
the rack to absorb any oxygen not successfully displaced.

3. The Absorption of the Atmospheric Oxygen. This

method was first suggested by Buchner, whose idea was to
absorb the atmospheric oxygen by alkaline pyrogallic acid
and permit the bacteria to develop in the indifferent nitro-
gen. Various methods have been suggested for achieving
this end, Buchner's own method consisting in the use of two
tubes, a small one to contain the culture (Fig. 68) and a larger
one to contain the absorbing fluid. A fresh solution of pyro-
gallic acid and sodium hy-
droxid were poured into
the large tube, the smaller
tube placed within it, upon
some appropriate sup-
port, and the whole tightly
corked.

Nichols and Schmitter,*
at the suggestion of Carroll,
have modified the method
by connecting the tube con-
taining the inoculated cul-
ture medium with a U-
shaped tube, to the other
end of which is attached
a tube to contain the pyro-
gallic acid solution. The
apparatus will at once be
understood by a glance at ]
the cut (Fig. 67). The mode
of employing it is as follows : ' ' After inoculating the culture-
tube the plug is pushed in a little below the lips of the tube ;
the ends of the U tube and the test-tubes are coated exter-
nally with vaselin, the rubber tubes are adjusted on the
U tube and a connection made with the culture-tube so that
the glass ends meet. One or two grams of pyrogallic acid
are put in the empty test-tube and packed down with a
little filter-paper over it; ten or twenty cubic centimeters,
respectively, of a 10 per cent, solution of sodium hydroxide
are then poured into the tube and the second connection
made before the acid and alkali react to any extent."
* "Jour, of Medical Research," July, 1906, p. 113.

66. — Bodkin's apparatus for
making anaerobic cultures.

264 The Cultivation of Anaerobic Organisms

Wright has given the most simple modification by sug-
gesting that the cotton stopper of the ordinary culture-tube

have its projecting part cut off
and the plug itself pushed down
the tube for a short distance.
Some alkaline pyrogallic acid so-
lution is poured upon the cotton,
to saturate it, and the tube
tightly corked.

Zinsser* has also adopted the
method so as to be very satis-
factory for use with Petri dishes.
The dishes selected should be
rather deeper than ordinary.
They are sterilized and inocu-
lated in the ordinary manner and
then inverted. The dish is cau-
tiously raised, and some pyro-
gallic acid carefully poured into
the lid and the dish gently
dropped into place again. The
alkaline solution is then poured
into the crevice between the
edges of the dish and the lid,
and then the remainder of the
space filled with melted albolene.
When these dishes are carefully
stood away, the alkaline pyro-
gallic acid absorbs all of the con-
tained oxygen and the anaerobic
cultures develop quite well. The
growing colonies can be exam-
ined as often as may be necessary

through the bottom of the dishes, which must, of course,
always be kept in the inverted position.

4. Reduction of Oxygen. — Pasteur and, later, Roux
have recommended the cultivation of anaerobic bacteria in
association with aerobic bacteria by which the oxygen was
to be absorbed. This method is too crude to be employed
at the present time, as it destroys the essential character-
istics of the cultures by mixing the products of the bacteria.
Chemic reduction of the oxygen has been attempted by
the addition of 2 per cent, of glucose, as suggested by Libo-
* "Journal of Experimental Medicine," 1906, vin, 542.

Fig. 67. — Spirillum ru-
brum. Glucose agar slant
culture of five days. Abun-
dant production of pigment
on the surface. (The U tube
was soiled by the reducing
fluid during handling by the
photographer.) (Nichols and
Schmitter.)

. Exclusion of Atmospheric Oxygen 265

rius, 0.3-0.5 per cent, of sodium formate, as suggested by
Kitasato and Weil, o.i per cent, of sodium sulphate, sug-
gested by the same authors, and various other chemicals.
None of these additions has been sufficiently successful to
merit continued favor, and at the present time this method
is not employed.

5. Exclusion of Atmospheric Oxygen by Means of
Various Physical Principles and Mechanical Devices.—
This has appealed to the ingenuity of many experimenters,
and many means of accomplishing the atmospheric exclusion
have been tried with success.

The most simple plan is that of Hesse, who made a deep
puncture in recently boiled and rapidly cooled gelatin or
agar-agar, then covered the surface of the medium with
sterile oil (Fig. 69). The so-called "shake culture" is an-

Fig. 68. — Buchner's method of
making anaerobic cultures.

Fig. 69. — Hesse's method of mak-
ing anaerobic cultures.

other very simple method, suggested by Liborius and Hesse.
The medium to be inoculated, contained in a well-filled tube
or flask, is boiled to displace the contained air, cooled so as
no longer to endanger the introduced bacteria, then inocu-
lated, the inoculated bacteria being distributed by gently
shaking. On cooling, the medium "sets," the organisms
below the surface remaining under anaerobic conditions.

266 The Cultivation of Anaerobic Organisms

Kitasato first used paraffin as a covering for the inoculated
medium, his recommendation having recently been revived
and made successful use of by Park for the cultivation of the
tetanus bacillus. The paraffin floats upon the surface of the
medium, melts during sterilization, but does not mix with
it, and "sets" when cool. The inoculation is to be made
while the culture medium is warm, after boiling and before
the paraffin sets.

Koch studied the colonies of anaerobic organisms by culti-
vating them upon a film of gelatin
covered by a thin sheet of steril-
ized mica, by which the air was
excluded.

Salomonsen has made use of a
pipet for making anaerobic cul-
tures. It is made of a glass tube
a . few millimeters in diameter,
drawn out to a point at each
end. The inoculated gelatin or
agar-agar is drawn in while lique-
fied and the ends sealed. The
tube, of course, contains no air,
and perfect anaerobiosis results.

Theobald Smith has found the
fermentation-tube and various
modifications of it excellently well
adapted to the growth of anae-
robes, which, of course, grow only
in the closed limb.

Hens' eggs have been used for
anaerobic cultures, and in them
the tetanus bacillus grows remark-
ably well. Conditions of anaero-
biosis are, however, not perfect,
as can be shown by the behavior
of the egg itself. If oxygen be
completely shut out by oiling or
varnishing the shell, a fertile egg
will not develop.

A quite satisfactory and simple
device for routine work with

anaerobic organisms has been invented by Wright * (Figs.

70 and 71). The essential feature consists of a pipet, D,

* "Jour. Boston Soc. of Med. Sci.," Jan., 1900.

Figs. 70, 71.— Wright's
method of making anaero-
bic cultures in fluid media
(Mallory and Wright).

Exclusion of Atmospheric Oxygen 267

with a rubber tube, E, at the end, and one interruption
connected by a rubber tube, C. The device will be made
clear at once by a glance at the accompanying illustration.
The method of employment is very simple. An ordinary
tube of bouillon or other fluid culture media receives the
pipet, the whole being sterilized, the cotton plug in place.
The bouillon being inoculated with the culture or secretion
to be studied is drawn up in the bulb of the pipet, A, by
suction, until it passes the rubber interruption, C. By
forcing the upper end of the pipet downward in the test-tube,
a kink is given each rubber tube and the fluid contained in
the bulbous part of the pipet becomes hermetically sealed.

In all cases where the presence of suspected micro-
organisms is to be demonstrated, it is necessary to make
both aerobic and anaerobic cultures. For routine work of
this kind, this method of Wright is probably the most con-
venient yet suggested.

CHAPTER X.
EXPERIMENTATION UPON ANIMALS*

THE principal objects of medical bacteriology are to dis-
cover the cause, explain the symptoms, and bring about the
cure and future prevention of disease. We cannot hope
to achieve these objects without experimentation upon
animals, in whose bodies the effects of bacteria and their
products can be studied.

No one should more heartily condemn wanton cruelty to
animals than the physician. Indeed, it is hard to imagine
men, so much of whose life is spent in relieving pain, and
who know so much about pain, being guilty of the butchery
and torture accredited to them by a few of the laity, whose
eyes, but not whose brains, have looked over the pages
of physiologic text-books, and whose "philanthropy has
thereby been transformed to zoolatry."

It is entirely through experimentation upon animals that
we have attained our knowledge of physiology, most of
our important knowledge of therapeutics, and most of our
knowledge of the infectious diseases. Without its aid we
would still be without one of the greatest achievements of
medicine, the serum therapy of diphtheria.

Experiments upon animals, therefore, must be made, and,
as the lower animals differ in their susceptibility to diseases,
large numbers and different kinds of animals must be em-
ployed.

The bacteriologic methods are fortunately not cruel, the
principal modes of introducing bacteria into the body being
by subcutaneous, intraperitoneal, and intravenous injec-
tion.

Any hypodermic syringe that can conveniently be cleaned
and disinfected may be employed for the purpose. Forms
expressly designed for bacteriologic work and most fre-

268

Animal Inoculations

269

quently employed are shown in figure 72. Those of Meyer
and Roux resemble ordinary hypodermic syringes; that of

Fig. 72. — i. Roux's bacteriologic syringe; 2, Koch's syringe; 3, Meyer's
bacteriologic syringe.

ig- 73- — Altmann syringes for bacteriologic and hematologic work.

Koch is supposed to possess the decided advantage of not
having a piston to come into contact with the fluid to

270 Experimentation upon Animals

be injected. This is, however, really disadvantageous,
inasmuch as the cushion of compressed air that drives out
the contents is elastic, and unless carefully watched will
follow the injection into the body of the animal. In making
subcutaneous injections there is no disadvantage or danger
from the entrance of air, but in intravenous injections it is
extremely dangerous.

Syringes with metal or glass pistons are excellent, though
not very durable. All syringes should be disinfected with 5
per cent, carbolic acid solutions before and after using, the
carbolic acid being allowed to act for some time and then
washed out with sterile water. Syringes should not often be

Fig. 74. — Method of making an intravenous injection into a rabbit.
Observe that the needle enters the posterior vein from the hairy
surface.

boiled, as it ruins the packings, whether of asbestos, leather,
or rubber.

The intravenous injections differ only in that the needle
of the syringe is introduced into a vein. This is easy to
achieve in a large animal, like a horse, but is very difficult
in a small animal, and well-nigh impossible in anything
smaller than a rabbit. Such injections, when given to rab-
bits, are usually made into the ear-veins, which are most

Animal Inoculations 271

conspicuous and accessible (Fig. 74). A peculiar and im-
portant fact to remember is that the less conspicuous poste-
rior -vein of the ear is much better adapted to the purpose
than the anterior. The introduction of the needle should be
made from the hairy external surface of the ear when the
vein is immediately beneath the skin.

If the ear be manipulated for a moment or two before the
injection, vasomotor dilatation occurs and the blood-vessels
become larger and more conspicuous. The vein should be
compressed at the root of the ear until the needle is intro-
duced, and the injection made as near the root as possible.
The fluid should be slowly injected.

Bacteria can be introduced into the lymphatics only by
injecting liquid cultures of them into some organ with com-
paratively few blood-vessels and large numbers of lym-
phatics. The testicle is best adapted to this purpose, the
needle being introduced deeply into the organ.

Sometimes subcutaneous inoculations are made by intro-
ducing the platinum wire through a small opening made in
the skin by a snip of the scissors. By this means solid
cultures from agar-agar, etc., can be introduced.

Intra-abdominal and intrapleural injections are some-
times made, and in cases where it becomes necessary to
determine the presence or absence of the bacilli of tuber-
culosis or glanders in fragments of tissue it may be neces-
sary to introduce small pieces of the suspected tissue
under the skin. To do this the hair is closely cut over
the point of election, which is generally on the abdo-
men near the groin, the skin picked up with forceps, a
snip made through it, and the points of the scissors intro-
duced for an inch or so and then separated. By this man-
oeuver a subcutaneous pocket is formed, into which the
tissue is easily forced. The opening should not be large
enough to require subsequent stitching.

When tissue fragments or collodion capsules are to be in-
troduced into the abdominal cavity, the animal should be
anesthetized and a formal laparotomy done, the wound being
carefully stitched together.

When, in studying Pfeiffer's phenomenon and similar con-
ditions, it is desirable occasionally to withdraw drops of
fluid from the abdominal cavity, a small opening can be
burned through with a blunt needle. This does not heal

272 Experimentation upon Animals

Fig. 75- — Latapie's animal holder for rabbits, guinea-pigs, and other
small animals. This form of holder is in general use at the Institute
Pasteur in Paris.

Fig. 76. — Guinea-pig confined in the holder.

Fig. 77. — Mouse-holder.

Securing Blood from Animals 273

readily, and through it, from time to time, a capillary pipet
can be introduced and the fluid withdrawn.

Small animals, such as rabbits and guinea-pigs, can be
held in the hand, as a rule. Guinea-pig and rabbit-holders
of various forms can be obtained from dealers in laboratory
supplies. The best of these is undoubtedly that of Latapie,
shown in the accompanying illustration (Fig. 75). Dogs,
cats, sheep, and goats can be tied and held in troughs. A
convenient form of mouse-holder, invented by Kitasato, is
shown in figure 77.

In all these experiments one must remember that the
amount of material introduced into the animal must be in
proportion to its size, and that injection experiments upon
mice are usually so crude and destructive as to warrant the
comparison drawn by Frankel, that the injection of a few
minims of liquid into the pleural cavity of a mouse is " much
the same as if one would inject through a fire-hose three or
four quarts of some liquid into the respiratory organs of a
man."

Method of Securing Blood from Animals. — For many
experimental purposes it becomes necessary to secure blood
in larger or smaller quantities from animals. For horses,
cattle, calves, goats, sheep, large dogs, etc., this is a simple
matter, all that is necessary being to restrain the animal,
make a minute incision in the skin over the jugular vein,
which is easily found by compressing it at the root of the
neck and noting where the vessel expands, and introducing
a canula when the vein is well distended. The trocar being
withdrawn, the blood at once flows. A sterile tube is
slipped over the cannula and the blood conducted into a
sterile bottle or flask.

For rabbits and guinea-pigs the technic is rather more
difficult because of the smaller size of the vessels. Drops
and small quantities of blood may be secured by opening
one of the ear veins, but when any quantity of blood is
required, the neatest operation is done by tapping the
common carotid artery by the method employed at the
Pasteur Institute at Paris.

The animal is restrained in a Latapie holder, with the
neck extended. Anesthesia can be used, but must be em-
ployed with great care. The hair on the front of the neck
is clipped and the neck shaved, or, as is easier, the hair is
18

274

Experimentation upon Animals

pulled out, -leaving a clean surface an inch square. The
skin is then washed with a disinfecting solution, an incision
one and a half inches long made through the skin and
superficial fascia in the middle line of the neck, the tissues
carefully separated, the deep fascia cautiously opened, the
tissues separated with the point of the forceps and a
grooved director, the sheath of the ves-
sels opened, and the artery completely
separated from its surrounding tissues
for a distance of at least an inch. A
ligature is now tightly tied about the
artery at the distal end of exposure, and
a ligature placed in position and loosely
looped ready to tie about the proximal
end. A tube with a sharp lateral tubu-
lature, as is shown in the illustration
(Fig. 78), is now made ready by break-
ing off the closed tip, the moistened
forefinger of the operator is placed be-
neath the artery, and the sharp tube
inserted (point toward the heart) into
the artery, through whose walls it cuts
its way easily. The moment the vessel
is entered the blood-pressure drives the
blood into the tube so that 20 c.c. may
be collected in about as many seconds.
An assistant now ties the artery at its
proximal end, the tube is withdrawn,

.- 7f-— Tube for holding it so that the blood does not
taking blood from the , .

carotid artery of a escape, and the end sealed in a name,
rabbit or guinea-pig. The ends of the ligatures are now cut
short and the external wound stitched.
The wound usually heals at once, and if subsequent study of
the blood is required, the other carotid and the femorals
can be similarly employed for obtaining it.

Small quantities of blood (drops) can be secured from
mice and rats by cutting off the tip of the tail, but to
secure a large quantity is difficult. One method that has
been recommended is to tie the animal to a tray or board,
on its back, anesthetize it, and, just before it dies, quickly
open the thoracic cavity, and cut through the heart with
scissors. The animal at once dies, the blood pouring out

Post-mortems upon Animals 275

into the pleural cavities. After coagulation the serum can
be secured by carefully pipetting it from the cavities.

- 79- — Showing the method of taking blood from the carotid artery
of a rabbit.

Post=mortems. — Observation of experiment animals by
no means ceases with their death. Indeed, he cannot be a
bacteriologist who is not already a good pathologist and ex-
pert in the recognition of diseased organs.

When an autopsy is to be made upon a small animal, it is
best to wash it for a few moments in a disinfecting solution,
to kill the germs present upon the hair and skin, as well as
to moisten the hair, which can then be much more easily
kept out of the incision.

Small animals can be tacked to a board or tied, by cords
fastened to the legs, to hooks soldered to the corners of an
easily disinfected tray. The dissection should be made with
sterile instruments. When a culture is to be made from the
interior of an organ, its surface should first be seared with a
hot iron, a puncture made into it with a sterile knife, and
the culture made by introducing a platinum wire.

276 Experimentation upon Animals

If the bacteriologic examination cannot be made at once,
the organs to be studied should be removed with aseptic pre-
cautions, wrapped in a sterile towel or a towel wet with a
disinfecting solution, and carried to the laboratory, where
the surface is seared and the necessary incisions made with
sterile instruments.

Fragments intended for subsequent microscopic examina-
tion should be cut into small cubes (of i c.c.) and fixed in
Zenker's fluid or absolute alcohol. (See page 179.)

Collodion capsules are quite frequently employed for the
purpose of cultivating bacteria in a confined position in the
body of an animal, where they can freely receive and utilize
the body- juices without being subjected to the action of the
phagocytes. In such capsules the bacteria usually grow
plentifully, and not rarely have their virulence increased.

The capsules can be made of any size, though they are
probably most easily handled when of about 5-10 c.c. capac-
ity. The size is always an objection, because of the dis-
turbance occasioned when they are introduced into the
abdominal cavity.

The capsules are made by carefully coating the outside of
the lower part of a test-tube with collodion until a suffi-
ciently thick, homogeneous layer is formed. During the
coating process the tube must be twirled alternately within
and without the collodion, so that it is equally distributed
upon its surface. When the desired thickness is attained,
and the collodion is sufficiently firm, the tube is plunged
under water and the hardening process checked.

A cut is next made around the upper edge of the collodion:
film, and it is removed by carefully turning it inside out. In
this manner an exact mold of the tube is formed. If a small
opening be made at the end of the tube over which the sac is
molded, and the tube filled with water after being properly
coated with collodion, a small amount of pressure, applied
by blowing gently into the tube, will force the water between
the collodion and glass and so detach it without inversion.
A test-tube of the same size is next constricted to a degree
that will not interfere with the future introduction of culture
media in a fine pipet or inoculation with a platinum loop,
and that will permit of ready sealing in a flame when neces-
sary ; the rounded end is cut off, and the edges are smoothed
in a flame. The upper open end of the collodion bag is care-
fully fitted over the end of the tube, shrunk on by a gentle

Collodion Capsules

277

heating, and cemented fast with a little fresh collodion
applied to the line of union. Novy recommends that
a thread of silk be wound around the
point of union, to hold the collodion in
place and to aid in handling the fin-
ished sac. It now appears as in figure
80, b. The sac is next filled with dis-
tilled water up to the thread, the tube
is plugged with cotton, and the whole
placed in a larger test-tube containing
distilled water, the cotton plug being
packed tightly around the smaller
tube, so that the collodion sac does
not reach the bottom of the large
tube, but hangs suspended in the
water it contains. The whole is now
carefully sterilized by steam.

When ready for use, a tube of
bouillon is inoculated with the culture
intended to be placed in the animal,
the water in the capsule is pipetted

out and replaced by the inoculated bouillon carefully intro-
duced with a pipet, the constricted portion is sealed in a
flame, and the capsule picked up with forceps and' intro-
duced into the peritoneum by an aseptic operation.

The collodion capsules may be made of any size. Those
for rabbit experiments should be of about id c.c. capacity,
those for guinea-pig experiments about 5 c.c. By coating
large glass tubes they can be made of 500 c.c. capacity,
the large bags being useful for chemic dialysis.

Fig. 80. — Prepara-
tion of collodion sacs:
a, Test-tube constric-
ted and cut; b, sac
attached to the tube.

CHAPTER XI.
THE DETERMINATION OF BACTERIA.

THE most difficult thing in bacteriology is the determina-
tion of the species of bacteria that come under observation.

A few micro-organisms are characteristic in morphology
and in their chemic and other products, and present no diffi-
culty. Thus, the tubercle bacillus is characteristic in its
reaction to the anilin dyes, and can usually be recognized by
this peculiarity. Some, as Bacillus mycoides, have charac-
teristic agar-agar growths. The red color of Bacillus pro-
digiosus and the blue of Bacillus janthinus speak almost
positively for them. The potato cultures of Bacillus mesen-
tericus fuscus and vulgatus are usually sufficient to enable
us to recognize them. Unfortunately, however, there are
several hundreds of described species that lack any one dis-
tinct characteristic that may be used for differential pur-
poses, and require that for their recognition we shall well-nigh
exhaust the bacteriologic technic in order to identify them.

Tables for the purpose have been compiled by Eisenberg,
Migula, Lehman and Neumann, Chester, and others, and are
indispensable to the worker. The most useful are probably
the "Atlas and Grundriss der Bakteriologie und Lehrbuch
der speziellen bakteriologischen Diagnostik," by Lehmann
and Neumann,* and the "Manual of Determinative Bacte-
riology," by F. D. Chester (1901), from which, through the
courtesy of the author and publisher, the following synopsis
of groups is taken. Unfortunately, in tabulating bacteria
we constantly meet species described so insufficiently as to
make it impossible to properly classify and tabulate them.

The only way to determine a species is to study it thor-
oughly, step by step, and compare it with the description
and tables. In this regard the differentiation of bacteria
resembles the determination of the higher plants with the
aid of a botanic key, or the qualitative analysis for the de-
tection of unknown chemic compounds. Such a key for
specific bacterial differentiation is really indispensable, even
though it be imperfect, and every student engaged in research

* J. F. Lehmann, Miinchen, 1907.
278

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Chester's Synopsis of Groups of Bacteria 279

work should have one. As Chester says, "probably nine-
tenths of the forms of bacteria already described might as
well be forgotten or given a respectful burial. This will then
leave comparatively few well-defined species to form the
nuclei of groups in one or another of which we shall be able
to place all new and sufficiently described forms." "That
typical forms or species of bacteria do exist, no one can deny.
These typical forms furthermore present certain definite
morphologic, biologic, cultural, and perhaps pathogenic
characters which establish the types independently of minor
variations.

"The most marked of these types we select to become the
centers of groups, around which are gathered all related
species or varieties." "The division of the bacteria into
groups, so far as grouping was possible, is outlined in the fol-
lowing tables:"

A PROPOSED SYNOPSIS OF GROUPS OF BACTERIA.

BACTERIUM.
I. Without endospores.

A. Aerobic and facultative anaerobic,
a. Gelatin not liquefied.

* Decolorized by Gram's method.

t Obligate aerobic. ACETIC FERMENT GROUP.
ft Aerobic and facultative anaerobic.
Gas generated in glucose bouillon.

Gas generated in lactose bouillon. BACT.

AEROGENES GROUP.
Little or no gas generated in lactose bouillon.

FRIEDLANDER GROUP.
No gas generated in glucose bouillon.

Milk coagulated. FOWL CHOLERA GROUP.
Milk not coagulated. SWINE PLAGUE GROUP.
** Stained by Gram's method.

f Gas generated in glucose bouillon. LACTIC FER-
MENT GROUP.
6. Gelatin liquefied.

* Colonies on gelatin ameboid or proteus-like. BACT.

RADIATUM GROUP.

** Colonies on gelatin round, not ameboid. BACT. AM-

BIGUUM GROUP.
II. Produce endospores.

1. No growth at room temperature, or below 22°-25° C. THER-

MOPHILIC GROUP.

2. Grow at room temperatures.

a. Gelatin liquefied. ANTHRAX GROUP.

6. Gelatin not liquefied. BACT. F^CALIS GROUP.

BACILLUS.
I. Without endospores.

A. Aerobic and facultative anaerobic.

280 The Determination of Bacteria

0. Gelatin colonies roundish, not distinctly ameboid.

* Gelatin not liquefied.

f Decolorized by Gram's method.

Gas generated in glucose bouillon.
Milk coagulated. COLON GROUP.
Milk not coagulated. HOG CHOLERA GROUP.
No gas generated in glucose bouillon. TYPHOID

GROUP.
tt Stained by Gram's method. B. MURIPESTIFER

GROUP.
** Gelatin liquefied.

f Gas generated in glucose bouillon. B. CLOACA GROUP.

ft No gas generated in glucose bouillon. Include a

large number of bacteria not sufficiently described

to arrange in groups.

b. Gelatin colonies ameboid, cochleate, or otherwise irregular.

* Gelatin liquefied. PROTEUS VULGARIS GROUP.
** Gelatin not liquefied. B. ZOPFI GROUP.

fl. Produce endospores.

A. Aerobic and facultative anaerobic.

1. Rods not swollen at sporulation.

a. Gelatin liquefied.

* Liquefaction of the gelatin takes place slowly. Fer-
ment urea, with strong production of ammonia.
URO-BACILLUS GROUP OF MIQUEL.
** Gelatin liquefied rather quickly.

t Potato cultures rugose. POTATO BACILLUS GROUP.
tf Potato cultures not distinctly rugose. B. SUBTILIS

GROUP.
b. Gelatin not liquefied. B. SOLI GROUP.

2. Rods spindle-shaped at sporulation. B. LICHENIFORMIS

GROUP.

3. Rods clavate at sporulation. B. SUBLANATUS GROUP.

B. Obligate anaerobic.

1. Rods not swollen at sporulation. MALIGNANT EDEMA

GROUP.

2. Rods spindle-shaped at sporulation. CLOSTRIDIUM GROUP.

3. Rods clavate-capitate at sporulation. TETANUS GROUP.

PSEUDOMONAS (Migula).

I. Cells colorless, without a red-colored plasma and without sulphur

granules.

A. Grow in ordinary culture media.
1. Without endospores.

a. Aerobic and facultative anaerobic.

* Without pigment.

t Gelatin not liquefied.

Gas generated in glucose bouillon. Ps. MONADI-

FORMIS GROUP.
No gas generated in glucose bouillon. Ps. AM-

BIGUA GROUP.
tt Gelatin liquefied.

Gas generated in glucose bouillon. Ps. COADU-

NATA GROUP.
No gas generated in glucose bouillon. Ps. FAIR-

MONTENSIS GROUP.

* Produce pigment on gelatin or agar.
t Pigment yellowish.

Chester's Synopsis of Groups of Bacteria 281

Gelatin liquefied. Ps. OCHRACEA GROUP.
Gelatin not liquefied. Ps. TURCOSA GROUP.
ft Pigment blue-violet.

Gelatin liquefied. Ps. JANTHINA GROUP.
Gelatin not liquefied. Ps. BEROLINENSIS GROUP.
** Produce a greenish-bluish fluorescence in culture media.

f Gelatin liquefied. Ps. PYOCYANEA GROUP.
ft Gelatin not liquefied. Ps. SYNCYANEA GROUP.
2. With endospores, aerobic and facultative anaerobic.

a. Non-chromogenic.

* Rods not swollen at sporulation. Ps. ROSEA GROUP.
** Rods swollen at one end at sporulation. Ps. TROM-

MELSCHLAGER GROUP.

b. Produce a greenish-bluish fluorescence in culture media.

* Gelatin liquefied. Ps. VIRIDESCENS GROUP.
** Gelatin not liquefied. Ps. UNDULATA GROUP.

B. Do not grow in nutrient gelatin or other organic media. Ni-

TRIMONAS GROUP.

II. Cell plasma with a reddish tint, also with sulphur granules. CHRO-
MATIUM GROUP.

MICROSPIRA (Migula).
I. Cultures show a bluish-silvery phosphorescence. PHOSPHORESCENT

GROUP.
II. Cultures not phosphorescent.

A. Gelatin liquefied.

1. Cultures show the nitro-indol reaction.

a. Very pathogenic to pigeons. MSP. METSCHNIKOVI

GROUP.

b. Not distinctly pathogenic to pigeons. CHOLERA GROUP.

2. Nitro-indol reaction negative or very weak, at least after

twenty-four hours. CHOLERA NOSTRAS GROUP.

B. Gelatin not liquefied or only slightly so. MSP. SAPROPHILA

GROUP.

MYCOBACTERIUM (Lehmann- Neumann).

I. Stain with basic anilin dyes, and easily decolorized by mineral
acids when stained with carbol-fuchsin.

A. Grow well on nutrient gelatin. Gelatin liquefied very slowly

or merely softened.

1. Stain by Gram's method. SWINE ERYSIPELAS GROUP.

2. Not stained by Gram's method. GLANDERS GROUP.

B. Little or no growth in ordinary nutrient gelatin.

1. Grow well in nutrient bouillon at body temperatures.

a. Stained by Gram's method. Rods cuneate — clavate —
irregularly swollen. DIPHTHERIA GROUP.

2. No growth in nutrient bouillon or on ordinary culture media.

Rods slender, tubercle-like,
a. Stain by Gram's method. LEPROSY GROUP.
6. Do not stain by Gram's method. INFLUENZA GROUP.

3. No growth in nutrient bouillon or on ordinary culture

media. Rods variable. ROOT-TUBERCLE GROUP.
II. Not stained with aqueous solutions of basic anilin dyes; not easily
decolorized by acids. TUBERCLE GROUP.

COCCACE^E.

Cells in their free condition globular, becoming slightly elongated
before division. Cell division in one, two, or three directions
of space.

282 The Determination of Bacteria

A. Cells without flagella.

1. Division in only one direction of space. Streptococcus

(Billroth).

2. Division in two directions of space. Micrococcus (Hallier).

3. Division in three directions of space. Sarcina (Goodsir).

B. Cells with flagella.

1. Division in two directions of space. Planococcus (Migula).

2. Division in three directions of space. Planosarcina

(Migula).

CHAPTER XII.

THE BACTERIOLOGY OF THE AIR,

MICRO-ORGANISMS are almost universally suspended in the
dust of the air, their presence being a constant source of con-
tamination in our bacteriologic researches and occasionally
a menace to our health.

Such aerial organisms are neither ubiquitous nor uni-
formly disseminated, but are much more numerous where
the air is polluted and dusty than where it is pure. The
purity of the atmosphere bears a distinct relation to the
purity of the surfaces over which its currents blow.

The micro-organisms of the air are for the most part harm-
less saprophytes taken up and carried about by the wind.
They are almost always taken up from dry materials, ex-
periment having shown that they arise from the surfaces of
liquids with much difficulty. Not all the micro-organisms
of the air are bacteria, and a plate of sterile gelatin exposed
to the air for a brief time will generally grow molds and yeasts
as well.

In some cases the bacteria are pathogenic, especially where
discharges from diseased animals have been allowed to collect
and dry. On this account the atmosphere of hospital wards
and of rooms in which infectious diseases are being treated is
more apt to contain them than the air of the street. How-
ever, because of the expectoration from cases of tuberculosis,
influenza, and pneumonia, which is often ejected upon the
sidewalks and floors of public places, the presence of occa-
sional pathogenic bacteria is far from uncommon in street-
dust.

Gunther points out that the greater number of the bacteria
which occur in the air are cocci, sarcina being particularly
abundant. Most of them are chromogenic and do not liquefy
gelatin. It is unusual to find more than two or three varie-
ties of bacteria at a time.

To determine whether bacteria are present in the air or
not, all that is necessary is to expose a film of sterile gelatin

283

284 The Bacteriology of the Air

on a plate or Petri dish to the air for a while, cover, and
observe whether or not bacteria grow upon it.

To make a quantitative estimation is, however, more diffi-
cult. Several methods have been suggested, of which the
most important may be briefly mentioned :

Hesse's method is simple and good. It consists in mak-
ing a measured quantity of the air to be examined pass
through a horizontal sterile glass tube about 70 cm. long and
3.5 cm. wide (Fig. 81), the interior of which is coated with a
film of gelatin in the same manner as an Ksmarch tube. The

Fig. 81. — Hesse's apparatus for collecting bacteria from the air.

tube is closed at both ends with sterile corks carrying small
glass tubes plugged with cotton. When ready for use the
tube at one end is attached to a hand-pump, the cotton re-
moved from the other end, and the air slowly passed through,
the bacteria having time to sediment upon the gelatin as
they pass. When the required amount has passed, the
tubes are again plugged, the apparatus stood away for a
time, and subsequently, when they have grown, the colonies
are counted. The number of colonies in the tube will repre-
sent pretty accurately the number of bacteria in volume of
air that passed through the tube.

Petri's Method

285

In such a tube, if the air pass through with proper slowness,
the colonies will be much more numerous near the point of
entrance than near that of exit. The first to fall will prob-
ably be those of heaviest specific gravity — i. e., the molds.

Petri's Method. — A more exact method is that of Petri,
who uses small filters of sand held in place in a wide glass
tube by small wire nets (Fig.
82). The sand used is made
to pass through a sieve whose
openings are of known size,
is heated to incandescence,
then arranged in the tube so
that two of the little filters,
held in place by their wire-

Fig. 82.— Petri's sand filter for
air-examination.

Fig. 83. — Sedgwick's expanded
tube for air-examination.

gauze coverings, are superimposed. One or both ends of the
tube are closed with corks having a narrow glass tube. The
apparatus is sterilized by hot air, and is then ready for use.
The method of employment is very simple. By means of
a hand-pump 100 liters of air are made to pass through the
filter in from ten to twenty minutes, the contained micro-
organisms being caught and retained by the sand. The sand

286 The Bacteriology of the Air

from the upper filter is then carefully mixed with sterile
melted gelatin and poured into sterile Petri dishes, where
the colonies develop and can be counted. Petri points out
in relation to his method that the filter catches a relatively
greater number of bacteria in proportion to molds than the
Hesse apparatus, which depends upon sedimentation. Stern-
berg points out that the chief objection to the method is the
presence of the sand, which interferes with the recognition
and counting of the colonies in the gelatin.

Sedgwick's Method. — Sedgwick and Miquel have recom-
mended the use of a soluble material — granulated or pulver-
ized sugar — instead of the sand. The apparatus used for
the sugar experiments differs a little from the original of
Petri, though the principle is the same, and can be modified
to suit the experimenter.

A particularly useful form of apparatus (Fig. 83), sug-
gested by Sedgwick and Tucker, has an expansion above the
filter, so that as soon as the sugar is dissolved in the melted
gelatin it can be rolled out into a film like that of an Esmarch
tube. This cylindric expansion is divided into squares
which make the counting of the colonies very easy.

Roughly, the number of germs in the atmosphere may be
estimated at from 100 to 1000 per cubic meter.

The bacteriologic examination of air is of very little im-
portance because of the numerous errors that must be met.
Thus, when the air of a room is quiescent it may contain
very few bacteria ; let some one walk across the floor so that
dust rises, and the number of bacteria becomes considerably
increased; if the room be swept, the increase is enormous.
From these and similar contingencies it becomes very diffi-
cult to know just when and how the air is to be examined,
and the value of the results is correspondingly lessened.

The most sensible studies of the air aim rather at the dis-
covery of some definite organism or organisms than at the
determination of the total number per cubic meter.

CHAPTER XIII.

BACTERIOLOGY OF WATER.

UNLESS water has been specially sterilized, received and
kept in sterile vessels, it always contains some bacteria, the
number usually bearing a distinct relationship to the quan-
tity of organic matter present.

The majority of the water bacteria are bacilli, and are as a
rule non-pathogenic. Wright,* in his examination of the
bacteria of the water from the Schuylkill River, found two
species of micrococci, two species of cladothrices, and forty-
six species and two varieties of bacilli. Pathogenic bacteria,
such as the spirillum of Asiatic cholera and the bacillus of

Fig. 84. — Wolfhiigel's apparatus for counting colonies of bacteria upon

plates.

typhoid fever, may occur in polluted water, but their occur-
rence is exceptional.

The method of determining the number of bacteria in
water is very simple, and can be accomplished with very
little apparatus. The method depends upon the equal dis-
tribution of a measured quantity of the water to be exam-
ined in some sterile liquefied medium, whose subsequent
solidification in a thin layer permits the colonies to be
counted.

The method originated with Koch, and may be performed
with plates, Petri dishes, or Esmarch rolls. It is always best

* "Memoirs of the National Academy of Sciences," vol. vn, Third
Memoir.

287

288

Bacteriology of Water

to make a number of cultures with different quantities of the
water, using, for example, o.oi, o.i, 0.5, and i.o c.c., re-
spectively, to a tube of liquefied gelatin, agar-agar, or gly-
cerin agar-agar.

The details of the method depend upon the quality of the
water to be examined. If the number of bacteria per cubic
centimeter be small, large quantities may be used ; but if
there be millions of bacteria in every cubic centimeter, it
may be necessary to dilute the water to be examined in the
proportion of i : 10 or i : 100 with sterile water, mixing well,
and making the plate cultures from the dilutions.

It is best to count all the colonies developed upon the

culture, if possible; but when
hundreds of thousands are scat-
tered over it, an estimate made
by counting and averaging the
number in each of the small
squares of some counting appa-
ratus, such as those devised by
Wolfhiigel (Fig. 84), Esmarch
(Fig. 85), or Frost (Fig. 86).
In counting the colonies a lens
is indispensable.

In some cases, as in the study
of sewage, badly contaminated
waters, inflammatory exudates,
and in the preparation of bac-
terial vaccines, it is expedient
to directly enumerate the bac-
teria without resorting to the cultivation method, where all
of the organisms may not grow.

Excellent methods for achieving this have been devised.
That of Winslow and Willcomb* being as follows:

"The cover-slips should be boiled in a 10 per cent, solution
of potassium bichromate in 50 per cent, sulphuric acid and
allowed to lie in this cleansing mixture. Just before using
they may be rinsed in 50 per cent, alcohol and dried on a
silk cloth, not in the flame. One- twentieth of a cubic centi-
meter of water placed on such a cover-slip spreads evenly
and should be allowed to dry in the air without sudden
heating. After drying it is fixed by passing through the
flame, covered with Ziehl-Neelson's carbol-fuchsin, warmed
* "Jour, of Infectious Diseases," Supplement No. i, May, 1905, p. 273.

Fig. 85. — Esm arch's instru-
ment for counting colonies of
bacteria in Esmarch tubes.

Determination of Bacteria in Water 289

until steam just rises, washed, dried, and mounted. For
counting the bacteria we use a Sedgwick-Rafter eye-piece
micrometer, made for the study of the larger micro-organisms
in drinking water." Very uniform results have followed.

The method of Wright* was devised for the computation
of bacteria in suspensions used in making tests of the opsonic
power of the blood and for use as bacterio-vaccines. The
bacteria containing fluid is diluted as may be necessary
with an equal volume or i : 2, 1:5, i : 10, with a fluid of which
one volume is normal human blood, then spread upon a
slide, fixed and stained by Irishman's or some other appro-
priate method, placed upon the stage of the microscope,
and the number of blood-corpuscles and bacteria counted
in each of a number of fields. To facilitate the counting a
cross is ruled with a writing diamond, upon a circular cover-
glass, which is then dropped into the eye-piece. If there
are, for example, ten bacteria to each corpuscle in a 1:5
dilution, the calculation is based upon the number of cor-
puscles. There being 5,000,000 red corpuscles in i c.mm. of
normal blood, there should be 50,000,000 bacteria in i c.mm.
of the suspension, but as it has been diluted 1:5, there are
only -J- of this, or 10,000,000.

The majority of the water bacteria rapidly liquefy gelatin,
on which account it is better to employ both gelatin and
agar-agar in making the cultures.

In ordinary city hydrant-water the bacteria number from
2 to 50 per cubic centimeter; in good pump- water, 100 to 500;
in filtered water from rivers, according to Giinther, 50 to 200;
in unfiltered river- water, 6000 to 20,000. According to the
pollution of the water the number may reach as many as
50,000,000.

The waters of wells and springs are dependent for their
purity upon the character of the earth or rock through which
they filter, and the waters of deep wells are much more pure
than those of shallow wells, unless contamination take place
from the surface of the ground.

Ice always contains bacteria if the water contained them
before it was frozen. In Hudson River ice Prudden found
an average of 398 colonies in a cubic centimeter.

A sample of water when collected for examination should
be placed in a clean sterile bottle or in a hermetically sealed
pre-sterilized glass bulb, and must be examined as soon as
possible, as the bacteria multiply rapidly in water which is

* "Lancet," July 5, 1902.
19

290 Bacteriology of Water

allowed to stand for a short time. If the water to be exam-
ined must be transported any considerable distance before
the manipulations are performed, it should be packed in ice.
The greatest care must always be exercised that the unnat-
ural conditions arising from the bottling of the water, the
changes of temperature, and the altered relationship to light
and the atmosphere, do not modify the number of contained
bacteria.

100

Fig. 86. — Frost's plate counter, for counting colonies of bacteria on
Petri dish or plate cultures. The cross-lines divide the figure into
square centimeters. The numbers at the top of the figure indicate the
area in centimeters of the various discs. The area of each sector (a and
b) is one-tenth of the whole area.

The detection of such important bacteria as the colon and
typhoid bacilli, and the cholera spirillum, will be considered
in the chapters treating of those respective organisms.

Unfortunately, the bacteriologic examination of waters
does not throw satisfactory light upon their exact hygienic
usefulness. Of course, if cholera or typhoid fever bacteria
are present, the water is dangerous, but the quality of the
water cannot always be gauged by the number of bacteria it
contains.

Determination of Bacteria in Water 291

Drinking-water, especially that furnished to large cities,
is not infrequently contaminated with sewage, and contains
intestinal bacteria — Bacillus coli communis. For the ready
determination of this organism, which is an important indi-
cation that the water is polluted, Smith * has made use of
the fermentation-tube in addition to the plate. His method
is to add to each of several fermentation-tubes containing
i per cent, dextrose-bouillon a certain quantity of water.
The evolution of 50-60 per cent, of gas by the third day is a
strong indication that the colon bacillus is present. Plates
may be used to confirm the presence of the bacillus, but are
hardly necessary, as there is scarcely another bacterium met
with in water that is capable of producing so much gas.

It was at one time thought that the occurrence of the colon
bacillus in water was sufficient to condemn its potability, but
the evidence accumulated in recent years, showing that this
organism may reach streams from manured soil, may enter
it with the dejecta of domestic animals, wild animals, birds,
and perhaps even of fishes, makes it doubtful whether any-
thing but an exceptionally large number of the organisms
should be looked upon as indicative of sewage pollution and
proof that the water is not potable.

In determining the species of bacteria found in the water
reference must be made to the numerous monographs upon
the subject and to special tables. An excellent table of this
kind, arranged by Fuller, f is given on pages 292, 293.

Filtration with sand, etc., diminishes the number of bac-
teria for a time, but, as the organisms multiply in the filter,
the benefit is not permanent and the filters must frequently
be subjected to bacteriologic tests and the sand washed,
spread out to dry and the filters renewed. Porcelain filters
seem to be the only positive safeguard, and even these, the
best of which seems to be the Pasteur-Chamberland, allow
the bacteria to pass through if used too long without proper
attention.

For those whose special line of work is the bacteriology of
water, the report of the Committee on Standard Methods
of Water Analysis to the Laboratory Section of the Ameri-
can Public Health Association, published in Supplement
No. i of the "Journal of Infectious Diseases," May, 1905,
will prove indispensable.

* " Amer. Jour. Med. Sci.," 1895, no, p. 301.

f " Public Health and Journal of Experimental Medicine,"

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CHAPTER XIV.
BACTERIOLOGY OF THE SOIL.

THE upper layers of the soil contain bacteria in proportion
to their richness in organic matter. Near the habitations of
men, where the soil is cultivated, the excrement of animals,
largely made up of bacteria, is spread upon it to increase its
fertility, this treatment not only adding new bacteria to
those already present, but also enabling those present to
grow much more luxuriantly because of the increased nour-
ishment they receive.

Where, as in Japan, human excrement is used to fertilize
the soil, or as in India, it is carelessly deposited upon the
ground, bacteria of cholera, dysentery, and typhoid fever
are apt to become disseminated by fresh vegetables, or
through water into which the soil drains. In such localities
fresh vegetables should not be eaten, and water for drinking
should be boiled.

The researches of Fliigge, C. Frankel, and others show that
the bacteria of the soil do not penetrate deeply, but gradu-
ally decrease in number until the depth of a meter is reached,
then rapidly diminish until at a meter and a quarter they
rather abruptly disappear.

The bacteria of soil are, for the most part, harmless sapro-
phytes, though a few highly pathogenic organisms, such as
the bacilli of tetanus and malignant edema, occur. Many
of them are anaerobic, and it is interesting to speculate upon
their biology. Whether they develop and multiply in the
soil in intimate association with strongly aerobic organisms
by which the free oxygen is absorbed, or whether they re-
main latent in the soil and develop only in the intestines of
animals, is not known.

The estimation of the number of bacteria in the soil seems
to be devoid of any practical importance. C. Frankel has,
however, originated an accurate method of determining it.
By means of a special boring apparatus (Fig. 87) earth can
be secured from any depth without digging and without
danger of mixing with that of the superficial strata. A

294

Bacteria in Soil

295

measured quantity of the secured soil is thoroughly mixed
with liquefied sterile gelatin and poured into a Petri dish
or solidified upon the walls of an Esmarch tube. The
colonies are counted with the aid of a lens. Fliigge found
in virgin earth about 100,000 colonies in a cubic centimeter.

Samples of earth, like samples of water, should be exam-
ined as soon as possible after being secured, for, as Gunther
points out, the number of bacteria changes because of the
unusual dryness, warmth, exposure to oxygen, etc.

The most important bacteria of the
soil are those of tetanus and malignant
edema, in addition to which, however,
there are a great variety of organisms
pathogenic for rabbits, guinea-pigs, and
mice.

In the "Bacteriological Examination
of the Soil of Philadelphia," Ravenel *
came to the conclusion that —

1. Made soils, as commonly found, are
rich in organic matter and excessively
damp through poor drainage.

2. They furnish conditions more suited
to the multiplication of bacteria than do
virgin soils, unless the latter are contami-
nated by sewage or offal.

3. Made soils contain large numbers
of bacteria per gram of many different
species, the deeper layers being as rich
in the number and variety of organisms
as the upper ones. After some years
the number in the deeper layers probably
becomes proportionally less. Made soils
are more likely than others to contain
pathogenic bacteria.

In seventy-one cultures that were iso-
lated and carefully studied by Ravenel,
there were two cocci, one sarcina, and
five cladothrices ; all the others were
bacilli.

Fig. 87.— Tip of
Frankel' s instru-
ment for obtaining
earth from various
depths for bacterio-
logic study. B
shows the instru-
ment with its cav-
ity closed, as it
appears during bor-
ing; A, open, as it
appears when twis-
ted in the other di-
rection to collect
the earth.

* "Memoirs of the National Academy of Sciences," First Memoir,
1896.

CHAPTER XV.

THE BACTERIOLOGY OF FOODS.

THE relation of bacteria to foods is an important one and
should be as thoroughly understood as possible by both the
profession and the laity. The relationship may be expressed
thus:

I. Foods serve as vehicles by which infectious agents are
conveyed to the body.

II. Foods are chemically changed and made unfit for use
by the bacteria.

I. Foods as Fomites. — In animal food the first source of
infection is the animal itself, danger of infection always
accompanying the employment of foods derived from dis-
eased animals. Thus, milk apparently normal in appear-
ance has been found to contain dangerous pathogenic bac-
teria. The tubercle bacillus is one of the most important of
these, and at the present time the consensus of opinion in-
clines toward the view that the great prevalence of tubercu-
losis among human beings depends partly upon the inges-
tion of tubercle bacilli in milk. It does not appear necessary
that the udder of the cow be diseased in order that the or-
ganisms enter the milk, as they seem to have been found in
milks derived from cows whose udders were entirely free
from demonstrable tuberculosis. It is, therefore, impera-
tive to retain only healthy cows in the dairy, and careful
legislation should provide for the detection and destruction
of all tuberculous animals. The detection of tubercle bacilli
in milk can only be certainly accomplished by the injection
of a few cubic centimeters of the fluid into guinea-pigs and
noting the results.

In addition to the tubercle bacillus, pyogenic streptococci
have been observed in enormous quantities and almost pure
culture in milk drawn from cows suffering from mastitis,
Stokes * has observed a remarkable case of this kind in which
the milk contained so much pus that it floated upon the top

* "Maryland Medical Journal," Jan. 9, 1897.
296

Foods as Fomites 297

like cream. Such seriously infected milk could not be used
with safety to the consumer.

In market milk one occasionally finds pathogenic organ-
isms, such as the diphtheria bacillus, typhoid bacillus, strep-
tococcus, etc., derived from human sources. Such polluted
milks have been known to spread epidemics of the respective
diseases whose micro-organisms are present. Bacteria may
enter milk from careless handling, from water used to wash
the cans or to dilute the milk, or from dust ; and as milk is
an excellent medium for the growth of bacteria, it should
always be treated with the greatest care to prevent such con-
tamination, as saprophytic bacteria produce chemical
changes in the milk, such as acidity and coagulation, which
destroy its usefulness or render it dangerous as food for in-
fants and invalids. Where the necessary precautions are
not or cannot be taken, Pasteurization of the milk as soon
after its reception as possible may act as a safeguard.

The student interested in the sanitary relations of milk
cannot do better than refer to Bulletin No. 35 of the Hy-
gienic Laboratory, Washington, D. C., 1907, "Upon the
Origin and Prevalence of Typhoid Fever in the District of
Columbia," and to Bulletin No. 41 of the same laboratory,
upon "Milk and its Relation to the Public Health" (1908);
also to the " Bacteriology of Milk," by Swithinbank and
Neuman, New York, E. P. Button & Co., 1903.

Meat from tuberculous animals might cause disease if
eaten raw or but partially cooked. As cooking suffices to
kill the organisms, the danger under ordinary conditions is
not great. Moreover, tuberculosis rarely affects the muscles,
the parts usually eaten.

Butter made from cream derived from tuberculous milk
may also contain tubercle bacilli, as has been shown by the
researches of Rabinowitsch.*

Foods may become polluted with bacteria in a variety of
ways that will suggest themselves to the reader. The com-
mon source is dust, which is more or less rich in bacteria
according to the soil from which it arises. The readiness
with which raw foods, such as meats, milk, etc., can be thus
contaminated in the barnyard, dairy, slaughter-house, and
shop, teaches but one lesson — that the greatest cleanliness
should prevail for the sake of the dealer, whose goods may be
*" Deutsche med. Wochenschrift," 1900, No. 26; abstract in the
"Centralbl. f. Bakt.," etc., xxix, 1901, p. 309.

298 The Bacteriology of Foods

spoiled by carelessness, and the consumer, who may be in-
jured by the food.

Any food may carry infectious organisms upon its surface,
such organisms being derived from the hands of the dealer,
from dust, from water, as when green vegetables are sprinkled
with impure water to keep them fresh, or from other sources.
The cleanliness of the merchant and the protection from
contamination that he bestows upon his goods should be
taken into consideration by his customers.

Shell-fish, especially oysters, seem to be common carriers
of infection, especially of typhoid fever. The oysters seem
to be contaminated with infected sewage carried to their
beds. It is not yet satisfactorily determined whether
typhoid bacilli multiply in the juices in the shells of the
oysters or not, but a number of epidemics of typhoid fever
have been very conclusively traced to the consumption of
certain oysters at a definite time and place. As cooking the
oysters will kill the contained bacilli, the prophylaxis of
disease in this case is very simple.

II. Food Poisons. — A new and useful nomenclature,
suggested by Vaughan and Novy,* contains the following
terms :

Bromatotoxismus — food-poisoning;

Galactotoxismus — milk-poisoning;

Tyrotoxismus — cheese-poisoning ;

Kreotoxismus — meat-poisoning ;

Ichthyotoxismus — fish-poisoning ;

Mytilotoxismus — mussel-poisoning;

Sitotoxismus — cereal-poisoning.

The most important chemic alterations effected by bac-
teria occur in milk and meat.

i. Milk-poisoning (Galactotoxismus). — Milk, even when
freshly drawn from the cow, always contains some bacteria,
whose numbers gradually diminish for a few hours, then rap-
idly increase until almost beyond belief. These organisms
are for the most part harmless to the consumer, but ulti-
mately ruin the milk. Although much attention has been
paid to the subject, bacteriologists are not agreed whether
the number of bacteria contained in milk is a satisfactory
guide as to its harmfulness.

The poisonous change in milk, cream, ice-cream, etc.,
has been shown by Vaughan to depend in part upon the
*" Cellular Toxins," Phila., 1902.

Food Poisons 299

presence of a ptomain known as tyrotoxicon, formed by
the growth of bacteria in the milk, but whether of any
particular bacterium is not known. The milk may become
poisonous during any time of the year, but chiefly in the
summer, when, because of the higher temperature, bacteria
develop most rapidly. The change takes place in stale milk,
and it is supposed that many cases of what was formerly
looked upon as "summer complaint" in infants were really
poisoning by this toxic ptomain.

Ice-cream poisoning depends upon the growth of the bac-
teria in the milk before it is frozen. In some cases the error
made has been to prepare the cream for freezing and then
keep or transport it, the freezing operation being delayed
until the development of the bacteria has led to the poison-
ous condition.

Cheese-poisoning (Tyroloxismus) is also thought to depend
upon tyrotoxicon at times, though it has been shown that
other cheese poisons exist. It is more or less a question
whether cases of milk- and cheese-poisoning do not depend
upon the toxic products of the colon bacillus growing in the
foods.

2. Meat-poisoning (Kreotoxismus). — Botulism or meat-
poisoning depends upon the growth of certain bacteria,
Bacillus botulinus of van Ermengem,* in the meat. The
symptoms following infection by the organism sometimes
closely resemble those of typhoid fever, and are characterized
by acute gastro-intestinal irritation, nervous disturbances,
and, in case of death, by fatty degenerations in the organs
and minute interstitial hemorrhages.

3. Fish-poisoning (Ichthyotoxismus) sometimes follows
the consumption of canned and presumably spoiled fish,
sometimes the consumption % of diseased fish. It is not
known whether it depends upon ptomains or upon toxico-
genic germs, though probably the latter, as Silber has
isolated a Bacillus piscicidus that is highly toxicogenic.

4. Mussel-poisoning (Mytilotoxismus) depends partly
upon irritating and nervous poisons in the mussel substance,
in part upon toxicogenic germs that they harbor.

5. Canned Goods. — Improperly preserved canned goods
not infrequently spoil because of the growth of bacteria, but
the occurrence of gas-formation, acidity, insipidity, etc.,
causes rejection of the product, and but few cases of poison-
ing from canned goods can be authenticated.

* "Zeitschrift fur Hygiene," Bd. xxvi, Heft 1.

CHAPTER XVI.

THE DETERMINATION OF THE THERMAL
DEATH-POINT OF BACTERIA.

SEVERAL methods may be employed for this purpose.
Roughly, it may be done by keeping a bouillon culture of
the micro-organism to be investigated in a water-bath whose
temperature is gradually increased, transplantations being
made from time to time until the temperature fatal to
the bacteria is reached.

It is economy to make the transplantations less frequently
at first than later in the experiment, when the ascending
temperature approaches a height dangerous to life. In or-
dinary determinations it is well to make a transfer at 40° C.,
another at 45°, another at 50°, still another at 55°, and then,
beginning at 60°, make one for every additional degree. The
day following the experiment it will be observed that all the
cultures grow except those heated beyond a certain point, say
62° C., when it can properly be concluded that 62° C. is the
thermal death-point. If all the transplantations grow, of
course the maximum temperature was not reached, and the
experiment must be repeated and the bacteria exposed to
still higher temperatures.

When more accurate information is desired, and one
wishes to know how long the micro-organism can endure
some such temperature as 60° C. without losing its vitality,
a dozen or more bouillon-tubes may be inoculated with the
organism to be studied, and stood in a water-bath kept at
the temperature to be investigated. The first can be re-
moved as soon as it is heated through, another in five min-
utes, another in ten minutes, or at whatever intervals the
thought and experience of the experimenter shall suggest,
the subsequent growth in each culture showing that the
endurance of the organism had not yet been exhausted.
By using gelatin and pouring each culture into a Petri dish,
and subsequently counting the colonies, it can be determined
whether many or only a few of the organisms in a culture

300

The Thermal Death-point 301

possess the maximum resisting power. To determine the
percentage, it is necessary to know how many bacteria were
present in the tubes before exposure to the destructive
temperature. Approximately the same number can be
placed in each tube by adding the same measured quantity
of a fluid culture to each.

In both of the procedures one must be careful that the
temperature of the fluid in the test-tube is identical with that
of the water in the bath. A sterile thermometer introduced
into an uninoculated tube exposed under conditions similar
to those of the experiment can be used as an index for the
others.

Another method of accomplishing the same end is by the
use of Sternberg's bulbs. These are small glass bulbs blown
on one end of a glass tube, drawn out to a fine point at the
opposite end. If such a bulb be heated so that the air is
expanded and partly driven out, its open tube, dipped into
inoculated bouillon, will in cooling draw the fluid in, so as
to fill it one-third or one-half. A number of these tubes are
filled in this manner with a freshly inoculated culture medium
and then floated, tube upward, upon a water-bath whose
temperature is gradually elevated, the bulbs being removed
from time to time as the required temperatures are reached.
As the bulbs are already inoculated, all that is necessary is
to stand them aside for a day or two, and observe whether
or not the bacteria grow, determining the death-point ex-
actly as in the other case.

CHAPTER XVII.

DETERMINATION OF THE VALUE OF ANTISEP-
TICS, GERMICIDES, AND DISINFECTANTS*

THE student must bear in mind that an antiseptic is a sub-
stance capable of restraining the growth of bacteria ; a germi-
cide, one capable of killing them. All germicides are anti-
septic in dilute solutions, but not -all antiseptics are germi-
cides. Disinfectants must be germicides.

Antiseptics are chiefly employed for purposes of preserva-
tion, and are largely used in the industries to protect organic
substances from the micro-organisms of fermentation and
decomposition. The problem is to secure a satisfactory
effect with the addition of the least possible preservative in
order that its presence shall not chemically destroy the good
qualities of the substances preserved. In the case of foods
it becomes necessary to use preservatives free from poisonous
properties.

Disinfectants and germicides are employed for the purpose
of destroying germs of all kinds, and the chief problem is to
secure efficiency of action, rather than to endeavor to save
on the reagent, which would be a false economy, in that the
very object desired might be defeated.

The following methods of determining the antiseptic and
germicidal values of various agents can be elaborated accord-
ing to the extent and thoroughness of the investigation to
be made.

I. The Antiseptic Value. — Remembering that an anti-
septic is a substance that inhibits bacterial growth, the de-
termination of its value can be made by adding varying
quantities of the antiseptic to be investigated to culture
media in which bacteria are subsequently planted. It is
always well to use a considerable number of tubes of bouillon
containing varying strengths of the reagent to be investi-
gated. If the antiseptic be non-volatile, it may be added
before sterilization, which is to be preferred ; but if volatile,
it must be added by means of a sterile pipet, with the greatest

302

The Germicidal Value 303

precaution as regards asepsis, after sterilization and imme-
diately before the test is made. Control experiments — i. e.,

bouillon cultures without the addition of the antiseptic

should always be made.

The results of antiseptic action are two: retardation of
growth and complete inhibition of growth. As the inoculated
tubes containing the antiseptic are watched in their devel-
opment, it will usually be observed that those containing
very small quantities develop almost as rapidly as the control
tubes; those containing more, a little more slowly; those
containing still more, very slowly, until at last there comes a
time when the growth is entirely checked.

Sternberg points out that the following conditions, which
must be avoided, may modify the results of experiment :

1 . The composition of the nutrient media, with which the
antiseptic may be incompatible (as bichloride of mercury
and albumin).

2. The nature of the test-organism, no two organisms
being exactly alike in their susceptibility.

3. The temperature at which the experiment is conducted,
a relatively greater amount of the antiseptic being necessary
at temperatures favorable to the organism than at tempera-
tures unfavorable.

4. The presence of spores which are always more resistant
than the asporogenous forms.

II. The Qermicidal Value. — Koch's original method of
determining this was to dry the micro-organisms upon sterile
threads of linen or silk, and then soak them for varying lengths
of time in the germicidal solution. After the bath in the
reagent the threads were washed in clean, sterile water,
transferred to fresh culture media, and their growth or fail-
ure to grow observed. This method also determines the
time in which a certain solution will kill micro-organisms, so
is advantageous.

Sternberg suggested a method by which the dilution nec-
essary to kill the bacteria could be determined, the time
remaining constant (two hours' exposure) in all cases.
"Instead of subjecting test-organisms to the action of the
disinfecting agent attached to a silk thread, a certain quan-
tity of a recent culture — usually 5 c.c. — is mixed with an
equal quantity of a standard solution of the germicidal
agent, . . . and after two hours' contact one or two
loopfuls are transferred to a suitable nutrient medium to
test the question of disinfection."

3°4

Value of Antiseptics

A very simple and popular method of determining the
germicidal value is to make a series of dilutions of the re-
agent to be tested ; add to each a small quantity of a fresh
liquid culture, and at varying intervals of time transfer a
loopful to fresh culture media. By a little ingenuity this

...A

Same rod immersed in broth after exposure
to disinfectant.

Fig. 88.— Glass rod in test-tube, for use in testing disinfectants.
Tube 6 in. by f in. ; rod 9 in. by £ in. Ring marked with diamond 1 in.
from lower end, to show upper limit of area on which the organisms
are dried. After exposure the rod is placed in a similar tube con-
taining broth, to test development, a, Cotton plug wrapped around
glass rod ; b, broth ; c, gummed label on handle of rod, for indenti-
fication; d, ring marked by diamond; e, dried organisms.

method may be made to yield information as to both time
and strength.

Hill * has suggested a convenient method of handling the
cultures, which are dried upon the ends of sterile glass rods
and can then be transferred from one solution to another or
otherwise manipulated (see Fig. 88).

* "Public Health," vol. xxiv, p. 246.

The Germicidal Value 305

One of the best methods for testing the germicidal value
of solutions is that suggested by Rideal and Walker.* To
use it, it is necessary to proceed with strict attention to
details.

The advantage of this method is that it expresses the
germicidal value in terms of carbolic acid, as the " carbolic
acid coefficient," and thus makes possible an accurate com-
parison of germicide with germicide.

The test culture should be grown in bouillon made accord-
ing to the same formula with exactly the same reaction.
The cultures should be grown in the thermostat at 37° C.
for just twenty-four hours, and, in order that they should
contain no clumps of bacteria, should be carefully filtered
through cotton-wool or glass wool just before using. The
transfer of the bacteria from the filtered culture to the diluted
germicide solutions is made with a large platinum loop,
about 6 mm. in diameter, slightly bent like a spoon, so as to
take up a large drop. Three drops are carried from the
culture into the germicide solution, which, no matter what
its dilution, should be of 5 c.c. volume, with great care that
the walls and mouth of the tube are not touched. The tube
is then well shaken from side to side, so as to distribute the
bacteria throughout the solution, and the tube set aside.
At intervals of one, three, five, ten, and fifteen minutes
respectively, three drops of the fluid are transferred with
the same platinum loop to fresh bouillon tubes. Failure
to grow upon such transplantation shows that the bacteria
have been killed.

Gaseous Disinfection. — If the germicide to be studied be
a gas, as in the case of sulphurous acid or formaldehyd, a
different method must, of course, be adopted.

It may be sufficient to place a few test-tube cultures of
various bacteria, some with plugs in, some with plugs out,
in a closed chamber in which the gas is evolved. The germi-
cidal action is shown by the failure of the cultures to grow
upon transplantation to fresh culture media. This crude
method may be supplemented by an examination of the dust
of the room. Pledgets of sterile cotton are rubbed upon the
floor, washboard, or any dust-collecting surface present, and
subsequently dropped into culture media. Failure of growth
under such circumstances is very certain evidence of good
disinfection. These tests are, however, very severe, for in
20

306 Value of Antiseptics

the cultures there are immense numbers of bacteria in the
deeper portions of the bacterial mass upon which the gas has
no opportunity to act, and in the dust there are many spor-
ogenous organisms of extreme resisting power. Failure to
kill all the germs exposed in such manner is no indication
that the vapor cannot destroy all the ordinary pathogenic
organisms.

A more refined method of making the tests consists in
saturating strips of blotting-paper, absorbent cotton, various
fabrics, etc., with cultures and exposing them, moist or dry,
to the action of the gas. Such materials are best made
ready in Petri dishes, which are opened immediately before
and closed immediately after the experiment. If, when
transferred to fresh culture media, the exposed objects fail
to give any growth, the disinfection has been thorough. If
the penetrating power of a gas, such as formaldehyd, is to
be tested, it can be done by inclosing the infected paper or
fabrics in envelopes, boxes perforated with small holes,
tightly closed pasteboard boxes, and by wrapping them in
towels, blankets, mattresses, etc.

Easier of execution, but rather more severe, is a method
in which cover-glasses are employed. A number of them
are sterilized, spread with cultures of various bacteria, al-
lowed to dry, and then exposed to the gas as long as re-
quired. They are subsequently dropped into culture media
to permit the growth of the organisms not destroyed.

Animal experiments may also be employed to determine
whether or not a germ that has survived exposure to the
action of reagents has its pathogenic power destroyed. An
excellent example of this is seen in the case of the anthrax
bacillus, a virulent form of which will kill rabbits, but after
being grown in media containing an insufficient amount of a
germicide to kill it will often lose its rabbit-killing power,
though still able to fatally infect guinea-pigs, or may lose its
virulence for both rabbits and guinea-pigs, though still able
to kill white mice.

CHAPTER XVIII

THE PHAGOCYTIC POWER OF THE BLOOD AND
THE OPSONIC INDEX.

FROM the time that Metschnikoff connected the phenomena
of phagocytosis with those of immunity until 1902, there
was no recognized technic for the observation and com-
parison of the bacteria-consuming and bacteria-destroying
power of the cells. In 1902 Leishman* suggested the
following simple technic:

A thin suspension of bacteria in normal salt solution is
mixed with an equal volume of blood by drawing in and
out of a capillary tube, then dropped upon a clean slide,
covered carefully, placed in a moist chamber, and incubated
at 37° C. for a half hour. The cover is then slipped off
carefully, as in making blood-spreads, dried, stained, and
the number of bacteria in each of 20 leukocytes counted
and averaged. For comparison with the normal the patient's
blood and normal blood are simultaneously examined.

This was greatly improved by Wright and Douglas, f the
accuracy of whose methods enabled them to discover the
"opsonins, " work out the "opsonic index," and formulate
methods by which sufficiently accurate observations could
be made for controlling the specific treatment of infectious
diseases.

The opsonic theory teaches that the leukocytes are
disinclined to take up bacteria unless they are prepared
for consumption or phagocytosis by contact with certain
substances in the serum that in some manner modify them.
This modifying substance is the opsonin (opsono, I cater to,
I prepare for).

To make a test of the opsonic value of the blood it is
necessary to prepare the following :

A uniform suspension of bacteria.

A suspension in salt solution of washed leukocytes.

The serum to be tested.

A normal serum for comparison.

* "British Medical Journal," 1902, I, Jan. n, p. 73.
t "Proc. Royal Soc. of London," 1904, LXXXII, p. 357.
307

308 The Phagocytic Power of the Blood

The Bacterial Suspension. — This is prepared like the
similar suspensions used for determining agglutination, but
with greater care, since the bacteria taken up by the cor-
puscles are to be counted, and any variation in the number
of bacteria with which they come into contact may modify
the count. It is also necessary to avoid all clumps of bac-
teria for the same reason.

The culture is best grown upon agar-agar for twelve to
twenty-four hours, the bacteria in young cultures being more
easy to separate than those in old cultures. Such a culture
may be taken up in a platinum loop, transferred to a test-
tube containing some 0.85 per cent, sodium chloride solution,

Fig. 89. — Grinding bacteria (Miller).

and gently rubbed upon the glass just above the fluid,
allowing the moistened and mixed bacterial mass to enter
the fluid little by little.

If the culture be older or of a nature that will not separate
in this manner (tubercle bacillus), it may be necessary to
rub it between two glass plates (Fig. 89), or in a small agate
mortar with a drop or two of salt solution, other drops being
added one at a time, until a homogeneous suspension is
secured. Such clumps of bacteria as may remain in the
suspension are easily removed by whirling for a few seconds
in a centrifuge.

The next step is the standardization of the suspension.
Wright recommends for this purpose and for the standard-
ization of the bacterio-vaccines that the number of bacteria
shall actually be counted. This he does by mixing one part
of the bacterial suspension with an equal volume of normal

The Bacterial Suspension

309

blood and three volumes of physiological salt solution.
After thorough mixing a smear is made upon a slide, the
smear stained, and the number of bacteria and corpuscles in
successive fields of the micro-
scope counted until at least
200 red blood-corpuscles have
been enumerated. As the
number of red corpuscles per
cubic millimeter of blood is
5,000,000, the number of bac-
teria per cubic centimeter can
be determined from the re-
sults of the counting by a
simple arithmetical process.
To facilitate the counting the
eye-piece of the microscope is prepared by the introduction
of a diaphragm shown in Fig. 90. The prepared suspen-
sion must usually be greatly diluted before using, but the

Fig. 90. — Diaphragm of eye-
piece showing hairs in position
(Miller).

Fig. 91. — Photomicrograph showing cross-hairs, bacteria, and red blood-
corpuscles (Miller).

reduction of bacteria is, of course, easily calculated. It re-
quires experience to determine the appropriate number of
bacteria to be employed. When this is once determined,

310 The Phagocytic Power of the Blood

future manipulations are made easy, because one first makes
his suspension, then enumerates the bacteria, and having
determined their number, immediately arrives at the
appropriate concentration by dilution (Fig. 91).

Fig. 92. — The nephelometer ; an instrument used for standardizing
bacterial suspensions used for the opsonic test or for vaccines, by com-
parison with precipitate of barium sulphate.

The writer* believes that equally accurate results can be
attained by means of a simple instrument which he has
called a " nephelometer" (Fig. 92). By the use of this

- 93- — Collecting blood for corpuscles (Miller).

instrument one arrives at the appropriate dilution of the

bacterial suspension by comparison with a tube containing

* " Jour. Amer. Med. Assoc.," Oct. 5, 1907, XLIX, p. 1176.

The Washed Leukocytes

barium sulphate precipitate. By careful comparison of the
suspension with the standard tube, and the addition of
more of the salt solution or of the bacteria, as may be
required, one can arrive at fairly uniform results in a few
moments.

The Washed Leukocytes. — It is not necessary to have
the leukocytes free from admixture with the erythrocytes,
but it is necessary to have large numbers of them. They
are collected by citrating the blood so as to prevent coagula-
tion, and then separating the citrated plasma from the
corpuscles by centrifugalization.

The hands of the patient are washed, and a piece of elastic
rubber tubing or some other convenient fillet wound about
the thumb or a finger to produce venous
congestion. With a convenient lancet
(Wright uses a pricker made by drawing
a bit of glass tubing or a glass rod to a
fine point in the flame) a prick is made
about a quarter inch from the root of
the nail (Fig. 93). From this the blood
is permitted to flow into small test-tubes
previously filled about three-fourths with
1.5 per cent, sodium citrate solution.
The blood and citrate solution are mixed,
and the tubes placed in a centrifuge,
balanced, and centrifugalized until the
corpuscles are collected at the bottom
of the tube (Fig. 94). The citrated
plasma is now withdrawn and replaced
with 0.85 per cent, sodium chloride solu-
tion, through which the corpuscles are
distributed by shaking. The tubes are
now again centrifugalized until the corpuscles are collected,
when the saline is removed carefully, the last drop from
the back of the meniscus (Fig. 95). In the corpuscular
mass that remains the leukocytes form a thin creamy layer
on the top.

The serum to be tested and the normal serum for
comparison are secured in the same manner, the former
from the patient, the latter from the operator. As it is
advisable to wound the patient but once, the tube for
obtaining the serum should be filled at the same time that
the citrated blood is taken.

Fig. 94. — Tube of
blood and citrate
solution before and
after centrifugaliz-
ing (Miller).

312 The Phagocytic Power of the Blood

The blood to furnish the serum is taken in a small bent
tube shown in Fig. 96.

The blood flowing from the puncture is allowed to flow
into the bent end of the tube, into which it enters by cap-
illary attraction and from which it descends to the body of
the tube by gravity. At least one cubic centimeter of the
blood is required to furnish the serum. The ends of the
tube are closed in the flame and the tube stood in the ther-
mostat for fifteen to thirty minutes. Coagulation takes
place almost immediately, and the serum usually separates
quickly. If it does not do so, Wright recommends hanging

Fig. 95. — Removing last drops of saline solution (Miller).

the curved arm of the tube over the centrifuge tube and
whirling it for a moment or two, when the clot is driven into
the straight arm of the tube and the clear serum appears
above. The tube is then cut with a file so that the serum can
be removed when needed. Mixing the factors concerned in
the test is a matter that requires practice and a steady hand.
It is best done, as recommended by Wright, in a capillary
tube controlled by a rubber bulb (Fig. 97). The object
of the experimenter is to take up into this pipette equal
quantities of the creamy layer of blood-corpuscles, of the
blood-serum, and of the bacterial suspension. Wright first
makes a mark with a wax pencil about i centimeter from the
end of the capillary tube. He first draws up the leukocytic

The Washed Leukocytes

layer of blood-corpuscles to this mark, then removing the
tube, permits the column to as-
cend a short distance. Next he
draws up the bacterial suspension
to the same point, withdraws the
tube, and permits the column to
ascend; then draws up the serum
to be taken to the same point;

Fig. 96.— Special blood pipette (Miller).

thus in the same capillary tube
he has three equal volumes of
three different fluids, separated by
bubbles of air. It is next neces-
sary to mix these, which is done
by repeatedly expelling them upon
a clean glass slide, and redrawing
them into the tube, as shown in
Fig. 98. After thus being thor-
oughly mixed, the fluid is once
more permitted to enter the capil-
lary tube and come to rest there.

Fig. 97. — Opsonizing pi-
pette containing blood-cor-
puscles, bacterial emulsion,
and blood-serum (Miller).

The end is now sealed in

314 The Phagocytic Power of the Blood

a flame, the rubber bulb removed and the tube placed in a
thermostat, or in case much work of the kind is being done,
to an opsonizing incubator (Fig. 99) in which the temperature
is not modified by opening and closing the doors. The tube
remains in the incubating apparatus at 37° C. for fifteen
minutes (some use twenty, some thirty, minutes as their
standard), is then removed, whirled about its long axis
between the thumbs and fingers a few times to mix the
contents from which the corpuscles have sedimented, its

Fig. 98.-

-Mixing liquids by repeatedly expelling on to slide and redraw-
ing into pipette (Miller).

end is broken off, and a good-sized drop is allowed to escape
upon a perfectly clean glass slide and spread over its surface.

The spreading is a matter of some importance, as an even
distribution of the leukocytes is desirable. The capillary
tube from which the drop has escaped will form a good
spreader if laid flat upon the glass and drawn along, but
the edge of another slide is better, and in distributing the
fluid, it is better to push than to pull it with the end of the
slide, rather than its side.

Miller* says that "a good smear should be uniform in
consistency and most of the leukocytes should be found
along the edges and at the end. For convenience in count-
ing, it is well to have the smear terminate abruptly and not
be drawn out into threads or irregular forms."

* "Therapeutic Gazette," March 15, 1907.

The Washed Leukocytes

This mixing, incubating, and spreading is done twice —
once with the serum of the patient, and once with the normal
serum of the operator. The technic is the same each time.

Fig- 99- — A small incubator of special design for opsonic work (Miller) .

In order that the enumeration of the bacteria taken up by
the leukocytes can be accomplished, it is next necessary to
stain the blood smears. This can be done by any method
that will demonstrate both
the bacteria and the cells.
For staphylococci and
similar organisms, Irish-
man's stain, Jenner's stain,
or J. H. Wright's stains
are appropriate. Marino's
stain, recommended by FiS- 100.— The smear (Miller).
Levaditi,* gives beautiful

results. For the tubercle bacillus the spreads may be stained

with carbol-fuchsin and counterstained with methylene-

* "Ann. de 1'Inst. Pasteur," 1904, xvm, p. 761.

3i 6 The Phagocytic Power of the Blood

blue, or perhaps better with gentian violet and counter-
stained with Bismarck brown or vesuvin.

The final step in the process is the enumeration of the
bacteria in the corpuscles by averaging the number taken
up by the cells. Only typical polymorphonuclear cells
should be selected for staphylococcic cases, and separate
averages made for polymorphonuclear and mononuclear
cells in tubercle bacillus cases. It is best to follow cer-
tain routine methods of enumeration. Some who content
themselves with a count of the number of bacteria in 20 cells,
secure less accurate results than those who count 50 cells.
It is usually best to count one-third of the cells in the central
portion of the spread, one- third at the edge, and one-third
at the end. In each portion no other selection of cells should
be made than the elimination of other than polymorpho-
nuclear cells and the elimination of all crushed or injured
cells ; the others should be taken one after the other, as they
are brought into the field with the mechanical stage. After
the bacteria included in each of the accepted number of
cells selected as the standard has been enumerated, an
average is struck.

The "opsonic index" is determined by dividing the
average number in the patient's serum preparation by the
average in the normal serum preparation.

Irishman's* studies of the phagocytic power of the blood
show that in cases of furunculosis, etc., with each recru-
descence of boils, there is a marked diminution of the pha-
gocytic power of the blood, and with each improvement,
a marked increase.

McFarland and 1'Englet found by an examination of the
blood of 24 supposedly healthy students and laboratory
workers that it was possible to prejudge, by the phagocytic
activity of the cells, the past occurrence of suppuration and
present liability to it.

Wright and Douglas use the opsonic index as a guide to
the specific therapy of the infectious diseases. If the
opsonic index is low they believe bacterio-vaccination is
indicated. In its administration, however, care must be
taken to administer a counted number of bacteria, and to
make frequent opsonic estimations to determine the good
or ill effects accomplished. Thus, the administration is

* "Lancet," 1902, i, p. 73.
f "Medicine," April, 1906.

The Washed Leukocytes 317

always followed by a temporary diminution (negative phase)
of the opsonic index, soon followed, if the dose be not too
large, by a marked increase (positive phase). It is sup-
posed, upon theoretic grounds, and proved by practical
experience, that the increase of phagocytic activity brings
about improvement. The care of the operator should be
to avoid giving so large a dose of the vaccine that the nega-
tive phase will be so long continued that harm instead of good
may be achieved.

Although Wright is said to cling to the study of the opsonic
index as a guide to bacterio-vaccination and the resulting
degree of immunity, the greater number of workers have
abandoned it upon grounds which the writer long ago ex-
pressed— " that the estimation of the value of bacterio-
vaccination by means of the opsonic index was a very com-
plicated way of finding out very little."

CHAPTER XIX.

THE WASSERMANN REACTION FOR THE
DIAGNOSIS OF SYPHILIS.

THIS now popular and fairly reliable method for assisting
in the diagnosis of atypical syphilitic infections was devised
by Wassermann, Neisser, and Bruck.* It is a method of
making the diagnosis of syphilis by demonstrating in the
blood (cerebrospinal fluid, milk, or urine) of the patient a
complement-fixing substance (antibody?) not present in nor-
mal blood.

The test is twofold: (i) A combination of syphilitic
antigen, complement, and suspected serum. (2) A subse-
quent addition to the mixture of blood-corpuscles and hemo-
lytic amboceptor. If the suspected serum contain the
syphilitic antibody the antigen and complement unite with
it, and the complement being thus " fixed," no hemolysis
can take place upon the subsequent addition of the blood-cor-
puscles and hemolytic serum. If, on the other hand, the sus-
pected serum contain no antibody, the complement cannot be
fixed, and is, therefore, free to act upon the subsequently
added blood-corpuscles in the presence of the hemolytic
serum, and hemolysis results.

It is thus seen that the first test is made for the purpose of
fixing the complement, and the second for the purpose of
finding out whether it has been fixed or not.

It is quite clear that such a test is very delicate, and can
only be successful when executed with great precision and with
reagents or factors titrated, so that their exact value may be
known.

CONSIDERATION OF THE REAGENTS EMPLOYED.

I. For the first, or fixation, test it is necessary to bring
together —

Syphilitic antigen.
Serum to be tested.
Complement.

* "Deutsch. Med. Wochenschr.," 1906, No. 19.

The Syphilitic Antigen 319

(i) The Syphilitic Antigen. — It was supposed by Wasser-
mann, Neisser, and Bruck, who first devised the test, that
the syphilitic antigen must contain the essential micro-
organisms of syphilis. No method for the cultivation of Tre-
ponema pallidum having at that time been devised, cultures
of the specific micro-organism could not be employed.
Histologists had, however, shown that greater numbers of
the organisms were to be found in the livers of the congeni-
tally syphilitic stillborn infants than anywhere else. With
the purpose, therefore, of securing the greatest possible num-
ber of micro-organisms for the antigenic function, such livers
were used. The tissue, having been cut into small fragments,
was spread out in Petri or other appropriate dishes and dried,
and the fragments rubbed to a fine powder with a mortar and
pestle. Such a powder can be kept indefinitely in an exsic-
cator over calcium chlorid if placed where it is cool and dark.
When the powder is to be used, 0.5 gin. is extracted either at
room temperature or in the ice-box with 25 c.c. of 95 per cent,
alcohol for twenty-four hours, filtered through paper, and the
filtrate used in quantities later to be mentioned.

Instead of drying the liver tissue, pulverizing, and then
extracting it, many investigators now prefer to cut it up, rub
it into a uniform paste with a mortar and pestle, and add 5
volumes of 95 per cent, or absolute alcohol, with which the
paste is thoroughly macerated and shaken many times or in
a shaking machine. The alcohol may then be filtered off,
or may be permitted to remain upon the sedimented liver
tissue remnants, and the clear supernatant fluid pipe ted off
and diluted, at the time of employment, with the isotonic
sodium chlorid solution. When this alcoholic extract is
added to the salt solution a turbidity occurs, but this must
not be filtered out, as it consists of the lipoids or other sub-
stances in the extract that are essential to the test, and the
quantity of the cloudy fluid in the final mixtures is so small
as not in any way to interfere with the results. The small
amount of alcohol in the diluted extract is negligible and has
no influence upon the reagents used for the test.

The mention of the lipoids now brings us to the point where
it seems advisable to state that one of the most interesting
facts about the Wassermann reaction is that its theoretic
basis was founded upon the erroneous assumption that the
essential antigenic substance consisted of the whole or frag-
mented treponemata in the liver extract. The method

320 Wassermann Reaction for Diagnosis of Syphilis

scarcely began to meet with practical application, however,
before it was discovered that the active antigenic substance
was soluble in alcohol, was present in other than syphilitic
livers, and could be extracted not only from human tissues,
but also from dogs' livers and from guinea-pigs' hearts.
Forges and Meier, indeed, found that lecithin could play the
role of syphilitic antigen, and Leviditi and Yamanonchi
place sodium glycocholate, sodium taurocholate, protogon,
and cholin among those bodies capable of acting as syphilitic
antigens, and Noguchi goes so far from the original that he
regularly employs an extract of the normal guinea-pig's heart
as the antigen to be employed in his modification of the test.

These discoveries now make it clear that the complement
fixation that takes place in syphilis is not identical with that
of the Bordet-Gengou reaction, in which it had its beginning.
Happily, however, the error does not destroy the usefulness
of the method for diagnosis.

The probable nature of the reaction will be described below.
For the present we must be content to follow the beaten path,
and for this purpose will use the congenitally syphilitic liver
extract as the antigen, preparing it as described above.

(2) The. Serum to be Tested. — Wassermann, Neisser, and
Bruck at first employed the cerebrospinal fluid, but now the
blood-serum of the suspected patient is almost universally
used. As is usual with antibodies, the substances engaging
in the complement-fixation test are widely distributed
throughout the body, and reach the cerebrospinal fluid, the
milk, the urine, and the other body fluids through the blood,
in which it exists in greatest concentration. The blood is,
moreover, readily obtainable for study, which is another
reason it is at present used for making the test under all
ordinary circumstances. Some workers who, like Noguchi,
work with very small quantities of the reagents, secure
the blood by obstructing the venous circulation of the
thumb or of a finger by means of a rubber band (see di-
rections for obtaining the blood for making the opsonic
index), but the greater number prefer to obtain it by in-
troducing a large hypodermic needle into one of the veins
near the bend of the elbow. The arm above the elbow
is compressed by a fillet, as though for the purpose of
performing phlebotomy, and a conspicuous vein selected
for the purpose. The skin is first carefully washed, then
treated with tincture of iodin. If the patient is nervous, a

The Complement 321

momentary spraying with chlorid of ethyl will make the
operation entirely painless. Some prefer to use the iodin
without the preliminary washing, believing that soap
makes it difficult for the iodin to effect satisfactory dis-
infection of the skin. The sterilized needle is thrust into
the vein, care being taken that the vein is not too com-
pressed and the point of the needle thrust entirely through
instead of into it. From 15 to 25 c.c. of blood may be with-
drawn into a large syringe or may be allowed to flow into a
sterile test-tube. The blood, however secured, is permitted
to coagulate and the clear serum removed by a pipet, or the
clotted blood is placed in a centrifuge tube and whined, so
that clear serum is secured in a few minutes.

As normal human blood-serum, when fresh, contains a
certain amount of complement which would interfere with
the success of the experiment, the serum is next placed in a
test-tube and kept in a water-bath between 55° to 58° C. for
a half -hour. This degree of heat destroys the complement
and leaves the complement-fixing substance uninjured. The
serum is now ready for use.

(3) The Complement. — The complement universally em-
ployed is contained in the blood of a healthy adult guinea-pig.
To obtain it a piece of cotton moistened with ether or chloro-
form is held to the guinea-pig's nose until it becomes uncon-
scious, when the head is forcibly extended and a longitudinal
incision made through the skin of the neck. The skin is then
drawn back between the finger, on the one side, and the thumb,
on the other side, of the operator's left hand, while, with a
sharp knife held in the right hand, he cuts through all of the
tissues of the neck down to the spinal column and thus opens
both carotid arteries. The spurting blood is caught in a
sterile Petri dish and the animal permitted to bleed to death.
The blood soon coagulates when undisturbed, and in a short
time clear serum exudes from the clot. As, however, the
complement seems to be at least in part derived from the
corpuscles, the serum should not be removed as soon as it
forms, but permitted to remain in contact with the clot for
three hours. If it is desired to save time, the clot, as soon as
formed, may be cut into strips and placed in the tubes of a
centrifuge and whirled for a half -hour. This secures a
greater quantity of the serum and at the same time gives it
its full value, probably by injuring the leukocytes.

Such serum containing the complement is useful for twenty-

322 Wassermann Reaction for Diagnosis of Syphilis

four hours. Longer it should not be kept or used, as it begins
to deteriorate almost at once, and the deterioration increases
in rapidity in proportion to the length of time it is kept. The
quantity of the complement in the serum of the guinea-pig
is fairly constant, when the animal is regularly fed, and fur-
nishes a fairly uniform reagent that requires no titration.

II. For the second, or hemolytic, test two additional re-
agents are required:

Blood-corpuscles to be dissolved.

Hemolytic amboceptors by which complement may be
united to them.

(4) The Blood-corpuscles. — It makes no difference what
kind of blood-corpuscles are employed. Ehrlich and Mor-
genroth, in their pioneer experiments into the mechanism of
hemolysis, used goat corpuscles. Bordet used rabbit cor-
puscles; Wassermann, Neisser, and Bruck, sheep corpuscles;
Detre, horse corpuscles; Noguchi, human corpuscles.

As those who do many tests require a considerable quantity
of blood, it seems wisest to make use of some kind that is
readily obtainable in any quantity, hence most investigators
now follow Wassermann and his collaborators and use sheep
blood, which is easily obtained at a slaughter-house or from
sheep kept for the purpose.

The flowing blood is caught in some open receptacle, stirred
until it is defibrinated (it must not be permitted to coagulate),
and then taken to the laboratory.

The corpuscles must next be washed with care, so as to
free them from all traces of amboceptors and complement be-
longing to the serum in which they are contained. For this
purpose a centrifuge is indispensable. The tubes of the
apparatus are filled with the defibrinated blood and then
whirled for fifteen minutes until the corpuscles form a com-
pact mass below a fairly clear serum. The serum is then
cautiously removed and replaced by 0.85 per cent, sodium
chlorid solution, the top of each tube closed by the thumb, and
vigorously shaken so as to distribute the corpuscles through-
out the newly added fluid. The tubes are next returned to
the centrifuge and again whirled until the corpuscles are
sedimented, when the fluid resulting from this first wash-
ing is removed and replaced by fresh salt solution, in which
the corpuscles are again thoroughly shaken up. They are
now again whirled until again sedimented, when the second
washing is removed, leaving the corpuscular mass undis-

The Hemolytic Amboceptor 323

turbed. Some prefer to give the corpuscles a third washing,
but it does not seem to be necessary. Of the remaining
corpuscular mass, 5 c.c. are added to 95 c.c. of salt solution
to make a 5 per cent, volume suspension, in which form they
are ready for use. As the corpuscles of healthy sheep thus
treated form a practically invariable unit, no titration or
other preliminary is needed before they are used. They
must, however, be used within twenty-four hours to secure
satisfactory results, as they tend to soften when kept and so
to lose their standard value.

(5) The Hemolytic Amboceptor. — As the validity of the
test depends upon the ability or inability of the complement
to dissolve the corpuscles, and as this can only be achieved
when appropriate amboceptors are added, the hemolytic
amboceptors must correspond to the kind of blood-corpuscles
employed in the experiment. As has been shown, the greater
number of investigators now employ sheep corpuscles, hence
must use such corpuscles as the antigen through whose stimu-
lation the amboceptors or antibodies are excited.

The usual method of obtaining the amboceptor is in the
blood-serum of an experimentally manipulated rabbit. A
large healthy rabbit is employed for the purpose, and is
given a series of intraperitoneal injections of the 5 per cent,
suspension of washed and sedimented sheep corpuscles pre-
pared as above described. These injections are usually given
about five days apart, and the dosage is usually 5, 10, 15, 20,
and 25 c.c. respectively.

A serum of higher amboceptor content may be prepared by
using a greater number of corpuscles, and for this purpose the
solid corpuscular mass thrown down by centrifugalization
after the second washing is employed. Of this, 2, 4, 8, and
12 c.c., diluted with just enough salt solution to make it
pass readily through the hypodermic needle, may be regarded
as appropriate doses, the intervals being the same, viz., five
days. The amboceptor content of the rabbit serum seems to
be greatest about the ninth or tenth day after the last injec-
tion. Much care must be taken to see that the injected fluid
is sterile and the operations performed under aseptic pre-
cautions, as the rabbits are easily infected and not infre-
quently die. They also seem prone to die after the last in-
jection, so that it is best to have more than one rabbit under
treatment at a time.

When the appropriate time has arrived the rabbit is bled

324 Wassermann Reaction for Diagnosis of Syphilis

from the carotid artery, according to the directions given in
the chapter upon Experiments upon Animals.

The blood thus obtained is permitted to coagulate, and the
serum, which should be clear, removed with a pipet. More
serum may be obtained from the clot by cutting it into strips,
placing these in a centrifuge tube, and whirling them for
fifteen minutes.

Having thus described the preparation of the reagents to be
employed in making the Wassermann test, the next step, that
of titrating them, becomes essential. One of the first ques-
tions that presents itself is how successful titration of reagents
that may all be more or less variable can be effected. To
achieve this it is necessary to begin with those that can be
assumed to present the least variation and work up those that
are most variable.

(1) The Sheep Corpuscles. — As these come from a healthy
animal, are always treated in precisely the same manner and
used under standard conditions of freshness, they can be
looked upon as an invariable factor : i c.c. of the 5 per cent,
suspension forms a good working quantity and constitutes
I unit.

(2) The Normal Guinea-pig Serum Containing the Comple-
ment.— As this also comes from a normal animal, is always
treated in precisely the same manner, and is also used under
standard conditions of freshness, etc., it may also be looked
upon as a factor subject to very slight variation. Of this
serum, o.i c.c. (i c.c. of a i : 10 dilution) forms the unit,
or working quantity.

These two reagents, therefore, may be regarded as the
standards of measurement through which the titer of a
third is made possible.

(3) The hemolytic serum from the rabbit treated with the
sheep corpuscles.

This is subject to very great variation, according to the
treatment of the rabbit, and apparently, also, according to
the ability of the individual rabbit to respond to the treat-
ment by the formation of hemolytic amboceptors. It is,
therefore, imperative to make a careful titration of this
reagent.

To do this we proceed as follows, the quantities recom-
mended being such as experience has proved most satis-
factory :

Into each of a series of common test-tubes or culture-

The Hemolytic Amboceptor -325

tubes i c.c. of the 5 per cent, suspension of sheep corpuscles
and i c.c. of the i : 10 dilution of the normal guinea-pig
serum (complement) are measured with graduated pipets, and
then to each of these tubes the rabbit serum (amboceptor)
is added in diminishing quantities for the purpose of de-
termining the least quantity that will bring about complete
hemolysis in two hours at the temperature of 37° C. The
occurrence of the hemolysis is shown by a very striking
change in the appearance of the fluids. The mixture is at
first opaque and pale red, but after hemolysis, or solution
of the red corpuscles, becomes a beautiful transparent
Burgundy wine red.

The actual " set-up " or working scheme for determining
the unit or least hemolyzing addition of the amboceptor
serum may be represented as follows, the tubes being placed
in a thermostat and observed every fifteen minutes:

Five per cent, suspen- Normal guinea-pig Hemolytic rabbit Result (final readings after
sion of corpuscles (c.c.). serum (c.c.). serum (c.c.). two hours).

o.oi Complete hemolysis.

0.005

0.002

O.OOI

0.0005

0.0003 Partial hemolysis.
0.0002 No hemolysis.

o.oooi "

After the reagents are added, enough 0.85 per cent, salt
solution is added to each tube to bring the total bulk of the
mixture up to 5 c.c.

From the results shown in the tubes it is evident that the
hemolyzing quantity of the rabbit serum lies between
0.0005 and 0.0003 c.c., and is probably 0.0004 c-c- To be
as accurate as possible, a second series of experiments should
be made with 0.0005, 0.00045, and 0.0004 c-c-> so that the
proportion of amboceptor serum necessary to effect hemolysis
be known within small limits. This least quantity, that will
certainly cause hemolysis in two hours at 37° C., is known
as the unit. The combination of the unit of corpuscular
suspension (i c.c.), the unit of complement (o.i c.c.), and the
unit of hemolytic amboceptor is known as the hemolytic
system.

As soon as this unit is known accurately, we are in posi-
tion to reverse the conditions of the test. Thus, if we
should desire to know how much variation there may be in
the complements from different animals under different

326 Wassermann Reaction for Diagnosis of Syphilis

conditions of age, feeding, health, etc., we can now do so by
determining whether, when i c.c. of the corpuscles, i unit
of amboceptor and varying quantities of complementary
serums are combined, any variation in the final results will
obtain.

Or, if we desire to know to what extent the sheep corpuscles
may change through prolonged keeping or other manipula-
tion, it can be done by maintaining the unit of amboceptor
and the unit of complement and adding larger or smaller
quantities of the corpuscles.

The conditions under which the unit of amboceptor is
titrated constitute the standard conditions of the Wasser-
mann reaction. In it are always employed i unit of sheep
corpuscle suspension, i unit of complement, and i unit of
amboceptor. Here, however, a slight difference of opinion
is reached, it being argued by many experimenters that such
exact proportions may make the test uncertain, because,
should there be the slightest tendency on the part of the
remaining reagents to inhibit hemolysis by means other
than complement fixation, it would result in positive readings
where the final result should be negative. To overcome this
possibility, they differentiate between the amboceptor
unit and the amboceptor dose, the latter being commonly
twice and sometimes four times the unit.

Now, though the amboceptor unit is determined by the
method given, it by no means follows that those proportions
are the only ones that will lead to hemolysis. By increasing
the amboceptor we can diminish the complement with the
same end-result, a matter that has been graphically shown
by Noguchi,* who says " that hemolysis is merely the rela-
tive expression of the combined action of amboceptor and
complement, and is not the absolute indication of the amount
of the hemolytic components present in the fluid. The
same amount of hemolysis can be produced by i unit of
complement and by i unit of amboceptor as by 20 units
of amboceptor and o.i unit of complement or any other
appropriate combination of these two components."

As in the performance of the test we work always with
i unit of complement, we do not want to unduly disturb its
proper proportional action by any excessive addition of am-
boceptor, but simply to increase the latter sufficiently to pro-
vide for the accidental presence, in the serum to be tested,
* "Serum Diagnosis and Syphilis," 1910, p. 13 et seq.

The Hemolytic Amboceptor 327

of substances affecting hemolysis. Fortunately, means are
provided for controlling this action, as will be shown below.

The amboceptor serum keeps indefinitely. When it is to be
kept and used from time to time, many experimenters prefer
to seal it in a number of small tubes, one of which is opened
when the serum is needed, the remainder being kept in an
ice-box. Others prefer a stoppered bottle that can be
opened and a measured quantity removed as needed. The
most convenient way of treating it seems to be Noguchi's
method of drying it upon filter-paper.

For this purpose a good quality of filter-paper is cut into
strips 10 to 20 cm. in length and 6 to 8 cm. in breadth, and
saturated with the serum, which is permitted to dry. It is
well to make a preliminary titration of the serum, for if
it be very active it may have to be diluted in order that the
piece of dry paper containing the dose be of a size convenient
to handle; i drop of serum usually covers about J sq. cm.,
which is about as small a piece as can be measured, cut, and
used with satisfaction if sufficient allowances are to be made
for variations in distribution and other conditions that may
modify the accuracy of the method. If the unit-strength of a
serum be, say, 0.00005 and the dose o.oooi, water should be
added to the extent of about 9 volumes and the mixture
gently agitated, so that diffusion may occur without frothing.
The diluted serum is poured into a large flat dish, and the
strips of paper passed lengthwise and slowly to and fro until
not only wet, but thoroughly saturated. Each strip, when
the dipping is finished, is held first by one end, then by the
other, to drain off the free drops, and then laid flat upon a
clean glass plate and permitted to dry. The use of an electric
fan is recommended to hasten drying. Paper so prepared
contains everywhere about the same quantity of serum.

The real titration of the serum now begins. With a ruler,
one piece of paper is divided into squares of, say, J cm.,
and a series of tubes prepared with corpuscle suspension and
complement and the paper added i square, 2 squares,
2j squares, and so on until the unit is determined. When
that is achieved, the exact size of the paper containing the
unit being known, one sheet of the paper can be ruled into
squares of that size or into squares of twice that size — since
the " dose " is two units — at the option of the investigator.

The sheets of paper are kept in a clean envelope, the quan-
tity for each test being cut off as needed. The dry serum

328 Wassermann Reaction for Diagnosis of Syphilis

changes so little that the dose once determined, the size of the
square of paper needed for the test remains about the same.

The method has the advantage that the amboceptor serum
cannot be spoiled or spilled. It has the disadvantage of
being slightly less accurate, though it must be admitted that
the chances of error in measuring and diluting the fluid
serum are probably as great as those arising from inequalities
in the distribution of the serum throughout the paper.

(4) The Antigen. — It has already been shown that com-
plement is labile, and it may have occurred to the reader
that its activity is similar to that of ferments. It is now
necessary to point out the many conditions (some of which
may arise in the performance of a test so delicate as the
Wassermann reaction) by which the complementary action
may be affected or set aside. Thus, temperature affects it,
and temperatures of o° C. suspend it. It is on this account
that the test is always made at 37° C. Like most of the
ferments of the living organism, salts affect it, and in salt-
free media its action ceases, to return when a small quantity
of an alkaline salt is added. Not only inorganic salts, but
salts of the fatty acids and the bile-salts may inhibit it.
Certain lipoids, such as lecithin, cholesterin, protogon and
tristearin, and neutral fats inhibit the complementary action.
Some of these substances are always present in the serum
containing the complement itself or in the other serums to
be tested by its use, and, as Wassermann and Citron have
pointed out, we really know nothing about complementary
action. Aleuronat, inulin, peptone, albumose, tuberculin,
natural and artificial aggressins, gelatin, casein, sitosterin,
coagulated serum-albumin, and albuminous precipitates all
act as inhibitives to complementary action.

Now, in all combinations of several serums and antigens it
is always possible that some of these complement-binding or
complement-inhibiting substances may be present, hence the
first thing that has to be done in the way of titrating the
antigen — which is a tissue extract, rich in lipoids which in-
hibit complementary action — is to determine how much of it
can be added to the " hemolytic system " without disturbing
hemolysis.

As, however, the antigen is not used by itself, but always
in combination with a serum to be tested, we must always
combine it with serum when making the titration, so that the
requirements of the test may be conformed with. In order

The Hemolytic Amboceptor 329

that the essential difference between the normal serum and
the syphilitic serum can be reduced to precise calculation it
is imperative that, in all the tests, the same quantity of
added serum be employed. Experience has shown this
quantity to be 0.2 c.c., and this we regard as the unit of
serum to be tested.

To titrate the antigen we require (i) a normal human
serum and (2) a known syphilitic serum, obtained from
blood drawn from the arm veins of cases known to be well
and cases known to be syphilitic respectively. These serums
should be kept on hand in the laboratory in consider-
able quantity, as they are constantly needed for making the
controls that must accompany each test, as well as for making
the preliminary titration of the antigen.

The " set-up " for the titration of antigen is fairly simple.
A series of tubes is prepared and divided into two groups.
Into each tube in each group is placed i unit of complement.
Each tube of one group receives the addition of 0.2 c.c. of the
normal serum; each tube of the other group, 0.2 c.c. of the
known syphilitic serum. All the tubes now receive additions
of antigen, so that one tube of each group contains the same
quantity. The quantity of antigen not being known, it is
only through the experience of others that we can guess where
to start. An idea can be formed through study of the follow-
ing tabulation:

TABLE I. — Series "with the Normal Serum,
Tubes.

1. i unit of + i unit of -f- antigen o.oi u &^ S = Complete
complement normal serum °«--5.c ^ hemolysis.

2. i unit of -|- i unit of + antigen 0.03 *»«ga 5 = Complete
complement normal serum 5^ •* hemolysis.

3. i unit of + i unit of + antigen 0.05 *1 §J % = Complete
complement normal serum |-^-^ hemolysis.

4. i unit of + i unit of + antigen 0.07 jg.« £ i = Complete
complement normal serum t^ 1 o hemolysis.

5. i unit of + i unit of + antigen 0.08 £ g ^~_ = Complete
complement normal serum •-w'S.tnlJ hemolysis.

6. i unit of + i unit of + antigen 0.09 "?•§ § 1-| = Complete
complement normal serum !~-3 jjj hemolysis.

7. i unit of + i unit of + antigen o.i B*i * = Complete
complement normal serum t<u<o^rt hemolysis.

8. i unit of + i unit of + antigen 0.12 J -3 03 ft— Complete
complement normal serum 'g g-cTg^g hemolysis.

9. i unit of + i unit of + antigen 0.15 «•£ M g g = Complete
complement normal serum |C ^ 1 2 «, hemolysis.

10. i unit of + i unit of + antigen 0.18 g.1 f f*l — Complete
complement normal serum £ „•§ S^ hemolysis.

i unit of -{- i unit of + antigen 0.2 bog.o^=No
complement normal serum jjj =3-5 hemolysis.

ii

330 Wassermann Reaction for Diagnosis of Syphilis

TABLE II. — Series with the Syphilitic Serum.
Tubes.

1. i unit of + i unit of •+- antigen o.oi «'2 jjjj = Complete
complement syphilitic serum «^° hemolysis.

2. i unit of + i unit of + antigen 0.03 g^ G~§ = Complete
complement syphilitic serum §-o|^^ hemolysis.

3. i unit of + i unit of + antigen 0.05 g% >- ^'5 — Sugges-
complement syphilitic serum sSftSj tion of

** <u g §S hemolysis.

4. i unit of + i unit of + antigen 0.07 .S^-2 •£ ° = Slight
complement syphilitic serum ^M *£ « hemolysis.

6. i unit of + i unit of + antigen 0.08 | g"3 £ | = Partial
complement syphilitic serum "o^^^ hemolysis.

6. i unit of + i unit of + antigen 0.09 H'^'S g a = No
complement syphilitic serum ^- ^ H a « hemolysis.

7. i unit of + i unit of + antigen o.i .*^| S'g = No
complement syphilitic serum 2 § <g H^ hemolysis.

8. i unit of + i unit of + antigen 0.12 ^Irf gt> :=^No
complement syphilitic serum J> g §.•§£ hemolysis.

9. i unit of + i unit of + antigen 0.15 „ g 8 « g = No
complement syphilitic serum Jt g-"5"! hemolysis.

10. i unit of + i unit of + antigen 0.18 b^-Siy £ = No

complement syphilitic serum °ST13'I j| hemolysis.

From this we find that the unit of antigen is 0.09 c.c.
The largest quantity of the antigen that can be added
without preventing hemolysis when the normal serum is
used is probably 0.18 c.c. At the same time 0.09 c.c. is
the smallest quantity that can be added, when the syph-
ilitic serum is used, to prevent it. In this case the dose
exactly fulfils Kaplan's requirement that " The unit dose of
antigen must completely inhibit hemolysis ... of a known
luetic serum, provided double the dose does not interfere
with the complete hemolysis of cells using a known normal
serum and complement."

We have now accomplished the titration of all five of the
factors involved in making the Wassermann reaction, but
we have done more, we have really done the test, and have
seen positive and negative results, for in titrating the anti-
gen we have developed the reaction by which we can confirm
the diagnosis of syphilis in the case from whom the syphilitic
serum was obtained, and have failed to develop it with the
known normal serum.

However, in order that those who perform the test may be
able to escape the numerous errors into which one may fall,
it will be necessary to point out the controls by which they
can be escaped.

A Wassermann reaction at the present time comprises not
only the test of the patient's serum, but simultaneously in-
cludes a long series of other tests by which the validity of

"S.3
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TEST.

Tube containing the serum to be tested.

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Control of serum to be

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CONTROL.

Control of the known positive to deter-
mine that it contains no recently developed
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«• Control of the test by the use of a known
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Control test to determine changes in the
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Control of the hemolytic system.

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331

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332 Wassermann Reaction for Diagnosis of Syphilis

every part of the test and the correct titer of all the reagents
employed can be simultaneously ascertained. Every one
who makes the test should practice some such systematic
method as is suggested by the following scheme for the " set-
up." Nine tubes are employed for the usual test. These are
stood in a rack in the same order for every test, and in the
course of time it becomes a matter of habit to know the tubes
by number, and to recall for what each stands.

If many tests are to be made at one time, it is, of course,
unnecessary to make more than one series of the various con-
trols.

Of the complementary serum we add i c.c. to 9 c.c. of 0.85
per cent, (physiologic) salt solution, making each cubic centi-
meter of the dilution of the fluid equal o.i c.c. This quan-
tity, carefully measured by the same volumetric pipet, is
dropped into each tube, and this pipet laid aside.

The serum to be tested is drawn into a second finely gradu-
ated pipet, and 0.2 c.c. added to tubes i, 2, and 9, and that
pipet laid aside.

The positive syphilitic serum used to control the test is
similarly drawn up in a fresh pipet and 0.2 c.c. of it measured
into tubes 3 and 4, and the pipet laid aside.

The normal serum used as a control is similarly drawn into
still another pipet and 0.2 c.c. measured into tubes 5 and 6,
and the pipet laid aside.

The alcoholic extract composing the antigen is next added,
either by diluting it so that i c.c. contains the unit, or measur-
ing the unit quantity directly into the tubes. The antigen is
added to tubes i, 3, 5, and 7, and the pipet laid aside.

Lastly, each tube receives a correctly measured quantity
of 0.85 per cent, sodium chlorid solution to bring the total
bulk of fluid up to exactly 3 c.c.

Each tube is now shaken carefully, so as not to cause froth-
ing of the fluid, and the rack is stood in a thermostat kept
at 37° C.

At the end of an hour the rack is removed, and every tube
receives the addition of i unit of the sheep corpuscle sus-
pension and, with the exception of tube 9, receives one dose
of amboceptor, either the serum measured by diluting so that
i c.c. equals the dose, or the necessary square of paper. This,
in the former case, brings the total bulk of fluid to 5 c.c., in
the latter makes it necessary to add i more cubic centimeter
of salt solution to each tube. We aim to have exactly 5 c.c.
of fluid in each tube.

The Hemolytic Amboceptor

333

The tubes are again stood in the thermostat, where they
are permitted to remain for an hour, when the readings are
taken and carefully noted. After this the rack and all the
tubes are placed in the ice-box until twenty-four hours' old,
when the final readings are taken and the conclusions are
reached.

As a rule, the readings taken after the second hour of in-
cubation and those taken after twenty-four hours correspond.

A valid test should show the following:

Test Controls.

Tubes.
I.

2.

3-
4-

6.

8.
9-

No hemolysis in syphilis.
Complete hemolysis.

Hemolysis in health.

No hemolysis (this is the standard of comparison).
Complete hemolysis.

No hemolysis, as a rule.

Fig. 101. — A typical positive Wassermann reaction with the recom-
mended controls as it appears after standing twelve hours. Corpus-
cular sedimentation without hemolysis is seen in tubes i, 3, and 9;
complete hemolysis in the others.

In the tubes in which hemolysis takes place the change is
very marked. The hemoglobin dissolves out of the corpus-

334 Wassermann Reaction for Diagnosis of Syphilis

cular stroma and saturates the fluid, transforming it from
the opaque pale red to a transparent Burgundy red. Some-
times the corpuscular stroma dissolves, sometimes it sedi-
ments as a colorless mass to the bottom of the tube.

In the tubes containing the positive or syphilitic serum, and
in which there is complete complement fixation, the unaltered
corpuscles sediment to the bottom of the tube, leaving a
colorless fluid above.

When the complement fixation is complete there is no so-
lution of the hemoglobin. Such a result has been described
by Citron as H — \- + + . When the sedimented corpuscles lie
at the bottom of a slightly reddened fluid, the result is said to
be + + + ; when at the bottom of a distinctly red fluid, + + ,
etc. Confusion will be avoided by making reports as positive
in all cases in which there is a distinct red corpuscular de-
posit, regardless of the state of the supernatant fluid, and
negative when there is no such deposit.

When we come to inquire why the supernatant fluid should
be red, we reach a question that is not quickly answered.
In order to be in a position to explain it in certain cases we
introduced in our series tube 9, by which to discover whether
the serum under examination contains, as is sometimes the
case in health as well as in syphilis, rabbit corpuscle ambo-
ceptors. If tube 9 shows such amboceptors to be in the
serum, it explains the redness of the fluid bathing the cor-
puscles, and does not invalidate the test. If no such ambo-
ceptors are present and the fluid is still red, it may indicate
that a little of the complement remained unfixed and acted
upon a few of the corpuscles.

The Validity of the Test. — The Wassermann reaction is not
a certain test for syphilis. It is an aid in making the diag-
nosis, especially in cases in which there are no symptoms.

Of thousands of bloods of normal persons examined, the
results are almost 100 per cent, negative. Basset-Smith has
had a positive reaction in a case of scarlet fever and one in a
case of malignant disease of the liver with jaundice ; Oppen-
heim, one in a case of tumor of the cerebellopontine angle;
Marburg, one in a similar case; Newmark reports 2 cases of
brain tumors with positive reactions; Cohn, a positive in a
patient with a cerebral tumor. These seem to form the greater
part of positive reactions in non-syphilitics thus far recorded.

In active syphilis Wassermann had 90 per cent, of positive
reactions in 2990 cases; and most others report about the

Noguchi 's Modification 335

same. Basset-Smith in 458 such cases found 94 per cent,
positive reactions.

In latent syphilis Wassermann found 50 per cent, positive
reactions; Basset-Smith, 46 per cent.

In chronic, presumably syphilitic, disease of the nervous
system, general paresis, and tabes dorsalis the positive reac-
tions vary. In the former disease some have found as high
as 90 per cent, positive; in the latter the usual figures vary
about 50 per cent.

It is thus seen that the occurrence of the reaction is much
more conclusive evidence of the presence of syphilitic infection
than the failure of the reaction is of its absence.

Treatment greatly influences the test. When under active
treatment, either with mercury and iodids or with salvarsan,
the reaction of the serums is usually negative.

Nature of the Reaction. — We now reach the point of con-
sidering the nature of the reaction. It is certainly not a vari-
ation of the Bordet-Gengou phenomenon. It does not occur
because of the presence in the blood of syphilitics of antibodies
which combine with the antigen and fix the complement.
It is probably not complement fixation so much as comple-
mentary inhibition, through the presence in the blood of
syphilitics of certain metabolic products, whose action inter-
feres with the complement in some entirely different manner.

NOGUCHFS MODIFICATION OF THE WASSERMAMN
REACTION.

Noguchi* has modified the Wassermann reaction, first by
employing as an antigen an extract of the heart of a normal
guinea-pig, and, second, by making use of human instead of
sheep corpuscles for the hemolytic test. The advantage
of the latter depends upon the fact, carefully determined by
Noguchi, that human blood-serum contains no amboceptors
active in effecting hemolysis of human blood-corpuscles,
though it not infrequently contains hemolytic amboceptors
for sheep corpuscles. In the directions for making the Was-
sermann test a control test for determining their presence or
absence was found expedient. It will also be remembered
that the presence of these amboceptors causes no invalidity
of the test, provided it be recognized.

* "Serum Diagnosis of Syphilis," Philadelphia, 1910, J. B. Lippin-
cott Co.

33 6 Wassermann Reaction for Diagnosis of Syphilis

Noguchi also varies the technic in such a manner that very
small quantities of the various reagents are employed — a
necessity that arises from the relatively small quantity of the
patient's blood obtainable according to the method he em-
ploys. The reagents employed are as follows:

(1) The Serum to be Tested. — To obtain this, Noguchi binds
the finger of the patient with a rubber band, makes a good-
sized puncture near the root of the nail with a Hagedorn
needle, and collects about 2 c.c. of the blood in a Wright tube
(see directions for making the opsonic index). The blood
soon coagulates in the tube, which is then scratched with
a diamond or file, broken, and the serum removed with a
capillary pipet. The serum may or may not be inactivated
by heat, according to the option of the experimenter. The
dose of the unheated serum is i drop; of the inactivated
serum, 4 drops. The same doses of the normal and syphilitic
control serums are used.

(2) The Complement. — This consists of fresh guinea-pig
serum. Of it he makes a 40 per cent, dilution in physiologic
salt solution by adding one part of the serum to i^ parts of
the salt solution; o.i c.c. is the unit. Two units constitute
the " dose."

(3) The Antigen. — The antigen is made, according to the
directions given in the description of the Wassermann
test, out of normal guinea-pig heart. The extract is dried
upon filter-paper, as has been recommended for the hemolytic
amboceptor, and titrated according to the size of the square
of paper needed, instead of the quantity of fluid to be added.

(4) The Corpuscle Suspension. — For this purpose either
normal human corpuscles or the corpuscles of the patient
whose blood is to be examined may be employed. Instead of
a 5 per cent, suspension a i per cent, suspension is recom-
mended. If normal corpuscles are employed, it is necessary to
wash them free of the normal serum or plasma, which Noguchi
accomplishes as follows: 8 c.c. of normal salt solution are
placed in a large test-tube, and the blood flowing from a punc-
ture (in the operator's own finger, for example) permitted to
drop in, the proportion being i drop to each 4 c.c. The fluid
is then shaken and stood on ice over night, when the corpuscle
sediment and the supernatant fluid containing the fibrin
factors and ferment is decanted and replaced by fresh salt
solution, and the suspension made by shaking. Or, in a
laboratory, the corpuscles can be washed as usual with the

Noguchi's Modification

337

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Addition of antihuman amboceptor, 2
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Incubation at 37° C. for 2 hours longer,
then at room temperature.

I!

338 Wassermann Reaction for Diagnosis of Syphilis

aid of the centrifuge. If the patient's own corpuscles are to
be employed, some of them may be distributed, without any
washing, through the serum by simply shaking it up a little
with the clot. It is not essential exactly to measure the
corpuscles, as after a few trials with the suspension of normal
corpuscles the eye becomes accustomed to the color, intensity,
and density corresponding to the requirement.

(5) The Antihuman Hemolytic Amboceptor. — This is pre-
pared by injecting rabbits, according to the method already
described, with washed human corpuscles obtained from fresh
human placentae or from the heart of a fresh cadaver come to
autopsy. The serum of the rabbit, when obtained, is dried
upon blotting-paper and titrated as already described.

The " set-up " for the test, as given by Noguchi, is less
cumbersome than that recommended for the Wassermann
test and includes six tubes. It can best be understood by
reference to the diagram on page 337.

The method recommends itself through its simplicity and
convenience, no sheep corpuscles being used, and through
the smaller quantity of blood required, it seeming to the
patient that less damage is done by pricking the finger than
by introducing a syringe needle into a vein. It is, moreover,
a very sensitive test, and gives very accurate results as far
as regards positive cases. Unfortunately, it seems to have
the demerit of occasionally finding the reaction in negative
cases, which is a serious defect.

Diagnosticians are still divided in opinion, some preferring
the Wassermann test, some the Noguchi test, and some always
doing both, permitting the one to control the other. In the
long run the Wassermann test seems to meet with most favor,
and in the hands of the majority leads to most satisfactory
results.

PART II.

THE INFECTIOUS DISEASES AND THE
SPECIFIC MICRO-ORGANISMS.

CHAPTER I.

SUPPURATION.

SUPPURATION was at one time looked upon as a normal
and inevitable outcome of the majority of wounds, and
although bacteria were early observed in the purulent dis-
charges, the insufficiency of information then at hand led to
the belief that they were spontaneously developed there.

From what has already been said about the evolution of
bacteriology and the biology and distribution of bacteria,
the relationship existing between bacteria and suppuration,
and, indeed, between bacteria and disease in general, is
found to be reversed. Instead of being the products of
disease, we now know that the micro-organisms are the
cause

With this altered point of view came the question, Whence
come the micro-organisms that cause disease? The wide
distribution of bacteria in the air naturally led surgeons to
look upon it as the source of all infection, and to make most
strenuous, though mistaken efforts to disinfect it, that it
might not contaminate wounds.

The development of antiseptic surgery, and the extremes
to which the application of germicides was carried, became
almost ridiculous, for not only were the hands of the opera-
tor, his instruments, sponges, sutures, ligatures, and dress-
ings kept constantly saturated with powerful and irritating
germicidal solutions, and the wound subsequently covered
by dressings saturated with germicides, but by means of

339

340 Suppuration

a steam atomizer the air over the wound was kept filled
with a disinfecting vapor during the whole operation.

More recent researches, however, have shown not only
that the atmosphere need not be disinfected, but also that the
air of ordinarily quiet rooms, while containing a few sapro-
phytic organisms, very rarely contains pathogenic bacteria,
and is rarely an important factor in wound infection. A
direct stream of air, such as is generated by an atomizer,
really directs more bacteria toward the wound than would
ordinarily fall upon it, thereby increasing, instead of lessen-
ing, the danger of infection.

The strong disinfecting solutions once employed have like-
wise been largely abandoned, the modern view being that it
is far wiser to prevent the entrance of organisms into wounds
than to destroy them by the application of strong and
irritating solutions.

Suppuration, while nearly always the result of micro-
organismal activity, is not a specific infectious process, but
the expression of a violent tissue-reaction that may result
from various injurious agents.

Being, therefore, but the expression of tissue irritation
arising through strong chemotactic influences, it is only to
be expected that as many bacteria may be associated with
it as can bring about the essential conditions. Bacteria with
which these qualities are exceptionally marked appear as the
common cause of the process; those with which it is less
marked, as exceptional causes.

Attention has already been called to the fact that certain
micro-organisms are so intimate in their relation to the skin
that it is almost impossible to get rid of them, and in this rela-
tion the experiments of Welsh, Robb, and Ghriskey upon
hand disinfection have been cited. These observers have
shown that, no matter how rigid the disinfection of the
patient's skin, the cleansing of the operator's hands, the
sterilization of the instruments, and the precautions exer-
cised, a certain number of wounds in which sutures are em-
ployed will always suppurate, the cause of the suppuration
being the skin cocci, all of which it is impossible to remove.
We thus carry infectious organisms constantly with us upon
our skins, and so pave the way for suppuration in wounds.
That all wounds do not suppurate probably depends largely
upon the local and general immunity of the individual,
rather than upon the absence of organisms from the wounds.

Staphylococcus Epidermic! is Albus 341

The relative frequency with which certain varieties of
bacteria are associated with suppuration is well shown in
the following table from Karlinski:*

Suppuration in man — Streptococci, 45 cases.

Staphylococci, 144

Other bacteria, 15

Suppuration in the lower animals — Streptococci, 23

Staphylococci, 45

Other bacteria, 15

Suppuration in birds — Streptococci, 1 1

Staphylococci, 40

Other bacteria, 20

Andrewes and Gordon, f after the examination of large
numbers of Staphylococci from lesions of the human skin
and mucous membranes, came to the conclusion that four
varieties are differentiable. Of these, the Staphylococcus
pyogenes is the most common and most important. When
typical, this produces an orange-colored pigment. When
atypical, it may be lemon yellow or white. Staphylococcus
epidermidis albus is a distinct species. The differences
between these cocci are shown in the table on page 342.

STAPHYLOCOCCUS EPIDERMIDIS ALBUS (WELCH).

General Characteristics. — A non-motile, non-flagellate, non-
sporogenous, slowly liquefying, non-chromogenic, aerobic and optionally
anaerobic, doubtfully pathogenic coccus, staining by the usual methods
and by Gram's method, and having its natural habitat upon the skin.

Under the name Staphylococcus epidermidis albus, Welch J
has described a micrococcus which seems to be habitually
present upon the skin, not only upon the surface, but also
deep down in the Malpighian layer. He believes it to be
Staphylococcus pyogenes albus in an attenuated condition,
and if this opinion be correct, and there is seated deeply
in the derm a coccus which may at times cause suppuration,
the conclusions of Robb and Ghirskey, that sutures of cat-
gut when tightly drawn may be a cause of skin-abscesses by
predisposing to the development of this organism, are cer-
tainly justifiable. As the morphologic and cultural char-
acteristics of the organism correspond fairly well to those of

* "Centralbl. f. Bakt.," etc., vn, S. 113, 1890.

t "Report of the Local Government Board of Great Britain," Sup-
plement; "Report of the Medical Officers," 1905-06, vol. xxxv, p. 543.
| "Amer. Jour. Med. Sci.," 1891, p. 439.

342

Suppuration

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Staphylococcus Pyogenes Aureus 343

the following species, no separate description of them seems
necessary.

STAPHYLOCOCCUS PYOGENES ALB us (ROSENBACH*).

General Characteristics.— A non-motile, non-flagellate, non-
sporogenous, liquefying, non-chromogenic, aerobic and optionally
anaerobic, mildly pathogenic coccus, staining by the ordinary methods
and by Gram's method.

Although, as stated, Staphylococcus pyogenes albus is
a common cause of suppuration, it rarely occurs alone,
Passet so finding it in but 4 out of 33 cases investigated.
When pure cultures of the coccus are subcutaneously in-
jected into rabbits and guinea-pigs, abscesses occasionally
result. Injected into the circulation, the staphylococci oc-
casionally cause septicemia, and after death can be found in
the capillaries, especially in the kidneys. From this it will
be seen that the organism is feebly and variably pathogenic.

In its morphologic and vegetative characteristics Sta-
phylococcus albus is almost identical with the species next
to be described, differing from it only in the absence of its
characteristic golden pigment.

STAPHYLOCOCCUS PYOGENES AUREUS (RosENBACHf).

General Characteristics. — A non-motile, non-flagellate, non-
sporogenous, liquefying, chromogenic, pathogenic, aerobic and option-
ally anaerobic coccus, staining by the ordinary methods and by Gram's
method.

Commonly present upon the skin, though in smaller
numbers than the organisms already described, is the more
virulent and sometimes dangerous Staphylococcus pyogenes
aureus (Fig. 102), or "golden Staphylococcus," first observed
by Ogston and cultivated by Rosenbach. As the mor-
phology and cultural characteristics of this organism are
identical with those of the preceding species, it seems con-
venient to describe them together, pointing out such minor
differences as occur. In doing this, however, it must not
be forgotten that, although Staphylococcus albus was first
mentioned, Staphylococcus aureus is the more common
organism of suppuration.

* " Wundinf ektionskrankheiten des Menschen," Wiesbaden, 1884.
f " Mikroorganismen bei Wundinf ektionskrankheiten des Men-
schen," Wiesbaden, 1884.

344 Suppuration

STAPHYLOCOCCI PYOGENES AUREUS ET ALBUS.

Distribution. — The cocci are not widely distributed in
nature, seeming not to find a purely saprophytic existence
satisfactory. They occur, however, upon man and the
lower animals, and can occasionally be found in the dusts of
houses and hospitals — especially in the surgical wards — if
proper precautions are not exercised. They are common
upon the skin, in the nose, mouth, eyes, and ears of man;
they are nearly always present beneath the finger-nails,
and sometimes occur in the feces, especially of children.

Fig. 102. — Staphylococcus pyogenes aureus (Gunther).

Morphology. — The cocci are small, measuring about
0.7 ^ in diameter. When properly stained, the organisms are
found to consist of hemispheres separated from one another
by a narrow interval, the approximated surfaces being flat-
tened. As observed in hastily stained preparations, they
are spheric. There is no definite grouping in either liquid
or solid cultures. It is only in pus or in the organs or tissues
of diseased animals that one can say that a true staphylo-
coccus grouping occurs.

The organisms are not motile and have no flagella.

Staining. — The organisms stain easily and brilliantly
with aqueous solutions of the anilin dyes and by Gram's
method.

Isolation. — Staphylococcus aureus is an easy organism to
isolate, and can be secured by plating out a drop of pus in

Staphylococci Pyogenes Aureus et Albus 345

gelatin or in agar-agar. Such preparations, however, gen-
erally do not contain Staphylococcus aureus by itself, but in
association with Staphylococcus albus.

The colonies of Staphylococcus aureus differ considerably
in color, some being much paler than others.

Cultivation. — The staphylococci grow well upon all the
standard culture-media either in the presence or in the ab-
sence of oxygen at temperatures above 18° C., the most rapid
development being at about 37° C.

Colonies. — Upon the surface of gelatin plates the colo-
nies appear as small whitish points after from twenty-

Fig. 103. — Staphylococcus pyogenes aureus. Colony two days old, seen
upon an agar-agar plate. X 40 (Heim).

four to forty-eight hours, rapidly extending to the sur-
face and causing extensive liquefaction of the medium.
The formation of the orange pigment can be best observed
near the center of the colonies. Under the microscope the
colonies appear as round disks with circumscribed, smooth
edges. They are distinctly granular and dark brown. When
the colonies are grown upon agar-agar plates, the formation
of the pigment is more distinct.

Gelatin Punctures. — In gelatin the growth occurs along
the whole length of the puncture, causing an extensive
liquefaction of the medium in the form of a long, narrow,
blunt-pointed, inverted cone, sometimes described as being
like a stocking, full of clouded liquid, at the apex of which
a collection of golden or orange-yellow precipitate is always

346

Suppuration

present in Staphylococcus aureus. It is this precipitate in
particular that gives the organism its name, " golden sta-
phylococcus."

Agar-agar. — The growth of the golden Staphylococcus
upon agar-agar is subject to considerable variation in the
quantity of pigment produced. Sometimes, perhaps rarely,
it is golden; more commonly it is yel-
low, often cream color. Along the
whole line of inoculation a moist,
shining, usually well-circumscribed
growth occurs. When the develop-
ment occurs rapidly, as in the incu-
bator, it exceeds the rapidity of color
production, so that the center of the
growth is distinctly colored, the edges
remaining white.

Potato. — Upon potato the growth
is luxuriant, Staphylococcus aureus
producing an orange-yellow coating
over a large part of the surface. The
potato cultures may give off a sour
odor.

Bouillon. — When grown in bouillon
the organism causes a diffuse cloudi-
ness, with a small quantity of slightly
yellowish sediment. The reaction of
the medium is increasingly alkaline.
Nitrates are reduced to nitrites.

Milk. — In milk, coagulation takes
place in about eight days, and is fol-
lowed by gradual digestion of the
casein.

Thermal Death Point. — Staphylococci are usually quite
susceptible to the effect of heat, though their resistance is
not uniform. Sternberg found them destroyed by an
exposure to 62° C. for ten minutes, and to 80° C. for one
and a half minutes, but three cultures studied by von Lin-
gelsheim were not killed by an exposure to 60° C. for an
hour, and one culture studied by him endured an exposure
to 80° C. for ten minutes.

Toxic Products. — Leber seems to have first conceived of
suppuration as a toxic process depending upon the soluble
products of parasitic fungi, and in 1888, through the action

Fig. 104. — Staphylo-
coccus pyogenes au-
reus. Puncture cul-
ture three days old in
gelatin (Frankel and
Pfeiffer).

Staphylococci Pyogenes Aureus et Albus 347

of alcohol upon staphylococci, prepared an acicular crystal-
line body soluble in alcohol and ether, but slightly soluble
in water, to which he gave the name phlogosin.

Mannatti found that pus has substantially the same toxic
properties as sterilized cultures of the staphylococcus ; that
repeated injections of sterilized pus induce chronic in-
toxication and marasmus; that injection of sterilized pus
under the skin causes a grave form of poisoning ; and that
the symptoms and pathologic lesions caused by these
injections correspond with those observed in men suffering
from chronic suppuration.

Van de Velde* found that the staphylococcus has some
metabolic products destructive to the leukocytes, which
he has called leukocidin. This poison causes the cells to
cease ameboid movement, become spheric, and gradually
to lose their granules, until they finally appear like empty
sacs containing shadow nuclei, which eventually disappear.
The leukolysis occurs in about two minutes. These ob-
servations have been abundantly confirmed. Kraussf first
observed that certain products of the staphylococcus were
hemolytic and destroyed red blood-corpuscles. This hemo-
lysin has been carefully studied by Neisser and Wechsberg, J
by whom it was called staphylolysin.

Durme§ found staphylolysin produced most abundantly
by virulent staphylococci.

Ribbertll found that both sterilized and unsterilized cul-
tures when intravenously injected into animals produced
definite changes in the heart, kidneys, lungs, spleen, and
bone-marrow, and attributed the action to the toxin.

Morse** found that the toxic products of Staphylococcus
aureus were capable of occasioning interstitial nephritis.

The staphylococci form very little extracellular toxin, as
filtered cultures provoke little local or general reaction in
animals, even when the staphylococcus is highly virulent.

Pathogenesis. — The virulent, golden staphylococcus is
a dangerous and often deadly organism. Its virulence is,

"La Cellule," xi, 1896, p. 349.
t "Wiener, klin. Wochenschrift," 1900.
| "Zeitschrift fur Hygiene," 1911, xxxvi, p. 330.
§ "Hyg. Rundschau," 1903, Heft 2, p. 66.

|| "Die pathologische Anatomie und die Heilung der durch den
Staphylococcus pyogenes aureus hervorgerufenen Erkrankungen."
** "Journal of Experimental Medicine," vol. I, 1896, p. 613.

348 Suppuration

however, very variable both for the lower animals and for
man. The classical test for virulence is to inject TV c.c. of
a twenty-four-hour old bouillon culture into the ear vein of a
middle-sized rabbit. If of the ordinary virulence, the
organism should kill the rabbit in from four to eight days.
During this time the animal suffers from fever and wasting,
and when examined postmortem almost invariably shows
small abscesses in the kidneys and heart. In cases in which
the rabbits are highly susceptible or the cocci virulent,
purulent arthritis may be found. Highly virulent cultures
kill the animal in from one to two days, commonly by
occasioning endocarditis.

When the cocci enter human beings subcutaneously, ab-
scesses commonly result, and occasionally lead to a fatal
generalization of the organisms. In such cases the organisms
may be cultivated from the streaming blood, though the
greater number collect in, and frequently obstruct, the capil-
laries. In the lungs and spleen, and still more frequently in
the kidneys, infarcts are formed by the bacterial emboli.
The Malpighian tufts of the kidneys are sometimes full of
cocci, and become the centers of small abscesses.

The coccus is almost equally pathogenic for man and the
lower animals, though the fatal outcome of human infection
is more rare, possibly because of the conditions of infection.
It enters the human system through scratches, punctures,
or abrasions, and when virulent usually occasions an ab-
scess. Garre* applied the organism in pure culture to the
uninjured skin of his arm, and in four days developed a large
carbuncle, with a surrounding zone of furuncles. Bockhartf
suspended a small portion of an agar-agar culture in salt
solution, and scratched it gently into the deeper layer of the
skin with his finger-nail; a furuncle developed. Bumm
injected the coccus suspended in salt solution beneath his
skin and that of several other persons, and produced an
abscess in every case.

Staphylococcus aureus is not only found in the great
majority of furuncles, carbuncles, abscesses, and other in-
flammatory diseases of the surface of the body, but also plays
an important role in a number of deeply seated diseases.
Becker and others obtained it from the pus of osteomyelitis,
demonstrating that if, after fracturing or crushing a bone,

* "Fortschritte der Med.," 1885, No. 6.

f "Monatschrift fur prakt. Dermatologie," 1887, iv, No. 10.

Staphylococci Pyogenes Aureus et Albus 349

the staphylococcus be injected into the circulation, osteo-
myelitis may occur. Numerous observers have demon-
strated its presence in ulcerative endocarditis. Rodet has
been able to produce osteomyelitis without previous injury
to the bones; Rosenbach was able to produce ulcerative
endocarditis by injecting some of the staphylococci into the
circulation in animals whose cardiac valves had been injured
by a sound passed into the carotid artery; and Ribbert has
shown that the injection of cultures of the organism may
cause valvular lesions without preceding injury.

Virulence. — Experiments have shown that both Staphy-
lococci aureus and albus exist in attenuated and virulent
forms, and there is every reason to believe that in the major-
ity of instances they inhabit the surface of the body in a
feebly virulent condition.

Agglutination.— Kolle and Otto* have found that im-
mune antistaphylococcic serums agglutinate the staphylo-
cocci. The reaction is not specific and is peculiar. All
pathogenic staphylococci are agglutinated; non-pathogenic
cocci are not agglutinated. The reaction cannot, therefore,
be used for specific differentiation.

Specific Therapy. — The treatment of staphylococcus in-
fections with immune serum has not met with encouraging
success. Viqueratf has experimented in this direction and
found that goats are best adapted to the manufacture of
the serum ; but the literature of medicine contains very little
mention of beneficial results following the employment of
antistaphylococcus serums.

Denys and van de Veldet and Neisser and Wechsberg§
also produced antileukocytic serum.

Bacterio= vaccination. — Although specific serums have
failed, a promising form of specific treatment for subacute
and chronic staphylococcic infections has been introduced by
A. B. Wright, || who first isolates from the lesion the partic-
ular strain of staphylococci by which it is caused, cultivates
this artificially, suspends the organisms in an indifferent
fluid, of which a given quantity contains a known (counted)

* "Zeitschrift fur Hygiene," etc., 1902, xu.
t Ibid., xvm, 1894, p. 483.
} "La Cellule," 1895, xi.
§ " Zeitschrift fur Hygiene," 1901, xxxn.

|| "Lancet," March 29, 1902, p. 874; "Brit. Med. Jour.," May 9,
1903, p. 1069.

35° Suppuration

number, kills the organisms by heating them for an hour at
60° C., and then uses them by subcutaneous injection for
producing increased resistance on the part of the patient.

The treatment is controlled by studying the " opsonic
index " (q.v.), the objects being the avoidance of the " nega-
tive phase " or condition of diminished resistance, and the
progressive establishemnt of the positive phase or stage of
increased resistance. As the resistance increases the patient
rapidly improves, and many cases of obstinate acne, furun-
culosis, and other pyogenic infections have quickly recovered
under this treatment.

STAPHYUDCOCCUS CITREUS (PASSET).

An organism identical in many respects with the pre-
ceding, except that its growth on agar-agar and potato is
of a brilliant lemon-yellow color and its pathogenicity for
animals doubtful, is Staphylococcus citreus of Passe t.* As
it is not common and is doubtfully pathogenic, it is of much
less importance than the previously described organisms.

STREPTOCOCCUS PYOGENES (ROSENBACH).

General Characteristics.— The streptococcus is a non-motile, non-
flagellate, non-sporogenous, non-liquefying, non-chromogenic, aerobic
and optionally anaerobic, spheric organism, infectious for man and the
lower animals. Its division in one direction of space leads to its asso-
ciation in the form of chains or "strings of beads." It stains by ordi-
nary methods and by Gram's method.

Streptococci were probably first seen by Kochf in 1878.
In 1 88 1 OgstonJ called attention to the fact that two distinct
kinds of cocci were to be found in pus, mentioning both
staphylococci and streptococci. The beginning of real
knowledge of the streptococci, however, dates from the time
of their isolation and cultivation by Fehleisen§ and of
Rosenbach, || who cultivated them from 1 8 of 33 suppurative

" Untersuchungen iiber die Aetiologie der eitrigen Phlegmone des
Menschen," Berlin, 1885, P- 9-

t " Untersuchungen iiber die Aetiologie der Wundinfektionskrank-
heiten," Leipzig, Vogel, 1878. ,

t "British Med. Jour.," March, 1881, p. 369.

§ "Aetiologie des Erysipels," Berlin, Fischer, 1883.

|| "Mikroorganismen bei Wundinfektionskrankheiten des Menschen,"
1884, p. 22.

Streptococcus Pyogenes 351

lesions, fifteen times alone and five times in association
with Staphylococcus aureus.

Morphology. — The organisms are spheric, of variable size
(0.4-1 [i in diameter), and are constantly associated in pairs
or in chains of from four to twenty or more individuals.
Special varieties, known as Streptococcus longus (chains of
more than one hundred members) and Streptococcus brevis
(chains of from four to ten), have been described by v.
Lingelsheim,* but do not hold as separate species.

It is not motile and does not form endospores, though
sometimes large individuals — much larger than the others in

Fig. 105. — Streptococcus pyogenes, from the pus taken from an ab-
scess. X looo (Frankel and Pfeiffer).

the chain — may be observed. Some believe these to be
arthrospores.

Staining. — The organisms stain well with ordinary
aqueous solutions of anilin dyes and by Gram's method.

Isolation. — The streptococcus can be isolated from pus
containing it by plating or by the inoculation of a mouse or
rabbit, from whose blood it may easily be secured after
death.

Cultivation. — The organism grows at both the room tem-
perature and that of incubation, its best and most rapid
development being at about 37° C.

* "Zeitschrift fur Hygiene," Bd. x, 1891, p. 331; xn, 1892, p. 308.

352 Suppuration

Colonies. — Upon gelatin plates very small, colorless, trans-
lucent colonies appear in from twenty-four to forty-eight
hours. When superficial, they spread out to form flat disks
about 0.5 mm. in diameter. The microscope shows them to
be irregular and granular, to have a slightly yellowish color
by transmitted light, and to have numerous irregularities
around the edges, due to projecting chains of the cocci. No
liquefaction of the gelatin occurs.

Gelatin Punctures. — In gelatin puncture cultures no
liquefaction is observed. The minute spheric colonies grow

Fig. 106. — Streptococcus colonies on serum agar (From Hiss and Zins-
ser, '• Text-Book of Bacteriology," D. Appleton & Co., Publishers).

along the whole length of the puncture and form a slightly
opaque granular line.

Agar-agar. — Upon agar-agar an exceedingly delicate
transparent growth develops slowly along the line of inocu-
lation. It consists of small, colorless, or slightly grayish
transparent colonies which do not readily coalesce.

Blood-serum. — The growth upon blood-serum resembles
that upon agar-agar. The colonies are small, white, dis-
crete, and do not affect the medium.

Potato. — The streptococcus does not seem to grow well
upon potato, the colonies being invisible.

Bouillon. — In bouillon the cocci develop slowly, seeming
to prefer a neutral or feebly alkaline reaction. The medium
remains clear, while numerous small flocculi are suspended
in it, sometimes adhering to the sides of the tube, sometimes
forming a sediment. When the flocculi formation is distinct,
the name Streptococcus conglomerate (Kurth) is sometimes
given to the organism ; when the medium is diffusely clouded,
it is called Streptococcus dijjusus.

Streptococcus Pyo genes 353

In mixtures of bouillon and blood-serum or ascitic fluid
the streptococcus grows more luxuriantly, especially at in-
cubation temperatures, distinctly clouding the liquid. As
the lactic acid which is rapidly formed inhibits the growth
of the cocci, Hiss recommends* that instead of eliminating
the sugars in the broth, upon which the streptococci are
nourished, i per cent, of sterile powdered CaCO3 be added
to the culture-media. This neutralizes the acid as rapidly as
it is formed. It also maintains the life of the culture for a
long time.

Milk. — The organism seems to grow well in milk, which is
coagulated and digested.

Reaction. — The streptococcus is sensitive to acids, and
can only grow well in media with a slightly acid reaction.
All streptococci produce acids and eventually acidulate the
media, thus checking their further development.

Vital Resistance. — The optimum temperature appears to
be in the neighborhood of 37° C. It grows well between
25° and 40° C., above 40.5° C. the growth is slowed. The
thermal death point is low. Sternberg found that the
streptococci succumb at temperatures of 52° to 54° C. if
maintained for ten minutes. Their vitality in culture is
slight, and unless frequently transplanted they die. Bouil-
lon cultures usually die in from five to ten days. On solid
media they seem to retain their vegetative and pathogenic
powers much longer, especially if kept cool and cultivated
beneath the surface of the medium in a deep puncture. They
resist drying well.

Differential Features. — It is not always easy to dif-
ferentiate Streptococcus pyogenes from other less impor-
tant forms of streptococci and from the pneumococcus.
One of the best methods is by the employment of blood-agar
plates, suggested by Schottmuller.t Such plates are easily
prepared by melting ordinary culture agar-agar, cooling to
about 45° C., and then adding about 0.5 c.c. of defibrinated
human or rabbit's blood to the tube. The blood is first
thoroughly mixed with the agar, then the tube inoculated,
and poured into a Petri dish. As the Streptococcus pyogenes
grows, it produces a hemolytic substance that destroys the
blood-corpuscles in the vicinity of the colony, thus sur-

*" Text-book of Bacteriology," p. 338.

f "Munch, med. Wochenschrift, " 1903, L, p. 909.

23

354 Suppuration

rounding each by a clear, pale halo that contrasts with the
red agar. The colonies themselves appear gray.

The test is not specific, and Ruediger* points out that the
diphtheria and pseudodiphtheria bacilli also produce
hemolyzing substance, so that the test cannot be used for
the immediate separation of streptococci from other bacteria
in cultures from the throat. Colonies of the pneumococcus
usually appear green and without hemolysis, but Ruediger
finds that they sometimes also cause solution of the hemo-
globin. The streptococci whose colonies are green and
without hemolysis are called Streptococcus viridans by Schott-
muller. They are practically without pathogenic power
for rabbits.

Pathogenesis. — The streptococcus has been found in
erysipelas, ulcerative endocarditis, periostitis, otitis, men-
ingitis, empyema, pneumonia, lymphangitis, phlegmons,
sepsis, puerperal endometritis, and many other forms of
inflammation and septic infection. In man it is usually
associated with active forms of suppuration and sepsis.

The relation of the streptococcus to diphtheria is of
interest, for, though in all probability the great majority of
cases of pseudomembranous angina are caused by the
Klebs-Loffler bacillus, yet a number are met with in which,
as in Prudden's 24 cases, no diphtheria bacilli can be found,
but which seem to be caused by the streptococcus.

There is no clinical difference between the throat lesions
produced by the two organisms, and the only positive
method of differentiating the one from the other is by means
of a careful bacteriologic examination. Such an examina-
tion should always be made, as it has much weight in con-
nection with the treatment; in streptococcus angina no
benefit can be expected from the administration of diphtheria
antitoxic serum.

Hirshf has shown that streptococci are by no means rare
in the intestines of infants, where they may occasion enter-
itis. In such cases the organisms are found in large num-
bers in the stomach and in the stools, and late in the course
of the disease in the blood and urine of the child. They
also occur in all of the internal organs of the cadaver.

The intestinal streptococci are often Gram-negative, when
they are usually non-virulent.

* "Jour. Amer. Med. Assoc.," 1906, XLVII, p. 1171.

t "Centralbl. f. Bakt. u. Parasit.," Bd. xxn, Nos. 14 and 15, p. 369.

Streptococcus Pyogenes 355

Libman* has reported 2 carefully studied cases of strep-
tococcic enteritis.

Flexner, f in a large series of autopsies, found the bodies
invaded by numerous micro-organisms, causing what he has
called " terminal infections," and hastening the fatal issue.
Of 793 autopsies at the Johns Hopkins Hospital, 255 upon
cases dying of chronic heart or kidney diseases, or both, were
sufficiently well studied, bacteriologically, to meet the re-
quirements of a statistical inquiry. Tuberculous infections
were not included. Of the 255 cases, 213 gave positive
bacteriologic results. ' The micro-organisms causing the in-
fections, 38 in all, were Streptococcus pyogenes, 16 cases;
Staphylococcus pyogenes aureus, 4 cases; Micrococcus lan-
ceolatus, 6 cases; gas bacillus (Bacillus aerogenes capsu-
latus), three times alone and twice combined with B. coli
communis; the gonococcus, anthrax bacillus, B. proteus, the
last combined with B. coli; B. coli alone; a peculiar capsu-
lated bacillus, and an unidentified coccus."

It is interesting to observe in how many cases the strepto-
coccus was present. All the streptococci found may not
have been Streptococcus pyogenes, but for convenience
in his statistics they were regarded as such.

The presence of streptococci in the blood in scarlatina has
been observed in 30 cases by Crooke, by Frankel and Tren-
denburg, Raskin, Leubarth, Kurth, and Babes. In 1 1 cases
of scarlatina studied by Wright { a general streptococcus
infection occurred in 4, a pneumococcus infection in i, and a
mixed infection of pyogenic cocci in i.

Lemoine§ found streptococci in the blood during life in 2
out of 33 cases of scarlet fever studied. Pearcell studied 17
cases of scarlatina and found streptococci in the heart's
blood and liver in 4, in the spleen in 2, in the kidney in 5
cases. In 2 of the cases Staphylococcus pyogenes aureus
was associated with the streptococcus.

The streptococcus is the most common organism found in
the suppurative sequelae of scarlatina, frequently occurring
alone; sometimes with the staphylococci ; sometimes with
the pneumococci. Councilman found secondary infection

* "Centralbl. f. Bakt. u. Parasit.," Bd. xxii, Nos. 14 and 15, p. 376.

f "Journal of Experimental Medicine," vol. I, No. 3, 1896.

J "Boston Med. and Surg. Jour.," March 21, 1895.

§ "Bull, et Mem. Soc. d'Hop. de Paris," 1896, 3 s., xm.

|| "Jour. Boston Soc. of Med. Sci.," March, 1898.

356 Suppuration

by the streptococcus more widespread in variola than in
any other disease.

Virulence. — In the great majority of cases, streptococci
isolated from human beings are pathogenic for rabbits and
mice. Rats become ill when injected with large doses, but
usually recover. Guinea-pigs, cats, and dogs are but
slightly susceptible. Large animals, like sheep, goats, cattle,
and horses, react very slightly to large doses, but sometimes
suffer from abscesses at the seat of injection. Mice die in
from one to four days from general infection. If the organ-
isms are less virulent, they die in from four to six days with
edema and abscess formation at the site of inoculation, and
subsequent invasion of the body. The streptococcus seems
to be most pathogenic for that species of animal from which
it has been isolated.

If the ear of a rabbit be carefully inoculated with a small
quantity of a pure culture, local erysipelas usually results,
the disturbance passing away in a few days and the animal
recovering. If, however, the streptococcus be highly viru-
lent, the rabbit dies of general septicemia in from twenty-
four hours to six days. The cocci may then be found in
large numbers in the heart's blood and in the organs. In
less virulent cases minute disseminated pyemic abscesses are
sometimes found.

According to Marmorek,* the virulence of the strepto-
coccus can be increased to a remarkable degree by rapid
passage through rabbits, and maintained by the use of a cul-
ture-medium consisting of 3 parts of human blood-serum
and i of bouillon. The blood of the ass or ascitic or pleu-
ritic exudates may be used instead of the human blood-serum
if the latter be unobtainable. By these means Marmorek
succeeded in intensifying the virulence of a culture to such
a degree that one hundred-thousand-millionth (un cent mil-
liardieme) of a cubic centimeter injected into the ear vein
was fatal to a rabbit.

Petruschkyf found the virulence of the culture to be well
retained when the organisms were planted in gelatin, trans-
planted every five days, and when grown, kept on ice.

Hoist J observed a virulent Streptococcus brevis that re-

* "Ann. de 1'Inst. Pasteur," t. ix, No. 7, July 25, 1895, p. 593.
t "Centralbl. f. Bakt. u. Parasitenk.," Bd. xvm, No. 16, May 4,
1895, P- 55i.

% Ibid., Bd. xix, No. n, March 21, 1896

Streptococcus Pyogenes 357

mained unchanged upon artificial culture -media for eight
years without any particular precautions having been taken
to maintain the virulence.

Dried streptococci are said by Frosch and Kolle * to retain
their virulence longer than those growing on culture-media.

Marmorek f and Lubenau J found that cultures of the strep-
tococcus when grown in bouillon containing glucose, produced
a hemolytic substance — streptokolysin — not seemingly pres-
ent in cultures grown in ordinary bouillon. Besredka§
found that streptokolysin was produced only by highly viru-
lent cultures of the streptococcus and not by saprophytic
organisms that have been for some time under cultivation in
the laboratory.

Levin |1 investigated the subject thoroughly and found
that different strains of streptococci produced strepto-
kolysin in varying quantities, that its production is entirely
independent of virulence, that it is destroyed by heat
(37° C. in some days; 55° C. in one-half hour); that acidity
of the nutrient media hinders its formation, and that it is
intimately associated with the bodies of the streptococci
by which it is produced, so that in the sediment obtained by
filtration or by centrifugation there is nearly one thousand
times as much as in the filtered fluid culture. The strepto-
kolysin is not destroyed by the death of the bacteria. An-
tistreptokolysin is present in antistreptococcus serum.

Toxic Products. — The toxic products of the strepto-
coccus are not well known. Cultures from different sources
vary greatly in the effects produced by hypodermic or intra-
venous injection after filtration through porcelain. Killed
cultures produce a much more marked effect than filtered
ones, so that the important product must be an endotoxin.

Simon** found that the toxic quality of the bodies of
streptococci of different stocks had nothing to do with their
virulence. Simon ft also found that the toxic products of
the streptococcus were diverse and peculiar. The bodies
of the cocci contained an intracellular toxin the activity of

* Flugge's "Die Mikroorganismen."

t "Annales de 1'Inst. Pasteur," 1895, 593.

t "Centralbl. f. Bakt.," etc., 1901, Bd. xxx, Nos. 9 and 10.

§ "Ann. de 1'Inst. Pasteur," 1901, p. 880.

|| "Nord. Med. Ark," 1903, n, No. 15, p. 20.
** "Centralbl. f. Bakt.," xxxv, No. 3, p. 308, Dec. 18, 1903.
tt Ibid., xxxv, No. 4, p. 350, Jan. 16, 1904.

358 Suppuration

which was independent of their virulence. This poison is
liberated only when the bactericidal activities of the body
act upon the cocci. The cocci also excrete a toxic substance
whose activity is greater than that of the intracellular toxin,
but whose production is subject to great variation and is
entirely independent of the intracellular toxin. The toxins
and hemolysins are entirely different bodies.

In general, the effects of streptococcus intoxication are
vague. The animals appear weak and ill, and have a
slight fever; but unless the virulence of the culture be
exceptional or the dose very large, they usually recover in
a short time.

Coley 's Mixture. — The clinical observation that occa-
sional accidental erysipelatous infection of malignant tumors
is followed by sloughing and the subsequent disappearance
of the tumor, suggested the experimental inoculation of
such tumors with Streptococcus erysipelatis as a therapeutic
measure. The danger of the remedy, however, caused many
to refrain from its use, for when one inoculates the living
erysipelas virus into the tissues it is impossible to estimate
the exact amount of disturbance that will follow.

To overcome this difficulty Coley* has recommended
that the toxin instead of the living coccus be used for in-
jection. A virulent culture of the streptococcus is obtained,
by preference from a fatal case of erysipelas, inoculated into
small flasks of slightly acid bouillon, and allowed to grow
for three weeks. The flask is then reinoculated with Bacil-
lus prodigiosus, allowed to grow for ten or twelve days at the
room temperature, well shaken up, poured into bottles of
about f5ss capacity, and rendered perfectly sterile by an
exposure to a temperature of 50° to 60° C. for an hour. It
is claimed that the combined products of the streptococcus
of erysipelas and Bacillus prodigiosus are much more active
than a simple streptococcus culture. The best effects follow
the treatment of cases of inoperable spindle-cell sarcoma,
where the toxin sometimes causes a rapid necrosis of the
tumor tissue, which can be scraped out with an appropriate
instrument. Numerous cases are on record in which this
treatment has been most efficacious; but, although Coley
still recommends it and Czerny upholds it, the majority of
surgeons have failed to secure the desired results.

* "Amer. Jour. Med. Sci.," July, 1894.

Streptococcus Mucosus 359

Antistreptococcus Serum. — Since 1895 considerable at-
tention has been bestowed upon the antistreptococcus serum
of Marmorek* and Gromakowsky, f which is said to act
specifically upon streptococcus infections, both general and
local. Numerous cases of suppuration, septic infection,
puerperal fever, and scarlatina are upon record in which
the serum seems to have exerted a beneficial action, and it
may be that antiphlogistic serums will occupy an important
place in the medicine of the future.

The serum is prepared by the injection of cultures of
living virulent streptococci, into horses, until a high degree
of immunity is attained. The serum is probably both
antitoxic and bactericidal in action.

The success following the serums of some experimenters
upon certain cases, and their occasional or constant failure
in other cases, have suggested that there is considerable
difference between different " strains " or families of strep-
tococci. To obviate this inequality Van de VeldeJ has made
a polyvalent antistreptococcus serum by using a number
of different cultures secured from the most diverse clinical
cases of streptococcus infection. Another serum, of Tavel§
and Moser, || is made by using cultures from different cases
of scarlatina. The use of these serums, however, has not
given the satisfaction expected, and at the present moment
the whole subject of antistreptococcus serums is debatable
both from the standpoint of its theoretic scientific basis
and its therapeutic application.

STREPTOCOCCUS Mucosus (HOWARD AND PERKINS).

This organism, described by Howard and Perkins,** was
isolated from a case of tubo-ovarian abscess with generalized
infection, and again later by Schottm tiller ft from a case
of parametritis, peritonitis, meningitis, and phlebitis.

It occurs as a rounded coccus in pairs and in short chains,
though sometimes long chains of a hundred were observed.

* "Ann. de 1'Inst. Pasteur," t. ix, No. 7, July 25, 1895, p. 593.

t Ibid.

J "Archiv. de. med. Exper.," 1897.

§ "Deutsche med. Wochenschrift," 1903, No. 50.

|| "Berliner klin. Wochenschrift," 1902, 13.
** "Journal of Medical Research," 1901, N. S. I, 163.
ft "Munch, med. Wochenschrift," 1903, xxi.

360 Suppuration

The pairs resemble gonococci. They measure i . 25 to i . 75^ in
length and 0.5 to 0.75 ^ in breadth. Each is surrounded by
a halo that varies in width from 1.5 to 3.0 (/, which shows
best in cultures grown on human blood-serum. The usual
capsule stains fail to color this halo when the organisms are
from artificial cultures, though they show it well when they
are in pus. The organisms stain with ordinary dyes and by
Gram's method.

The cultures resemble those of Streptococcus pyogenes,
but are rather more luxuriant, the colonies having a bluish

* •

/ -

Fig. 107. — Streptococcus mucosus, from peritoneal exudate. X 1200
(Howard and Perkins, in " Journal of Medical Research ").

cast. The organism ferments inulin, which makes Hiss think
it related to the pneumococcus.

The organism taken at autopsy and inoculated into the
peritoneum of a guinea-pig caused the animal to die, coma-
tose, in thirty-six hours with peritonitis. There were 15 to
20 c.c. of peculiar viscid fluid in the peritoneal cavity. It
had a grayish purulent character and contained numerous
flakes of fibrin. There was no generalized infection. Mice
and rabbits were susceptible and died of generalized in-
fection.

The organism is not infrequently found as an apparently
harmless tenant of the human mouth, where it maybe con-
fused with the pneumococcus. It has also turned up un-
expectedly in a variety of inflammatory diseases.

Micrococcus Tetragenus 361

STREPTOCOCCUS ERYSIPELATIS (FEHLEISEN).

The streptococcus of Rosenbach is generally thought to
be identical with a streptococcus described by Fehleisen* as
Streptococcus erysipelatis .

The streptococcus of erysipelas can be obtained in almost
pure culture from the serum which oozes from a puncture
made in the margin of an erysipelatous patch. They are
small cocci, usually forming chains of from six to ten indi-
viduals, but sometimes reaching a hundred or more in num-
ber. Occasionally the chains occur in tangled masses.

They can be cultivated at the room temperature, but grow
much better at 30° to 37° C. They are not particularly sensi-
tive to the presence or absence of oxygen, but perhaps de-
velop a little more rapidly in its presence. The cultural
appearances are identical with those of Streptococcus
pyogenes.

When injected into animals Fehleisen 's coccus behaves
exactly like Streptococcus pyogenes.

MICROCOCCUS TETRAGENUS (GAFFKY).

General Characteristics. — Large, round, encapsulated cocci, regu-
larly associated in groups of four, forming tetrads. They are non-
motile, non-flagellated, non-sporogenous, non-liquefying, non-chromo-
genic, non-aerogenic, aerobic and optionally aerobic, pathogenic
for mice and other small animals, and stain well by all methods, in-
cluding that of Gram.

A large micrococcus grouped in fours and known as Micro-
coccus tetragenus can sometimes be found in normal saliva,
tuberculous sputum, and more commonly in the contents of
the cavities of tuberculosis pulmonalis. It sometimes occurs
in the pus of acute abscesses, and may be of importance in
connection with the pulmonary abscesses which complicate
tuberculosis. It was discovered by Gaffky.f

Morphology. — The cocci are rather large, measuring
about i u in diameter. In cultures they do not show the regu-
lar arrangement in tetrads as constantly as in the blood and
tissues of animals, where they occur in groups of four sur-
rounded by a transparent gelatinous capsule.

* " Verhandlungen der Wiirzburger med. Gesellschaft," 1881.
t "Archiv. f. Chirurgie," 28, 3.

362

Suppuration

Staining. — The organisms stain well by ordinary methods
and beautifully by Gram's method, by which they can be
best demonstrated in tissues.

Isolation. — The organism can be isolated by inoculating
a white mouse with sputum or pus containing it. After
death it can be recovered from the blood.

Cultivation. — It grows readily upon artificial media.
Upon gelatin plates small white colonies are produced in

Fig. 108. — Micrococcus tetragenus in spleen of infected mouse.
(From Hiss and Zinsser "Text-Book of Bacteriology," D. Appleton &
Co., Publishers.)

from twenty-four to forty-eight hours. Under the micro-
scope they appear spheric or elongate (lemon shaped), finely
granular, and lobulated like a raspberry or mulberry. When
superficial they are white and elevated, i to 2 mm. in diam-
eter.

Gelatin. — In gelatin punctures a large white surface
growth takes place, but development in the puncture is very
scant, the small spheric colonies usually remaining isolated.
The gelatin is not liquefied.

Micrococcus Tetragenus 363

Agar-agar. — Upon agar-agar spheric white colonies are
produced. They may remain discrete or become confluent.

Potato. — Upon potato a luxuriant, thick, white growth
is formed.

Blood-serum. — The growth upon blood-serum is also
abundant, especially at the temperature of the incubator.
It has no distinctive peculiarities.

Pathogenesis. — The introduction of tuberculous sputum
or of a minute quantity of a pure culture of this coccus into
white mice usually causes a fatal bacteremia in which these

Fig. 109. — Micrococcus tetragenus; colony twenty-four hours old upon
the surface of an agar-agar plate. X 100 (Heim).

organisms are found in small numbers in the heart's blood,
but are numerous in the spleen, lungs, liver, and kidneys.

Japanese mice and white mice are highly susceptible to the
organism and die three or four days after inoculation.

House-mice, field-mice, and rabbits are comparatively
immune. Guinea-pigs may die of general septic infection,
though local abscesses result from subcutaneous inoculation.

The tetracocci, when present, probably hasten the tissue-
necrosis in tuberculous cavities, aid in the formation of ab-
scesses of the lung, and contribute to the production of the
hectic fever.

An interesting contribution to the relationship of this
coccus to human pathology has been made by Lartigau,*
who succeeded in demonstrating that the tetracoccus may

*<<Phila. Med. Jour.," April 22, 1899.

364 Suppuration

be the cause of a pseudomembranous angina, 3 cases of
which came under his observation.

Bezancon* has isolated this organism from a case of menin-
gitis. Forneacaf has reported a case of generalized tetra-
genous septicemia.

BACILLUS PYOCYANEUS (GESSARD).

General Characteristics. — A minute, slender, actively motile, flag-
ellated, non-sporogenous, chromogenic and feebly pathogenic, aerobic
or facultative anaerobic, liquefying bacillus, staining by ordinary
methods, but not by Gram's method.

In some cases pus has a peculiar bluish or greenish color,
which depends upon the presence of Bacillus pyocyaneus
of Gessard.l

Distribution. — The bacillus appears to be a rather com-
mon saprophyte, being found in feces, manure, and water.

Fig. no. — Bacillus pyocyaneus, from an agar-agar culture. X 1000
(Itzerott and Niemann).

It easily takes up its residence upon the skin and mucous
membranes, and has been found in the perspiration. It
sometimes occurs as a saprophyte upon the surgical dressings
applied to wounds, and sometimes invades the tissues
through wounds, to occasion dangerous infections.

*"Semaine Medicale," 1898.

t"Riforma Medica," 1903.

J "De la Pyocyanine et de son Microbe," These de Paris, 1882.

Bacillus Pyocyaneus 365

Morphology. — It is a short, slender organism with rounded
ends, measuring 0.3 x i to 2 ^, according to Fliigge; 0.6 x
2 to 6 /w, according to Ernst, and 0.6 x i ^, according to
Charrin. It is quite pleomorphous, which probably accounts
for the difference in measurements. It is frequently united
in chains of four or six. It is actively motile, has one
terminal flagellum, and does not form spores. It can exist
without free oxygen, though it is an almost purely aerobic
organism.

It closely resembles a harmless bacillus found in water,
and known as Bacillus fluorescens liquefaciens, from which
Ruzicka* thinks it has probably descended

vi

Fig. in. — Bacillus pyocyaneus. Colonies upon gelatin (Abbott).

Staining. — It stains well with the ordinary staining solu-
tions, but not by Gram's method.

Isolation. — The isolation of the organism is simple, the
ordinary plate method being a satisfactory means of securing
it from pus or other discharges.

Cultivation. — Colonies. — The superficial colonies upon
gelatin plates are small, irregular, slightly greenish, ill-
defined, and produce a distinct fluorescence of the neigh-
boring gelatin.

Microscopic examination shows the superficial colonies to
be rounded and coarsely granular, with serrated or slightly
filamentous borders. They are distinctly green in the center
and pale at the edges. The colonies sink into the gelatin as

* "Centralbl. f. Bakt. u. Parasitenk.," July 15, 1898, p. n.

366 Suppuration

the liquefaction progresses. Four or five days must elapse
before the medium is all fluid.

Gelatin Punctures. — In gelatin puncture cultures the
chief development of the organisms occurs at. the upper
part of the tube, where a deep saucer-shaped liquefaction
forms, slowly descending into the medium, and causing a
beautiful fluorescence. At times a delicate scum forms
on the surface, sinking to the bottom as the culture ages,
and ultimately forming a slimy sediment.

Agar-agar. — Upon agar-agar the growth developing all
along the line of inoculation at first appears bright green.
The green color depends upon a soluble pigment (fluorescein)
which soon saturates the culture-medium and gives it the
characteristic fluorescent appearance. As the culture ages,
or if the medium upon which it grows contains much pep-
tone, a second blue pigment (pyocyanin) develops, and
the bright green fades to a deep blue-green, dark blue, or
in some cases to a deep reddish-brown color. This pigment
has been made the subject of a careful investigation by Jor-
dan.* Its formula, according to Ledderhose, f is C14H14N2O.

A well-known feature of the growth upon fresh agar-
agar, upon which much stress has recently been laid by
Martin, J is the formation of crystals in fresh cultures.
Crystal formation in cultures of other bacteria usually takes
place in old, partially dried agar-agar cultures, but Bacil-
lus pyocyaneus often produces crystals in a few days upon
fresh media. In my experience freshly isolated bacilli show
this power more markedly than those which have been for
some time part of the laboratory stock of cultures and
frequently transplanted.

Bouillon. — In bouillon the organism produces a diffuse
cloudiness, a fluorescence, and sometimes an indefinite thin
pellicle on the surface.

Potato. — Upon potato a luxuriant greenish or brownish,
smeary layer is produced.

Milk. — Milk is coagulated and peptonized. It is slightly
acid for the first day or two, then becomes alkaline again.

Metabolic Products. — Apart from the pyocyanin and
fluorescin, the former blue, the latter green, cultures of this
organism frequently turn red brown. This suggested the

* "Journal of Experimental Medicine," vol. iv, 1899.
t" Deutsche Zeitschr. f. Chirurgie," 1888, Bd. xxvm.
t "Centralbl. f. Bakt.," xxi, April 6, 1897, p. 473.

Bacillus Pyocyaneus 367

formation of a third pigment, but the work of Boland * has
shown this to be a transformation product of pyocyanin
common in old cultures.

The organism produces a curdling ferment, a fibrin- and
casein-dissolving ferment, a gelatin-dissolving ferment,
and a bacteriolytic ferment, the pyocyanase of Emmerich
and Low.

It also produces, under favorable conditions, a toxin
which has been studied by Wassermann, who found it fatal
in doses of 0.2 to 0.5 c.c. when intraperitoneally injected into
guinea-pigs. The animals show peritonitis and punctiform
hemorrhages on the serous membranes.

Bullock and Hunter f found that Bacillus pyocyaneus also
produces a hemolytic substance, pyocyanolysin, by which
corpuscles of man, oxen, sheep, apes, rabbits, cats, rats,
dogs, and mice are dissolved. The peculiar substance was
produced in greatest quantity in virulent cultures three or
four weeks old. JordanJ believes that this hemolytic
property of the cultures depends solely upon the intense
alkali formed in old pyocyaneus cultures. Gheorghewski §
found a leukocyte-destroying substance in the cultures.

In addition to the metabolic pigments mentioned, the or-
ganism produces toxins. Wassermann || found that filtrates
of old cultures were more toxic for guinea-pigs than the endo-
toxins made by lysis of dead bacteria. The organism thus
produces both endo- and exotoxins.

Pathogenesis. — The bacillus is pathogenic for the small
laboratory animals, but different cultures differ greatly
in virulence. One c.c. of a virulent bouillon culture, injected
into the subcutaneous tissue of a guinea-pig or a rabbit,
caused rapid edema, suppurative inflammation, and death
in a short time (twenty-four hours). Sometimes the animal
lives for a week or more, then dies. There is a marked
hemorrhagic subcutaneous edema at the seat of inoculation.
The bacilli can be found in the blood and in most of the
tissues. The guinea-pig is most susceptible.

Doses too small to prove fatal sometimes lead to suppu-
ration, and the injection of sterilized cultures leads to simi-
lar results, a relatively larger quantity being required.

* "Centralbl. f. Bakt.," Bd. xxv, 1899, p. 897.

t Ibid., xxviu, 1900, p. 865. | Ibid., Bd. xxxin, Ref. 1903.

§ "Ann. de 1'Inst. Pasteur," 1899, xin.

|| "Zeitschrift fur Hygiene, 1896, xxn.

368 Suppuration

Intraperitoneal injections cause purulent peritonitis.

Blum* reports a case of pyocyaneus infection with endo-
carditis in a child.

Lartigau,f in his study of " The Bacillus Pyocyaneus as
a Factor in Human Pathology," sums up what is known
about this role of the organism as follows: " The Bacillus
pyocyaneus, like many pathogenic micro-organisms, is
occasionally found in a purely saprophytic role in various
situations in the human economy. It has been found in the
saliva by Pansini, in sputum by Frisch, and in the sweat by
Kberth and Audanard. Abelous demonstrated its presence
in the stomach as a saprophyte. Its existence in suppurat-
ing wounds has long been known, and Koch early detected
its presence in tuberculous cavities, regarding it as an
organism incapable of playing any pathologic role. The
etiologic relation of the organism to certain cases of purulent
otitis media in children was pointed out by Martha, Mag-
giora and Gradenigo, Babes, Kossel, and others. H. C.
Ernst obtained it from a pericardial exudate during life. G.
Blumer demonstrated its presence in practically pure cul-
tures in a case of acute angina simulating diphtheria;
Jadkewitsch, B. Motz, and Le Noir obtained the bacillus
in cases of urinary infection. The cases of Triboulet,
Karlinski, Oettinger, Bhlers, and Barker are interesting
instances of its role in cutaneous lesions.

" In addition to these lesions, other morbid processes
have been associated in some cases with the bacillus of
blue pus, such as meningitis and bronchopneumonia, by
Monnier; diarrhea of infants, by Neumann, Williams, Thier-
celin and Lesage, and other observers; dysentery, by Cal-
mette and by Lartigau; and general infection, by Khlers,
Neumann, Oettinger, Karlinski, Monnier, Krannhals, Cal-
mette, Finkelstein, and I,. F. Barker."

Nine additional cases of human infection are reported by
Perkins. {

Immunity. — Immunity against pyocyaneus infection de-
velops after a few inoculations with attenuated or sterilized
cultures. These are easily prepared, the thermal death-point
determined by Sternberg being 56° C. It also follows in-
jection of either the endotoxin or the exotoxin. In the

* "Centralbl. f. Bakt. u. Parasitenk.," Feb. 10, 1899, xxv, No. 4.

t "Phila. Med. Jour.," Sept. 17, 1898.

t "Jour, of Med. Research," vol. vi, 1901, p. 281.

Bacillus Proteus Vulgaris 369

immunity resulting from the treatment with killed cultures
the serum of the animal becomes agglutinative and bac-
tericidal ; in the immunity resulting from treatment with the
exotoxin, antitoxin is produced.

BACILLUS PROTEUS VULGARIS (HA USER).

General Characteristics. — An actively motile, flagellated, non-
sporogenous, non-chromogenic, liquefying, aerobic and optionally
anaerobic, doubtfully pathogenic, aerogenic bacillus, easily cultivated
on artificial media and readily stained by the ordinary methods, though
not by Gram's method.

This bacillus was first found by Hauser* in decomposing
animal infusions, usually in company with two closely allied
forms, Proteus mirabilis and Proteus zenkeri, which, as the
experiments and observations of Sanfelice and others show,
may be identical with it. According to Kruse, it is quite
probable that the mixed species formerly called Bacterium
termo was largely made up of the proteus.

Distribution. — The organism is a common saprophyte
and is very abundant in water, earth, and air. It is to be
expected wherever putrefactive change is in progress. It is
a common mistake for the novice to look upon it as a member
of the Bacillus coli group.

Morphology. — The bacilli are variable in size and shape —
pleomorphic — and are named proteus from this peculiarity.
Some differ very little from cocci, some are more like the
colon bacillus in shape, others form long filaments, and oc-
casional spirulina forms are met with. True spirals are
never found. All of the forms mentioned may be found in
pure cultures of the same organism. The diameter of the
bacillus is usually about 0.6 /w, but the length varies from 1.2
fi or less to 4 f* or more. No spores are formed. The organ-
isms are actively motile. The long filaments frequently form
loops and tangles. Flagella are present in large numbers.
Upon one of the long bacilli as many as one hundred have
been counted. Involution forms are frequent in old cultures.

Staining. — The bacilli stain well by the ordinary methods.
Gram's method usually fails.

Cultivation. — The proteus is easily cultivated and grows
well in all the artificial media.

Colonies. — Upon gelatin plates a typical phenomenon is

*"Ueber Faulnissbakterien," Leipzig, 1885.
24

370

Suppuration

observed in connection with the development of the colonies,
for the most advantageous observation of which the medium
used for making the cultures should contain 5 instead of 10
per cent, of gelatin. Kruse * describes the phenomenon as
follows: "At the temperature of the room, rounded, saucer-
shaped depressions, with a whitish central mass surrounded
by a lighter zone, are quickly formed. Under low magni-
fication the center of each is seen to be surrounded by
radiations extending in all directions into the solid gelatin,
and made up of chains of bacilli. Between the radiations

Fig. 112. — Swarming islands of proteus bacilli on the surface of gelatin;
X 650 (Hauser).

and the granular center bacteria are seen in active motion.
Upon the surface the colony extends as a thin patch, con-
sisting of a layer of bacilli arranged in threads, sending
numerous projections from the periphery. Under certain
conditions the wandering of the processes can be directly
observed under the microscope. It depends not only upon
the culture-medium, but, in part, upon the culture itself.
Entire groups of bacilli or single threads, by gradual exten-
sion and circular movement, detach themselves from the
colony and wander about upon the plate. From the radi-

* Flugge's " Die Mikroorganismen."

Bacillus Proteus Vulgaris 371

ated central part of the colony peculiar zooglea are formed,
having a sausage or screw shape, or wound in spirals like
a corkscrew. The younger colonies, which have not yet
reached the surface of the gelatin, are more compact, rounded
or nodular, later covered with hair-like projections, and
becoming radiated like the superficial colonies."

If the culture-medium be concentrated, or the culture
has been frequently transplanted, the phenomenon is less
marked and may not occur.

Bouillon. — In this medium the organism grows rapidly,
and quickly clouds the fluid. A pellicle soon forms upon the
surface and a mucilaginous sediment occurs later.

Gelatin Punctures. — Puncture cultures in gelatin are not
characteristic. A stocking-like liquefaction of the gelatin
extends so rapidly that the entire gelatin is liquefied in a few
days. Anaerobic cultures do not liquefy.

Agar-agar. — Upon agar-agar the bacillus forms a moist,
thin, transparent, rapidly extending layer which rarely
reaches the sides of the tube. Upon agar-agar plates ame-
boid movement of the colonies may also occur.

Potato. — Upon potato the growth occurs in the form of
a smeary patch of soiled appearance.

Milk is coagulated.

Metabolic Products. — The bacillus usually produces
alkalies. Indol and phenol are formed from the peptone of
the culture-media. Nitrates are reduced to nitrites, and
then partly reduced to ammonia. In most culture-media
not containing sugar the bacillus produces a very disagree-
able odor.

In culture-media containing either grape- or cane-sugar
fermentation occurs both in the presence and in the absence
of oxygen. Milk-sugar is not decomposed.

Pathogenesis. — It is a question whether or not Bacillus
proteus is to be ranked among the pathogenic bacteria.
Small doses are harmless for the laboratory animals; large
doses produce abscesses. A toxic substance resulting from
the metabolism of the organism seems to be the cause of
death when considerable quantities of a culture are injected
into the peritoneal cavity or blood-vessels. The bacilli do
not seem able to multiply in the healthy animal body, but
can do so when previous disease or injury of its tissues has
taken place.

The proteus has been secured in cultures from wound and

372 Suppuration

puerperal infections, purulent peritonitis, endometritis, and
pleurisy. When the local lesion is limited, as in endometritis,
the danger of toxemia is slight ; but when widespread, as the
peritoneum, it may prove serious. Bacillus proteus has also
been found in acute infectious jaundice and in acute febrile
icterus, or Weil's disease.

Bordoni-Uffredizzi has shown that the proteus quite
regularly invades the tissues after death, though it ap-
pears unable to maintain an independent existence in the
tissues during life, and is probably of importance only when
present in association with other bacteria. It at times
grows abundantly in the urine, and may produce primary
inflammation of the bladder. The inflammatory process
may also extend from the bladder to the kidney, and so
prove quite serious.

Epidemics of meat-poisoning have been thought to depend
upon Bacillus proteus. One of them was studied by Wesen-
berg, * who cultivated the organism from the putrid meat by
which 63 persons were made ill. Sil verschmidt f and PfuhlJ
have made similar investigations with similar results.

AND SUPPURATION.

The process of suppuration is not confined to bacterial
micro-organisms, but is shared to a limited extent by the
protozoa. Thus, Bntamoeba histolytica (q. v.) is, to all
appearances, the sole excitant of the abscesses of the liver
secondary to dysentery. It is true that these are cold
abscesses and necrotic rather than distinctly purulent in
character, yet it seems best to speak of the organism in this
connection.

Bntamoeba buccalis (Prowazek§) is a small ameba that
has been found in purulent exudates in the oral tissues of
persons with carious teeth.

Amoeba kartulisi (Doflein||) appears to be capable of ex-
citing suppuration. It was found by Kartulis in the pus
from an abscess of the right side of the lower jaw. The
patient was a man aged forty-three years who had been

* "Zeitschrift fur Hygiene," etc., 1898, xxvm.

t Ibid., 1899, xxx.

J Ibid., 1900, xxxv.

§ "Arbeitena. d. Kaiserl. Gesundh.-Amt.," xxi, i, Bull. 1904, p. 42.

|| "Die Protozoa als Krankheitserreger," Jena., 1901, p. 30.

Miscellaneous Organisms Described Elsewhere 373

operated upon for the removal of a piece of bone. It is 30
to 38 {*. in diameter, is actively motile. Its coarse protoplasm
contains red and white blood-corpuscles. Kartulis* found
the same organism five times in other cases, and Flexnerf
found it also.

Amoeba mortinatalium, described by Smith and Weidman,|
was found in distributed small purulent foci in the kidneys
and other organs of a still-born fetus.

MISCELLANEOUS ORGANISMS OF SUPPURATION DESCRIBED
MORE FULLY ELSEWHERE.

Before leaving the subject, attention must be directed
to other bacteria that under exceptional circumstances
become the cause of suppuration. Among these are the
pneumococcus of Frankel and Weichselbaum, the typhoid
bacillus, and the Bacillus coli communis. These organisms
are considered under separate and appropriate headings, to
which the reader is advised to refer.

* "Centralbl. f. Bakt. u. Parasitenk." 1903, xxxni, p. 471.

t "Bulletin of the Johns Hopkins Hospital," 1892, xxv.

I "University of Pennsylvania Medical Bulletin," Sept., 1910.

CHAPTER II.
MALIGNANT EDEMA.

BACILLUS (EDEMATIS MALIGNI (KOCH).

General Characteristics. — A motile, flagellated, sporogenous, an-
aerobic, liquefying, aerogenic, non-chromogenic, pathogenic bacillus of
the soil, readily stained by the ordinary methods, but not by Gram's
method.

This organism was originally found by Pasteur* in putres-
cent animal infusions and called -by him (1875) Vibrion
septique. It was later more carefully studied and described
by Koch.f

Distribution. — The organism is widely distributed in
nature, being commonly present in garden earth. It is also
found in dust, in waste water from houses, and sometimes in
the intestinal canals of animals.

Morphology. — The bacillus of malignant edema is a large
rod-shaped organism with rounded ends, measuring 2 to 10 /w
by 0.8 to i.o [i. It is usually motile, and possesses many
flagella. It produces oval endospores centrally situated and
giving a barrel shape to the parent bacillus.

Staining. — The bacillus stains well with ordinary cold
aqueous solutions of the anilin dyes, but not by Gram's
method.

Cultivation. — The organism is a strict anaerobe, but under
conditions by which provision is made for the removal of
oxygen, grows well both at the room temperature and at that
of the incubator. It is not difficult to secure in pure culture,
being most easily obtained from the edematous tissues of
guinea-pigs and rabbits inoculated with garden earth.

The colonies which develop upon the surface of gelatin
kept under anaerobic conditions appear to the naked eye
as small shining bodies with liquid, grayish-white contents.
Under the microscope they appear filled with a tangled
mass of long filaments which under a high power exhibit

* "Bull. Acad. Med.," 1877 and 1881.

t "Mittheilungen aus dem kaiserl. Gesundheitsamte," i, 53.
374

Metabolic Products

375

active movement. The edges of the colony have a fringed
appearance, much like the colonies of the hay or potato ba-
cillus.

In gelatin and agar-agar tube cultures the characteristic
growth cannot be observed in a puncture, because of the air
which remains in the path of the wire, unless the tube be
placed under anaerobic conditions. The best preparation,
therefore, is made by heating the gelatin to expel any air it
may contain, inoculating it while still liquid, and solidifying
it in cold (iced) water. In such a tube the bacilli develop
in globular circumscribed areas of cloudy liquefaction

: •>

Fig. 1 13. — Bacillus of malignant edema, from the body- juice of a guinea-
pig inoculated with garden earth. X 1000 (Frankel and Pfeiffer).

which contain a small amount of gas. In gelatin to which a
little grape-sugar has been added the gas production is
marked. The gas is partly inflammable, partly not. A dis-
tinct odor accompanies the gas production, and is especially
noticeable in agar-agar cultures. In bouillon diffuse cloud-
ing occurs, followed by the formation of a sediment. No
surface growth occurs. Milk is slowly coagulated. It grows
well upon the surface of potato and blood-serum under con-
ditions of strict anaerobiosis.

Metabolic Products. — Of the toxic products of the organ-
ism nothing definite is known. It decomposes albumin, form-
ing fatty acids, leucin, hydroparacumaric acid, and an oil

376

Malignant Edema

with an offensive odor. Among the gases formed, carbonic
acid, hydrogen, and marsh gas have been detected.

Pathogenesis. — When introduced beneath the skin, the
bacillus is pathogenic for a large number of animals — mice,
guinea-pigs, rabbits, horses, dogs, sheep, goats, pigs, calves,
chickens, and pigeons. Cattle seem to be immune.

Giinther points out that the simple inoculation of the
bacillus upon an abraded surface is insufficient to produce
infection, because the presence of oxygen is detrimental to
its growth. When the bacilli are deeply introduced beneath
the skin, infection occurs.

Mice, guinea-pigs, and rabbits sicken and die in about
forty-eight hours.

Fig. 114. — Bacillus cedematis, dextrose gelatin culture (Giinther).

Lesions. — In the blood the bacilli are few because of the
loosely combined oxygen it contains. The great majority
of the bacilli occupy the subcutaneous tissue, where very
little oxygen is present and the conditions of growth are
good. The autopsy shows a marked subcutaneous edema
containing immense numbers of the bacilli. If the animal
be permitted to remain undisturbed for some time after
death, the bacilli spread to the circulatory system and reach
all the organs.

Brieger and Ehrlich* have reported 2 cases of malignant
edema in man. Both occurred in typhoid fever patients
subcutaneously injected with musk, the infection no doubt
resulting from impurities in the therapeutic agent.

* "Berliner klin. Wochenschrift," 1882, No. 44.

I

Immunity 377

Grigorjeff and Ukke* have observed another interesting
case of typhoid fever with intestinal ulcerations, through
which infection by the bacillus of malignant edema took place.
The case was characterized by interstitial emphysema of
the subcutaneous tissue of the neck and breast, gas bubbles
in the muscles, and a transformation of the entire liver into
a spongy porous mass of a grayish-brown color. The spleen was
enlarged and soft, and contained a few gas-bubbles. Though
the writers consider this organism to be the bacillus of malig-
nant edema, the general impression one receives from the
description of the lesions suggests that it was Welch's Bacillus
aerogenes capsulatus.

No case is reported in which healthy men have been in-
fected with malignant edema.

Immunity. — Cornevin found that the passage of the bacil-
lus through white rats diminished its virulence, and that the
animals of various species that recovered were immune
against the virulent organisms. Roux and Chamberlandf
found that the filtered cultures were toxic and that animals
could be immunized by injection with this toxic filtrate.

GASEOUS EDEMA.

BACILLUS AEROGENES CAPSULATUS (WELCH).

General Characteristics. — A large, stout, non-motile, non-flagel-
lated, sporogenous, non-chromogenic, purely anaerobic, markedly aero-
genie, doubtfully pathogenic bacillus, easily cultivated in artificial
media, readily stained by the ordinary methods and by Gram's method.

This disease is caused by an interesting micro-organism
described by Welch, and subsequently studied by Welch
and Nuttall, { Welch and Flexner,,§ and others. Welch said
at the meeting of the Society of American Bacteriologists held
at Philadelphia, December 30, 1904, that he believed this or-
ganism to be identical with Kline's Bacillus enteritidis sporo-
genes, and that it belongs to the butyric acid group. It is
probably also identical with Bacillus phlegmone emphysema-
tose of Frankel.il It was first secured by Welch from the

* " Militar-medizin. Jour.," 1898, p. 323.
t "Ann. de 1'Inst. Pasteur," 1887.

f'Bull. of the Johns Hopkins Hospital," July and Aug., 1892,
vol. viii, No. 24.

§ "Jour, of Experimental Medicine," Jan., 1896, vol. i, No. i, p. 6.
|| "Centralbl. f. Bakt.," etc., Bd. xm, p. 13.

378 Gaseous Edema

body of a man dying suddenly of aortic aneurysm with a pe-
culiar gaseous emphysema of the subcutaneous tissues and in-
ternal organs, and a copious formation of gas in the blood-
vessels. The blood was thin and watery, of a lac color, and
contained many large and small gas bubbles, and many
bacilli, which were also obtained from it and the various
organs, especially in the neighborhood of the gas bubbles,
in nearly pure culture. The coloring-matter of the blood
was dissolved out of the corpuscles and stained the tissues
a deep red.

Distribution. — The organism is apparently of wide dis-
tribution. It is believed that the natural habitat of the

Fig. 115. — Bacillus aerogenes capsulatus (from photograph by Prof.
Simon Flexner).

bacillus is the soil, but there is reason to think that it com-
monly occurs in the intestine, and may occasionally be found
upon the skin.

Morphology. — The bacillus is a large organism, measuring
3-5 p. in length, about the thickness of the anthrax bacillus,
with ends slightly rounded, or, when joined, square (Fig.
115). It occurs chiefly in pairs and in irregular groups, but
not in chains, in this particular differing from the anthrax
bacillus. In culture media it is usually straight, with slightly
rounded ends. In old cultures the rods may be slightly
bent, and involution forms occur. The bacillus varies some-
what in size, especially in length, in different culture-media.

Staining 379

It usually appears thicker and more variable in length in
artificial cultures than in the blood of animals.

The bacillus is not motile and has no flagella. Hndo-
spores are formed upon Loffler's blood-serum.

It was at first thought that the bacillus produced no spores,
but Dunham* found that spores were produced upon blood-
serum, and especially upon Loffler's blood-serum bouillon
mixture. The spores resist desiccation and exposure to the
air for ten months. They stain readily in hot solutions of
fuchsin in anilin water, and are not decolorized by a mod-
erate exposure to the action of 3 per cent, solution of
hydrochloric acid in absolute alcohol. They are oval, and
are usually situated near the middle of the bacillus, which
is distended because of the large size of the spore and bulges
at the sides.

Staining. — The organism stains well with the ordinary
stains, and retains the color well in Gram's method. When
stained with methylene-blue a granular or vacuolated ap-
pearance is sometimes observed, due to the presence of un-
stained dots in the cytoplasm.

Usually in the body-fluids and often in cultures the bacilli
are surrounded by distinct capsules — clear, unstained zones.
To demonstrate this capsule to the best advantage, Welch
and Nuttall devised the following special stain:

A cover is thinly spread with the bacilli, dried, and fixed
without overheating. Upon the surface prepared, glacial
acetic acid is dropped for a few moments, then allowed to
drain off, and at once replaced by a strong aqueous solution
of gentian violet, which is poured off and renewed several
times until the acid has been replaced by the stain. The
specimen is then examined in the coloring solution, after
soaking up the excess with filter-paper, the thin layer of
coloring fluid not interfering with a clear view of the bac-
teria and their capsules. After mounting in Canada balsam
the capsules are not nearly so distinct. The width of the
capsule varies from one-half to twice the thickness of the
bacillus. Its outer margin is stained, leaving a clear zone
immediately about the bacillus.

The bacillus is anaerobic and aerogenic. It grows upon
all culture-media at the room temperature, though better
at the temperature of incubation.

* "Bull, of the Johns Hopkins Hospital," April, 1897, p. 68.

38o

Gaseous Edema

r

Cultivation. — Gelatin. — It grows in ordinary neutral or
alkaline gelatin, but better in gelatin
containing glucose, in which the char-
acteristic gas production is marked.
Soft media, made with 5 instead ot
10 per cent, of the crude gelatin, is
said to be better than the standard
preparation.

There is no distinct liquefaction of
the medium, but in 5 per cent, gelatin
softening can sometimes be demon-
strated by tilting the tube and observ-
ing that the gas bubbles change their
position, as well as by noticing that
the growth tends to sediment.

Agar-agar. — In making agar-agar
cultures careful anaerobic precautions
must be observed. The tubes should
contain considerably more than the
usual quantity of the medium, which
should be boiled and freshly solidified
before using. The implantation should
be deeply made with a long wire. The
growth takes place slowly unless such
tubes are placed in a Buchner's jar or
other anaerobic device. The deeper
colonies are the largest. Sometimes
the growth only takes place within
10— 12 mm. of the surface; at others,
within 3-4 cm. of it. After repeated
cultivation the organisms seem to
become accustomed to the presence
of oxygen, and will grow higher up in
the tube than when freshly isolated.

Colonies. — The colonies seen in the
culture-media are grayish-white or
brownish- white by transmitted light,
and sometimes exhibit a central dark
dot. At the end of twenty-four hours
the larger colonies do not exceed 0.5-
i.o mm. in diameter, though they
may subsequently attain a diameter
of 2—3 mm. or more. Their first appearance is as little

Fig. 1 1 6. — Bacillus
aerogenes capsulatus,
with gas production
(from photograph by
Prof. Simon Flexner).

Cultivation 381

spheres or ovals, more or less flattened, with irregular con-
tours, due to the presence of small projecting prongs, which
are quite distinct under a lens. The colonies may appear as
little irregular masses with projections.

After several days or weeks, single, well-shaped colonies
may attain a large size and be surrounded by projections,
either in the form of little knobs or spikes or of fine branch-
ings— hair-like or feathery. Their appearance has been
compared to thistle-balls or powder-puffs and to thorn-apples.
When the growth takes place in the puncture, the feathery
projections are continuous. Bubbles of gas make their ap-
pearance in plain agar as well as in sugar-agar, though, of
course, less plentifully. They first appear in the line of
growth; afterward throughout the agar, often at a distance
from the actual growth. Any fluid collecting about the bub-
bles or at the surface of the agar-agar may be turbid from
the presence of bacilli. The gas-production is more abun-
dant at 37° C. than at the room temperature.

The agar-agar is not liquefied by the growth of the bacillus,
but is often broken up into fragments and forced into the
upper part of the tube by the excessive gas-production.

In its growth the bacillus produces considerable acid.

Bouillon. — In bouillon growth does not occur in tubes
exposed to the air, but when the tubes are placed in Buchner's
jars, or kept under anaerobic conditions, it occurs with abun-
dant gas-formation, especially in glucose-bouillon, with the
formation of a frothy layer on the surface. The growth is
rapid in development, the bouillon becoming clouded in two
to three hours. After a few days the bacilli sediment and
the bouillon again becomes clear. The reaction of the bouil-
lon becomes strongly acid.

Milk. — In milk the growth is rapid and luxuriant under
anaerobic conditions, but does not take place in cultures
exposed to the air. The milk is coagulated in from twenty-
four to forty-eight hours, the coagulum being either uniform
or firm, retracted, and furrowed by gas bubbles. When
litmus has been added to the milk, it becomes decolorized
when the culture is kept without oxygen, but turns pink
when it is exposed to the air.

Potato. — The bacillus will also grow upon potato when the
tubes are inclosed in an anaerobic apparatus. There is a
copious gas-development in the fluid at the bottom and sides
of the tube, so that the potato becomes surrounded by a

382 Gaseous Edema

froth. After complete absorption of the oxygen a thin,
moist, grayish-white growth takes place upon the surface of
the medium.

Vital Resistance. — The vital resistance of the organism
is not great. Its thermal death-point was found to be 58°
C. after ten minutes' exposure. Cultures made by displacing
the air with hydrogen are less vigorous than those in which
the oxygen is absorbed from the air by pyrogallic acid. It
was found that in the former class of cultures the bacillus
died in three days, while in the absorption experiments it
was kept alive at the body temperature for one hundred and
twenty-three days. It is said to live longer in plain agar
than in sugar-agar. To keep the cultures alive it has been
recommended to seal the agar-agar tube after two or three
days' growth.

Pathogenesis. — The pathogenic powers of the bacillus are
limited, and while in some infected cases it seems to be the
cause of death, its power to do mischief in the body seems to
depend entirely upon the pre-existence of depressing and
devitalizing conditions predisposing to its growth.

Being anaerobic, the bacilli are unable to live in the cir-
culating blood, though they grow in old clots and in cavities,
such as the uterus, etc., where little oxygen enters, and from
which they enter the blood and are distributed.

In support of these views Welch and Nuttall show that
when a healthy rabbit is injected with 2.5 c.c. of a fresh
sugar-bouillon into the ear-vein, it usually recovers without
any evident symptoms. After similar injection with but i
c.c. of the culture, a pregnant rabbit carrying two dead
embryos, died in twenty-one hours. It seems that the
bacilli were first able to secure a foothold in the dead em-
bryos, and there multiplied sufficiently to bring about the
subsequent death of the mother.

After death, when the blood is no longer oxygenated, the
bacilli grow rapidly, with marked gas-production, which in
some cases is said to cause the body to swell to twice its
natural size. The effect upon guinea-pigs does not differ
from that upon rabbits, though gaseous phlegmons are
sometimes produced.

Pigeons, when subcutaneously inoculated in the pectoral
region, frequently succumb. Following the injection gas-
production causes the tissues of the chest to become emphy-

Pathogenesis 383

sematous. The birds usually die in from seven to twenty-
four hours, but may recover.

Intraperitoneal inoculation of animals sometimes causes
fatal purulent peritonitis.

Sources of Infection. — The infection seen in man usually
occurs from wounds into which earth has been ground,
as in the case of a compound, comminuted fracture of the
humerus, with fatal infection, reported by Dunham, or in
wounds and injuries in the neighborhood of the perineum.

Among the twenty-three cases reported by Welch and
Flexner * we find wounds of the knee, leg, hip, and forearm,
ulcer of the stomach, typhoid ulcerations of the intestine,
strangulated hernia with operation, gastric and duodenal
ulcer, perineal section, and aneurysm, as conditions in which
external or gastro-intestinal infection occurred.

Dobbin, f P. Ernst, { Graham, Stewart and Bald win, § and
Kronig and Menge|| have studied cases of puerperal sepsis
and sepsis following abortion either caused by the bacillus,
or in which it played an important role.

Williams ** has found the bacillus in a case of suppura-
tive pyelitis.

The symptoms following infection are quite uniform, con-
sisting of redness and swelling of the wound, with rapid
elevation of temperature and rapid pulse. The wound usu-
ally becomes more or less emphysematous, and discharges a
thin, dirty, brownish, offensive fluid that contains gas bubbles
and is sometimes frothy. The patients occasionally recover,
especially when the infected part can be amputated, but
death is the common outcome. After death the body begins
to swell almost immediately and may attain twice its normal
size and be unrecognizable. Upon palpation a peculiar crep-
itation can be felt in the subcutaneous tissue nearly every-
where, and the presence of gas in the blood-vessels is easy of
demonstration. The gas is inflammable, and as the bubbles
ignite explosive sounds are heard.

At the autopsy the gas bubbles are found in most of the
internal organs, sometimes so numerously as to justify the

* "Journal of Experimental Medicine," vol. I, No. 1, Jan., 1896.

t"Bull. Johns Hopkins Hospital/' Feb., 1897, No. 71, p. 24.

J "Virchow's Archiv," Bd. cxxxm, Heft 2.

§ "Columbus Med. Jour.," Aug., 1893.

|| " Bakteriologie des weiblichen Genitalkanals," Leipzig, 1897.

** "Bull. Johns Hopkins Hospital," April, 1896, p. 66.

384 Gaseous Edema

German term " Schaumorgane " (frothy organs). The liver
is especially apt to show this condition. When such tissues
are hardened and examined microscopically, the bubbles
appear as spaces in the tissue, their borders lined with large
numbers of the bacillus. There are also clumps of bacilli
without gas bubbles, but surrounded by tissue, whose
nuclei show a disposition to fragment or disappear, and
whose cells and fibers show signs of disintegration and
fatty change. In discussing these changes Ernst concluded
that they were ante-mortem and due to the irritation caused
by the bacillus. The gas-production he regards as post-
mortem.

In the internal organs the bacillus is usually found in pure
culture, but in the wound it is usually mixed with other bac-
teria. On this account it is difficult to estimate just how
much of the damage before death depends upon the activity
of the gas bacillus. That gas-production after death has
nothing to do with pathogenesis during life is shown by
injecting into the ear-vein of a rabbit a liquid culture of the
gas bacillus, permitting about five minutes' time for the dis-
tribution of the bacilli throughout the circulation, and then
killing the rabbit. In a few hours the rabbit will swell and
its organs and tissues be riddled with the gas bubbles.

At times, however, as in a case of Graham, Stewart and
Baldwin, there is no doubt but that the bacillus produces
gas in the tissues of the body during life. These observers,
in a case of abortion with subsequent infection, found the
patient "emphysematous from the top of her head to the
soles of her feet" several hours before death.

In this case, in which the bacillus was found in pure culture,
it would indeed be difficult to doubt that the fatal issue was
due to Bacillus aerogenes capsulatus.

Probably the best review of the subject is to be found in
"A Contribution to the Knowledge of the Bacillus Aerog-
enes Capsulatus," by W. T. Howard, Jr.*

* "Contributions to the Science of Medicine by the Pupils of W. H.
Welch," 1900, p. 461.

CHAPTER III.
TETANUS*

BACILLUS TETANI (FLUGGE).

General Characteristics. — A motile, flagellated, sporogenous,
liquefying, obligatory anaerobic, non-chromogenic, toxic, pathogenic
bacillus of the soil, staining by ordinary methods and by Gram's
method. Its chief morphologic characteristic is the occurrence of a
large round spore at one end.

The bacillus of tetanus was discovered by Nicolaier * in
1884, and obtained in pure culture by Kitasatof in 1889.
It is universally acknowledged to be the cause of tet-
anus.

Distribution. — The tetanus bacillus is a common sapro-
phyte in garden earth, dust, and manure, and is a constant
parasite in the intestinal canal of herbivorous animals.

The relation of the bacillus to manure is interesting, but
it is most probable that manured ground, because it is
richer, permits the bacilli to flourish better than sterile
ground. The common occurrence of the bacilli in the
excrement of herbivorous animals is to be explained through
the accidental ingestion of earth with the food cropped from
the ground. The spores of the bacillus thus reaching the
intestine, seem able to develop because of appropriate
anaerobic conditions.

Verneuil has observed that tetanus rarely occurs at sea
except upon cattle transports, where there is likely to be
considerable earth and dust from hay, straw, etc., which
may carry the bacilli.

Le Dantec t has shown that the tetanus bacillus is a
common organism in New Hebrides, where the natives poison
their arrows by dipping them into a clay rich in tetanus
bacilli.

*" Deutsche med. Wochenschrift," 1884, 42.

t/Wd., 1889, No. 31.

J See abstracts in the "Centralbl. f. Bakt. u. Parasitenk.," IX.
286; xm, 351.

25 385

386 Tetanus

Morphology. — The tetanus bacillus is a long, slender
organism measuring 0.3 to 0.5 X 2 to 4^ (Fliigge). Its most
striking characteristic is an enlargement of one end, which
contains a large round spore. The bacilli in which no
spores are yet formed have rounded ends and seldom unite
in chains or pairs. They are motile and have many flagella
arising from all parts of the surface (petrichia).

Staining. — The bacilli stain readily with ordinary aqueous
solutions of the anilin dyes and by Gram's method.

Fig. 117.— Bacillus tetani. X 1000 (Frankel and Pfeiffer).

Isolation. — The method usually employed for the isolation
of the tetanus bacillus was originated by Kitasato, and based
upon the observation that its spores can resist exposure to
high temperatures for considerable periods of time. After
finding by microscopic examination that the bacilli were
present in pus, Kitasato spread it upon the surface of an
ordinary agar-agar tube and incubated it for twenty-four
hours, during which time all of the contained micro-organ-
isms, including the tetanus bacillus, increased in number.
He then exposed it for an hour to a temperature of 80° C.,
by which all fully developed bacteria, tetanus as well as the
others, and the great majority of the spores, were destroyed.
As scarcely anything but the tetanus spores remained alive,
their subsequent growth gave a fairly pure culture.

Cultivation. — The tetanus bacillus is difficult to culti-

Cultivation

387

vate because it will not grow where the smallest amount
of free oxygen is present. It is hence a typical obligatory
anaerobe. Farran * and Grixoni believe it to have originally
been an optional anaerobe, and it is said by these writers
that the organism can gradually be accustomed to oxygen
so as to grow in its presence. When this is achieved, it
loses its virulence.

Fig. 1 1 8.— Bacillus tetani; six- Fig. 119. — Bacillus tetani; cul-

days-old puncture culture in glu- ture four days old in glucose-
cose-gelatin(FrankelandPfeiffer). gelatin (Frankel and Pfeiffer).

The methods for excluding the oxygen from the cul-
tures and replacing it by hydrogen, as well as other methods
suggested for the cultivation of the strictly anaerobic
organisms, are given under the appropriate heading (Anae-
robic Cultures), and need not be repeated here.

* "Centralbl. f. Bakt. u. Parasitenk.," July 15, 1898, p. 28.

388 Tetanus

Park,* following the suggestion of Kitasato, covers the
surface of the bouillon with a layer of paraffin about i to 2 cm.
thick. This melts in the sterilization and forms a firm layer,
through which the bouillon is inoculated, warmed until
the paraffin melts again, then stood away until develop-
ment in the air-free bouillon occurs. If the paraffin be
found too brittle, some albalene may be mixed with it until
it is flexible when cool.

The colonies of the tetanus bacillus, when grown upon
gelatin plates in an atmosphere of hydrogen, resemble those

Fig. 120. — Bacillus tetani; five-day-old colony upon gelatin-containing
glucose. X 1000 (Frankel and Pfeiffer).

of the well-known hay bacillus. There is a rather dense,
opaque central mass surrounded by a more transparent
zone, the margins of which consist of a fringe of radially
projecting bacilli. Liquefaction occurs slowly.

Bouillon. — The organism can be grown in bouillon, and
attains its maximum development at a temperature of
37° C. Gas is given off from the cultures, and they have a
peculiar odor, very characteristic, but difficult to describe,
The bouillon is clouded and contains a sediment.

* "Jour. Med. Research," N. S., vol. i, No. i, p. 298.

Vital Resistance

389

In bouillon containing sugar considerable gas is formed in
the fermentation tube. Both CO2 and
H2S are formed

Gelatin. — The growth occurs deep
in the puncture, and is arborescent.
Liquefaction begins in the second
week and causes the disappearance of
the radiating filaments. The lique-
faction spreads slowly, but may in-
volve the entire mass of gelatin and
resolve it into a grayish-white syrupy
liquid, at the bottom of which the
bacilli accumulate. The growth in
gelatin containing glucose is rapid.

Agar-agar. — The growth in agar-
agar punctures is slower, but similar
to the gelatin cultures except for the
absence of liquefaction.

Milk is favorable for the develop-
ment of the tetanus bacillus. There
is no coagulation. Litmus milk is
acidified.

Potato. — Upon potatoes under strict
anaerobic conditions the bacilli grow
but slightly.

Vital Resistance. — The tetanus
spores may remain alive in dry earth
for many years. Sternberg says they
can resist immersion in 5 per cent,
aqueous carbolic acid solutions for ten
hours, but fail to grow after fifteen
hours. A 5 per cent, carbolic acid
solution, to which 0.5 per cent, of
hydrochloric acid has been added,
destroys them in two hours. They
are destroyed in three hours by
i : looo bichlorid of mercury solu-
tion, but when to such a solution
0.5 per cent, of hydrochloric acid is
added, its activity is so increased that
the spores are destroyed in thirty
minutes. According to Kitasato,* ex-
posure to streaming steam for from five to eight minutes is
* "Zeitschrift fur Hygiene," xn, p. 225.

Fig. 121 . — Tetanus
bacillus ; glucose-agar
culture, five months old
(Curtis).

390 Tetanus

certain to kill tetanus spores, and this statement has found
its way into most of the text-books without discussion.
Theobald Smith,* however, has studied several cultures
of the organism and finds that its resistance to heat is much
greater, and that in one case seventy minutes' exposure to
streaming steam did not kill all of the spores.

Metabolic Products. — Bouillon cultures of the tetanus
bacillus contain acids, proteolytic ferment, and several toxic
substances, of which tetanospasmin and tetanolysin are best
known. The toxic products are apparently all soluble.
No endotoxin is known to be formed.

The most ready method of preparing the toxins for experi-
mental study is to cultivate the bacilli in freshly prepared
neutral or slightly alkaline sugar-free bouillon under condi-
tions of most strict anaerobiosis, at a temperature of 37°
C., and then filter the culture through porcelain. Field f
found the highest degree of toxicity about the sixth or
seventh day. It may attain a toxicity so great that
0.000005 c-c- will cause the death of a mouse. I found the
average toxicity such that o.ooi c.c. was fatal to a guinea-
pig. KnorrJ gives some interesting comparisons of the
susceptibility of different animals, as follows:

i gram of horse is destroyed by x toxin

i gram of goat is destroyed by 2x toxin

i gram of mouse is destroyed by 13 x toxin

i gram of rabbit is destroyed by 2,000 x toxin

i gram of hen is destroyed by 200,000 x toxin

The toxin is very unstable, and is easily destroyed by
heat above 60° C. It is also quickly destroyed by light,
especially direct sunlight. Flexnerand Noguchi§ found that
5 per cent, of eosin added to the toxin destroyed it through
the photodynamic power of the stain. The toxin is also
easily destroyed by electric currents. It is also decomposed
by exposure to the air and light, so that it is difficult to pre-
serve it for many days. The best method of keeping it is to
add 0.5 per cent, of phenol, and then store it in a cool, dark
place, in bottles completely filled and tightly corked. It will
not keep its strength in liquid form under the best conditions.

* "Jour. Amer. Med. Assoc.," March 21, 1908, vol. L, No. 12, p. 931.
t "Proc. N. Y. Path. Soc.," March, 1904, p. 18.
J "Munch, med. Wochenschrift," 1898, p. 321.
§ "Studies from the Rockefeller Institute," 1905, v.

Metabolic Products 391

To keep it for experimental purposes it is advisable
to precipitate it by supersaturation with ammonium sul-
phate, which causes it to float upon the liquid in the form
of a sticky brown scum. It can be skimmed oft7 and dried.
Such dry precipitate will retain its activity for months
with but little deterioration.

From cultures of tetanus bacilli grown in various media,
and from the blood and tissues of animals affected with the
disease, Brieger succeeded in separating " tetanin," " tetano-
toxin," " tetanospasmin," and a fourth substance to which
no name is given. All were very poisonous and productive
of tonic convulsions. Later Brieger and Frankel isolated an
extremely poisonous toxalbumin from sugar-bouillon cul-
tures of the bacillus. Ehrlich* later discovered a new poison-
ous element to which he applied the name tetanolysin.

The purified toxin of Brieger and Cohn was surely fatal
to mice in doses of 0.00000005 gram. The work of these
older writers is now so completely superseded by that of
others as to be of historic interest only. Lambert f considers
the tetanus toxin to be the most poisonous substance that
has ever been discovered.

Fermi and PernossJ found most toxin produced in agar-
agar cultures, less in gelatin cultures, and least in bouillon
cultures.

Ehrlich § found two poisons in the tetanus toxin, one of
which was convulsive and was in consequence called tetano-
spasmin, the other hemolytic and called tetanolysin. When
tetanus toxin is added to defibrinated blood, the tetano-
lysin is absorbed by the corpuscles, many of which are
dissolved, while the tetanospasmin remains unchanged.

D6nitz|| and Wassermann and Takaki** have found that
the tetanus toxin has a specific affinity for the central
nervous system, with whose cells it combines in vitro and
becomes inert.

Roux and Borreljt have also found that when tetanus
toxin is injected into the brain substance a very much

* "jBerliner klin. Wochenschrift," 1898.

f "New York Med. Jour.," June 5, 1897.

t "Centralbl. f. Bakt.," etc., xv, p. 303.

§ "Berliner klin. Wochenschrift," 1898, No. 12, p. 273.

§ "Deutsche med. Wochenschrift," 1897, p. 428.
** "Berliner klin. Wochenschrift," 1898, 35.
ft "Ann. de 1'Inst. Pasteur," t. xn, 1898.

39 2 Tetanus

smaller dose will cause death than is necessary when the
poison is absorbed from the subcutaneous tissues.

Like most of the bacterial toxins, the tetanus poison is
only effective when produced in or injected into the tissues
and absorbed into the circulation. It is harmless when
given by the digestive tract, Ramon* having adminis-
tered by the mouth 300,000 times the fatal hypodermic
dose without producing any symptoms. The toxin seemed
to pass out with the feces.

One of the most interesting peculiarities about the toxin
is the comparative uniformity of the period intervening
between its administration and the appearance of the
symptoms — erroneously called the incubation period. This
varies within a narrow margin, inversely, with the size of
the dose. Thus, according to Behring, the effect of varying
doses of the toxin upon mice becomes evident according
to the size of the dose in from twelve to thirty-six hours,
thus:

13 lethal doses symptoms in 36 hours

1 10 lethal doses symptoms in 24 hours

333 lethal doses symptoms in 20 hours

1300 lethal doses symptoms in 14 hours

3600 lethal doses symptoms in 12 hours

The local action of the toxin is very painful and asso-
ciated with spasm of the muscular fibers with which it
comes in contact. Pitfield,f thinking that it might be
useful in the treatment of certain paralytic affections,
injected a minute quantity of it into the calf of his leg
and experienced the severe spasmodic local effects of the
poison for twelve hours.

It has been the belief of most physiologists that tetanus
toxin acts solely upon the motor cells of the spinal cord, and
produced the tonic spasms as strychnin does. The affinity
of the toxin for the nervous tissues has been made the
subject of careful investigations by Marie and Moraxf and
Meyer and Ransom. § The former found that the absorption
of tetanus toxin took place partly through the peripheral
nerves because of specific affinity between the toxin and

* " Deutsche med. Wochenschrift," Feb. 24, 1898.
t "Therapeutic Gazette," March 15, 1897.

J "Ann. de 1'Inst. Pasteur," 1902, xvi, p. 818; and "Bull, de 1'Inst.
Past.," 1903, i, p. 41.

§ "Arch. f. exper. Path. u. Pharmak.," 1903, xux.

Metabolic Products 393

the axis cylinder substance; the latter found the toxin car-
ried to the central nervous system solely by the motor
nerves, the action depending upon the integrity of the
axis cylinder. They believe that the toxin is absorbed
by the axis cylinder endings, and reaching the correspond-
ing spinal nerve center by that route spreads to the cor-
responding center in the other half of the cord and out-
ward, resulting in generalized tetanus. When intoxication
is produced through the circulation, the poison is taken
up by the nerve endings in all parts of the body, and the
disease is not localized, but general. Antitoxin, unlike the
toxin, does not travel by the nerve route, but is found only
in the blood and lymph. Zupnik* has brought forward
evidence that this view is incorrect and that there are
two distinct actions caused by the toxin. He differentiates
between tetanus ascendens and tetanus descendens. The
former always succeeds the intramuscular introduction of
the toxin, and depends upon its direct action upon the
muscle itself. It explains the familiar phenomenon of
rigidity making its first appearance in that member into
which the inoculation was made. The ascending tetanus
gradually ascends from muscle to muscle. He thinks the
absorption of the poison by the muscle-cells depends upon
their normal metabolic function, as when their nerves are
severed, the fixation of the toxin and the occurrence of the
tonic spasm does not occur.

Tetanus descendens results from the entrance of the toxin
into the circulation from the cellular tissue and its distribu-
tion in the blood. Under these conditions Zupnik believes
it acts upon the central nervous system, especially upon the
spinal cord, manifesting itself in extreme reflex excitability
with irregular motor discharges resulting in clonic spasms.

There are, therefore, two forms of spasm in tetanus:
the tonic convulsions, seeming to depend upon local action
and fixation of the toxin, and the clonic convulsions, de-
pending upon the centric action. The latter are the more
dangerous for the sufferer.

The lockjaw or trismus and the opisthotonos that are so
characteristic of the affection depend, according to Zupnik's
view, upon a loss of equilibrium among the muscles affected.
They occur only in descending tetanus and depend upon
spasm of muscle without equally powerful opposing groups.
* "Wiener klin. Wochenschrift," Jan. 23, 1902.

394 Tetanus

The stronger muscles of the jaw are those that close it; the
stronger muscles of the back, those of the erector group.
This view is exactly the opposite of Meyer and Ransom,*
who believe that the tetanus toxin is absorbed only along
the nerve trunks, and found that section of the spinal cord
prevented the ascent of tetanus from the lower extremities.
Injection of the toxin into a posterior nerve-root produced
tetanus dolorosus. Injection of the toxin into a posterior
nerve-root together with section of the spinal cord produced
exaltation of the reflex irritability — "Jactationstetanus."
Injection in sensory nerves does not produce tetanus doloro-
sus because the transportation of the poison along these
trunks is so slow.

The tetanolysin is a hemolytic component of the toxic
bouillon, and is entirely separate and distinct from the tetano-
spasmin or convulsive poison. It probably takes no part in
the usual clinical manifestations of tetanus.

Pathogenesis. — The work of Kitasato has given us very
complete knowledge of the biology of the tetanus bacillus
and completely established its specific nature:

When a white mouse is inoculated with an almost in-
finitesimal amount of tetanus culture, or with garden earth
containing the tetanus bacillus, the first symptoms come
on in from one to two days, when the mouse develops
typical tetanic convulsions, first beginning in the neighbor-
hood of the inoculation, but soon becoming general. Death
follows sometimes in a very few hours. In rabbits, guinea-
pigs, mice, rats, and other small animals the period of
incubation is from one to three days. In man the period
of incubation varies from a few days to several weeks, and
averages about nine days.

The disease is of much interest because of its purely
toxic nature. There is usually a small wound with a slight
amount of suppuration and at the autopsy the organs of the
body are normal in appearance, except the nervous system,
which bears the greatest insult. It, however, shows little else
than congestion either macroscopically or microscopically.

The conditions in the animal body are in general un-
favorable to the development of the bacilli, because of the
loosely combined oxygen contained in the blood, and they
grow with great slowness, remaining localized at the seat
of inoculation, and never entering the blood. Doubtless

t " Archiv. f. exper. Path. u. Pharmak.," Bd. xux, 1903, p. 396.

Pathogenesis 395

most cases of tetanus are cases of mixed infection in which
the bacillus enters with aerobic bacteria, which aid its
growth by absorbing the oxygen in the neighborhood. The
amount of poison produced must be exceedingly small and
its power tremendous, else so few bacilli growing under
adverse conditions could not produce fatal toxemia. The
toxin is produced rapidly, for Kitasato found that if mice
were inoculated at the root of the tail, and the skin and
the subcutaneous tissues around the inoculation afterward
either excised or burned out, the treatment would not save
the animal unless the operation were performed within
an hour after the inoculation.

Some incline to the view that the toxin is a ferment,
and the experiments of Nocard* might be adduced in
support of the theory. He says: "Take three sheep with
normal tails, and insert under the skin at the end of each
tail a splinter of wood covered with the dried spores of the
tetanus bacillus"; watch these animals carefully for the first
symptoms of tetanus, then amputate the tails of two of
them 20 cm. above the point of inoculation, . . . the
three animals succumb to the disease without showing any
sensible difference."

The circulating blood of diseased animals is fatal to
susceptible animals because of the toxin which it contains;
and the fact that the urine is also toxic to mice proves that
the toxin is excreted by the kidneys.

The organisms usually enter the body through a wound
caused by some implement which has been in contact with
the soil, or enter abrasions from the soil directly. Doubt-
less many of the wounds are so small that their existence is
overlooked, and this, together with the fact that the period
of incubation of the disease, especially in man, is of con-
siderable duration (three to nine days), and at times permits
the wound to heal before any symptoms of intoxication
occur, serves to explain the occurrence of some of the
reported cases in which no wound is said to have existed.

There are two classes of infected wounds particularly prone
to be followed by tetanus — namely, those into which soil
has been carried by the injuring implement and those of
considerable depth. The infecting organism reaches the
first class in large numbers, but finds itself under aerobic
and other inappropriate conditions of growth. It reaches
* Quoted before the Academic de Medicine, Oct. 22, 1895.

396 Tetanus

the second class in smaller numbers, but finds the conditions
of growth better because of the depth of the wound.

The severity of the wound has nothing whatever to do
with the occurrence of tetanus, pin -pricks, nail punctures,
insect stings, vaccination, and a variety of other mild
injuries sometimes being followed by it.

An interesting fact has been presented by Vaillard and
Rouget,* who found that if the tetanus spores were intro-
duced into the body freed from their poison, they were
unable to produce the disease because of the promptness
with which the phagocytes took them up. If, however,
the toxin was not removed, or if the body-cells were injured
by the simultaneous introduction of lactic acid or other
chemic agent, the spores would immediately develop into
bacilli, begin to manufacture toxin, and produce the disease.
This suggests that many wounds may be infected by the
tetanus bacillus though the surrounding conditions rarely
enable it to develop satisfactorily and produce enough
toxin to cause disease.

In very rare cases tetanus may possibly occur without
the previous existence of a wound, as in the case reported
by Kamen, who found the intestine of a person dead of
the disease rich in Bacillus tetani. Kamen is of the opinion
that the bacilli can grow in the intestine and be absorbed,
especially where imperfections in the mucosa exist. It is
not impossible, though he does not think it probable, that
the bacteria growing in the intestine can elaborate enough
toxin to produce the disease by absorption.

A peculiar observation has been made by Montesano and
Montesson, * who unexpectedly found the tetanus bacillus
in pure culture in the cerebro-spinal fluid of a case of par-
alytic dementia that died without a tetanic symptom.

Immunity. — All animals are not alike susceptible to
tetanus. Men, horses, mice, rabbits, and guinea-pigs are
susceptible; dogs much less so. Cattle suffer chiefly after
accouchement, and after abortion. Most birds are scarcely at
all susceptible either to the bacilli or to their toxin. Am-
phibians and reptiles are immune, though it is said that frogs
can be made susceptible by elevation of their body-tempera-
ture.

* See " Centralbl. f. Bakt., Infekt., u. Parasitenk.," vol. xvi, p. 208.
t"Centralbl. f. Bakt. u. Parasitenk.," Dec., 1897, Bd. xxn, Nos.
22, 23, p. 663.

Antitoxin 397

The injection of the toxic bouillon or of the redis-
solved ammonium sulphate precipitate, in progressively
increasing doses, into animals, causes the formation of anti-
bodies (antitoxin) by which the effects of both the tetano-
spasmin and the tetanolysin are destroyed. The purely toxic
character of the disease makes it peculiarly well adapted for
treatment with antitoxin, and at the present time our sole
therapeutic reliance is placed upon it. The mode of prepar-
ing the serum and the system of standardization are discussed
in the section upon Antitoxins in the part of this work that
treats of the Special Phenomena of Infection and Immunity.

Antitoxin. — Numerous cases of the beneficial action of
antitoxin are on record, but, as Welch* has pointed out,
the antitoxin of tetanus has proved a disappointment in
the treatment of tetanus. Moschcowitz,f in his excellent
literary review of the subject, has shown that its use has
reduced the death-rate from about 80 to 40 per cent., and
that it therefore cannot be looked upon as a failure. The
result of its experimental injection, in combination with
the toxin, into mice, guinea-pigs, rabbits, and other animals
is perfectly satisfactory, and affords protection against
almost any multiple of the fatal dose, but the quantity
needed, in proportion to the body-weight, to save an animal
from the unknown quantity of toxin being manufactured
in its body increases so enormously with the day or hour
of the disease as to make the dose, which increases millions
of times where that of diphtheria antitoxin increases but
tenfold, a matter of difficulty and uncertainty. Nocard
also called attention to the fact that the existence of tetanus
cannot be known until a sufficient toxemia to produce
spasms exists, and that therefore it is impossible to attack
the disease in its inception or to begin the treatment until too
late to effect a cure. At this point it is well to recall
Nocard's experiment with the sheep, in whose blood so much
toxin was already present when symptoms first appeared
that the amputation of their infected tails could not save
them.

The explanation of this inability of the antitoxin to effect
a cure when administered after development of the symp-
toms of tetanus is probably found in a ready fixation of the

* "Bulletin of the Johns Hopkins Hospital," July and August,
1895.

f "Annals of Surgery," 1900, xxxn, 2, pp. 219, 416, 567.

398 Tetanus

toxin in the bodies of the infected animals. This is well
shown by the experiments of Donitz,* who found that if a
mixture of toxin and antitoxin were made before injection
into an animal, twelve minimum fatal doses were neutralized
by i c.c. of a i : 2000 dilution of an antitoxin. If, however,
the antitoxin was administered four minutes after the
toxin, i c.c. of a i : 600 dilution was required; if eight
minutes after, i c.c. of a i : 200 dilution ; if fifteen minutes
after, i c.c. of a i : 100 dilution. He found that similar
but slower fixation occurred with diphtheria toxin.

It was found by Roux and Borrel f that doses of tetanus
antitoxin absolutely powerless to affect the progress of the
disease, when administered in the ordinary manner by
subcutaneous injection, readily saved the animal if the
antitoxin were injected into the brain substance.

Chauffard and Quenu,{ who injected the antitoxin into
the cerebral substance, found that such administration
brought about an apparent cure in one case.

Their observations were followed by an attempt to apply
the method in human medicine, and patients with tetanus
were trephined and the antitoxin injected beneath the dura
and into the cerebral substance. The results have not, how-
ever, been satisfactory, and as the method cannot be looked
upon as itself free from danger, it has been abandoned.

The only means of treating the disease that can be recom-
mended at present is by intravenous and subcutaneous injec-
tion of large and frequently repeated doses of the antitoxic
serum. There can be little doubt but that the administra-
tion must be so free as to load up the patient's blood with the
antitoxin in hopes that its presence there may be able to de-
tach the toxic molecules from their anchorage to the nerve
cells and form an inert union.

Prophylactic Treatment. — While tetanus antitoxin is
extremely disappointing, in practice, for the cure of tetanus,
it is most satisfactory for its prevention. " An ounce of
prevention is better than a pound of cure," and if the sur-
geon would administer a prophylactic injection of tetanus
antitoxin in every case in which the occurrence of tetanus
was at all likely, the disease would rarely develop.

* Reference 18, in "Jour, of Hygiene," vol. n, No. 2, in Ritchie's
article.

t "Ann. de 1'Inst. Pasteur," 1898, No. 4.
t " La Presse med.," No. 5, 1898.

Bacilli Resembling the Tetanus Bacillus 399

BACILLI RESEMBLING THE TETANUS BACILLUS.

Tavel * has called attention to a bacillus commonly found
in the intestine, sometimes in large numbers in the appendix
in cases of appendicitis, and looked upon by one of his
colleagues, Fraulein Dr. von Mayer, as the probable common
cause of appendicitis. He calls it the "Pseudo-tetanus-
bacillus."

The bacillus is slender and measures 0.5 by 5-7 /*, is
rather more slender than the tetanus bacillus, and its spores
are oval, situated at the end of the rod, and cause a slight
bulging rather pointed at the end. The bacillus is provided
with not more than a dozen flagella, — usually only four to
eight,— thus differing markedly from the tetanus bacillus,
which has many. The flagella are easily stained by Loffler's
method without the addition of acid or alkali. The organ-
ism does not stain so well by Gram's method as the true
tetanus bacillus. The bacillus is a pure anaerobe.

The growth in bouillon is rather more rapid than that of
the tetanus bacillus. It will not grow in gelatin. The
growth in agar-agar is very luxuriant and accompanied
by the evolution of gas. Upon obliquely solidified agar-
agar the colonies are round, circumscribed, and often en-
compassed by a narrow, clear zone, which is often notched.
The organism grows in serum only in a vacuum. The spores
are killed at 80° C.

The organism produced no symptoms in mice, guinea-
pigs, and rabbits even when 2-5 c.c. of a culture were
subcutaneously introduced.

Sanfelice f and L/ubinski { have observed a bacillus in
earth and meat-infusions that is morphologically and cul-
turally like the tetanus bacillus, but differs from it in not
possessing any pathogenic powers.

Kruse § has also described a bacillus much like the tetanus
micro-organism that grows aerobically. It is not patho-
genic.

* "Centralbl. f. Bakt.," etc., March 31, 1898, xxm, No. 13, p. 538.

f "Zeitschrift fur Hygiene," vol. xiv.

} "Centralbl. f. Bakt. u. Parasitenk.," xvi, 19.

§ Haggis, " Die Mikroorganismen," vol. n, p. 267.

CHAPTER IV.
ANTHRAX.

BACIIXUS ANTHRACIS (KOCH).

General Characteristics. — A non-motile, non-flagellated, spor-
ogenous, liquefying, non-chromogenic, pathogenic, aerobic bacillus
staining by the ordinary methods and by Gram's method.

The disease of herbivora known as anthrax, "splenic fever,"
"Milzbrand," and "charbon," of infrequent occurrence in
this country and England, is a dreaded and common malady
in France, Germany, Hungary, Russia, Persia, and the
East Indian countries. In Siberia the disease is so common

Fig. 122. — Bacillus anthracis; colony three days old upon a gelatin
plate; adhesive preparation. X 1000 (Frankel and Pfeiffer).

and malignant as to deserve its popular name, "Siberian
pest." Certain districts, as the Tyrol and Auvergne, in
which it seems to be endemic, serve as foci from which the
disease spreads in summer, afflicting many animals, and
ceasing its depredations only with the advent of winter. It
is not rare in the United States, where it seems to be chiefly
a disease of the summer season.

400

Bacillus Anthracis 401

The animals most frequently affected are cows and sheep.
Among laboratory animals, white mice, house-mice, guinea-
pigs, and rabbits are highly susceptible; dogs, cats, most
birds, and amphibians are immune. White rats are infected
with difficulty. Man is slightly susceptible, the disease in
the human species usually being a local affection — "malig-
nant carbuncle" — commonly succeeded by a general fatal
infection.

Anthrax was one of the first infectious diseases proved to
depend upon a specific micro-organism. As early as 1849

Fig. 123. — Bacillus anthracis; showing the capsules. From a case of
human infection. Magnified 1000 diameters (Schwalve).

Pollender* discovered small rod-shaped bodies in the blood
of animals suffering from anthrax, but the exact relation
which they bore to the disease was not pointed out until
1863, when Davaine,| by a series of interesting experiments,
proved their etiologic significance to most unbiased minds.
The final confirmation of Davaine's conclusions and actual
proof of the matter rested with Koch,J who, observing that
the bacilli bore spores, cultivated them successfully outside
the body, and produced the disease by the inoculation of
pure cultures.

* "Vierteljahrsschr. fur ger. Med.," Bd. vin, 1855.
t "Compte-rendu," Ivii, 1863.
t "Beitrage zur Biol. d. Blauzen," 1876. n.
26

402 Anthrax

Morphology. — The anthrax bacillus is a large 'rod-shaped
organism, of rectangular form, with slightly rounded cor-
ners. It measures 5 to 20 ^ in length and from i to
1.25 ^ in breadth. It has a pronounced tendency to
form long threads, in which, however, the individuals can
usually be made out, the lines of junction of the com-
ponent bacilli giving the thread somewhat the appear-
ance of a bamboo rod. In preparations made by staining
blood or other animal juices the bacilli often appear sur-
rounded by transparent capsules. Such are not found in
specimens made from artificial cultures.

'

'.

..

-y*;#

,,; <T,
I '

\ *»

Fig. 124. — Bacillus anthracis, stained to show the spores. X 1000
(Frankel and Pfeiffer).

Sporulation. — The formation of endospores is prolific:
each spore has a distinct oval shape, is transparent, situated
at the center of the bacillus in which it occurs. It does not
alter the contour of the bacillus. The spores are formed
only in the presence of oxygen upon the surfaces of the
culture-media. When a spore is placed under conditions
favorable to its development, it increases in length and
ruptures at the end, from which the new bacillus escapes.
The spores of the anthrax bacillus, being large and readily
obtainable, form excellent subjects for the study of spore-
formation and germination, for the study of the action of
germicides and antiseptics, and for staining.

Cultivation 403

Motility. — The bacilli are not motile and have no flagella.

Staining. — They stain well with ordinary solutions of the
anilin dyes, and can be beautifully demonstrated in the
tissues by Gram's method and by Weigert's modification of
it. Picrocarmin, followed by Gram's stain, gives a beau-
tiful, clear picture. The spores can be stained by any of the
special methods for staining spores (q. v.).

Isolation. — The bacillus of anthrax is one of the easiest
organisms to secure in pure culture from the tissues and
excreta of diseased animals. Its luxurious vegetation, the
typical appearance of its colonies, and its infectivity for the
laboratory animals combine to make possible its isolation
either by direct cultivation from the tissues, by the plate
method, or by the inoculation into animals and recovery
of the micro-organisms from their blood.

Fig. 125. — Bacillus anthracis; colony upon a gelatin plate. X 100
(Frankel and Pfeiffer).

Cultivation. — Colonies. — Upon the surface of a gelatin
plate the bacillus forms beautiful and highly characteristic
colonies (Fig. 125). To the naked eye they appear first as
minute round, grayish-white dots upon the surface. They
early begin liquefaction of the gelatin, which progresses
rapidly as they increase in size. Under the microscope the
smallest colonies are egg-shaped, slightly brown and granular.
They do not attain their full development except upon the

404

Anthrax

Fig. 126.— Bacillus an-
thracis ; gelatin stab cul-
ture, showing character-
istic growth with com-
mencing liquefaction and
cupping (from evapora-
tion) at the surface of
the medium (Curtis).

surface of the medium, where they
spread out into flat, irregular,
transparent tufts like curled wool.
From a tangled center large num-
bers of curls, made up of parallel
threads of bacilli, extend upon the
gelatin. As soon as the colony at-
tains to any considerable size lique-
faction becomes rapid. Beautiful
adhesion preparations can be made
if a perfectly clean cover-glass be
passed once through a flame and
laid carefully upon the gelatin, the
colonies being picked up entire as
the glass is carefully removed. Such
a specimen can be dried, fixed, and
stained in the same manner as an
ordinary cover-glass preparation.

Gelatin Punctures. — In gelatin
puncture cultures the growth is
even more characteristic than are
the colonies. The bacilli begin to
grow along the entire track of the
wire, but develop most luxuriantly
at the surface, where oxygen is
plentiful and where a distinct
shaggy pellicle is formed. From the
deeper growth, fine filaments extend
from the puncture into the sur-
rounding gelatin, with a beautiful
arborescent effect (Fig. 126).

Liquefaction progresses from
above downward until ultimately
the entire gelatin is fluid and the
growth sediments.

Agar-agar. — Upon agar-agar
characteristic appearances are few.
The growth takes place along the
line of inoculation, forming a
grayish-white, translucent, slightly
wrinkled layer with irregular edges,
from which curls of bacillary threads
extend upon the medium. When

Metabolic Products 405

the culture is old, the agar-agar usually becomes brown in
color. Spore formation is luxuriant.

Bouillon. — In bouillon the anthrax bacillus, because of its
marked affinity for oxygen, grows chiefly upon the surface,
where a thick felt-like pellicle forms. From this, fuzzy ex-
tensions descend into the clear bouillon below. After a few
days some wooly aggregations can be seen in the bottom of
the tube. In the course of time the growth ceases and the
surface pellicle sinks. If, by shaking, it is caused to sink
prematurely, a new, similar surface growth takes its place.
Spore formation is rapid at the surface.

Potato. — Upon the potato the growth is white, creamy,
and rather dry. Sporulation is marked.

Blood-serum. — Blood-serum cultures lack characteristic
peculiarities; the culture-medium is slowly liquefied.

Milk. — The anthrax bacillus grows well in milk, which
it coagulates and acidulates. Later the coagulum is pep-
tonized and dissolved, leaving a clear whey. The reaction
is not altered. Iwanow* found that the organism forms
acetic, formic, and caproic acids.

Thermic Sensitivity. — The bacillus grows between the
extremes of 12° and 45° C., best at 37° C. The exposure of
the organism to the temperature of 42° to 43° C. slowly di-
minishes its virulence.

When dried upon threads, the spores retain their vital-
ity for years, and are highly resistant to heat .and disinfec-
tants. The spores of anthrax are killed by five minutes'
exposure to 100° C. It is said by some that spores sub-
jected to 5 per cent, carbolic acid can subsequently ger-
minate when introduced into susceptible animals, their
resistance to this strength carbolic solution being so great
that they are not destroyed by it under twenty-four hours.
They are killed in a short time by exposure to i : 1000
bichlorid of mercury solution.

Metabolic Products. — The anthrax bacillus produces a
curdling ferment. It produces no important change of
reaction in the medium in which it grows, and generates no
indol. Its proteolytic enzyme is active, digesting both
casein and fibrin.

It is doubtful whether the anthrax bacillus produces any
important toxic substance. Hoffaf isolated a basic sub-
* "Ann. de 1'Inst. Pasteur," 1892.
t "Ueber die Natur. des Milzbrandgifts," Wiesbaden, 1886.

406

Anthrax

Fig. 127. — Bacillus an-
thracis; glycerin agar-
agar culture (Curtis).

stance from anthrax cultures and
called it anthracin; Hankin and Wes-
brook,* an albumose fatal in large
doses and immunizing in small ones.
Brieger and Frankel f isolated a tox-
albumin from the tissues of animals
dead of anthrax. Martin J separated
protalbumose, deuteroalbumose,
peptone, an alkaloid, leucin, and
tyrosin. The albumoses were not
very poisonous, but the alkaloid was
capable of producing death after the
development of somnolence. The
animals were edematous. Marmier §
isolated a toxin of non-albuminous
nature and immunizing power. Con-
radi || in an elaborate research failed
to find that the anthrax bacillus pro-
duces any soluble extracellular or in-
tracellular poison capable of affect-
ing susceptible animals, and con-
cludes that it is highly improbable
that the anthrax bacillus produces
any toxic substance at all.

Pathogenesis. — Avenues of In-
fection.— Infection usually takes
place through the respiratory tract,
through the alimentary canal, or
through wounds. It may take
place through the placenta.

When the bacilli are taken into
the stomach they are probably de-
stroyed by the acid gastric juice.
The spores, however, are able to en-
dure the acid gastric juice, and pass
into the intestine, where the suitable
alkalinity enables them to develop
into bacilli, surround the villi with

*"Ann. de 1'Inst. Pasteur," 1892, No. 9.
f'Ueber Ptomaine," Berlin, 1885-1886.
t "Proceedings of the Royal Society," May 22, 1890.
§ "Ann. de.l'Inst. Pasteur," 1895, p. 533.
||"Zeitschrift fur Hygiene," June 14, 1899.

Pathogenesis 407

thick networks of bacillary threads, separate the covering
epithelial cells, enter the lymphatics, and then the blood,
from which a general infection occurs.

The bacillus frequently enters the body through wounds,
cuts, scratches, and perhaps occasionally fly-bites. Under
these conditions the organisms at once find themselves in
the lymphatics or capillaries, and may cause immediate
general infection. In human beings a " malignant pustule "
is apt to follow local infection, and may recover or ulti-
mately cause death by general infection. Those whose
occupations bring them in contact with the skins and
hair from animals dead of anthrax are liable to the infection.

Anthrax in cattle probably results from the inhalation
or ingestion of the spores of the bacilli from the pasture.
From the work of Nuttall * it is pretty clear that flies
play little part in the transmission of the disease. Inter-
esting discussions arose concerning the infection of the
pastures. It was argued that, the bacilli being inclosed in
the tissues of the diseased animals, infection of the pasture
must depend upon the distribution of the germs from buried
cadavers, either through the activity of earthworms, which
ate of the earth surrounding the corpse and deposited the
spores in their excrement (Pasteur), or to currents of mois-
ture in the soil. Koch seems, however, to have demon-
strated the fallacy of both theories by showing that the
conditions under which the bacilli find themselves in buried
cadavers are opposed to fructification or sporulation, and
that in all probability the bacteria suffer the same fate as
the cells of the buried animals, and disintegrate, especially
if the animal be buried at a depth of two or three meters.

Frankel points out particularly that no infection of the
soil by the dead animal could be worse than the pollution
of its surface by the bloody stools and urine, rich in bacilli,
discharged upon it by the animal before death, and that
it is the live, and not the dead, animals that are to be blamed
for the infection.

Lesions. — The disease as seen in the laboratory is
accompanied by few marked lesions. The ordinary ex-
perimental inoculation is made by cutting away a little
of the hair from the abdomen of a guinea-pig or rabbit, or
at the root of a mouse's tail, making a little subcutaneous
pocket by a snip with sterile scissors, and introducing the
* "Johns Hopkins Hospital Reports," 1899.

408

Anthrax

spores or bacilli with a heavy platinum wire, the end of which
is flattened, pointed, and perforated. An animal inoculated
in this way dies, according to the species, in from twenty
four hours to three days, suffering from weakness, fever,
loss of appetite, and a bloody discharge from nose and
bowels. There is much subcutaneous gelatinous edema
near the inoculation wound. The abdominal viscera are
injected and congested. The spleen is enlarged, dark in
color, and of mushy consistence. The liver is also some-
what enlarged. The lungs are usually slightly congested.

r

Fig. 128. — Anthrax bacilli in glomeruli of kidney.

When organs which present no appreciable changes to the
naked eye are subjected to a microscopic examination, the
appropriate staining methods show the capillary and
lymphatic systems to be almost universally occupied by
bacilli, which extend throughout their meshworks in long
threads. Most beautiful bacillary threads can be found in
the glomeruli of the kidney and in the minute capillaries
of the intestinal villi. In the larger vessels, where the
blood-stream is rapid, no opportunity is afforded for the
formation of the threads, and the bacteria are relatively

Vaccination 409

few. so that the burden of bacillary obstruction is borne by
the minute vessels. The condition is thus a pure bacteremia.

Death from anthrax seems to depend essentially upon
the obstruction of the circulation by the multitudes of
bacilli in the capillaries, upon the appropriation of the
oxygen destined to support the tissues, by the bacilli,
leaving the tissues to be poisoned by the carbon* dioxid,
rather than upon intoxication by metabolic products of
bacillary growth.

Vaccination. — Pasteur * early realized the importance
of some practical measure for the protective vaccination
of cattle against the disease, and devoted himself to inves-
tigating the problem. He found that the inoculation of
attenuated bacilli into cows and sheep, and their subse-
quent reinoculation with mildly virulent bacilli, afforded
them immunity against highly virulent organisms. Loffler,
Koch, and Gaffky, however, found that these immunized
animals were not absolutely protected against intestinal
anthrax.

The means of diminishing the virulence of the anthrax
bacillus are numerous. Toussaint f first produced im-
munity in animals by injecting them with sterile cultures of
the bacillus, and found that the addition of i per cent, of
carbolic acid to blood of animals dead of anthrax destroyed
the virulence of the bacilli ; Chamberland J and Roux found
the virulence destroyed when 0.1-0.2 per cent, of bichro-
mate of potassium was added to the culture medium;
Chauveau used atmospheric pressure to the extent of six to
eight atmospheres and found the virulence diminished;
Arloing § found that direct sunlight operated similarly;
Lubarsch, that the inoculation of the bacilli into immune
animals, such as the frog, and their subsequent recovery
from its blood, diminishes the virulence of the bacilli mark-
edly.

The protective inoculations prepared by Pasteur consisted
of two cultures of increasing virulence, to be employed one
after the other, rendering the vaccinated animals more and
more immune. The cultures were prepared, that is, at-

* "Rec. de Med. vet.," Paris, 1879, p. 193.

t " Compte-rendu Acad. des Sci. de Paris," xci, 1880, p. 135.

t "Ann. de 1'Inst. Pasteur," 1894, p. 161.

\ "Compte-rendu de 1'Acad. des Sci.," Paris, 1892, cxiv, p. 1521.

410 Anthrax

tenuated by cultivation at 42° C. for a sufficient length of
time, the bacilli forming no spores and gradually losing
their virulence at this temperature. The first vaccine was kept
from fifteen to twenty days at 42° C. It killed mice and
guinea-pigs one day old, but was without action on guinea-
pigs of adult size. The second vaccine only remained at
the temperature of 42° C. for from ten to twelve days
and killed mice, guinea-pigs, and occasionally rabbits.

The second vaccine is administered from two to three
weeks after the first is given, by hypodermic injection into
the tissues of the neck or flank. Of each broth culture
about i c.c. is administered. The animals frequently be-
come ill.

Pasteur demonstrated the value of his method in 1881 at
Pouilly-le-Fort, in a manner so convincing to the entire
world that it was immediately put into practice in France.
Roger* says that between 1882 and 1894 there were
1,788,677 sheep vaccinated, with a mortality of 0.94 per
cent., the previous death-rate having been 10 per cent.
There were also 200,962 cattle vaccinated, with a reduction
of the death-rate from 5 per cent, to 0.34 per cent.

Chamberland has shown that protective inoculation by
Pasteur's method has diminished the death-rate from 10
per cent, for sheep and 5 per cent, for cattle to about 0.94
per cent, for sheep and 0.34 per cent, for cattle, so that
the utility of the method is scarcely questionable. The
method has been less successful elsewhere than in France,
and has sometimes caused the death of the animals to be
protected.

Protection against anthrax can be afforded in other ways.
Hiippe found that the simultaneous inoculation of bacteria
not at all related to anthrax will sometimes cause the
animal to recover. Hankin found in the cultures chemic
substances, especially an albuminose, that exerted a pro-
tective influence. Rettgerf prepared ' ' prodigiosus powder ' '
from potato cultures of B. prodigiosus, which when injected
into guinea-pigs during experimental anthrax infection pro-
longed life or induced recovery.

Serum Therapy. — In 1890 Ogata and Jasuhara showed
that experiment animals convalescent from anthrax pos-
sessed an antitoxic substance in the blood of such strength

*"Les Maladies Infectieuse," n, p. 1489.

f "Jour, of Infectious Diseases," vol. n, No. 4, p. 562, Nov. 25, 1905.

Bacilli Resembling the Anthrax Bacillus 411

that i : 800 parts per body-weight of dog's serum contain-
ing the antitoxin would protect a mouse. Similar results
have been attained by Marchoux. * Serum therapy in
anthrax is, however, of no practical importance either for
prophylaxis or treatment, as vaccinating the animals is
far cheaper and more satisfactory.

Bacteriologic Diagnosis. — When it is desired to have
a bacteriologic diagnosis of anthrax made where no labora-
tory facilities are at hand, an ear of the dead animal can
be inclosed in a bottle or fruit jar and sent to the nearest
laboratory where diagnosis can be made. The ear contains
so little readily decomposable tissue, that it keeps fairly well,
drying rather than rotting. It contains enough blood to
enable a bacteriologist to make a successful examination.

Sanitation. — As every animal affected with anthrax is a
menace to the community in which it lives, — to the men who
handle it as well as the animals who browse beside it, —
such animals should be killed as soon as the diagnosis is
made, and, together with the hair and skin, be burned,
or if this be impracticable, Frankel recommends that they
be buried to a depth of at least 1^-2 meters, so that the
sporulation of the bacilli is made impossible. The dejecta
should also be carefully disinfected with 5 per cent, carbolic
acid solution.

BACILLI RESEMBLING THE ANTHRAX BACILLUS.
Bacilli presenting the 'morphologic and cultural char-
acteristics of the anthrax bacillus, but devoid of any disease-
producing power, are occasionally observed. Of these,
Bacillus anthracoides of Hiippe and Wood,f Bacillus anthra-
cis similis of McFarland,J and Bacillus pseudoanthracis§
have been given special names. What relationship they
bear to the anthrax bacillus is uncertain. They may be
entirely different organisms, or they may be individuals
whose virulence has been completely lost through un-
favorable environment.

* "Ann. de 1'Inst. Pasteur," November, 1895, t. ix, No. 11, pp.
50-75.

f "Berliner klin. Wochenschrift," 1889. 16.

t "Centralbl. f. Bakt.," vol. xxiv, No. 26, p. 556.

\ "Hygienische Rundschau," 1894, No. 8.

CHAPTER V.

HYDROPHOBIA, LYSSA, OR RABIES.

HYDROPHOBIA, lyssa, or rabies is a specific infectious
toxic disease to which dogs, wolves, skunks, and cats are
highly susceptible, and which, through their saliva, can be
communicated to men, horses, cows, and other animals.
The means of communication is almost invariably a bite,
hence the specific organism must be present in the saliva.

The infected animals manifest no symptoms during a vary-
ing incubation period in which the wound heals kindly.
This period may be of twelve months' duration, but in rare
cases may be only a few days. The average duration of the
period of incubation is about six weeks.

Toward the close of the incubation period an observable
alteration occurs in the wound, which becomes reddened,
may suppurate, and is painful. The victim has a sensation
of horrible dread, which passes into wild excitement, with
paralysis of the pharyngeal muscles and inability to swallow.
The wild delirium ends in a final stage of convulsion or
palsy. The convulsions are tonic, rarely clonic, and finally
cause death by interfering with respiration.

During the convulsive period much difficulty is experi-
enced in swallowing liquids, and it is supposed that the
popular term " hydrophobia " arose from the reluctance of
the diseased to take water because of painful spasms caused
by the attempt.

Pasteur* and his co-workers, Chamber-land and Roux,f
found that in animals that die of rabies the salivary glands,

* "Compte-rendu Acad. des Sciences," Paris, 1889, cvm, p. 1228.
t Ibid., Oct. 26, 1885.

412

Pasteur's Treatment 413

the pancreas, and the nervous system contain the infection,
and are more appropriate for experimental purposes than
the saliva, which is invariably contaminated with accidental
pathogenic bacteria.

The introduction of a fragment of the medulla oblongata
of a dog dead of rabies, beneath the dura mater of a rabbit,
causes the development of typical rabies in the rabbit in
about six days. It is only by such an inoculation that a posi-
tive diagnosis of the disease can be made. The operation
must be performed with the greatest care in order to avoid
septic infection with meningitis. The technic is simple, a
small trephine for opening the rabbit's skull being obtainable
from the dealers, though in its absence the thin bone of the
cranial cavity may be cut with a heavy scalpel. The material
to be inoculated should be crushed to a fine pulp in sterile
physiologic salt solution, and introduced beneath the dura
with a hypodermic syringe. The tissue of the medulla of a
rabid rabbit introduced beneath the dura mater of a second
rabbit produces a more violent form of the disease in a shorter
time, and by frequently repeated implantations Pasteur
found that an extremely virulent material could be ob-
tained.

Pasteur observed that the virulence of the poison was less
in animals that had been dead for some time than in those
just killed, and by experiment found that when the ner-
vous system of an infected rabbit was dried in a sterile at-
mosphere its virulence attenuated in proportion to the
length of time it was kept. A method of attenuating the
virulence was thus suggested to Pasteur, and the idea of
using it as a protective vaccination soon followed. After
careful experimentation he found that by inoculating a
dog with much attenuated, then with less attenuated, then
with moderately strong, and finally with a strong, virus, it
developed an immunity that enabled it to resist infection
with an amount of virulent material that would certainly
kill an unprotected dog.

It is remarkable that this theory, based upon limited accu-
rate biologic knowledge, and upon experience with very few
bacteria, should find absolute confirmation as our knowledge
of immunity, toxins, and antitoxins progressed. Pasteur
introduced the unknown poison-producers, attenuated by
drying, and capable of generating only a little poison, accus-

414 Hydrophobia, Lyssa, or Rabies

tomed the animal first to them and then to stronger and
stronger ones until immunity was established.

For the treatment of infected cases exactly the same method
is followed as for the production of immunity. Indeed, the
treatment of a patient bitten by a rabid animal is simply the
production of immunity during the prolonged incubation
period of the affection, so that the disease may not develop.
The patient, to be successfully treated, must come under
observation early.

Method.— To protect human beings from the development
of hydrophobia after they have been bitten by rabid animals,
it is necessary to use material of standard or known virulence.
This can be prepared, according to the directions of Hogyes,*
by the passage of virus from a rabid animal through from
21 to 30 rabbits. For this purpose some of the hippocampal
tissue of the dog is made into an emulsion with sterile salt
solution and injected subcutaneously into a rabbit. As soon
as this animal dies, its spinal cord is removed, a similar emul-
sion made with a fragment of it, and a second rabbit inoculated,
and so on through the series until a standard virulence is
attained and the virus is said to be " fixed." It has a much
higher degree of virulence than the " street virus " taken from
the rabid dog, but its virulence does not vary. In most
laboratories the "fixed virus" is obtained from other labora-
tories and kept passing through rabbits. In this manner
uniformity of dosage and virulence is most easily main-
tained.

The technic of obtaining the rabbit's cord given by Oshidaf
is the one now generally employed. As given by Stimson, J it
is performed at the Hygienic Laboratory as follows: "The
rabbit, when completely paralyzed, is killed with chloroform
and nailed to a board, back uppermost, and thoroughly
wetted down with an aseptic solution (i per cent, trikresol).
An incision is made through the skin from the forehead nearly
to the tail and the skin laid back on each side, the ears
being cut close to the head. An area i inch wide is seared
with a hot iron around the occiput and nuchal region and ear
openings. The skin is then transversely divided in the center

* See Kraus and Levaditi, "Handbuch der Immunitatsforschung," i.
t "Centralbl. f. Bakt. u. Parasitenk.," 1901, xxix, Orig., 988.
t" Facts and Problems of Rabies," Hygienic Laboratory Bulletin,
No. 65, June, 1910, Washington, D. C.

Method

of the seared areas by means of bone-cutting forceps. The
neck is dissected loose from the skin and a large square of
sterile gauze is inserted beneath it. The lumbar region is
dissected up for a few inches and a similar piece of gauze
placed beneath it. Then a piece of telegraph wire about 14
inches long, bent into a handle at one end and having a small
wisp of cotton twisted about the other end, is used to push the
cord out of its canal. The spine is steadied by a pair of lion-
jawed forceps.

Fig. 129. — Removal of the spinal cord from a rabbit (Stimson, Bull. No.
65, Hygienic Laboratory).

An assistant catches the cord with forceps as it emerges
from the cervical opening and lifts it out. The spinal nerves
are torn off during this procedure, and the membranes stripped
off, leaving a clean sterile cord. A silk ligature with one long
end is placed around the upper end, and another, just below the
middle of the cord, which is then cut into two pieces just above
the lower ligature. A small piece is cut off of the upper end of
the upper portion and placed in a tube of bouillon, which is
incubated as a test for sterility. The cords are hung in the
drying bottle.

The cords thus secured are used for perpetuating the

4i 6 Hydrophobia, Lyssa, or Rabies

" fixed virus " by inoculating other rabbits, and after the
virus has become " fixed " are also used for treating the
bitten patient.

For this purpose, however, Pasteur attenuated the virus
by drying the cords, placing them in a bottle containing
caustic potash.

The longer the cord dries, the more the virulence of the
micro-organisms attenuates, and as in beginning the immuni-

Fig. 130. — Method of drying the spinal cord of a rabbit for the purpose of
attenuation (Stimson, Bull. No. 65, Hygienic Laboratory).

zation of human beings it is essential to use very attenuated
material, the first cord used to furnish inoculation material
must have dried about ten days.

The cord, when it has reached the necessary attenuation,
is transferred, and i cm. of it is emulsified with 3 c.c. of sterile
0.8 per cent, salt solution. There can be no absolute ac-
curacy of dosage. The injection material made in the
laboratory under strict aseptic precautions can be used with
perfect safety for many hours subsequently if kept cold, and

Pasteur's Original Schemata

can be packed in ice and sent by express to the physician to
use at the home, of his patients.

As the transfer of the cord to glycerin preserves the viru-
lence at whatever degree it had when so transferred, it is now
customary to keep on hand, in glycerin, in the laboratory,
spinal cords of rabbits dried one, two, three, four days, and
so on through the whole series.

This makes it possible to keep on hand, always available,
material for furnishing all the vaccines at any time, indepen-
dently of the experimental rabbits, and also makes it possible
for one rabbit cord to serve several cases. The cords in
glycerin, if kept in the cold, do not change in virulence for
several weeks.

The following table shows Pasteur's original schemata:

PASTEUR'S ORIGINAL SCHEMATA (Marx).

Light schema.

Intense schema.

Day of treatment.

!

Age of Amount of
dried injected
cord. | emulsion.

Day of treatment.

Age of
dried
cord.

Amount of
injected
emulsion.

First.

Days .

l«4
1i3

"

h

I;'!

{^

5

4
3

5
5
4
4
3
3
5
4
3

c.c.

3
3
3
3
3
3
3
3

First

Days.

M
»3

12
II
IO

9

8

I

\ 6
5

1

4
3
3

4
3
5
4
3

c.c.

3
3
3
3
3
3
3
3

Second

Second

Third

Fourth
Fifth

Third

Sixth.

Fourth
Fifth

Eighth
Ninth

Sixth
Seventh

Tenth
Eleventh
Twelfth

Eighth . .

Ninth
Tenth

Thirteenth
Fourteenth
Fifteenth
Sixteenth
Seventeenth
Eighteenth

Eleventh...
Twelfth
Thirteenth

Fourteenth

Fifteenth
Sixteenth

Seventeenth
Eighteenth
Nineteenth.

Twentieth

Twenty-first. . .

(From Bulletin No. 65, Hygienic Laboratory, June, 1910, U. S. Public

Health and Marine-Hospital Service.)
27

4i8

Hydrophobia, Lyssa, or Rabies

The system of treatment at present used at the Hygienic
Laboratory is shown in the following tables:

SCHEME FOR MILD TREATMENT.

Amount injected.

Amount injected.

Day.

Cord.

Five

One

Day.

Cord.

Five

One

Adult.

to ten

to five

Adult, to ten

to five

years.

years.

years.

years.

Injections.

c.c.

c.c.

c.c.

Injections.

c.c.

c.c.

c.c.

j

8—7—6=3

2.5

2-5

2.O

12

2

5—4=2

2-5

2-5

i-5

13

4=

•5

•5

-5

3

4 — 3—2

2-5

2-5

2.O

14

4

5=

2-5

2.5

2.5

1C

5
6

4=

2.5

2.5

2-5

ii:::::::::

2 =

•5

.O

•5

3=

2.5

2.5

2.0

IS:::::::::

4=

2-5

•5

•5

2 —

9 •.

2 =

2.5

2 O

i 5

20

10

5=

2-5

2.5

2-S

21

2=

2-5

2-5

.O

ii

5=r

2.5

2.5

2-5

SCHEME FOR INTENSIVE TREATMENT.

Day.

Cord.

Amount injected.

Day.

Cord.

Amount injected.

Adult.

Five
to ten
years.

One

to five
years.

Adult.

Five
to ten
years.

One
to five
years.

i

Injections.
8—7—6=3

c.c.

2-5

c.c.

2-5

c.c.
2.5

il2 .

Injections.

3=
3=

2 =
2 =

4=

3 =

2 =

3=

2 =
1 =

c.c.

2-5
•5
•5
•5
•5
• 5
•5
•5
•5
-5

c.c.
2-5

2.5

• 5
• 5
-5
• 5
•5
•5
2-5

2-5

c.c.

2.0
2.0

2.O
2.0
2-5
2-5
2.0
2.O
2-5
2.0

2 ...

4—3=
5—4=
3=
3=

2 =
2 =

5=
4=
4=

2.5
2.5

2-5

2.5
2.5
2.5

2.5

2.5

2.5

2-5

s-5
2-5

2-5

2-5
2.O
2-5

i-5
2-5
2-5
2-5

2.0

2-5
2 0
2.0

i-5

2.0
I.O

2-5
2-5
2-5

13

3
4

14

15

16

1:::
1

17
18

19

20
21

9
10

ii

(From Bulletin No. 65, Hygienic Laboratory, June, 1910, U. S. Public
Health and Marine-Hospital Service.)

This, in brief, is the theory and practice of Pasteur's
system of treating hydrophobia. It is entirely in keeping
with the ideas of the present time. When wef remember
that the first application of the method to human medicine
was made October 26, 1885, six years before the time we
began to understand the production and use of antitoxins,

The Dilution Method 419

it becomes one of the most remarkable achievements of
medicine.

Hogyes states that 50,000 persons in danger of developing
rabies have received the treatment in the last ten years and
that only i per cent, have died.

The Dilution Method. — Hogyes, of Budapest,* believes
that Pasteur was mistaken in supposing that the drying was
of importance in attenuating the virus, and thinks that
dilution is the chief factor. He makes an emulsion of rabbit's
medulla (i gram of medulla to 10 c.c. of sterile broth) as a
stock solution, to be prepared freshly every day, and uses it
for treatment, the first dilution used being i : 10,000; then
on succeeding days i : 8000, i : 6000, i : 5000, i : 2000,
i : looo, i : 500, i : 250, i : 200, i : 100, and finally the full
strength, i : 10.

Cabot t prepared a stock solution of 8 parts of rabbit's
brain and 80 parts of glycerin and water. The quantity
of glycerin added comprised one-fifth of the total bulk.
After the emulsion was made it was filtered through sterile
cheese-cloth. This emulsion containing the glycerin, if
kept in the ice-chest, will be of standard virulence during
the entire period of- immunization. As the result of his
experiments, Cabot found the dilution method attended
with danger to the animal immunized, which is not true of
the dried-cord method of Pasteur. The latter method is,
therefore, the one to be preferred.

Though the essential cause of rabies has not yet been dis-
covered, its lesions can be found in the medulla oblongata
and in the spinal ganglia. These consist in certain cellular ag-
gregations known as the " tubercles of Babes, "t and in certain
degenerative changes in the ganglionic nerve-cells. The regu-
larity with which these changes were observed by Babes led
him to regard them as useful for diagnosticating the disease,
and Van Gehuchten and Nelis§ and Ravenel and McCarthy
have confirmed this opinion. Ravenel and McCarthy || think
that Babes gives undue prominence to the rabic tubercle,
which consists of an aggregation of embryonal cells about the

*"Acad. des Sciences de Buda-Pest," Oct. 17, 1897; "Centralbl. f.
Bakt. u. Parasitenk.," 1887, n, 579.

t "Journal of Experimental Medicine," 1899, vol. iv, No. 2.

t "Virchow's Archives," ex; "Ann. de 1'Inst., Pasteur," 1892, vi.

§ "Archiv. de Biologic," 1900, xvi.

|| "Trans. Phila. Pathological Society," N. S., vol. m, 1900, p.
231; "University Medical Magazine," Jan., 1901.

42O

Hydrophobia, Lyssa, or Rabies

central canal of the cord, about the ganglionic nerve-cells,
and about the capillary blood-vessels, but that the lesions of
the nerve-cells are pathognomonic if taken in connection with
the clinical manifestations of the disease. The ganglion-cell
changes consist of degeneration, chromatolysis, and even
total disappearance of the nuclei, a dilatation of the peri-
cellular space, and an invasion not only of this space, but
also of the nerve-cells by embryonal cells, and at the same
time the appearance of small corpuscles which are hyaline,

$£

k - ' Vr, ** :-/x -

Fig. 131. — Normal sympathetic
ganglion from a normal dog.
(Modified from Crocq.)

Fig. 132. — Sympathetic ganglion
of a rabbit with experimental rab-
ies. (Modified from Ravenel and
McCarthy.)

brownish, and in parts metachromatic. Spiller* does not
regard the lesions as pathognomonic of rabies.

If an accurate diagnosis of rabies can be made by a sim-
ple histologic examination, in cases where animals thought
to be mad have bitten human beings, much time can be
saved in beginning the Pasteur treatment, and probably a
number of cases saved.

Negrif found that by staining sections of the cerebrum,
cerebellum, pons, basal ganglia, and sometimes even the

* "Pathological Society of Philadelphia," March, 1901.
f'Soc. Med.-Chirurg. di Pavia," 24, in, 1903; "Zeitschrift fiir
Hygiene," etc., xun and xuv.

The Bodies of Negri 421

spinal ganglia of human beings or animals dead of rabies,
by Mann's methylene-blue and eosin method, it was possi-
ble to demonstrate in the interior of the nerve-cells of their
protoplasmic process red colored, rounded bodies, measuring
4 to 10 [i, as a rule, though varying from i to 25 /u. In ex-
perimental infections under the dura they were most num-

Fig. 133. — Negri bodies. Cells from Ammon's horn impression of
cow 133, dried and fixed with gentle heat and stained with saturated
alcoholic eosin and Lomer's alkaline methylene-blue. Drawn with one-
twelfth oil immersion. Oc. 3 (Frothingham).

erous in the hippocampal convolution. These bodies have
now become known as Negri bodies.

The careful studies of Williams and Lowden* convince
them that the Negri bodies are protozoan organisms and
that their presence is pathognomonic of rabies.

At the State Live-stock Sanitary Board of Pennsylvania,
Reichel and Englej stain Negri bodies with the following:

* ''Jour. Infectious Diseases," in, 452, 1906.
t Personal communication.

422 Hydrophobia, Lyssa, or Rabies

Sat. ale. sol. methylene violet 10 c.c.

Sat. ale. sol. fuchsin 7 drops

Sterile water 40 c.c.

The smears of cerebellum or hippocampus are fixed with
absolute alcohol and ether and the stain poured on, heated,
poured back into the bottle, again poured on, heated and
poured back into the bottle, this being done three times, each
time for about half a minute. Then wash in water, blot, and
examine. To examine, a nerve-cell is found with the low
power and then examined with the high power. The Negri
bodies are brick red. The stain soon fades. Smears kept
for any length of time lose the staining reaction.

Antirabic Serum. — Babes and Lepp* thought that the
serum of animals that have received repeated injections of
the crushed nervous tissue of rabid animals was neutralizing
or destructive to the rabies virus in vitro, called it "anti-
rabic serum," and believed that it conferred a defensive
power upon other animals. Marie t found it to be a simple
neurotoxic serum and inert in its. action upon the virus.

* "Ann. de 1'Inst. Pasteur," 1889, m.

t " Compt.-rendu Soc. Biol.," t. i,vi, June 18, 1904, p. 1030.

CHAPTER VI.

CEREBROSPINAL MENINGITIS.

DIPLOCOCCUS INTRACELLULARIS MENINGITIDIS
(WEICHSELBAUM) .

General Characteristics. — A minute non-motile, non-flagellate,
non-sporogenous, non-chromogenic, non-liquefying, aerobic and op-
tionally anaerobic, pathogenic coccus, staining by ordinary methods,
but not by Gram's method.

Acute cerebrospinal meningitis may be secondary to
various more or less well-localized infections when it depends
upon such micro-organisms as may be carried by accident
to the meninges. Among these may be mentioned pneumo-
cocci, staphylococci, streptococci, Bacillus influenzae, B.
typhosus, B. coli, B. mallei, B. pestis, and others.

In addition to these cases, however, there are numerous
cases of primary infection of the membranes, either sporadic
or epidemic in occurrence. Such constitute the disease
known as cerebrospinal fever, epidemic cerebrospinal menin-
gitis, or " spotted fever." It is a very dangerous febrile
malady, characterized by high temperature, an irregular
exanthem, early meningitis, a moderate degree of contagion,
and a high mortality. The cause of this infection is a specific
organism known as the meningococcus, or Diplococcus intra-
cellularis meningitidis.

As early as 1887 Weichselbaum* carefully described a
diplococcus found in 6 cases of cerebrospinal meningitis
that may have been identical with one found by L/eichten-
stern| in 1885 in the purulent exudate of a case of meningitis,
and with a coccus observed as early as 1884 by Celli and
Marchiafava. J Weichselbaum 's studies and description of
this coccus seem to have attracted but little attention at first,
and references to them are but brief in most of the text-
books. The prevailing opinion was that its occurrence in

*"Fortschritte der Med.," x, 18 and 19.
t "Deutsche med. Wochenschrift," 1885.
t "Gazette degli Ospedali," 1884, vm.
423

424 Cerebrospinal Meningitis

cerebrospinal meningitis was accidental, as inoculations
into animals showed its pathogenic power to be very limited.
The careful studies of Jager,* Scherer,f Councilman, and
Mallory and Wright J (embracing 55 cases, in which the
cocci were found by culture or by microscopic examina-
tion in 38), and of Flatten, § Schneider, § Rieger,§ Schmidt,!
G6ppert,§ Flugge,§ von Lingelsheim, § and others. Bes-
redka|| and Flexner** have, however, shown the diplococcus
of Weichselbaum to be, without doubt, the specific organism.

;*e

*

Fig. 134. — Meningococcus in spinal fluid (from Hiss and Zinsser,
" Text-Book of Bacteriology," D. Appleton & Co., Publishers).

Distribution. — The distribution of Diplococcus intra-
cellularis in nature is as yet unknown. It has been found
in cerebrospinal meningitis by those who have looked for
it, twice has been found in the nose in coryza by Scherer,
has been found in the conjunctiva by Carl Frankelf f and
Axenfeld, J t and in the purulent discharges of rhinitis and
otitis by Jager. §§

Morphology. — The micro-organism is a biscuit-shaped
diplococcus having a great resemblance to the gonococcus.

* "Zeitschrift fur Hygiene," xix, 2, 351.

t "Centralbl. f. Bakt. u. Parasitenk.," 1895, xvn, 13 and 14.

J "Amer. Jour. Med. Sci.," March, 1898, vol. cxv, No. 5.

§ "Klinisches Jahrbuch," 1906.

|| "Annales de 1'Inst. Pasteur," 1906, xx, 4.
** "Jour. Kxp. Med.," 1906-07.
ft "Zeitschrift fur Hygiene," June 14, 1899.

\l Lubarsch and Oestertag, "Ergebnisse der allg. Path. u. path.
Anat.," in, S. 573-

§§ "Deutsche med. Wochenschrift," 1894, S. 407.

Staining 425

This resemblance is further increased by the fact that the
cocci are usually found inclosed in the protoplasm of the
leukocytes. Weichselbaum, by whom this was first ob-
served, found it constant in sections of the brain and its
membranes, though in the exudate of the disease a good
many free cocci may be observed. It was this peculiar
relationship to the cells that led Weichselbaum to name
the organism Diplococcus intracellularis. Many of the cocci
inclosed in the cells are apparently dead and degenerated,
as they stain badly and do not grow when the pus is trans-
ferred to culture-media.

Identification. — Carl Frankel, in discussing the micro-
organism, points out that its morphologic peculiarities have
much in common with the pneumococcus, so that the most
refined methods of differentiation should always precede a
positive determination. Its resemblance to the gonococcus
should also be kept in mind.

Perhaps the greatest difficulty obtains in making a
certain differentiation between the meningococcus and
Micrococcus catarrhalis (q. v.), especially when such investi-
gations are intended as discovering the former organism in the
nasal discharges. This cannot be done by microscopic ex-
amination, but must be achieved through cultivation of the
organisms and observation of the cultures. Micrococcus
catarrhalis grows well upon nearly all culture-media; menin-
gococci, very sparsely except upon special media. The former
organism grows fairly well at room temperatures (20° C. or
less) ; the latter, only at 25° C. and above. The colonies of the
former are coarsely granular ; those of the latter, finely granu-
lar.

Staining. — The organism is easily stained with the usual
aqueous solutions of the anilin dyes. According to Weich-
selbaum, Mallory, and Wright it does not stain by Gram's
method.

For staining the meningococcus the method of Pick and
Jacobsohn* is highly praised by Carl Frankel, who modifies
it by adding three times as much carbol-fuchsin as is recom-
mended in the original instructions, which are as follows:
Mix 20 c.c. of water with 8 drops of saturated methylene-blue
solution; then add 45 to 50 drops of carbol-fuchsin. Allow
the fluid to act upon the cover-glass for five minutes. The
cocci alone are blue, all else red.

* "Berliner klin. Wochenschrift," 1896, S. 811.

426 Cerebrospinal Meningitis

Isolation. — The organism can be secured for cultivation
either from the purulent matter of the exudate found at
autopsy, or from the fluid obtained by lumbar puncture. To
obtain this fluid Park * gives the following directions: " The
patient should lie on the right side with the knees drawn up
and the left shoulder depressed. The skin of the patient's
back, the hands of the operator, and the large antitoxin
syringe should be sterile. The needle should be 4 cm. in
length, with a diameter of i mm. for children, and larger for
adults. The puncture is generally made between the third
and fourth lumbar vertebrae. The thumb of the left hand
is pressed between the spinous processes, and the point of
the needle is entered about i cm. to the right of the median
line and on a level with the thumb-nail, and directed slightly
upward and inward toward the median line. At a depth of
3 or 4 cm. in children and 7 or 8 cm. in adults the needle
enters the subarachnoid space, and the fluids flow out in
drops or in a stream. If the needle meets a bony obstruc-
tion, withdraw and thrust again rather than make lateral
movements. Any blood obscures microscopic examination.
The fluid is allowed to drop into sterile test-tubes or vials
with sterile stoppers. From 5 to 15 c.c. should be with-
drawn. No ill effects have been observed from the opera-
tion."

In making a culture from this fluid Park points out that,
as many of its contained cocci are dead, a considerable
quantity of the fluid (say about i c.c.) must be used.

The cocci have also been cultivated from the nasal dis-
charges in the 6 cases studied by Weichselbaum and in
1 8 studied by Scherer. Elserf has isolated the organism
from the circulating blood of patients suffering from epi-
demic cerebrospinal fever. To determine the presence of
the coccus in the nasal discharges where other similar cocci
may be present, Gram's stain may be used and followed by
an aqueous solution of Bismarck-brown. The meningococci
will be brown.

Cultivation. — The organism was successfully cultivated
by Weichselbaum, but does not readily adapt itself to
artificial media. It develops upon agar-agar and glycerin
agar-agar, upon Loffler's blood-serum mixture, and, accord-

* "Bacteriology in Medicine and Surgery," Philadelphia, 1899,
p. 364.

t "Jour. Medical Research," 1906, xiv, 89.

Vital Resistance 427

ing to Goldschmidt,* upon potato. Wiechselbaum did not
find that it developed upon potato. It does not grow in
bouillon or gelatin. The cultures are usually scanty and
without characteristic features.

Flexnerf found that the difficulties of cultivation were
greatly reduced by the employment of sheep-serum instead
of human serum. Sheep-serum water was prepared accord-
ing to the method of Hiss (sheep-serum i part, water 2
parts, sterilized in the autoclave) and mixed with a beef-
infusion agar-agar containing 2 per cent, of glucose. The
quantity of sheep-serum need not exceed 7V to •£$ of the vol-
ume of the agar-agar. It is added to the sterile melted
agar, which is afterward slanted in test-tubes or allowed to
congeal on the expanded surface of i6-ounce Blake bottles
when mass cultures are to be used. There is nothing charac-
teristic about the cultures. The cocci grow only at the tem-
perature of the body, attain only a sparse development,
and form a more or less confluent line of minute, rounded,
grayish colonies which are easily overlooked upon opaque
media like blood-serum. The general characteristics of the
growth are not unlike those of the pneumococcus, strep-
tococcus, and gonococcus.

Colonies. — When grown upon agar-agar plates, the deep
colonies scarcely develop at all, appearing under the low-
power lens as minute, irregularly rounded granular masses.
The surface colonies are larger, and consist of an opaque
yellowish-brown nucleus about which a flat, rounded disk
spreads out. The edges may be dentate; the color is grayish
or yellowish near the center, becoming less intense as the
thin edges are reached ; the structure is finely granular.

Vital Resistance.— The vitality of the culture is low,
and the cocci die out readily, ceasing to grow when trans-
planted after eight or ten days. It becomes necessary,
therefore, when studying the organism to transplant it
frequently — ParkJ says every two days. Flexner§ found
that they do not survive beyond two or three days and that
transplantations do not succeed unless considerable quanti-
ties of the culture are placed upon the surface of the fresh
medium, showing that many of the organisms were already

* "Centralbl. f. Bakt. u. Parasitenk.," n, 22, 23.

t "Jour. Experimental Med.," 1907, ix, p. 105.

| "Bacteriology in Medicine and Surgery," 1899, p. 362.

§ Loc. cit.

428 Cerebrospinal Meningitis

dead. This is confirmed by the microscopic appearance of
the cultures. Those sixteen to twenty-four hours old stain
sharply and uniformly; on the second day many of the cocci
show irregularities of size and staining, and after several
days no normal-looking cocci can be found. It was found,
however, that in carefully preserved cultures of certain
strains a few cocci might survive for many months. Vitality
is preserved longest when the cultures are kept in the
thermostat and not taken out when grown, and kept at
room temperature or in a refrigerator. The addition of a
small quantity of a calcium salt favors prolonged vitality
and will sometimes maintain it for four or five weeks in cul-
tures that would otherwise die in a few days. Sodium
chlorid is injurious to the cocci. Flexner attributed the
autolysis of the cultures to an enzyme.

The organism is soon killed by drying, by exposure to the
sun, and by quite moderate variations of temperature. It
succumbs to very high dilutions of most germicides in a very
short time.

Agglutination. — When animals are immunized by repeated
injections of the Diplococcus intracellularis, their blood-
serum and body- juices become agglutinative. Such serums
kept in the laboratory can be used for the identification of
the coccus in fresh culture, though the reaction is not exact,
since the agglutinability of different strains of cocci is differ-
ent. The serums have an agglutinating power that varies
from i : 500 to i : 3000 in the hands of different observers.

Metabolic Products. — The meningococcus produces an
endotoxin. Albrech and Ghon* were able to kill white mice
with dead cultures. Lepierre | obtained a toxin from bouillon
cultures by precipitating them with alcohol.

Pathogenesis. — The results of animal inoculations made
with Diplococcus intracellularis meningitidis are disappoint-
ing. Subcutaneous inoculations into the lower animals are
continually without effect. Intrapleural and intraperito-
neal injections of cultures of the organism into mice and
guinea-pigs are sometimes fatal, the dead animals showing
a serofibrinous inflammation with the presence of the cocci.
The intravenous injection of the coccus into rabbits is
followed by death without important or conclusive symp-
toms, and usually without the presence of cocci in the blood.

* "Wiener klin. Wochenschrift," 1901.

t " Jour, de phys. et de path, gen.," v, No. 3.

Pathogenesis 429

Weichselbaum endeavored to reproduce the original
cerebrospinal meningitis in animals by trephining and in-
jecting the cocci beneath the dura. In this manner he
inoculated three rabbits and three dogs. Two of the rabbit
injections failed, probably because the injected material
escaped at once from the wound. The third rabbit died,
and showed marked congestion of the membranes of the
brain and a minute softened and hemorrhagic area. In
these the cocci were found by culture to be abundant.
The three dogs all died with congestion and pus-formation
in the membranes and areas of softening in the brain sub-
stance. The cocci were recovered from two of the dogs,
but the lesions of the third animal, which lived twelve days,
contained none.

Flexner* found that in large doses the coccus was always
capable of killing small guinea-pigs and mice when injected
intraperitoneally. To achieve this, however, the organisms
should be suspended in sheep-serum water, not in salt
solution, which is an active poison to the coccus.

Bettencourt and Franca f tried to infect monkeys by
trephining, by injecting into the spinal canal, and by rubbing
the cocci upon the nasal mucous membranes, but without
success. Von Lingelsheim and Leuchs| and Flexner § were
more successful. Flexner 's method was to introduce a hypo-
dermic needle into the spinal canal, wait until a few drops of
cerebrospinal fluid had escaped, and then inject the culture.
When thus introduced at a low level of the spinal canal, the
diplococci distribute themselves through the meninges in a
few hours and excite an acute meningitis, the exudate of
which accumulates chiefly in the lower spinal meninges and
the meninges of the base of the brain. The inflammation
extends, in monkeys, into the membranes covering the
olfactory lobes and along the dura mater into the ethmoid
plate and nasal mucosa.

The nasal mucous membrane is found in many instances
to be inflamed and beset with hemorrhages. Smear prepara-
tions from the nasal mucosa show many polymorphonuclear
leukocytes containing the cocci in a degenerated form. The
cocci were not cultivated from the nasal exudates.

* Loc cit.

t "Zeitschr. f. Hyg. u. Infekt.," XL,VI, p. 463.

J "Klin. Jahrbuch," 1906, xv, p. 489.

§ Loc. cit.

430 Cerebrospinal Meningitis

Mode of Infection. — It is not known by what channels
infection with Diplococcus intracellularis meningitidis takes
place. Weichselbaum supposed it might enter by the nasal,
auditory, or other passages, especially the nose, where he
constantly found it, and the more recent studies of Goodwin
and Sholly* have shown the organisms to be of frequent
occurrence in the nasal cavities of meningitis patients as
well as occasionally in those associated with them. It thus
becomes evident that association with the diseased may
lead to the infection of the well, and that the cases should
be isolated. The same conclusions were reached by Kolle
and Wassermann,f who studied the nasal secretions of 112
healthy individuals, not exposed to the disease, without
finding any cocci, but found them in the nasopharynx of
the father of a child suffering from the disease, and that of
another child with suspicious symptoms.

Steel J has found what may be a variety of the meningo-
coccus in the simple posterior basic meningitis of infants.
The organism differs from that of Weichselbaum in having
a greater longevity upon culture-media, where it often lives
as long as thirty days. It is easily stained by methylene-
blue, but not by Gram's method. Another similar organism
has been described by Elser and Huntoon.§

Specific Therapy. — Kolle and Wassermann|| carefully
studied antimeningococcus sera for specific opsonins, for
bacteriotropic substances, and for other evidences of favor-
able therapeutic action, but came to no definite conclusions.
Flexner and Jobling ** had better success both in developing
the experimental and practical knowledge of the serum.
The serum was prepared first with goats and then with
horses, the animals being injected with suspensions of the
meningococci. The serum is used by injecting it into the
spinal canal through a lumbar puncture. The precaution
must be taken to permit some of the fluid to escape first, and
then replace it by the antiserum, of which not more than
30 c.c. must be injected. Tabulations of the results follow-
ing the employment of Flexner 's serum show a large per-
centage of recoveries.

* ''Journal of Infectious Diseases," 1906, Supplement No. 2, p. 21.
f "Klinisches Jahrbuch," xv, 1906.
t " Pediatrics, " Nov. 15, 1898.
§ "Journal of Medical Research," 1909, xx, 377.
|| Loc. cit.
** "Jour. Experimental Medicine," 1907, ix, p. 168, and 1908, x, p. 141.

CHAPTER VII.
GONORRHEA.

MICROCOCCUS GONORRHOEA (NEISSER).

, General Characteristics.— A minute, biscuit-shaped, non-motile,
non-sporogenous, non-liquefying, non-chromogenic, non-flagellate, aero-
bic, strictly parasitic coccus, not stained by Gram's method, cul-
tivable upon special media, and pathogenic for man only.

All authorities now accept the " gonococcus " as the
specific cause of gonorrhea. It was first observed in the
urethral and conjunctival secretions of gonorrhea and
purulent ophthalmia by Neisser* in 1879.

Bumm| found other cocci closely resembling the gono-
coccus in the inflamed urethra, and points out that neither
its shape nor its position in the cells can be regarded as
characteristic, but that failure to stain by Gram's method
can alone enable us to say with certainty that biscuit-shaped
cocci found in urethral pus are gonococci.

Distribution. — The gonococcus is a purely parasitic
pathogenic organism. It can be found in the urethral dis-
charges of gonorrhea from the beginning until the end of the
disease, and often for many months and even years after re-
covery from it. After the period of creamy pus has passed, its
numbers are usually outweighed by other pyogenic organisms.
WertheimJ cultivated the gonococcus from a case of chronic
urethritis of two years' standing, and proved its virulence
by producing experimental gonorrhea in a human being.
The organisms are chiefly found within the pus-cells or
attached to the surface of epithelial cells, and should
always be sought for as diagnostic of gonorrhea, as purulent
urethritis is sometimes caused by other organisms, as
Bacillus ooli communis§ and Staphylococcus pyogenes.

* "Centralbl. f. d. med. Wissenschaft," 1879, No. 28.

t"Der Mikroorganismus der gonorrhoischen Schleimhauterkrank-
ungen," "Gonococcus Neisser," second edition, 1887.

t "Archiv f. Gynakologie," Bd. xui, 1892, Heft i.

§ Van der Pluyn and Loag, "Centralbl. f. Bakt. u. Parasitenk.," Bd.
xvn, Nos. 7, 8, Feb. 28, 1895, p. 233.

432 Gonorrhea

Morphology. — The organisms occur in pairs. Bach pair
of young cocci is composed of two spherical organisms, but
as they grow older the inner surfaces become flattened and
separated from one another by a narrow interval, so that
they somewhat resemble a coffee-bean. Sometimes tetrads
are seen, the group no doubt resulting from the division of
a pair. A pair of the cocci resembles the German biscuit,
and is described by the Germans as semmelfdrmig.

The gonococci are small, the length of one of the coffee-
bean cocci being about 1.6 ^, its breadth about 0.8 ^. They
are not motile, nor provided with flagella, and are without
spores.

Quite as characteristic as the form of the organism is its
relation to the cells. In most of the inflammatory exudates

Fig- 135- — Gonococci in urethral pus.

the gonococci are contained either in epithelial cells or in
leukocytes, very few of them lying free. This intracellular
position is supposed to depend upon active phagocytosis of
the cocci by the cells. It may not obtain in old lesions.

Staining. — They stain readily with all the aqueous solu-
tions of the anilin dyes — best with rather weak solutions,
but not by Gram's method.

The organisms contained in pus can be beautifully shown
by first treating the prepared film with alcoholic eosin, and
then with LofHer's alkaline methylene-blue. A differential
color test can be made by staining the film by Gram's method
and then with aqueous Bismarck-brown, or, what may be still
better, with 3 per cent, aqueous solution of pyronin. Ordi-

Isolation and Cultivation 433

nary pus cocci, taking the Gram's stain, appear blue-black;
the gonococci are dark brown.

Isolation and Cultivation. — The cultivation of the gono-
coccus is difficult and requires considerable bacteriologic
skill.

The organism does not grow upon any of the ordinary
culture-media, and grows very scantily upon any artificial
medium. Wertheim* succeeded in cultivating it by diluting
a drop of gonorrheal pus with human blood-serum, mixing
this with an equal part of melted 2 per cent, agar-agar at
40° C., and pouring the mixture into Petri dishes, which,
as soon as the medium became firm, were stood in the
incubator at 37° C. or, preferably, 40° C. In twenty-four
hours the colonies could be observed. Those upon the sur-
face showed a dark center, surrounded by a delicate granu-
lar zone.

Young f had excellent success with a hydrocele-agar pre-
pared as follows: " The fluid (hydrocele or ascitic) is ob-
tained sterile, the locality of the puncture being carefully
sterilized by modern surgical methods, the sterile trocar
covered at its external end with sterilized gauze so as not to
be infected by the operator's hand, and the fluid collected in
sterile flasks, the sterile stoppers being then replaced. Col-
lecting the fluid in .this way we have very rarely had it con-
taminated, often keeping it several months before using it.
The fluid is mixed with ordinary nutrient agar. A number
of common slants are put in the autoclave for five minutes.
This liquefies the agar and at the same time thoroughly
sterilizes the tubes and cotton stoppers. The slants are
then put in a water-bath at 55° C. so as not to coagulate the
albumin when mixed with the agar. The stopper having
been removed from a small flask of hydrocele fluid, the top
of the flask is flamed and the albuminous fluid is then poured
into an agar tube (the top of which has also been flamed) in
proportions a little more than one to two." The medium
can be allowed to solidify in tubes or can be poured into
Petri dishes.

When one of the colonies was transferred to a tube of
human blood-serum, or of one of the above-described mix-
tures obliquely coagulated, isolated little gray colonies occur,

* "Archiv. fur Gynakologie," 1892.

f "Contributions to the Science of Medicine by the Pupils of Wil-
liam M. Welch," Baltimore, 1900, p. 677.

28

434 Gonorrhea

later becoming confluent and producing a delicate smeary
layer upon the medium. The main growth is surrounded by
a thin, veil-like extension which gradually fades away at
the edges. A slight growth also occurs in the water of
condensation.

Heiman* found that the gonococcus grows best in a
mixture of i part of pleuritic fluid and 2 parts of 2 per
cent. agar. Wright t prefers a mixture of urine, blood-
serum, peptone, and agar-agar.

Wassermann t used a mixture of 15 c.c. of pig-serum,
35 c.c. of water, 3 c.c. of glycerin, and 2 per cent, of nutrose.
The nutrose is dissolved by boiling and the solution sterilized.
This is then added to agar, in equal parts, and used in plates. §

Laitinen|| found agar-agar mixed with one-third to one-
half its volume of cyst or ascitic fluid, and bouillon con-
taining i per cent, of peptone and 0.5 per cent, of sodium
chlorid, mixed with one-third to one-half its volume of
cyst or ascitic fluid, very satisfactory. The gonococcus
could be kept alive upon these media for two months.,
Laitinen found that the gonococcus produces acids in the
early days of its development, and alkalies subsequently.
He was unable to isolate any toxin from the cultures.

Vital Resistance. — Authorities agree that the gonococ-
cus has very slight power of heat endurance. Wertheim
found the optimum temperature of cultivation to be 39° to
40° C., and saw no harm result from exposure to 42° C. It
is, however, doubtful whether this temperature can be long
survived and whether higher temperatures can be endured.
The gonococci, though not easily cultivated, are said to
resist unfavorable conditions, especially drying, very well.
Kratter was able to demonstrate their presence upon washed
clothing after six months, and found that they still stained
well. This may not mean that the organisms were still
alive.

In artificial culture the gonococcus soon dies, though
cultures from different sources differ considerably in this
regard. As a rule they survive but a few transplantations,

* "Medical Record," Dec. 19, 1886.
f "Amer. Jour. Med. Sci.," Feb., 1895.
t "Berliner klin. Wochenschrift," 1897.

§ See " Text-Book of Bacteriology," by Hiss and Zinsser, 1910, p. 383,
|| "Centralbl. f. Bakt. u. Parasitenk.," June i, 1898, vol. xn, No. 20,
p. 874-

Toxic Products 435

though Young found that one culture had been kept alive
by students in his laboratory for more than three months.

Diagnosis. — The diagnosis of gonorrhea by finding the
diplococci in the pus and epithelial cells is a very simple
matter. The certain recognition of the micro-organisms
under other conditions is by no means an easy matter.
Thus, when gonorrhea becomes chronic and the cocci are
no longer taken up by the phagocytes, and so lose their
intracellular occurrence, it raises a little doubt whether
Gram-negative cocci may be true gonococci or not, yet it is
at precisely this time when a patient getting over gleet and
wanting to marry desires to know definitely whether gono-
cocci are any longer present in his urethra or not. Again,
when the gonococcus-like organisms occur upon the conjunc-
tiva, in the pus taken from joints, from the valves of the
heart, or from the Fallopian tubes, the same difficulty is met.
Probably the greatest perplexity arises when the conjunctiva
is called in question, for here there can come about a confu-
sion of the gonococcus, the pneumococcus, and Micrococcus
catarrhalis (q. v.) which only careful staining and culture
experiments can solve. The pneumococcus may be readily
separated if its lanceolate form and capsules can be observed,
but it is only by seeing that Micrococcus catarrhalis grows
readily and luxuriantly upon all the laboratory media,
and the gonococcus with difficulty and very sparingly upon
any media, that the diagnosis can be made with certainty.

The method of diagnosis by staining and looking for
Gram-negative diplococci in the cells is only a " rough and
ready " one and is not dependable.

Toxic Products. — The toxic metabolic products of the
gonococcus appear to be contained within the bodies of the
bacteria and disseminated but slightly throughout the
culture-media. Christmas,* Nicolaysen,f and Wasser-
mannj have studied gonotoxin, and have all found that it
remains in the bodies of the bacteria. The toxin seems
to be quite stable and is not destroyed by temperatures
fatal to the cocci. Wassermann obtained some cultures of
which o.i c.c. would kill mice; others, of which i.o c.c.

* ''Ann. de 1'Inst. Pasteur," 1897.

t "Centralbl. f. Bakt. u. Parasitenk.," 1897, Bd. xxn, Nos. 12
and 13, p. 305.

I "Zeitschrift fiir Hygiene," 1898, and "Berliner klin. Wochen-
schrift," 1897, No. 32, p. 685.

436 Gonorrhea

was required. The poison can be precipitated with absolute
alcohol. Small quantities of the toxin introduced into the
urethra cause suppuration at the point of application, fever,
swelling of the neighboring lymphatic nodes, and muscular
and articular pains,

Pathogenesis. — It is generally believed that gonorrhea
cannot be communicated to animals.

There is no doubt but that the gonococcus causes gonor-
rhea, as it has on several occasions been intentionally and
experimentally inoculated into the human urethra with
resulting typical disease. It is constantly present in the
disease, and very frequently in its sequelae, though it not
infrequently happens that the lesions secondary to gonor-
rhea are caused by the more common organisms of suppu-
ration that have entered through the surface denudations
caused by the gonococcus.

The mucous membranes, especially those covered with
squamous epithelium, are the appropriate portals for gonor-
rheal infection.

The injection of gonococci into the subcutaneous tissue is
not followed by either abscess formation or septic infection.

Gonococci may enter the circulation of human beings and
occasion a peculiar septic condition with irregular tempera-
ture, apt to be followed by invasion of the cardiac valves,
joints, or other tissues. P. Kraus* has twice succeeded in
cultivating the gonococcus from the blood of patients in the
stage of septic infection.

The deep lesions caused by the gonococcus are, however,
numerous, and in Young's paper (loc. cit.) its widespread
powers of pyogenic infection are well shown in a collection
of the cases recorded in the literature, and some original
observations showing the undoubted occurrence of the gono-
coccus in gonorrhea, ophthalmia neonatorum, arthritis, ten-
dosynovitis, perichondritis, subcutaneous abscess, intra-
muscular abscess, salpingitis, pelvic peritonitis, adenitis,
pleuritis, endocarditis, septicemia, acute cystitis, chronic
cystitis, pyonephrosis, and diffuse peritonitis.

In the beginning of the inflammatory process the cocci
grow in the superficial epithelial cells, but soon penetrate
between the cells to the deeper layers, where they continue
to keep up the irritation as the superficial cells desquamate.
Opinions differ as to whether the gonococci can, with equal
* "Berliner klin. Wochenschrift," No. 19, p. 494, May 9, 1904.

Immunization 437

facility, penetrate squamous and columnar epithelium.
Their attacks are usually made upon surfaces covered with
squamous epithelium.

All urethral inflammations, and in gonorrhea all of the
inflammatory symptoms, do not depend upon the gonococcus.
The periurethral abscess, salpingitis, etc., not infrequently
depend upon ordinary pus cocci, and I remember having
seen a case of gonorrhea with double orchitis, general septic
infection, and endocarditis in which the gonococci had no
role in the sepsis, which was caused by a large dumb-bell
coccus that stained beautifully by Gram's method.

In the remote secondary inflammations the gonococci
disappear after a time, and the inflammation either subsides
or is maintained by other bacteria. In synovitis, however,
the inflammation excited may last for months.

So long as the gonococci persist in his urethra or other
superficial tissues the patient may spread the contagion,
and after apparent recovery from gonorrhea the cocci may
remain latent in the urethra for years, not infrequently
causing a relapse if the patient partake of some substance,
as alcohol, irritating to the mucous membranes. Bearing
this in mind, physicians should be careful that their patients
are not too soon discharged as cured and permitted to
marry.

Immunization against the gonococcus has not yet been
successfully achieved. Wassermann failed altogether ; Christ-
mas claims to have immunized goats, but the serum of these
animals could not be shown to contain any antitoxin or to
be bacteriolytic.

Torrey* prepared an antigonococcus serum by immunizing
rabbits with gonotoxin. The culture used was isolated from
a case of acute gonorrhea in a medium of rich ascitic fluid
and slightly acid beef infusion, peptone broth, equal parts.
In speaking about this mixture Dr. Torrey told me that
the exact reaction was its most important feature, as other-
wise the gonococci soon died. Tubes of about 12 cm. of
the mixture were heated to about 60° C. for several hours
and then tested for sterility. The cocci were cultivated at
36° to 37° C. After eighteen to twenty-four hours' incuba-
tion a slight granular growth appears near the surface and on
the sides of the tube. This slowly increases until after six
days the medium is well clouded on shaking. Large rab-
* "Journal Amer. Med. Assoc.," Jan. 27, 1906, XL,VI, p. 261.

438 Gonorrhea

bits were used for making the serum, and were intraperi-
toneally inoculated with 10 c.c. of an entire culture. The
first inoculation resulted in a loss of weight, sometimes
amounting to one-fourth of the body-weight. After an
interval of five or six days a second injection is given, then
after a similar interval, a third, and so on. The best results
were obtained when cultures from six to fifteen days old
were employed. The rabbits were bled for the first time
after the sixth dose, as if the treatment be pushed they
soon fall into a state of cachexia, rapidly emaciate, and die.
Each animal furnishes 70 to 90 cm. of the serum, which was
inclosed in 2 -cm. bulbs, hermetically sealed, and kept without
any preservative.

With serum made in this way by Torrey, Rogers* treated
a number of obstinate cases of gonorrheal rheumatism, with
most satisfactory results.

Good results in gonorrheal arthritis and in gleet are also
claimed for treatment with gonococco-vaccines prepared as
suggested by A. E. Wright.

* "Jour. Amer. Med. Assoc.," XLVI, p. 261, Jan. 27, 1906.

CHAPTER VIII.

CATARRHAL INFLAMMATION.

MICROCOCCUS CATARRHALIS (SEIFERT).

General Characteristics.— A small, slightly ovoid, non-motile,
non-sporulating, non-flagellated, non-liquefying aerobic and optionally
anaerobic, non-chromogenic coccus, pathogenic for man, and not for the
lower animals, cultivable upon the ordinary media, staining by the
ordinary methods, but not by Gram's method.

This micro-organism, which seems to be closely related to
the staphylococci, was first observed, in sections of the lung
of a case of influenza, by Seifert.* It was successfully culti-
vated in 1890 by Kirchnerf from 10 cases of an influenza-
like affection. It has since been frequently demonstrated
in the exudates from various inflammatory conditions of the
respiratory tract and conjunctiva, and seems to be a not
uncommon organism of superficial inflammations. It is
a rather troublesome organism, causing some confusion
because of its disposition to occur in pairs, which gives it a
close resemblance to the pneumococcus except in cases in
which the capsules of the latter are distinct. It is also
readily taken up by the leukocytes, and may so resemble
the gonococcus; and it is not always easy, perhaps not
always possible, to distinguish it from the Diplococcus
intracellularis meningitidis.

Morphology. — The organism is spheric or slightly ovoid,
may occur singly, though usually appears in pairs or clus-
ters. Large numbers are enclosed in the leukocytes or other
cells. The spheric organisms have a diameter of about

1 ^; the ovoid organisms may measure as much as 1.5 by

2 li. The relation of the cocci to the cells seems to have
something to do with the course of the inflammatiory con-
ditions with which they are associated. During the activity
of the process large numbers of the cocci may be free ; toward
its close they may all be enclosed in the leukocytes.

* "Volkmann's klin. Vortr.," Nr. 240.
t "Zeitschr. f. Hyg.," Bd. 9.
439

44° Catarrhal Inflammation

The organisms are not motile and they have no flagella.

Staining. — The cocci stain by ordinary methods, but not
by Gram's method.

Cultivation. — The organism can be easily cultivated,
and thus differentiates itself from the fastidious gonococcus.
The colonies are large, white, irregular in outline, elevated at
the center, not viscid, and grow readily at room temperatures
upon all the culture-media, the best upon blood agar-agar.
The vitality of the organism in culture is not great. Very
often transplantations made after from four to six days fail

Fig. 136. — Micrococcus catarrhalis in smear from sputum (F. T. Lord;
photo by L. S. Brown).

to grow; and in the cultures one usually finds many deeply
staining, supposedly living cocci, and many poorly staining,
supposedly dead organisms.

Agar=agar. — The culture in general resembles that of
Staphylococcus albus. When blood is added to the agar-
agar, the growth is more luxuriant, whitish, and usually
consists of closely approximated colonies which do not
become confluent.

Gelatin. — This medium is not liquefied.

Bouillon. — At the end of the first day no growth seems

Pathogenesis

441

to have taken place, but at the end of the second day there
is a slight clouding and a meager precipitate. The organism
seems to maintain its vitality somewhat longer in bouillon
than in other culture-media.

Pathogenesis. — The organism seems to be scarcely patho-
genic for animals. Kirchner was able to kill a guinea-
pig by intrapleural injection, and Neisser, who performed
numerous experiments upon mice, guinea-pigs, and rabbits,

Fig. 137. — Micrococcus catarrhalis colonies on agar (F. T. Lord; photo
by L. S. Brown).

only once succeeded in producing a fatal infection, by the
intraperitoneal injection of 0.4 c.c. of bouillon culture. In
this animal the cocci were found in all the internal organs.
As has already been said, the organism is found associated
with superficial inflammatory conditions of the mucous
membrane. It is probably most common in influenza. It
has also been found in conjunctivitis, in bronchitis, in whoop-
ing-cough, and in pneumonia.

CHAPTER IX.
CHANCROID.

THE BACILLUS DUCREYI.

General Characteristics. — A small, ovoid streptobacillus, with
rounded, deeply staining ends, non-motile, non-flagellate, non-sporog-
enous; aerobic and optionally anaerobic, non-chromogenic, staining
by ordinary methods, but not by Gram's method, cultivable on special
media only and pathogenic only for man and certain monkeys.

The chancroid, soft chancre, or non-specific sore, as it is
called, is a common venereal affection of both sexes, most
frequent among those who give little attention to cleanli-
ness. It is characterized by the appearance of a soft reddish
papule, which makes its appearance usually upon the genital
organs, rarely upon other parts of the body, soon after the
infection, and soon becomes transformed to an ugly ulcera-
tion whose usual tendency is toward slow and persistent
enlargement, though in different cases it may be indolent,
active, phagedenic, or serpiginous. The inguinal or other
nearby lymph-nodes early enlarge and soon soften and
ulcerate. The disease is, therefore, extremely destructive
to the tissues invaded, though no constitutional involvement
ever takes place.

Specific Organism. — In 1889 Ducrey* described a peculiar
organism whose presence he was able to demonstrate with
great constancy, sometimes in pure culture, in the lesions
of chancroid, and which he believed to be the specific or-
ganism of the affection. Unnaf later described an organ-
ism resembling that of Ducrey, and the later observa-
tions of Krefting, { Peterson, § Nicolle,|| Cheinisse,** and

*"Congres. Inter, de Dermatol. et de Syphiligr.," Paris, 1889;
"Compt rendu," p. 229.

f "Monatschr. f. praktische Dermatologie," Bd. xiv, 1892, p. 485.
J "Archiv. f. Dermatol. u. Syphilol.," 1897, p. 263; 1897, p. 41.
§ "Centralbl. f. Bakt.," etc., 1893, Xm, p. 743.
§ "Med. Moderne," Paris, 1893, iv, p. 735.
** "Ann. de Dermat. et de Syphil.," Par., 1894, P- 272-

442

Cultivation 443

Davis* have abundantly confirmed the observations of
Ducrey and Unna, and proved the identity of the two
micro-organisms and their specificity for the disease.

Morphology. — The organism is commonly described as
a " strep tobacillus." It is very small, short, and ovoid in
shape, and occurs habitually in longer or shorter chains.
Each organism measures about 1.5 + 0.5 fi. The ends are
rounded and stain deeply. In pure cultures long undivided
filaments, at least twenty times as long as the individual
bacilli, are not uncommon. There seems to be no relation
between the cells and the bacilli. As a rule, they are free,
sometimes they are inclosed in leukocytes.

•7 -*--•<

^r;*

Fig. 138. — Smear of pus of chancroid of penis stained with carbol-
fuchsin and briefly decolorized by alcohol. X 1500 (Davis). (Photo-
micrograph by Mr. L. S. Brown.)

Staining. — The organisms are somewhat difficult to stain,
as they do not retain the color well, giving it up quickly
when washed. They do not stain by Gram's method.

Cultivation. — The first successful isolation and cultiva-
tion of the organism seems to have been by Benzancon,
Griffon and Le Sours f upon a culture-medium consisting of
rabbits' blood i part, and agar-agar 2 parts. Davis J has
been equally successful in cultivating the organism upon this
medium. His method was as follows:

"Tubes of 2 per cent, agar, reaction + 1.5, were melted

* " Jour. Med. Research," 1893, IX» P- 4QI-

t ''Ann. de Dermat. et de Syphiligr.," 1901, n, p. i.

J Loc. cit.

444 Chancroid

and mixed with fresh rabbits' blood drawn under aseptic
precautions, in the proportion of two-thirds agar to one-
third blood, and slanted while in a fluid state. At a later
period tubes of rabbits' blood-serum uncoagulated, also
rabbits' blood bouillon, one-third blood to two-thirds bouil-
lon, were used, and gave equally satisfactory results. By
employing small tubes of freshly drawn human blood pure
cultures were obtained in several instances from genital
lesions direct without any special cleansing of the ulcerated
surface. This, I believe, is the best medium for obtaining
cultures from a source open to contamination, the fresh

Fig. 139. — Culture from ulceration on monkey resulting from inocu-
lation of culture from a case of chancroid of finger, first generation.
Stained with carbol-fuchsin and briefly decolorized by alcohol. Cul-
ture of twenty-four-hours' growth in rabbit's bouillon. X 1500 (Davis).
(Photomicrograph by Mr. L. S. Brown.)

blood apparently inhibiting to a certain extent the growth
of extraneous organisms."

No growth takes place upon ordinary culture-media under
either aerobic or anaerobic conditions.

Cultures are best obtained by puncturing an unopened
bubo with a sterile needle and planting the pus directly and
immediately upon the special medium which should have
been warmed in the incubator so that the pus is not chilled.
In this way pure cultures may be secured, which are difficult
to get from the soft sore itself.

Colonies. — The colonies appear upon the appropriate
media in about twenty-four hours, and attain their complete

Pathogenesis 445

development in about forty-eight hours. They are at first
round bright globules, and later become grayish and opaque.
They measure i to 2 mm. in diameter and never become
confluent. They are difficult to pick up with the platinum
wire, tending to slide over the smooth surface of the medium.

Vital Resistance. — The organisms seem to possess little
vitality, their life in artificial culture being limited to a few
days. Frequent transplantation enabled Davis to carry
them on to the eleventh cultural generation.

Pathogenesis. — The organism is pathogenic for man
and certain monkeys (macacus), but not for the ordinary
laboratory animals. The organism can be found in large
numbers in both the genital and extragenital chancroidal
lesions, and usually in small numbers in the pus from chan-
croidal buboes. It has not been encountered elsewhere.
Lenglet* isolated the organism in pure culture, and by
inoculation with his cultures reproduced the lesions in man.

*"Bull. Med.," 1898, p. 1051; "Ann. de Dermatol. et de Syph.,"
1901, t. n. p. 209.

CHAPTER X.

ACUTE CONTAGIOUS CONJUNCTIVITIS.

THE KOCH-WEEKS BACILLUS.

General Characteristics. — A minute, slender bacillus, non-motile,
non-flagellated, non-sporogenous, non-liquefying, non-chromogenic,
aerobic, and optionally anaerobic, staining by the ordinary methods
but not by Gram's method, susceptible of cultivation upon special
media only, and specific for acute contagious conjunctivitis.

Acute contagious conjunctivitis is a common and world-
wide affection, sometimes called "pink eye," and sometimes
erroneously called catarrhal conjunctivitis. All its char-
acteristics, and especially its contagiousness, point to its
being a specific disease due to a specific cause, and thus
entirely different from ordinary non-specific catarrh.

Specific Micro=organism. — The first bacteriologic inves-
tigation of acute contagious conjunctivitis was made by
Robert Koch,* when in Egypt investigating a cholera
epidemic. While in Alexandria he examined the secretions
from 50 cases of conjunctivitis, finding the gonococcus,
or an organism closely resembling it. In a less severe form
of the disease, however, he found a peculiar small bacillus.
He seemed satisfied with this observation, or had no time to
pursue the matter farther, for no cultivation or other ex-
periments are mentioned.

The organism was observed from time to time, but no
serious consideration seems to have been devoted to it until
Weeks f published an account of what seemed to be the identi-
cal organism, which he not only observed, but alsp cultivated,
and eventually successfully inoculated into the human con-
junctiva. In the same year Kartulis| in Alexandria suc-
ceeded in cultivating the same organism. In 1894 Morax
published a brochure in Paris in which he says that "the
disease [which he describes under the name of acute con-

* "Wiener klin. Wochenschrift," 1883, p. 1550.
t "N. Y. Med. Record," May 21, 1887.
t "Centralbl. f. Bakt. u. Parasitenk.," 1887, p. 289.
446

Staining 447

junctivitis] is characterized by the constant presence in the
conjunctival secretions of a small bacillus seen for the first
time by Koch, but studied some years later by Weeks, and
now known as the bacillus of Weeks."

Further descriptive and clinical information can be found
in a paper by Weeks, "The Status of our Knowledge of the
J^tiological Factor in Acute Contagious Conjunctivitis."*

Morphology. — The organism is very tiny and is said to
bear some resemblance to the bacillus of mouse-septicemia.
It measures i to 2 X 0.25 ^ (Weeks). The length is more
constant in individuals found in the pus than those taken

Fig. 140. — The Koch- Weeks bacillus in conjunctival secretion. Magni-
fied 1000 diameters (Rymowitsch and Matschinsky).

from cultures. In cultures the organisms are longer and
more slender. Involution forms of considerable length and
of irregular shape also occur. No spores are observed. The
organism has no flagella and is not motile.

Staining. — Weeks found that the organism stained well
with watery solutions of methylene-blue, basic fuchsin, or
gentian violet. The color is fainter than that of the nuclei
of the associated pus-corpuscles, and is much less intense in
old than in fresh cultures. It is readily given up when
treated with alcohol or acids. Morax found that the bacilli
did not retain the color in Gram's method.

''New York Eye and Ear Infirmary Reports," vol. in, Part i,
Jan., 1895, p. 24.

448 Acute Contagious Conjunctivitis

Cultivation. — The organism refuses to grow upon any of
the ordinary culture-media. Weeks found, however, that if
the percentage of agar-agar used was reduced to 0.5 per
cent., growths could be secured by incubation at 37° C., and
successful transplantations carried on to the sixteenth gen-
eration. Abundant moisture was essential. The method
of isolation adopted by Weeks was as follows:

"The conjunctival sacs were thoroughly washed with clean water,
removing the secretion present by means of absorbent cotton. The
patient was then directed to keep the eyes closed. After five or ten
minutes had elapsed, the eyes were opened, and the secretion that had
formed, lying at the bottom of the cul-de-sac, was removed by means
of a sterilized platinum rod and transferred to the surface of the agar.
The mass of tenacious secretion was drawn over the surface of the
organ and left there, the platinum being thrust into the agar two or
three times before removal."

At the end of forty-eight hours a slight haziness appears
along the path of the wire, and on the surface of the agar
a very small patch is noticeable ; this is of a pearly color and
possesses a glistening surface. By the formation of small
concentric colonies the growth extends for a short distance.
At the end of the fourth or fifth day the growth ceases to
advance; it is never abundant. The culture dies in from
one to three weeks.

Pathogenesis. — Both Weeks and Morax have tested the
organism for pathogenic activity, and in every case in which
pure cultures of it were placed upon the human conjunctiva,
typical attacks of the acute conjunctivitis resulted. The
organism fails to infect any of the lower animals.

Association. — Both Weeks and Morax found the organ-
ism in intimate association with a larger club-shaped bacillus,
which was regarded as the pseudodiphtheria bacillus. It
seems to be of no pathogenic significance.

THE MORAX-AXENFELD BACILLUS.

In 1896 Morax* found a new bacillus in certain cases of
epidemic subacute conjunctivitis. Immediately afterward
Axenfeldf presented to a congress in Heidelberg cultures of
the same bacillus that he had isolated from 5 1 cases of what
he called " diplobacillenconjunctivitis" that occurred a few
months before as an epidemic in Marburg. De Schweinitz

* "Ann.de 1'Inst. Pasteur," June, 1896; "Ann. d'Oculist," Jan., 1897.
f "Heidelberg, Congress," 1896; "Centralbl. f. Bakt.," etc., 1897, xxi.

Cultivation 449

and Veasy,* Alt,f and others found the same diplobacillus in
America, and many others confirmed the observations in
various parts of Europe. It has also been found in Egypt.
There is no doubt, therefore, but that this is a widely distrib-
uted organism. Morax produced the disease by placing a
pure culture of the organism upon the human conjunctiva.
He was unable to infect any of the lower animals.

In this subacute form of conjunctivitis there is very little
secretion, and to secure the micro-organism either for micro-
scopic examination or for cultivation recourse must be had to
minute flakes of grayish mucus that collect upon the caruncle.

Fig. 141. — The Morax-Axenfeld diplobacillus of conjunctivitis. Magni-
fied 1000 diameters (Rymowitsch and Matschinsky.

Morphology. — The bacillus is small, commonly occurs in
pairs or chains. It measures approximately 2 [i in length.
It is not motile, has no flagella, and forms no spores. It is
somewhat pleomorphous. Involution forms soon appear in
artificial cultures.

Staining. — The organism stains by ordinary methods, but
does not stain by Gram's method.

Cultivation. — The organism grows only upon alkaline
blood-serum or upon culture-media containing blood-serum.
Morax made his original observation by using L°ffler's
blood-serum mixture. The colonies appear in twenty-four
hours at 37° C. The blood-serum is almost immediately

* " Ophthalmological Record," 1899.
t "Amer. Jour, of Ophthalmology," 1898, p. 171.
29

45° Acute Contagious Conjunctivitis

liquefied, so that the growing colonies appear to be sinking into
the medium after thirty-six hours. The entire tube of medium
may eventually be liquefied.

Upon agar-agar containing serum, grayish-white colonies
of small size, resembling colonies of gonococci, are formed.
Growth is slow. Bouillon is slowly clouded.

Pathogenesis. — The pathogenic and specific nature of
the diplobacillus was made clear by Morax, who produced the
disease in man by placing a pure culture upon the human con-
junctiva.

ZUR NEDDEN'S BACILLUS.

This bacillus was the only organism that Haupt* was able
to isolate from a neuroparalytic with confluent peripheral
ulcer ations of the cornea. It seemed to be identical with an
organism that zur Nedden had found previously in a case of
corneal ulceration in the clinic at Bonn.

Morphology. — It is a tiny bacillus, less than i u in length,
slightly curved, generally single, but sometimes in pairs and
short chains. It is not motile, has no flagella, forms no spores.

Staining. — It stains ordinarily, but not by Gram's
method.

Cultivation. — It is easily cultivated upon the ordinary
laboratory media, the cultures being without characteristic
peculiarities. Gelatin is not liquefied. Milk is coagulated.
Acid but no gas is formed in glucose media. A thick yellow-
ish growth appears upon potato. No indol is formed.

Pathogenesis. — Corneal ulcers were formed in a guinea-
pig after artificial implantation in the corneal tissue.

MISCELLANEOUS ORGANISMS IN CONJUNCTIVITIS.

In addition to the foregoing organisms, others not infre-
quently make their appearance as excitants of conjunctivitis.
The most frequent of these is the pneumococcus, the most
dangerous, the gonococcus. The former produce a severe
conjunctivitis, with the formation of a false membrane, the
latter the well-known blenorrhea and ophthalmia neonatorum.
Streptococci, diphtheria bacilli, staphylococci, meningococci,
colon bacilli, Bacillus pneumonia (Friedlander), and other
organisms have been found and appear to be responsible for
conjunctivitis.

*" Inaugural Dissertation," Bonn, 1902.

CHAPTER XI.

DIPHTHERIA.

BACILLUS DIPHTHERIA (KLKB

General Characteristics. — A non-motile, non-flagellate, non-spor-
ogenous, non-chromogenic, non-liquefying, aerobic, purely parasitic,
pathogenic, toxicogenic bacillus, cultivable upon the ordinary culture
media, staining by the ordinary methods and by Gram's method.

In 1883 Klebs * demonstrated the presence of a bacillus
in the pseudo-membranes upon the fauces of patients
suffering from diphtheria, but it was not until 1884 that
Loffler f succeeded in isolating and cultivating it. The
organism is now known by both their names, and called the
Klebs-Loffler bacillus.

Morphology. — The bacillus is about the length of the
tubercle bacillus (1.5-6.5 /*), but about twice its diameter
(0.4-1.0 /*), has a slight curve similar to that which char-
acterizes the tubercle bacillus, and has rounded and usu-
ally clubbed ends. It does not form chains, though
two, three, and rarely four individuals may be found con-
joined; usually the individuals are separate from one an-
other. The bacillus is peculiar in its pleomorphism, for
among the well-formed individuals which abound in fresh
cultures a large number of peculiar organisms are to be
found, much larger than normal, some with one end en-
larged and club shaped, some greatly elongated, with both
ends similarly and irregularly expanded. These bizarre
forms probably represent an involution form of the organ-
ism, for, while present in perfectly fresh cultures, they
are much more abundant in old cultures where scarcely a
single well-formed bacillus can be found. Distinct polar

* " Verhandlungen des Congresses fur innere Med.," 1883.
f " Mittheilungen aus dem kaiserlichen Gesundheitsamte," 2.

45i

45 2 Diphtheria

granules can be denned at the ends of the bacilli. Occa-
sional branched forms are observed, and the diphtheria
bacillus probably belongs to the higher bacteria, though
Abbott and Gildersleeve* do not regard branching as a phase
of the normal development of the organism, do not find it.
common upon the standard culture media, and so do not
think that it is properly classified elsewhere than among the
bacilli.

No flagella have been demonstrated upon the bacillus,
and it is non-motile. It is almost purely aerobic.

The involution of the diphtheria bacillus seems to occur
in proportion to the rapidity of its growth. Upon Loffler's
serum mixture, which seems best adapted for its cultivation,
the involution of the organism takes place with great
rapidity, so that large clubbed organisms and large or-
ganisms with polar granules are very common. On the
other hand, upon agar and glycerin agar-agar, where the
organism grows very slowly, it usually appears in the form
of short spindle and lancet shapes. So different are these
forms that a beginner would certainly fail to recognize them
as identical. The small short forms also stain much more
uniformly than the large club-shaped bacilli.

Wesbrook f has established certain morphologic types of
the bacillus (see illustration), and from the appearances
presented draws conclusions regarding their virulence,
which are confirmed by Gorham,J but disputed by Denny. §
The rapidly growing bacilli with clubbed ends and polar
granules are supposed to be virulent forms; the slowly
growing, uniformly staining forms, non-virulent bacilli.
Park and Denny believe that the uniformly staining bacil-
lus, when it develops in blood-serum cultures, is the pseudo-
diphtheria bacillus, an entirely different organism.

Staining. — The bacillus can readily be stained with aque-
ous solutions of the analin colors, but more beautifully
and characteristically with Loffler's alkaline methylene-
blue:

* "Centralbl. f. Bakt.," etc., Dec. 18, 1903, Bd. xxxv, No. 3.

t "Trans. Assoc. Amer. Phys.," 1900, and "Trans, of the Amer.
Public Health Assoc.," 1900.

t "Journal of Medical Research," N. S., vol. i, p. 201, 1901.

§ American Public Health Association (New Orleans) Meeting,
1902.

Staining 453

Saturated alcoholic solution of methylene-blue . . 30
1 : 10,000 aqueous solution of caustic potash . . 100

Emery prefers Hanson's borax methylene-blue. A stock
solution which keeps well is prepared by dissolving 2 grams
of methylene-blue and 5 grams of borax in 100 c.c. of water.
This is diluted with from five to ten times its volume of
water for ordinary use. An aqueous solution of dahlia
is recommended by Roux.

The Neisser method of staining the diphtheria bacillus,
which met with a very cordial reception, is as follows:

The prepared cover-glass is immersed for from two to
three seconds in

Alcohol (96 per cent.) 20 parts

Methylene-blue 1 part

Distilled water 950 parts

Acetic acid (glacial) 50 "

Then for three to five seconds in

Bismarck brown 1 part

Boiling distilled water 500 parts

The true diphtheria bacilli appear brown, with a dark blue
body at one or both ends; the pseudo-diphtheria .bacilli
usually exhibit no polar bodies.

Park * in his large experience found that neither the
Neisser nor the Roux stain gave any more information
concerning the virulence of the bacilli than the Loffler
alkaline methylene-blue.

When cover-glass preparations are stained with these
solutions, the bizarre forms already mentioned are par-
ticularly obvious, and the contrast between the polar
granules, which color intensely, and the cytoplasm of the
bacillus, which tinges slightly, is marked. Through good
lenses the organisms are always distinct bacilli, notwith-
standing the fact that the ends stain more deeply than the
centers, and it is only through poor lenses that the organ-
isms can be mistaken for diplococci.

The bacilli stain well by Gram's method, which is ex-
cellent for their definition in sections of tissue, though

* "Bacteriology in Medicine and Surgery," 1900.

454 Diphtheria

Welch and Abbott found that Weigert's fibrin method and
picrocarmin gave the most beautiful results.

Cultivation. — The diphtheria bacillus grows readily upon
all the ordinary media, and is very easy to obtain in pure
culture, plates not being necessary. Material from the in-
fected throat can be taken with a swab or platinum loop
and spread upon the surface of several successive tubes
of Loffler's blood-serum media. Upon the first a confluent
growth of the bacillus usually occurs; but upon the third,
scattered colonies suitable for transplantation can usually
be found.

Loftier has shown that the addition of a small amount
of glucose to the culture medium increases the rapidity of
growth, and suggests a special medium which bears his
name — Loffler's blood-serum mixture:

Blood-serum . % 3

Ordinary bouillon + 1 per cent, of glucose .... 1

This mixture is filled into tubes, coagulated, and sterilized
like blood-serum, and is one of the best known media to be
used in connection with the study of diphtheria.

The studies of Michel * have shown that the development
of the culture is much more luxuriant and rapid when
horses' serum instead of beef or calves' serum is used.
Horse's blood can easily be secured by the introduction of
a trocar into the jugular vein ; 5 liters of it can be withdrawn
without causing the animal inconvenience or symptoms of
weakness.

Westbrook suggested that the addition of a small amount
of glycerin to the preparation of blood-serum would prevent
it from drying so rapidly as usual and would have the added
advantage of preventing the growth of certain varieties
of bacteria not desired. Duboisf carried out a series of
observations upon this question and found that 3 to 5
per cent, of glycerin makes a very valuable addition, as
the diphtheria bacilli grow very rapidly and almost in

* "Centralbl. f. Bakt. u. Parasitenk.," Sept. 24, 1897, Bd. xxn,
Nos. 10 and u.

t "Seventeenth Annual Report of the Department of Health and
Charities," Indianapolis, Ind., 1907.

Fig. 142. — Bacillus diphtheriae, five hours Fig. 143. — Bacillus diphtheriae, same cul-

at 36° C. This shows only solid staining ture, eight hours at 36° C. This also shows

forms.

solid forms, many of them with parallel ar-
rangement.

\/i <*'*

N ' » %**

.'•-.'**V' '•
' YX *T* -VJ

A . J +-~^'

-':'fr'\?~4^
,/^lAr/r

* fc'^y N \

m •> '-arVrA

Fig. 144. — Bacillus diphtheriae, same cul-
ture, twelve hours at 36° C. The bacilli
stain faintly at their ends, and in some small
granules are seen at the tip of the faintly
stained portions.

Fig. 145. — Bacillus diphtheriae, same cul-
ture, fifteen hours at 36° C. The bacilli
stain more unevenly and the granules are
larger.

r^- ^

v-^

Fig. 146. — Bacillus diphtheriae, same cul-
ture, twenty-four hours at 36° C. This
shows clubbed and barred forms as well as
granular forms. At the lower part of the
field is a paired form which shows the char-
acteristic clubbing of the distal ends.

(Photomicrographs by Mr. Louis Brown.

All of the preparations were made from growth on blood-serum.)
" Jour, of Med. Research.")

455

Fig. 147. — Bacillus diphtheriae, forty-
eight hours at 36° C. This is the same
bacillus as in the preceding figures, but from
a culture where the colonies were discrete.
It shows long filamentous forms.

The magnification is the same in all — X 2000.
(Francis P. Denny, in

456

Diphtheria

pure culture upon the blood-serum mixture to which it is
added.

The impossibility of
making an accurate
diagnosis of diph-
theria without a bac-
teriologic examination
has caused many pri-
vate physicians and
many medical socie-
ties and boards of
health to equip labor-
atories where bacte-
riologic examinations
can be made. The
method requires some
apparatus, though a
competent bacteriolo-
gist can often make
shift with a bake-
oven, a wash-boiler,
and other household
furniture, instead of
the regular sterilizers
and incubators, which
are expensive.

Bacteriologic Di-
agnosis.— When it is
desired to make a
bacteriologic diagno-
sis in suspected diph-
theria, or to secure the

bacillus in pure culture, a sterile platinum wire having
a small loop at the end, or a swab made by wrapping a
little absorbent cotton about the end of a piece of wire
and carefully sterilizing it in a test-tube, is introduced
into the throat and touched to the false membrane, after
which it is carefully smeared over the surface of at least
three of the blood-serum mixture tubes, without either
again touching the throat or being sterilized. The tubes
thus inoculated are stood away in an incubating oven at
the temperature of 37° C. for twelve hours, then exam-

Fig. 148. — The Providence Health De-
partment outfit for diphtheria diagnosis,
consisting of a pasteboard box containing a
swab-tube and a serum-tube, both with
etched surface on which to write the name
and address of patients, etc.

Cultivation

457

istf

ined. If the diphtheria bacillus be present, a smeary,
yellowish- white layer will be present upon the first tube, a
similar layer with outlying colonies on the second tube,
while the third tube will show rather
large, isolated, whitish or slightly
yellowish, smooth colonies. The
colonies may be china-white in ap-
pearance. These colonies, if found
by microscopic examination to be
made up of diphtheria bacilli, will
confirm the diagnosis of diphtheria,
and will at the same time give
pure cultures of the bacillus when
transplanted. There are very few
other bacilli that grow so rapidly
upon Loffler's mixture, and scarcely
any other is found in the throat.

When no tubes of the blood-
serum mixture are at hand, the
swab can be returned to its tube
after having been wiped over the
throat of the patient, and can be
shipped to the nearest laboratory.

When an early diagnosis is re-
quired, Ohlmacher recommends
that the microscopic examination
of the still invisible growth be made
in five hours. A platinum loop is
rubbed over the inoculated surface ;
the small amount of material thus
secured is mixed with distilled
water, spread on a cover-glass,
dried, fixed, stained with methy-
lene-blue, and examined. An abun-
dance of the organisms are usually
found and valuable time is saved preparatory to the use of
the antitoxin.

The presence of diphtheria bacilli in material taken from
the throat does not necessarily mean that the person has
diphtheria. Virulent bacilli can occasionally be discovered
in the throats of healthy persons who have knowingly or
unknowingly come in contact with the disease, but whose

Fig , 1 49 . — Sterilized
test-tube and swab for
collecting pus and fluids
for bacteriologic examina-
tion (Warren).

458

Diphtheria

vital resistance is such that the bacilli grow scantily without
producing disease of the throat. The bacteriologic examin-
ation is, therefore, only an adjunct to the clinical diagnosis,
and must never be taken as positive in itself.

Fig. 150. — Diphtheria bacilli (from photographs taken by Prof. E.
K. Dunham, Carnegie Laboratory, New York): a, Pseudobacillus ;
b, true bacillus; c, pseudobacillus.

Gelatin. — Gelatin is not an appropriate medium for the
cultivation of the bacillus. Upon the surface of gelatin
plates the colonies attain but a small size and appear to
the naked eye as whitish points with smooth contents and
regular, though sometimes indented, borders. Under the
microscope they appear granular and yellowish-brown, with
irregular borders (Fig. 151). The growth in gelatin punc-
ture is characterized by the occurrence of small spheric
colonies along the line of inoculation. The gelatin is not
liquefied.

Cultivation 459

Agar-agar. — Upon agar-agar and glycerin agar-agar the
colonies are slower to develop, larger, more translucent,
without the yellowish-white or china-white color of the
blood-serum cultures, and are more or less distinctly divided
into a small elevated center and a flat surrounding zone
with indented edges, and a radiated appearance. When
transplantations are made from blood-serum to agar-agar,
the resulting growth is usually meager, but the oftener the
organism is transplanted to fresh agar-agar, the more luxu-
riant its growth becomes.

Bouillon. — When planted in bouillon a distinct, whitish,

Fig. 151. — Bacillus diphtheriae ; colony twenty-four hours old, upon
agar-agar. X 100 (Frankel and Pfeiffer).

granular pellicle forms upon the surface of the medium.
The pellicle appears quite uniform when the flask is un-
disturbed, but it is so brittle that it at once falls to pieces if
the flask be moved, the minute fragments slowly sedimenting
and forming a miniature snow-storm in the flask or tube.
The organism at times also causes a diffuse cloudiness of the
medium, but, not being motile, soon settles to the bottom
in the form of a flocculent precipitate which has a tendency
to cling to the sides of the glass, and leave the bouillon
clear.

460 Diphtheria

Spronk * found that the characteristics of the growth of
the diphtheria bacillus in bouillon, as well as the amount
of toxin produced, vary according to the amount of glucose
in the bouillon.

Zinnof found that digested brain added to the culture
bouillon greatly facilitated the growth of diphtheria and
tetanus bacilli and increased the toxin- product ion.

Blood-serum. — The bacillus grows similarly upon blood-
serum and Loffler's mixture.

Potato. — Upon potato it develops only when the reaction
is alkaline. The potato growth is not characteristic.

Milk. — Milk is an excellent medium for the cultivation of
Bacillus diphtherise, and is possibly at times a medium of
infection. Litmus milk is useful for detecting the changes
of reaction brought about by the alkalinity, which at first
favors the development of the bacillus, being soon replaced
by acidity. When the culture becomes old, the reaction
again becomes strongly alkaline. This variation in reaction
seems to depend entirely on the transformation of the sugars.

Vital Resistance. — The diphtheria bacillus does not
form spores. It possesses very little vital resistance and
is delicate in its thermic sensitivity. LofHer found that it
could not endure a temperature of 60° C., and Abbott has
shown that a temperature of 58° C. is fatal to it in ten
minutes. The organism can sometimes be kept alive for
several weeks after being dried upon shreds of silk or when
surrounded by dried diphtheria membrane.

Metabolic Products. — The earliest researches upon the
nature of the poisonous products of the diphtheria bacillus
seem to have been made in 1887 by Loffler, { who came to
the conclusion that they belonged to the enzymes. The
credit of removing the bacteria from the culture by filtration
through porcelain and the demonstration of the soluble
poison in the filtrate belongs to Roux and Yersin.§ Toxic
bouillon prepared in this manner was found to cause serous
effusions into the pleural cavities, acute inflammation of
the kidneys, fatty degeneration of the liver, and edema
of the tissue into which the injection was made. In some
cases palsy subsequently made its appearance, usually in the

* "Ann. del'Inst. Pasteur," Oct. 25, 1895, vol. ix, No. 10, p. 758.
t "Centralbl. f. Bakt.," Jan. 4, 1902, xxxi, No. 2, p. 42.
J "Centralbl. f. Bakt.," etc., 1887, n, p. 105.
§ " Ann. de 1'Inst. Pasteur," 1888-1889.

Metabolic Products 461

hind quarters. The effect of the poison was slow and death
took place days or weeks after injection, sometimes being
preceded by marked emaciation. Temperatures of 58° C.
lessened the activity of the toxin and temperatures of 100°
C. destroyed it. It was precipitated by absolute alcohol
and mechanically carried down by calcium chlorid. Brieger
and Frankel * confirmed the work of Roux and Yersin, and
concluded that the poison was a toxalbumin. Tangl f
was able to extract the toxin from a fragment of diphtheria
pseudo-membrane macerated in water.

The nature of the diphtheria toxin has been studied by
Khrlich { and found to be extremely complex. As it
exists in cultures it is composed of equal parts of toxin and
toxoid. Of these, the former is poisonous, the latter harm-
less for animals — or at least not fatal to them. The toxoids
have equal or greater affinity for combining with antitoxin
than the toxin and cause confusion in testing the unit value
or strength of the antitoxin. In old or heated toxin all
of the toxin molecules become changed into toxons or tox-
oids and the poisonous quality is lost though the power of
combining with antitoxin remains.

The toxin is intensely poisonous, and a filtered bouillon
containing it may be fatal to a 3oo-gram guinea-pig in doses
of only 0.0005 c-c- It is thought not to be an albuminous
substance, as it can be elaborated by the bacilli when
grown in non-albuminous urine, or, as suggested by Uschin-
sky, in non-albuminous solutions whose principal ingredient
is asparagin. The toxic value of the cultures is greatest
in the second week.

This soluble toxin so well known in bouillon cultures is
probably only one of the poisonous substances produced by
the bacillus. An intracellular, insoluble toxic product
seems to have been discovered by Rist,§ who found it in
the bodies of dried bacilli, and observed that it was not
neutralized by the antitoxin.

Palmirski and Orlowski|| assert that the bacillus pro-
duces indol, but only after the third week. Smith,** how-

*" Berliner klin. Wochenschrift," 1890, 11-12.
f "Centralbl. f. Bakt.," etc., Bd. xi, p. 379.
J "Klinisches Jahrbuch," 1897.
§ "Soc. de Biol. Paris," 1903, No. 25.
|| "Centralbl. f. Bakt. u. Parasitenk.," March, 1895.
** "Jour. Exp. Med.," Sept., 1897, vol. n, No. 5, p. 546.

462 Diphtheria

ever, found that when the diphtheria bacillus grew in
dextrose-free bouillon no indol was produced.

The acidity of the culture media depends upon the
formation of lactic acid.

Pathogenesis. — Diphtheria in man is characterized by a
pseudo-membranous inflammation of the mucous mem-
branes, particularly of the fauces, though it may occur upon
other parts of the body and is not infrequent in the nose, in
the mouth, upon the genital organs, or upon wounds. Wil-
liams * has reported a case of diphtheria of the vulva, and
Nisot and Bumm have reported cases of puerperal diphtheria
from which the bacilli were cultivated. It is in nearly
all cases a purely local infection, depending upon the
presence and development of the bacilli upon the diseased
mucous membrane, but is accompanied by a serious intoxi-
cation resulting from the absorption from the local lesions
of a poisonous metabolic product of the bacilli. The bacilli
are found only in the membranous exudation, and are
most plentiful in its older portions.

The entrance of the diphtheria bacillus into the internal
organs can scarcely be regarded as a frequent occurrence,
though metastatic occurrence of the organism with and
without associated staphylococci and streptococci, and
with and without purulent inflammations have from time
to time been reported. Such a case of septic invasion by
the diphtheria bacillus, with a synopsis of the literature to
date, is given by Ucke. f

The disease pursues a course of variable length, in favor-
able cases the patient recovering gradually, the pseudo-
membrane first disappearing, leaving an inflamed mucous
membrane behind it, upon which virulent diphtheria bacilli
persist, always for weeks and sometimes for months. Smith J
describes the bacteriologic condition of the throat in diph-
theria as follows: "The microscope informs us that during
the earliest local manifestations the usual scant miscel-
laneous bacterial flora of the mucosa is quite suddenly
replaced by a rich vegetation of the easily distinguishable
diphtheria bacillus. Frequently no other bacteria are found

* " Amer. Jour, of Obstet. and Dis. of Women and Children," Aug.,
1898.

t "Centralbl. f. Bakt. u. Parasitenk.," original, XLVI, Heft 4, March
10, 1908, p. 292.

J "Boston Med. and Surg. Jour.," 1898, I, p. 157.

Pathogenesis 4.63

in the culture-tube. This vegetation continues for a few
days, then gradually gives way to another flora of cocci and
bacilli, and finally the normal condition is reestablished."

Diphtheria bacilli were first found in the heart's blood,
liver, spleen, and kidney, by Frosch.* Kolisko and Pal-
tauff had already found them in the spleen, and other
observers in various lesions of the deeper tissues and occa-
sionally in the organs. In the blood and organs it is com-
monly associated with Streptococcus pyogenes and some-
times with other bacteria. While present in nearly all of
the inflammatory sequelae of diphtheria, the Klebs-Loffler
bacillus probably has very little influence in producing them,
the conditions being almost invariably associated with the
pyogenic cocci, either the streptococci or staphylococci.

Howard} studied a case of ulcerative endocarditis caused
by the diphtheria bacillus, and Pearce § has observed
it in i case of malignant endocarditis, 19 out of 24 cases of
broncho-pneumonia, i case of empyema, 16 cases of middle-
ear disease, 8 cases of inflammation of the antrum of High-
more, i case of inflammation of the sphenoidal sinuses, i
case of thrombosis of the lateral sinuses, 2 cases of abscesses
of the cervical glands, and in esophagitis, gastritis, vulvo-
vaginitis, dermatitis, and conjunctivitis following or asso-
ciated with diphtheria.

In animals artificially inoculated with the diphtheria
bacillus the resulting lesions resemble those seen in the
human subject, in that they consist of a local infection
with a general toxemia.

Human beings, horses, rabbits, guinea-pigs, mice, kittens,
and young pups are susceptible ; rats are immune. When half
a cubic centimeter of a twenty-four-hour-old bouillon culture
is injected beneath the skin of a susceptible animal, the bacilli
multiply at the point of inoculation, producing a fibrinous in-
flammation with edema. The animal dies about the third
day. When examined post-mortem the liver is found en-
larged and sometimes shows minute whitish points, which
upon microscopic examination prove to be necrotic areas in
which the cells are completely degenerated, and the chrom-
atin of their nuclei scattered about in granular form. Similar

* "Zeitschrift fur Hygiene," etc., 1893, XIII» Heft i.

t "Wiener klin. Wochenschrift," 1889.

i "Amer. Jour. Med. Sci.," Dec., 1894.

§ "Jour. Boston Soc. of Med. Sci.," March, 1898.

464 Diphtheria

necrotic foci, to which attention was first called by Oertel,
are present in nearly all the organs in cases of death from
diphtheria intoxication. No bacilli are present in these
lesions. Welch and Flexner * have shown these foci to
be common to numerous intoxications, and not peculiar to
diphtheria.

The lymphatic glands are usually enlarged, and the adre-
nals enlarged and hemorrhagic. The kidneys show paren-
chymatous degeneration. There is no inflammation of the
fauces.

Roux and Yersin found that when the bacilli were intro-
duced into the trachea of animals opened by operation, a
typical pseudo-membrane was formed, and that diphtheritic
palsy sometimes followed.

Associated Bacteria. — Streptococcus pyogenes and Staphy-
lococci pyogenes aureus and albus are, in many cases,
found in association with the diphtheria bacillus, especially
when severe lesions of the throat exist.

In a series of 234 cases carefully and statistically studied
by Blasi and Russo-Travali,f it was found that in 26 cases
of pseudo-membranous angina due to streptococci, staphy-
lococci, colon bacilli, and prieumococci, 2 patients died,
the mortality being 3.84 per cent. In 102 cases of pure
diphtheria, 28 died, a mortality of 27.45 per cent. Seventy-
six cases showed diphtheria bacilli and staphylococci; of
these, 25, or 32.89 per cent., died. Twenty cases showed
the diphtheria bacilli and Streptococcus pyogenes, with 6
deaths — 30 per cent. In 7 cases, of which 3, or 43 per
cent., were fatal, the diphtheria bacillus was in combination
with streptococci and pneumococci. The most dangerous
forms met were 3 cases, all fatal, in which the diphtheria
bacillus was found in combination with Bacillus coli.

In 157 cases of diphtheria and scarlatina studied at the
Boston City Hospital by Pearce,{ there were 94 cases of
diphtheria, 46 cases of complicated diphtheria (29 with
scarlet fever, n with measles, and 5 with measles and
scarlet fever), and 17 cases of scarlet fever (in 3 of which
measles was also present).

Of the 94 cases of uncomplicated diphtheria, the Klebs-
Loffler bacilli were present in the heart's blood in 4, twice

* " Bull, of the Johns Hopkins Hospital," Aug., 1901.

f "Ann. del'Inst. Pasteur," 1896, p. 387.

% "Jour. Boston Soc. of Med Sci.," March, 1898.

Pathogenesis 465

alone and twice with streptococci. In 9 cases the strepto-
coccus occurred alone ; in i case the pneumococcus occurred
alone. In the liver the bacillus was found in 24 cases,
alone in 1 2 and together with the streptococcus in 12; the
streptococcus occurred in 27 cases, alone in 14, with the
Klebs-Loffler bacillus in 12, and with Staphylococcus
pyogenes aureus in i. Staphylococcus pyogenes aureus
occurred in 4 cases, alone in 3 and associated with the
streptococcus in i. The pneumococcus occurred alone in
i case.

In the spleen the Klebs-Loffler bacillus occurred eighteen
times, fifteen times alone and three times associated with
the streptococcus. The streptococcus occurred in 24 cases,
alone in 21, associated with the Klebs-Loffler bacillus twice,
and with Staphylococcus pyogenes aureus once. Staphy-
lococcus pyogenes occurred twice, once alone and once with
the streptococcus. The pneumococcus occurred twice alone.

In the kidney the Klebs-Loffler bacillus occurred in 23
cases, in 15 alone, in 5 associated with the streptococcus,
and in 2 with Staphylococcus pyogenes aureus. The
streptococcus occurred in 26 cases, in 19 of which it was the
only organism present. Staphylococcus pyogenes aureus
occurred in 8 cases, in 4 of which it was in pure culture.
The pneumococcus occurred four times, three times in pure
culture and once with the Klebs-Loffler bacillus.

In the 46 cases of complicated diphtheria, the heart's
blood showed pure cultures of the streptococcus nine times
and the streptococcus associated with the Klebs-Loffler
bacillus once. The diphtheria bacillus occurred alone once.

In the liver, in 10 cases streptococcus occurred alone, in
7 cases associated with the Klebs-Loffler bacillus, and in 3
cases with Staphylococcus pyogenes aureus. The diphtheria
bacillus occurred in pure culture in 5 cases.

The spleen contained streptococci only thirteen times and
mixed with the diphtheria bacillus twice. The diphtheria
bacillus was found in pure culture in 5 cases.

The kidney contained pure cultures of streptococci in
10 cases, streptococci associated with diphtheria bacilli five
times, and with Staphylococcus pyogenes aureus three
times. The diphtheria bacillus occurred alone in 7 cases.
Staphylococcus pyogenes aureus and the pneumococcus
each alone once and both together once.

"The clinical significance of this general infection with the
30

466 Diphtheria

Klebs-Loffler bacillus is not apparent. It occurred gener-
ally, but not always, in the gravest cases, or those known as
'septic' cases. It is probable that it may be due to a
diminished resistance to the tissue-cells, or of the germicidal
power of the blood. In this series of fatal cases the number
of infections with the streptococcus and with the Klebs-
Loffler bacillus was about even, though slightly in favor of
the streptococcus."

The mixed infections add to the clinical diphtheria the
pathogenic effects of the associated bacteria. The diphtheria
bacillus probably begins the process, growing upon the
mucous membrane, devitalizing it by its toxin, and pro-
ducing coagulation-necrosis. Whatever pyogenic germs
happen to be present are thus afforded an opportunity to
enter the tissues and add suppuration, gangrene, and re-
mote metastatic lesions to the already existing ulceration.

Diphtheritic inflammations of the throat are not always
accompanied by the formation of the pseudo-membrane,
but in some cases a rapid inflammatory edema in the larynx,
without a fibrinous surface coating, may cause fatal suffoca-
tion, only a bacteriologic examination revealing the true
nature of the disease.

Lesions. — The pseudo-membrane characterizing diph-
theria consists of a combined necrosis of the tissues acted
upon by the toxin and coagulation of an inflammatory
exudate. When examined histologically it is found that
the surface of the mucous membrane is chiefly affected.
The superficial layers of cells being embedded in coagulated
exudate- — fibrin — and show a peculiar hyaline degeneration.
Sometimes the membrane seems to consist exclusively of
hyaline cells ; sometimes the fibrin formation is secondary to
or subsequent to the hyaline degeneration. Leukocytes
caught in the fibrin also become hyaline. From the super-
ficial layer the process may descend to the deepest layers,
all of the cells being included in the coagulated fibrin and
showing more or less hyaline degeneration. The walls of the
neighboring capillaries also become hyaline, and the necrotic
mass forms the diphtheritic membrane. The laminated
appearance of the membrane probably depends upon the
varying depths affected at different periods, or upon differ-
ences in the process by which it has been formed. The
pseudo-membrane is continuous with the subjacent tissues
by a fibrinous reticulum, and is in consequence removed

Specificity 467

with difficulty, leaving an abraded surface. When the mem-
brane is divulsed during the course of the disease, it imme-
diately forms anew by the coagulation of the inflammatory
exudate.

The coagulation -necrosis seems to depend upon the local
effect of the toxin. Morax and Elmassian * found that
when strong diphtheria toxin is applied to the conjunctiva
of rabbits every three minutes for eight or ten hours,
typical diphtheritic changes are produced.

Flexnerf has made a study of the minute lesions caused
by bacterial toxins and especially of the diphtheria toxin,
and Councilman, Mallory, and Pearce,J of both gross and
minute lesions, that the thorough student should read.

Specificity. — Herman Biggs, § in an interesting discus-
sion of the occurrence of the diphtheria bacillus and its
relation to diphtheria, comes to the following conclusions:

1. "When the diphtheria bacillus is found in healthy
throats, investigation almost always shows that the indi-
viduals have been in contact with cases of diphtheria.
The presence of the bacillus in the throat, without any
lesion, does not, of course, indicate the existence of the
disease."

2. "The simple anginas in which virulent dipntheria
bacilli are found are to be regarded from a sanitary stand-
point in exactly the same way as the cases of true diph-
theria."

3. "Cases of diphtheria present the ordinary clinical
features of diphtheria, and show the Klebs-Loffler bacilli."

4. "Cases of angina associated with the production of
membrane in which no diphtheria bacilli are found might
be regarded from a clinical standpoint as diphtheria, but
bacteriological examination shows that some other organ-
ism than the Klebs-Loffler bacillus is the cause of the pro-
cess."

Any skepticism of the specificity of the diphtheria bacillus
on my own part was dispelled by a somewhat unique ex-
perience. Without having been previously exposed to
diphtheria while experimenting in the laboratory I acciden-

* "Ann. de 1'Inst. Pasteur," 1898, p. 210.
f" Johns Hopkins Hospital Reports," vi, 259.
J " Diphtheria : A Study of the Bacteriology and Pathology of
Two Hundred and Twenty Fatal Cases," 1901.

§ "Amer. Jour. Med. Sci.," Oct., 1896, vol. xxn, No. 4, p. 411.

468 Diphtheria

tally drew a living virulent culture of the diphtheria bacillus
through a pipet into my mouth. Through carelessness no
precautions were taken to prevent serious consequences, and
two days later my throat was filled with typical pseudo-
membrane which private and Health Board bacteriologic
examinations showed to contain pure cultures of the Klebs-
Loffler bacilli.

Some have been led to doubt the specificity of the diphtheria
bacillus because of the existence of what is called the pseudo-
diphtheria bacillus or bacillus of Hofmann (q. v.). Bomstein*
found that though it was possible to modify the activity of
virulent bacilli, and bring back the virulence of non- virulent
diphtheria bacilli, it was impossible to make the pseudo-
diphtheria bacillus virulent. Denny f also found that the
morphology of the two organisms was continually different
when they were grown upon the same medium for the same
length of time, and that the short pseudodiphtheria bacillus
never showed any tendency to develop into the large clubbed
forms characteristic of the true diphtheria organism. The
chief points of difference between the bacilli are that the
pseudodiphtheria bacillus, when grown upon blood-serum, is
short and stains uniformly; that cultures grown in bouillon
develop more rapidly at a temperature of from 20° to 22° C.
than those of the true bacillus; and that the pseudobacillus
is not pathogenic for animals.

Contagion. — The diphtheria bacilli, being always present
in the throats of patients suffering from diphtheria, con-
stitute the element of contagion, and by being accidentally
discharged from the nose and mouth during coughing,
sneezing, vomiting, etc., endanger whoever comes in contact
with the patient.

The results obtained by Biggs, Park, and Beebe in New
York are of great interest. Bacteriologic examinations
conducted in connection with the Health Department of
New York city show that virulent diphtheria bacilli may
be found in the throats of convalescents from diphtheria,
as long as five weeks after the discharge of the membrane
and the commencement of recovery, and that they exist
not only in the throats of the patients themselves, but also
in those of their caretakers, who, while not themselves
infected, may be the means of conveying the disease germs
* "Archiv Russes de Path.," etc., Aug. 31, 1902.
t American Public Health Association, 1902.

Specificity 469

from the sick-room to the outer world. Still more extra-
ordinary are the observations of Hewlett and Nolen,* that
the bacilli remained in the throats of patients seven,
nine, and in one case twenty-three weeks after convalescence.
The hygienic importance of this observation must be ap-
parent to all readers, and serves as further evidence why
thorough isolation should be practised in connection with the
disease.

Neumann f found that virulent diphtheria bacilli may
occur in the nose with the production of what seems to be
a simple rhinitis as well as a pseudo-membranous rhinitis.

Fig. 152. — Wesbrook's types of Bacillus diphtheriae: a, c, d, Granular
types; a1, c1, d1, barred types; a2, c2, d2, solid types. X 1500.

Such cases, not being segregated, may easily serve to spread
the contagion of the disease.

Wesbrook, and Wilson and McDaniel J have found it con-
venient to describe three chief types of the diphtheria
bacillus as it occurs in twenty-four-hour-old cultures on
Loffler's blood-serum, sent to the laboratory for diagnosis.
The classification places all types in three general groups:
(a) granular, (6) barred, and (c) solid or evenly staining
forms. Each group is subdivided into types based on the

*"Brit. Med. Jour.," Feb. i, 1896.

t "Centralbl. f. Bakt. u. Parasitenk.," Jan. 24, 1902, Bd. xxxi,
No. 2, p. 41.

J "Trans. Assoc. Amer. Phys.," 1900.

47° Diphtheria

shape and size of the bacilli. A study of variations in the
sequence of types in series of cultures derived from clinical
cases of diphtheria shows that (a) granular types are usually
the most predominant forms at the outset of the disease;
(6) the granular types usually give place wholly or in part
to barred and solid types shortly before the disappearance
of diphtheria-like organisms; (c) solid types, by many
observers called "pseudo-diphtheria bacilli," may cause
severe clinical diphtheria. Solid types may sometimes be
replaced by granular types when convalescence is established
and just before the throat is cleared of diphtheria-like bacilli.

From these data the writers conclude that it is not safe
to base an opinion regarding the maintenance of quarantine
upon the bacterioscopic findings independently of the clinical
history of the case.

The occurrence of true diphtheria bacilli in the throats
of healthy persons has been a stumbling-block to many
practitioners uninformed upon bacteriologic subjects, who
fail to account for its presence and also fail to realize how
rare its appearance under such circumstances really is.

Park * found virulent diphtheria bacilli in about i per
cent, of the healthy throats examined in New York city,
but diphtheria was prevalent in the city at the time, and
no doubt most of the persons in whose throats they existed
had been in contact with cases of diphtheria. He very
properly concludes that the members of a household in
which a case of diphtheria exists, though they have not
the disease, should be regarded as possible sources of
danger, until cultures made from their throats show that
the bacilli have disappeared.

In connection with the contagiousness of diphtheria the
recent experiments of Reyes are interesting. He has
demonstrated that in absolutely dry air diphtheria bacilli
die in a few hours. Under ordinary conditions their vitality,
when dried on paper, silk, etc., continues for but a few days,
though sometimes they can live for several weeks. In sand
exposed to a dry atmosphere the bacilli die in five days
in the light ; in sixteen to eighteen days in the dark. When
the sand is exposed to a moist atmosphere, the duration
of their vitality is doubled. In fine earth they remained

* "Report on Bacteriological Investigations and Diagnosis of Diph-
theria, from May 4, 1893, to May 4, 1894," "Scientific Bulletin No. i,"
Health Department, city of New York.

Diphtheria Antitoxin 471

alive seventy-five to one hundred and five days in dry air,
and one hundred and twenty days in moist air.

Diphtheria Antitoxin. — Behring * discovered that the
blood of animals rendered immune against diphtheria by
inoculation, first with attenuated and then with virulent
organisms, contained a neutralizing substance (Anti-korper]
capable of annulling the effects of the bacilli or the toxin when
simultaneously or subsequently inoculated into susceptible
animals. This substance, held in solution in the blood-
serum of the immunized animals, is the diphtheria antitoxin.
For method of preparing the antitoxins see Antitoxins.
The serum may be employed for purposes of prophylaxis or
for treatment.

Prophylaxis. — The serum can be relied upon for prophy-
laxis in cases of exposure to diphtheria infection. In most
cases a single dose of 500 units is sufficient for the purpose.
The protection thus afforded does not continue longer than
about three months. The transitory nature of the immun-
ity afforded by prophylactic injections of antitoxin is prob-
ably dependent upon the fact that the antitoxin is slowly
excreted through the kidneys.

Treatment. — Diphtheria antitoxin is always to be admin-
istered by the hypodermic method at some point where the
skin is loose. Some clinicians prefer to inject into the ab-
dominal wall; some, into the tissues of the back. A slightly
painful swelling is formed, which usually disappears in a short
time. In a few cases sudden death, with symptoms sug-
gesting anaphylaxis (q. v.), has followed the injection.

Ehrlich asserts that a dose of 500 units is valueless for the
treatment of diphtheria, 2000 units being probably an aver-
age dose for an adult and 1000 units for a child. As the
remedy is practically harmless, it is far better to err on the
side of administering too much than on that of not enough.
Forty thousand units have been administered to a moribund
child with resulting cure. The administration of the
remedy should be repeated in twelve hours if the disease
is one or two days old, in six hours if three or four days old,
in four hours if still older. The serum may have to be given
two, three, four, or even more times, according to the case.
Occasionally there is an outbreak of local urticaria — rarely
general urticaria. Sometimes considerable local erythema

*" Deutsche med. Wochenschrift," 1890, Nos. 49 and 50; "Zeit-
schrift fiir Hygiene," xn, i, 1892.

472 Diphtheria

results. Fever and pain in the joints (serum disease of von
Pirquet) also occur, especially if the patients have been pre-
viously treated with horse-serum. Serums of high unit
strength can be given with the ordinary hypodermic syringe ;
those of lower strength, of which a larger quantity is required,
must be given with a special "antitoxin syringe." The syr-
inge should always be carefully sterilized by boiling, and the
packings, etc., found to be in good condition before it is filled
with antitoxin.

Diphtheria paralysis is said to be more frequent after the
use of antitoxin than in cases treated without it. In a paper
upon this subject I* have shown that this is to be expected,
as the palsies usually occur after bad cases of the disease,
of which a far greater number recover when antitoxin is used
for treatment. The subject has been worked over in an in-
teresting manner, from the experimental side, by Rosenau. f

An interesting collection of statistics upon the antitoxic
treatment of diphtheria in the hospitals of the world has
been published by Professor Welch, t who, excluding every
possible error in the calculations, "shows an apparent re-
duction of case-mortality of 55.8 per cent."

Nothing should so impress the clinician as the necessity
of beginning the antitoxin treatment early in the disease.
Welch's statistics show that 1115 cases of diphtheria treated
in the first three days of the disease yielded a fatality of
8.5 per cent., whereas 546 cases in which the antitoxin
was first injected after the third day of the disease yielded
a fatality of 27.8 per cent.

On the other hand, it can scarcely be said that any time
is too late to begin the serum treatment, for the experiences
of Burroughs and McCollum in the Boston City Hospital
show that by immediate and repeated administration of
very large doses of the serum, apparently hopeless cases
far advanced in the disease, may often be saved.

After the toxin has occasioned destructive organic lesions
of the nervous system and in the various organs and tissues
of the body, no amount of neutralization can restore the
integrity of the parts, and in such cases antitoxin must
fail.

* "Medical Record," New York, 1897.

t "Bulletin No. 38 of the Hygienic Laboratory, U. S. Public Health
and Marine Hospital Service," Washington, D. C., 1907.

t "Bull, of the Johns Hopkins Hospital," July and Aug., 1895.

Diphtheria Antitoxin 473

The occurrence of tetanus following the employment of
serum drawn from a horse suffering from tetanus was ob-
served in a number of cases in St. Louis. In rare cases
in which local and metastatic abscesses have been observed,
the condition is probably correctly attributable to infection
from the patient's skin or from the syringe.

I have found that the serums are by no means regular
in the rapidity of deterioration, so that no very old serum
should be used.

Freezing is without effect upon the serum and ordinary
temperature changes are harmless to it. The antitoxic
power is destroyed at 60° C., the point at which the serum
coagulates. The antitoxin is precipitated with the globulins.*

The serums from different horses probably vary much
in both their irritant and globulicidal .properties, so that
mixed serums from a number of horses may be preferable to
that from a single horse.

A very interesting paper by Parkf shows the effect of the
introduction of antitoxin upon the death-rate from diphtheria
and the advantages of its employment. From it the follow-
ing table is taken:

" Combined statistics of deaths and death-rates from diphtheria and
croup in New York, Brooklyn, Boston, Pittsburgh, Philadelphia, Berlin,
Cologne, Breslau, Dresden, Hamburg, Konigsberg, Munich, Vienna,
Soudan, Liverpool, Glasgow, Paris, and Frankfort:

Year. population. Deathsjromdiphtheria Deaths,per

1890 .16,526,135 H,059 66.9

1891 17,689,146 12,389 70

1892 18,330,787 14,200 77.5

1893 18,467,970 15,726 80.4

1894 19.033,902 15,125 79-9

i«95J 19,143,188 10,657 55.6

1896 19,489,682 9,651 49.5

1897 19,800,629 8,942 45.2

1898 20,037,918 7,170 35.7

1899 20,358,837 7,256 35.6

1900 20,764,614 6,791 32.7

1901 20,874,572 6,104 29.2

1902 21,552,398 5,630 26.1

1903 .21,865,299 5,117 23.4

1904 22,532,848 4,917 21.8

1905 22,790,000 4,323 19.0

* See paper by J. P. Atkinson, ''Journal of Experimental Medicine,"
Sept. and Nov., 1899, vol. iv, Nos. 5 and 6.

t" Journal of the Amer. Med. Assoc.," Feb. 17, 1912, LVIII, No. 7,
P- 453-

J Introduction of antitoxin treatment.

474

Diphtheria

BACILLI RESEMBLING THE DIPHTHERIA BACILLUS.
BACILLUS HOFMANNI.

The pseudodiphtheria bacillus (bacillus of Hofmann-
Wellenhof*) — Bacillus pseudodiphthericus — was first found
by L/offlerf in diphtheria pseudomembranes and in the
healthy mouth and pharynx. Ohlmacher has found it with
other bacteria in pneumonia ; Babes, in gangrene of the lung ;
and Howard, t in a case of ulcerative endocarditis not suc-
ceeding diphtheria.

"x\

*"*4

J*< v ' '

„ #

%»»' .

•> ^

.\ - . *,

*. ^-""V:"6 ' ^

' ' -•

Fig- 153- — Pseudodiphtheria bacilli.

Park§ found that all bacilli with the typical morphology
of the diphtheria bacillus, found in the human throat, are
virulent Klebs-L/ofrler bacilli, while forms found in the throat
closely resembling them, but more uniform in size and shape,
shorter in length, and of more homogeneous staining proper-
ties with Loffler's alkaline methylene-blue solution, can with
reasonable safety be regarded as pseudodiphtheria bacilli,
especially if it be found that they produce an alkaline rather
than an acid reaction by their growth in bouillon. The
pseudodiphtheria bacilli were found in about i per cent, of

* "Wiener klin. Woch.," 1888, No. 3.
t "Centralbl. f. Bakt. u. Parasitenk.," n, 105.
t "Bull, of the Johns Hopkins Hospital," 1893, 30.
§ "Scientific Bulletin No. i," Health Department, city of New
York, 1895.

Bacilli Resembling the Diphtheria Bacillus 475

throats examined in New York; they seem to have no rela-
tionship to diphtheria, and are never virulent.

Morphology. — This micro-organism bears a more or less
marked resemblance to Bacillus diphtherias, but differs in
certain particulars that usually make it possible to recognize
and identify it. It is shorter and stouter than its relative,
is straight, usually slightly clubbed. It usually stains in-
tensely, and commonly shows but one unstained transverse
band. When the bacilli are short and have a single band,
they may resemble cocci. When longer they may show two
transverse bands.

There are no flagella and no spores.

Staining. — The organism stains intensely and more uni-
formly than Bacillus diphtherias . When colored by Neisser's
or Roux's method, no metachromatic end bodies can be
defined.

Cultivation. — The organism is usually discovered in
smears made for the diagnosis of diphtheria, and sometimes
occasions considerable confusion through its cultural simi-
larities and morphologic resemblances to Bacillus diphtherias.
It grows more luxuriantly upon the ordinary culture-media
than B. diphtherias. The colonies are larger, less transpar-
ent and whiter, as seen upon agar-agar. In bouillon there is
more marked clouding and less marked pellicle formation.
Upon Loffler's blood-serum the cultures are too much alike
to be easily differentiated.

G. F. Petri * found no substances in filtrates of cultures of
Hofmann's bacillus capable of neutralizing diphtheria anti-
toxin; he also found that horses immunized with large
quantities of filtrates of the Hofmann bacillus did not pro-
duce any antitoxin to diphtheria toxin. Eleven different
cultures were studied and the results are very important.

Cobbettf and Knapp| show that there is a chemicobio-
logic difference between the true and pseudodiphtheria
bacilli, in that the pseudobacillus does not ferment dextrin
or any of the sugars as the true bacillus does.

Chemistry. — The chemical peculiarities of the culture
serve to make certain that Bacillus hofmanni is an independ-
ent micro-organism. Under no circumstances does it pro-
duce or can it be made to produce toxin. Under no circum-

* "Jour, of Hygiene," vol. v, No. 2, April, 1905, p. 134.
f "Centralbl. f. Bakt. u. Parasitenk.," 1898, xxm, 395.
t "Jour, of Med. Research," xn (N. S., vol. vn), 1904, p. 475.

476 Diphtheria

stances can it be made to produce acid through the decompo-
sition of sugars.

Pathogenesis. — Bacillus hofmanni is not pathogenic for
any of the laboratory animals. Animals immunized to
repeated injections of its cultures acquire no resistance to
Bacillus diphtheriae.

Dr. Alice Hamilton* carefully studied 29 organisms, of
which 26 corresponded fully with the pseudodiphtheria
bacilli. They were divisible into three groups: I, Those
non-pathogenic for guinea-pigs; II, those that produce
general bacteremia in guinea-pigs, and are neutralized by
treatment with the serum of a rabbit immunized against a
member of the group; III, organisms which form gas in
glucose media, produce bacteremia in guinea-pigs, and are
neutralized neither by diphtheria nor by pseudodiphtheria
antitoxin. Some of the organisms of the second group are
also pathogenic for man. Instead of regarding the pseudo-
diphtheria bacillus as a harmless saprophyte, Dr. Hamilton
believes it an important organism explaining some of the
paradoxes that we find at hand. Thus, cases of supposed
diphtheria irremediable by or deleteriously affected by anti-
toxic serum may depend upon one of these organisms. It
is also probably one of them that Councilman found in his
case of "general infection by, Bacillus diphtheriae," and that
Howard encountered in his case of acute ulcerative endo-
carditis without diphtheria, from the valves of whose heart
cultures of a diphtheria-like organism not pathogenic for
guinea-pigs were isolated.

The still more recent and comprehensive work of Clark |
shows that no kind of manipulation is capable of so modifying
Bacillus hofmanni as to make its identity with B. diphtheriae
in the least likely. Clark is, however, willing to admit the
probability that the organisms may have descended from a
common stock.

BACILLUS XEROSIS.

This bacillus was first described in 1884 by Kutschbert and
Neisser,t who regarded it as the cause of xerosis conjunctivae,
having found it upon the conjunctiva in that disease. It has,
however, been so frequently found upon the normal conjunc-

* "Jour. Infectious Diseases," 1904, i, p. 690.

t "Journal of Infectious Diseases," vn, 1910, 335.

t "Deutsche med. Wochenschrift," 1884, Nos. 21, 24.

Bacillus Xerosis 477

tiva that it can no longer be looked upon as pathogenic. It
is also found upon other mucous membranes than the con-
junctiva; thus, Leber found it in the mouth, the pelvis of
the kidney, and in intestinal ulcers. From the investigations
of Sattler, Frankel and Franke, Schleich, Weeks, Fick, Baum-
garten, and others it appears that Bacillus xerosis is a harm-
less saprophyte that is occasionally found upon the con-
junctiva. Happening to be found in xerosis it was accorded
undue distinction.

Morphology. — It resembles Bacillus diphtheriae very
closely, but is probably a little shorter. The ends are clubbed,
and in them metachromatic bodies are stained by Neisser's
and Roux's methods.

There is no motility; there are no flagella and no spores.

Cultivation. — Upon Loffler's medium and other media
commonly used for the diagnosis of diphtheria, the organism
grows with so close resemblance to the Bacillus diphtheriae as
to make the differentiation difficult. Transplanted to other
media, it continues to resemble B. diphtheriae.

Chemistry. — The organism is incapable of forming any
toxin. It ferments sugars like Bacillus diphtheriae, with the
exception of saccharose, which B. xerosis ferments, but which
B. diphtheriae cannot ferment. B. xerosis also fails to fer-
ment dextrin, which B. diphtheria ferments.

These sugar-decomposing properties form the most reliable
methods of differentiating Bacillus diphtheriae, B. hofmanni,
and B. xerosis.

Pathogenesis. — The organism is not pathogenic for
man and is certainly not the cause of xerosis. It is not toxi-
cogenic and is not known to be pathogenic for any animal.

CHAPTER XII.

VINCENT'S ANGINA.

VINCENT'S angina is an acute, specific, infectious, pseudo-
membranous form of pharyngitis or tonsillitis characterized
by the formation of a soft yellowish -green exudate upon the
mucous membranes, which, when removed, leaves a bleeding
surface which remains an ulcer. Sometimes these ulcers are
superficial, sometimes they are deep, necrotic, and fetid.
There is considerable pain on swallowing, some fever, and
some prostration. The patient not infrequently keeps up and
about, though feeling very badly. The ulcerations sometimes
persist for several months. As there is considerable swelling
of the glands of the neck and as the pseudomembrane is
sometimes quite distinct, the disease is apt to be mistaken for
diphtheria, and may be differentiated from it only by a bacteri-
ologic examination. When such an examination is made two
apparently different micro-organisms may be found. The
first is the Bacillus fusiformis; the second, Spirochaeta vin-
centi.

BACILLUS FUSIFORMIS (BABES (?)).

In 1882 Miller* described a fusiform bacillus that occurred
in small numbers between the gums and the teeth and in
cavities in carious teeth in the human mouth. In 1884
Cornil and Babes f also described a fusiform bacillus which
seems to be somewhat different, that occurred in a ne-
crotic exudation from a pseudomembranous — diphtheritic
— pharyngitis in school children. Lammershirt, Vincent,
Nicolle, Plaut, and others observed similar cases. Later
Lichtowitz and Sabrazes observed great numbers of fusiform
bacilli in the pus of a maxillary empyema. Elders and Matz-
enauer observed similar organisms in noma. Fusiform
bacilli are, therefore, not infrequently associated with ne-
crotic processes of various kinds. Similar but not identical
bacilli were found by Babes in the gums of scorbutic patients.

* "Micro-organisms of the Human Mouth."
t "Les Bacteries," 1884.
478

Relation of the Organisms to One Another 479

SPIROCH^TA VINCENTI (PLAUT- VINCENT).

Plaut* and Vincent t observe that in the ulcerative and
necrotic pharyngitis described, together with the fusiform
bacilli, there were varying numbers of spiral organisms.
These were difficult to stain, always took faint but uniform
coloring, varied in length, and showed such irregular and
non-uniform undulations as to appear more serpentine than
"corkscrew-like." They seem never to occur without as-
sociated fusiform bacilli. The writers believed these organ-
isms and not the bacilli to be the cause of the angina, but the
relation of the organisms to one another and to the morbid
conditions with which they were associated was a point long
under debate, since none of those studying either organism
succeeded in artificially cultivating them.

RELATION OF THE ORGANISMS TO ONE ANOTHER.

We have, in Vincent's angina, to do with two micro-organ-
isms that occur in habitual association. Neither was found to
be cultivable by the earlier writers. The spirochaeta could
not be cultivated by Vincent, and of the various fusiform
bacilli, one found by Babes in scurvy, which was obviously
different from the others, was alone susceptible of cultivation.
Later, however, reports were made of the growth of the organ-
isms in mixed cultures. Still later, Veillon and Zuber,
Ellermann, Weaver, and Tunnicliff were able to secure pure
cultures of the fusiform bacillus. Quite a number of writers
reached the conclusion that the organisms were not different,
but were different stages of the same organism. This was
finally proved by Tunnicliff, J who found that in pure cultures
of Bacillus fusiformis, after forty-eight hours, spiral organisms
resembling those seen in smear preparations from the original
source were found. From Tunnicliff 's results it must be
concluded that Bacillus fusiformis and Spirochaeta vincenti
are .identical organisms in different stages of their life-history.
Which, however, is the perfect form is not known, what the
true nature of the organism is, is not known, nor can it be
determined at present whether it is more correctly classified
among the bacteria or among the protozoa.

* "Deutsche Med. Wochenschrift," 1894, xux.

f "Ann. de 1'Inst. Pasteur," 1896, 488.

J "Journal of Infectious Diseases," 1906, in, 148.

480 Vincent's Angina

Cultivation. — The organisms were cultivated by Ttmni-
cliff upon the surface of ascitic fluid agar-agar (i 13) under
strictly anaerobic conditions at 37° C. After two or three
days the fusiform bacillus appeared in the form of delicately
whitish colonies, 0.5 to 2 mm. in diameter, resembling colonies
of streptococci. By transplanting these, pure cultures of
Bacillus fusiformis were obtained. In the transplantation
tubes the organism again grew in the form of similar whitish
colonies, a flocculent deposit accumulating at the bottom of
the water of condensation.

Loffler's Blood-serum Mixture. — After twenty-four to forty-
eight hours similar colonies appear and a similar flocculent
deposit collects in the condensation water.

Rabbit's Blood Agar-agar. — The growth is similar, but
brownish in color.

Glycerin Agar-agar. — No growth.

Glucose Agar-agar Stab. — A delicate whitish growth with
small lateral prolongations develops along the path of the
wire in twenty-four to forty-eight hours. Some gas is
formed.

Litmus Milk. — In forty-eight hours there is a moderate
growth. The litmus becomes decolorized. There is no
coagulation. When oxygen is admitted the medium re-
gains its lost color.

Potato. — No growth.

Bouillon and Dextrin-free Bouillon. — No growth.

Glucose-bouillon. — No growth when more than i per cent, of
glucose is present. The medium is clouded with some sedi-
ment.

From all of the cultures a somewhat offensive odor is
given off.

Morphology. — The Bacillus fusiformis presents the same
appearances, no matter what medium it grows upon. It
measures 3 to 10 ^ in length, 0.3 to 0.8 /w in thickness. The
greatest diameter is at the center, from which the organisms
gradually taper to blunt or pointed extremities.

The organisms stain with Loffler's alkaline methylene-blue,
with diluted carbol-fuchsin, by Gram's method, and by
Giemsa's method. The staining is intense, but is rarely uni-
form, the substance usually being interrupted by vacuoles
or fractures, reminding one of those seen in the diphtheria and
tubercle bacilli. The organism forms endospores sometimes
situated at the center, but more frequently toward one end.

Morphology

481

In twenty-four to forty-eight hours filaments are seen.
These are of the same diameter throughout, and usually con-
tain deeply staining bodies, sometimes round, oftener in bands.

Fig. 154. — Bacillus fusiformis. Pure culture grown forty-eight hours
anaerobically on Loffler's blood-serum. (Ruth Tunnicliff in "Journal
of Infectious Diseases.")

Fig. 155. — Bacillus fusiformis. Pure culture grown forty-eight hours
anaerobically in the fluid of condensation of Loffler's blood-serum.
(Ruth Tunnicliff in " Journal of Infectious Diseases.")

Most of the filaments are made up of strings of bacilli, but
some stain uniformly. About the fourth or fifth day the
31

482

Vincent's Angina

spirals make their appearance, sometimes in enormous
numbers. As a rule, they stain uniformly, some show the
dark bodies seen in the bacilli and filaments. They form from

Fig. 156. — Bacillus fusiformis. Pure culture grown four days in ascites
broth. (Ruth Tunnicliff in " Journal of Infectious Diseases.")

Fig. 157. — Bacillus fusiformis. Smear from gum in normal mouth.
(Ruth Tunnicliff in "Journal of Infectious Diseases.")

one to twenty turns, which are not uniform. The spirals
are flexible, the ends pointed. The spirals persist in the
cultures, at times for fifty-five days.

Pathogenesis 483

Neither the bacilli nor the spirals show any progressive
movement, though with the dark-field illuminator they show
a slight vibratile and rotary movement. No flagella were
observed.

Pathogenesis. — Pure cultures of the organisms were in-
oculated into guinea-pigs without result. As in Vincent's
angina the throat always contains staphylococci and strep-
tococci, and not infrequently diphtheria bacilli, it is thought
by many that Bacillus fusiformis does not initiate the morbid
process, but is a secondary invader, by which simpler inflam-
mations are intensified and made necrotic.

This seems to be particularly true of diphtheria, and may
account for the occurrence of noma, in which gangrenous
condition of the mouth and genitals the organisms have been
found in great numbers.

CHAPTER XIII.

THRUSH.

OIDIUM ALBICANS (ROBIN).

THRUSH, Soor (German), Muguet (French), or parasite
stomatitis is an affection of marasmatic infants and adults
characterized by the occurrence of peculiar whitish patches
upon an inflamed oral mucous membrane. The white of the
patches consists of material that is not easily removed, but
which when detached leaves a bleeding surface upon which it
forms again. Upon microscopic examination the white sub-
stance proves to be composed of masses of mycelia with en-
larged epithelial cells and leukocytes. The affection is far
more frequent in children than in adults. It seems not to
occur among healthy children, but among those suffering from
marasmus, and particularly among those whose mouths have
already become sore through neglect. It is usually confined
to the mouth, but may spread to the pharynx, to the larynx,
in rare cases to the esophagus, in very rare cases to the
stomach and intestines, and in exceptional cases, both in
adults and children, may become a generalized disease through
hematogenous distribution, and be attended by mycotic in-
flammatory lesions in the kidneys, the liver, and the brain.

The specific micro-organism seems to have been discovered
in 1839 by Langenbeck* and Berg, f Langenbeck missed the
significance of the organism altogether, for, finding it in a
case of typhoid fever, he conceived it to be the cause of that
disease. Berg, on the other hand, regarded it as the cause
of the thrush. Robin { furnished the first correct description
of the organism and gave it its name, O'idium albicans. Many
systematic writers have exercised themselves concerning the
exact place in the botanical system in which the organisms
should be placed. Thus, Gruby and Heim regarded it as a

v * See Kehrer, "Ueber den Soorpilz," etc., Heidelberg, 1883.

fSee Behrend, "Deutsche med. Wochenschrift," 1890.

| "Histoire naturelle des vegetaux parasites qui croissent sur 1'homme
et sur les animaux vivants," Paris, 1853.

484

Morphology 485

sporotrichum ; Robin, as an oidium; Quinquaud, as a syringo-
spora; Hallein called it Stemphylium polymorpha; Grawitz,
as Mycoderma vini; Plaut, as Monilia Candida; Guidi, Ress,
Brebeck-Fischer, as a saccharomyces ; Laurent, as Dematium
albicans; Linossier and Roux, as a mucor, and Alav, Olsen,
and Vuillemin, as Endomyces albicans. The matter is still
undecided and until it is finally agreed upon it seems best to
resort to the original name, Oidium albicans.

Morphology. — The organism consists of elements that
bear a close resemblance to yeast cells and multiply by bud-
ding, of hyphae and mycelial threads into which these grow,
and of chlamydospores and conidia.

Fig. 158. — Oidium. (Kolle and Wassermann.)

The yeast-like elements measure 5 to 6 [i in length and 4 {i
in breadth. They have an oval form and cannot be distin-
guished from yeast cells. The mycelia are formed by elon-
gation of these elements, some of which appear slightly elon-
gate, some greatly elongate and slender and more or less
septate, like those of the true molds. They are refractile,
doubly contoured, and contain droplets, vacuoles, and gran-
ules. In the interior of the hyphae conidia-like organs often
appear, and chlamydospores appear. The latter are large,
oval, doubly contoured, highly refracting, and have been seen
by Plaut to germinate.

The morphology is, however, extremely varied, and the
greatest differences of interpretation have been expressed re-
garding the different elements.

486

Thrush

Cultivation. — The organism grows readily in artificial
media, both with and without free access of oxygen. An acid
reaction is most appropriate.

Colonies. — The superficial colonies upon gelatin plates are
rounded, waxy, and coarsely granular. The deep colonies are
irregular in shape and show feathery processes extending into
the medium. The color varies according to the composition
of the medium, from snow white on ordinary gelatin to meat-
red on beet-root gelatin. A sour odor is
given off from the cultures.

Gelatin Punctures. — Along the line of
puncture there is a slow formation of
rounded, feathery, colorless colonies, not
unlike those shown by many molds.
The gelatin is slowly liquefied only when
it contains sugar. In such cultures
chlamydospores are abundant.

Agar-agar. — Cultures are similar to
those in gelatin.

Bouillon. — The organism grows only at
the bottom of the tube in the form of
yellowish- white flocculi.

Potato. — Various in different cases.
Often floury.

Milk. — The organism grows very poorly
in milk, which is not coagulated or fer-
mented.

Fermentation. — The organism utilizes
dextrin, mannite, alcohol, lactose, and
glycerin without fermentation. Saccha-
rose is destroyed without invertin for-
mation. Glucose, levulose, and maltose
are fermented very slowly.

Metabolic Products. — In addition to the ferments that
act upon the sugars, etc., and soften the gelatin, the organ-
ism forms alcohol, aldehyd, and acetic acid.

Pathogenesis. — Animals are not known to suffer from
spontaneous infection. Grawitz was able to induce thrush in
puppies. Stooss inoculated the scarified vaginas of rabbits
with mixed cultures of pyogenic cocci and o'idium and ob-
tained thrush plaques. The oidi'um alone was unable to
secure a foothold. Doderlein, Grosset, and Stooss all suc-
ceeded in producing abscesses, sometimes by subcutaneous

Fig- 159. — Oidium
albicans. Culture
in gelatin (Hansen).

Immunity 487

injection of the oidium, but usually only when it was combined
with pus cocci. In such abscesses the cocci are killed off by
phagocytes, and when cultures are made only the oi'dium
grows. Plaut points out that this is exactly the reverse of
what happens in artificial cultures of the two organisms
where the cocci outgrow and kill off the oidium.

Intravenous injection sometimes causes generalized oidium
infection, with colonies of the micro-organism in the kidneys,
heart-muscle, peritoneum, liver, spleen, stomach, and in-
testines. The central nervous system may also show small
foci of the infection.

Immunity. — Roger* and Noissette f were able to immunize
animals against oidium.

*"Compt.-rende de la Societe de Biologic," Paris, 1896.
t "These de Paris," 1898.

CHAPTER XIV.

WHOOPING-COUGH.

THE; BORDET-GENGOU BACILLUS.

THE subacute, contagious, undoubtedly infectious disease
of childhood, characterized by periodic attacks of spasmodic
cough and laryngeal spasm, terminating in a prolonged
crowing inspiration and frequently followed by vomiting
and prostration, known as pertussis, or whooping-cough, has
long been subject to bacteriologic investigation. Deichler,
Kurloff, Szemetzchenko, Cohn, Neumann, Ritter, and
Afanassiew have all written upon bacteria which they sup-
posed to be the causal factors of the disease, but which time
has consigned to oblivion. Koplik* and Czaplewski and
Henself described micro-organisms that for some years at-
tracted attention and caused more or less discussion as to
which might be the real excitant of the disease or whether
they were identical organisms. As time passed, both obser-
vations lacked sufficient confirmation to carry conviction
of their importance, and they, too, fell into oblivion. A still
different organism was described by VincenziJ, but also
failed to meet sufficient confirmatory evidence to prevent
it from meeting the fate of its predecessors.

Spengler,§ Kraus and Jochmann,|| and Davis** showed the
frequent presence of minute bacilli in the sputum and also in
the lesions of the disease. They were, almost beyond doubt,
influenza bacilli.

In 1906 Bordet and Gengouft described a new organism
whose importance was supported by such weighty evidence

* "Centralbl. f. Bakt.," etc., Sept. 15, 1897, xxn, 8 and 9, p. 222.
t "Deutsch. med. Wochenschrift," 1897, No. 57, p. 586; " Centralbl.
f. Bakt.," etc., Dec. 22, 1897, xxn, Nos. 22 and 23, p. 641.

t"Alti della Accademia di Medicina in Torino," LXI, 5-7; "Cen-
tralbl. f. Bakt.," etc., Jan. 19, 1898, xxm, p. 273.
§"Deutsch. med. Wochenschrift," 1897, 830.
|| "Zeitschrift fur Hygiene," etc., 1901, xxxvi, 193.
** "Jour. Infectious Diseases," 1906, in, i.
ft "Ann. de 1'Inst. Pasteur," 1906, xx, 731.

488

Staining 489

as the formation of an endotoxin sufficiently active to ex-
plain the symptoms, and the fixation of complement by the
serum of the infected animal. This organism, therefore,
presents itself as sufficiently meritorious to maintain the
field for the present.

Morphology. — The organisms, as found in the sputum, oc-
cur as very minute ovoid rods of about the same size as the
influenza bacillus. They measure approximately 1.5 fi in
length by 0.3 u in breadth. They do not remain united
as chains or rods, but separate as individuals. They are

Fig. 1 60. — The Bordet-Gengou bacillus of whooping-cough. Twenty-
four-hour-old culture upon solid media containing blood (Bordet-
Gengou).

somewhat pleomorphous, yet the variations are not con-
siderable. Involution forms are not common. There are
no spores, no flagella, no motility.

Staining. — The organisms do not hold the stain well.
Most of the bacilli are pale, some contain uncolored areas
or vacuoles. In some cases the ends of the bacilli appear
more deeply stained than the middle. They do not stain
by Gram's method. The discoverers recommend that the
organism be stained with —

Toluidin blue 5 ^ Dissolve and add 500 of 5 per cent.

Alcohol 100 X aqueous carbolic acid. After two

Water 500 j days filter.

Isolation. — The organisms occur in almost pure cultures
in the whitish expectoration which escapes from the bronchi

49° Whooping-Cough

in the beginning of the disease. Later they become few and
may disappear, though the symptoms of the disease persist.
Cultivation. — The cultures were secured upon a special
medium made as follows:

I.

l}**. Pour off the fluid.

II. Potato extract (made as above) . . 50 c.c. ] Boil, dissolve, filter,

0.6 per cent, aqueous NaCl 150 c.c. >• and tube; 2 to 3

Agar-agar 5 gm. J c.c. to a tube.

III. To each tube add an equal volume of defibrinated rabbits' (or,
better, human) blood before cooling to the point of coagulation. Permit
the tubes to solidify in the oblique position.

At first the growth is scant, but upon transplantation
grows better and better, until finally it may be made to
grow upon other media, such as blood-agar, ascitic agar, or
broth to which blood or ascitic fluid has been added. The
organism is a strict aerobe. It grows best at 37° C., but also
grows at temperatures as low as 5° to 10° C. On appropriate
culture-media Wollstein found it might remain alive for two
months.

Metabolic Products. — An endotoxin was found by Bordet
and Gengou, the method of preparing which was improved
by Besredka* as follows: The growth upon agar-agar is
removed with a small quantity of salt solution, dried in
vacuo, and ground in a mortar with a small measured
quantity of salt. Enough distilled water is then added to
make a 0.75 per cent, solution, after which the mixture is
centrifugalized and decanted. Of this preparation i to 2
c.c. usually killed a rabbit about twenty-four hours after
intravenous injection. Subcutaneous injection caused a
necrosis without suppuration and without constitutional
symptoms. Small quantities of the toxin placed in the
rabbit's eye caused local necrosis, with little inflammatory
reaction. The introduction of dead or living cultures into
the peritoneal cavity of guinea-pigs caused death with great
effusion and hemorrhage in the peritoneal tissues.

Pathogenesis. — Inoculation of monkeys with cultures of
the bacillus failed to produce the disease. Klimenko,t
however, succeeded in infecting monkeys and pups by intra-
tracheal introduction of pure cultures. After a period of

* Bordet, "Bull, de la Soc. Roy. de Bruxelles," 1907.
t "Centralbl. f. Bakt.," etc. (Orig.), XLVIII. 64.

Pathogenesis 491

incubation an illness came on, the most marked symptoms
being pyrexia and pulmonary irritation. After two or three
weeks the dogs died. Postmortem examination showed
catarrh of the respiratory tissues with patches of broncho-
pneumonia. Healthy dogs contracted the disease by con-
tact with those suffering from the infection. Frankel* ob-
tained similar results.

The differences between the Bordet-Gengou bacillus and
the influenza bacillus are not great. In size, mode of oc-
currence, grouping and staining there is much resemblance,
between the two. Culturally, however, they differ because
the influenza bacillus grows best upon hemoglobin or blood
agar-agar, which is less adapted for the isolation of the Bor-
det-Gengou bacillus than the culture-medium given for its
cultivation, upon which the influenza bacillus does not
grow well. Further, we have as differential the peculiar
endotoxin of the Bordet-Gengou bacillus, the successful
infection of dogs and monkeys with the disease resembling
whooping-cough, and the transmission of this infection
from animal to animal by natural means.

The subject of complement deviation as a proof of the
specific nature of the organism is still under consideration.
Bordet and Gengou found that the serum of convalescent
patients fixed complement when applied to the bacilli;
Frankel and Wollstein,f that it did not. It is claimed by
Bordet and Gengou that the difference in results came about
through the employment of different culture-media in per-
forming the Complement fixation tests.

* "Mtinchener med. Wochenschrift," 1908, p. 1683.
t "Journal of Exp. Med.," 1909, xi, 41.

CHAPTER XV.

PNEUMONIA.
LOBAR OR CROUPOUS PNEUMONIA,

DIPLOCOCCUS PNEUMONIA (WEICHSEXBAUM).

General Characteristics.— A minute, spheric, slightly elongate
or lancet-shaped, non-motile, non-flagellate, non-sporogenous, aerobic
and optionally anaerobic, non-chromogenic, non-liquefying diplococ-
cus, pathogenic for man and the lower animals, staining by ordinary
methods and by Gram's method.

" Pneumonia," while generally understood to refer to the
lobar form of the disease particularly designated as croupous
pneumonia, is a vague term, comprehending a number of quite
dissimilar inflammatory conditions of the lung. This being
true, no single micro-organism can be " specific " for all.
Indeed, pneumonia must be conceived of as a group of dis-
eases, and the various micro-organisms associated with it
must be separately considered in connection with the
particular varieties of the disease in which they occur.

The micro-organism, that can be demonstrated in at least
75 per cent, of cases of lobar pneumonia, which is almost
universally accepted to be the cause of the disease, and about
whose specificity very few doubts can now be raised, is the
Diplococcus pneumoniae, or pneumococcus of Frankel and
Weichselbaum.

Priority of discovery of the pneumococcus seems to be in
favor of Sternberg,* who as early as 1880 described an
apparently identical organism which he secured from his
own saliva. Pasteur | seems to have cultivated the same
micro-organism, also from saliva, in the same year. The
researches of the observers whose names are now attached
to the organism were not completed until five years later.
It is to Telamon, f Frankel,§ and particularly to Weichsel-

* ''National Board of Health Bulletin," 1881, vol. n.
t "Compte-rendus Acad. des Sciences," 1881, xcn, p. 159.
t "Societe anatom. de Paris," Nov. 30, 1883.
§ "Deutsche med. Wochenschrift," 1885, 31.
492

Morphology 493

baum,* however, that we are indebted for the discovery of
the relation which the organism bears to pneumonia.

Distribution. — The pneumococcus is one of a group of
widely disseminated organisms of the respiratory tract. It
is characterized by certain peculiarities of morphology,
certain metabolic peculiarities, a definite pathogenesis, and
a distinct agglutinative reaction with immune serum.
From such typical individuals a number of irregular depart-
ures are known, and recent researches make it certain that
many of the organisms formerly looked upon as pneumococci
are different and perhaps harmless. The pneumococcus
is a purely parasitic, pathogenic organism, best known to
us in its relation to croupous pneumonia, where it is present
in the lungs, sputum, and blood. It may be found in the
saliva of a large number of healthy persons, Parke and
Williams, f especially during the winter months, Longcope
and Fox, J and the inoculation of human saliva into rabbits
frequently causes septicemia in which the pneumococci are
abundant in the blood and tissues. Its frequent occurrence
in the saliva led Flugge to describe it as Bacillus septicus
sputigenus. It is occasionally found in inflammatory lesions
other than pneumonia, as will be pointed out below.

Morphology. — The organism is variable in morphology.
When grown in bouillon it appears oval, has a pronounced
disposition to occur in pairs, and not infrequently forms
chains of five or six members, so that some have been dis-
posed to look upon it as a streptococcus (Gamaleia) . In the
fibrinous exudate from croupous pneumonia, in the rusty
sputum, and in the blood of rabbits and mice containing
them, the organisms occur in pairs, have a lanceolate shape,
the pointed ends usually being approximated, and are
usually surrounded by a distinct halo or capsule of clear,
colorless, homogeneous material, thought by some to be a
swollen cell- wall, by others a mucus-like secretion given off
by the cells. When grown in culture-media, especially
upon solid media, the capsules are not apparent. The
elongate form has led Migula§ to describe it under the name
Bacterium pneumoniae.

The organism measures about i ^ in greatest diameter,

* "Wiener med. Jahrbuch," 1886, p. 483.

t "Jour. Exp. Med.," vn, Aug. 7, 1905, p. 403.

t Ibid., p. 430.

§ "System der Bakterien," Jena, 1900, p. 347.

494 Pneumonia

is without motility, has no flagella, forms no spores, and
seems unable long to resist unfavorable conditions when
grown artificially.

Staining. — It stains well with the ordinary solutions of
the anilin dyes, and gives most beautiful pictures in blood
and tissues when stained by Gram's and Weigert's methods.

To demonstrate the capsules, the glacial acetic acid method
of Welch* may be used. The cover-glass is spread with
a thin film of the material to be examined, which is dried
and fixed as usual. Glacial acetic acid is dropped upon it

Fig. 161. — Capsulated pneumococci in blood from the heart of a rabbit;
carbol-fuchsin, partly decolorized. X 1000.

for an instant, poured (not washed) off, and at once followed
by anilin-water gentian violet, in which the staining con-
tinues several minutes, the stain being poured off and
replaced several times until the acid has all been removed.

Finally, the preparation is washed in water containing i or
2 per cent, of sodium chlorid, and may be examined at once
in the salt solution, or mounted in balsam after drying.
The capsules are more distinct when the examination is
made in water.

Hissf recommends the following as an excellent method

* "Bull, of the Johns Hopkins Hospital," Dec., 1892, p. 128.

t Abstract, "Centralbl. f. Bakt. u. Parasitenk.," Bd. xxxi, No. 10, p.
302, March 24, 1902. More complete details appear in a later paper in
the "Journal of Experimental Medicine," vi, p. 338.

Isolation 495

of staining the capsules of the pneumococcus : The organism
is first cultivated upon ascites serum-agar to which i per
cent, of glucose is added. The drop containing the bacteria
to be stained is spread upon a cover-glass mixed with a
drop of serum or a drop of the fluid culture-medium, and
dried and fixed. A half-saturated aqueous solution of gen-
tian violet is applied for a few seconds and then washed
off in a 25 per cent, solution of carbonate of magnesium.
The preparation is then mounted in a drop of the latter
solution and examined.

If it is desired to stain the capsules and preserve the
specimens permanently in balsam, Hiss employs a 5 or 10
per cent, solution of fuchsin or gentian violet (5 c.c. saturated
alcoholic solution of dye in 95 c.c. of distilled water). The
stain is applied to the fixed specimen and heated until it
begins to steam, when the stain is washed off in a 20 per
cent, solution of crystals of sulphate of copper. The prepara-
tion is then dried and mounted in balsam.

Hiss finds this stain a useful aid in differentiating the
pneumococcus from the streptococcus, with which it is
easily confounded if the capsules are not distinct, and to
which it is probably closely related.

To demonstrate the organisms in sections of tissue either
Gram's or Weigert's methods may be used, with beautiful
results.

Isolation. — When desired for purposes of study, the
pneumococcus may be obtained by inoculating rabbits with
pneumonic sputum and recovering the organisms from the
heart's J^lood, or it may be obtained from the rusty sputum
of pneumonia by the method employed by Kitasato for
securing tubercle bacilli from sputum: A mouthful of fresh
sputum is washed in several changes of sterile water to free
it from the bacteria of the mouth and pharynx, carefully
separated, and a minute portion from the center transferred
to an appropriate culture- medium.

Buerger,* in conducting a research upon pneumococcus
and allied organisms with reference to their occurrence in the
human mouth, under the auspices of the Rockefeller Insti-
tute, used a 2 per cent, glucose-agar of a neutral, or, at most,
0.5 per cent, phenolphthalein acid titer.

* "Jour. Exp. Med.," Aug. 25, 1905, vn, No. 5.

496 Pneumonia

"The medium was usually made from meat infusion and contained
1.5 to 2 per cent, peptone and 2.4 per cent. agar. Stock plates of
these media (serum-agar and 2 per cent, glucose-serum-agar) were
poured. The agar or glucose-agar was melted in large tubes and
allowed to cool down to a temperature below the coagulation point of
the serum. One-third volume of rich albuminous ascitic fluid was
added, and the resulting media poured into Petri plates. These were
tested by incubation and stored in the ice-chest ready for use. . . .

"The plan finally adopted [for inoculating the plates] was as fol-
lows: A swab taken from the mouth was thoroughly shaken in a tube
of neutral bouillon. From this primary tube dilutions in bouillon
with four, six, and eight loops may be made. A small portion of the
dilute mixture was poured at a point near the periphery of the pre-
pared plates. By a slight tilting motion the fluid was carefully dis-
tributed over -the whole surface of the plates. Care must be taken
to avoid an excess of fluid. It was found that plates made in this
way gave a sufficiently thick and discrete distribution of surface col-

Cultivation. — The organism grows upon all the culture-
media except potato, but only between the temperature
extremes of 24° and 42° C., the best development being at
about 37° C. The growth is always meager, probably be-
cause of the metabolic formation of formic acid. The addi-
tion of alkali to the culture-medium favors the growth of
the pneumococcus by neutralizing this acid. Hiss and
Zinnser* advise that the culture-media used for the pneumo-
coccus be made with 3 to 4 per cent, of peptone.

Colonies. — The colonies which develop at 24° C. upon
gelatin plates (15 per cent, of gelatin should be used to
prevent melting at the temperature required) are described
as small, round, circumscribed, finely granular white points
which grow slowly, never attain any considerable size,
and do not liquefy the gelatin.

If agar-agar be used instead of gelatin, and the plates
kept at the temperature of the body, the colonies appear
transparent, delicate, and dewdrop-like, scarcely visible to
the naked eye, but under the microscope appear distinctly
granular, a dark center being surrounded by a paler marginal
zone.

Upon the medium recommended by Buerger for isolating
the pneumococcus, the colonies appear in from eighteen to
twenty-four hours, the surface colonies being circular and
disk-like. When viewed from above, the surface appears
glassy with a depressed center. When viewed from the side
or by transmitted light, they appear as distinct milky rings
with a transparent center. This " ring type " is regarded as

* "Text-book of Bacteriology," 1910, p. 356.

Vital Resistance 497

characteristic and enables the organism to be separated
without difficulty from the streptococcus.

Gelatin Punctures. — In gelatin puncture cultures, made
with 15 instead of the usual 10 per cent, of gelatin, the
growth takes place along the entire puncture in the form
of minute whitish granules distinctly separated from one
another. The growth in gelatin is always very meager.

Agar-agar and Blood-serum. — Upon agar-agar and blood-
serum the growth consists of minute, transparent, semi-
confluent, colorless, dewdrop-like colonies, which die before
attaining a size which permits of their being seen without
careful inspection. Upon glycerin agar-agar the growth is
more luxuriant. The addition of a very small percentage
of blood-serum greatly facilitates the growth.

Bouillon. — In bouillon the organisms grow well, slightly
clouding the medium. With the death of the organisms and
their sedimentation, the medium clears again after a few
days.

Milk. — Milk is an appropriate culture-medium, its casein
being coagulated. Alkaline litmus milk is slowly acidified.

Potato. — The pneumococcus does not grow upon potato.*

Vital Resistance. — The organism usually dies after a few
days of artificial cultivation, and so must be transplanted
every three or four days. In rabbit's blood, in sealed tubes
kept cold, it can sometimes be kept alive for several weeks.
Hiss and Zinnserf find that when the organism is planted in
" calcium-carbonate-infusion broth" and kept in the ice-chest,
the cultures often remain alive for several months. Bordoni-
UffreduzziJ found that when pneumococci were dried in
sputum attached to clothing, and were exposed freely to the
light and air, they retained their virulence for rabbits for
from nineteen to ninety-five days. Direct sunlight destroyed
their virulence in twelve hours. Guarniere§ found that dried
blood containing pneumococci remained virulent for months.

The pneumococcus is destroyed in ten minutes by a tem-
perature of 52° C. It is highly sensitive to all disinfectants,
weak solutions quickly killing it.

* Ortmann asserts that the pneumococcus can be grown on potato at
37° C., but this is not generally confirmed. The usual acid reaction of
the potato would indicate that it was a very unsuitable culture-medium.

t Loc. cit.

I "Arch. p. 1. Sc. Med.," 1891, xv.

§ "Atti della R. Acad. Med. di Roma," 1888, iv.
32

498 Pneumonia

Metabolic Products. — Hiss * found that the pneumococcus
produces acid with ease from monosaccharids, disaccharids,
and such complex saccharids as dextrin, glycogen, starch,
and inulin. The fermentation of inulin is most important as
a means of differentiating pneumococci from streptococci,
which cannot ferment it.

Toxic Products. — Nothing definite is known about the
metabolic toxic products of the pneumococcus. That the
symptoms of pneumonia are not entirely dependent upon
the disturbance of respiration is clearly shown by the fact
that the patients suffer from high fever and have marked
leukocytosis with enlargement of the spleen. The cases in
which the cocci invade the blood are usually more serious
than those in which their operations are restricted to the
lung.

The toxin must be purely or almost purely intracellular,
however, as filtered cultures are scarcely at all toxic.

Auldf found that if a thin layer of prepared chalk were
placed upon the bottom of the culture-glass, it neutralized
the lactic acid produced by the pneumococcus, and enabled
it to grow better and produce much stronger toxin. Mal-
fadyenj found that by freezing cultures of the pneumococcus
with liquid air and then destroying them by trituration in the
frozen state and then extracting the fragments with i : 1000
caustic potash solution, a toxin whose activity corresponded
fairly well with the virulence of the culture could be secured.
This toxin killed rabbits and guinea-pigs in doses varying
from 0.5 to i c.c.

Pathogenesis. — If a small quantity of a pure culture
of the virulent organism be introduced into a mouse, rabbit,
or guinea-pig, the animal dies in one or two days. Exactly
the same result can be obtained by the introduction of a piece
of the lung-tissue from croupous pneumonia, by the intro-
duction of some of the rusty sputum, and frequently by the
introduction of human saliva. Postmortem examination
of infected animals shows an inflammatory change at the
point of subcutaneous inoculation, with a fibrinous exudate
similar to that succeeding subcutaneous inoculation with
the diphtheria bacillus. At times, and especially in dogs,
a little pus may be found. The spleen is enlarged, firm, and

* "Jour. Exp. Med.," vn, No. 5, Aug. 25, 1905.
f "Brit. Med. Jour.," Jan. 20, 1900.
J Ibid., 1906, ii.

Pathogenesis 499

red-brown. The blood with which the cavities of the heart
are filled is firmly coagulated, and, like that in other organs
of the body, contains large numbers of the bacteria, most of
which exhibit a lanceolate form and have distinct capsules.
The disease is thus shown to be a bacteremia unassociated
with conspicuous tissue changes.

In such cases the lungs show no consolidation. Even if
the inoculation be made by a hypodermic needle plunged
through the breast-wall into the pulmonary tissue, pneu-
monia rarely results. Gamaleia* reported that pneumonic
consolidation of the lungs of dogs and sheep could be brought
about by injecting the pneumococcus through the chest-
wall into the lung. Tchistowitsch f stated that by intra-
tracheal injections of cultures into dogs he succeeded in
producing in 7 out of 19 experiments typical pneumonic
lesions. Monti { claimed to have found that a characteristic
croupous pneumonia results from the injection of cultures
into the trachea of susceptible animals. A very interesting
review of the literature of the experimental aspects of the
subject, embracing 198 references, will be found in Wads-
worth's paper upon " Experimental Studies on the Etiology
of Acute Pneumonitis."§

The final proof that true pulmonary consolidation, i. e.,
pneumonia, can be produced experimentally by cultures of
the pneumococcus is to be found in a paper by Lamar and
Meltzer.|| These investigators etherized dogs, kept the
mouth open by means of a large wooden gag, drew the tongue
forward by means of hemostatic forceps, and then, seizing the
median glosso-epiglottic fold, pulled it forward so that the
posterior aspect of the epiglottis presented an inclined
plane. Into this concavity one end of a tube is placed.
Under the protection of the left index-finger the tube was
directed into the larynx and pushed down slowly and gently
through the trachea until a resistance was met with. The
inner end of the tube was then found to engage in a bronchus
— usually the right bronchus. A pipet containing a liquid
culture of the pneumococcus was next attached to the
external end of the tube, and by means of a syringe the

* "Ann. de 1'Inst. Pasteur," 1888, u, 440.

t Ibid., 1890, m, 285.

J "Zeitschrift fur Hygiene," etc., 1892, xi, 387.

\ "Amer. Med. Jour. Sciences," 1904, cxxvn, p. 851.

|| "Jour. Exp. Med.," 1912, xv, No. 2, p. 133.

500

Pneumonia

culture (about 6 c.c.) was injected into the bronchus. The
syringe was then removed, the piston withdrawn, and the
syringe again attached to the pipet. By the injection of
air the culture was driven deeper into the bronchi. The tube
was then clamped and withdrawn and the animal released.
By these means experimental pneumonia, with the typical

Fig. 162. — Lung of a child, showing the appearance of the organ in
the stage of red hepatization of croupous pneumonia. The pneu-
monia has been preceded by chronic pleuritis, which accounts for the
thickened fibrous trabeculae extending into the tissue, and which may
have had something to do with the peculiarly prominent appearance
of the bronchioles throughout the lung.

consolidation and lobar distribution, was produced in 42
successive cases. The course of the inflammatory disturb-
ance thus produced was rapid, and in one case nearly com-
plete consolidation had occurred in seven hours.

Pathogenesis 501

Lesions. — The lesions of croupous pneumonia of man are
almost too well known to need descripion. The distribution
of the disease conforms more or less perfectly to the divisions
of the lung into lobes, one or more lobes being affected.
An entire lung may be affected, though, as a rule, the apex
escapes consolidation and is simply congested. The in-
vaded portion of the lung is supposed to pass through a
succession of stages clinically described as (i) congestion,
(2) red hepatization, (3) gray hepatization, and (4) resolu-
tion. In the first stage bloody serum is poured out into the
air-cells, filling them with a viscid reddish exudate. In
the second stage this coagulates so that the tissue becomes
solid, airless, and approximately like liver tissue in appear-
ance. The third stage is characterized by dissolution of
the erythrocytes and invasion of the diseased air-cells by
leukocytes, so that the color of the tissue changes from
red to gray. At the same time the coagulated exudate be-
gins to soften and leave the air-cells by the natural pas-
sages, and the stage of resolution begins.

In more rare cases circumscribed areas of consolidation
occur in the lung tissue. The inflammatory lesions of other
organs present nothing characteristic by which they can be
recognized by macroscopic examination.

The pneumococcus is not infrequently discovered in dis-
eased conditions other than croupous pneumonia; thus, Foa,
Bordoni-Uffreduzzi, and others found it in cerebrospinal
meningitis; Frankel, in pleuritis; Weichselbaum, in perito-
nitis; Banti, in pericarditis; numerous observers, in acute
abscesses; Gabbi isolated it from a case of suppurative
tonsillitis; Axenfeld observed an epidemic of conjunctivitis
caused by it; Zaufal, Levy, and Schroder and Netter have
been able to demonstrate it in the pus of otitis media, and
Fouler ton and Bonney* isolated it from a case of primary
infection of the puerperal uterus. It has also been found
in arthritis following pneumonia, and in primary arthritis
without previous pneumonia by Howard.f

Interesting statistics concerning the relative frequency of
pneumococcus infections in adults given by Netter { are as
follows :

* "Trans. Obstet. Soc. of London," 1903, part n, p. 128.
t "Johns Hopkins Hospital Bulletin," Nov., 1903.
$ "Compte-rendu," 1889.

502 Pneumonia

Pneumonia 65.95

Bronchopneumonia 15.85

Meningitis 13.00

Empyema 8.53

Otitis media 2.44

Endocarditis 1.22

Hepatic abscess 1.22

In 46 consecutive pneumococcus infections of children
he found :

Otitis media 29

Bronchopneumonia 12

Meningitis 2

Pneumonia i

Pleurisy : i

Pericarditis i

Susceptibility. — Not all animals are equally susceptible to
the action of the pneumococcus. Mice and rabbits are
highly sensitive, dogs, guinea-pigs, cats, and rats are much
less susceptible, though they may also succumb to the in-
oculation of large doses.

Specificity. — The etiologic relationship of the pneumo-
coccus to pneumonia is based chiefly upon the frequency of
its presence in croupous pneumonia. Netter* found it 82
times in 82 autopsies upon such cases; Klemperer, 21 times
out of 2 1 cases studied by puncturing the lung with a hypo-
dermic syringe. Weichselbaum obtained it in 94 out of 129
cases; Wolf, in 66 out of 70; and Pierce, in no out of 121
cases. In about 5 per cent, of the cases it remains localized
in the respiratory apparatus; in 95 per cent., it invades the
blood. An interesting paper upon this subject has been
written by E. C. Rosenow.f

The conditions under which it enters the lung to produce
pneumonia are not known. It is probable that some sys-
temic depravity is necessary to establish susceptibility, and
in support of this view we may point out that pneumonia is
very frequent, and exceptionally severe and fatal, among
drunkards, and that it is the most frequent cause of death
among the aged. Whether, however, any particular form of
vital depression is necessary to predispose to the disease,
further study will be required to tell.

Virulence. — Pneumococci vary greatly in virulence, and
rapidly lose this quality in artificial culture. When it is

* "Compte-rendu," 1889.

f "Jour. Infectious Diseases," 1904, i, p. 280.

Bacteriologic Diagnosis 503

desired to maintain or increase the virulence, a culture must
be frequently passed through animals. Washbourn found,
however, that a pneumococcus isolated from pneumonic
sputum and passed through one mouse and nine rabbits
developed a permanent virulence when kept on agar-agar
so made that it was not heated beyond 100° C., and alka-
linized 4 c.c. of normal caustic soda solution to each liter
beyond the neutral point determined with rosolic acid.
The agar-agar is first streaked with sterile rabbit's blood,
then inoculated. The cultures are kept at 37.5° C. Ordi-
narily pneumococci seem unable to accommodate them-

Fig. 163. — Diplococcus pneumoniae. Colony twenty-four hours old
upon gelatin. X 100 (Frankel and Pfeiffer).

selves to a purely saprophytic life, and unless continually
transplanted to new media die in a week or two, some-
times sooner. Lambert found, however, that in Marmorek's
mixture (bouillon 2 parts and ascitic or pleuritic fluid i
part) the organisms would sometimes remain alive as long
as eight months, preserving their virulence during the entire
time.

Virulence can also be retained for a considerable time by
keeping the organisms in the blood from an infected rabbit,
hermetically sealed in a glass tube, on ice.

Bacteriologic Diagnosis. — It is usually unnecessary to
call upon the bacteriologist to assist in making the diagnosis

504 Pneumonia

of pneumonia. If necessary, the expectoration can be
examined by the methods already given for staining the
pneumococcus, or rabbits may be inoculated and the organ-
ism recovered from the blood. Caution must be exercised
in using this means of diagnosis, however, as the organ-
ism sometimes occurs in normal saliva, and is a common
associated organism in tuberculosis and other respiratory
diseases. Wadsworth* has been able to show that agglu-
tination reactions can be obtained by concentrating the
pneumococci in isotonic solution and adding the serum.
The method does not seem easily applicable for diagnosis.
Neufeld f and Wadsworth J have also found that when
rabbit's bile is added to a pneumococcus culture so as to pro-
duce lysis of the organisms, the addition of pneumococcus-
immune serum to the clear fluid so obtained results in a
specific precipitation. This seems to have little practical
importance, however, for purposes of diagnosis. It is,
however, of some importance in assisting in the recognition
of the pneumococcus and differentiating it from the strepto-
coccus, for when the latter organisms are similarly treated no
precipitate takes place.

Buerger§ found that all pneumococci, irrespective of
source, were agglutinated by pneumococcus immune serum,
that such serum was capable of agglutinating various pyo-
genic streptococci, certain atypical organisms, and certain
strains of Streptococcus mucosus capsulatus. The sera of
pneumonia patients varies in its power to agglutinate dif-
ferent pneumococci; some strains were agglutinated, others
not. The sera of normal individuals and of normal rab-
bits possess no agglutinating power for pneumococci, the
atypical organisms, certain streptococci, and the Strepto-
coccus mucosus capsulatus.

As pneumococci sometimes grow in chains instead of in
pairs, and as the capsules are not always more distinct than
the capsules that sometimes surround streptococci, it may
be necessary to resort to special methods of cultivation for
the final determination of the organism. One of the first
to be recommended is the use of the blood-agar plate, to

* "Jour. Med. Research," vol. x, p. 228, 1904.

t "Zeitschrift fiir Hygiene," 1902, xi.

J Loc. cit.

§ "Jour. Exp. Med.," Aug. 25, 1905, vn, No. 5.

Immune Serum 505

which reference has been made in the section upon Strepto-
coccus pyogenes.

A second important method, and one that not only
differentiates the pneumococcus from the streptococcus, but
from the common organisms of similar morphology that
infect the mouth, is the inulin-serum water fermentation
test of Hiss.* In using this medium, Ruedigerf found it
best prepared as follows: Dissolve 5 gm. of NaCl, 20
gm. of Witte's peptone, and 20 gm. of pure inulin in 1000
c.c. of distilled water. Add 20 c.c. of a 5 per cent, solu-
tion of pure litmus, and tube, putting 2 c.c. of the mixture
into each tube, and sterilize in the autoclave. After steril-
ization add (with a sterile pipet) 2 c.c. of sterile, heated
ascitic fluid, or, preferably, heated beef-serum, to each tube,
and incubate twenty-four hours before using. Great care
must be taken not to use ascitic fluid that contains ferment-
able carbohydrates. Each lot must be tested with some
strongly fermentative bacterium, and the absence of ferment-
able carbohydrates proved. Ruediger prefers this prepa-
ration to the original solution of Hiss because he found that
some pneumococci would not grow on the latter. Fermenta-
tion of the inulin is regarded as characteristic of the pneumo-
coccus.

The pneumococcus produces red colonies upon litmus-
inulin-agar plates, which makes their use desirable when
pneumococci are to be isolated from saliva, throat secretions,
or other material in which similar appearing organisms
are apt to occur. Ruediger found no other mouth bacteria
that produced red colonies on these plates.

Immunity. — Pneumonia is peculiar in that recovery is
followed by immunity of such brief duration as to permit
the occurrence of frequent relapses; and it is well known
that many cases show a subsequent predisposition to fresh
attacks of the disease.

Immune Serum. — G. and F. Klemperert have shown
that the serum of rabbits immunized against the pneumo-
coccus protects animals infected with virulent cultures.
When applied to human medicine, the serum failed to
do good.

The treatment of pneumonia by the injection of blood-

* "Jour. Amer. Med. Assoc.," 1906, vol. XLVII, p. 1171.

t "Jour, of Exp. Med.," 1905, vol. vi, p. 317.

J "Berliner klin. Wochenschrift," 1891, Nos. 34 and 35.

506 Pneumonia

serum from convalescent patients, tried by Hughes and
Carteras,* has been abandoned as useless and dangerous.

More recent antipneumococcic sera have been experi-
mentally investigated by De Renzi,f Washbourn, t and
Pane.§

Washbourn prepared an antipneumococcus serum that
protected rabbits against ten times the fatal dose of live
pneumococci, in doses of 0.3 c.c. In general, the lines upon
which he operated were those of Behring, Marmorek's work
with the streptococcus furnishing most of the details.
Two cases of human pneumonia seem to have derived some
benefit from large doses of this serum. The sera of Pane
and De Renzi were not so powerful as those of Washbourn,
requiring about i c.c. to protect a rabbit.

McFarland and Lincoln || succeeded in immunizing a
horse against large doses of a virulent culture of the pneumo-
coccus, and obtained a serum of which 0.5 to 0.25 c.c. pro-
tected rabbits from many times the fatal dose.

The experiments by Passler** showed some gain over the
earlier work.

The antipneumococcic sera thus far produced have given
disappointing results in clinical application.

A leukocytic extract prepared by Hiss and Zinsser|t
from an aleuronat exudation in the rabbit's pleura has led
to results sufficiently encouraging in the treatment of
pneumonia in man to warrant further investigation along
similar lines.

RosenowJI found that pneumococci suspended in sodium
chlorid solutions autolyse rapidly. By means of this au-
tolysis it is possible to separate, at least to a large degree, the
toxic from the antigenic parts of the pneumococcus, as the
toxic part goes into solution. The injection of the non-
toxic and, as it appears, antigenic portion — autolyzed pneu-
mococci— causes a marked increase in the immunity curve

* "Therapeutic Gazette," Oct. 15, 1892.
t "II Policlinico," Oct. 31, 1896, Supplement.
I "Brit. Med. Jour.," Feb. 27, 1897, p. 510.

§ "Centralbl. f. Bakt. u. Parasitenk.," May 29, 1897, xxi, 17 and 18,
p. 664.

|| "Jour. Amer. Med. Assoc.," Dec. 16, 1899, p. 1534.
** "Deutsches Archiv fur klin. Med.," Bd. LXXXII, Nos. 3, 4, 1905;
"Jour. Amer. Med. Assoc.," May 13, 1905, p. 1538.
ft "Jour. Med. Research," 1908, xix, 323.
tJ "Jour. Amer. Med. Assoc.," June 10, LIV, No. 24, p. 1943-

Sanitation 507

as measured by the specific increase in pneumococcus
opsonin. The injection of such autolyzed pneumococci into
25 patients with lobar pneumonia seemed to have a marked
beneficial effect.

Sanitation. — Pneumonia is undoubtedly a contagious
disease. Exactly how infection takes place is not known,
but seeing that the infectious agent is in the respiratory tract,
from which it is easily discharged into the atmosphere during
cough, etc., and the facility with which it can then be inhaled
by those nearby, it seems justifiable to conclude that the
primary entrance of the organism into the body is through
the respiratory tract. Wood* has shown that " the organ-
isms in the sputum do hot remain long in suspension and die
off rapidly under the action of light and desiccation. In
sunlight or diffuse daylight the bacteria in such powder die
within an hour, and in about four hours if kept in the dark.
The danger of infection from powdered sputum may, there-
fore, be avoided by ample illumination and ventilation of the
sick-room in order to destroy or dilute the bacteria, and by
the avoidance of dry sweeping or dusting. Articles which
may be contaminated and which cannot be cleaned by cloths
dampened in a suitable disinfectant should be removed from
the patient's vicinty.

PNEUMOCOCCUS (FRIEDLANDER) — BACTERIUM PNEUMONIA
(ZOPFJ) — BACILLUS CAPSULATUS Mucosus (FASCHINGJ).

General Characteristics. — An encapsulated, non-motile, non-flag-
ellated, non-sporogenous, non-liquefying, aerobic and optionally anaer-
obic, non-chromogenic, aerogenic and pathogenic bacillus, staining by
ordinary methods, but not by Gram's method.

This organism was discovered by Friedlander§ in 1883 in
the pulmonary exudate from a case of croupous pneumo-
nia, and, being thought by its discoverer to be the cause of
that disease, was called the pneumococcus, and later the
pneumobacillus. The grounds upon which the specificity of
the organism was supposed to depend were soon found to
be insufficient, and the organism of Friedlander is at pres-
ent looked upon as one whose presence in the lung is, in

* "Jour. Exp. Med.," Aug. 25, 1905, vn, No. 5, p. 624.

f "Spaltpilze," 1885, p. 66.

t "Centralbl. f. Bakt.," etc., xn, 1892, p. 304.

§ "Fortshritte der Medizin," 1883, 22, 715.

508

Pneumonia

most cases, unimportant, though it is sometimes asso-
ciated with and is probably the cause of a special form
of lobular pneumonia, which, according to Stiihlern,* is
clinically atypical and usually fatal. Frankel points out that
Friedlander's error in supposing his organism to be the chief
parasite in pneumonia depended upon the fact that his studies
were made by the plate method, which permitted the dis-
covery of this bacillus to be made more easily than that of
the slowly growing and more delicate pneumococcus. In
the light of present knowledge Friedlander's bacillus must be

Fig. 164. — Bacterium pneumonicum (modified after Migula).

looked upon as the type of a group of organisms varying among
themselves in many minor particulars.

Distribution. — The organism is sometimes found in
normal saliva; it is a common parasite of the respiratory
apparatus, not infrequently occurs in purulent accumula-
tions, is occasionally found in feces, and sometimes occurs
under external saprophytic conditions. Thus it is probably
identical with that described as the " capsulated canal-water
bacillus " by Mori,f and may be identical with or at least
belong to the same group in which we find Bacillus aerogenes
capsulatus.

*"Centralbl. f. Bakt.," etc. (Originale), Bd. xxxvi, No. 4, p. 493,
July 21, 1904.

t "Zeitschnft fur Hygiene," iv, 1888, p. 53.

Cultivation

509

Morphology. — Though usually dis-
tinctly bacillary in form, the organism
is of variable length and when paired
sometimes bears a close resemblance
to the pneumococcus of Frankel and
Weichselbaum It measures 0.5 to
1.5 ^ in breadth and 0.6 to 0.5 [A in
length. It frequently occurs in
chains of four or more elements and
occasionally appears elongated. It is
these variations in form that have
led to the description of the organism
by different writers as a coccus, a
bacterium, and a bacillus. It is com-
monly surrounded by a distinct trans-
parent capsule, hence its name " cap-
sule bacillus" and Bacillus capsulatus
mucosus. The organism is non-
motile, has no spores, and no flagella.
It stains well with the ordinary anilin
dyes, but does not retain the color
when stained by Gram's method.

Cultivation. — Colonies. — If pneu-
monic exudate be mixed with gelatin
and poured upon plates, small white
spheric colonies appear at the end of
twenty-four hours, and spread out
upon the surface of the gelatin to
form whitish masses of a considerable
size. Under the microscope these
colonies appear irregular in outline
and somewhat granular.

Bouillon. — There is nothing char-
acteristic about the bouillon cultures
of Friedlander's bacillus. The me-
dium is diffusely clouded. A pellicle
usually forms on the surface and a
viscid sediment soon accumulates.

Gelatin Puncture. — When a colony
is transferred to a gelatin puncture
culture, a luxuriant growth occurs.
Upon the surface a somewhat ele-
vated, rounded white mass is formed,

Fig. 165. — Friedlan-
der's pneumobacillus ;
gelatin stab culture,
showing the typical
nail-head appearance
and the formation of
gas bubbles, not always
present (Curtis).

510 Pneumonia

and in the track of the wire innumerable little colonies
spring up and become confluent, so that a ' ' nail-growth ' '
results. No liquefaction of the gelatin occurs. Gas bubbles
not infrequently appear in the wire track. The cultures
sometimes become brown in color when old.

Agar-agar. — Upon the surface of agar-agar at ordinary
temperatures a luxuriant white or brownish-yellow, smeary,
viscid, circumscribed growth occurs.

Blood-serum. — The blood-serum growth is similar to that
upon agar.

Potato. — Upon potato the growth is luxuriant, quickly
covering the entire surface with a thick yellowish -white
layer, which sometimes contains bubbles of gas.

Milk is not coagulated as a rule. Litmus milk is reddened.
Vital Resistance. — The bacillus grows at a temperature
as low as 16° C., and, according to Sternberg, has a thermal
death-point of 56° C.

Metabolic Products. — Friedlander's bacillus ferments
nearly all the sugars, with the evolution of much gas. It
generates alcohol, acetic and other acids, and both CO2 and
H. According to the best authorities the organism does not
form indol. There is, however, some difference of opinion
upon the subject.

Perkins* divides the organisms of this group into three
chief types according to their reactions toward carbohy-
drates :

I. Bacillus aerogenes type ferment all carbohydrates,

with the formation of gas.

II. Bacillus pneumoniae (Friedlander) type ferment all
carbohydrates except lactose, with formation of
gas.

III. Bacillus lactis aerogenes type ferment all carbo-
hydrates except saccharose, with formation of
gas.

Pathogenesis. — Friedlander found considerable difficulty
in producing pathogenic changes by the injection of his
bacillus into the lower animals. Rabbits and guinea-pigs
were immune to its action, and the only important patho-
genic effects that Friedlander observed occurred in mice,
into whose lungs and pleura he injected the cultures, with
resulting inflammatory lesions.

That Friedlander's bacillus may be the cause of true lobar
* ''Jour, of Infect. Dis.," 1904, i, No. 2, p. 241.

Pathogenesis 511

pneumonia there can be no room for doubt after the demon-
strations of Lamon and Meltzer,* who found that its experi-
mental introduction into the bronchi of dogs was followed by
true lobar pneumonia. The lesions in these dogs, like those
in human beings, were paler in color, the lung tissue less
friable, and the exudate more viscid than those caused by
the pneumococcus.

Pneumonia in man, caused by Bacillus mucosus capsulatus,
is atypical clinically, very severe, and often fatal.

Curryt found Friedlander's bacillus in association with the
pneumococcus in acute lobar pneumonia; in association with
the diphtheria bacillus in otitis media associated with croup-
ous pneumonia; and in the throat in diphtheria. In pure
culture it was obtained from vegetations upon the valves of
the heart in a case of acute endocarditis with gangrene of the
lung; from the middle ear, in a case of fracture of the skull
with otitis media ; and from the throat in a case of tonsillitis.
Zinsser has twice cultivated Friedlander's bacillus from in-
flamed tonsils in children.

AbelJ cultivated it from the discharges of fetid ozena,
and supposed it to be the specific cause.

Occasionally Friedlander's bacillus bears an important
relationship to lobular or catarrhal pneumonia, an interesting
case having been studied by Smith. § The histologic changes
in the lung were remarkable in that the " alveolar spaces
of the consolidated areas were dilated and for the most
part filled with the capsule bacilli." In some alveoli there
seemed to be pure cultures of the bacilli; others contained
red and white blood-corpuscles; in some there was a little
fibrin. The bacillus obtained from this case, when injected
into the peritoneal cavity of guinea-pigs, produced death
in eleven hours. The peritoneal cavity after death con-
tained a large amount of thick, slimy fluid; the intestines
were injected and showed a thin fibrinous exudate upon the
surface; the spleen was enlarged and softened, and the
adrenals much reddened. Cover-glass preparations from
the heart, blood, spleen, and peritoneal cavity showed large
numbers of the capsule bacilli.

* "Jour. Exp. Med.," 1912, xv, 133.

f "Jour. Boston Soc. of Med. Sci.," March, 1898, vol. n. No. 8, p. 137.

t "Zeitschrift fur Hygiene," xxi.

§ "Jour. Boston Soc. of Med. Sci.," May, 1898, vol. n, No. 10, p. 174.

512 Pneumonia

Howard* has also called attention to the importance of
this bacillus in connection with numerous acute and chronic
infectious processes, among which may be mentioned
croupous pneumonia, suppuration of the antrum of High-
more and frontal sinuses, endometritis, perirenal abscesses,
and peritonitis.

Virulence. — The virulence of the organism seems to vary
under different conditions. It is sometimes harmless for
the experiment animals, but when injected into mice and
guinea-pigs usually produces local inflammatory lesions, and
sometimes invasion of the circulation and death from sepsis.

CATARRHAL PNEUMONIA OR BRONCHOPNEUMONIA.

This form of pulmonary inflammation occurs in local
areas, commonly situated about the distribution of a bron-
chiole. It cannot be said to have a specific micro-organism,
as almost any irritating foreign matter accidentally inhaled
may cause it. The majority of the cases, however, are
infectious in nature and result from the inspiration, from
higher parts of the respiratory apparatus, of the staphylo-
cocci and streptococci of suppuration, Friedlander's bacillus,
the bacillus of influenza, and other well-known organisms.

TUBERCULAR PNEUMONIA.

The progress of pulmonary tuberculosis is at times so
rapid that the tubercle bacilli are distributed with the
softened infectious matter throughout the entire lung or to
large parts of it, and a distinct pneumonic inflammation
occurs. Such a pneumonia may be caused by the tubercle
bacillus, but more frequently depends upon accompanying
staphylococci, streptococci, tetragenococci, pneumococci,
pneumobacilli, and other organisms accidentally present in
a lung in which ulceration and cavity formation are ad-
vanced.

PLAGUE PNEUMONIA.

The pneumonic form of plague is characterized by con-
solidation of the lung histologically and anatomically, indis-
tinguishable from pneumococcal and other extensive pul-
monary infections.

* "Phila. Med. Jour.," Feb. 19, 1898, vol. i, No. 8, p. 336.

Mixed Pneumonias 513

MIXED PNEUMONIAS.

It frequently happens that pneumonia occurs in the course
of influenza or shortly after convalescence from it. In these
cases a mixed infection by the influenza bacilli and pneu-
mococci is commonly found. Sometimes pneumococci and
staphylococci simultaneously affect the lung, purulent pneu-
monia with abscess formation being the conspicuous feature.
Almost any combination of the described bacteria may occur
in the lungs, producing varying inflammatory conditions,
so that it must be left for the student to work out what the
particular characteristics of each may be.

Among the mixed forms of pneumonia may be mentioned
those called by Klemperer and Levy " complicating pneu-
monias," occurring in the course of typhoid fever, etc.

33

CHAPTER XVI.

INFLUENZA.
BACILLUS INFLUENZA (R. PFEIFFER).

General Characteristics. — A minute, non-motile, non-flagellated,
non-sporogenous, non-liquefying, non-chromogenic, aerobic, pathogenic
bacillus, staining by the ordinary methods, b.ut not by Gram's method,
and susceptible of artificial cultivation, chiefly through the addition
of hemoglobin to the culture-media.

Notwithstanding the number of examinations conducted
to determine the cause of influenza, it was not until 1892,
after the great epidemic, that Pfeiffer* found, in the blood
and purulent bronchial discharges, a bacillus that con-
formed, in large part, to the requirements of specificity.

Morphology. — The bacilli are very small, having about
the same diameter as the bacillus of mouse septicemia, but
only half its length (0.2 by 0.5 fi). They are usually soli-
tary, but may be united in chains of three or four.

They are non-motile, have no flagella, and, so far as is
known, do not form spores.

Staining. — They stain rather poorly except with such
concentrated and penetrating stains as carbol-fuchsin and
Loffler's alkaline methylene-blue, and even with these more
deeply at the ends than in the middle, so that they appear
not a little like diplococci. They do not stain by Gram's
method.

Canon f recommends a rather complicated method for the
demonstration of the bacilli in the blood. The blood is
spread upon clean cover-glasses in the usual way, thoroughly
dried, and then fixed by immersion in absolute alcohol for
five minutes. The best stain is Czenzynke's:

Concentrated aqueous solution of methylene-blue 40

0.5 per cent, solution of eosin in 70 per cent, alcohol. .20
Distilled water 40

.* " Deutsche med. Wochenschrift," 1892, 2 ; "Zeitschrift fur Hygiene,"
13-

t "Centralbl. f. Bakt.," etc., Bd. xiv, p. 860.

Isolation

The cover-glasses are immersed in the solution, and kept
in the incubator for from three to six hours, after which they
are washed in water, dried, and mounted in Canada balsam.
By this method the erythrocytes are stained red, the leuko-
cytes blue; and the bacilli, also blue, appear as short rods
or as dumb-bells.

Large numbers of bacilli may be present, though some-
times only a few can be found after prolonged search, as they
are prone to occur in widely scattered but dense clusters.
They are frequently inclosed within the leukocytes. It is

Fig. 166. — Bacillus of influenza. Smear from sputum. (After Heim.)

scarcely necessary to pursue so tedious a staining method for
demonstrating the bacilli, for they stain well enough for recog-
nition by ordinary methods.

Isolation. — The influenza bacillus grows poorly upon
artificial culture-media, and is not easy to isolate, because
the associated bacteria tend to outgrow it. When isolated
it is difficult to keep, as it soon dies in unnatural environ-
ment.

Pfeiffer found that the organism grew when he spread pus
from the bronchial secretions upon serum-agar. Subcul-
tures made from the original colonies did not "take." It
therefore seemed as though it might depend upon the absence
of some ingredient that the bronchial secretions contained.

Influenza

By a series of experiments he was able to make the organism
grow when he transferred it to agar-agar, the surface of which
was coated with a film of blood taken, with precautions as to
sterility, from the finger-tip. Later it was found that the
addition of hemoglobin to the culture-medium was equally
efficacious. By the use of such blood-smeared agar and gly-
cerin-agar the organism can now be successfully cultivated.
The isolation is best achieved through the use of bronchial
secretions, carefully washed in sterile water or salt solution
to remove contaminating organisms from the mouth.

Fig. 167. — Bacillus of influenza; colonies on blood agar-agar. Low
magnifying power (Pfeiffer).

Cultivation. — Upon blood-spread glycerin agar-agar,
after twenty-four hours in the incubator, minute colorless,
transparent, dewdrop-like colonies may be seen along the line
of inoculation. They look like condensed moisture, and Kita-
sato makes a special point of the fact that they never become
confluent. The colonies may at times be so small as to
require a lens for their detection.

No growth takes place at room temperature. The organ-
isms die quickly and must be transplanted every three or four
days if they are to be kept alive.

Immunity 517

The organism is aerobic and scarcely grows at all where the
supply of oxygen is not free.

In bouillon a scant development occurs, small whitish
particles appearing upon the surface, subsequently sinking
to the bottom and causing a "wooly" deposit there. The
bacillus grows more luxuriantly upon culture-media contain-
ing hemoglobin or blood, and can be transferred from culture
to culture many times before losing vitality.

Vital Resistance. — Its resisting powers are very re-
stricted, as it speedily succumbs to drying, and is certainly
killed by an exposure to a temperature of 60° C. for five
minutes. It will not grow at any temperature below 28° C.

Specificity. — From the fact that the bacillus is found
chiefly in cases of influenza, that it is present as long as the
purulent secretions of the disease last, and then disappears,
and that Pfeiffer was able to demonstrate its presence in all
cases of uncomplicated influenza, it seems that his conclu-
sion that the bacillus is specific is justifiable. It is also
found in the secondary morbid processes following influenza,
such as pneumonia, endocarditis, middle-ear disease, menin-
gitis, etc. Horder* has cultivated it from the valvular vege-
tation of 2 cases of endocarditis following influenza.

Davis f found the influenza bacillus in the respiratory
passage of a large number of patients suffering from whoop-
ing-cough.

Pathogenesis. — The bacillus is pathogenic for very few
of the laboratory animals, the guinea-pig being susceptible
of fatal infection. The dose required to cause death of a
guinea-pig varies considerably.

Pfeiffer and BeckJ produced what may have been influenza
in monkeys by rubbing their nasal mucous membranes with
pure cultures.

Immunity. — As influenza is a disease that commonly re-
lapses, and from which one rarely seems to acquire protection
against future attacks, there must be scarcely any immunity
induced through ordinary infection. Moreover, the organism
once finding its way into the body seems to remain almost in-
definitely, especially when, as in pulmonary tuberculosis,
there is already present an abnormal condition furnishing
discharges or exudates in which it can thrive.

* "Path. Soc. of London," "Brit. Med. Jour.," April 22, 1905.
t "Jour. Infectious Diseases," in, 1906, i.
J" Deutsche med. Wochenschrift," 1893, xxi.

Influenza

In the immunization experiments of Delius and Kolle*
one-twentieth of a twenty-four-hour-old culture was fatal in
twenty-four hours. They found that the toxicity of the cul-
ture does not depend upon a soluble toxin, but upon an intra-
cellular toxin. The outcome of the researches, which were
made most painstakingly, was total failure to produce experi-
mental immunity.

Increasing doses of the cultures, injected into the perito-
neal cavity, enabled the animals to resist more than a fatal

Fig. 1 68. — Bacillus of influenza; cover-glass preparation of sputum
from a case of influenza, showing the bacilli in leukocytes. Highly
magnified (Pfeiffer).

dose, but never enabled them to maintain vitality when large
doses of living cultures were administered. This observation
is in exact harmony with the familiar clinical observation
that, instead of an individual remaining immune after an
attack of influenza, he is quite as susceptible as before.

A. Catanni, Jr.,f trephined rabbits and injected influenza
toxin into their brains, at the same time trephining control
animals, into some of whose brains he injected water. The
animals receiving 0.5 to i mg. of the living culture died in

* "Zeitschrift fur Hygiene," etc., Bd. xxiv, 1897, Heft 2.
t Ibid., Bd. xxm, 1896.

The Pseudo-Influenza Bacillus 519

twenty-four hours with all the nervous symptoms of the dis-
ease, dyspnea, paralysis beginning in the posterior extremities
and extending over the whole body, clonic convulsions, stiff-
ness of the neck, etc. Control animals injected in the same
manner with water, and with a variety of other pathogenic
bacteria never manifested similar symptoms. The viru-
lence of the bacillus increased rapidly when transplanted
from brain to brain.

Diagnosis of Influenza. — Wynekoop* employs for diag-
nosticating influenza and isolating the bacillus, a culture
outfit similar to that used for diphtheria diagnosis, except
that the serum contains more hemoglobin. The swab is
used to secure secretions from the pharynx and tonsils, and
from the bronchial secretions of patients with influenza, then
rubbed over the blood-serum. In many such cultures
minute colonies corresponding to those of the influenza
bacillus were found. Those most isolated were picked up
with a wire and transplanted to bouillon, from which fresh
blood-serum was inoculated and pure cultures secured.

Carbol-fuchsin was found most useful for staining the
bacilli. Wynekoop observed that influenza and diphtheria
bacilli sometimes coexist in the throat, and that influenza
bacilli are present in the sore eyes of those in the midst
of household epidemics of influenza.

THE PSEUDO-INFLUENZA BACILLUS.

Pfeiffer f has also described a pseudo-influenza bacillus — a
small, non-motile, non-flagellated, non-sporogenous, Gram-
negative bacillus — that he found in certain cases of broncho-
pneumonia in children. It differed from the influenza bacillus
by a slightly greater size, a tendency to grow in chains, and
to undergo involution. Martha Wollsteinf believes that
they are influenza bacilli.

* ''Bureau and Division Reports," Department of Health, city of
Chicago, Jan., 1899.

t " Zeitschrif t fur Hygiene," etc., 1892, xm.
t "Jour. Exp. Med.," 1906, vm.

CHAPTER XVII.
MALTA OR MEDITERRANEAN FEVER.

MICROCOCCUS MEWTENSIS (BRUCE); BACILLUS MEUTENSIS

(BABES).

General Characteristics. — A non-motile, non-flagellate, non-spo-
rogenous, non-chromogenic, 'non-liquefying, pathogenic coccus, staining
by the ordinary methods, but not by Gram's method; characterized
by remarkably slow growth and by pathogenic action upon monkeys.

In 1877, while working in Malta, Bruce* succeeded in
finding in every fatal case of Malta fever a micrococcus
which could be isolated in pure cultures from the spleen,
liver, and kidney, which grew readily on artificial media, and
which, when injected into monkeys, produced the disease.

Morphology. — Micrococcus melitensis, as Bruce called it,
is a round or slightly oval organism measuring about 0.3 ^
in diameter. It is usually single, sometimes in pairs, but
never in chains. When viewed in the hanging drop it is
said to exhibit active "molecular" movements, but is not
motile and has no flagella. Babes f declares it to be a bacillus.

Staining. — It stains well with aqueous solutions of the
anilin dyes, but not by Gram's method.

Thermal Death Point. — This has been fixed by Dalton
and Eyre J at 57.5° C.

Cultivation. — The best medium for its cultivation is
said to be ordinary agar-agar. After inoculating, by a punc-
ture, from the spleen of a fatal case of Malta fever, the tubes
should be kept at 37° C. The growth first appears after
several days, in the form of minute pearly white spots scat-
tered around the point of puncture and along the needle
path. After some weeks the colonies grow larger and join
to form a rosette-like aggregation, while the needle tract be-
comes a solid rod of yellowish-brown color. After a lapse of
months the growth still remains restricted to the same area
and its color deepens to buff.

* ''Practitioner," xxxiv, p. 161.

t Kolle and Wassermann "Die Pathogenic Mikroorganisms," m, p.
443-

t "Jour, of Hygiene," iv., 1904, p. 157.

520

Bacteriologic Diagnosis

When the sloping surface of inoculated agar-agar is ex-
amined by transmitted light, the appearance of the colonies
is somewhat different. At the end of nine or ten days, if
kept at 37° C., some of the colonies have a diameter of 2 to 3
mm. They are round in form, have an even contour, are
slightly raised above the surface of the agar-agar, and are
smooth and shining in appearance. On examining the
colonies by transmitted light, the center of each is seen to
be yellowish, while the periphery is bluish-white in color.
The same colonies by reflected light appear milky white in
color. Colonies on the surface of the agar-agar are found
to be no larger than hemp-seed after a couple of months
of cultivation.

When kept at 25° C., no colonies become visible to the

* -

Fig. 169. — Micrococcus melitensis.

naked eye before the seventh day; at 37° C., before the
third or fourth day.

The growth in gelatin takes place at room temperature
with great slowness, first appearing in about a month,
and no liquefaction of the medium occurs.

No growth takes place on boiled potato.

Plate cultures are not adapted to the study of the organ-
ism because of its extreme slowness of growth.

Bacteriologic Diagnosis. — The specific agglutinative
effect of the serum can be made use of for the purpose

522 Malta or Mediterranean Fever

of diagnosis. This has been studied by Wright,* Birt and
Lamb,f and later by Bassett-Smith.J

All of the observers have shown that the agglutinative
reaction takes place both with living and dead cultures of
the Micrococcus melitensis, but that to make the diagnosis
dilutions of serum equal to about i : 30, never greater than
i : 50, must be used. Birt and Lamb also arrive at certain
conclusions regarding the prognosis based upon a study of
the agglutinative phenomena. Their conclusions are :

1. Prognosis is unfavorable if the agglutinating reaction is per-

sistently low.

2. Also if the agglutinating reaction rapidly fall from a high figure

to almost zero.

3. A persistently high and rising agglutinating reaction sustained

into convalescence is favorable.

4. A long illness may be anticipated if the agglutination figure, at

first high, decreases considerably.

The agglutination reaction appears early, and is available
by the end of the first week, and persists often for years
after convalescence.

The organisms may sometimes be cultivated from the
blood taken from a vein, but are more certainly to be secured
by splenic puncture.

Pathogenesis. — The micro-organism is not pathogenic
for mice, guinea-pigs, or rabbits, but is fatal to monkeys
when agar-agar cultures suspended in water are injected
beneath the skin.

The micro-organism usually seems to be absent from the
circulating blood, though Hughes has cultivated it from the
heart's blood of a dead monkey.

Bruce not only succeeded in securing the micro-organism
from the cadavers of Malta fever, but has also obtained it
during life by splenic puncture.

Accidental inoculation with micrococcus melitensis, as by
the prick of a hypodermic needle, is almost invariably fol-
lowed by an attack of the disease. Six cases of this kind
occurred in connection with bacteriologic work on Malta
fever at Netley and two additional at the Royal Naval
Hospital at Haslar and in the Philippines. §

Treatment — The treatment of Mediterranean fever by

* "Lancet," 1897, March 6; "Brit. Med. Jour.," 1897, May 15.

f Ibid., 1899, n, p. 701.

J "British Med. Jour.," 1902, n, p. 861.

§ See Wright and Windsor, "Jour, of Hygiene," n, 1902, p. 413.

Goats' Milk and Malta Fever 523

means of bacterio-vaccines has been attempted with what
seems to be glittering results by Bassett-Smith.*

The report of " British Government Commission for the
Investigation of Mediterranean Fever," published by the
Royal Society, April, 1907, has greatly elucidated our knowl-
edge of the pathogen y of the disease by showing that the
Micrococcus melitensis leaves the body of the patient in the
urine and in the milk. It has not been found in the saliva,
sweat, breath, or feces. The discovery of the organism in
the milk suggested that it might be through milk that the
disease organisms were disseminated, and an investigation of
the goats at Malta, where the disease is most prevalent, and
their milk most generally used, showed that a large per-
centage of the animals were infected with the specific cocci.
The commission has, therefore, concluded that it is by
goats' milk that the disease is commonly disseminated,
though they point out that fly-transmission is also possible.
In the Colonial Office Report on Malta in 1907 it was shown
that over 40 per cent, of the goats of Malta gave the serum
reaction, showing that they had had the disease, while 10 per
cent, of them were actually secreting the cocci in their milk.
The authorities permit no milk to be used in the garrison
unless it is boiled, and notice that by this simple measure
the incidence of the disease, which was 9.6 in 1905, had
fallen to 2 in the corresponding month of 1906. In Report
VII. of the Mediterranean Fever Commission (1906-07) we
read:

"The epidemiologists are led to believe that quite 70 per cent, of
the cases are due to the ingestion of goat's milk." In their opinion
ordinary contact with the sick, conveyance of infection by biting insects,
house flies, dust, drain emanations, food (other than milk), and water,
play a very subordinate part, if any, in setting up Mediterranean fever
in man. The excellent results following the preventive measures di-
rected against goat's milk in barracks and hospitals also point to goat's
milk as being the chief factor. Among the soldiers this resulted in a
diminution of about 90 per cent.

"For example, in the second half of 1905 there were 363 cases of
Mediterranean fever, whereas in the corresponding part of 1906 there
were only 35 cases. Among the sailors there was also as marked a fall
in the number of cases. The Naval Hospital had a bad reputation, as
about one-third of the cases of fever occurring in the fleet at Malta
could be traced to residence in this hospital, either as patients suffering
from other diseases or among the nursing staff. The goats supplying
the hospital were found to be infected, and since their milk was absolutely
forbidden, not a single case of Malta fever has occurred in or been traced
to residence in this hospital."

* "Journal of Hygiene," vn, 1907, p. 115.

CHAPTER XVIII.

MALARIA.

MALARIA, or paludism, has been known since the days of
ancient medicine, and has always been regarded as the
typical miasmatic disease. Its name, mala aria, means
" bad air," and is Italian derived from the Latin, mains and
aer, coming from the Greek dyp, air, from dew, to blow.
The other name, paludism, from the Latin pains, a " marsh,"
refers the disease to the bad air coming from marshes.

It is a disease of extremely wide geographic distribution,
and since the supposed requirement, marshy ground, is
found in nearly all countries, and the disease is particularly
prevalent in the marshy districts of those countries in which
it occurs, the connection between the marshes and the disease
seemed clear. Indeed, the two are intimately connected,
but not in the original sense, as will be shown below.

Both hemispheres, all of the continents, and most of the
islands of the sea suffer more or less from malaria, and in
many places, especially in the tropics, it is so pestilential as
to make the country uninhabitable. Probably no better
idea of the wide distribution and severity of the disease can
be obtained than by reference to Davidson's " Geographical
Pathology." *

The disease assumes the form of a fever of intermittent or
remittent type, characterized by certain peculiar paroxysms.
When typical, as in well-marked intermittent fever, these are
ushered in by depression, headache, and chilly sensations,
which are soon followed by pronounced rigors in which the
patient shivers violently, his teeth chattering. The tem-
perature soon begins to rise and attains a height of 102°, 104°,
or even 106° F., according to the severity of the case. As
the temperature rises the sense of chilliness disappears and
gives place to burning sensations. The skin is flushed, hot,
and dry. After a period varying in length the skin begins
to break out into perspiration, which is soon profuse, the

* D. Appleton & Co., New York, 1892.
524

Malaria 525

fever and headache disappear and the patient commonly
sinks into a refreshing sleep. The frequency of the paroxysms
varies with the type of the disease, which, in its turn, can be
referred to the kind of infection by which it is caused. The
paroxysms exhaust the patient and incapacitate him and
may eventually prove fatal, though in by far the greater
number of cases the disease gradually expends itself and a
partial or complete recovery ensues. Some cases, known
as pernicious, are rapidly fatal, others develop into a
chronic cachexia, with profound anemia and complete in-
capacitation for physical or mental effort. The discovery
of Peruvian or Jesuits' bark, and its introduction into Europe
by the Countess del Cinchon, the wife of the Viceroy of Peru,
about 1639, marked an important epoch in the study of
malarial fever. The isolation of its alkaloids, quinin and
cinchona, begun in 1810 by Gomez and perfected in 1820
by Pelletier and Coventou, a second great epoch. But the
most important epoch began in 1880, when Charles Louis
Alphonse Laveran,* a French physician engaged in the study
of malarial fever in Algeria, announced the discovery of a
parasite, to which he gave the name Plasmodium malariae,
in the blood of patients suffering from the disease. His ob-
servations were immediately confirmed, Biitschli recognizing
the parasitic nature of the bodies observed. For the discov-
ery he was awarded the Breant prize.

Laveran, however, threw no light upon the source of in-
fection, and malaria continued to be described as a mias-
matic disease.

It was, however, recognized that there were different
types of parasites corresponding to the different clinical
forms of the disease, and Golgif succeeded in correlating
the various appearances of the parasites so as to express
their life cycles. But in spite of the interesting and im-
portant work of Golgi, Celli, Bignami and Marchiafava, and
many others no progress was made in accounting for the
entrance of the parasites into the human body.

This problem had long interested Sir Patrick Manson,
who had devised a theory which, though wrong in de-
tail, proved in the end to open the door to the next im-
portant discovery. Finding that the malarial parasites
could not be shown to leave the body in any of its elimina-

* "Acad. d. Med.," Paris, Nov. 28 and Dec. 28, 1880.
t "R. Acad. di Medicina di Torino," 1885, xi, 20.

526 Malaria

tions, and remembering that the same was true of the filarial
worms and their embryos, Manson came to the conclusion
that they must be taken out of the blood by some suctorial
insect. The one naturally first considered was the mosquito,
which was known to abound wherever malaria prevailed.
Examining mosquitoes that had been permitted to distend
themselves with the blood containing the parasites, Manson
found that in the stomach of the insect the peculiar phenom-
enon known as " flagellation," long before observed by Lav-
eran, took place in the parasites, giving rise to long, slender,
lashing, and, finally, free-swimming filaments. These, he
conjectured, might be the form in which the parasites left
the mosquito to infect the swamp water, with which human
infection eventually was brought about. Here Manson
failed, but while he was investigating he explained the
whole matter to Major Ronald Ross, who was soon to go to
India, and whom he advised to make the matter a subject for
study when he arrived at his destination. Ross* accepted
the opportunity that soon presented itself, and, after a most
painstaking investigation, the details of which are given in a
paper which can be found in the International Medical An-
nual, f 1890, made the second great discovery in the parasit-
ology of malarial ever. He found that, as Manson thought,
the mosquito is the definitive host of the parasite, but that the
matter is much less simple than was imagined, for the organ-
isms taken up by the mosquito undergo a complicated life
cycle requiring about a fortnight for completion, after which,
not the water into which the mosquito might fall and into
which its contained organisms might escape, but the mosquito
itself becomes the agent of infection. In other words, the
parasites taken up by the mosquito, after the completion of
the necessary developmental cycle, are returned by the mos-
quito to new human beings, who thus become infected. Thus
it was shown that malaria is not a miasmatic disease at all,
but that it is an infectious disease whose parasites divide their
life cycle between man and the mosquito, each becoming
infected by the other. The only role of the swamp is to
furnish the mosquitoes, and since these are only more numer-
ous, where swamps are numerous, but may occur without
swamps, the not infrequent occurrence of malarial fevers
apart from swamps is also explained. Ross further discov-

* "Indian Medical Gazette," xxxui, 14, 133, 401, 448.
t E. B. Treat & Co., New York.

Malarial Parasites 527

ered that all mosquitoes are not equally susceptible of infec-
tion, and, therefore, not all able to spread the infection.
Indeed, he so carefully studied the mosquitoes as to narrow
the infectability and infectivity of mosquitoes down to one
single family, the Anophelinae, and to one single genus,
Anopheles.

There remained, however, one more important fact to be
elucidated, and one more mysterious body to be accounted
for, viz., the " flagellated " body that had misled Manson.
This was found by MacCallum* to be but the spermatozoit
of the male parasite. While observing one of the malarial
parasites of birds — Plasmodium danliewskyi — he saw one of
these " flagella " swimming away from its parent parasite, and
followed it carefully, moving the slide upon the stage of the
microscope. It, and others of its kind, approached a large
globular parasite, to which one effected an attachment and
into which it entered. MacCallum realized that he had
observed the sexual fertilization of the organism. Thus
from its time-honored place as the typical miasmatic disease,
full of mystery and obscurity, malarial fever suddenly had
a flood of light thrown upon it by which every peculiarity was
fully illuminated.

In summarizing the knowledge thus set forth we find the
following facts :

1880 — Discovery of the Plasmodium malarise by Laveran.

1890 — Discovery of its human developmental cycle by
Golgi.

1895 — Discovery of the mosquito cycle and mode of trans-
mission by Ross.

iSgS: — Discovery of the sexual fertilization of the parasite
by MacCallum.

The interest aroused by Laveran's original discovery gave
a great impetus to the study of hematology with special refer-
ence to parasites, and it soon became evident that the plas-
modium was but one of a group of similar parasites. Of
these we have now become acquainted with the following:

Parasite. Disease. Host. Insect host.

Plasmodium Quartan fever. Man. Anopheles, My-

malariae. zorrhynchus,

Myzomyia.

Plasmodium Tertian fever. Man. Anopheles, My-

vivax. zorrhynchus,

Myzomyia.

*"Jour. of Exper. Med.," 1898, in, 117.

528

Malaria

Parasite. Disease.

Plasmodium fal- Aestivo - autum-
ciparum nal fever.

Plasmodium

kochi.
Plasmodium

inui.

Plasmodium
pitheci.

Plasmodium

brazilianum.
Plasmodium

cynomolgi.

Plasmodium
grassii (Pro-
teosoma gras-
sii).

Plasmodium
danliewskyi
(Halteridium
danliewskyi) .

Host.

Man.

Cercopithicus.

Macacus (In-
uus cyno-
molgus).

Orang - outang
(Pithecus sa-
tyrus) .

Brachyrus cal-
ores.

Inuus cynomol-
gus and Inuus
nemistrinus.

Sparrows, can-
ary birds, and
other small
birds.

Owls, hawks,
crows, and
other large
birds.

Insect host.

Anopheles, My-
zorrhynchus,
Myzomyia.

Unknown.

Unknown.

Unknown.

Unknown.
Unknown.

Culex pipens.
Unknown.

These micro-organisms correspond in all essentials. They
are protozoan parasites belonging to the sporozoa and live
in the blood (hematozoa) as parasites of the red corpuscles.
They all have two life cycles, one which is asexual in the
intermediate warm-blooded host, and one that is sexual in
the definitive cold-blooded (insect) host. Though the inter-
mediate hosts vary and may be birds or mammals, the
insect hosts, so far as known, are always mosquitoes. The
mosquitoes become infected by biting and sucking the
blood of infected animals; the warm-blooded animals be-
come infected by being bitten by infected mosquitoes, and so
on, in endless cycles.

The parasites differ but little in the details of structure
and development, so that the following description may serve
as a type for all :

From the proboscis of the mosquito, with its saliva, from
cells in the salivary glands where they have been harbored,
tiny elongate spindles, measuring about 1.5 ^ in length and
0.2 fi in breadth, and know as sporozoits, enter the blood of
the individual bitten. These sporozoits attach themselves
to the red blood-corpuscles, gradually lose their elongate
form, and become irregularly spherical. There is some
difference of opinion as to whether the little bodies are simply
upon the corpuscles, as Koch believed, or in the corpuscles,

Asexual Life Cycle

529

as the majority of writers believe, but it is an immaterial
difference, for the parasite soon makes clear that it is con-
suming the corpuscle. This little body is known as a
schizont. When stained with polychrome methylene-blue,
and examined under a high power of the microscope, it ap-

Fig. 170. — Plasmodium falciparum. Ookinetes in the stomach of An-
opheles (Grassi).

pears as a little ring with a dark chromatin dot upon one
side. It grows steadily, feeding upon the hemoglobin, which
seems to be chemically transformed into fine or coarse gran-
ules of a bacillary or rounded form, presumably melanin.
In a length of time that varies —
twenty-four to forty-eight hours
(Plasmodium falciparum), forty-
eight hours (Plasmodium vivax),
seventy-two hours (Plasmodium
malariae) — the schizonts mature,
becoming nearly as large or quite
as large as the corpuscles. The
pigment granules now collect at
the center and the substance of
the parasite divides into a group
of equal-sized merozoits, com-
monly known as spores. Of these cipafum. Transverse section
there are usually eight in the °J the stomach of Anopheles,
,, f *r*t ,. showing the ookmetes of the

meroblasts of Plasmodium ma- parasite in various stages of
lariae, from fifteen to twenty-five development attached to the
in those of Plasmodium vivax, outer surf ace (Grassi) .
and from eight to twenty-five

in Plasmodium falciparum. As the spores become fully
formed and ready to separate, the paroxysm of the disease
begins. It ends as the spores are freed and enter new
corpuscles to begin the cycle over again. After a good many
34

Fig. 171. — Plasmodium fal-

22

Fig. 172. — Developmental cycle of plasmodium vivax, the tertian
malarial parasite. Figures i to 17 are magnified 1200 diameters; 18 to
27, only 600 diameters: i, Sporozoit; 2, penetration of a sporozoit into a
red blood-corpuscle; 3 and 4, schizont developing in the red blood-cor-
puscles; 5 and 6, nuclear division of the schizont; 7, free merozoits; 8
(following the arrows to the left to 3), merozoits entering red blood-cor-
puscles, and multiplying by schizogony 3 to 7 ; after longer continuance
of the disease the sexual forms arise; ga to 12 a, macrogametocytes; 9b

Sexual Life Cycle 531

paroxysms have occurred it may be observed that not all of
the schizonts change to meroblasts and form spores. Some
remain large spheroidal bodies or, as in Plasmodium falci-
parum, assume a peculiar crescentic form and remain un-
changed in the blood. These are the sexual parasites. The
female is usually the larger and is known as the makrogame-
tocyte, the male, the smaller, the micro gametocyte. These
are the bodies which when removed by the mosquito
lay the foundation of its infection. When they are with-
drawn for microscopic examination or exposed to the in-
testinal juices of the mosquito, the microgametocyte be-
comes tumultuous, its granules are observed to be in a state
of active cytoplasmic streaming, and suddenly there burst
forth long slender filaments, the micro gametes or sporozoits.
These correspond with the flagella of Laveran and others,
and are the same bodies that Manson thought might be
the form in which the parasite leaves the insect's body.
The microgametes lash vigorously for a time, then, breaking
loose, swim away, and, as MacCallum observed, conjugate
with macrogametes sexually perfect cells formed from the
macrogametocytes, thus fertilizing them. As the result of
this fertilization a zygote or ookinete is formed. It assumes
a somewhat elongate pointed form and attaches itself
to the wall of the mosquito's stomach. In the course
of time it penetrates and appears upon the outside, pro-
jecting into the body cavity. It grows larger and rounder,
divides into several segments, and eventually forms an
oocyst with many small cells, which break up into myriads
of tiny elongate fusiform bodies, the sporozoits. These,
in the course of time, seem to find their way to the sali-

to i2b, microgametocytes still in the circulatory blood of man. If the
macrogametocytes (i2a) are not taken into the alimentary canal of the
mosquito, they multiply parthenogenetically (i2a, I3C to lye) and the
resulting merozoits (lye) become schizonts (3 to 7). The figures below
the dotted line represent what takes place in the alimentary canal of
anopheles (13 to 17); i3b and i^b the formation of microgametocytes;
i3a and i3b, maturation of the macrogametes; isb, a microgamete;i 6,
fertilization; 17, ookinete; 18, ookinete on the wall of the mosquito's
stomach; 19, penetration of the gastric epithelium by the ookinetes; 20
to 25, stages of sporogenesis on the outer wall of the mosquito's stomach;
26, migration of the sporozoits to the salivary glands of the mosquito; 27,
salivary gland with sporozoits in the epithelial cells, and escape of the
sporozoits from the salivary glands through the insect's proboscis at
the time a human host is bitten; i, free sporozoit from the mosquito's
saliva in the human blood; 2, penetration of the sporozoit into a red
blood-corpuscle, beginning the human cycle again (Liihe).

532 Malaria

vary glands, entering into the epithelial cells and taking
radial positions about the nuclei, where they remain for a
time. Later, they leave the cells with the saliva, and when
the mosquito again bites, enter the warm-blooded host to
infect it, if of the appropriate species.

The whole cycle in the mosquito varies, according to the
external temperature, from ten days to a fortnight. The
mosquito may remain alive for more than one hundred days,
and must bite frequently to satisfy its needs. It remains
infective so long as the sporozoits remain in the saliva, which
is usually as long as the insect is alive. Here it may be
remarked that as it is only the female mosquitoes that bite,
it is only by them that the infection can be spread. It is
an interesting question, not yet solved, whether any of the
sporozoits entering into the mosquito's ovaries can infect
its eggs so that a new generation of mosquitoes may be born
infective. The longer the human infection persists, the
greater the number of gametocytes formed, until sometimes,
especially in aestivo-autumnal malaria, no schizonts are any
longer found, though the blood contains large numbers of
gametocytes. In such cases the gametocytes, especially the
crescents of sestivo-autumnal fever, but sometimes also those
of tertian and quartan fever undergo regressive schizogony in
the patient's blood, and without fertilization suddenly break
up into spores which enter the red blood-corpuscles and
occasion a relapse of the infection that had apparently spent
itself.

THE HUMAN MALARIAL PARASITES.

There are three known forms of human malarial parasites:
Plasmodium malariae, Plasmodium vivax, and Plasmodium
falciparum.

I. Plasmodium Malariae (Laveran,* 1880). — This is the
smallest of the human malarial parasites. Its occurrence is
relatively infrequent, as is that of the quartan fever that
it occasions. The schizogonic period is seventy-two hours
long, and as each is completed, a paroxysm of the disease
occurs.

The parasite, in the red blood-corpuscle, first appears as
a tiny ring, at one side of which there is a chromatin dot.
At this time the organism cannot be differentiated from

* "Acad. de Med.," Nov. 23, Dec. 28, 1880.

The Human Malarial Parasites

533

Plasmodium vivax. At the end of twenty-four hours the
organism seems to extend itself more or less linearly, and
sometimes appears as a long drawn band which crosses
the substance of the unchanged corpuscle. In another
twenty-four hours the breadth of the parasite is two or
three times as great, and it has become pigmented. The
corpuscle itself is still unchanged. In the last twenty-
four hours the parasite enlarges, becomes more or less
quadrilateral, finally rounds up, shows depressions upon
the surface corresponding to the divisions into which it . is
to segment, the pigment gathers at the center, and the
substance undergoes cleavage resulting in the formation of
from six to fourteen, but usually eight, spores. It is to be

Fig. 173. — Parasite of quartan malarial fever: a, b, c, d, Enlarging
intracellular parasites; e, }, g, h, segmentating parasites forming a dis-
tinct rosette from which the spores separate; i, macrogametocyte; ;,
microgametocyte; k, flagellum.

noticed that it is not until a few hours before segmentation
that the parasite becomes as large as the corpuscle, and that
the corpuscle is never enlarged or bleached by the presence
of the parasite. The meroblasts form regular rosettes, or
"daisy-heads," within the corpuscles.

In single infections the parasites are all of the same age
and all mature at the same time, so that in any examination
of the blood they will all appear uniform. It is, however,
sometimes true that the patient may have been infected one
day by one mosquito bite, and again infected the next day
or the third day by a second mosquito bite, so that his blood
contains two crops of the microparasites, arriving at maturity
at different times. This perplexes the clinician through the
variety of parasitic forms in the blood and the abnormal fre-
quency of the paroxysms.

534 Malaria

The gametocytes of the parasite remain for some time in
the red corpuscles without division, but, finally, become free
spherical bodies. Two sizes can be made out, one a little
larger, the macrogametocyte or female, the other, the
microgametocyte or male. Bach has protoplasm, with a tend-
ency to take a blue-gray color and appear uniformly granu-
lar, except that at some part of the periphery of each there is
a circular or semicircular area that is free from granules.
This area is larger in the microgametocyte.

Fig. 174- Fig- J75-

Figs. 174, 175. — Gametocytes of plasmodium malariae: 85, The macro-
gametocyte; 86, the microgametocyte (Kolle and Wassermann).

II. Plasmodium Vivax (Grassland Feletti,* 1890).— This
is the most common of the malarial parasites of man, and
occasions the " benign " tertian fever. It is a large parasite,
the full-grown schizont (meroblast) , ready to form merozoits,
and the gametocytes all exceeding the size of the red blood-
corpuscles. It matures in forty-eight hours, but not with
mathematic precision. In single infections the greater
number of the parasites are of the same age and present the
same appearance, but various shapes and ages may be found
together. In double infections, with paroxysms every day,
parasites of different ages may be found.

The youngest form in which the parasite can be observed
is that of a tiny ring in a red blood-corpuscle. The periphery
of this ring (when the blood is stained with polychrome
methylene-blue) is outlined with blue, at one side there is

* " Centralbl. f. Bakt. u. Parasitenk.," 1890, vn, 396; 1891, x, 449,
481, 517.

PLATE I

o

•

w

o

10

11

12

PLATE II

13

mm

15

-•.

16

17

18

19

o

o

20

21

22

*.*-
>

m

.

25

26

The Human Malarial Parasites

535

a distinct blue dot, and the center appears colorless and like
a vacuole. The dot is usually on the side of the vacuole that
has the thinner protoplasmic outline. The smallest such
rings usually have a diameter equal to about ^ the diameter
of the blood-corpuscle. The tiny ring-form, or, as it might
better be called, the "seal-ring form," continues until the
schizont becomes half the diameter of the blood-corpuscle,
when its protoplasm has begun to increase so rapidly that
the vacuole no longer appears to be so conspicuous. The
organism also becomes irregular in shape and is actively

Fig. 176. — Parasite of tertian malarial fever: a, b, c, d, e, f, g, Grow-
ing pigmented parasite in the red blood-corpuscles; h, spores formed
by segmentation of the parasite — no rosette is formed, but concentric
rings of the cytoplasm divide; i, macrogametocyte; ;, microgametocyte
with flagella.

ameboid, its protoplasm streaming this way and that when
examined in fresh blood. At this time it may be noticed
that the infected blood-corpuscle is increasing in volume,
sometimes becoming twice the normal size, and also becoming
pale in color. It seems also as though the disk shape of the
corpuscle was lost, and it had become swollen into a more
spherical — sometimes irregular — form. The parasite, which
may still show a relic of its original ring- form, now shows plen-
tifully throughout its protoplasm exceedingly fine granules of
yellow-brown pigment. When from thirty-six to forty hours
old, all trace of the " seal-ring " form disappears, the ame-

536

Malaria

boid action becomes less marked, and the parasites (now
three-quarters the size of the enlarged pale and misshapen
corpuscles in which they are contained) appear as irregular,
ragged, protoplasmic bodies filled with fine pigment granules.
In about forty-five hours they completely fill the enlarged
corpuscles, and begin to gather their protoplasm into rounded
formations in which the pigment is no longer distributed,
but occurs in irregular stripes or gathers together into a
rounded clump. In a couple of hours the blood-corpuscle
has disappeared and the rounded parasite, larger than
normal red corpuscles, with a tabulated surface, and with its
pigment granules collected to form one or two rounded

Q&

Fig. 177. Fig. 178.

Figs. 177, 178. — Gametocytes of plasmodium vivax: 87, The micro-
gametocyte; 88, the macrogametocyte (Kolle and Wassermann).

masses, is seen to have reached the stage of the meroblast.
This does not form the rosette or " daisy-head " shown by
the quartan parasite, but might better be compared to a
mulberry, and eventuates in the formation of from fifteen
to twenty-five small, rounded or ovoid, pale, unpigmented
bodies, the merozoits or spores. These become freed from
the pigment and attached to new red corpuscles, in which
they are easily recognized as the " tiny rings " that begin the
schizogonic cycle. The gametocytes of the tertian parasite,
the " free spheres," as they are sometimes called, are large,
rounded or slightly ovoid bodies, with a uniformly dull
bluish-gray or grayish-green protoplasm, in the interior of
which there is always a circular or semicircular area periph-
erally or centrally situated, and colorless. Except in this

The Human Malarial Parasites 537

area the pigment is distributed throughout the parasite.
The larger or macrogametocyte, the female parasite, measures
10 to 14 H in diameter. It has a greenish or grayish-green
or almost colorless protoplasm, containing an oval or bean-
shaped colorless area almost half as large as the organism
itself. Yellowish-brown pigment in short broad rods is spar-
ingly scattered throughout the substance elsewhere.

The microgametocyte or male form is approximately the
size of a red blood-corpuscle — 8 to 9 ft in diameter. It
stains more deeply than its mate and contains more and
coarser pigment granules.

III. Plasmodium Falciparum (Blanchard, 1897). — This
is the parasite of estivo-autumnal or malignant tertian

Fig. 179. — Parasite of estivo-autumnal fever: a, b, c, Ring-like and
cross-like hyaline forms; d, e, pigmented forms; /, g, segmentary forms;
h, i, j, crescents.

malarial fever. It is a very small parasite, whose occur-
rence, even multiple occurrence, in the corpuscles does not
change their size or shape. It does, however, quickly change
the appearance of the corpuscles, which become polychro-
matophilic, and frequently show numerous small dots — the
granulations of Schuffner — in the corpuscular substance.

The first appearance of the schizont is in the form of tiny
rings, which appear to lie upon rather than in the corpuscles,
and are first seen at the edges. The rings are outlined by
extremely fine lines and sometimes seem to be incompletely
closed, so that they are like horseshoes rather than circles.
They increase to several times the original size without losing
the ring shape, and are variously known as "middle-sized
rings " and " large rings." They are with difficulty differ-
entiated from the " tiny rings " of the tertian parasite. As

538 Malaria

the " large ring " stage is reached the parasites begin to
disappear from the peripheral blood to complete their growth
and undergo meroblast formation in the capillaries of the
spleen, the brain, and the bone-marrow. Here the full-
grown parasites — meroblasts — appear as irregular disks,
resembling those of the quartan parasite, but smaller in size.
The pigment is gathered toward the center in a little mass,
and eight to twenty-five merozoits are formed in a morula
or mulberry-like mass similar to those of the tertian para-
site. Two or three parasites to the corpuscle are frequent.
They are actively ameboid, do not mature simultaneously,
and hence there are no regularly occurring paroxysms. The

Fig. 1 80. Fig. 181.

Figs. 1 80, 181. — Gametocytes of plasmodium falciparum: 91, The mi-
crogametocyte; 92, the macrogametocyte (Kolle and Wassermann).

duration of the asexual cycle is from twenty-four to forty-
eight hours.

The gametocytes are striking and characteristic ovoid and
crescentic bodies — crescents — i J times the diameter of a red
blood-corpuscle in length, and about half the diameter of the
corpuscle in breadth. The ends color more intensely with
methylene-blue than the middle portion, and the bacillary
pigment granules are collected toward the centers. The
longer and more slender crescents are usually bent, and the
relic of the corpuscle in which they have formed can often
be seen forming a line connecting the ends on the concave
side. These are the microgametocytes or male elements.
The macrogametocytes are broader, not curved, and some-
times are ovoidal or prolate spheroidal in shape. The pig-

Pathogenesis 539

ment granules are more widely scattered throughout the sub-
stance. The crescents are most numerous after the fever
has lasted for some time or in recurrences of the fever.

The fever in this form of malarial infection may be inter-
mittent with daily — quotidian — paroxysms, or with irregu-
lar paroxysms, or the fever may be remittent. The infec-
tion is sometimes mild, but may be so severe as to be rapidly
fatal. In such cases the number of parasites is enormous,
the cerebral capillaries become filled with them, and coma
quickly comes on and is soon followed by death. Such
cases are described as " congestive chills " or " algid "
cases.

Cultivation of the Parasites. — The parasites have not
been successfully cultivated, though they have been kept
alive for some time in blood, prevented from coagulation, by
Bass.

Animal Inoculation. — The human malarial parasites can-
not be successfully transmitted by experimental inoculation
to any of the lower animals.

Human Inoculation. — The blood of one human being con-
taining schizonts, when experimentally introduced into an-
other human being in doses of i to 1.5 c.c. transmits the dis-
ease. When thus transmitted, an incubation period of
from seven to fourteen days intervenes before the disease,
which is of the same type as that from which the blood was
taken, makes its appearance.

Pathogenesis. — The pathogenic effects wrought by the
malarial parasite are imperfectly understood. The syn-
chrony of the segmentation of the parasite and the occur-
rence of the paroxysms seems to indicate that a toxic sub-
stance saturates and disturbs the economy at that time.
Whether it be an endotoxin liberated by the dividing parasite
is not, however, known.

The anemia that follows infection can be referred to the
destruction of the red blood-corpuscles by the parasites which
feed upon them and transform the hemoglobin into melanin
(?). When great numbers of the parasites are present the
destruction is enormous, and the number of corpuscles and
the quantity of hemoglobin in the blood sink far below the
normal. Leukopenia instead of leukocytosis is the rule, and
while the leukocytes have an appetite for the spores of the
parasites and often phagocyte and destroy them, their activ-

54° Malaria

ity is not sufficiently rapid or universal to check their rapid
increase.

The melanin granules set free during sporulation are also
taken up by the leukocytes and endothelial cells, the latter
becoming deeply pigmented at times.

The spleen enlarges as the disease continues until it forms
the " ague-cake." The enlargement may cause the organ
to weigh 7 to 10 pounds. It appears to result from hyper-
trophy. The tissue is pigmented. The liver and kidneys
are also enlarged and pigmented.

Prophylaxis. — With the knowledge of the role of the
mosquito in the transmission of malaria, its prophylaxis be-
comes a matter of simplicity when certain measures can be
systematically carried out. There are two equally import-
ant factors to be considered — the human being and the mos-
quito. The measures must be directed toward preventing
each from infecting the other.

1 . The Human Beings. — In districts where malarial fever
prevails, the first part of the campaign had perhaps best be
directed toward finding and treating all cases of malarial
fever, so that the parasites in their blood may be destroyed
and the infection of mosquitoes prevented. This is done by
the systematic and general use of quinin.

All cases of malarial fever should be required to sleep in
mosquito-proof houses under nets, and as the mosquitoes are
nocturnal and begin to fly at dusk, the patients should shut
themselves in at that time. By thus killing the parasites in
the blood, and keeping the mosquitoes from the patients in
the meantime, much can be done. But where malarial
fever prevails, the mosquitoes are already largely infected,
hence the healthy population should also learn to respect the
habits of the insects and not expose themselves to their bites,
should screen their houses and their beds, and should take
small prophylactic doses of quinin to prevent the develop-
ment of the parasites when exposure cannot be prevented.

2. The Mosquitoes. — It is not known that the parasites
can pass from one generation of mosquitoes to another,
hence the mosquitoes to be feared are those that are already
infected. By making the houses mosquito-proof most of
the insects can be kept out, while those that get in can be
caught and killed.

By draining the swamps and destroying all the breed-
ing places in and near human habitations, the number

Mosquitoes and Malarial Fever 541

of mosquitoes can be greatly diminished. Fortunately
this is particularly true with reference to the mosquitoes
most concerned — the anopheles — which fly but short dis-
tances. By closing all the domestic cisterns and reservoirs,
cesspools, etc., so that no mosquitoes can get in to breed
or get out to bite, and by draining the pools for half a mile
in all directions from human habitations, the number of

Fig. 182. — Anopheles maculipennis : Adult male at left, female at right

(Howard) .

anopheles mosquitoes can be made almost negligible. If at
the same time no mosquitoes are any longer permitted to
infect themselves by biting infected human beings, the
spread of the disease must be greatly restricted or checked.

MOSQUITOES AND MALARIAL FEVER.

In order that the student may be able to differentiate
with reasonable accuracy such mosquitoes as come under
his observation, use must be made of tabulations, to cor-
rectly use which, however, the student should have some
familiarity with insect structure and the general principles

542

Malaria

of entomology. The mosquitoes, or culicidae, must be rec-
ognized first by their well-known general form, and second
by the presence of scales upon some part of the head, thorax,
abdomen, and wings.

Fig. 183 — Various mosquitoes in attitudes of repose: a, Culex pipiens;
b, Myzorrhynchus pseudopictus ; c, Anopheles maculipennis (Manson).

CLASSIFICATION (Stitt).

There are four subfamilies of CULICIDJS, differentiated according
to the palpi:

I. Palpi as long or longer than the proboscis in the

male.

i . Palpi as long as the proboscis in the fe-
male ; proboscis straight

2. Palpi as long or shorter than the pro-

boscis; proboscis curved

3. Palpi shorter than the proboscis

II. Palpi shorter than the proboscis in the male
and female

Of these the Anophelinae is the one family concerned in the transmis-
sion of malarial fever, so that it is important to be able to differentiate
the genera included in the family.

1. Scales on head only; hairs on thorax and abdo-

men.

1. Scales on wings large and lanceolate.

Palpi only slightly scaled ............. Anopheles.

2. Wing scales small, narrow, and lanceolate.

Only a few scales on palpi .............. Myzomyia.

3. Large inflated wing scales .............. Cycloleppteron.

2. Scales on head and thorax. Scales narrow and

curved. Abdomen with hairs, not scales.
i . Wing scales small and lanceolate ........ Pyretophorus.

3. Scales on head, thorax, and abdomen. Palpi

covered with thick scales.
i. Abdominal scales on ventral surface only.
Thoracic scales like hairs. Palpi rather
heavily scaled ...................... Myzorrhynchus.

Mosquitoes and Malarial Fever 543

2. Abdominal scales narrow, curved or spindle

shaped, in tufts and dorsal patches. . . Nyssorrhynchus.

3. Abdomen almost completely covered with

scales and also having lateral tufts .... Cellia.

4. Abdomen completely scaled ............. Aldrichia.

Species of the genera Anopheles, Myzomyia, and Myzorrhynchus, are
known to transmit malarial parasites. The culicinae include Stegomyia
and Culex, which have some medical interest, as the former transmits
yellow fever; the latter, filarial worms.

I. Posterior cross-vein nearer the base of the

wing than the mid-cross-vein.
i . Proboscis curved in the female ........ Psorophora.

2. Proboscis straight in the female:

A. Palpi with three segments in the

female.

a. Third segment somewhat

longer than the first two . . Culex.

b. The three segments are equal

in length ................ Stegomyia.

B. Palpi with four segments in the

female.

a. Palpi shorter than the third

of the proboscis. Spotted

wings ................... Theobaldia.

b. Palpi longer than the third of

the proboscis. Irregular

scales on the wings ....... Mansonia.

C. Palpi with fine segments in the

female ............. .......... Taniorrhynchus.

II. Posterior cross- vein in line with the mid-cross-

vein ................................. Joblotina.

III. Posterior cross-vein further from the base of

the wing than the mid-cross-vein ...... Mucidus.

The mosquitoes used for study and for classification
should be mounted dry in the usual way well known to all
entomologists. Fine entomologic pins (oo-ooo) should be
employed for the purpose. The insects should be caught in
a wide-mouth bottle containing some fragments of cyanid of
potassium, covered with a layer of saw-dust, over which a
thin layer of plaster of Paris is allowed to solidify. The
insects die in a moment or two, can be emptied upon a table,
and the pin carefully thrust through the central part of the
thorax. As soon as the insect is impaled, the pin should be
passed through an opening in a card or between the blades of
a forceps until the insect occupies a position at the junction
of the middle and upper third. The insect should not be
touched with the fingers, as the scales will be brushed off and
the limbs broken. Mounted insects must be handled with

544

Malaria

entomologic forceps, touching the pins only. Every in-
sect thus mounted should have placed upon the pin, at the
junction of the middle and lower thirds, a small bit of card or

Fig. 184. — Method of withdrawing the digestive tube of the mosquito
for study (Blanchard).

paper, telling where and when and under what circumstances
it was taken.

The dissection of fresh mosquitoes for determining whether
or not they are infected with malarial organisms must be
made with the aid of needles mounted in handles. The
position of the stomach, intestines, and the salivary glands,

Fig. 185.-

-Method of withdrawing the salivary glands of the mosquito
for study (Blanchard).

and the mode of pulling the insect apart to show them can be
learned from the diagram. The organs thus withdrawn and
separated from the unnecessary tissue can be fixed to a slide
with Meyer's glycerin-albumin or other albuminous matter,
and then stained like a blood-smear, but should be cleared

Mosquitoes and Malarial Fever 545

after staining and washing, and mounted in Canada balsam
under a cover-glass.

A more certain and more elegant manner of showing the
parasites in infected mosquitoes is by pulling off the legs and
wings and then embedding the insect in paraffin and cutting
serial longitudinal vertical sections.

To infect mosquitoes and study the development of the
malarial parasites in their bodies, the insects should be
bred from the aquatic larva in the laboratory, to make sure
that they do not already harbor parasites. The mosquitoes
are allowed to enter a small cage made with mosquito netting,
and are taken to the bedside of the malarial patient, against
whose skin the cage is placed until the insects have bitten
and distended themselves with blood, when they are taken
back to the laboratory and kept as many days as may be
desired, then killed and sectioned. In this way, remember-
ing that the entire mosquito cycle of development takes
about a fortnight, any stage of the cycle may be observed.

35

CHAPTER XIX.

RELAPSING FEVER.

SPIROCH^TA

IN 1868 Obermeier* first observed the presence of actively
motile spiral organisms in the blood of a patient suffering
from relapsing fever. Having made the observation, he
continued to study the organism until 1873, when he made
his first publication. From 1873 until 1890 it was supposed
that spirochaeta rarely played any pathogenic role. Miller
had, indeed, called attention to the constant presence of
Spirochaeta dentinum in the human mouth, but it had not
been connected with any morbid condition. In 1890
Sacharofff discovered a spirillary infection of geese in the
Caucasus, caused by an organism much resembling Spiro-
chaeta obermeieri and called Spirochaeta anserinum. In
1903 Marchoux and Salimbenif found a third disease,
fatal to chickens, caused by Spirochaeta gallinarum, and
found that the spread of the disease was determined by
the bites of a tick, Argas miniatus. In 1902 Theiler,§
in the Transvaal, observed a spiral organism in a cattle
plague. This has been named after him by Laveran,
Spirochaeta theileri. It was found to be disseminated by
the bites of certain ticks — Rhipicephalus decolor atus.
Later, what was probably the same organism, was found in
the blood of sheep and horses. In 1905 Nicolle and Comte||
found a spiral organism infecting certain bats. By this
time, therefore, it became evident that spirochaetal infections
were fairly well disseminated among the lower animals and
that the spirochaeta were of different species with different
hosts and intermediate hosts.

*"Centralbl. f. d. med. Wissenschaft," 1873.
t "Ann. de 1'Inst. Pasteur," 1891, xvi, No. 9, p. 564.
J Ibid, 1903, xvii, p. 569.

§ "Jour. Comp. Path, and Therap.," 1903, XLVII, p. 55.
|| "Compt.-rendu de la Soc. de Biol. de Paris," July 22, 1905, Lix,
p. 200.

546

Relapsing Fever 547

In 1904 Ross and Milne* and Button and Toddf studied
a peculiar African fever which they were able to refer to a
spirochaeta for which NovyJ has proposed the name Spiro-
chaeta duttoni in memory of Dutton, who lost his life while
studying it. It was found that this organism, like most of
the others described, was transmitted by a tick, Ornitho-
doros moubata.

With the work of Schaudinn and his associate, Hoffmann, §
the spirochaeta came to be regarded as protozoan parasites
because of the presence of an undulating membrane; the
refusal of most of the organisms to grow upon artificial media,

Fig. 1 86. — Spirochaeta obermeieri from human blood (Kolle and Was-

sermann).

the role of an intermediate host (ticks, etc.) in transmitting
them, and the longitudinal mode of division.

Fevers characterized by relapses and by the presence of
spirochaeta in the blood have been found in northern and
northeastern Europe (true relapsing fever with Spirochaeta
obermeieri), in various parts of Africa (African relapsing
fever with Spirochaeta duttoni), in Bombay, and in America.
The question, therefore, arises whether these similar diseases
are slight modifications of the same thing caused by the same

* ''British Med. Jour.," Nov. 26, 1904, p. 1453.

t" Memoir xvu, Liverpool School of Tropical Medicine," "Brit.
Med. Jour.," Nov. n, 1905, p. 1259.

J "Jour. Infectious Diseases," 1906, m, p. 295.

§" Deutsche med. Wochenschrift," Oct., 1905, xxxi, p. 1665;
"Arbeiten aus dem kaiserlichen Gesundheitsamte," 1904, xx, pp. 387-
439-

548 Relapsing Fever

parasite, or whether they are different diseases caused
by slightly different parasites. Fulleborn, Mayer, and
Martin* consider them to be four different organisms:

Spirochaeta obermeieri, of the European relapsing fever.

Spirochaeta duttoni, of the African relapsing fever.

Spirochaeta novyi, of American relapsing fever.

Spirochaeta carteri, of Bombay relapsing fever.

As the differences between these organisms are minute, it
scarcely seems well to devote space to the consideration of

Fig. 187. — Spirochaeta obermeieri (Novy). Rat blood No. 32 la.

X 1500.

each, but better to select the oldest and the best known—
Spirochaeta obermeieri — as the type, describe it, and point out
such variations as are shown by its close relations.

General Characteristics. — An elongate, flexible, flagellated, non-
sporogenous, actively motile spiral organism, pathogenic for man and
monkeys, not susceptible of cultivation in artificial media, stained by
ordinary methods, but not by Gram's method.

Morphology. — The Spirochaeta obermeieri is extremely

slender, flexible, spirally coiled, like a corkscrew, and pointed

at the ends. It measures approximately i ^ in breadth and

10, 20, or even 40 ^ in length. The number of spiral coils

* "Med. Klinik," No. 17, April, 1907.

Morphology 549

varies from 6 to 20; the diameter of the coils varies so greatly
that scarcely any two are uniform. Wladimiroff* doubts the
existence of a flagellum, but flagella-like appendages are
usually to be seen at one or both ends of the organisms.
An undulating membrane attached nearly the entire length
of the organism, very narrow, and inconspicuous, forms the
chief means of locomotion. The organism is actively motile,
and darts about in fresh blood with a double movement, con-
sisting of rotation about the long axis and serpentine flexions.
No structure can be made out by our present methods of

Fig. 1 88. — Spirochaeta duttoni (Novy;. Tick fever, No. 520. Rat
blood. X 1500.

staining and examining the spirochaeta. No spores are
found. Multiplication is thought to take place by longitudi-
nal division, though some believe the division to be trans-
verse.

The Spirochaeta duttoni is said by Koch,f in his interest-
ing studies of "African Relapsing Fever," to resemble the
Spirochaeta obermeieri in all particulars.

The Spirochaeta novyi with which Novy and Knapp t exper-
imented, and which they believed to be identical with

"Kolle and Wassermann's Handbuch der pathogene Mikroorgan-
ismen," 1903, in, p. 82.

f ''Berliner klin. Wochenschrift," Feb. 12, 1906, xxxiv, No. 7, p. 185.
J "Jour. Infectious Diseases," 1906, in, p. 291.

550 Relapsing Fever

Spirochaeta obermeieri, measured 0.25 to 0.3 f* in breadth by
7 to 19 ^ in length. The number of coils varies from three
to six. The shorter forms are pointed, with a long flagel-
lum at one end and a short one at the other.

Staining. — The spirochaeta can be stained with ordinary
anilin dye solutions, by the Romanowsky and Giemsa
methods, and by the silver methods (see Treponema palli-
dum). It does not stain by Gram's method.

Cultivation. — The organism will not grow upon any
known culture-medium. Following the suggestion of
Levaditi, Novy and Knapp* cultivated Spirochaeta ober-
meieri in collodion sacs in the abdominal cavity of rats,
and succeeded in maintaining it alive in this way through
twenty consecutive passages during sixty-eight days. They
were able to do this in rat serum from which all corpuscles
had been removed by centrifugation, so that it is proved
that no intercellular developmental stage of the organism
takes place. Organisms thus cultivated are attenuated in
virulence.

Norris, Pappenheimer, and Flournoyf believe that they
succeeded in securing multiplication of the spirochaeta by
placing several. drops of blood containing them in 3 to 5 c.c. of
citrated rat or human blood. A third generation always
failed.

Mode of Infection. — The means by which Spirochaeta
obermeieri is transmitted from individual to individual is not
definitely known. TictinJ seems to have been the first to
believe that the transmission of the disease was accomplished
through the intermediation of some blood-sucking insect.
He investigated lice, fleas, and bed-bugs, in the latter of
which he was able to find the organisms, and through blood
obtained from which he was able to transmit the disease to
an ape. He was not able to infect apes by permitting infected
bed-bugs to bite them. Breinl and Kinghorn and Todd§
made a careful study of the subject, but, like Tictin and
their other predecessors, were unable to infect monkeys by
permitting infected bed-bugs to bite them.

This leaves the transmission of the micro-organism un-
accounted for. When we come to consider Spirochaeta

* "Jour. Amer. Med. Assoc.," Dec. 29, 1906, XLVII, p. 2152.
t "Journal of Infectious Diseases," 1906, in, 266.
J "Centralbl. f. Bakt. u. Parasitenk.," i Abt., xv, 1894, P- 840.
§ Ibid., Oct., 1906, xui, Heft 6, p. 537.

Mode of Infection 551

duttoni, however, we find our knowledge much further
advanced. On Nov. 26, 1904, Button and Todd announced
that they had discovered a spirillum to be the specific
agent in the causation of tick fever in the Congo, and on
the same date Ross and Milne* published the same fact.
Button and Todd subsequently withdrew their claim to
priority of the discovery. On Feb. 4, 1905, Ross published
in the "British Medical Journal" the following cablegram
from Button and Todd, then working on the Congo: "Spirilla
cause human 'tick fever; naturally infected ornithodoros
infect monkey." It was not until Nov. n, 1905, that the
paper upon the subject was read and published in the same

Fig. 189. — Ornithodorus moubata. Tick that transmits African
relapsing fever: a, Viewed from above; b, viewed from below (Murray
from Doflein).

journal by Button and Todd, and the etiology of the disease
made clear. These observers found that the horse- tick,
Ornithodoros moubata (Murray) is the intermediate host of
the spirilla or spirochaeta causing the disease, and that when
these ticks were permitted to bite infected human beings,
and then subsequently transferred to monkeys, the latter
sickened with the typical infection.

The matter received confirmation and addition through
the studies of Koch,f who studied the ticks, observed the
distribution of the micro-organisms in their bodies, and
found that they collected in large numbers in the ovaries,

* "British Medical Journal," Nov. 26, 1904.
t "Berliner klin. Wochenschrift," Feb. 12, 1906.

552 Relapsing Fever

so that the eggs were commonly infected and the embryo
hexapod ticks hatched from them were infective. Thus,
in regard to Spirochaeta duttoni we are able to say quite
definitely that the tick is the usual if not the only means of
dissemination.

Pathogenesis. — The spirochaeta of relapsing fever are
pathogenic for man and monkeys, some of them for smaller
animals. Novy and Knapp* found their organism and Spiro-
chseta duttoni to be infectious for mice and rats, and attribute
the failure of others to discover this to their failure to exam-
ine the blood during the first and second days. Fulleborn
and Meyer and Martin f were able successfully to transmit
the spirochaeta of Russian relapsing fever to mice after first
passing it through apes. Rabbits and guinea-pigs seem
to be refractory; white mice susceptible. Man, monkeys,
and mice suffer from infection characterized by relapses,
and in them the disease may be fatal. Rats never die and
rarely have relapses.

The micro-organisms are free parasites of the blood in
which they swim with a varying rapidity, according to the
stage of the disease. They are present during the febrile
paroxysms only, disappearing completely as soon as the
crisis is reached.

The course of relapsing fever in man is peculiar and char-
acteristic. After a short incubation period the invasion
comes on with chill, fever, headache, pain in the back,
nausea and vomiting, and sometimes convulsions. The tem-
perature rises rapidly and there are frequent sweats. The
pulse is rapid. By the second day the temperature may be
104° to 105° F. and the pulse 1 10 to 130. There is enlargement
of the spleen. Icteroid discoloration of the conjunctiva may
be observed. The fever persists with severity and the
patient appears very ill for five or six days, when a crisis occurs,
and the temperature returns to normal; there is profuse
sweating and sometimes marked diarrhea, and the patient at
once begins to improve. So rapid is the convalescence that
in a few days he may be up and may desire to go out. The
disease is, however, not at an end, for on or about the four-
teenth day the relapse characteristic of the affection makes
its appearance as an exact repetition of what has gone be-
fore. This is followed by another apyretic interval, and
then by another relapse, and so on. The patient usually re-
* Loc. cit. t Loc. cit.

Immunity 553

covers, the mortality being about 4 per cent. The fatal
cases are usually old or already infirm patients. The Indian,
African, and American varieties present variations of no great
importance. The European fever usually ends after the sec-
ond or third relapse, the African not until after a greater
number.

The spirochaeta are present in the blood in great numbers
during the febrile stages, but entirely disappear during the
intervals:

Lesions. — There are no lesions characteristic of relapsing
fever.

Bacteriologic Diagnosis. — This should be quite easily
made by an examination of either the fresh or stained
blood, provided the blood be secured during a febrile parox-
ysm. The readiness with which the organisms take the stain
leaves little to be desired.

Novy and Knapp have found that the serum of recovered
cases can be used to assist in making diagnosis because of its
agglutinating, germicidal, and immunizing powers.

Immunity. — The phenomena of immunity are vivid and
important. At the moment of decline of the fever a power-
ful bacteriolytic substance appears in the blood and dis-
solves the organisms. At the same time an immunizing
substance appears. The two do not appear to be the same.

The immunizing body affords future protection to the
individual for an indefinite length of time. It can be
increased by rapidly injecting the animal with blood con-
taining spirochaeta. Serum containing the immunizing body
imparts passive immunity to other animals into which it is
injected, and, according to Novy and Knapp, establishes a
solid basis for the prevention and cure of relapsing fever in
man.

CHAPTER XX.

SLEEPING SICKNESS.

TRYPANOSOMA GAMBIENSE (BUTTON, 1902).

SLEEPING sickness, African lethargy, Maladie du sommeil,
Schlafkrankheit, or human trypanosomiasis is a specific, in-
fectious, endemic disease of equatorial Africa characterized
by fever, lassitude, weakness, wasting, somnolence, coma,
and death. The first mention of the disease seems to have
been made by Winterbottom.*

Sir Patrick Mansonf says that "For upward of a century
students of tropical pathology have puzzled over a peculiar
striking African disease, somewhat inaccurately described by
its popular name, the sleeping sickness. Its weirdness and
dreadful fatality have gained for it a place not in medical
literature only, but also in general literature. The mystery
of its origin, its slow but sure advance, the prolonged life in
death that so often characterizes its terminal phases, and its
inevitable issue, have appealed to the imagination of the
novelist, who more than once has brought it on his mimic
stage, draping it, perhaps, as the fitting nemesis of evil-doing.
The leading features of the strange sickness are such as might
be produced by a chronic meningo-encephalitis. Slow irreg-
ular febrile disturbance, headache, lassitude, deepening into
profound physical and mental lethargy, muscular tremor,
spasm, paresis, sopor, ultimately wasting, bed-sores, and
death by epileptiform seizure, or by exhaustion, or by some
inter current infection.

" In every case the lymphatic glands, especially the cer-
vical, are enlarged, though it be but slightly. In many cases
pruritus is marked. In all, lethargy is the dominating feature.

"In some respects this disease, which runs its course in
from three months to three years from the oncoming of the

* "An Account of Native Africans in the Neighborhood of Sierre
Leone," 1803.

t "The Lane Lectures for 1905," Chicago, 1905.

554

Specific Organism 555

decided symptoms, resembles the general paralysis of the
insane. It differs from this, however, in the absence, as a
rule, of the peculiar psychic phenomenon of that disease.
There are exceptions, but generally, though the mental fac-
ulties in sleeping sickness are dull and slow acting, the
patient has no mania, no delusions, no optimism. So far
is the last from being the case, that he is painfully aware
of his condition and of the miserable fate that is in store for
him; and he looks as if he knew it."

Specific Organism. — The discovery of the specific organ-
isms was foreshadowed by Nepveu,* who recorded the exist-
ence of trypanosomes in the blood of several patients coming
from Algeria, by Barron, f and by Brault.f In 1901 Forde
received under his care at the hospital in Bathurst (Gambia),

i

>K

t

<*:

Fig. 190. — Trypanosoma gambiense (Todd).

a European, the captain of a steamer on the River Gambia, who
had navigated the river for six years, and who had suffered sev-
eral attacks of fever that were looked upon as malarial. The
examination of his blood revealed the presence not of malarial
parasites, but of small worm-like bodies, concerning the nature
of which Forde was undecided. § Later, Button, in conjunc-
tion with Forde, examined this patient, whose condition had
become more serious, and recognized that these worm-like
bodies seen by Forde were trypanosomes. Of these parasites

* "Memoirs, Soc. de Biol. de Paris," 1891, p. 49.
t "Transactions of the Liverpool Medical Institute," Dec. 6, 1894.
t "Janus," July to August, 1898, p. 41.

§ "Trypanosomes and Trypanosomiasis," Laveran and Mesnil,
1907.

556

Sleeping Sickness

he has written an excellent description, calling them Try-
panosoma gambiense.* The patient thus studied by Forde

Fig. 191. — Various species of trypanosoma: i, Trypanosoma lewisi
of the rat; 2, Trypanosoma lewisi, multiplication rosette; 3, Trypano-
soma lewisi, small form resulting from the disintegration of a rosette;
4, Trypanosoma brucei of nagana; 5, Trypanosoma equinum of caderas;
6, Trypanosoma gambiense of sleeping sickness; 7, Trypanosoma gam-
biense, undergoing division; 8, Trypanosoma theileri, a harmless trypano-
some of cattle; 9, Trypanosoma transvaliense, a variation of T. theileri;
10, Trypanosoma amum, a bird trypanosome; 1 1, Trypanosoma damonice
of a tortoise; 12, Trypanosoma solece of the flat fish; 13, Trypanosoma
granulosum of the eel; 14, Trypanosoma rajas, of the skate; 15, Trypano-
soma rotatorium of frogs; 16, Cryptobia borreli of the red-eye (a fish).
(From Laveran and Mesnil.)

* See Forde, "Jour. Trop. Med.," Sept. i, 1902; Button, Ibid., Dec.
i, 1902; Button, "Thompson Yates' Laboratory Reports," 1902, v, 4,
part n, p. 455.

Specific Organism 557

and Dutton died in England January i, 1903. In 1903
Button and Todd* examined 1000 persons in Gambia and
found similar trypanosomes in the bloods of 6 natives and
i quadroon. In the same year Mansonf discovered 2 cases
of trypanosomiasis in Europeans that had become infected
upon the Congo. Brumptj also observed T. gambiense at
Bounba at the junction of the Ruby and the Congo, and
Baker observed 3 cases at Entebbe in Uganda. §

During all this time no connection was suspected between
these micro-organisms and African lethargy, and much inter-
est was being taken in a coccus — the hypnococcus — that
was being studied by Castellani in Uganda. As Castellani
was prosecuting the investigation of this organism, he chanced
to examine the cerebrospinal fluid of several negroes in
Uganda who were suffering from sleeping-sickness, and in it
found trypanosomes. Even then, though Castellani || real-
ized that these organisms were connected with sleeping-sick-
ness, he did not identify them in his mind with the Trypano-
soma gambiense discovered in the blood by Forde and Dut-
ton, and described the newly discovered organism as Try-
panosoma ugandense. Kruse,** thinking to honor the dis-
coverer, called it Trypanosoma castellani. Bruce and
Nabarroft found the new trypanosome in each of 38 cases of
sleeping sickness in the cerebrospinal fluid, and 12 out of 13
times in the blood. These observers also found that 23 out
of 28 natives from parts of Uganda where sleeping sickness
is endemic had trypanosomes in their blood, while in 117
natives from uninfected areas the blood examination was neg-
ative in every case. They also declared that, contrary to
what had been stated, there were no appreciable morphologic
differences between Trypanosoma gambiense and Trypano-
soma ugandense. Dutton, Todd, and Christy tJ arrived at
the same conclusion. The matter was finally settled by

* "First Report of the Trypanosomiasis Expedition to Senegambia,"
1902, Liverpool, 1903.

t"Jour. Trop. Med.," Nov. i, 1902, and March 16, 1903; "Brit.
Med. Jour.," May 30, 1903.

I "Acad. de Med.," March 17, 1903.

§ "Brit. Med. Jour.," May 30, 1903.

|| Ibid., May 23, 1903; June 20, 1903.

**"Gesell. f. natur. Heilkunde," 1903.

ft "Brit. Med. Jour.," Nov. 21, 1903.

Jt Ibid., Jan. 23, 1904, also " Thompson- Yates and Johnson Lab.
Reports," v, 6, part i, 1905, pp. 1-45.

558 Sleeping Sickness

Thomas and Linton* and Laveran,| who, by means of
animal experiments, determined not only the complete
identity of the organisms, but their uniform virulence.

Morphology. — Trypanosoma gambiense is a long, slender,
spindle-shaped, flagellate micro-organism that measures 17
to 28 ^ in length and 1.4 to 2 ^ in breadth. From the ante-
rior end (that which moves forward as the organism swims)
a whip-like flagellum projects about half the length of the
organism. The terminal third of the flagellum is free in
most cases. The proximal two-thirds are connected with a
band of the body substance, which is continued like a ruffle
along one side of the organism to within a short distance of
its blunt posterior end, where the flagellum abruptly ends
at the blepharoplast. This thin ruffle is known as the un-
dulating membrane. By means of the flagellum and the
undulating membrane the organism swims rapidly with a
wriggling and rotary movement that give it the name Try-
panosome, which means " boring body."

The protoplasm is granular and often contains chromatin
dots that are remarkable for their size and number. There
is a distinct nucleus of ovoid form. There is also a cen-
trosome or blepharoplast, which appears as a distinct deeply
staining clot near the posterior blunt end and from which the
flagellum appears to arise. Near this a vacuole is sometimes
situated.

Staining. — The organisms are best observed when stained
with one of the polychrome methylene-blue combinations —
Irishman's, Wright's, Jenner's, Romanowsky's, Marino's.
To stain them a spread of the blood or cerebrospinal fluid is
made and treated precisely as though staining the blood for
the differential leukocyte count or for the malarial parasite.

Cultivation. — The cultivation of Trypanosoma gam-
biense has not yet been achieved. This seems singular, as
Trypanosoma lewisi of the rat and Trypanosoma brucei of
"nagana" or "tsetse-fly" disease of Africa have been culti-
vated by Novy and McNealf in mixtures composed of
ordinary culture agar-agar and defibrinated rabbit-blood,
combined as necessary, 1:1, 2:1, 1:2, or 2 13, etc. The

* "Lancet," May 14, 1904, pp. 1337-1340.

t "Compt.-rendu de 1'Acad. des Sciences," v, 142, 1906, p. 1065.

J "Contributions to Medical Research dedicated to Victor Clarence
Vaughan," Ann Arbor, Michigan, 1903, p. 549; "Journal of Infectious
Diseases," 1904, i, p. i.

Transmission 559

actual culture was made chiefly in the water of condensation
collected at the bottom of obliquely congealed media.

Laveran and Mesnil found that when blood containing
Trypanosoma gambiense was mixed with salt solution or
horse-serum, the trypanosomes remain alive for five or six
days at the temperature of the laboratory. They live much
longer in tubes of rabbit's blood and agar, sometimes as long
as nineteen days, and during this time many dividing forms
but no rosettes were observed. But subcultures failed, and
eventually the original culture died out.

Reproduction. — Multiplication takes place by binary
division, the line of cleavage being longitudinal. The cen-
trosome and nucleus divide, then the flagellum divides longi-
tudinally, and finally the protoplasm divides.

In addition to this simple longitudinal fusion, the trypan-
osomes seem to possess a sexual mode of reproduction. When
the well-stained organisms are carefully studied, it is possible
to divide them into three groups — those that are peculiarly
slender, those that are peculiarly broad, and those of ordinary
breadth. The fact that conjugation takes place between the
first two has led to the opinion that they represent the male
and female gametocytes respectively, while the others are
asexual. All forms multiply by fission, and conjugation
between the gametes is observed to take place only in the
body of the invertebrate host. It has not yet been accurately
followed in the case of Trypanosoma gambiense, but there is
no reason to think that the organism differs in its method of
reproduction from Trypanosoma lewisi. Prowazek found
that when rat blood containing the latter organism was
taken into the stomach of the rat louse, Hematopinus spinu-
losus, the male trypanosome enters the female near the
micronucleus and the various parts of the two individuals
become fused. A non-flagellate ookinete results, and, after
passing through a spindle-shaped gregarine-like stage, can
develop into an immature trypanosome-like form in the cells
of the intestinal epithelium, after which the parasite is
thought to enter the general body cavity, and, migrating to
the pharynx, enter the proboscis, through which it is trans-
mitted to a fresh host.

Transmission. — It is well known that the disease does
not spread from person to person. In the days when African
negroes were imported into America as slaves the disease
often reached our shores, and though freshly arrived negroes

560 Sleeping Sickness

and those in the country less than a year frequently died of it,
there was no spread of the affection to those that were ac-
climated. The Europeans that carried the disease from
Africa to England and were the first in whose bloods the
trypanosomes were found, did not spread it among their fel-
low countrymen. A case from the Congo that died in a
hospital in Philadelphia and came to autopsy at my hands,
did not spread the disease in this city.

Yet the disease is infectious, and the transfer of a small
quantity of the parasite-containing blood to appropriate
experiment animals perfectly reproduces it.

The present knowledge of the mode of transmission came
about through the knowledge of other trypanosome infections
that had already been carefully studied and understood.
In speaking of nagana or tsetse-fly disease Livingstone, as
early as 1857, recognized that the flies had to do with it.
For years, however, the supposition was that the fly was
poisonous and that its venom was responsible for the disease.
In 1875 Megnin stated that the tsetse -fly carries a virus, and
does not inoculate a poison of its own. In 1879 Drysdale sug-
gested that the fly might be an intermediate host of some
blood parasite, or the means of conveying some infectious
poison. In 1884 Railliet and Nocard, who suspected the
same thing, proved that inoculations with the proboscis of
the tsetse-flies were harmless. The exact connection be-
tween the flies and the disease was worked out by Bruce,*
who found, first, that flies fed on infected animals, kept in
captivity for several days, and afterward placed upon two
dogs, did not infect; second, that flies fed on a sick dog,
and immediately afterward on a healthy dog, conveyed the
disease to the latter. The flies were infectious for twelve,
twenty-four, and even forty-eight hours after having fed
on the infected animal. It was, therefore, shown that the
flies could and did infect, not through something of which they
were constantly possessed, but through something taken from
the one animal and put into the other ; this, of course, proved
to be the trypanosome. Further, it was shown that where
there were no tsetse -flies, there never was nagana.

So soon as African lethargy was shown to be a form of
trypanosomiasis, the question arose, Was it spread by tsetse-

* "Preliminary Report on the Tsetse-fly Disease or Nagana in
Zululand, Ubombo, Zululand," Dec., 1895; "Further Report," etc.,
Ubombo, May 29, 1896; London, 1897.

Transmission

561

flies ? Sambon* and Brumpt f both suggested it, but it was
soon discovered that the geographic distribution of the tsetse-
fly, Glossina morsitans, that distributes nagana, does not co-
incide with the geographic distribution of sleeping sickness.
There are, however, different kinds of tsetse-flies, and Bruce
and NabarroJ first showed that it was not Glossina morsi-
tans, but a different tsetse-fly, Glossina palpalis, that is the
most important, if not the only source of the spread of human
trypanosomiasis. They submitted a black-faced monkey
(Cercopithicus) to the bites of numerous tsetse-flies caught
in Entebbe, Uganda, and found trypanosomes in its blood.

Fig. 192. — Glossina pal-
palis. A perfect insect just
escaped from the pupa. B,
pupa; a, b, valves; c, body
of the pupa (Brumpt) .

Fig. 193. — Glossina palpalis before
and after feeding (Brumpt).

Bruce, Nabarro, and Greig§ allowed Glossina palpalis to suck
the blood of negroes affected with sleeping sickness and
afterward to bite five monkeys (Cercopithicus). At the end
of about two months trypanosomes appeared in the blood
of these monkeys. They also made maps showing the geo-
graphic distribution of African lethargy and of Glossina
palpalis, which were found perfectly to correspond.

It is, of course, not impossible that other flies, especially
other species of tsetse-flies, may act as distributing hosts of

c "Jour. Trop. Med.," July i, 1903.
t "C. R. Soc. de Biol.," Jan. 27, 1903.

t ''Reports of the Sleeping Sickness Commission of the Royal Society,"
1903, i, ii, ii.

§ Ibid., 1903, No. 4, vin, 3.
36

562 Sleeping Sickness

the trypanosomes, but there is no doubt about the chief
agent being Glossina palpalis. With increased entomologic
and geographic information it has been found that there
are certain districts where these flies abound though the dis-
ease is unknown, but that only shows that in those districts
the flies are not infected. Tsetse-flies are not, as was for-
merly supposed, peculiar to Africa, but have been found in
Arabia, where African lethargy could no doubt spread should
the flies become infected through imported cases of the dis-
ease. The inability of the disease to spread in England and
America depends upon the absence of tsetse-flies from those
countries.

According to recent experiments of Bruce, Harrison, Hamer-
ton, Batement, and Mackie, and especially of Kleine, only a
small proportion of the tsetse-flies are capable of spreading
the infection, because the transmission is not merely the
mechanical transplantation of the trypanosomes, but the
implantation of the parasites after they have completed a
certain developmental cycle in the body of the fly. Thus,
the insect does not become infective until about eighteen
hours after infecting itself, and remains infective seventy-
five days (Brumpt). Of course, to be infected, one must be
bitten by a fly itself infected and in the infective stage.

It is possible for the disease tb be transmitted from human
being to human being through such personal contacts as may
afford opportunity for interchange of blood. Thus, Koch
observed that in certain parts of Africa where there were no
tsetse-flies the wives of men that had become infected in
tsetse-fly countries sometimes developed the disease, prob-
ably through sexual intercourse, a probable explanation when
one remembers that it is solely or chiefly by such means that
a trypanosome disease of horses — Dourine or Maladie du
coit, caused by Trypanosoma equiperdum — is transmitted.

Transmission to Lower Animals. — Try panosoma gambiense
is infectious for monkeys as well as for human beings. In the
monkeys a disease indistinguishable from the sleeping sickness
is brought about. It is also infective for dogs, cats, guinea-
pigs, rabbits, rats, mice, marmots, hedgehogs, goats, sheep,
cattle, horses, and asses. The lower animals are not, how-
ever, so far as is known, subject to natural infection.

Pathogenesis. — The first effect of human trypanosomiasis
seems to be fever of an irregular and atypical type, occurring
in irregular paroxysms. It was in such cases that Forde and

Prophylaxis 563

Dutton first found the parasites in the blood. As the para-
sites increase in number the somnolence begins to show itself.
The lymph-nodes enlarge at this time and become easily
palpable, and their enlargement is looked upon as one of the
most ready means for confirming the diagnosis in suspicious
cases.

In the bodies of those that die there are few changes vis-
ible to the naked eye, but the microscope re veals that through-
out the body and especially in the nervous system there are
perivascular collections of lymphocytes and many trypano-
somes.

Prophylaxis. — This must be partly based upon measures
taken to prevent the infection of men by the flies, and partly
upon those preventing the infection of the flies by the men.
Its success must depend upon the probability that there is
no other host — wild animals — in which the parasites are kept
alive where there are no men.

The tsetse-fly — Glossina palpalis — is a fairly large brown-
ish insect, easily recognized when once pointed out. It makes
a loud humming sound and thus attracts attention to itself.
In the larval and pupal stages it lives in the soft mud along
the banks of the streams and rivers, and its natural prey is
thought to be the crocodile, though it readily turns to other
animals and to human beings. In the adult state it still
remains more or less confined to the streams, though it has
been found to fly as far as a mile.

To prevent the infection of men by the flies is extremely
difficult where naked or half -naked savages are to be dealt
with. For Europeans, the customary dress, the avoidance of
exposure in bathing, the use of mosquito guards, etc., are to
be recommended, as well as the erection of habitations and the
building of roads, etc., as far as possible from the fly districts.
The destruction of the grass and reeds along the river banks,
the use of drainage, and the introduction of chickens, to pick
up the larvae and pupae, have been recommended.

To prevent infection of the flies is impossible where, as in
some sections of Africa, 50 per cent, of the population of
some of the villages already harbor the parasites. A means
to this end is, however, being tried, and the entire population
of a district has been removed from the fly country and the
land abandoned, in the hope that when, after the lapse of
some time, only healthy persons are permitted to return, the
absence of infected flies in the country, and the absence of

$64 Sleeping Sickness

infective organisms from the blood of the healthy persons
newly entering it, may remove most of the danger.

The importance of undertaking radical measures for the
suppression of the disease may be imagined when it is under-
stood that in the last few years no less than a half -million of
the natives of the infected districts have died of sleeping
sickness.

AMERICAN TRYPANOSOMIASIS.
TRYPANOSOMA CRUZI (CHAGAS).

Human trypanosomiasis in America is extremely rare and
does not assume the form of sleeping sickness. The occur-
rences, thus far reported, have been in Brazil, where a disease

Fig. 194. — Conorhinus megistus (female), the insect host and distributing
agent of Trypanosoma cruzi (Chagas).

of childhood, characterized by fever, anemia, edema, enlarge-
ment of the lymphatic nodes, liver, and spleen, and ending in
convulsions and death, goes by the name of "Apilacao."
In the peripheral blood of i case of this affection Chagas*
found a trypanosome which he describes as Trypanosoma

* "Archives fur Schiffs u. Trop. Hyg.," 1909, H. 4; "Abstract Cen-
tralblatt. f. Bakt. etc. Ref.," 1909, xuv, 639; also "Ref. Bull. del'Inst.
Pasteur," 1910, vin, 373.

American Trypanosomiasis 565

cruzi. It proved upon experiment to be infective for Brazil-
ian monkeys, dogs, guinea-pigs, rabbits, and other small
animals.

It has been successfully cultivated upon the special blood
agar-agar of Novy and McNeal.

The developmental cycle of this trypanosome as worked
out by Chagas is peculiar, and not only takes place by the
usual asexual fission, but also is apparently in some way
associated with an intracorpuscular infection, in which fig-
ures resembling the merozoits of the malarial plasmodium
are formed. These subsequently grow into trypanosomes.
In the lungs, also, special forms are said to develop, con-
taining light bodies resembling the merozoits seen in the
peripheral circulation, and probably corresponding to the
bodies seen by Schaudinn in his studies of Trypanosoma
noctua, in the stomach of the mosquito.

Transmission. — The transmitting agent was found by
Chagas to be a rather large hemipterus insect, Conorhinus
megistus. A special cycle of development of the parasite
in the body of this bug was described, but has not been
confirmed. Chagas was able to transmit the parasites to
monkeys by permitting the infected bugs to bite them.

CHAPTER XXI.

KALA-AZAR (BLACK FEVER).

LEISHMANIA DONOVANI (LAVERAN AND MESNIL).

" KALA-AZAR," " Dumdum fever," "Febrile tropical spleno-
megaly," " Non-malarial remittent fever," is a peculiar, fatal,
infectious disease of India, Assam, certain parts of China, the
Malay Archipelago, North Africa, the Soudan, and Arabia,
caused by a protozoan micro-organism known as Leishmania
donovani, and characterized by irregular fever, great enlarge-
ment of the spleen, anemia, emaciation, prostration, not in-
frequent dysentery, occasional ulcerations of the skin and
mucous membranes, and sometimes cancrum oris.

Because of its protean manifestations the disease has been
given many names, and has been confused with the various
diseases which its symptoms may resemble. It was not
until 1900 that it was finally differentiated from malarial
fever and came to be regarded as a distinct entity.

In 1900 Leishman* noticed in the spleen of a soldier re-
turned from India and suffering from " dumdum fever " —
a fever acquired at Dumdum, an unhealthy military can-
tonment not far from Calcutta — certain peculiar bodies. He
reserved publishing the observation until 1903, so that it
appeared almost simultaneously with a paper upon the same
subject by Donovan, f As the publications came from men
in different parts of the world, appeared so nearly at the* same
time, and showed that they had independently arrived at the
same discovery, the parasite they described became known
as the Leishman- Donovan body. For a long time its nature
was not known and its proper classification impossible, but
after it had been carefully studied by Rogers J Ross,§ and

*"Brit. Med. Jour.," 1903, i, 1252.

t Ibid., 1903, n, 79.

J "Quarterly Jour. Microscopical Society," XLVIII, 367; "Brit. Med.
Jour.," 1904, i, 1249; n, 645; "Proceedings of the Royal Society," LXXVII,
284.

§ "Brit. Med. Jour.," 1903, n, 1401,

566

Leishmania Donovan!

567

others, and its developmental forms observed, it was agreed
that it belonged in a new genus of micro-organisms, not far

• *% f9 ••

% !• »-

•, .fl

V- '*

• f

A*

rSi^-

»

Fig. 195. — Evolution of the parasite of kala-azar: i to 5. Parasites
of kala-azar. i, Isolated parasites of different forms in the spleen and
liver; 2, division forms from liver and bone-marrow; 3, mononuclear
spleen cells containing the parasites; 4, group of parasites; 5, phagocy-
tosis of a parasite by a polynuclear leukocyte. 6 to 15. Parasites from
cultures. 6, First changes in the parasites. The protoplasm has in-
creased in bulk and the nucleus has become larger; 7, further increase
in size; vacuolization of the protoplasm; 8, division of the enlarged
parasite; 9, evolution of the flagella; 10, small piriform parasite show-
ing flagellum; n, further development and division of the parasite; 12,
flagellated trypanosoma-like form; 13, 14, flagellated forms dividing by
a splitting off of a portion of the protoplasm; 15, narrow flagellated
parasites which have arisen by the type of division shown in 13 and 14.
(From Mense's " Handbuch," after Leishman.)

removed from the trypanosomes, and eventually Ross, and
then Laveran and Mesnil, honored both of its discoverers by

568 Kala-Azar

calling it Leishmania donovani, which name has been gener-
ally accepted.

Morphology. — As seen in a drop of splenic pulp the organ-
ism is a minute round or oval intracellular body measuring
2.5 by 3.5 (A. When properly stained with polychrome
methylene-blue (Wright's, Irishman's, or Jenner's stains) and
examined under a high magnification, it is found that the
protoplasm takes a pinkish color and contains two well-
defined bright red bodies. The larger of these is ovoid and lies
excentrically, its long diameter corresponding to the long di-
ameter of the organism. This is regarded as the nucleus.

Fig. 196. — Leishman-Donovan bodies from the spleen of a case of
kala-azar. X about 1000. (From Seattle and Dickinson's "A Text-
Book of General Pathology," by kind permission of Rebman, Limited,
publishers.)

The second body is smaller and of bacillary shape, and usu-
ally lies with its long diameter transverse to the nucleus.
This is looked upon as a blepharoplast. It stains more in-
tensely than the nucleus. In addition to these bodies the
protoplasm may contain one or two vacuoles.

All of the bodies are intracellular, as can easily be deter-
mined by examining sections of tissue, but in smears of splenic
pulp the cells are broken and many free bodies may appear.
The cells in which they occur are lymphocytes, endothelial
cells, and peculiar large cells whose histogenesis is obscure.
They are rarely to be found in polymorphonuclear leukocytes,
and though there has been much discussion upon this point,
probably never appear in the red blood-corpuscles.

Cultivation

569

The bodies divide by binary and multiple fission, without
recognizable mitotic changes. When multiple fission occurs,
the nucleus divides several times before the protoplasm
breaks up. The organism is not motile and at this stage has
no flagella.

Cultivation. — The organism was first cultivated artificially
by Rogers in citrated splenic juice at 17° to 24° C. It can
also be cultivated in the blood-serum agar medium used
by Novy, MacNeal, and Hall for trypanosomes.

Under conditions of cultivation the appearance of the or-
ganism undergoes a complete change. It enlarges, the
nucleus increases greatly in size, and a pink vacuole appears
near the blepharoplast. In the course of twenty-four to

Fig. 197. — Leishmania donovani. Flagellated forms obtained in pure
cultures (Irishman) .

forty-eight hours the organism elongates, the blepharoplast
moves to one end, and from the vacuole near it a flagellum is
developed, and the organism becomes in about ninety-six
hours a flagellate protozoan resembling herpetomonas. It
now measures about 20 p in length and 3 to 4 {i in breadth, its
whip or flagellum measuring about 3 /w additional. It is also
motile and, like the trypanosomes, swims with the flagellum
anteriorly. There is no undulating membrane.

This may be regarded as the perfect or adult form of the
organism. It multiplies by a peculiar mode of division first
observed by Leishman. Chromatin granules, a larger and
a smaller, appear in the protoplasm in pairs, after which,
through unequal longitudinal cleavage, long, slender, almost
hair-like individuals, containing one of the pairs of chromatin
granules, are separated. These were serpentine at first, but

570 Kala-Azar

later, as they grew larger, a flagellum was thrust out at one
end.

Distribution. — The Leishman-Donovan body is widely
distributed throughout the body of the patients suffering
from kala-azar. It occurs in great numbers in the cells of the
spleen, of the liver, of the bone-marrow, and in the ulcerations
of the mucous membranes and skin. In the peripheral blood
they are few and only in the leukocytes. They are always
intracellular, or when in the circulating blood may be found
in indefinite albuminous masses, probably destroyed cells.
The number in a cell varies up to several hundred, such great
aggregations only being found in the peculiar large cells of
the spleen.

Lesions. — The splenomegaly is the most striking lesion.
The change by which the enlargement is effected is not spe-
cific. The organ is not essentially changed histologically, but
seems to be merely hyperplastic. The liver is enlarged, but
here, again, specific changes may be absent. In some cases
a pallor of the centers of the lobules may depend upon
numbers of parasite-containing cells, partly degenerated.

The yellow bone-marrow becomes absorbed and red tissue
takes its place, as in most profound anemias.

Transmission. — Roger's observation, that the round
bodies grew into flagellate bodies at temperatures much below
that of the human body, led Manson to conjecture that the
extra-human phase of the life of the organism took place at
similar low temperatures in the soil or in water. Patton*
found that a number of cases sometimes occurred in the same
house, while neighboring houses were free, and thought this
suggested that a domestic insect might be the distributing
host. He made some unconvincing experiments on the sub-
ject. Rogers tried to convict the bedbug, but also failed.
The rarity of the Leishmania in the peripheral blood seems
opposed to its transmission by insects.

On the other hand, the organisms leave the body in con-
siderable numbers in the dejecta of patients suffering from
ulcerative lesions of the intestines and in the discharged pus
from the ulcerations of the skin.

However, in the present state of knowledge there can be
nothing but speculation upon the subject. The disease ap-
pears not to be transmissible to any of the laboratory animals,

* "Scientific Memoirs of the Government in India," 1907, No. 27.

Infantile Kala-Azar 571

hence great difficulties surround all investigation of the
problem of transmission.

Diagnosis. — The anemia of kala-azar is usually not pro-
found. The erythrocytes number about 3,000,000 in ordi-
nary cases and the hemoglobin is correspondingly diminished.
As in malaria, there is leukopenia, but it is usually more severe,
the white corpuscles sometimes being as few as 600 to 650 per
cubic millimeter of blood. The enlargement of the spleen
and liver suggest malaria.

The only certain way to make a diagnosis, except in those
rare cases where one has the good fortune to find occasional
parasites in the leukocytes of the circulating blood, is by
splenic culture. A large hypodermic needle should be used,
should be carefully sterilized, thrust into the spleen, and a
bit of splenic pulp secured by firmly withdrawing the piston.

Before making such a puncture, leukemia should be ex-
cluded, lest hemorrhage occur.

INFANTILE KALA-AZAR.
' LEISHMANIA INFANTUM

Nicolle,* while in Tunis, observed a form of kala-azar that
was peculiar to childhood and most frequent in babies of
about two years of age. Mesnil has identified the affection
with a disease known as "ponos" in Greece. In the spleens
of such patients Nicolle found an organism that was not dis-
tinguishable either by microscopic examination or by culti-
vation from Leishmaniadonovani, but, finding that it was in-
fectious for dogs, he came to the conclusion that it was a
separate species, and called it Leishmaniainfantum. He also
found that the dogs in Tunis frequently suffered from spon-
taneous infection from this parasite, and it is possible that the
dogs are the source from which the children become infected.

Pianesef found infantile kala-azar in Italy, and in the
children suffering from it he was able to find the Leishmania
infantum.

Further experiments with this parasite by Nicolle and
Comte have shown that in the form in which it occurs in the
human spleen it is capable of infecting monkeys, and Novy
has succeeded in cultivating the organism and infecting dogs

* "Ann. de 1'Inst. Pasteur," 1909, xxm, 361, 441.
f'Gaz. Intern, di Medicin," 1905, vm, 8.

572 Kala-Azar

with the artificial cultures containing the flagellate forms of
the organism

TROPICAL ULCER.
LEISHMANIA FURUNCULOSA (FIRTH).

In India, northern Africa, southern Russia, parts of China,
the West Indies, South America, and, indeed, most tropical
countries, a peculiar intractable chronic ulceration is occa-
sionally observed, and is variously known as Tropical ulcer,
Oriental sore, Biscra boil, Biscra button, Aleppo boil, Delhi

i

Fig. 198. — Helcosoma tropicum, from a case of tropical ulcer ("Delhi
sore") smear preparation from the lesion stained with Wright's Roman-
owsky blood-staining fluid. The ring-like bodies, with white central
portions and containing a larger and a smaller dark mass, are the micro-
organisms. The dark masses in the bodies are stained a lilac color,
while the peripheral portions of the bodies, in typical instances, are
stained a pale robin's egg blue. The very dark masses are nuclei of
cells of the lesion. X 1500 approx. (Wright). (From photograph by
Mr. L. S. Brown.)

boil, Bagdad boil, and Buton d'Orient. It has long been
known as a specific ulcerating granuloma. The lesions, which
begin as red spots, develop into papules which become covered
with a scaly crust which separates, leaving an ulcer upon which
a new crust develops. The lesion spreads and is much larger
when the crust again separates. A purulent discharge is
given off in moderate quantities and the ulcer becomes deep
and perpendicularly excavated. It lasts for months — some-
times a year or more — and gradually cicatrizes, forming a con-
tracting scar that is quite disfiguring when upon the face.

Histoplasmosis 573

The lesions may be single, though they are commonly mul-
tiple, as many as twenty sometimes occurring simultaneously.
It is thought that recovery is followed by immunity.

In 1885 Cunningham* described a protozoan organism
found in the tropical ulcer, the observation being confirmed
by Firth, f who called the bodies Sporozoa furunculosa.
Later, J. H. Wright J studied a case of tropical ulcer and found
bodies precisely like the Leishmania donovani. He gave it the
name Helcosoma tropicum. The great similarity to the other
organisms has led more recent writers to identify it with
Leishmania, but as it induces a local and not a general infec-
tion like kala-azar, it is now known as Leishmania furunculosa.

The organism has not been cultivated. It can undoubtedly
be transmitted by inoculation from human being to human
being, and Jackson § is authority for the statement that " the
Jews of Bagdad recognized that tropical ulcer is inoculable
and autoprotective years ago, and practiced vaccination of
their children upon some portion of the body covered by
clothing, in order that their faces and other exposed parts of
the body be not disfigured by the ulcers and the resultant
scars."

The mode of transmission is not known, but as the lesions
usually occur where the body surface is uncovered, it may
be that flies or other insects are concerned in the transmission
of the parasites.

HISTOPLASMOSIS.
HISTOPLASMA CAPSULATUM (DARLING).

In 1906 Darling, || working at the Isthmus of Panama, ob-
served certain cases presenting pyrexia, anemia, leukopenia,
splenomegaly, and emaciation, and bearing a close resem-
blance to kala-azar. The disease was quite chronic, and it
terminated fatally. When examined at autopsy, these cases
showed necrosis with cirrhosis of the liver, splenomegaly,
pseudogranulomata of the lungs, small and large intestines,

*" Scientific Memoirs by Medical Officers of the Army in India,"
1884, i.

t "British Med. Journal," 1891, Jan. 10, p. 60.

t ''Jour, of Med. Research," x, 1904, 472.

§ "Tropical Medicine," Phila., P. Blakiston's Son & Co., 1907, p.
478.

|| "Jour. Amer. Med. Assoc.," 1906, XLVI, 1283; "Archiv. f. Int. Med."
1908, n, 107; "Jour. Exp. Med.," 1909, xi, 515.

574

Kala-Azar

ulceration of the intestines, and necrosis of the lymph-nodes
draining the injected viscera. The lesions seemed to depend
upon the invasion of the endothelial cells of the smaller
lymph- and blood-vessels by enormous numbers of a small
encapsulated micro-organism.

The organism is small, round or oval in shape, and meas-
ures i to 4 ft in diameter. It possesses a polymorphous,

Fig. 199. — Histoplasma capsulatum. Mononuclear cells from the
lung containing many parasites (Darling). (Samuel T. Darling in
"Journal of Experimental Medicine.")

chromatin nucleus, basophilic cytoplasm, and achromatic
spaces all enclosed within an achromatic refractile capsule.
The micro-organism differs from the Leishman-Donovan
body of kala-azar in the form and arrangement of its chro-
matin nucleus and in not possessing a chromatin rod. The
distribution of the parasite in the body is accomplished by
the invasion of the contiguous endothelial cells of the smaller

Histoplasmosis 575

blood- and lymph-vessels and capillaries, and by the infec-
tion of distant regions by the dislodgment of infected endo-
thelial cells and their transportation thither by the blood- and
lymph -stream. Thus the skin, intestinal, and pulmonary
nodules may be due to secondary distribution of the parasite.
The micro-organism apparently lives for a considerable
period of time iin the tissues, because in the older areas of
necrosis there are myriads of parasites all staining well.

The mode of infection and portal of entry are unknown.
The parasite has neither been cultivated nor transmitted by
inoculation.

Believing it to be a new parasite, Darling has suggested
that it be called Histoplasma capsulatum.

CHAPTER XXII

YELLOW FEVER.

THE bacteriology of yellow fever has been studied by
Domingos Freire,* Carmona y Valle,f Sternberg,J Havel-
burg^ and Sanarelli,|| but all of their work has been shown
to be incorrect by the interesting researches and very con-
clusive results of Finlay,** Carter, ff Reed, Carroll, Lazear,
and Agramonte, J { and Reed and Carroll, §§ which have
proved the mosquito to be the definitive host of an invis-
ible micro-organism.

Reed, Carroll, Lazear, and Agramonte, |||| constituting a
Board of Medical Officers "for the purpose of pursuing scien-
tific investigations with reference to the acute infectious dis-
eases prevalent on the island of Cuba," began their work in
1900, at Havana, by a careful investigation of the relationship
of Bacillus icteroides to yellow fever. By a most careful tech-
nic they withdrew and examined the blood from the veins of
the elbow of 1 8 cases of yellow fever, making 48 separate ex-
aminations on different days of the disease, and preparing
115 bouillon cultures and 18 agar plates, every examination
being negative so far as Bacillus icteroides was concerned.

*" Doctrine microbienne de la fievre jaune et ses inoculation pre-
ventives," Rio Janeiro, 1885.

f "Lemons sur 1'etiologie et la prophylaxie de la fievre jaune," Mexico,
1885.

t "Report on the Etiology and Prevention of Yellow Fever," Wash-
ington, 1891; "Report on the Prevention of Yellow Fever by Inocula-
tion," Washington, 1888.

§ "Ann. de 1'Inst. Pasteur," 1897.

|| "Brit. Med. Jour.," July 3, 1897; "Ann. de 1'Inst. Pasteur,"
June, Sept., and Oct., 1897.

** "Amer. Jour. Med. Sci.," 1891, vol. en, p. 264; "Ann. de la Real
Academia," vol. xvm, 1881, pp. 147-169; "Jour. Amer. Med. Assoc.,"
vol. xxxvm, April 19, 1902, p. 993.

ft "New Orleans Med. Jour.," May, 1890.

U "Phila. Med. Jour.," Oct. 27, 1900; "Public Health," vol. xxvi,
1900, p. 23.

§§ "Public Health," vol. xxvn, 1901, p. 113.

Ill) "Phila. Med. Jour.," Oct. 27, 1900.

576

Mosquitoes and Yellow Fever 577

They were entirely unable to confirm the findings of Wasdin
and Geddings,* that Bacillus icteroides was present in blood
obtained from the ear in 13 out of 14 cases, and concluded
that both Sanarelli and Wasden and Geddings were mistaken
in their deductions.

In lieu of the remarkably interesting discoveries of Ronald
Ross concerning the relation of the mosquito to malarial in-
fection, the commissioners, remembering the theory of
Finlay,t who in 1881 published an experimental research
showing that mosquitoes spread the infection of yellow fever,
and the interesting and valuable observations of Carter J
upon the interval between infecting and secondary cases of
yellow fever, turned their attention to the mosquito. Secur-
ing mosquitoes from Finlay and continuing the work where
he had left it, they found that when mosquitoes (Stegomyia
fasciata sen calopus} were permitted to bite patients suffering
from yellow fever, after an interval of about twelve days
they became able to impart yellow fever with their bites.
This infectious character of the bite, having once developed,
seems to remain throughout the subsequent life of the
insect. So far as it was possible to determine, only one
species of mosquito, Stegomyia calopus, served as a host
for the parasite whose cycles of development in the mosquito
and in man must explain the symptomatology of yellow
fever.

In order to establish these observations, experimental
inoculations were made upon human beings in sufficient
number to prove their accuracy. Unfortunately, Dr.
Lazear, one of the victims of the experiment, lost his life
from an attack of yellow fever.

Reed, Carroll, and Agramonte§ came to the following
conclusions :

1 . The mosquito C. fasciatus (Stegomyia calopus) serves
as the intermediate host of the yellow fever parasite.

2. Yellow fever is transmitted to the non-immune indi-
vidual by means of the bite of the mosquito that has pre-
viously fed on the blood of those sick with the disease.

* " Report of the Commission of Medical Officers Detailed by the
Authority of the President to Investigate the Cause of Yellow Fever,"
Washington, D. C., 1899.

f "Annales de la Real Academia," vol. xvm, 1881, pp. 147-169.

t "New Orleans Med. Jour.," May, 1900.

\ Pan-American Medical Congress, Havana, Cuba, Feb. 4-7, 1901;
Sanitary Department, Cuba, series 3, 1902.

37

578

Yellow Fever

3. An interval of about twelve days or more after con-
tamination appears to be necessary before the mosquito is
capable of conveying the infection.

Fig. 200. — Stegomyia fasciata (Stegomyia calopus): a, female; b, male
(after Carroll) .

4. The bite of the mosquito at an earlier period after
contamination does not appear to confer any immunity
against a subsequent attack.

Mosquitoes and Yellow Fever 579

5. Yellow fever can be experimentally produced by the
subcutaneous injection of blood taken from the general
circulation during the first and second days of the disease.

6. An attack of yellow fever produced by the bite of a
mosquito confers immunity against the subsequent injection
of the blood of an individual suffering from the non-experi-
mental form of the disease.

7. The period of incubation in 13 cases of experimental
yellow fever has varied from forty -one hours to five days and
seventeen hours.

8. Yellow fever is not conveyed by fomites, and hence
disinfection of articles of clothing, bedding, or merchandise,
supposedly contaminated by contact with those sick with the
disease, is unnecessary.

9. A house may be said to be infected with yellow fever
only when there are present within its walls contaminated
mosquitoes capable of conveying the parasite of this disease.

10. The spread of yellow fever can be most effectually
controlled by measures directed to the destruction of mos-
quitoes and the protection of the sick against the bites of
these insects.

11. While the mode of propagation of yellow fever has
now been definitely determined, the specific cause of the
disease remains to be discovered.

The probability that Bacillus icteroides is the specific
cause and is transmitted by the mosquito is so slight that it
need scarcely be considered. All analogy points to the
organism being an animal parasite similar to that of malarial
fever.

With this positive information before us, the prophylaxis
of yellow fever and the prevention of epidemics of the disease
where sporadic cases occur becomes very simple and may be
expressed in the following rules:

1. Whenever yellow fever is likely to occur, the breeding
places of mosquitoes should be destroyed by drainage. Cis-
terns and other necessary collections of standing water should
be covered or secured.

2. Houses should have the windows and doors screened
and the inhabitants should use bed nets.

3. So soon as a case of fever appears it should be removed in
a mosquito-proof ambulance to a mosquito-proof apartment
in a well-screened hospital ward and kept there until con-
valescent.

580 Yellow Fever

4. The premises where such a case has occurred should be
fumigated by burning pyre thrum powder (i pound per 1000
cubic feet) to stun the mosquitoes, which fall to the floor and
must afterward be swept up and destroyed.

By these means Major W. C. Gorgas,* without expensive
disinfection and without regard for fomites, has virtually
exterminated yellow fever from Havana and from the Canal
Zone, Panama, where it was for many years endemic.

A practical point connected with the screens is given in the
work of Rosenau, Parker, Francis, and Beyer, | who found that
to be effective the screens must have 20 strands or 19 meshes
to the inch. If coarser than this the stegomyia mosquitoes
can pass through.

Reed and Carroll { were the first to filter the blood of yellow
fever patients and prove that after it had passed through a
Berkefeld filter that kept back Staphylococcus aureus, it
still remained infective and capable of producing yellow fever
in non-immune human beings.

This subject was further investigated by Rosenau, Parker,
Francis and Beyer, § who found that the virus was even smaller
than the first experiment would suggest, as it not only passed
through the Berkefeld filter, but also through the Pasteur-
Chamberland filter. The filtrates always remained sterile
when added to culture-media.

The virus has not been artificially cultivated.

Prophylaxis. — Guiteras|| has studied the effect of inten-
tionally permitting non-immunes who are to be exposed to the
disease to be experimentally infected by being bitten by in-
fected mosquitoes, after which they are at once carefully
treated. His first conclusion was that "the intentional inoc-
ulation gives the patient a better chance of recovery," but the
danger of death from the experimental injection was later
shown to be so great that it had to be abandoned.

* International Sanitary Congress held at Havana, Cuba, Feb. 16,
1902; Sanitary Department, Havana, series 4.

f Report of Working Party No. 2, Yellow Fever Institute, Bull. 14,
May, 1904.

| "Am. Med.," Feb. 22, 1902.

§ "Bull. No. 14, U.S. Public Health and Marine Hospital Service,"
Washington, D. C., May, 1904.

|| "Revista de Medicina Tropical," Havana, Cuba, 1902.

CHAPTER XXIII. •

PLAGUE.

BACILLUS PESTIS (YERSIN, KITASATO).

General Characteristics. — A minute, pleomorphous, diplococcoid
and elongate, sometimes branched, non-motile, non-flagellated, non-
sporogenous, non-chromogenic, aerobic and optionally anaerobic,
pathogenic organism, specific for bubonic plague, easily cultivated
artificially, and susceptible of staining by ordinary methods, but not
by Gram's method.

Plague, bubonic plague, pest, black plague, " black death,"
or malignant polyadenitis is an acute epidemic infectious
febrile disease of an intensely fatal nature, characterized by
inflammatory enlargement and softening of the lymphatic
glands, marked pulmonary, cerebral, and vascular disturb-
ance, and the presence of the specific bacillus in the lymphatic
glands and blood.

The history of plague is so full of interest that many ref-
erences to it appear in popular literature. The student can
scarcely find more profitable reading than the "History of the
Plague Year in London," by DeFoe, and readers of Boccacio
will remember that it was the plague epidemic then raging in
Florence that led to the isolation of the group of young
people by whom the charming stories of the Decameron were
told.

During the reign of the Emperor Justinian the plague is
said to have carried off nearly half of the population of the
Roman Empire. In the fourteenth century it is said to have
destroyed nearly twenty-five millions of the population of
Europe. Epidemics of less severity but attended with great
mortality appeared in the sixteenth, seventeenth, and eight-
eenth centuries. In 1894 an epidemic broke out in the
western Chinese province of Yunnan and reached Canton in
January, 1894, thus escaping from its endemic center and
began to spread. It can be traced from Canton to Hong-
kong. In 1895 it appeared also in Amoy, Macao, and
Foochoo. In 1896 it had reached Bombay and reappeared in
Hongkong. In 1897 Bombay, the Madras Presidency, the

581

582

Plague

Punjab, and Madras were visited. In 1898 the disease spread
greatly throughout India and into Turkestan, and by sea went
to Madagascar and Mauritius. In 1899 it extended still
more widely in India and China, Japan and Formosa, and
succeeded in disseminating as widely as the Hawaiian Islands
and New Caledonia on the east, Portugal, Russia, and
Austria on the west, and Brazil and Paraguay on the south.
In 1900 it had spread to nearly every part of the world. In
these places when sanitary measures could not be carried into
effect the people died in great numbers — thus in India in
1901 there were 362,000 cases and 278,000 deaths. Where

•I

Fig. 201. — Axillary bubo. (Reproduced from Simpson's "A Treatise
on Plague," 1905, by kind permission of the Cambridge University
Press.)

the precautions were possible and co-operation between the
people and the authorities could be brought about, as in New
York, San Francisco, and other North American and Euro-
pean ports, the disease remained confined pretty well within
limits and did not spread. An interesting account of "The
Present Pandemic of Plague" by J. M. Eager, was published
in 1908 in Washington, D. C., by the U. S. Public Health and
Marine Hospital Service.

Plague is an extremely fatal affection, whose ravages in the
hospital at Hongkong, in which Yersin made his original
observations, carried off 95 per cent, of the cases. The
death-rate varies in different epidemics from 50 to 90 per

Plague 583

cent. In the epidemic at Hongkong in 1894 the death-rate
was 93.4 per cent, for Chinese, 77 per cent, for Indians, 60
per cent, for Japanese, 100 per cent, for Eurasians, and 18.2
per cent, for Europeans. It affects both men and animals,
and is characterized by sudden onset, high fever, prostration,
delirium, and the occurrence of exceedingly painful lymphatic
swellings — buboes — affecting chiefly the inguinal glands,
though not infrequently the axillary, and sometimes the cer-
vical, glands. Death comes on in severe cases in forty-eight
hours. The pneumonic form is most rapidly fatal. The
longer the duration of the disease, the better the prognosis.
Autopsy in fatal cases reveals the characteristic enlargement
of the lymphatic glands, whose contents are soft and some-
times purulent.

Wyman,* in his very instructive pamphlet, "The Bu-
bonic Plague," finds it convenient to divide plague into (a)
bubonic or ganglionic, (6) septicemic, and (c) pneumonic
forms. Of these, the bubonic form is most frequent and the
pneumonic form most fatal.

The infection usually takes place through some peripheral
lesion, but may occur by inhalation of the specific organ-
isms.

The bacillus of bubonic plague was independently dis-
covered by Yersinj and KitasatoJ in the summer of 1894,
during an epidemic of the plague then raging at Hongkong.
There seems to be little doubt but that the micro-organisms
described by the two observers are identical.

Ogata § states that while Kitasato found the bacillus in the
blood of cadavers, Yersin seldom found it in the blood, but
always in the enlarged lymphatic glands; that Kitasato's
bacillus retains the color when stained by Gram's method;
Yersin's does not; that Kitasato's bacillus is motile; Yersin's
non-motile; that the colonies of Kitasato's bacillus, when
grown upon agar, are round, irregular, grayish white, with
a bluish tint, and resemble glass-wool when slightly mag-
nified; those of Yersin's bacillus, white and transparent,
with iridescent edges. Ogata, in his investigations, found
that the bacillus corresponded with the description of

* Government Printing Office, Washington, D. C., 1900.

t "Ann. de 1'Inst. Pasteur," 1894, 9.

t Preliminary notice to the bacillus of bubonic plague, Hongkong,
July 7, 1894-

§"Centralbl. f. Bakt. u. Parasitenk.," Sept. 6, 1897, Bd. xxn,
Nos. 6 and 7, p. 170.

584 Plague

Yersin rather than that of Kitasato, and it is certain that
the description given by Yersin is the more correct of the
two.

In the "JaPan Times," Tokio, November 28, 1899, Kita-
sato explains that, his investigations being made upon
cadavers that were partly putrefied, he was led to believe
that the bacillus first invaded the blood. Later studies
upon living subjects showed him the error of this view and
the correctness of Yer sin's observation that the bacilli first
multiply in the lymphatics.

Both Kitasato and Yersin showed that in blood drawn
from the finger-tips and in the softened contents of the
glands the bacillus may be demonstrable.

Fig. 202. — Bacillus of bubonic plague (Yersin).

Morphology. — The bacillus is quite variable. Usually it
is short and thick — a "coco-bacillus," as some call it —
with rounded ends. Its size is small (1.5 to 2 ^ in length)
and 0.5 to 0.75 ^ in breadth. It not infrequently occurs in
chains of four or six or even more, and is occasionally en-
capsulated. It shows active Brownian movements, which
probably led Kitasato to consider it motile. Yersin did not
regard it as motile, and was correct. Gordon* claims that
some of the bacilli have flagella. No spores are formed.

Staining. — It stains by the usual methods; not by
Gram's method. When stained, the organism rarely

* "Centralbl. f. Bakt. u. Parasitenk.," June 24, 1897, Bd. xxi, Nos.
20 and 2 1 .

Cultivation 585

appears uniformly colored, being darker at the ends than
at the center, so as to resemble a dumb-bell or diplococcus.
The bacilli sometimes appears vacuolated, and nearly all
cultures show a variety of involution forms. Kitasato has
compared the general appearance of the bacillus to that of
chicken-cholera.

Involution forms on partly desiccated agar-agar not
containing glycerin are said by Haffkine to be characteris-
tic. The microbes swell and form large, round, oval, pea-
shaped, spindle-shaped or biscuit-like bodies which may at-
tain twenty times the normal size, and gradually lose the

Fig. 203. — Bacilli of plague and phagocytes, from human lymphatic
gland. X 800 (Aoyama).

ability to take the stain. Such involution forms are not
seen in liquid culture.

Cultivation. — Pure cultures may be from the blood or
from the softened contents of the buboes, and develop well
upon artificial media. The optimum temperature is about
30° C. The extremes at which growth occurs are 20° and
38° C.

Bouillon. — In bouillon a diffuse cloudiness was ob-
served by Kitasato, though Yersin observed that the cul-
tures resembled erysipelas cocci, and contained zooglea
attached to the sides and at the bottom of the tube of
nearly clear fluid.

586 Plague

Haffkine* found that when an inoculated bouillon cul-
ture is allowed to stand perfectly at rest, on a firm shelf

Fig. 204. — Bacillus pestis. Highly virulent culture forty-eight
hours old, from the spleen of a rat. Unstained preparation (Kolle and
Wassermann).

or table, a characteristic appearance develops. In from
twenty-four to forty-eight hours, the liquid remaining limpid,
flakes appear underneath the surface, forming little islands

Fig. 205. — Bacillus pestis. Involution forms from a pure cul-
ture on 3 per cent, sodium chlorid agar-agar. Methylene-blue (Kolle
and Wassermann).

of growth, which in the next twenty-four to forty-eight
hours grow into a jungle of long stalactite-like masses, the

* "Brit. Med. Jour.," June 12, 1897, p. 1461.

Cultivation 587

liquid remaining clear. In from four to six days these
islands become still more compact. If the vessels be dis-
turbed, they fall like snow and are deposited at the bottom,
leaving the liquid clear.

Colonies. — Upon gelatin plates at 22° C. the colonies may
be observed in twenty-four hours by the naked eye. They
are pure white or yellowish white, spheric when deep in the
gelatin, flat when upon the surface, and are about the size
of a pin's head. The gelatin is not liquefied. Upon micro-
scopic examination the borders of the colonies are found to

Fig. 206. — Stalactite growth of bacillus pestis in bouillon. (Repro-
duced from Simpson's ''A Treatise on Plague," 1905, by kind permission
of the Cambridge University Press.)

be sharply defined. The contents become more granular as
the age increases. The superficial colonies are occasionally
surrounded by a fine, semi-transparent zone.

Klein* says that the colonies develop quite readily upon
gelatin made from beef bouillon (not infusion), appearing
in twenty-four hours, at 20° C., as small, gray, irregularly
rounded dots. Magnification shows the colonies to be
serrated at the edges and made up of short, oval, some-
times double bacilli. Some colonies contrast markedly
with their neighbors in that they are large, round, or oval,
and consist of longer or shorter, straight or looped threads
of bacilli. The appearance was much like that of the young

*"Centralbl. f. Bakt. u. Parasitenk.," July 10, 1897, xxi, Nos. 24
and 25.

588 Plague

colonies of Proteus vulgaris. At first these were regarded
as contaminations, but later he was led to believe that their
occurrence was characteristic of the plague bacillus. The
peculiarities of these colonies cannot be recognized after
forty-eight hours.

Gelatin Punctures. — In gelatin puncture cultures the
development is scant. The medium is not liquefied; the
growth takes place in the form of a fine duct, little points
being seen on the surface and in the line of puncture. Some-
times fine filaments project into the gelatin from the central
puncture.

Abel found the best culture-medium to be 2 per cent, alka-
line peptone solution containing i or 2 per cent, of gelatin,
as recommended by Yersin and Wilson.

Agar-agar. — Upon agar-agar the bacilli grow freely, but
slowly, the colonies being whitish in color, with a bluish tint
by reflected light, and first appearing to the naked eye when
cultivated from the blood of an infected animal after about
thirty-six hours' incubation at 37° C. Under the micro-
scope they appear moist, with rounded uneven edges.
The small colonies are said to resemble tufts of glass-wool.
Microscopic examination of the agar-agar culture shows the
presence of chains resembling streptococci.

Upon glycerin-agar the development of the colonies is
slower, though in the end the colonies attain a larger size
than those grown upon plain agar.

Hankin and Leumann* recommended for the differential
diagnosis of the plague bacillus a culture-medium prepared
by the addition of 2.5 to 3.5 per cent, of salt to ordinary
culture agar-agar. When transplanted from ordinary agar-
agar to the salt agar-agar, the involution forms so charac-
teristic of the bacillus are formed with exceptional rapidity.
In bouillon containing this high percentage of salt the stalac-
tite formation is beautiful and characteristic.

Blood-serum. — Upon blood-serum, growth, at the tem-
perature of the incubator, is luxuriant and forms a moist
layer, of yellowish-gray color, unaccompanied by lique-
faction of the serum.

Potato. — Upon potato no growth occurs at ordinary
temperatures. When the potato is stood in the incubator
for a few days a scanty, dry, whitish layer develops.

*"Centralbl. f. Bakt. u. Parasitenk.," Oct., 1897, Bd. xxn, Nos.
16 and 17, p. 438.

Metabolism 589

Vital Resistance. — Kitasato found that the plague
bacillus did not seem able to withstand desiccation longer
than four days; but Rappaport* found that they remained
alive when kept dry upon woolen threads at 20° C. for
twenty-three days, and Yersin found that although it could
be secured from the soil beneath an infected house at a depth
of 4 to 5 cm., the virulence of such bacilli was lost.

Kitasato found that the bacillus was killed by two hours'
exposure to 0.5 per cent, carbolic acid, and also by exposure
to a temperature of 80° C. for five minutes. Ogata found
the bacillus instantly killed by 5 per cent, carbolic acid, and
in fifteen minutes by 0.5 per cent, carbolic acid. In o.i per
cent, sublimate solution it is killed in five minutes.

According to Wyman, the bacillus is killed by exposure
to 55° C. for ten minutes. The German Plague Commission
found that the bacilli were killed by exposure to direct sun-
light for three or four hours; and Bowhillf found that they
are killed by drying at ordinary room temperatures in about
four days.

Wilson I found the thermal death-point of the organism
one or two degrees higher than that of the majority of
non-sporulating pathogenic bacteria, and that the influ-
ence of sunlight and desiccation cannot be relied upon to
destroy it.

Rosenaui found temperature the most important factor,
as it dies quickly when kept dry at 37° C., but remains alive
for months when kept dry at 19° C. Sunlight kills it in a
few hours. A temperature of 70° C. is invariably fatal in
a short time.

Metabolism. — The bacillus develops under conditions of
aerobiosis and anaerobiosis. In glucose-containing media
it does not form gas. No indol is formed. Ordinarily the
culture-medium is acidified, the acid reaction persisting for
three weeks or more.

Ghon,|| Wernicke,** and others who have studied the toxic

* Quoted by Wyman.

f "Manual of Bacteriological Technique and Special Bacteriology,"
1899, p. 197.

t "Journal of Medical Research," vol. vi, No. i, p. 53, July, 1901.
§ Bulletin No. 4 of the Hygienic Laboratory of the U. S. Marine
Hospital Service, 1901.

|| Wien, 1898.
** "Centralbl. f. Bakt.," etc., 1898, xxiv.

590 Plague

products of the bacillus all incline to the belief that it forms
only endotoxin.

Kossee and Overbeck,* however, believe that there is, in
addition, a soluble exotoxin that is of importance.

Experimental Infection. — Mice, rats, guinea-pigs, rab-
bits, monkeys, dogs, and cats are all susceptible to experi-
mental inoculation. During epidemics the purely herbiv-
orous animals usually escape, though oxen have been known
to die of the disease. When blood, lymphatic pulp, or
pure cultures are inoculated into them, the animals be-
come ill in from one to two days, according to their size and
the virulence of the bacillus. Their eyes become watery,
they show disinclination to take food or to make any bodily
effort, the temperature rises to 41.5° C., they remain quiet
in a corner of the cage, and die with convulsive symptoms
in from two to seven days. If the inoculation be made
intravenously, no lymphatic enlargement occurs; but if it
be made subcutaneously, the nearest lymph-nodes always
enlarge and suppurate if the animal live long enough. The
bacilli are found everywhere in the blood, but not in very
large numbers.

Rats seem to suffer from a chronic form of the disease,
and sometimes can be found to have encapsulated caseous
nodules in the submaxillary glands, caseous bronchial glands,
and fibroid pneumonia, months after inoculation. In all
such cases virulent plague bacilli are present.

In and about San Francisco the extermination of rats for
the eradication of the plague was unexpectedly complicated
by the discovery that other rodents with which the rats came
into contact also harbored the plague bacilli. McCoy and
Smith f found this to be true of the prairie dog, the desert
wood rat, the rock squirrel, and the brush rat. To insure
security against the recurrence of the disease among men
necessitates continued observation of these animals and the
extermination of diseased colonies, as well as their complete
extermination in the neighborhood of human habitations.

According to Yersin, an infiltration or watery edema can
be observed in a few hours about the point of inoculation.
The autopsy shows the infiltration to be made up of a yellow-
ish gelatinous exudation. The spleen and liver are enlarged,
the former often presenting an appearance similar to that

* "Arbeiten aus d. Kaiserl. Gesundheitsamte," 1901, xvm.
t "Journal of Infectious Diseases," 1910, vn, p. 374.

Experimental Infection 591

observed in miliary tuberculosis. Sometimes there is uni-
versal enlargement of the lymphatic glands. Bacilli are
found in the blood and in all the internal organs. Skin
eruptions may occur during life, and upon the inner abdom-
inal walls petechiae and occasional hemorrhages may be
found. The intestine is hyperemic, the adrenals congested.
Serosanguinolent effusions may occur into the serous
cavities.

Devell* has found frogs susceptible to the disease.

Wyssokowitsch and Zabolotnyf found monkeys highly
susceptible to plague, especially when subcutaneously in-
oculated. When an inoculation was made with a pin dipped
in a culture of the bacillus, the puncture being made in the
palm of the hand or sole of the foot, the monkeys always
died in from three to seven days. In these cases the local
edema observed by Yersin did not occur. They point out
the interest attaching to infection through so insignificant a
wound and without local lesions.

Klein { found that intraperitoneal injection of the bacillus
into guinea-pigs was of diagnostic value, producing a thick,
cloudy, peritoneal exudate rich in leukocytes and containing
characteristic chains of the plague bacillus, occurring in
from twenty-four to forty-eight hours.

The plague bacillus may enter the body by inhalation from
an atmosphere through which it is disseminated, under
which circumstances it is usually of the pneumonic type, or
it may enter through the skin. Lesions too small to be seen
with the naked eye may suffice, and some have shown that
when the organisms are vigorously rubbed into the unbroken
skin, they may succeed in penetrating it. The pulmonary
or pneumonic form is not unlike other forms of pneumonia.
The lung is consolidated, enormous numbers of plague bacilli
occur in the sputum, the fever is high, and death occurs in a
few days.

Cases in which the infection takes place through the skin
soon show swelling of the adjacent lymph-nodes. These
become very large and tense, occasion great suffering, and
finally may soften and evacuate if death from blood invasion
does not intervene.

* "Centralbl. f. Bakt. u. Parasitenk.," Oct. 12, 1897.
t "Ann. de 1'Inst. Pasteur," Aug. 25, 1897, xi, 8, p. 665.
} "Centralbl. f. Bakt. u. Parasitenk.," xxi, No. 24, July 10, 1897,
p. 849.

592 Plague

Mode of Infection. — Klein found that animals fed upon
cultures of the bacillus or upon the flesh of animals dead
of the disease became ill and died with typical symptoms.
When he inoculated animals with the dust of dwelling-houses
in which the disease had occurred, some of them died of
tetanus, one from plague. Many rats and mice died spon-
taneously in Hongkong, examination showing the character-
istic bacilli. Such infected animals carry the cause of the
disease from place to place in their migrations.

Yersin showed that flies may die of the disease. Macer-
ating and crushing a fly in bouillon, he not only succeeded
in obtaining the bacillus, but infected an animal with it.

Nuttall,* in repeating Yersin 's fly experiment, found his
observation correct, and showed that flies fed with the ca-
davers of plague -infected mice die in a variable length of time.
Large numbers of plague bacilli were found in their intes-
tines. He also found that bed-bugs allowed to prey upon
infected animals took up large numbers of the plague bacilli
and retained them for a number of days. These bugs did
not, however, infect healthy animals when allowed to bite
them; but Nuttall was not satisfied that the number of his
experiments upon this point was great enough to prove
that plague cannot be spread by the bites of suctorial
insects.

Ogata found plague bacilli in fleas taken from diseased
rats. He crushed some fleas between sterile object-glasses
and introduced the juice into the subcutaneous tissues of
a mouse, which died in three days with typical plague, a
control-animal remaining well. Some guinea-pigs taken for
experimental purposes into a plague district died sponta-
neously of the disease, presumably because of insect in-
fection.

The animal most prone to spontaneous infection seems to
be the rat, and there is much evidence in support of the view
that it aids in the spread of epidemics. In several of the
Asiatic plague districts and at Santos the appearance of
plague among the inhabitants was preceded by a large mor-
tality among the rats, which examination showed had died
of plague. Dead rats are usually to be found in plague-
infected houses in India.

Galli- Valerio f and others think that the fleas of the mouse

* "Centralbl. f. Bakt. u. Parasitenk.," xxi, No. 24, Aug. 13, 1897.
t Ibid., xxvii, No. i, p. i, Jan. 6, 1900.

Diagnosis 593

and rat are incapable of living upon man and do not bite
him, and that it is only the Pulex irritans, or human flea,
that can transmit the disease from man to man. Tidswell,*
however, found that of 100 fleas collected from rats, there
were four species, of which three — the most common kinds
— bit men as well as rats. Lisbon f found that of 246 fleas
caught on men in the absence of plague, only one was a rat
flea, but out of 30 fleas caught upon men in a lodging-house,
during plague, 14 were rat fleas. This seems to show that
as the rats die off their fleas seek new hosts, and may thus
contribute to the spread of the disease.

That fleas can cause the transmission of plague from animal
to animal has been proved by experiments made in India.
These experiments, which are published as "Reports on Plague
Investigations in India," issued by the Advisory Committee
appointed by the Secretary of State for India, the Royal
Society, and the Lister Institute, appear in the Journal of
Hygiene from 1 906 onward, t It seems from these experiments
that human fleas (Pulex irritans) do not bite rats, but that
the rat fleas of all kinds do, though not willingly, bite men.
By placing guinea-pigs in cages upon the floor of infected
houses, the fleas of all kinds quickly attack them with result-
ing infection, but if the guinea-pigs are kept in flea-proof
cages, or if the cages are surrounded by "tangle-foot," or
"sticky fly-paper," the fleas, not being able to spring over the
barrier, are caught on the sticky surfaces and do not reach
the guinea-pigs, which then remain uninfected. What is
true of the guinea-pigs, is undoubtedly true of the rats; the
disease is transmitted from rat to rat by the fleas (Ceratophyl-
lus fasciatus, Ctenopsylla musculi, and Pulex cheopis, which
appears to be most common).

M. Herzog§ has shown that pediculi may harbor plague
bacilli and act as carriers of the disease.

The plague bacilli can also be transmitted from place to
place by fomites, from which they may reach man or rats.

Diagnosis. — It seems possible to make a diagnosis of the
disease in doubtful cases by examining the blood, but it is

*" British Medical Journal," June 27, 1903.

f "Times of India," Nov. 26, 1904.

t "Journal of Hygiene," Sept., 1906, vol. vi, p. 421; July, 1907, vol.
vn, p. 324; Dec., 1907, vol. vn, p. 693; May, 1908, vol. vin, p. 162;
1909, vol. ix; 1910, vol. x; 1911, vol. xi.

§ "Amer. Jour. Med. Sci.," March, 1895.
38

594 Plague

admitted that a good deal of bacteriologic practice is neces-
sary for the purpose.

Abel found that blood-examinations may yield doubt-
ful results because of the variable appearance of the con-
tained bacilli, which may easily be mistaken for other bac-
teria. He deems the best tests to be the inoculation of
broth cultures and the subsequent inoculation into animals,
which, he advises, should have been previously vaccinated
against the streptococcus.

Kolle* has suggested a method valuable both for the
diagnosis of the disease and for estimating the virulence of
the bacillus. It is as follows: "The skin over a portion of
the abdominal wall of the guinea-pig is shaved, care being
taken to avoid the slightest injury of the skin. The infective
material is carefully rubbed into the shaved skin. Im-
portant, in order rightly to understand the occurrence of
plague infection, is the fact disclosed here in the case of
guinea-pigs, that by this method of inoculation the animals
present the picture of true bubonic plague — that is to say,
the production of nodules in the various organs, principally
in the spleen. In this manner guinea-pigs, which would
not be affected by large subcutaneous injections, even
amounting to 2 mg. of agar culture (equal to a loop) of low-
virulence plague bacillus, may be infected and eventually
succumb."

Virulence. — By frequent passage through animals of the
same species the bacillus can be much increased in virulence.
Kolle recommends rats for this purpose, and, indeed, declares
that without the use of rats it is impossible to keep cultures
at a high grade of virulence. Batzaroff found that the most
virulent plague bacilli were to be obtained from the pneu-
monic lungs of rats that had been infected through the nasal
aperture with cotton-wool saturated with a culture of the
bacillus. This is not, however, a reliable method of inocu-
lation.

Yersin found that when cultivated for any length of time
upon culture-media, especially agar-agar, the virulence was
rapidly lost and the bacillus eventually died. On the other
hand, when constantly inoculated from animal to animal,
the virulence of the bacillus is much increased.

* See Havelburg, "Public Health Reports," Aug. 15, 1902, vol.
xvn, No. 33, p. 1863.

Sanitation 595

Knorr, Yersin, Calmette, and Borrel* have shown that
the bacillus made virulent by frequent passage through mice
is not increased in virulence for rabbits.

This no doubt depends upon the sensitivity of the bacillus
to the protective substances of the body juices, immuniza-
tion against those of one animal not necessarily protecting
the organism against those of other animals.

Bielonovskyf finds that broth, agar, and serum cultures
of the plague bacillus possess the property of hemolyzing
the blood of normal animals. The hemolytic power of fil-
trates of plague cultures increases up to the thirteenth or
fourteenth day, then gradually diminishes, but without
completely disappearing. The hemolysins are notably re-
sistant to heat, not being destroyed below 100° C.

Sanitation. — A disease that may be transmitted from
man to man by atmospheric infection and inhalation, that can
be transported from place to place by fomites, that occurs in
epidemic form among the lower animals as well as among men,
and that can be transmitted from man to man and from lower
animals to man by biting insects, must inevitably become a
source of anxiety to the sanitarian.

The preventive measures must take account of men, rats,
and goods. If vessels are permitted to visit and leave plague-
stricken ports, means must be taken to see that all passengers
are healthy at the time of leaving and have remained so
during the voyage, and provision should be made at the port
of entry for the disinfection of the cargo before the foods are
landed. But the rats must be given special consideration,
for so soon as the vessel reaches port some of them jump over-
board and swim to the shore, carrying the disease with them.
When a vessel visits a plague port, every precaution should be
taken to orevent the entrance of rats, first by anchoring
in the stream instead of tying to the dock ; by carefully scru-
tinizing the packages taken from the lighters to see that there
are no rats hidden among them ; by placing large metal shields
or reversed funnels about all anchor chains, hawsers, and
cables so that no rats can climb up from the water in which
they are swimming at night. Arrangements should also be
made for rat destruction on board the ship by means of sul-
phurous oxid or other poisonous vapors to rid the ship of
rats before the next port is reached. Passengers and crew

* "Ann. de 1'Inst. Pasteur.," July, 1895.

t "Arch, des Sci. Biol.," Tome x, No. 4, St. Petersb., 1904.

596 Plague

should also be kept in quarantine before mingling with
society. It is much more easy to keep plague out of a
port than to combat it when it has entered, for under the
latter condition are involved the isolation of the patients
in rat-free and vermin-free quarters, the disinfection of
the premises and goods where the case arose, and an im-
mediate warfare upon the rats and other small animals of the
neighborhood. To emphasize how difficult the latter may be
it is only necessary to point out that plague reached San
Francisco in May, 1907, during which year there were 156
cases and 76 deaths. Every precaution was taken to prevent
its spread, and extermination of rats, many being found
infected, practised. Though at great expense and with the
utmost thoroughness the rats were destroyed, the plague
spread to the ground squirrels and other small rodents, and in
1911 plague-infected rodents were still to be found in the
outskirts of the city.

Immunity. — An attack of plague usually exempts from
future attacks. Kitasato's experiments first showed that it
was possible to bring about immunity against the disease, and
Yersin, working in India, and Fitzpatrick, in New York,
have successfully immunized large animals (horses, sheep,
and goats). The serum of the immunized animals contains
specific agglutinins and bacteriolysins as well as an antitoxin,
capable not only of preventing the disease, but also of curing
it in mice and guinea-pigs and probably in man.

Haffkine's Prophylactic. — Haffkine* followed his plan
of preventive inoculation as employed against cholera, and
has invented a mode of prophylaxis based upon the use
of devitalized cultures. Bouillon cultures are used, and
small floating drops of butter are employed to make the
"islands" of plague bacilli float. The cultures are grown
for a month or so, successive crops of the island-stalactite
growth being precipitated by agitating the tube. In this
manner an "intense extracellular toxin" containing large
numbers of the bacilli is prepared. The culture was killed
by exposure to 70° C. for one hour, and used in doses of i to 3
c.c. as a preventive inoculation.

An interesting collection of statistics, showing in a con-
vincing manner the value of the Haffkine prophylactic, is
published of Leumann, of Hubli. The figures, together

* "Brit. Med. Jour.," June 12, 1897.

Haffkine Prophylactic 597

with a great deal of interesting information upon the subject,
can be found in the paper upon "A Visit to the Plague Dis-
tricts in India," by Barker and Flint.*

The immunity conferred by the Haffkine prophylactic
lasts about a month. The preparation must never be used
if the person has already been exposed to infection, and is
in the incubation stage of the disease, as it contains the
toxins of the disease, and therefore greatly intensifies the
existing condition. When injected into healthy persons
it always produces some fever, slight local swellings, and
malaise.

Wyssokowitsch and Zabolotny,f whose studies have al-
ready been quoted, used 96 monkeys in the study of the value
of the "plague serums," and found that when treatment is
begun within two days from the time of inoculation the
animals can be saved, even though symptoms of the disease
are marked. After the second day the treatment cannot
be relied upon. The dose necessary was 20 c.c. of a serum
having a potency of i : 10. If too little serum was given,
the course of the disease was retarded and the animal im-
proved for a time, then suffered a relapse, and died in from
thirteen to seventeen days. The serum also produced
immunity, but of only ten to fourteen days' duration. Im-
munity lasting three weeks was conferred by inoculating
a monkey with an agar-agar culture heated to 60° C. If
too large a dose of such a culture was given, however, the
animal was enfeebled and remained susceptible.

Of Yersin's serum, which is prepared by immunizing
horses against the toxins and cultures of the bacillus in the
usual manner, 5 c.c. doses have been found to confer an im-
munity lasting for about a fortnight. Larger doses confer a
longer immunity. For the treatment of the developed
disease in man, doses of 50 and even 100 to 200 c.c. seem
necessary to produce the desired effect.

OTHER MICRO-ORGANISMS OF THE PLAGUE GROUP.

The Bacillus pestis is a member of a group of organisms
collectively known as the bacilli of hemorrhagic septicemia.
Two of these organisms are of sufficient interest to deserve
special mention.

* "New York Med. Jour.," Feb. 3, 1900.
t Loc. cit.

598 Micro-organisms of the Plague Group

BACILLUS CHOLERA GALLINARUM (PERRONCITO) ; BACILLUS

CHOLERA; BACILLUS AVICIDUM; BACILLUS Avi-

SEPTICUS; BACILLUS OF RABBIT SEPTI-

CEMIA; BACILLUS CUNICULICIDA.

General Characteristics.— A non-motile, non-flagellated, non-
sporogenous, non-liquefying, non-chromogenic, aerobic bacillus, patho-
genic for birds and mammals, staining by the ordinary methods, but
not by Gram's method, producing acids, indol, and phenol, and co-
agulating milk.

The barnyards of both Europe and America are occa-
sionally visited by an epidemic disease known as " chicken-
cholera," Huhner cholera, or cholera de poule, which rapidly de-
stroys pigeons, turkeys, chickens, ducks, and geese. Rabbit-
warrens are also at times affected and the rabbits killed.

The bacillus responsible for this disease was first observed
by Perroncito* in 1878, and afterward thoroughly studied
by Toussaint and Pasteur, f

Morphology. — The organisms are short and broad, with
rounded ends, measuring i X 0.4 to 0.6 ^, sometimes joined
to produce chains. Pasteur at first Regarded them as diplo-
cocci, because the poles stain intensely, a narrow space
between them remaining almost uncolored. This pecu-
liarity is very marked, and careful examination is required
to detect the intermediate substance. The bacillus does not
form spores, is not motile, and has no flagella. |

Staining. — The organism stains with ordinary anilin dye
solutions, but not by Gram's method.

Cultivation. — Colonies. — Colonies upon gelatin plates
appear after about two days as small, irregular, white points.
The deep colonies reach the surface slowly, and do not attain
to any considerable size. The gelatin is not liquefied. The
colonies appear under the microscope as irregularly rounded
yellowish-brown disks with distinct smooth borders and
granular contents. Sometimes there is a distinct concentric
arrangement.

Gelatin. — In gelatin puncture cultures a delicate white
line occurs along the entire path of the wire. Upon the
surface the development is much more marked, so that the
growth resembles a nail with a good-sized flat head. If

* "Archiv. f. wissenschaftliche und praktische Thierheilkunde," 1879.
t " Compte-rendu de 1'Acad. de Sci. de Paris," vol. xc.
J Thoinot and Masselin assert that the organism is motile. "Precis
de Microbie," 2d ed., 1893.

Chicken Cholera 599

the bacilli be planted upon the surface of obliquely solidi-
fied gelatin, a much more pronounced growth takes place,
and along the line of inoculation a dry, granular coating is
formed. There is no liquefation of the medium.

Bouillon. — The growth in bouillon is accompanied by a
slight cloudiness.

Agar. — This growth, like that upon agar-agar and blood-

Fig. 207. — Bacillus of chicken-cholera, from the heart's blood of a
pigeon. X 1000 (Frankel and Pfeiffer).

serum, is white, shining, rather luxuriant, and devoid of
characteristics .

Potato. — Upon potato no growth occurs except at 37° C.
It is a very insignificant, yellowish-gray, translucent film.

Milk is acidulated and slowly coagulated.

Vital Resistance. — The bacillus readily succumbs to the
action of heat and dry ness. The organism is an obligatory
ae'robe.

Metabolic Products. — Indol and phenol are formed,
Acids are produced in sugar-containing media, without gas
formation.

6oo Micro-organisms of the Plague Group

Pathogenesis. — The introduction of cultures of this
bacillus into chickens, geese, pigeons, sparrows, mice, and
rabbits is sufficient to produce fatal septicemia. Feeding
chickens, pigeons, and rabbits with material infected with
the bacillus is also sufficient to produce the disease. Guinea-
pigs, cats, and dogs seem immune, though they may succumb
to large doses if given intraperitoneally. The organism is
probably harmless to man.

Fowls ill with the disease fall into a condition of weakness
and apathy, which causes them to remain quiet, seemingly
almost paralyzed, and the feathers ruffled up. The eyes
are closed shortly after the illness begins, and the birds
gradually fall into a stupor, from which they do not awaken.
The disease is fatal in from twenty-four to forty -eight hours.
During its course there is profuse diarrhea, with very fre-
quent fluid, slimy, grayish-white discharges.

Lesions. — The autopsy shows that when the bacilli are
introduced subcutaneously a true septicemia results, with
the formation of a hemorrhagic exudate and gelatinous
infiltration at the seat of inoculation. The liver and spleen
are enlarged; circumscribed, hemorrhagic, and infiltrated
areas occur in the lungs; the intestines show an intense
inflammation with red and swollen mucosa, and occasional
ulcers following small hemorrhages. Pericarditis is fre-
quent. The bacilli are found in all the organs. If, on the
other hand, the disease has been produced by feeding, the
bacilli are chiefly to be found in the intestine. Pasteur
found that when pigeons were inoculated into the pectoral
muscles, if death did not come on rapidly, portions of the
muscle (sequestra) underwent degeneration and appeared
anemic, indurated, and of a yellowish color.

Immunity. — Pasteur* discovered that when cultures are
allowed to remain undisturbed for several months, their
virulence becomes greatly lessened, and new cultures trans-
planted from them are also attenuated. If chickens be
inoculated with such attenuated cultures, no other change
occurs than a local inflammatory reaction that soon disap-
pears and leaves the birds protected against future infec-
tion with virulent bacilli. From these observations Pasteur

* An interesting account of Pasteur's experiments upon chicken-chol-
era can be found in the "Life of Pasteur, " by Vallery-Radot, translated
by Mrs. R. S. Devonshire, 1909. Popular Edition, New York, Double-
day, Page and Co.

Swine-plague 60 1

worked out a system of protective vaccination in which the
fowls are first inoculated with attenuated, then with more
active, and finally with virulent, cultures, with resulting
protection and immunity.

Use has been made of this bacillus to kill rabbits in Aus-
tralia, where they are pests. It is estimated that two gallons
of bouillon culture will destroy 20,000 rabbits, irrespective
of infection by contagion.

The bacillus of chicken-cholera may be identical with
organisms found in various epidemic diseases of larger
animals, and, indeed, no little confusion has arisen from
the description of what is now pretty generally accepted to
be the same organism as the bacillus of rabbit septicemia
(Koch), Bacillus cuniculicida (Fliigge), bacillus of swine-
plague (Loffler and Schiitz), bacillus of "Wildseuche"
(Hiippe), bacillus of "Biiffelseuche" (Oriste-Armanni), etc.

BACILLUS SUISEPTICUS (LOFFLER AND SCHUTZ).

General Characteristics. — A non-motile, non-flagellated, non-
sporogenous, non-liquefying, non-chromogenic, aerobic and optionally
anae'robic bacillus, pathogenic for hogs and many other animals, stain-
ing by the ordinary methods, but not by Gram's method. It produces
a slight acidity in milk, but does not coagulate it.

The bacillus of swine-plague, or Bacillus suisepticus of
Loffler and Schiitz* and Salmon and Smith, f but slightly
resembles the bacillus of hog-cholera (q. v.), though it was
formerly confounded with it and at one time thought to be
identical with it. The species have sufficient well-marked
characteristics, however, to make their differentiation easy.

Swine-plague is a rather common and exceedingly fatal
epidemic disease. It not infrequently occurs in association
with hog-cholera, and because of the lack of sufficiently well-
characterized symptoms — sick hogs appearing more or less
alike — is often mistaken for it. The confusion resulting from
such faulty diagnosis makes it difficult to determine exactly
how fatal either may be in uncomplicated cases.

Morphology. — The bacillus of swine-plague much re-
sembles that of hog-cholera, and not a little that of chicken-
cholera. It is a short organism, rather more slender than

: "Arbeiten aus den kaiserlichen Gesundheitsamte," I.
t "Zeitschrift f. Hygiene," x.

602 Micro-organisms of the Plague Group

the related species, not possessed of flagella, incapable of
movement, and producing no spores.

It is an optional anaerobe.

Staining. — The bacillus stains by the ordinary methods,
sometimes only at the poles, then closely resembling the
bacillus of chicken-cholera. It is not colored by Gram's
method.

Cultivation. — In general, the appearance in culture-media
is very similar to that of the hog cholera bacillus. Kruse,*
however, points out that when the bacillus grows in bouillon
the liquid remains clear, the bacteria gathering to form a
flocculent, stringy sediment. The organism does not grow
upon ordinary acid potato, but if the reaction of the me-
dium be alkaline, a grayish-yellow patch is formed. In milk
a slight acidity is produced, but the milk is not coagu-
lated.

Vital Resistance. — The vitality of the organism is low,
and it is easily destroyed. Salmon says that it soon dies in
water or when dried, and that the temperature for its growth
must be more constant and every condition of life more
favorable than for the hog-cholera germ. The organism is
said to be widely distributed in nature, and is probably
present in every herd of swine, though not pathogenic except
when its virulence becomes increased or the vital resistance
of the animals diminished by some unusual condition.

Pathogenesis. — While similar to hog-cholera, swine-
plague presents some marked differences, especially in
regard to the seat of the local manifestations, and in its
duration, which is much shorter. There is also considerable
resemblance to chicken-cholera, but the local reaction fol-
lowing the injection of the micro-organisms partakes of the
nature of a hemorrhagic edema, which is not present in
chicken-cholera, and rabbits commonly exhibit fatty meta-
morphosis of the liver.

Rabbits, mice, and small birds are very susceptible to
the disease, usually dying of septicemia in twenty-four
hours; guinea-pigs are less susceptible, except very young
animals, which die without exception. Chickens are more
immune, but usually succumb to large doses. Hogs die of
septicemia after subcutaneous injection of the bacilli. There
is a marked edema at the point of injection. If injected
into the lung, a pleuropneumonia follows, with multiple
* Fliigge's "Die Mikroorganismen," p. 419, 1896.

Swine-plague 603

necrotic areas in the lung. In these cases the spleen is not
much swollen, there is slight gastro-intestinal catarrh, and
the bacilli are present everywhere in the blood.

Animals can be infected only by subcutaneous, intra-
venous, and intraperitoneal inoculation, not by feeding.

As seen in hogs, the symptoms of swine-plague closely
resemble those of hog-cholera, but differ in the existence of
cough, swine-plague being prone to affect the lungs and
oppress the breathing, which becomes frequent, labored,
and painful, while hog-cholera is chiefly characterized by
intestinal symptoms.

The course of the disease is usually rapid, and it may be
fatal in a day or two.

Lesions. — At autopsy the lungs are found to be inflamed,
and to contain numerous small, pale, necrotic areas, -and
sometimes large cheesy masses one or two inches in diameter.
Inflammations of the serous membranes affecting the pleura,
pericardium, and peritoneum, and associated with fibrinous
inflammatory deposits on the surfaces, are common. There
may be congestion of the mucous membrane of the intes-
tines, particularly of the large intestine, or the disease in
this region may be an intense croupous inflammation with
the formation of a fibrinous exudative deposit on the surface.
A hemorrhagic form of the disease is said to be common
in Europe, but, according to Salmon, is rare in the United
States.

CHAPTER XXIV.
ASIATIC CHOLERA.

SPIRILLUM CHOLERA ASIATICS (Kocn*).

General Characteristics. — A motile, flagellated, non-sporogenoust
liquefying, non-chromogenic, non-aerogenic, parasitic and saprophytic,
pathogenic, aerobic and optionally anaerobic spirillum, staining by
ordinary methods, but not by Gram's method.

Cholera is a disease endemic in certain parts of India
and probably indigenous in that country. Though early
mention of it was made in the letters of travelers, and though
it appeared in medical literature and in governmental
statistics more than a century ago, we find that little
attention was paid to the disease, except in its disastrous
effect upon the armies, native and European, of India and
adjacent countries. The opening up of India by Great
Britain in the last half century made scientific observation
of the disease possible and permitted us to determine the
relation its epidemics bear to the manners and customs
of the people.

The filthy habits of the Oriental people, their poverty,
crowded condition, and peculiar religious customs, are all
found to aid in the distribution of the disease. Thus, the
city of Benares drains into the Ganges River by a mcst
imperfect system, which distributes the greater part of
the sewage immediately below the banks upon which the
city is built and along which are the numerous "Ghats"
or staircases by which the people reach the sacred waters.
It is a matter of religious observance for every zealot who
makes a pilgrimage to the " sacred city" to take a bath in
and drink a quantity of this sacred but polluted water,
and it may be imagined that the number of pious Hindoos
who leave Benares with "comma bacilli" in their intestines
or upon their clothes must be great, for there are few months
in the year when the city is exempt from cholera.

* "Deutsche med. Wochenschrift," 1884-1885, Nos. 19, 20, 37, 38,
and 39.

604

Distribution 605

The pilgrimages and great festivals of the Hindoos and
Moslems, by bringing together enormous numbers of people
to crowd in close quarters where filth and bad diet prevail,
cause a rapid increase in the number of cases during these
periods and facilitate the distribution of the disease when
the festivals break up. Probably no more favorable con-
ditions for the dissemination of a disease can be imagined
than occurs with the return of the Moslem pilgrims from
Mecca. The disease extends readily along the regular lines
of travel, visiting town after town, until from Asia it has
frequently extended into Europe, and by steamships plying
foreign waters has several times been carried to our own
continent. Many cases are on record which show conclu-
sively how a single ship, having a few cholera cases on
board, may be the starting-point of an outbreak of the
disease in the port at which it arrives.

The most recent great epidemic of cholera began in 1883.
From Asia it spread westward throughout Europe, extended
by means of the steamship lines to numerous of the large
ports, of which Hamburg in Germany suffered most acutely,
and even extended to some of the ports of Africa and America.
Russia probably suffered more than any other European
country, and it is estimated that in that country there were
no less than 800,000 deaths. During 1911 the disease
again appeared in Europe and invaded the countries along
the Mediterranean coasts.

Specific Organism. — The discovery of the spirillum of
cholera was made by Koch while serving as a member of a
German commission appointed to study the disease in Egypt
and India in 1883-84. Since its discovery the spirillum has
been subjected to much careful investigation, and an im-
mense amount of literature, a large part of which was stim-
ulated by the Hamburg epidemic of 1892, has accumulated.

Distribution. — The cholera spirilla can be found with
great regularity in the intestinal evacuations of cholera
cases, and can often be found in drinking-water and milk,
and upon vegetables, etc., in cholera-infected districts.
There can be little doubt that they find their way into
the body with the food and drink. Cases in the literature
show how cholera germs enter drinking-water and are thus
distributed ; how they are sometimes thoughtlessly sprinkled
over green vegetables offered for sale in the streets, with
infected water from polluted gutters; how they enter milk
with water used to dilute it ; how they appear to be carried

6o6

Asiatic Cholera

about in clothing and upon food-stuffs; how they can be
brought to articles of food by flies that have preyed upon
cholera excrement ; and other interesting modes of infection.
The literature is so vast that it is scarcely possible to men-
tion even the most instructive examples. A bacteriologist
became infected while experimenting with the cholera spirilla
in Koch's laboratory. It is commonly supposed that the
cholera organism may remain alive in water for an almost
unlimited length of time, but experiments have not shown
this to be the case. Thus, Wolffhiigel and Riedel have
shown that if the spirilla be planted in sterilized water they
grow with great rapidity after a short time, and can be found
alive after months have passed. Frankel, however, points
out that this ability to grow and remain vital for long peri-
ods in sterilized water does not guarantee the same power
of growth in unsterilized water, for in the latter the simul-
taneous growth of other bacteria serves to extinguish the
cholera spirilla in a few days.

J

J

Fig. 208. — Cholera spirilla.

Morphology. — The micro-organism described by Koch,
and now generally accepted to be the cause of cholera, is
a short rod i to 2 ^ in length and 0.5 ^ in breadth, with rounded
ends, and a distinct curve, so that the original name by which
it was known, the " comma bacillus," applies very well. One
of the most common forms is that in which two short curved
individuals are conjoined in an S-shaped curve.

When the conditions of nutrition are good, multipli-

The Comma Bacillus 607

cation by fission progresses with rapidity; but when ad-
verse conditions arise, long spiral threads — unmistak-
able spirilla — develop. Frankel found that the exposure
of the cultures to unusually high temperatures, the addition
of small amounts of alcohol to the culture-media, and other
unfavorable conditions lead to the production of spirals
instead of "commas."

The cholera spirilla are actively motile, and in hanging-
drop preparations can be seen to swim about with great
rapidity. Both comma- shaped and spiral organisms move
with a rapid rotary motion.

The presence of flagella can be demonstrated without

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Coa*« <i.t\

s*-* ;%%•

Fig. 209. — Spirillum of Asiatic cholera, from a bouillon culture three
weeks old, showing long spirals. X 1000 (Frankel and Pfeiffer).

difficulty. Each spirillum possesses a single nagellum
attached to one end (spiromonas).

Involution-forms of bizarre appearance are common in
old cultures of the spirillum, and sometimes in fresh cultures
many individuals show by granular cytoplasm and irregular
outline that they are degenerated. Cholera spirilla from
various sources differ in the extent of involution.

In partially degenerated cultures containing long spirals
Hiippe observed, by examination in the "hanging-drop,"
certain large spheric bodies which he described as spores
(arthrospores). Koch and, indeed, all other observers fail

608

Asiatic Cholera

to find spores in the cholera organism, and the nature of
the bodies described by Hiippe must be regarded as doubtful.
Staining. — The cholera spirillum stains well with the
ordinary aqueous solutions of the anilin dyes, especially
fuchsin. At times the staining must be continued for from
five to ten minutes to secure homogeneity. The organism
does not stain by Gram's method. It may be colored and
examined while alive; thus, Cornil and Babes, in demon-
strating it in the rice-water discharges, "spread out one
of the white mucous fragments upon a glass slide and allow
it to dry partially; a small quantity of an exceedingly

Fig. 210. — Cover-glass preparation of a mucous floccule in Asiatic
cholera. X 650 (Vierordt).

weak solution of methyl violet in distilled water is then
applied to it, and it is flattened out by pressing down
a cover-glass, over which is placed a fragment of filter
paper, which absorbs any excess of fluid at the margin of
the cover-glass. The characteristics of comma bacilli so
prepared and examined with an oil-immersion lens (X 700-
800) are readily made out because of the slight stain they
take up, and because they still retain the power of vigor-
ous movement, which would be entirely lost if the specimen
were dried, stained, and mounted in the ordinary fashion."

Isolation of the Organism.— One of the best methods
of securing a pure culture of the cholera spirillum, and also

Cultivation

609

of making a bacteriologic diagnosis of the disease in a
suspected case, is probably that of Schottelius.

A small quantity of the fecal matter is mixed with bouillon and
stood in an incubating oven for twenty-four hours. If the cholera
spirilla are present they will grow most rapidly at the surface of the
liquid where the supply of air is good. A pellicle will be formed, a
drop from which, diluted in melted gelatin and poured upon plates,
will show typical colonies.

Cultivation. — The cholera organism is easily cultivated,
and grows luxuriantly upon the usual laboratory media.

Fig. 211. — Spirillum of Asiatic cholera; colonies two days old upon
a gelatin plate. X 35 (Heim).

Colonies. — The colonies grown upon gelatin plates are
characteristic and appear in the lower strata of the gelatin
as small white dots, which gradually grow out to the surface,
effect a slow liquefaction of the medium, and then appear
to be situated in little pits with sloping sides (Fig. 211).
This appearance suggests that the plate is full of little holes
or air-bubbles, and is due to the slow evaporation of the
liquefied gelatin.

Under the microscope the colony of the cholera spirillum
is fairly well characterized. The little colonies that have
39

6io

Asiatic Cholera

not yet reached the surface of the gelatin soon show a
pale yellow color and an irregular contour. They are
coarsely granular, the largest granules being in the center.
As the colony increases in size the granules do the same
and attain a peculiar transparent character suggestive of
powdered glass. The slow liquefaction causes the colony to
be surrounded by a transparent halo. As the liquefied
gelatin evaporates, the colony begins to sink, and also to
take on a peculiar rosy color.

Gelatin. — In puncture cultures in gelatin the growth is

Fig. 212. — Spirillum cholerae asiaticae; gelatin puncture cultures aged
forty-eight and sixty hours (Shakespeare).

again quite characteristic (Fig. 212). It occurs along
the entire puncture, best at the surface, where it is in
contact with the atmosphere. Liquefaction of the medium
begins almost at once, keeps pace with the growth, but
is always more marked at the surface than lower down.
The result of this is the formation of a short, rather wide
funnel at the top of the puncture. As the growth continues,
evaporation of the medium takes place slowly, so that the
liquefied gelatin is lower than the solid surrounding portions,
and the growth appears to be surmounted by an air-bubble.
The luxuriant development of the spirilla in the liquefying

Vital Resistance 611

gelatin is followed by the formation of considerable sediment
in the lower third or half of the liquefied area. This solid
material consists of masses of spirilla which have probably
completed their life-cycle and become inactive. Under the
microscope they exhibit the most varied involution-forms.
The liquefaction reaches the sides of the tube in from five
to seven days, but is not complete for several weeks.

Agar-agar. — When planted upon the surface of agar-
agar the spirilla produce a grayish-white, shining, translu-
cent growth along the entire line of inoculation. It is in
no way peculiar or characteristic. The vitality of the
organism is retained much better upon agar-agar than upon
gelatin, and, according to Frankel, the organism can be
transplanted and grown when nine months old.

Blood-serum. — The growth upon blood-serum is also
without distinct peculiarities; gradual liquefaction of the
medium occurs.

Potato. — Upon potato the spirilla grow well, even when
the reaction is acid. In the incubator, at a temperature of
37° C., a transparent, slightly brownish or yellowish-brown
growth, somewhat resembling that of glanders, is produced.
It contains large numbers of long spirals.

Bouillon. — In bouillon and in peptone solution the
cholera organisms grow well, especially upon the surface,
where a folded, wrinkled pellicle is formed, the culture
fluid remaining clear.

Milk. — In milk the growth is luxuriant, but does
not visibly alter its appearance. The existence of cholera
organisms in milk is, however, rather short-lived, for the
occurrence of acidity at once destroys them.

Vital Resistance. — Although an organism that multiplies
with great rapidity under proper conditions, the cholera
spirillum does not possess much resisting power. Stern-
berg found that it was killed by exposure to 52° C. for
four minutes, but Kitasato found that ten or fifteen
minutes' exposure to 55° C. was not always fatal to it. In
a moist condition the organism may retain its vitality for
months, but it is very quickly destroyed by desiccation, as
was found by Koch, who observed that when dried in a thin
film its power to grow was destroyed in a few hours. Kita-
sato found that upon silk threads the vitality might be
retained longer. Abel and Claussen * have shown that it

* "Centralbl. f. Bakt. u. Parasitenk.," Jan. 31, 1895, vol. xvn, No. 4.

612 Asiatic Cholera

does not live longer than twenty or thirty days in fecal
matter, and often disappears in from one to three days.
The organism is very susceptible to the influence of carbolic
acid, bichlorid of mercury, and other germicides, and is
also destroyed by acids. Hashimoto * found that it could
not live longer than fifteen minutes in vinegar containing
2.2-3.2 per cent, of acetic acid.

According to Frankel, in eight weeks the organisms in
the liquefied cultures all die, and cannot be transplanted.
Kitasato, however, has found them living and active on
agar-agar after from ten to thirty days, and Koch was
able to demonstrate their vitality after two years.

This low vital resistance of the microbe is very fortunate,
for it enables us to establish satisfactory quarantine for the
prevention of the spread of the disease. Excreta, soiled
clothing, etc., are readily rendered harmless by the proper
use of disinfectants. Water and food are rendered innocu-
ous by boiling or cooking. Vessels may be disinfected by
thorough washing with jets of boiling water discharged
through a hose connected with a boiler, and baggage can
be sterilized by superheated steam.

Metabolic Products. — Indol is one of the characteristic
metabolic products of the cholera spirillum. As the cholera
organisms also produce nitrites, all that is necessary to
demonstrate its presence in a colorless solution is to add a
drop or two of chemically pure sulphuric acid, when the
well-known reddish color will appear.

The organism also produces acid in milk and other
media. Bitter has also shown that the cholera organism
produces a peptonizing and probably also a diastatic
ferment.

Toxic Products.— Rietsch thinks the intestinal changes
depend upon the action of the peptonizing ferment. Can-
tani, Nicati and Rietsch, Van Ermengem, Klebs, and others
found toxic effects from cultures administered to dogs
and other animals. Several toxic metabolic products of
the spirilla have been isolated. Brieger, f Brieger and
Frankel, { Gamaleia, § Sobernheim, || and Villiers have

* "Kwai Med. Jour.," Tokyo, 1893.

f "Berliner klin. Wochenschrift," 1887, p. 817.

J " Untersuchungen iiber die Bakteriengifte," etc., Berlin, 1890.

§ "Archiv de Med. exp.," iv, No. 2.

|| "Zeitschrift fur Hygiene," xiv, 145, 1893.

Pathogenesis 613

studied more or less similar toxic products. The real toxic
substance is, however, not known.

Pathogenesis. — Through what activity the cholera or-
ganism provokes its pathogenic action is not yet deter-
mined. The organisms, however, abound in the intestinal
contents, penetrate sparingly into the tissues, but slightly
invade the lymphatics, and almost never enter the circula-
tion ; hence it is but natural to conclude that the first ac-
tion must be an irritative one depending upon toxin-for-
mation in the intestine.

In the beginning of the disease the small and large intes-
tines are deeply congested, almost velvety in appearance,
and contain liquid fecal matter. The patient suffers from
diarrhea, by which the feces are hurried on and be-
come extremely thin from the admixture of a copious
watery exudate. As the feces are hurried out, more and
more of the aqueous exudate accumulates, until the intes-
tine seems to contain only watery fluid. The solitary
glands and Peyer's patches are found enlarged and the
mucosa becomes macerated and necrotic, its epithelium
separating in small shreds or flakes. The evacuations of
watery exudate rich in these shreds constitute the char-
acteristic "rice-water discharges" of the disease. As the
disease progresses, the denudation of tissue results in the
formation of good-sized ulcerations. Perforations and deep
ulcerations are rare. Pseudo-membranous formations not
infrequently occur upon the abraded and ulcerated surfaces.
The other mucous membranes of the alimentary apparatus
become congested and abraded; the parenchyma of the
liver, kidneys, and other organs becomes markedly de-
generated, so that the urine becomes highly albuminous
and very scanty in consequence of the anhydremia. The
cardio-vascular, nervous, and respiratory systems present
no characteristic changes.

So far as is known, cholera is a disease of human beings only,
and never occurs spontaneously in the lower animals.

Intraperitoneal injection of the virulent cultures produces
fatal peritonitis in guinea-pigs.

Supposing that the lower animals were immune against
cholera because of the acidity of the gastric juice, Nicati
and Rietsch,* Van Ermengem, and Kochf have suggested
methods by which the micro-organisms can be introduced

*"Deutsch. med. Wochenschrift," 1884.
t Ibid., 1885.

6 14 Asiatic Cholera

directly into the intestine. The first-named investigators
ligated the common bile-duct of guinea-pigs, and then
injected the spirilla into the duodenum with a hypodermic
needle, with the result that the animals usually died, some-
times with choleraic symptoms. The excessively grave
nature of the operation upon such a small and delicately
constituted animal as a guinea-pig, however, greatly lessens
the value of the experiment. Koch's method of infection
by the mouth is much more satisfactory. By injecting
laudanum into the abdominal cavity of guinea-pigs the
peristaltic movements of the intestine can be checked. The
amount necessary for the purpose is large and amounts to
about i gram for each 200 grams of body-weight. It
completely narcotizes the animals for a short time (one to
two hours), but they recover without injury. The contents
of the stomach are neutralized after administering the
opium, by introducing 5 c.c. of a 5 per cent, aqueous solution
of sodium carbonate through a pharyngeal catheter. With
the gastric contents thus alkalinized and the peristalsis
paralyzed, a bouillon culture of the cholera spirillum is
introduced through the stomach-tube. The animal recovers
from the manipulation, but shows an indisposition to eat,
is soon observed to be weak in the posterior extremities,
subsequently is paralyzed, and dies within forty-eight hours.
The autopsy shows the intestine congested and filled with
a watery fluid rich in spirilla — an appearance which Frankel
declares to be exactly that of cholera. In man, as well
as in these artificially infected animals, the spirilla are
never found in the blood or tissues, but only in the intestine,
where they frequently enter between the basement mem-
brane and the epithelial cells, and aid in the detachment of
the latter.

Issaeff and Kolle found that when virulent cholera spirilla
are injected into the ear- veins of young rabbits the animals
die on the following day with symptoms resembling the
algid state of human cholera. The autopsy in these cases
showed local lesions of the small intestine very similar to
those observed in cholera in man.

Guinea-pigs are also susceptible to intraperitoneal injec-
tions of the spirillum, and speedily succumb. The symp-
toms are rapid fall of temperature, tenderness over the
abdomen, and collapse. The autopsy shows an abundant
fluid exudate containing the micro-organisms, and injection
and redness of the peritoneum and viscera.

Detection of the Organism 615

Specificity. — The cholera spirillum is present in the de-
jecta of cholera with great regularity, and as regularly absent
from the dejecta of healthy individuals and those suffering
from other diseases. There is no satisfactory proof of the
specific nature of the organisms to be obtained by experi-
mentation upon animals. Animals are never affected by
any disease similar to cholera during epidemics, nor do foods
mixed with cholera discharges or with pure cultures of the
cholera spirillum affect them. Subcutaneous inoculations
do not produce cholera.

Detection of the Organism. — It often becomes a matter
of importance to detect the cholera spirilla in drinking-
water, and, as the number in which the bacteria exist in
such a liquid may be very small, difficulty may be experienced
in finding them by ordinary methods. One of the most
expeditious methods is that recommended by LofHer, who
adds 200 c.c. of the water to be examined to 10 c.c. of
bouillon, allows the mixture to stand in an incubator for
from twelve to twenty-four hours, and then makes plate
cultures from ^the superficial layer of the liquid, where, if
present, the development of the spirilla will be most rapid
because of the free access of air.

Gordon * employs a medium composed of Lemco i gram,
peptone i gram, sodium bicarbonate o. i gram, starch i
gram, and distilled water 100 c.c. for the differentiation
of the cholera and Finkler-Prior spirilla. If the medium
be tinted with litmus and the cultures grown at 37° C.,
a strongly acid change is produced by the true cholera
organism in twenty-four hours. The Finkler-Prior spirillum
produces but slight acidity, which first appears about the
third day.

The identification of the cholera spirillum, and its differen-
tiation from spiral organisms of similar morphology obtained
from feces or water in which no cholera organisms are
expected, is becoming less and less easy as our knowledge
of the organisms increases. The following points may be
taken into consideration: (i) The typical morphology.
The true cholera organism is short, has a single curve, is
rounded at the ends, and possesses a single flagellum. (2)
The infectivity. Freshly isolated cultures should be patho-
genic for guinea-pigs and harmless to pigeons. (3) Vegeta-

* "British Medical Journal," July 28, 1906.

616 Asiatic Cholera

live: The organism should liquefy 10 per cent, gelatin and
should not coagulate milk. (4) Metabolic: Indol reaction
should be marked. (5) Immunity reactions: The organism
when injected into guinea-pigs in ascending doses should
occasion immunity against the typical cholera organism,
and the serum of the immunized guinea-pig, when intro-
duced into a new guinea-pig, should protect it from infection
and produce Pfeiffer's phenomenon. The blood-serum of
animals immunized against the cholera organism should
agglutinate the doubtful organism in approximately the
same dilution, and that of animals immunized to the doubtful
organism should agglutinate the cholera organism recipro-
cally. Both organisms should have equal capacity for
absorbing complements and amboceptors from blood-serum.
(6) The true cholera organism should not be hemolytic.
Too much reliance must not be placed upon the agglu-
tination tests alone, as will be made clear by a perusal of
the paper upon Bacteriological Diagnosis of Cholera by
Ruffer.*

Immunity. — One attack of cholera usually leaves the vic-
tim immune from further attacks of the disease. Gruber
and Wiener,! Haffkine, } Pawlowsky, § and Pfeiffer|| have im-
munized animals against toxic substances from cholera cul-
tures or against living cultures.

Pfeiffer and Vogedes** have applied the "immunity re-
action" to the differentiation of cholera spirilla in cultures.
A hanging drop of a i : 50 mixture of a powerful anticholera
serum and a particle of cholera culture is made and ex-
amined under the microscope. The cholera spirilla at once
become inactive, and are in a short time converted into
little rolled-up masses. If the culture added be a spirillum
other than the true cholera spirillum, instead of being
destroyed the micro-organisms multiply and thrive in the
mixture of serum and bouillon.

Sobernheimft found the Pfeiffer reaction specific against

* "British Medical Journal," March 30, 1907, i, p. 735.
t "Centralbl. f. Bakt.," etc., 1892, xiv, p. 76.

J"L,e Bull, med.," 1892, p. 1113, and "Brit. Med. Jour.," 1893,
p. 278.

§ "Deutsche med. Wochenschrift," 1893, No. 22.
|| "Zeitschrift fur Hygiene," Bd. xvm and xx.

** "Centralbl. f. Bakt. u. Parasitenk.," March 21, 1896, Bd. xix,
No. 1 1 .

t| "Zeitschrift fur Hygiene," xx, p. 438.

Serum Therapy and Prophylaxis 617

cholera alone, and thought the protection not due to the
strongly bactericidal property of the serum, but to its
stimulating effect upon the body-cells; for if the serum be
heated to 6o°-7o° C., and its bactericidal power thus de-
stroyed, it is still capable of producing immunity; This,
of course, is in keeping with our present knowledge of
the immune body, which is not destroyed by such tem-
peratures.

The immunity produced by the injection of the spirilla
into guinea-pigs continues in some cases as long as four and
a half months, but the power of their serum to confer
immunity is lost much sooner.

Serum Therapy and Prophylaxis. — Of the numerous
attempts to produce immunity against cholera in man or
to cure cholera when once established in the human organ-
ism, nothing very favorable can be said. Experiments in
this field are not new. As early as 1885 Ferran, in Spain,
administered hypodermic injections of pure virulent cul-
tures of the cholera spirillum, in the hope of bringing
about immunity. The work of Haffkine,* however, is the
chief important contribution, and his method seems to be
followed by a positive diminution of mortality in protected
individuals. Haffkine uses two vaccines — one mild, the
other so powerful that it would bring about extensive tissue-
necrosis and perhaps death if used alone. His studies
embrace more than 40,000 inoculations performed in India.
The following extract will show results obtained in 1895:

"1. In all those instances where cholera has made a large number
of victims, — that is to say, where it has spread sufficiently to make
it probable that the whole population, inoculated and uninoculated,
were equally exposed to the infection, — in all these places the results
appeared favorable to inoculation.

"2. The treatment applied after an epidemic actually breaks out
tends to reduce the mortality even during the time which is claimed
for producing the full effect of the operation. In the Goya Garl,
where weak doses of a relatively weak vaccine had been applied, this
reduction was to half the number of deaths ; in the coolies of the Assam-
Burmah survey party, where, as far as I can gather from my pre-
liminary information, strong doses have been applied, the number
of deaths was reduced to one-seventh. This fact would justify the
application of the method independently of the question as to the
exact length of time during which the effect of this vaccination lasts.

"3. In Lucknow, where the experiment was made on small doses
of weak vaccines, a difference in cases and deaths was still noticeable
in favor of the inoculated fourteen to fifteen months after vaccination

*"Le Bull, med.," 1892, p. 1113; "Indian Med. Gazette," 1893,
p. 97; "Brit. Med. Jour.," 1893, p. 278.

618 Asiatic Cholera

in an epidemic of exceptional virulence. This makes it probable
that a protective effect could be obtained even for long periods ol
time if larger doses of a stronger vaccine were used.

"4. The best results seem to be obtained from application of middle
doses of both anticholera vaccines, the second one being kept at the
highest possible degree of virulence obtainable.

"5. The most prolonged observations on the effect of middle doses
were made in Calcutta, where the mortality from the eleventh up to
the four hundred and fifty-ninth day after vaccination was, among
the inoculated, 17.24 times smaller, and the number of cases 19.27
times smaller than among the not inoculated."

Pawlowsky and others have found the dog susceptible to
cholera, and have utilized it in the preparation of an anti-
toxic serum. The dogs were first immunized against
attenuated cultures, then against more and more virulent
cultures, until a serum was obtained whose value was
estimated at i : 130.000 upon experimental animals.

Freymuth * and others have endeavored to secure favor-
able results from the injection of blood-serum from con-
valescent patients into the diseased. One recovery out
of three cases treated is recorded.

In all these preliminaries the foreshadowing of a future
therapeusis must be evident, but as yet nothing satis-
factory has been achieved.

One of the chief errors made in the experimental prepara-
tion of anticholera serums is that efforts have been directed
toward endowing the blood with the power of resisting
and destroying the bacteria that rarely, if ever, reach it.
The two essentials to be aimed at are an antitoxin to neu-
tralize the depressing effects of the toxalbumin, and some
means of destroying the bacteria in the intestine.

The cholera spirillum is one of a considerable-sized group
of closely related organisms, from some of which it is dif-
ferentiated with difficulty.

Sanitation. — The first appearance of cholera may depend
upon the introduction of the micro-organisms upon fomites,
hence to avoid epidemics it is necessary to disinfect all such
coming from cholera infected localities. Especially are food-
stuffs and particularly raw vegetables and fruits to be con-
sidered. All such should either be avoided or only eaten after
cooking.

So soon as cholera asserts itself, the chief danger lies in the
probable contamination of the water-supply. To prevent
this the utmost effort must be made to locate all cases and see

* "Deutsche med. Wochenschrift," 1893, No. 43.

Cultivation 619

that the dejecta are thoroughly disinfected, and as the micro-
organisms persist in the intestinal discharges for some weeks
after convalescence, the patients should not too soon be dis-
charged from the hospital, but should be retained until a
bacteriologic examination shows no more comma bacilli in
the feces. During an epidemic the water consumed should all
be boiled, raw milk should be avoided, and no green or un-
cooked vegetables or fruits eaten. Foods should be carefully
defended from flies, which may carry the organisms to them
and infect them. The intestinal evacuations and all the
clothing, bedding, and other articles used by the patients
should be carefully disinfected.

SPIRILLA RESEMBLING THE CHOLERA SPIRILLUM.
THE FINKLER AND PRIOR SPIRILLUM (VIBRIO PROTEUS).

Similar in morphology to the spirillum of cholera, and in
other respects closely related to it, is the spirillum obtained
from the feces of a case of cholera nostras by Finkler and
Prior.*

Morphology. — It is shorter and stouter, with a more
pronounced curve than the cholera spirillum, and rarely
forms long spirals. The central portion is also somewhat
thinner than the ends, which are a little pointed and give
the organism a less uniform appearance. Involution
forms are common in cultures, and appear as spheres,
spindles, clubs, etc. Like the cholera spirillum, each organ-
ism is provided with a single flagellum situated at its end,
and is actively motile.

Staining. — The organism stains readily with the ordinary
solutions, but not by Gram's method.

Cultivation. — Colonies. — The growth upon gelatin
plates is rapid, and leads to such extensive liquefaction
that four or five dilutions must frequently be made to se-
cure few enough organisms to enable one to observe the
growth of a single colony. To the naked eye the deep colo-
nies appear as small white points. They rapidly reach the
surface, begin liquefaction of the gelatin, and by the sec-
ond day appear about the size of lentils, and are situated
in little depressions. Under the microscope they are
yellowish brown, finely granular, and are surrounded by

*"Centralbl. fur allg. Gesundheitspflege," Bd. I, Bonn, 1885;
"Deutsche med. Wochenschrift," 1884, p. 632.

620 The Finkler and Prior Spirillum

a zone of sharply circumscribed liquefied gelatin. Careful
examination with a high-power lens shows rapid movement
of the granules in the colony.

Gelatin Punctures. — In gelatin punctures the growth
takes place rapidly along the whole length of the puncture,
forming a stocking-shaped liquefaction filled with cloudy
fluid which does not precipitate rapidly; a rather smeary,
whitish scum is usually formed upon the surface. The
more extensive and more rapid the liquefaction of the
medium, the wider the top to the funnel, the absence of the
air-bubble, and the clouded nature of the liquefied material,

^HP**

Fig. 213. — Spirillum of Finkler and Prior, from an agar-agar culture.
X 1000 (Itzerott and Niemann).

all serve to differentiate the culture from the cholera spiril-
lum.

Agar-agar. — Upon agar-agar the growth is also rapid,
and in a short time the whole surface of the culture medium
is covered with a moist, thick, slimy coating, which may
have a slightly yellowish tinge.

Bouillon. — In bouillon the organism causes a diffuse
turbidity with a more or less distinct pellicle on the surface.
In sugar-containing culture media it causes no fermenta-
tion and generates no gas.

Potato. — The cultures upon potato are also different
from those of the cholera organism, for the Finkler and Prior
spirilla grow rapidly at the room temperature, and produce

Cultivation

621

Fig. 214. — Spirillum of Finkler and Prior; colony twenty-four hours
old, as seen upon a gelatin plate. X 100 (Frankel and Pfeiffer).

Fig. 215. — Spirillum of Finkler and Prior; gelatin puncture cultures
aged forty-eight and sixty hours (Shakespeare).

622 Spirillum of Denecke

a grayish-yellow, slimy shining layer, which may cover the
whole of the culture medium.

Blood-serum. — Blood-serum is rapidly liquefied by the
organism.

The spirillum does not grow well in milk, and speedily dies
in water.

Metabolic Products. — The organism does not produce
indol. Buchner has shown that in media containing some
glucose an acid reaction is produced. Proteolytic enzymes
capable of dissolving gelatin, blood-serum, and casein are
formed.

Pathogenesis. — It was at first supposed that if not the
spirillum of cholera itself, this was a very closely allied
organism. Later it was supposed to be the cause of cholera
nostras. At present it is a question whether the organism
has any pathologic significance. It was in one case secured
by Knisl from the feces of a suicide, and has been found in
carious teeth by Miiller.

When injected into . the stomach of guinea-pigs treated
with tincture of opium according to the method of Koch,
about 30 per cent, of the animals die, but the intestinal
lesions produced are not identical with those produced by
the cholera spirillum. The intestines in such cases are pale
and filled with watery material having a strong putrefactive
odor. This fluid teems with the spirilla.

It seems unlikely, from the evidence thus far collected,
that the Kinkier and Prior spirillum is pathogenic for the
human species. As Krankel points out, it is probably a
frequent and harmless inhabitant of the human intestine.

THE SPIRILLUM OF DENECKE (VIBRIO TYROGENUM).

Another organism with a partial resemblance to the
cholera spirillum was found by Denecke * in old cheese.

Morphology. — Its form is similar to that of the cholera
spirillum, the shorter individuals being of equal diameter
throughout. The spiral forms are longer than those of the
Kinkier and Prior spirillum, and are more tightly coiled than
those of the cholera spirillum.

Like its related species, tLis micro-organism is actively
motile and possesses a terminal flagellum.

* "Deutsche med. Wochenschrift," 1885.

Cultivation 623

Cultivation. — It grows at the room temperature, as well
as at 37° C., in this respect, as in its reaction to stains, much
resembling the other two.

Colonies. — Upon gelatin plates the growth of the colonies
is much more rapid than that of the cholera spirillum,
though slower than that of the Kinkier and Prior spirillum.
The colonies appear as small whitish, round points, which

Fig. 216. — Spirillum of Denecke, from an agar-agar culture. X 1000
(Itzerott and Niemann).

soon reach the surface of the gelatin and commence lique-
faction. By the second day each is about the size of a
pin's head, has a yellow color, and occupies the bottom of
a conical depression. The appearance is much like that
of colonies of the cholera spirillum.

The microscope shows the colonies to be of irregular
shape and coarsely granular, pale yellow at the edges,
gradually becoming intense toward the center, and at first
circumscribed, but later surrounded by clear zones, resulting
from the liquefaction of the gelatin. These, according to
the illumination, appear pale or dark. The colonies differ
in appearance from those of cholera in the prompt lique-
faction of the gelatin, their rapid growth, yellow color,
irregular form, and distinct lines of circumscription.

Gelatin Punctures. — In gelatin punctures the growth
takes place all along the track of the wire, and forms a
cloudy liquid which precipitates at the apex in the form of

624 Spirillum of Denecke

a coiled mass. Upon the surface a delicate, imperfect,
yellowish scum forms. Liquefaction of the entire gelatin
generally requires about two weeks.

Agar-agar. — Upon agar-agar this spirillum forms a thin
yellowish layer which spreads quickly over most of the sur-
face.

Bouillon. — In bouillon the growth of the organism is
characterized by a diffuse turbidity. No gas-formation
occurs in sugar-containing media.

Fig. 217.— Spirillum of Denecke; gelatin puncture cultures aged forty-
eight and sixty hours (Shakespeare).

Potatoes. — The culture upon potato is luxuriant if
grown in the incubating oven. It appears as a distinct
yellowish, moist film, and when examined microscopically
is seen to contain beautiful long spirals.

Metabolic Products. — The organism produces no indol.

Pathogenesis. — The spirillum of Denecke is mentioned
only because of its morphologic resemblance to the cholera
spirillum. It is not associated with any human disease.
Experiments, however, have shown that when the spirilla
are introduced into guinea-pigs whose gastric contents are
alkalinized and whose peristalsis is paralyzed with opium,
about 20 per cent, of the animals die.

Cultivation 625

THE SPIRILLUM OF GAMAL£IA* (VIBRIO METSCHNIKOVI).

Resembling the cholera spirillum in morphology and
vegetation, and possibly, as has been suggested, a de-
scendant of the same original stock, is a spirillum which
Gamaleia cultivated from the intestines of chickens affected
with a disease similar to chicken-cholera.

Morphology. — This spirillum is a trifle shorter and
thicker than the cholera spirillum. It is a little more

Fig. 2 1 8. — Spirillum metschnikovi, from an agar-agar culture. X 1000
(Itzerott and Niemann)

curved, and has similar rounded ends. It forms long spirals
in appropriate media, and is actively motile. Bach spi-
rillum is provided with a terminal flagellum. No spores
have been demonstrated.

Staining. — The organism stains easily, the ends more
deeply than the center. It is not stained by Gram's method.

Cultivation. — It grows well both at the temperature of
the room and at that of incubation.

Colonies. — The colonies upon gelatin plates have a
marked resemblance to those of the cholera spirillum, yet
there is a difference; and as Pfeiffer says, "it is compara-
tively easy to differentiate between a plate of pure cholera
spirillum and a plate of pure Spirillum metschnikovi, yet it

* "Ann. de 1'Inst. Pasteur," 1888.
40

626 Spirillum of Gamaleia

is almost impossible to pick out a few colonies of the latter
if mixed upon a plate with the former."

Frankel regards this organism as a species intermediate
between the cholera and the Kinkier- Prior spirilla.

The colonies upon gelatin plates appear in about twelve
hours as small whitish points, and rapidly develop, so that
by the end of the third day large saucer-shaped liquefactions
resembling colonies of the Finkler- Prior spirilla occur. The
liquefaction of the gelatin is quite rapid, the resulting fluid
being turbid. Usually, upon a plate of Vibrio metschnikovi
some colonies are present which closely resemble those of

Fig. 219.— Spirillum metschnikovi; puncture culture in gelatin forty-
eight hours old (Frankel and Pfeiffer).

the cholera spirillum, being deeply situated in conical de-
pressions in the gelatin. Under the microscope the contents
of the colonies, which appear of a brownish color, are ob-
served to be in rapid motion. The edges of the bacterial
mass are fringed with radiating organisms

Gelatin Punctures. — In gelatin tubes the growth closely
resembles that of the cholera organism, but develops more
slowly.

Agar-agar. — Upon the surface of agar-agar a yellowish-
brown growth develops along the whole line of inoculation.

Potato. — On potato at the room temperature no growth
occurs, but at the temperature of the incubator a luxuriant

Metabolic Products 627

yellowish-brown growth takes place. Sometimes the color
is quite dark, and chocolate-colored potato cultures are not
uncommon.

Bouillon. — In bouillon the growth which occurs at the
temperature of the incubator is quite characteristic, and
very different from that of the cholera spirillum. The
entire medium becomes clouded, of a grayish-white color,
and opaque. A folded and wrinkled pellicle forms upon the
surface.

Milk. — When grown in litmus milk, the original blue
color is changed to pink in a day, and at the end of another
day the color is all destroyed and the milk coagulated.
Ultimately the clots of casein sediment in irregular masses,
from the clear, colorless whey.

Vital Resistance. — The organism, like the cholera
vibrio, is very susceptible to the influence of acids, high
temperatures, and drying. The thermal death-point is
50° C., continued for five minutes.

Metabolic Products. — The addition of sulphuric acid to
a culture grown in a medium rich in peptone produces the
same rose color observed in cholera cultures and shows the
presence of indol. When glucose is added to the bouillon,
no fermentation or gas-production results. The organism
produces acids and curdling enzymes.

Pathogenesis. — The organism is pathogenic for animals,
but not for man. Pfeiffer has shown that chickens and
guinea-pigs are highly susceptible, and when inoculated under
the skin usually die. The virulent organism is invariably
fatal for pigeons. W. Rindfleisch has pointed out that this
constant fatality for pigeons is a valuable criterion for the
differentiation of this spirillum from that of cholera, as the
subcutaneous injection of the most virulent cholera cultures
is never fatal to pigeons, the birds only dying when the
injections are made into the muscles in such a manner that
the muscular tissue is injured and becomes a locus minoris
resistenti<B. When guinea-pigs are treated by Koch's
method of narcotization and cholera infection, the tem-
perature of the animal rises for a short time, then ab-
ruptly falls to 33° C. or less. Death follows in from twenty
to twenty-four hours. A distinct inflammation of the intes-
tine, with exudate and numerous spirilla, may be found.
The spirilla can also be found in the heart's blood and in
the organs of such guinea-pigs. When the bacilli are intro-

628 Vibrio Schuylkiliensis

duced by subcutaneous inoculation, the autopsy shows a
bloody edema and a superficial necrosis of the tissues.

The organisms can be found in the blood and all the
organs of pigeons and young' chickens, in such large numbers
that Pfeiffer has called the disease Vibrionensepticcemia. In
the intestines very few alterations are noticeable, and very
few spirilla can be found.

Immunity. — Gamaleia has shown that pigeons and
guinea-pigs can be made immune by inoculating them with
cultures sterilized for a time at a temperature of 100° C.
Mice and rabbits are immune, except to very large doses.

VIBRIO SCHUYLKILIENSIS (ABBOTT).

Morphology. — This micro-organism, closely resembling
the cholera spirillum, was found by Abbott * in sewage-
polluted water from the Schuylkill River at Philadelphia.

Cultivation. — Colonies. — The colonies developed upon
gelatin plates very closely resemble those of the Spirillum
metschnikovi.

Gelatin Punctures. — In gelatin puncture cultures the
appearance is exactly like the true cholera spirillum. At
times the growth is a little more rapid.

Agar-agar. — The growth on agar is luxuriant, and gives
off a pronounced odor of indol.

Blood-serum. — Loffler's blood-serum is apparently not
a perfectly adapted medium, but upon it the organisms
grow, with resulting liquefaction.

Potato. — Upon potato, at the point of inoculation a thin,
glazed, more or less dirty yellow growth, shading to brown
and sometimes surrounded by a flat, dry, lusterless zone, is
formed.

Milk. — In litmus milk a reddish tinge develops after
the milk is kept twenty-four hours at body-temperature.
After forty-eight hours this color is increased and the milk
coagulates.

Metabolic Products. — In peptone solutions indol is
easily detected. No gas is produced in glucose-containing
culture media. Acids and coagulating enzymes are formed.
The organism is a facultative anaerobe.

* "Journal of Experimental Medicine," vol. I, No. 3, July, 1896,
p. 419.

Immunity 629

Vital Resistance. — The thermal death-point is 50° C.
maintained for five minutes.

Pathogenesis. — The organism is pathogenic for pigeons,
guinea-pigs, and mice, behav-ing much like Spirillum
metschnikovi. No Pfeiffer's phenomenon was observed
with the use of serum from immunized animals.

Immunity. — Immunity could be produced in pigeons,
and it was found that the serum was protective against both
Vibrio schuylkiliensis and Spirillum metschnikovi, the im-
munity thus produced being of about ten days' duration.

In a second paper by Abbott and Bergey * it was shown
that the vibrios occurred in the water during all four seasons
of the year, and in all parts of the river within the city, both
at low and at high tide. They were also found in the
sewage emptying into the river, and in the water of the
Delaware River as frequently as in that of the Schuylkill.

One hundred and ten pure cultures were isolated from
the sources mentioned and subjected to routine tests. It
was found that few or none of them were identical in all
points. There seems to be, therefore, a family of river
spirilla, closely related to one another, like the different
colon bacilli.

The opinion expressed is that "the only trustworthy
difference between many of these varieties and the true
cholera spirillum is the specific reaction with serum from
animals immune against cholera, or by Pfeiffer's method of
intraperitoneal testing in such animals."

In discussing these spirilla of the Philadelphia waters
Bergey f says :

' ' The most important point with regard to the occurrence
of these organisms in the river water around Philadelphia
is the fact that similar organisms have been found in the
surface waters of the European cities in which there had
recently been an epidemic of Asiatic cholera, notably at
Hamburg and Altona. . . . The foremost bacteriolo-
gists of Europe have been inclined to the opinion that the
organisms which they found in the surface waters of the
European cities were the remains of the true cholera organ-
ism, and that the deviations in the morphologic and biologic
characters from those of the cholera organism were brought
about by their prolonged existence in water. No such

* "Journal of Experimental Medicine," vol. n, No. 5, p. 535.
t "Journal Amer. Med. Assoc.," Oct. 23, 1897.

630 Vibrio Schuylkiliensis

explanation of the occurrence of the organisms in Phila-
delphia waters can be given."

A number of interesting spirilla, more or less closely
resembling that of Asiatic cholera, have been described
from time to time. Their variation from the true cholera
organism can best be determined by an examination of the
following table, though for precise information the student
will do well to look up the original descriptions, references
to which are given in each case.

tZ=3?*?*

COC/5C/)C/2C/5CCtflC/5t/5CDCfl

•O'O'O'C'O'O'OXJ'U'O'O

U3C/2 CflC/5

•CT3 y-UTJ

n>s o

sxil?=

ccccccccccc
33==53==333

c c ITJS c

3 3 -t 3 3

icr klin. Wochenschrift,"
de 1'Inst. Pasteur," t. n,
iv fiir Hygiene," xxi, 189
albl. f. Bakt.," etc., Bd.
jienische Rundschau," 18

dunbarensis (Dunbar ||)
danubicus (Heider**)
I (Wernickett) • • •
II (Wernickett) - - .
liquefaciens (Bonhoff g§
weibeli (Weibel ||||) . .
milleri (Miller***) . .
terrigenus (Giintherttt
berolinensis (Neisser|t^
aquatilis (Guntherggg)
schuylkiliensis (Abbott

Intestinal Orou

cholerae asiaticae (Koch
i choleras nostras (Vibrio
orf)
tyrogenum (Denecke t)

metschnikovi (Gamaleif
Water Group.

S J* - °° "

C. > — '

^ . XJ3?

« "O j» 88

a

ri 3

§^'

w

n

^"S^SS

• • • c •

?' t>8

= ^ yy

><

T1

= £ ^

J2

3

cJi P

-^

PT

? 5.

(t

5 w

•

i

ooooooooooo

+ 4-4- +

Found in Intesti-
nal Diseases.

m 3*t~=~"=

-|- + -f 0 0-f + + + + +

000 +

Found in Water.

1 #87

00000000000

000 0

Stain by Gram's
Method.

2. C 3-t/i x

rr 3-o o

-i- T H- -f -f + + -i- -f + +

+ + -T -t-

Comma Shape.

1 g^JfJf

-f + + + + + + + +o o

4-4-+ 4-

Thick Spirals.

5 !*ll

ooooooooo+o

000 0

Slender Spirals.

5 I5&S-

ooooooooooo

0 C 0 0

Spores.

1 -INs-

+ -f + + -f + + + -l- + +

4-4 -t- +

Active Motility.

« ?i*f

+ + + + + + + + -t- + 4-

4-44- +

Terminal Flagella.

=• S--??1

«> ir -a 8

~- £ X on 35

+0+^+++++++

o++ +

Scum and Slight
Turbidity.

5?

^ 5*34

£ r~n

000-^0000000

+ 00 0

Scum and Marked
Turbidity.

z£
' 7

a r?X=

+ 4- 00000 + 000

+ 00 0

Very Slow Lique-
faction.

O

N

j> M?*=§g

QPW(B j?

00 + 000000 + 0

04-0 +

Slow Liquefaction.

>

•o IRS - ^

^ ^fO-e

0000 ++ 00 + 0 +

OO+ 0

Rapid Liquefac-
tion.

s

*> 7 3#

. i-t vO W

+000+00 00

+ + + o

Yellowish.

AGAR-

£.£*••

0+++0+0 +

000 +

Grayish.

AGAR.

„

+ + 4-4-

+ +

Yellowish.

7

^E

00 00+ 00

+ 00 0

Brown.

%

•° ^^$-

o o o o o o o

00+ +

Colorless.

H

^° - = 'M

in ' >~ ~ O

0 + O + 0 00

o o o o

No Growth.

O

I^3?S

+ +004- o +

+ 00 +

Nitrites.

§£?.§£

+ +00 + + + o +

+ 00 +

Indol.

« S'<?LS-

+ 0 + 0 ++0 + +

4-4-4- +

STr-i

GELATIN.

3 5. c-Zn
n % -« ' 3

0

0

l£

CASEIN.

a 2. >r r> ^D

• sr»E o.

^rt«< W-

<?o*? » <•

4-

4-. 4-

11

BLOOD-
SERUM.

8§|'rf

o o o o o o o

o o o o

Fermentation.

||.«; 4

0 0

Alkalinized.

K

is-xsSi

+ +

4_ _L

Acidified.

r

S-gs" o

•^ c X 3-

+ 4- 4-

+ o

Coagulated.

X

Ji;":' X g

0000000 0 +

000 0

Phosphorescent.

"s^J*^."
M - c/j ^ •:

4- o +

4- o

PIGEONS.

f|?^|

+ +00 +++++

+ 4-+ +

n v

s-f

GUINEA-
PIGS.

?^"fe' '

o +

0 0

RABBITS.

H~M

ooooo oo o

000 0

Chromogenic.

$r

00000 00 0

o o o o

631

Fluorescent.

CHAPTER XXV.
TYPHOID FEVER.

BACILLUS TYPHOSUS (EBERTH-GAFFKY).

General Characteristics.— A motile, flagellated, non-sporogenous,
non-liquefying, nonrchromogenic, non-aerogenic, aerobic and optionally
anaerobic, pathogenic bacillus, staining by ordinary methods, but not
by Gram's method. It does not form indol, acids from sugars, or coag-
ulate milk.

Typhoid fever, " typhus abdominalis," enteric fever,
"la fievre typhique," is a disease so well known and of
such universal distribution, that no introductory remarks
concerning it are necessary.

The bacillus of typhoid fever (Bacillus lyphosus) was
discovered in 1880 by Eberth* and Koch,f and was first
secured in pure culture from the spleen and lymphatic
glands four years later by Gaffky.J

Fig. 220. — Bacillus typhosus, from twenty-four-hour culture on
agar. (From Hiss and Zinsser, "Text-Book of Bacteriology," D. Ap-
pleton & Co., publishers.)

Distribution. — The bacillus is both saprophytic and
parasitic. It finds abundant opportunity, in nature, for
growth and development, and, enjoying strong resisting
powers, can accommodate itself to its environment much
better than the majority of pathogenic bacteria, and can

* "Virchow's Archiv," 1881 and 1883.

f " Mittheilungen aus dem kaiserl. Gesundheitsamt," 1, 45,
id., 2.

632

Morphology — Staining

633

be found in water, air, soiled clothing, dust, sewage, milk,
etc., contaminated directly or indirectly with the intestinal
discharges of diseased persons.

Morphology. — The organism is a short, stout bacillus,
about i to 3 ft (2 to 4 ft — Chantemesse, Widal) in length and
0.5 to 0.8 U broad (Sternberg). The ends are rounded, and
it is exceptional for the bacilli to be united in chains. The
size and morphology vary with the nature of the culture-
medium and the age of the culture. Thoinot and Masselin,*
in describing these morphologic variations, point out that

Fig. 221. — Bacillus typhi.

when grown in bouillon the typhoid bacillus is very slender;
in milk it is stouter; upon agar-agar and potato it is thick
and short; and in old gelatin cultures it forms long fila-
ments.

Flagella. — The organisms are actively motile and are
provided with numerous flagella, which arise from all parts
of the bacillus (peritricha) , and are 10 to 20 in number.
They stain well by Loffler's method. The movements of
the short bacilli are oscillating; those of the longer bacilli,
serpentine and undulating.

Staining. — The organism stains quite well by the ordinary
methods, but loses the color when stained by Gram's method.

* "Precis de Microbie," Paris, 1893.

034 Typhoid Fever

The bacillus gives up its color in the presence of almost any
solvent, so that it is particularly difficult to stain in tissue.

When sections of tissue containing the typhoid bacilli
are to be stained, the best method is to allow them to remain
in Loffler's alkaline methylene-blue for from fifteen minutes
to twenty-four hours, then wash in water, dehydrate rapidly
in alcohol, clear up in xylol, and mount in Canada balsam,
Ziehl's method also gives good results: The sections are
stained for fifteen minutes in a solution of distilled water
100, fuchsin i, and phenol 5. After staining they are
washed in distilled water containing i per cent, of acetic
acid, dehydrated in alcohol, cleared, and mounted. In such
preparations the bacilli are always found in scattered groups,
which are easily discovered, under a low power of the mi-
croscope, as reddish specks, and readily resolved into bacilli
with the oil-immersion lens.

In bacilli stained with the alkaline methylene-blue solu-
tion, dark-colored dots (Babes-Ernst or metachromatic
granules) may sometimes be observed near the ends of the
rods.

The typhoid bacillus produces no endospores.

Isolation^ — The bacillus can be secured in pure culture
from an enlarged lymphatic gland or from the splenic pulp
of a case of typhoid. To secure it in this way the autopsy
should be made as soon after death as possible, lest the
colon bacillus invade the tissues, and cause confusing con-
taminations.

As the groups of bacilli are sometimes widely scattered
throughout the spleen, B. Frankel recommends that as soon
as the organ is removed from the body it be wrapped in
cloths wet with a solution of bichlorid of mercury and kept
for three days in a warm room, in order that a considerable
and massive development of the bacilli may take place. The
surface is then seared with a hot iron and material for cultures
obtained by introducing a platinum loop into the substance
of the organ through the sterilized surface.

Cultures of typhoid bacillus may be more easily obtained
from the blood of the living patients. (See "Blood culture, *
under the section "Bacteriologic Diagnosis.")

The bacilli can also be secured, but with much less certainty,
from the alvine discharges of typhoid patients during the
second and third weeks of the disease.

Cultivation 635

Cultivation. — The bacillus grows well upon all culture-
media under both aerobic and anaerobic conditions.

Colonies. — The deep colonies upon gelatin plates appear
under the microscope of a brownish-yellow color and spindle -
shape, and are sharply circumscribed. When superficial,
however, they become larger and form a thin, bluish, iri-
descent layer with notched edges. The superficial colonies
are often described as resembling grapevine leaves in shape.
The center of the superficial colonies is the only portion
which shows the yellowish-brown color. The. gelatin is not
liquefied.

Gelatin Punctures. — When transferred to gelatin punc-
ture cultures, the typhoid bacilli develop along the entire

Fig. 222. — Bacillus typhi abdominalis; superficial colony two days
old, as seen upon the surface of a gelatin plate. X 20 (Heim).

track of the wire, with the formation of minute, confluent,
spheric colonies. A small, thin, whitish layer develops
upon the surface near the center. The gelatin is not lique-
fied, but is sometimes slightly clouded in the neighborhood
of the growth.

Agar-agar. — The growth upon the surface of obliquely
solidified gelatin, agar-agar, or blood-serum is not luxuriant.
It forms a thin, moist, shining, translucent band with smooth
edges and a grayish-yellow color.

Potato. — When a potato is inoculated and stood in the
incubating oven, the typical growth cannot be detected
even at the end of the second day, unless the observer be
skilled and the examination thorough. If, however, the

636 Typhoid Fever

surface of the medium be touched with a platinum wire, it
is found that its entire surface is covered with a rather
thick, invisible layer of a sticky vegetation which the mi-
croscope shows to be made up of bacilli. This is described
as the invisible growth. Unfortunately, it is not a constant
characteristic, for occasionally a typhoid bacillus will show
a distinct yellowish or brownish color. The typical growth
seems to take place only when the reaction of the potato
is acid.

Bouillon. — Jn bouillon the only change produced by
the growth of the bacillus is a diffuse cloudiness. Rarely a
pellicle is formed. When sugars are added to the bouillon
the typhoid bacillus is found to form acid from dextrose,
levulose, galactose, mannite, maltose, and dextrin, but not to
form acid from lactose or saccharose. No gas is formed in
the fermentation tube with any of the sugars.

Milk. — In milk containing litmus a very slight and slow
acidity is produced, which later gives rise to a distinct alka-
linity. The milk is not coagulated.

Vital Resistance. — The organisms grow well at all
ordinary temperatures. The thermal death-point is given
by Sternberg as 56° C., destruction being effected in ten
minutes. Upon ordinary culture-media, the organisms re-
main alive for several months if drying is prevented. In
carefully sealed agar-agar tubes Hiss found the organism
still living after thirteen years. According to Klemperer and
Levy,* the bacilli can remain vital for three months in dis-
tilled water, though in ordinary water the commoner and
more vigorous saprophytes outgrow them and cause their dis-
appearance in a few days. There seems to be some doubt,
however, on this point, as Tavelf found that it lived for
six months in the blind terminal of a water-supply pipe,
and Hofmann, { after planting it in an aquarium containing
fish, snails, water-plants, and protozoa, was able to recover
it from the water after thirty-six days, and from the mud
in the bottom after two months. In elaborate experimental
studies of this question Jordan, Russell, and Zeit§ found
its longevity to be only three or four days under conditions

*" Clinical Bacteriology." Translated by A. A. Eshner, Phila.j
W. B. Saunders Co., 1900.

t "Centralbl. f. Bakt. u, Parasitenk.," xxxm, p. 166, 1903.

t "Archiv. f. Hyg.," 1905, LII, 2, 208.

§ "Journal of Infectious Diseases," 1904, i, p. 641.

Toxic Products 637

simulating as nearly as possible those found in nature
When buried in the upper layers of the soil the bacilli
retain their vitality for nearly six months. Robertson*
found that when planted in soil and occasionally fed
by pouring bouillon upon the surface, the typhoid bacillus
maintained its vitality for twelve months. He suggests
that it may do the same in the soil about leaky drains.

Cold has little effect upon typhoid bacilli, for some can
withstand freezing and thawing several times. Observing
that epidemics of typhoid fever had never been traced to
polluted ice, Sedgwick and Winslowf made some investiga-
tions to determine what quantitative reduction might be
brought about by freezing, and accordingly experimentally
froze a large number of samples of water intentionally in-
fected with large numbers of typhoid bacilli from different
sources. It was found that the typhoid bacilli disappeared
in proportion to the length of time the water was frozen,
and that the reduction averaged 99 per cent, in two weeks.
The last two or three germs per thousand appeared very
resistant and sometimes remained alive after twelve weeks.

They have been found to remain alive upon linen from
sixty to seventy-two days, and upon buckskin from eighty
to eighty-five days.

The typhoid bacillus resists the action of chemic agents
rather better than most non-sporogenous organisms. The
addition of from o.i to 0.2 per cent, of carbolic acid to the
culture-media being without effect upon its growth. At
one time the tolerance to carbolic acid was thought to be
characteristic, but it is now known to be shared by other
bacteria (colon bacillus) . It is killed by i : 500 bichlorid of
mercury solutions and 5 per cent, carbolic acid solutions in
five minutes.

Metabolic Products. — The typhoid bacillus does not
produce indol. It produces a small amount of acid when
grown in sugar-containing media, but its regular tendency is to
form alkalies, as is shown by the reactions in litmus milk.
It forms no coagulating or proteolytic enzymes.

Toxic Products. — The disproportion of local to consti-
tutional disturbance in typhoid fever and the irritative
and necrotic character of its lesions suggest that we have

* "Brit. Med. Jour.," Jan. 8, 1898.

f"Jour. Boston Soc. of Med. Sci.," vol. iv, No. 7, p. 181, March
20, 1900.

638 Typhoid Fever

to do with a toxic organism. Brieger and Frankel have,
indeed, separated a toxalbumin, which they thought to be
the specific poison, from bouillon cultures. When injected
into guinea-pigs the typhotoxin of Brieger 'causes salivation,
accelerated respiration, diarrhea, mydriasis, and death in
from twenty-four to forty-eight hours. Klemperer and Levy
also point out, as affording clinical proof of the presence of
toxin, the occasional fatal cases in which the typical picture
of typhoid has been without the characteristic postmortem
lesions, the diagnosis being made by the discovery of the
bacilli in the spleen.

Pfeiffer and Kolle* found toxic substance in the bodies
of the bacilli only. It was not, like the toxins of diph-
theria and tetanus, dissolved in the culture -medium. This
was an obstacle to the immunization experiments of both
Pfeiffer and Kolle and Lofner and Abel, f for the only method
of immunizing animals was to make massive agar-agar
cultures, scrape the bacilli from the surface, and distribute
them through an indifferent fluid before injecting them into
animals.

If the bacilli grown upon ordinary culture-media are
several times washed in distilled water, and then allowed
to macerate in normal salt solution, autolysis takes place
and a toxin is liberated, showing that the toxin is intracel-
lular. Macfadyen and Rowland t liberated an intracellular
toxin from cultures of the typhoid bacilli by freezing them
with liquid air and grinding them in an agate mortar. Ani-
mals immunized with this poison produced an antiserum
active against it, but useless against infection with typhoid
bacilli. Wright, of Netley, § observes that Macfadyen's
method of securing this intracellular toxin was unneces-
sarily cumbersome, as the body juices of animals injected
with dead cultures of the bacilli dissolve them at once and
thus liberate the same toxic product.

Besredka|| and Macfadyen** think that exotoxin is also
forrned. Vaughanff has obtained poisonous and non-

* "Deutsche med. Wochenschrift," Nov. 12, 1896.

t "Centralbl. f. Bakt. u. Parasitenk.," Jan. 23, 1896, Bd. xix, No.
23, P- Si-

J "Brit. Med. Jour.," 1903.

§ Ibid., April, 4 1903, i, p. 786.

|| "Ann. de 1'Inst. Pasteur," 1895, x, 1896, xi.
** "Centralbl. f. Bakt.," etc., 1906, i.
ft "Amer. Jour. Med. Sci.," 1908, cxxxvi.

Pathogenesis 639

poisonous fractions by extracting massive cultures of typhoid
bacilli with 2 per cent, solutions of sodium hydrate in abso-
lute alcohol at 78° C.

Mode of Infection. — The typhoid bacillus enters the body
by way of the alimentary tract with infected foods and drinks.

Rosenau, Lumsden, and Kastle* were able to connect 10
per cent, of the cases occurring in the District of Columbia
with infection through milk. Interesting additional facts
upon the subject can be found in Bulletin No. 41 of the
Hygienic Laboratory upon "Milk in its Relation to the
Public Health." The bacillus occasionally enters milk in
water used to dilute it or to wash the cans.

The occurrence of typhoid fever among the soldiers of the
United States Army during the Spanish-American War in
1898 was shown by Reed, Vaughan, and Shakespeare f to
be largely the result of the pollution of the food of the soldiers
by flies that shortly before had visited infected latrines.

The bacillus is also occasionally present upon green vege-
tables grown in soil fertilized with infected human excre-
ment or sprinkled with polluted water, and epidemics are
reported in which the occurrence of the disease was traced
to oysters infected through sewage. NewsholmeJ found
that in 56 cases of typhoid fever about one-third were at-
tributable to eating raw shell-fish from sewage-polluted beds.

Pathogenesis. — The primary activities of the typhoid
bacillus are unknown. It is supposed that it passes uninjured
through the acid secretions of the stomach to enter the intes-
tine, where local disturbances are set up. Whether dur-
ing an early residence in the intestine its metabolism is
accompanied by the formation of a toxic product, irritating
to the mucosa, and affording the bacilli means of entrance
to the lymph-vessels, through diminutive breaches of con-
tinuity, is not known. We usually find it well established
in the intestinal and mesenteric lymphatics at the time we
are able to recognize the disease, though in rare cases it
appears able to reach the blood through other than the
customary channels and occasion an entirely different
pathologic picture.

It is quite certain that the chief operations of the typhoid

* "Hygienic Laboratory Bulletin No. 33," Washington, D. C., 1907.
t "Report on Typhoid Fever in the U. S. Military Camps in the Span-
ish War," vol. I.

J"Brit. Med. Jour.," Jan. 1895.

640 Typhoid Fever

bacillus are in the tissues and not in the intestine, as seems
to be a widely prevalent error. It is contrary to most of
our knowledge of the organism that it should easily adapt
itself to saprophytic existence among the more vigorous
intestinal organisms. Those who look for it in the feces
are usually surprised at the difficulty of finding it, or at the
small numbers they succeed in isolating. It is far more
easy to isolate the organism from the blood than from the
feces, and much greater numbers occur in the urine than in
the feces. It probably escapes from the blood into the
bile, where it grows luxuriantly, and entering the gall-bladder
may take up permanent residence there, escaping into the
intestine each time the gall-bladder is emptied. Many
bacilli thus discharged probably meet with destruction in the
intestine, though some convalescents from typhoid fever for
years have a periodic appearance of bacilli in the feces.
Such individuals have become known as "typhoid carriers"
and are a menace to the public.

In a case studied by Miller* they were found in the gall-
bladder seven years after recovery from typhoid fever; in a
case studied by Drobaf bacilli were found in both the gall-
bladder and a gall-stone seventeen years after recovery from
typhoid fever ; in a case of Humer, J in the gall-bladder of a
patient suffering from cholecystitis, eighteen years after re-
covery from an attack of typhoid fever, and in a case studied
by Dean,§' in the stools of a man twenty-nine years after
he had had typhoid fever, and who had probably been
carrying them about in his gall-bladder ever since. Gush-
ing || invariably found the bacilli in the bile in clumps re-
sembling the agglutinations of the Widal reaction. He
thinks it probable that these clumps form nuclei upon which
bile salts can be precipitated and calculous formation begun.
The presence of gall-stones, together with the long-lived
infective agents, may at any subsequent time provoke a
cholecystitis. Gushing collected 6 cases of operation for
cholecystitis with calculi in which typhoid bacilli were
present, and 5 in which Bacillus coli communis was present
in the gall-bladder.

* "Bull, of the Johns Hopkins Hospital," May,i898.

t "Wiener klin. Wochenschrift," 1899, xn, p. 1141.

J"Bull. of the Johns Hopkins Hospital," Aug. and Sept., 1899.

§ "British Medical Journal," March 7, 1908, I, p. 562.

|| "Bull, of the Johns Hopkins Hospital," ix, No. 86.

Pathogenesis

641

With the most approved methods yet devised, Peabody
and Pratt* were unable to recover the micro-organism from
the intestinal contents in more than 21 per cent, of febrile
cases, and only in small numbers as a rule. The greatest
number was obtained when there was much blood in the
stool.

There is always well-marked blood-infection during the
first couple of weeks of the disease, and upon this depends
the occurrence of the rose-colored spots.

The bacilli enter the solitary glands and Peyer's patches,
and multiply slowly during the incubation period of the

Fig. 223. — Intestinal perforation in typhoid fever. Observe the
threads of tissue obstructing the opening. (Museum of the Penn-
sylvania Hospital.) (Keen, "Surgical Complications and Sequels of
Typhoid Fever.")

disease — one to three weeks. The immediate result of their
activity in the lymphatic structures is an increase in the
number of cells, and ultimately necrosis and sloughing of
the Peyer's patches and solitary glands (Fig. 223). From
the intestinal lymphatics the bacilli pass, in all probability,

* "Journal of the American Medical Association," Sept. 7, 1907,
xux, p. 846.
41

642 Typhoid Fever

to the mesenteric glands, which become enlarged, softened,
and sometimes rupture. They also invade the spleen, liver,
and sometimes the kidneys, and other organs where they
may be found always aggregated in small clusters in properly
stained specimens. The occurrence of the bacilli in the
tissues in clumps or clusters may depend upon the presence
of agglutinating substances in the blood.

Mallory * found the histologic lesions of typhoid fever to
be widespread throughout the body and not limited to the
Peyer's patches of the intestine, where they are most
evident. His conclusions regarding the pathogenesis of the
disease are briefly: "The typhoid bacillus produces a mild
diffusible toxin, partly within the intestinal tract, partly
within the blood and organs of the body. This toxin pro-
duces proliferation of the endothelial cells, which acquire
for a certain length of time malignant properties. The new-
formed cells are epithelioid in character, have irregular,
lightly staining, eccentrically situated nuclei, abundant,
sharply defined, acidophilic protoplasm, and are charac-
terized by marked phagocytic properties. These phago-
cytic cells are produced most abundantly along the line of
absorption from the intestinal tract, both in the lymphatic
apparatus and in the blood-vessels. They are also produced
by distribution of the toxin through the general circulation,
in greatest numbers where the circulation is slowest. Finally,
they are produced all over the body in the lymphatic spaces
and vessels by absorption of the toxin eliminated from the
blood-vessels. The swelling of the intestinal lymphoid tissue
of the mesenteric lymph-nodes and of the spleen is due
almost entirely to the formation of phagocytic cells. The
necrosis of the intestinal lymphoid tissue is accidental in
nature and is caused through occlusion of the veins and
capillaries by fibrinous thrombi, which owe their origin to
degeneration of phagocytic cells beneath the lining endo-
thelium of the vessels. Two varieties of focal lesions occur
in the liver : one consists of the formation of phagocytic cells
in the lymph-spaces and vessels around the portal vessels
under the action of the toxin absorbed by the lymphatics;
the other is due to obstruction of liver capillaries by phago-
cytic cells derived in small part from the lining endothe-
lium of the liver capillaries, but chiefly by embolism through
the portal circulation of cells originating from the endo-
* "Journal of Experimental Medicine," vol. m, 1898, p. 611.

Pathogenesis 643

thelium of the blood-vessels of the intestine and spleen.
The liver-cells lying between the occluded capillaries undergo
necrosis and disappear. Later the foci of cells degenerate
and fibrin forms between them. Invasion by polymorpho-
nuclear leukocytes is rare."

". . . Histologically the typhoid process is prolifera-
tive and stands in close relationship to tuberculosis, but the
lesions are diffuse and bear no intimate relation to the
typhoid bacillus, while the tubercular process is focal and
stands in the closest relation to the tubercle bacillus."

The growth of the bacilli in the kidneys causes albu-
minuria, and the bacilli can be found in the urine in about
25 per cent, of the cases. Smith * found them in the
urine in 3 out of 7 cases which he investigated; Richardson, f
in 9 out of 38 cases. They did not occur before the third
week, and remained in one case twenty-two days after
cessation of the fever. Sometimes they were present in
immense numbers, the urine being actually clouded by their
presence. Petruschky J found that albuminuria sometimes
occurs without the presence of the bacilli; that their
presence in the urine is infrequent; that the bacilli never
appear in the urine in the early part of the disease, and
hence are of little importance for diagnostic purposes.
Gwyn § has found as many as 50,000,000 typhoid bacilli
per cubic centimeter of urine, and mentions a case of Gush-
ing's in which the bacilli persisted in the urine jor six years
after the primary attack of typhoid fever. Their occurrence,
no doubt, depends primarily upon a typhoid bacteremia,
by which they are brought to the kidney. After recovery
from typhoid fever, their persistence in the urine depends
upon continued growth in the bladder and not upon con-
tinuous escape from the blood. It is of importance from
a sanitary point of view to remember that the urine as well
as the feces is infectious.

The bacilli pass from the lymphatics to the general
circulation, so that all cases of typhoid fever are true
bacteremias during part or all of their course.

Ordinarily few bacilli can be found in the circulatory
blood, but blood from the roseolas contains them, and the

* "Brit. Med. Jour.," Feb. 13, 1897.

f "Journal of Experimental Medicine," May, 1898.

% "Centralbl. f. Bakt. u. Parasitenk.," May 13, 1898, No. 13, p. 577,

\ "Phila. Med. Jour.," March 3, 1900.

644 Typhoid Fever

eruption may be regarded as one of the local irritative
manifestations of the bacillus.

Particularly careful work upon this subject has been
done by Richardson,* who found that by carefully disin-
fecting the skin, freezing it with chlorid of ethyl, making
a crucial incision, and cultivating from the blood thus
obtained, he was able to secure the typhoid bacillus in
13 out of 14 cases examined. It was, however, necessary to
examine a number of spots in each case.

As a means of diagnosis the matter is of some importance,
as the rose spots may precede the occurrence of the Widal
reaction by a number of days.

In rare instances the bacillus may be found in the expec-
toration, especially when pulmonary complications arise in
the course of the disease. Cases of this kind have been re-
ported by Chantemesse and Widal f and Frankel.f

The pyogenic power of the typhoid bacillus was first
pointed out by A. Frankel, who observed it in a suppuration
that occurred four months after convalescence. Low§ found
virulent typhoid bacilli in the pus of abscesses occurring
from one to six years after convalescence.

Weichselbaum has seen general peritonitis from rupture
of the spleen in typhoid fever with escape of the bacilli.
Otitis media, ostitis, periostitis, and osteomyelitis are very
common results of the lodgment of the bacilli in bony tis-
sue. Ohlmacherll has found the bacilli in suppurations of
the membranes of the brain. The bacilli are also encoun-
tered in other local suppurations occurring in or following
typhoid fever. Flexner and Harris** have seen a case in
which the distribution of the bacilli was sufficiently wide-
spread to constitute a real septicemia, the bacillus being
isolated from various organs of the body.

Lower Animals. — Typhoid fever is communicable to
animals with difficulty. They are not infected by bacilli
contained in fecal matter or by the pure cultures mixed with
the food, and are not injured by the injection of blood from
typhoid patients. Gaffky failed completely to produce any

"Phila. Med. Jour.," March 3, 1900.
t "Archiv. de physiol. norm. et. path.," 1887.
J "Deutsche med. Wochenschrift," 1899, xv, xvi.
§ "Sitz. der k. k. Gesellschaft d. Aerzt. in Wien," "Aerztl. Central-
Anz.," 1898, No. 3.

|| "Jour. Amer. Med. Assoc.," Aug. 28, 1897.
** "Bull. Johns Hopkins Hospital," Dec., 1897.

Prophylaxis 645

symptoms suggestive of typhoid fever in rabbits, guinea-
pigs, white rats, mice, pigeons, chickens, and calves, and
found that Java apes could feed daily upon food polluted
with typhoid bacilli for a considerable time, yet without
symptoms. Grunbaum* produced typhoid fever in the
chimpanzee by inoculation with the bacillus. This seems
to prove its specific nature. The introduction of virulent
cultures into the abdominal cavity of animals is followed by
peritonitis.

Germano and Maureaf found that mice succumbed in
from one to three days after intraperitoneal injection of
i or 2 c.c. of a twenty-four-hour-old bouillon culture. Sub-
cutaneous injections in rabbits and dogs caused abscesses.

Losener found the introduction of 3 mg. of an agar-agar
culture into the abdominal cavity of guinea-pigs to be
fatal.

Petruschky t found that mice convalescent from sub-
cutaneous injections of typhoid cultures frequently suffered
from a more or less widespread necrosis of the skin at the
point of injection.

Prophylaxis. — One of the most important and practical
points for the physician to grasp in relation to the subject of
typhoid fever is the highly virulent character of the dis-
charges, both feces and urine. In every case the greatest
care should be taken for their proper disinfection, a rigid
attention paid to all the details of cleanliness in the sick-
room, and the careful sterilization of all articles which are
soiled by the patient. If country practitioners were as
careful in this particular as they should be, the disease
\\ould be much less frequent in regions remote from the
filth and squalor of large cities with their unmanageable
slums, and the distribution of the bacilli to villages and
towns, by milk and by watercourses polluted in their in-
fancy, might be checked.

In large cities where typhoid fever has been endemic the
incidence of the disease has been enormously reduced by
purification of the water-supply. Where this measure is not
possible, the safety of the individual citizens can be promoted
by using bottled pure waters for drinking purposes or by
boiling the water for domestic consumption.

* "Brit. Med. Jour.," April 9, 1904.

f "Ziegler's Beitrage," Bd. xn, Heft, 3, p. 494.

% "Zeitschrift fur Hygiene," 1892, Bd. xn, p. 261.

646 Typhoid Fever

In military camps, etc., the fly as a carrier of the infection
must first be excluded from the latrines and then as well from
the kitchens and mess tents. When epidemics are in prog-
ress, green vegetables and oysters that may be polluted by
infected water must be guarded against.

Prophylactic Vaccination. — Following the principle of
Haffkine's anticholera inoculations, Pfeiffer and Kolle,*
Wright,! and Wright and SempleJ have used subcutaneous
injections of sterilized cultures as a prophylactic measure.
One cubic centimeter of a bouillon culture sterilized by heat
was used.

The "Indian Medical Gazette" gives the following im-
portant figures showing what was accomplished in 1899:
Among the British troops in India there were 1312 cases of
typhoid fever, with 348 deaths (25 per cent.). The ratio of
admissions to the total strength was 20.6 per 1000. There
were 4502 inoculations, and among them there were only 9
deaths from typhoid fever — 0.2 per cent, of the strength.
There were 44 admissions, giving 0.98 per cent, of the
strength. Among the non-inoculated men of the same
corps and at the same stations, of 25,851 men there were
657 cases and 146 deaths, giving the relative percentages
of admissions and deaths as 2.54 and 0.56. §

In a later contribution, Wright || showed that the pro-
phylactic vaccination against typhoid fever reduced the
number of cases and diminished the death-rate among the
inoculated, and also called attention to the slight risk the
inoculated run of being injured in case their vital resistance
is below normal, or they are already in the early stages of
the disease, or where the dose administered is too large or
the second vaccination given too soon after the first.

In 1903 Wright** published new statistics on the subject,
and between 1903 and 1908 numerous references to the
subject appear in the "British Medical Journal," in the
"Lancet," and in the "Journal of the Royal Army Medical
Corps," all favorable in their general attitude.

During the Mexican Revolution of 1911, the United States

*" Deutsche med. Wochenschrift," 1896, xxn; 1898, xxiv.
t "Lancet," Sept., 1896.
J "Brit. Med. Jour.," 1897, i, p. 256.
§ "Phila. Med. Jour.," Oct. 13, 1900, p. 688.
|| "The Lancet," Sept. 6, 1902.
** "Brit. Med. Jour.," Oct. 10, 1903.

Specific Therapy 647

Government began, on March 10, 1911, the mobilization of
regiments of the United States Army on the Mexican fron-
tier near San Antonio, Texas. In order to prevent the sad
experiences of the Spanish-American War, in which the
troops suffered terribly from typhoid fever, the Secretary
of War determined that the entire command should be im-
munized against the disease. Many of the soldiers arriving
on the ground had already been immunized, the remainder
were at once given the necessary injections upon arrival.
The mean strength of the command at San Antonio was 12,000
up to June 30, 1911, a period approximating four months.
During all that time there were only 2 cases of typhoid fever
in the encampment, i in an uninoculated civilian teamster
and i in an inoculated soldier. Both cases recovered. The
soldier suffered from so mild an attack that it was over in
sixteen days and probably would not have been diagnosed had
not a blood-culture been made. During the four months
from March loth to June 3oth the typhoid fever was
prevalent in San Antonio, there being 49 cases with 19
deaths.*

The prophylactic used was prepared from a specially se-
lected strain of Bacillus typhosis grown on agar-agar in Kolle
flasks for twenty-four hours. The growth was washed off
with normal salt solution, killed by heating at 55° to 56° C. in
a water-bath, standardized by counting the bacteria accord-
ing to the method of Wright, and after being diluted with salt
solution, 0.25 per cent, of trikresol was added. One cubic cen-
timeter of the finished prophylactic contained i ,000,000,000
bacilli. The first dose injected contained 500,000,000 bacilli,
the second and third, given after ten and twenty days, con-
tained 1,000,000,000 each. The injections caused little in-
convenience either locally or constitutionally. Only i case
had fever, chills, and sweats, and this was the only case requir-
ing treatment in the hospital. It subsequently developed
that this soldier was suffering from early tuberculosis,
which may explain the untoward symptoms from which he
suffered.

Specific Therapy. — Animals can be immunized to this
bacillus, and then, according to Chantemesse and Widal, de-
velop antitoxic blood capable of protecting other animals.

* ''Report of the Surgeon-General of the United States Army to the
Secretary of War," 1911, Washington, D. C.

648 Typhoid Fever

Stern* found in the blood of human convalescents a substance
thought to have a protective effect upon infected guinea-pigs.
His observation is in accordance with a previous one by
Chantemesse and Widal, and has recently been abundantly
confirmed.

The immunization of dogs and goats by the introduction
of increasing doses of virulent cultures has been achieved
by Pfeiffer and Kolle f and by Loffler and Abel. | From these
animals immune serums were secured.

Walger§ reported 4 cases treated successfully with a serum
obtained from convalescent patients. Ten cubic centimeters
were given at a dose, and the injection was repeated in i case
with relapse.

Rumpfll and Kraus and Buswell** report a number of
cases of typhoid favorably influenced by hypodermic injec-
tions of small doses of sterilized cultures of Bacillus pyo-
cyaneus.

Jez ft believes that the antitoxic principle in typhoid fever
is contained in some of the internal organs instead of the
blood, and claims to have obtained remarkable results in
1 8 cases treated with extracts of the bone-marrow, spleen,
and thymus of rabbits previously injected with the typhoid
bacillus.

Chantemesse, it Pope,§§ and Steele|| || have all used serums
from animals immunized against typhoid cultures for the
treatment of typhoid fever, with more or less success. An
analysis of the results will, however, show them to be very
inconclusive.

The serum prepared by Macfadyen,*** by crushing cul-
tures frozen with liquid air and injecting animals with the
thus liberated intracellular toxin, seems to be no improve-
ment upon others.

* " Zeitschrif t fur Hygiene," 1894, xvi, p. 458.

t "Centralbl. f. Bakt. u. Parasitenk.," Jan., 23, 1896, Bd. xix, No.
23, P- 51-

J Ibid, 1896.

§ "Miinchener med. Wochenschrift," Sept. 27, 1898.

|| "Deutsche med. Wochenschrift," 1893, No. 41.
** "Wiener klin. Wochenschrift," July 12, 1894.
ft "Med. moderne," March 25, 1899.
tJ "Gaz. des Hopitaux," 1898, LXXI, p. 397.
§§"Brit. Med. Jour.," 1897, i, 259.
Illl Ibid., April 17, 1897.
*** "Brit. Med. Jour.," April, 3, 1903.

Bacteriologic Diagnosis 649

Meyer and Bergell* prepared a serum by injecting horses
with a new typhoid toxin. After two years' treatment
they were able to demonstrate its value in curing infection
in laboratory animals, von Leydenj speaks in favorable
terms of this serum.

The typhoid immune (bacteriolytic) serum is specific, but
its action requires the presence of additional complementary
substance, and by itself it is useless. Indeed, it may do
harm by causing the formation of anti-immune bodies.

So far no serum has been produced that is of any value
in therapeutics.

Bacteriologic Diagnosis. — There are four means by
which bacteriologic methods may assist the clinician in corn-

Fig. 224. — Typhoid bacilli, tmag- Fig. 225. — Typhoid bacilli, show-
glutinated (Jordan). ing typical clumping by typhoid

serum (Jordan).

pleting the diagnosis of typhoid fever. In the order of their
general usefulness and practicability these are:

1. The Widal reaction of agglutination.

2. The blood-culture.

3. The isolation of the bacillus from the feces.

4. Conjunctival and dermal reactions.

Widal Reaction of Agglutination. — This very valuable
adjunct to our means of making the diagnosis of atypical and
obscure cases of typhoidal infection was discovered in 1896
when Widal and Griinbaum, { working independently, ob-

* "Med. Klinik," m, No. 31, p. 917, Aug. 4, 1907.
f'Berl. klin. Wochenschrift," 1907, No. 18.
t "La Semaine Medicale," 1896, p. 295.

650 Typhoid Fever

served that when blood-serum from typhoid fever patients
is added to cultures of the typhoid bacillus a definite reactive
phenomenon occurs. The phenomenon, now familiarly
known as the "Widal reaction," consists of complete loss
of the motion so characteristic of the typhoid bacillus, and
the collection of the micro-organisms into clusters or groups
— agglutination. The bacteria frequently appear shrunken
and partly dissolved.

The technic of the test is outlined in the section upon
Agglutination (q. v.). For the use of the practising physician,
commercial houses now furnish various outfits known as
"agglutometers," in which are found such simple apparatus
and directions as will enable those inexpert in laboratory
manipulations to arrive at very accurate results.

The Blood-culture. — The technic of this operation is sim-
ple. The skin of the fold of the elbow is thoroughly cleansed,
a fillet put about the arm, and as the veins become promi-
nent, a sterile hypodermic needle is introduced into one and
about 10 c.c. of blood drawn into the syringe. Before clot-
ting can take place, this is discharged into a small flask con-
taining loo c.c. of bouillon, mixed, and stood away to incu-
bate. After twenty-four hours the bacilli can usually be
found in pure culture.

In case the culture is not pure, the typhoid bacillus can
be separated from contaminating organisms by plating.

The Isolation of the Bacillus from the Feces. — This
method of making the diagnosis has practically been aban-
doned because of its uncertainty, its cumber someness, its
tediousness, and because the preceding methods suffice in all
cases.

An excellent resume of the many methods employed for
isolating the bacillus from the stools has been published by
Peabody and Pratt,* and is appropriate reading for those
interested in this subject.

The Conjunctival Reaction. — An additional aid to the di-
agnosis of typhoid in doubtful cases based upon the Wolff-
Eisner- Calmette reaction in tuberculosis is the "ocular ty-
phoid reaction" of Chantemesse. f This test consists in the
instillation into the eye of a solution made by extracting the
typhoid bacillus as follows: "Gelatin plates covered with an

* "Boston Medical and Surgical Journal," 1907.

t " Deutsche med. Wochenschrift," 1907, No. 31, p. 1264.

Differentiation of Typhoid and Colon Bacilli 651

eighteen- to twenty-hour-old culture of virulent typhoid bacilli
were washed with 4 to 5 c.c. of sterile water. The suspension
thus obtained was heated to 60° C., centrifugated, and the
supernatant fluid withdrawn. The centrifugated organisms
were then dried and triturated. A second suspension of these
broken up bacillary bodies was then made, and allowed to
stand for from two to three days at 60° C. The extract
thus obtained, after removing the disintegrated and digested
remnants, was precipitated with alcohol, forming a fine coag-
ulum. This was subsequently dried and powdered and dis-
solved in sterile water in the proportion of 0.02 mg. to a
drop."*

When one drop of this is placed upon the conjunctiva
of a patient in the early days of typhoid fever, diffuse
redness increases and becomes marked in two or three hours.
There is also some feeling of heat in the eye. Tears flow
freely, and there is a slight mucopurulent exudate in some
cases. The reaction persists about ten hours and then
declines, usually disappearing in twenty-four hours. Ham-
burger f confirmed the results of Chantemesse. It is too
early to say how useful the reaction is, but it seems to
promise aid in diagnosing difficult cases.

Differential Diagnosis of the Typhoid and Colon
Bacilli. — This constitutes the chief perplexity of bacterio-
logic work with the typhoid bacillus, and is the great bugbear
of beginners. A great deal of energy has been expended upon
it, a considerable literature has been written about it, and
much still remains to be learned by which it may be simplified.

Two chief methods are in vogue at present:

1. The serum differentiation.

2. The culture differentiation.

Serum Differentiation. — The specific agglutinating action
of experimentally prepared serums can be used to differentiate
cultures of the colon, paracolon, typhoid, and paratyphoid
bacilli, the typhoid bacilli alone exhibiting the specific effect
of the typhoid serum. This is a very reliable means of differ-
entiation when the cultures have already been isolated. The
method is described under the heading "Agglutination," in
the section devoted to the "Special Phenomenon of Infection
and Immunity."

* See Hamburger, "Jour. Amer. Med. Assoc.," L, 17, p. 1344, April 25,
1908.

f Loc. cit.

652 Typhoid Fever

Richardson* has found it very convenient to saturate filter-paper
with typhoid serum, dry it, cut into 0.5 cm. squares, and keep it on
hand in the laboratory for the purpose of making this differentiation.
To make a test, one of these little squares is dropped in 0.5 c.c. of a
twenty-four-hour-old bouillon culture of the suspected bacillus and
allowed to stand for five minutes. A drop .of the fluid placed upon
a slide and covered will then show typical agglutinations if the culture
be one of the typhoid fever bacillus. In a second mention of this
methodf he has found its use satisfactory in practice and the paper
serviceable after fourteen months' keeping.

The Cultural Differentiation.— When the typhoid bacilli
are to be isolated from the blood of living patients, they are
so likely to be obtained in pure culture that little trouble is
experienced. If they are to be isolated from the pus of a
posttyphoidal abscess, or from viscera at autopsy, from
water suspected of pollution, and especially when they are to
be isolated from the intestinal contents, with its rich bacterial
flora, the matter becomes progressively complicated.

As the colonies of the typhoid bacilli closely resemble those
of Bacillus coli, etc., special media have, from time to time,
been devised for the purpose of emphasizing such differences
as rapidity of growth, acid production, etc. Thus, Eisner t
has suggested the employment of a special medium made as
follows :

One kilogram of grated potatoes (the small red German potatoes
are best) is permitted to macerate over night in i liter of water. The
juice is carefully pressed out and filtered cold, to get rid of as much starch
as possible. The filtrate is boiled and again filtered. The next step is a
neutralization, for which Eisner used litmus as an indicator, and added
2.5 to 3 c.c. of a TV normal sodium hydrate solution to each.,10 c.c. of the
juice. Abbott prefers to use phenolphthalein as an indicator. The
final reaction should be slightly acid. Ten per cent, of gelatin (no pep-
tone or sodium chlorid) is dissolved in the solution, which is boiled, and
must then be again neutralized to the same point as before. After filtra-
tion the medium receives the addition of i per cent, of potassium iodid;
then it is filled into tubes and sterilized like the ordinary culture-media.

When water or feces suspected of containing the typhoid bacillus are
mixed in this medium and poured upon plates, no bacteria develop well
except the typhoid and colon bacilli.

These, however, differ markedly in appearance, for the colon colonies
appear of the usual size in twenty-four hours, at which time the typhoid
bacillus, if present, will have produced no colonies discoverable by the
microscope.

It is only after forty-eight hours — long after the colon colonies have
become conspicuous — that little colonies of the typhoid bacillus appear
as finely granular, small, round, shining, dew-like points, in marked con-
trast to their large, coarsely granular predecessors.

* "Centralbl. f. Bakt. u. Parasitenk.," 1897, p. 445.

f "Journal of Experimental Medicine," May, 1898, p. 353, note.

J "Zeitschrift fur Hygiene," xxn, Heft i, 1895; Dec. 6, 1896.

Differentiation of Typhoid and Colon Bacilli 653

Unfortunately, many of the small colonies that develop
in Eisner's medium subsequently prove to be those of the
colon bacillus, and the method is thus rendered unreli-
able.

Remy* prefers to make an artificial medium approxi-
mating a potato in composition, but without dextrin or glu-
cose. The composition is as follows :

Distilled water 1000.0 grains

Asparagin 6.0

Oxalic acid 0.5 gram

Lactic acid 0.15

Citric acid 0.15

Disodic phosphate 5.0 grams

Magnesium sulphate 2.5

Potassium sulphate 1.25

Sodium chlorid 2.0

All the salts excepting the magnesium sulphate are powdered in a
mortar and introduced into a flask with the distilled water. Thirty
grams of Witte's or Grubler's peptone are then added and the mixture
heated in the autoclave under pressure for one-quarter hour. As soon
as removed, the contents are poured into another flask into which 120 to
150 grams of gelatin had previously been placed. The flask is shaken to
dissolve the gelatin, and the contents then made slightly alkaline with
soda solution. The mixture is again heated in the autoclave at 1 10° C.
for one-quarter hour, then acidified with a one-half normal solution of
sulphuric acid, so that 10 c.c. have an acidity neutralized by 0.2 c.c. of
one-half normal soda solution. This acidity is equal to 0.5 c.c. sulphuric
acid per liter. After shaking, place the flask in a steam sterilizer for ten
minutes, then filter. When filtered, verify the acidity of the medium,
correcting if necessary. Finally, add the magnesium sulphate, dissolve,
dispense in tubes, and sterilize by the intermittent method.

At the moment of using, put into each tube i c.c. of a 35 per cent,
solution of lactose and o.i c.c. of a 2.5 per cent, solution of carbolic acid.

Upon this medium the colonies of the typhoid and colon
bacilli show marked differences. The colon colonies are
yellowish brown, the typhoid colonies bluish white and
small. Fine bubbles of gas from the fermentation of the
lactose often occur about the colon colonies.

By this method Remy was able to isolate the typhoid
bacillus from the stools in 23 cases which he studied. He
believes that the constant presence of the typhoid bacillus
in the stools of typhoid fever, and its absence from them
under all other conditions, is a far more important and
valuable method of diagnosis than even the Widal re-
action.

* ''Ann. de 1'Inst. Pasteur," Aug., 1900.

654 Typhoid Fever

Wiirtz* and Kashidaf make the differential diagnosis by
observing the acid production of Bacillus coli in a medium
consisting of bouillon containing i .5 per cent, of agar, 2 per
cent, of milk-sugar, i per cent, of urea, and 30 per cent, of
tincture of litmus. This is the so-called litmus-lactose-agar-
agar. The culture-medium should be blue. When liquefied,
inoculated with the colon bacillus, poured into Petri dishes,
and stood for from sixteen to eighteen hours in the incubator,
the blue color passes off and the culture-medium becomes
red. If a glass rod dipped in hydrochloric acid be held
over the dish, vapor of ammonium chlorid is given off. The
typhoid bacillus produces no acid in this medium, and there
is consequently no change in its color. Upon plates with
colonies of both bacilli, the typhoid colonies produce no
change of color, while the colon colonies at once redden the
surrounding medium.

Rothberger J first employed neutral red for the differentia-
tion of the typhoid and colon bacilli. When grown in fluid
media containing it, the colon bacillus produces a yellowish
fluorescence, while the typhoid bacillus does not destroy the
port wine color. Savage § and Irons || have made use of the
color reaction for the routine detection of the colon bacillus
in water. The best adaptation of the method is by Stokes,**
who adds it to the various sugar bouillons in the proportion
of o.i gram per liter, and uses the medium in the fermen-
tation tube. The colon bacillus always ferments the sugars
and produces a typical color reaction.

Hiss ft recommends the use of two special media — one
for plates, the other for tube-cultures. The first consists
of 5 grams of agar-agar, 80 grams of gelatin, 5 grams of
Liebig's beef -extract, 5 grams of sodium chlorid, and 10
grams of glucose to the liter. The agar is dissolved in the
1000 c.c. of water, to which have been added the beef-
extract and sodium chlorid. When the agar is completely
melted, the gelatin is added and thoroughly dissolved by

* "Archiv. de med. Experimentale," 1892, iv, p. 85.

t "Centralbl. f. Bakt. u. Parasitenk.," Bd. xxi, Nos. 20 and 21,
June 24, 1897.

t "Centralbl. f. Bakt.," 1893, p. 187.

§ "Journal of Hygiene," 1901, i, p. 437.

|| Ibid., 1902, n, p. 437.

** "Jour, of Infectious Diseases," 1904, i, p. 341.
ft "Jour, of Experimental Medicine," Nov., 1897, vol. n. No. 6.

Differentiation of Typhoid and Colon Bacilli 655

a few minutes' boiling. The medium is then titrated to
determine its reaction, phenolphthalein being used as the
indicator, and enough HC1 or NaOH added to bring it to
the- desired reaction — i. e., a reaction indicating 1.5 per
cent, of normal acid. To the clear medium add one or two
eggs, well beaten in 25 c.c. of water; boil for forty-five
minutes, and filter through a thin layer of absorbent cotton.
Add the glucose after clearing.

This medium is used in tubes, in which the culture is
planted by the ordinary puncture. The typhoid bacillus alone
has the power of uniformly clouding this medium without
showing streaks or gas-bubbles.

The second medium is used for plating. It contains 10
grams of agar, 25 grams of gelatin, 5 grams of beef -extract,
5 grams of sodium chlorid, and 10 grams of glucose. The
method of preparation is the same as for the tube-medium,
care always being taken to add the gelatin after the agar
is thoroughly melted, so as not to alter this ingredient by
prolonged exposure to high temperature. The preparation
should never contain less than 2 per cent, of normal acid.
Of all the organisms upon which Hiss experimented with
this medium, Bacillus typhosus alone displayed the power of
producing thread- forming colonies.

The colonies of the typhoid bacillus when deep in Hiss'
medium appear small, generally spheric, with a rough,
irregular outline, and, by transmitted light, of a vitreous
greenish or yellowish-green color. The most characteristic
feature consists of well-defined filamentous outgrowths,
ranging from a single thread to a complete fringe about the
colony. The young colonies are, at times, composed solely
of threads. The fringing threads generally grow out nearly
at right angles to the periphery of the colony.

The colonies of the colon bacillus appear, on the average,
larger than those of the typhoid bacillus; they are spheric
or of a whetstone form, and by transmitted light are darker,
more opaque, and less refractive than the typhoid colonies.
By reflected light they are pale yellow to the unaided eye.

Surface colonies are large, round, irregularly spreading,
and are brown or yellowish-brown in color. Hiss claims
that by the use of these media the typhoid bacillus can
readily be detected in typhoid stools.

Piorkowski* recommends a culture-medium composed of
* "Berliner klin. Wochenschrift," Feb. 13, 1899.

656 Typhoid Fever

urine two days old, to which 0.5 per cent, of peptone and 3.3
per cent, of gelatin have been added. Colonies of the ty-
phoid bacillus appear radiated and filamentous; those of the
colon bacillus, round, yellowish, and sharply defined at the
edges. The cultures should be kept at 22° C., and the colo-
nies should appear in twenty-four hours.

Adami and Chapin* have suggested what seems to be a
promising method for the isolation of typhoid bacilli from
water, making use of the agglutination of the bacilli by
immune serum. Two quart bottles (Winchester quarts)
are carefully sterilized and filled with the suspected water
with an addition of 25 c.c. of nutrient broth and incubated
for eighteen to twenty-four hours at 37° C. By this time
the typhoid bacillus grows abundantly in spite of the small
amount of nourishment the water contains. At the end of
the incubation, 10 c.c. of the fluid is filled into each of a
number of long narrow (7 mm.) test-tubes made by sealing
a glass tube one-half meter long at one end. About one inch
from the bottom the tube is filed completely round so as to
break easily at that point. The different tubes next receive
additions of typhoid immune serum sufficient to make the
dilutions i : 60, i : TOO, i 1150, and i 1200. If typhoid
bacilli are present, within a quarter of an hour beginning
agglutination can be seen, and by the end of two to five hours
flocculent masses collect at the bottom of the tube, forming
a flocculent precipitate. The next procedure should be
with the tube showing agglutination with the greatest dilu-
tion, as the more concentrated preparations carry down not
only the typhoid bacilli, but also closely related organisms.
After the sedimentation of the agglutinated bacilli is com-
plete, the tube is broken at the file mark, and the sediment
contained in the short tube washed with two or three changes
of distilled water, being allowed to settle each time. This
removes many of the organisms not agglutinated. A loop-
ful of the washed sediment is transferred to a tube of nutrient
broth, and finally from this tube plate cultures are made upon
Eisner's or Hiss's media.

A culture medium for isolating the typhoid bacillus from
feces is recommended by Drigalski-Conradif and by Petko-
witsch.J It is made as follows:

* "Journal of Medical Research," May, 1904, vol. xi, No. 2, p. 469.

t "Zeitschrift f. Hygiene," Bd. xxix.

t "Centralbl. f. Bakt.," etc., May 28, 1904, Bd. xxxvi, No. 2, p. 304.

Differentiation of Typhoid and Colon Bacilli 657

Horse-meat infusion (3 pounds of horse meat

to 2 liters of water) 2 liters

Witte's peptone 20 grams

Nutrose 20 grams

Sodium chloride 10 grams

Agar-agar 60 grams

Litmus solution (Kubel and Tiemann) 260 c.c.

Lactose 30 grams

Crystal- violet solution (0.01 per cent.) 20 c.c.

Before adding the crystal- violet solution render feebly alkaline to
litmus (about 0.04 per cent, of pure soda).

Colon colonies upon this medium appear in fourteen to sixteen hours
to be red and opaque. Typhoid colonies blue or violet, transparent
and drop-like.

Beckman * modifies the preparation, making it as follows :

"(a) Add i liter of water to 680 grams of finely chopped lean beef
and place in the cold for twenty-four hours. Express the juice and
make up to i liter. Coagulate the albumin, either by boiling for ten
minutes or by heating to 120° C. in the autoclave. Filter. Add 10
grams of Witte's peptone, 10 grams of nutrose, and 5 grams of sodium
chloride. Heat in the autoclave at a temperature of 120° C. for thirty
minutes, or boil vigorously for fifteen minutes. Render slightly alkaline
to litmus-paper. Filter. Add 30 grams of agar. Heat in the auto-
clave at a temperature of 1 20° C. for one-half hour, or heat over the gas-
flame until the agar is dissolved. Render slightly alkaline to litmus-
paper while hot, if necessary. Filter through glass wool into a sterile
vessel.

" (6) To 130 c.c. of litmus solution (Kubel and Tiemann' s) add 15
grams of chemically pure lactose. Boil for ten minutes.

" (c) Mix (a) and (6) while hot. Render slightly alkaline to litmus,
if necessary. To the mixture add 2 c.c. of hot sterile solution of 10 per
cent, sodium hydrate in distilled water and 10 c.c. of a fresh solution of
Hochst's crystal violet (o.i gram of crystal violet to 100 c.c. of sterile
water) .

" The medium is now poured into Petri dishes and is of a deep pur-
ple color. So much water of condensation forms on the solidified surface
that it is an advantage to use porous clay covers (Hill) for the Petri
dishes instead of the ordinary glass covers. The medium keeps well
but dries up rapidly."

A very ingenious method of isolating the typhoid and
colon bacilli from drinking water has been suggested by
Starkey,f who uses a tubular labyrinth of glass into which
a nutrient medium, ordinary bouillon containing 0.05 per
cent, of carbolic acid, or Pariette's bouillon:

i. Measure out pure hydrochloric acid, 4 c.c., and add it to carbolic
acid solution (5 per cent.), 100 c.c. Allowr the solution to
stand at least a few days before use.

* See F. F. Wesbrook, "Jour. Infectious Diseases," May, 1905, Sup-
plement, No. i, p. 319.

f " Amer. Jour. Med. Sci.," July, 1906, cxxxn, No. i, No. 412, p. 109.
42

658

Typhoid Fever

2. This solution is added in quantities of o. i, 0.2, and 0.3 c.c.

(delivered by means of a sterile graduated pipette to tubes,
each containing 10 c.c. of previously sterilized nutrient bouil-
lon).

3. Incubate at 37° C. for forty-eight hours to eliminate contami-

nated tubes.

as recommended by Somers.* In these labyrinths the
motility of the organisms will separate the colon and
typhoid bacilli from non-motile organisms, and from those
less motile than themselves. The restraining medium pre-
vents the ready growth of most
organisms except colon and
typhoid bacilli. The anaerobic
conditions prevent the devel-
opment of aerobic organisms
which form the majority of
bacteria with which one comes
in contact in ordinary bac-
teriological examinations. The
typhoid bacillus, being more
motile than the colon, travels
more quickly through the coils
of the tube and first arrives
at its end, where it can be
found in pure or nearly pure
culture after about forty-eight
hours.

Somers has improved the
instrument by bending it in a
circular form, so that it can

stand alone, and by adapting its size to the Novy jar, so
that satisfactory anaerobic conditions can easily be attained.
Hesse f has recommended the following medium:

Agar-agar 5 grams (4.5 grams absolutely dry).

Witte's peptone 10

Liebig's beef -extract 5 "

Sodium chlorid 8.5 "

Distilled water. . . . 1000 "

Fig. 226. — Starkey's labyrinth
as modified by Somers.

Dissolve the agar-agar in 500 c.c. of the water over a free flame,
making up the loss by evaporation. Dissolve the other ingredient, in the
remaining 500 c.c. of water, heat until dissolved, replacing the loss by

* "Trans. Phila, Path. Soc.," 1906.

t "Zeitschrift fur Hygiene," 1908, i,vmf 441.

Differentiation of Typhoid and Colon Bacilli 659

evaporation. Pour the two solutions together and heat for thirty minutes
and add distilled water to replace loss by evaporation. Filter through
cotton until clear. Adjust reaction to i per cent, acidity. Tube — 10
c.c. to a tube. Sterilize in the autoclave.

The medium is used for plating. The material containing
the micro-organisms must be so dilute that only a few colonies
will develop upon the plates. The typhoid colonies greatly
outgrow the colon colonies and may attain to a diameter of
several centimeters. They show a small opaque center and
an opalescent body and appear circular.

Capaldi* recommends the following medium for plating
typhoid and colon colonies:

Witte's peptone 20 grams

Gelatin 10

Agar-agar 20

Dextrose or mannite 10

Sodium chlorid 5

Potassium chlorid 5

Distilled water 1000

Dissolve the agar in 500 c.c. of water, the other ingredients in the
other 500 c.c. of water. Pour together, add 10 c.c. of NaOH, filter, and
tube.

Upon this medium the typhoid colonies are small, glisten-
ing, bluish, and translucent. Colon colonies are larger,
opaque, and brownish.

Endo f recommends the employment of the following me-
dium upon which colonies of the typhoid bacillus grow large
and remain colorless, while those of the colon bacillus remain
small and red:

1000 c.c. of meat infusion.
30 grams of agar-agar.
10 grams of peptone (Witte's).
5 grams of sodium chlorid.

Neutralize and clear by filtration, then add 10 c.c. of a 10 per cent,
solution of NaOH to alkalinize, 10 grams of chemically pure lactose and
5 c.c. of a filtered, saturated, alcoholic solution of fuchsin. Next add
25 c.c. of a 10 per cent, sodium sulphate solution, by which the intense
red given by the fuchsin is entirely bleached by the time the agar-agar
is cold. After adding the necessary reagents and while still warm and
perhaps red, tube the medium. The tubes should be kept in the dark.

* "Zeitschrift fur Hygiene," 1896, xxm, 475.
t "Centralbl. f. Bakt.," etc., 1904, xxxv.

66o Typhoid Fever

Loffler* has found malachite green a very useful adjunct
to our means of differentiating the typhoid from other simi-
lar bacilli.

For the purpose, 2^ to 3 per cent, of a 2 per cent, solution of malachite
green are added to the culture-medium. The preparation given the
preference consists of i pound of meat macerated in i liter of water,
neutralized with potassium, with the addition of 2 per cent, of peptone, 5
per cent, of lactose, i per cent, of glucose, 0.5 per cent, of sodium sul-
phate, 2 per cent, of nitrate of potassium, and 3 per cent, of a 2 per cent,
solution of malachite green.

In the medium the ordinary cocci and bacilli do not grow,
Gartner's bacillus and the paratyphoid bacillus b leave the
medium clear, but grow as a deposit at the bottom of the
tube; the typhoid bacillus destroys the green. If agar-agar
be added, the colonies are surrounded by a clear yellow zone.
The colon and other organisms grow slowly if at all.

Not many workers were satisfied with the results obtained
by malachite green, nor were the results obtained uniform.
A careful study of the subject was made by Peabody and
Pratt, f who found great differences in the quality and reac-
tions of different malachite greens in the market. That with
which LofBer worked was commercially known as " 120."
They obtained three samples of this dye, which varied in
acidity between wide margins (0.2-1.0). Experimenting
with the different preparations, they found that the least
acid was the most useful preparation. The success of the
method, therefore, depends upon the adjustment of the
concentration of the dye to the reaction of the medium.
When this is done, malachite green becomes a valuable
adjunct to specific differentiation. Their studies of the
media led Peabody and Pratt to the invention of a new
method of isolating typhoid bacilli from the feces. Instead
of employing malachite green agar-agar directly for this
purpose, they first employ malachite green bouillon as an
" enriching" culture, and after eighteen to twenty-four hours'
growth in the incubator inoculate one or two large (20 cm.
diameter) Drigalski-Conradi plates, from which the colonies
can subsequently be picked out.

Bile salts were first employed in culture-media by Lim-
bourgt and have been more or less popular ever since,

| "Inst. hyg. Univers. Griefswald," see "Bull. Inst. Past.," iv, No. 9,
May 15, 1906, p. 393.

* "Boston Med. and Surg. Journal," Feb. 13, 1908, CLVIII, p. 213.
f "Zeitschrift f. physiol. Chemie," 1889, m, p. 196.

Differentiation of 'Typhoid and Colon Bacilli 66 1

though for differentiation of typhoid and colon bacilli they
cause occasional disappointment.

Buxton and Coleman* prepare a medium composed of:

Ox-bile 900 c.c.

Glycerin 100 c.c.

Peptone 20 grams

This was placed in a number of 100 c.c. flasks, sterilized in
the Arnold sterilizer, and employed chiefly for blood-culture.
The typhoid bacillus grows well in it.

Jackson f prepares a medium for water examination when
typhoid and colon bacilli are suspected. It consists of un-
diluted ox-bile to which i per cent, of peptone and i per cent,
of lactose are added. It is filled into fermentation-tubes of
40 c.c. capacity and sterilized in the Arnold apparatus. If
fresh ox-bile cannot be secured, an 1 1 per cent, solution of
fresh dry ox-bile can be made; 10 c.c. of suspected water or
milk are planted in the tubes of this medium. The contained
micro-organisms grow rapidly, typhoid bacilli outgrowing
all others, and not fermenting the sugar ; rapid fermentation
and copious gas-formation take place if colon bacilli are
present.

An excellent medium suggested by MacConkeyJ has the
following composition:

Agar 1.5 grams

Sodium taurocholate 0.5 gram

Peptone 2.0 grams

Water ico.o c.c.

It is boiled, clarified, and filtered as usual, then receives an addition
of i.o gram of lactose, is tubed, and then sterilized three times on suc-
cessive days.

For determining fermentation by colon bacilli the same
investigator advises a broth composed of:

Sodium taurocholate (pure) 0.5 gram

Peptone 2.0 grams

Glucose 0.5 gram

Water . 100.0 c.c.

Boil, filter, add sufficient neutral litmus, fill into fermentation-tubes,
and sterilize at 100° C. Colon colonies appear red; typhoid, blue.

* "Journal of Infectious Diseases," 1909, vi, No. 2, p. 194.
t "Biological Studies of the Pupils of W. T. Sedgwick," 1906, Uni-
versity of Chicago Press.

t "The Thompson- Yates Laboratory Reports," in, p. 151.

662 Typhoid Fever

In a careful study of the bile-salt media MacConkey*
points out an error, first discovered by Theobald Smith, that
depends upon the alkali production of the colon bacillus in
the absence of sugar. If too little sugar be added to the
medium, the alkali production masks the acid production
unless the oxygen be removed, and red colonies of the colon
bacillus grown upon the medium may in time turn dis-
tinctly blue. It becomes obvious, therefore, that the
medium should be as neutral as possible to the indicator
used. After trial he found neutral red preferable to litmus,
and makes the medium as follows:

i. A stock solution is made:

Sodium taurocholate (commercial from ox-bile

and neutral to neutral red) 0.5 per cent.

Peptone (White's) 2.0

Water (distilled or tap) 100.0 c.c.

(As calcium 0.03 per cent, is favorable to the growth of the

organisms, it should be added if distilled water is used.)

The ingredients should be mixed, steamed in a steam sterilizer for
one to two hours, filtered while hot, allowed to stand twenty-four to
forty-eight hours, then filtered cold through paper. A clear solution
should then result, which will keep indefinitely under proper conditions.
The various bile-salt media are prepared from this stock solution by
adding glucose, 0.5 per cent. ; lactose, i per cent. ; cane-sugar, i per cent. ;
dulcit, 0.5 per cent.; adonit, 0.5 per cent., or inulin, i per cent.; and
neutral red (i per cent, solution), 0.25 per cent., distributing into fer-
mentation-tubes and sterilizing in the steamer for fifteen minutes on
each of three successive days.

Bile-salt agar-agar is made by dissolving 2 per cent, of agar-agar in
the stock fluid, either in the steamer or in the autoclave. The mixture
is cleared with an egg, filtered, neutral red added in the same proportion
as for the broth, and distributed into flasks in quantities of 80 c.c.
When required for use, the fermentable substance is added to the agar
in the flask, and the whole placed in a water-bath or steamer (care must
be taken not to heat either the fluid or solid medium beyond 100° C.).
When melted, the agar preparation is poured into Petri dishes, allowed
to solidify, and then dried in an incubator or warm room, the plate being
placed upside down with the bottom detached and propped up on the
edge of the cover. It is necessary that the surface of the agar-agar
should not be too wet, lest the colonies become confluent, nor too dry,
lest the growth be stunted. Inoculations are made by placing a loop-
ful of the material to be examined on the center of one plate, and
rubbed over the surface with a bent glass-rod; the same rod, without
recharging, being used to inoculate the surface of two other plates.
The plates are then incubated upside down. The colonies of the colon
bacillus appear yellow.

* ''Journal of Hygiene," 1908, vm, p. 322.

Bacilli Resembling the Typhoid Bacillus 663

BACILLI RESEMBLING THE TYPHOID BACILLUS.

Bacillus typhosus is one of a group of organisms pos-
sessing a considerable number of common characteristics,
each member of which, however, can be differentiated by
some one fairly well-marked peculiarity. At one end of
the series is the typhoid bacillus, which we conceive to be
devoid of the power to liquefy gelatin, ferment sugars, form
indol, coagulate milk, or progressively form acids. At the
other extreme stands Bacillus coli, an organism whose
typical representatives coagulate milk, form indol, ferment
dextrose, lactose, saccharose, and maltose with the forma-
tion of hydrogen 'and carbon dioxid in the proportion of

H 2^

coT ~ i-

Between these extremes are numerous organisms known
as "intermediates." It is usually a simple matter to dif-
ferentiate these forms from the typical species at the two
ends of the series, but it is quite difficult to differentiate
them from one another. Whether they are of sufficient
importance to make it worth while to pay much attention
to them is, as yet, uncertain; and, indeed, we do not know
whether they are to be regarded as variations from the
type species or separate and distinct organisms. The fact
that some of them are associated with serious and fatal
disorders — paracolon bacillus and bacillus of psittacosis —
proves them, at least, to be important.

In his careful review of the intermediate forms thus far
described, Buxton* summarizes the main points of difference
as follows:

B. coli com-

munis. Intermediates. B. typhosus.

Coagulation of milk -+-

Production of indol -j-

Fermentation of lactose with gas .... -j-

Fermentation of glucose with gas. . . -j- +

Agglutination by typhoid serum .... -f-

The characteristics of the three groups as shown by the
fermentation-test stand thus:f

Gas upon Gas upon Gas upon

dextrose. lactose. saccharose.

Bacillus typhosis

Intermediates +

Bacillus coli communis -j- -f-

Bacillus coli communior -j- -j- +

c "Journal of Medical Research," vol. vm, No. i, June, 1902, p. 201.
t Hiss and Zinsser, "Text-book of Bacteriology," 1910, p. 429.

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664

Bacilli Resembling the Typhoid Bacillus 665

Buxton finds those pathogenic for man clinically divisible
into three groups, as follows:

(a) The Meat- poisoning Group. — This includes Bacillus
enteritidis of Gartner and others. The symptoms begin soon
after eating the poisonous meat, and are toxic. Bacilli
quickly invade the body. The illness continues four or
five days, after which recovery is quick. In a few cases
death has occurred on the second or third day.

(b) The Pneumonic or Psittacosis Group. — Psittacosis is
an epidemic infectious disease with pneumonic symptoms
and a high mortality. Its origin has been traced to dis-
eased parrots, and from them Nocard isolated Bacillus
psittacosis, supposed to be the cause of the disease in man.
Later epidemics were studied by Achard and Bensaude.

(c) The Typhoidal Group. — The organisms to be included
in this group occasion symptoms closely resembling typhoid
fever, though they differ biologically from the typhoid
bacillus, and do not agglutinate with typhoid serums.

It is thus evident that some of the intermediates occasion
symptoms resembling typhoid fever, while others occasion
symptoms widely differing from it. It is suggested that to
the former the term paratyphoid bacilli be applied, while the
latter are known as paracolon bacilli.

Although Achard and Bensaude,* and Johnson, Hewlett,
and Longcope f have studied the paratyphoid infections,
Gwyn,t Libman,§ and others the paracolon bacilli, and
Gushing || and Durham ** have made comparative studies
of the members of the group, it is still too soon to regard
the knowledge attained sufficient to warrant particular
mention of the various intermediate and related organisms
in a work of this kind. In the following pages, therefore,
attention will be devoted only to the more important
organisms of the group and to a few belonging to offshoots
from the parent stem.

* "Soc. Med.," Nov., 1896.

t " Amer. Jour. Med. Sci.," Aug., 1902.

J "Johns Hopkins Bulletin," vol. ix, 1898.

§ "Journal of Medical Research," 1902, p. 168.

|| "Johns Hopkins Bulletin," vol. xi, 1900.

** "Journal of Experimental Medicine," vol. v, p. 353, 1901.

666 Bacillus Coli

BACILLUS COLI COMMUNIS (ESCHERICH).

General Characteristics. — A motile, flagellated, non-sporogenous,
aerobic and optionally anaerobic, non-chromogenic, non-liquefying,
aerogenic, saprophytic, occasionally pathogenic bacillus, staining by
the ordinary methods, but not by Gram's method. It produces indol,
coagulates milk, and produces acids and gases from dextrose, lactose,
and sucrose.

This micro-organism was first isolated from human feces by
Emmerich,* in 1895, who thought it to be the specific cause
of Asiatic cholera, and called it Bacillus neapolitanus.
Many investigators have since studied its peculiarities, until
it has become one of the best known bacteria.

Fig. 227. — Bacillus coli (Migula).

Distribution. — It is habitually present in the feces of
animals, and in water and soil contaminated with them.
Soon after birth the organism finds its way into the alimen-
tary canal and permanently establishes itself in the intes-
tine, where it can be found in great numbers throughout
the entire life of the individual. It is almost certainly
identical with Bacillus pyogenes foetidus of Passet, and so
closely resembles B. acidi lactici that Prescott f believes
them to be identical. It may also be identical with Ba-
cillus lactis aerogenes, Bacillus cavicida, and other described
species.

*" Deutsche med. Wochenschrift," 1885, No. 2.

t Society of American Bacteriologists, Dec. 31, 1902.

Bacilli Resembling the Typhoid Bacillus 667

Morphology. — The bacillus is rather variable, both size
and form depending to a certain extent upon the culture
medium on which it grows. It measured about 1-3 X 0.4-
0.7 fj.. It usually occurs in the form of short rods, but
coccus-like individuals and elongate individuals may be
found in the same culture. The bacilli are usually separate
from one another, though occasionally joined in pairs, are
actively motile, and provided with flagella, which are vari-
able in number, usually from four to a dozen. The organ-
isms from some cultures swim actively, even when the cul-
ture is some days old ; others are sluggish even when young

Fig. 228. — Bacillus coli communis; superficial colony two days old
upon a gelatin plate. X 21 (Heim).

and actively growing, and still other cultures consist of
bacilli that scarcely move at all. It forms no endospores.

Staining. — The bacillus stains well with the aqueous
solutions of the anilin dyes, but not by Gram's method.

Cultivation. — It is readily cultivated upon the ordinary
media.

Colonies. — Upon gelatin plates the colonies are visible
in twenty-four hours. Those situated below the surface
appear round, yellow-brown, and homogeneous. As they
increase in size they become opaque. The superficial
colonies are larger and spread out upon the surface. The
edges are dentate and slightly resemble grape-vine leaves,
often showing radiating ridges suggestive of the veins of a
leaf. They may have a slightly concentric appearance.
The colonies rapidly increase in size and become more and
more opaque. The gelatin is not liquefied.

668 Bacillus Coli

Gelatin Punctures. — Development in gelatin punctures
occurs upon the surface, and also in the needle's track, caus-
ing the formation of a nail-like growth. The head of the
nail may reach the walls of the test-tube. No gas is formed
in ordinary gelatin, but should any dextrose be present in it,
gas-production may occur and irregularly break up the me-
dium. The gelatin may become slightly clouded as the
bacilli grow, but is not liquefied.

Agar-agar. — Upon agar-agar along the line of inocula-
tion a grayish- white, translucent, smeary growth devoid of
any characteristics takes place. The entire surface of the
culture-medium is never covered, the growth remaining con-
fined to the inoculation line, except where the moisture of
condensation allows it to spread out at the bottom. Kruse
says that crystals may form in old cultures.

Bouillon. — Bouillon is densely clouded by the growth of
the bacteria, a delicate pellicle at times forming upon the sur-
face. There is usually considerable sediment in the culture.

Potato. — Upon potato the growth is luxuriant. The
bacillus forms a yellowish -brown, glistening layer spreading
from the line of inoculation over about one-half to two-
thirds of the potato. The color varies considerably, some-
times being pale, sometimes quite brown, sometimes green-
ish. It cannot, therefore, be taken as a characteristic of
much importance. The growth on potato may be almost
invisible.

Milk. — In milk rapid coagulation and acidulation occur,
with the evolution of gas. The culture gives off a fecal odor.
Litmus added to the culture media is first reddened, then
decolorized by the bacilli.

Vital Resistance. — It is quite resistant to antiseptics
and germicides, and grows in culture media containing
from 0.1-0.2 per cent, of carbolic acid. It is, however,
easily killed by heat, and is destroyed by exposure to 60°
C. for ten minutes.

Metabolic Products. — Wiirtz found that Bacillus coli
produced ammonia in culture media free from sugar, and
thus caused an intense alkaline reaction in the culture
media. The cultures usually give off an odor that varies
somewhat, but is, as a rule, unpleasant.

Nitrates are reduced to nitrites by the growth of the
bacillus.

In bouillon containing i per cent, of dextrose, lactose,
levulose, galactose, and mannite, the colon bacillus splits up

Bacilli Resembling the Typhoid Bacillus 669

the sugar, liberating CO2 and H, the gas formula being
^ = ~. This gas formula is very constant for the micro-
organisms of the colon group and forms one of their most
important differential characteristics. In sugar-containing
bouillon, acetic, lactic, and formic acids are produced. It
does not ferment saccharose. When a similar bacillus is
found to ferment saccharose it is best regarded as a sub-
species or separate type, for which Dunham has introduced
the name Bacillus coli communior.

The bacillus requires very little nutriment. It grows in
Uschinsky's asparagin solution, and is frequently found living
in river and well waters.

Indol is formed in both bouillon and peptone solutions,
but phenol is not produced. The presence of indol is best
determined by Salkowski's method (q. V.).

Toxic Products. — Vaughan and Cooley* have shown
that the toxin of the colon bacillus is contained in the
germ-cell and under ordinary conditions does not diffuse
from it into the culture medium. The toxin may be heated
in water to a very high temperature without injuring its
poisonous nature. They have devised an apparatus in
which enormous cultures can be prepared and the bacteria
pulverized. f Of such a preparation 0.0002 gram will kill a
2oo-gram guinea-pig.

Pathogenesis. — The bacillus begins to penetrate the
intestinal tissues almost immediately after death, and is the
most frequent contaminating micro-organism met with in
cultures made at autopsy. It may spread by direct con-
tinuity of tissue, or via the blood-vessels.

Although under normal conditions a saprophyte, the
colon bacillus is not infrequently found in the pus in sup-
purations connected with the intestines — as, for example,
appendicitis — and sometimes in suppurations remote from
them.

In intestinal diseases, such as typhoid, cholera, and
dysentery, the bacillus not only seems to acquire an unusual
degree of virulence, but because of the existing denudation
of mucous surfaces, etc., finds it easy to enter the general
system, with the formation of remote secondary suppura-
tive lesions in which it is the essential factor. When ab-
sorbed from the intestine, it frequently enters the kidney

* "Jour. Amer. Med. Assoc.," 1901; "American Medicine," 1901.
f "Trans. Assoc. Amer. Phys.," 1901.

670 Bacillus Coli

and is excreted with the urine, causing, incidentally, local
inflammatory areas in the kidney, and occasionally cystitis.
A case of urethritis is reported to have been caused by it.

In infants cholera infantum may not infrequently be
caused by the colon bacillus, though probably in this disease
other bacteria play an important role (B. dysenteriae?).

The bile-ducts are sometimes invaded by the bacillus,
which may lead to inflammation, obstruction, suppuration,
or calculus formation.

The colon bacillus has also been met with in puerperal
fever, Winckel's disease of the newborn,* endocarditis, men-
ingitis, liver-abscess, bronchopneumonia, pleuritis, chronic
tonsillitis, urethritis, and arthritis.

An interesting summary of the pathogenic effect of Bacillus
coli can be found in Rolleston's paper in the "British Medi-
cal Journal" for Nov. 4, 1911, p. 1186.

In a certain number of cases general hemic infection may
be caused by Bacillus coli. In 1909 Jacob t published an
analysis of 39 such cases, and in 1910 Draper! increased the
number to 43. Wiens§ also reported 6 cases and Maher|| i
case, so that the total now stands 50.

Virulence. — It is a question whether the colon bacillus is
always virulent, or whether it becomes so under abnormal
conditions. Klencki** found it very virulent in the ileum,
and less so in the colon and jejunum of dogs. He also
found that the virulence was greatly increased in a strangu-
lated portion of intestine. Dreyfus ft found that the colon
bacillus as it occurs in normal feces is not virulent. Most
experimenters believe that pathologic conditions, such as
disease of the intestine, strangulation of the intestine, etc.,
increase its virulence.

Frequent transplantation lessens the virulence of the
bacillus; passage through animals increases it.

It has been observed that cultures of the bacillus obtained
from cases of cholera, cholera nostras, and other intestinal
diseases are more pathogenic than those obtained from
normal feces or from pus.

"Kamen-Ziegler's Beitrage," 1896, 14.

t "Deutsch. Archiv. f. Klin. Med.," 1909, xcvii, 303.

t "Bull, of the Ayer Clin. Lab. of the Penna. Hosp.," 1910, No. 6, p. 21.

§ "Munch, med. Woch.," 1909, LVI, 962.

|| "Med. Record," 1909, LXXV, 482.
** "Ann. de 1'Inst. Pasteur," 1895, No. 9.
ft "Centralbl. f. Bakt.," etc., xvi, p. 581

Bacilli Resembling the Typhoid Bacillus 671

Adelaide Ward Peckham,* in an elaborate study of the
"Influence of Environment on the Colon Bacillus," con-
cludes that while the conditions of nutrition and develop-
ment in the intestine seem to be most favorable, the colon
bacillus is ordinarily not virulent. She says:

"Its first force is spent upon the process of fermentation, and as
long as opportunities exist for the exercise of this function the affinities
of this organism appear to be strongest in this direction.

"Moreover, the contents of the intestine remain acid until they
reach the neighborhood of the colon, and by that time the tryptic
peptones have been formed and absorbed to a great extent.

"During the process of inflammation in the digestive tract a very
different condition may exist. The peptic and tryptic enzymes may
be partially suppressed. Fermentation of carbohydrates and proteid
foods then begins in the stomach, and continues after the mass of
food is passed on into the intestine. The colon bacillus cannot, there-
fore, spend its force upon fermentation of sugars, because they are
already broken up and an alkaline fermentation of the proteids is in
progress. It also cannot form peptones from the original proteids,
for it does not possess this property, and unless trypsin is present it
must be dependent upon the proteolytic activity of other bacteria for
a suitable form of proteid food. Perhaps these bacteria form an albu-
minate molecule which, like leucin and tyrosin, cannot be broken up
into indol, and thus there might be caused an important modification
of the metabolism of the colon bacillus, which might have either an
immediate or remote influence upon its acquisition of disease-producing
properties, for our own experiments indicate that the power to form
indol, and the actual forming of it, are to some extent an indication
of the possession of pathogenesis."

For the laboratory animals the colon bacillus is patho-
genic in varying degree. Intraperitoneal injections into
mice cause death in from one to eight days if the culture be
virulent. Guinea-pigs and rabbits also succumb to intra-
peritoneal and intravenous injection. Subcutaneous injec-
tions are of less effect, and in rabbits seem to produce
abscesses only.

When injected into the abdominal cavity, the bacilli
set up a sero-fibrinous or purulent peritonitis, and are very
numerous in the abdominal fluids.

Cumston, f from a careful study of thirteen cases of summer
infantile diarrheas, comes to the following conclusions:

Bacterium coli seems to be the pathogenic agent of the greater
number of summer infantile diarrheas.

The organism is often associated with Streptpcoccus pyogenes.

The virulence, more considerable than in the intestine of a healthy
child, is almost always in direct relation to the condition of the child

* "Journal of Experimental Medicine," Sept., 1897, vol. n, No. 4,
P- 549-

t "International Medical Magazine," Feb., 1897.

672 Bacillus Coli

at the time the culture is taken, and does not appear to be propor-
tionate to the ulterior gravity of the case.

The mobility of Bacterium coli is, in general, proportionate to its
virulence. The jumping movement, nevertheless, does not correspond
to an exalted virulence in comparison with the cases in which the
mobility was very considerable, without presenting these jumping
movements.

The virulence of Bacterium coli found in the blood and other organs
is identical with that of Bacterium coli taken from the intestines of the
individual.

Lesage,* in studying the enteritis of infants, found that
in 40 out of 50 cases depending upon Bacillus coli the blood
of the patient agglutinated the cultures obtained, not only
from his own stools, but from those of all the other cases.
From this uniformity of action Lesage suggests that the
colon bacilli in these cases are all of the same species.

The agglutinating reaction occurs only in the early stages
and acute forms of the disease.

Immunization. — It is not difficult to immunize an
animal against the colon bacillus. Loffler and Abel im-
munized dogs by progressively increased subcutaneous
doses of live bacteria, grown in solid culture and suspended
in water. The injections at first produced hard swellings.
The blood of the immunized animals possessed an active
bactericidal effect upon the colon bacteria. The serum
was not in the correct sense antitoxic.

Differential Diagnosis. — This problem is considered at
greater length under the heading "Cultural Differentiation
of the Bacillus Typhosus" (q.v.). For the recognition of the
colon bacillus the most important points are the motility,
the indol-formation, the milk-coagulation, and the active
gas-production. As, however, most of these features are
shared by other bacteria to a greater or less degree, the most
accurate differential point is the immunity reaction with the
serum of an immunized animal, which protects susceptible
animals from the effects of inoculation, and produces a simi-
lar agglutinative reaction to that observed in connection with
the blood and serum of typhoid patients, convalescents, and
immunized animals.

The fact that, with rare exceptions, the typhoid serum
produces a specific reaction with the typhoid bacillus, and
the colon serum with the colon bacillus, should be the most
important evidence that they are entirely different species.

What is commonly known as Bacillus coli communis is,
no doubt, not a single species, but a group of bacilli too
* "La Semaine Medicale," Oct. 20, 1897.

Bacilli Resembling the Typhoid Bacillus 673

similar to be differentiated into groups, types, or families
by our present methods.

In order to establish a type species of Bacillus coli com-
munis, Smith * says :

"I would suggest that those forms be regarded as true to this species
which grow on gelatin in the form of delicate bluish or more opaque,
whitish expansions with irregular margin, which are actively motile
when examined in the hanging drop from young surface colonies taken
from gelatin plates, which coagulate milk within a few days ; grow upon
potato, either as a rich pale or brownish-yellow deposit, or merely
as a glistening, barely recognizable layer, and which give a distinct
indol reaction. Their behavior in the fermentation-tube must conform
to the following scheme:

" Variety a:

"One per cent, dextrose-bouillon (at 37° C.). Total gas approx-
imately i; H — COa = approximately 2 : 1 ; reaction strongly acid.

"One per cent, lactose-bouillon: as in dextrose-bouillon (with slight
variations).

"One per cent, saccharose-bouillon; gas-production slower than
the preceding, lasting from seven to fourteen days. Total gas about
$; H — CO2 = nearly 3:2. The final reaction in the bulb may be
slightly acid or alkaline, according to the rate of gas-production (B. coli
communior, Dunham).

" Variety /3:

"The same in all respects, excepting as to its behavior in saccharose-
bouillon; neither gas nor acids are formed in it."

DIFFERENTIAL CHARACTERISTICS.
TYPHOID BACILLUS. COLON BACILLUS.

Bacilli usually slender. Bacilli a little thicker and shorter.

Flagella numerous (10-20), long, Flagella fewer (8-10) (peritricha).

and wavy (peritricha).

Growth not very rapid, not par- Growth rapid and luxuriant. This

ticularly luxuriant. character is by no means con-
stant.

Upon Eisner's, Hiss', Piorkow- Upon Eisner's, Hiss', Piorkow-

ski's, and other media gives ski's, and other media gives

characteristic appearances. characteristic appearances.

Upon fresh acid potato the so- Upon potato a brownish-yellow

called "invisible growth" form- distinct pellicle.

erly thought to be differential.

Acid-production in whey not ex- Acid-production well marked

ceeding 3 per cent. Sometimes throughout.

slight in ordinary media, and

succeeded by alkali-production.

Grows in media containing sugars Fermentation with gas-produc-

without producing any gas. tion well marked in solutions

containing dextrose, lactose,
etc., the usual formula being
H — C02 = 2 : i.

Produces no indol. Indol-production marked.

Growth in milk unaccompanied Milk coagulated.

by coagulation.

Gives the Widal reaction with the Does not react with typhoid

serum of typhoid blood. blood.

* " Amer. Jour. Med. Sci.," 1895, 110, p. 287.
43

674 Bacillus Coli

Colon Bacillus in Drinking Water. — Much importance
attaches to the presence or absence of colon bacilli in judging
the potability of drinking waters.

It is a speculation whether the colon bacilli were originally
micro-organisms of the soil that accidentally found their
way into the congenial environment of the intestine and
there took up permanent residence, or whether they have
always been intestinal parasites and have been discharged
with the excrement of animals until the soil has become
generally infected with them. However this may be,
they are at present found in the intestinal canals of all
animals, and in pretty much all soils, their number being
greatest in manured soils. From the soil it is inevitable
that the organisms shall pass into the surface waters, which
with few exceptions will be found to contain them. The
numbers, however, can be made use of to indicate the quality
of the water, a few organisms indicating that the water is
pure, many that it is freely mixed with surface washings.

As sewage contains as many as i ,000,000 colon bacilli per
cubic centimeter and pure water very often o per cubic
centimeter (only i cubic centimeter being examined at a
time), the number of bacilli per cubic centimeter can be
expressed as indicating the amount of sewage pollution.
The number of colon bacilli in the water is, therefore, of
importance in determining its potability, and in cases in
which the quality of the water is doubtful, should always
be employed. There is no infallible criterion for judging
the quality of water, but most American bacteriologists
are in accord in concluding that when the repeated examina-
tion of i c.c. samples shows the presence of numerous colon
bacilli, the water is seriously polluted and doubtfully potable,
but when samples of i c.c. are without colon bacilli or
contain very few, the water is safe.

Another important matter in regard to the colon bacillus
in water is the presence or absence of certain characters
by which one can judge how recently it has ended its intes-
tinal parasitism and taken up a saprophytic life. The
chief of these characters is the ability to ferment lactose.
Only recently isolated organisms manifest this fermentative
power in the laboratory, so that when organisms capable
of fermenting lactose are found, one can suppose that they
result from recent sewage pollution.

Many media have been recommended for the rapid detec-

Bacilli Resembling the Typhoid Bacillus 675

tion of the colon bacilli in water, the favorite at the present
time probably being the litmus-lactose-agar plate (q. v.) of
Wiirtz.* This depends upon the fermentative and acid-
producing power of the bacillus, which is shown through
the presence of red colonies (acid producers) on the else-
where blue plate. These red colonies are then fished up
and transplanted to appropriate media for further study.

Kline f substitutes lactose for the glucose in this medium,
pointing out that by so doing one at once differentiates
between typical colon bacilli which ferment lactose and
atypical varieties which do not.

Other media and methods useful in studying the colon
bacilli are also discussed in the chapter upon Typhoid Fever
(q. v.).

BACILLUS ENTERITIDIS (GARTNER).

General Characteristics.— A motile, flagellated, non-sporogenous,
non-chromogenic, non-liquefying, aerogenic, aerobic and optionally
anaerobic, pathogenic bacillus staining by the ordinary methods, but
not by Gram's method.

This bacillus was first cultivated by A. Gartner { from the
flesh of a cow slaughtered because of an intestinal disease,
and from the spleen of a man poisoned by eating meat
obtained from it. The bacillus was subsequently found by
Karlinski and Lubarsch in other cases of meat-poisoning.

Morphology. — The bacillus closely resembles Bacillus
coli communis. It is short and thick, is surrounded by a
slight capsule, is actively motile, and has flagella.

Staining. — It stains irregularly with the ordinary solu-
tions, but not by Gram's method. It has no spores.

Cultivation. — Upon gelatin plates it forms round, pale
gray, translucent colonies. It does not liquefy the gelatin.
The deep colonies are brown and spheric. The growth on
agar-agar is similar to that of the colon bacillus. The
organism produces no indol, coagulates milk in a few days,
and reduces litmus. Its fermentative powers have not been
sufficiently studied, but is known to ferment dextrose media.
Upon potato it forms a yellowish- white, shining layer.

* "Archiv. de med. Experimental, " iv, p. 85, 1892.
t "British Medical Journal," Oct. 27, 1906, p. 1090.
J "Korrespond. d. allg. arztl. Ver. von Thuring," 1888, 9.

676 Bacillus Faecalis

Pathogenesis. — The bacillus is pathogenic for mice,
guinea-pigs, pigeons, lambs, and kids, but not for dogs,
cats, rats, or sparrows. The infection may be fatal for
mice and guinea-pigs, whether given subcutaneously, intra-
peritoneally, or by the mouth.

Lesions. — The bacilli are found scattered throughout the
organs in small groups, resembling those of the typhoid
bacillus.

At the autopsy a marked enteritis and swelling of the
lymphatic follicles and patches, with occasional hemor-
rhages, are found. The bacilli occur in the intestinal con-
tents. The spleen is somewhat enlarged.

The bacillus is differentiated from the colon bacillus
chiefly by the absence of indol-production, by its ability to
produce infection when ingested, and by the fact that it
elaborates a toxic substance capable of producing symp-
toms similar to those seen in the infection.

It may be distinguished from Bacillus lactis aerogenes
by its motility. It closely resembles certain water bacteria;
but its pathogenesis can be made use of for assisting in its
differentiation in doubtful cases.

BACILLUS F^CALIS ALKALIGENES (PETRUSCHKY).

General Characteristics.— A motile, flagellated, non-sporogenous,
non-liquefying, non-chromogenic, non-aerogenic, aerobic and option-
ally anaerobic, non-pathogenic bacillus of the intestine, staining by
ordinary methods, but not by Gram's method.

This bacillus has occasionally been isolated by Petruschky *
and others from feces. It closely resembles the typhoid
bacillus, being short, stout, with round ends, forming no
spores, staining with the usual dyes, but not by Gram's
method, being actively motile, and having numerous flagella.
It does not liquefy gelatin, does not coagulate milk, produce
gas, or form indol. Its pathogenic powers for the lower ani-
mals are similar to those of the typhoid bacillus.

It grows more luxuriantly than the typhoid bacillus upon
potato, producing a brown color, and generates a strong
alkali when grown in litmus- whey. Its cultures are not
agglutinated by the typhoid serums.

*"Centralbl. f. Bakt. u. Parasitenk.," xix, 187.

Bacilli Resembling the Typhoid Bacillus 677

BACILLUS PSITTACOSIS (NOCARD).

General Characteristics.— A motile, flagellated, non-sporogenous,
aerobic, optionally anaerobic, non-chromogenic, aerogenic, pathogenic,
non-liquefying bacillus, staining by the ordinary methods, but not
by Gram's method.

This micro-organism was discovered by Nocard,* who first
observed it in 1892 in certain cases of psittacosis, or epidemic
pneumonia, traceable to infection from diseased parrots. The
original paper contained an excellent account of the specific
organism.

The subsequent work of Gilbert and Fournierf shows the
specificity of the micro-organism to be quite well established
and Nocard's characterizations accurate.

Morphology. — The bacillus is short, stout, rounded at
the ends, and actively motile. It is provided with flagella,
but forms no spores. It resembles the typhoid and the
colon bacilli and is evidently a form intermediate between
the two.

Isolation. — Gilbert and Fournier succeeded in isolating
it from the blood of a patient dead of psittacosis, and from
parrots, by the use of lactose-litmus agar. The organism
does not alter the litmus, and if a small percentage of carbolic
acid be added to the culture-media, it grows as does the
typhoid bacillus.

Cultivation. — The colonies, agar-agar and gelatin cul-
tures, closely resemble those of the typhoid fever organism.
Upon potato it more closely resembles the colon bacillus.
Bouillon becomes clouded.

Metabolic Products. — In bouillon containing sugars the
micro-organism is found to ferment dextrose, but not lactose.
Milk is not coagulated and not acidulated. No indol is
formed.

Pathogenesis. — Bacillus psittacosis can be immediately
differentiated from the typhoid and colon bacilli by its
peculiar pathogenesis. It is extremely virulent for parrots,
producing a fatal infection in a short time. White and
gray mice and pigeons are equally susceptible. Ten drops
of a bouillon culture injected in the ear-vein of a rabbit

*" Seance du Conseil d'hygiene publique et Salubrite du Departe-
ment de la Seine," March 24, 1893.

f "Comptes de la Societe de Biologic," 1896; "LaPresse medicate,"
Jan. 16, 1897.

678 Bacillus Suipestifer

kill it in from twelve to eighteen hours. Guinea-pigs are
more resistant. Subcutaneous injection of dogs produces a
hard, painful swelling, which persists for a short time and
then disappears without suppuration. It is also infectious
for man, a number of epidemics of peculiar pneumonia, char-
acterized by the presence of the bacillus in the blood, traceable
to diseased parrots, having been reported.

Differentiation. — Bacillus psittacosis can best be differ-
entiated from the typhoid and the colon bacilli and others
of the same group by its pathogenesis and by the reaction
of agglutination. Typhoid immune serum produces some
small agglutinations, but a comparison between these and
the agglutinations formed by cultures of the typhoid bacillus
shows immediately that the micro-organisms are dissimilar.
Differentiation is best made out when the prepared hanging-
drop specimens of serums and cultures are kept for some
hours in an incubating oven. It is not known whether the
bacillus is peculiar to the intestines of parrots, invading their
tissues when they become ill, or whether it is a purely patho-
genic micro-organism found only in psittacosis.

BACILLUS SUIPESTIFER (SALMON AND SMITH).

General Characteristics.— An actively motile, flagellated, non-
sporogenous, non-chromogenic, non-liquefying, aerobic and optionally
anaerobic, aerogenic bacillus pathogenic for hogs and other animals.
It stains by the ordinary methods, but not by Gram's method. It
ferments dextrose, lactose, and sucrose, but does not form indol or
coagulate or acidulate milk.

Hog-cholera, or "pig typhoid," as the English call it, is a
common epidemic disease of swine, which at times kills 90
per cent, of the infected animals, and thus causes immense
losses to breeders. Salmon estimates that the annual losses
from this disease in the United States range from $10,000,000
to $25,000,000. For years it was thought to be caused by the
Bacillus suipestifer, but DeSchweinitz and Dorset* were
able to transmit the disease from one hog to another in certain
of the body fluids that had been passed through the finest
porcelain filters and were shown by inoculation and cultiva-
tion to be free of bacilli. It therefore depends upon a fil-
terable and unknown virus.

* "Circular No. 41 of Bureau of Animal Industry," U.S. Dept. of
Agriculture, Washington, D. C.

Bacilli Resembling the Typhoid Bacillus 679

This observation was received with approval by those who
had any experience with the effect of hog-cholera bacilli
upon hogs, all of whom must have observed that though
infection with the bacilli occasionally caused the death of an
animal, the dead animal usually did not show the typical
lesions of the disease and never infected other animals
with which it was kept. The papers upon the subject by
Dorset, Bolton, and McBryde* and by Dorset, McBryde, and
Nilesf are worth reading.

These investigations entirely change our ideas of the im-
portance of the hog-cholera bacillus, whose relation to the
disease now comes to resemble that of Bacillus icteroides
to yellow fever.

The bacillus of hog-cholera was first found by Salmon and
Smith,| but was for a long time confused with the bacillus
of "swine-plague," which it closely resembles, and in associa-
tion with which it frequently occurs. It is a member of
the group of bacteria to which Bacillus icteroides and B.
typhi murium belong. The organism was secured by
Smith from the spleens of more than 500 hogs. It occurs
in the blood and in all the organs, and has also been culti-
vated from the urine.

Morphology. — The organisms appear as short rods with
rounded ends, 1.2 to 1.5 ^ long and 0.6 to 0.7 ^ in breadth.
They are actively motile and possess longflagella (peritrichia) ,
easily demonstrable by the usual methods of staining. No
spore production has been observed. In general the bacillus
resembles that of typhoid fever. It stains readily by the
ordinary methods, but not by Gram's method.

Cultivation. — No trouble is experienced in cultivating
the bacilli, which grow well in all the media under aerobic
and anaerobic conditions.

Colonies. — Upon gelatin plates the colonies become
visible in from twenty-four to forty-eight hours, the deeper
ones appearing spheric with sharply defined borders. The
surfaces are brown by reflected light, and without markings.

* "Bull. No. 72 of Bureau of Animal Industry," U. S. Dept. Agricul-
ture, Washington, D. C., 1905.

t "Bull. No. 102 of Bureau of Animal Industry," U. S. Dept. Agri-
culture, Washington, D. C., Jan. 18, 1908.

t" Reports of the Bureau of Animal Industry," 1885-91; and
"Centralbl. f. Bakt. u. Parasitenk.," Bd. ix, Nos. 8, 9, and 10, March
2, 1891.

68o Bacillus Suipestifer

They are rarely larger than 0.5 mm. in diameter and are
homogeneous throughout. The superficial colonies have
little tendency to spread upon the gelatin. They rarely
reach a greater diameter than 2 mm. The gelatin is not
liquefied.

Upon agar-agar they attain a diameter of 4 mm. and
have a gray, translucent appearance with polished surface.
They are round and slightly arched.

Gelatin. — In gelatin punctures the growth takes the form
of a nail with a flat head. There is nothing characteristic
about it. The medium is not liquefied.

Agar-agar. — Linear cultures upon agar-agar present a
translucent, circumscribed, grayish, smeary layer without
characteristic appearances.

Potato. — Upon potato a yellowish coating is formed,
especially when the culture is kept in the thermostat.

Bouillon. — Bouillon made with or without peptone is
clouded in twenty-four hours. When the culture is allowed
to stand for a couple of weeks without being disturbed, a
thin surface growth can be observed.

Milk is an excellent culture-medium, but is not visibly
changed by the growth of these bacteria. Its reaction re-
mains alkaline.

Vital Resistance. — The bacillus is hardy. Smith found
it vital after being kept dry for four months. It ordinarily
dies sooner, however, and I have experienced difficulty in
keeping it in the laboratory for any length of time unless
frequently transplanted. The thermal death-point is 54° C.,
maintained for sixty minutes.

Metabolic Products. — Gas Production. — The hog-chol-
era bacillus is a copious gas-producer, capable of break-
ing up dextrose and lactose into CO2, H, and an acid, which,
formed late, eventually checks its further development. It
does not ferment saccharose.

Indol. — No indol and no phenol are formed in the culture-
media.

Toxin. — In pure cultures of the hog-cholera bacillus
Novy* found a poisonous base with the probable composition
C16H26N2, which he gave the provisional name "susotoxin."
In doses of 100 mg. the hydrochlorid of this base causes con-
vulsive tremors and death within one and one-half hours in

* "Medical News," 1900, p. 231.

Bacilli Resembling the Typhoid Bacillus 68 1

white rats. He has also obtained a poisonous protein of
which 50 mg. were fatal for white rats, and which immu-
nized them against highly virulent hog-cholera organisms
when administered by repeated subcutaneous injection.

De Schweinitz* has also separated a slightly poisonous
base which he calls "sucholotoxin," and a poisonous protein
that crystallizes in white, translucent plates when dried over
sulphuric acid in vacua, forms needle-like crystals with pla-
tinic chlorid, and was classed among the albumoses.

Pathogenesis. — The bacillus is disappointing in its
effects upon hogs. When it is subcutaneously or intra-
venously introduced into such animals or fed to them, they
sometimes show no signs of disease; sometimes show fever
and depression, but rarely sicken enough to die. Animals
thus made ill do not communicate the disease to others.

Smith found that 0.75 c.c. of a bouillon culture injected
into the breast muscles of pigeons would kill them.

In Smith's experiments one four-millionth of a cubic
centimeter of a bouillon culture injected subcutaneously
into a rabbit was sufficient to cause its death. The tem-
perature abruptly rises 2° to 3° C., and remains high until
death. Subcutaneous injection of larger quantities may
kill in five days. Injected intravenously in small doses the
bacillus may kill rabbits in forty-eight hours.

Agglutination. — Pitfieldf found that after a single in-
jection of a killed bouillon culture of the bacillus into a
horse, the serum, which originally had very slight agglutina-
tive power, showed a decided increase. If the horse be
immunized to large doses of such sterile cultures, the serum
becomes so active that with a dilution of i : 10,000 a typical
agglutination occurs in sixty minutes.

McClintock, Boxmeyer and SifferJ found that the serum
of normal hogs agglutinates strains of ordinary hog-cholera
bacilli in dilutions occasionally as high as i : 250, and con-
sider reaction in a dilution of less than i : 300 without
diagnostic value.

"Medical News," 1900, p. 237.
f "Microscopical Bulletin," 1897, p. 35.
J "Jour, of Infectious Diseases," March i, 1905, vol. n, No. 2, p. 351.

682 Bacillus Icteroides

BACILLUS ICTEROIDES (SANARELU).

General Characteristics.— An actively motile, flagellated, non-
sporogenous, non-liquefying, non-chromogenic, aerogenic, aerobic and
optionally anaerobic, pathogenic bacillus which stains by the ordinary
method, but not by Gram's method. It produces indol, but does not
coagulate milk.

Sanarelli regarded this bacillus as the specific organism of
yellow fever. He found it in n autopsies upon yellow
fever cases, but always in association with streptococci,
colon bacilli, proteus, and other organisms. It is found in
the blood and tissues, and not in the gastro-intestinal tract,
and isolation of the organism was possible in only 58 per
cent, of the cases, and only in rare instances was accom-
plished during life.

Distribution. — By suitable methods it can be found in
the organs of yellow fever cadavers, usually aggregated in
small groups, in the capillaries of the liver, kidneys, and
other organs. The best method of demonstration is to
keep a fragment of liver, obtained from a body soon after
death, in the incubator at 37° C. for twelve hours, and
allow the bacteria to multiply in the tissue before exam-
ination.

Morphology. — The bacillus presents nothing morpho-
logically characteristic. It is a small pleomorphous bacillus
with rounded ends, usually joined in pairs. It is 2 104 ^ in
length, and, as a rule, two or three times longer than broad.
It is actively motile and has flagella. It does not form spores.

Staining. — It stains by the usual methods, but not by
Gram's method.

Cultivation. — The bacillus can be grown upon the usual
media. It grows readily at ordinary room temperatures,
but best at 37° C.

Colonies. — Upon gelatin plates it forms rounded, trans-
parent, granular colonies, which during the first three or
four days somewhat resemble leukocytes. The granular
appearance becomes continuously more marked, and usu-
ally an opaque central or peripheral nucleus is seen. In
time the entire colony becomes opaque, but does not liquefy
gelatin.

Gelatin. — Stroke cultures on obliquely solidified gelatin
show brilliant, opaque, white colonies resembling drops of
milk. The medium is not liquefied.

Bouillon. — In bouillon it develops slowly, without either
pellicle or flocculi.

Bacilli Resembling the Typhoid Bacillus 683

Agar-agar. — The culture upon agar-agar is said to be
characteristic.

The peculiar and characteristic appearances of the colonies
do not develop if grown at 37° C.; but at 20° to 22° C. the
colonies appear rounded, whitish, opaque, and prominent,
like drops of milk. This appearance of
the colonies also shows well if the cul-
tures are kept for the first twelve to six-
teen hours at 37° C., and afterward at
the room temperature, when the colonies
will show a flat central nucleus, trans-
parent and bluish, surrounded by a
prominent and opaque zone, the whole
resembling a drop of sealing-wax. Sana-
relli refers to this appearance as consti-
tuting the chief diagnostic feature of
Bacillus icteroides. It can be observed
in twenty-four hours.

Blood-serum. — Upon blood-serum the
growth is very meager.

Potato. — The growth upon potato cor-
responds with that of the bacillus of
typhoid fever.

Vital Resistance. — It strongly resists
drying, but dies when exposed in cultures
to a temperature of 60° C. for a few
minutes, and is killed in seven hours by
the solar rays. It can live for a consider-
able time in sea- water.

Metabolism. — The bacillus is an op-
tional anaerobe. It slowly ferments dex-
trose, lactose, and saccharose, forming
gas only in dextrose solutions in which
there are no other sugars. It does not
coagulate milk. In the cultures a small
amount of indol is formed.

Pathogen esis. — The bacillus is patho-
genic for the domestic animals, all mammals seeming to be
more or less sensitive to it. Birds are often immune.
White mice are killed in five days, guinea-pigs in from eight
to twelve days, rabbits in from four to five days, by virulent
cultures. The morbid changes present include splenic tumor,
hypertrophy of the thymus, and adenitis. In the rabbit

Fig. 229. — Cul-
ture of bacillus
icteroides on agar
(Sanarelli).

684 Bacillus Murium

there are, in addition, nephritis, enteritis, albuminuria,
hemoglobinuria, and hemorrhages into the body cavities.

Sanarelli states that the dog is the most susceptible animal.
When this animal is injected intravenously, symptoms ap-
pear almost immediately and recall the clinical and anatomic
features of yellow fever in man. The most prominent symp-
tom in the dog is vomiting, which begins directly after the
penetration of the virus into the blood, and continues for a
long time. Hemorrhages appear after the vomiting, the
urine is scanty and albuminous, or is suppressed shortly
before death. Grave jaundice was once observed.

BACILLUS TYPHI MURIUM (Z,OFF%ER).

General Characteristics. — A motile, flagellated, non-sporogenous,
non-liquefying, non-chromogenic, non-aerogenic, aerobic and optionally
anaerobic bacillus, pathogenic for mice and other small animals, stain-
ing by the ordinary methods, but not by Gram's method. It acidulates
but does not coagulate milk.

Bacillus typhi murium was discovered by Loffler* in
1889, when it created havoc among the mice in his labora-
tory at Greifswald.

Morphology. — The organism bears a close resemblance
to that of typhoid fever, sometimes appearing short, some-
times long and flexible. There are many long and curly
flagella with peritrichial arrangement, and the organism is
actively motile. It does not produce spores.

Staining. — It stains with the ordinary dyes, but rather
better with Loffler's alkaline methylene-blue, not by Gram's
method.

Isolation. — The bacilli were first isolated from the blood
of dead mice.

Cultivation. — Their cultivation presents no difficulties.

Colonies. — Upon gelatin plates the deep colonies are at
first round, slightly granular, transparent, and grayish.
Later they become yellowish brown and granular. Super-
ficial colonies are similar to those of the typhoid bacillus.

Gelatin. — In gelatin punctures there is no liquefaction.
The growth takes place principally upon the surface, where
a grayish-white mass slowly forms, and together with the
growth in the puncture suggests a large flat-headed nail.

Agar-agar. — Upon agar-agar a grayish-white growth de-
void of peculiarities occurs.

* "Centralbl. f. Bakt. u. Parasitenk.," xi, p. 129.

Bacilli Resembling the Typhoid Bacillus 685

Potato. — Upon potato a rather thin whitish growth may
be observed after a few days.

Milk. — The bacillus grows well in milk, causing acid reac-
tion, without coagulation.

Bouillon. — In bouillon it produces clouding. There is no
fermentation of saccharose, dextrose, lactose, or levulose.

Pathogenesis. — The organism is pathogenic for mice of
all kinds, which succumb in from one to two days when
inoculated subcutaneously, and in from eight to twelve
days when fed upon material containing the bacillus. The

Fig. 230. — Bacillus typhi murium (Migula).

bacilli multiply rapidly in the blood- and lymph-channels,
and cause death from septicemia.

Loffler expressed the opinion that this bacillus might be
of use in ridding infested premises of mice, and its use for
this purpose has been satisfactory in many places. He has
succeeded in ridding fields so infested with mice as to be
useless for agricultural purposes, by saturating bread with
bouillon cultures of the bacillus and distributing it near their
holes. The bacilli not only killed the mice that had eaten
the bread, but also infected others which ate their dead
bodies, the extermination progressing until scarcely a mouse
remained in the field.

In discussing the practical employment of this bacillus

686 Bacillus Murium

for the satisfactory destruction of field-mice, Brunner* calls
attention to certain conditions that are requisite: (i) It is
necessary, first of all, to attack extensive areas of the in-
vaded territory, and not to attempt to destroy the mice of a
small field into which an indefinite number of fresh animals
may immediately come from surrounding fields. The
country people, who are the sufferers, should combine their
efforts so as to extend the benefits widely. (2) The prepa-
ration of the cultures is a matter of importance. Agar-agar
cultures are most readily transportable. They are broken up
in water, well stirred, and the liquid poured upon a large
number of small pieces of broken bread. These are then
distributed over the ground with care, being dropped into
the fresh mouse-holes, and pushed sufficiently far in to
escape the effects of sunlight upon the bacilli. Attention
should be paid to holes in walls, under railway tracks, etc.,
and other places where mice live in greater freedom from
disturbance than in the fields. (3) The destruction of
the mice should be attempted only at a time of the year
when their natural food is not plenty. By observing these
precautions the mice can be eradicated in from eight to
twelve days. In the course of two years no less than 250,000
cultures were distributed from the Bacteriological Labora-
tory of the Tierarznei Institut in Vienna, for the purpose of
destroying field-mice.

The bacilli are not pathogenic for animals, such as the fox,
weasel, ferret, etc., that feed upon the mice, do not affect
man in any way, and so seem to occupy a useful place in
agriculture by destroying the little but almost invincible
enemies of the grain.

A similar organism, secured from an epidemic among
field-mice and greatly increased in virulence by artificial
manipulation, has been recommended by Danyszf for the
destruction of rats. When subjected to a thorough study
by RosenauJ this organism was found to be identical with
Bacillus typhi murium. It is, however, too uncertain in
action to be relied upon for the destruction of rats in plague-
threatened cities for which it was suggested.

* "Centralbl. f. Bakt.," etc., Jan. 19, 1898, Bd. xxm, No. 2, p. 68.
t "Ann. de 1'Inst. Pasteur," April, 1900.

I "Bulletin No. 5 of the Hygienic Laboratory of the U.S. Marine
Hospital Service," Washington D. C., 1901.

CHAPTER XXVI.

DYSENTERY.

DYSENTERY is an acute, subacute, or chronic, infectious coli-
tis, usually characterized by an acute onset, mild fever,
pain in the abdomen, rectal tenesmus, and the passage of
frequent, usually small, mucous and bloody evacuations
from the rectum.

The disease was known to the ancients. It was probably
dysentery that is meant by "emerods" in describing an
epidemic that took place among the people of Israel during
the time of the Judges. Hippocrates differentiated between
diarrhea and dysentery.

Sporadic cases of the disease occur in almost all countries,
the number of such increasing as the equator is approached.
In addition to these sporadic cases epidemics not infrequently
appear. Though such may break out at any time in towns
or cities, they are more apt to occur when unusual activities
of the people are in progress. The most frequent of these
is military, and armies are apt to be the greatest sufferers.
The incidence of dysentery in the Federal Army during the
War of the Rebellion was appalling. Woodward* states
that there were 259,071 cases of acute and 28,451 of chronic
dysentery.

Endemics also occur from time to time and assume devas-
tating proportions, as in Japan, where between 1878 and 1899
there were 1,136,096 cases, with 275,308 deaths — a mortality
of 25.23 per cent.f Osier quotes Macgregor as saying, "In
the tropics dysentery is a destructive giant compared to
which strong drink is a mere phantom. It is one of the
great camp diseases and has been more destructive to armies
than powder and shot."

The disease early came under the observation of the bac-
teriologists, and Klebs, Ziegler, Ogata, Grigorieff, de Silvestri,

* "Medical and Surgical History of the War of the Rebellion," Medi-
cal, n.

t "Public Health Reports," Jan. 5, 1900, xv, No. i.

687

688 Dysentery

Maggiora, Arnaud, Celli and Fiocca, Galli-Valerio, Vala-
gussa, Deycke, and others published descriptions of various
micro-organisms isolated from dysenteric stools, and looked
upon by their discoverers as the cause of the disease. The
results were, however, so discordant that none of the described
micro-organisms could be agreed upon as the excitant of the
disease.

In 1 860 Lambl* published a description of an ameba found
in the human intestine. No one seemed inclined to believe
that it might have any significance until much later.

In 1875 Loschf described an ameba which he found in
great numbers in the colon of a case of dysentery occurring
in St. Petersburg. Not much notice was taken of his paper
or much made of his observation until eight years later, when
Koch and Gaffky, { in studying the cholera in Egypt, also
observed amebas in the intestinal discharges in certain cases,
and Kartulis§ wrote upon the "Etiology of the Dysentery in
Egypt," which he referred to these amebas. In America the
study of these amebas was quickly taken up. Osier || discov-
ered the organisms in the evacuations of a case of dysentery
contracted by a patient during a visit to Panama. Council-
man and Lafleur** wrote a fine monograph upon "Amebic
Dysentery," while Quincke and Roosff and Kruse and Pas-
qualeJt confirmed the observations and results in Europe.

Thus it came to be recognized that an ameba was the
cause of dysentery. It was soon pointed out, however, that
there were cases of dysentery in which no ameba could be
found in the intestinal discharges, or in which they were so
few that it seemed impossible that they could be the cause of
the disease. This was particularly impressive throughout
the years of the endemic dysentery in Japan, already referred
to. Great numbers of cases occurred, great numbers of
people died, no amebas were found to account for the disease.
It therefore occurred to Kitasato that some other causal agent
must be looked for, and Shiga took up the problem, which was
* "Aus. d. Franz Joseph Kinderspital zur Prague," 1860, I, 326.
f'Virchow's Archives," 1875, Bd. LXV.

t "Behricht iiber die Erforschung du Cholera," 1883; "Arbeiten aus
d. Kaiserl. Gesundheitsamt.," in, 65.
§ "Virchow's Archives," 1886, cv.
|| "Centralbl. f. Bakt. u. Parasitenk.," 1890, vu, 736.

** "Johns Hopkins Hospital Reports," 1891, n.

ft "Berliner klin. Wochenschrift," 1893,

it " Zeitschrift f. Hygiene," etc., 1894, xvi.

Amebic Dysentery 689

a difficult one, and might not have been solved had he not
made use of a new means of investigation, viz., the phenom-
enon of agglutination. By studying such bacteria as could
be cultivated from the intestinal discharges, with particular
reference to the effect of the blood of dysenteric patient's
blood in agglutinating them, Shiga * succeeded in discovering
a new micro-organism which he called Bacillus dysenteriae.
Two years afterward Krusef investigated an outbreak of
dysentery in an industrial section of Westphalia and found
the same bacillus, and FlexnerJ showed the same bacillus to
be present in the epidemic dysentery of the Philippine
Islands.

Thus the discovery of Shiga became thoroughly substanti-
ated, and it became evident that in addition to theameba there
was a bacillus to be reckoned with in the etiology of dysen-
tery. It soon became evident that there are two forms of
dysentery, one amebic, the other bacillary. Both occur spo-
radically and endemically in the tropics and in temperate
climates, and both may occur epidemically, though of the
two the bacillary form is much more liable to do so. Of the
chronic cases of dysentery 90 per cent, are amebic.

L AMEBIC DYSENTERY.

AMOEBA COLI (Loscn, 1875); AMCEBA DYSENTERIC (COUN-
CILMAN AND LAFLEUR, 1893); ENTAMCEBA HisToivYTicA

(SCHAUDINN, 1903.)

As has been shown, amebas were first found in the human
intestine by Lambl; in dysentery, by Losch, Koch, Gaffky,
Kartulis, Osier, Councilman and Lafleur, and many others.
The welcome finally accorded to the organisms as excitants
of dysentery was sufficiently enthusiastic to compensate for
the neglect of a quarter of a century. In all countries intes-
tinal ameba was looked for, and while it was becoming clear
in the minds of many pathologists that there could be no
doubt about the role played by the ameba in dysentery, it
was becoming equally clear to others that amebas were not
infrequently present in intestines in which there was not and
had not been dysenteric infection, and many cases of dysentery

* "Centralbl. f. Bakt. u. Parasitenk.," 1898, xxiv, 817.
t "Deutsche med. Wochenschrift," 1900, No. 40.
t "Centralbl. f. Bakt. u. Parasitenk.," 1900, xxvm, No. 19.
44

6go

Dysentery

in which there were no ameba. The lapse of time and the
thoroughness of investigation that came with it solved both
of the difficulties, for it was shown that there were several
varieties of intestinal amebas and also that there was a
bacillary as well as an amebic form of dysentery.

Celli and Fiocca* were the first to study the amebas sys-
tematically and to cultivate them upon artificial media.
Councilman and Lafleur pointed out that there were two
varieties of amebas which they called Amoeba coli and Amoeba
dysenteriae. The former was supposed to be a harmless
commensal, the latter a pathogenic organism and the cause
of dysentery. As, however, Losch had called the organism

Fig. 231. — Amoeba coli in intestinal mucus, with blood-corpuscles and
bacteria (Losch).

found in dysentery the Amoeba coli, Stiles declared the nomen-
clature faulty, and pointed out that Amoeba coli, variety dys-
enteriae, must be the name of the pathogenic form. Schau-
dinn f reviewed the subject and grouped all of the intestinal
amebas under the following:

I. Chlamydophrys stercorea (Cienkowsky).

II. Amoeba coli rhizopodia.

1. Entamoeba coli (Losch) (Schaudinn).

2. Entamoeba histolytica (Schaudinn).
To these has been since added in 1907:

Entamoeba tetragena (Viereck).

* "Centralbl. f. Bakt. u. Parasitenk.," 1894, xv, 470.

t "Arbeiten aus d. Kaiserl. Gesundheitsamt.," 1903, xix, No. 3.

Amebic Dysentery 691

1. Entamoeba Coli (Losch, 1875). — This organism seems
to be a harmless commensal, living in the intestines of man,
many domestic, and many wild animals. It may be abundant
when the reaction of the intestinal contents is neutral or al-
kaline. It usually measures between 10 and 20 ^ in diameter
when free, but when encysted from 15 to 50 p. It is sphe-
roidal when not in motion, and under these conditions it is
difficult to differentiate endoplasm and ectoplasm. The
ameboid movement is sluggish and the pseudopods are rather
short, broad, and blunt. As they are protruded the clear
ectoplasm becomes visible. The organism has a grayish
color, a finely granular cytoplasm, and usually only a single
vacuole. The nucleus is usually fairly well defined and
spherical, and, in addition to the chromatin, contains several
nucleoli. When stained with polychrome methylene-blue
the ectoplasm stains blue; the endoplasm, violet; and the
nucleus, red.

Reproduction usually takes place by simple division, but
a form of autogamous sporulation also takes place, the organ-
ism first becoming encysted, the nucleus dividing into eight
segments, and the whole process eventuating in the formation
of eight young organisms.

This ameba is easily cultivated upon artificial media
according to methods to be described below.

It is not pathogenic, and all attempts to make it damage the
the intestine of experiment animals have failed.

2. Entamoeba Histolytica (Schaudinn). — This is now
recognized as the organism seen by Losch, Koch, Kartulis,
Councilman and Lafleur, and accepted as the cause of the
amebic form of dysentery. It is found in all parts of the
world, but more frequently in tropical than colder climates,
and is present only in the intestines of those suffering from
dysentery. When present it is usually in great numbers, so
that its discovery in the evacuations is usually easy.

Morphology. — It is usually considerably larger than Enta-
moeba coli and varies in diameter up to 50 /w. When at rest
it is spherical, when active it is very irregular. Its move-
ment is active and the pseudopodia are larger and more
numerous than in the other species. The differentiation of
ectoplasm and endoplasm is usually distinct. The former is
hyaline, the latter granular. The protoplasm has a greenish
or yellowish color. The nucleus is small, not very distinct.
There are numerous vacuoles. When examined in the in-

69 2 Dysentery

testinal evacuations of dysentery the protoplasm commonly
contains many red blood-corpuscles, upon which the organ-
ism seems to feed.

Staining. — When stained with polychrome methylene-blue
the ectoplasm stains more deeply than the endoplasm. The
nucleus contains relatively little chromatin.

Reproduction. — Multiplication takes place by binary divis-
ion after karyokinesis and by encystment and sporulation.
The sporulation is quite different from that seen in Enta-
moeba coli, and only takes place when conditions are unfavor-
able to continued division. It is accomplished by a peculiar
nuclear budding, by which chromatin granules or chronidia
are pushed out from the nucleus toward the ectoplasm, where
they develop into new nuclei, about which the cytoplasm
collects until a distinct bud is formed and cast off as a small
but distinct new organism — a spore or bud. These when
separated are round or oval, measure 3 to 6 p in diameter,
and are surrounded by a yellowish envelope, which resists
drying and the penetration of stains and chemicals.

Craig gives (see page 693) a tabulation of the differential
features of Bntamoeba coli, Bntamoeba hystolytica, and
Entamoeba tetragena.

3. Entamceba Tetragena. — This organism resembles En-
tamoeba hystolytica more than Amoeba coli, but differs from
it in the mode of reproduction. The sporocysts contain but
four instead of eight spores.

Isolation and Cultivation. — Many experimenters have made
more or less successful attempts to cultivate ameba. Mus-
grave and Clegg,* whose interesting paper the student will
do well to read, and in which he will find a complete review of
all antecedent work, were able to cultivate a considerable
variety of amebas upon agar-agar made of :

Agar 20.

Sodium chlorid 0.3-0.5

Extract of beef 0.3-0.5

Water 1000.0

Prepare as ordinary culture agar, and render i per cent,
alkaline to phenolphthalein. The finished medium is poured
into Petri dishes. To obtain the greatest number of most
active amebas the patient should be given a dose of a saline
purgative, and the fluid evacuation resulting from its action
employed for inoculating the media. The cultures are, natu-

* "Department of the Interior, Bureau of Government Laboratories,
Biological Laboratory," Manila, Oct., 1904, No. 8.

Amebic Dysentery

693

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694 Dysentery

rally, not pure; they contain various amebas and numerous
bacteria.

To isolate and cultivate a single kind of ameba Musgrave
and Clegg have recommended an ingenious technic. A plate
is selected upon which the desired amebas are so widely sep-
arated from one another that not more than one is in a
microscopic field of a low-power objective. The microscope
used should have a double or triple nose-piece. With a low-
power (Zeiss A A) objective a well isolated organism is brought
to the center of the field. The lens is then swung out and a
perfectly clean higher power lens (Zeiss DD) swung in and
racked down until it touches the surface of the agar-agar, when
it is quickly elevated again. In three out of five cases the
ameba adheres to the objective and is so picked up. Whether
it has done so or not can be determined by swinging in the
low-power lens again and looking for the organism. If it
has disappeared, it is attached to the objective. It is now
planted upon a fresh plate by depressing the high-power lens
until it touches the surf ace of the culture-medium, when, upon
elevating it again, it usually leaves the ameba behind. Ob-
servation with the low power will enable one to determine
whether it be successfully planted or not.

Naturally the organisms cannot be thus transplanted
without some bacteria falling upon the plate, but this is not
very material, for in the first place they do not grow very
rapidly upon the medium used for culture, and in the
second, they are essential to the nourishment of the ameba,
which is holophagous, and cannot live by the absorption
of nutritious fluids. Later it was by Tsugitani* shown
that killed cultures of bacteria could supply the necessary
nourishment. All cultures of amebas must contain the
symbiotic organisms upon which the amebas live. It can-
not always be foretold what symbiotic organisms are needed.
When planted as above suggested a variety of organisms
grow, and as the amebas multiply and gradually extend
over the plate, their preference for one or other of the
associated bacteria may be determined in part by placing a
drop of the ameba culture upon a plate of sterile media, and
then with the platinum wire, dipped in a culture of the bac-
teria, drawing concentric circles about the drop further and
further apart. As the amebas move about over the plate,
passing through the growing circles of bacteria, they soon
* "Centralbl. f. Bakt. u. Parasitenk.," Abt. 5, xxiv, 666.

Amebic Dysentery 695

loose the miscellaneous bacteria and come to contain the one
variety planted with them, or if several have been used in
drawing different circles, that one which they prefer to feed
upon. By transplanting the amebas from plate to plate with
suitable symbiotic bacteria for them to feed upon, the cul-
tures may be kept growing almost indefinitely.

Anna Williams* has been able to grow ameba in pure
culture without symbiotic bacteria, either dead or alive, by
smearing the surface of a freshly prepared agar-agar plate
with a fragment of freshly removed rabbit's or guinea-pig's
brain, kidney, or liver, held in a pair of forceps. The
ameba gladly take up and live upon the cells left behind
upon the surface of the agar.

Vital Resistance. — The free amebas in the intestinal dis-
charges are easily destroyed by dilute germicides and by
drying. Encysted amebas are, however, more difficult to
kill. They resist drying well and also resist the penetra-
tion of germicides. Direct sunlight inhibits the activities
of the organisms, but does not kill them. Losch was the
first to observe that quinin was destructive to intestinal
amebas, and his observations have been reviewed by many
others. Musgrave and Clegg found that active cultures of
one ameba were killed in ten minutes by a i : 2500 solution
of quinin hydrochlorate. The exposed organisms quickly
encysted themselves, and in from five to eight minutes many
of them had broken up and disappeared. After ten minutes
all were dead. Cultures of another ameba similarly treated
gave a scanty growth after ten minutes.

Exposure to i : 1000 solution of formalin did not kill en-
cysted amebas in twenty-four hours. Acetozone did not kill
amebas in i : 1000 dilutions. If, however, the acetozone was
made i per cent, acid to phenolphthalein the amebas were all
killed by i : 5000 solutions in ten minutes.

Metabolic Products. — It seems as though Entamoeba his-
tolytica must produce some metabolic product that exerts an
enzymic action upon the human tissues and thus account for
the destructive nature of the lesions. This has not, however,
been demonstrated as yet.

Pathogenesis. — Schaudinn was the first to prove the patho-
genic action of the organism. He inspissated the evacuations
of a case suffering from dysentery, so that it contained con-
siderable numbers of encysted amebas. When this was fed
! "Journal of Medical Research," xxv, No. 2, Dec.. 1911, p. 263.

696

Dysentery

to kittens they died in two weeks with the typical lesions of
dysentery. Musgrave and Clegg had less satisfactory re-
sults with cats, dogs, and other laboratory animals, but were
quite satisfied of the results secured with monkeys, which
took the disease and sometimes died. The lesions resembled,

but were less severe than, those in man. Musgrave and Clegg
would not admit that there were non-pathogenic intestinal
amebas, but this was not in accord with the work of any other
investigators, and was strongly opposed by Craig,* who found
both varieties, and though he was never able to infect ani-
* "Journal of Infectious Diseases," v, 1908, p. 324.

Amebic Dysentery 697

mals with Emtamoeba coli, was successful with the pathogenic
varieties, and succeeded in infecting 50 per cent, of the kittens
he experimented upon by injecting the amebas into the
rectum.

Lesions. — The gross morbid appearances of the intestinal
lesions in both forms of dysentery are sufficiently distinct in
typical cases to enable an experienced pathologist to differ-
entiate them, yet not sufficiently distinct to make them easy
of description. The one great characteristic feature of the
amebic dysentery is abscess of the liver which occurs in
nearly 25 per cent, of the cases, but which never occurs in
bacillary dysentery.

The distinct and somewhat rigid ectoplasm of the Enta-
moeba histolytica is supposed to make it easy for the organism,
which it will be remembered are actively motile, to penetrate
between the epithelial cells of the intestinal mucosa to the
lymph-spaces of the submucosa below. Here the amebas
multiply in large numbers, and by the enzymic action of their
metabolic products produce necrosis of the suprajacent tis-
sues with resulting exfoliation and the production of round,
oval, or ragged ulcerations with markedly infiltrated and un-
dermined edges. As the amebas continue to increase and
fill up the lymphatics, and as bacteria add their effects to
those occasioned by the amebas, the ulcers increase in extent
and depth until the mucosa and submucosa may be almost
entirely destroyed, leaving the entire large intestine denuded,
except for occasional islands of much congested, inflamed, and
partly necrotic mucous membrane. The diseased wall is the
seat of much congestion and is much thickened. The
amebas not only occur in great numbers in the interstices
of the tissues about the base of the ulcers and in the lym-
phatics, but also enter the capillaries, through which they are
carried to the larger vessels, and eventually to the liver, where
their activities continue and give rise to the amebic abscess.
The first expression of their injury to the liver parenchyma
is shown by focal necroses. In each of these the organisms
multiply and the lesion extends until neighboring necroses are
brought into union, and eventuate in great collections of
colliquated necrotic material which may be so extensive as
to involve the entire thickness of the organ. There is usually
one large abscess, but there may be several small ones, or
the liver may be riddled with minute lesions. The contents
of the abscess is pinkish necrotic material in which amebas are
few. The walls are of semi- necrotic material, in which great

698 Dysentery

numbers of amebas abound. The liver sometimes becomes
adherent to the diaphragm, may perforate it, and after ad-
hesion of the lung to the diaphragm may evacuate through
the lung, the pinkish abscess contents with amebas being
expectorated.

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wall showing the amebas at the base of a dysenteric ulcer: A, A, A,
Amebas, some of which are in blood-vessels, Gf (Harris).

Sections of the intestinal wall and of the liver near the
border of the abscess show the amebas well when stained
with iron-hematoxylon, or perhaps still better by Mallory's
differential method.*

1. Harden the tissue in alcohol.

2. Stain sections in a saturated aqueous solution of thionin three to
five minutes.

3. Differentiate in a 2 per cent, aqueous solution of oxalic acid for
one-half to one minute.

4. Wash in water.

5. Dehydrate in absolute alcohol.

6. Clear in alcohol.

7. Xylol-balsam.

The nuclei of the amebas and the granules of the mast-cells are
stained brownish red ; the nuclei of the mast-cells and of all other cells
are stained blue.

*" Pathological Technic," 1911, p. 434.

Bacillary Dysentery 699

IL BACILLARY DYSENTERY.

BACILLUS DYSENTERIC (SHIGA),

General Characteristics. — A non-motile, non-flagellated, non-
sporogenous, non-liquefying, aerobic and optionally anaerobic, non-
chromogenic, non-aerogenic, pathogenic bacillus of the intestine,
staining by ordinary methods, but not by Gram's method. It does
not produce mdol. It first acidifies, then alkalinizes milk, but does not
coagulate it.

After considerable investigation of the epidemic dysentery
prevalent in Japan, Shiga* has come to the conclusion that
a bacillus which he calls Bacillus dysenteriae is its specific
cause.

It is not improbable that the bacillus of Shiga is identical
with Bacterium coli, variety dysenteries, of Celli, Fiocca, and
Scala,t a view that has been further confirmed by Flexner.J
It may also be identical with an organism described in 1888
by Chantemasse and Widal.§

In 1899 Flexner,|| while visiting the Philippine Islands, iso-
lated a bacillus from the epidemic dysentery prevailing there,
which he regarded as identical with Shiga's organism. In
1890 Strong and Musgrave** isolated what appeared to be
the same organism, also from cases of dysentery in the
Philippines. Almost at the same time Kruseft was inves-
tigating an epidemic of dysentery in Germany, and suc-
ceeded in isolating a bacillus that also bore fair correspond-
ence to that of Shiga. In 1901 SpronckJt found a bacillus in
cases of dysentery occurring in Utrecht, Holland, that cor-
responded with a slightly different organism first found and
described by Kruse§§ as a " pseudodysentery bacillus."

In 1902 Park and Dunham |||| investigated a small epi-
demic of dysentery in Maine, and there found a bacillus
similar to those already described. In 1903 Hiss and Rus-

* "Centralbl. f. Bakt. u. Parasitenk.," 1898, xxiv, Nos. 22-24.

f'Hygien. Institut. Rom. Univ.," 1895, and "Centralbl. f. Bakt.
u. Parasitenk.," 1899.

| "Univ. of Penna. Med. Bulletin," Aug., 1901.

§ "See Deutsche med. Wochenschrift," 1903, No. 12.

|| "Bulletin of the Johns Hopkins Hospital," 1900, ix.
** "Report Surg. Gen. U. S. Army," Washington, 1900.
ff " Deutsche med. Wochenschrift," 1900, xxvi.
tt " Ref. Baumgarten's Jahresberichte," 1901.
§§ " Deutsche med. Wochenschrift," 1901, Nos. 23 and 24.
|||| "New York Bull, of Med. Sciences," 1902.

700 Dysentery

sell described a bacillus " Y " from a case of fatal diarrhea in
a child.

Bacillus dysenteriae was also found by Vedder and Duval*
in the epidemic and sporadic dysentery of the United States.
Duval and Bassettf and Martha WollsteinJ found Bacillus
dysenteriae in cases of the summer diarrheas of infants, especi-
ally when such diarrheas were epidemic.

About this time Lentz§ published an interesting and im-
portant paper in which such dysentery and pseudodysentery
bacilli as he could secure were found to present differences
in their behavior toward sugars. Other observers were care-
fully comparing the behavior of the various bacilli by means
of the agglutination by artificially prepared immune serum.
The outcome of these investigations is the discovery that
Bacillus dysenteriae is a species in which there are a number
of different varieties well characterized, but by differences
too slight to permit them to be regarded as separate species.
This thought — that we are dealing with a group of varieties
and not a single well-defined organism — is essential to an
intelligent understanding of the bacteriology of dysentery.

Before taking up the variations, the characters common to
all and constituting those of the type species must be
described.

Morphology. — The organism is a short rod with rounded
ends, generally similar to the typhoid bacilli. It usually
occurs singly, but may occur in pairs. It is frequently sub-
ject to involutional changes. It is doubtfully motile and is
probably without flagella.

Staining. — When stained with methylene-blue the ends
color more deeply than the middle; and organisms from
old cultures show numerous involution forms and irregu-
larities. It stains with ordinary solutions, but not by
Gram's method. It has no spores.

Cultivation. — The organism grows well in slightly al-
kaline media under aerobic conditions.

Colonies. — The colonies upon gelatin plates are small and
dewdrop-like in appearance. Upon microscopic examination
they are seen to be regular and of spheric form. By trans-

* "Journal of Experimental Medicine," vol. vi, No. 2, 1902; "Amer-
ican Medicine," 1902.

t "American Medicine," Sept. 13, 1902, vol. iv, No. n, p. 417.
J "Jour. Med. Research," x, p. u, 1904.
§ "Zeitschrift f. Hygiene," etc., 1902, xu.

Bacillary Dysentery 701

mitted light they appear granular and of a yellowish color.
They do not spread out in a thin pellicle like those of the
colon bacillus, and there are no essential differences between
superficial and deep colonies.

Gelatin Punctures. — The growth in the puncture culture
consists of crowded, rounded colonies along the puncture.
A grayish- white growth forms upon the surface. There is
no liquefaction of the gelatin.

Agar-agar. — Upon the surface of agar-agar, cultures
kept in the incubating oven show large solitary colonies at
the end of twenty-four hours. They are bluish- white in
color and rounded in form. The surface appears moist.
In the course of forty-eight hours a transparent border is
observed about each colony, and the bacilli of which it is
composed cease to stain evenly, presenting involution forms.

Glycerin agar-agar seems less well adapted to their growth
than plain agar-agar. Blood-serum is not a suitable medium.

Litmus Milk. — Milk is not coagulated. As the growth
progresses there is slight primary acidity, which later gives
place to an increasing alkalinity.

Potato. — Upon boiled potato the young growth resembles
that of the typhoid bacillus, but after twenty-four hours it
becomes yellowish brown, and at the end of a week forms
a thick, brownish-pink pellicle.

Bouillon. — In bouillon the bacillus grows well, clouding
the liquid. No pellicle forms on the surface.

Metabolic Products. — The organism does not form in-
dol, does not ferment dextrose, lactose, saccharose, or other
carbohydrates. Acids are produced in moderate quantities
after twenty-four hours. Milk is not coagulated. Gelatin
is not liquefied.

Toxins, chiefly endotoxins, are produced. They may best
be prepared by making massive agar-agar cultures in Kitasato
flasks or flat-sided bottles, and after growth is complete
washing off the bacillary mass with a very small quantity of
sterile salt solution, and after killing the bacilli by exposure
to 60° C. for fifteen to thirty minutes, permitting the rich
suspension to autolyze for three days. The toxins may be
precipitated from the sodium chlorid solution by ammonium
sulphate.

Vital Resistance. — The thermal death-point is 68° C.
maintained for twenty minutes. It grows slowly at ordi-
nary temperatures, rapidly at the temperature of the body.

702 Dysentery

The Different Varieties of the Dysentery Bacillus.—

Three varieties of the dysentery bacillus may now be de-
scribed :

1. The Shiga-Kruse variety.

2. The Flexner variety.

3. The Hiss-Russell variety.

The differences by which they are separated are to be
found in the agglutinability by artificially prepared immune
serum, each of which exerts a far more pronounced effect
upon its own variety than upon the others, and in the be-
havior toward sugars with reference to acid formation and
gas production. It seems not improbable that the future
will have much to say about the dysentery bacillus, and
that the validity of much that is accepted at present may
have to be amended. This seems to be particularly true
with regard to the matter of fermentation, the details of
which are displayed in the following table taken from Muir
and Ritchie's "Manual of Bacteriology."

Pathogenesis. — Shiga and Flexner found that infection
of young cats and dogs could be effected by bacilli introduced
into the stomach, and that lesions suggestive of human dys-
entery were found in the intestines. Kazarinow * found that
when guinea-pigs and young rabbits were narcotized with
opium, the gastric contents alkalinized with 10 c.c. of a 10
per cent. NaOH solution, and a quantity of Shiga bacilli
introduced into the stomach with an esophageal bougie, it
was possible to bring about diarrhea and death with lesions
similar to those described by Vaillard and Dopter.

In these experiments it was found that rapid passage
through animals greatly increased the virulence of the
bacilli, and it was also observed that though 0.0005 c.c. of
a virulent culture introduced into the peritoneal cavity
would cause fatal infection, to produce infection by the
mouth as above stated required the entire mass of organisms
grown in five whole culture- tubes.

The virulent organisms are infectious for guinea-pigs and
other laboratory animals, and cause fatal generalized infec-
tion without intestinal lesions.

Lesions. — The lesions found in human dysentery are
usually fairly destructive. They consist of a severe catarrhal
and pseudomembranous colitis, which later passes into a

* " Archiv. f. Hyg.," Bd. L, Heft i, p. 66; see also "Bull, de 1'Inst.
Past.," 15 Aout, 1904, p. 634.

Bacillary Dysentery

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704 Dysentery

stage of marked ulceration. There is great thickening of the
submucosa and the whole of the intestinal lining is corru-
gated. For the most part the ulcerations are more superfi-
cial than those of the amebic dysentery, and the edges of the
ulcerations show less tumefaction and less undermining.
Abscess of the liver does not occur in bacillary dysentery.

Diagnosis. — The blood-serum of those suffering from
epidemic dysentery or from those recently recovered from it
causes a well-marked agglutinative reaction. This agglutina-
tion was first carefully studied by Flexner, and is peculiar,
in that the serums prepared from the different varieties of
the bacillus, while they exert some action upon all varieties
of the organism, exert a much more powerful influence upon
the particular variety used in their preparation. The same
is true of the patient's serum, hence, in making use of the
agglutination reaction for the diagnosis of the disease, the
blood of the patient should be tested by contact with all of
the different cultures.

Serum Therapy. — By the progressive immunization of
horses to an immunizing fluid, the basis of which is a twenty-
four-hour-old agar-agar culture dried in vacua, Shiga has
prepared an antitoxic serum with which, in 1898, in the
Laboratory Hospital, 65 cases were treated, with a death-rate
of 9 per cent.; in 1899, in the Laboratory Hospital, 91 cases,
with a death-rate of 8 per cent.; in 1899, in the Hirowo
Hospital, no cases, with a death-rate of 12 per cent. These
results are very significant, as the death-rate in 2736 cases
simultaneously treated without the serum averaged 34.7
per cent., and in consideration of the frequency and high
death-rate of the disease, Japan alone, between the years
1878 and 1899, furnishing a total of 1,136,096 cases, with
275,308 deaths (a total mortality for the entire period of
24.23 per cent.).*

III. BALANTIDIUM DIARRHEA.
BALANTIDIUM Cou (MALMSTEN).

In certain rare cases a severe form of diarrhea, or a mild

form of dysentery appears to depend neither upon Entamoeba

histolytica nor Bacillus dysenteriae, but upon an infusorian

parasite known as Balantidium coli. This organism was

* "Public Health Reports," Jan. 5, 1900, vol. xv, No. i.

Balantidium Diarrhea 705

first observed by Malmsten* in 1857 in the intestines of a
man who had suffered from cholera two years before and had
ever since suffered from diarrhea. Upon investigation an
ulceration was found in the rectum just above the internal
sphincter. In the bloody pus from this ulcer numerous
balantidia were seen swimming about. Although the ulcer
healed, the diarrhea did not cease. Since this original ob-
servation and up to 1908 Braunf had been able to collect
142 cases of human infection. In all of these cases the pres-
ence of the balantidium was accompanied by obstinate diar-
rhea with bloody discharges (dysentery) in some, and many
of the cases ended in death.

Morphology. — The Balantidium coli is an infusorian
micro-organism of ovoid or ellipsoidal form, measuring from
30 to 200 fi in length and from 20 to 70 [i in breadth. The
body is surrounded by a distinct ectosarc completely covered
by short fine cilia. The anterior end, which is usually a little
sharper than the posterior, presents a deep indentation, the
peristome, which continues, in an infundibuliform manner,
deeply into the endosarc. The peristome is surrounded by a
circle of longer cilia — adoral cilia — than those elsewhere
upon the body. At the opposite pole there is a small open-
ing in the ectosarc, the anus. The mouth is the simple ter-
mination of the infundibuliform extension of the peristome
and opens directly into the endosarc, so that the small bodies
upon which the organism feeds, and which are continually
being caught in the vortex caused by the rapidly vibrating
adoral cilia are driven down the short tubulature directly
into the endosarc.

The endosarc is granular and contains fat and mucin
granules, starch grains, bacteria, and occasionally red and
white blood-corpuscles.

There are usually two contractile vacuoles, sometimes more,
and as the quiet organism is watched these large clear spaces
can be seen alternately to contract and expand.

There are two nuclei. The larger, or macronucleus, is bean-
shaped, kidney-shaped, or, more rarely, oval. The smaller,
the micronucleus, is spherical. There is no digestive tube;
the nutritious particles are directly in the endosarc, in which
they are digested, any residuum being extruded from the
anus.

! "Archiv. f. pathologische Anatomic," etc., xn, 1857, p. 302.
t "Tierische Parasiten des Menschen," Wiirzburg, 1908.
45

706

Balantidium Coli

Motility. — The organism is actively motile, swimming
rapidly at a steady pace or darting here and there.

Staining. — The organism can be most easily and satisfac-
torily studied while alive. To stain it a drop of the fluid
containing the balantidia is spread upon a slide and per-
mitted to dry. Just before the moisture disappears from the
film, methyl alcohol may be poured upon it to kill and fix
the organisms. The staining may then be performed with
Giemsa's polychrome methylene-blue or iron-hematoxylon.
The cilia usually do not show.

Fig. 234. — Reproduction of Balantidium coli: 1-5, Asexual reproduc-
tion by division; 6, encysted form of single individuals; 7, conjugation
of two individuals; 8, reproductive cyst; 9, cyst with peculiar contents
whose further development, has not been followed (Brumpt).

Reproduction. — This commonly takes place by karyoki-
nesis, followed by transverse division, and in cases of ex-
perimental infection so rapidly that the organisms have not
time to grow to the full size before dividing again. The result
is that many appear that are no more than 30 (* in length.
In addition to multiplication by division, there is a sexual
cycle of development with conjugation. This was first pointed

Balantidium Diarrhea 707

out by Gourvitsch,* studied by Leger and Duboscq,f and
further confirmed by BrumptJ. In the process of conjuga-
tion two individuals come together, become attached length-
wise, and fuse into a single large organism that forms a cyst
several times as large as a balantidium, and with contents no
longer recognizable as such. The contents of this cyst
eventually divide into a number of spheres, but how these
subsequently develop appears not to have been deter-
mined.

Habitat. — The balantidium is unknown except as a para-
site of the colon. It is very common in hogs and has been
found in the orang-outang, in certain lower monkeys
(Macacus cynomolgus), and in man.

Cultivation. — The organism quickly dies when trans-
planted to artificial media and has not yet been cultivated
artificially.

Pathogenesis. — The presence of the organisms, in whatever
kind of animal, gives rise to colitis, which is at first catarrhal,
but soon becomes more or less ulcer ative. Some doubt has
been expressed as to the exact role of the balantidia in
the causation of the inflammation, some believing them to be
rather accidental factors than the true etiologic excitants.
As the organisms descend into the ulcerated tissues and from
the denuded surfaces invade the lymphatics, there seems to
be little doubt of their pathogenic activity.

Animal Inoculation. — Experiments made by Casagrandi
and Barbagallo,§ Klimenko, || and others upon kittens and
pups have failed to produce the disease even when the colon
was already inflamed. Brumpt,** on the contrary, succeeded
in reproducing it in monkeys and pigs by introducing the
encysted organisms into the already inflamed intestine via the
anus.

Lesions. — In the majority of fatal cases postmortem ex-
amination of the colon shows it to be in a state of catarrhal
inflammation with numerous superficial ulcerations with con-
siderable surrounding infiltration of the mucosa. Twenty-

*"Russ. Archiv. f. Path. klin. Med. u. Bact. St. Petersb.," 1896,
quoted by Braun.

t "Archiv. de Zool. Exper.," 1904, n, No. 4.
J "Compt.-rendu de la Soc. de Biol.," July 10, 1909.
§ "Bal. coli," etc., Catania, 1896, quoted by Braun.
|| "Beitrage zur. Path. Anat. u. allg. Path.," 1903, xxxn, 281.
** "Precis de Parasitology," 1910, 152.

Balantidium Coli

four hours from the time of the death of the patient the
balantidia are all dead. Strong and Musgrave,* Solowiew, f
Klimenko, t and others have shown that in microscopic sec-
tions of the inflamed tissues the micro-organisms could be
found deep down in the blood-vessels and lymphatic spaces
about the ulcerated areas, sometimes penetrating as deeply
as the serous coat of the bowel. Metastatic abscess of the
liver may be caused by balantidia, and has been reported

Fig- 235. — Balantidium coli deeply situated in the interglandular tissue
of the intestinal mucosa (Brumpt).

by Manson,§ and a case of abscess of the lung caused by
the organism by Winogradow and Stokvis.H

Transmission. — The transmission of the disease can only
come about through the encysted form of the parasites.
Great numbers are passed in the fecesof the infected animals,

* "Bulletin of the Johns Hopkins Hospital," 1901, xn, 31.
t "Centralbl. f. Bakt.," etc., i Abl., 1901, xxix, 821, 849.
t Loc. cit.

§ "Tropical Diseases," 1900, p. 394.

|| "Niederl. Tijdschr. v. Geneeskde.," 1884, xx, No. 2, quoted by
Braun.

Balantidium Diarrhea 709

but except the encysted forms all die very quickly as the
fecal matter dies. Unfortunately the further life-history of
the encysted forms is unknown.

FLAGELLATES IN THE HUMAN INTESTINES.

In certain cases of diarrhea, flagellates — Trichimonas in-
testinalis, Cercomonas intestinalis, and Lamblia (Megasto-
mum) intestinalis have been discovered. As, however, they
seem to be frequent denizens of normal intestines, it is
doubtful whether their presence is more than incidental.

CHAPTER XXVII.
TUBERCULOSIS.

BACILLUS TUBERCULOSIS (KOCH).*

General Characteristics. — A non-motile, non-flagellate, non-spor-
ogenous, non-liquefying, non-chromogenic, non-aerogenic, distinctly
aerobic, acid-resisting, purely parasitic, highly pathogenic organism,
staining by special methods and by Gram's method. Commonly oc-
curring in the form of slender, slightly curved rods with rounded ends,
not infrequently showing branches, hence probably not a bacillus, but
an organism belonging to the higher bacteria. It does not produce
indol or acidulate or coagulate milk.

Tuberculosis is one of the most dreadful and, unfor-
tunately, one of the most common diseases. It is no re-
specter of persons, but affects alike the young and old, the
rich and poor, the male and female, the enlightened and
savage, the human being and the lower animals. It is the
most common cause of death among human beings, and is
common among animals, occurring with great frequency
among cattle, less frequently among goats and hogs, and
sometimes, though rarely, among sheep, horses, dogs, and
cats.

Wild animals under natural conditions seem to escape
the disease, but when caged and kept in zoologic gardens,
even the most resistant of them — lions, tigers, etc. — are
said at times to succumb to it, while it is the most common
cause of death among captive monkeys.

The disease is not limited to mammals, but occurs in a
somewhat modified form in birds, and it is said even at
times to affect reptiles, batrachians, and fishes.

The disease has been recognized for centuries ; and though,
before the advent of the microscope, it was not always clearly
differentiated from cancer, it has not only left unmistakable
signs of its existence in the early literature of medicine, but
has also imprinted itself upon the statute-books of some
countries, as the kingdom of Naples, where its ravages were
great and the means taken for its prevention radical.

* "Berliner klin. Wochenschrift," 1882, 15.
710

Distribution 711

Specific Organism. — Although the acute men of the early
days of pathology clearly saw that the time must come
when the parasitic nature of tuberculosis would be proved,
and Klebs, Villemin, and Cohnheim were " within an ace "
of its discovery, and Baumgarten* probably saw it in tissues
cleared with lye, it remained for Robert Koch to demon-
strate and isolate the Bacillus tuberculosis, the specific
cause of the disease, and to write so accurate a description
of the organism, and the lesions it produces, as to be almost
without a parallel in medical literature.

Distribution. — So far as is known, the tubercle bacillus is
a purely parasitic organism. It has never been found

,*• r'.<F£m

*fr /-Jfjtu $/£ W,

>:'*•( \ .'•% -; »f /••'/ ; %-

"/'^A'^^^'

&-'& $ #>*'./••.; •

Fig. 236. — Tubercle bacillus in sputum (Frankel and Pfeiffer).

except in the bodies and discharges of animals affected with
tuberculosis, and in dusts of which these are component
parts. This purely parasitic nature interferes with the
isolation of the organism, which cannot be grown upon
the ordinary culture-media.

The widespread distribution of tuberculosis at one time
suggested that tubercle bacilli were ubiquitous in the at-
mosphere, that we all inhaled them, and that it was only
our vital resistance that prevented us all from becoming its
victims. Cornet, f however, showed the bacilli to be present

* "Virchow's Archives," Bd. LXXXII, p. 397.

t "Zeitschrift fur Hygiene," v, 1888, pp. 191-331.

712

Tuberculosis

only in dusts with which pulverized sputum was mixed, and
to be most common where the greatest uncleanliness pre-
vailed.

Morphology. — The tubercle bacillus is a slender, rod-
shaped organism with slightly rounded ends and a slight
curve. It measures from 1.5 to 3.5 [A in length and from
0.2 to 0.5 fJ- in breadth. It commonly occurs in pairs, which
may be associated end to end, but generally overlap some-
what and are not attached to each other. Organisms found
in old pus and sputum show a peculiar beaded appearance
caused by fragmentation of the protoplasm and the presence

Fig. 237. — Bacillus of tuberculosis, showing branched forms with invo-
lution (Migula).

of metachromatic granules. These fragmented forms have
been thought to be bacilli in the stage of sporulation, and
Koch originally held this view himself, though later researches
have not confirmed it.

The tubercle bacillus forms no endospores. The frag-
ments thought by Koch to be spores are irregular in shape,
have ragged surfaces, and are without the high refraction
peculiar to spores. Spores also resist heat strongly, but the
fragmented bacilli are no more capable of resisting heat
than others.

The bacilli not infrequently present projecting processes
or branches, this observation having changed our views

Staining 713

regarding the classification of the organism, which is prob-
ably erroneously placed among the bacilli, belonging more
properly to the higher bacteria.

The organism is not motile, and does not possess flagella.

Staining. — The tubercle bacillus belongs to a group of
organisms which because of their peculiar behavior toward
stains is known as " saurefest " or acid-proof. It is difficult
to stain after it has lived long enough to invest itself with a
waxy capsule, requiring that the dye used shall contain a
mordant (Koch). It is also tenacious of color once assumed,
resisting the decolorizing power of strong mineral acids
(Ehrlich).

The peculiarity of staining the bacillus delayed its dis-
covery for a considerable time, but, now that we are familiar
with it, gives us a most valuable differential character, few
other organisms reacting in the same way.

Koch* first stained the bacillus with a solution consisting
of i c.c. of a concentrated solution of methylene-blue mixed
with 20 c.c. of distilled water, well shaken, and then, before
using, receiving an addition of 2 c.c. of a 10 per cent, solu-
tion of caustic potash. Cover-glasses were allowed to
remain in this for twenty-four hours and subsequently
counterstained with vesuvin. Ehrlich subsequently modified
Koch's method, showing that pure anilin was a better
mordant than potassium hydrate, and that the use of a
strong mineral acid would remove the color from everything
but the tubercle bacillus. This modification of Koch's
method, given us by Ehrlich, probably remains the best
method of staining the bacillus.

Nearly all of the recent methods of staining are based
upon the impenetrability of the bacillary substance to
mineral acids which characterizes the acid-fast or acid-proof
(saurefest) micro-organisms. But it is not improbable that
we have been led into error by the assumption, upon inade-
quate grounds, that this is a constant and uniform quality
of the tubercle bacillus and similar micro-organisms. The
interesting observations of Muchf have shown that many of
the paradoxes of tuberculosis can be accounted for by the
fact that during certain stages, or in certain conditions, the
bacilli are not acid-proof at all. Thus, examinations of
caseous masses from the lungs of cattle show complete

"Mittheilungen aus dem Gesundheitsamte," n, 1884.
t "Berliner klin. Wochenschrift," April 6, 1908, p. 691.

714 Tuberculosis

absence of tubercle bacilli when examined by the usual
method, yet cause typical tuberculosis when implanted into
guinea-pigs, with typical bacilli, recoverable upon culture-
media, in the lesions. This is certainly due to the inability
of the bacilli in the bovine lesions mentioned to endure the
acids, for when the same tissues are stained by Gram's
method many organisms can be found. This shows that
Gram's method is really a more useful method for demon-
strating the bacillus than those in which acids are employed.
Naturally, Gram's method, not being differential, is inappro-
priate for sputum, cavity contents, and tissues in which many
other species of bacteria might be present. Much has found
two forms of the tubercle bacillus, one rod-like, the other
granular, that are not acid-proof, and has succeeded in
changing one into the other by experimental manipulation.
He believes that the acid-proof condition has some bearing
upon virulence, and speculates that the more acid-proof
the organisms are, the less virulent they will be found.

In this connection the work of Maher,* who claims to be
able, by appropriate methods of cultivation, to make many
of the ordinary saprophytic bacteria (Bacillus coli, B. subtilis,
etc.) thoroughly acid-proof, must be mentioned.

Staining the Bacillus in Sputum. — As the purpose for
which the staining is most frequently performed is the
diagnosis of the disease through the demonstration of the
bacilli in sputum, the method by which this can be accom-
plished will be first described.

When the sputum is mucopurulent and nummular, any
portion of it may suffice for examination, but if the patient
be in the early stages of tuberculosis, and the sputum is
chiefly thin, seromucus, and flocculent, care must be exercised
to see that such portion of it as is most likely to contain the
micro-organisms be examined.

If one desires to make a very careful examination, it is
well to have the patient cleanse the mouth thoroughly upon
waking in the morning, and after the first fit of coughing
expectorate into a clean, wide-mouthed bottle, the object
being to avoid the presence of fragments of food in the
sputum.

The best result will be secured if the examination be
made on the same day, for if the bacilli are few they occur
most plentifully in small flakes of caseous matter, which are

* " International Conference on Tuberculosis," Philadelphia, 1907.

Staining 715

easily found at first, but which break up and become part
of a granular sediment that forms in decomposed sputum.

The sputum should be poured into a watch-glass and
held over a black surface. A number of grayish-yellow,
irregular, translucent fragments somewhat smaller than the
head of a pin can usually be found. These consist prin-
cipally of caseous material from the tuberculous tissue,
and are the most valuable part of the sputum for exam-
ination. One of the fragments is picked up with a pointed
match-stick and spread over the surface of a perfectly
clean cover-glass or slide. If no such fragment can be found,
the purulent part is next best for examination.

The material spread upon the glass should not be too
small in amount. Of course, a massive, thick layer will be-
come opaque in staining, but should the layer spread be,
as is often advised, " as thin as possible," there may be so
few bacilli upon the glass that they are found with diffi-
culty.

The film is allowed to dry thoroughly and is then passed
three times through the flame for fixation.

Ehrlich's Method, or the Kock-Ehrlich Method. — Cover-
glasses thus prepared are floated, smeared side down, or
immersed, smeared side up, in a small dish of Ehrlich's
anilin-water gentian violet solution:

Anilin 4

Saturated alcoholic solution of gentian violet 1 1

Water 100

and kept in an incubator or paraffin oven for about twenty-
four hours at about the temperature of the body. Slides
upon which smears have been made can be placed in Coplin
jars containing the stain and stood away in the same manner.
When removed from the stain, they are washed momentarily
in water, and then alternately in 25 to 33 per cent, nitric acid
and 60 per cent, alcohol, until the blue color of the gentian
violet is entirely lost. A total immersion of thirty seconds is
enough in most cases. After final thorough washing in 60
per cent, alcohol, the specimen is counterstained in a dilute
aqueous solution of Bismarck brown or vesuvin, the excess of
stain washed off in water, and the specimen dried and mounted
in balsam. The tubercle bacilli are colored a fine dark blue,
while the pus-corpuscles, epithelial cells, and other bacteria,
having been decolorized by the acid, will appear brown.

716 Tuberculosis

This method, requiring twenty-four hours for its com-
pletion, has fallen into disuse, as it is desirable to know in
the briefest possible time whether bacilli are present in the
sputum or not.

Ziehl's Method. — Among clinicians, Ziehl's method of
staining with carbol-fuchsin has met with just favor. It is
as follows: After having been spread, dried, and fixed,
the cover-glass is held in the bite of an appropriate forceps
(cover-glass forceps), or the slide spread at one end is held
by the other end as a handle, and the stain (fuchsin, i ;
alcohol, 10; 5 per cent, phenol in water, 100) dropped upon it
from a pipet. As soon as the entire smear is covered with
stain, it is held over the flame of a spirit lamp or Bunsen
burner until the stain begins to volatilize a little. When
vapor is observed the heating is sufficient, and the tem-
perature can be maintained by intermittent heating.

If evaporation take place, a ring of encrusted stain at the
edge prevents the prompt action of the acid. To prevent
this, more stain should now and then be added. The stain-
ing is complete in from three to five minutes, after which
the specimen is washed off with water, and then with a 3
per cent, solution of hydrochloric acid in 70 per cent, alco-
hol, 25 per cent, aqueous sulphuric, or 33 per cent, aqueous
nitric acid solution dropped upon it for thirty seconds, or
until the red color is extinguished. The acid is carefully
washed off with water, the specimen dried and mounted
in Canada balsam. Nothing will be colored except the
tubercle bacilli, which appear red.

Gobbet's Method. — Gabbet modified the method by
adding a little methylene-blue to the acid solution, which
he makes according to this formula:

Methylene-blue 2

Sulphuric acid '. . 25

Water 75

In Gabbet 's method, after staining with carbol-fuchsinr.
the specimen is washed with water, acted upon by the
methylene-blue solution for thirty seconds, washed again
with water until only a very faint blue remains, dried, and
finally mounted in Canada balsam. The tubercle bacilli
are colored red; the pus-corpuscles, epithelial cells, and
unimportant bacteria, blue.

Staining 717

Pappenheim,* having found bacilli stained red by Ziehls'
method in the sputum of a case which subsequent post-
mortem examination showed to be one of gangrene of the
lung without tuberculosis, condemns that method as not
being sufficiently differential, and recommends the following
as superior to methods in which the mineral acids are em-
ployed :

1. Spread the film as usual.

2. Stain with carbol-fuchsin, heating to the point of steaming for a

few minutes.

3. Pour off the carbol-fuchsin and without washing —

4. Dip the spread from three to five times in the following solution,

allowing it to run off slowly after each immersion:

Corallin i

Absolute alcohol 100

Methylene-blue . ad sat.

Glycerin 20

5. Wash quickly in water.

6. Dry.

7. Mount.

The entire process takes about three minutes. The tubercle
bacilli alone remain red.

Where examination by this means fails to reveal the
presence of bacilli because of the small number in which they
occur, recourse may be had to the use of caustic potash or,
what is better, antiformin (q. v.) for digesting the sputum.
A considerable quantity of sputum is collected, receives the
addition of an equal volume of the antiformin, is permitted
to stand until the formed elements and pus-corpuscles have
been dissolved, is then shaken and poured into centrifuge
tubes and whirled for fifteen to thirty minutes. The sedi-
ment at the bottom of the tubes will then reveal the bacilli
which, having been freed from the viscid materials in the
sputum, have been thrown down by the centrifuge. When
the number is still smaller, it may be possible to show their
presence by guinea-pig inoculation though staining methods
all fail.

The possible relation that the number of bacilli in the
expectoration of consumptives might bear to the progress
of the disease was investigated by Nuttall.f

But a glance down the columns of figures in the original
article is sufficient to show that the number of bacilli is devoid

*"Berl. klin. Wochenschrift," 1898, No. 37, p. 809.

f "Bull, of the Johns Hopkins Hospital," May and June, 1891, n, 13.

7i8

Tuberculosis

of any practical interest, as is only to be expected when one
considers the pathology of the disease and remembers that
accident may cause wide variations in the quality, if not in
the quantity of the sputum.

Staining the Bacillus in Urine. — The detection of tuber-
cle bacilli in the urine is sometimes easy, sometimes difficult.
The centrifuge should be used and the collected sediment
spread upon the glass. If there be no pus or albumin in
the urine, it is necessary to add a little white of egg to

Fig. 238. — Bacillus tuberculosis in sputum, stained with carbolic fuchsin
and aqueous methylene-blue. X 1000 (Ohlmacher).

secure good fixation of the urinary sediment to the glass.
The method of staining is the same as that for sputum.
The smegma bacillus (q. v.) is apt to be present in the urine,
and the precaution must be taken to wash the specimen with
absolute alcohol, so that it may be decolorized and confusion
avoided.

Staining the Bacillus in Feces. — It is difficult to find
tubercle bacilli in the feces because of the relatively small
number of bacilli and large bulk of feces.

In all cases where the detection of tubercle bacilli in pus
or secretions is a matter of clinical importance, it must be

Staining 719

remembered that the quantity of material examined by the
staining method is extremely small, so that a few bacilli in
a relatively large quantity of matter can easily escape dis-
covery.

Staining the Bacillus in Sections of Tissue. — Ehrlich's
Method for Sections. — Ehrlich's method must be recom-
mended as the most certain and best. The sections of
tissue, embedded in paraffin, should be cemented to the slide
and then freed from the embedding material. They are then
placed in the stain for from twelve to twenty-four hours and
kept at a temperature of 37° C. Upon removal they are
allowed to lie in water for about ten minutes. The washing
in nitric acid (20 per cent.) which follows may have to be
continued for as long as two minutes. Thorough washing in
60 per cent, alcohol follows, after which the sections can be
counterstained, washed, dehydrated in 96 per cent, and abso-
lute alcohol, cleared in xylol, and mounted in Canada balsam.
Unna's Method for Sections. — Unna's method is as follows :
The sections are placed in a dish of twenty-four-hour-old,
newly filtered Ehrlich's solution, and allowed to remain
twelve to twenty-four hours at the room temperature
or one to two hours in the incubator. From the stain
they are placed in water, where they remain for about
ten minutes to wash. They are then immersed in acid
(20 per cent, nitric acid) for about two minutes, and be-
come greenish black. From the acid they are placed
in absolute alcohol and gently moved to and fro until the
pale-blue color returns. They are then washed in three
or four changes of clean water until they become almost color-
less, and then removed to the slide by means of a section-
lifter. The water is absorbed with filter-paper, and then the
slide is heated over a Bunsen burner until the section be-
comes shining, when it receives a drop of xylol balsam and
a cover-glass.

It is said that sections stained in this manner do not
fade so quickly as those stained by Ehrlich's method.

Gram's Method. — The tubercle bacillus stains well by
Gram's method and by Weigert's modification of it, but
these methods are ill adapted for differentiation. They
should not be neglected when no tubercle bacilli are demon-
strable by the other methods, as they are particularly well
adapted to the demonstration of such of the organisms as
may not be acid-proof.

720 Tuberculosis

Isolation. — Piatkowski* has suggested that the cultiva-
tion of the tubercle bacillus and other " acid-proof " organ-
isms may be achieved by taking advantage of their ability to
resist the action of formaldehyd. The material containing
the acid-proof organism is mixed thoroughly with 10 c.c. of
water or bouillon, which receives an addition of 2 or 3
drops of 40 per cent, formaldehyd or " formalin." After
standing from fifteen to thirty minutes transfers are made to
appropriate culture-media, when the acid-proof organisms
may develop, the others having been destroyed by the for-
maldehyd.

Still further improvement in the means by which the tuber-
cle bacilli can be secured free from contamination with other
organisms and from surrounding unnecessary and undesirable
materials, has accrued from the use of antiformin. This
commercial product, patented in 1909 by Axel Sjoo andTor-
nell, consists of Javelle water to which sodium hydrate is
added. To make it in the laboratory one first makes the
Javelle water as follows:

K2CO3 58

CaO(OCl)2 80

Water, q. s. 1000

and after dissolving the salts add an equal volume of 15
per cent, aqueous solution of caustic soda.

Uhlenhuth andXylanderf investigated its usefulness and
recommend it highly for assisting in manipulating the tuber-
cle bacillus. The sputum or tissue supposed to contain these
organisms receives an addition of antiformin, by which the
tissue elements, the pus cells, the mucous and other objec-
tionable substances, and bacteria are quickly dissolved, leav-
ing the tubercle bacilli uninjured. It is then centrifugal-
ized, the fluid poured off and replaced by sterile water or
salt solution, and the bacilli washed, after which they
are again centrifugalized and caught at the bottom of the
tube. This sediment, rich in bacilli, may be immediately
transferred to appropriate culture-media, where the organ-

* " Deutsche med. Wochenschrift," June 9, 1904, No. 23, p. 878.
f "Arbeiten a. d. Kaiserlichen Gesundheilsamt," 1909, xxxi, 158;
"Centralbl. f. Bakt. u. Parasitenk.," Referata, 1910, xi,v, 686.

Isolation 721

isms infrequently grow quite well, or can be used for the
inoculation of guinea-pigs.

The most certain method of obtaining a culture of the
tubercle bacillus from sputum, pus, etc., is to inoculate a
guinea-pig, allow artificial tuberculosis to develop, and make
cultures from one of the tuberculous lesions.

To make such an inoculation with material such as
sputum, in which there are many associated micro-organisms
that may destroy the guinea-pig from septicemia, Koch ad-
vised the following method, with which he never experienced
an unfavorable result.

With a sharp-pointed pair of scissors a snip about J cm.
long is made in the skin of the belly- wall. Into this the
points of the scissors are thrust, between the skin and the
muscles for at least i cm., and the scissors opened and
closed so as to make a broad subcutaneous pocket. Into
this pocket the needle of the hypodermic syringe contain-
ing the injection, or the slender glass point of a pipet con-
taining it, is introduced, a drop of fluid expressed and gently
rubbed about beneath the skin. When the inoculating in-
strument is withdrawn, the mouth of the pocket is left open.
A slight suppuration usually occurs and carries out the or-
ganisms of wound infection, while the tubercle bacilli are
detained and carried to the inguinal nodes, which usually en-
large during the first ten days. The guinea-pigs usually die
about the twenty-first day after infection.

The guinea-pig is permitted to live until examination shows
the inguinal glands are well enlarged, and toward the middle
of the third week is chloroformed to death. The exterior of
the body is then wet with i : 1000 solution of bichlorid of
mercury and the animal stretched out, belly up, and tacked
to a board or tied to an autopsy tray. The skin is ripped up
and turned back. The exposed abdominal muscles are now
washed with bichlorid solution and a piece of gauze wrung
out of the solution temporarily laid on to absorb the excess.
With fresh sterile forceps and scissors the abdominal wall is
next laid open and fastened back. With the same instru-
ments or, preferably, with fresh sterile instruments the
spleen, which should be large and full of tubercles, is
drawn forward and, one after another, bits the size of a
pea cut or torn off and immediately dropped upon the sur-
face of appropriate culture-media in appropriate tubes. The
fragments of tissue from the spleen of the tuberculous
46

722

Tuberculosis

guinea-pig are not crushed or comminuted, but are simply
laid upon the undisturbed surface of the blood-serum and
then incubated for several weeks. If no growth is apparent
after this period, the bit of tissue is stirred about a little
and the tube returned to the incubator, where growth al-
most immediately begins from bacilli scattered over the
surface as the bit of tissue was moved. As the appropriate
media, blood-serum was recommended by Koch; glycerin
agar-agar, by Roux and Nocard; glycerinized potato, by

Nocard; coagulated dogs' blood-
serum, by Smith, or coagulated
egg, by Dorset, may be men-
tioned. The most certain results
seem to follow the employment
of the dogs' serum and egg media.
Cultivation. — Blood-serum.—
Koch first achieved artificial cul-
tivation of the tubercle bacillus
upon blood-serum, upon which
the bacilli are first apparent to
the naked eye in about two
weeks, in the form of small, dry,
whitish flakes, not unlike frag-
ments of chalk. These slowly
increase in size at the edges, and
gradually form small scale-like
masses, which under the micro-
scope are found to consist of tan-
gled masses of bacilli, many of
which are in a condition of invo-
lution. The medium is so ill
adapted to the requirements of
the tubercle bacillus and gives
such uncertain results that it is no longer used.

Glycerin Agar-agar. — In 1887 Nocard and Roux* gave a
great impetus to investigations upon tuberculosis by the dis-
covery that the addition of from 4 to 8 per cent, of glycerin
to bouillon and agar-agar made them suitable for the devel-
opment of the bacillus, and that a much more luxuriant de-
velopment could be obtained upon such media than upon
blood-serum. The growth upon "glycerin agar-agar" re-
sembles that upon blood-serum. A critical study of the
* "Ann. de 1'Inst. Pasteur," 1887, No. i.

Fig. 239. — Bacillus tuber-
culosis on "glycerin agar-
agar."

Isolation

723

relationship of massive development and glycerin was made
by Kimla, Poupe, and Vesley,* who found that the most lux-
uriant growth occurred when the culture-media contained
from 5 to 7 per cent, of glycerin.

Dogs' Blood-serum. — A very successful method of isolating
the tubercle bacillus has been published by
Smith, f A dog is bled from the femoral ar-
tery, the blood being caught in a sterile flask,
where it is allowed to coagulate. The serum
is removed with a sterile pipet, placed in
sterile tubes, and coagulated at 75° to 76° C.
Reichel has found it advantageous to add
to each 100 c.c. of the dogs' serum 25 c.c.
of a mixture of glycerin i part and distilled
water 4 parts. The whole is then carefully
shaken without making a froth, and dis-
pensed in tubes, 10 c.c. to a tube. The
coagulation and sterilization he effects by
once heating to 90° C. for three to five
hours. At the Henry Phipps Institute in
Philadelphia I employed this medium with
thorough satisfaction for the isolation of
many different tubercle bacilli. Smith pre-
fers to use a test-tube with a ground cap,
having a small tubular aperture at the end,
instead of the ordinary test-tube with the
cotton-plug. The purpose of the ground
glass cap is to prevent the contents of the
tube from drying during the necessarily long
period of incubation; that of the tubula-
ture, to permit the air in the tubes to enter
and exit during the contraction and expan-
sion resulting from the heating incidental
to sterilization.

To the same end the ventilators of the
incubator are closed, and a large evapo-
rating dish filled with water is stood inside,
so that the atmosphere may be constantly
saturated with moisture

Egg Media. — DorsetJ recommends the isolation of the

* "Revue de la Tuberculose," 1898, vi, p. 25.

t " Transactions of the Association of American Physicians," 1898,
vol. xm, p. 417.

I "American Medicine," 1902, vol. in, p. 555.

Fig. 240. — Glass
capped culture-
tube used by
Theobald Smith
for the isolation
of the tubercle
bacillus.

724

Tuberculosis

tubercle bacillus upon an egg medium, which has the advan-
tage of being cheap and easily pre-
pared, while eggs are always at hand,
and can be made into an appropriate
medium in an hour or two. He also
claims that the chemic composition
of the eggs makes them particularly
adapted for the purpose. The me-
dium is prepared by carefully open-
ing the egg and dropping its contents
into a wide-mouth sterile receptacle.
The yolk is broken with a sterile
wire and thoroughly mixed with the
white by gentle shaking. The mix-
ture is then poured into sterile tubes,
about 10 c.c. in each, inclined in a
blood-serum sterilizer, and sterilized
and coagulated at 70° C. for two
days, the temperature being main-
tained for four or five hours each
day. The medium appears yellow-
ish and is usually dry, so that before
using it is well to use a few drops of
water to make conditions appropri-
ate for the growth of the tubercle
bacillus.

Potato. — Pawlowski* was able to
isolate the bacillus upon potato.
Sander found that it could be
readily grown upon various vege-
table compounds, especially upon
acid potato mixed with glycerin.
Rosenauf has shown that it can
grow upon almost any cooked and
glycerinized vegetable tissue. Ac-
cording to French writers, the viru-
lence of the bacillus is not dimin-
ished when it grows upon potato.
It has also been said that the con-
tinued cultivation of the tubercle
bacillus upon culture-media lessens

Fig. 241. — Bacillus tu-
berculosis; glycerin agar-
agar culture, several
months old (Curtis).

' ''Ann. de 1'Inst. Pasteur," 1888, t. vi.
t " Jour. Amer. Med. Assoc.," 1902.

Isolation 725

its parasitic nature, so that in the course of time it can be
induced to grow feebly upon the ordinary agar-agar, and that
prolonged cultivation destroys its virulence.

Animal Tissues. — Frugoni* recommends that the tubercle
bacillus be isolated and cultivated upon animal tissue and
organs used as culture-media. He especially recommends
rabbit's lung and dog's lung for the purpose. The tissues
are first cooked in a steam sterilizer, then cut into prisms,
placed in a Roux tube, an addition .of 6 to 8 per cent,
glycerin- water added, so as to bathe the lower part of the tis-

Fig. 242. — Bacillus tuberculosis; adhesion cover-glass preparation
from a fourteen-day-old blood-serum culture. X 100 (Frankel and
Pfeiffer).

sue and keep it moist, and the whole then sterilized in the
autoclave.

The organisms are planted upon the tissue, the top of the
tube closed with a rubber cap, and the culture placed in the
thermostat. The tubercle bacilli grow quickly and luxuriantly.

Bouillon. — Upon bouillon to which 6 per cent, of glycerin
has been added the bacillus grows well, provided the trans-
planted material be in a condition to float. The organism
being purely aerobic, grows only at the surface, where a
much wrinkled, creamy white, brittle pellicle forms.

* "Centralbl. f. Bakt. u. Parasitenk.," I. Abl. Orig., 1910, un, 553.

726 Tuberculosis

Non-albuminous Media. — Instead of requiring the most
concentrated albuminous media, as was once supposed,
Proskauer and Beck* have shown that the organism can be
made to grow in non-albuminous media containing asparagin,
and that it can even be induced to grow upon a mixture of
commercial ammonium carbonate, 0.35 per cent.; primary
potassium phosphate, 0.15 per cent.; magnesium sulphate,
0.25 per cent.; glycerin, 1.5 per cent. Tuberculin was pro-
duced in this mixture.

Gelatin. — The tubercle bacillus can be grown in gelatin
to which glycerin has been added, but as its development
takes place only at 37° to 38° C., a temperature at which
gelatin is always liquid, its use for the purpose has no
advantages.

Fig. 243. — Bacillus tuberculosis: a, Source, human; b, source, bovine.
Mature colonies on glycerin-agar. Actual size (Swithinbank and
Newman) .

Appearance of the Cultures. — Irrespective of the media
upon which they are grown, cultures of the tubercle bacil-
lus present certain characteristics which serve to separate
them from the majority of other organisms, though insuffi-
cient to enable one to certainly recognize them.

The bacterial masses make their appearance very slowly.
As a rule very little growth can be observed at the end of a
week, and sometimes a month must elapse before the cul-
tures can be described as well grown.

They usually develop more rapidly upon fluid than upon
solid media. The growth is invariably and purely aerobic,

* "Zeitschrift fur Hygiene," Aug. 10, 1894, xvm, No. i.

Pathogenesis 727

and the surface growth formed upon liquids closely resembles
that upon solids.

The growth is dry and lusterless, coarsely granular,
wrinkled, slightly yellowish, and does not penetrate into the
substance of the culture-medium. It sometimes extends
over the surface of the medium and spreads out upon the
contiguous surface of moist glass.

When the medium is moist, the bacterial mass may in
rare instances be shining in spots, but it is usually lusterless.
When the medium is dry, it is apt to be scaly and almost
chalky in appearance.

The organism grows well when once successfully isolated,
and, when once accustomed to artificial media, not only
lives long (six to nine months) without transplantation, but
may be transplanted indefinitely without variation.

Reaction. — The tubercle bacillus will grow upon other-
wise appropriate media whether the reaction be feebly
acid or feebly alkaline. Human tubercle bacilli scarcely
change the reaction of the media in which they grow, but
bovine bacilli produce a slight acidity.

Relation to Oxygen. — The tubercle bacillus requires con-
siderable oxygen and, therefore, grows only upon the surface
of the culture-media.

Temperature Sensitivity. — The bacillus is sensitive to
temperature variations, not growing below 29° C. or above
42° C. Rosenau* found .that an exposure to 60° C. for
twenty minutes destroys the infectiousness of the tubercle
bacillus for guinea-pigs.

Effect of Light. — It does not develop well in the light,
and when its virulence is to be maintained should always
be kept in the dark. Sunlight kills it in from a few minutes
to several hours, according to the thickness of the mass of
bacilli exposed to its influence.

Pathogenesis. — Channels of Infection. — The channels by
which the tubercle bacillus enters the body are numerous. A
few cases are on record where the micro-organisms have
passed through the placenta, a tuberculous mother infecting
her unborn child. It is not impossible that the passage of
bacilli through the placenta in this manner causes the rapid
development of tuberculosis after birth, the disease having
remained latent during fetal life, for Birch-Hirschfeld has
shown that fragments of a fetus, itself showing no tuberculous
* "Hygienic Laboratory," Bulletin No. 24, Jan., 1908.

728 Tuberculosis

lesions, but coming from a tuberculous woman, caused fatal
tuberculosis in guinea-pigs into which they were inoculated.

The most frequent channel of infection is the respiratory
tract, into which the finely pulverized pulmonary discharges
of consumptives and the dusts of infected rooms and streets
enter. Fliigge, Laschtschenko, Heyman-Sticher, and Be-
ninde* found that the greatest danger of infection was
from the atomized secretions, discharged during cough, from
the tuberculous respiratory apparatus. Nearly every one
discharges finely pulverized secretions during coughing and
sneezing, as can easily be determined by holding a mirror
before the face at the time. Even though discharged by
consumptives, these atoms of moisture are not infectious
except when tubercle bacilli are present in the sputum.
Experiment showed that they usually do not pass further
than 0.5 meter from the patient, though occasionally they
may be driven 1.5 meters. A knowledge of these facts
teaches us that visits to consumptives should not be pro-
longed; that no one should remain continually in their
presence, nor habitually sit within 2 meters of them; also
that patients should always hold a handkerchief before
the face while coughing. The rooms occupied by con-
sumptives should also be frequently washed with a dis-
infecting solution.

Probably all of us at some time in our lives inhale living
virulent tubercle bacilli, yet not all suffer from tubercu-
losis. Personal variations in predisposition seem to account
in part for this, as it has been shown that without the
formation of tubercles virulent bacilli may sometimes be
present for considerable lengths of time in the bronchial
lymphatic glands — the dumping-ground of the pulmonary
phagocytes.

In order that infection shall occur, it does not seem
necessary that the least abrasion or laceration shall exist
in the mucous lining of the respiratory tract.

Infection also commonly takes place through the gastro-
intestinal tract from infected food. At present evidence
points to danger from the presence of tubercle bacilli in the
milk of cattle affected with tuberculosis. It does not seem
necessary that tuberculous ulcers shall be present in the
udders; indeed, the bacilli have been demonstrated in

* " Zeitschrif t fur Hygiene," etc., Bd. xxx, pp. 107, 125, 139, 163,
193-

Pathogenesis 729

considerable numbers in milk from udders without tuber-
culous lesions discoverable to the naked eye.

The meat from tuberculous animals is less dangerous than
the milk, because it is nearly always cooked before being
eaten, while the milk is generally consumed in the raw state.

The ingested bacilli may enter the tonsils and be carried
to the cervical lymph-glands, but seem more commonly
to reach the intestine, from which they enter the lymphatics,
sometimes to produce lesions immediately beneath the
mucous membrane, and lead to the later formation of
ulcers; but usually to invade the more distant mesenteric
lymphatic glands. Nicolas and Descos* and Ravenel f found
that when fasting dogs were fed upon soup containing
large quantities of tubercle bacilli, they were able to dis-
cover the bacilli a few hours afterward in the contents
of the thoracic duct. The thoracic duct is sometimes
affected, and from such a lesion it is easy to understand
the development of general miliary tuberculosis through
systemic distribution of bacilli thrown into the circulation.
The occasional absorption of tubercle bacilli by the lacteals,
and their immediate entrance into the systemic circulation
and subsequent deposition in the brain, bones, joints, etc.,
are supposed to explain primary lesions of these tissues.

Kochf believes that human beings are infected only by
bacilli from other human beings, and his paper upon this
subject has stimulated extensive experimentation on the
problem. Most authorities believe both human and bo-
vine bacilli to be equally infectious for man. Behring§
believes that nearly all children become infected by ingest-
ing tubercle bacilli in milk, though a certain predisposition
is necessary before the disease can develop. Baumgarten
believes that all children harbor bacilli taken in the food,
but that the disease does not develop until a certain sus-
ceptibility occurs.

Infection also occasionally takes place through the sexual
apparatus. In sexual intercourse tubercle bacilli from
tuberculous testicles can enter the female organs, with
resulting bacillary implantation. Sexual infections are

' "Jour, de Phys. et Path, gen.," 1902, iv, 910.
t "Jour. Med. Research," x, p. 460, 1904.

t " International Congress on Tuberculosis," London, 1901, and
Washington, 1908.

§ "Deutsche med. Wochenschrift," 1903, No. 39.

730 Tuberculosis

usually from the male to the female, primary tuberculosis of
the testicle being more common than of the uterus or ovaries.

Wounds are also occasional avenues of entrance for
tubercle bacilli. Anatomic tubercles are not uncommon
upon the hands of anatomists and pathologists, most of
these growths being tuberculous in nature. Such dermal
lesions usually contain few bacilli.

Lesions. — The macroscopic lesions of tuberculosis are too
familiar to require a description of any considerable length.
They consist of nodules, or collections of nodules, called
tubercles, irregularly scattered through the tissues, which
are more or less disorganized by their presence and retro-
gressive changes.

When tubercle bacilli are introduced beneath the skin of
a guinea-pig, the animal shows no sign of disease for a
week or two, then begins to lose appetite, and gradually
diminishes in flesh and weight. Examination usually shows
a nodule at the point of inoculation and enlargement of the
neighboring lymphatic glands. The atrophy increases, the
animal shows a febrile reaction, and dies at the end of a
period of time varying from three to six weeks. Post-
mortem examination usually shows a cluster of tubercles
at the point of inoculation, tuberculous enlargement of
lymphatic glands both near and remote from the primary
lesion, and a widespread tuberculous invasion of the lungs,
liver, kidneys, peritoneum, and other organs. Tubercle
bacilli are demonstrable in immense numbers in all the
invaded tissues. The disease in the guinea-pig is usually
more widespread than in other animals because of its greater
susceptibility, and the death of the animal occurs more
rapidly for the same reason. Intraperitoneal injection of
tubercle bacilli in guinea-pigs causes a still more rapid dis-
ease, accompanied by widespread lesions of the abdominal
organs. The animals die in from three to four weeks. In
rabbits the disease runs a longer course with similar lesions.
In cattle and sheep the infection is commonly first seen in
the alimentary apparatus and associated organs, and may
be limited to them though primary pulmonary disease also
occurs. In man the disease is chiefly pulmonary, though
gastro-intestinal and general miliary tuberculosis are com-
mon. The development of the lesions in whatever tissue or
animal always depends upon the distribution of the bacilli
by the lymph or the blood.

Lesions 731

The experiments of Koch, Prudden, and Hodenpyl,* and
others have shown that when dead tubercle bacilli are in-
jected into the subcutaneous tissues of rabbits, small local
abscesses develop in the course of a couple of weeks, show-
ing that the tubercle bacilli possess chemotactic properties.
These chemotactic properties ' seem to depend upon some
other irritant than that by which the chief lesions of tuber-
culosis are caused. When the dead tubercle bacilli, instead
of being injected en masse into the areolar tissue, are intro-
duced by intravenous injection and disseminate themselves
singly or in small groups, the result is quite different, and
the lesions closely resemble those caused by the living or-
ganisms.

Baumgarten, whose researches were made upon the iris,
found that the first irritation caused by the bacillus is
followed by multiplication of the fixed connective-tissue
cells of the part. The cells increase in number by karyo-
kinesis, and form a minute cellular collection or primitive
tubercle. Such leukocytes as occur are of the small mono-
nuclear variety, are of secondary importance, appear later,
and are no doubt attracted both by the chemotactic sub-
stance shown by Prudden and Hodenpyl to exist in the
bodies of the dead bacilli and by the necrotic changes already
affecting the tissue in which the tubercle occurs. For rea-
sons not understood, the number of lymphocytes varies con-
siderably in different cases. Sometimes there will be enough
to justify the name " tuberculous abscess "; sometimes they
will be completely absent.

The essential toxic substance of the bacillus does not
cause the chemotaxis, for when the lymphocytes are absent
the characteristic coagulation-necrosis persists.

The group of epithelioid cells and lymphocytes constituting
the primitive tubercle scarcely reaches visible proportions
before coagulation-necrosis begins. The cytoplasm of the
cells takes on a hyaline character, and appears to become
abnormally viscid, contiguous cells tending to fuse. The
chromatin of the nuclei becomes dissolved in the nuclear
juice and gives a pale but homogeneous appearance to the
stained nuclei. Sometimes this nuclear change is only
observed very late. There is little karyorrhexis. As the
necrosis advances, some of the cells flow together and form
large protoplasmic masses — giant-cells — which contain as
* "New York Med. Jour.j" June 6-20, 1891.

732

Tuberculosis

many nuclei as there were component cells. It may be
that the nuclei of the giant-cells multiply by karyokinesis
after the protoplasmic coalescence, but only one observer,
Baumgarten, has found signs of this in giant-cells.

Different writers hold varying opinions concerning the
formation and office of the giant cells. Thus, while I* re-
gard them as degenerative formations, and unimportant

•*•:<

A> •,/vV/U*

/: a

:im

Fig. 244. — Miliary tubercle of the testicle: a, Zone of epithelioid cells
and leukocytes; b, area of coagulation-necrosis; c, giant cell with its
processes; peripherally arranged nuclei and necrotic center; d, semi-
niferous tubule (Cameron, in "International Text-book of Surgery").

entities, there are many who believe, with Metchnikoif,
that they are enormous phagocytes. Hektoenf believes
that they are active bodies from which cells split off.

Giant cells are not always formed in tubercles, as the

* "International Medical Magazine," vol. i, No. 10, 1892; vol. m,
No. 2, 1894.

f "Journal of Experimental Medicine," vol. in, 1898, p. 21.

Lesions 733

necrotic changes are sometimes so rapid and widespread
as to convert the whole into a mass of unrecognizable frag-
ments.

Tubercles are constantly avascular — i. e., in them no
new capillary blood-vessels form, as in other inflammatory
tissues — and the coagulation-necrosis soon destroys pre-
existing capillaries; the avascularity may be a factor in the
necrosis of the larger tuberculous masses, though probably
playing no important part in the degeneration of the small
tubercles, which is purely toxic.

The minute primitive tubercle was first called a miliary
tubercle, and small aggregations of these, "crude tubercles,"
by Laennec. Tubercles may be developed in any tissue
and in any organ. In whatever situation they occur, the
component cells are either pushed aside or included in the
lesion. In miliary tuberculosis of the kidney it is not un-
usual to find a tubercle including a glomerule, and resolving
its component capillaries and epithelium into necrotic frag-
ments. In this way the tissues become disorganized and
disintegrated.

As almost all tissues contain a supporting connective-
tissue framework, its fibers must be embodied in the new
growth. These possess little vitality, but are more resistant
than the cells, and, after the cells of a tubercle have been
destroyed, may be distinctly visible among the granules,
giving the tubercle a reticulated appearance.

As a rule, tubercles progressively increase in size by the
invasion of fresh tissue. The tubercle bacillus does not
seem to find the necrotic centers of the tubercles adapted
to its growth, and most of the bacilli are usually observed
at the edges, among the healthy cells, where the nutri-
tion is good. From this position they are occasionally
picked up by leukocytes and transported through the
lymph-spaces, until the phagocyte falls a prey to its pris-
oner, dies, and sows the seed of a new tubercle. However,
for the spread of tubercle bacilli from place to place phago-
cytes may not always be necessary, for the bacilli can
probably be transported by streams of lymph. It is by
the steady advance in necrosis and consolidation that the
tissues invaded are destroyed, becoming cheesy and crumbly
and forming necrotic masses which, in the lungs, gradually
crumble away, the detritus escaping through the air-tubes,
thus forming cavities. From the beginning of pulmonary

734

Tuberculosis

tuberculosis the process of destruction is greatly accelerated
by inspired saprophytic bacteria that live in the necrotic

Fig. 245. — Tuberculosis of the lung: the upper lobe shows advanced
cheesy consolidation with cavity-formation, bronchiectasis, and fibroid
changes; the lower lobe retains its spongy texture, but is occupied by
numerous miliary tubercles.

tissue. The patient also suffers from secondary infections,
especially by the streptococcus and pneumococcus.

Virulence 735

Most cases of tuberculosis steadily advance, but a certain
number may recover.

About the center of a typical tubercle there is a zone of re-
action in which the reparative tendency of the tissue is
usually but slightly outweighed by the invasive power of the
bacilli. If the vital condition of the individual becomes so
changed that the invasive activity of the bacilli is checked
or their death brought about, the tubercle begins to cica-
trize, and becomes surrounded by a zone of newly formed
contracting fibrillar tissue, by which it is circumscribed
and isolated. This constitutes recovery from tuberculosis.
Sometimes the process of repair is accomplished without
the destruction of the bacilli, which are incarcerated and
retained. Such a condition is called latent tuberculosis, and
may at a future time be the starting-point of a new in-
fection.

Virulence. — The virulence of tubercle bacilli varies
considerably according to the sources from which they are
obtained. Bacilli from different cases are of different
degrees of virulence, and bacilli from different animals vary
still more. Lartigau,* in an instructive paper upon " Varia-
tion in Virulence of the Bacillus Tuberculosis in Man,"
found much variation among bacilli secured from the lesions
of human tuberculosis. The virulence was tested by em-
ploying cultures only for inoculation, and taking of each
bacillary mass exactly 5 mg. by weight, suspending it in 5 c.c.
of an indifferent fluid until the density was uniform and
the microscope showed no clumps, and injecting into rab-
bits and guinea-pigs, pairs of animals being injected in
the same manner, with the same material, at the same
time, and being subsequently kept under similar conditions.
The occurrence of tuberculosis in the inoculated animals
was decided by both macroscopic and microscopic tests.

Lartigau found that human tubercle bacilli from different
sources produced varying degrees of tuberculosis in animals ;
that the injection of the same culture in different amounts
produces different results; that the extent and rapidity of
development usually corresponds to the virulence of the
culture; that doses of i mg. of a very virulent culture may
induce general tuberculosis in rabbits in a very short time;
that 20 mg. of a bacillus of low virulence may fail to pro-

c "Journal of Medical Research," vol. vi, No. i; N. S., vol. i, No. i,
p. 156, July, 1901.

736 Tuberculosis

duce any lesion in rabbits or guinea-pigs ; that no morphologic
relationship could be observed between the bacilli and their
virulence; that highly virulent bacilli grew scantily on
culture-media and were short lived; that bacilli of widely
different virulence may be present in any one of the various
human tuberculous lesions ; that in scrofulous lymphadenitis
the bacilli are usually of low virulence; the bacilli in pul-
monary tuberculosis with ulcer ation are of feeble virulence,
those of miliary tuberculosis of very great virulence; that
the so-called " healed tubercles " of the lung may contain
virulent or attenuated bacilli; that individuals suffering
from infection with a bacillus of a low grade of virulence
may be again infected with extremely virulent tubercle
bacilli; that chronic tuberculosis of the bones may contain
bacilli of high or low virulence, and that variations in
virulence among human tubercle bacilli may possibly some-
times depend, like many other qualities among tubercle
bacilli, on peculiarities inherited through serial trans-
missions in other than human hosts.

Chemistry of the Tubercle Bacillus. — Klebs* found
that the tubercle bacillus contains two fatty bodies, one
of which, having a reddish color and melting at 42° C.,
can be extracted with ether. It forms about 20 per cent,
by weight of the bacillary substance. The other is in-
soluble in ether, but soluble in benzole, with which it can
be extracted. It melts at about 50° C. and constitutes
1.14 per cent, of the bacillary substance. After removing
these fatty bodies the bacilli fail to resist the decolorant
action of acids when stained by ordinary methods, so that
it seems probable that their acid-resisting power depends
upon them.

De Schweinitzf showed that it was possible to extract
from the tubercle bacillus an acid closely resembling, if
not identical with, teraconic acid. It melts at 161° to 164° C.
and is soluble in ether, water, and alcohol. He thinks the
necrotic changes caused by the organism depend upon it.

RuppelJ believes that three different fatty substances
are present in the tubercle bacillus, making up from 8 to
26 per cent, by weight. The first can be extracted with

* "Centralbl. f. Bakt.," 1896, xx, p. 488.

t "Trans. Assoc. of Amer. Phys.," 1897; "Centralbl. f. Bakt.," etc.,
Sept. 15, 1897, Bd. xxn, p. 200.

J "Zeitschrift fur physiol. Chemie," 1899, xxvi.

Toxic Products 737

cold alcohol, the second with hot alcohol, the third with
ether. In addition to the fatty substance Ruppel also
found what he believes to be a protamin, and calls tuber -
culosamin. It seems to be combined with nucleinic acid,
and, indeed, from it he isolated an acid for which he proposes
the name tuber culinic acid.

Behring* found that this acid contained a histon-like
body whose removal left chemically pure tuber culinic acid.
One gram of this acid is capable of killing a 6oo-gram guinea-
pig when administered beneath the skin. One gram is
fatal to 90,000 grams of guinea-pig when introduced into
the brain. If injected into tuberculous guinea-pigs it is
much more fatal, i gram destroying 60,000 when injected
subcutaneously and 40,000,000 when injected into the
brain.

Levenef also found free and combined nucleinic acid
varying in phosphorus content from 6.58 to 13.19 per cent.
He also found a glycogen-like substance that reduced
Fehling's solution when heated with a mineral acid.

Toxic Products. — In 1890 Koch I announced some ob-
servations upon the toxic products of the tubercle bacillus
and their relation to the diagnosis and treatment of tubercu-
losis, which at once aroused an enormous though transitory
enthusiasm. The observations are, however, of great
importance. Koch found that when guinea-pigs are
inoculated with tubercle bacilli, the wound ordinarily heals
readily, and soon all signs of local disturbance other than
enlargement of the lymphatic glands of the neighborhood
disappear. In about two weeks, however, there appears,
at the point of inoculation a slight induration, which develops
into a hard, nodule, ulcerates, and remains until the death
of the animal. If, however, in a short time the animals be
reinoculated, the course of the local lesion is changed, and,
instead of healing, the wound and the tissue surrounding it
assume a dark color, become obviously necrotic, and ulti-
mately slough away, leaving an ulcer which rapidly and
permanently heals without enlargement of the lymph-
glands.

This observation was made by injecting cultures of the
living bacillus, but Koch observed that the same changes

*" Berliner klin. Wochenschrift," xxxvi.
t " Jour, of Med. Research," I, 1901.
J "Deutsche med. Wochenschrift," 1891, No. 343.
47

738 Tuberculosis

also occur when the secondary inoculation is made with
killed cultures of the bacilli.

It was also observed that if the material used for the
secondary injections was not too concentrated and the
injections not too often repeated (only every six to forty-
eight hours), the animals treated improved in condition,
and continued to live, sometimes (Pfuhl) as long as nineteen
weeks.

Tuberculin. — Koch also discovered that a 50 per cent,
glycerin extract of cultures of the tubercle bacillus — tuber-
culin— produced the same effect as the dead cultures orig-
inally used, and announced the discovery of this substance
to the scientific world, in the hope that the prolongation of
life observed to follow its use in the guinea-pig might also
be true of man.

The active substance of the " tuberculin " seems to be
an albuminous derivative (bacterioprotein) insoluble in
absolute alcohol. It is a protein substance and gives all
the characteristic reactions. It differs from the toxalbumins
in being able to resist exposure to 120° C. for hours without
change. Tuberculin is almost harmless for healthy animals,
but extremely poisonous for tuberculous animals, its injec-
tion into them being followed either by a violent febrile
reaction or by death, according to the extent of the dis-
ease and size of the dose administered.

Preparation of Tuberculin. — The preparation of tuberculin is simple.
Flasks made broad at the bottom so as to expose a considerable sur-
face of the contained liquid are filled to a depth of about 2 cm. with
bouillon containing 4 to 6 per cent, of glycerin, and preferably made
with veal instead of beef infusion. They are inoculated with pure cul-
tures of the tubercle bacillus, care being taken that the bacillary mass
floats upon the surface, and are kept in an incubator at 37° C. In the
course of some days a slight surface growth becomes apparent about the
edges of the floating bacillary mass, which in the course of time develops
into a firm, coarsely granular, wrinkled pellicle. At the end of some
weeks development ceases and the pellicle sinks, a new growth some-
times occurring from floating scraps of the original.

Some bacteriologists prefer to use small Erlenmeyer flasks for the
purpose, but large flasks, which contain from 500 c.c. to i liter, are more
convenient. The contents of a number of flasks of well-grown cultures
are poured into a large porcelain evaporating dish, concentrated over a
water-bath to one-tenth their volume, and filtered through a Pasteur-
Chamberland filter. This is crude tuberculin.

When doses of a fraction of a cubic centimeter of crude tuberculin
are injected into tuberculous animals, an inflammatory and febrile
reaction occurs. Superficial tuberculous lesions (lupus) sometimes
ulcerate and slough away. The febrile reaction is sufficiently character-
istic to be of diagnostic value, though tuberculin can only be used with
perfect safety as a diagnostic agent upon the lower animals.

Toxic Products

739

From the "crude" or original tuberculin Koch prepared a purified or
"refined" tuberculin by adding one and one-half volumes of absolute
alcohol, stirring thoroughly, and standing aside for twenty-four hours.
At the end of this time a flocculent deposit will be seen at the bottom of
the vessel. The supernatant fluid is carefully decanted and an equal
volume of 60 per cent, alcohol poured into the vessel for the purpose of
washing the precipitate, which is again permitted to settle, the fluid
decanted, and the washing thus repeated several times, after which it is

Fig. 246. — Massive culture of the tubercle bacillus upon the surface of
glycerin-bouillon, used in the manufacture of tuberculin.

finally washed in absolute alcohol and dried in a vacuum exsiccator.
The white powder thus prepared is fatal to tuberculous guinea-pigs in
doses of 2 to 10 mg. It is soluble in water and glycerin and gives the pro-
tein reactions. The tuberculin as Koch prepared it is now known as
"concentrated" or "Koch's tuberculin," to differentiate it from the
"diluted tuberculin" sometimes sold in the shops, which is the same
thing so diluted with i per cent, aqueous carbolic acid solution that i c.c.
equals a dose. The dose of the concentrated tuberculin is 0.4 to 0.5 c.c. ;
that of the diluted tuberculin, i c.c.

740 Tuberculosis

Tuberculin does not exert the slightest influence upon the
tubercle bacillus, but acts upon the tuberculous tissue, aug-
menting the poisonous influence upon the cells surrounding
the bacilli, destroying their vitality, and removing the condi-
tions favorable to bacillary growth, which for a time is
checked. This action is accompanied by marked hyperemia
of the perituberculous tissue, with transudation of serum,
softening of the tuberculous mass, and its absorption into
the blood, a marked febrile reaction resulting from the
intoxication.

Virchow, who well understood the action of the tuberculin,
soon showed that as a diagnostic and therapeutic agent in
man its use was attended by grave dangers. The destroyed
tissue was absorbed, but with it some of the bacilli, which,
being transported to new tissue areas, could occasion a
widespread metastatic invasion of the disease. Old tuber-
culous lesions which had been encapsulated were sometimes
softened and broken down, and became renewed sources of
infection to the individual, so that, a short time after an
enthusiastic reception, tuberculin was placed upon its proper
footing as an agent valuable for diagnosis in veterinary
practice, but dangerous in human medicine, except in cases
of lupus and other external forms of tuberculosis where the
destroyed tissue could be readily discharged from the surface
of the body.

Many, however, continued to use it, and Petruschky* has
reported, with careful details, 22 cases of tuberculosis which
he claims have been cured by it.

Recently there has been a return to the use of tuberculin
for the diagnosis of tuberculosis, it being claimed that by the
use of minute doses, several times repeated, the charac-
teristic reaction and a positive diagnosis can be obtained
without danger.

von Pirquetf found that if a drop or two of Koch's (old)
tuberculin is placed upon the skin of a tuberculous child,
and a small scarification made through the drop with a
sterile lancet, a small papule develops at the point of inocula-
tion that is not unlike a vaccine papule. It is at first bright,
later on dark red, and remains for a week. Out of 500 tests
made, the results were positive in nearly every case of
clinical tuberculosis. The most characteristic reactions

* "Berliner klin. Wochenschrift," 1899, Dec. 18-25.
t Ibid., May 20, 1907.

Toxic Products 741

were obtained in tuberculosis of the bones and glands, and
the method is recommended chiefly for the diagnosis of
tuberculosis during the first year of life. This method of
testing is called the dermotuberculin reaction.

A modification of this method by Lignieres* is called by
him the cutituberculin reaction. Lignieres soaps and shaves
the skin with a safety razor, avoiding scarification, but
removing the superficial epidermal cells by scraping, and
then applies 6 large drops of undiluted tuberculin, rubbing
the reagent in with a pledget of cotton. The reaction
obtained is purely local and without fever.

Morrof has improved upon von Pirquet's method by using
the tuberculin in the form of a 50 per cent, ointment made by
mixing equal parts of "old tuberculin" and lanolin, which is
rubbed into the skin without previous scarification.

HissJ says that "it is more simple and equally efficient
to massage into the skin a drop of undiluted ' old tuberculin.' "
• Calmette§ suggested the " ophthalmo-tuberculin reaction,"
which consists of dropping i drop of a solution of prepared
tuberculin into the eye of the suspect. If no tuberculosis ex-
ists, no reaction follows, but if the patient be infected with
tuberculosis, the eye becomes reddened in a few hours and
soon shows all of the appearances of a more or less pronounced
acute mucopurulent inflammation of the conjunctiva. This
attains its maximum in six or seven hours, and entirely
recovers in three days. It usually causes the patient very
little discomfort, but a number of patients have been un-
fortunate enough to suffer from supervening corneal ulcera-
tion and other destructive lesions of the eye, so that the
test is now rarely used, having been superseded by the dermal
methods.

The method of preparing the solution employed by Cal-
mette is to precipitate the tuberculin with alcohol, dry the
precipitate, and dissolve it in 100 parts of distilled water.
One or two drops may be used. Ordinary tuberculin must
be avoided, as the glycerin it contains causes too much
irritation and masks the reaction.

Priority in regard to the theoretic aspects of these

* "Centralbl. f. Bakt. u. Parasitenk.," orig., XLVI, Hft. 4, March 10,
1908, p. 373.

t "Munch, med. Wochenschrift," 1906, p. 216.
% "Text-book of Bacteriology," 1910, p. 489.
§ "La Presse Medicale," June 19, 1907.

742 Tuberculosis

reactions seems to belong to Wolff-Eisner,* who was the
first to point out that the injection of all albuminous sub-
stances resulted in hypersensitivity instead of immunity
unless certain precautions were observed. Upon this ground
Levyf gives him credit as the founder of the method. The
reaction is undoubtedly one of anaphylaxis (q. v.).

KlebsJ has made strong claims for his own modifications
of tuberculin, known as antiphthisin and tuber culocidin.
According to the experimental studies of Trudeau and Bald-
win, however, antiphthisin is only much diluted tuberculin,
and exerts no demonstrable influence upon the tubercle
bacillus in vitro, does not cure tuberculosis in guinea-pigs, and
probably inhibits the growth of the tubercle bacillus upon
culture-media to which it has been added only by its acid
reaction. The preparations are no longer mentioned in the
literature except as having failed to cure tuberculosis.

The " bouillon-filtrate " (Bouillon filtre) of Denys§ is a
porcelain filtrate of bouillon culture of the tubercle bacillus
and corresponds to Koch's original tuberculin before con-
centration, except in that it has not been subjected to
heat.

Tuberculin-R. — What appears to be an important modi-
fication of tuberculin has been made by Koch,|| in the
TR or tuberculin-R.

All attempts to produce immunity against the tubercle
bacillus by the injection of attenuated cultures, whether
dead or alive, fail because of the invariable occurrence of
abscesses following their introduction into the cellular
tissue, and of nodular growths in the lungs succeeding their
injection into the circulation. It seemed as if the fluids of
the body could not effect the solution of the bacteria and the
liberation of their essential toxic and immunizing con-
stituents.

Koch, therefore, endeavored to bring about artificial con-
ditions advantageous to the absorption of the bacilli, and
for the purpose tried the solvent action of diluted mineral
acids and alkalies. The changes thus brought about facili-

*"Centralbl. f. Bakt. u. Parasitenk.," orig., xxxvn, 1904.
f "Verein fur innere Medizin zu Berlin," Dec. 16, 1907.
t "Die Behandlung der Tuberculose mit Tuberculocidin," 1892.
§ "Acad. royale de med. de Belgique," Feb. 22, 1902; abst. "Cen-
tralbl. f. Bakt. u. Parasitenk.," Ref., 1902, xxxi, p. 563.
|| "Deutsche med. Wochenschrift," 1897, No. 14.

Toxic Products 743

tated absorption, but the absorption of bacilli in this chem-
ically altered condition was not followed by immunity, prob-
ably because the chemic composition of tubercle toxin (or
whatever one may name the poisonous product of the
bacillus) was altered by the reagents.

Tuberculin, with which Koch performed many experi-
ments, was found to produce immunity only against tuber-
culin, not against bacillary infection.

Pursuing the idea of fragmenting the bacilli, or treating them chem-
ically to increase their solubility, Koch found that a 10 per cent, sodium
hydrate solution yielded an alkaline extract of the bacillus, which, when
injected into animals, produced effects similar to those following the
administration of tuberculin, except that they were more brief in dura-
tion and more constant in result ; but the disadvantage of abscess for-
mation following the injections remained. The fluid, when filtered,
possessed the properties of tuberculin.

Mechanical fragmentation of bacilli had been employed by Klebs
in his studies of antiphthisin and tuber culocidin, and Koch now used it
with advantage. He pulverized living, virulent, but perfectly dry
bacilli in an agate mortar, in order to liberate the toxic substance from its
protecting envelop of fatty acid, triturating only very small quantities
of the bacteria at a time.

Having thus reduced the bacilli to fragments, he removed them from
the mortar, placed them in distilled water, washed them, and collected
them by centrifugation, as a muddy residuum at the bottom of an opal-
escent, clear fluid. For convenience he named the clear fluid TO; the
sediment, TR. TO was found to contain tuberculin. In order to
separate the essential poison of the bacteria as perfectly as possible from
the irritating tuberculin, the TR fragments were again dried perfectly,
triturated once more, re-collected in fresh distilled water, and recen-
trifugated. After the second centrifugation microscopic examination
showed that the bacillary fragments had not yet been resolved into a
uniform mass, for when TO was subjected to staining with carbol-fuchsin
and methylene-blue it was found to exhibit a blue reaction, while in TR
a cloudy violet reaction was obtained.

The addition of 50 per cent, of glycerin had no effect upon TO, but
caused a cloudy white deposit to be thrown down from TR. This last
reaction showed that TR contained fragments of the bacilli insoluble in
glycerin.

In making the TR preparation Koch advises the use of a fresh, highly
virulent culture not too old. It must be perfectly dried in a vacuum
exsiccator, and the trituration, in order to be thorough, should not be
done upon more than 100 mg. of the bacilli at a time. A satisfactory
separation of the TR from TO is said to only occur when the perfectly
clear TO takes up at least 50 per cent, of the solid substance, as otherwise
the quantity of TO in the final preparation is so great as to produce un-
desirable reactions.

The fluid is best preserved by the addition of 20 per cent, of glycerin,
which does not injure the TR and prevents its decomposition.

The finished fluid contains 10 mg. of solid constituents to the cubic
centimeter, and before administration should be diluted with physiologic
salt solution (not solutions of carbolic acid). When administering the
remedy to man the injections are made with a hypodermic syringe into
the tissues of the back. The beginning dose is 7£7 mg., rapidly increased
to 20 mg., the injections being made daily.

744 Tuberculosis

Experiment showed that TR had decided immunizing
powers. Injected into tuberculous animals in too large a dose
it produces a reaction, but its immunizing effects were entirely
independent of the reaction. Koch's aim in using this
preparation in the therapeutic treatment of tuberculosis was
to produce immunity against the tubercle bacillus without
reactions by gradual but rapid increase of the dose. In so
large a number of cases did Koch produce immunity to
tuberculosis by the administration of TR, that he believes
it proved beyond a doubt that his observations are correct.

By proper administration of the TR he was able to render
guinea-pigs so completely immune that they were able
to withstand inoculation with virulent bacilli. The point
of inoculation presents no change when the remedy is ad-
ministered; and the neighboring lymph-glands are generally
normal, or when slightly swollen contain no bacilli.

In speaking of his experiments upon guinea-pigs, Koch
says:

"I have, in general, got the impression in these experiments that full
immunization sets in two or three weeks after the use of large doses. A
cure in tuberculous guinea-pigs, animals in which the disease runs, as is
well known, a very rapid course, may, therefore, take place only when
the treatment is introduced early — as early as one or two weeks after the
infection with tuberculosis.

''This rule avails also for tuberculous human beings, whose treat-
ment must not be begun too late. ... A patient who has but a few
months to live cannot expect any value from the use of the remedy, and
it will be of little use to treat patients who suffer chiefly from secondary
infection, especially with the streptococcus, and in whom the septic
process has put the tuberculosis entirely in the background."

One very serious objection, first urged against commer-
cially prepared TR by Trudeau and Baldwin,* is that it is
possible for it to contain unpulverized, and hence still living,
virulent tubercle bacilli. Thellingf could not observe any
good effect to result from the use of Koch's new tuberculin,
and, like Trudeau, found living, virulent bacilli in the prepa-
ration secured from Hochst. Many others have since dis-
covered the same danger. In the preparation of the remedy
it will be remembered that no antiseptic or germicide was
added to the solutions by which the effects of accidental fail-
ure to crush every bacillus could be overcome, Koch having
specially deprecated such additions as producing destruc-
tive changes in the TR. Until this possibility of danger can
be removed, and our confidence that attempts to cure

* "Medical News," Aug. 28, 1897.

t "Centralbl. f. Bakt.," etc., July 5, 1902, xxxn, No. i, p. 28.

Agglutination 745

patients may not result in their infection be restored, it be-
comes a question whether TR can find a place in human
medicine, or must remain an interesting laboratory product.

Baumgarten and Walz* find that the administration of
tuberculin-R to guinea-pigs is without curative effect.
They insist that the results obtained are like those of the
old tuberculin; that " small doses are of no advantage,
while the larger the doses one employs, the greater are the
disadvantages that result from their employment."

During his experiments upon the agglutination of tubercle
bacilli, to be described below, Kochf found that animals
injected with an emulsion of tubercle bacilli showed great
increase in the agglutinative power of the blood. This
led him to suggest that a new preparation, " bacillary emul-
sion " [Bazillen emulsion] be investigated for its immunizing
and curative properties. Many are still using it and some
claim good results.

It is almost impossible to make an accurate estimation
of the usefulness or uselessness of therapeutic preparations
of tubercle bacilli at the present time, not only because of
their diversity of composition and the enthusiasm with
which many have been exploited, but also because of our
inability to compare the results attained with any definite
standard. The advantages or disadvantages of any prepara-
tion, therefore, depend upon the personal opinions of those
employing them rather than upon any demonstration re-
garding them — a very unscientific state of knowledge.

The suggestion of A. K. Wright that the administration
of all such products should be governed by an examination
of the opsonic power of the blood, the remedy being withheld
if this was high and applied if low, the utmost care being
taken not to prolong the " negative phase," seemed to be an
excellent one, affording the beginning of a scientific method
of studying the disease, but unfortunately it seems not to
have been successful in practice, and the tedium and ex-
pense of the examinations makes them impracticable.

Agglutination. — Arloingi and Courmont§ found it pos-

* "Centralbl. f. Bakt. und Parasitenk.," April 12, 1898, xxm, No.
14, P- 593-

f "Deutsche med. Wochenschrift," 1901, No. 48, p. 829.

% "Congress de med. int. Montpellier," 1898; "Compt. rendu Acad.
de Sciences de Paris," 1898, T. cxxvi, pp. 1319-1321.

§ "Compt. rend. Soc. de Biol. de Paris," 1898, No. 28, v; "Congr.
pour 1'etude de la Tuberculose," Paris, 1898.

746 Tuberculosis

sible to prepare homogenized cultures of the tubercle bacil-
lus, and saw them agglutinated by the serum of immu-
nized animals and by the serum of tuberculous patients.
The subject was investigated by Koch,* who carefully
reviews the details of technic and investigates the method,
which, he concludes, is valueless for the diagnosis of human
infection, though a good guide to the extent of immunization
achieved by the therapeutic administration of tuber culin-R.
Thellingf has also shown the reaction to be too irregular
to be of practical diagnostic importance.

The technic of the agglutination test as given by KochJ
is as follows :

Any culture of the tubercle bacillus can be made useful by the fol-
lowing treatment: Collect the bacillary masses upon a filter-paper
and press between layers of filter-paper to remove the fluid. Weigh
out, say, 0.2 gm. of the solid mass and rub it in an agate mortar, adding,
drop by drop, a ^V normal sodium hydroxid solution until the proportion
of i part of the culture to 100 parts of the solution is reached.

It is necessary that the rubbing be thorough in order that the firm
connection between the bacilli shall be broken up and the organisms
distributed throughout the fluid. The operation usually lasts fifteen
minutes. The fluid is then placed in a hand centrifuge and whirled
for six minutes, then pipeted off, and rendered feebly alkaline by adding
diluted hydrochloric acid solution. The fluid thus obtained is too con-
centrated to be used in this form, so must be diluted with 0.5 per cent,
carbolic acid in 0.85 per cent, sodium chlorid solution. This solution
should be repeatedly filtered before receiving the bacillary suspension.
The quantity of bacillary suspension to be added should make the final
product a 3000 dilution of the original. It should look like water by
transmitted light, but slightly opalescent by reflected light.

The serum to be tested is added in proportions of i : 10, i : 25, i : 50,
i : 75, i : 100, i : 200, i : 300, etc., and is to stand for twenty-four hours.
By inclining the tube and looking through a thin stratum of the fluid
the agglutinations can be at once detected.

Antitubercle Serums. — Tizzoni and Centanni,§ Bern-
heim,|| Paquin,** Viqueratff and others have experimented
in various ways, hoping that the principles of serum therapy
might apply to tuberculosis. Nothing has, however, been
achieved. Maragliano's|t antitubercle serum has been used

* "Deutsche med. Wochenschrift," 1901, No. 48, p. 829.

f Loc. cit.

t "Deutsche med. Wochenschrift," 1901, No. 48, p. 829.

§ "Centralbl. f. Bakt.," etc., Bd. xi, p. 82, 1892.

|| Ibid., Bd. xv, p. 654, 1894.
** "New York Med. Record," 1895.

ft "Zur Gewinnung von Antituberkulin, Centralbl. f. Bakt.," etc.,
Nov. 5, 1896, xx, Nos. 1 8, 19, p. 674.

it "Berliner klin. Wochenschrift," 1895, No. 32.

Prophylaxis 747

in a very large number of cases in human medicine, but the
glittering results reported by its author have not been con-
firmed. Behring* comments upon it by saying that " Ma-
ragliano's tubercle antitoxin contains no antitoxin."

Babes and Proca, | Maffucci and di Vestea, { McFarland, §
De SchweinitzJI Fisch,** and Patersonff have all endeavored
to obtain serums of therapeutic value by immunizing animals
against living or dead tubercle bacilli or their products, but
without success.

From these discordant observations, the more favorable
of which are probably the hasty records of inadequate or
incomplete experiments, the conclusion that little is to be
hoped from immune serums in the treatment of tuberculosis
is inevitable.

Prophylaxis. — It is the duty of every physician to
use every means in his power to prevent the spread of
tuberculous infection in the households under his care.
To this end patients should cease to kiss the members of
their families and friends; should have individual knives,
forks, spoons, cups, napkins, etc., carefully kept apart —
secretly if the patient be sensitive upon the subject — from
those of the family, and scalded after each meal; should
have their napkins and handkerchiefs, as well as whatever
clothing or bed-clothing is soiled by them, kept apart from
the common wash, and boiled; and should carefully col-
lect the expectoration in a suitable receptacle, that is ster-
ilized or disinfected, without being permitted to dry, as
it has been shown that the tubercle bacillus can remain
alive in dried sputum as long as nine months. The phys-
ician should also give directions for disinfecting the bed-
room occupied by a consumptive before it becomes the
chamber of a healthy person, though this should be as
much the function of the municipality as the disinfec-
tion practised after scarlatina, diphtheria, and smallpox.

Boards of health are now becoming more and more in-

* "Fortschritte der Med.," 1897.
t "La Med. Moderne," 1896, p. 37.
j "Centralbl. f. Bakt.," etc., 1896, Bd. xix, p. 208.
§ "Jour. Amer. Med. Assoc.," Aug. 21, 1897.

|| "Centralbl. f. Bakt. und Parasitenk.," Sept. 15, 1897, Bd. xxn,
Nos. 8 and 9.

** "Jour. Amer. Med. Assoc.," Oct. 30, 1897.
ft "Amer. Medico-Surg. Bull.," Jan. 25, 1898.

748 Tuberculosis

terested in tuberculosis, and, though exceedingly slow and
conservative in their movements, are disseminating litera-
ture with the hope of achieving by volition that which might
otherwise be regarded as cruel compulsion.

So long as tuberculosis exists among men or cattle, it
shows that existing hygienic precautions are insufficient.
While condemning any unreasonable isolation of patients,
we should favor the registration of tuberculous cases as a
means of collecting accurate data concerning their origin ; in-
sist upon the careful domestic sterilization and disinfection
of all articles used by the patients; recommend public disin-
fection of the houses they cease to occupy; and approve of
special hospitals for as many (especially of the poorer classes,
among whom hygienic measures are almost always opposed)
as can be persuaded to occupy them.

BOVINE TUBERCULOSIS.
BACILLUS TUBERCULOSIS Bovis.

The tuberculous diseases of the lower animals and espe-
cially cattle have lesions closely resembling those of human
tuberculosis, and containing bacilli similar both in morphol-
ogy and in staining reaction to those found in human tuber-
culosis. The conclusion that they are identical seems in-
evitable, but in his monograph upon tuberculosis Koch
called attention to certain morphologic and cultural differ-
ences that exist between bacilli obtained from human and
from animal tuberculosis. Unfortunately, very little atten-
tion was paid to the subject until Theobald Smith* care-
fully compared a series of bacilli obtained from human
sputum with another series obtained from cattle, horses,
hogs, cats, dogs, and other animals.

His observations form the foundation of the following
description of the bovine tubercle bacillus:

Morphology. — The size of the bovine bacillus is quite
constant, the individuals being quite short (1-2 ^). They
are straight, not very regular in outline, and sometimes
of a spindle, sometimes a barrel, and sometimes an oval
shape. The human bacilli, on the other hand, are prone to
take an elongate form under artificial cultivation.

* ''Trans. Assoc. Amer. Phys.," 1896, xi, p. 75, and 1898, xm, p. 417;
"Jour, of Experimental Medicine," 1898, in, 495.

Bovine Tuberculosis 749

Staining. — The bovine bacillus usually stains homoge-
neously; the human bacillus commonly shows the so-called
" beaded appearance."

Vegetation. — The human bacillus grows upon dogs' serum
much more luxuriantly and rapidly than the bovine bacillus.

Metabolic Products. — An important variation in the
metabolism of the human and bovine tubercle bacilli has been
pointed out by Theobald Smith,* who observed that cultures
of the two organisms upon glycerin bouillon differed in the
induced reaction of the media. The cultures of the bovine
bacillus tend toward neutrality, those of the human bacillus
toward acidity. This chemical difference is an adjunct to-
our means of differentiating the two organisms.

Pathogenesis. — (a) Guinea-pigs. — The bovine bacilli are
more virulent than those of human tuberculosis, intraperi-
toneal inoculation of the former producing death in adult
animals in from seven to sixteen days; of the latter, in from
ten to thirty-eight days. Subcutaneous inoculation of the
bovine bacillus causes death in less than fifty days; of the
human bacillus, in from fifty to one hundred days.

(b) Rabbits. — Rabbits inoculated into the ear vein with
the bovine bacillus die in from seventeen to twenty-one days.
Those receiving human bacilli sometimes live several months.

(c) Cattle. — Cows and heifers receiving intrapleural
and intra-abdominal injections of the human bacilli usually
gain in weight and show no symptoms. When examined
postmortem, circumscribed chronic lesions were found.
Those inoculated with the bovine bacillus lose weight, suffer
from constitutional symptoms, and show extensive lesions
at the necropsy. Two-thirds of the cattle inoculated experi-
mentally with the bovine bacillus die.

Lesions — In general the lesions produced by the bovine
bacillus were rapid, extensive, and necrotic. Many bacilli
are present. Those produced by the human bacillus are
more apt to be productive, chronic, and contain relatively
few bacilli. The bacilli of human tuberculosis produce lesions
with many giant cells; those of bovine tuberculosis, lesions
with rapid coagulation necrosis. The lesions resulting from
the intravenous injection of human bacilli into rabbits re-
sembled those observed by Prudden and Hodenpyl f after the
intravenous injection of boiled, washed tubercle bacilli.

* "Trans. Assoc. Amer. Phys.," 1903, vol. xvm, p. 109.
f "New York Med. Jour.," June 6-20, 1891.

750 Tuberculosis

From these data it is evident that the bovine bacillus
is by far the more virulent and dangerous organism.

At the International Congress on Tuberculosis, held in
London, 1901, Koch expressed the opinion that bovine
tuberculosis was not communicable to man. The matter
is of the utmost importance to the medical profession and
of far-reaching influence upon many important sanitary
measures that bear directly upon the public health.

Koch's opinion, being opposed to all that had been believed
before, received almost universal disapproval. The papers
by Arloing,* Ravenel,| and Salmon J contain evidence show-
ing that under certain conditions bovine tuberculosis can
be communicated to man.

Ravenel§ has reported 3 cases of accidental cutaneous
inoculation of bovine tuberculosis in man. All were veter-
inary surgeons who became infected through wounds ac-
cidentally inflicted during the performance of necropsies
upon tuberculous cattle. The tubercle bacilli were demon-
strated in some of the excised cutaneous nodules.

Theobald Smith, || in studying 3 cases of supposed food
infection, found what corresponded biologically with the
human rather than the bovine bacillus.

In a later paper Koch** analyzed the cases usually se-
lected from the literature to prove the communicability of
bovine tuberculosis to man, and showed that not one of
the cases really proves what is claimed for it, and that the
subject requires further careful investigation and demon-
stration before it will be possible to express any positive
opinion in regard to it.

During the years that have elapsed since 1901 and the
present time sentiment has been almost uniformly against
Koch, and an enormous literature has accumulated that in
reality means very little. The most important is probably
the Royal Commission on Tuberculosis of Great Britain, ft

*"Lyon Med.," Dec. i, 1901.

t"Univ. of Pa. Bulletin," xiv, p. 238, 1901; "Lancet," Aug. 17
and 19, 1901; "Medicine," July and Aug., 1902, vol. vm.

I " Bull. No. 33, Bureau of Animal Industry," U. S. Dept. of Agricul-
ture, 1901.

§ "Phila. Med. Jour.," July 21, 1900.

|| "Amer. Jour. Med. Sciences," Aug. 1904, vol. cxxvni, No. 389,
p. 216.

** Eleventh International Congress for Tuberculosis, Berlin, 1902.
ft "See the "British Medical Journal," 1907 and 1908.

Bovine Tuberculosis 751

The general tenor of this report is contrary to Koch's views,
and many believed it settled the subject. At the Inter-
national Congress on Tuberculosis in Washington, 1908,
Koch reviewed the subject and stated his continued belief
in the principle he had enumerated seven years before.
Practically the same contentions were raised against him
by much the same group of men, but the controversy was
more bitter than before. Koch,* however, leaves us in no
doubt upon the subject, summarizing his views in these
words :

1. The tubercle bacilli of bovine tuberculosis are different from

those of human tuberculosis.

2. Human beings may be infected by bovine tubercle bacilli, but

serious diseases from this cause occur very rarely.

3. Preventive measures against tuberculosis should, therefore,

be directed primarily against the propagation of human
tubercle bacilli.

He weighed the contrary evidence that had been col-
lected during seven years, showed how errors had crept
into the investigations, and laid down certain rules to be
observed before the experiments could be accepted. At the
close of the congress the matter remained unsettled, Koch
appearing to have the best of the argument.

The opponents of Koch based their opinions upon the sup-
posed modifiability of the tubercle bacillus in different en-
vironments. When it lived in man, it was by virtue of the
contact with the human juices and their chemical peculiarities
compelled to assume the human form ; in the cow, by virtue of
the different chemical conditions, the bovine form, etc.
Proofs of this were, however, wanting, and have not yet been
published. On the other hand, Moriyaf seems to have shown
that such changes are either purely hypothetic or come
about with great difficulty. He succeeded in keeping human
and also bovine types of tubercle bacilli alive in tortoises
for twelve months, at the end of which period each was found
unmodified and possessed of its original characteristics.

It was Koch's hope to be able to finally settle the whole
matter, and to this end he asked the cooperation of many
laboratories throughout different parts of the world. Un-
fortunately he died before the results could be compiled, but
much work had been done and much support thereby given

* "Jour. Amer. Med. Assoc.," Oct. 10, 1908, u, No. 15, p. 1256.
t "Centralbl. f. Bakt. u. Parasitenk.," i. Abt. Orig., u, 1909, 460.

752

Tuberculosis

his views. A most fertile research, the results of which form
a valuable addition to our knowledge of the problem, has
been published by Park and Krumwiede,* who, basing their
opinions upon the following tabulation of 1224 cases,

COMBINED TABULATION CASES REPORTED AND OWN SERIES OF CASES.

Diagnosis.

Adults 1 6 years
and over.

Children 5 to 16
years.

Children under
5 years.

H.

B.

H.

B.

H.

B.

Pulmonary tuberculosis
Tuberculous adenitis, axillary
or inguinal

644

2
27
14

6
29

5

27
17
3

_!

(I?)

I

4

i

i

i

1 1

4
36

8

2

4

i

7
3

38

2

I

21
7

3
i

3
i

23

2
15
9

13

43

3

52
27

26

21
13

12

5

8

i

4

Tuberculous adenitis, cervical
Abdominal tuberculosis
Generalized tuberculosis, ali-
mentary origin.

Generalized tuberculosis
Generalized tuberculosis in-
cluding meninges, aliment-
ary origin

Generalized tuberculosis in-
cluding meninges
Tubercular meningitis
Tuberculosis of bones and
joints

Genito-urinary tuberculosis. .
Tuberculosis of skin
Miscellaneous cases:
Tuberculosis of tonsils ....
Tuberculosis of mouth and
cervical nodes
Tuberculous sinusor abscess
Sepsis, latent bacilli

Totals . .

777

10

117

36

2I.S

65

Mixed or double infections, 4 cases.

Total cases, 1224.

come to the following conclusions : —

Conclusions. — Bovine tuberculosis is practically a negligi-
ble factor in adults. It very rarely causes pulmonary tuber-
culosis or phthisis which causes the vast majority of deaths
from tuberculosis in man, and is the type of disease respon-
sible for the spread of the virus from man to man.

In children, however, the bovine type of tubercle bacillus
causes a marked percentage of the cases of cervical adenitis,
leading to operation, temporary disablement, discomfort, and

* "Journal of Medical Research," 1910, xxin, No. 2, p. 205; 191 1,
xxv, No. 2, p. 313.

Bovine Tuberculosis 753

disfigurement. It causes a large percentage of the rarer
types of alimentary tuberculosis requiring operative inter-
ference or causing the death of the child directly or as a
contributing cause in other diseases.

In young children it becomes a menace to life and causes
from 6 J to 10 per cent, of the total fatalities from this disease.

Prophylaxis. — The prevention of tuberculosis in cattle is
a matter of vast sanitary importance. Not only have we
to consider the danger of infection from milk containing
tubercle bacilli, but also the inferior quality and diminished
usefulness of milk and flesh coming from animals that are
diseased. The extermination of bovine tuberculosis, there-
fore, becomes imperative, and the utmost efforts should be
made to bring it about. Several separate measures must
be considered:

1. Improvement in the methods of diagnosis, by which
the recognition of the disease is made possible before its
ravages are great. This is rapidly coming about with in-
creasing information regarding the use and abuse of tu-
berculin, etc.

2. Means by which infected animals shall be destroyed.
Here the municipal and state governments furnish inade-
quate funds to make possible the destruction of diseased
cattle without adequate compensation — an injustice to the
unfortunate owner.

3. Means of preventing the infection of healthy ani-
mals. In many places this is being achieved with brilliant
success by separation of the herd, healthy and newly born
animals constituting one part, suspicious animals the other.
By these means valuable breeding animals can be kept for
a time, at least, in usefulness. A second and less successful
means of preventing infection is by means of prophylactic
vaccination of the healthy animals with dead cultures,
modified living cultures, or by bacteriotoxins made by com-
minuting them.

Experiments of this kind have been conducted by Mc-
Fadyen,* on a large scale by von Behring, f by Pearson
and Gilliland, t Calmette and Guerin,§ and by Theobald

c "Jour. Comp. Path, and Therap.," June, 1901.

f "Beitrage zur experimentellen Therapie," 1902, Hft. 5.

| "Jour, of Comp. Med. Vet. Archiv," Nov. 1902, "Univ. of Penna.
Med. Bull.," April, 1905.

§ "Ann. de 1'Inst. Pasteur.," Oct., 1905, May, 1906, and July, 1907;
and "International Congress on Tuberculosis," Washington, 1908.
48

754 Tuberculosis

Smith,* all of whom think distinct resisting power against
infection by the tubercle bacillus can thus be brought about.

Tuberculin Test for Tuberculosis of Cattle. — The febrile
reaction caused by the injection of tuberculin into tubercu-
lous animals is an important adjunct to our means of diag-
nosticating the disease. For the recognition of tuberculosis
in cattle it is easily carried out.

To make a satisfactory diagnostic test the temperature of
the animal should be taken every few hours for a day or two
before the tuberculin is administered, in order that the nor-
mal diurnal and nocturnal variations of temperature shall
be known. The tuberculin is then administered by hypoder-
mic injection into the shoulder or flank, and the tem-
perature subsequently taken every two hours for the next
twenty-four hours. A reaction of two degrees beyond that
normal to the individual animal is positive of tuberculosis.
After one reaction of this kind the animal will not again
react to an equal dose of tuberculin for a number of weeks.

FOWL TUBERCULOSIS.
BACILLUS TUBERCULOSIS AVIUM.

The occasional spontaneous occurrence of tuberculosis in
chickens, parrots, ducks, and other birds, observed as early
as 1868 by Rolofff and Paulicki, { was originally attributed
to Bacillus tuberculosis hominis, but the work of Rivolta,§
Mafucci,|| Cadio, Gilbert and Roger,** and others has shown
that, while similar to it in many respects, the organism
found in the avian diseases has distinct peculiarities which
make it a different variety, if not a separate species. Cadio,
Gilbert, and Roger succeeded in infecting fowls by feeding
them upon food containing tubercle bacilli, and keeping
them in cages in which dust containing tubercle bacilli was
placed. The infection was aided by lowering the tempera-
ture of the birds with antipyrin and lessening their vitality
by starvation.

* ''Journal of Medical Research," June, 1908, xvm, No. 3, p. 451.
f'Mag. f. d. ges. Tierheilkunde," 1868.
t "Beitr. zur vergl. Anat.," Berlin, 1872.
§ "Giorn. anat. fisiol. e. path.," Pisa, 1883.
|| "Zeitschrift fur Hygiene," Bd. xi.
** "La Semaine medicale," 1890, p. 45.

Fowl Tuberculosis 755

Morphologic Peculiarities. — Morphologically, the organ-
ism found in avian tuberculosis is similar to that found
in the mammalian disease, but is a little longer and more
slender, with more marked tendency to club and branched
forms. Fragmented and beaded forms occur as in the
human tubercle bacilli.

Staining. — The avian bacillus stains in about the same
manner as the human and bovine bacilli and has an equal
resistance to the decolorant effect of acids.

Cultivation. — Marked rapidity and luxuriance of growth
are characteristic of the avian bacillus, which grows upon
agar-agar and ordinary bouillon prepared without glycerin.

Fig. 247. — Bacillus tuberculosis avium.

The growth also lacks the dry quality characteristic of
cultures of the human and bovine bacilli. Old cultures of
the bacillus of fowl tuberculosis turn slightly yellow.

Thermic Sensitivity. — The bacillus also differs in its
thermic sensitivity and will grow at 42° to 45° C. quite as
well as at 37° C., while the growth of the human and mam-
malian bacilli ceases at 42° C. Moreover, growth at 43° C.
does not attenuate its virulence. The thermal death-
point is 70° C. Upon culture-media it is said to retain its
virulence as long as two years.

Pathogenesis. — Birds are the most susceptible animals
for experimental inoculation, the embryos and young being

756 Tuberculosis

more susceptible than the adults. Artificial inoculation
can be made in the subcutaneous tissue, in the trachea, and
in the veins; never through the intestine. After inoculation
the birds die in from one to seven months. The chief seat
of the disease is the liver, where cellular (lymphocytic) nodes,
lacking the central coagulation and the giant-cell formation
of mammalian tuberculosis, and enormously rich in bacilli,
are found. The disease never begins in the lungs, and the
fowls that are diseased never show bacilli in the sputum or
in the dung.

Guinea-pigs are quite immune, or after inoculation develop
cheesy nodes, but do not die.

Rabbits are easily infected, an abscess forming at the
seat of inoculation, nodules forming later in the lungs, so
that the distribution is quite different from that seen in
birds. It is possible that the avian bacillus occasionally
infects man.

The possibility that this bacillus is derived from the same
stock as the tubercle bacillus is strengthened by the experi-
ments of Fermi and Salsano,* who succeeded in increasing
its virulence until it became fatal to guinea-pigs by adding
glucose and lactic acid to the cultures inoculated.

FISH TUBERCULOSIS.

Dubarre and Terref isolated a bacillus having the tinctorial
and morphologic characteristics of the tubercle bacillus
from carp suffering from a tubercle-like affection. In respect
to cultivation, however, it was unlike tubercle bacilli, growing
readily upon simple culture-media at 15° to 30° C., and not
at 37° C.

Weber and TaubeJ found the same organism, or what
seemed to be the same organism, in mud and in a healthy frog.

BACILLI RESEMBLING THE TUBERCLE BACILLUS.

It is not improbable that the bacilli of human, bovine,
and avian tuberculosis are closely related to one another, and,
together with a few other micro-organisms of similar mor-
phology and staining peculiarities, have a common ancestry

""Centralbl. f. Bakt.," etc., xn, 750.
t "Compt. rendu de la Soc. de Biol. de Pari," 1897, 446.
I "Tuberkulose Arbeiten aus dem Kaiserlichen Gesundheitsamte,"
1905.

Bacillus Smegmatis 757

and are descended from the same original stock. The
most important of these similar organisms are Bacillus
leprce (q. v.), B. smegmatis, and Moeller's grass bacillus.

BACILLUS SMEGMATIS.

Alvarez and Tavel,* Matterstock, f Klemperer and Bittu, J
Cowie, § and others have described peculiar bacilli in smegma
taken from the genitals of man and the lower animals, as well
as from the moist skin in the folds of the groin, the axillae,
and the anus. They are also sometimes found in urine, and
occasionally in the saliva and sputum.

Morphology and Staining. — The organisms are of some-
what variable morphology, but in general resemble the tuber-
cle bacillus, stain with carbol-fuchsin, as does the tubercle
bacillus, and resist the decolorant action of acids. They are,
however, decolorized by absolute alcohol, though Moeller de-
clares the smegma bacillus to be absolutely alcohol-proof as
well as acid-proof, and admits no tinctorial difference between
it and the tubercle bacillus. The bacillus, being about the
size and shape of the tubercle bacillus, is very readily mis-
taken for it, and its presence in cases of suspected tubercu-
losis of the genito-urinary apparatus, and in urine and other
secretions in which it is likely to be present, may lead to
considerable confusion. The final differentiation may have
to rest upon animal inoculation.

Cultivation. — The cultivation of the smegma bacillus is
difficult and was first achieved by Czaplewski.|| Doutrele-
pont and Matterstock cultivated it upon coagulated hydro-
cele fluid, but were unable to transplant the growth suc-
cessfully.

Novy** recommends the cultivation of the smegma
bacillus by inoculating a tube of melted agar-agar cooled
to 50° C. with the appropriate material, and mixing with
it about 2 c.c. of blood withdrawn from a vein of the arm
with a sterile hypodermic syringe. The blood-agar mixture
is poured into a sterile Petri dish and set aside for a day

*"Archiv de Physiol. norm, et Path.," 1885, No. 7.
f "Mittheil. aus d. med. Klin. d. Univ. d. Wiirzburg," 1885, Bd. VI.
J "Virchow's Archives," v, 103.

§ "Journal of Experimental Medicine," vol. v, 1900-01, p. 205.
|| "Miinchener med. Wochenschrift," 1897.
** " Laboratory Work in Bacteriology," 1899.

758 Tuberculosis

or two at 37° C. The colonies that form are to be examined
for bacilli that resist decolorization with acids.

Moeller* found it comparatively easy to secure cultures
of the smegma bacillus by a peculiar method. To secure
small quantities of human serum for the purpose of investi-
gating the phenomena of agglutination he applied small
cantharidal blisters to the skins of various healthy and
other men, and found large numbers of acid-proof bacilli in
the serum saturated with epithelial substance, that re-
mained after most of the serum had been withdrawn. He
removed the skin covering from the blister, placed it in the
remaining serum, and kept it in the incubator for three or
four days, after which he found a dry, floating scum, which
consisted of enormous numbers of the bacilli, upon the serum.
From this growth he was subsequently able to start cultures
of the smegma bacillus upon glycerin agar-agar. Human
blood-serum is thus found to be the best medium upon
which to start the culture.

Agar. — A culture thus isolated grew upon all the usual
culture-media. Upon glycerin-agar, at 37° C., the colonies
appeared as minute, dull, grayish- white, dry, rounded scales,
which later became lobulated and velvety. At room tem-
perature the dry appearance of the growth was retained.
The water of condensation remains clear.

Potato. — On potato the growth was luxuriant, grayish,
and dull.

Milk. — Milk is said to be an exceptionally good medium,
growth taking place in it with rapidity and without coagu-
lation.

Bouillon. — The growth forms a dry white scum upon the
surface, the medium remaining clear.

Pathogenesis. — So far as is known, the smegma bacillus
is a harmless saprophyte.

MOELLER'S GRASS BACILLUS.

Bacilli found in milk, butter, timothy hay, cow-dung, etc.,
which stain like the tubercle bacillus and may be mistaken
for it, have been described by Moeller. f The organisms so
closely resemble the tubercle bacillus that guinea-pig inocu-

*"Centralbl. f. Bakt. u. Parasitenk" (Originale), Bd. xxxi, No. 7,
p. 278, March 12, 1902.

t "Deutsche med. Zeitung," 1898, p. 135; "Deutsche med. Wochen-
schrift," 1898, p. 376, etc.

Bacilli Resembling the Tubercle Bacillus 759

lations must be resorted to in cases of doubt, but as some of
these organisms sometimes kill the guinea-pigs after a
month or two, and as small nodules or tubercles may be
present in the mesentery, peritoneum, liver, lung, etc., of
such animals, the diagnosis may have to be subjected to the
further confirmation of a histologic examination of the
lesions in order to exclude tuberculosis. In cases of this
kind it should not be forgotten that the tubercle bacillus
can be present in the substances mentioned, so that the
exact differentiation becomes a very fine one. An instruc-
tive study of these organisms has been made by Abbott
and Gildersleeve,* who, in an elaborate work upon the
" Etiological Significance of the Acid-resisting Group of
Bacteria, and the Evidence in Favor of their Botanical
Relation to Bacillus Tuberculosis," a work that gives com-
plete references to the literature of the subject, come to the
following conclusions :

1. That the majority of the acid-resisting bacteria may be dis-
tinguished from true tubercle bacilli by their inability to resist decolor-
ization by a 30 per cent, solution of nitric acid in water.

2. That some of the acid-resisting bacteria are capable of causing
in rabbits and guinea-pigs nodular lesions suggestive of tubercles;
that these lesions, while often very much like tubercles in their histo-
logic structure, may nevertheless usually be distinguished from them by
the following peculiarities:

(a) When occurring as a result of intravenous inoculation, they are
always seen in the kidneys, only occasionally in the lungs, and practi-
cally not at all in the other organs.

(b) They constitute a localized lesion, having no tendency to dis-
semination, metastasis, or progressive destruction of tissue by casea-
tion.

(c} They tend to terminate in suppuration or organization rather than
in progressive caseation, as is the case with true tubercles.

(d) They are more commonly and conspicuously marked by the ac-
tinomyces type of development of the organisms than is the case with
true tubercles, and these actinomycetes are less resistant to decoloriza-
tion by strong acid solutions than are those occasionally seen in tubercles.

3. That by subcutaneous, intravenous, and intrapulmonary inocula-
tion of hogs (4) and calves (15) the typical members of the acid-resisting
group are incapable of causing lesions in any way suggestive of those re-
sulting from similar inoculations of the same animals with true tubercle
bacilli.

4. That though occasionally present in dairy products, they are to be
regarded as of no significance, etiologically speaking, but may be con-
sidered as accidental contaminations from the surroundings, and not as
evidence of disease in the animals.

5. That the designation "bacillus" as applied to this group of bac-
teria and to the exciter of tuberculosis is a misnomer; they are more cor-
rectly classified as actinomyces.

*"Univ. of Penna. Bulletin," June, 1902.

760 Tuberculosis

THE BUTTER BACILLUS.

Petri,* Rabinovitsch, | and KornJ have described, as
Bacillus butyricus, an acid-fast organism morphologically
like the tubercle bacillus, which may at times be found in
butter. Its chief importance lies in the confusion that may
arise through mistaking it for the tubercle bacillus where
attention is paid to the morphologic and tinctorial characters
only, as tubercle bacilli may be found in butter made from
cream from the milk of tuberculous cattle.

Isolation and cultivation of these organisms is easy, and
more than any other measure serves to differentiate them
from the tubercle bacillus, as they grow upon nearly all the
culture-media with rapidity and luxuriance.

PSEUDOTUBERCULOSIS.
BACILLUS PSEUDOTUBERCULOSIS.

Pfeiffer,§ Malassez and Vignal,|| Eberth,** Chantemesse,tt
Charrin, and Roger it have all reported cases of so-called
pseudotuberculosis occurring in guinea-pigs, and character-
ized by the formation of cellular nodules in the liver and
kidneys much resembling miliary tubercles. Cultures made
from them showed the presence of a small motile bacillus
which could easily be stained by ordinary methods. When
introduced subcutaneously into guinea-pigs, the original dis-
ease was reproduced.

Morphology and Cultivation. — Bacillus pseudotuber-
culosis is characterized by Pfeiffer as follows : The organism is
rod-shaped, the rods varying in length (0.4 to 1.2 {i} and
sometimes united in chains. They may be almost round,
and then resemble diplococci. They stain by ordinary
methods, but not by Gram's method. They are motile and
have flagella like the typhoid and colon bacilli. They form

* "Arbeiten aus dem Kaiselichen Gesundheitsamt," 1897.

t "Zeitschrift fur Hygiene," etc., 1897.

t "Centralbl. f. Bakt." etc., 1899.

\ "Bacillare tuberculose, u. s. w.," Leipzig, 1889.

j| "Archiv. de Physiol. norm, et Path.," 1883 and 1884.
** "Virchow's Archiv," Bd. en.
ft ''Ann. de 1'Inst. Pasteur," 1887.
tJ "Compte-rendu de 1'Acad. des Sci.," Paris, t. cvi.

Pseudotuberculosis 761

no spores. Upon gelatin and agar-agar circular colonies
with a dark nucleus surrounded by a transparent zone are
formed. In gelatin punctures the bacilli grow all along the
line of puncture and form a surface growth with concentric
markings. The gelatin is not liquefied. The bacilli grow
readily upon agar and on potato, but without character-

Fig. 248. — Bacillus pseudotuberculosis from agar-agar. X 1000
(Itzerott and Niemann).

istic appearances. In bouillon a diffuse turbidity occurs,
with floating and suspended flakes. Milk is not altered.

Pathogenesis. — The bacillus is fatal to mice, guinea-
pigs, rabbits, hares, and other rodents in about twenty
days after inoculation. At the seat of inoculation an abscess
develops, the neighboring lymphatic glands enlarge and
caseate, and nodules resembling tubercles form in the
internal organs. Similar bacilli studied by Pfeiffer were
isolated from a horse supposed to have glanders.

CHAPTER XXVIII.
LEPROSY.

BACILLUS LEPR^ (HANSEN).*

General Characteristics. — A non-motile, non-flagellate, non-spor-
ogenous, chromogenic, non-liquefying, non-aerogenic, distinctly aerobic,
parasitic and highly pathogenic, acid-resisting bacillus, staining by
Gram's method, and cultivable upon specially prepared artificial media.
It does not form indol, or acidulate or coagulate milk.

Leprosy very early received attention and study. Moses
included in the laws to the people of Israel rules for its diag-
nosis, for the isolation of the sufferers, for the determination
of recovery, and for the sacrificial observances to be fulfilled
before the convalescent could once more mingle with his peo-
ple. The Bible is replete with miracles wrought upon lepers,
and during the times of biblical tradition it seems to have
been an exceedingly common and malignant disease. Many
of the diseases called leprosy in the Bible were, however, in
all probability, less important parasitic skin affections.

Distribution. — At the present time, although we hear
very little about it in the northern United States, leprosy is
a widespread disease and exists much the same as it did
several thousand years ago in Palestine, Syria, Egypt,
and the adjacent countries, and is common in China, Japan,
and India. South Africa has many cases, and Europe, es-
pecially Norway, Sweden, and parts of the Mediterranean
coast, a considerable number. In certain islands, especially
the Sandwich and Philippine Islands, it is endemic. In
the United States the disease is uncommon, the Southern
States and Gulf coast being chiefly affected.

A commission of the Marine- Hospital Service, formed for
the purpose of investigating the prevalence of leprosy, in
1902 reported 278 existing cases in the United States. Of
these, 155 occurred in the State of Louisiana. The other
States with numerous cases were California, 24; Florida, 24;
Minnesota, 20; and North Dakota, 16. No other State
had more than 7 (New York). Of the cases, 145 were
American born, 120 foreign born, the remainder uncertain

* "Virchow's Archives," 1879.
762

Staining

763

Etiology. — The cause of leprosy is, without doubt, the
lepra bacillus, discovered by Hansen in 1879.

Morphology. — The bacillus is about the same size as
the tubercle bacillus. Its protoplasm commonly presents
open spaces of fractures, giving it a beaded appearance, like
the tubercle bacillus. It occurs singly or in irregular groups.
There is no characteristic grouping and filaments are un-
known. It is not motile and has no flagella and no spores.

Duval found that the cultivated bacilli are longer, more
curved, and show a greater irregularity in the distribution of
the chromatin than those in the tissues where they are short,
slender, and slightly curved. In artificial cultures there is a

Fig. 249. — Lepra bacilli. Smear from a lepra node stained with carbol-
fuchsin (Kolle and Wassermann).

delicate filamentous arrangement of the bacilli, especially
where they have become accustomed to a saprophytic exist-
ence. They often contain distinct metachromatic granules
analogous to those met with in certain forms of the diphtheria
bacillus. They are quite pleomorphous, and in the same cul-
ture all forms occur, from solidly staining coccoid shapes to
slender slightly curved filaments, with numerous chromatic
segments and occasionally metachromatic granules. Some-
times the organisms are pointed at the ends.

Staining. — It stains in very much the same way as the
tubercle bacillus, but permits of a more ready penetra-
tion of the stain, so that the ordinary aqueous solutions

764

Leprosy

of the anilin dyes color it quite readily. The property of
retaining the color in the presence of the mineral acids also
characterizes the lepra bacillus, and the methods of Ehrlich,
Gabbet, and Unna for staining the tubercle bacillus, can
be used for its detection. It stains well by Gram's method
and by Weigert's modification of it, by which beautiful
tissue specimens can be prepared.

Fig. 250. — Section of one of the nodules from the patient shown in
Fig. 252, stained by the Weigert-Gram method to show the lepra ba-
cilli scattered through the tissue and inclosed in the large vacuolated
"lepra-cells." Magnified 1000 diameters.

Czaplewski found that the lepra bacilli in his cultures
colored uniformly when young, but were invariably granular
when old. The more rapidly the organism grew, the more
slender it appeared.

Cultivation 765

Cultivation. — Many endeavors have been made to culti-
vate this bacillus upon artificially prepared media, but in
1903 Hansen,* who discovered the organism, declares that
no one had yet cultivated it.

Bordoni-Uffredozzif was able to cultivate a bacillus which
partook of the staining peculiarities of the lepra bacillus as
it appears in the tissues, but differed in morphology.

CzaplewskiJ confirmed the work of Bordoni-Uffredozzi,
and described a bacillus supposed to be the lepra bacillus,
which he 'succeeded in cultivating from the nasal secretions
of a leper.

The bacillus was isolated upon a culture-medium consist-
ing of glycerinized serum without the addition of salt,
peptone, or sugar. The mixture was poured into Petri
dishes, coagulated by heat, and sterilized by the inter-
mittent method.

The secretion, being rich in lepra bacilli, was taken up with
a platinum wire and inoculated upon the culture-medium
by a series of linear strokes. The dishes were then sealed
with paraffin and kept in the incubating oven at 37° C.

Numerous colonies, chiefly of Staphylococcus pyogenes
aureus and the bacillus of Friedlander, developed, and in
addition a number of strange colonies, composed of slender
bacilli about the size and form of the lepra bacillus.

These colonies were grayish yellow, humped in the middle,
i to 2 mm. in diameter, irregularly rounded, and uneven at
the edges. They were firm and could be entirely inverted
with the platinum wire, although the consistence was
crumbly. They were excavated on the under side.

The colonies that form upon agar-agar are much like those
described by Bordoni-Uffredozzi, and appear as isolated,
grayish, rounded flakes, thicker in the center than at the
edges, and characterized by an irregular serrated border
from which a fine irregular network extends upon the
medium. These projections consist of bundles of the bacilli.

When a transfer was made from one of these colonies
to fresh media, the growth became apparent in a few days

* Kolle and Wassermann's "Handbuch der pathogenen Mikroorgan-
ismen," n, p. 184, 1903.

t "Zeitschrift f. Hygiene," etc., 1884, in.

t "Centralbl. f. Bakt. und Parasitenk.," Jan. 31, 1898, vol. xxm, Nos.
3 and 4, p. 97.

766 Leprosy

and assumed a band-like form, with a plateau-like elevation
in the center.

The bacillus thus isolated grew with moderate rapidity
upon all the ordinary culture-media except potato. Upon
blood-serum the growth was more luxuriant and fluid
than upon the solid media. Upon coagulated serum the
growth was somewhat dry and elevated, and was frequently
so loosely attached to the surface of the medium as to
be readily lifted up by the platinum wire.

The growth was especially luxuriant upon sheep's blood-
serum to which 5 per cent, of glycerin was added. The
growth upon the Loffler mixture was also luxuriant.

Upon agar-agar the growth is more meager; it is more
luxuriant upon glycerin agar-agar than upon plain agar-
agar, the bacterial mass appearing grayish and flatter than
upon blood-serum. The growth never extends to the
water of condensation to form a floating layer.

The bacillus develops well upon gelatin after it has grown
artificially for a number of generations and become accus-
tomed to a saprophytic existence. Upon the surface of gela-
tin the growth is, in general, similar to that upon agar-agar.
In puncture cultures most of the growth occurs upon the
surface to form a whitish, grayish, or yellowish wrinkled
layer. Below the surface of the gelatin the growth occurs as
a thick, granular column. The medium is not liquefied.

In bouillon, growth occurs only at the bottom of the tube
in the form of a powdery sediment.

Spronck* believed that he had successfully cultivated the
organism upon glycerinized, neutralized potatoes, first seeing
the growth after the lapse of ten days. Cultures thus pre-
pared were found to be agglutinated by the blood-serum of
lepra cases, and he recommends the agglutination test for
the diagnosis of obscure cases of the disease.

Ducrey seems to have cultivated the lepra bacillus in
grape-sugar, agar, and in bouillon in vacuo. His results
need confirmation.

Rostf claimed to isolate and cultivate the lepra bacil-

* "Weekblad van het Nederlandsch Tijdschrift voor geneeskunde,"
Deel n, 1898, No. 14; abstract "Centralbl. f. Bakt.," etc., xxv, p. 257,
1899-

t"Brit. Med. Jour.," Feb. 22, 1905, and "Indian Med. Gazette,"
1905.

Cultivation 767

lus upon media free from sodium chlorid. The technic of his
method is thus described by Rudolph*: "Small lumps of
pumice stone are washed and then dried in the sun, and then
allowed to absorb a mixture of i ounce of meat extract and
2 ounces of water. This pumice stone is then placed in
wide-mouthed bottles and placed in the autoclave. Each
bottle is provided with a stopper through which pass two
tubes, the one tube opening into the autoclave and reaching
nearly to the bottom of the bottle, and the other leading from
the top of the bottle into a condenser adjoining. When the
cover of the autoclave is adjusted and the steam admitted,
then in the case of each bottle, the steam passes by the one
tube to the bottom of the bottle, and rising through the
pieces of pumice stone, the steam, carrying with it the volatile
constituents of the meat-extract, reaches the condenser by
the second tube. The vapor in the condenser yields the
salt-free nutrient medium in the proportion of 2 liters to
each ounce of meat-extract originally used. The medium is
collected from the condenser in sterilized Pasteur flasks which
are kept plunged during the process in a freezing mixture
in order to condense some of the volatile alkaloids from the
beef that would otherwise escape. The nutrient fluid is
now inoculated with the bacillus of leprosy and the flasks
kept at 37° C. for from four to six weeks; at the end of this
period when examined the flasks should present a turbid
appearance with a stringy white deposit."

Cleggt announced the cultivation of lepra bacilli from
human leprous tissue in symbiosis with ameba and other
bacteria. The organisms thus cultured he kept alive in
subcultures. The method devised by Clegg was the starting-
point of a more extended research by Duval, J who, after con-
firming the work of Clegg, found that the bacillus could be
cultivated directly from human lesions upon culture-media
containing tryptophan, without the symbiotic ameba or other
bacteria. The initial culture was somewhat difficult to
secure, but once the bacilli grewT, transplantation was easily
and successfully carried on for indefinite generations. He
further found that the lepra bacillus could be successfully
started to grow upon the ordinary laboratory media if bits
of leprous tissue were placed upon them, and at the same time

* "Medicine," March, 1905, p. 175.

f "Philippine Journal of Science," 1909, iv, 403.

J "Journal of Experimental Medicine," 1910, xn, 649; 1911, xm, 365.

768 Leprosy

some symbiotic organism, such as the colon, typhoid, proteus,
or other bacilli, added. Or if the tissue were already con-
taminated the lepra bacilli proceeded to multiply. Duval
interprets this to mean that the lepra bacillus is unable to
effect the destruction of the albumin molecule alone, and hence
explains the advantage of adding tryptophan. The medium
most successfully employed by Duval is as follows :

" Egg- albumen or numan blood-serum is poured into
sterile Petri dishes and inspissated for three hours at 70° C.
The excised leprous nodule is then cut into thin slices, 2 to 4
mm. in breadth and 0.5 to i mm. in thickness, which are
distributed over the surface of the coagulated albumin.
By means of a pipet the medium thus seeded with bits of
tissue is bathed in a i per cent, sterile solution of trypsin, care
being taken not to submerge the pieces of leprous tissue.
Sufficient fluid is added to moisten thoroughly the surface of
the medium. The Petri dishes are now placed in a moist
chamber at 37° C., and allowed to incubate for a week or ten
days. They are removed from the plates from time to time,
as evaporation necessitates, for the addition of more trypsin.
It will be noted that after a week or ten days the tissue bits
are partially sunken below the surface of the medium and are
softened to a thick, creamy consistence, fragments of which
are readily removed with a platinum needle. On microscopic
examination of this material it is noted that the leprosy bacilli
have increased to enormous numbers and scarcely a trace of
the tissue remains. Separate lepra bacillus colonies are also
discernible on and around the softened tissue masses. . . .
The colonies are at first grayish white, but after several days
they assume a distinct orange-yellow tint. . . . Subcultures
may be obtained by transferring portions of the growth to a
second series of plates or to slanted culture- tubes that contain
the special albumin- trypsin medium. After the third or
fourth generation the bacilli may be grown without difficulty
upon glycerinated serum agar prepared in the following
manner :

' Twenty grams of agar, 3 gm. of sodium chlorid, 30 c.c.
of glycerin, and 500 c.c. of distilled water are thoroughly
mixed, clarified, and sterilized in the usual way. To tubes
containing 10 c.c. of this material is added in proper propor-
tion a solution of unheated turtle muscle infusion. Five
hundred grams of turtle muscle are cut into fine pieces and
placed in a flask with 500 c.c. of distilled water. This is

Cultivation 769

kept in the ice-chest for forty-eight hours and then filtered
through gauze to remove the tissue. The filtrate is then
passed through a Berkefeld filter for purposes of sterilization.
By means of a sterile pipet, 5 c.c. of the muscle filtrate is
added to the agar mixture which has been melted and cooled
to 42° C. The tubes are now thoroughly agitated and
allowed to solidify in the slanted position.

" This medium is perfectly clear or of a light amber color,
and admirably suited to the cultivation of the Bacillus lepra,
once the initial culture has been started. Growth is luxuriant
and reaches its maximum in forty -eight to sixty hours. On
the surface of this medium the growth is moist and orange-
yellow in color, while in the water of condensation, though
growth apparently has not occurred, the detached bacilli col-
lect in the dependent parts in the form of feathery masses
without clouding the fluid.

" Ordinary nutrient agar may be used with trypsin as a
plating medium instead of the inspissated serum where bits
of tissue are employed. With the addition of i per cent, of
tryptophan it answers every purpose, whether the bacilli are
planted with tissue or alone. It also serves to start multipli-
cation of lepra bacilli that are contaminated at the time of
plating. In the latter case the medium is ' surface seeded '
with an emulsion of the tissue juices in the same manner as
in preparing ' streak ' plates. The leprosy colonies in the
thinner parts of the loop track are well separated and easily
distinguished from those of other species by their color and
by their appearance only after two to five days.

" In using an agar medium it is well to leave out the pep-
tone and to titrate the reaction to 1.5 per cent, alkaline in
order to prevent too profuse growth of the associated bacteria ;
besides, an alkaline medium seems best adapted for the mul-
tiplication of the lepra bacillus.

" Bacillus leprse will also grow on the various blood-agar
media once they are accustomed to artificial conditions. The
Novy-McNeal agar for the cultivation of trypanosomes gives
a luxuriant growth of the organism if 2 per cent, glycerin has
been added; without the glycerin, growth is very scant.
Fluid media are not suited for the artificial cultivation of
leprosy bacilli unless they are kept upon the surface. Like
the tubercle bacilli they require abundant oxygen. . . .

" Ordinarily the growth of Bacillus leprae is very moist, and
in this respect unlike that of Bacillus tuberculosis, except
49

770 Leprosy

possibly the avian stain. Sometimes when the medium is
devoid of water of condensation, the growth is dry and oc-
casionally wrinkled, though it is easily removed from the
surface of the medium.

"The chromogenic property of lepra cultures is a constant
and characteristic feature of the rapidly growing strains.
The color varies in the degree of intensity depending upon the
medium employed. If glycerinated agar (without peptone) is
used, the colonies are faint lemon, while on inspissated blood-
serum they are deep orange. It is noteworthy that the
growth in the tissues and in the first dozen or so generations
on artificial media is entirely without pigment."

Pathogenesis. — Melcher and Ortmann* introduced frag-
ments of lepra nodules into the anterior chambers of the
eyes of rabbits, and observed the death of the animals after
some months, with what they considered to be typical lep-
rous lesions of all the viscera, especially the cecum ; but the
recent careful experiments of Tashiroj show that most of the
lower animals are entirely insusceptible to infection with the
lepra bacillus, and that when they are inoculated the bacilli
persistently diminish in numbers and finally disappear.

Nicolle | found it possible to infect monkeys with material
rich in lepra bacilli taken from human beings. The lesions
appeared only after an incubation period that was in some
cases prolonged from twenty-two to ninety-four days. The
lesions persisted but a short time and the monkeys recovered
in from thirty to one hundred and fifty days.

Clegg§ and Sugai|| found Japanese dancing mice suscep-
tible to infection with leprous material, the micro-organisms
not remaining localized at the seat of inoculation, but dissem-
inating throughout the animal's body. Their observation
has been confirmed by Duval,** who later ff was also able to
infect monkeys — Macacus rhesus — with pure cultures of the
organism and produce the typical disease.

Very few instances are recorded in which actual inocula-

* "Berliner klin. Wochenschrift," 1885-1886.

f "Centralbl. f. Bakt. u. Parasitenk." Originale), xxxi, No. 7, p.
276, March 12, 1902.

f "Semaine medicale," 1905, No. 10, p. no.

§ "Philippine Journal of Science," 1909, iv, 403.

|| "Lepra," 1909, vin, 203.

** "Journal of Experimental Medicine," 1910, xn, 649.
tt Ibid., 1911, xiu, 374.

Lesions 771

tion has produced leprosy in man. Arning* was able to
experiment upon a condemned criminal, of a family entirely
free from the disease, in the Sandwich Islands. Fragments
of tissue freshly excised from a lepra nodule were introduced
beneath his skin and the man was kept under observation.
In the course of some months typical lesions began to
develop at the points of inoculation and spread gradually,
ending in general leprosy in about five years.

Sticker | is of the opinion that the primary infection in
lepra takes place through the nose, supporting his opinion
by observations upon 153 accurately studied cases, in which —

1. The nasal lesion is the only one constant in both the
nodular and anesthetic forms of the disease.

2. The nasal lesion is peculiar — i. e., characteristic — and
entirely different from all other lepra lesions.

3. The clinical symptoms of lepra begin in the nose.

4. The relapses in the disease always begin with nasal
symptoms, such as epistaxis, congestion of the nasal mucous
membrane, a sensation of heat, etc.

5. In incipient cases the lepra bacilli are first found in
the nose.

Lesions. — The lepra nodes in general resemble tubercu-
lous lesions, but are superficial, affecting the skin and sub-
cutaneous tissues. Rarely they may also occur in the
organs. VirchowJ has seen a case in which lepra bacilli
could be found only in the spleen.

Once established in the body, the bacillus may grow in
the connective tissues and produce chronic inflammatory
nodes — the analogues of tubercles; or in the nerves, causing
anesthesia and trophic disturbances. On this account two
forms of the disease — lepra nodosa (elephantiasis graecorum)
and lepra an&sthetica — are described. These forms may
occur independently of one another, or may be associated
in the same case.

The nodes consist of lymphoid and epithelioid cells and
fibers, and are vascular, so that much of the embryonal
tissue completes its transformation to fibers without necrotic
changes. This makes the disease productive rather than
destructive, the lesions resembling new growths. The

* "Centralbl. f. Bakt.," etc., vi, p. 201, 1889.

t "Mittheilungen und Verhandlungen der internationalen wissen-
schaftlichen Lepra-Konferenz zu Berlin," Oct., 1897, 2, Theil.
J Ibid.

772

Leprosy

bacilli, which occur in enormous numbers, are often found
in groups inclosed within the protoplasm of certain large
vacuolated cells — the " lepra cells " — which seem to be
partly degenerated endothelial cells. Sometimes they are
anuclear; rarely they contain several nuclei (giant cells).
Bacilli also occur in the lymph-spaces and in the nerve-sheath.

;

Fig. 251. — Lepra nervorum (McConnell).

Lepra nodules do not degenerate like tubercles, and the
ulceration, which constitutes a large part of the pathology
of the disease, seems to be largely due to the injurious action
of external agencies upon the feebly vital pathologic tissue.

According to the studies of Johnston and Jamieson,* the
bacteriologic diagnosis of nodular leprosy can be made by
spreading serum obtained by scraping a leprous nodule upon
a cover-glass, drying, fixing, and staining with carbol-
fuchsin and Gabbet's solution as for the tubercle bacillus.

Montreal Med. Journal." Jan., 1897.

Varieties

773

In such preparations the bacilli are present in enormous
numbers, thus forming a marked contrast to tuberculous
skin diseases, in which, very few can be found.

In anesthetic leprosy nodules form upon the peripheral
nerves, and by connective-tissue formation, as well as by
the entrance of the bacilli into the nerve-sheaths, cause
irritation, followed by degeneration of the nerves. The

Fig. 252. — A case of lepra nodosa treated in the Medico- Chirurgical
Hospital of Philadelphia.

anesthesia following the peripheral nervous lesions pre-
disposes to the formation of ulcers, etc., by allowing injuries
to occur without detection and to progress without observa-
tion. The ulcerations of the hands and feet, with frequent
loss of fingers and toes, follow these lesions, probably in the
same manner as in syringomyelia.

The disease usually first manifests itself upon the face,
extensor surfaces, elbows, and knees, and for a long time
confines itself to the skin. Ultimately it sometimes invades

774 Leprosy

the lymphatics and extends to the internal viscera. Death
ultimately occurs from exhaustion, if not from the frequent
intercurrent affections, especially pneumonia and tuberculo-
sis, to which the conditions predispose.

Specific Therapy. — Carrasquilla's* "leprosy serum" is
prepared by injecting the serum separated from blood with-
drawn from lepers, into horses, mules, and asses, and, after
a number of injections, bleeding the animals and sepa-
rating the serum. There is no reason for thinking that such
a product could have therapeutic value.

Rostf prepares massive cultures of the lepra bacillus,
niters them through porcelain, concentrates the nitrate to
one- tenth of its volume, and mixes the nitrate with an equal
volume of glycerin. The resulting preparation is called
leprolin and is supposed to be analogous to tuberculin.
With it he has treated a number of lepers at the Leper
Hospital at Rangoon, Burmah, many of whom have greatly
improved and some of whom seem to be cured. Confirma-
tion of the work by others is greatly desired, and it is too
early to judge the merits of the treatment. It is, however,
the most promising method yet published.

Sanitation. — While not so contagious as tuberculosis,
it has been proved that leprosy is transmissible, and it may
be regarded as an essential sanitary precaution that lepers
should be segregated and mingle as little as possible with
healthy persons. The disease is not hereditary, so that
there is no reason why lepers should not marry among
themselves. The children should, however, be taken from
the parents lest they be subsequently infected.

*" Wiener med. Wochenschrift," No. 41, 1897.
f'Brit. Med. Jour.," Feb. n, 1905.

CHAPTER XXIX.

GLANDERS.
BACILLUS MALLEI (LOFFLER AND SCHUTZ).*

General Characteristics. — A non-motile, non-flagellate, non-spor-
ogenous, non-liquefying, non-chromogenic, non-aerogenic, aerobic and
optionally anaerobic, acid-forming and milk coagulating bacillus, patho-
genic for man and the lower animals, staining by ordinary methods,
but not by Gram's method.

Glanders is an infectious mycotic disease which, for-
tunately, is almost entirely confined to the lower animals.
Only occasionally does it secure a victim among hostlers,
drovers, soldiers, and others whose vocations bring them in
contact with diseased horses. Several bacteriologists have
succumbed to accidental laboratory infection.

Glanders was first known to us as a disease of the horse
and ass, characterized by the formation of discrete, cleanly
cut ulcers upon the mucous membrane of the nose. The
ulcers in the nose of the horse and ass are formed by the
breaking down of inflammatory nodules which can be detected
in all stages upon the diseased membranes. The ulcers,
having once formed, show no tendency to recover, but
slowly spread and persistently discharge a virulent pus.
The edges of the ulcers are indurated and elevated, their
surfaces often smooth. The disease does not progress to
any great extent before the submaxillary lymphatic glands
begin to enlarge, soften, open, and become discharging
ulcers. The lungs may also become infected by inspiration
of the infectious material from the nose and throat, and
contain small foci of bronchopneumonia not unlike tubercles
in their early appearance. The animals ultimately die of
exhaustion.

Specific Organism. — In 1882, shortly after the dis-
covery of the tubercle bacillus, Loffler and Schiitz discovered
in the discharges and tissues of the disease the specific
micro-organism, the glanders bacillus (Bacillus mallei).

*" Deutsche med. Wochenschrift," 1882, 52.
775

776 Glanders

Distribution. — The glanders bacillus does not seem to
find conditions outside the animal body suitable for its
growth, and probably lives a purely parasitic existence.

Morphology. — The glanders bacillus is somewhat shorter
and distinctly thicker than the tubercle bacillus, and has
rounded ends. It measures about 0.25 to 0.4 X 1.5 to 3 [*,
and is slightly bent; coccoid and branched forms sometimes
occur. It usually occurs singly, though upon blood-serum,
and especially upon potato, conjoined individuals may occa-
sionally be found. Long threads are never formed.

Fig- 253. — Bacillus mallei, from a culture upon glycerin agar-agar.
X looo (Frankel and Pfeiffer).

When stained with ordinary aqueous solutions of the
aniline dyes, or with Loffler's alkaline methylene-blue, the
bacillary substance does not usually appear homogeneous,
but, like that of the diphtheria bacillus, shows marked in-
equalities, some area being deeply, some faintly, stained.

The bacillus is non-motile, has no flagella, and does not
form spores.

Staining. — The organism can be stained with the watery
anilin-dye solutions, but not by Gram's method. The bacil-
lus readily gives up the stain in the presence of decolorizing
agents, so is difficult to stain in tissues. Loffler accomplished
the staining by allowing the sections to lie for some time
(five minutes) in the alkaline methylene-blue solution, then

Isolation 777

transferring them to a solution of sulphuric and oxalic
acids —

Concentrated sulphuric acid 2 drops

Five per cent, oxalic acid solution i drop

Distilled water 10 c.c.

for five seconds, then to absolute alcohol, xylol, etc. The
bacilli appear dark blue upon a paler ground. This method
gives very good results, but has been largely superseded by
the use of Kiihne's carbol-methylene-blue :

Methylene-blue i .5

Alcohol 10.0

Five per cent, aqueous phenol solution 100.0

Kiihne stains the section for about half an hour, washes it
in water, decolorizes it carefully in hydrochloric acid (10
drops to 500 c.c. of water), immerses it at once in a solution
of lithium carbonate (8 drops of a saturated solution of lithium
carbonate in 10 c.c. of water), places it in a bath of distilled
water for a few minutes, dips it into absolute alcohol col-
ored with a little methylene-blue, dehydrates it in anilin
oil containing a little methylene-blue in solution, washes
it in pure anilin oil, not colored, then in a light ethereal oil,
clears it in xylol, and finally mounts it in balsam.

Vital Resistance. — The organism grows only between
25° and 42° C. It is killed by exposure to 60° C. for two
hours, or to 75° C. for one hour. Sunlight kills it after
twenty-four hours' exposure. Thorough drying destroys it in
a short time. When planted upon culture-media, sealed, and
kept cool and in the dark, it may be kept alive for months
and even years. Exposure to i per cent, carbolic acid de-
stroys it in about half an hour; i : 1000 bichlorid of mercury
solution, in about fifteen minutes. According to Hiss and
Zinsser, it may remain alive in the water of horse-troughs
for seventy days.

Isolation. — Attempts at the isolation of the glanders
bacillus from infectious discharges by the usual plate method
are apt to fail, on account of the presence of other more
rapidly growing organisms.

The best method of isolation seems to be by infecting an
animal and recovering the bacillus from its tissues.

The guinea-pig, being a highly susceptible as well as a
readily procurable animal, is appropriate for the detection

778 Glanders

and isolation of the bacillus. When a subcutaneous inocu-
lation of some of the infectious pus is made, a tumefaction
can be observed in guinea-pigs in from four to five days.
Somewhat later this tumefaction changes to a caseous
nodule, which ruptures and leaves a chronic superficial ulcer
with irregular margins. The lymph-glands speedily become
invaded, and in four or five weeks signs of general infec-
tion appear. The lymph-glands, especially of the inguinal
region, suppurate, and the testicles frequently undergo the
same process. Later the joints are affected with a suppura-
tive arthritis, the pus from which contains the bacilli. The
animal eventually dies of exhaustion. No nasal ulcers
form in guinea-pigs.

In field-mice the disease is much more rapid, no local
lesions being visible. For two or three days the animal
seems unwell, its breathing is hurried, it sits with closed
eyes in a corner of the cage, and finally, without any other
preliminaries, tumbles over on its side, dead.

From the tissues of the inoculated animals pure cultures
are easily made. Perhaps the best places from which to
secure a culture are the softened nodes which have not
ruptured, or the joints.

Diagnosis of Glanders. — Straus* has given us a method
which is of great use, both for isolating pure cultures of the
glanders bacillus and for making a diagnosis of the disease.
But a short time is required. The material suspected to
contain the glanders bacillus is injected into the peritoneal
cavity of a male guinea-pig. In three or four days the
disease becomes established and the testicles enlarge; the
skin over them becomes red and shining; the testicles
themselves begin to suppurate, and often evacuate through
the skin. The animal dies in about two weeks. If, how-
ever, it be killed and its testicles examined, the tunica
vaginalis testis will be found to contain pus, and some-
times to be partially obliterated by inflammatory exudation.
The bacilli are present in this pus, and can be secured from it
in pure cultures.

The value of Straus' method has been somewhat lessened
by the discovery by Kutcher,f that a new bacillus, which
he has classed among the pseudo- tubercle bacilli, produces
a similar testicular swelling when injected into the abdominal

* "Compt. rendu Acad. d. Sciences," Paris, cvm, 530.
t "Zeitschrift fur Hygiene," Bd. xxi, Heft i, Dec. 6, 1895.

Cultivation 779

cavity; also by Levy and Steinmetz,* who found that
Staphylococcus pyogenes aureus was also capable of provok-
ing suppurative orchitis. However, the diagnosis is certain
if a culture of the glanders bacillus be secured from the
pus in the scrotum.

As the purulent discharges from the noses of horses and
other large animals commonly contain very few bacilli,
their detection by the use of the guinea-pig inoculation is
much simplified.

For the diagnosis of the disease in living animals, sub-
cutaneous injections of mallein (q. v.) are also employed.

McFadyenj was the first to recommend agglutination of
the glanders bacillus by the serum of supposedly infected
animals as a test of the existence of glanders. The subject
has been somewhat extensively tried and officially adopted
by the Prussian government. Moore and Taylor, J in a recent
review and examination of the test, conclude that it is easier
and quite as accurate as the mallein method and is applicable
in cases where fever exists. The maximum dilution of
normal horse-serum that will macroscopically agglutinate
glanders bacilli is i : 500, but occurs in very few cases. The
maximum agglutinative power of the serum of diseased horses
not suffering from glanders is not higher than that of normal
serum. The diagnosis is usually not difficult to make, but
requires much care. Cultures of the glanders bacillus some-
times unexpectedly lose their ability to agglutinate.

The diagnosis of glanders by means of the complement-
fixation method has been tried with glittering results by
Mohler and Eichhorn.§

Cultivation. — The bacillus is an aerobic and optionally
anaerobic organism, and can be grown in bouillon, upon
agar-agar, better upon glycerin agar-agar, very well upon
blood-serum, and quite characteristically upon potato. The
optimum temperature is 37.5° C.

Colonies. — Upon 4 per cent, glycerin agar-agar plates
the colonies appear upon the second day as whitish or pale
yellow, shining, round dots. Under the microscope they
are brownish yellow, thick and granular, with sharp borders.

Bouillon. — In broth cultures the glanders bacillus causes

* "Berliner klin. Wochenschrift," March 18, 1895, No. n.
t "Jour. Comp. Path, and Therap.," 1896, p. 322.
J "Jour. Infectious Diseases," iv, 1907, p. 85, supplement.
§ "Report of the Bureau of Animal Industry," 1910.

780

Glanders

turbidity, the surface of the culture being covered by a
slimy scum. The medium becomes brown in color.

Gelatin is not liquefied. The growth upon the surface is
grayish white and slimy, never abundant.

Agar-agar. — Upon agar-agar and glycerin agar-agar the
growth occurs as a moist shining layer.

Blood-serum. — Upon blood-serum the growth is rather
characteristic, the colonies along the line of inoculation
appearing as circumscribed, clear, transparent drops, which
later become confluent and form a transparent layer un-
accompanied by liquefaction.

Fig. 254. — Culture of glanders bacillus upon cooked potato (Loffler).

Potato. — The most characteristic growth is upon potato.
It first appears in about forty-eight hours as a transparent,
honey-like, yellowish layer, developing only at incubation
temperatures, and soon becoming reddish-brown in color.
As this brown color of the colony develops, the potato for a
considerable distance around it becomes greenish brown.
Bacillus pyocyaneus sometimes produces somewhat the same
appearance.

Milk. — In litmus milk the glanders bacillus produces acid.
A firm coagulum forms and subsequently separates from
the clear reddish whey.

Metabolic Products. — The organism produces acids and
curdling ferments. It forms no indol, no liquefying or pro-

Pathogenesis 781

teolytic ferments. There is no exotoxin. All the poisonous
substances seem to be endotoxins.

Mallein. — Babes,* Bonome,f Pearson, J and others have
prepared a substance, mallein, from cultures of the glanders
bacillus, and have employed it for diagnostic purposes. It
seems to be useful in veterinary medicine, the reaction fol-
lowing its injection into glandered animals being similar to
that caused by the injection of tuberculin into tuberculosis
animals. The preparation of mallein is simple. Cultures of
the glanders bacillus are grown in glycerin bouillon for sev-
eral weeks and killed by heat. The culture is then filtered
through porcelain, to remove the dead bacteria, and evapo-
rated to one-tenth of its volume. Before use the mallein is
diluted with nine times its volume of 0.5 per cent, aqueous
carbolic acid solution. The dose for diagnostic purposes is
0.25 c.c. for the horse. It has also been prepared from
potato cultures, which are said to yield a stronger product.
The agent is employed exactly like tuberculin, the tem-
perature being taken before and after its hypodermic injec-
tion. A febrile reaction of more than 1.5° C. is said to be
indicative of the disease.

Pathogenesis. — That the bacillus is the cause of glanders
there is no room to doubt, as LofHer and Schutz have suc-
ceeded, by the inocultion of horses and asses, in producing
the well-known disease.

The goat, cat, hog, field-mouse, wood-mouse, marmot,
rabbit, guinea-pig, and hedgehog all appear to be susceptible.
Cattle, house-mice, white mice, rats, and birds are immune.

Infection may take place through the mucous membranes
of the nose, mouth, or alimentary tract, and apparently
without pre-existing demonstrable lesions.

The disease assumes either an acute form, characterized by
destructive necrosis and ulceration of the mucous membranes
with fever and prostration, terminating in pneumonia, or,
as is more frequent, a chronic form (" farcy "), in which the
lesions of the mucous membranes are less destructive and in
which there is a generalized distribution of the micro-organ-
isms throughout the body, with resulting more or less wide-
spread nodular formations (farcy-buds) in the skin. The

* "Archiv. de Med. exp. et d'Anat. patholog.," 1892, No. 4.
t "Deutsche med Woch.," 1894, Nos. 36 and 38, pp. 703, 725, and 744.
t "Jour, of Comp. Med. and Vet. Archiv.," Phila., xn, 1891, pp.
411-415.

782

Glanders

acute form is quickly fatal, death sometimes coming on in
from four to six weeks ; the chronic form may last for several
years and end in complete recovery.

Lesions. — When stained in sections of tissue the bacilli
are found in small inflammatory areas. These nodules can
be seen with the naked eye scattered through the liver,
kidney, and spleen of animals dead of experimental glanders.
They consist principally of leukocytes, but also contain
numerous epithelioid cells. As is the case with tubercles,

Fig- 255. — Pustular eruption of acute glanders as exhibited on the day
of the patient's death, twenty-eight days after initial chill (Zeit).

the centers of the nodules are prone to necrotic changes, but
the cells show marked karyorrhexis, and the tendency is more
toward colliquation than caseation. The typical ulcera-
tions depend upon retrogressive changes occurring upon
mucous surfaces, the breaking down of the nodules per-
mitting the softened material to escape. At times the
lesions heal with the formation of stellate scars.

Baumgarten* regarded the histologic lesions of glanders
as much like those of the tubercle. He first saw epithe-
* " Pathologische Mykologie," Braunschweig, 1890.

Pathogenesis 783

lioid cells accumulate, followed by the invasion of leuko-
cytes. Tedeschi* was not able to confirm Baumgarten's
work, but found the primary change to be necrosis of the
affected tissue followed by invasion of leukocytes. The
observations of Wright | are in accord with those of Tedeschi.
He first saw a marked degeneration of the tissue, and then
an inflammatory exudation, amounting in some cases to
actual suppuration.

Glanders in Human Beings. — Human beings are but
rarely infected. The disease has, however, occurred among

Fig. 256. — Farcy affecting the skin of the shoulder (Mohler and
Eichhorn, in Twenty-seventh Annual Report of the Bureau of Animal
Industry, U. S. Department of Agriculture, 1910).

those in frequent contact with many horses and among
bacteriologists. It occurs either in an acute form in which,
from whatever primary focus may have been its starting-
point, the distribution of micro-organisms may be so rapid
as to induce an affection with skin lesions resembling small-
pox and terminating fatally in eight or ten days.

The chronic form in man is chiefly confined to the nasal
and laryngeal mucosa. It is commonly mistaken for more
simple infections, and though it sometimes shows its character
by generalizing, it not infrequently recovers.

* "Ziegler's Beitrage z. path. Anat.," Bd. xm, 1893.

t "Journal of Experimental Medicine," vol. I, No. 4, p. 577.

784

Glanders

Virulence. — The organism is said to lose virulence if
cultivated for many generations upon artificial media.
While this is true, attempts to attenuate fresh cultures by
heat, etc., have usually failed.

Immunity. — Leo has pointed out that white rats, which
are immune to the disease, may be made susceptible by
feeding with phloridzin and causing glycosuria.

Babes has asserted that the injection of mallein into sus-
ceptible animals will immunize them against glanders.
Some observers claim to have seen good therapeutic results

I

Fig. 257. — Lesions of glanders in the nasal septum of a horse (Mohler
and Eichhorn, in Twenty-seventh Annual Report of tthe Bureau of
Animal Industry, U. S. Department of Agriculture, 1910).

follow the repeated injection of mallein in small doses.
Others, as Chenot and Picq,* find blood-serum from im-
mune animals like the ox to be curative when injected
into guinea-pigs infected with glanders.

Pseudoglanders Bacillus. — A bacillus similar in its tinc-
torial and cultural peculiarities, but not pathogenic for mice,
guinea-pigs, or rabbits, was isolated from pus by Selter. f The
organism was called the pseudoglanders bacillus. A similar
one had previously been described by JJabes.f

* " Compte-rendu de la Soc. de Biol.," March 26, 1892.

t "Centralbl. f. Bakt.," etc., Feb. 18, 1902, xxxv, 5, p. 529.

I "Archiv. de med. exp. et d'anat. path.," 1891.

CHAPTER XXX.
RHINOSCLEROMA.

BACILLUS RHINOSCLEROMATIS (VON FRISCH *) .

General Characteristics. — A non-motile, non-flagellate, non-
sporogenous, non-chromogenic, aerobic and optionally anaerobic, cap-
sulated bacillus, pathogenic for man and identical with Bacillus pneu-
moniae of Friedlander, except that it stains by Gram's method.

A peculiar disease of the nares, characterized by the
formation of circumscribed nodular tumors, and known
as rhinoscleroma, is occasionally seen in Austria-Hungary,
Italy, and some parts of Germany. A few cases have been
observed among the foreign-born residents of the United
States. The nodular masses are flattened, may be discrete,
isolated, or coalescent, grow with great slowness, and recur
if excised. The disease commences in the mucous mem-
brane and the adjoining skin of the nose, and spreads to the
skin in the immediate neighborhood by a slow invasion,
involving the upper lip, jaw, hard palate, and sometimes
even the pharynx. The growths are without evidences of
acute inflammation, do not ulcerate, and upon microscopic
examination consist of an infiltration of the papillary layer
and corium of the skin, with round cells which in part change
to fibrillar tissue. The tumors possess a well-developed
lymph-vascular system. Sometimes the cells undergo hya-
line degeneration.

In the nodes von Frisch discovered bacilli closely re-
sembling the pneumobacillus of Friedlander, both in mor-
phology and vegetation, and, like it, surrounded by a
capsule. The only differences between the bacillus of
rhinoscleroma and Bacillus pneumoniae of Friedlander are
that the former stains well by Gram's method, while the
latter does not; that the former is rather more distinctly
rod-shaped than the latter, and more often shows its capsule
in culture media.

The bacillus can be cultivated, and cultures in all media

* "Wiener med. Wochenschrift," 1882, 32.
50 785

786 Rhinoscleroma

resemble those of the bacillus of Friedlander (q. v.) so closely
as to be indistinguishable from it. Even when inoculated
into animals the bacillus behaves much like Friedlander 's
bacillus.

Inoculation has, so far, failed to reproduce the disease
either in man or in the lower animals.

,%•!

Fig. 258. — Bacillus rhinoscleromatis. Pure culture on glycerin agar-
agar. Magnified 1000 diameters (Migula).

Pathogenesis. — The bacillus is said to be pathogenic for
man only, producing granulomatous formations of the skin
and mucous membranes of the anterior and posterior nares.
These vary in structure according to age. The young nodes
consist of a loose fibrillar tissue composed of lymphocytes,
fibroblasts, and fibers. Some of the cells are large and have a
clear cytoplasm and are known as the cells of Mikulicz. In
and between them the bacilli are found in considerable num-
bers. The older lesions consist of a firm sclerotic cicatricial
tissue.

CHAPTER XXXI.

SYPHILIS.

ALTHOUGH syphilis has been well known for centuries, its
specific cause has only recently been discovered. The fact
that the disease had not been successfully communicated
to any of the lower animals was supposed to be a sufficient
explanation of the delay in recognizing it. Such has not,
however, proved to be the case, for in spite of the discovery
by Metschnikoff and Roux* that chimpanzees could be suc-
cessfully inoculated with virus from a human lesion, and the
confirmation of their work by Lassarf and others, and the
additional discovery of Metschnikoff and Roux, | that it is
also possible to infect macaques with syphilis, the specific
organism was, after all, discovered for the first time in
matter secured from human lesions.

TREPONEMA (SPIROCH^TA) PALUDUM (SCHAUDINN AND
HOFFMANN).

General Characteristics.— A minute, slender, spiral organism,
closely coiled, flexible, non-chromogenic, non-aerogenic, anaerobic, non-
liquefying, motile, flagellated, cultivable upon specially prepared media,
pathogenic for man and certain of the lower animals, staining by cer-
tain methods only and not by Gram's method. •

It has been known for a long time that preputial smegma
and various ulcerative lesions of the generative organs con-
tain certain spiral organisms. Bordet studied these with
some care, expecting to prove that they were concerned
with the etiology of syphilis, but it remained for Schaudinn
and Hoffmann § to point out that there were two separate
species — one, which they call Spirochaeta refringens, com-
monly found in ulcerative lesions of the genitalia, and
another, called Spirochaeta pallida, later, and more correctly,
Treponema pallidum, found only in syphilitic lesions — and,

* "Ann, de 1'Inst. Pasteur," Dec., 1903, p. 809.
t "Berliner klin. Wochenschrift," 1903, p. 1189.
J "Annales de 1'Inst. Pasteur," Jan., 1904.
§ "Deutsche med. Wochenschrift," May 4, 1905.
787

788 Syphilis

therefore, their probable cause. The observations of Schau-
dinn and Hoffmann, quickly confirmed by Metschnikoff,*
have now been universally accepted.

Morphology. — The organism is a slender, flexible, closely
coiled spiral, usually showing from eight to ten uniform un-
dulations, but occasionally being so short as to show only
two or three, or so long as to show as many as twenty.

It is very slender, measuring from 0.33 to 0.5 [A in breadth
to 3.5 to 15.5 11 in length (Levaditi and Mclntosh).

It forms no spores. Multiplication seems to take place by
longitudinal division.

It is motile, and when observed alive with a dark field
illuminator, can be seen to rotate slowly about its longitu-
dinal axis at the same time that it slowly sways from side
to side with a serpentine movement. The organisms are
provided with flagella at one end, sometimes one at each
end.

Noguchif observed two types of treponema, one slender,
one stouter. When carried through culture and used to in-
oculate rabbits their differences were found to be fairly con-
stant. The lesions produced in rabbit's testicles varied with
the variety of organism inoculated, one causing a diffuse, the
other a nodular, orchitis. He conjectures that the distinction
may be of value in explaining certain obscure points in human
syphilis.

Staining. — The original discovery of the organism was
achieved through the employment of Giemsa's stain — a
modification of the Romanowsky method. But by this
method the organisms appeared very pale and not very
numerous. GoldhornJ improved the method as follows:

In 200 c.c. of water, 2 grams of lithium carbonate are dissolved and
2 grams of Merck's medicinal, Griibler's BX, or Koch's rectified methy-
lene-blue added. This mixture is heated moderately in a rice boiler
until a rich polychrome has formed. To determine this a sample is
examined in a test-tube every few minutes by holding it against an
artificial light. As soon as a distinctly red color is obtained, the desired
degree of heating has been reached. After cooling it is filtered through
cotton in a funnel. To one-half of this polychrome solution 5 per cent,
of acetic acid is gradually added until a strip of litmus-paper shows
above the line of demarcation a distinct acid reaction, when the re-
maining half of the solution is added, so as to carry the reaction back
to a low degree of alkalinity. A weak eosin solutioa is now prepared,

* "Bull. Acad. de med. de Paris," May 16, 1905.

t "Journal of Experimental Medicine," 1912, xv, No. 2, p. 201.

{Ibid., 1906, vin, p. 451.

Staining 789

approximately 0.5 per cent. French eosin, and this is added gradually
while the mixture is being stirred until a filtered sample shows the filtrate
to be of a pale bluish color with a slight fluorescence. The mixture
is allowed to stand for one day and then filtered. The precipitate
which has separated is collected on a double piece of filter-paper and
dried at room temperature (heating spoils it). When completely
dried it can easily be removed from the paper and may then be dissolved
without further washing in commercial (not pure) wood alcohol. The
solution should be allowed to stand a day, then filtered. The strength
of this alcoholic solution is approximately i per cent. To use the stain,
one drops upon an unfixed spread enough dye to cover it, permits it
to act for three or four seconds, and then pours it off and introduces the
glass slowly, spread side down, into clean water, where it is held for
another four or five seconds, after which it is shaken to and fro in the
water to wash it. It is next dried and examined at once or after mount-
ing in balsam. The spirochaetes appear violet in color.

Ghoreyeb* recommends the following rapid method of
staining the organism in smears. A thin spread is to be
preferred. No heat fixation is necessary:

1. Cover the smear with a i per cent, aqueous solution of osmic acid,
and permit it to act for thirty seconds. This solution acts as a fixative
and mordant.

2. Wash thoroughly in running water.

3. Cover the smear with a i : 100 dilution of Liquor plumbi subace-
tatis (freshly prepared). Permit it to act for ten seconds. The lead
unites with the albumin to form lead albuminate which is insoluble in
water.

4. Wash thoroughly in running water.

5. Cover the smear with a 10 per cent, aqueous solution of sodium
sulphid. This is to act ten seconds, during which the salt transforms
the lead albuminate into lead sulphid and causes the preparation to turn
brown. The osmic acid when reapplied causes it to become black.

6. Wash thoroughly in running water.

The whole process is to be repeated in exactly the same man-
ner three times, the washings all being very thorough. The
preparation is then dried and mounted in Canada balsam.
The micro-organisms and cellular detritus are stained black.

When serum from a primary sore or other syphilitic lesion
is treated by these methods, a number of the spirochaeta ap-
pear well stained and a number very palely stained, so that
one is in doubt whether there may be many others un-
stained, and this seems to be the case, for when similar
smears are treated by other methods many more can be
found.

The method of silver incrustation was first employed for
the demonstration of the organism in tissues, but Sternt

" Jour. Amer. Med. Assoc.," May 7, 1910, liv., No. 19, p. 1498.
t "Berliner klin. Wochenschrift," 1907, No. 14.

790

Syphilis

has applied it with great success to the examination of
fluids by the following simple procedure: Spreads are made
in the usual manner, dried in the air, and then for a few
hours in an incubating oven at 37° C. They are next
placed in a 10 per cent, solution of nitrate of silver in a
colorless glass receptacle and allowed to rest in the diffused
daylight of a comfortably lighted room for a few hours, until
they become brownish metallic in appearance, when they
are thoroughly washed in water. The spirochaeta appear
black, the background brownish.

Fig. 259. — Treponema pallidum in the periosteum near an epiphysis

(Bertarelli).

Staining the organism in the tissues was found to be a more
difficult matter, for the Giemsa stain scarcely showed it at all.
Bertarelli and Volpino* endeavored to stain sections by a
modification of the van Ermengen method for flagella and
had some success, but the demonstration of the organisms
in tissue was not really successful until Levaditif devised
the method of silver impregnation. This consists in hard-
ening pieces of tissue about i mm. in thickness in 10 per
cent, formol for twenty-four hours, rinsing in water, and
immersing in 95 per cent, alcohol for twenty-four hours.

' "Centralbl. f. Bakt. u. Parasitenk.," Orig., 1905, XL, p. 56.
f "Compt.-rendu de la Soc. de Biol. de Paris," 1905, ux, p. 326.

Staining

791

The block is then placed in diluted water until it sinks to
the bottom of the container, and then transferred to a
1.5 to 3 per cent, aqueous solution of nitrate of silver in a blue
or amber bottle and kept in a dark incubating oven at 37° C.
for from three to five days. Finally, it is washed in water and
placed in a solution of pyrogallic acid 2 to 4 grams; formol,
5c.c. ; distilled water, 100 c.c., and kept in the dark, at room
temperature, from twenty-four to seventy-two hours, then

Fig. 260. — Treponema pallidum impregnated with silver. Film
prepared from the skin of a macerated, congenitally syphilitic fetus.
X 750 diameters (Flexner). The dense aggregation of organisms may
indicate agglutination.

washed in distilled water, embedded in paraffin, and cut.
The treponemata are intensely black, the tissue yellow brown.
The sections are finally stained with — (a) Giemsa's stain for
a few minutes, then washed in water, differentiated with
absolute alcohol containing a few drops of oil of cloves,
cleared with oil of bergamot or xylol, or (6) concentrated
solution of toluidin blue, differentiated in alcohol containing
a few drops of Unna's glycerin-ether mixture, cleared in oil
of bergamot, then in xylol, and mounted in Canada balsam.

792 Syphilis

This method was later improved by Levaditi and Mamou-
elian* by the addition of 10 per cent, of pyridin to the
silver bath just before the block of tissue is put in, and
by using for the reducing bath a mixture of pyrogallic
acid, acetone, and pyridin. The details are as follows:
Fragments of organs or tissues i to 2 mm. in thickness are
fixed for twenty-four to forty-eight hours in a solution of
formalin 10 : 100, then washed in 96 per cent, alcohol for
twelve to sixteen hours, then in distilled water until the
blocks fall to the bottom of the container. They are
then impregnated by immersion in a bath composed of
a i per cent, solution of nitrate of silver, to which, at the
moment of employment, 10 per cent, of pyridin is added.
Keep the blocks immersed in this solution at room temper-
ature for two or three hours, and at 50° C. for four or six
hours, then wash rapidly in a 10 per cent, solution of py-
ridin, and reduce in a bath composed of 4 per cent, pyro-
gallic acid, to which, at the moment of using, 10 per cent, of
pure acetone and 15 per cent, (total volume) of pyridin are
added. The reduction bath must be continued for several
hours, after which the tissue goes through 70 per cent,
alcohol, xylol, paraffin, and sections are cut. The sections,
fastened to the slide, are stained with Unna's blue or tolu-
idin blue, differentiated with glycerin-ether, and finally
mounted in Canada balsam.

Burrif has recommended a simple and rapid method of
demonstrating the treponema and other similar organisms by
the use of India ink. A drop of juice is squeezed from a
chancre or mucous patch and mixed with a drop of India ink
and then spread upon a glass slide as in making a spread of
a drop of blood. As the ink dries it leaves a black or dark
brown field upon which the spiral organisms stand out as
shining, colorless, and hence conspicuous objects. Williams
uses Higgins' water-proof ink, and Hiss recommends "chin-
chin," Giinther- Wagner liquid pearl ink for the purpose.

The method is fairly satisfactory for diagnosis and can be
applied in a few moments.

Distribution. — The Treponema pallidum is not known in
nature apart from the lesions of syphilis. It has now been
found in all the lesions of this disease and in the blood of
syphilitics in larger or smaller numbers. The discovery has

* "Compt.-rendu de la Soc. de Biol. de Paris," 1906. LVIII, p. 134.
f "Wiener klin. Wochenschrift," July i, 1909.

Cultivation 793

greatly modified our ideas of the tertiary stage, for the
demonstration of the organisms in its lesions shows them to
be undoubtedly contagious. The greatest number of the
organisms are found in the tissues — especially the liver — of
still-born infants with congenital syphilis.

Cultivation. — The cultivation of the treponema was first
attempted by Levaditi and Mclntosh,* who, deriving the
organism from an experimental primary lesion in a monkey
(Macacus rhesus), carried it through several generations
in collodion sacs inclosed in the peritoneal cavity of other
monkeys (Macacus cynomolgus) and in the peritoneal
cavity of rabbits. They were unable, however, to secure
the treponema in pure culture, having it continually mixed
with other organisms from the primary lesion. In the
mixture, however, they were able to cultivate it for genera-
tions and study its morphology and behavior. During
cultivation its virulence was lost.

Schereschewsky f endeavored to cultivate the treponema
by placing a fragment of human tissue containing it deep
down into a high layer of gelatinized horse -serum. The
treponema grew together with the contaminating organism,
and no pure culture was secured. Muhlens J and Hoffmann, §
using the same method, succeeded in securing pure cultures
of the treponema, but found them avirulent.

Noguchi,|| taking advantage of the observations of Bruck-
ner and Galasesco ** and Sowade, ft that an enormous multi-
plication of treponema occurred when material containing it
was inoculated into the rabbit's testis, performed a lengthy
series of cultivation experiments with the enriched material
thus obtained. The only suitable culture-medium in these
earlier experiments proved to be a " serum water," composed
of i part of the serum of the sheep, horse, or rabbit and 3 parts
of distilled water; 16 c.c. of this mixture was placed in test-
tubes 20 cm. long and 1.5 cm. in diameter and sterilized for
fifteen minutes at 100° C. each day for three days.

To each of a series of such tubes a carefully removed frag-

* "Ann. de 1'Inst. Pasteur," 1907, p. 784.

t" Deutsche med. Wochenschrift," 1909, xxxv, 835, 1260, 1652.

t Ibid., 1909, xxxv, 1261.

§ "Zeitschrift fur Hygiene und Infektionsk.," 1911, LXVIII, 27.

|| "Journal of Experimental Medicine," 1911, xiv, 99.
** "Compt.-rendu de la Soc. de Biol. de Paris," 1910, LXVIII, 648.
ft "Deutsche med. Wochenschrift," 1911, xxxvii, 682.

794 Syphilis

ment of sterile rabbit's testis was added, after which the tubes
were incubated at 37° C. for two days to determine their
sterility. To each tube the material from the inoculated
rabbit's testis, rich in the treponema, is added, after which the
surface of the medium in each receives a thick layer of sterile
paraffin oil. As the most strict anaerobiosis is necessary, the
tubes are placed in a Novy jar, the bottom of which contains
pyrogallic acid. Noguchi first passes H gas through the
jar, permitting it to bubble through the pyrogallic acid solu-
tion for ten minutes. He then uses a vacuum pump to ex-
haust the atmosphere in the jar, and lastly permits the alka-
line solution (KOH) to flow down one of the tubes to mix with
the pyrogallic acid.

In these cultures the pallidum grows together with such
bacteria as may have been simultaneously introduced. To
secure the cultures free from these bacteria Noguchi permitted
the treponema to grow through a Berkefeld filter, which for
a long time held back the other organisms. Later it was found
that both bacteria and treponema grow side by side in a deep
stab in a serum-agar-tissue medium. Under these conditions
the bacteria grow only in the stab or puncture, but the trepo-
nema grow out into the medium as a hazy cloud. By cau-
tiously breaking the tube and securing material for transplan-
tation from such a scarcely visible cloud, the organisms may
be transplanted from the new media and pure cultures thus
obtained.

In a later paper, Noguchi* details the cultivation of the
treponema from fragments of human chancres, mucous
patches, and other cutaneous lesions. The medium employed
is a mixture of 2 per cent, slightly alkaline agar and i part of
ascitic or hydrocele fluid, at the bottom of which a fragment
of rabbit kidney or testis is placed. The medium is prepared
in the tubes, after the addition of the tissue, by mixing 2
parts of the melted agar at 50° C. with i part of the ascitic
or hydrocele fluid. After solidification a layer of paraffin oil
3 cm. deep is added.

A considerable number of tubes should be prepared at the
same time and incubated for a few days prior to use to deter-
mine sterility. The bits of human tissue are snipped up with
sterile scissors in salt solution containing i per cent, of sodium
citrate and should be kept immersed in this fluid from the time
of securing to the time of planting, so as not to become dried.
* "Journal of Experimental Medicine," 1912, xv, i, p. 90.

Pathogenesis and Specificity 795

A bit of the tissue should be emulsified in a mortar with citrate
solution and examined with a dark field illuminator to make
sure that the organisms to be cultivated are present.

If they are found, and the material shown to be adapted
to cultivation, each of the remaining bits of tissue is taken up
by a thin blunt glass rod and pushed to the bottom of a cul-
ture-tube and into each tube several drops of the emulsion
examined are introduced by means of a capillary pipet, also
inserted deeply into the medium. The tubes are next incu-
bated at 37° C. for two or three weeks. In successful tubes,
in which the medium has not been broken up by gas-producing
bacteria, there is a dense opaque growth of bacteria along the
line of puncture, and a diffuse opalescence of the agar-agar
caused by the extension into it of the growing treponema.
A capillary tube cautiously inserted into the opalescent me-
dium withdraws a particle that can be examined with dark field
illuminator. When such observation shows the cause of the
opalescence to be, in fact, the treponema, the tube can be
cautiously broken at some appropriate part and the trans-
plantation made from the opalescent part of the medium to
fresh appropriate culture-media. By these means, after a few
trials, pure cultures of treponema were secured.

The colonies were said never to be sharp, but always faintly
visible. There is no color and no odor.

By inoculating the organisms recently secured from human
lesions (by the method given) into monkeys (Macacus rhesus
and Cereopithicus callitrichus) Noguchi was able to produce
typical syphilis of the monkey, thus showing that the virulence
of the organisms was not lost in the cultivation.

Pathogenesis and Specificity. — There can be no doubt
about the causal relation of Treponema pallidum to syphilis.
It is unknown in every other relation; it has appeared in
every required relation, and thus has completely fulfilled the
laws of specificity laid down by Koch. Treponema pallidum
is not only pathogenic for man, but, as has already been
shown, can also be successfully implanted into chimpanzees,
macaques, rabbits, guinea-pigs, and other experiment animals.
As syphilis is, however, unknown under natural conditions,
except in man, it may be looked upon as a human disease.

The organism enters the body through a local breach of
continuity of the superficial tissues, except in experimental
and congenital infections, where it may immediately reach
the blood.

796 Syphilis

In ordinary acquired syphilis the point of entrance shows
the first manifestations of. the disease after a period of pri-
mary incubation about three weeks long, in what is known
as the primary lesion or chancre. This appears as a papule,
grows larger, undergoes superficial indolent ulceration,
and eventually heals with the formation of an indurated
cicatrix. It is in this lesion that the treponema first makes
its appearance. From this lesion, where it multiplies
slowly, it enters the lymphatics and soon reaches the lymph-
nodes, which swell one by one as its invasion progresses.
During this stage of glandular enlargement the organisms
can be found in small numbers in juice secured from a punc-
ture made in the gland with a hollow needle. This period
of primary symptoms (chancre and adenitis) includes part
of what is known as the period of secondary incubation,
which intervenes between the appearance of the chancre and
that of the secondary symptoms. It usually lasts about six
weeks. During this time the organisms are multiplying
in the lymph-nodes and occasionally entering the blood.
What fate the organisms meet when they reach the blood
in small numbers is not yet known, but the slow inva-
sion suggests that those first entering are destroyed, and that
it is only when their numbers are great and their viru-
lence increased that they suddenly become able to over-
come the defenses and permit the development of the
secondary symptoms. This period of secondary symptoms
corresponds to the invasion of the blood by the parasite.
It may continue from one to three years, during which time
the patient suffers from general symptoms, fever, etc., proba-
bly due to intoxication and local symptoms, such as alopecia,
exanthemata, etc., due to local colonization of the organ-
isms. At the end of this period a partial immunity, such as
is seen in other infectious diseases (malaria), develops, the
organisms disappear from the blood, the general local and
constitutional disturbances recover, and the patient may
be well. Should he continue to harbor some of the micro-
parasites, however, there may be an insidious sclerosis of
the blood-vessels and parenchymatous organs consequent
upon the growth and multiplication of the parasites, or
there may be after many years a period of tertiary symptoms
characterized by the sudden appearance of severe lesions
in which the parasites are very few in number.

The specific organisms are present in juice expressed

Diagnosis 797

from the primary lesion, in juice from the bubos and en-
larged lymph-nodes; in the blood, in the roseola, and all of
the secondary lesions, and sparingly in the tertiary lesions.

In congenital syphilis they reach the fetus from either the
ovum, the spermatozoon, or from the blood of the mother.
Prenatal death from syphilis is accompanied by lesions in
which enormous numbers of the organisms can be found,
and furnishes the best tissues for their experimental demon-
stration and study.

Lesions. — The lesions of syphilis are so numerous that
the reader is referred to works on pathology and dermatology
for satisfactory descriptions. Here it may suffice to say
that though diverse in appearance and location, they have
certain features in common. The first of these, and that
which naturally places syphilis among the infectious granu-
lomata, is the lymphocytic infiltration of the tissues, with
which all of the lesions begin. The second is a peculiar
form of necrosis — slimy when superficial, gummy when
deep — with which they terminate. The third is a tendency
toward excessive cicatrization.

Diagnosis. — It is now possible to make a certain and
early diagnosis of syphilis by the recognition of the specific
organisms, and as the difficulty of treatment is in proportion
to the stage at which the disease arrives before treatment,
it should never be neglected.

I. Staining. — The expressed lymph from a carefully
cleaned freshly abraded primary lesion can be stained by
Giemsa's method, or, as is much better and more certain, by
Stern's method, with nitrate of silver, or by the use of India
ink.

II. Dark-field Examination. — For those who possess the
" dark-field illuminator " or some similar apparatus with
the proper lamp, direct examination of the fluid expressed
from the lesions can be made, and the living, moving organ-
isms recognized. This should be the quickest method of
diagnosis, though it takes practice.

III. Serum Diagnosis. — Wassermann and Bruck have
devised a laboratory method of making the diagnosis of
syphilis by testing the complement fixing power of the
patient's serum. This method, now known as the " Was-
sermann reaction," is given in complete detail under a more
appropriate heading. (See Wassermann Reaction.)

The success of the von Pirquet cutaneous tuberculin re-

798 Syphilis

action in assisting the diagnosis of tuberculosis led to ex-
periments on the part of a number of investigators — Mei-
rowsky, Wolff-Eisner, Tedeschi, Nobe, Ciuffo, Nicholas,
Favre, and Gauthier and Jodasshon — to obtain analogous
reaction in syphilis by applying extracts of syphilitic tissues
to the scarified epiderm of syphilitics. Some reactions were
observed, but Neisser and Bruck found that similar reactions
occurred when a concentrated extract of normal liver was
applied, and to such reactions which could not be looked upon
as specific, Neisser applied the term " Umstimmung."

After having successfully achieved the cultivation of Tre-
ponema pallidum, Noguchi* resolved to try the effect of an
application of an extract of the organisms applied to the skin,
in the hope that it might provoke a reaction useful for diag-
nosis. To this end he prepared two cultures, one in ascitic
fluid containing a piece of sterile placenta, the other in ascitic
fluid agar also containing a piece of placenta. After per-
mitting them to grow under strictly anaerobic conditions at
37° C. until luxurient development occurred, the lower part
of the solid culture was carefully cut off, the tissue fragment
removed, and the rich culture carefully ground in a sterile
mortar, the thick paste being diluted from time to time by
adding a little of the fluid culture. The grinding was con-
tinued until the emulsion became perfectly clear, when it
was heated to 60° C. for one hour upon a water-bath and 0.5
per cent, of carbolic acid added. When examined with the
dark-field illuminator, 40 to 100 dead treponemata could be
seen in every field. Cultures made from the suspension re-
mained sterile and inoculation into rabbits' testicles was with-
out result.

This extract of the treponema cultures he calls luetin.
When it was applied to the ear of a normal rabbit, by means of
an endermic injection with a fine needle, an erythema ap-
peared, but faded within forty-eight hours, the skin resuming
its normal appearance, but when it was applied to the ear of a
syphilized rabbit, at the end of the forty-eight hours the red-
ness developed into an induration the size of a pea and per-
sisted from four to six days, disappearing in ten days. In one
case a sterile pustule developed.

Luetin was tested by Noguchi and his colleagues upon 400
cases: 146 of these were controls, 177 syphilitics, and 77
parasyphilitics. In the controls there was erythema without
* "Journal of Experimental Medicine," 1911, xm, p. 557.

Spirochaeta Refringens 799

pain or itching, which disappeared without induration within
forty-eight hours. In the syphilitics at the end of forty-eight
hours there was an induration in the form of a papule 5 to
10 mm. in diameter, surrounded by a zone of redness and
telangiectasis. This slowly increased for three or four days
and became dark bluish red. It usually disappeared in about
a week. Sometimes the papule underwent vesiculation and
sometimes pustulation. It always healed kindly without in-
duration. In certain cases described as torpid, the erythema
cleared away and a negative result was supposed to have re-
sulted, when suddenly the spots lighted up again and pro-
gressed to vesiculation or pustulation. In 3 cases there were
constitutional symptoms — malaise, loss of appetite, and diar-
rhea. Noguchi found that the reaction is specific, that it is
most striking and most constantly present in tertiary,
latent tertiary, and congenital syphilis. It, therefore, forms
a valuable adjunct to diagnosis, seeing that it is most evident
in precisely those cases in which the Wassermann reaction is
most apt to fail. A few early cases energetically treated with
mercury and salvarsan give marked reactions. A few old
cases fail to give it.

SPIROCH^TA REFRINGENS (SCHAUDINN AND HOFFMANN).

This spiral organism, though given the name by which it is
now known by Schaudinn and Hoffmann, was probably first
described by Donne under the name Vibrio lineola. It is
probably a frequent organism of the skin and mucous mem-
branes, and occurs in greatest numbers in lesions of the geni-
talia because of the smegma upon which it customarily lives.
It is present in most primary lesions of syphilis, but is no less
frequently found in non-syphilitic lesions, such as balanitis,
venereal warts, and genital carcinoma. It is also found in the
mouth and on the tonsils. According to Hoffmann and
Prowazek* it is not entirely harmless, but has a pathogenic
action, and some of the complicating lesions of syphilis as
well as some of the destructive diseases of the genitals may be
intensified by it. They found it pathogenic for apes.

Morphologically, it is much broader than Treponema palli-
dum, its spiral waves are much coarser and less regular. It
is easy to stain by all methods and is hence easily found. It
has been cultivated by Noguchi. f

* "Centralbl. f. Bakt.," etc., 1906, xu.

t "Journal of Experimental Medicine," May i, 1912, xv.

CHAPTER XXXII.
FRAMBESIA TROPICA (YAWS).

TREPONEMA PERTENUE (CASTELLANI).

THIS peculiar, specific, infectious, contagious, chronic fe-
brile disease of the tropics is characterized by the appearance
of one or more primary papular lesions — the yaws — bearing
some resemblance to raspberries, upon the skin, malaise,
fever, and other constitutional disturbances. These are
later followed by the appearance of a second crop of small
papules which grow to the size of a pea or a small nut or may
grow to be as large as apples, which become covered with
firm scabs and gradually cicatrize. The patient either re-
covers or suffers from relapses and the appearance of further
crops of the lesions. The duration of the disease varies
from a few weeks to several years. In most cases the con-
stitutional disturbances occur only at the period preceding
the development of the eruptions and for a short time after-
ward. Little children frequently die; older children and
adults may die of exhaustion in case extensive lesions with
marked ulcer ations develop.

The patients usually recover and pigmented areas remain
for some time where the lesions have occurred.

The disease appears to have been known since 1525, when
Oviedo became acquainted with it in St. Domingo. It has
always been very puzzling because it bears so many resem-
blances to syphilis; but the peculiar raspberry-like character
of the primary lesion, its disposition to occur upon the face,
mouth, nose, eyes, neck, limbs, fingers, and toes, as well as
upon the genitals, seem to point in another direction, and
all authorities now admit that it is not syphilis, but an
independent disease.

It occurs only in tropical countries, and is most frequent in
equatorial Africa on the west coast, from Senegambia to
Angola. It also occurs in West Soudan, Algeria, the Nile
Valley, and in the islands about the east coast of Africa. It
has been seen rarely in South Africa. In Asia it occurs in

800

Morphology — Staining

801

Malabar, Assam, Ceylon, Burraah, Siam, Malay Peninsula,
the Indian Archipelago, Moluccas, and China. It is also
endemic in many of the islands and archipelagos of the
southern Pacific.

The cause of the disease was unknown until the discovery of
Treponema pallidum, which opened a way for its investiga-
tion. Castellani* was quick to seize the opportunity, and in
the same year in which Schaudinn and Hoffmann discovered
the cause of syphilis, announced a similar organism as the
cause of yaws. At the time of discovery he called it Spiro-

Fig. 261. — Yaws (photograph by P. B. Cousland, M. B., Swatow,

China).

chaeta pertenuis and Spirochaeta pallidula, but it is now recog-
nized as a treponema and is called Treponema pertenue.

Morphology. — The organism so closely resembles Tre-
ponema pallidum that it is rather by knowing the source
from which the organism was derived than by any morpho-
logic distinctions that the two are separated. It measures
7 to 20 ft in length, is closely and regularly coiled, and is said
to have rounded ends.

Staining. — It stains like its close relative, palely with
most of the dyes. The silver nitrate, the India ink methods,
and the other methods of staining Treponema are all ap-

*"Brit. Med. Jour.," 1905, n, 282, 1280, 1330.

802 Frambesia Tropica

propriate, both for demonstrating it in smears from the le-
sions or in sections of tissue.

Cultivation. — Up to the beginning of 1912 the organism
had not yet been cultivated.

Pathogenesis. — Castellani* has succeeded in infecting
monkeys with the scrapings from yaws papules. The infec-
tion usually resulted in a local lesion, though there was also
a generalized infection, for he found treponemata everywhere
in the lymph-nodes. When the inoculation material was
filtered and all of the organisms removed, the infectivity was
destroyed. Blood and splenic substance from the infected
monkey, containing no organisms other than the treponemata,
was infective for other monkeys. When monkeys success-
fully inoculated with yaws are afterward infected with syphil-
itic virus they are not immune. On the other hand, monkeys
that have successfully been inoculated with syphilis are not
immune against yaws. Levaditi and Nattan-Larrier f differ
from Castellani in this particular, and found that monkeys
infected with syphilis are refractory to yaws. Castellani
was able, by means of complement-fixation tests, to detect
different specific antibodies for syphilis and yaws. Halber-
stadterj has successfully infected orang-outangs.

There is no doubt but that in their clinical manifestations
arid in their etiology frambesia and syphilis are closely
related.

* "Jour, of Hygiene," 1907, vn, p. 558.

t "Ann. de 1'Inst. Pasteur.," 1908, xxu, 260.

t "Arbeiten a. d. Kaiserl. Gesund.," 1907, xxvi, 48.

CHAPTER XXXIII.
ACTINOMYCOSIS.

ACTINOMYCES BOVIS

General Characteristics.— A parasitic, pathogenic, aerobic and
optionally anaerobic, non-motile, non-flagellate, non-sporogenous (?),
liquefying, pathogenic, branched micro-organism, belonging to the higher
bacteria, staining by ordinary methods and by Gram's method.

In 1845 Langenbeck discovered that an infectious disease
of cattle known as " wooden tongue " and " lumpy jaw," and
later as actinomycosis, could be communicated to man.
The observation, however, was not published until 1878,

Fig. 262. — Bovine actinomycosis.

one year after Bollinger* had discovered the actinomyces, the
specific cause of the disease.

Israeli wrote the first important paper upon actino-
mycosis as a disease of man, though the best paper on the
subject is probably that by Bostrom,J who made a careful
study of the microscopic lesions of the disease.

Its first manifestations are usually found either about the
jaw or in the tongue, and consist of considerable sized en-
largements which are sometimes dense and fibrous (wooden
tongue), sometimes suppurative in character. In sections
of tissue containing these nodular formations, small yellow-

*" Deutsche Zeitschrift fur Thiermedizin," 1877.
t "Virchow's Archives," 1874-78.
% "Zeitschrift fur Hygiene," 1889.
803

804

Actinomycosis

ish granules surrounded by some pus can usually be found.
These granules, when examined beneath the microscope,
consist of peculiar rosette-like bodies — the " ray-fungi " or
actinomyces.

Distribution. — The actinomyces is best known as a
parasitic organism associated with actinomycosis. That it
occurs rather widely in nature seems to be indicated by the

Fig. 263. — Colony or granule of actinomyces in a section through a
lesion, showing the Gram-stained filaments and hyaline material and
also the pus-cells surrounding the colony (Wright and Brown).

fact that cases of infection have been known to occur from the
spines of barley and other cereals. Berestnew* succeeded
in isolating the organisms from hay and straw.

Morphology. — A complete ray-fungus consists of several
distinct zones composed of different elements. The center
is composed of a granular mass containing numerous bodies
resembling micrococci or spores. Extending from this cen-
ter into the neighboring tissue is a radiating, branched,
* "Centralbl. f. Bact.," etc., Ref., 1898, No. 24.

Morphology 805

tangled mass of mycelial threads. In an outer zone these
threads are seen to terminate in conspicuous, club-shaped,
radiating forms which give the colonies their rosette-like
appearance. The clubs are inconspicuous in the human
lesions of the disease.

The pleomorphism of the organism and the branched
network it forms class it among the higher bacteria in the
genus Actinomyces. When the clumps formed in artificial
cultivations of the parasite are properly crushed, spread

Fig. 264. — Actinomyces granule crushed beneath a cover-glass,
showing radial striations in the hyaline masses. Preparation not
stained; low magnifying power (Wright and Brown).

out, and stained, the long mycelial threads, 0.3-0.5 p. in
thickness, frequently show flask- or bottle-like expansions
— the clubs — at the ends. These probably depend upon
gelatinization of the cell-membrane of the degenerating
parasite. The club is one of the chief characteristics of the
organism. In sections of tissue the radiating filaments are
very distinct, and the terminal clubs are all directed outward,
closely packed together, and making the whole mass form
a rounded little body often spoken of as an "actinomyces
grain." When tissues are stained first with carmin and then
by Gram's method, the fungous threads appear blue-black,
the clubs red. The cells of the tissues affected and a larger
or smaller collection of leukocytes form the surrounding
resisting tissue-zone.

806 Actinomycosis

The fungus is of sufficient size to be detected in pus,
etc., by the naked eye. It can be colored, in sections of
tissue, by the use of Gram's or Weigert's stain. Tissues
pre-stained with carmin, then by Weigert's method, show
beautifully.

Cultivation. — The actinomyces fungus may be grown
upon all the artificial culture media, as has been fully shown
by Israel,* Wolff, and others.

To obtain a pure culture, material containing the actino-
myces granules, secured so as to be as free as possible from
contaminating micro-organisms, is crushed between glass
plates or in a mortar, and the crushed fungi transferred
to plates or tubes as desired. The colonies appear as small
gray dots, and consist of a translucent, radiating filamentous
network. If kept for a few days at 37° C. they become
opaque and nodular, with radiating processes about the
periphery. Still later they develop a whitish downy ap-
pearance from the formation of short aerial hyphae. The
best growth occurs when free access of oxygen is permitted.

Blood-serum. — Upon blood-serum the nodular growths
present a yellowish or rust-red color, and are surrounded
with a whitish down of fine threads. The colonies adhere
closely to the culture media and are so firm that they crush
with difficulty. If the surface be scraped, spores and fine
threads may be secured. If the mass be crushed, branched
filaments may be secured. The colonies become confluent
in the course of time, and a thick wrinkled membrane is
produced. The growth liquefies blood-serum.

Gelatin. — In gelatin puncture cultures an arborescent
growth occurs and the gelatin is liquefied.

Agar-agar. — Upon agar-agar and glycerin agar-agar the
growth is similar to that upon blood-serum. The agar-agar
turns brown as the culture ages.

Bouillon. — In bouillon the growth occurs in the form
of large granules if allowed to stand quietly; of numerous
small granules if frequently shaken up. The granules are
similar in structure to those formed upon the dense media.
The bouillon does not become clouded.

Potato. — Upon potato the growth resembles that upon
blood-serum, but is slower in developing. The color is red-
dish-yellow and the white down early makes its appearance.

Eggs. — The organism can also be grown in raw eggs, into
* "Virchow's Archives," cxv.

Cultivation

807

Fig. 265. — Colony of actinomyces with well-developed "clubs" at
the periphery in a nodule in the peritoneal cavity of a guinea-pig in-
oculated with a culture from another guinea-pig. Paraffin section.
Low magnification (Wright). (Photograph by Mr. L. S. Brown.)

Fig. 266. — A colony of actinomyces in a nodule twenty-eight days
old in the peritoneal cavity of a guinea-pig inoculated with a culture
from another guinea-pig (Bovine case). The "clubs" are well devel-
oped and show some indications of stratification. Paraffin section.
X 750aprox. (Wright). (Photograph by Mr. L. S. Brown.)

8o8

Actinomycosis

ABC

Fig. 267. — Actinomycosis; glycerin-agar cultures: A, Discrete
rounded colonies after about ten days' incubation at 37° C. ; B,
limpet-shaped colonies three and a half months old ; C, lichen-like
appearance frequently seen ; the growth is three and a half months
old (Curtis).

Pathogenesis 809

which it is carefully introduced through a small opening
made under aseptic precautions. In the eggs long,
branched mycelial threads quite unlike the bacillary forms
that grow upon agar-agar are formed.

The characteristic rosettes so constantly found in the tis-
sues are never seen in artificial cultures.

Virulence. — When the actinomyces is grown upon artifi-
cial media the virulence is retained for a considerable time.

Pathogenesis. — Actinomycosis is almost peculiar to bo-
vine animals, but sometimes occurs in hogs, horses, and
other animals, and rarely in human beings. The disease
can with difficulty be inoculated into experiment animals,
the introduced fungi either becoming absorbed or encap-
sulated by connective tissue and not growing. In the
abdominal cavities of rabbits the peritoneum, mesentery, and
omentum show typical nodules containing the actinomyces
rays in cases of successful inoculation.

Mode of Infection. — The manner by which the organ-
ism enters the body is not positively known. In some cases
it may be by direct inoculation with infectious pus, but
there is some reason to believe that the organism occurs in
nature as a saprophyte, or as an epiphyte upon the hulls
of certain grains, especially barley. Woodhead has recorded
a case where a primary mediastinal actinomycosis in the
human subject was apparently traced to perforation of the
posterior pharyngeal wall by a barley spikelet accidentally
swallowed by the patient.

Cases of actinomycosis are fortunately somewhat rare in
human medicine, and do not always occur in those brought in
contact with the lower animals. The fungi may enter the or-
ganism through the mouth and pharynx, through the respira-
tory tract, through the digestive tract, or through wounds.

The invasion has been known to take place at the roots
of carious teeth, and is more liable to occur in the lower
than in the upper jaw. Israel reported a case in which
the primary lesion seemed to occur external to the bone
of the lower jaw, as a tumor about the size of a cherry,
with an external opening. Two cases of the disease
observed by Murphy, of Chicago, began with toothache and
swelling of the jaw. A few cases of dermal infection are
recorded. Elsching * has seen a case in which calcified
actinomyces grains were observed in the tear duct.
* "Centralbl. f. Bakt. u. Parasitenk.," xvm, p. 7.

8io Actinomycosis

When inhaled, the organisms enter the deeper portions
of the lung and cause a suppurative broncho-pneumonia
with adhesive inflammation of the contiguous pleura. After
the formation of the pleuritic adhesions the disease may

Fig. 268. — Section of liver from a case of actinomycosis in man
(Crookshank).

penetrate the newly formed tissue, extend to the chest-
wall, and ultimately form external sinuses; or, it may
penetrate the diaphragm and invade the abdominal organs,
causing interesting and characteristic lesions in the liver
and other large viscera.

Lesions 811

Lesions. — The degree of chemotactic influence exerted
by the organism seems to depend upon the tissue affected,
upon the peculiarity of the animal, and upon the virulence
of the organism. When an animal is but slightly suscepti-
ble, and especially when the tongue is affected, the disease
is characterized by the formation of cicatricial tissue —
"wooden tongue." If, on the other hand, the animal be
highly susceptible and the jaw-bone affected, suppuration,
with the formation of abscesses, osteoporotic cavities, and
sinuses, are apt to be noticed. This form of the disease is
called "lumpy jaw" in cattle.

Before the nature of the affection was understood it
was confounded with diseases of the bones, especially osteo-
sarcoma.

From the tissues primarily affected the disease spreads
to the lymphatic glands, and eventually to the lungs. Israel
has pointed out that certain cases of human actinomycosis
begin in the peribronchial tissues, probably from inhalation
of the fungi.

But few cases recover, the disease terminating in death
from exhaustion or from complicating pneumonia or other
organic lesions.

CHAPTER XXXIV.

MYCETOMA, OR MADURA-FOOT*

ACTINOMYCES MADURA (VINCENT).

General Characteristics.— A non-motile, non-flagellate, sporogen-
ous (?), non-liquefying, non-aerogenic, chromogenic, aerobic and option-
ally anaerobic, branched, parasitic organism belonging to the higher
bacteria, staining by ordinary methods and by Gram's method, and
pathogenic for man.

A curious disease of not infrequent occurrence in the
Indian province of Scinde and of rare occurrence in other
countries is known as mycetoma, Madura-foot, or pied de
Madura. Although described as peculiar to Scinde, the dis-
ease is not limited to that province,
but has been met with in Madura,
Hissar, Bicanir, Delhi, Bombay,
Baratpur, Morocco, Algeria, and in
Italy. In America less than a
dozen cases of the disease have been
placed on record. In India it al-
most invariably affects natives of
the agricultural class, and in nearly
all cases is referred by the patient
to the prick of a thorn. It usually
affects the foot, more rarely the
hand, and in one instance was seen
by Boyce to affect the shoulder and
hip. It is more common in men
than in women, individuals between

^^ and forty years of ^ suffer-
ing most frequently, though persons
of any age may suffer from the

disease. It is insidious in onset, no symptoms being ob-
served in what might be called the incubation stage of a
couple of weeks' duration, except the formation of a nodular
growth which gradually attains the size of a marble. Its deep
attachments are indistinct and diffuse. The skin over it
becomes purplish, thickened, indurated, and adherent. The

812

Fig. 269. — Madura-foot
— mycetoma (Musgrave
and Clegg).

Morphology

813

ball of the great toe and the pads of the fingers and toes
are the points most frequently invaded. The lesions pro-
gress very slowly, and in the course of a few months form
distinct inflammatory nodes. After a year or two the
nodes begin to soften, break down, discharge necrotic and
purulent material, occasioning the formation of ulcers and
sinuses. The matter discharged from the lesions at this
stage of the disease is a thin seropus, and contains occasional
fine round pink or black bodies, similar to actinomyces
" grains," described, when pink, as resembling fish-roe;
when black, as resembling gunpowder. It is upon the de-
tection of these particles that the diagnosis rests. Accord-
ing to the color of the bodies found, cases are divided into the
pale or ochroid, and melanoid varieties.

The progress of the disease causes an enormous enlarge-
ment of the affected part.
The malady is usually pain-
less.

The micro-organismal na-
ture of the disease was early f|r
suspected. In spite of the
confusion caused by some
who confounded the disease
with "guinea- worm," Carter
held that it was due to some
indigenous fungus as early
as 1874. Boyce and Sur-
veyor found that the black
particles of the melanoid
variety consisted of a large
branching septate fungus.

Pale Variety. -- Kan-
thack was the first to prove
the identity of the fungus
with the well-known actino-
myces, but there seems to be
considerable doubt about the
identity of the species:

Morphology. — Under the
microscope the organism is
found by Vincent* to be

branched and belong to the higher bacteria. It consists of
* "Ann. de 1'Inst. Pasteur," 94, 3.

Fig. 270. — Streptothrix mad-
urae in a section of diseased tis-
sue (Vincent).

8 14 Mycetoma, or Madura-foot

long, branched bacillary threads forming a tangled mass.
In many of the threads spores could be made out. He
was unable to communicate the disease to animals by in-
oculation.

Cultivation. — Vincent succeeded in isolating the specific
micro-organism by puncturing one of the nodes with a
sterile pipet, and cultivated it upon artificial media, acid
vegetable infusions seeming best adapted to its growth.
It develops scantily at the room temperature, better at
37° C. — in from four to five days. In twenty to thirty
days a colony attains the size of a little pea.

Bouillon. — In bouillon and other liquid media the
organisms form little clumps resembling those of actino-
myces. They cling to the glass, thus remain near the
surface of the medium, and develop a rose- or bright-red
color. Those which sink to the bottom form spheric balls
devoid of the color.

Gelatin. — The growth in gelatin is not very abundant,
and forms dense, slightly reddish, rounded clumps. Some-
times there is no color. There is no liquefaction.

Agar-agar. — Upon the surface of agar-agar beautiful
rounded, glazed colonies are formed. They are at first
colorless, but later become rose-colored or bright red.
The majority of the clusters remain isolated, some of them
attaining the size of a small pea. They are usually
umbilicated like a variola pustule, and present a curious
appearance when the central part is pale and the periphery
red. As the colony ages the red color is lost and the colony
becomes dull white or downy from the formation of aerial
hyphae. The colonies are very adherent to the surface of the
medium, and are almost of cartilaginous consistence.

Milk. — The organism grows in milk without causing
coagulation.

Potato. — Upon potato the growth of the organism is
meager and slow, with very little chromogenesis. The
color-production is more marked if the potato be acid in
reaction. Some of the colonies upon agar-agar and potato
have a powdery surface, either from the formation of spores
or of aerial hyphae.

Lesions. — Microscopic study of the diseased tissues in
mycetoma is not without interest. The healthy tissue is
sharply separated from the diseased areas, which appear
like large degenerated tubercles, except that they are ex-

Lesions

815

tremely vascular. The mycelial or filamentous mass occu-
pies the center of an area of degeneration, where it can
be beautifully demonstrated by the use of appropriate
stains, Gram's and Weigert's methods being excellent for

Fig. 271. — Melanoid form of mycetoma. Section showing black
granules and general features of the lesions as they appear under a
low-magnifying power. Zeiss 0,1 (James H. Wright).

Fig. 272. — Melanoid form of mycetoma. showing structure and ap-
pearance of the hyphae of the mycelium obtained from the granules.
Zeiss apochromat; 4 mm. (James H. Wright).

the purpose. The tissue surrounding the nodes is infiltrated
with small round cells. Tne youngest nodules consist of
granulation-tissue, whose development is checked by early
coagulation-necrosis. Giant-cells are few.

8i6

Mycetoma, or Madura-foot

Not infrequently small hemorrhages occur from the
ulcers and sinuses of the diseased tissues; the hemorrhages
can be explained by the abundance of small blood-vessels
in the diseased tissue.

Fig. 273. — Melanoid form of mycetoma. Two bouillon cultures
showing the powder-puff ball appearance. In one the black granule is
seen in the center of the growth (James H. Wright).

Fig. 274. — Melanoid form of mycetoma. Potato culture of the
hyphomycete obtained from the granules. The black globules are
composed of a dark brown fluid (James H. Wright).

The Melanoid Form of mycetoma has been carefully
investigated by Wright* and appears to depend upon an
entirely different micro-organism properly classed among the
* "Journal of Experimental Medicine," vol. m, 1898, p. 421.

The Melanoid Form 817

hyphomycetes. It is probably identical with the organism
described by Boyce and Surveyor.

In the case studied, Wright found the diseased tissues,
consisting of several of the pads of the toes, to be either
translucent and myxomatous or yellowish and necrotic in
appearance. The black granules were embedded in the
tissue and appeared mulberry-like and less than i mm. in
diameter. They were firm, and when enucleated and
pressed between cover and slide did not crush. Only after
digestion with a solution of caustic potash and careful
teasing could the granules be resolved into the hyphse of
the mold. The central part of the granule formed a reticu-
lum, with radiating, somewhat clavate elements projecting
from it.

In sections of tissue it was found possible to stain the
fungus with Gram's and Weigert's stains, though prolonged
washing removed most of the dye.

Cultural Characteristics. — Enucleated granules carefully
washed in sterile bouillon and then planted upon agar-agar
afforded cultures of the mold in 25 out of 65 attempts.

The growth began in five or six days, appearing on solid
media as a tuft of delicate whitish filaments, springing from
the black grain, and in a few days covering the entire surface
of the medium with a whitish or pale brown felt-work.
Upon potato this felt-work supports drops of brownish
fluid. The long branched hyphae thus formed were from
3 to 8 /J. in diameter, with transverse septa in the younger
ones. The older hyphae were swollen at the ends. No buds
were observed. No fruit organs were detected. In fluid
media the filaments radiated from the central grain with the
formation of a kind of puff-ball. Eventually the whole
medium becomes filled with mycelia and a definite surface
growth forms.

The general characteristics of the fungus are well shown
in the accompanying illustrations from Wright's paper.

52

CHAPTER XXXV.
BLASTOMYCOSIS.

BLASTOMYCES DERMATITIDIS (GILCHRIST AND STOKES).

THE first case in Which yeasts or blastomycetes were defi-
nitely connected with disease seems to have been published by
Busse.* He observed a case of tibial abscess in a woman
thirty-one years of age, who died about a year after coming
under observation. Postmortem examination showed num-
bers of broken-down nodular formations upon the bones, and
in the spleen, kidneys, and lungs. In all of these lesions he
found, and from them he cultivated, an yeast, which, when in-
troduced in pure culture into animals — mice and rats —
proved infective for them. He called the organism Sac-
charomyces hominis, and the affection in which it was found
"Saccharomycosis hominis."

In May, 1904, three months before the appearance of
Busse's paper, Gilchrist exhibited to the American Dermato-
logical Association in Washington microscopic sections from
a case of cutaneous disease in which peculiar bodies, recognized
as plant forms, were found. After the appearance of Busse's
papers, Gilchrist f more fully described and illustrated his
findings, calling the lesions " blastomycetic dermatitis."
Though much work upon pathogenic blastomycetes has been
published and pathogenic forms of these micro-organisms
have been described by Sanfelice,J by Rabinowitsch,§ and
others, the chief and almost the sole form in which these
infections make their appearance is a dermal infection known
as "blastomycetic dermatitis."

The infection usually begins with the formation of a papule
upon the face or one of the extremities, which suppurates and
evacuates minute quantities of viscid pus. The lesion crusts

* "Centralbl. f. Bakt. u. Parasitenk.," 1894, xvi, 175.
f "Johns Hopkins Hospital Reports," I, 269, 291.
t "Centralbl. f. Bakt. u. Parasitenk.," 1895, xvn, 113, 625; xvm, 521;
xx, 219.

§ "Zeitschrift fur Hygiene," etc., 1896, xxi, n.

818

Blastomycosis 819

and begins to heal, but at the periphery new and usually mi-
nute foci of suppuration occur, so that while the original
lesion tends to heal very slowly, with much cicatricial for-
mation, it is always spreading. The progress is usually
slow, and Gilchrist's first case spread only two inches in four
years.

Though the progress is slow, it is sure, and there is no tend-
ency to spontaneous recovery in most cases, nor is the condi-
tion modified by treatment. The patients may die from in-
tercurrent disease or from a generalization of the blastomy-
cetic infection, which not infrequently happens.

After the work of Gilchrist had made clear the symptoma-
tology and parasitology of the disease, a number of other

Fig. 275. — Cutaneous blastomycosis (Montgomery).

cases were reported, and Ricketts* published an excellent
and lengthy summary of all the cases with references to all
of the literature up to that date. Another very interesting
paper by Montgomery , f published in 1902, contains a splendid
atlas of photographs of the various lesions and of the cultures.
In addition to the cutaneous blastomycosis, a second form
is also occasionally seen, and is known as Coccidioidal granu-
loma. It seems to have been first reported by Posadas and
WernickeJ and has been carefully studied by Ophiils.§ In

* "Jour. Med. Research," 1901, i, 373.
t "Jour. Amer. Med. Assoc.," June 7, 1902, i, 1486.
J "Jour, de Microorganismen," 1891, xv, 14.

§ "Jour. Experimental Medicine," 1905, vi, 443. Ophiils and Moffit,
"Phila, Med. Jour.," 1900, v, 1471.

820

Blastomycosis

this form of the disease the lesions are in the internal organs,
macroscopically and microscopically resemble tubercles, and
can only be differentiated from them by the presence of the
blastomyces and the absence of tubercle bacilli. The lungs
may be affected, and Walker and Montgomery* mistook a
case for miliary tuberculosis of the lungs. They also seem,
according to Evans f to have a predilection for the central
nervous system.

There seems to be little reason for believing that there is
any other difference than that of distribution between the

Fig. 276. — Giant cell from a cutaneous lesion in blastomycosis, showing
a group of blastomyces (Montgomery).

blastomycetic dermatitis and the blastomycetic granuloma,
or that they are caused by different micro-organisms. Re-
garding the organisms, however, we are by no means sure
that there are not several species.

Specific Organism. — The organism presents a variety
of appearances which may be thus brought together: First,
there are round and elliptical disk-like bodies that some regard
as spores, others as the primitive or yeast form. These
measure 10 to 30 {* in greatest diameter, are distinctly doubly
contoured, highly refracting, and, though sometimes clear

* "Jour. Amer. Med. Assoc.," 1902, xxxvni, 867.
t "Jour, of Infectious Diseases," 1909, vi, 535.

Cultivation

821

and transparent, as frequently granular and vacuolated.
From these buds may grow, as in the yeasts ; or hypha may
form, as in oi'dium. In artificial cultivations the hypha may
form a tangled mycelium.

Staining. — The organisms are usually better found with-
out staining. They do not stain with aqueous anilin dyes,
but are penetrated by warm thionin, alkaline methylene-blue,
and polychrome methylene-blue. In sections of tissue
stained with hematoxylon and eosin they show as uncolored

Fig. 277. — Blastomyces dermatitidis. Budding forms and mycelial
growths from glucose agar (Irons and Graham, in " Journal of Infectious
Diseases").

circles; with thionin and alkaline methylene-blue they may
take a blue color.

Cultivation. — The organism grows readily upon artificial
media when once started, but the primitive culture is difficult
to secure, because the cocci and other associated organisms
are more numerous than the blastomyces and outgrow it.
It seems most satisfactory to first infect a guinea-pig with the
organism from the skin, and then start the cultivation from
its lesions than to attempt it directly from the pus from
human dermal lesions. When the human lesions are internal,
pure cultures are easily started.

Gilchrist and Stokes* were able to start cultures directly

* "Journal of Experimental Medicine," 1898, in, 53.

822

Blastomycosis

from the dermal lesions. Hiss and Zinsser recommended
that this be done by greatly diluting the culture material, so
as to separate the contained organisms widely.

Many culture-media prove appropriate, glycerin agar-agar
and agar-agar containing i per cent, of dextrose being ex-
cellent. When once isolated the organism is easily kept
growing by transplanting every month or two.

Fig. 278. — Cultures of Blastomyces dermatitidis upon solid culture-
media (Montgomery).

The colonies appear in a few days as small round hemi-
spheric dots with numerous prickles about the surfaces.
Later they have a moldy appearance from the develop-
ment of aerial hypha. They are almost purely aerobic,
those on the surface growing well, those deeply seated in the
medium scarcely at all.

Agar-agar Slants. — These at first show a creamy white
layer that becomes quite thick, and is moldy and fluffy on the
surface. After a few weeks the agar-agar begins to turn
yellow and later may become brown, though the growth itself

Lesions 823

remains white and unchanged. The growth is firmly at-
tached to the agar. When old, the growth wrinkles.

Bouillon. — The growth is not luxuriant. The medium
is not clouded and contains fluffy flocculi of stringy viscid
material. Sugars added to the medium may be fermented.

Gelatin. — Growth takes place with aerial hypha. Lique-
faction does not occur or is very slow.

Potato. — Abundant growth with aerial hypha.

Milk. — Not coagulated, not acidified, slowly digested.

There is some difficulty in describing the cultures, as differ-
ent authors describe them quite differently, evidently having
different organisms or different strains under observation.

Pathogenesis. — The organisms are pathogenic for guinea-
pigs, rabbits, and dogs, in which an abscess, not infrequently
followed by a generalized infection, takes place.

Lesions. — The human lesions vary somewhat. Gilchrist's
first case resembled lupus vulgar is, other cases present an
exaggeration of the ulcerative element. Cases have also
been mistaken for syphilis. The intractable character of the
lesions is suggestive, and the finding of the micro-organisms in
the viscid pus is pathognomonic.

Upon section the lesions still resemble lupus and other tu-
berculous lesions, but here again the absence of tubercle
bacilli and the presence of the blastomyces enable diagnosis
to be made.

Transmission. — The disease is transmissible. The source
of infection is not known.

CHAPTER XXXVI.
RINGWORM.

TRICHOPHYTON TONSURANS (MALMSTEN).

TINKA trichophytina, ringworm of the scalp, herpes ton-
surans, tinea circinata, ringworm of the body, herpes cir-
cinatus, tinea unguium, onychomycosis, tinea imbricata,
herpes desquamans, tinea versicolor, .pityriasis versicolor,
erythrasma, etc., are diseases with well-marked clinical
manifestations and differences, all of which may be compre-
hended under the general term dermatomycosis, and are
caused by closely related forms of parasitic fungi, whose
generic and specific differences are matters of considerable
confusion.

That certain of the diseases affect hairy parts and others
hairless parts of the body, that still others occur about the
nails, and that some are superficial and practically sapro-
phytic, while others penetrate more deeply and are undoubt-
edly parasitic, do not necessarily point any more conclusively
to essential differences in the infecting organisms than to
accidents of infection and variations in resisting power. A
review of the literature leaves the student with a deplorable
confusion of ideas, and a feeling that the synonomy is too
complicated and the use of terms too loose to permit of sys-
tematic reconstruction.

The discovery of micro-organisms in these lesions seems to
have been made in 1842 by Gruby,* who found mycelial
threads and spores on and in the hairs, and in 1860 by Hebra,f
between the epithelial cells. The organism appears to have
been called Trichophyton tonsurans in 1845 by Malmsten.
The parasitology of all of the trichophyton infections was
thoroughly studied by Sabouraud,J and the old species,
Trichophyton tonsurans, divided into eleven new species, to

* "Compt.-rendu," Paris, 1842, xv.

t "Handbuch der spezullen Path. u. Therapie von Virchow," in, 1860.

J"Ann. de dermat. et de syphilis," m, 1892; iv, 1893; v, 1894;
"Monatshefte," 1896, 576; "La Practique dermatologique. Tricho-
phytie," 1900.

824

Morphology

825

/*

which four others have since been added, so that there are
now described, with or without justification, Trichophyton
crateriforme, T. acuminatum, T. violaceum, T. effractum,
T. fulmatum, T. umbilicatum, T. regulare, T. pilosum, T.
glabrum, T. sulphureum, T. polygonum, T. exsiccatum, T.
circonvulatuni, T. flavum, and T. plicatili.

. In general it is customary to divide the organisms into two
groups, Trichophyton microsporon and T. megalosporon,
the former having large, the latter small, spores.

Morphology. — The trichophyton parasites form delicate
mycelia composed of somewhat slender septate hypha.
They can best be observed by ex-
tracting one of the hairs, including
its root, from the diseased area,
or if the affection be upon a hair-
less part of the body, by scraping
off some of the epiderm, and
mounting the material between a
slide and cover in a drop of caustic
potash solution (20 per cent.).
Under these circumstances the
spores are conspicuous and so
numerous as to give the impression
that they occur in rows in a kind
of structureless zooglea upon the
outside of the hair. In some cases,
however, especially in Trichophy-
ton megalosporon, the hypha may
be observed with the spores inside.
The hypha measure from 2 to 8 ^
in diameter, are usually simple,
and rarely divide. The spores are
from 2 to 3 ^ in diameter in the
Trichophyton microsporon and 7
to 8 li in T. megalosporon. The former is the more com-
mon upon the hairless, the latter upon the hairy, portions of
the skin.

Cultivation. — The organisms may be secured in pure
culture without much difficulty, except for the annoying and
almost constant presence of the associated bacteria of the
skin. By crushing the hairs and scales in a mortar with some
dilute KOH solution, and then, after thoroughly distributing
the spores through the alkaline medium which dissolves many

I

fl

! il

Fig. 279. — Invasion of a
human hair by trichophy-
ton : A, Points at which the
parasitic fungi coming from
the epidermis are elevating
the cuticle of the hair and
entering into its substance.
Magnified 200 diameters
(Sabouraud).

826

Ringworm

of the bacteria, plates can be made with high dilutions, or
drops of the fluid may be spread over potato, which is an
excellent medium for the culture.

The culture, whether upon agar-agar, glycerin agar-agar,
glucose agar-agar, gelatin, or potato, occurs in the form of a
tuft of white mycelial filaments with aerial hypha, looking
like a tiny white powder-puff. Upon the surface of liquid cul-
ture-media the growth appears as a thick wrinkled pellicle
with aerial hypha of velvety appearance. As the cultures

Fig. 280. — Trichophyton tonsurans. Primary cultures twenty days'
old on maltose agar-agar. Natural size (Sabouraud).

grow older the lower mycelial growth becomes yellowish and
wrinkled, but the aerial hypha maintain the velvety white ap-
pearance. Some of the colonies are mammillated, some are
crateriform. Gelatin is liquefied, the growth floating upon
the surface of the fluid. As the cultures become very old and
dry the velvety appearance is lost and the surface becomes
powdery. The powder detaches only when the growth is
touched, and does not shake off.

Pathogenesis. — The trichophytons are pathogenic for
man and for the lower animals. They spread from animal to

Pathogenesis 827

animal by contact and by inoculation. Men, dogs, cats,
horses, sheep, goats, and swine all suffer from the infection.
The growth of the hypha between the epidermal layers causes
a chronic inflammation, with hyperemia, desquamation, the
formation of some papules, and occasional pustules. The
invasion of the hair-follicles and the growth of the fungi into
the hairs cause them to become fragile and break off, as well
as to loosen and drop out.

The name "barber's itch" results from the frequent trans-
mission of the infection by the barber's razors. The disease
is easily transmissible and precautions should always be
taken to prevent its dissemination.

CHAPTER XXXVII.
FAVUS.

ACHORION SCHONLEINII (REMAK).

FAVUS, or tinea favosa, is a chronic and destructive form of
dermatomycosis occurring in man and animals, caused by a
fungus discovered in 1839 by Schonlein,* and called in his
honor Achorion schonleinii by Remak in 1845. This fungus
is widely distributed and affects mice, cats, dogs, rabbits,
fowls, and men. Among human beings it usually occurs upon
the scalp and other hairy parts of the body, though it may
also affect the hairless portions and even attack the roots of
the nails. It is more frequent in children than in adults.
The fungus grows vigorously and usually forms a small
sulphur yellow disk about the base of a hair. The edges of
this detach, become everted, and the whole eventually sepa-
rates, forming the "scutulum," or characteristic lesion of the
disease. The reaction is more marked, the damage done
greater, and the disease less tractable than in other forms of
dermatomycosis .

The infection seems to take place in most cases by way of
the hair-follicles, and the mycelia of the fungi grow into and
about the hairs, invading the epiderm, and causing atrophy
of the hair-follicles by pressure. Beneath and around the
scutulum, which consists chiefly of the fungi, an inflamma-
tory reaction takes place, and leukocytic invasion and ulcer-
ation cause the scutulum to separate.

Although usually confined to the skin , the favus infection
may extend to the mucous membranes, and Kaposi and
Kundratf have reported a case in which favus fungi were
found to have invaded the stomach and intestines.

The disease runs a course sometimes extending over many
years. Crocker J mentions a case that recovered after thirteen
years. It may remain localized upon the scalp or may spread

* Muller's "Archiv.," 1839.
f "Ann. de Dermat. et de Syph.," 1895, p. 104.
J "Diseases of the Skin," Phila., 1903, p. 1276.
828

The Specific Organism

829

itself over much of the skin surface. When the lesions are
large they give off an odor suggesting that peculiar to white
mice. In recovering, the lesions leave considerable cicatricial

, -

Fig. 281 . — Favus. Hairs of a child infected with Achorion schonleinii.
A, Magnified 260 diameters; B, 75 diameters. The large rounded bodies
are droplets of air which always appear after treatment with 40 per cent,
potash solution. The linear threads are the fungi. Some are without
spores, others contain rows of spores (Sabouraud).

scarring, and atrophy of hair-follicles, sweat, and sebaceous
glands is inevitable.

The Specific Organism. — The Achorion schonleinii is
probably better regarded as a group of closely related organ-

830 Favus

isms than as a single one. Indeed, Quincke has described
three species, though they are not yet generally accepted.

The organism can be studied by extracting a hair and exam-
ining it in KOH or NaOH solution (20 per cent.), or by
teasing a scutulum in the same medium and examining with
a low power. Sections of the skin may also be made when
possible.

The fungus resolves itself into mycelial threads and spores.
The scutulum consists of masses of spores at the center and
about the hair, with mycelia containing spores at the edges.

Fig. 282. — Achorion schonleinii. Fig. 283. — Achorion schonleinii.

Four weeks' old culture upon beer- Pure culture, four weeks old, on

wort agar-agar (Kolle and Wasser- beerwort agar-agar (Kolle and

mann). Wassermann).

From the mycelium hypha are given off, the ends being
knobbed or clavate.

The mycelial threads are highly refractile, contain granular
protoplasm, and are of varying thickness. Sometimes the
terminal hypha are simple, sometimes they fork, the ends are
.always clavate. The hypha give off buds at right angles
along their course.

The spores are oval, doubly contoured, as a rule, but may be
round or pointed and more or less polyhedral. They measure
3 to 8 ft in length and 3 to 4 f* in breadth. They form the
great central mass of the scutulum, which is the oldest part.
Together with them one finds a number of detritus granules,
fat-droplets, and occasional swollen epidermal cells.

Cultivation. — The cultivation of the achorion is quite
easy if care be used, for the central part qf each scutulum
contains pure cultures of the organism. The best method is

Pathogenesis 831

probably that of Krai,* which is as follows: "A good deal of
the material from the scutula is rubbed up in a porcelain
mortar dish with previously heated diatomaceous earth, with
a porcelain pestle, without exerting too much pressure.
Melted agar-agar tubes are then inoculated with two or three
loopfuls of the crushed material and poured into Petri dishes.
Greater dilution can be made if desired. The plates are ex-
amined after forty-eight hours.

Cultures may, however, be directly made with material
from the center of a scutulum. Agar-agar should be used, as
the cultures grow best at the body temperature. The
young colonies that appear in forty -eight hours can easily be
transplanted by fishing under a lens.

The best medium was found by Sabouraud to consist of
maltose, 4; peptone, 2; fucus crispi, 1.5; water, 100.

As the colonies eventually become quite large it is recom-
mended that, instead of tubes, they be made in Erlenmeyer
flasks, the transplanted little colonies being placed at the
center of the medium congealed upon the bottom of the flask.

The appearance of the cultures varies considerably.
Plaut gives two principal varieties : ( i ) The waxy type — a yel-
lowish mass of a waxy character with radiating folds and a
central elevation. As a rule no aerial hypha, but occasionally
short aerial hypha.

(2) The downy type — this forms a white disk with a velvety
or plush-like covering of white aerial hypha. Sometimes in-
stead of white the color is yellowish or reddish. A marked
dimple with a smaller elevation usually occurs in the middle,
and there may be radial folds.

Pathogenesis. — The micro-organism is pathogenic for
mice, rabbits, cats, dogs, hens, and men, in all of whom typical
scutula form. Scutulum formation has not been observed in
guinea-pigs. The disease readily spreads from animal to
animal by direct contact, and by indirect contact by the use
of combs, hair-brushes, and similar objects. On account of
its chronicity, its obstinacy, its disfigurement, and its trans-
missibility it is a dangerous disease, and one that requires
prompt isolation of the patient and the utmost care for the
prevention of contagion.

*See Plaut, in Kolle and Wassermann's "Pathogene Mikroorgan-
ismen," i, p. 608.

BIBLIOGRAPHIC INDEX

ABBOTT, 101, 102, 187, 188, 238,
250, 365, 454. 460, 628, 652

Abbott and Bergey, 629, 631

Abbott and Gildersleeve, 452, 759

Abel, 130, 5*11, 588, 672

Abel and Claussen, 611

Abel and Loffler, 638, 648

Abelous, 368

Achard, 665

Adami, 78, 86, 87

Adami and Chapin, 656

Afanassiew, 488

Agramonte, 576

Agramonte, Reed, and Carroll, 577

Agramonte, Reed, Carroll, and
Lazear, 576

Alav, 485

Albrech and Ghon, 428

Alessi, 101

Alt, 449

Altmann, 269

Alvarez and Tavel, 757

Anaximander, 17

Anderson and Rosenau, 123, 161

Andrewes and Gordon, 341

Andrews, 211

Anjeszky, 188

Aoyama, 585

Aristotle, 17

Arloing, 114, 409, 745, 75O

Arnaud, 688

Arning, 771

Arnold, 205

Arrhenius, 26

Arustamoff and Vignal, 42

Aschoff, 131

Aschoff and Gaylord, 200

Atkinson, 473

Audanard, 368

Auld, 498

Axenfeld, 424, 448, 501

BABES, 355, 368, 419, 474, 478, 479,

520, 784, 781

Babes and Cornil, 478, 608
Babes and Ernst, 34, 175
Babes and Lepp, 117, 422
53

Babes and Proca, 747

Bail, 146, 150

Baker, 557

Baldwin, 384

Baldwin and Stewart, 383

Baldwin and Trudeau, 742, 744

Banti, 256, 501

Barbagallo and Casagrandi, 707

Barker, 368

Barron, 555

Bass, 539

Bassett and Duval, 700

Bassett-Smith, 334, 335, 522, 523

Batement, 562

Batzaroff, 594

Baumgarten, 89, 477, 729, 731, 732,

782, 783

Baumgarten and Walz, 745
Bauzhaf and Steinhardt, 160
Beattie and Dickinson, 568
Beck and Pfeiffer, 517
Beck and Proskauer, 59, 726
Becker, 348
Beckman, 657

Beebe, Biggs, and Park, 468
Behring, 26, 116, 138, 155, 157,

214, 392, 471, 506, 729, 737, 747,

753

Behring and Kitasato, 117, 160
Behring and Nissen, 129
Behring and Nocht, 213
Belfanti and Carbone, 140, 163
Beninde, 728
Bensaude, 665
Benzancon, 443
Berestneff, 43
Berestnew, 804
Berg, 484

Bergell and Meyer, 649
Bergey, 629

Bergey and Abbott, 629, 631
Bergholm, 85
Berkefeld, 208
Bernheim, 746

Bernheim and Hildebrand, 82
Bertarelli, 790
Bertarelli and Bocchia, 211
Bertarelli and Volpino, 790
833

834

Bibliographic Index

Bertrand and Phisalix, 117, 118,161

Besredka, 357, 424, 490, 638

Besredka and Steinhardt, 124

Bettencourt and Franca, 429

Beyer, Rosenau, Parker, and Fran-
cis, 580

Beyerinck, 76

Bezancon, 364

Bielonovsky, 595

Biggs, 467

Biggs, Park, and Beebe, 468

Bignami, 525

Billroth, 24, 282

Biondi, 82, 199

Biondi and Heidenhain, 199

Birch-Hirschfeld, 727

Birt and Lamb, 522

Bitter and Sternberg, 70

Bittu and Klemperer, 757

Blanchard, 27, 537, 544

Blase and Russo-Travali, 464

Blum, 368

Blumer, 368

Boas-Oppler, 83

Bocchia and Bertarelli, 2 1 1

Bockhart, 86, 348

Boehm, 23

Boland, 367

Bellinger, 803

Bolton, 130, 1 68

Bolton and Globig, 237

Bolton and McBryde, 679

Bolton and Pease, 62

Bomstein, 468

Bonhoff, 631

Bonney and Foulerton, 501

Bonome, 116, 781

Bonome and Gros, 63

Bonome and Viola, 62

Bordet, 26, 120, 140, 141, 144, 146,
163, 166, 170, 322, 787

Bordet and Gay, 167

Bordet and Gengou, 119, 320, 335,
488, 489, 490, 491

Bordoni-Uffreduzzi, 372, 497, 501
765

Borrel, 595

Borrel and Roux, 391, 398

Bostrom, 803

Botkin, 261, 262, 263

Bowhill, 589

Boxmeyer, McClintock, and Siffer,
68 1

Boyce, 812

Boyce and Surveyor, 813, 817

Brault, 555

Braun, 705, 708

Brebeck-Fischer, 485

Brefeld, 46, 48

Breinl, 550

Brieger, 391, 612, 638

Brieger and Cohn, 391

Brieger and Ehrlich, 128, 376

Brieger and Frankel, 91, 391, 406,

461, 612, 638
Brown, 440, 441, 443, 444, 455, 572,

807

Brown and Wright, 804, 805
Bruce, 520, 522, 560, 562
Bruce and Nabarro, 557, 561
Bruce, Nabarro, and Greig, 561
Bruck and Neisser, 798
Bruck and Wassermann, 797
Bruck, Wassermann, and Neisser,

318, 319, 320, 322
Bruckner and Galasesco, 793
Brumpt, 557, 561, 562, 706, 707, 708
Brunner, 686
Buchner, 128, 129, 165, 263, 265,

622

Buchner and Daremberg, 141
Buchner and Nuttall, 128
Buerger, 495, 496, 504
Bujwid, 159

Bullock and Hunter, 367
Bumm, 348, 431, 462
Burri, 792

Burroughs and McCollum, 472
Busse, 818

Buswell and Kraus, 648
Biitschli, 525
Buxton, 663

Buxtori and Coleman, 66 1
Buxton and Torry, 126
Buxton and Vaughan, 151

CABOT, 419

Cadio, Gilbert, and Roger, 754

Calkin, 30

Calmette, 117, 161, 162, 368, 595,
650, 741

Calmette and Guerin, 753

Cameron, 732

Camus and Gley, 163

Canon, 514

Canon and Pfeiffer, 26

Cantani, 612

Capaldi, 659

Carbone and Belfonti, 140

Cardan, 18

Carmona y Valle, 576

Carrasquilla, 774

Carroll, 263, 576, 578

Carroll and Reed, 576, 580

Carroll, Reed, and Agramonte, 577

Carroll, Reed, Lazear, and Agra-
monte, 576

Bibliographic Index

835

Carter, 576, 577, 813
Carteras and Hughes, 506
Casagrandi and Barbagallo, 707
Castellani, 26, 557, 800, 80 1, 802
Catanni (Jr.), 518
Celli, 525, 688, 699
Celli and Fiocca, 690
Celli and Marchiafava, 423
Celli-Shiga, 664
Centanni and Tizzoni, 746
Chagas, 564, 565
Chamberland, 208, 409, 410
Chamberland and Roux, 116, 377,

412
Chantemesse, 633, 644, 648, 650,

651, 760
Chantemesse and Widal, 647, 648,

699

Chapin and Adami, 656
Charrin, 116, 365
Charrin and d'Arsonval, 62
Charrin and Roger, 102, 149, 760
Chauffard and Quenu, 398
Chauveau, 116, 409
Cheinisse, 442
Chenot and Picq, 784
Chester, 32, 41, 278, 279
Chevreul and Pasteur, 21
Cheyne, 98
Christmas, 435, 437
Christy, Button, and Todd, 557
Cienkowsky, 690
Citron, 334

Citron and Wassermann, 328
Ciuffo, 798
Clark, 476

Clarke and Miller, 211
Claudius, 211
Claussen and Abel, 611
Clegg, 767, 770
Clegg and Musgrave, 692, 694, 695,

696, 812

Cobbett, 1 06, 130, 475
Cohn, 36, 334, 448
Cohn and Brieger, 391
Cohnheim, 711
Colbach, 23

Coleman and Buxton, 66 1
Coley, 63, 358
Colla, 103

Comte and Nicolle, 546, 571
Comus and Gley, 169
Conn, 95
Conradi, 406

Conradi and Drigalski, 656
Cooley and Gelston, 90
Cooley and Vaughan, 669
Coplin, 181, 715
Cornet, 711

Cornevin, 114, 377
Cornil and Babes, 478, 608
Councilman, 355, 424, 476
Councilman and Lafleur, 27, 688,

689, 690, 691
Councilman, Mallory, and Pearce,

467

Countess del Cinchon, 525
Courmont, 745
Cousland, 80 1
Cowie, 757
Craig, 692, 693, 696
Crandenigo, 380
Creite, 163
Crocker, 828
Crocq, 420
Crooke, 355
Crookshank, 810
Cruveilhier, 160
Cumston, 671
Cunningham, 573
Curry, 511
Curtis, 44, 389, 4°4. 406, 509, 724,

808

Gushing, 640, 643, 664, 665
Czaplewski, 757, 764, 765
Czaplewski and Hensel, 488
Czenzynke, 514
Czerny, 358

D ALTON and Eyre, 520

Daniels, 196

Danysz, 686

Daremberg and Buchner, 141

Darling, 573, 574, 575

d'Arsonval and Charin, 62

Davaine, 24, 401

Davaine and Pollender, 25

Davidson, 524

Davis, 443, 444, 445, 488, 51?

Dean, 640

De Foe, 581

Deichler, 488

Delage, 30

Delepine, 214

Delezene, 121, 165

Delius and Kolle, 518

Denecke, 622, 623, 624, 631

de Mondeville, 22

Denny, 452, 455, 468

Denys, 742

Denys and van de Velde, 349

De Renzi, 506

De Schweinitz, 68 1, 736, 747

De Schweinitz and Dorset, 678

De Schweinitz and Veasy, 448, 449

Descos and Nicholas, 87

de Silvestri, 687

836

Bibliographic Index

Detre, 322

Detweiler, 90

Deutsch, 115

Deutsch and Feistmantel, 115

Devell, 591

Deycke, 237, 688

Dickinson and Beattie, 568

Dineur, 150

di Vestea and Maffucci, 747

Dobbin, 383

Doderlein, 486

Doderlein and Winterintz, 85

Doflein, 372, 551

Donitz, 132, 391, 398

Donne, 799

Donovan, 566

Donovan and Leishman, 27

Dopter and Vaillard, 702

Dorset, 679, 722, 723

Dorset and De Schweinitz, 678

Dorset, McBryde, and Niles, 679

Douglas and Wright, 127, 307, 316

Doutrelepont and Matterstock,

757

Draper, 670
Dreyfus, 670

Drigalski and Conradi, 656
Droba, 640
Drysdale, 560
Dubarre and Terre, 756
du Bary, 49
Dubois, 454
Du Bois-Reymond, 62
Duboscq and Leger, 707
Ducrey, 88, 442, 443, 766
Dujardin and Ehrenberg, 29
Dunbar, 631
Dunham, 240, 241, 379, 383, 458,

662, 669, 673
Dunham and Park, 699
Durham, 140, 665
Durham and Gruber, 149
Durme, 347

Dutton, 547, 554, 555, 556, 557
Dutton and Forde, 27, 557, 562,

563

Dutton and Todd, 547, 551, 557
Dutton, Todd, and Christy, 557
Duval, 763, 767, 768, 770
Duval and Bassett, 700
Duval and Vedder, 700

EAGER, 582

Eberth, 26, 293, 368, 632, 644, 760

Effront, 68

Ehlers, 368

Ehrenberg, 19, 292, 293

Ehrenberg and Dujardin, 29

Ehrlich, 26, 109, 117, 118, 131, 132,
136, 138, 143, 144, 145, 150, 153,
157, 158, 159, 162, 170, 182, 183,
185, 186, 187, 190, 391, 461, 471,
713, 7i5, 719, 764

Ehrlich and Brieger, 128, 376

Ehrlich and Morgenroth, 120, 122,
131, 163, 166, 322

Eichhorn and Mohler, 779, 783, 784

Eisenberg, 150, 278, 292, 293

Eisner, 650

Elders, 478

Ellermann, 479

Elmassian and Morax, 467

Elsching, 809

Elser, 426

Elser and Huntoon, 430

Eisner, 652, 653, 656, 673

Emery, 453

Emmerich, 666

Emmerich and Low, 77, 367

Emory, 83

Empedocles, 17

Endo, 659

Engle and Reichel, 421

Eppinger, 43

Ernst, 159, 365, 368, 383, 384

Ernst and Babes, 34, 175

Ernst and Robey, 150

Escherich, 37, 293, 664, 666

Esmarch, 223, 237, 246, 250, 251,
261, 288

Evans, 219, 820

Evans and Russell, 218

Eyre, 50

Eyre and Dalton, 520

FARRAN, 387

Fasching, 507

Favre, 798

Fehleisen, 350, 361

Fehling, 240, 737

Feistmantel and Deutsch, 115

Feletti and Grassi, 534

Fermi, 71

Fermi and Pernoss, 391

Fermi and Salsano, 756

Ferran, 617

Fick, 477

Ficker, 152

Field, 390

Finkelstein, 368

Finkler and Prior, 619, 620, 621,

622, 623, 631
Finlay, 576, 577
Fiocca, 189, 688, 699
Fiocca and Celli, 690
Firth, 572, 573

Bibliographic Index

837

Fisch, 747

Fischel and Wunschheim, 1 30

Fischer, 135

Fish, 147

Fitzpatrick, 596

Flatten, 424

Flexner, 43, 355, 373, 378, 380, 424,

427, 428, 429, 430, 467, 689, 699,

702, 704, 791
Flexner and Harris, 644
Flexner and Jobling, 430
Flexner and Noguchi, 162, 163, 390
Flexner and Welch, 377, 383, 464
Flournoy, Norris, and Pappen-

heimer, 550
Fliigge, 64, 94, 127, 129, 181, 216,

217, 292, 293, 294, 295, 365, 370,

385, 386, 424, 493, 601, 602, 728
Foa, 501
Fodor, 127
Forde, 555, 556
Forde and Button, 27, 557, 562,

563

Forneaca, 364
Forssner, 146
Foulerton, 42

Foulerton and Bonney, 501
Fournier and Gilbert, 677
Fox and Longcope, 493
Fracastorius, 22
Franca and Bettencourt, 429
Frances, Beyer, Rosenau, and

Parker, 580
Francis and Grubs, 73
Franke, 477
Frankel, 64, 106, 261, 262, 273, 294,

295, 355, 377, 407, 4ii, 424, 425,
477, 491, 492, 501, 508, 606, 607,
611, 612, 614, 622, 626, 634, 644

Frankel and Brieger, 91, 391, 406,
461, 612, 638

Frankel and Pfeiffer, 346, 351, 386,
387, 388, 400, 402, 403, 459, 503,
599, 607, 621, 626, 711, 725, 776

Frankel and Weichselbaum, 88,
373, 492, 509

Frankel and Wollstein, 491

Frankforter, 219

Frankland, 292, 293

Fredericq, 127

Freire, 576

Freymuth, 618

Friedlander, 85, 183, 450, 507, 508,
509, 510, 511, 512, 785, 786

Frisch, 368

Frosch, 463

Frosch and Kolle, 357

Frost, 64, 251, 252, 254, 255, 288,
290

Frothingham, 421

Frugoni, 725

Fuller, 225, 291

Fulleborn, 552

Fulleborn, Mayer, and Martin, 548

Funck and Metschnikoff, 121

GABBET, 716, 764, 772
Gabbi, 501

Gaffky, 361, 409, 632, 644, 689
Gaffky and Koch, 688
Galasesco and Bruckner, 793
Galeotti, 72

Galli-Valerio, 63, 592, 688
Gamaleia, 493, 499, 612, 625, 628,

631

Garini, 240
Garre, 86, 348
Gartner, 660, 664, 665, 675
Gaspard, 21

Gauss and Schumburg, 35
Gauthier, 798
Gay, 123

Gay and Bordet, 167
Gay and Southard, 123, 124
Gaylord and Aschoff, 200
Geddings and Wasdin, 577
Gelston, 90

Gelston and Cooley, 90
Gelston and Marshall, 90
Gengou, 170
Gengou and Bordet, 119, 320, 335,

488, 489, 490, 491
Germano and Maurea, 645
Gessard, 72, 292, 364
Gheorghiewski, 119, 367
Ghon, 589

Ghon and Albrech, 428
Ghoreyeb, 789
Ghriskey, 340, 341
Ghriskey and Robb, 81
Gibier, 102
Gibson, 159
Giemsa, 197, 480, 550, 706, 788,

790, 79i, 797
Gilbert and Fournier, 677
Gilbert, Cadio, and Roger, 754
Gilchrist, 44, 818, 819, 823.
Gilchrist and Stokes, 818, 821
Gildersleeve and Abbott, 452, 759
Gilliland and Pearson, 753
Gley and Camus, 163, 169
Globig and Bolton, 237
Goldhorn, 788
Goldschmidt, 427
Golgi, 525, 527
Goodby, 82
Goodsir, 282
Goodwin and Sholly, 430

838

Bibliographic Index

Goppert, 424

Gorden, 192

Gordon, 584, 615

Gordon and Andrewes, 341

Gorgas, 580

Gorham, 73, 452

Gottschalk and Immerwahr, 85

Gottstein, 122

Gourvitsch, 707

Graham, 383, 384

Graham and Irons, 44, 821

Gram, 182, 183, 184, 185, 198, 343,
350, 35i, 360, 361, 362, 364, 365,
369, 374. 377, 379, 385, 386, 399,
400, 403, 423, 425, 426, 430, 431,
432, 433, 437, 439, 442, 443, 446,
447, 449, 450, 451, 453, 480, 489,
492, 494, 495, 507, 509, 514, 520,
548, 550, 581, 583, 584, 598, 601,
602, 604, 608, 619, 625, 631, 632,
633, 675, 667, 676, 677, 678, 679,
682, 684, 699, 710, 714, 719, 760,
762, 764, 775, 776, 785, 787, 803,
805, 806, 812, 815, 817

Gram and Weigert, 185

Grassi, 529

Grass! and Feletti, 534

Grawitz, 45, 485, 486

Gregorieff, 687

Greig, Bruce, and Nabarro, 561

Griffon and Le Sours, 443

Grigorjeff and Ukke, 377

Grimme, 175

Grixoni, 387

Grohmans, 127

Gromakowsky, 359

Gros and Bonome, 63

Grosset, 486

Gruber, 140, 260

Gruber and Durham, 149

Gruber and Wiener, 616

Griibler, 175, 187, 653, 788

Grubs and Francis, 73

Gruby, 484, 824

Griinbaum, 645

Griinbaum and Widal, 649

Grysez and van Steenberghe, 87

Gscheidel and Traube, 127, 166

Guarniere, 497

Guerin and Calmette, 753

Guidi, 485

Guiteras, 580

Gunther, 231, 283, 289, 295, 344,
376, 631

Gwyn, 643, 665

HAECKEL, 29

Haffkine, 114, 585, 586, 596, 597,
616, 617, 646

Halberstadter, 802

Hall, 569

Hallein, 485

Hallier, 282

Hamburger, 651

Hamerton, 562

Hamilton, 476

Hankin, 64, 102, 128, 410

Hankin and Leumann, 588

Hankin and Wesbrook, 406

Hansen, 26, 68, 486, 762, 763, 765

Harris, 698

Harris and Flexner, 644

Harrison, 562

Harvey, 18

Hashimoto, 612

Hasslauer, 85

Haupt, 450

Hauser, 369, 370

Havelburg, 576, 594

Hebra, 824

Hegar, 85

Heidenhain, 199

Heidenhain and Biondi, 199

Heider, 631

Heim, 345, 363, 484, 515, 635, 609,

667

Heiman, 434
Hektoen, 732
Henle, 23

Hensel and Czaplewski, 488
Herman, 98
Herzog, 593
Hesse, 265, 284, 658
Hesse and Liborius, 265
Hewlett, 132, 136, 665
Hewlett and Nolen, 469
Heyman-Sticher, 728
Heymans, 132
Hildebrand, 118
Hildebrand and Bernheim, 82
Hill, 174, 250, 304, 657
Hippocrates, 687
Hirsch, 354
Hiss, 50, 216, 353, 427, 494, 495,

498, 505, 636, 654, 655, 656, 673,

702, 741, 792
Hiss and Russell, 699
Hiss and Zinsser, 41, 225, 352, 362,

424, 496, 497, 506, 632, 663, 777,

822

Hochst, 657, 744
Hodenpyl, 731
Hodenpyl and Prudden, 749
Hoffa, 405

Hoffmann and Prowazek, 799
Hoffmann and Schaudinn, 26, 82,

787, 788, 799, 801
Hofmann, 468, 474, 547, 636, 793

Bibliographic Index

839

Hogyes, 414, 419

Holmes, 23

Hoist, 95, 356

Horder, 517

Howard, 463, 474, 501, 512, 541

Howard and Perkins, 359, 360

Howard, Jr., 384

Hiickel and Losch, 48

Hughes, 522

Hughes and Carteras, 506

Humer, 640

Hunter and Bullock, 367

Huntoon and Elser, 430

Hunziker, 260

Hiippe, 293, 410, 60 1, 607, 608

Hiippe and Wood, 411

Huxley, 30

IMMERWAHR and Gottschalk, 85

Inchley and Nuttall, 148

Irons, 654

Irons and Graham, 44, 821

Israel, 803, 806, 809

IssaefF, 149

Itzerott and Niemann, 364, 620,

623, 625, 761
Iwanow, 405

JACKSON, 573, 66 1

Jacob, 670

Jacobsohn and Pick, 425

Jacoby, 147

Jadkewitsch, 368

Jager, 293, 424

Jamieson and Johnston, 772

Jasuhara and Ogata, 117, 410

Jenner, no, 113, 197, 315, 558, 568

Jez, 648

Jobling and Flexner, 430

Jochmann and Kraus, 488

Jodasshon, 798

Johnson, 665

Johnston and Jamieson, 772

Joos, 150

Jordan, 293, 366, 367, 649, 664

Jordan, Russell, and Zeit, 636

Jorgensen, 68

Justinian, 581

KAENSCHE, 69
Kamen, 396
Kanthack, 813
Kaplan, 330

Kaposi and Kundrat, 828
Karlinski, 341, 368, 675
Kartulis, 372, 373, 446, 688, 689,
691

Kashida, 654

Kastle, Rosenau, and Lumsden, 639

Kazarinow, 702

Keen, 641

Kerr, MacNeal, and Latzer, 84

Kimla, Poupe, and Vesley, 723

Kinghorn and Todd, 550

Kircher, 18, 22

Kirchner, 439, 441

Kitasato, 26, 116, 117, 208, 266,
273, 385, 386, 388, 389, 394, 395,
495, 58i, 583, 584, 585, 589, 596,
6n, 612, 688, 701

Kitasato and Behring, 160

Kitasato and Weil, 265

Kitasato and Yersin, 26

Kitt, 114

Klebs, 24, 451, 612, 687, 711, 736,
742

Klebs and Pasteur, 125

Klein, 587, 591, 592

Kleine, 562

Klemperer, 502

Klemperer and Bittu, 757

Klemperer and Levy, 513, 636, 638

Klemperer (G. and F.), 505

Klencki, 670

Klimenko, 490, 707, 708

Knapp, 475

Knapp and Novy, 549, 550, 552,
553

Knisl, 622

Knorr, 390, 595

Kny, 47

Koch, 23, 25, 26, 58, 125, 214, 217,
235, 236, 242, 246, 247, 249, 266,
269, 287, 303, 350, 368, 374, 400,
401, 407, 409, 446, 447, 528, 549,
551, 562, 601, 604, 605, 606, 607,
611, 612, 613, 614, 622, 627, 631,
632, 689, 691, 710, 711, 712, 713,
715, 721, 722, 729, 731, 737, 738,
739, 740, 742, 743, 744, 745, 746,
748, 750, 751, 788, 795

Koch and Gaffky, 688

Kohlbrugge, 84

Kolisko and Paltauf, 463

Kolle, 181, 594, 614

Kolle and Frosch, 357

Kolle and Otto, 349

Kolle and Pfeiffer, 638, 646, 648

Kolle and Wassermann, 42, 45, 48,
131, 430, 485, 520, 534, 536, 538,
547,586,763,765,830,831

Koplik, 488

Korn, 760

| Kossee and Overbeck, 690
I Kossel, 117, 118, 163, 169, 368
, Krai, 831

840

Bibliographic Index

Krannhals, 368

Kraus, 119, 140, 146, 147, 436

Kraus and Buswell, 648

Kraus and Jochmann, 488

Krauss, 347

Krefting, 442

Kronig, 211

Kronig and Menge, 383

Kronig and Paul, 213

Kruger, 62

Krumwiede and Park, 752

Kruse, 94, 369, 370, 399, 557, 602,

667, 689, 699, 702
Kruse and Pasquale, 688
Kubel and Tiemann, 657
Kuhne, 777

Kundrat and Kaposi, 828
Kurloff, 488
Kurth, 352, 355
Kutcher, 778
Kutschbert-and Neisser, 476

LAENNEC, 732

Lafleur and Councilman, 27, 688,

689, 690, 691
Laitinen, 434
Lamar and Meltzer, 499
Lamb and Birt, 522
Lambert, 391, 503
Lambl, 688, 689
Lammershirt, 478
Lamon and Meltzer, 511
Landois, 163
Landsteiner, 121
Langenbeck, 484, 803
Laplace, 213
Larkin, 43

Lartigau, 363, 368, 735
Laschtschenko, 728
Lassar, 787
Latapie, 165, 272, 273
Latour, 20

Latour and Schwann, 20
Latzer, MacNeal, and Kerr, 84
Laurent, 485
Laveran, 27, 525, 526, 527, 531,

532, 546, 558
Laveran and Mesnil, 555, 556, 559,

566, 567

La Wall and Leffmann, 231
Lazear, 576, 577
Lazear, Carroll, Reed, and Agra-

monte, 576
Leach, 90
Leber, 50, 346
Le Dantec, 385
Ledderhose, 366
Leffmann and La Wall, 231

Leger and Duboscq, 707

Lehman and Neumann, 278, 281

Leichtenstern, 423

Leishman, 197, 289, 307, 315, 316,
558, 566, 567, 568, 569

Leishman and Donovan, 27

Lemoine, 355

1'Engle and McFarland, 316

Lenglet, 445

Lennholm and Miller, 68

Le Noir, 368

Lentz, 700

Leo, 1 02, 784

Lepierre, 428

Lepp and Babes, 117, 422

Lesage, 292, 368, 672

Le Sours and Griffon, 443

Leubarth, 355

Leuchs and Von Lingelsheim, 429

Leumann, 596

Leumann and Hankin, 588

Levaditi, 315, 550, 790

Levaditi and Mamouelian, 792

Levaditi and Mclntosh, 788, 793

Levaditi and Nattan-Larrier, 802

Levaditi and Yamanonchi, 320

Levene, 737

Levin, 357

Levy, 501, 742

Levy and Klemperer, 513, 636, 638

Levy and Steinmetz, 779

Lewis, 27

Libman, 355, 665

Liborius, 262, 264

Liborius and Hansen, 261

Liborius and Hesse, 265

Lichtowitz, 477

Liebig, 20

Ligniere, 741

Limbourg, 660

Lincoln and McFarland, 506

Lindemann, 121

Linossier, 485

Linton and Thomas, 558

Lisbon, 593

Lister, 25, 209

Livingstone, 560

Lock wood, 210

Lofner, 26, 182, 189, 190, 236, 237,
379, 399, 409, 426» 432, 45i, 452,
453, 454, 457, 460, 469, 474, 475,
477, 480, 481, 514, 615, 628, 633,
634, 660, 664, 672, 684, 685, 776,
780

Loffler and Abel, 638, 648

Loiner and Shutz, 26, 601, 775, 781

Longcope, 665

Longcope and Fox, 493

Lord, 440, 441

Bibliographic Index

841

Losch, 27, 688, 689, 690, 691, 695
Losch and Hiickel, 48
Losener, 645
Low, 644

Low and Emmerich, 77, 367
Lubarsch, 129, 409, 675
Lubbert, 214
Lubenau, 357
Lubinski, 399
Lugol, 183
Liihe, 531

Lumsdcn, Rosenau, and Kastle,
639

MACCOLLUM, 527, 531

MacConkey, 66 1, 662

Macfadyen, 638, 648

Macfadyen and Rowland, 90, 638

Macgregor, 687

Mackie, 562

MacNeal, Latzer, and Kerr, 84

Madsen, 26, 120, 121, 131, 160

Madsen and Noguchi, 162

Madsen and Salmonson, 139

Maffucci and diVestea, 747

Mafucci, 754

Magendie, 122

Maggiora, 81, 368, 688

Maher, 670, 714

Malassez and Vignal, 760

Malfadyen, 498

Mallory, 186, 642, 698

Mallory and Wright, 197, 199, 424,

425
Mallory, Councilman, and Pearce,

467

Malmsten, 704, 705, 824
Malvoz, 150

Mamouelian and Levaditi, 792
Mann, 421
Mannatti, 347
Manson, 453, 525, 526, 527, 531,

542, 554, 557, 570, 708
Maragliano, 746, 747
Marburg, 334
Marchiafava, 525
Marchiafava and Celli, 423
Marchoux, 411

Marchoux and Salimbeni, 546
Marie, 422

Marie and Morax, 392
Marino, 197, 198, 199, 315, 558
Marmier, 406
Marmorek, 97, 356, 357, 359, 503,

506

Marshall and Gelston, 90
Martha, 368
Martin, 155, 162, 366, 406

I Martin and Meyer, 552

Martin and Roux, 130

Martin, Fulleborn, and Mayer, 548

Marx, 175
j Masselin and Thoinot, 598, 633

Matschinsky and Rymowitsch,
447, 449

Matterstock, 757

Matterstock and Doutrelepont, 757

Mattson, 122

Matzenauer, 478

Maurea and Germano, 645

Mayer, Fulleborn, and Martin, 548

McBryde and Bolton, 679

McBryde, Dorset, and Niles, 679

McCarthy and Ravenel, 419, 420

McClintock, 68 1

McCollum and Burroughs, 472

McConkey, 703

McConnell, 772

McCoy and Smith, 590

McDaniel and Wilson, 469

McFadyen, 753, 779

McFarland, 310, 319, 411, 437, 467,
472, 473, 479, 732, 747

McFarland and 1'Engle, 316

McFarland and Lincoln, 506

McFarland and Small, 73

Mclntosh and Levaditi, 788, 793

Mclntyre, 90

McNeal, 569

McNeal and Novy, 558, 565

Megnin, 560

Meier and Forges, 320
i Meirowsky, 798

Melcher and Ortmann, 770

Meltzer and Lamar, 499
| Meltzer and Lamon, 511
I Menge and Kronig, 383

Mense, 567

Mesnil, 127, 571

Mesnil and Laveran, 555, 556, 559,
566, 567

Messea, 35

Metalnikoff, 165

Metschnikoff, 26, 105, 107, 120,
121, 125, 126, 127, 128, 129, 130,
141, 144, 145, 149, 150, 169, 307,
732, 788

Metschnikoff and Funck, 121
i Metschnikoff and Metalnikoff, 121

Metschnikoff and Roux, 787

Meunier, 63
I Meyer, 269, 544

Meyer and Bergell, 649

Meyer and Martin, 552

Meyer and Ransom, 392, 394

Meyers, 119, 180

Michel, 454

842

Bibliographic Index

Migula, 32, 35, 38, 39, 40, 41, 84,

278, 280, 281, 282, 493, 508, 666,

685, 712, 786
Mikylicz, 786
Miller, 82, 83, 308, 309, 310, 311,

312, 313, 314, 315, 478, 546, 631,

640,

Miller and Clarke, 211
Miller and Lennholm, 68
Milne and Ross, 547, 551
Miquel, 286
Mitchell, 24

Mitchell and Stewart, 163
Mithridates, 116
Mittman, 81
Moeller, 188, 757, 758
Mohler and Eichhorn, 779, 783, 784
Monnier, 368

Montesano and Montesson, 396
Montesson and Montesano, 396
Montgomery, 87, 819, 820, 822
Montgomery and Walker, 820
Monti, 499

Moore and Taylor, 779
Morax, 446, 447, 448, 449, 450
Morax and Elmassian, 467
Morax and Marie, 392
Morgenroth, 119, 131, 141, 147
Morgenroth and Ehrlich, 120, 122,

131, 163, 166, 322
Mori, 508
Moriya, 751
Moro, 128
Morro, 741
Morse, 347
Moschowitz, 397
Moser, 359
Moses, 762
Mosso, 163
Motz, 368
Mouton, 127
Much, 713, 714
Muhlens, 793
Muir and Ritchie, 184, 188, 193,

555

Muller, 179, 259
Murphy, 809
Murray, 551
Musgrave and Clegg, 692, 694, 695,

696, 812

Musgrave and Strong, 699, 708
Myers, 118, 120, 121, 131, 147

NABARRO and Bruce, 557, 561
Nabarro, Bruce, and Greig, 561
Nattan-Larrier and Levaditi, 802
Neelow, 88
Negri, 420, 421, 422

Neisser, 26, 431, 441, 453, 475, 477,

631, 798

Neisser and Bruck, 798
Neisser and Kutschbert, 476
Neisser and Sachs, 171
Neisser and Wechsberg, 167, 168,

347, 349
Neisser, Wassermann, and Bruck,

318, 319, 320, 322
Nelis and Van Gehuchten, 419
Nepveu, 555
Nessler, 75
Netter, 501, 502
Neufeld, 504

Neuman and Swithinbank, 297
Neumann, 368, 469, 488
Neumann and Lehman, 278, 281
Newman, 194

Newman and Swithinbank, 726
Newmark, 334
Newsholme, 639
Nicati and Rietsch, 612, 613
Nicholas, 798
Nicholas and Descos, 87
Nicholls, 87

Nichols and Schmitter, 263, 264
Nicolaier, 26, 385
Nicolas and Descos, 729
Nicolaysen, 435

Nicolle, 185, 442, 478, 571, 770
Nicolle and Comte, 546, 571
Niemann and Itzerott, 364, 620,

623, 625, 761

Niles, Dorset, and McBryde, 679
Nisot, 462
Nissen, 129, 214
Nissen and Behring, 129
Nobe, 798

Nocard, 43, 395, 397, 665, 677, 722
Nocard and Railliet, 560
Nocard and Roux, 234, 722
Nocht and Behring, 213
Noguchi, 27, 162, 320, 322, 326,

327, 335, 336, 337, 338, 788, 793,

794, 795, 798, 799
Noguchi and Flexner, 162, 163, 390
Noguchi and Madsen, 162
Noisette, 487
Nolen and Hewlett, 469
Nolf, 147
Norris, 43

Norris and Oliver, 72
Norris, Pappenheimer, and Flour-

noy, 550
Novy, 177, 260, 261, 277, 547, 548,

569, 680, 757
Novy and Knapp, 549, 550, 552,

553
Novy and McNeal, 558, 565

Bibliographic Index

843

Novy and Vaughan, 68, 298
Nuttall, 127, 128, 148, 174, 175,

407, 592, 717
Nuttall and Buchner, 128
Nuttall and Inchley, 148
Nuttall and Welch, 377, 379, 382

OBERMEIER, 24, 26, 546

Oertel, 464

Oetinger, 368

Ogata, 583, 589, 592, 687

Ogata and Jasuhara, 117, 410

Ogston, 343, 350

Ohlmacher, 457, 474, 644, 718

Oliver and Norris, 72

Olsen, 485

Ophuls, 44, 819

Oppenheim, 334

Oppler-Boas, 83

Oriste-Armanni, 60 1

Orlowski and Palmirski, 461

Orth, 185

Ortmann, 497

Ortmann and Melcher, 770

Oshida, 414

Osier, 687, 688, 689

Otto, 122

Otto and Kolle, 349

Overbeck and Kossee, 590

Ovid, 17

Oviedo, 800

PALMIRSKE and Orlowski, 461

Paltauf, 48

Paltauf and Kolisko, 463

Pane, 506

Panfili, 213

Pansini, 368

Pappenheim, 717

Pappenheimer, Norris, and Flour-

noy, 550
Paquin, 746
Pariette, 657
Park, 97, 266, 388, 426, 427, 452,

453, 470, 473, 474
Park and Dunham, 699
Park and Krumwiede, 752
Park, Briggs, and Beebe, 468
Parke and Williams, 493
Parker, Rosenau, Francis, and

Beyer, 580

Pasquale and Kruse, 688
Passet, 343, 350, 666
Passler, 506
Pasteur, 19, 20, 25, 26, 102, 113,

114, 135, 260, 264, 374, 407, 409,

410, 412, 413, 416, 417, 419, 420,

492, 598, 600

Pasteur and Chevreul, 21

Pasteur and Klebs, 125

Pasteur and Toussaint, 598

Pasteur- Chamberland, 207, 208

Paterson, 747

Patton, 570

Paul and Kronig, 213

Paulicki, 754

Pawlowski, 63, 724

Pawlowsky, 616, 618

Peabody and Pratt, 641, 650, 660

Pearce, 355, 463, 464

Pearce, Councilman, and Mallory,

467

Pearson, 781

Pearson and Gilliland, 753
Pease and Bolton, 62
Peckham, 671
Perkins, 368, 510
Perkins and Howard, 359, 360
Pernoss and Ferni, 391
Perroncita, 664
Perroncito, 598
Peterson, 442
Petkowitsch, 656
Petri, 246, 249, 285, 475, 760
Petruschky, 41, 43, 240, 356, 643,

645, 664, 676, 740
Pfeiffer, 120, 140, 141, 142, 166,

181, 234, 271, 514, 515, 516, 517,

518, 519, 616, 625, 627, 628, 629,

760, 761

Pfeiffer and Beck, 517
Pfeiffer and Canon, 26
Pfeiffer and Frankel, 346, 351, 375,

386, 387, 388, 400, 402, 403, 459,

503, 599, 607, 621, 626, 711, 725,

776

Pfeiffer and Kolle, 638, 646, 648
Pfeiffer and Vogedes, 616
Pfuhl, 372, 738
Phisalix and Bertrand, 117, 118,

161

Pianese, 571
Piatkowski, 720
Pick and Jacobsohn, 425
Picq and Chenot, 784
Pictet and Yung, 65
Pierce, 502
Piorkowski, 655, 673
Pitfield, 191, 392, 68 1
Platania, 102

Plaut, 48, 478, 479, 485, 487, 831
Plencig, 23
Plett, no
Pohl, 293
Pollender, 24

Pollender and Davaine, 25
Pope, 648

844

Bibliographic Index

Forges and Meier, 320

Portier and Richet, 122

Posadas and Wernicke, 819

Pott, 63

Poupe, Kimla, and Vesley, 723

Pratt and Peabody, 641, 650, 660

Preindelsberger, 81

Prescott, 68, 666

Prescott and Winslow, 239

Prior and Kinkier, 619, 620, 621,

622, 623, 631
Proca and Babes, 747
Proskauer and Beck, 59, 726
Prowazek, 372, 559
Prowazek and Hoffmann, 799
Prudden, 289, 354, 731
Prudden and Hodenpyl, 749

QUENU and Chauffard, 398
Quincke, 830
Quincke and Roos, 688
Quinquaud, 485

RABINOWITSCH, 297, 760, 818

Railliet and Nocard, 560

Ramon, 392

Ransom and Meyer, 392, 394

Rappaport, 589

Raskin, 355

Ravenel, 64, 87, 233, 237, 238, 243,
261, 292, 293, 295, 729, 750

Ravenel and McCarthy, 419, 420

Redi, 1 8

Reed, 576

Reed and Carroll, 576, 580

Reed, Carroll, and Agramonte, 577

Reed, Carroll, Lazear, and Agra-
monte, 576

Reed, Vaughan, and Shakespeare,

639

Reichel, 208, 723
Reichel and Kngle, 421
Remak, 828
Remy, 653
Ress, 485
Rettger, 85, 410
Reyes, 470
Ribbert, 347, 349
Richardson, 643, 644, 652
Richet and Portier, 122
Ricketts, 819
Rideal, 214

Rideal and Walker, 305
Riedel, 606
Rieder, 63
Rieger, 424
Rietsch and Nicati, 612, 613

Rindfleisch, 627

Rist, 461

Ritchie and Muir, 184, 188, 193,

555

Ritter, 488
Rivolta, 27, 754
Robb, 340, 341
Robb and Ghirskey, 81
Robb and Welch, 210
Robertson, 637
Robey and Ernst, 150
Robin, 484, 485
Robinson, 218
Rodet, 349

Roger, 98, loo, 410, 487
Roger and Charrin, 102, 149, 760
Roger, Cadio, and Gilbert, 754
Rogers, 438, 566, 569, 570
Rogone, 211
Rolleston, 670
Roloff, 754
Romanowsky, 197, 199, 550, 558,

788

Roos and Quincke, 688
Rosenau, 124, 159, 218, 472, 589,

686, 724, 727

Rosenau and Anderson, 123, 161
Rosenau, Lumsden, and Kastle,

639
Rosenau, Parker, Francis, and

Beyer, 580

Rosenbach, 343, 349, 350, 361
Rosenberger, 250
Rosenow, 502, 506
Roser, 125

Ross, 200, 526, 527, 566, 567, 577
Ross and Milne, 547, 551
Rossi, 193
Rost, 766, 774
Rothberger, 654
Roudoni and Sachs, 337
Rouget and Vaillard, 396
Roux, 26, 116, 156, 204, 260, 264,

269, 409, 453, 475, 477, 485
Roux and Borrel, 391, 398
Roux and Chamberland, 116, 377,

412

Roux and Martin, 130
Roux and Metschnikoff, 787
Roux and Nocard, 234, 722
Roux and Yersin, 91, 1 16, 460, 461,

464

Rowland and Macfadyen, 90, 638
Rudolph, 767
Ruediger, 354, 505
Ruffer, 616
Rumpf, 648
Ruppel, 736, 737
Russell, 702

Bibliographic Index

845

Russell and Evans, 218
Russel and Hiss, 699
Russell, Jordan, and Zeit, 636
Russo-Travali and Blasi, 464
Ruzicka, 365

Rymowitsch and Matschinsky,
447, 449

SABOURAUD, 824, 825, 826, 829, 831

Sabrazes, 478

Sacharoff, 546

Sachs and Neisser, 171

Sachs and Roudoni, 337

Salant, 103

Salimbeni and Marchoux, 546

Salkowski, 73, 669

Salmon, 602, 603, 664, 678, 750

Salmon and Smith, 116, 601, 678,

679

Salmonson and Madsen, 139
Salomonsen, 266
Salsano and Fermi, 756
Sambon, 561
Sanarelli, 64, 576, 577, 664, 682,

683, 684
Sander, 724
Sanfelice, 369, 399, 818
Sattler, 477
Savage, 654
Scala, 699
Schaudinn, 547, 565, 689, 690, 691,

693, 695
Schaudinn and Hoffmann, 26, 82,

787, 788, 799, 80 1
Scherer, 424, 426
Schereschewsky, 793
Schering, 179, 215
Schick and von Pirquet, 122
Schleich, 477
Schmidt, 424

Schmitter and Nichols, 263, 264
Schneider, 214, 424
Schonlein, 828
Schottelius, 609
Schottmuller, 353, 354, 359
Schroder, 501

Schroder and Van Dusch, 25, 204
Schroter, 293
Schuffner, 537
Schulze, 19

Schumburg and Gauss, 35
Schutz and Loffler, 26, 601, 775,

781

Schutze and Wassermann, 119, 148
Sch waive, 401
Schivam and Latour, 20
Sedgwick, 285, 286, 661
Sedgwick and Tucker, 286

Sedgwick and Winslow, 65, 637
Seifert, 439
Selter, 241, 784
Semmelweiss, 23
Semple and Wright, 646
Shakespeare, 610, 621, 624
Shakespeare, Vaughan, and Reed,

639

Shaw, 97

Shiga, 26, 688, 689, 699, 702, 704
Sholly and Goodwin, 430
Sievenmann, 50
Siffer, McClintock, and Boxmeyer,

68 1

Silber, 299
Silverschmidt, 372
Simon, 357
Simpson, 582, 587
Sjoo and Tornell, 720
Small and McFarland, 73
Smirnow, 62
Smith, 192, 228, 230, 291, 461, 5.1 1,

643, 664, 673, 679, 680, 681, 722
Smith (L.)» 194. 237
Smith (T.), 70, 122, 151, 156, 241,

266, 390, 662, 723, 748, 749, 750,

754

Smith and McCoy, 590
Smith and Salmon, 116, 601, 678,

679

Smith and Weidman, 373
Sobernheim, 612, 616
Solowiew, 708
Somers, 658

Southard and Gay, 123, 124
Sowade, 793
Soyka, 58
Spallanzani, 19
Spengler, 488
Spiller, 420

Spronck, 460, 699, 766
Starkey, 657, 658
Steel, 430
Steele, 84, 648

Steinhardt and Bauzhaf, 160
Steinhardt and Besredka, 124
Steinmetz and Levy, 779
Stern, 130, 648, 789, 797
Sternberg, 125, 301, 303, 346, 353,

368, 389, 492, 576, 611, 636, 633
Sternberg and Bitter, 70
Stewart, 178, 384
Stewart and Baldwin, 383
Stewart and Mitchell, 163
Sticker, 50, 771
Stiles, 690

Stimson, 414, 415, 416
Stitt, 542
Stokes, 296, 654

846

Bibliographic Index

Stokes and Gilchrist, 818, 821

Stokvis and Winogradow, 708

Stooss, 486

Strasburger, 84

Straus, 778

Strehl, 222

Strong and Musgrave, 699, 708

Stiihlerm, 508

Sugai, 770

Surveyor and Boyce, 813, 817

Swithinbank and Neuman, 297

Swithinbank and Newman, 726

Szemetzchenko, 488

TAKAKI and Wassermann, 118, 391

Tangl, 461

Tashiro, 770

Taube and Weber, 756

Tavel, 47, 359, 399, 636

Tavel and Alvarez, 757

Taylor and Moore, 779

Tchistowitch, 119, 146, 499

Tedeschi, 783, 798

Telamon, 492

Terre and Bugarre, 756

Theiler, 546

Thelling, 744, 746

Theodoric, 22

Thiercelin, 368

Thoinot and Masselin, 598, 633

Thomas, 114

Thomas and Linton, 558

Thompson, 696

Thompson- Yates, 556, 661 r

Tinctin, 550

Tidswell, 593

Tiemann and Kubel, 657

Timpe, 228

Tizzoni and Centanni, 746

Todd, 555

Todd and Button, 547, 551, 557

Todd and Kinghorn, 550

Todd, Button, and Christy, 557

Tornell and Sjoo, 720

Torrey, 437, 438

Torrey and Buxton, 126

Toussaint, 409

Toussaint and Pasteur, 598

Trambusti, 65

Traube and Gscheidel, 127, 166

Trendenburg, 355

Treskinskaja, 61

Triboulet, 368

Trillat, 218

Trudeau, 744

Trudeau and Baldwin, 742, 744

Tsiklinsky, 64

Tsugitani, 694

Tucker and Sedgwick, 286
Tunnicliff, 479, 480, 481, 482
Tyndall, 1 8, 19, 58

, 462
Uhlenhuth, 147
Uhlenhuth and Xylander, 720
Ukke and Grigorjeff, 377
Unna, 81, 442, 443, 719, 791, 792
Uschinsky, 461

VAIU,ARD and Bopter, 702

Vaillard and Rouget, 396

Valagussa, 688

Van de Velde, 347, 359

Van de Velde and Benys, 349

Van Busch and Schroeder, 25, 204

Van Ermengem, 192, 299, 612, 613,

790

Van Gehuchten and Nelis, 419
Van Helmont, 18
Van Leeuwenhoek, 18, 20, 29
Van Steenberghe and Grysez, 87
Varro, 21

Vaughan, 90, 124, 298, 558, 638
Vaughan and Buxton, 151
Vaughan and Cooley, 669
Vaughan and Novy, 68, 398
Vaughan, Reed, and Shakespeare,

639

Veasy and de Schweinitz, 448, 449
Vedder and Buval, 700
Veillon, 479
Verneuil, 385

Vesley, Kimla, and Poupe, 723
Viereck, 690, 693
Vierordt, 608

Vignal and Arustamoff, 42
Vignal and Malassez, 760
Villemin, 711
Villiers, 612
Vincent, 478, 479, 483, 812, 813,

814

Vincentini, 82
Vincenzi, 488
Viola and Bonome, 62
Viquerat, 349, 746
Virchow, 740, 771
Virgil, 17

Vogedes and Pfeiffer, 616
Voll, 150

Volpino and Bertarelli, 790
Von Bungern, 119, 120, 121, 140,

165

Von Frisch, 785
Von Ley den, 649
Von Lingelsheim, 346, 351, 424

Bibliographic Index

847

Von Lingelsheim and Leuchs, 429

Von Mayer, 399

Von Pirquet, 740, 741, 797

Von Pirquet and Schick, 122

Von Szekely, 37

Vuillemin, 485

WADSWORTH, 499, 504

Wagner, 102

Walger, 648

Walker, 145

Walker and Montgomery, 820

Walker and Dideal, 305

Walz and Baumgarten, 745

Warren, 230, 457

Wasdin and Geddings, 577

Washbourn, 503, 506

Wassermann, 26, n 8, 147, 171, 318,
319, 322, 324, 326, 328, 330, 331,
333, 334, 335, 336, 338, 367, 434,
435, 437, 797, 799

Wassermann and Bruck, 797

Wassermann and Citron, 328

Wassermann and Kolle, 42, 45, 48,
131, 430, 485, 520, 534, 536, 538,
547, 586, 763, 765, 830, 831

Wassermann and Schiitze, 119, 148

Wassermann and Takaki, 118, 391

Wassermann, Neisser, and Bruck,
318, 319, 320, 322

Weaver, 479

Weber and Traube, 756

Wechsberg and Neisser, 167, 168,

347, 349

Weeks, 446, 447, 448, 477
Weibel, 631
Weichselbaum, 293, 423, 424, 425,

426, 429, 430, 492, 501, 502, 644
Weichselbaum and Frankel, 88,

373, 492, 509
Weidman and Smith, 373
Weigert, 25, 137, 172, 174, 403, 454,

494, 495, 719, 764, 806, 815, 817
Weigert and Gram, 185
Weil and Kitasato, 265
Weinzirl, 61
Welch, 81, 131, 145, 341, 377, 384,

397, 454, 472, 494
Welch and Flexner, 377, 383, 464
Welch and Nuttall, 377, 379, 382
Welch and Robb, 210
Wellenhof, 474
Welsh, 340

Wernich and Chauveau, 125
Wernicke, 26, 589, 631
Wernicke and Posadas, 819
Wertheim, 431, 433, 434
Wesbrook, 452, 454, 469, 657
Wesbrook and Hankin, 406

Wesenberg, 372

Wheeler, 90

Widal, 149, 633, 640, 644, 649, 650,

653, 673
Widal and Chantemesse, 647, 648,

699

Widal and Griinbaum, 649
Wiener and Gruber, 616
Wiens, 670
Wigura, 81

Willcomb and Winslow, 288
Williams, 368, 383, 462, 695, 792
Williams and Lowden, 421
Williams and Parke, 493
Wilson, 588, 589
Wilson and McDaniel, 469
Windsor and Wright, 522
Winkler, 259

Winogradow and Stokvis, 708
Winogradsky, 74
Winslow, 8 1

Winslow and Prescott, 239
Winslow and Sedgwick, 65, 637
Winslow and Willcomb, 288
Winterbottom, 554
Winterintz and Doderlein, 85
Witte, 228, 229, 241, 505, 653,657,

659, 662

Wladimiroff, 549
Wolf, 502
Wolff, 650, 806
Wolff-Eisner, 742, 798
Wolffhugel, 287, 288, 606
Wolfhugel, 287, 288
Wollstein, 490, 519, 700
Wollstein and Frankel, 491
| Wood, 177, 507
Wood and Hiippe, 411
Woodhead, 36, 809
Woodward, 687
Wright, 1 15, 197, 264, 266, 267, 287,

289, 292, 293, 308, 311, 312, 315,

317, 349, 355, 434, 438, 522, 558,

568, 572, 638, 646, 647, 745, 783,

807, 815, 816, 817
Wright (J. H.), 573
Wright and Brown, 804, 805
Wright and Douglas, 127, 307, 316
Wright and Mallory, 197, 199, 424,

425

Wright and Semple, 646
Wright and Windsor, 522
Wschinsky, 669
Wunschheim and Fischel, 130
Wiirtz, 653, 668, 675
Wyman, 583, 589
Wynekoop, 519
Wyssokowitsch and Zabolotny,

591, 597

848

Bibliographic Index

XYLANDER and Uhlenhuth, 720

YAMANONCHI and Levaditi, 320
Yersin, 581, 582, 583, 584, 585, 588,

589, 590, 59i, 592, 594, 595, 596,

597

Yersin and Kitasato, 26
Yersin and Roux, 91, 116, 460, 461,

464

Young, 433, 435, 436
Yung and Pictet, 65

ZABOLOTNY and Wyssokowitsch,

591, 597
Zaufal, 501

Zeit, 62, 782

Zeit, Jordan, and Russell, 636

Zenker, 179, 186, 199

Ziegler, 687

Ziehl, 181, 189, 634, 716, 717

Zieler, 186

Zimmermann, 292, 293

Zinno, 460

Zinsser, 264, 511

Zinsser and Hiss, 41, 225, 352, 362,

424, 496, 497, 506, 632, 663, 777,

822

Zopf, 37, 293, 507
Zuber, 479
Zupinski, 260
Zupnik, 393
Zur Nedden, 450

INDEX

ABBOTT'S method of staining spores,

1 88

Abscess of liver in amebic dysen-
tery, 697
tuberculous, 731
Acetic fermentation, 67
Achorion, 46
schonleinii, 828
cultivation, 830

Krai's method, 831
pathogenesis, 831
Acid, carbolic, as disinfectant, 215

tuberculinic, 737
Acids as germicides, 213

production of, 71
Actinodiastase, 127
Actinomyces, 43, 803, 804
bovis, 803

cultivation, 806
distribution, 804
general characteristics, 803
lesions, 811
morphology, 804
pathogenesis, 809
virulence, 809
grain, 805
madurae, 812
cultivation, 814
cultural characteristics, 817
general characteristics, 812
lesions, 814
morphology, 813
Actinomycosis, 803

communication of, to man, 809
Acute contagious conjunctivitis,

446

Adami and Chapin's method for
isolation of typhoid bacillus, 656
Addiment, 120, 144
Adhesion preparations, 256
Aerobes, 59
Aerogens, 66
African lethargy, 554
Agar-agar as culture-media, 232
bile-salt, 662

blood, as culture-media, 234
culture, 255

glycerin, as culture-media, 234
54

Agar-agar, litmus-lactose, 654
preparation of, 233

Ravenel's method, 233
Agglutination, 149

test, Koch's, for tubercle bacillus,

746

technic of, 151
Widal reaction of, 649
Agglutinins, 140
Agglutometers, 650
Aggressins, 146
Ague-cake, 540
Air, bacteria in, 58, 283

quantitative estimation, by

Hesse's method, 284
by Petri's method, 285
by Sedgwick's method,

286

bacteriology of, 283
displacement of, by inert gases,

in anaerobic cultures, 260
of sick-room, disinfection, 211
withdrawal of, in anaerobic cul-
tures, 260
Air-examination, Petri's sand filter

for, 285
Sedgwick's expanded tube for,

285

Alcoholic fermentation, 67
Aleppo boil, 572
Alexin, 128, 144, 166
Algid cases of malaria, 539
Alkali-albuminate, Deycke's, as

culture- media, 237
Alkalies, production of, 71
Alkaline blood-serum as culture-
media, 237
Allergia, 123
Altmann's syringes, 269
Amboceptor dose in Wassermann

reaction, 326

hemolytic, for Wassermann re-
action, 323
unit in Wassermann reaction,

325, 326

Amboceptors, 144, 145
Amebadiastase, 127
i Amebae and suppuration, 372

849

850

Index

Amebic dysentery, 689

abscess of liver in, 697
lesions, 697
American trypanosomiasis, 564

transmission, 565
Amoeba coli, 689

rhizopodia, 690
dysenteriae, 689
kartulisi as cause of suppuration,

372

martinatalium as cause of suppu-
ration, 373
Anaerobes, 59
facultative, 59
optional, 59
Anaerobic bacteria, cultivation of,

260
by absorption of atmospheric

oxygen, 263

by displacement of air, 259
by exclusion of atmospheric

oxygen, 265

by formation of vacuum, 260
by reduction of oxygen, 264
by withdrawal of air, 260
cultures, Botkin's apparatus for

making, 262
Buchner's method of making,

263
Frankel's method of making,

261

Hesse's method of making, 265
Koch's method of making, 266
Liborius' tube for, 261
Nichols and Schmitter's meth-
od of making, 263
Novy's jars for, 261
Salomonsen's method of mak-
ing, 266
Wright's method of making,

264, 267
Zinsser's method of making,

264

Anaphylactin, 124
Anaphylaxis, 122

passive, 124

Anesthetic leprosy, 771, 773
Angina, Vincent's, 478
Animal holder, Latapie's, 272, 273

inoculations, 271
Animalculae, 29
Animals, experimentation upon,

269
method of securing blood from,

273

postmortems on, 275
Anjeszky's method of staining

spores, 1 88
Anopheles maculipennis, 541, 542

Anthracin, 406
Anthrax, 400

bacillus of, 400

bacteriologic diagnosis, 411

causes of death from, 409

distribution, 400

in cattle, how acquired, 407

lesions, 407

means of protecting animals
against, 410

Pasteur's protective inoculation
against, 409

sanitation in, 411

serum therapy in, 410

vaccination in, 409
Antibiosis, influences on growth of

bacteria, 63

Antibodies, miscellaneous, 163
Anticholera serum, 617
Antiferments, 140
Antiformin for isolation of tubercle

bacillus, 720
Antigen, 115

syphilitic, 319

titration of, 328
Antigonococcus serum, 437
Anti-immune bodies, 169
Antikorper, 140, 471
Antimeningococcus serum, 430
Antiphthisin, 742, 743
Antipneumococcus serum, 505, 506
Antirabic serum, 422
Antisepsis, 25

early, 25

Antiseptic action, results of, 303
Antiseptics, 201

determination of value, 302

influence on growth of bacteria,

6.5 . .
inhibition strengths of, 216

Antispermotoxin, 121

Antistaphylococcus serum, 349

Antistreptococcus serum, 359

Antistreptokolysin, 357

Antitoxin of diphtheria, 155, 471
bleeding, 156
effect on death-rate, 473
immunization of animals, 156
method of administration, 471
paralysis after use of, 472
potency of serum, 157
preparation of serum, 157

of toxin, 156
prophylaxis, 471
tetanus after use of, 473
treatment with, 471
tetanus, 160, 397

Antitoxins, 153

Antitubercle serums, 746

Index

851

Antityphoid serums, 648

Antivenene, 161

Antivenomous serum, 161

Apilacao, 564

Apparatus, complete leveling, for

pouring plate cultures, 247
Hesse's, for collecting bacteria

from air, 284

Koch's, for coagulating and
sterilizing blood-serum, 235,
236
Wolfhiigel's, for counting colonies

of bacteria upon plates, 287
Aqueous solution for staining, 177
Argas miniatus, 546
Arnold's steam sterilizer, 205
Aromatics, production of, 73
Arthrospores, 38
Ascococcus, 39
Asiatic cholera, 604

immunity against, 616

prophylaxis, 617

rice-water discharges of, 613

sanitation in, 618

specific organism of, discovery,

605

Aspergillus, 48
flavus, 50
fumigatus, 49
glaucus, 49
malignum, 49
nidulans, 49
niger, 50
subfuscus, 50
Association, influence on growth

of bacteria, 63

Atmospheric oxygen, absorption
of, in anaerobic cultures, 263
exclusion of, in anaerobic cul-
tures, 265

Auditory meatus, external, bac-
teria in, 82
Autoclave, 206

sterilization in, 206
Axenfeld, bacillus of, 448

BABES and Cornil's method of stain-
ing spirillum cholerae Asiaticae,
608

Babes-Ernst granules, 34
Babes' tubercles, 419
Bacillary dysentery, 699
diagnosis, 704
lesions, 702
serum therapy, 704
emulsion, 745
Bacilli, paracolon, 665
paratyphoid, 665

Bacilli resembling Bacillus diph-

theriae, 474
of anthrax, 411
typhosus, 663

meat-poisoning group, 665
pneumonic group, 665
psittacosis group, 665
table for differentiation,

664

typhoidal group, 665
tetanus bacillus, 399
tubercle bacillus, 756
Bacillus, 39

acidi lactici, 666
aerogenes capsulatus, 377
cultivation, 380
distribution, 378
morphology, 378
pathogenesis, 382
sources of infection, 383
staining, 379

Welch and Nuttall's meth-
od, 379

vital resistance, 382
anthracis, 400

avenues of infection, 406

bacilli resembling, 411

cultivation, 403

isolation, 403

means of diminishing virulence,

409

metabolic products, 405
morphology, 402
pathogenesis, 406
similis, 411
staining, 403
thermic sensitivity, 405
anthracoides, 411
avicidum, 598
avisepticus, 598
Bordet-Gengou, 488

and influenza bacillus, differ-
ences between, 491
cultivation, 490
isolation, 489
metabolic products, 490
morphology, 489
pathogenesis, 490
staining, 489
butter, 760
butyricus, 760
canal-water, capsulated, 508
capsulated canal-water, 508
capsulatus mucosus, 507
capsule, 509
cavicida, 666
choleras, 598
gallinarum, 598
cultivation, 598

852

Index

Bacillus choleras gallinarum, gen-
eral characteristics, 598
immunity against, 600
lesions, 600

metabolic products, 599
morphology, 598
pathogenesis, 600
staining, 598
vital resistance, 599
coli communior, 669
communis, 666

bacillus typhosus and, differ-
entiation, 651
cultural, 652
serum, 651
cultivation, 667
diagnosis, differential, 672
distribution, 666
general characteristics, 666
immunization against, 672
in summer infantile diarrhea,

671
in water, 291, 674

MacConkey's medium for

detecting, 66 1
Wiirtz's medium for de-
tecting, 675
influence of environment on,

671

metabolic products, 668
morphology, 667
pathogenesis, 669
staining, 667
toxic products, 669
virulence, 670
vital resistance, 668
comma, 606
cuniculicida, 598, 601
diphtherias, 451

bacilli resembling, 474
bacteria associated with, 464
bacteriologic diagnosis, 456
chief types, 469
classification of types, 469
contagion from, 468
cultivation, 454

lyoffler's method, 454
differentiation of, from pseu-
dodiphtheria bacillus, 468,

474

general characteristics, 451
metabolic products, 460
morphology, 451
pathogenesis, 462
relation of, to diphtheria, 467
seats of infection by, 464, 465
specificity, 467
staining, 452

Neisser's method, 453

Bacillus diphtherias, staining with
Lofner's alkaline methylene-
blue, 452

vital resistance, 460

Wesbrook's types of, 452, 469
dysenteriae, 699

cultivation, 700

Flexner variety, 702

Hiss-Russel variety, 702

metabolic products, 701

morphology, 700

pathogenesis, 702

Shiga-Kruse variety, 702

staining, 700

varieties, 702

vital resistance, 701
enteritidis, 675

cultivation, 675

general characteristics, 675

lesions, 676

morphology, 675

pathogenesis, 676

staining, 675
faecalis alkaligenes, 676
fluorescens liquefaciens, 365
fusiformis, 478

and spirochaeta Vincenti, re-
lation, 479

cultivation, 480

morphology, 480

pathogenesis, 483
Gartner's, 675
geniculatus in carcinoma of

stomach, 83
icteroides, 682

cultivation, 682

distribution, 682

metabolism, 683

morphology, 682

pathogenesis, 683

staining, 682

vital resistance, 683
influenzas, 514

and Bordet-Gengou bacillus,
differences between, 491

cultivation, 516

immunity against, 517

isolation, 515

morphology, 514

pathogenesis, 517

pseudo-, 519

specificity, 517

staining, 514

vital resistance, 517
Klebs-Loffler, 45 1 . See also Ba-
cillus diphtheria.
lactis aerogenes, 666
leprae, 762

cultivation, 765

Index

853

Bacillus leprae, cultivation, Clegg's

method, 767

Duval's method, 767, 768
Rost's method, 766
distribution, 762
general characteristics, 762
lesions, 771
morphology, 763
pathogenesis, 770
staining, 763
mallei, 775

cultivation, 779
distribution, 776
general characteristics, 775
immunity against, 784
isolation, 777
metabolic products, 780

mallein, 781
morphology, 776
pathogenesis, 781
staining, 776

Kiihne's method, 777
Loffler's method, 776
virulence, 784
vital resistance, 777
melitensis, 520. See also Micro-
coccus melitensis.
Moeller's grass, 758
neapolitanus, 666
Nocard's, 677
cedematis maligni, 374
cultivation, 374
distribution, 374
immunity against, 377
lesions, 376

metabolic products, 375
morphology, 374
pathogenesis, 376
staining, 374

of anthrax, 400. See also Bacil-
lus anthracis.
of Axenfeld, 448
of Buffelseuche, 601
of chicken-cholera, 598. See also

Bacillus cholera gallinarum.
of Ducrey, 442
cultivation, 443

Davis' method, 443
morphology, 443
pathogenesis, 445
staining, 443
vital resistance, 445
of Fasching, 507
of gaseous edema, 377. See also

Bacillus aerogenes capsulatus.
of hemorrhagic septicemia, 597
of Hoffmann, 474. See also

Bacillus, pseudodiphtheria.
of Koch- Weeks, 446

Bacillus of Koch- Weeks, associa-
tion, 448
cultivation, 448
morphology, 447
pathogenesis, 448
staining, 447

of malignant edema, 374. See
also Bacillus cedematis maligni.
of Morax-Axenfeld, 447
cultivation, 449
morphology, 449
pathogenesis, 450
staining, 449

of rabbit septicemia, 598, 601
of Shiga, 699. See also Bacillus

dysenteric.
of swine-plague, 60 1
of syphilis, 787

of tetanus, 385. See also Bacil-
lus tetani.
of Weeks, 446. See also Bacillus

of Koch-Weeks.
of Wildseuche, 60 1
Oppler-Boas, in carcinoma of

stomach, 83
pestis, 581

cultivation, 585

diagnosis, 593

experimental infection with,

590

immunity against, 596
metabolism, 589
mode of infection with, 592
morphology, 584
staining, 584
virulence, 594

Kolle's method of estimat-
ing, 594

vital resistance, 589
Petruschky's, 676
proteus vulgaris, 369
cultivation, 369
distribution, 369
metabolic products, 371
morphology, 369
pathogenesis, 371
staining, 369
pseudo-anthracis, 411
pseudodiphtheria, 468, 470, 474
chemistry, 475
cultivation, 475
differentiation from bacillus

diphtheriae, 468, 474
morphology, 475
pathogenesis, 476
staining, 475
pseudodysentery, 699
pseudoglanders, 784
pseudo-influenza, 519

854

Index

Bacillus, pseudotetanus, 399
pseudotuberculosis, 760

cultivation, 760

morphology, 760

pathogenesis, 761
psittacosis, 677

cultivation, 677

differentiation, 678

isolation, 677

metabolic products, 677

morphology, 677

pathogenesis, 677
pyocyaneus, 364

cultivation, 365

distribution, 364

immunity against, 368

isolation, 365 •

metabolic products, 366

morphology, 365

pathogenesis, 367

staining, 365
pyogenes foetidus, 666
rhinoscleromatis, 785

general characteristics, 785

pathogenesis, 786
Sanarelli's, 682
septicus sputigenus, 493
smegmatis, 757

cultivation, 757

Moeller's method, 758
Novy's method, 757

in urine, 718

morphology, 757

pathogenesis, 758

staining, 757
suipestifer, 678

agglutination, 68 1

cultivation, 679

metabolic products, 680

morphology, 679

pathogenesis, 68 1

toxin of, 680

vital resistance, 680
suisepticus, 60 1

cultivation, 602

general characteristics, 60 1

lesions from, 603

morphology, 60 1

pathogenesis, 602

staining, 602

vital resistance, 602
tetani, 385

antitoxin against, 397

bacilli resembling, 399

cultivation, 386

Park's method, 388

distribution, 385

immunity against, 396

isolation, 386

Bacillus tetani, metabolic products,

390

morphology, 386
staining, 386
toxic products, 390
vital resistance, 389
tuberculosis, 710
agglutination, 745
appearance of cultures, 726
avium, 754

cultivation, 755
morphologic peculiarities,

755

pathogenesis, 755
staining, 755
thermic sensitivity, 755
bacilli resembling, 756
bovis, 748

lesions produced by, 749
metabolic products, 749
morphology, 748
pathogensis, 749
staining, 749
vegetation, 749
channels of infection for, 727
gastro-intestinal tract, 728
placenta, 727
respiratory tract, 728
sexual apparatus, 729
wounds, 730
chemistry, 736
cultivation, 722

Koch's method, 721
distribution, 711
effect of light on, 727
general characteristics, 710
in feces, staining, 718
in sections of tissue, Ehrlich's
method of staining, 719
Gram's method of stain-
ing, 719
Unna's method of stain?

ing, 719

in sputum, staining, 714
in urine, staining, 718
isolation, 720

antiformin for, 720
Dorset's method, 723
Frugoni's method, 725
Smith's (T.) method, 723
morphology, 712
pathogenesis, 727
reaction, 727
relation to oxygen, 727
staining, 713

Ehrlich-Koch method, 715
Ehrlich's method, 713, 715

for sections, 719
Gabbet's method, 716

Index

855

Bacillus tuberculosis, staining,
Gram's method, for sec-
tions, 719
in feces, 718
in sputum, 714
in urine, 718
Koch method, 713
Pappenheim's method, 717
Unna's method, for sections,

719

Ziehl's method, 716
temperature sensitivity, 727
toxic products, 737
virulence, 735
typhi murium, 684
cultivation, 684
isolation, 684
morphology, 684
pathogenesis, 685
staining, 684
typhosus, 632

bacilli resembling, 663

meat-poisoning group, 665
pneumonic group, 665
psittacosis group, 665
table for differentiation,

664

typhoidal group, 665
Buxton and Coleman's me-
dium for, 66 1
Capaldi's medium for plating,

659

colon bacillus and, differentia-
tion, 651
cultural, 652
serum, 651
cultivation, 635

Eisner's method, 652
Hiss' method, 654
. Kashida's method, 654
Piorkowski's method, 655
Remy's method, 653
Rothberger's method, 654
distribution, 632
effect of chemic agents on,

637

of cold on, 637
general characteristics, 632
Hesse's medium for plating,

658

in blood, 643, 644
in feces, isolation, 650
in gall-bladder, 640
in lower animals, 644
in oysters, 298
in sputum, 644
in urine, 643
invisible growth, 636
isolation, 634

Bacillus typhosus, isolation, Adami
and Chapin's method,
656

Beckman's method, 657
Drigalski-Conradi method,

656

Endo's method, 659
Petkowitsch's method, 656
Starkey's method, 657
Jackson's culture-medium for,

66 1

MacConkey's medium for, 66 1
metabolic products, 637
mode of infection, 639
morphology, 633
pathogenesis, 639
prophylactic vaccination with

cultures of, 646
specific therapy, 647
staining, 633

Ziehl's method, 634
thermal death-point, 636
toxic products, 637
vital resistance, 636
xerosis, 476
chemistry, 477
cultivation, 477
morphology, 477
pathogenesis, 477
Y, 700

zur Nedden's, 450
Bacteremia, 94
Bacteria, 29

anaerobic, cultivation of, 260.
See also Anaerobic bacteria,
cultivation of.
associated with Bacillus diph-

theriae, 464
with suppuration, 341
biology, 58

Brownian movement, 36
capsule, 34
cell-walls, 34
Chester's synopsis of groups of,

279

chromogenic, 71
classification, 29, 32
colonies of, 246, 251

in Esmarch's tubes, Esmarch
instrument for counting,

288

types, 251

colors produced by, 71
cultivation, 224
determination, 278
distribution of, 58
fission of, 36
flagella of, 35
grouping of, 32

856

Index

Bacteria, groups of, Chester's
synopsis of, 279

higher, 41

in air, 58, 283

quantitative estimation, by

Hesse's method, 284
by Petri's method, 285
by Sedgwick's method,
286

in bladder, 85

in body, 80

in butter, 297

in conjunctiva, 82

in dental caries, 82

in external auditory meatus, 82

in feces, 84, 85

in foods, 296

in ice, 289

in intestine, 83

in larynx, 85

in lungs, 85

in meat, 297

in milk, 296

in mouth, 82

in nose, 85

in oysters, 298

in sections of tissue, method of
observing, 178

in shell-fish, 298

in skin and adjacent mucous
membranes, 80

in soil, 294

Frankel's method of estimat-
ing number, 294

in stomach, 83

in trachea, 85

in urethra, 85

in uterus, 85

in vagina, 85

in water, 287

method of determining num-
ber, 287

Winslow and Willcomb's direct
method of enumeration of,
288

influences of antibiosis on growth

of, 63

of antiseptics on growth of, 65
of association on growth of, 63
of chemic agents on growth of,

65

of electricity on growth of, 62
of food on growth of, 59
of light on growth of, 61
of moisture on growth of, 59
of movement on growth of, 63
of oxygen on growth of, 59
of reaction on growth of, 61
of symbiosis on growth of, 63

Bacteria, influences of temperature

on growth of, 64
of x-rays on growth of, 62, 63

invasive power, 89, 94

isolation of, 246

Koch's law of specificity, 23

liquefaction of gelatin by, 70

living, study of, 172

measurement of, 200

metabolism of, 66

methods of observing, 172

morphology of, 38

motility, 35

non-chromogenic, 71

non-pathogenic, 76

nucleus, 34

of plague group, 597

parasitic, 79

pathogenic, 76, 79

peptonization of milk by, 76

photographing, 200

polar granules, 34

production of acids by, 71
of alkalies by, 71
of aromatics by, 73
of disease by, 76
of enzymes by, 77
of fermentation by, 67
of gases by, 69
of nitrates by, 74
of odors by, 73
of phosphorescence by, 73
of putrefaction by, 68

reproduction of, 36

saprophytic, 79

size of, 36

specific, 339

sporulation of, 36

staining, 174. See also Staining.

structure, 29, 34

thermal death-point of, deter-
mination, 300

thermophilic, 64

toxic power, 89

transplantation of, from culture-
tube to culture-tube, method
of, 245

virulence of, 95. See also Viru-
lence.

Bacterial suspension in testing op-
sonic value of blood, 308
Bactericidal strength of common

disinfectants, 217
Bacterination, 113
Bacteriologic syringes, 269
Bacteriology, evolution, 17

of air, 283

of foods, 296

of soil, 294

Index

857

Bacteriology of water, 297
Bacteriolysins, 166
Bacteriolysis, 164, 166
Bacterio-vaccination in staphylo-

coccic infections, 349
Bacterium, 40

coli dysenteriae, 699
pneumonias, 507
termo, 369
Bagdad boil, 572
Bain fixateur, 192

reducteur et reinforcateur, 192
sensibilisateur, 192
Balantidium coli, 704

animal inoculation with, 707
cultivation, 707
habitat, 707

lesions produced by, 707
morphology, 705
motility, 706
pathogenesis, 707
reproduction, 706
staining, 706
transmission, 708
diarrhea, 704
lesions of, 707
transmission of, 708
Barber's itch, 827
Beckman's method of isolating

typhoid bacillus, 657
Behring's method of determining
potency of diphtheria serum, 157
Bench, glass, 248
Bergell and Meyer's typhoid serum,

649

Berkefeld filter, 208
Bichlorid of mercury as germicide,

212

Bile-salt agar-agar, 662
Biologic contributions, 17
Biology of bacteria, 58
Biondi-Heidenhain method of

staining protozoa in tissue, 199
Biscra boil, 572

button, 572
Black death, 581
fever, 566
molds, 47
plague, 581

Bladder, bacteria in, 85
Blastomyces dermatitis, 818
cultivation, 821
lesions, 823
pathogenesis, 823
staining, 821
transmission, 823
Blastomycetes, 43

dermatitis, 44
Blastomycetic dermatitis, 818

Blastomycosis, 818

specific organism, 820

transmission, 823
Blenorrhea, 450
Blood agar-agar as culture-media,

234

Bacillus typhosus in, 643, 644
method of securing, from animals,

273.

opsonic value of, bacterial sus-
pension in testing, 308
serum in testing, 311
washed leukocytes in test-
ing, 3 1 1

phagocy tic -power of, 307
streptococcus in, in scarlatina,

355
Blood-corpuscles for Wassermann

reaction, 322
titration of, 324
preparation of, 164
Blood-culture in typhoid fever, 650
Blood-serum, alkaline, as culture-
media, 237
as culture-media, 234
mixture, Loffler's, as culture-
media, 236
for cultivation of Bacillus

diphtheria, 454
therapy, 26
Boil, Aleppo, 572
bagdad, 572
biscra, 572
Delhi, 572
Bordet-Gengou bacillus, 488

and influenza bacillus, differ-
ences between, 491
cultivation, 490
isolation, 489
metabolic products, 490
morphology, 489
pathogenesis, 490
staining, 489
phenomenon, 170
Botkin's apparatus for making

anaerobic cultures, 262
Botulism, 299
Botulismus, 69

Bouillon as culture-media, 227
preparation of, from fresh meat,

227

from meat extract, 229
sugar, 230

Bouillon-filtrate, Denys', 742
Bovine actinomycosis, 803
tuberculosis, 748

communicability to man, 750
prophylaxis, 753
tuberculin test for, 754

858

Index

Bromatotoxismus, 298

Bronchopneumonia, 512

Broth, nitrate, 74

Brownian movement of bacteria,
36

Buboes in plague, 583

Bubonic plague, 581. See also
Plague.

Buchner's method of making anae-
robic cultures, 263

Buerger's medium for isolating
diplococcus pneumoniae, 495, 496

Buret for titrating media, 225

Burri's India ink method of identi-
fying treponema p'allidum, 792

Buton d'Orient, 572

Butter bacillus, 760
bacteria in, 297

Butyric fermentation, 68

Buxton and Coleman's culture-
medium for typhoid bacillus, 66 1

CABOT'S method of treatment of

hydrophobia, 419
Calmette's ophthalmo-tuberculin

reaction, 741
Canal-water bacillus, capsulated,

508

Canned goods, poisoning from, 299
Capaldi's medium for plating ty-
phoid bacillus, 659
Capillary glass tubes, 243, 244
Capsulated canal-water bacillus,

508
Capsule bacillus, 509

of bacteria, 34
Capsules, collodion, 276
Carbolic acid as disinfectant, 215
Carbuncle, malignant, 401
Carcinoma of stomach, Oppler-

Boas bacillus in, 83
Caries, dental, bacteria in, 82
Carrasquilla's leprosy serum, 774
Carriers, typhoid, 640
Catarrhal inflammation, 439

pneumonia, 512
Catgut, sterilization of, 210
Claudius' method, 211
cumol method, 211
Celloidin embedding, 179
Cells, giant-, in tuberculosis, 732

lepra, 772

specific affinity of, for toxins, 93
Cell-walls of bacteria, 34
Ceratophyllus fasciatus, 593
Cercomonas intestinalis, 709
Cerebrospinal fever, 423

meningitis, 423

Cerebrospinal meningitis, diplococ-
cus of, 423

Chamberland filter, 208

Chancroid, 442

Chantemesse's ocular typhoid re-
action for diagnosis of typhoid
fever, 650

Chapin and Adami's method for
isolation of typhoid bacillus, 656.

Charbon, 400

Cheese-poisoning, 299

Chemic agents, influence on growth

of bacteria, 65
contributions, 20

Chester's synopsis of groups of
bacteria, 279

Chicken-cholera, 598

Chlamydophrys stercorea, 690

Chlorin as germicide, 214

Cholera, Asiatic, 604

immunity against, 616
prophylaxis, 617
rice-water discharges of, 613
sanitation in, 618
specific organism of, discovery,

605

chicken-, 598
de poule, 598
hog-, 678
nostras, 619

Chromogenesis, 71

Chromogenic bacteria, 71

Chromogens, 66

Cilia of protozoa, 56

Cladothrix, 42

Claudius' method of sterilization
of catgut, 211

Clegg and Musgrave's agar-agar
for cultivating amebas, 692, 694

Clegg's method of cultivation of
lepra bacillus, 767

Clonic convulsions in tetanus, 393

Clostridium, 37

Clothing, etc., disinfection of, 221

Coagulins, 140

Cobralysin, 120

Cocci, 38

Coccidioidal granuloma, 819

Coccus, diagram illustrating mor-
phology, 39

Cold, effect of, on Bacillus typho-

sus, 637
influence on growth of bacteria,

65
Coleman and Buxton's medium for

typhoid bacillus, 66 1
Coley's mixture, 358
Collodion capsules, 276

sacs, preparation of, 276, 277

Index

859

Colonies, 246, 251

in Esmarch's tubes, Esmarch in-
strument for counting, 288

types of, 251

Colors produced by bacteria, 71
Comma bacillus, 606
Complement, 120, 141, 142, 144,

145
deviation of, phenomenon result

of, 167
fixation, 170
for Wassermann reaction, 321

titration of, 324
Complicating pneumonias, 513
Concentrated tuberculin, 739
Congestive chills of malaria, 539
Conjunctiva, bacteria in, 82
Conjunctival reaction in typhoid

fever, 650
Conjunctivitis, acute contagious,

446

miscellaneous organisms in, 450
Conorhinus megistus, 565
Conradi-Drigalski method of iso-
lation of typhoid bacillus, 656
Contagion from Bacillus diph-

theriae, 468
Contagious conjunctivitis, acute,

446

Contractile vacuoles, 54
Convulsions, clonic, in tetanus, 393

tonic, in tetanus, 393
Coplin's staining jar, 181
Copper sulfate as germicide, 213
Corks, sterilization of, 204
Cornil and Babes' method of stain-
ing spirillum cholerae Asiaticee,
608
Corpuscles, blood-, for Wasser-

mann reaction, 322
titration of, 324
preparation of, 164
Cover-glass forceps, 177

preparations for general exami-
nation, 175, 176
for staining protozoa, 196
Gram's method, 185
Creolin, 215
Croupous pneumonia, 492

lesions, 501
Crude tubercles, 733

tuberculin, 738
Cryptobia borreli, 556
Ctenopsylla musculi, 593
Culex pipiens, 542
Culicidae, classification, 542
Culture-media, 224
agar-agar, 232
alkaline blood-serum, 237

Culture - media, blood - agar - agar,

234

- blood-serum, 234
bouillon, 227

Deycke's alkali-albuminate, 237
Dunham's solution, 240
glycerin agar-agar, 234
litmus milk, 239
Loffler's blood-serum mixture,

236

milk, 239

peptone solution, 240
Petruschky's whey, 240
potatoes, 237
potato-juice, 238
standard reaction, 227
sterilization and protection, 205
Cultures, 242
agar-agar, 255
anaerobic, Botkin's apparatus

for making, 262
Buchner's method of making,

263
Frankel's method of making,

261

Hesse's method of making, 265
Koch's method of making, 266
Liborius' tube for, 261
Nichols and Schmitter's meth-
od of making, 263
Novy's jars for, 261
Salomonsen's method of mak-
ing, 266
Wright's method of making,

264, 267
Zinsser's method of making,

264
freshly isolated, standardizing,

259

gelatin culture, 254
in fluid media, 256
manipulation, technic, 243
microscopic study, 259
plate, 242, 246

apparatus for, 247
Esmarch's tubes for making,

250

method of, 247
Petri's dishes for making, 249
pure, 242, 252

special methods of securing,

256

shake, 265
standardizing freshly isolated,

259

study of, 242
upon potato, 256

Cumol method of sterilization of
catgut, 211

86o

Index

Cup, pasteboard, for receiving in-
fectious sputum, 220

Cutituberculin reaction, 741

Cytase, 144

Cytolysins, 164

Cytolysis, 164

Cytoplasm, 34
of protozoa, 53

Cytotoxins, 163

Czenynke's stain for Bacillus in-
fluenzae, 514

DAVIS' method of cultivation of

Ducrey's bacillus, 443
Death, black, 581
Death-point, thermal, of bacteria,

determination of, 300
Defensive proteids, 128
Dejecta, disinfection of, 220
Delhi boil, 572
Dematium albicans, 485
Denecke, spirillum of, 622
Dental caries, bacteria in, 82
Deny's tuberculin, 742
Dermatitis, blastomycetic, 818
Dermatomycosis, 824
Dermatotuberculin reaction, 741
Desmon, 144

Deviation of complement, phenom-
enon result of, 167
Deycke's alkali-albuminate as cul-
ture-media, 237
Diarrhea, balantidium, 704
lesions of, 707
transmission of, 708
Digestive apparatus, infection

through, 86

Diluted tuberculin, 739
Diphtheria, 451
antitoxin, 155, 47 1
bleeding, 156
effect on death-rate, 473
immunization of animals, 156
method of administration, 471
paralysis after use of, 472
potency of serum, 157
preparation of serum, 157

of toxin, 156
prophylaxis, 471
tetanus after, use of, 473
treatment with, 471
bacillus of, 45 1 . See also Bacillus

diphtheria.
bacteriologic condition of throat

in, 462

contagion from, 468
diagnosis, 456

bacteriologic, 456

Diphtheria, diagnosis, outfit for, 456
lesions, 466
mixed infections, 466
paralysis after, 472
pseudomembrane of, 466
relation of Bacillus diphtherias

to, 467
of streptococcus pyogenes to,

. 354

toxin, 460

Diplobacillenconjunctivitis, 448
Diplococcus, 38

intracellularis meningitidis, 423
agglutination, 428
cultivation, 426
distribution, 424
identification, 425
isolation, 426
metabolic products, 428
micrococcus catarrhalis and,

differentiation, 425
mode of infection with, 430
morphology, 424
meningitidis, pathogenesis, 428
specific therapy with, 430
staining, 425
vital resistance, 427
of Weichselbaum, 492
pneumonias, 492

animals susceptible to, 502
bacteriologic diagnosis, 503
cultivation, 496
distribution, 493
general characteristics, 492
immune serum against, 505
immunity against, 505
isolation, 495
metabolic products, 498
morphology, 493
pathogenesis, 498
specificity of, 502
staining, 494
toxic products, 498
virulence, 502
vital resistance, 497
Diseases, infectious, 339

study of, 21
production of, 76
Dishes, Petri's, 249
Disinfectants, common, bacterici-
dal strength, 217
determination of value, 302
inorganic, 213
organic, 215

Disinfection and sterilization, 201
gaseous, 305
of air of sick-room, 2 1 1
of bodies dead of infectious dis-
eases, 223

Index

86 1

Disinfection of clothing, etc., 221

of dejecta, 211, 220

of furniture, etc., 222

of hands, 209

of instruments, 209

of ligatures, 209

of patient, 222

of sick-chambers, 211

of sutures, 209

of wound, 211

use of sulphur in, 218

with formaldehyd, 218
Displacement of air by inert gases

in anaerobic cultures, 260
Donovan-Leishman body, 566
Dorset's method of isolation of

tubercle bacillus, 723
Dose, amboceptor, in Wassermann

reaction, 326
Dourine, 562

Drigalski-Conradi method of iso-
lation of typhoid bacillus, 656
Drumstick, 37
Ducrey's bacillus, 442. See also

Bacillus of Ducrey.
Dumdum fever, 566
Dunham's solution as culture-me-
dium, 240
Duval's method of cultivation of

lepra bacillus, 767, 768
Dyscrasia, 101
Dysentery, 687

amebic, 689

abscess of liver in, 697
lesions, 697

bacillary, 699
diagnosis, 704
lesions, 702
serum therapy, 704

bacillus of, 699. See also Bacillus
dysentericB.

endemics of, 687

epidemics of, 687

EBERTH-GAFFKY bacillus, 632 . See

also Bacillus typhosus.
Edema, gaseous, 377

bacillus of, 377 See also Bacil-
lus aerogenes capsulatus.
malignant, 374

bacillus of, 374. See also Bacil-
lus cedematis maligni.
Ehrlich-Koch method of staining

tubercle bacillus, 715
Ehrlich's lateral-chain theory of

immunity, 131

method of determining potency
of diphtheria serum, 158

Ehrlich's method of staining tuber-
cle bacillus, 713, 715
in sections, 719
solution, 182
Electricity, influence on growth of

bacteria, 62
Electrozone, 214
Elephantiasis graecorum, 771
Eisner's method of cultivation of

typhoid bacillus, 652
Embedding, 119
celloidin, 179
glycerin-gelatin, 180
paraffin, 180
Emulsion, bacillary, 745
Encystment of protozoa, 57
Endogenous infections, 80
Endomyces albicans, 485
Endo's method of isolation of ty-
phoid bacillus, 659
! Endospores, 37
I Endotheliolysis, 164
l Engle and Reichel's stain for Negri

bodies, 421
Entamreba buccalis as cause of

suppuration, 372
coli, 691

table of differential features,

693
histologica, 689, 691

morphology, 691

reproduction, 692

staining, 692

table of differential features,

693

lesions produced by, 697
Mallory's differential stain for,

698

metabolic products, 695
pathogenesis, 695
tetragena, 690, 692

isolation and cultivation, 692

metabolic products, 695

pathogenesis, 695

table of differential features,

693

vital resistance, 695
vital resistance, 695
Enteric fever, 632
Enzymes, production of, 77

tryptic, 71
Eosin and methylene-blue stain,

1 86
I Epidemic cerebrospinal meningitis,

423

Epitheliolysins, 121
I Epitheliolysis, 164
Ernst-Babes granules, 34
Erysipelas, streptococcus of, 361

862

Index

Erythrasma, 824

Esmarch's instrument for counting

colonies of bacteria in Esmarch

tubes, 288
tubes, 250
Estivo-autumnal fever, parasite of,

537.

Eurotium, 48
Exhaustion theory of immunity,

125

Exogenous infections, 79
Experimentation upon animals, 268
Extracellular toxins, 89, 91

FACULTATIVE anaerobes, 59
Farcin du boeuf, 43
Farcy, 781
Farcy-buds, 781
Faulnisszymoid, 24
Favus, 828

scutulum formation, 828

specific organism of, 829
Febrile tropical splenomegaly, 566
Feces, Bacillus typhosus in, isola-
tion, 650

bacteria in, 84, 85

spirillum cholerae Asiaticae in,
Schottelius' method of detect-
ing, 609

tubercle bacillus in, staining, 718
Ferment, putrefactive, 24
Fermentation, 20, 67

acetic, 67

alcoholic, 67

butyric, 68

lactic acid, 68

Fermentation-tube, Smith's, 69
Filter, Berkefeld's, 208

Chamberland, 208

Kitasato's, 208

Pasteur-Chamberland, 207

Petri's sand, for air-examination,
285

Reichel's, 208

Filtration, sterilization by, 208
Finkler and Prior spirillum, 619
Fiocca's method of staining spores,

189

Fish tuberculosis, 756
Fishing, 252
Fish-poisoning, 299
Fission, 36

results, 36
Fixateur, 144, 145
Fixation of complement, 170
Fixed virus in hydrophobia, 414
Flagella, 35

staining of, 189. See also Stain-
ing flagella.

Flagellates in intestines, 709
Fleas, plague and, 592, 593
Fleischner-giftung, 69
Flexner variety of dysentery bacil-
lus, 702
Flexner's antimeningococcus serum,

430

Flies, plague and, 592
Fluid media, cultures in, 256

Miiller's, 259

Zenker's, 179
Fluorescein, 366
Fomites, 80

foods as, 296
Food as fomites, 296

bacteria in, 296

bacteriology of, 296

influence on growth of bacteria,

59

of molds, 60
of protozoa, 60
of yeasts, 60

poisons, 298
Food-poisoning, 298
Forceps, cover-glass, 177

Petri dish, 249

sterilization of, 204
Formaldehyd, 218

as germicide, 218
Formalin, 215, 218
Fowl tuberculosis, 754
Frambesia tropica, 800
Frankel's instrument for obtaining
earth for bacteriologic study,

295
method of estimating number of

bacteria in soil, 294
of making anaerobic cultures,

261
of staining diplococcus intra-

cellularis meningitidis, 425
Friedlander's Bacillus pneumoniae,

507. See also Pneumococcus.
Frost's plate counter, 290
Frothy organs, 384
Frugoni's method of isolation of

tubercle bacillus, 725
Fungi, ray-, 804

Funnel for rilling tubes with culture-
media, 230

Furniture, etc., disinfection of, 222
Fusiform bacillus, 478

GABBET'S method of staining tuber-
cle bacillus, 716

GafTky-Eberth bacillus, 632. See
also Bacillus typhosus.

Galactotoxismus, 298

Index

863

Gall-bladder, Bacillus typhosus in,

640

Gamaleia, spirillum of, 625
Gametocytes of plasmodium falci-

parum, 538
malariae, 534
vivax, 536

Gartner's bacillus, 675
Gaseous disinfection, 305

edema, 377

Gases, production of, 69
Gastro-intestinal tract as avenue of

infection for tubercle bacillus,

728
Gelatin as culture-media, 231

liquefaction of, 70

puncture culture, 254
Gelatin-media, gelatin, 231
Generation, spontaneous, doctrine

of, 17
Gengou-Bordet bacillus, 488

phenomenon, 170
Genital apparatus, infection

through, 88

Germ theory of disease, 23
Germicidal value of solutions,

method of testing, 304
Germicide, 201

determination of value, 303
Germination of spores, 38
Ghoreyeb's method of staining

treponema, 789
Giant-cells in tuberculosis, 731
Gibson's globulin precipitation for

concentration of diphtheria

serum, 159
Glanders, 775

bacillus of, 775. See also Bacillus
mallei.

diagnosis, 778

in human beings, 783

specific organism, 775
Glass bench, 248

tubes, capillary, 243, 244
Glassware and instruments, ster-
ilization of, 203

protection of, 203
Globulin precipitation for concen-
tration of diphtheria serum, 159
Glossina morsitans, 561

palpalis, 561, 563

Glycerin agar-agar as culture-
media, 234

Glycerin-gelatin, embedding, 180
Glycoproteids, 134
Golden staphylococcus, 343, 346
Goldhorn's method of staining tre-
ponema pallidum, 788
Gonococcus, 431

Gonorrhea, 431

communication of, to animals,

436

diagnosis, 435
Gonotoxin, 435

Gordon's medium for differentia-
tion of cholera and Kinkier-
Prior spirilla, 615
Gram's method for staining tuber-
cle bacillus in sections, 719
of staining, 182, 183, 184
Nicolle's modification, 185
tubercle bacillus in sections,

719
Gram-Weigert method of staining,

185

Granulations of Schiiffner, 537
Granules, Babes-Ernst, 34
metachromatic, 34
polar, 34

Granuloma, coccidioidal, 819
Grass bacillus, Moeller's, 758

HAFFKINE'S prophylactic, 596, 617

Halogens and compounds as germi-
cides, 214

Hands, disinfection of, 209

Hanging block, directions for pre-
paring, 174
drop, 173

Hankin and Leumann's method for
differential diagnosis of plague
bacillus, 588

Haptophore group, 131

Hardening, 178

Healed tubercles, 736

Heat, influence on growth of bac-
teria, 64

Heidenhain-Biondi method of stain-
ing protozoa in tissue, 199

Heidenhain's method of staining
protozoa in tissue, 199

Heiman's method of cultivation of
micrococcus gonorrhoeas, 434

Helcosoma tropicum, 572, 573

Hematozoa, 528

Hemolysins, 163

Hemolysis, 163

Hemolytic amboceptor for Wasser-

mann reaction, 323
serum for Wassermann reaction,

titration of, 324
system in Wassermann reaction,

325
Hemorrhagic septicemia, bacilli of,

597, 60 1

Hemorrhagin, 162
Herpes circinatus, 824

864

Index

Herpes desquamans, 824

tonsurans, 824
Hesse's apparatus for collecting

bacteria from air, 284
culture-medium for typhoid ba-
cillus, 658

method for quantitative estima-
tion of bacteria in air, 284
of making anaerobic cultures,

265

Higher bacteria, 41
Hill's hanging block, 174
Hiss' inulin-serum-water test for
determining pneumococcus,
505
method of cultivation of typhoid

bacillus, 654
of staining diplococcus pneu-

monise, 494, 495
Hiss-Russell variety of dysentery

bacillus, 702

Histoplasma capsulatum, 573
Histoplasmosis, 573
Historical introduction, 17
Hoffmann's bacillus, 474. See also

Bacillus pseudodiphtheria.
Hog-cholera, 678

Hogyes' method of treating hydro-
phobia, 419
Host, 78

susceptibility of, 101. See also

Susceptibility.
Hot-air sterilizar, 203
Hiihnercholera, 598
Hydrogen peroxid as germicide, 216
Hydrophobia, 412

communication to man, 412

fixed virus in, 414

scheme for intensive treatment,

418

for mild treatment, 418
street virus in, 414
treatment, 414

Cabot's method, 419
dilution method, 419
Hogyes' method, 419
Pasteur's method, 114, 413,

414,417
Hypnococcus, 557

ICE, bacteria in, 289

Ice-cream poisoning, 299

Ichthyotoxismus, 299

Immune body, 120, 141, 142, 143,

144, 145
Immunity, 105
acquired, 108
accidentally, 109

Immunity, acquired, active, 109
experimentally, 109
passive, 115
through accidental infection,

109

through bacterination, 113
through infection, 109
through inoculation, 109
through intoxication, 1 1 5
through vaccination, no
active, 106

acquired, 109
Ehrlich's lateral-chain theory,

3i

exhaustion theory, 125
explanation of, 125
Metschnikoff's theory, 126
natural, 107
passive, 106

acquired, 116
relative, 106
retention theory, 125
special phenomena of, 146
synopsis of experimental studies

of, 118

to vaccination, 112
variations in, 112
Incubating oven, 258
Incubator for opsonic work, 315
Index, opsonic, 307, 316
India ink method of identifying

treponema pallidum, 792
Indol, 73

Salkowski's test for, 73
Infantile kala-azar, 571
Infection, 78

avenues of, 86, 98

cardinal conditions, 95

endogenous, 80

exogenous, 79

experimental, 109

mixed, 103

proteus, 369

sources of, 79

special phenomena of, 146

sub-, 78

terminal, 355

through digestive apparatus, 86

through genital apparatus, 88

through placenta, 88

through respiratory apparatus,

88

through skin, 86
virulence of, 95. See also Viru-
lence.
Infectious diseases, 339

study, 21

Inflammation, catarrhal, 439
Influenza, 514

Index

865

Influenza, diagnosis, 519

Infusoria, 53

Infusorial life, 19

Injection, intra-abdominal, 271

intrapleural, 271

intravenous, 270

into rabbit, method of making,
270

subcutaneous, 271
Inoculation, 109

advantages of vaccination over,
in

animal, 271

subcutaneous, 271
Inorganic disinfectants, 213
Instruments and glassware, steril-
ization, 203

disinfection of, 209

protection of, 203

surgical sterilization of, 211
Intermediate body, 120
Intermittent sterilization, 205
Intestine, bacteria in, 83

flagellates in, 709
Intoxication, immunity acquired

by, 115

Intra-abdominal injection, 271
Intracellular toxins, 90
Intrapleural injections, 271
Intravenous injections, 270

into rabbit, method of making,

270

Introduction, historical, 17
Invasive power of bacteria, 89, 94
Inulin-serum-water test of Hiss for

determining pneumococcus, 505
lodin terchlorid as germicide, 215
Iron-hematoxylin stain for proto-
zoa, 199

Isolation of bacteria, 246
Itch, barber's, 827

JACKSON'S medium for typhoid

bacillus in water, 66 1
Jactationstetanus, 394
Jars, Novy's, for anaerobic cultures,

261

Javelle water, 720
Jaw, lumpy, 803, 811
Jennerian vaccination, no

KALA-AZAR, 566

diagnosis, 571

infantile, 571

lesions, 570

transmission, 570
Kashida's method of cultivation of

typhoid bacillus, 654

55

Kitasato's filter, 207
mouse-holder, 273
Klatschpraparat, 256
Klebs-Loffler bacillus, 451. See

also Bacillus diphtheria.
Knives, sterilization of, 204
Koch-Ehrlich method of staining

tubercle bacillus, 715
Koch's agglutination test for tuber-
cle bacillus, 746

apparatus for coagulating and
sterilizing blood-serum, 235,
236

law of specificity of bacteria, 23
method of isolation of tubercle

bacillus, 721
of making anaerobic cultures,

266
of staining tubercle bacillus,

7i3

syringe, 269
tuberculin, 739

Koch-Weeks bacillus, 446. See
also Bacillus of Koch-Weeks.

Kolle's method for diagnosis of
plague, 594

Krai's method of cultivation of
Achorion schonleinii, 831

Kreotoxismus, 299

Kiihne's method of staining Bacil-
lus mallei, 777

LA FIEVRE typhique, 632
Lactic acid fermentation, 68
Laitinen's method of cultivation of

micrococcus gonorrhoeas, 434
Lamblia intestinalis, 709
Larynx, bacteria in, 85
Latapie's animal holder, 272, 273

instrument for preparing tissue

pulp, 165

Latent tuberculosis, 735
Lateral chain-theory of immunity,

Ehrlich's, 131

Law, Koch's, of specificity of bac-
teria, 23

Leishman- Donovan body, 566
Leishmania donovani, 566
cultivation, 569
distribution, 570
morphology, 568
transmission, 570

furunculosa, 572

infantum, 571
Lepra anaesthetica, 771, 773

cells, 772

nodosa, 771
Leprolin, 774

866

Index

Leprosy, 762

anesthetic, 771, 773
etiology, 763
nodular, 771
sanitation in, 774
serum, 774
therapy in, 774
Leptothrix, 41
Lethargy, African, 554
Leuconostoc, 39
Leukocidin, 347

Leukocytes, washed, in testing op-
sonic value of blood, 211
Leumann and Hankin's method for
differential diagnosis of plague
bacillus, 588
Levaditi's method of staining tre-

ponema pallidum, 790
Liborius' tube for anaerobic cul-
tures, 261
Life, infusorial, 19

spontaneous generation of, doc-
trine, 17
Ligatures, disinfection of, 209

sterilization of, 210
Light, effect of, on tubercle bacillus,

727
influences on growth of bacteria,

61

Liquefaction of gelatin, 70
Listerism, 25
Litmus, method of preparing, 239

milk as culture-media, 239
Litmus-lactose-agar-agar, 654
Liver, abscess of, in amebic dysen-
tery, 697

Lobar pneumonia, 492
Lockjaw, 385. See also Tetanus.
Loffler-Klebs bacillus, 451. See

also Bacillus diphtheria.
Loffler's alkaline methylene-blue,
staining Bacillus diphtherias with,

452

blood-serum mixture as culture-
media, 236
method for detecting spirillum

cholerae Asiaticse, 615
of cultivation of Bacillus diph-
therias, 454

of differentiating typhoid bacil-
lus by means of malachite
green, 660
of staining, 182

Bacillus mallei, 776
flagella, 189
Luetin, 798

Lugol's solution, dilute, 183
Lumpy jaw, 803, 811
Lungs, bacteria in, 85

Lupus, 99
Lysin, 120
Lysol, 215
Ly ssa, 412. See also Hydrophobia.

MAcCoNKEY's medium for ty-
phoid bacillus, 66 1

method for detecting colon bacil-
lus in water, 66 1
Macfadyen's typhoid serum, 648
Macrocytase, 127, 145
Macrogametocyte, 531
Macrophages, 126
Madura-foot, 812
Maladie du coit, 562

du sommeil, 554
Malaria, 524

ague-cake of, 540

algid cases, 539

and mosquitoes, relation, 526,

54i

congestive chills of, 539
enlargement of spleen in, 540
estivo-autumnal parasite of, 537
geographic distribution, 524
history, 524
parasites of, 525, 532
animal inoculation, 539
cultivation, 539
human, 532

inoculation, 539
pathogenesis, 539
paroxysms of, 524
prophylaxis, 540

human beings, 540
' mosquitoes, 540
quartan, parasite of, 532
temperature in, 524
tertian, parasite of, 534
Malignant carbuncle, 401
edema, 374
polyadenitis, 581
pustule, 407
Mallein, 781
Mallory's differential stain for en-

tamoeba, 698
method of staining, 186
Malta fever, 520

bacteriologic diagnosis, 521
treatment, 522
Mastigophora, 51
Matino's method of staining proto-
zoa, 198
Measurement of micro-organisms,

200
Meat, bacteria in, 297

extract, preparation of bouillon
from, 229

Index

867

Meat, fresh, preparation of bouillon

from, 227
Meat-infusion, 228
Meat-poisoning, 69, 299
Meatus, external auditory, bac-
teria in, 82
Medical and surgical contributions

to history of bacteria, 21
Mediterranean fever, 520
Megastomum intestinalis, 709
Meningitis, cerebrospinal, 423

diplococcus of, 423
Meningococcus, 423
Mercuric chlorid as germicide, 213
Mercury, bichlorid of, as germicide,

212

Merismopedia, 38
Merozoits, 529
Metabolism of bacteria, 66
Metachromatic granules, 34
Metschnikoff 's theory of immunity,

126
Meyer and Bergell's typhoid serum,

649

Meyer's syringe, 269
Micrococcus, 38
catarrhalis, 439
cultivation, 440
diplococcus intracellularis and,

differentiation, 425
morphology, 439
pathogenesis, 441
staining, 440
gonorrhoeas, 431
cultivation, 433

Heiman's method, 434
Laitinen's method, 434
Wassermann's method, 434
Wertheim's method, 433
Wright's method, 434
Young's method, 433
diagnosis of gonorrhea from,

435

distribution, 431

immunization against, 437

isolation, 433

morphology, 432

pathogenesis, 436

staining, 432

toxic products, 435

vital resistance, 434
melitensis, 520

cultivation, 520

morphology, 520

pathogenesis, 522

staining, 520

thermal death-point, 520
pasteuri, 26
tetragenus, 361

Micrococcus tetragenus, cultiva-
tion, 362
isolation, 362
morphology, 361
pathogenesis, 363
staining, 362
Microcytase, 127, 145
Microgametocyte, 531
Micromillimeter, 36
Micro-organisms, anaerobic, cul-
tivation of, 260. See also An-
aerobic bacteria, cultivation of.
cultivation of, 224
in air, 283

measurement of, 200
methods of observing, 172
of plague group, 597
photographing, 200
specific, 339

structure and classification, 29
Microphages, 126
Microscopic study of cultures, 259
Microspira, 40
Microsporon, 46
Migula's classification of bacteria,

32

Miliary tubercle, 733
Milk as culture-media, 239
bacteria in, 296
litmus, as culture-media, 239
peptonization of, 76
Milk-poisoning, 298
Milzbrand, 400
Mixed infections, 103

pneumonias, 513
Mixture, Coley's, 358
Moeller's grass bacillus, 758

method of cultivation of smegma

bacillus, 758
of staining spores, 188
Moisture, influence on growth of

bacteria, 59
Molds, 46
black, 47
influence of food on growth of, 60

of light on growth of, 61
Monilia caudida, 485
Morax-Axenfeld, bacillus of, 448
cultivation, 449
morphology, 449
pathogenesis, 450
staining, 449

Morphology of bacteria, 38
Morro's method of diagnosis of

tuberculosis, 741
Mosquitoes and malaria, relation,

526, 541

and yellow fever, 577
classification, 542

868

Index

Mosquitoes, destruction of, in pre-
vention of malaria, 540
Motility of bacteria, 35
Mouse-holder, 272, 273
Mouth, bacteria in, 82
Movement, influence on growth of
bacteria, 63

of protozoa, 55
Mucor, 47

conoides, 48

corymbifer, 47, 48

mucedo, 46, 47, 48

mycosis, 48

pusillus, 48

ramosus, 47

rhizopodiformis, 47

septatus, 48
Muguet, 484

Muir and Ritchie's method of stain-
ing spores, 1 88
Miiller's fluid, 259
Musgrave and Clegg's agar-agar

for cultivation of amebas, 692,

694

Mussel-poisoning, 299
Mycetoma, 812

melanoid form, 813, 816

ochroid variety, 813

pale variety, 813
Mycoderma vini, 485
Mycophylaxis, 129
Mycosis, mucor, 48
Mycosozins, 129
Mytilotoxismus, 299
Myzorrhynchus psueudopictus, 542

NAGANA, 560

Needles, platinum, for transferring
bacteria, 243

Negri bodies, 421

Neisser and Wechsberg's phenome-
non, 167, 168

Neisser's method of staining Bacil-
lus diphtherias, 453

Nephelometer, 310

Nephrolysins, 121

Nephrotoxins, 121

Nessler's solution, 75

Nichols and Schmitter's method of
making anaerobic cultures, 263

Nicolle's modification of Gram's
method of staining, 185

Nitrate broth, 74

Nitrates, formation of, 74
reduction of, 74

Nitrobacter, 74

Nitrogen, combination of, 75

Nitrosococcus, 74

Nitroso-indol reaction, 73

Nitrosomonas, 74

Nocard's bacillus, 677

Nodular leprosy, 771

Noguchi's cutaneous reaction in

diagnosis of syphilis, 798
method of cultivation of trepo-

nema pallidum, 793
modification of Wassermann re-
action, 335

Non-chromogenic bacteria, 71
Non-malarial remittent fever, 566
Non-pathogenic bacteria, 76
Nose, bacteria in, 85
Novy's jars for anaerobic cultures,

261
method of cultivation of smegma

bacillus, 757
Nucleus of bacteria, 34

of protozoa, 55

Nuttall and Welch's method of
staining bacillus aerogenes cap-
sulatus, 380

OBERMEIER'S spirillum, 546
Ocular typhoid reaction of Chante-

messe, 650

Odors, production of, 73
Oidia, 44

Oidium albicans, 484
cultivation, 486
fermentation, 486
immunity, 487
metabolic products, 486
morphology, 485
pathogenesis, 486
Onychomycosis, 824
Oocysts, 56, 531
Ookinetes, 56, 531
Ophidiomonas, 40
Ophthalmia neonatorum, 450
Ophthalmo-tuberculin reaction of

Calmette, 741
of Wolff-Eisner, 742
Opisthotonos in tetanus, 393
Oppler-Boas bacillus in carcinoma

of stomach, 83
Opsonic index, 307, 316

value of blood, bacterial suspen-
sion in testing, 308
serum in testing, 311
washed leukocytes in test-
ing, 311

Opsonins, 127, 307
Opsonizing pipette, 313
Optional anaerobes, 59
Organic disinfectants, 215
Oriental sore, 572

Index

869

Ornithodoros moubata, 547, 551
Oshida's method of obtaining rab-
bit's cord for use in hydrophobia,
414

Oven, incubating, 258
Oxygen, atmospheric, absorption
of, in anaerobic cultures, 263
exclusion of, in anerobic cul-
tures, 265
influence on growth of bacteria,

59

reduction of, in anaerobic cul-
tures, 264
relation of tubercle bacillus to,

727
Oysters, bacteria in, 298

PALUDISM, 524

Pappenheim's method of staining
tubercle bacillus, 717

Paracolon bacilli, 665

Paraffin embedding, 180

Paralysis after use of diphtheria
antitoxin, 472

Parasite, 78
stomatitis, 484

Parasites of malaria, 525, 532
animal inoculation, 539
cultivation, 539
human, 532

inoculation, 539
pathogenesis, 539

Parasitic bacteria, 79

Paratyphoid bacilli, 665

Pariette's culture fluid, 657

Park's method of cultivating teta-
nus bacillus, 388

Paroxysms of malaria, 524

Pasteboard cup for receiving in-
fectious sputum, 220

Pasteur- Chamberland filter, 207

Pasteurian vaccination, 113

Pasteurization, 206

Pasteur's protective inoculation

against anthrax, 409
treatment of hydrophobia, 114,
413, 414, 417

Pathogenesis, 89

Pathogenic bacteria, 76, 79
protozoa, classification, 51

Pathogens, 66

Patient, disinfection of, 222

Penicillium, 50
crustaceum, 50
glaucum, 50
minimum, 50

Peptone solution as culture-me-
dium, 240

Peptonization of milk, 76
Peroxid of hydrogen as germicide,

216

Pertussis, 488
Pest, 581
Petkowitsch's method of isolation

of typhoid bacillus, 656
Petri dish forceps, 249

dishes, 249

method for quantitative estima-
tion of bacteria in air, 285

sand filter for air-examination,

285
Petruschky's bacillus, 676

whey as culture-media, 240
Pfeiffer's method of staining, 181

phenomenon, 120, 142, 166
Phagocytes, 126
Phagocytic power of blood, 307
Phagocytosis, theory of, 125
Phagolysis, 126
Phenomenon, Bordet-Gengou, 170

Neisser and Wechsberg's, 167,
1 68

Pfeiffer's, 120, 142, 166

Theobald-Smith, 122
Phlogosin, 347

Phosphorescence, production of, 73
Photogens, 66
Photographing micro-organisms,

200

Phylaxins, 128
Pied de Madura, 812
Pig typhoid, 678
Pink eye, 446
Piorkowski's method of cultivation

of typhoid bacillus, 655
Pipette, opsonizing, 313
Pirquet's method of cutaneous di-
agnosis of tuberculo-
^3,^740
Lignieres' modification,

74i
Pitfield's method of staining fla-

gella, 191

Smith's modification, 191
Pityriasis versicolor, 824
Placenta as avenue of infection for

tubercle bacillus, 727
infection through, 88
Plague, 581
buboes in, 583
diagnosis, 593
fleas and, 592, 593
flies and, 592

group micro-organisms of, 597
history, 581
immunity against, 596
mode of infection, 592

87o

Index

Plague pneumonia, 5 1 2
sanitation in. 595
serum, 596, 597
swine, bacillus of, 601
Planococcus, 38
Planosarcina, 39
Plasmodium falciparum, 529, 537

gametocytes of, 538
malariae, 525, 529, 532
gametocytes of, 534
spores of, 429
vivax, 529, 534

developmental cycle, 530
gametocytes of, 536
Plasmolysis, 35
Plate cultures, 242, 246
apparatus, 247
Esmarch's tubes for making,

250

method, 247

Petri's dishes for making, 249
Platinum needles for transferring

bacteria, 243
wires for bacteriologic use, 243

sterilization of, 203
Pneumobacillus, 507
Pneumococcus, 492, 507
cultivation, 509
distribution, 508
Hiss' inulin-serum-water test for

determining, 505
infections in adults, statistics,

501, 502

metabolic products, 510
morphology, 509
pathogenesis, 510
virulence, 512
vital resistance, 510
Pneumonia, 492
broncho-, 512
catarrhal, 512
complicating, 513
croupous, 492
lesions, 501
lobar, 492
mixed, 513
plague, 512
sanitation in, 507
tubercular, 512
Pneumonias, complicating, 513

mixed, 513

Pneumonic plague, 512
Poisoning, food-, 298
from canned goods, 299
cheese-, 299
fish-, 299
ice-cream, 299
meat-, 69, 299
milk-, 298

Poisoning, mussel-, 299

Poisons, food, 298

Polar granules, 34

Polyadenitis, malignant, 581

Polyceptor, 147

Ponos, 571

Postmortems, 275

Potassium permanganate as germi-
cide, 214

Potato as culture-media, 237
cultures upon, 256
cutter, Ravenel's, 238

Potato-juice as culture-media, 238

Precipitate, specific, 146

Precipitation, globulin, for concen-
tration of diphtheria serum,

159

specific, 146

Precipitins, specific, 140
Predisposition, 101
Prescott and Winslow's method of

preparing litmus, 239
Prior and Kinkier 's spirillum, 619
Prodigiosus powder, 410
Proteids, defensive, 128
Proteus infection, 369
Protista, 29
Protozoa, 51

cilia of, 56

classification, 51

cytoplasm of, 53

encystment, 57

influence of food on growth of,

60
of light on growth of, 61

living, observation of, 194

movements of, 55

nucleus of, 55

pathogenic, classification of, 51

reproduction of, 56

size of, 56

staining, 195. See also Staining
protozoa.

structure, 53
Pseudodiphtheria bacillus, 468, 470,

474. See also Bacillus, pseudo-
diphtheria.

Pseudodysentery bacillus, 699
Pseudoglanders bacillus, 784
Pseudo-influenza bacillus, 519
Pseudomonas, 40
Pseudotetanus bacillus, 399
Pseudotuber.culosis, 760
Psittacosis, 677
Ptomains, 68

definition of, 68
Pulex cheopis, 593

irritans, 593
Pure culture, 242, 252

Index

Pure culture, special methods of Rossi's method of staining flagella,

securing, 256 193

Pustule, malignant, 407
Putrefaction, 20, 68
Putrefactive ferment, 24
Pyemia, 94
Pyocyanase, 77, 367
Pyocyanin, 366
Pyocyanolysin, 367

QUARTAN malarial fever, parasite
of, 532

RABBIT septicemia, bacillus of,

598, 601

Rabies, 412. See also Hydrophobia.
Ravenel's method of preparation of

agar-agar, 233
potato cutter, 238
Ray-fungi, 804
Receptors, 132, 153
Refined tuberculin, 739
Regressive schizogony, 532
Reichel and Engle's stain for Negri

bodies, 421
Reichel's filter, 208
Relapsing fever, 546

bacteriologic diagnosis, 553
course, 552
immunity to, 553
lesions of, 553

Remittent fever, non-malarial, 56
Remy's method of cultivation of

typhoid bacillus, 653
Reproduction of bacteria, 36

of protozoa, 56
Respiratory apparatus, infection

through, 88
tract as avenue of infection for

tubercle bacillus, 728
Retention theory of immunity, 125
Rhinoscleroma, 785
Rhipicephalus decoloratus, 546
Rhizopoda, 51
Rice-water discharges of Asiatic

cholera, 613

Richardson's method of differenti-
ating typhoid bacillus, 652
Ringworm, 824
of body, 824
of scalp, 824

Ritchie and Muir's method of stain-
ing spores, 1 88

Romanowsky's method of staining
* protozoa, 197
Ross' method of staining protozoa,

Rost's method of cultivation of

lepra bacillus, 766
Rothberger's method of cultivation

of typhoid bacillus, 654
Rouget du Pore, 26
Roux's syringe, 269
R tuberculin, 742
Rubber stoppers, sterilization of,

204

SACCHAROMYCES hominis, 44, 818

Saccharomycosis hominis, 818

Salkowski's test for indol, 73

Salomonsen's method of making
anaerobic cultures, 266

Salts as germicides, 213

Sanarelli's bacillus, 682

Sand filter, Petri's, for air-examina-
tion, 285

Sapremia, 94

Saprogens, 66

Saprophytic bacteria, 79

Sarcina, 39

Sarcoma, Coley's mixture in, 358

Scalp, ringworm of, 824

Scarlatina, streptococcus in blood
in, 355

Schaumorgane, 384

Schizogony, regressive, 532

Schizont, 529

Schlafkrankheit, 554

Schmitter and Nichols' method of
making anaerobic cultures, 263

Schottelius' method of detecting
spirillum cholerae Asiaticae in
feces, 609

of securing pure cultures of
spirillum cholerae Asiaticae,
609

Schuffner's granulations, 537

Scissors, sterilization of, 204

Scutulum, 828

Sedgwick's expanded tube for air-
examination, 285
method for quantitative estima-
tion of bacteria in air, 286

Seitenkettentheorie, 131

Semmelformig, 432

Septicemia, 94

hemorrhagic, bacilli of, 597
rabbit, bacillus of, 598, 601

Serum, anticholera, 617
antigonococcus, 437
antimeningococcus, 430
antipneumococcus, 505, 506
antirabic, 422

872

Index

Serum, antistaphylococcus, 349

antistreptococcus, 359

antitubercle, 746

antityphoid, 648

anti venomous, 161

diagnosis of syphilis, 797

disease, 123

hemolytic, for Wassermann re-
action, titration of, 324

immune, against pneumococcus,
505

in testing opsonic value of blood,

3H

leprosy, 774
plague, 596, 597
tetanus antitoxic, 160
therapy in anthrax, 410
in Asiatic cholera, 617
in bacillary dysentery, 704
to be tested in Wassermann re-
action, 320
Yersin's, 597

Sexual apparatus as avenue of in-
fection for tubercle bacillus, 729
Shake culture, 265
Sheep corpuscles, titration of, for

Wassermann reaction, 324
Shell-fish, bacteria in, 298
Shiga-Kruse variety of dysentery

bacillus, 702
Shiga's bacillus, 699. See also

Bacillus dysenteric.
Siberian pest, 400
Sick-chambers, disinfection of, 211
Sickness, sleeping-, 554. See also

Sleeping-sickness.

Sick-room, air of, disinfection, 211
Silver nitrate as germicide, 214
Size of bacteria, 36

of protozoa, 56

Skin and adjacent mucous mem-
branes, bacteria in, 80
infection through, 86
Sleeping-sickness, 554
prophylaxis, 563
specific organism, 555
transmission, 559

to lower animals, 562
Smegma bacillus, 757. See also

Bacillus smegmatis.
Smith's fermentation-tube, 69
method for determining bacillus
coli communis in water,

291

nature of gases, 70
for isolation of tubercle bacil-
lus, 723

modification of Newman's meth-
od of staining flagella, 194

Smith's modification of Pitfield's
method of staining flagella, 191
phenomenon, 122
tube for isolation of tubercle

bacillus, 723
Soil, bacteria in, 294

Frankel's method of estimat-
ing number, 294
bacteriology of, 294
Soluble toxins, 89, 91
Solvent, 120
Somers' modification of Starkey's

labyrinth, 658
Soor, 484
Sozins, 128
Spasm in tetanus, 393
Specific micro-organisms, 339
Spermatolysis, 164
Spermatoxin, 121
Spermotoxin, 121

anti-, 121

Spinal cord of rabbit, attenuation,
for use in hydrophobia,
416
method of obtaining, for

use in hydrophobia, 414
Spirilla resembling cholera spiril-
lum, 619
table for differentiating,

631
Spirillum, 38, 40

cholerae Asiaticae, cultivation,

609
detection, 615

Loffler's method, 615
distribution, 605
general characteristics, 604
immunity against, 616
in feces, Schottelius' method

of detecting, 609
isolation, 608

Loffler's method of detect-
ing, 615

metabolic products, 612
morphology, 606
pathogenesis, 613
Schottelius' method of mak-
ing pure cultures of, 609
serum therapy of, 617
specificity, 615
spirilla resembling, 619

table for differentiating,
. 631
staining, 608

Cornil and Babes' method,

608

toxic products, 612
vital resistance, 611
of Denecke, 622

Index

873

Spirillum of Denecke, cultivation,

623

metabolic products, 624
morphology, 622
pathogenesis, 624
of Kinkier and Prior, 619
cultivation, 619
metabolic products, 622
morphology, 619
pathogenesis, 622
staining, 619
of Gamaleia, 625
cultivation, 625
immunity against, 628
metabolic products, 627
morphology, 625
pathogenesis, 627
staining, 625
vital resistance, 627
of Obermeier, 546
Spirochaeta, 40
anserinum, 546
carteri, 548
dentinum, 546
duttoni, 547, 548
gallinarum, 546
novyi, 548

obermeieri, 546, 547, 548
cultivation, 550
general characteristics, 548
mode of infection, 550
morphology, 548
pathogenesis, 552
staining, 550
pallida, 787. See also Treponema

pallidum.
pallidula, 801
pertenuis, 80 1
refringens, 787, 799
theileri, 546
vincenti, 479

and Bacillus fusiformis, re-
lation, 479
cultivation, 480
morphology, 480
pathogenesis, 483
Spiromonas, 40
Spirosoma, 40
Spirulina, 40
Spleen, enlargement of, in malaria,

540

Splenic fever, 400
Splenomegaly, febrile tropical, 566
Spontaneous generation, doctrine

of, 17
Spores, 36

germination of, 38
method of staining, 187. See also
Staining spores.

Spores of plasmodium malarise, 529
Sporocysts, 56
Sporozoa, 52

furunculosa, 573
Sporozoits, 528, 531
Sporulation, 36
Spotted fever, 423
Sputum, Bacillus typhosus in, 644
infectious, pasteboard cup for

receiving, 220

tubercle bacillus in, staining, 714
Stain, eosin and methylene-blue,

1 86
iron-hematoxylin, for protozoa,

199
Staining, 174

aqueous solution, 177
flagella, Loffler's method, 189
method of, 189
Pitfield's method, 191

Smith's modification, 191
Rossi's method, 193
Smith's modification of New-
man's method, 194
Van Ermengem's method, 192
Gram's method, 182, 183, 184

Nicolle's modification, 185
Gram-Weigert method, 185
jar, Coplin's, 181
Loffler's method, 182
Mallory's method, 186
Pfeiffer's method, 181
preparations for general examin-
ation, 975
protozoa, 195

Biondi-Heidenhain method,

199

cover-glasses, 196
Heidenhain's method, 199
in tissue, 199
Marino's method, 198
Romanowsky's method, 197
Ross' method, 200
slides, 196

Wright's method, 197
simple method, 176, 181
spores, Abbott's method, 188
Anjeszky's method, 188
Fiocca's method, 189
method of, 187
Moller's method, 188
Muir and Ritchie's method,

1 88

stock solutions, 177
Zieler's method, 186
Standard reaction of culture-media,

227

Standardizing freshly isolated cul-
tures, 259

Index

Staphylococci, chief types, table

of, 342

Staphylococcus, 39
citreus, 350

epidermiditis albus, 341
golden, 343, 346
pyogenes albus, 343
aureus, 343
et albus, 344

agglutination, 349
bacterio-vaccination, 349
cultivation, 345
distribution, 344
isolation, 344
morphology, 344
pathogenesis, 347
staining, 344
thermal death-point, 346
toxic products, 346
treatment with serum, 349
virulence, 349
Staphylolysin, 347
Starkey's labyrinth modified by

Somers, 658
method of isolation of typhoid

bacillus, 657

Steam sterilizer, Arnold's, 205
Stegomyia calopus, 578
fasciata, 578

seu calopus, 577
Stemphylium polymorpha, 485
Sterilization and disinfection, 201
and protection of culture-media,

205

by filtration, 208
in autoclave, 206
intermittent, 205
of catgut, 210

Cladius' method, 211
cumol method, 211
of instruments and glassware, 203
of ligatures, 210
of platinum wires, 203
of surgical instruments, 211
Sterilizer, hot-air, 203
Stewart's cover-glass forceps, 177
Stock solutions, 177
Stomach, bacteria in, 83

carcinoma of, Oppler-Boas bacil-
lus in, 83

Stomatitis, parasite, 484
Street virus in hydrophobia, 414
Streptobacillus, 443
Streptococcus, 39
brevis, 351
conglomeratus, 352
diffusus, 352
erysipelatis, 361
in blood in scarlatina, 355

Streptococcus longus, 361
mucosus, 359
pyogenes, 350
cultivation, 351
differential features, 353
isolation, 351
morphology, 351
pathogenesis, 354
relation to diphtheria, 354
staining, 351
toxic products, 357
virulence, 356
vital resistance, 353
viridans, 354
Streptokolysin, 357
Streptothrix, 42
enteola, 42
farcinica, 43
Structure of bacteria, 34

of protozoa, 53
Subcutaneous injection, 271

inoculation, 271
Subinfection, 78

Substance sensibilisatrice, 144, 145
Sucholotoxin, 68 1
Sugar bouillon, 230
Suppuration, 339
amebae and, 372
amoeba kartulisi as cause, 372

mortinatalium as cause, 373
bacteria associated with, 341
entamoeba buccalis as cause, 372
miscellaneous organisms of, 373
Surgical contributions to history of

bacteria, 21

instruments, sterilization of, 211
Susceptibility, 101
diet as cause, 102
exposure to cold as cause, 102
fatigue as cause, 101
inhalation of noxious vapors as

cause, 1 01

intoxication as cause, 102
morbid conditions in general as

cause, 103

traumatic injury as cause, 103
Susotoxin, 680
Sutures, disinfection of, 209
Swine-plague, bacillus of, 60 1
Symbiosis, influence on growth of

bacteria, 63
Synopsis, proposed, of groups of

bacteria, 279
Syphilis, 787
bacillus of, 787
diagnosis, 797
lesions of, 797

Noguchi's cutaneous reaction in
diagnosis of, 798

Index

875

Syphilis, serum diagnosis, 797
Wassermann reaction for diag-
nosis of, 318. See also Wasser-
mann reaction.
Syphilitic antigen, 319

titration of, 328
Syringe, Altmann's, 269
bacteriologic, 269
Koch's, 269
Meyer's, 269
Roux's, 269

TEMPERATURE in malaria, 524
influence on growth of bacteria,

64

Terminal infections, 355
Tertian malarial fever, parasite of,

^534.
Tetamn, 391

Tetanolysin, 91, 120, 131, 391, 394
Tetanospasmin, 91, 391
Tetanotoxin, 391
Tetanus, 385

after use of diphtheria antitoxin,

4.73
antitoxic serum, 160

antitoxin, 160, 397

ascendens, 393

bacillus, 385. See also Bacillus
tetani.

clonic convulsions in, 393

descendens, 393

opisthotonos in, 393

pathogenesis, 394

prophylactic treatment, 398

spasm in, 393

tonic convulsions in, 393
Tetracoccus, 38

Theobald-Smith phenomenon, 122
Theory, Ehrlich's lateral-chain,
of immunity, 131

MetschnikofP s, of immunity, 126
Thermal death-point of bacteria,

determination, 300
Thermophilic bacteria, 64
Thrush, 484
Tinea circinata, 824

favosa, 828

imbricata, 824

trichophytina, 824

unguium, 824

versicolor, 824
Tongue, wooden, 803, 811
Tonic convulsions in tetanus, 393
Torrey's antigonococcus serum,

437

Toxemia, 94
Toxic power of bacteria, 89

Toxins, extracellular, 89
intracellular, 89, 91
soluble, 89, 91
specific action, 92

affinity of cells for, 93
Toxophile groups, 133
Toxophore group, 131
Toxophylaxins, 129
Toxosozins, 129
T R tuberculin, 742
Trachea, bacteria in, 85
Treponema, 40
pallidum, 787

Burri's India ink method of

identifying, 792
cultivation, 793

Noguchi's method, 793
distribution, 792
general characteristics, 787
morphology, 788
pathogenesis and specificity.

795
staining, 788

Ghoreyeb's method, 789
Goldhorn's method, 788
Levaditi's method, 790
pertenue, 800
cultivation, 802
morphology, 801
pathogenesis, 802
staining, 80 1

Trichomonas intestinalis, 709
Trichophyton, 46
acuminatum, 825
circonvulatum, 825
crateriforme, 825
effractum, 825
exsiccatum, 825
flavum, 825
fulminatum, 825
glabrum, 825
megalosporon, 825
microsporon, 825
pilosum, 825
plicatili, 825
polygonum, 825
regulare, 825
sulphureum, 825
tonsurans, 824
cultivation, 825
morphology, 825
pathogenesis, 826
umbilicatum, 825
violaceum, 825
Trikresol, 215
Trommelschlager, 37
Tropical splenomegaly, febrile, 566

ulcer, 572
Trypanosoma avium, 556

876

Index

Trypanosoma brucei, 556
castellani, 557
cruzi, 564

transmission, 565
damoniae, 556
equinum, 556
equiperdum, 562
gambiense, 554
cultivation, 558
morphology, 558
pathogenesis, 562
reproduction, 559
staining, 558
transmission, 559

to lower animals, 562
lewisi, 556.
rajae, 556
rotatorium, 556
theileri, 556
transvaliense, 556
ugandense, 557
various species, 556
Trypanosomiasis, American, 564
human, 554
transmission, 565
Tryptic enzymes, 71
Tse-tse-fly disease, 560
Tubercle bacillus, 710. See also

Bacillus tuberculosis.
Tubercles, 733
crude, 733
healed, 736
miliary, 733
of Babes, 419
Tubercular abscess, 731

pneumonia, 512
Tuberculin, 738
concentrated, 739
crude, 738
Denys', 742
diluted, 729
influence on tuberculous tissue,

740

Koch's, 739
preparation of, 738
refined, 739

test for tuberculosis of cattle, 754
Tuberculin-R, 742
Tuberculin-T R, 742
Tuberculinic acid, 737
Tuberculocidin, 742, 743
Tuberculosamin, 737
Tuberculosis, 710

bacillus of , 7 1 o. See also Bacillus

tuberculosis.
bovine, 748

communicability to man, 750
prophylaxis, 753
tuberculin test for, 754

Tuberculosis, diagnosis, Calmette's
ophthalmo-tuberculin reac-
tion, 741

Morro's method, 741
von Pirquet's cutaneous meth-
od,- 740
Lignieres' modification,

74i

Wolff-Eisner ophthalmo-tu-
berculin method, 742
fish, 756
fowl, 754
giant-cells in, 731
latent, 735
lesions of, 730
of cattle, tuberculin test for,

754

prophylaxis, 747
pseudo-, 760
specific organism, 711
Tuberculous abscess, 731
Tubes, capillary glass, 243, 244
Esmarch's, 250

method of holding, during inocu-
lation, 245

Typhoid carriers, 640
fever, 632

bacillus of, 632
bacteriologic diagnosis, 649
blood-culture in, 650
carriers, 640

conjunctival reaction in, 650
histologic lesions, 642
in lower animals, 644
isolation of bacillus from feces

in, 650

pathogenesis, 639
prophylactic vaccination

against, 646
prophylaxis, 645
specific therapy, 647
Widal reaction in, 649
pig, 678

reaction of Chantemesse, 650
Typhus abdominalis, 632
Tyrotoxicon, 68, 299
Tyrotoxismus, 299

ULCER, tropical, 572

Umstimmung, 798

Unna's method for staining tuber-
cle bacillus in sections, 719

Urethra, bacteria in, 85

Urine, Bacillus tuberculosis in,

staining, 718
Bacillus typhosus in, 643
smegma bacillus in, 718

Uterus, bacteria in, 85

Index

877

VACCINATION, no
accidents of, 112
advantages of, over inoculation,

in
bacterio-, in staphylococcic in-

fections, 349
efficient, 112
immunity to, 112
in anthrax, 409
inefficient, 112
Jennerian, no
Pasteurian, 113
prophylactic, against typhoid

fever, 646

Vaccine, nature of, in
Vaccines, 113
Vaccinia, 112
Vacuoles, contractile, 54
Vacuum, formation of, in anaerobic

cultures, 260
Vagina, bacteria in, 85
Van Brmengem's method of stain-

ing flagella, 192
Vibrio, 40
lineola, 799
Metschnikovi, 625
proteus, 619
schuylkiliensis, 628
cultivation, 628
immunity against, 629
metabolic products, 628
morphology, 628
pathogenesis, 629
vital resistance, 629
tyrogenum, 622
Vibrion septique, 374
Vibrionensepticaemia, 628
Vincent's angina, 478
Virulence, 95
decrease of, 95
increase of, 96

by addition of animal fluids

to culture-media, 97
by passage through animals, 96
by use of collodion sacs, 96
number of bacteria influencing,

97
Virus, fixed, in hydrophobia, 414

street, in hydrophobia, 414
von Pirquet's method of cutaneous
diagnosis of tubercu-
losis, 740
Lignieres' modification,

WASHED leukocytes in testing op-
sonic value of blood, 311

Wassermann method of cultivation
of micrococcus gonorrhoeae, 434

Wassermann reaction, 318
amboceptor dose in, 326

unit in, 325, 326
antigen in, 319

titration of, 328
blood-corpuscles for, 322

titration of, 324
complement for, 321

titration of, 324
hemolytic amboceptor for,

323
serum for, titration of,

324

system in, 325
nature of, 335
Noguchi's modification, 335
reagents employed, 318
serum to be tested, 320
validity of, 334
Water, Bacillus coli communis in,

291
bacteria in, 287

method of determining num-
ber, 287

Winslow and Willcomb's di-
rect method of enumeration
of, 288

bacteriology of, 287
colon bacillus in, 674

MacConkey's medium for

detecting, 66 1

Wiirtz's medium for detect-
ing, 675
typhoid bacillus in, Starkey's

method of isolating, 657
Wechsberg and Neisser's phenome-
non, 167, 168
Weeks-Koch bacillus, 446. See

also Bacillus of Koch-Weeks.
Weigert-Gram method of staining,

185

Welch and Nuttall's method of
staining bacillus aerogenes cap-
sulatus, 379
Welch's method of staining Dip-

lococcus pneumonias, 494
Wertheim's method of cultivation

of micrococcus gonorrhoeae, 433
Wesbrook's types of Bacillus dip-

theriae, 452, 469

Whey, Petruschky's, as culture-
medium, 240
Whooping-cough, 488
Widal reaction, 149, 649
Willcomb and Winslow's direct
method of enumeration of bac-
teria in water, 288
Williams' method for cultivation of
ameba, 695

Index

Winslow and Prescott's method of

preparing litmus, 239
and Willcomb's direct method
of enumeration of bacteria in
water, 288
Wolff-Bisner ophthalmo-tubercu-

lin reaction, 742

Wolfhtigel's apparatus for counting
colonies of bacteria upon plates,
287
Wooden apparatus, sterilization of,

204

tongue, 803, 811
Wounds as avenue of infection for

tubercle bacillus, 730
disinfection of, 211
Wright's method of cultivation of
micrococcus gonorrhoeae, 434
of enumerating bacteria in

fluid, 289
of making anaerobic cultures,

264, 267

of staining protozoa, 197
Wiirtz's method for detecting colon
bacillus in water, 675

X-RAYS, influence on growth of
bacteria, 62, 63

YAWS, 800

Y bacillus, 700

Yeasts, 43, 818

influence of food on growth of, 60

of light on growth of, 61
Yellow fever, 576

mosquitoes and, 577

prophylaxis, 580
Yersin's serum, 597
Young's method of cultivation of
micrococcus gonorrhreae, 433

fluid, 179
Ziehl's method of staining Bacillus

typhosus, 634
tubercle bacillus, 716
Zieler's method of staining, 186
Zinsser's method of making anae-

robic cultures, 264
Zopf's bacterium pneumoniae, 507
Zur Nedden's bacillus, 450
Zygote, 531

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Haab and DeSchweinitzV
Ophthalmoscopy

Atlas and Epitome of Ophthalmoscopy and Ophthalmoscopic
Diagnosis. By Dr. O. HAAB, of Zurich. Edited, with additions, by
G. E. DESCHWEINITZ, M. D., Professor of Ophthalmology, University
of Pennsylvania. With 152 colored lithographic illustrations and 92
pages of text. Cloth, $3.00 net. In Sannders' Hand- Atlas Series.

THE NEW (2d) EDITION

The great value of Prof. Haab's Atlas of Ophthalmoscopy and Ophthalmo-
scopic Diagnosis has been fully established and entirely justified an English
translation. Not only is the student made acquainted with carefully prepared
Ophthalmoscopic drawings done into well-executed lithographs of the most im-
portant fundus changes, but, in many instances, plates of the microscopic lesions
are added. The whole furnishes a manual of the greatest possible service.

The Lancet, London

"We recommend it as a work that should be in the ophthalmic wards or in the library of
every hospital into which ophthalmic cases are received."

SAUNDERS* BOOKS ON

Cradle's
Nose, Pharynx, and Ear

Diseases of the Nose, Pharynx, and Ear. By HENRY GRADLE,
M.D., late Professor of Ophthalmology and Otology, Northwestern
University Medical School, Chicago. Octavo of 547 pages, illustrated,
including two full-page plates in colors. Cloth, $3.50 net.

INCLUDING TOPOGRAPHIC ANATOMY

This volume presents diseases of the Nose, Pharynx, and Ear as the author
has seen them during an experience of nearly twenty-five years. In it are
answered in detail those questions regarding the course and outcome of diseases
which cause the less experienced observer the most anxiety in an individual case.
Topographic anatomy has been accorded liberal space.

Pennsylvania Medical Journal

"This is the most practical volume on the nose, pharynx, and ear that has appeared
recently. ... It is exactly what the less experienced observer needs, as it avoids the confusion
incident to a categorical statement of everybody's opinion."

Kyle's
Diseases of Nose and Throat

Diseases of the Nose and Throat. By D. BRADEN KYLE, M. D.,
Professor of Laryngology in the Jefferson Medical College, Phila-
delphia. Octavo, 797 pages; with 219 illustrations, 26 in colors.
Cloth, $4.00 net; Half Morocco, $5.50 net.

THE NEW (4th) EDITION

Four large editions of this excellent work fully testify to its practical value.
In this edition the author has revised the text thoroughly, bringing it absolutely
down to date. With the practical purpose of the book in mind, extended con-
sideration has been given to treatment, each disease being considered in full, and
definite courses being laid down to meet special conditions and symptoms.
Pennsylvania Medical Journal

" Dr. Kyle's crisp, terse diction has enabled the inclusion of all needful nose and throat
knowledge in this book. The practical man, be he special or general, will not search in vain
for anything he needs."

GENITO-URINARY AND NOSE, THROAT, ETC. 9

Greene and Brooks'
Genito-Urinary Diseases

Diseases of the Genito=Urinary Organs and the Kidney. By

ROBERT H. GREENE, M. D., Professor of Genito-Urinary Surgery at
Fordham University; and HARLOW BROOKS, M. D., Assistant Pro-
fessor of Clinical Medicine, University and Bellevue Hospital Medical
School. Octavo of 639 pages, illustrated. Cloth, $5.00 net; Half
Morocco, $6.50 net.

THE NEW (3d) EDITION

This new work presents both the medical and surgical sides. Designed as a
work of quick reference, it has been written in a clear, condensed style, so that
the information can be readily grasped and retained. Kidney diseases are very
elaborately detailed.

New York Medical Journal

" As a whole the book is one of the most satisfactory and useful works on genito-urinary
diseases now extant, and will undoubtedly be popular among practitioners and students."

Gleason on Nose, Throat,
and Ear

A Manual of Diseases of the Nose, Throat, and Ear. By E.

BALDWIN GLEASON, M. D., LL. D., Clinical Professor of Otology,
Medico-Chirurgical College, Philadelphia. I2mo of 556 pages, pro-
fusely illustrated. Flexible leather, $2.50 net.

THE NEW (2d) EDITION

Methods of treatment have been simplified as much as possible, so that in
most instances only those methods, drugs, and operations have been advised
which have proved beneficial. A valuable feature consists of the collection of
formulas.

American Journal of the Medical Sciences

" For the practitioner who wishes a reliable guide in laryngology and otology there are few
books which can be more heartily commended."

American Text-Book of Genito-Urinary Diseases, Syphilis, and
Diseases of the Skin. Edited by L. BOLTON BANGS, M. D., and
W. A. HARDAWAY, M. D. Octavo, 1229 pages, 300 engravings, 20
colored plates. Cloth, $7.00 net.

io SAUNDERS' BOOKS ON

Goepp's
Dental State Boards

Dental State Board Questions and Answers — By R. MAX GOEPP,
M. D., author " Medical State Board Questions and Answers." Octavo
of 428 pages. Cloth, $2.75 net.

COMPLETE AND ACCURATE

This new work is along the same practical lines as Dr. Goepp's successful work
on Medical State Boards. The questions included have been gathered from reliable
sources, and embrace all those likely to be asked in any State Board examination
in any State. They have been arranged and classified in a way that makes for a
rapid resume of every branch of dental practice, and the answers are couched in
language unusually explicit — concise, definite, accurate.

The practicing dentist, also, will find here a work of great value — a work
covering the entire range of dentistry and extremely well adapted for quick
reference.

Haab and deSchweinitz's
Operative Ophthalmology

Atlas and Epitome of Operative Ophthalmology. By DR. O.

HAAB, of Zurich. Edited, with additions, by G. E. DE SCHWEINITZ,
M. D., Professor of Ophthalmology in the University of Pennsylvania.
With 30 colored lithographic plates, 1 54 text-cuts, and 375 pages of
text. In Sounders' Hand- Atlas Series. Cloth, $3.50 net.

Dr. Haab's Atlas of Operative Ophthalmology will be found as beautiful and
as practical as his two former atlases. The work represents the author' s thirty
years' experience in eye work. The various operative interventions are described
with all the precision and clearness that such an experience brings. Recognizing
the fact that mere verbal descriptions are frequently insufficient to give a clear
idea of operative procedures, Dr. Haab has taken particular care to illustrate
plainly the different parts of the operations.

Johns Hopkins Hospital Bulletin

" The descriptions of the various operations are so clear and full that the volume can well
hold place with more pretentious text-books."

CHEMISTRY AND

Holland's Medical
Chemistry and Toxicology

A Text=Book of Medical Chemistry and Toxicology. By JAMES
W. HOLLAND, M. D., Professor of Medical Chemistry and Toxicology,
and Dean, Jefferson Medical College, Philadelphia. Octavo of 6j$
pages, fully illustrated. Cloth, $3.00 net.

THE NEW (3d) EDITION

Dr. Holland's work is an entirely new one, and is based on his forty years'
practical experience in teaching chemistry and medicine. It has been subjected to
a thorough revision, and enlarged to the extent of some sixty pages. The additions
to be specially noted are those relating to the electronic theory, chemical equilib-
rium, Kjeldahl's method for determining nitrogen, chemistry of foods and their
changes in the body, synthesis of proteins, and the latest improvements in urinary
tests. More space is given to toxicology than in any other text-book on chemistry.

American Medicine

" Its statements are clear and terse ; its illustrations well chosen; its development logical,
systematic, and comparatively easy to follow. . . . We heartily commend the work."

Ivy's Applied Anatomy and

Oral Surgery for Dental Students

Applied Anatomy and Oral Surgery for Dental Students. By

ROBERT H. IVY, M.D., D.D.S., Assistant Oral Surgeon to the Philadel-
phia General Hospital. I2mo of 280 pages, illustrated. Cloth, $1.50
net.

FOR DENTAL STUDENTS

This work is just what dental students have long wanted— a concise, practical
work on applied anatomy and oral surgery, written with their needs solely in
mind. No one could be better fitted for this task than Dr. Ivy, who is a graduate
in both dentistry and medicine. Having gone through the dental school, he
knows precisely the dental student's needs and just how to meet them. His
medical training assures you that his anatomy is accurate and his technic modern.
The text is well illustrated with pictures that you will find extremely helpful.

H. P. Kuhn, M.D., Western Dental College, Kansas City.

" I am delighted with this compact little treatise. It seems to me just to fill the bill."

12 SAUNDERS* BOOKS ON

Wells' Chemical Pathology

Chemical Pathology. Being a discussion of General Path-
ology from the Standpoint of the Chemical Processes Involved.
By H. GIDEON WELLS, PH. D., M. D., Assistant Professor of
Pathology in the University of Chicago. Octavo of 549 pages.
Cloth, $3.25 net; Half Morocco, $4.75 net.

Wm. H. Welch, M. D., Professor of Pathology, Johns Hopkins University.

" The work fills a real need in the English literature of a very important subject, and
I shall be glad to recommend it to my students."

The New (2d) Edition

Saxe's Urinalysis

Examination of the Urine. By G. A. DE SANTOS SAXE, M. D.,
formerly Instructor in Genito-Urinary Surgery, New York Post-
graduate Medical School and Hospital. I2mo of 448 pages, fully
illustrated. Cloth, $1.75 net.

Francis Carter Wood, M. D., Adjunct Professor of Clinical Pathology, Columbia Uni-
versity.

"It seems to me to be one of the best of the smaller works on this subject ; it is,
indeed, better than a good many of the larger ones."

deSchweinitz and Randall on the Eye, Ear,
Nose, and Throat

American Text-Book of Diseases of the Eye, Ear, Nose, and
Throat, Edited by G. E. DE SCHWEINITZ, M.D., and B. ALEX-
ANDER RANDALL, M.D. Imperial octavo, 1251 pages, with 766
illustrations, 59 of them in colors. Cloth, $7.00 net; Half Mo-
rocco, $8.50 net.

Griinwald and Grayson on the Larynx

Atlas and Epitome of Diseases of the Larynx. By Dr. L.

GRUNWALD, of Munich. Edited, with additions, by CHARLES P.
GRAYSON, M.D., University of Pennsylvania. With 107 colored
figures on 44 plates, 25 text-cuts, and 103 pages of text. Cloth,
$2.50 net. In Saunders1 Hand-Atlas Scries.

Mracek and Stelwagon's Atlas of Skin

Atlas and Epitome of Diseases of the <Skin. By PROF. DR.
FRANZ MRACEK, of Vienna. Edited, with additions, by HENRY
W. STELWAGON, M.D., Jefferson Medical College. With 77 col-
ored plates, 50 half-tone illustrations, and 280 pages of text. In
Saunders' Hand-Atlas Series. Cloth, $4.00 net.

CHEMISTRY, SKIN, AND VENEREAL DISEASES. 13

American Pocket Dictionary New (7th) Edition

THE AMERICAN POCKET MEDICAL DICTIONARY. Edited by W. A,
NEWMAN BORLAND, M. D., Editor " American Illustrated Medical
Dictionary." Containing the pronunciation and definition of the
principal words used in medicine and kindred sciences. 610 pages.
Flexible leather, with gold edges, $1.00 net; with thumb index,
#1.25 net.
James W. Holland, M. D.,

Professor of Medical Chemistry and Toxicology, and Dean, Jefferson Medical College,
Philadelphia,

" I am struck at once with admiration at the compact size and attractive exterior. ]
can recommend it to our students without reserve."

Stelwagon's Essentials of Skin 7th Edition

ESSENTIALS OF DISEASES OF THE SKIN. By HENRY W. STEL-
WAGON, M. D., PH.D., Professor of Dermatology in the Jeffer-
son Medical College, Philadelphia. Post-octavo of 291 pages,
with 72 text-illustrations and 8 plates. Cloth, $1.00 net. In
Saunders' Question- Compend Series.
The Medical News

" In line with our present knowledge of diseases of the skin. . . . Continues to main-
tain the high standard of excellence for which these question compends have been noted."

Wolffs Medical Chemistry New (7th) Edition

ESSENTIALS OF MEDICAL CHEMISTRY, ORGANIC AND INORGANIC.
Containing also Questions on Medical Physics, Chemical Physiol-
ogy, Analytical Processes, Urinalysis, and Toxicology. By LAW-
RENCE WOLFF, M. D., Late Demonstrator of Chemistry, Jefferson
Medical College. Revised by A. FERREE WITMER, PH. G., M. D.,
Formerly Assistant Demonstrator of Physiology, University of
Pennsylvania. Post-octavo of 222 pages. Cloth, $1.00 net. In
Sounder^ Question- Compend Series.

Martin's Minor Surgery, Bandaging, and the Venereal

Diseases Second Edition, Revised

ESSENTIALS OF MINOR SURGERY, BANDAGING, AND VENEREAL
DISEASES. By EDWARD MARTIN, A. M., M. D., Professor of Clin-
ical Surgery, University of Pennsylvania, etc. Post-octavo, 166
pages, with 78 illustrations. Cloth, $1.00 net. /// Saunders*
Question- Compend Series.

Vecki's Sexual Impotence New (4th) Edition-just Ready

SEXUAL IMPOTENCE. By VICTOR G. VECKI, M. D., Consulting
Genito-Urinary Surgeon to Mt. Zion Hospital, San Francisco.
I2mo of 400 pages. Cloth, $2.25 net.

Johns Hopkins Hospital Bulletin

" A scientific treatise upon an important and much neglected subject. . . . The
treatment of impotence in general and of sexual neurasthenia is discriminating and
judicious."

14 EYE, EAR, NOSE. AND THROAT.

deSchweinitz and Holloway on Pulsating Exoph-
thalmos

PULSATING EXOPHTHALMOS. An analysis of sixty-nine cases not pre-
viously analyzed. By GEORGE E. DESCHWEINITZ, M. D., and THOMAS
B. HOLLOWAY, M. D. Octavo of 125 pages. Cloth, $2.00 net.

This monograph consists of an analysis of sixty-nine cases of this affection
not previously analyzed. The therapeutic measures, surgical and otherwise,
which have been employed are compared, and an endeavor has been made
to determine from these analyses which procedures seem likely to prove of
the greatest value. It is the most valuable contribution to ophthalmic liter-
ature within recent years.

British Medical Journal

"The book deals very thoroughly with the whole subject and in it the most complete account of
the disease will be found."

Jackson on the Eye The New (2d) Edition

A MANUAL OF THE DIAGNOSIS AND TREATMENT OF DISEASES OF THE
EYE. By EDWARD JACKSON, A. M., M. D., Professor of Ophthalmology,
University of Colorado. i2mo volume of 615 pages, with 184 beautiful
illustrations. Cloth, $2.50 net.

The Medical Record, New York

" It is truly an admirable work. . . . Written in a clear, concise manner, it bears evidence of the
author's comprehensive grasp of the subject. The term ' multum in parvo ' is an appropriate one to
apply to this work."

Grant on Face, Mouth, and Jaws

A TEXT-BOOK OF THE SURGICAL PRINCIPLES AND SURGICAL DISEASES
OF THE FACE, MOUTH, AND JAWS. For Dental Students. By H. HORACE
GRANT, A. M., M. D., Professor of Surgery and of Clinical Surgery,
Hospital College of Medicine, Louisville. Octavo of 231 pages, with
68 illustrations. Cloth, $2.50 net.

Preiswerk and Warren's Dentistry

ATLAS AND EPITOME OF DENTISTRY. By PROF. G. PREISWERK, of
Basil. Edited, with additions, by GEORGE W. WARREN, D.D.S., Pro-
fessor of Operative Dentistry, Pennsylvania College of Dental Surgery,
Philadelphia. With 44 lithographic plates, 152 text-cuts, and 343 pages
of text. Cloth, $3.50 net. /// Saunters' Atlas Series.

Friedrich and Curtis on Nose, Larynx, and Ear

RHINOLOGY, LARYNGOLOGy, AND OTOLOGY, AND THEIR SIGNIFICANCE
IN GENERAL MEDICINE. By DR. E. P. FRIEDRICH, of Leipzig. Edited
by H. HOLBROOK CURTIS, M. D., Consulting Surgeon to the New York
Nose and Throat Hospital. Octavo volume of 350 pages. Cloth,
$2.50 net.

SAUNDERS' BOOKS ON

Wolfs Examination of Urine

A LABORATORY HANDBOOK OF PHYSIOLOGIC CHEMISTRY AND
URINE-EXAMINATION. By CHARLES G. L. WOLF, M. D., Instructor in
Physiologic Chemistry, Cornell University Medical College, New
York. 1 2mo volume of 204 pages, fully illustrated. Cloth, $1.25 net.
British Medical Journal

" The methods of examining the urine are very fully described, and there are at the
end of the book some extensive tables drawn up to assist in urinary diagnosis."

Jackson's Essentials of Eye Third Revised Edition

ESSENTIALS OF REFRACTION AND OF DISEASES OF THE EYE. By
EDWARD JACKSON, A. M., M. D., Emeritus Professor of Diseases of
the Eye, Philadelphia Polyclinic. Post-octavo of 261 pages, 82 illus-
trations. Cloth, $1.00 net. In Saunders1 Question- Compend Series.
Johns Hopkins Hospital Bulletin

" The entire ground is covered, and the points that most need careful elucidation
are made clear and easy."

Gleason's Nose and Throat Fourth Edition, Revised

ESSENTIALS OF DISEASES OF THE NOSE AND THROAT. By E. B.
GLEASON, S. B., M. D., Clinical Professor of Otology, Medico-
Chirurgical College, Philadelphia, .etc. Post-octavo, 241 pages, 1 12
illustrations. Cloth, $1.00 net. In Saunders' Question Compends.
The Lancet, London

" The careful description which is given of the various procedures would be sufficient
to enable most people of average intelligence and of slight anatomical knowledge to
make a very good attempt at laryngoscopy."

Gleason's Diseases of the Ear Third Edition, Revised

ESSENTIALS OF DISEASES OF THE EAR. By E. B. GLEASON, S. B.,
M. D., Clinical Professor of Otology, Medico-Chirurgical College,
Phila., etc. Post-octavo volume of 214 pages, with 114 illustra-
tions. Cloth, $ i. oo net. In Saunders1 Question- Compend Series.
Bristol Medico-Chirurgical Journal

" We know of no other small work on ear diseases to compare with this, either in
freshness of style or completeness of information."

Wilcox on Genito-Urinary and Venereal Diseases

The New (2d) Edition

ESSENTIALS OF GENITO-URINARY AND VENEREAL DISEASES. By
STARLING S. WILCOX, M. D., Lecturer on Genito-Urinary Diseases
and Syphilology, Starling-Ohio Medical College, Columbus. I2mo
of 321 pages, illustrated. Cloth, $1.00 net. Saunders' Compends.

Stevenson's Photoscopy

PHOTOSCOPY (Skiascopy or Retinoscopy). By MARK D. STEV-
ENSON, M. D., Ophthalmic Surgeon to the Akron City Hospital.
I2mo of 126 pages, illustrated. Cloth, $1.25 net.

Edward Jackson, M. D., University of Colorado.

" It is well written and will prove a valuable help. Your treatment of the emergent
pencil of rays, and the part falling on the examiner's eye, is decidedly better than any
previous account."