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BOOKS

JOSEPH McFARLAND, M.D., Sc. D.

Pathogenic Bacteria and Protozoa

Octavo of 858 pages, illustrated.
Ninth Edition

Pathology

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

Biology: General and Medical

1Izmo of 457 pages, with 160 illustra-
tions. Cloth, #1.75 net. Third Edition

A TEXT-BOOK

UPON THE

PATHOGENIC BACTERIA
AND PROTOZOA

FOR STUDENTS OF MEDICINE AND PHYSICIANS

JOSEPH McFARLAND, M. D., Sc. D.

Professor of Pathology and Bacteriology in the University of Pennsylvania; Pathologist to the Philar
delphia General Hospital; Fellow of the College of Physicians of Philadelphia, Etc.

NINTH EDITION, REVISED
WITH 330 ILLUSTRATIONS
A NUMBER IN COLORS

PHILADELPHIA AND LONDON

W. B. SAUNDERS COMPANY

[eh E

att a {
A9ID.
eee

Copyright, 1896, by W. B. Saunders. Reprinted September, 1896. Re-
vised, reprinted, and recopyrighted August, 1898. Reprinted November,
1898, Revised, reprinted, and recopyrighted August, z900. Reprinted
June, 1901. Revised, entirely reset, reprinted, and recopyrighted May,
1903. eprinted 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. Reprinted May, 1914. Revised, entirely reset, re-
printed, and recopyrighted November, 1915. Revised. entirely reset, re-
printed, and recopyrighted August, 1919.

Yeo) be SS Oe
PRINTED IN AMERICA

TO
MY HONORED AND BELOVED GRANDFATHER
five. Jacob Grim

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

THIS BOOK IS AFFECTIONATELY DEDICATED

PREFACE TO THE NINTH EDITION

Tuis ninth edition of the “ Pathogenic Bacteria and Protozoa,” has
been prepared under conditions as unfavorable for literary study
and compilation as can easily be conceived. The countries of
Europe were engaged in a terrible war into which the United
States had been drawn as a participant and every resource of
our country was requisitioned in order that tyranny might be
overcome and “‘democracy made safe for the world.”

The scholastic quiet of the author’s university was invaded by
the call of the bugle, the voice of command and the tramp of march-
ing feet as the students were called from class-rooms and laboratories
to engage in military training.

The author himself was called to serve in the army, and the actual
work of revision was accomplished “in the field.” The “copy”
was prepared from memorandum notes, during long evening hours,
at the Base Hospital at Camp Beauregard, Alexandria, La.; the
“galley sheets” were read and corrected at the U. S. A. General
Hospital at Lakewood, N. J., and the finished pages were read at
the U. S. A. General Hospital No. 14 at Fort Oglethorpe, Ga.
and the U. S. A. General Hospital No. 19 at Azalea, N. C.

Amid these distracting surroundings, away from books and jour-
nals, the old fabric was unravelled and rewoven to an extent that
necessitated the resetting of the type of the entire volume. It is,
however, hoped that old errors have been corrected, new ones
avoided, and enough new matter introduced to bring the whole
work up to date and greatly increase its practical value.

It is with deep regret that we find, upon looking over the biblio-
graphic index, that more than two hundred and fifty of our friends
in science, whose work has been mentioned in the text, have become
our enemies in politics. That such a circumstance should arise is
deplorable; that it shall persist is inconceivable.

It has always been the author’s proud contention that men of
science formed a kind of international brotherhood, distinguished by
an unwearied ambition to free human existence of unnecessary
suffering and untimely death and to provide it with means of pro-
moting happiness and increasing longevity. With his vision
focused upon the work of the physician and sanitarian intent upon

se) . Preface to the Ninth Edition

the preservation of life, he neglected to observe the equally enthu-
siastic endeavors of the physicist, the chemist and the engineer to
provide means for its destruction. The war has been the rude
means of falsifying his delusion and of dispelling his dream of
idealism and internationalism. Men of science are like other men!
But as these words are being written the world rejoices in an armis-
tice that promises peace—a peace longed for and prayed for by
hundreds of millions of troubled mortals! One turns his eyes
toward the eastern horizon a few days ago reddened by the flames
of burning villages and the flashing of the guns, and sees the eternal
serenity of a clear, bluesky. May it presage the end of war, a clear
understanding among nations and the return of men to useful and

creative labor!
THe AUTHOR.

August, 1919.

CONTENTS

PART I.—GENERAL

HIsTorIcAL INTRODUCTION. . ... Bg Sees sea ak Hae! Oa

CHAPTER I

ee Y

STRUCTURE AND CLASSIFICATION OF THE MICRO-ORGANISMS .......

CHAPTER II

BIoLoGy oF MICRO-ORGANISMS. . .......
INFECTION. ........ tle? ae. ets

TRIMUNTE: 551.) Bat ale a ve ee a eS Got

CHAPTER V

METHODS OF OBSERVING MICRO-ORGANISMS. . .

CHAPTER VI

STERILIZATION AND DISINFECTION. ......

CHAPTER VII

CULTURE-MEDIA AND THE CULTIVATION OF MICRO-ORGANISMS .

CHAPTER VIII

CULTURES, AND THEIR STUDY. ........

CHAPTER IX

Tue CULTIVATION OF ANAEROBIC ORGANISMS. . .

CHAPTER X
EXPERIMENTATION UPON ANIMALS. ...... .
CHAPTER XI

Tue IDENTIFICATION OF SPECIES. .......

CHAPTER XII

Tut BACTERIOLOGY OF THE AIR. .......

CHAPTER XIII

Tae BACTERIOLOGY OF WATER. ... 2...

Pace
17

147

. 189

203

217

227

235

239

. 242

12 Contents

CHAPTER XIV

Pace
Tue BACTERIOLOGY OF THE SOIL... ee ee ee ee et 249
CHAPTER XV
THE BacTERIOLOGY OF Foops .... 2... 6 ee ee et ee 251
CHAPTER XVI
Tue DETERMINATION OF THE THERMAL DEATH-POINT OF BACTERIA. . . . 257
CHAPTER XVII
Tue DETERMINATION OF THE VALUE OF ANTISEPTICS, GERMICIDES, AND Dis-
INFECTANTS ou Gb kon we RE we RR A RE Ee we we 259
CHAPTER XVIII a
BACTERIO-VACCINES:..0. .0 6 2 AE RE ee EE we BR ew we 271
CHAPTER XIX
THe PHAGOCYTIC POWER OF THE BLOOD AND THE OPSONIC INDEX ... . 278
; CHAPTER XX
THE WASSERMANN REACTION FOR THE DIAGNOSIS OF SYPHILIS ..... 287

PART II.—_THE INFECTIOUS DISEASES AND THE
SPECIFIC MICRO-ORGANISMS

CHAPTER I

SUPPURATION® «@ @ #4) @ Gf ee Ww ® ww WR RE Awe wo awa 307
CHAPTER II

Maticnant EpEMA AND Gasgous EpeEMA.............. 339

SPETAN USS 2c: Ges) ie! a Gahiatcig Jat HS eo a ey er ee Se Se Oe a 352
CHAPTER IV
CHAPTER V

HypropuosiA, Lyssa, oR RABIES... ......., 2 AR OS, ae 375
CHAPTER VI

Acute ANTERIOR POLIOMYELITIS ...... SPR al tee ie + 6 + 6 303

Contents 13

CHAPTER VII

Paan
CEREBRO-SPINAL MENINGITIS. ©. 6 1 ee ee 398
CHAPTER VIII
(GONORRHEA: 6: 2S so2 siete chad Ges Sate) AY AP aL Bae on ee BLS De Gee 410
CHAPTER IX
CATARRHAL INFLAMMATION. 2. 0 0 ee ee ee 417
CHAPTER X
CHANCROID 46/32) Ai AO ey wa SS Re SSE EP ee a 420
CHAPTER XI
Acute Contacious CONJUNCTIVITIS. .. 2.2. 2 ee eee ee 423
CHAPTER XII
DIPHTHERIA... . 2... Eitan co Beef anieteattl fo, blac hone (1 428
. CHAPTER XIII
VINCENT’S‘ANGINA, 6 joa 2k ee a eae ee ee 451
CHAPTER XIV
THRUSH Ts Bg ey ghee ea ee ee Oy a. ges) BA eae 457
CHAPTER XV
WHOOPING-COUGH. . 2. 2 1. ee 460
CHAPTER XVI
PNEUMONIA’ ¢ (s.cy-a ge Go RO RR REA eG ee eS 464
CHAPTER XVII
INELUENZAL ie OE ee ae te ee BE PA 486 -
CHAPTER XVIII
Matta oR MEDITERRANEAN FEVER. . . 1. 2. ee et ee et 491
CHAPTER XIX
IMALARUAL C4 8k! ede, 2 we RE hw. oe ee 495
CHAPTER XX

RELAPSING PRVER.c.«. a. go) ade are aes at ea ee a 520

CHAPTER XXI

INFECTIVE JAUNDICE; WEIL’s DISEASE; SPIROCHZTOSIS ICTEROHEMOR-
RHAGICA; RAT-BITE FEVER. .....- ck: Gp eer ele de gw & We 4532

14 Contents

: CHAPTER XXII

Pace

SLEEPING SICKNESS. . 2. 6) 2 ee tt ee ee i Bee 544
CHAPTER XXIII

Kata-azar (BLACK SICKNESS) .. 2. 6 6 2 ee et 563
CHAPTER XXIV

YELLOW FEVERS ¢ 3 & & Bo SR Be we es ee 1. 574
CHAPTER XXV

TypHus FEVER. ...........- a ee ee 578
CHAPTER XXVI

PUAG UR fe ee tee eae os Goda, Se ee ay ROE ae A er Se ; 582
CHAPTER XXVII

ASTATIC: CHOLERAS. 2.6: 2c oe oe RR Re ee 608
CHAPTER XXVIII

IVPHOW: FEVER: © s% a 406 Ge HR SR a Se eee ke as 629
CHAPTER XXIX

DOVSENDER VY) 2 lx erin oe let el ea Oh aden, Ue sal eres Waersed ses GU peter eat 671
CHAPTER XXX

{DUBERCULOSIS: ei 08 <i ne Ye apse. A RS Sl SS ny & ot BB AR 699
CHAPTER XXXI

Leprosy........... Neto apes Eh Goes WOU ee eae ee 739
CHAPTER XXXII

GUANDERS 50 3 ide Aa ho, Be Bes Ss @ eR Re 749
CHAPTER XXXTII

RHINOSCLEROMA . 2 2... 758
CHAPTER XXXIV

DSVPHILIB Ge Swe, A se conerag iD ae cae es A a kee UB eo bee ae 761
CHAPTER XXXV

FrampesiaA Tropica (YAWs)........20..02..2.2.2.2.24.4 772
CHAPTER XXXVI

ACTINOMYCOSIS!s, 62. ceo me ae ae RR ee a a ew a 775

Contents 15

CHAPTER XXXVII : e
AGB
MycreToMA, OR MADURA-FOOT . 2... ee ee ee es 786
CHAPTER XXXVIII
BLASTOMYCOSIS. . 2. 2 1 2 ee ee ee Te a (ae ae sO 793
CHAPTER XXXIX
RINGWORM. «65 2 Se RE ERR NS Oe a 798
CHAPTER XL
AVUS§. we. fod ai hy Ae Ga ge, wel Ue a ep eS co anertee ease 801
CHAPTER XLI
SPOROTRICHOSIS 2.5. 6% 4 i ee ee a eG HR we 805
Breuiocrapuic INDEX... 1. tt oe ee 813
INDEX OF “SUBJECTS? .5: eo aoc Ao ee es en ee es BS HG, 831

PART I. GENERAL

HISTORICAL INTRODUCTION

BioLocy, chemistry, medicine, and surgery, in their evolution,
contributed to a new branch of knowledge, Bacteriology, whose
subsequent development has become of inestimable importance to
each. Indeed, bacteriology illustrates 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; THE DOCTRINE OF SPONTANEOUS
GENERATION

Among the early Greeks we find that Anaximander (43d Olym-
piad, 610 B. C.) of Miletus held the theory that animals were
formed from moisture. Empedocles of Agrigentum (450 B. C.)
attributed to spontaneous generation 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 sometimes in the
fluids of other animals.”

Three centuries later, in his disquisition upon the Pythagorean
philosophy, we find Ovid defending the same doctrine 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 generation. In 1542, in his
treatise called ‘‘De Subtilitate,” we find Cardan asserting that
water engenders fishes, and that many animals spring from fermenta-
tion. 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:
* See Tyndall: “Floating Matter in the Air.”

2 : 17

18 Introduction

“Watching meat in its passage from freshness to decay, prior to
the appearance of maggots, he invariably observed flies buzzing
around the meat and frequently alighting 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 putrefied 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 circulation of the blood; discovered
bacteria, seeing them first in saliva, discovered the rotifers, and first
saw the little globules in yeast which Latour and Schwann subse-
quently proved to be plants.

Leeuwenhoek involuntarily reopened the old controversy 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. ;

Abbé Lazzaro Spallanzani (1777) filled flasks with organic in-
fusions, sealed their necks, and, after subjecting their contents 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 vegetable 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 destroyed. 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

The History of the Subject - 19

Schulze worked, for, as was shown by those who repeated 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 recognized at the
present time. He, however, looked upon all forms of “infusorial
life” as animalcule or minute animals, and Dujardin who wrote
about them in 1845 continued to do the same. The first to regard
some of them, notably the vibrios and spirilla of Ehrenberg, as
plant organisms, seems to have been Joseph Leidy who published a
paper in the Proceedings of the Academy of Natural Sciences of
Philadelphia in 1849 upon “Entophyta or vegetal parasites infest-
ing the intestinal canal of animals,” in which he held that the
organisms mentioned should be classed among the alge and placed
in the vegetable kingdom.

Tyndall, stimulated by the work of Pasteur, conclusively 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 chambers, 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 optical test, to determine the purity of the contained
atmosphere, by passing a powerful ray of light through the side
windows. When viewed through the front, this ray was visible
only so long as there were particles suspended in the atmosphere to
reflect it. When the dust had completely settled and the light ray
had become invisible because of the purity of the contained atmos-
phere, 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 PUTREFACTICN

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 inexplicable 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

. 20 Introduction

the time of Leeuwenhoek were living organisms—vegetable forms— 2
capable of growth. ——

So long as yeast was looked upon as an inert substance it was
impossible to understand how it could impart fermentation to other
substances; but when it was shown by Latour that the essential
element of yeast was a growing plant, the phénomenon became a
perfectly natural consequence of life. Not only the alcoholic, but
also the acetic, lactic, and butyric fermentations have been shown
to result 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 fermentation was the result of the
internal molecular movements which a body in the course of de-
composition communicates to other matter whose elements are |
connected by a very feeble affinity. ‘

Pasteur was the first to prove that fermentation is an ordinary
chemic transformation of certain substances, taking 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 Corpuscles Exist-

ing in the Atmosphere,” in which he showed that many of the
floating particles collected from the atmosphere 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
infusions came from the air, but was not evenly distributed
through it.”
_ Three years later he showed that the organized corpuscles 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:

The History of the Subject 21

“Tt 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 success upon
exclusion of the air, and of course, incidentally if unknowingly,
exclusion of bacteria, seem to have been practised quite early.
Theodoric, of Bologne, about 1260 taught that the action of the air
upon wounds induced a pathologic conditions predisposing to sup-
puration. 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 Mondeville 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 com-
pounded 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).

“Tn 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 infecting 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 fomites. Germs
transmitting disease at a distance must be able to live in the air a
certain length of time, and this condition he 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 dissolution, 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 contemporaries to be founded
upon too little evidence, and were not received.

* “Brit. Med. Jour.,” May 7, 1910, p. 1122.

92 Introduction

Plencig, of Vienna, became convinced that there was an undoubted
connection between the microscopic animalcules exhibited by the
microscope and the origin of disease, and advanced this opinion as
early as 1762.

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 occurrence of
yeast plants in the stools of cholera, and conjectured 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 infectious diseases was to be
sought for in minute living organisms or fungi. He may be looked ~
upon as the real propounder 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 formulated 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 elaborated, 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 micro-organisms and
disease has been determined.

Briefly summarized, these requirements are as follows:

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

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

3. When introduced into healthy animals it must produce the
disease, and in the animal in which the disease has been experiment-
ally 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 the breasts, and announced
his belief that the disease depended upon poisons carried by the
fingers 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 “ Cryptogamous
Origin of Malarious and Epidemic Fevers,” foreshadowed 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 History of the Subject 23

the presence of the anthrax bacillus in the blood of animals suffering
from and dead of that disease. 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. Billroth, 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’’ (putrefactive ferment),
or through inflammation a “Phlogistischezymoid”’ (inflammatory
ferment), supplying the necessary 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 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 artifi-
cially prepared media was unquestionable, were spontaneously gen-
erated there.

Before many of the important bacteria had been discovered, and
while ideas upon the relation of micro-organisms to disease were
most crude, some practical measures were suggested that produced
greater agitation and incited more observation and experimentation
than anything suggested in surgery since the introduction of anes-
thetics—namely, antisepsis.

“Tt 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 suppuration, 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, instruments, fingers, etc., into
wounds, suggested the employment of carbolic-acid for the purpose
of f keeping sterile the hands of the operator, the skin of the patient,

* Agnew’s “Surgery,” vol. 1, chap. 11.

24 | Introduction

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 “ Wundinfectionskrankheiten”
(Traumatic Infectious Diseases), in 1878, and spread slowly at first,
‘but surely in the end, to all departments 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 otherwisé
have failed. Among them may be mentioned 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 Schréder 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 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 discoveries.

Thus, in 1873, Obermeier discovered Spirillum obermeieri 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 Eberth and
independently by Koch, Pasteur published his work upon “ Chicken-
cholera,” and Sternberg described the pneumococcus, calling it
Micrococcus pasteurt.

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 porc,” 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 Léffler 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 triumphant
achievement of scientific medicine, was inaugurated 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, Ehrlich, Metchnikoff, Bordet,
Wassermann, Shiga, Madsen, and Arrhenius taking front rank.

The discovery of the Treponema pallidum, the specific organism

The History of the Subject . 25

of syphilis, was made in 1905 by Schaudinn and Hoffmann, long
after clinical study of the disease had anticipated 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 Treponema
pertenue, the cause of frambesia or yaws.

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

In 1913 Flexner and Noguchi appear to have been successful { in
cultivating the virus of acute anterior poliomyelitis, in vitro.

During the time that so much investigation of the problems 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 purpose 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 Liésch discovered the Amceba coli; in 1878 Rivolta de-
scribed the Coccidium cuniculi of the rabbit; in 1879 Lewis first
saw Trypanosoma lewisi in the blood of the rat; in 1881 Laveran
discovered Plasmodium malaria in the blood of cases of human
paludism; in 1885 Blanchard described the Sarcocystis in muscle-
fibers; in 1893 Councilman and Lafleur studied Amceba dysenteriz
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 in-
dependently, observed trypanosomes—the Trypanosoma gambiense
of African lethargy—in the blood of human beings.

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 ultra-
microscope, that shows the existence of things too small to be defined,
and still more to the adaptation of the method of filtration to
the study of the diseases in question, we realize that the “viruses”’

‘of disease may be visible or invisible and that they have no limita-
tions of size. Just as bacteria readily find their way through paper
filters, so the invisible and hence undescribed viruses—i.e., micro-
organisms—of yellow fever, pleuro-pneumonia of cattle, foot-and-
mouth disease, rinderpest, hog-cholera, African horse-fever, infec-
tious 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 un-
glazed porcelain or Chamberland filters. Thus there is opened a new
world that is ultramicroscopic, but still teems with invisible living
organisms.

CHAPTER I

STRUCTURE AND CLASSIFICATION OF
THE MICRO-ORGANISMS

BACTERIA

WueEn Leeuwenhoek with his improved microscope discovered
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 animalculea. The early systematic writers,
Ehrenberg and Dujardin, fell into the same error, and although Leidy —
in 1849 looked upon them as alge, and as belonging to the plant
kingdom, it was many years before biologists were satisfied as to
their true position in nature. Indeed, no less an authority than
Haeckel, as late as 1878, suggested that they form a group by them-
selves neither animal nor vegetable, but intermediate between the
two, to be known as Protista. This, however, was unsatisfactory
alike to botanists and zodlogists, and did not become popular.

It was evident that structure could not be looked upon as a satis-
factory 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 difference was, however, noted: plants possess the power
of nourishing themselves upon purely inorgariic compounds, while
animals are unable to do so and cannot live except upon complex
molecular combinations synthesized by the plants. In this meta-
bolic difference we find the present criterion for the separation of the
living organisms into the two main groups. But this does not dis-
pose of all of the difficulties, for there are certain small groups to
which it 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 un-
hesitatingly assigns most of the known organisms to one or the
other groups, and that occasional mistakes may be-made, and

Bacteria 27

organisms, like the spirocheta, appear sometimes in the group of
plants among the bacteria, and in other writings in the group of ani-
mals among the protozoa, is a matter of small consequence so long
as the knowledge of the organisms themselves is in no particular
diminished by the method of classifying them.

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 another 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 meaningless, 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
_ animals 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 relation-
ship between the lowest alge and some of the flagellates, and it is
the general opinion that no one feature separates 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 now accepted relative position of the pathogenic vegetable
micro-organisms to the other vegetable organisms can be determined
by reference to the following table. The wide separation of the bac-
teria in Group II. and all of the others, which appear in Group X.,
should be noted.

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:

Size.— Bacteria are so minute that a special unit has been adopted
for their measurement. This is the micron, micromillimeter or
u, and is the one-thousandth part of a millimeter, equivalent to the
one-twenty-five-thousandth (145000) of an inch.

There is no limit to the minuteness of micro-organisms. Visibility
is no longer a criterion. There are micro-organisms that can be
seen with low powers, others that can only be seen with high
powers, and a few that probably cannot be seen with any power of
the microscope. These are called “invisible viruses.” They are

_ known to us through the biological quality of filtrates in which they
are present because of their ability to pass through the pores of the
filters.. For this reason they are also called “filterable viruses.”
As they cannot be seen, we have no way of classifying them; they
may be bacteria or protozoa, or neither or both.

* Calkins’, ‘The Protozoa,” p. 23.

icro-organisms

Structure and Classification of M

TABLE I
THE PLANT KINGDOM

He Phytosarcodina; myxothallophyta; myxomycetes (slime moulds).

: , - Schizophyta (oxifew to cleave or split; ¢urov plant). Plants repro-
Cryptogamia (xpumros hidden, yapos ; ducing by division.

marriage). Plants without flowers or Class 1. Schizomycetes (Bacteria).
seeds, reproducing by spores. Class 2. Schizophycee (blue-green alge).

eee III. Flagellata Flagellates; organisms claimed equally by botanists
8 FY 3 IV. Dinoflagellata { and zoologists.
on V. Zygophycee—conjugate alge.
& as Alge VI. Chlorophycee—green alge.
ow VII. Charales—Stoneworts.
4 0o8 Thallophyta— ‘VIII. Pheophycee—brown seaweeds.
ee ats (@adXos a young shoot, IX. Rhodophycee—red seaweeds. _
gare guroy a plant). Plants with- ;
Qe. out differentiation into| Fungi— X. Eumycetes—fungi, moulds, yeasts, smuts, rusts, mildews, etc. (See
‘4:32 root, stem, leaf, flower, etc. Table II.) |
2 so 3 XI. Embryophyta asiphonogama. :
we ag | Bryophyta (Spvoy mossy seaweed, ¢uroy
>Eao ; Archegonite— plant). Liverworts and mosses.
Saye (apxeyovos, the first of the | Pteridophyta (mrepis fern, dvroy plant).
gas.8 race), Plantsshowing.a Ferns, horse-tails, club-mosses, ground
Ed we regularalternationoftwo pine, etc.
a8 a8 3 generations in the life i
ay Sa history. - The asexual
sao. generation multiplies by
aase spores.
nas
Phanerogamia (¢avepos visible, yapos XII. Embryophyta siphonogama.
Biarneee), Plants having flowers {Gymnosperme (yuurds naked, oreppa
and seeds.

{
(oreppa seed, dutos cach _ginkos, etc.

Plants with true flowers | Angiosperme (4yyetoy a tube or vessel,
and true seeds. omepna a seed).

Monocotyledons.

Dicotyledons. ~

Bacteria 29

TABLE II

X. Eumycetes (ev good, puxnros fungus). The true fungi: plants without
chlorophyl.
Class 1. Phycomycetes (duxos seaweed), alga-like fungi.
Order 1. Zygomycetes.
Sub-order—M ucorinez.
Family—Mucoracez.
Genus—Mucor.
Order 2. Odmycetes.

Class 2. Hemiascomycetes.
Order 1. Hemiascales.
Family—Saccharomycetacee.
Genus—Saccharomyces.
«« —Blastomyces (?).

Class 3. Euascomycetes. f Fungi imperfecti.
Order 1. Euascales (contains 45 families). | This is a large sup-
Family—Aspergillacee. plementary group, of
Genus—Aspergillus. imperfectly known
“ —Penicillium. fungi not included in

the tabulation.
Class 4. Laboulbeniomycetes. In it we find Oidium.

Order 1. Laboulbeniales.

Class 5. Basidiomycetes.
Sub-class—Hemibasidii.
Order 1. Hemibasidiales.
Family—Ustilaginacee (smuts). _
Sub-class—Eubasidii.
Order 1. Protobasidiomycetes.
Family—Uredineinez (rusts).
Order 2. Autobasidiomycetes (mushrooms, toad-stools, etc.).

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

A. SUB-oRDER: Haplobacteria (Lower Bacteria)

I. Family Coccace#. Cells globular, becoming slightly elongate before
division. Division in one, two, or three directions of space. Forma-
tion 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.
(B) With flagella.
1. Planococcus. Division in two directions of space, like micrococcus.
2. Planosarcina. Division in three directions, like sarcina.

II. Family Bactertacez. 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) With flagella.
2. Bacillus. Flagella arising from any part of the surface. Endo-
spore-formation common.
3. Pseudomonas. Flagella attached only at the ends of the cell.

; Endospores very rare.

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

30 Structure and Classification of Micro-organisms

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 undulat-
ing flagella at the ends.

4. Spirocheta.* Serpentine and flexible. Fiagella not observed;
probably swim by means of an undulating membrane.

B. SuB-oRDER: Trichobacteria (Higher Bacteria)

IV. Family Mycopactertacez. 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 filament. Filaments not
surrounded by a sheath as in Chlamydobacteriacee.

1. Mycobacterium. 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 fila-
ments; growth coherent, dry or crumpled. Produce gonidia-
like bodies. Cultures generally have a moldy appearance, due
to the development of aérial hyphe. No flagella.

V. Family CHLAMYDOBACTERIACE2. Forms that vary in different stages
of their development, but all characterized by a surrounding sheath
about both branched and unbranched threads. Division transverse
to the length of the filaments.

1. Cladothrix. Characterized by pseudo-dichotomous branchings.

ivision only transverse. Multiplication by the separation of
whole branches. Transplantation by means of polar flagellated
swarm-spores.

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 BrcciaToace&. 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 accomplished through
the presence of an undulating. membrane. Cells contain sulphur
grains.

There are two families, numerous sub-families, 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.—Wucleus.—When subjected to the action of nuclear
stains, large vague nuclear formations are usually observed in the
bacterial cells. t

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

{For literature upon the nucleus of the bacteria, see the lengthy paper by
Douglas and Distaso (‘‘Centralbl. fiir Bakt.,” etc., I. Abt. Orig., rxvi, p. 321).

Bacteria 31

Cytoplasm.—The cytoplasm, of which very little exists between the
large nucleus and cell-wall, is sometimes granular, as in Bacillus
megatherium, and sometimes contains fine granules of chlorophy]l,
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 surrounded by a halo or capsule.
Such capsules are seen about the pneumococcus as found in blood
or sputum, Friedlinder’s bacillus, as seen in sputum, Bacillus
aérogenes 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

2b ;
Fig. 1.—Flagella (Kolle and Wassermann).

distinct, though it could not be demonstrated at all when the organ-
isms 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 granules” (metachromatic or
Babes-Ernst granules)—rounded or oval -bodies—situated at the
ends of the cell. Their significance is unknown. They have been
supposed to bear some relationship to the biologic activity of the
organism, especially its pathogenesis, but this is uncertain, and
Gauss* and Schumburgf believe that they vary with the reaction
of the culture-media upon which the bacteria grow and have
nothing to do with virulence. 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 degener-
ated organisms (involution forms) which, because of ia sae or
solution, can no longer stain uniformly.

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

*“Centralbl. f. Bakt.,” etc., Feb. 5, 1902, xxxi, No. 3, p. 106.
+ Ibid., June 3, 1902, xxx1, No. 14, p. 694.

32 Structure and Classification of Micro-organisms

Messea* has suggested that the bacteria be classified, according to _
the arrangement of the flagella, into:

I. Gymnobacteria (forms without fegell):
II. Trichobacteria (forms with flagella). :
1. Monotricha (with a single flagellum at one end).
2. Lophotricha (with a bundle of flagella at one end).
3. Amphitricha (with a flagellum at each end). .
4. Bearer (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 imply
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.

Bacillus megatherium has a distinct but limited ameboid move-
ment.

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 tendencies upon 5 per cent.
gelatin. The active movement of the bacteria composing the
colony causes its shape constantly to change, so that it bears a
faint resemblance to an ameba, and moves about from place to
place upon the surface of the gelatin.

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 nutrition are good, fission progresses with
astonishing rapidity. Buchner and others have determined the
length of a generation 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

* “Rivista d’igiene e sanata publica,” 1890, 11.

Bacteria : 330

be reckoned 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
becatise of the presence of other conditions unfavorable to their
existence.”

Sporulation.—When the conditions for rapid multiplicatian by
fission are no longer good, many of the organisms guard against
extinction by the formation of spores.

Endospores, or spores developed within the cells, are generally
formed in the elongated bacteria—Bacillus and Spirillum—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 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

pee

J ca Rg a . Sa ty
o va . wf z * {
i » ‘ , - A ae Ss |
v/ | -N oN A i 4
‘ ; <
. on | ee i ° ,
ota Af
a b , c

Fig. 2—Spores, showing their various positions in the microirganismal cells
(Kolle and Waeeeeeenny

greater than that of the bacillus, it causes a peculiar barrel shape
bulging of the organism, described as Clostridium. When the dis-
tending spore is at the end, a “Trommelschlager,” or ‘“drum-
stick,” is formed. End-spores are almost characteristic of anaérobic
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 applica-
tion of a considerable degree of heat. Very few adult bacteria are
able to resist temperatures above 70°C. Spores are, however,
uninjured by such temperatures, 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
temperature of 150°C. will invariably destroy them, as will also
steam under pressure. Not only can the spores successfully resist

34 Structure and Classification of Micro-organisms

_a considerable degree of heat, but they are also unaffected by cold
of almost any intensity. Von Szekely* found anthrax spores ca-
pable of germination after eighteen years 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. x

Though the cell-wall of the adult bacterium is easily penetrated ~
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 germinate, 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

c
a b dad e vi
CSD =) ,] Oo oes Com)

Fig. 3——Diagram illustrating sporulation: a, Bacillus inclosing a small oval
spore; 6, drumstick bacillus, with the spore at the end; c, clostridum; d, free
spores; e and f, bacilli escaping from spores.

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 increase in size,
develops a characteristic capsule, and presently begins the propaga-
tion 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 Coccus or Micrococcus. When they divide,
and the resulting organisms remain attached to one another, a
Diplococcus is produced. Diplococci may consist of two attached
spheres, though each half commonly shows flattening of the con-
tiguous surfaces. In a few cases, as the gonococcus, the approxi-
mated surfaces may be slightly concave, causing the organism to
resemble the German biscuit called a “Semmel.’’ When a second
binary division occurs, and four resulting individuals remain at-
tached to one another, without disturbing the arrangement of the
first two, a tetrad, or Tetracoccus, 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

* “Zeitschr. fiir Hygiene,” 1903, XLIV, 3.

Bacteria 35

been given. Migula uses the term Micrococcus for the unflagellated
tetrads, and Planococcus for the flagellated forms.

If division takes place in three directions of space, so as to pro-
duce a cubic “package” of. cocci, the resulting aggregation is de-
scribed as a Sarcina. This form resembles a dice or a miniature
bale of cotton. Few sarcine have flagella, similar flagellated
organisms being called by Migula Planosarcina. OO

If division always take place in the same direction, so that the

Fig. 4.—Various forms of bacilli (Kolle and Wassermann).

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 fancied re-
semblance to bunches of grapes. Such are called Staphylococcus,
and most organisms not finding a place in the varieties already de-
scribed 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

36 Structure and Classification of Micro-organisms

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.

ke ae oe
a Se a » fae ms a:
7 = ea Fond -
oe oe
I 2
oF es
BS OL ht ery
eC «6
OF
%. "3 fa
FH Te
Pr oad
3 : ;

Fig. 5.—Various forms of spiral organisms (Kolle and Wassermann).

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 described under the
generic term Bacterium. Migula now employs the term to include
only bacillary forms without flagella. “ A Pseudomonas isa bacillary _
form with polar: flagella. Some of the flexile bacilli have sinuous —

. movements resembling the swimming of a snake or an eel, and are

sometimes described as Vibrio; but this name also has passed into
disuse, except in France.

The Higher Bacteria 37

Spirilla.—If a rod-shaped bacterium is spirally twisted and re-
sembles a corkscrew, it is called Spirillum. The rigid forms without
flagella are known as Spirosoma; rigid forms with flagella, Spirillum
and Microspira,

A spiral organism of ribbon shape is called Spiromonas, while a
similar organism of spindle shape is called a Spirulina. One species
of spiral bacteria in whose cytoplasm sulphur granules have been
detected has been called Ophidiomonas.

Fig. 6.—Various forms of cocci: 1, Staphylococci; 2, Streptococci; 3, Diplococci;
4, Sarcina (Kolle and Wassermann).

Spiral organisms with undulating membranes are known as
Spirocheta, but these and the similar genus Treponema are now
regarded as more correctly placed among the protozoan organisms.

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 Myco-

38 Structure and Classification of Micro-organisms

bacteriaceg and the Chlamydobacteriaceez. 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. 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 réle, though those inhabiting
the mouth occasionally secure a foothold upon the edges of the
tonsillar crypts, where they grow, with the formation of persistent

ry. *

ie, al

Fig. 7.—Cladothrix, showing false branching. (From Hiss and Zinsser, ‘‘Text-
Book of Bacteriology,” D. Appleton & Co., publishers:)

white patches. This form of leptothrix mycosis is chronic and diffi-
cult to treat. The leptothrix is a very difficult organism to secure
in culture. The attempts of Vignal* and of Arustamoff{ 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 branching; that is, —
branches seem to originate from the threads, but no distinct connec-
tion between the thread and the apparent branch obtains. None of
the cladothrices is known to be pathogenic. They are frequent
organisms of the atmospheric dust, and not infrequently appear as

*“ Annales de physiologie,” 1886.

{ Kolle and Wassermann, “‘Handbuch der Pathogenen Mikroorganismen,”
1903, U, p. 851; Wratsch, 1889. so &

The Higher Bacteria 39

“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 powdery (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 pathogenic action. For along
time there has been a disposition to regard Bacillus tuberculosis as a
form of streptothrix, 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

oy )

g \\

Fig. 8.—Streptothrix enteola. Film preparation from peptone-beef-broth cul-
ture, fourteen days at 37° C. X 1000 (Foulerton).

Bollinger is called Stréptothrix actinomyces, the Actinomyces
madure, Streptothrix madure, and the organism found by Nocard
in the disease known as “‘farcin du beuf,’ Streptothrix farcinica.
There seems, however, no adequate ground for this arrangement,
and the old genus Actinomyces should be kept. Eppinger 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 prob-
ably 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.

40 Structure and Classification of Micro-organisms

Actinomyces.—The chief characterization of the organisms of this
group is a clavate expansion of the terminal ends of radiating fila-
ments. 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 organisms 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 multiplication by
gemmation or budding.
Each organism is surrounded by a sharply defined, doubly
contoured, highly refracting, transparent cellulose envelope. Com-

Fig. 9.—Blastomycetes ‘dermatitidis. Budding forms and mycelial growth
from glucose agar (Irons and Graham, in “Journal of Infectious Diseases’).
monly 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 CO: and alcohol, and are largely cultivated 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 no pigment at all, are
known. They 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,
Ophiils, and others have seen localized human infections by blasto-
mycetes. (See Blastomycetic dermatitis.)

Ashford* has described a pathogenic yeast to which Andersont has

* “Journal of Tropical Diseases and Preventive Medicine,” 1915, m1, No. 1,

. 32.
T “Journal of Infectious Diseases,” 1917, xx1, No. 4, p. 341.

The Oidia 41

given the name Parasaccharomyces. Both authors regard it as
the cause of the tropical diarrhoea known as “Sprue.”’

KEY TO THE GENERA OF BUDDING FUNGI *

I. Ascospores known:
Vegetative cells single or attached in irregular colonies, my-
celium not developed, ascospores formed within
isolated vegetative cells............. (Saccharomycetacez.*)
*This genus, which does not bud, and the relatively unimportant genera,
Monospora and Nematospora, are not included in this key.
Spores globose or ovoid:
Spores on germination forming typical yeast cells:
Ascus formation preceded by the conjugation of

PAMELES oie tn Faw aie tek new cae e s00-% 1. Zygosaccharomyces.
’ Ascus formation not preceded by ene conjugation of
gametes:
Spore membrane single..............000005 2 ee
Spore membrane double................ 3. Saccharomycopsis.
Spores on germination forming a poorly developed pro-
WAY CONLUME ice os Sear ce ccm ard weld ss ecmirshanle dd eee 4. Subchpretyeod ss
Spores pileiform or limoniform, costate.. ; weeeee 5. Willia.

Spores hemispheric, angular or irregular | in form, on
germination forming an extended promycelium... 6. Pichia.

Vegetative, cells produced predominently by budding, but

forming a mycelium under some conditions, asci

terminal or intercalary, differentiated from the

MY COMI sas sierste ced Bar eee Belt ae meus eee aaa 7. Endomyces.
II. Ascospores not known, i.e., Fungi imperfecti:
Heavy dry pellicle formed on liquid mediums..... Pea abs 8. Mycoderma.

No distinct pellicle formed:
Vegetative cells forming a septate mycelium under excep-
tional conditions but predominently budding.
9. Parasaccharomyces.
Vegetative cells formed only by budding: f

Cells eae limoniform............. ro. Pseudosaccharomyces.
Cells frequently elongate into narrow non-septate
hyphal threads.......... 00... cceeeeeees rz. Pseudomonilia.

Cells typically yeast-like............cceeeeeeeee 12. Cryptococcus.

THE OIDIA

These organisms seem to occupy a place intermediate between the

- yeasts and the molds—the Blastomycetes and the Hyphomycetes.

In certain stages they appear as oval cells which multiply by gem-

mation, but instead of becoming separated, hang together. At a

later stage of development they grow into long filamentous forma-

tions suggesting the mycelia of molds, but being less regular.
Certain cells also develop as reproductive organs.

They are common micro-organisms of the air and appear as
frequent causes of contamination in culture-media, upon all 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 albicans, which causes the
common disease of childhood known as thrush (q. 2.).

* Anderson, “Jour. Infectious Diseases,” xx1, No. 4, p. 376, 1917.

42 Structure and Classification of Micro-organisms

THE MOLDS, BRANCHED FUNGI OR HYPHOMYCETES

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 respects as to place them
in widely separated groups in the systematic arrangement of the

X 350 (Grawitz).

fungi. Some are correctly placed among the “Imperfect fungi,”
some among the Ascomycetes, and some among the Phycomy-

Fig. 11.—Oidium (Kolle and Wassermann).

cetes. They are all active enzymic agents and produce fer-

mentative and putrefactive changes.

1. Achorion.—The organisms of this genus are characterized by
a more or less branched hypha, 3 to su in diameter, which breaks up
after a time into rounded or cuboidal spores. The Achorion

Fig. 10.—Oidium, showing the various vegetative and reproductive elements. |

The Molds . 43

schénleini is highly pathogenic and will be described in the section
upon Favus.

2. Tricophyton and Microsporon.—These names are applied some-
what loosely to organisms affecting skin and hair follicles of men and
animals. They form tangled slender mycelia with many spores of
varying size. They occasion “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 Phyco-
mycetes. They forma thick, tangled mycelium, in and above which
the rounded black sporangia can be seen with the naked eye. The
mycelium becomes divided at the time of reproduction. Multiplica-
tion takes place asexually through conidia-spores which develop

Fig. 12.—Mucor mucedo: 1, 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).

within sporangia, and sexually by the conjugation of specialized
terminal septate branches of the mycelium, which conjugate with
similar cells, belonging to other colonies, to form zygospores.

The sporangia form upon the ends of aérial hypha and consist of a
smooth spherical capsule within which the spores develop, to become
liberated only when the membrane ruptures. 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

44 Structure and Classification: of Micro-organisms

occasionally appear as contaminations upon solid culture-media.
About 130 species are known. Of these, Mucor corymbifer, Mucor
rhizopodiformis, Mucor ramosus, Mucor 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
Paltauft to result from the presence of the same organism.

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 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

Fig. 13—Mucor mucedo. Single-celled mycelium with three hyphe and one
developed sporangium (After Kny, from Tavel).

day or two, when tangled mycelial growths with rapidly spreading
hyphez will usually be discovered. The recognition is easily made
_ when the sporangia appear. These are well shown in the accom-
panying illustration. The mycelium is divided into many cells.
Reproduction is asexual and takes place through conidia spores.
The fruit hyphe, which are aérial, terminate in rounded extremities

which are known as columella, from which many radiating sterig-' —

mata 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.

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

* Kolle and Wassermann, “Die Pathogenen Mikroorganismen,” 1903, 1, «52
t Hiickel-Lésch in Fliigge, ‘Die Mikroorganismen.” ; t Ibid. i

The Molds 45

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, diaphragm, 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 infrequently
pathogenic form. Its most common lesion is a pneumomycosis, in
which the lung is riddled with small inflammatory necrotic and
cavernous areas containing the molds. Though of frequent occur-
rence in cattle it is but occasionally observed in human beings,
Sticker having collected 39 cases.*

Fig. 14—Muctr mucedo. Different stages in the formation and germination
of the zygospore: 1, Two conjugating branches.in contact; 2, septation of the
conjugating cells (a) from the suspensors (b); 3, more advanced stage in the
development of the conjugating cells (a); 4, ripe zygospore (b) between the
suspensors (a); 5, germinating zygospore with a germ-tube bearing a sporangium
(After Brefeld).

Leber and others have observed keratitis following corneal infec-
tion 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. Moist bread
exposed to the atmosphere soon becomes covered with them. They

= Nothnagel’s Spezielle Path. u. Therap., x1v, 1900.

46 Structure and Classification of Micro-organisms

are included in the group of fungi imperfecti, and are characterized by
a luxuriant tangled septate mycelium, with aérial fruit hyphe,
ending in conidiophores, each of which divides into two or three
sterigmata, the tip of which forms a chain of rounded spores. The

Fig. 15.—Aspergillus glaucus: A, A portion of the mycelium m, with a con-
idiaphore 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).

whole germinal organ thus comes to resemble a whisk-broom or, as
Hiss describes it, a skeleton hand, in which the conidiaphore cor-

Fig. 16.—Penicillium: a, Mycelium; b, conidiaphores; c, d, sterigmata; ¢,
spores (Eyre).

responds to the wrist; the sterigmata, to the metacarpal bones; the
chains of spores, to the phalanges.

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

The Protozoa 47

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.

THE PROTOZOA

The Protozoa are unicellular animal organisms as differentiated
from the Metazoa which are multicellular animal organisms. The
restriction, implied by the term unicellular is, however, too narrow,
for there are colonial protozoa that consist of many cells, yet share
other protozoan characters.

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

Classification. Many schemes have been devised for systematic-
ally arranging the protozoa, that which follows being an abbrevia-
tion 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 (aparos first, ¢wov animal). Unicellular animal
organisms. :

Class Rhizopoda (gl{a root, mwéos foot). Having soft plasmic bodies
with or without . external protecting shells. The contour 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 GYMNAMGBA (yuuvds naked). Rhizopoda without external

shells or coverings.
Genus Ameceba (apol8a to change).
Genus Entameeba.
Genus Chlamydophrys.
Genus Leydenia. :

Class Mastigophora (yaorvyos whips, gépos to bear). Organisms of
well-defined form, naked or surrounded by a well-defined membrane.
Nutrition is holozoic, holophytic, parasitic, or saprophytic. Mouth,
contractile vesicle, and nucleus usually present.

Order FLAGELLATA (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, holophytic, parasitic, or saprophytic.

Family Cercomonide. Body pyriform with several anterior flagella
and an undulating membrane.

Genus Cercomonas.
Genus Trichomonas.
Genus Monas.

Genus Plagiomonas.

Family Lambliade. Body pyriform, very manele 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).

Family Trypanosomide. Body delicately fusiform. Contains a
nucleus, a bleptaroplast or centrosome, and an undulating mem-
brane. A single wavy flagellum arises in the posterior part
of the body close to the centrosome, passes along the edge of the

48 - Structure and Classification of Micro-organisms

undulating membrane to the anterior extremity, where it continues
free for some distance. Nutrition parasitic. Reproduces by
division. —

Genus Trypanosoma.

Genus Leishmania.

Genus Babesia. : .

Family Spirochetide. Organisms very long and spirally twisted.
Nucleus indistinct. Multiplication probably by longitudinal
division only. Nutrition is parasitic or saprophytic. :

Genus Spirocheta. 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 (orépos a spore, {Gov 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 sporozoites
cither being produced by the parent or indirectly from spores, into
which the parent divides. ete: Ore

Subclass Telosporidia. Spore-formation ends the individual life, the
entire organism being transformed to spores. .

Order GrecGARinipa. Possess distinct membrane with myonemes
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 CoccipmpaA. Spherical or ovoid in form, without a free and
motile adult stage. Never ameboid. Sporulation takes place
within cysts formed while the organism is an intracellular parasite.

Genus Coccidium.
Genus Eimeria.

Order HaMosporipmpa. 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 ex-
ogenous sporulation after conjugation.

Genus Plasmodium.
Subclass Neosporidia. Organisms that form sporocysts throughout life,
the entire cell not being used up in the formation of the spores.

Order Sarcosporipi1A. 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 sporozoites are formed.

Genus Sarcocystis.
Genus Miescheria.
Genus Balbiania. :
Subclass Haplosporidia. Spores provided with large round nuclei. No
polar capsules.
Genus Rhinosporidium.

Class Infusoria (Latin, infusus, 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. Reproduc-
tion is effected by simple transverse division or by budding. Nutrition -
is holozoic or parasitic.

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

Order Hototricnipa. 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.
Genus Chilodon.

The Protozoa 49

Order HEeTERoTRICHIDA. 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 Bursaride. The body is usually short and pocketlike,
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 organ-
isms, such as Ameba, it presents the appearance which some describe
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 carried 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—accumulations of digested but not
yet assimilated food, etc.; but others, seen in Ameba and in the
ciliata, are large, permanent, and characterized by rhythmical
contractions through which they disappear from one part of the
body substance to appear in another. These are known as “‘con-
tractile vacuoles,” and are supposed to subserve the useful purpose of
assisting in maintaining cytoplasmic currents and so distributing the
nourishing juices.

The cytoplasm also contains remnants of undigested or indigest-
ible foods which constitute the paraplasm or deuteroplasm. In a
few cases granules of chlorophyl are also to 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 masti-
gophora and infusoria 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 of 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 body substance may show no morphologic differentiation in

50 Structure and Classification of Micro-organisms

Rhizopoda, but in the corticata there may not only be a permanent
form, but there may be adaptations, such as an oral aperture, some-
times infundibular in shape and communicating 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 pos- -
teriorly 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—Spirocheta 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.

Fig. 17.—Internal parasites: A, Amceba coli, Lésch; B, Monocystis agilis,
Leuck., a gregarine; C, Megastoma entericum, Grassi, a flagellate; D, Balantidium
coli, Ehr., a ciliate. Under very different magnifications.

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 phe-
nomena supervening upon conjugation both participate. The nuc-
lei of the protozoa are, therefore, extremely diversified, and vary
from the most simple collections of granules of nuclear substance
to large well-formed fantastically 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 pseudopodia, or else a
continuous flowing, by which the upper surface continually coming

The Protozoa 51

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 the presence of longer or shorter, flexile or rigid, coarse
threads or ‘“‘whips.”” These usually project anteriorly—Trypano-
soma—and by means of a spiral movement draw the cell along with
a propeller-like action; symmetrically arranged flagella may operate
more like oars.

The Sporozoa usually manifest very little movement, yet their
sporozites are motile, and the spermatozoites are also motile and
commonly flagellated.

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 movement, propelling the
organisms forward or backward or making 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 Sarcosporidium 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 ac-
quaintance 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 con-
jugation 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 multiplication 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 sometimes
contrary to expectation, as in the cases of Treponema and
Spirocheta.

The multitudinous sporozoites into which the zygotes of the
sporozoa divide are commonly the result of anterior division into
intermediate bodies know as oécysts, odkinetes, sporocysts, etc.
The nuclear substance is first divided so as to be’ uniformly dis-

52 Structure and Classification of Micro-organisms

tributed among these, then further divided so thdt some of it reaches
each sporozoite. .

In the process of sporulation the entire parent may be used up,
as in Coccidium and: Plasmodium or the parent 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, 7.e., surrounding themselves with dense
capsules by which life may be preserved for some time amid such
unfavorable surroundings as excessive cold, excessive dryness, 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 comparatively
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 distribution
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 plants live, die, and decompose,
they are sure to be.

Their presence in the air generally depends upon their previous
existence in the soil, its pulverization, and distribution by currents
of the atmosphere. Koch has shown that the upper stratum of the
soil is exceedingly rich in bacteria, but that their numbers decrease
as the soil is penetrated, 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 de-
composing materials are allowed to penetrate to a considerable
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 successful 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 tkat 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 un-
distributed by currents of air unless the surface of the colony becomes
roughened or broken.

Most of the organisms carried about ty 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

* “Floating Matter in the Air.”
53 .

54 Biology of Micro-organisms

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

Anaérobes, which do not grow in the presence of uncombined

oxygen.
As, however, some of the aérobic forms grow almost as well with-
out free oxygen as with it, they are known as optional (facultative)
‘anaérobes.
As examples of strictly aérobic bacteria Bacillus subtilis, Bacillus
aérophilus, Bacillus tuberculosis, and Bacillus diphtheria 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 free oxygen, and
hence belong to the optional anaérobes. 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 anaérobic.

The higher bacteria, oidia, molds and protozoa, are for the most
part aérobes and optional anaérobes.. Treponema pallidum seems to
be a strictly anaérobic protozoan.

Food.—The bacteria grow best where diffusible albumins are
present, the ammonium salts being less fitted to support them than
their organic compounds. Proskauer and Beck* have succeeded
in growing the tubercle bacillus in a mixture containing ammonium
carbonate 0.35 per cent., potassium phosphate o.15 per cent., mag-
‘nesium sulphate 0.25 per cent., and glycerin 1.5 per cent.. Some of
the water microbes can live in distilled water to which the smallest
amount of organic matter has been added; others require so con-
centrated a medium that only blood-serum can be uséd for their
cultivation. The statement that certain forms of bacteria 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 bacteria, the distilled water ceases to be such, and
becomes a diluted bouillon fitted to support bacterial life for a time.
Sometimes aspecies 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 influence, 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

* “Zeitschrift ftir Hygiene,” etc., Aug. to, 1894, vol. xvi, No. 1.

Conditions Prejudicial to Growth of Bacteria 55

through the ectosarc such substances as are capable of assimilation
and nutrition. These forms are cultivable only upon media con-
taining the same or approximately the same proteins as those to
which they have been accustomed. Thus, to cultivate Trypanosoma,
blood-serum must be added to the media.

The larger protozoa live upon smaller animal and vegetable organ-
isms, which they ingest entire. Such can only be artifically culti-
vated provided the attempt be made under conditions of symbiosis
with some other and smaller organism that may constitute the
food.

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

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 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 non-spore-bearing bacteria, including Bacillus
tuberculosis, B. diphtheria, B. typhosus, S. cholere asiatice, B.
coli, B. prodigiosus, and others are killed in from two to ten minutes.
Certain colors are distinctly inhibitory to the growth, blue being
especially prejudicial.

Treskinskaja } found that sunlight had a marked destructive 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 thanin 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.

*“Centralbl. f. Bakt. u. Parasitenk. Ref.,”’ xtvu1, Nos. 22-24, p. 681.
t “Jour. Infectious Diseases,” 1907, vol. 1v, Supplement, No. 3, p. 128.

56 Biology of Micro-organisms

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
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 organisms and change
the reaction of the culture-medium; rapidly reversed currents of high
intensity, to destroy the pathogenesis of the bacteria and transform
their toxic products into neutralizing bodies (antitoxin?). Atten-
tion has been called to this subject by Smirnow, d’Arsonval and
Charin, Bolton and Pease, Bonome and Viola, and others. a

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

Sse

1. A continuous current of 260 to 300 milliampéres passed through bouillon
cultures kills bacteria of low thermal death-points in ten minutes by the pro-
duction 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 milliampéres 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 roo milliampéres 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
aS hours. Subtilis spores were still alive after the current was passed for three

ours. ;

4. A continuous current passed through bouillon cultures of bacteria 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 liberation of hydrogen in gas bubbles. With a current
of 100 milliampéres for two hours it required 8.82 milligrams of H,SO, to neutral-
ize 1 cc. 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 antiseptic properties, but can destroy bacteria only
by its physical effects (heat) or chemic effects (the production of bactericidal
substances by electrolysis).

* “Your. Amer. Med. Assoc.,” Nov. 30, 1901.

Conditions Prejudicial to Growth of Bacteria 57

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.

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 bacterial suspension
within a solenoid. When exposed to the brush discharges, ozone is produced
ge kills the bacteria.

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

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

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

12. Réntgen rays have no direct bactericidal properties. The clinical results
must be explained by other factors, possibly the production of ozone, hypochlor-
ous 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 currents are interrupted by falls and
rapids, should, other things being equal, furnish a better drinking-
water than a deep, still-flowing river.

Galli-Valeriof has shown, however, that agitation does not in-
hibit the growth of the anthrax, typhoid or colon bacilli or the
pheumococcus, 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
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.

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

+ “Lancet,” 1897, vol. 11, No. 21.

t “Centralbl. f. Bakt.,” etc., Sept. 23, 1904, Orig., XXXVI, Pp. I51.

§ Société de PEpIRR IE Séance du rz Juin, 1898, “La Semaine médicale,’”’ June
15, 1808.

58 Biology of Micro-organisms

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

An interesting experimental study of the bacterial antagonisms
with special reference to Bacillus typhosus, that the student should

read, is by W. D. Frost, and appeared in the “Journal of Infectious

Diseases,” 1904, 1, 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 (60°
70°C.), and are described as thermophilic. They are found in
manure piles and in hot springs. Tsiklinsky{ 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{ 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 temperature of the cultures being
reduced to about—140°C., yet in no case was the vegetative ca-
pability 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 sub-
sequent development of the frozen cultures takes place from the few
surviving organisms.

Bacteria usually grow best at the temperature of a comfortably

* “Brit. Med. ie

t “Russ. Rete 6 ta ae, Te eee a v.

{ “The Medical News,” June 10, 1899.

§ ae f. Bakt. u. Parasitenk.,” etc., May 26, 1900, Bd. xxvu, Nos. 18,
19, p. 684.

Conditions Prejudicial to Growth of Bacteria 59

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 tu-
bercle bacillus, grow best at a niperare 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 oidia, 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
protozoa endure greater variations than the active organisms.

Effect of Chemic Agents.—The presence of chemic agents, espe-
cially certain of the mineral salts, in an otherwise perfectly suitable
medium may completely inhibit the development of bacteria, and
if added to grown cultures in greater concentration, destroy them.
Such substances are spoken of as antiseptics in the former, germi-
cides 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 concentra-
tions with almost unvarying result, Trambusti* found it possible to
produce a tolerance to a certain amount of bichlorid of mercury by
cultivating Friedlinder’s bacillus upon culture-media containing
gradually increasing amounts of the salt, until from 1-15,000, which
inhibit ordinary cultures, it could accomodate itself to 1-2000.

The various chemic agents act in different ways upon the micro-
organisms. Thus, they may combine with the protoplasm to make
a new and no longer vital compound; or, they may coagulate or
dissolve or dehydrate or oxidize the protoplasm 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. Ifnow 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 embarassed, made impotent, or destroyed.

On the other hand, when micro-organisms have enjoyed a con-
centrated medium like blood-serum and are suddenly transferred to
distilled water, so much water may be suddenly 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.

* “Lo Sperimentale,” 1893-94.

60 Biology of Micro-organisms |

Metabolism.—According = 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.

Aérogens, 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 con-
demnation ultimately effect its purification by exhausting the
organic matter it contains in their own nutrition. In the treat-
ment of sewage by the “septic:tank” method, the organic matter
contained in the water is consumed through the agency of anaérobic
and aérobic bacteria, until the water once more becomes clear and
pure, the bacteria dying out as the nutrition becomes exhausted.

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, advantageous,
though the fact that they are not infrequently accompanied by
chemic changes, some of which are poisonous, make it necessary to
watch and time their operations lest acridity, acidity, insipidity, or
toxicity of the food replace the desired effect.

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

Fermentation.—Fermentation is catalysis of carbon compounds
caused by catalysts or ferments resulting from micro-organismal _
metabolism. 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 saccharo-
myces 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:
C6Hi120¢ = 2C.H;,OH + 2CO,
Sugar Alcohol Carbon dioxid
There are also several bacteria which produce the acetic fermenta-
tion, though it is generally attributed to Bacillus aceticus. There
are two equations to express this fermentation:

Putrefaction 61

I. CH:CH.OH + O = CH;CHO + 4H2.20
Alcohol Oxygen Aldehyd Water
II. CH;CHO + O = CH;COOH
Aldehyd Oxygen Acetic acid

A number of different bacilli seem capable of converting milk-sugar
into lactic acid, though Bacillus acidi lactici is 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 fermentation, have the following equations:

i. CieH 2011 + H.0 = Ce6H1202 + C6Hi206
Lactose or milk sugar Galactose Dextrose
II. Ce6Hi206 _ 2C3H.O3
Galactose Lactic acid :
III. Co6H1206 = C.4HsO, ; + CO2 + 2He
Galactose Butyric acid

Putrefaction.—Putrefaction is a catalysis of proteins resulting
from the activity of micro-organismal catalysts or enzymes. It is
associated with the evolution of a vile odor. 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, amino-acids, bases,
and salts. In the process innocuous albumins are frequently
changed to toxalbumins, and sometimes to peculiar putrefactive
alkaloids known as piomains.

Vaughan and Novy define a ptomain as “a chemical compound,
basic in character, formed by the action of bacteria on organic maiter.”
The chemistry of these bodies is very complex, and for a satisfac-
tory 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 substances ofthe milk before it is frozen into ice-cream or
made into cheese. The safeguard is to freeze the milk only when
perfectly fresh and avoid mixing the milk, cream, sugar, and flavor-
ing substances, and allowing the mixture to stand for some time
beforehand.

The occasional cases of ‘‘Fleischvergiftung,” ‘‘meat-poisoning,”

r “Botulismus,” are due to the development of toxic ptomains in
consequence of the growth of certain bacteria (Bacillus botulinus) in

0 06

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

t “Ptomaines and Leucomaines,” 1888; ‘‘Cellular Toxins,” 1902.

62 Biology of Micro-organisms

the meat. Kaensche* has carefully investigated the subject, and
given 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 meat to become poisonous.

With the increase of knowledge upon the toxic character of the
bacteria themselves, the importance of the toxic ptomains 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 decom-
position and fermentation, among them being COs, H2S, NH,
H, CH,. Gases produced by aérobic bacteria
usually fly off from the surface of the
culture unnoticed, but if the bacterium
= be anaérobic and develop in the lower part of
a tube of solid culture media, a visible bubble
of gas is usually formed about the colonies.
Such gas bubbles are almost invariably pres-
ent in cultures of the bacilli of tetanus. and
malignant edema.

To quantitatively determine the gas-produc-
tion, 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.

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

Fig. 18.—Smith’s fer-
mentation tube.

“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 CO:
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 COz 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 differ-
ence between this amount and that measured before shaking with the sodium
hydroxid solution gives the proportion of CO, absorbed. The explosive charac-
ter 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 proportion. of gases resulting [rom the
fermentation is frequently of importance for the differential diagnosis of related

* “Zeitschrift fiir Hygiene,” etc., June 25, 1896, Bd. xxu, Heft 1.

Liquefaction of Gelatin | 63

bacteria. Smith has designated this relation of a as the ‘gas formula.’ The
2

colon bacillus has a gas formula corresponding to ae = :. Other aérogenic -
2

bacilli sometimes show a formula a =i”
CO, 2

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 pathogenic and non-pathogenic forms.
The liquefaction is supposed to be dependent upon a form of pepto-
nization. Bitter* and Sternberg t 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. 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. Formalin 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 differen-
tiation of species. ;

Production of Acids and Alkalies.—Under the head of “‘Fermen-
tation’ the formation of acetic, lactic, and butyric acids has been dis-
cussed. Formic, propionic, baldrianic, 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 de-
tecting 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 em-
ployed for the purpose. The contents of the bulb and branch should
be shaken together, a measured quantity withdrawn, and titration
with = sodium hydroxid, or = hydrochloric acid, performed.

The alkali most frequently formed by bacterial growth is ammo-
nium, 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 alkalies. Both acids and the alkalies, when in excess,
serve to check the further activity of the micro-organisms.

* “Archiv fiir Hygiene,” 1886, Heft 2.
+ “Medical News,’ 1887, No. 14.
t “Centralbl. f. Bakt.,” etc., 1891, Bd. x, p. 4or-

64 Biology of Micro-organisms

Chromogenesis.—Bacteria that produce colored colonies or impart
color to the medium in which they grow are called chromogenic;
those producing no color, non-chromogenic. Most chromogenic
bacteria are saprophytic and non-pathogenic. Some of the patho-
genic forms, as Staphylococcus pyogenes aureus, are, however, color
producers. It seems more likely that certain chromogenetic sub-
stances unite with constituents of the culture medium to produce the
colors than that the bacteria form the actual pigments; but, as Gale-
otti* has shown, there are two kinds of pigment, one being soluble,
readily saturating the culture medium, as the pyocyanin and
fluorescin of Bacillus pyocyaneus, the other insoluble, not tingeing
the solid culture media, but retained in the colonies, like the pigment
of Bacillus prodigiosus. The pigments are found in greatest in-
tensity near the surface of a bacterial mass. The coloring matter
never occupies the cytoplasm of the bacteria (except Bacillus
prodigiosus, in whose cells occasional pigment-granules may be
seen), but occurs as an intercellular 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 fluorescin, both being soluble
pigments—one blue, the other green. Gessardt has shown that
when Bacillus pyocyaneus is cultivated upon white of egg, it produces
only the green fluorescent 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 fluorescin
it is necessary that the culture medium contain a definite amount
of a phosphatic salt. Sometimes, an organism produces two pig-
ments, one is soluble, the other insoluble, so that the colony will
appear one color, the medium upon which it grows another. The
author once found an interesting coccus,! with this peculiarity, upon
the conjunctiva. It formed a brilliant yellow colony upon the sur-
face of agar-agar, but colored the agar-agar itself a beautiful violet.
In this case the yellow pigment was insoluble, the violet pigment
soluble and diffusible through the jelly. Some organisms will
only produce 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, re-
fuse to produce pigments at so high a temperature. Thus, Bacillus
prodigiosus produces a brilliant red color when growing at the tem-
perature of the room, but is colorless when grown in the incubator.
The reaction of the culture medium is also of much importance in
this 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—

* “Lo Sperimentale,” 1892, xLv1, Fasc. 11, p. 26r.

{ “Ann. de l’Inst. Pasteur,” 1892, pp. 810-823.

t See Norris and Oliver, ‘System of Diseases of the Eye,” vol. 11, p. 480, and
“University Medical Magazine,” Philadelphia, Sept., 1895.

Production of Odors 6 5

perhaps most of them—are formed only in the presence of
oxygen.

Production of Odors.—Gases, such as H»S and NHy, and acids, :
butyric and acetic acids, have sufficiently characteristic odors.
There are, however, a considerable number of pungent odors which
seem to arise from independent odoriferous principles. Many of
them are extremely unpleasant, as that of the tetanus bacillus. The
odors seem to be peculiar individual characteristics of the organisms.

Production of Phosphorescence.—Cultures of Bacillus phos-
phorescens and numerous other organisms are distinctly phosphor-
escent. 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 phosphorescent 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 stale meats.

Production of Aromatics.—Pnenol, kresol, hydrochinon, hydro-
paracumaric acid, and paroxyphenylic-acetic acid are by no means
uncommon products ot 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
nitrosoindol reaction. To perform it, 10 cc. of the fluid to be tested
receive an addition of 10 drops of concentrated sulphuric acid. The
mixture is shaken in a test-tube. A few cubic centimeters of a 0.02
per cent. solution of potassium 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.t| McFarland and Small{ 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.

The Formation of Nitrates.—A process of fundamental 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 putrefactive changes have occurred, ap-
pears 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 extensive

* “Zeitschrift. f£. physiol. Chemie,” vim, p. 4 : :
t See Grubs and Francis, “Bull. of the Hyg. Laboratory, > 1902, No. 7.
} “Trans. of the American Public Health Association,” 1905.

66 Biology of Micro-organisms

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 vegetation.

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 com-
pounds of any sort. Their supplies of carbon are obtained by the
dissocfation 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 bacteria are
able to reduce nitrogen compounds in the soil or in culture media,
prepared for them, intoammonia. 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 ordi-
nary 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 cc. of water 1 gram
of peptone and o.2 gram of potassium nitrate. The ingredients are
dissolved, filtered, then filled into tubes, 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 employedf{ for testing the culture:

I. Naphthylamin, 0.1 gram, Boil, cool, filter, and add 156 cc. of
Distilled water, 20.0 grams, dilute (1:16) hydric acetate.

II. Sulphanilic acid, 0.5 gram.
Hydric acetate, diluted, 150.0 cc.

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

About 3 cc. of the culture and an equal quantity of the uninocu-
lated culture fluid are placed in test-tubes and about 2 cc. of the
test fluid slowly added to each. The development 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 produced by the
growth of the bacteria.

* Ann, de l’Inst. Pasteur,” 1891; “‘La Semaine médicale,” 1892.
} “Journal of the American Public Health Association,” 1888, p. 92.

Combination of Nitrogen 67

The presence of ammonia in either fluid is easily determined by
the immediate development of a yellow color or precipitate when a
few drops of Nessler’s solution* are added.

Failure to determine either ammonia or nitrites may ant 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 phenolsulphonic acid and sodium hydroxid,
which in the presence of nitrates give a yellow color.

Combination of Nitrogen.—Not only do bacteria destroy or re-
duce nitrogen compounds, but some of them are also able to assimi-
late nitrogen from the air and so combine it as to be useful for the
nourishment of vegetable and animal life. The most interesting
organisms of this 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 agriculturist knows how sterile soil is
improved by turning under one or two crops of clover with me
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 appar-
ent change in the milk, some producing coagulation, some gelatiniza-
tion 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 possessing 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 similar 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. Be-
tween 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
determine that a micro-organism is possessed of pathogenic
powers, the committee of bacteriologists of the American Public
Health Associationt recommends that: (1) When a given form

* Nessler’s solution consists of potassium iodid, 5 grams, dissolved in hot
water, 5 cc. Add mercuric chlorid, 2.5 grams, dissolved in 10 cc. of water, then
to the mixture add potassium hydrate, 16 grams, dissolved in water, 4o cc. and
dilute the whole to 1000 cc.

+ “Centralbl. f. Bakt.,” etc., Bd. vir, p. 338.

t “Jour. Amer. Public Health Assoc.,” Jan., 1898.

68 Biology of Micro-organisms

grows only at or below 18° to 20°C., inoculation of about 1 per cent
of the body-weight with a liquid culture seven days old should bi
made into the dorsal lymph-sac of a frog. (2) When a specie:
grows at 25°C. and upward, an inoculation should be made into th
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 1 per cent,
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 unnecessary
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 men-
tioned as causing fermentation and putrefaction, coagulating milk,
dissolving gelatin, etc. There are, however, others which have
interesting and important actions upon both animal and vegetable
substances.

Emmerich and Léw* observed that in old cultures of Bacillus
pyocyaneus the bacteria become transformed into a gelatinous mass,
and were led to experiment with old and degenerating cultures con-
densed to }{9 volume in a vacuum apparatus. The bacteriolytic
powers were then found to be much increased, and they. were sub-
sequently able to precipitate from the concentrated culture an
enzyme, which they called pyocyanase. The authors reached the
rather hasty conclusions 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, appesen 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 fir Hygiene,” 1899.

CHAPTER Til
INFECTION

INFECTION is the successful invasion of an organism by micro-
parasites. 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 harm-
less bacteria into the deeper tissues, are all said by the surgeon to be
“infected,’’ when, in fact, it would be more correct to describe them
as infective.

The term infection should imply an abnormal state resulting
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 “‘in-
fected,” but infested with it, and it is only when abnormal or un-
natural 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 appropriate 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 customary association, because of newly acquired power of
invasion on the one hand, or diminished vital resistance on the
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 parasitic when they live in or
upon them. Saprophytic bacteria when accidentally transplanted
from their natural environment 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 multi-
ply rapidly, invade the tissues in all directions, eliminate their met-

70 Infection

abolic products into the juices, and occasion varying 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 qeldeitiel 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 condi-
tion. Indeed, although we are acquainted with a 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-organ-
isms 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 agents belonging to the external world.

A polluted atmosphere may carry into the respiratory passages
micro-organisms capable of colonizing there. From the respiratory
passages, minute drops of secretion may be coughed or sneezed into
the atmosphere to be inhaled by neighboring persons and infect
them. Such ‘drop infection’ has been studied in reference to
tuberculosis and diphtheria, and doubtless explains the transmission
of whooping-cough, pneumonia, and other respiratory disturbances.
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, as in typhoid fever, cholera and
dysentery.

Wounds inflicted by the teeth of animals, by weapons, by imple-
‘ments, 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. Examples are to be found in rabies,
‘tetanus, anthrax, malignant and gaseous cedema, suppuration, etc.

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 conveyed to the well with resulting
infection.

Contact with unclean objects of various kinds—spoons, knives,
cups, blow-pipes, catheters, syringes, dental instruments, etc.—

Bacterial Tenants of the Normal Human Body qI

may serve to transfer disease-producing organisms from one person
to another who might otherwise never come in contact with them.

Attention should be called to the facility with which the diseases
of childhood may be spread through the thoughtless or ignorant
custom of many adults and children of using handkerchiefs, napkins,
forks, cups, spoons, etc., in common; in having wash-rags, towels,
hair-brushes and combs in common; cultivating the ‘habit of putting
lead-pencils, etc., in the mouth, and then passing them on to others
who will do the same, and to many other relations of every-day life by
which infectious agents maybe spread. Scarlatina, measles, mumps,
acute anterior poliomyelitis, ophthalmia, tuberculosis, ringworm,
fevers, syphilis, etc., may all be spread through such means.

Suctorial insects seem occasionally to act as the medium by which
micro-organisms withdrawn in blood from one person may be in-
troduced 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 trypanosomiasis; the tick, of relapsing fever, the louse
of typhus fever, etc.

Endogenous infections arise through the activity of micro-organ-
isms habitual to the body. They indicate morbid conditions of the
body by which the defensive mechanisms are disturbed, so that
organisms harmless under normal conditions become invasive.

MICRO-ORGANISMAL TENANTS OF THE NORMAL HUMAN BODY

All normal animals are presumably born free of parasitic micro-
organisms, 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-organisms, 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.

The Skin and Adjacent Mucous Membranes.—The slightly moist
warm surface of the skin is well adapted to bacterial life, and its un-
avoidable contact with surrounding objects determines that a variety
of organisms shall adhere to it. Of these, we can differentiate be-
tween forms whose presence is unexpected and temporary; others
whose 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, ft who studied the finger-nails, under
which he found no less than seventy-eight different species;
Maggiora,{t who isolated twenty-nine forms from the skin of the foot,

* “ Monatshefte fiir prakt. Dermatol.,”’ 1888, vu, p. 817; 1889, VIII, pp. 293,
562; 1880, Ix, p. 49; 1890, X, p. 485; 1890, XI, Pp. 471; 1891, XI, p. 249.

t ‘Archiv f. path. Anat. u. Phys. u. f. klin. Med.,” 1888, cximI, p. 203.

I “Giornale della R. Societé d’Igiena,”’ 1889, Fasc. 5, p. 335-

72 Infection

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 Winslowt
has found the colon bacillus upon the hands of ten out of one hundred
and eleven persons examined. Wigurat also examined the hands
of forty persons in hospitals, finding tubercle bacilli in two out of
ten persons from phthisical wards, 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. Welch§ and
Robb and Ghriskey|| seem to have been the first to make a clear
differentiation between the accidentally present bacteria and the
permanently parasitic organisms of the skin, and to show that cer-
tain 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 described the most common as
Staphylococcus epidermidis albus, are universally and invariably .
present upon the human skin, and must be regarded as habitual
parasites.

Where the skin is peculiar in its moisture and greasiness, however,
additional forms are found. Thus, in preputial smegma, in the
axille, and sometimes about the lips and nostrils, a bacillary organ-
ism, Bacillus smegmatis, is invariable, and Schaudinn and Hoff-
mann** have shown that the skin of the genitalia harbors a spiral
organism which they call Spirocheta 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. They do not
remain, however, but are quickly wiped off by the lids and driven
into the lachrymal sac. The researches of Hildebrand and
Bernheim and others seemed 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,{t who found six organisms—Leptothrix innominata,

*“Samml. medic. Schriften,” herausg. von der “Wiener klin. Wochenschrift,”
1891; xxi, Wien, ‘Rev. Jahresbericht iiber die Fortschritten in der Lehre von
den pathogenen Mikroorganismen,” 1891, VII, p. 619.

I “Jour. Med. Research,” vol. x, p. 463.

‘Wratsch,” 1895, No. 14.
3 “Tansactions of the Congress of American Physicians and Surgeons.”
1801, Il, p. I.

| ‘Bulletin of the Johns Hopkins Hospital,” 1892, m1, p. 37.

** “Teutsche re wie Sy ee on ee

tt “ Micro-organisms of the Human Mouth,” Phila., 1800.

Micro-Organismal Tenants of the Normal Human Body 73

Bacillus buccalis maximus, Leptothrix buccalis maxima, Iodococcus
vaginatus, Spirillum sputigenum and Spirocheta dentinum (denti-
cola)—in every mouth. Practically the same conclusions were
reached by Vincentini.* 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.

In studying the micro-organisms of dental caries Goodby} found
a large number of organisms which he divided iato three groups:
A. Those that produce acids, including Streptococcus brevis, Ba-
cillus necrodentalis (Goodby), Sarcina alba, Sarcina lutea, Sarcina
aurantiaca, Staphylococcus pyogenes aureus, and Staphylococcus
pyogenes salivarius (Biondi). B. Those that liquefy blood-serum:
Bacillus mesentericus rubra, B. mesentericus vulgatus, B. mesenteri-
cus fuscus, Bacillus fuscus, a yellow bacillus, probably B. gingive
pyogenes (Miller), and Bacillus liquefacium 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 bactericidal action of the
saliva. In addition to the bacteria, representatives of the sac-
charomycetes and hyphomycetes, are commonly found in normal
mouths.

Amoebe are also commonly found in the gingival grooves and
in the crevices between the teeth. These have been called Amoeba
gingivalis and are regarded by some as harmless commensals of
the normal mouth, by others as pathogenic parasites responsible
for the suppuration of the tooth follicles in pyorrhcea alveolaris.

The crypts of the tonsils regularly harbor a miscellaneous collec-
tion of bacteria, among which staphylococci and streptococci, chiefly
of the non-hemolyzing varieties may be found. The unprotected
surfaces of the crypts doubtless permit these organisms to penetrate
to the deeper lymphatic structures of the neck where they are usually
destroyed.

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-organisms
of fermentation make their appearance. They are, however, acci-
dental 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 i is of some diagnostic im-

* “Bacteria of the Sputa and Cryptogamic Flora of the Mouth,” London, 1897.
{ Transactions of the Odontological Society, June, 1899.

74 _ Infection

portance. It is called after its discoverer the Oppler-Boas bacillus,* © ©

also on account of angulations found in its threads, Bacillus gen-

iculatus. 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 Emoryt

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, increased 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 lactis aérogenes and bacilli of the colon
groups are found. These increase in number as the iliocecal valve
is reached. The cecum shows large numbers 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 the organisms of the bowel
and their distribution has been made by Kohlbrugge.{ Kendall§
finds the meconium contained in the intestine of the new-born to be
sterile, the first bacteria making their appearance in the course of
eighteen or twenty-four hours. The initial flora is not characteristic.
The most interesting early organism in the meconium is a large ba-
cillus with a terminal spore, and resembling the Bacillus tetani. It is
supposed to be Bacillus putrificus of Bienstock. ‘‘Other spore-
bearing bacteria both aérobic and anaérobic are usually present
in the meconium at this period. Of these Bacillus aérogenes
capsulatus and members of the Bacillus mesentericus group are the
best known. Bacillus coli, Bacillus proteus, Bacillus lactis aérogenes
and Micrococcus ovalis also commonly occur.”

As the infant nourished at the breast becomes accustomed to the
new condition, and settles down to its milk diet the bacteria through-
out the alimentary canal become more numerous, the spore-bearing
types disappear rather abruptly, and the coccal forms and Gram-
negative bacilli of the B. coliand B. aérogenes types diminish relatively,
though they never entirely disappear. At the same time a long
slender bacillus occurring singly and in pairs, or in groups with their
axes parallel becomes strikingly prominent. They are slightly

* “Deutsche med. Wochenschrift,” 1905, No. 5.

t “Bacteriology and Hematology,” p. 114.

{ Centralbl. f. Bakt.,” etc., r901, Bd. xxx, pp. 10 and 7o.

§ “Bacteriology, General, Pathological and Intestinal,” Phila., 1916.

Micro-Organismal Tenants of the Normal Human Body 75

curved, are attenuated at the ends and when typical are Gram-
negative and stain uniformly. Sometimes they show a Gram-
positive granule, sometimes a punctate appearance and may
resemble a chain of cocci. This organism first described by Escherich
and later isolated and cultivated by Tissier is Bacillus bifidus.
It is an obligatory anaérobe, capable of fermenting lactose and other
sugars with the production of considerable acid, but no gas. In
culture it shows a peculiarity not shared by other known bacilli,
namely, a bifid or divided appearance of its ends. Bacillus acido-
philus, Micrococcus ovalis, Bacillus coli, Bacillus lactis aérogenes
and other bacteria are also found.

In adult life the acidity of the gastric contents, that continues into
the duodenum, and the more rapid passage of food through the upper
as contrasted with the lower bowel, determines that the bacteria
shall increase in number as the ileocecal region is approached.
Staphylococci and streptococci may occur high up, with a few Gram-
positive bacilli; lower down Gram-negative bacilli of the B. coli group
and some of the B. proteus group are the chief organisms. About 75
per cent. of the bacteria of the normal adult feces are B. coli, and
of those in the colon about 90 per cent. are dead or incapable of
growing in artificial media.

The total bacteria that finally appear in the feces, according to the
studies of Strasburger* and Steele,t 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.

Rettger§ found the Bacillus enteritidis sporogenes regularly pres-
ent in the human feces and believes it to be responsible for some of
the putrefactive processes that occur there. Yeasts of various kinds
are commonly present in the intestinal contents and have been care-
fully studied by Anderson.|| Moldsalsooccur occasionally. Amoeba
are not infrequent in occurrence, and flagellate and ciliate pro-
tozoan organisms sometimes occur in considerable numbers in the
intestinal contents of apparently normal human beings.

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** found but few organisms of limited variety.
A flagellate organism, Trichomonas vaginalis is somtimes found in
the vagine of apparently healthy women.

_ The uterus harbors no bacteria in health, and but few in disease.

* “Zeitschrift fiir klin. Med.,”’ 1902, XLIv, 5 and 6; 1903, XLVIII, 5 and 6.
tT “Jour. Amer. Med. Assoc.,” Aug. 24, 1907, Pp. 647.
t ‘Journal of Infectious Diseases,” 1909, VI, Pp. 132, 571.
§ “Jour. of Biological Chemistry,” Aug., 1906, 11, 1 and 2, p. 71.
M ‘Journal of Infectious Diseases,” 1917, XxI, No. 4, p. 341.
** “ Archiv f. Gynak.,” Bd. Lxiv, Heft 3.

76 " Infection

The intervening acidity of the vagina makes it difficult for bacteria
from the surface to penetrate so deeply, and the tenacious alkaline
mucus of the cervix is an additional barrier to their progress.
Careful studies of the bacteriology of the uterine secretions have
been made by Gottschalk and Immerwahr* and Déderlein and
Winterintz.t

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 bacteria in the
dust of the inspired atmosphere. Thesé organisms are too numer-
ous and too various to enumerate, and might, indeed, comprehend
the entire bacterial flora.. But in spite of the large numbers of organ-

isms 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 pseudo-
diphtheria bacillus, streptococci, pneumococci, staphylococci, Ba-
cillus pneumonie (Friedlander), Bacillus subtilis and sarcina. A
complete review of the subject with references to the literature has
been made by Hasslauer.t

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.

AVENUES OF INFECTION

The skin seems to form an effectual barrier against the entrance.
of bacteria into the deeper tissues. A few higher fungi—Tryco-.

phyton, Microsporon, Achorion, etc.—seem able to establish them-

selves 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. Garré applied a pure
culture of Staphylococcus pyogenes aureus to the skin of his fore-
arm, and found that furuncles developed in four days, though the
skin was supposed to be uninjured. Bockhart moistened his skin
with a suspension of the same organism, 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
toywhat depth the lesion extends—abrasions, punctures, lacerations,
incisions—the protective covering is gone and the infecting organ-

* Ibid., 1896, Bd. 1, Heft 3.

} “Beitrage fiir Geburtshiilfe und Gynikologie,” Bd. mm, Heft 2.

t “Centralbl. f. Bakt. u. Parasitenk. I. Abt. Referata,” Bd. xxxvir, Nos. 1-3,
p. 1, and Nos. 4-6, p. 97. ,

Avenues of Infection q7

isms find themselves in the tissues, surrounded by the tissue lymph,
under conditions appropriate for growth and multiplication, provided
no inhibiting or destructive mechanism be called into action.

The digestive apparatus is the portal through which many infec-
tions take place. The Bacillus diphtheria, finding its way to the
pharynx, speedily establishes itself upon the surface, producing
pseudomembranous inflammation there. Typhoid bacilli, dysentery
amoeba, and 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 injury of the host, who
may suffer 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,* the intestine is responsible for a
condition of sub-infection depending upon the constant entrance
of colon bacilli 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.
Nichollst confirms Adami by finding similar dumbbell or diplococcoid
bodies in the epithelial denuded tissues of the mesentery of normal
animals.

Nicholas and Descos{ and Ravenel§ 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 venous circulation, and so carried to the lung,
where anthracosis was produced.

In a subsequent paper** they believe that they have demonstrated
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. Montgomery ff re-
peated the work of van Steenberghe and Grysez at the Henry Phipps
Institute, Philadelphia, but though many attempts were made by

* “Tour. of the American Medical Association,” Dec. 16 and 23, 1899, vol.
xxximl, Nov. 25 and 26.
t “Jour. Med. Research,” vol. x1, No. 2.
t “Jour. de Phys. et Path. gén.,” 1902,-IV, 910-912.
_ §* Tour. Med. Research,” 1904, X, p. 460.
|| Ann. de l’Inst. Pasteur,” Dec. 25, 1905, Tome xrx, No. 12, p. 787.

| ** Tbid., 1910, XXIV, 316.
TT “Jour. of Med. Research,” Aug., 1910, vol. xx1u, No. 1.

78 Infection

various methods, no carbon particles seemed to be transported from
the alimentary to the pulmonary 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 retaine
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.

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 controversial. Undoubtedly in the final dis-
solution some change takes place in the constitution of the individual
by which general invasion by bacteria is made more easy than under
aormal conditions.

The respiratory apparatus affords admission to a few micro-organ-
isms whose activities seem more easily carried on there than else-
where. 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 pneu-
mococcus of Fraenkel and Weichselbaum, probably results from the
entrance of the organism into the respiratory 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, vesiculitis, cystitis, odphoritis,
sometimes peritonitis, and rarely endocarditis; the bacillus of
Ducrey, that causes the chancroid 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
Neélow* shows that the non-pathogenic organisms do not pass
from the mother through the placenta to the fetus. Some patho-
genic micro-organisms, however, readily pass through, and a few

*“Centralbl. f. Bakt.,” etc., Aug., 1902, I. Abt., Bd. xxx1, Orig., p. 691.

Pathogenic Bacteria 79

diseases, such as syphilis, are well known in the congenital form.
Pregnant women 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, Baum-
garten having reached the opinion, exactly the opposite of what
is commonly believed, that many children are subject to antenatal
infection, though the bacilli subsequently develop and cause disease
in only a few of them.

PATHOGENIC BACTERIA HARBORED WITHIN THE SEEMINGLY
HEALTHY BODY. CRYPTOGENETIC INFECTION

In the section upon “The Micro-organismal Tenants of the
Human Body” it has been shown that a considerable number of
micro-organisms among which are quite a number of pathogenic
species, are commonly to be found in relation with the outer and inner
surfaces of the body.

So long as these maintain this purely superficial position and are ©
excluded from the tissues and circulating fluids by the surface
coverings, all goes well, and if the occasional penetration of a few
takes place, all may still go well, provided that the defensive mechan-
isms, later to be described, succeed in effecting their destruction.
Many persons presumably in perfect health are deceived as to their
true condition, and careful examination reveals the fact that not a
few, though unconscious of it, are, suffering from inconspicuous,
inconsequential or latent foci of infection. To these much attention
has recently been directed with the result that is now recognized
that the crypts of enlarged tonsils, the gall-bladder, the vermiform
appendix, the tooth sockets, the apices of the roots of the teeth,
the follicles of the urethra, the recesses of the prostate gland, the
Fallopian tube, neglected inflammatory tracts and the scar tissue
found in the healing of infectious lesions like carbuncles, may all
harbor pathogenic bacteria that are effecting disturbances too
trivial to call attention to themselves, but really constituting
limited invasions of the body of the host.

Such latent foci of disease constitute a constant menace to the
patient’s health because should the conditions that determine their
latency become disturbed, they may suddenly flare up and produce
active and destructive local lesions—as, for example, when the
vermiform appendix, long the seat of unimportant subacute
disturbance, suddenly becomes invaded with resulting suppuration
and the occurrence of fetid pus. Or, if no such sudden aggravation
of the local disturbance occurs, they constitute more numerous
and more fruitful opportunities for the admission of bacteria to
the blood than when a few pathogenic Decveta are situated upon
the undisturbed surfaces.

Such pathogenic organisms, whether from the surface or from these

80 Infection

latent foci of disease, when they succeed in penetrating into the
blood and escaping the destructive effects of the defensive mechan-
ism, if transported to distant parts may initiate new morbid proc-
esses formerly known as idiopathic, but in the light of present
knowledge better known as cryptogenic infections. Such are
‘exemplified by primary endocarditis, pericarditis, pleuritis, arthritis,
meningitis, etc.

HUMAN CARRIERS OF INFECTION

In some cases pathogenic micro-organisms, falling upon the
surfaces of the body, find the conditions suitable for life and multi-
plication, though unsuited for invasion, and remain indefinitely,
though apparently doing no harm. More frequently, the recovery
from an infectious disease is attended by a form of immunity that
determines that the infectious agent can no longer do the patient harm,
though it is not necessarily extinguished from all parts of the body.

In either case the individual becomes a “‘carrier”’ of infectious
agents that may be transmitted to others. Thus, the examination
of the nasal secretions of large numbers of persons show that a few
harbor meningococci though they may never have had meningitis, -
but that more harbor meningococci who have had ‘meningitis.
But in either case, the carrier may transfer the meningococci to
others, through the indiscriminate use of handkerchiefs, wash-rags,
towels, etc., who may readily become infected with the disease.

A nurse that has attended a child through diphtheria or a doctor
that has visited a case of diphtheria, or other healthy children in a
household in which there has been diphtheria, may have a few
diphtheria bacilli in the nasal, tonsillar or pharyngeal mucose, that
may not be able to induce disease because the individuals are not
receptive, and which may not die out for a long time. The child
recovered from diphtheria, though entirely well, frequently carries
large numbers of diphtheria bacilli upon the formerly diseased
membrane, for weeks. Under such circumstances, the healthy persons
who have not had the disease and the well child that has recovered
from it are alike “‘carriers’’ of diphtheria and may spread the micro-or-
ganisms to new and susceptible persons who quickly become diseased.

Though the patients seem entirely to have recovered from gonor-
rhea, gonococci frequently remain alive in the previously inflamed
passages, so that such persons are “carriers, ’’ and though seemingly
entirely free of disease readily transmit it to others.

The typhoid bacilli escaping from the blood of the patient in
the bile and urine, remain alive in the urinary bladder for many
weeks and in the gall-bladder for many months after complete
recovery. As they grow readily in both locations, the numbers that
are discharged with each emptying of the receptacles in which they
are multiplying may be enormous and makes the individual a _
“carrier” until they are no longer present.

Pathogenesis 81

Plasmodia of malaria may cease any longer to appear in the
peripheral blood and the patient may cease to have any paroxysms
of the disease, yet a few of the parasites in the bone-marrow or
spleen continuing to multiply, may suffice to keep a few gametes
in the blood from which they may be taken by a mosquito, to be ©
passed after the necessary cycle of development in its body, to .
other human beings subsequently bitten.

Much of the future of sanitary science will have to do with the
discovery and proper treatment of “carriers’’ of the infectious agents,
from whom the public must be defended until the infectious agents
can be eradicated from their bodies.

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 phenomena of infection, for in many cases the in-
toxication 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 anthracis,
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, ex-
cite actively destructive reactions in the tissues with which they
come incontact. Still others, with limited invasive powers, elimi-
nate active toxic substances, soluble in nature, that enter the cir-
culation 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 defensive mechanism. The
relation of these factors is exceedingly complex, only partly under-
stood, and will be fully discussed in the chapter upon Immunity.

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

The Intracellular Toxins.—Until the investigations of Vaughan,
Cooley and Gelston,* and later Vaughan and his associates, Det-
weiler,t} Wheeler,t Leach,§ Marshall and Gelston,|| Gelston,** J.
V. Vaughan,{t Wheeler,{{ Leach,§§ MclIntyre,|||| and others, it

* Journal of the’ American Medical Association,” Feb. 23, 1901; ‘Trans.

Assoc. Amer. Phys.,” 1901; “American Medicine,”’ May, 1901.
{ “Trans. Asso. Amer. Phys.,” Igo2. Ibid.

§ Ibid. {| Ibid. ** Thid.

tt Ibid.

tt “Jour. Amer. Med. Assoc.,” 1904, XLII, p. 1000. ;

§§ Ibid., p, 1003. |||] Ibid., p. 1073.

82 Infection

seemed remarkable that micro-organisms whose filtered cultures
contained 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 quan-
tities, 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 sub-
stances 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 com-
minution of culture masses made solid and brittle by exposure to
liquid air, as suggested by Macfadyen and Rowland; the autolytic

digestion of bacteria 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 i is usually 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.

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 reactions of varying intensity result.

The extracellular toxins, of which those of Bacillus tetani and
Bacillus diphtheriz can be taken as types, have been known since
the early work of Brieger and Frankel 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 agar-
agar.

The soluble toxin is but one of numerous metabolic products of

Specific Action of Toxins 83

the bacteria. Thus in culture filtrates of the tetanus bacillus there
are at least two very different active substances, the ¢etano-spasmin
that acts upon the nervous system with convulsive effect, and the
tetano-lysin that is solvent for erythrocytes.

Tn 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 ab-
straction of those molecular constituents upon which the bacteria
have fed. This complexity makes:it difficult to accurately study
the toxins, which we scarcely know apart from their assomated
products.

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

The variations observed in toxicogenesis under experimental
conditions in the test-tube indicate that similar variations occur
in the bodies of animals, and a few experiments conducted with
slight variations in the composition and reaction of the media in

_which the bacteria grow will 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 eciinpelidlas soluble
toxins differs from that of the intracellular and difficultly soluble
toxins in that it is more easily diffused throughout the animal juices,
and that its diffusion is independent of the invasiveness of the bac-
teria, 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 bac-
teria 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.

84 , Infection

Fever and suppuration are, therefore, non-specific actions, be-
cause numerous micro-organisms: share in common the qualities

productive of these conditions.

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 typical effect, differing from the total effect
of the operation of other micro-organisms, and a recognized type
of disease results, it becomes possible to say that the micro-organism
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 profoundly 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 controlled 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 in-
fections, 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—pneu-
mococcus, streptococcus, etc. Neither of these reactions is specific,

The Invasion of the Body by Micro-Organisms 85

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.

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, occasionally get a
foothold, in diseased tissues and assist in morbid changes. This
is seen in what is described as sapremia, in which various sapro-
phytic bacteria, possessing no invasive powers, by growing in the
putrefying tissues of a gangrenous part, give rise to poisonous sub-
stances which when absorbed by the adjacent healthy tissues pro-
duce such constitutional disturbances as depression, fever, and the
like.

Bacteria with limited invasive powers and intracellular toxins
can at best occasion local 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
characterized by the presence of bacteria in the circulating blood.
When bacteria entering the circulation are unable to pervade the
entire organisms, they may collect in the capillaries of the less re-
sisting tissues, producing local metastatic lesions, usually purulent
in character. This 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 some-
times are passively forced through the stomata of the vessels when
the pressure of the inflammatory exudate is greater than that of the
blood within them; that they may sometimes enter into the bodies
of leukocytes that have incorporated them; that they may actually
grow through the capillary walls, or that they reach the blood cir-
‘culation 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.

THE CARDINAL CONDITIONS OF INFECTION

Infection can take place only when the micro-organisms are
sufficiently virulent, when they enter in sufficient number, 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

* Fliigge, ‘‘Die Mikroorganismen,” vol. 1, p. 271.

86 Infection

upon the invasiveness of the micro-organisms, or the eaey 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 Holst observed a culture whose virulence
was unaltered after eight years of 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 of yellow cocci from milk.

Decrease of virulence under artificial conditions probably depends.
upon artificial selection of the organisms in transplantation from
culture to culture. When planted upon artificial media, the vege-
tative members of the bacterial 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
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 pathogenic 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 experi-
mental manipulation of the culture is directed toward gradual im-
munization 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

The Cardinal Conditions of Infection 87

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 can be explained by the
“Jateral-chain theory of immunity,’’ where it will again be
considered.

The Use of Collodion Sacs—When cultures of bacteria are en-
closed 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 considerably 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
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 eliminating those that cannot
endure the defensive agencies of those juices with which the organ-
isms come in contact.*

The addition of animal fluids to the iulbite pelle sometimes
enables the investigator to increase, and usually enables him to
maintain, the virulence of bacteria. A series of generations in
gradually increasing concentrations of the body fluid should be
employed, until the organism becomes thoroughly accustomed to it.

In some cases it may be sufficient to use a single standard mixture,
thus: Shawt. found that he could exalt the virulence 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 of virulence under such conditions probably depends
upon the immunization of the bacteria to the body juices of the
animals, and this whole matter will be understood after the subject
“Immunity” has been considered.

Number.—The number of bacteria entering the infected animal
has a very important bearing upon infection.

The entrance of a single micro-organism of any kind is senda

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

88 Infection

ever able to cause infection because of the uncertainty of its being
able to withstand the changed conditions to which it is subjected.
In most cases a considerable number of organisms is necessary in
order that some may survive. 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, there-
fore, 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 through rabbits and jin- |
termediate 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 cc. of a culture of Staphylococcus pyogenes to produce suppura-
tion in the peritoneal cavity of an animal; 0.75 cc. to produce it
beneath the skin; 0.25 cc. in the pleura; 0.05 cc. in the veins and
0.0001 cc. 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 phlegmon 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, and furnishes
the typical picture of the infections. :

Thus, gonococci usually reach the body through the urogenital
mucous membranes, where they set up the various inflammatory
reactions collectively known as gonorrhea—7.e., urethritis, vaginitis,
prostatitis, orchitis, cystitis, etc. These constitute thé typical
picture of the infection. The organism may also successfully invade
the conjunctiva, producing blennorrhea, but there is no evidence
that gonococci can successfully invade the body through the skin,
the respiratory, or alimentary mucous membranes.

The Cardinal Conditions of Infection 89

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 to take place ai
the urogenital system, the conjunctiva, or the skin.

The avenue of entrance not only determines infection, but ae
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 tuberculosis. Bacilli reaching the cervical or other
lymph-nodes by entrance through the tonsils, may remain localized,
producing 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 inflammation with necrosis
and ultimate evacuation or exfoliation of the diseased mass, after
which the patient may recover. Bacilli entering the intestine in
many cases produce 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 pulmonary
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. In 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 some 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 in-
halation into the lungs, or by way of the lymphatics through super-
ficial 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 trans-
ported to 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 capillaries, where
they are apt to be caught and destroyed. Finally, should any es-
cape 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

90 Infection

also defended against such micro-organisms as might reach the veins
through lesions or accidents of the abdominal viscera, by the inter-
position of the portal capillary network of the liver, where the bac-
teria 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 phago-
cytes for the destruction 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 foxins 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 introduced through other
channels. :

The conditions differ, however, in different infections, for when
Roger experimented with streptococci instead of anthrax bacilli,
he found that if they were inoculated into the portal vein the
animals died more quickly than when they were injected into the
aorta, and that when they were injected into the peripheral veins
the animals lived longest, the liver seeming to be far less destruc-
tive to streptococci than the lungs.

The Susceptibility of the Host.—Susceptibility is liability to in-
fection. It is a condition in which the host is unable to defend itself.
against invading micro-organisms. Unusual or unnatural suscep-
tibility 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 physio-
logic changes may lose the defensive power.

In general, it is true that any condition that depresses or dimin-
dishes 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

*“Tntroduction to the Study of Medicine,” p. rr. - r

The Cardinal Conditions of Infection gI

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 in-
fectious 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 open-
ing of a privy, while in others the excreta of the animals were allowed
to accumulate, suffered from a pronounced diminution of the re-
sisting powers. This would seem to be inconsistent with the
habits of rats, many of which live in sewers. Abbottt 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 susceptibility to
disease, and experimental evidence of its correctness is not wanting.
Charrin and Roger{t 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 diminishes the resisting power of the
warm-blooded animals. It is an everyday experience 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
Gibier§ 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 disorders of the
mouth, the frequency with which epidemics of infectious disease fol-
low 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.

“* “Centralbl. f. Bakt.,” etc., 1894, XV, p. 228.

+ ‘Trans. Assoc. Amer. Phys.,”’ 1895.

t‘Compte rendu Soc. de Biol. de Paris,” Jan. 24, 1890. .

§ ‘““Compte rendu Acad. des Sciences de Paris,” 1882, t. XCrx, p. 1605.

92 ~*~ Infection

Intoxication .of all kinds’ predisposes to infection. Platania*
found that such animals as frogs, pigeons, and dogs became sus-
ceptible to anthrax when under the influence of curare, chloral,
and alcohol. Leot found that white rats fed upon phloridzin: be.
came susceptible to anthrax. Wagnert found that pigeons become
susceptible to anthrax when under the influence of chloral.
Abbott§ found the resisting powers of rabbits against Streptococcus
pyogenes and 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 combating’ the
infectious diseases because it diminished the glycogen content of the .
liver which Colla** had found an important adjunctin supporting the
resisting power. -

It is a common clinical observation that excessive indulgence
in alcohol predisposes to certain infections, notably. ‘pneumonia,
and every surgeon knows the danger of pneumonia.after Bea
tion 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 infection, 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 fre-
quency 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 accompany-
ing 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 suipestifer in the bodies of
hogs suffering with hog cholera, all show the diminution in the gen-
eral 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 association of other

*See Sternberg’s “Immunity and Serum Therapy,” p. 10; “Centralbl. f.
Bakt.,” etc., Bd. vit, p. 40 5:
t “ Zeitschrift fiir Hyg.,” 1889, Bd. vu, p. sos.
t “Wratsch,” 1890, 39, 40.
§ “Jour. of Exp. Med.,”’ 1896, vol. 1, No. 3.
al Jour. Amer. Med. Assoc. ,” 1906, “XLVI, 18, Nov. 3, p. 1467.
‘Archiv Ital. de Biologie,” xxv1.

Mixed Infections 93

kinds. Therefore their operation in the body is subject to modifi-
cations produced in them or in the host by these. associated
organisms.

In experimental investigations this fact is not infrequently for-
gotten 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 anaérobic 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 organisms, having different
chemical. composition, may neutralize one another, or combine to
form some entirely new substance 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 mech-
anism of immunity is complicated or otherwise according to cir-
cumstances. When the invasive action of non-toxicogenic bacteria
is to be overcome, certain reactions, mostly on the part of the phago-
cytic cells, are called into action; when the toxic products of bacteria
are to be deprived of injurious effects, the reaction seems totake
place between the toxin and certain combining 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 com-
plex, according to the conditions involved. The more involved
the conditions of infection become, the more complicated the de-
fensive reactions become, until it may no longer be possible accu-
rately 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 un-
necessary. Metchnikoff* found that frogs injected with 0.5 cc.
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 pre-
venting the production of the toxin to which the frog was susceptible.

Immunity must not be conceived as something inseparably as-
sociated 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 bacteriotoxins
are identical with those toward other toxic substances, so that the
only way by which a satisfactory 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.

*“Tmmunite dans les Maladies Infectieuses,” Paris, 1901, p. 150.
94

Natural Immunity 95

Immunity is called active when the animal protects itself through
its own activities, passive when the protection depends upon de-
fensive substances prepared by some other animal entering into
it. Thus, if a frog be injected with anthrax bacilli, its leukocytes
devour the bacteria, destroy them, and so protect the frog from
infection; the immunity is active because it depends upon the ac-
tivity of the frog’s phagocytes. But if a guinea-pig previously
given antitetanic serum be injected with tetanus toxin, and so re- ~
covers from the toxin, the resisting power, conferred by the antitoxin
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 in-
oculation. If, however, a large quantity of tetanotoxin produced
in a test-tube be introduced 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 infection with Bacillus diphtheriz,
and Cobbett* found that they could endure from 1500 to 1800 times
as much diphtheria toxin as guinea-pigs, though more than that would
kill them. ;

‘Carl Frankel has expressed the whole matter very forcibly when
he says: “A white rat is immune against anthraxin doses suff-
ciently large to kill a rabbit, but not necessarily against a dose suffi-
ciently large to kill an elephant.”

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 infecting all kinds of animals;
indeed, it is doubtful whether any known organism possesses such
power.

The micro-organisms of suppuration seem able to infect animals
_ of many different kinds, sometimes producing local lesions, some-
times invading rapidly with resulting bacteremia. 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

* “Brit. Med. Jour.,” April 15, 1899.

96 Immunity

have more or less resemblance to one another; thus, anthrax is
essentially a disease of warm-blooded animals, though certain ex-
ceptions are observed, and Metchnikoff has found that hippo-
campi (sea-horses), perch, crickets, and certain mussels are.
susceptible. Among the warm- -blooded animals anthrax is most
frequent among the herbivora, 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 susceptible 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 different in their susceptibility to the malarial parasite. Among
the members of the human species, it has been asserted that Mon-
golians, and especially Japanese, are immune against scarlatina, and
that negroes are immune against yellow fever, but increasing in-
formation is to the contrary.

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, 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 larve 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 j 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, deter-
mines that the micro-organism, though it may be able to invade
the body, can do very little harm.

- Acquired Immunity | 97

ACQUIRED IMMUNITY

Acquired immunity is resistance against infection or intoxication
possessed by certain animals, of a naturally susceptible 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
invasion of pathogenic organisms, for neutralizing or destroying
their toxins or for both. As an acquired character and an individual
peculiarity it is not transmitted to the offspring, though these some-
times also acquire immunity through the parents. Thus in study-
ing 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 her milk.

Acquired immunity differs from natural immunity in being more
variable in degree and 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 intoxication, and
in either case may be accidental or experimental.

(A) Active Acquired Immunity.—1. Immunity Acquired 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 respective
disease. Immunity thus acquired is not transmissible to the off-
spring. 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 observable and the patient may suffer from relapses. Later
it becomes well-established and no repetition of a 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
days the when smallpox was iroquent, and has now become
extremely rare.

(b) Experimental Infection—1. Inoculation: This is an attempt
to prevent the occurrence of a fatal attack of an infectious disease,
by inducing a mild attack of the same disease when the individual
is in good health, and at his maximum resisting power. The oldest

98 - ' Immunity

experiments date from unknown antiquity and were practised in
China and other Oriental countries for the 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. The following
extract is from a letter of Lady Montague,* a wife of the British
Ambassador to Turkey, who brought the so-called “inoculation”
method from Turkey in the early part of the eighteenth century
(1718):

“ . . . . Apropos of distempers, I am going to tell you a thing that I am
sure will make you wish yourself here. The smallpox, so fatal, and so general
amongst us, is here entirely harmless by the invention of ingrafting, which is
the term they give it. There is a set of old women who make it their business
to perform the operation every autumn, in the month of September, when the
great heat is abated. People send to one another to know if any of their family
has a mind to have the smallpox; they make parties for this purpose, and when
they are met (commonly fifteen or sixteen together), the old woman comes with
a nut-shell full of the matter of the best sort of smallpox, and asks what vein
you please to have opened. She immediately rips open that you offer to her
with a large needle (which gives you no more pain than a common scratch), and
puts into the vein as much venom as can lie upon the head of her needle, and
after binds up the little wound with a hollow bit of shell; and in this manner
opens four or five veins. The Grecians have commonly the superstition of open-
ing one in the middle of the forehead, in each arm, and on the breast, to mark
the sign of the cross; but this has a very ill effect, all these wounds leaving little
scars, and is not done by those that are not superstitious, who choose to have
them in the legs, or that part of the arm that is concealed. The children of young
patients play together all the rest of the day, and are in perfect health to the
eighth. Then the fever begins to seize them, and they keep their beds two days,
very seldom three. They have very rarely above twenty or thirty [pocks] in
their faces, which never mark; and in eight days’ time they are as well as before
their illness. Where they are wounded, there remain running sores during the
distemper, which I don’t doubt is a great relief to it. Every year thousands
undergo this operation; and the French embassador says pleasantly, that they
take the smallpox here by way of diversion,.as they take the waters in other
countries. There is no example of any one that has died in it; and you may
believe I am very well satisfied of the safety of this experiment, since I intend to
try it on my dear little son.

“T am patriot enough to take pains enough to bring this useful invention into
fashion in England; and I should not fail to write to some of our doctors very
particularly about it, if I knew any one of them that I thought had virtue enough
to destroy such a considerable branch of their revenue for the good of mankind. :
But that distemper is too beneficial to them not to expose to all their resentment
the hardy wight that should undertake to put an end to it.’”’

By both methods the very disease, variola, against which protection
was desired, was induced, 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 4
time when no epidemic of the disease was raging, and by doing it at

* See the “Letters of Lady Mary Wortley Montague;” letter to Miss Sarah
Chisives dated Adrianople, April 1 (O. S.), 1717.

Vaccination 99

a time when the person infected was in the most perfect physical
condition, the dangers of the malady might be mitigated.

There was always danger, however, that the induced disease being
true variola might prove unexpectedly severe, or even fatal, and
that each inoculated individual, suffering from the contagious disease,
might start an epidemic.

2. Jennerian 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 subse-
quently become infected with smallpox, to experimental demonstra-
tion, 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 insusceptible to the disease. This led
Jenner to perform many other experiments, and record his observa-
tions in numerous scientific memoirs. The success of his work
immediately attracted the attention of both scientific investigators
and sanitarians, and its outcome has been the establishment of
compulsory vaccination 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 immunities, these variations ex-
plaining 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 exceptions 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 trans-
planted micro-organisms are only capable of inducing a local
instead of general disease, or whether it is an independent affection
natural to the cow.

100 Immunity

In reality the matter is 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 variations in the sus- :
ceptibility of different vaccinated individuals determine that a few
persons cannot be successfully vaccinated, being immune to the
mildly invasive organisms of vaccinia, though perhaps susceptible
to the actively invasive 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 incubation, vesieulaicisn, pustu-

-lation, and cicatrization.

The occasional variations in immunity of diffierent. individuals
also determine that having been vaccinated once an individual
may not again become susceptible to vaccination, 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 permanent after vaccina-
tion 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 under-
stood 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 effi-
ciently vaccinated by a single operation. The term is usually
interpreted 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 scarifica-
tion has been performed upon a child, and that a scar has remained
thereafter may mean nothing. It is not the operation but the dis-
ease that achieves the result, and if the operation be improperly
done, poor—.e., old or inert—matter introduced, or if after intro-
duction it be destroyed by the application of antiseptics, no effect
can be expected. Hence ali 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

Vaccination IOL

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’ pustula-
tion, and subsequent cicatrization with the formation of a punctate

scar. ;

An arm may be made very sore, may suppurate or even become
gangrenous, without vaccinia having occurred or the desired benefit
attained.

The accidents of vaccination were formerly numerous and some-
times 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 treatment of the resulting lesions.
When human virus was-used, that is, matter taken from a vaccinia
lesion from a human being, the transmission of human diseases,
such as syphilis and erysipelas, occasionally took place; now these
are rare accidents indeed, because no virus is employed except
that taken from carefully selected and treated calves or heifers.
When no attention was paid to the quality of the bovine virus, and
no governmental inspection of laboratories required, the accidental
contamination of the virus occasioned a small number of accidental
infections of the wound. There are a good many cases of phleg-~
mon, gangrene and tetanus in the older literature. But these evils
are becoming less and less as greater attention is given to the selection
and preparation of the virus. Some accidents and some few deaths
there will probably always be, just as there are occasional accidents
and occasional fatal results following all kinds of trivial injuries,
though care will eliminate them as the sources of accident are bet-
ter 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 cul-
ture 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 were 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 achiévement of great success

* “ Compte rendu de la Soc. de Biol.,’’ 1880, 239; 315 et Seq.

102 Immunity

in preventing epidemics by which great numbers of the barnyard
fowls of France were being destroyed.

In 1881 Pasteur,* in experimenting with Bacillus anthracis, ob-
served that if the organism were cultivated at unusually high temper-
atures it lost the power of producing spores, and diminished in viru-
lence. Healsofound that when the organisms 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 increas-
ing 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 differ-
ent parts of the world.

Arloing, Cornevin and Thomas,f and Kittt 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 calcium
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 followed by the develop-
ment of a certain degree of immunity. By repeated inoculation of
more and more active viruses animals acquired complete immunity
against street virus. These experiments form 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
incubation period of the disease.

Haffkine|| found that the introduction of killed cultures of virulent
cholera spirilla produced immunity against the living micro-organ-.
isms, and used the method with considerable success for preventing |.
the disease. Later** he applied the same method, also with consider- ©

* “Compte rendu de la Soc. de Biol. de Paris,’ 1881, xc1, pp. 662-665.
tLe Charbon Symptomatique du Beeuf,” Paris, 1887.
t“Centralbl. f. Bakt.,” etc., 1, p. 684.
§ ““Compte rendu de la Soc. de Biol. de Paris,’ 1881, cvit, p. 1228.
| “Brit. Med. Jour.,” 1891, 11, p. 1278.
** «Brit. Med. Jour.,”? 1895, 1, p. 1541.

Immunity Acquired by Intoxication 103

able success, for the prevention of bubonic plague, and A. E. Wright*
followed pretty much the same method for the prevention of typhoid
fever.

In all these cases the immunity induced by the experimental
manipulations is specific in nature, and variable in intensity, ac-
cording to the method of treatment adopted and the thoroughness
with which it is carried out.

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 bacterio-proteins. The true nature of
the greater number of these bodies is unknown, but study of their
physiologic 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 substances
by which they are induced have been called by Deutscht antigens.
Since its introduction the precise meaning given the word by Deutsch
has been slightly changed. An antigen is any substance which
when injected into the body of a living organism is capable of pro-
ducing a chemicophysiologic reaction resulting in the appearance of
a neutralizing, precipitating, agglutinating, dissolving, or other-
wise 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 habituated or
tolerant to a certain quantity of mercury or arsenic, and to certain
alkaloids, such as morphin, caffein, nicotin, cocain, etc., but he
does ‘not react as to them as to antigens and no antibodies antago- ©
nistic to them are formed. To these various substances he really
acquires only a slight degree of tolerance; to the effects of injurious
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
consume small quantities of poisons become irresponsive to their
action, and it is well known that Mithridates attempted this mode of
defending himself from his enemies.

* Ibid., Jan. 30, 1897, I, p. 256.
+ Deutsch und Feistmantel, ‘‘ Die Impfstoffe und Sera,”’ 1903, Leipzig, Thieme.

104 Immunity

Chauveau* believed that the immunity conferred by inoculations
of bacteria was due to the presence of their soluble products, but the
first direct demonstration of the fact was by Salmon and Smith, f 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.
Charrint found it possible to immunize rabbits against Bacillus
pyocyaneus by injecting them with the filtered products of cultures
of that organism, and Bonome§ similarly to immunize animals
against Bacillus proteus, B. cholera gallinarum and the pneu-
mococcus. Roux and Chamberland|| and Roux** were able by the
use of boiled cultures of the bacilli of malignant edema, and of
quarter evil, similarly to immunize animals against these respective
infections.

The subject was much further elaborated by Roux and Yersin}}
in their experiments with diphtheria toxin; Behring{{ in his early
studies of diphtheria, and by Kitasato$§ in his experiments with
tetanus. ;

These early experiments opened a wide field, through the investiga. .
tion 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 alway.
acquired, never natural. It depends upon defensive factors not
originating in the animal protected, but artificially or experimentally
supplied to it. The fundamental principle is simple and has become -
the basis of serum therapeutics. 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 factors
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 Jasuhara*** found that the subcutaneous
injection of blood-serum from an animal immunized against anthrax
enabled the injected animals successfully to resist infection. Beh-

*« Ann. de l’Inst. Pasteur,”’ 1888, 2.
t “Centralbl. f. Bakt.,” etc., 1887, 11, No. 18, p. 543.
t “Compte rendu,” de la Soc. de Biol., cv, p. 756.
§ “Zeitschrift f. Hyg.,” v, p. 415.
|| “Ann. de l’Inst. Pasteur,” 1887, 12.

** Thid., 1888, 2.

TT Ibid., 1888, 11, p. 269.

ti ‘Deutsche med. Wochenschrift,’? 1890, No. so.

§§ “Zeitschrift fiir Hygiene,” 1891, x, p. 267.

||| ‘Annales de l’Inst. Pasteur,’ 1889, vol. 111.

*** “Centralbl. f. Bakt.,” etc., 1890, Ix, p. 25.

Passive Acquired Immunity 105

ring andKitasato* found that the blood-serums of animals immun-
ized against diphtheria and tetanus, when mixed with cultures of
these respective bacilli, neutralized their power to produce disease.
Kitasatot found that if mice were inoculated with tetanus bacilli,
they could be saved from the fatal infection by the intra-abdominal
injection 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; Phisalix and Bertrand,§ and, later, Cal-
mette|| found thé blood-serum of animals, immunized against the
venoms of serpents, similarly possessed the power of neutralizing
the poisonous effects of the venoms. Kossel** 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 reactions
are stimulated in experiment animals, the reactions are accompanied
by the appearance in the blood-serum of those animals of factors
that can be utilized to defend other animals in whose bodies no
similar reactions have taken place.

Passive immunity may also be brought about in a few cases
by the injection into the intoxicated animal of substances, other
than immunity products, that have a specific affinity for the poison.
Thus Wassermann and Takakift found that when the crushed spinal
cord of a rabbit was mixed im 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. Wasser-
mann also found that the same effects could be produced in the
bodies of animals, and that when the crushed spinal cord was in-
jected into an animal a few hour previously, or a few hours after
a fatal dose of tetanus toxin, enough of the combining elements re-
mained in the blood to fix the toxin before it anchored itself to the
central nervous system of the intoxicated animal. Myers{{ 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 accomplished
or initiated by factors prepared experimentally, and forced upon
the animal in whose body their activities are manifested.

* “Deutsche med. Woch.,’’ 1890, No. 49.
t “Zeitschrift fiir Hygiene,” 1892, X11, p. 256.
t “Deutsche med. Wochenschrift,’’ 1891, Nos. 32 and 44.
§“Compte rendu Acad. des Sciences de Paris,” Cxvitt, p. 556.
\|‘‘Ann. de l’Inst. Pasteur,” 1894, VIII, p. 275.
** Berliner klin. Woch.,” 1898, p. 152.
+t “Berliner klin. Wochenschrift,” Jan. 3, 1898.
tt “Lancet,” July 2, 1898.

106 Immunity

EXPERIMENTAL INVESTIGATION OF THE PROBLEMS OF
IMMUNITY

Very important contributions were made by Ehrlich,* in his
work upon the vegetable toxalbumins, ricin, abrin, and robin, that
were found to be antigens capable of producing anti-ricin, anti-
abrin and anti-robin respectively, each antibody being capable
of neutralizing the effect of its specific antigen. Kossel} investigated.
the reactions produced by toxic eels’ blood and found that im-
munity could be established against its hemolytic action, and that
specific antibodies were formed. Phisalix and Bertrandt showed
that immunity could also be produced in guinea-pigs against the
action of viper venom, and that a specific antibody, “ antivenene”
was the source of the immunity.

The investigation of other active bodies was soon begun. In
1893 Hildebrand§ studied emulsin and found that it produced a
definite reaction with the formation, in animals injected, of an anti-
emulsin. v. Diingern|| 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. Gheorghiewski** immunized animals to
cultures of Bacillus pyocyaneus, and found that the reaction pro-
voked caused the appearance in the serum of some body that pre-
vented the formation of the blue pigment so characteristic of the
organism. Morgenrotht} applied the same principle to rennet,
finding that it produced definite reactions, with the formation of
an antibody inhibiting the coagulation of milk. Bordet and Gen- |
gouft 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 Kraus§§ 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, specific
precipitate.

Wassermann and Schiitzel||| found that when cow’s milk was
repeatedly injected into rabbits, their serum acquired the property
of occasioning a precipitate when added to cow’s milk, but not when -

* “Deutsche med. Woch.,” 1891, Nos. 32 and 44.
t “Berliner klin. Wochenschrift,”’ 1898. :
t Atti d XI Congr. med: internaz. Roma, 1894, 11, 200-202.
§ “Virchow’s Archives,’’ Bd. cxxxi. ,
|| ‘Miinchener med. Woch.,” Aug. 15, 1898.
** “ Ann, de l’Inst. Pasteur,” 1899.
tt “Centralbl. f. Bakt.,”’ etc., 1899, XXVI, p. 349.
ti ‘Ann. de l’Inst. Pasteur,” 1903, XVII, p. 822.
§§ “Wien. klin. Woch.,”’ 1897.
||| ‘Deutsche med. Woch.,”’ 1900.

Experimental Investigation of the Problems of Immunity 107

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 cow’s milk.
The reaction was thus shown to be specific.

Myers* found that the repeated intraperitoneal injection of
egg-albumen into rabbits caused their serum to give a dense pre-
cipitate when added to solutions of egg-albumen.

Tchistowitcht found that eels’ serum injected into animals
produced a reaction in which immunity to its poisonous action was
associated with the ability of their serum to produce a precipitate
when added to the eels’ serum.

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,t 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 disappear. 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 Metchnikoff|| that the living animal was not a fac-
tor in the process, but that what was seen in the peritoneal cavity
could be reproduced in a test-tube, though not quite as well.

Bordet§ made frequent injections of defibrinated rabbit’s blood
into guinea-pigs, and obtained a serum that had a solvent action
upon the rabbit’s corpuscles i# 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 de-
fibrinated blood of sheep and goats, and were able to point out the
mechanism of the corpuscle solution or hemolysis. 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

* “Tancet,’’ 1900, II. ;

t “Ann. de l’Inst. Pasteur,”’ vol. XII, 406.

t “Deutsche med. Wochenschrift,” 1896, No. 7.
§ “Ann, de l’Inst. Pasteur,’’ 1895.

|| Ibid., 1898, x12.
** © Berliner klin. Wochenschrift,”’ 1899.

108 Immunity

direct solution of the corpuscles 7” vitro by cobralysin, which was
studied by Myers,* and tetanolysin, studied by Madsen, in that it
is intermediate, and only brought about by the codperation of two
factors, while the action of the lysins of venom, the tetanus bacillus,
the steptococcus, 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 pro-
duce reactions in animals, and that when successful immunization
against them was accomplished, the serums of the experiment animals
became antidotal or inhibiting to the action of the respective lysins.

Von Diingernt found that by injecting dissociated epithelial
cells from the trachea of oxen into the peritoneal cavity of guinea-
pigs, it was possible to produce epitheliolysins; Lindemann,§ that
emulsions of kidney substance injected into animals caused them to
form nephrolysins or nephrotoxins; Landsteiner|| and Metchnikoff**
in the same manner successfully prepared spermatoxin by injecting
the spermatozoa of one animal into the peritoneal cavity of another.
Metalnikoff}{ found that if he introduced the spermatozoa of a
guinea-pig into the peritoneum of another, the spermatoxic serum
produced was solvent for the spermatozoa of both. Both Metch-
nikoff and Metalnikoff also found that the spermatoxin when
introduced into animals was active in producing anti-spermatoxin
by which the destructive action of the serum upon spermatozoa
could be inhibited. :

Metchnikofff{ and Funck§§ 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|||| found that dissociated
liver cells injected into animals similarly caused the formation of a
specific cytotoxic serum.

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. a

Thus 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 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

* “Trans. Path. Soc. of London,” 11.
{ “Zeitschr. f. Hyg.,”? 1899, XxxIII, p. 239.
t ‘Miinchener med. Wochenschrift,” 1899.
§“ Ann. de l’Inst. Pasteur,” 1900. ;
|| Centralbl. f. Bakt.,”’ etc., 1899, xxv.
** © Ann. de l’Inst. Pasteur,’ 1899. * :
tt Ibid., 1900. tt Ibid., 1899.

i “Centralbl. f. Bakt.,”’ etc., 1900, XXVIT.
[||| Compte rendu de l’Acad: des Sciences,”’ 1900, cxxx, pp. 938, 1488.

Allergia or Anaphylaxis 10g .

bodies, which inhibited the activities of the respective immune
bodies by whose stimulation 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 symptoms 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 in private
conversation called the author’s attention to the fact that when
guinea-pigs used for testing antitoxic serums were subsequently
injected with another dose of serum, they commonly 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,t 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 negative, guAaés protection, destroying protection or break-
ing 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 Gottsteint was able to
collect eight deaths following the use of the serum, four 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-nodes, edema and albuminuria, these symptoms
usually appearing after an incubation period of eight to thirteen
days, and constituting what they call the “serum disease,”’ or allergic.
Sometimes these reactions 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 interest and industry,
by Gay,** Gay and Southard, {f and others.

* von Lenthold, “ Gedenkschrift,’’ Bd. 1, pp. 9, 16, 18.
+ “Compte rendu de la Soc. de Biol. de Paris,’’ 1902.
t “Therap. Monatschrift,”’ 1896.
§ “Die Serumkrankheit,’”’ Leipzig and Wien, 1905. !
|| “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,’”’ 1907, xv1, No. 3, p. 381; ‘‘Jour. Infectious Diseases,’’
1907, Iv, No. 1, p. 1, “Jour. Infectious Diseases,” 1907, vol. Iv, Pp. 552.
** “Tour. Med. Research,’’ May, 1907, xv1, No. 2, p. 143.
tt Ibid., June, 1908 x,vm, No. 3, p. 385.

IIO Immunity

Experimental study shows that when an animal is injected with
an alien protein. of almost any kind, a reaction takes place 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 proceeds 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 char-
acteristic. 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 1459 cc. It is necessary to wait until the effects of this
first injection are completely 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 149 cc., is given. Both
doses are given by injection into the peritoneal cavity.

The symptoms come on almost immediately after the second
dose. 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 Southardf found
hemorrhages in most of the organs, and believe anaphylaxis to
depend upon the presence, in the blood of the sensitized animal, of
a substance to which they have given the name anaphylactin..
Besredka and Steinhardt{ found that by the repeated injection of
horse-serum into guinea-pigs, the intervals being too short to permit
anaphylaxis, antianaphylactin could be prepared. It seems difficult,
however, to imagine how such a substance could remain in the blood
throughout the entire subsequent life of the animal.

Vaughan has endeavored to explain anaphylaxis by assum-
ing that when the strange protein in the blood reaches the cells

Sees No. 32 of the Hygienic Laboratory,’ Washington, D. C., October,
1906.

t “Jour. Med. Research,” July, 1908, xrx, No. 1, pp. 1, 5, 17.

ft ‘Ann. de l’Inst. Pasteur,” February 25, 1907, xx1, No. 2, pp. 117-127.

Explanation of Immunity riz

it is slowly broken down by enzymic action, but that the cells,
having once acquired the property of destroying it, seize eagerly
upon the protein the next time it is offered, disintegrate it rapidly,
and so disseminate throughout the body the degradation products,
some of. which may be toxic and account for the reaction.

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 compo- |
sition of the blood, as can be shown by the occurrence of what is
known as passive anaphylaxis. If the blood-serum of a senitized
animal be withdrawn and injected 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.

Anaphylaxis may play a réle 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 Klebst endeavored
to explain acquired immunity 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.”” Wer-
nicht 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 cytoplasm. Koch,
Sternberg, and others, confirmed the observation, but no attention
was paid to it until Metchnikoff** correlated it with other known

*“Compte rendu de la Soc. de Biol. de Paris,’’ xct.

+ “Arch. f. experimentelle Path. u. Pharmak.,’’ x11.

Tt “‘Virchow’s Archives,” Bd. LxxviItI.

§ ““Compte rendu de la Soc. de Biol. de Paris,’”’ xc and xct.

|| “Beitrage zur Biologie niederster Organismen,”’ Inaugural Dissertation,
Marburg, 1881.

** “Virchow’s Archives,” Bd. xcv, p. 177; ‘Ann. de l’Inst. Pasteur,” 1887,
t. I, p. 321.

112 Immunity

facts and original observations, 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 phagocytosis’? 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. These devouring cells Metchnikoff |

F 4
a a pee
3 cn, DB

L Tip yeu estes Pars Eeee eer |

Fig. 19.—Phagocytosis; the omentum immediately after injection of typhoid
bacilli into a rabbit. Meshwork showing a macrophage, intermediate form and
a trailer, all containing intact bacilli (Buxton and Torry).

the body from infectious organisms, is at least an important
one. Many of the most interesting facts are described in Metchni-
koff’s books, “Etudes sur l’Inflammation” and “Immunite dans les
Maladies Infectieuses,”’ which every interested student of the subject
should read. j
These studies show that in nearly all cases in which animals are
naturally immune against infection, the leukocytes are active in
their phagocytic behavior toward them; that in acquired immunity,
the leukocytes previously 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

Phagocytosis—Opsonins . 113

that when the toxicogenic power of the bacteria is great, many of
the phagocytes are killed and dissolved—phagolysis. Study of the
primitive forms of animal life shows that amebe constantly 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
ceelenterate animals is accomplished by means of actinodiastase, an
enzyme discovered by Fredericq, and studied by Mesnil. It seems
to be related to papine and digests albuminoids. . The digestion of
erythrocytes and tissue fragments is accomplished through an en-
zyme of the macrophages, which Metchnikoff calls macrocytase,
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 bacterio-
lytic action similar to that normally peculiar to the cytoplasm of the
cells. The dissemination of the enzymes in phagolysis, with re-
sulting bacteriolytic power of the blood plasma and serum, is a_
later modification of the original conception of Metchnikoff, 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 Metchnikoff began his studies of the phagocytes
Traube and Gscheidelt 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 extravascular
blood possessed this property. Further studies of the subject were
made by von Fodor.|| The systematic investigation of the bac-
tericidal activity of blood-serum im vitro was next taken up by
Fligge,** and more particularly by Nuttall,t{ who found that dif-
ferent blood-serums possessed the power of killing bacteria in larger
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. Metchnikoff
objected to the observations, declaring that all the phenomena were
ultimately referable to the leukocytes, so Nuttall investigated

*“Compte rendu de l’Acad. des Sciences de Paris,’”? 1901, CXXXIII, p. 244.
“Proc. Royal Society of London,’’ 1904, LXXXI, p. 357.

{“Jahresberichte der schles. Ges. f. vaterl. Kultur,” 1874.
§“Untersuchungen aus dem physiol. Institut zu Dorpat,” Dorpat, 1884;
tiger. ;

|| “Centralbl. f. Bakt.,” etc., 1890, VIL, p. 753-
** “Zeitschrift fiir Hygiene,” Bd. rv, S. 208.
tt Ibid., Bd. rv, 353.

II4 Tmmunity

pericardial fluid and the aqueous humor of the eye, which were also
found to possess bactericidal powers.

The matter was next taken up by Buchner and his associates, *
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 ofthe bactericidal fluid
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.- Neutralization 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.

Moro{ showed that alexin was proportionally more active in
sucking infants than in adults, and Ehrlich and Brieger{ 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 im-
munity 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 acting upon their
toxic products. To the whole group he applied the term defensive
proteins. Those present in natural immunity he called sozins,
those found in acquired immunity phylaxins. Sozins with bacteri-
cidal activity he further described as mycosozins, those with toxin-
destroying activities as foxosozins. Phylaxins with bactericidal —
action were called mycophylaxins; those with toxin-destroying
properties toxophylaxins.

Metchnikoff found it unnecessary to modify his ideas, but
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 indubitable evidence that
both the body cells and humors are important factors in defending
the body against invading organisms, and that in each we see mechan- ©
isms operative in certain cases. But we have seen that both
Metchnikoff and Buchner are obliged to strain a point in order to
meet the requirement of increasing knowledge to the subject of
immunity. :

*“Centralbl. f. Bakt.,” etc., 1889, Bd. v, 817; vi, 1; “Archiv fiir Hyeet
1891, X, S. 727; “Centralbl. £. Bakt.,’ ” etc., 1890, VII, 76.

+ ‘Jahresb. . Kinderheilkunde,”” v, 306.

{ “Zeitschrift fiir Hyg.,’’ 1893, x11, 336.

t econ med. Woch.,” 1899.

|| ‘‘Centralbl. f. Bakt.,” etc., x1, Nos. 22, 23; xiv, No. 25.

Defensive Proteins, etc. 115

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 zm vivo, and that the invasion of the
animal’s body by bacteria should be accompanied by diminution
of the bactericidal substance in its blood, which should be used
up before the bacteria can be successful in their invasion. Experi-
mental evidence is, however, at hand to show that this is not always
true.

Behring and Nissen* found that there was a definite relation be-
tween the bactericidal power of the blood ém vitro and the resisting
powers of a large number of animals studied, but Lubarscht 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.

Fligget 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 bacteri-
cidal power was shown to occur both in the animal and in the test-
tube. He also showed that the reactions of the bactericidal serums
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 bactericidal energy was shown
by him to depend upon the presence of the bacteria, as the injection
of filtrates of bacterial cultures did not affect the bactericidal prop-
erties 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 organ-
ism 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,||
and Bolton** have shown that horses that cannot be supposed ever
to have come into contact with diphtheria bacilli, vary considerably

* “Zeitschrift fiir Hygiene,” 1890, VIII, 412.
1 “Centralbl. f. Bakt.,” etc., 1889, v1, fie
t “Zeitschrift fiir Hygiene,” rv, 208.
§ “Lancet,” Aug. 5, 1899, II, p- 532.
| “Ann. de ’Inst. Pasteur,’”’ 1894, VIII, p. 615.
** “Tour, of Experimental Medicine,” July, 1896, 1, No. 5.

116 Immunity

in their resistance to diphtheria toxin, and that the serum of the
resisting horses contains something that destroys or neutralizes 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 Metch-
nikoff one -that protected against cholera infection. Fischel
and Wunschheimt found newly born babies immune against diph-
theria, presumably because of the presence of a small quantity of

demonstrable protective substance in the blood. These are, how- »

ever, peculiar and exceptional cases.

The most suggestive and fascinating explanation of immunity is
that of Paul Ehrlich, known as the “‘Seitenkettentheorie,” or the
“Lateral-Chain Theory of Immunity.’’t

It was the outgrowth of philosophic speculation concerning the
mechanism of cell-nutrition, of observation of the behavior of certain
anilin dyes when brought into contact with living cells, of studies of
the composition of diphtheria toxin, and the application of Carl
Weigert’s law of regeneration applied to the requirements of cell
life. Like all great theories it has been the subject of much contro-
versy, and not a few of its adversaries believe that they have com-
pletely disproved it. Whether it be adequate to meet the require-
ments of increasing knowledge of immunity the future must decide.
Of its present usefulness there can be no doubt. It has been to the
investigation of the problems of immunity, like the theory of evolu-
tion has been to the biological sciences, a most convenient and sug-
gestive method of reasoning and deduction.

The theory begins with the supposition that all living cells possess
certain functions that are individual, fundamental and indispensable
and that such cells as are components of multicellular organisms

* “Centralbl. £. Bakt.,” etc.,.1895, XVII, p. 36.

+ “Zeitschr. fiir Heilkunde,” 1895, XvI, p. 429-482. ;

t 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 consult
the original articles can best do so by making use of the following: Ehrlich,
“Die Werthbemessung des Diphtherie Heilserums,”’ Klinisches Jahrbuch, 18973
Ehrlich, ‘Die Konstitution des Diphtheriegiftes,’? Deutsche med. Woch., 1898}
“Gesammelte Arbeiten zur Immunitatsforschung,” August Hirschwald, Berlin,
. I904—this work contains the collected papers of Ehrlich and his associates;
Aschoff, “Ehrlich’s Seitenkettentheorie und ihre Anwendung auf die Kiinst-
lichen Immunusirungs-prozesse,” Jena, 1902, and the chapter upon “Wirkung
und Entstehung der Aktiven Stoffe im Serum noch der Seitenkettentheorie,”
by Ehrlich and Morgenroth in Kolle and Wassermann’s “‘Handbuch der -Patho-
gene 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, 1900, txv1, p. 424, and in
Welch’s “Huxley Lecture,” Medical News, 1902, LXXXI, 2, p. 72I.

‘The ‘‘Lateral-chain Theory”? of Immunity 117

have additional functions that are special and somatic. As examples
of the first, nutrition and reproduction may be suggested, of the
second glandular secretion, nervous impulse transmission, muscular
contraction, bone and pigment formation.

The nutrition of each cell in a composite organism, therefore, re-
quires material with which to meet two demands, first the sustenance
’ of its own substance, second the supply of those special or particular

substances through which its special functions are to be performed.

According to its particular necessities each cell is undoubtedly
endowed with selective affinities by which these appropriate sub-
stances are caught, held and brought finally into molecular composi-
tion with the cell substance. This ‘is, naturally, a chemical problem,
but one of such complexity that no symbols used in chemical science
enable us to follow it either accurately or adequately.

To arrive at a clear comprehension of the matter, and to progress
from this comparatively simple beginning to the more involved
problems to come later, conventional chemical expressions and sym-
bols are laid aside, and new and simple symbols introduced.

The cell is conceived to consist of an executive center (Leistenkern)
surrounded by numerous conductors (Seitenketten) or “side-chains.”
It is by the latter that molecules brought to the cell by the inter-

‘cellular lymph are caught and held when of a quality necessary to
the requirements of the cell and of a composition adapted to one or
more of the side-chains.

The side-chains are known as receptors or haptophiles (arrev
to bind and @:Aey to love). A chemist is apt to picture to himself
a benzine ring with its various possibilities of combination and sub-
stitution, but, as has been said it seems better to avoid this form
of symbol. Ehrlich pictures.the cell as a sphere the surface of which
is covered by nipple-like processes; the receptors.

The molecules in the body fluids are conceived to be or not to be
provided with adaptations to these receptors, according to their
nature. The adaptations go by the name of haptophores (Array, to
bind and ¢épey, to bear) and are graphically represented as small |
figures, excavated at one end so as to fit on the receptors. Under
normal conditions during which cell nutrition and cell function
progress regularly one conceives that useful molecular groups with
haptophores adapted to the receptors are constantly brought to the
cell, and are seized and held until incorporated into the composition
‘of the cell itself.

Under abnormal conditions, however, new substances appear in
the tissue fluids, among which are toxic products of micro-organis-
mal metabolism, should these have haptophores adopted to the re-
ceptors of the cell, they may be caught and held with disastrous
results for if they are in sufficient number to immediately appropriate
all of the receptors so as to exclude the necessary molecules, and be
of themselves of no nutritive value, the cells may die of starvation.

Immunity

118

or should they possess toxic properties the cells may be poisoned.

It is doubtless thus that death frequently occurs through the destruc-
tion of cells essential to somatic life in the infectious diseases.

Butif the number of receptors appropriated by the abnormal hapto-

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, it reacts under the stimulus

and according to the theory of Wei

present impediment and future men

phores is not sufficient to destroy the cell

of necessity,
against the

gert, provides itself
ace to its activities

by regenera

ting new receptors to take the place of these thrown out

of use, and by regenerating them in excess of the usual numbers.

The ‘“Lateral-chain Theory” of Immunity 119

Tf now these should also be appropriated, a further regeneration
occurs, and so on and on until, the cell continuing to live and more
and more receptors thus being formed, their number eventually
becomes so excessive that the cell is encumbered with them and
throws them off into the surrounding juices where they continue
to circulate as “free receptors” or “‘haptines” for a considerable
time, retaining the same combining power for circulating haptophores,
that they possessed on the cells.

In this statement may be found an explanation of several facts
in regard to immunity:

1. The injury effected by micro-organismal products i is in part due
to the adaptations between their haptophores and the receptors by
which the cells are starved or poisoned. This only applies to those
products that correspond to what are known as antigens, and not
to acids, alkalies, etc.

2. The increase in the resisting power of the animal during disease
or after repeated experimental administration of sub-lethal doses
of a toxin, is due to the regeneration of receptors.

3. The appearance of substance (antibody-antitoxin) in the blood
of the animal, is due to the presence in the blood of the haptines or
free receptors, cast off by the cells after excessive regeneration.

This is the general statement and is the foundation of the theory,
but to fulfil all of the requirements of the complicated facts of im-
munity, it is necessary to amplify the matter by modifying the nature
of the receptors. Ehrlich therefore supposes the existence of re-
ceptors of three orders:

I, Receptors of the First Order (Antitoxins and Anti-enzymes).—
- These have just been discussed in making plain the general prin-
ciples of the theory. Every cell is conceived to have innumerable
receptors of many orders, with many different adaptations, so the
student must not conceive that the condition is simple. Receptors
of the first order are regarded as adapted to food molecules. The
haptophores fit on directly, and may be simple or complex. In the
case of the micro-organismal enzymes and toxins, Ehrlich describes
such of the molecular groups as composed of a haptophore and a
toxophore. By the attachment of the haptophore, the toxin may be
brought into the cell and its health or life disturbed, but the dis-
turbance effected by the toxins is'unessential to the regeneration of
teceptors and the formation of haptines, as was shown by Ehrlich*
who found that when diphtheria toxin is kept,.it undergoes a change
into toxoids and loses its poisonous quality, though its haptophores
being unchanged, it still attaches to the receptors and stimulates
their regeneration, thus bringing about antitoxin formation, and also
still attaches to haptines when brought into contact with ‘them by
mixing arititoxic serum and old toxin in vitro. Further it has been
found by Metchnikoff that the cells of the central nervous system

* Klinisches Jahrbuch, 1897.

120 Immunity

of alligators are insusceptible to the action of tetanus toxin, though
the haptophores of that toxin attach to the cells and occasion the
formation of tetanus antitoxin.

All antitoxins and anti-enzymes may be accounted for as resulting
from the excessive regeneration of receptors of the first order and
their appearance in such of the body juices as possess the antitoxic
or anti-enzymic quality.

II. Receptors of the Second Order (Agglutinins and Precipitins).—
It is assumed by Ehrlich that some of the nutritive molecular
groups anchoring to the cells are not in condition to be utilized until
they have beén subjected to preliminary treatment effected by
the receptor itself. To achieve this purpose another kind of recep-
tor, providing the means for such treatment, had to be imagined.
These are supposed to consist of two portions, one adapted to union
with the antigen (haptophore group) the other providing the means
of preliminary treatment (zymophore group). The appropriation

of the receptors by antigenic molecules of abnormal character being
of no benefit to the cells, new receptors of the second order are
regenerated in precisely the same manner as were those of the first
order, and similarly appear in the juices when their excessive num-
ber causes them to be thrust off from the cells. The quality imparted
to the serum by these haptines is, however, different from that
occasioned by haptines of the first order. The serum and juices
become agglutinating and precipitating, and in this group fall the
specific agglutinins and the specific precipitins.

III. Receptors of the Third Order (Hemolysins, Bacteriolysins, Cyto-
toxins)—When the antigen is still more complex it seems as though
the preparation for admission to the cell composition required other
substances than could be furnished by the cell itself, and must be
caught from the juices surrounding it. To meet this requirement,
Ehrlich conceived receptors with two adaptations—i.e., two separate
haptophore groups—one fitting to the molecular group to be
utilized, the other to some other molecular group (enzymic substance)
by which its utilization was to be made possible. In applying this
principle to the reactions of immunity, the antigen attaching to the
one side of the receptor, is brought into relation with the enzymic
substance called complement, attaching to the other end of the re-
ceptor, by the receptor itself, which then becomes the intermediate
body or amboceptor. Ehrlich was at first of the opinion that there
were as many different receptors of the third order—i.e., amboceptors
——as there were antigens, and that there was also a considerable
number of complements. It is now believed that he was correct
in the former assumption, but incorrect as to the latter, there being
but a single complement.

The excessive regeneration and liberation of receptors of the third
order into the body juices is presupposed to occur just as in the case
of the receptors of the first and second order. These receptors or

The “Lateral-chain Theory” of Immunity 121

amboceptors have no specific action by themselves, and effect no
visible changes when serum containing them is mixed in vitro with
the corresponding antigen, for their only function is to form a bond
between the antigen and the complement. To determine their
presence in any serum or other body juice, it is therefore necessary
‘ tosupply the complement, when dissolution of the antigen is quickly
effected.

In connection with the factors here involved it is interesting to
observe that under certain experimental, and perhaps also under
certain natural conditions both amboceptor and complement may give
rise to antigenic reactions in the animal body, so that anti-ambocep-
torand anti-complement may beformed. The former, when brought
into contact with immune body and antigen, may substitute itself
for the antigen, thus preventing the attachment of the amboceptor
to the antigen; the latter may substitute itself for the complement,
thus preventing the complement from attaching to the amboceptor,
and in either case making impossible the antigen-amboceptor-

complement combination by which alone the dissolution of the an-
tigen can be effected:

Such antigens as heterologous red blood corpuscles, spermatozoa,
dissociated tissue cells, bodies of micro-organisms destroyed by heat
‘etc., all bring about reactions tending to increase the number of
receptors of the third order and occasion the presence of ambo-
ceptors in the body juices.

An analysis of this theory shows complete natural immunity
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; comparative insensitivity or insusceptibility upon
the fact that 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 (antitoxin) 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 hapto-
phores 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 com-
plement by which it is to be dissolved. Antibodies having this dou-
ble combining affinity have been called “amboceptors”” by Ehrlich.
_ They are variously known in different writings as “immune bodies,”
amboceptors, substance sensibilisatrice, desmon, and fixateur. The

’

122 Immunity

“complement” ot “addiment” of Ehrlich is also called alexin and
cytase. Ehrlich conceives every amboceptor and every comple-
ment 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 said that Metchnikoff’s primitive con-
ception of the body being defended against infection through the
phagocytic incorporation and digestion of the microparasites, has
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 con-
ception of “phagocytosis” to make it applicable to the new require-
ments. He now teaches that when invasive micro-organisms enter
the body, chemotactic influences determine that they shall be met
by phagocytes. If the invading micro-organisms are too powerful
and the phagocytes are killed, phagolysis or dissolution of the phago-
cytes liberates their enzymes into the blood. These liberated
enzymes still act deleteriously upon the invaders, tending to ag-
glutinate—aggregate them in clumps—and sensitize them to the
future action of other phagocytes by which they may be taken
up. Through extensive phagolysis, and the liberation of large
quantities of the enzyme contents of the phagocytes into the blood,

the plasma and serum acquire a “fixing” or “‘sensitizing” quality '

- from the macrocytase of the macrophages, which is the ‘‘fixeteur”
or “substance sensibilisatrice,”’ and a bacteria-dissolving quality
forms another enzyme, microcytase, from the microphages. Thus,
we find that Metchnikoff is prepared to account for the “ambo-
ceptor”? or “immune body” of Ehrlich, which is the macrocytase,
and the “complement,” which is the “microcytase.” In cases
where the bacteria exert a negatively chemotactic influence upon
the leukocytes, no immunity exists.

The antitoxins are similarly accounted for by Metchnikoff: the
cellular digestive enzymes exert their action not only upon the
microparasites, but also upon their products, fixing or otherwise
altering them until they can be finally destroyed.

It will thus be seen that the two chief theories of immunity, though
they appear discordant when explained independently of one
another, can be fairly well harmonized. Ehrlich believes the im-
mune bodies to be the products of those cells of the body with whose
haptophile combining groups the haptophore groups of the antigen
engaged, and does not attribute the function to any particular
group of cells; Metchnikoff attributes all the activities to the
phagocytes, and especially the leukocytes. Ehrlich looks upon the
phenomena as chemical and pictures them as taking places inde-
pendently of the cells; Metchnikoff looks upon them as vital and
brought about by the agency of living cells. Both theories are
ultimately chemical.

AEbbbbbbe bbb bbND

Special Phenomena of Infection and Immunity 123

The fundamental ideas embodied in the “‘lateral-chain theory” of immunity
may, by reversing the hypothesis and considering the bacterial instead of the
body:cells to be upon the defensive, be made to explain 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.t The micro-
organismal cells must be regarded as endowed with receptors of their own, fitted
for combinaton with adapted haptophorous elements in the juices reaching
them, and therefore capable of reacting toward such 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 facili-
tate bacteriolysis, so the latter are stimulated to antagonize their action by pro-
ducing neutralizing bodies. These neutralizing bodies by which the defenses of
the host are broken down are among those described by Bailt 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 reacting toward the
defensive products of the former. If the reactive processes of the host predomi-
nate, 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 infection
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 interesting experimental demonstra-
tion by Forssner§ 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
casually made, must now be considered in detail.

SPECIFIC PRECIPITATION

Specific precipitation is the coagulation or precipitation of an anti-
gen by its specific antibody. In 1897 Kraus|| while 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.”

*«“TJour. of Path. and Bact.,’’ March, 1902, vit, No. 1, p. 34.
Fl “British Medical Journal,’’ Oct. 11, 1902, p. 1105; ‘‘ Medical News,” Oct.
18, 1902.

t Wiener klin. Woch.,’’ 1905, Nos. 9, 14, 16, 17; “Berl. klin. Woch.,” 1905,
No. 15; “Zeitschr. f. Hyg.,”” 1905, Bd. 1, No. 3.

§“Nordiskt Medicinskt Archiv,” 1902, Bd. xxxv, p. I.

| “Wiener klin. Woch.,’’ 1897, No. 32.

I24 Immunity
Bordet* and Tchistowitcht showed that the phenomenon was’
of wide occurrence and had a broad significance, for they discoyered °
that when the serum of one animal was injected into another ani-
mal of different kind, some reaction took place in the injected ani-
mal, which caused a precipitate to form whenever the serums ‘of
the two animals were subsequently brought together in a test-tube.
The same was found true of milk. When an animal was injected
with the milk of a different kind of animal, its serum acquired the
property of causing a precipitate to form when
its serum and filtered milk were mixed together
a inatest-tube. The substance or factor inducing
4 the precipitation was called “precipitin” or
. “coagulin.”” Myers,t Jacoby,§ Nolf, || and others
showed that the faculty of provoking specific
precipitins was common to many albuminous
bodies—albumen, globulin, albumose, peptone, —
ricin, etc. Kraus in his original communication _

REET dwelt upon the specific nature of the precipita-
21.—Poly-

F ig.

: tion, and was corroborated by Fish,**
ceptor (Ehrlich and
Marshall) such as Wassermann,{{ Morgenroth, and others, by

can be conceived to
occur in hemolysis
and _bacteriolysis
where various com-
plements are en-
gaged. a, Receptor
of bacterial cell; 6,
cytophil group of
the amboceptor; c,

dominating comple-’

ment; d, subordinate
complement; a, 8,
complementophil
groups of the ambo-
ceptor, @ for the
dominating, 8 for
the subordinate
complements.

whom it has been shown that the reaction is
sufficiently accurate to make possible the
differentiation of human and goat’s milk. The
most important practical application of the |
specific character of the precipitins, however,
came through Uhlenhuth{t and Wassermann,§§
who made use of it for the differentiation of
bloods for forensic purposes.
Uhlenhuth gave rabbits intraperitoneal in-
jections of to cc. of defibrinated blood at
intervals of from six to eight days and found
the blood-serum strongly precipitant after the
fifth. He used such serum for testing the re- _
action with the bloods of oxen, horses, donkeys,

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 carelessness of
detail will interfere with the accuracy of the result. The blood to be
tested is diluted about 1:100, or until it has a feeble red color, with

*“ Ann. de l’Inst. Pasteur,” 1899, p. 173. :
ie Ann. de l’Inst. Pasteur,” 1899, p. 406.
i“ Centralbl.f. Bakt.,” etc., 1900, Bd. xxx1ur, and “The Lancet,’’ 1900, 11, p. 98.

§ “Archiv fiir exper.

Path. u. Pharmak.,” r900.

{| “Ann. de l’Inst. Pasteur,’’ 1900, p. 297.
** “ Courier of Medicine,” St. Louis, Feb., 1900.

tt “Verhandl. d. Kong. f. innere Med.,”’ 190 Wiesbad
tf “Deutsche med. Woch.,’’ 1900 and Say ice aleeieneans
§§ “Samm. klin, Vortr. von Volkman,” Leipzig, Verlag von Breitkopf and

Hartel, rgo2.

The Agglutinins | 125

tap water, and then freed from 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
solution, from six to eight drops of the immune serum are added. If
the diluted blood come from the same kind of animal as that whose
blood was 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.

Wassermann* and Schutze prepared a test serum by injecting
rabbits with human blood. They tested its precipitating powers
upon twenty-three other kinds of blood and found no precipitate
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 biological
applications of this phenomenon is by Nuttall, whose work, “Blood
Immunity and Blood Relationship” (Cambridge, 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 medicolegal importance in recognizing blood-stains.
Nuttall comes to the following conclusions:

(rt) The investigations we have made confirm and extend the observations
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 sub-
stance in serum which brings about the formation of a precipitin, as also the pre-
cipitin 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. 3

Further perfection in the technic of the precipitation experiments
can be found in a paper by Nuttall and Inchley.f |
The precipitinogen is capable of acting as an antigen and the
injection into animals of serum containing it results in the formation
of anti-precipitins.
AGGLUTINATION

Agglutination is a phenomenon of infection and immunity in
which the serum or other body juice of the infected animal so acts
upon the infecting micro-organism as to destroy its power of move-
ment, and cause it to sediment in clusters in the liquid in which it
is suspended. This phenomenon was first observed by Charrin
and Roger{ in the course of experiments with Bacillus pyocyaneus.
They found that when Bacillus pyocyaneus was introduced into a
test-tube containing the diluted serum of an animal infected with or

* “Deutsche med. Wochenschrift,” 1900, No. 30.
tT “Journal of Hygiene,” 1904, Iv, p. 201.

126 Immunity

immunized against it, the bacilli ceased their active movements,
became aggregated in clusters and settled to the bottom of the tube,
leaving the supernatant fluid clear. Observations confirming and
enlarging upon the subject were made by Metchnikoff,* Issaefft
and others. Gruber and Durhamt made an elaborate and now
classic study of the subject, first employing the term ‘‘agglutina-
tion” to the phenomenon, and “agglutinins” to the substances in
the serum by which 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 aggre-
gated in clusters, masses or “clumps.” They further showed the
reaction to be specific within certain limitations, 7.e., typhoid im-
mune serum agglutinated typhoid-like bacilli but no others, etc.,
and they saw in the phenomenon a practical means for the dif-
ferentiation 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 impor-
tant application, in that the micro-organism being known, the effect
produced by a serum upon it would be an indication of the infec-
tion of the animal from which the serum was secured. The first
practical application was made in connection with the diagnosis of
typhoid fever, and the brilliant success attending it has led to the
test being known as the “‘ Widal reaction.”

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 chlorid that
throws down fibrinogen and further precipitated with magnesium
sulphate, which throws down globulins. They therefore thought
them to be intimately related to the globulins and to fibrinogen. A
temperature of 60°C. diminishes their activity, but they are not |
destroyed below 80°C. Sunlight has no effect upon them.

Metchnikoff looks upon agglutination as preliminary to phagocy-
tosis 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 denominates the II order, each of which possesses
a zymophore and an agglutinophore group.

Malvoz** found that the addition of chemical substances, such as
safranin, vesuvin, and corrosive sublimate, to cultures of the typhoid —
bacilli would cause their agglutination. Typhoid bacilli retained —
on the Chamberland filter and washed for a long time, could no

*“Compte rendu de la Soc. de Biol.,” 1899, p. 667.

t Ibid., 1893, vi.

t “Miinchener med. Woch.,”’ 1896, No. 9.

§ “Société Médicales des Hopitaux,’’ June 26, 1806.

|| See Nothnagel’s “Specielle Pathologie und Therapie,” r901, vin.
** “Ann. de l'Inst. Pasteur,” 1897, No. 6.

The Agglutinins 127

longer be agglutinated, and were found to have lost their flagella
and to be without motion. This led Dineur,* who made additional
experiments, to conclude that agelutination depended upon the
flagella. Malvozt} 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 thoroughly distributing
it through 34 c.c of distilled water, and then added a loopful of a
six-day-old culture. After standing for a few hours typical agglu-
tinations were observed under the microscope.

H. C. Ernst and Robeyt found that flagella have nothing to do
with agglutination, which subsequent experiment has shown to be
correct, as non-flagellated bacteria can be agglutinated by their
respective serums quite as well as the flagellated forms.

Bail,§ Joos,|| Eisenberg and Voll** 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 temperature 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 pre-
venting the action of unheated agglutinogenic serum.

Buxton and Vaughanff found that bacteria differ both in then
agelutinogenic powers and their agglutinability, both of which must
be taken into account in studying the subject.

Theobald Smitht{ 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 reaction is one of the most delicate known to us for the
identification of bacteria. It is so specific that, in the case of many
organisms, it is even possible to tell from what original source they
may have come, and always to tell to what variety they belong.
It is, moreover, a comparatively simple method that can be used by
physicians with little technical skill. The various serums necessary
can be obtained from the large public and commercial laboratories
where animals immunized against various cultures can always be
kept on hand and periodically bled. The serums, sealed in small
tubes, can be kept an almost unlimited length of time and shipped
to any distance ready for use when opened and diluted.

There is no uniform technic by which to apply the test. Scarcely
any two laboratories employ the same method, but the results are

* “Bull. de PAcad. de Med. de Belgique,’’ 1898, Iv, p. 705.
{ “Ann. de l’Inst. Pasteur,’’ Aug. 25, 1899.
t “Trans. Cong. Amer. Phys. and Surg.,”’ 1900, p. 26.
§ “Archiv f. Hyg.,”’ 1902, XLII, Heft 4.
|| “‘Zeitschr. f. Hyg.,”’ ai XXXVI, p. 422.
** Thid., 1902, XL, p. 15
tt “Jour. Med. Resa ,’ July, 1904.
tf Ibid., 1904, vol. x, p. 89.

128 Immunity

uniform and the method to be employed, provided it is free from
error, is that found most convenient to the individual operator.
The agglutination test now subserves two important functions:
1, the diagnosis of any infectious disease, provided the infecting organ-
ism be at hand; 2, the recognition of any micro-organism, provided
specific serum be at hand.

Technic of Agglutination Tests

If possible, a culture of the micro-organism, grown upon agar-agar, is to be
selected for the purpose. A good-sized platinum loopful ofthe culture is taken
up and distributed as uniformly as possible throughout 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 cc.),
to make the fluid opalescent by reflected light though transparent by trans-
mitted light. The same quantity should be added to each, so that they form
a uniform series. The patient’s blood or serum is next diluted and added so
that the watch-glasses or tubes receive’ a 1:10, 1:20, 1:30, 1:40, 1:50,
1:60, 1 : 80, I : 100, I : 150, I : 200, I : 300, etc., respectively, or if an experi-
mental laboratory serum of high agglutinative value be used, 1 : 1000, 1 : 2000,
I 15000, I : 10,000, I : 50,000, and 1 : 100,000 respectively.

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 aggre-
gate in clumps.

If test-tubes are employed, they are best observed by tilting them and look-
ing 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 ex-
periments. Thus, with typhoid bloods, the agglutination of the typhoid bacillus
usually occurs within an hour in dilutions of 1:50, but the agglutinability of
the culture employed should be known before the experiment is undertaken.

Similarly, when the method is employed for the differentiation of bacteria the
agglutinative value of the serum should be known to begin with.

The agglutinins are capable of acting as antigens and when injected into animals
effect reactions followed by the formation of antibodies inhibiting their own
activity. P

ANTITOXINS

Antitoxins are immunity products by which the injurious actions
of toxins are annulled. In the synopsis of immunity experiments’
already given, the history of the discovery and development of the
antibodies 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 occurrence of antitoxins
is afforded by Ehrlich in the lateral-chain theory. He regards them
as cell haptophiles—receptors—that are formed in excess of the re-
quirements, by cells frequently stimulated by the presence of bacte-
rial products possessing adapted haptophores. The receptors are

The Antitoxins - 129

under normal conditions engaged in maintaining the proper nutrition
of the cell; under abnormal conditions (as when preempted by the
inert or injurious haptophores of the bacterial products) are obliged
to increase in number to compensate for the damage done the cell.
Antibody formation can be induced only by antigens or bodies that
bear a resemblance to the normal nutrient substances absorbed
by the cells in that they are provided with haptophore groups
corresponding with the haptophile groups of the cells and so adapted
for union with them. Mineral and alkaloidal substances have
no such adaptations, but bacterial products, the toxalbumins of
various higher plants, venoms, enzymes, and other protein com-
binations have. The possession of the haptophile groups determines
whether or not the cell can stimulate antibody formation, and the
ability to produce antibodies 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 as-
sociated toxophore and toxophile groups, as in the case of the bacterio-
toxins, but may not be discovered if there are none. The combina-
tion of the toxin-haptophores with the cell-haptophiles 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 haptophiles or receptors, loses its further
combining powers and fails to affect animals into which it is sub-
sequently injected. The increased formation of receptors in con-
’ sequence 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—z.e.,
fixing—the abrin 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 haptophiles 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 antibodies is entirely
. independent of any toxic action and is entirely the work of the hap-
tophiles. This is best shown in the fact that diphtheria toxin that
has been heated or otherwise manipulated until its toxic action is
lost, still retains the power of combining with antitoxin, or of
producing antibodies.

The cells furnishing the haptophile 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

130 Immunity

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 antibody forming substance
is followed by an outpouring of the antibody far in excess of what
would neutralize it, so that after a systematic treatment has been
carried out for some time, the neutralizing value of the blood may be
a thousand times what would be necessary to neutralize the total
quantity of active substance introduced into the animal.

Each antibody is specific in action, as must be evident from its
mode of formation. Should it be found, however, that several active
bodies possessed haptophores 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 antibodies enjoys immunity
from the active body by which they were formed only so long as
they are preserit. 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 treat-
ment by which the antitoxins are produced is interrupted, they im-
mediately begin to lessen in quantity, and eventually disappear.
Their occurrence in the blood determines that they should be found
in all the body juices, though in varying quantity.

Their chemical composition, which experiment shows to be of
protein nature, determines that when practical use is to be made
.of them, they must not be administered by the stomach, as diges-
tion is usually followed by their destruction. In infants, the pro-
tein digestion being feeble, antitoxins pass from the mother’s milk to
the blood of the sucking offspring without digestion, but the ad-
ministration of antitoxins by this method at later periods of life is
followed by effects too uncertain to be depended upon. For practi-
cal therapeutic purposes, therefore, the administration must always
be made hypodermically or intravenously.

Diphtheria Antitoxin——This was first utilized for practical
therapeutic purposes by Behring.* As usually prepared by the
administration of the toxin, it is essentially an antitoxin and has
no destructive action upon the diphtheria bacilli. In therapeutics

* “Deutsche med. Wochenschrift,” 1890, Nos. 49 and 50; “Zeitschrift fir
Hygiene,” etc., 1892, x11, p. 1; ‘Die Blutserumtherapie,” Berlin, 1902.

The Antitoxins 131

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 injuriously acted upon by the toxin. Martin is of the
opinion that such purely antitoxin serums are inferior to those con-
taining other immunity products, such as bacteriolysins, and recom-
mends 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 antitoxic effects of the serum.

The serum may be used to prevent or to cure diphtheria.

The antitoxin is commercially manufactured at present by im-
munizing horses against increasing quantities of diphtheria toxin
until the proper degree of immunity has been attained, then with-
drawing the antitoxic blood. The details are as follows:

I. The Preparation of the Toxin.—The toxic metabolic products of the Bacillus
diphtheriz 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 phenol-
phthalein. It then receives an addition of 0.2 per cent. of dextrose, and is-sterilized
in the autoclave. To secure the best 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
‘9,001 cc. given hypodermically.

II. The Immunization of the Animals.—All commercial manufacturers of
diphtheria antitoxic serums now use horses, as recommended 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 cc.—should be given in the begin-
ning, as one occasionally finds exceptionally susceptible animals that will suc-
cumb 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 in-
terrupted for a day or two. The animal yields good antitoxic serum when it
can endure several doses of 500 cc. of the strong toxin mentioned above.

Il. Bleeding— When the withdrawal of a small quantity of blood by a
hypodermic needle introduced into the jugular vein shows that the serum con-
tains 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

* “Journal of Experimental Medicine,’ May and July, 1899, p. 373.

132 Immunity

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 precautions. It is variously prepared
for the market. Some manufacturers 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 contamination
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 filtration 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 in .
use at the present time, though to understand it, it seems wise to mention the
. original method from which it was derived.

(A) Behkring’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 circumstances 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 fen times the above determined least fatal dose of toxin.

3. Express the required dose of antitoxic serum as a fraction of a cubic centi-
meter and multiply by 10; the result is one unit.

Example: It is found that 0.01 cc. 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—say 14900 cc., 34500 cc., }Z000cc. The first
two live. The fraction 145090 is now multiplied by 10; }4500 X 10 = 1450 = 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 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 epitoxoids have a greater anti-
toxin-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 important modification of
the method, by which the standard of measurement is a special antitoxin of
known strength, by which the antitoxin-combining power of the test toxic bouil-
lon 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

* “Klinisches Jahrbuch,” 1897.

The Antitoxins 133

of these tubes was opened and the contained powder dissolved in 200 cc. of a
mixture of to per cent. aqueous solution of sodium chlorid 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, x cc. represented 17 units; }47 cc., one unit.

Having by dilution—1 cc. 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 or L +
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 test-
ing, 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 quantity 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 cc. -+ water 16 cc. = 1 cc. is the unit. 1 cc. of
the dilution containing one antitoxic unit is mixed with 0.01, 0.025, 0.05, 0.075,
o.1 cc. of the toxic bouillon. All the animals receiving less than o.1 cc. 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 days,
but that the animal receiving that dose, though very ill, lives longer. The test
oA may then be assumed to be o.1, or it may be calculated more closely if

esired. ,

To test the serum itself, guinea-pigs weighing exactly 250 grams are now all
iven toxic bouillon 0.1 cc. plus varying quantities of the serum—}400, 3400,
40, etc. All live except those receiving less than 1499, 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 was first shipped at small expense from the Kaiser-
liches Institut fiir Serum-Therapie at Hichst-on-the-Main. At
present the Hygienic Laboratory of the United States Public Health
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 standard
test serum, similar to that of Ehrlich, to those entitled to receive it.
. A full description of “The Immunity Unit for Standardizing
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 therapeutic serums be as strong
as possible. Various methods of concentration have been sug-
gested. Bujwid* and H. C. Ernst} found that when an antitoxic

oe f. Bakt. u. Parasitenk.,” Sept., 1897, Bd. xxur, Nos. ro and 11,
p. 2

7.
Tt “Jour. Boston Soc. of Med. Sci.,’? May, 1898, vol. 11, No. 8, p. 137.

134 Immunity

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 concentration
is by the employment of the globulin precipitation as recommended
by Gibson,* which is briefly as follows: The diluted citrated plasma
is precipitated with an equal volume of saturated ammonium sul-
phate solution and the antitoxic proteins separated by extracting
the precipitate with saturated sodium chlorid solution. The solu-
ble antitoxic proteins are then reprecipitated from the saturated
sodium chlorid solution with aceticacid. 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 Bauzhaff 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 Cruveilhier{ on this point.

Tetanus antitoxin was first prepared by Behring and Kitasato.§
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 administered in frequently repeated large doses by
hypodermic or intravenous injection. The results are less brilliant
than those attained with diphtheria antitoxin because of the avidity
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 fol-
lowing the free administration of the serum exceed those effected by
other methods of treatment by about 4o per cent.

By the gradual introduction of tetanus toxin Behring and Kita-
sato|| have been able to produce a powerful antitoxic 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 produced, the one annul-
ling the convulsive, the other the hemolytic, properties of the toxin.
The usual therapeutic serums contain both of these.

* “Tour. Biol. Chem.,’’ I, p. 161; III, p. 253. :
} “Jour. Infectious Diseases,’’ March, 1908, vol. 11, pp. 202 and 264.
{ ‘Ann. de l’Inst. Pasteur,’’ 1904, XVIII, p. 249.

§ “Deutsche med. Wochenschrift,’’ 1890, No. 49. || Ibid.
** “Zeitschrift fiir Hygiene,’ 1899, XXXII, p. 239.

The Antitoxins 135

Different standards for measuring the strength of the tetanus
toxin and different definitions of the unit of measurement are given
in different countries, so that great confusion and dissatisfaction
were experienced until a special committee of the Society of Ameri-
can Bacteriologists met in New York, Dec. 27 and 28, 1906, and in
collaboration with the United States Public Health and Marine
Hospital Service, Hygiene Laboratory, formulated a standard
unit which has become the legal unit of measurement 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 350-gram 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 defined, 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 the injection of 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 Laboratory upon “The
Standardization of Tetanus Antitoxin,’’ by Rosenau and Anderson.

Antivenene or Antivenomous Serum.—This was discovered
by Phisalix and Bertrand* and made practical for therapeutic
purposes by Calmette.f Calmette found that cobra venom con-
tained two principles, one of which, labile in nature and readily
destroyed by heat, was destructive 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
heating 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 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 modi-
fied and unmodified cobra venom, and was used successfully in
the treatment of a number of human beings who had been bitten
by cobras. Calmette, however, erroneously concluded that be-
cause 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 antagonized. Arguing from this stand-

ry “Compt. rendu de l’Acad. des Sciences de Paris,’ Feb. 5, 1894, CXVIII, p.

t “Compt. rendu de la Soc. de Biol. de Paris,” Feb. 10, 1894, 10 Series, 1,
Pp. 120,

% 36 Immunity

point, 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 antivenéne. was
useless in the treatment of the bites of the Australian serpents,
and the experiments of the author 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 pdison
is essentially locally destructive in action, the fatal influence upon
the respiratory centers being of secondary importance. Flexner and
Noguchi,f 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—hemorrhagin
—of the Crotalus venom. »

Antivenene is useful in the treatment of cobra invenomation,
as Calmette has shown by cases treated in his own laboratory.
The serums of Noguchi and others are equally useful in their re-
spective invenomations, but the opportunity for successfully em-
ploying antivenenes is very small. Few persons are bitten 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 zodlogical gardens where
venomous serpents are kept, and in laboratories where the snakes
are kept for experimental purposes, it is well to be provided with a
supply of the serum, but it has no wide sphere of usefulness.

CYTOTOXINS

Cytotoxins are immunity products that exert a specific destructive
action upon cellular antigens. They are essentially cell-dissolving
products of immunity. The solution of the cells, of whatever kind,
takes place through the complement, native to the blood, fixed to
the cells by the specific amboceptor. The complement is pre-
sumably always the same and is present in all normal blood; the
amboceptor is an “immune body” susceptible of artificial produc-
tion or increase, and specifically differs according to the particular
cell through whose antigenic activity it was produced.

Hemolysis.—The phenomena of hemolysis or the solution of
erythrocytes, caused by heterologous serums were first studied by
Creite|] and Landois,** who studied hemoglobinuria following trans-
fusion. Subsequent observations were made upon corpuscular
agglutination and solution by venoms by Mitchell and Stewart{t

* “Tntercolonial Medical Journal of Australia,” 1897, II, p. 537.

} “Journal of Experimental Medicine,” IQOI—1905, VI, Pp. 277.

{ Tbid., 1906, vir, p. 614.

* §Tbid., 1907, Ix, p. 18.

|| ‘Zeitschrift f. ration. Med.,” 1869, Bd. Xxxvi—quoted by Nuttall in his -
“Blood Immunity and Relationships.” ; :

** Tur Lehre von der Bluttransfusion,” Leipzig, 1875.
tt “Transactions of the College of Physicians of Philadelphia,” 1897, p. 105.

The Cytotoxins 137

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,t and Kossel.§ Theserious considera-
tion of the subject was, however, deferred until Belfanti and Carbonell
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 cc. 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 Morgenrothtt had shown the
mechanism of the hemolytic action. From this time on the litera-
ture of hemolysis rapidly grew and the subject-assumed a more:
and more important place in the domain of chemico-physiological
research.
The technic of hemolysis 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.

For the study of hemolysis and hemo-agglutination it is necessary to prepare
a § per cent. suspension of the blood-corpuscles in an isotonic salt (NaCl) solu-
tion. 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 thoroughly
_ shaken to distribute the corpuscles. This slightly greenish-red fluid is the 5 per

cent. solution of corpuscles. -It is, of course, not permanent, and easily spoils
if bacteria enter. It also gradually deteriorates through changes in the corpus-
cles, 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 corpus-
cles 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 re-
quirement 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 increasing doses of the washed blood corpuscles of the animal
for whose corpuscles the serum is to be made hemolytic, the doses being given
intraperitoneally about 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 cc. 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.,

ig ‘Journal of Exp. Med.,” 1901-1905, VI, p. 277.
} “Archiv f. Exp. Path. and Pharmak.,” xxv, pp. r1z and 145.
t “Compt. rendu de la Soc. de Biol. de Paris,’’ 1898, p. 129.
§ “Berliner klin. Wochenschrift.,’’ 1898. fi
|| “Jour. de la R. Acad. d. Med. de Torino,” 1898, No. 8.
** “Ann de V’Inst. Pasteur,” 1898, XI, 688.
tt “Berliner klin. Wochenschrift,” 1899.

138 Immunity

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 beauti-
ful clear red color of the formerly cloudy greenish suspension. One must notice
the difference between partial and complete hemolysis, different additions of the
hemolytic substance being required for these results.

Cytolysis——-The phenomena of hemolysis correspond to those
by which many other cells, vegetable and animal, are destroyed and
dissolved through the activity of immunity product. Delezene*
first produced a leukolytic or leukocyte-destroying serum by in-
jecting animals with the leukocytes of a heterologous species;
Metalnikoff,t by injecting the spermatozoa of one animal into
another of different species, produced a spermatoxic or spermalytic
‘serum; von Diingern,{ a serum capable of dissolving the ciliated
epithelium scraped from the trachea of an ox by injecting the dis-
sociated epithelial cells into an animal. Delezene§ found that

a ee y

Fig. 22.—Latapie’s instrument for preparing tissue pulp.

by injecting an animal with the dissociated liver cells of a heter-

ologous animal, a hepatolytic serum could be produced.

The technic of these investigators is not difficult. It is, however, first neces-
sary to prepare a homogeneous tissue pulp for injection into the animal that is
to furnish immune serum. For this purpose it is necessary to grind the tissues,
when solid, in some kind of mill, one of the best froms of apparatus being that

of Latapie.|| After the pulp is made, it is diluted to a convenient extent with |

physiological salt solution and then injected into the experiment animal in the
same manner as is the blood for making the hemolytic serum. After the animal
has received a number of injections made at intervals of a few days and is thought
to be “immunized” it is bled and the serum separated. The remaining steps
in the experiment do not differ essentially from those of hemolytic experiments.
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 tissue suspension or antigen,

* “Compt. rendu de l’Acad. de Sciences de Paris,”’ 1900.

+ “Ann. de l’Inst. Pasteur,” 1899.

tf “Miinchener med. Wochenschrift,”’ 1899.

nu rendu de l’Acad. de Sciences de Paris,” 1900, Cxxx, pp. 938 and
1488.
|| ‘Ann. de l’Inst. Pasteur,”’ 1902, XVI, p. 947.

BRRARR RRR BR

Bacteriolysis 139

the immune serum or amboceptor, and the complement in normal guinea-pig
serum are brought into contact in small test-tubes, kept for twenty-four hours
in the refrigerator, and the amount of solution gauged by the naked eye supple-
mented by microscopical examination of the tissue elements.

Bacteriolysis—The first observations upon bacteriolysis were
made in 1874 by Traube and Gscheidel,* who found that freshly
drawn blood was destructive to bacteria. The matter was pur-
sued by numerous subsequent investigators and was explained by
Buchner as depending upon alexins. Pfeiffert described the
peculiar reaction known as “Pfeiffer’s phenomenon.” Ehrlich and
Morgenrotht and Bordet§ described 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 quan-
tities of both factors involved—i.e, amboceptor and complement.
For the usual bacteriolytic investigations it is, therefore, necessary
to consider three factors: 1, The bacteria to be destroyed; 2, the
serum furnishing the complement; and 3, the serum furnishing the
immune body. ,

Technic.—1,. The bacteria to be destroyed should be prepared in the form of a
homogeneous suspension in physiological salt solution, similar to that employed
for making the agglutination tests (g. v.). It is best to use the surface growths
from agar-agar, 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 sus-
pension 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. The guinea-
pig is the animal preferred. , :

3. The serum containing the amboceptor or the immune body is obtained
from an animal that has been given a high degree of immunization against the
bacterium to be destroyed or dissolved. The complement contained in this
serum should be destroyed by heating for a short time to 55°C.

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
diluted normal serum added. To this mixture of two constants varying quanti-
ties of the immune serum are added and the tube stood away for twenty-four
hours on ice. In almost every case it will be found that the immune serum con-
tains a great quantity of agglutinating 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 or by their failure to
grow when transplanted to appropriate culture media.

_ By making the bacterial suspension and complementary serum constant quan-
tities (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 complement) as constants and varying the addi-
tion of complementary serum, one can estimate the respective values of several
complementary serums. By using both serums as constant factors and varying

*“Tahresb. der schles. Ges. f. vaterl. Kultur,’ 1874.
+ “Deutsche med. Wochenschrift,” 1896, No. 7.

t “Berliner klin. Wochenschrift,’’ 1899.

§ “Ann, de l’Inst. Pasteur,” 1898, x1.

140 : Immunity

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. — :

THE DEVIATION OF THE COMPLEMENT, OR THE “NEISSER-WECHSBERG
PHENOMENON”

A peculiar phenomenon has been observed and studied by Neisser
and Wechsberg.* When an animal whose blood-serum is normally

possessed of a high degree of germicidal power is immunized by
repeated injections of a bacterial antigen, its serum when examined
by the usual methods fails to show the usual increase in the specific

* “Miinch. med. Wochenschrift,” April 30, 1901, xLv11I, No. 13, p. 697.

Therapeutic Uses of Bacteriolytic Serums 141

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 com-

plements in the serum, some of the former satisfy A B
their combining affinities by attaching themselves 4

to the bacteria, some by attaching themselves to c
the complement, instead of forming combinations ac
of all three. If under these circumstances the

serum containing the amboceptors is diluted until am

their number becomes approximately equal to
the number of complements introduced, any
’ deviation resulting from inequality of the com-
bining affinities becomes improbable. Bordet

and Gay,* however, have performed experi- —

ments tending to show that these elements do
not really unite, this seeming to controvert the
theory of Neisser and Wechsberg, and Boltont
has shown that normal serum may kill rela-
tively more bacteria when diluted than when
undiluted. .

THERAPEUTIC USES OF BACTERIOLYTIC SERUMS

It was at first hoped that some of these
serums and especially the bacteriolytic serums
would have a wide therapeutic application in
cases in which non-toxicogenic bacteria were in-
vading the body, but experiment and ex-

Fig. 24.—Schemat-
ic representation of
the interfering ac-
tion of anti-ambo-
ceptors, and anti-
complements. A,
Anti-amboceptor
action: c, Comple-
ment; am, ambo-
ceptor; aa, anti-am-
boceptor preventing
theamboceptor from
connecting with the -
cell. B: c, Com- |
plement; ac, anti-
complement pre-
venting the comple-
ment from connect-
ing with the ambo-

A “ ceptor, am.
perience have shown that the laws governing ae

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.

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 com-
plement. To give injections of active bodies that cannot be utilized
is shown by Comus and Gleyt and Kossel§ to be followed’ by the
formation of antibodies—in this case “anti-immune bodies”—
by which their effect is neutralized. Should anti-immune bodies be

* “Ann, de l’Inst. Pasteur,’’ June 25, 1906, xx, No. 6, pp. 267-408.

+ “The Bacteriolytic Power of the Blood-serum of Hogs,” Bull. No. 95 of the
Bureau of Animal Industry, U. S. Dept. of Agriculture.

{“Compte rendu de Acad. de Sciences de Paris,” Jan. 1, 1898, 126.
§ “Berl. klin. Woch.,”’ 1898, S. 152.

I42 Immunity

formed by this meddlesome medication, the state of the infected:
animal would be worse than before, because it would now be pre-
paring 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 activating the complements.

No satisfactory method of experimentally increasing the comple-
ment has been devised. If, as Metchnikoff supposes, the comple-
ment is microcytase derived from disintegrated leukocytes, aseptic
suppurations with active phagolysis 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 pro-
portions that, regardless of other elements in the blood, they can
exercise their combining and dissolving functions.

We are unable experimentally to accomplish these prerequisites,
therefore are not in the position to accurately apply bacteriolytic _
serums in practice. :

COMPLEMENT FIXATION

In 1901 Bordet and Gengou* while investigating 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.” The method of
procedure was as follows: Blood-corpuscles were sensitized with
appropriate amboceptors and then treated with freshly drawn nor-
mal 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.

They next found that if, instead of employing blood-corpuscles for
the first test, they used sensitized bacteria—.e., bacteria treated with
an immune serum containing the amboceptors appropriate for effect-
ing their solution—the complement would similarly be used up,
“fixed,” so that when they subsequently added sensitized red blood-
corpuscles there was no hemolysis.

This reaction was naturally quantitative, the result as described
depending upon the fact that no more complement (normal serum)
was used in the original hemolysis or bacteriolysis than was necessary
and so none left “unfixed” to effect the lysis or solution of the second ,
factor introduced.

They interpreted the results as indicating that there was only
one complementary or solvent substance, and though Ehrlich sub-
sequently published what he looked upon as proofs to the contrary,
the opinion of Bordet and Gengou prevails.

* Ann. de l’Inst. Pasteur, 1901, Xv, 290.

Defensive Ferments 143

In addition, however, the experiments have been of practical
use. As affording a means of quantitative experimentation they
have enabled investigators to measure the quantity of complement
in normal bloods and in immunized bloods, and so led to the discov-
ery 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 substances. Thus,
when a certain antibody and its complement 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 Jed 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 meesetenaints reac-
tion for the diagnosis of syphillis (g.v.). By careful perusal of the
chapter upon the method of performing the Wassermann reaction
the student will learn the general details of the technic of comple-
ment fixation, and can modify them to correspond to the requirements
of other cases in which complement fixation is to be studied.

DEFENSIVE FERMENTS

Defensive ferments are enzymic substances that make their
appearance in the body juices in a short time after any unusual
protein substance is intentionally or accidentally thrown into the
blood. They were discovered by Abderhalden* who found that
when substances capable of digestive transformation in the animal
economy, by any means obtain access to the blood, ferments capable
of effecting such transformations also quickly appear in the blood’
in increased quantity, effect the transformation and then quickly
disappear. The appearance and disappearance of the enzymes is
supposed to depend upon “mobilization” of defensive ferments, of
which the body presumably has reserve supplies. ‘The most common
source of supply is supposed to be the leukocytes.

The Abderhalden Reaction——The subject was first investigated
with reference to the presence of a proteolytic ferment in the blood
of pregnant woman, whose office was the defense of the mother
against the syncytial and chorionic cells of the offspring which with
their products may occasionally get into the circulation.

If such a ferment were present in the blood, it ought to be demon-
strably capable of effecting transformations in the sub-stratum by
whose presence it has been called forth. To determine it, therefore,
it should only be necessary to apply the blood serum to the sub-
stratum for a brief time, and then determine by sufficiently delicate

* “Schtitzfermente des tierische Organismus,”’ Berlin, 1912; Berlin, 1913.

144 Immunity

tests that some transformation has been effected. For the latter
Abderhalden has made use of two separate tests:

The first of these is rarely employed, the second is now regularly
employed.

I. The Optical Test—This depends upon the fact that in the
transformation of protein substances, aminoacids may be formed,
some of which are optically active. The contact of the enzymic

serum and the appropriate sub-stratum is permitted to take place,

then after the appropriate length of time, the polariscope is employed
to determine whether rotation differences obtain because of the
presence of transformation products.

II. The Dialysis Test.—This test not requiring apparatus or skill
of unusual or special kind, has met with greater favor. Its first
employment was for the demonstration of the presence, in the blood,
of an enzyme that would transform placental tissue. As no such
enzyme appeared in the blood except placental tissue was in the
body, it became a test for the determination of the existence of preg-
nancy. The method required but little in the way of special appar-
atus or reagents. The chief requirements being small “ dialyzing
shells” or thimbles, which are made by Schleichter and Schull, and
are commercially known as No. 579a. They are procurable through
importing agents dealing in laboratory apparatus. These shells
must be tested before using, and it is best to test a large number at
the same time. Each must be impervious to albumen, but readily
permeable to peptones, aminoacids and other cleavage products of
protein digestion. .

The shells or “thimbles” are tested thus by Kolmer:*

They are first soaked in sterile distilled water for half an hour or more, until
they are softened. Each then receives about 2.5 cc. of a 5 per cent. solution of
egg-albumen in distilled water, thoroughly mixed and freed from flakes or shreds.
In filling the shell, care should be exercised that none of the albumen solution by
any chance falls upon the outside. The shell is then picked up with forceps and
transferred to a short tube containing about 20 cc. of sterile distilled water.
This tube should be so wide that the column of water is not so deep as the shell is
high, and not so broad that the shell is in danger of oversetting. . As bacteria
may not have been successfully excluded and by multiplying may cause proteo-
lytic cleavage of the albumen, it is well to cover the fluid in the thimble and that
in the tube outside of it, with a thin layer of toluol. The outer tube is plugged

or corked, and the whole is stood in the incubating oven where it is kept at 37°C.

for sixteen to eighteen hours. At the end of this time, ro cc. of the water in the
outer tube is removed by a pipette, and tested by the biuret reaction to determine
whether any albumen has penetrated the thimble. For this purpose the fluid,
in a test-tube, receives 2.5 cc. of a 33 per cent. solution of sodium hydroxid andis
shaken gently. One cubic centimeter of a 0.2 per cent. cupric sulphate solution
is permitted to trickle down the side of the tube and overlie the contents. Ifa
delicate voliet is produced at the line of junction of the two liquids, albumen has
escaped from the thimble into the water outside. Under such circumstances the
thimble is, of course, useless and should be thrown away. If there is any uncer-
tainty about the reaction, the tube can be stood away for eight hours or so longer
(twenty-four hours in all) and the remaining water subjected to the ninhydrin
test (see below).

* “Infection, Immunity and Specific Therapy.’’ Phila., 1915; p. 253.

‘Defensive Ferments 145

The good shells or thimbles are next to be tested for permeability to peptones.
Before this they should be carefully washed in running water and boiled for
thirty seconds. ; on

At per cent. solution of Héchst “‘silk peptone” is made in distilled water, and
of it 2.5 cc. is pipetted into each thimble to be tested, taking care, as before, that
none of the solution by accident drops on the outside of the shell. The shell is
now placed in the 20 cc. of sterile distilled water in the wide tube such as was
used before, covered with toluol and stood in the incubator at 37°C. After
twenty-four hours, a pipette is thrust through the toluol and 10 cc. of the water
taken up. The finger being held over the top of the pipette, the tube is wiped
outside with care, so as to get off any toluol, and the fluid then delivered into a
test-tube. Here it receives 0.2 cc. of a 1 per cent. solution of ninhydrin, and-is
boiled for exactly one minute. If the peptone has dialyzed, a deep blue color
develops after standing for a short time. Good thimbles should be equally per-
meable to peptone. The thimble that permits no transfusion of peptone is
worthless and should be thrown away.

The good thimbles are now again thoroughly washed in running water for a
minute, or so, and are then transferred to a vessel of stecile distilled water con-
taining chloroform to saturation and covered with toluol. j

In making the Abderhalden test it is imperative that the glass-
ware used should be chemically clean, that the reagents be pure,
that the preparations be kept sterile and that the thimbles and sub-
strata should be handled with forceps, not with the fingers.

To make the test for pregnancy known as the “‘ Abderhalden reac-
tion,” the foundation of all the other tests of the protective or defen-
sive ferments, it is necessary to prepare a substratum upon which
the enzyme in the blood may act.

To do this one obtains a healthy placenta, removes the blood clots, cord and
membranes, and washes it in running water. When it is clean on the outside,
it is cut into small pieces—z cm. cubes—which are placed upon a towel or on a
wire sieve and washed in running water. The purpose of the washing is to re-
move every trace of blood serum and of blood pigment. From time to time the
bits of tissue are moved about and squeezed by the fingers, and occasionally they
are crushed together in a towel. The process is completed when the tissue has
become perfectly white in color. It now receives 100 times its weight of distilled
water (I gram-z cc.), to which are added five drops of glacial acetic acid per
1000 cc., and is boiled for ten minutes. The fluid is then thrown away, the tissue
fragments are caught in a sieve or cloth, more distilled water added, this time
without the acetic acid, and it is boiled again. This is repeated for six times.
After the sixth boiling, some of the water is transferred to a tube and tested for
proteins with ninhydrin. If the faintest blue color develops upon boiling, the
process of washing the tissue by boiling it with clean water, must be repeated
again and again until the ninhydrin produces no discoloration after boiling for a
minute, and standing for one-half hour. The tissue is then caught on acloth,
finally looked over for any objectionable components, and transferred to a jar of
sterile distilled water saturated with chloroform and covered with toluol.

The blood of the patient is obtained with a Keidel tube or with
a sterile syringe from which latter it is at once transferred to a sterile
test-tube. When the blood has firmly coagulated, the expressed
serum is removed by a sterile pipette to a sterile centrifuge tube and
any cells it may still contain are thrown out by centrifugation.

The technic of the test is more simple than the preparation and
preliminary tests it entailed. The glassware being chemically clean
and sterile, the thimbles all tested and sterile, and the substratum
(placental tissue) ready, one proceeds as follows:

146 Immunity

A fragment of the placental tissue is removed from the container with sterile
forceps and blotted with sterile filter or blotting paper to absorb the toluol and
chloroform, It is then placed upon a sterile filter paper and weighed; about
o.5 gram should be placed in each of two thimbles. 1.5 cc. of the serum to be
tested is cautiously pipetted into one thimble; 1.5 cc. of sterile distilled water
into the other. Each is then transferred with forceps to a large tube containing
20 cc. of sterile distilled water, and the surface of each fluid is covered with
toluol. The tubes are now stood in the thermostat at 37°C. for twenty-four
hours, at the end of which time a sample of the fluid in each outer tube is tested
by boiling for one minute with ninhydrin (0.2 cc. of a 1 per cent. solution, to
ro cc. of the fluid). The reaction is not read for thirty minutes after boiling. If
the conditions are all favorable, i.e., the serum used be from a pregnant woman,
the tissue used as substratum be placenta, the enzyme in the serum acts upon
the substratum and transforms its albumins to peptones and amino-acids; if the
transfusion is perfect in both thimbles, and neither thimble leaks (this has, of
course, been previously tested and security can be counted upon now) the fluid
surrounding the thimble containing the serum should give a bright blue color or
positive reaction, and that surrounding the thimble containing the water no
color or a negative reaction.

By the test we are then able to determine, the substratum being
known, whether the serum contains an enzyme capable of acting
upon or transforming it; or the enzymic character of the serum being
known, it may be possible to tell something about the substratum.
The general consensus of opinion is in favor of this reaction as being
a useful adjunct in making the diagnosis of pregnancy. But its — .
applicability may not be limited to the diagnosis of pregnancy for
Freund and Abderhalden,* Frank and Heimant{ and many others
have used it as an adjunct in the diagnosis of cancer, and various
other investigators have shown that modifications of the method
makes it applicable for purposes of diagnosis or investigation of other
conditions in which defensive enzymes may be present in the blood.
For each of these investigations the specific substratum must be pre-
pared, and in making each test, the application of the enzyme-con-
taining serum to the sterile and appropriate substratum must be
made in the tested thimbles with the precautions given above.

The method is not exclusively adapted for investigation of proteo-
lytic enzymes in the serum, but to diastatic and lipolytic ferments
as well and Abderhalden has shown that it has uses in these fields.
How much importance attaches to the enzymes thus mobilized in
the blood in the conditions comprehended in the studies of immunity
‘is as yet uncertain. That there is some bearing of the one upon the
other cannot be. doubted. The Abderhalden reactions seem to be
less specific than the immunity reactions and appear more as reac-
tions en gros, while the immunity reactions previously studied
were reactions en detail, but it may well be that this apparent differ-
ence depends upon the newness of the former reactions and the
crudity of the methods employed’ as contrasted with the more
elaborate study of the latter and the more delicate methods used. : : :

* Miinch. med Wochenschrift, 1913, XIV, 763.
t Berl. klin. Wochenschrift, 1913, L, No. 14.

CHAPTER V
METHODS OF OBSERVING MICRO-ORGANISMS

- Iris of the utmost importance to examine micro-organisms alive,
aid 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, reproduction, and natural
history. It has the disadvantage of being somewhat difficult be-
cause of its small size and transparency.

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 the 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 disturbed must be the structure
of an organism after it has been dried, fixed, boiled, or steamed,
passed through several chemic reagents, dehydrated and impreg-
nated with stains, etc., to suggest how totally unnatural its appear-
ance 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.

I. 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.

While this method is ile: it cannot be Sees uneuaeny as evapo-
ration 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 ex-
amination 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.

147

148 Methods of Observing Micro-organisms

The best way to examine living micro-organisms is in what is
called the hanging drop. 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. A drop of the material to be
examined is placed in the center of a large clean cover-glass and
then placed upon the slide so that the drop hangs in the concavity,
but does not touch the glass. The micro-organisms are thus her-
metically 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 ap-
paratus in which the microscope is placed, the growing bacteria
may be watched at any temperature, and exact observations 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,

Fig. 25—The ‘“‘hanging drop”’ seen from above and in profile.

grouping, division, sporulation, and motility of the organism under
observation.

Care should be exercised to use a rather small drop, especially for
the detection of motility, as a large one vibrates 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 thickness 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

* Journal of Medical Research,” March, 1¢02, vol. vir, No. 2; new series,
vol. 1,

Staining Bacteria 149

if making an ordinary cover-slip preparation. Place the slide and block in a
37°C. incubator 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, carefully
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 chamber 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 ac-
quired interesting knowledge of the fission and budding of Bacillus
diphtheriz.

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. .

II. STAINING BACTERIA

In the early days of bacteriology efforts were made to facilitate
the observation of bacteria by the use of nuclear dyes. Both carmin
and hematoxylin tinge the nuclei of the bacteria a little, but so un-
satisfactorily that since Weigert introduced the anilin dyes for the
purpose, all other stains have been abandoned. The affinity be-
tween the bacteria and the anilin dyes is peculiar, and in certain
cases can be used for the differentiation of species.

Readers interested in the biochemistry of the subject will do well
to refer to the excellent papers by Arnold Grimme,* upon ‘The
Important Methods of Staining Bacteria, etc.,” and Marx,f upon
“The Metachromatic and Babes-Ernst Granules.’

In this work special methods for staining such bacteria as have
peculiar reactions will be given together with the description of the
particular organisms, general methods only being discussed in this
chapter.

Preparations for General Examination.—For bacteriologic pur-
poses thin covers (No. 1) are required, because thicker glasses may
interfere with the focussing of the oil-immersion lenses. Where
cover-glasses are not employed, and the staining is done upon the
slide, only the best quality of thin glass slides free from bubbles
should be used. The cover-glasses must be perfectly clean. It is
therefore best to clean a large quantity in advance of use by immers-
ing 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 edges, a convenient method of preparing cover-glasses is to
wipe them as clean as possible with a soft cotton cloth, seize them
with fine-pointed forceps, and pass them repeatedly through a small

*“Centralbl. f. Bakt.,”’ etc., 1902, Bd. xxx, Nos. 2, 3, 4, and 5.
Tbid., 1902, xxxu, Nos. to and 11, p. 108.

150 Methods of Observing Micro-organisms

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 em-

scopic objects.

The fragility of the
covers and their likeli-
hood to be broken or
dropped at the critical mo-

prefer to stain directly upon
the slide. The slide should
be thoroughly cleaned, and
if the material to be ex-
amined 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 Stain-
ing—The material to be
: examined must be spread
Pa bee gue for keeping objects jn the thinnest possible
Poe ean at constant 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 accomplished. 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 révolution
occupying a second 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.

ment, make most workers

ployed for ordinary micro-..

Simple Method of Staining Ba

Inequality in the size of various flames may make it desirable
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 labora-
tory should be provided with “stock solutions,” which are saturated
solutions of the ordinary dyes. For preparing them Woodf gives
the following parts per roo as being sufficiently accurate:

Alcoholic solutions (96 per cent. alcohol) Aqueous solutions (distilled water)
Fuchsin................. 3.0 grams.
Gentian violet........... 48 Gentian violet........... I.5 grams,
Methylene-blue.......... mo. Methylene-blue.......... 6.7 “
(70 per cent. alcohol)
Scharlach R............. 3:2 © ;
Soudan JIT.............. 0.2 *

(50 per cent. alcohol)
(T3 oes

Thionin............ Leal OO SERIO HIM 0.5 cos 40 aGoveaue disies 1.2

Of these it is well to have fuchsin, gentian violet, and methylene-
blue always made up. The stock solutions will not stain, but form
the basis of the staining solutions. For ordinary staining an ague-
ous 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 objects through it. For exact work it is probably best to give
these stains a standard composition, using 5 cc. of the saturated
alcoholic solution to 95 cc. of water. Such a watery solution pos-
sesses 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
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 at-
traction 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
shown in the cut. This altogether prevents capillary attraction
between the blades. When the material is spread upon the slide
no forceps are needed, and the method correspondingly 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

* “Laboratory Work in Bacteriology,” 1890.

et: Fj chamical and Microscopical Diagnosis,’ N. Y., 1905, D. Appleton & Co.,
Pp: 003.

152 Methods of Observing Micro-organisms

upon a slide with a drop of water between, and then examined,
though this is less satisfactory than examination after drying and
mounting it in Canada balsam.

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 anda 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 mixture contains no bacteria to cause confusion with
those in the material to be studied.

The entire process is, in brief: (1) 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.

Fig. 27.—Stewart’s cover-glass forceps.

To Observe Bacteria in Sections of Tissue.—Hardening.—It
not infrequently happens that the bacteria to be examined are scat-
tered among or inclosed in the cells of tissues. ‘The demonstration
then becomes a matter 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 go per cent. alcohol, which does not shrink the tissue. Solutions
of bichlorid of mercury* may also be used and are particularly useful
when the bacteria are to be studied in relation to the cells of the
tissues. ,

Tissues preserved in 95 per cent. alcohol, Miiller’s fluid, 4 per

* Zenker’s fluid:

Bichromate of potassium..................... 2.5 grams
Sulphate of sodium........................5. TO ©
Bichlorid of mercury..............0.00020005 5.0 “
Water....... Ioo.o “

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.

Embedding 153

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:

I. Celloidin (Schering).—The solutions of celloidin are made in
equal parts of absolute alcohol and ether and should have the thick-
ness 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 wood, allow the ether
to evaporate until the block can be overturned without dislodging
the specimen; then place in 80 per cent. alcohol 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 accomplished 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.

II. Parafin—Pure paraffin having a melting-point of about
52°C. is used. The hardened blocks of tissue are passed through:

Ninety-five per cent. alcohol, twelve to twenty-four hours;

Absolute alcohel, six to twelve hours;

Chloroform, benzole, or xylol, four hours;

A ce solution of paraffin in one of the above reagents, four to eight

The block is then placed in melted paraffin in an oven or paraffin
water-bath, at 50°—55°C., until the volatile reagent is all evaporated,
and the tissue impregnated with paraffin (four to twelve hours),
and is finally embedded in freshly melted paraffin, in any convenient
mold, and rapidly solidified in ice water. 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-albumin (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
Ae 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
accomplished by dropping the reagents upon the slide.

154 Methods of Observing Micro-organisms

ITI. Glycerin-gelatin—As the penetration of the tissue by cel-
loidin is attended with deterioration in the staining qualities of
the tubercle bacillus, it has been recommended by Kolle* that the
tissue be saturated with a mixture of glycerin, 1 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 ee work the following
simple method can be recommended: After the sections are cut
and cemented to the slide, the paraffin and celloidin should be re-
moved 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 1 per cent
acetic acid solution. The acid re-
moves 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 toabsolutealcohol. At
this point the process may be in-

SHOW r .
wosiron. taents to be countercolored with

Fig. 28—Coplin’s staining jar. alum-carmin or any stain not re-

quiring 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 exposure to the reagents is to be prolonged, the Coplin jar
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 (q.v.), then transferred to absolute
alcohol made feebly acid with acetic acid. The sections must
be carefully watched, and so soon 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.

Léffler’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 Léffler’s alkaline methylene-blue:

Saturated alcoholic solution of methylene-blue.......... 30
I : 10,000 aqueous solution of caustic potash......... 1. roc

* Fliigge’s “Die Mikroorganismen,”’ vol. 1, page 534.

caoss-secroy tetrupted to allow the tissue ele-

prays

Staining 155

The cut sections of tissue are stained for a few minutes and then
differentiated in.a 1 per cent. solution of hydrochloric acid for a
few seconds, after which they are dehydrated in alcohol, cleared in
xylol, and mounted in balsam.

Some bacteria, such as the typhoid fever bacillus, decolorize
readily so that the use of acid should be avoided, 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 Ehrlich,
is known as Ehrlich’s solution. The ordinary method of preparing
it is to mix the following:

Pure anilin.. seeeene A
Saturated alcoholic solution of gentian violet........... a1
Water.. de git ibedle Pn Song Bed dle tes ands Aan ce us thE cn thc STOO)

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 1 cc. 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 appearance.

Friedlander recommends that the section remain from fifteen to
thirty minutes in warm stain, and in many cases the prolonged proc-
ess 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):

TOMER YS tals: <i adnan aca ad HA mNAe wameeeniain deus dno I
Potassium iodid. 2.0.0... ccc cece eee ee een eens 2
Wateliiciscie vay wanes duping auewnnben shuiiian stent eaeniten: OO

The specimen while in the Gram solution turns a dark blackish-
brown color, but when removed and carefully washed in 9s 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

{

156 Methods of Observing Micro-organisms

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 bac-
terial substance, anilin dye, and the todids 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.

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
(r) by making the staining solution with 1 : 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.

The Gram-Weigert Stain can be employed with beautiful results
for staining many micro-organisms in tissue. It differs from the
Gram method in that anilin oil instead of alcohol is used for decolor-
izing. To secure the most brilliant results it is best first to stain
the tissue with alum, borax, or lithium carmin, and then—

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 (iodine 1, iodide 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,

Gram’s Method as an Aid in the Identification of Species.—
Gram’s method does not stain all bacteria, hence can be used to
aid in the differentiation of species. The following lists show the
reaction of the well-known species to the stain.

Staining 157

Gram-negative : Gram-positive
Bacillus anthracis symptomatici; Bacillus aérogenes capsulatus;
Bacillus coli (whole group); Bacillus anthracis;
Bacilltis ducreyi; Bacillus botulinus;
Bacillus dysenterie; Bacillus diphtheriz;
Bacillus icteroides; Bacillus subtilis (whole group);
Bacillus influenzze; Bacillus tetani;
Bacillus mallei; Bacillus tuberculosis (whole acid-fast
Bacillus oedematis maligni; (group);
Bacillus pestis bubonica; Diplococcus pneumoniz;
Bacillus pneumoniz (Friedlander); Micrococcus tetragenus;
Gram-negative Gram-positive
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;
Micrococcus catarrhalis;
Micrococcus flavus;

Micrococcus crassus;
Micrococcus pharyngis siccus;
Micrococcus gonorrhoee (Neisser)
Micrococcus melitensis;
Spirillum cholere asiatice
Spirillum cholerz gallinarum;
Spirillum cholerz nostras;
Spirillum metschnikovi;
Spirillum tyrogenum;

Spirochzte duttoni;

Spirochete obermeieri;
Spirochzte refringens;
Spirochzte icterohemorrhagie;
Spirochzte morsus muris;
Treponema pallidum;
Treponema pertenue.

To apply the test to micro-organisms in culture or in morbid
fluids, the following simple method may be employed: 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.

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 pyronin, 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;

158 Methods of Observing Micro-organisms

Counterstain if desired; wash off the counterstain math water;
Dry;
Mount in Canada balsam.

Nicolle* suggests the following modification of the Gram
technic:

(a) For Cover-glass Specimens:

1. Stain for one to five minutes in a warm solution made as follows: 10 cc.
of saturated alcoholic solution of gentian violet, 100 cc. of a I per cent,
aqueous solution of carbolic acid.

2. Immerse from four to six seconds in the iodine-iodide of potassium solu-

tion,

3. Decolorize in a mixture of 3 parts of absolute alcohol and I part of acetone.

4. Counterstain if desired.

(b) For Sections:

1. Stain the nuclear elements of the tissue with carmine. For this Nicolle
prefers Orth’s carmine solution (§ parts of Orth’s carmine with 1 part
of 95 per cent. alcohol).

. Stain in the carbol-gentian violet, as indicated above.

. Immerse for four te six seconds in the iodine-iodide of potassium solution.

. Differentiate with absolute alcohol containing 0.33 per cent. (by volume)
of acetone.

. Treat with 95 per cent. alcohol containing some picric acid until the
tissue is greenish yellow (one to five seconds).

. Dehydrate with absolute alcohol.

. Clear with xylol or other abprepeate reagent.

. Mount in balsam.

Cont DH nm WN

Eosin and Methylene-blue (Mallory) make a beautiful contrast _

tissue stain for routine work, and also demonstrate 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

from 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 1, car-
bonate of potassium 1, water roo) diluted 1 : 10 with water, from one- »
halt to one hour, or use a stronger solution and stain for a few minutes
only;

. Wash in water;

. Differentiate and dehydrate in 95 per cent. alcohol, followed by. absolute
alcohol until the pink color returns in the section;

. Clear with xylol;.

. Mount in xylol balsam.

NIA NP

The nuclei and micro-organisms will be colored blue, the cyto-
plasm, etc., red.

Zieler} recommends for the staining of the typhoid, glanders and
other difficultly stainable bacteria, the following method of demon-
stration in the tissues:

*“ Ann, de l’Inst. Pasteur,” 1895, i
t “Centralbl. f. allg. Path. u. path, - Anat.” Bd. xiv, No. 14, p. 561.

Staining a 59

x, Fix and harden in Miiller-formol solution.
Paraffin imbedding.

Orcein D.. iwapigsapecese “Ost
2. Staining overnight in ; Officinal sulphuric ‘acid.. sieahiap eens bu 2.

70 per cent. alcohol.. biniecis sva9g 4 OO:
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.

g. Seventy per cent. alcohol.

10. Absolute alcohol.

11, Xylol.

12, Balsam.

Glanders bacilli appear dark violet on a colorless background;
typhoid bacilli intense dark red violet.

Method of Staining Spores.—It has already been pointed out that
the peculiar quality of the spore capsules protects them to a certain
extent from the influence of stains and disinfectants. On this ac-
count 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 solution and
gently agitated for exactly one minute, removed, washed in water,
and counterstained with an aqueous:solution of methyl or methylene-
blue.

In such a specimen the spores should appear red, and the adult
organisms blue.

A good simple method is to place the prepared cover-glass in a
test-tube half full of carbol-fuchsin:

Fuchsin I
PI COMON sy -onscacsrete ieaivcncton stan sav ac td scythe ianiatsn oh afecali lenis ay Io
Five per cent. aqueous solution of phenol crystals....... 100

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 prepared and
stained as for tubercle bacilli (g.v.), decolorized with a 1 per cent.
sulphuric acid solution in water or methyl alcohol, then washed in °

* “Manual of Bacteriology,” London, 1897.

160 Methods of Observing Micro-organisms

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:

. Stain deeply with methylene-blue, heating repeatedly until the stain
reaches the boiling point—one minute.

. Wash in water. m
. Wash in gs per cent. alcohol containing 0.2 to 0.3 per cent. of hydrochloric

acid.

. Wash in water.

. Stain for eight to ten seconds in anilin-fuchsin solution.
. Wash in water.

Dry.

. Mount in balsam.

The spores are blue; the bacteria, red.

COonr Dun Wn

Moller* finds it advantageous to prepare the films, before staining,
by immersion in chloroform for two minutes, following this by
immersion in’s per cent. chromic acid solution for one-half to two

minutes.
The exact technic is as follows:

. Treat the spread with chloroform for two minutes.

. Wash with water.

. Treat with 5 per cent. solution of chromic acid for one-half to two minutes.
. Wash in water.

. Stain with carbol-fuchsin, slowly heating until the fluid boils.

. Decolorize in 5 per cent. aqueous sulphuric acid.

. Wash well with water. :

8: Stain in a 1:100 aqueous solution of methylene-blue for thirty seconds.
The spores should be red and the bacilli blue. :

NAN BW NH

Anjeszkyt 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
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. sul-
phuric acid solution, and counterstained for a minute or two with malachite
green or methylene-blue. The whole procedure should not take longer than
eight or ten minutes.

Fioccat suggests the following rapid method:

“About 20 cc. of a 10 per cent. aqueous solution of ammonium are poured!
into a watch-glass, and 1o 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 counterstained with an

* “Centralbl. f. Bakt. u. Parasitenk.,”’ Bd. x, p. 273.
{ Ibid., Feb. 27, 1898, xx1m, No. 8, p. 329.
¢“Centralbl. f. Bakt. u. Parasitenk.,’’ July 1, 1893, xiv, No. 1.

Staining 161

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 more difficult than the
staining of the bacteria or the spores.

Léffler’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 acid........... Io
Cold saturated aqueous solution of ferrous sulphate........ 5
Alcoholic solution of fuchsin or methyl violet............... 2

(B) One per cent. aqueous solution of caustic soda.
(C) An aqueous solution of sulphuric acid of such strength that 1 cc. will
exactly neutralize an equal quantity of solution B.

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 plac-
ing 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 Léffler 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—
Léffler recommends an anilin-water fuchsin (Ehrlich’s solution)—which should
have a neutral reaction, is next dropped on so as to cover the film, and heated
for a minute until vapor begins to rise, after which it is washed off carefully,
dried, and mounted in Canada balsam. To obtain the neutral reaction of the
stain, enough of the 1 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 1 drop to 1 cc. of solution C in 16 cc. 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 flagella.
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. Léffler fortunately worked out
the amounts required for some species, and of the more important ones the fol-
lowing solutions of B and C must be added to 16 cc. of solution A to attain the
desired effect:

Cholera spirillum.................. 14-1 drop of solution C

_ Typhoid fever..................... 1c. 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 using a very young
culture and having the bacteria thinly spread upon the glass, so
as to beas free from albuminous and gelatinous materials as possible.

* Ibid., 1890, Bd. vu, p. 625.
It

162 Méthods of Observing Micro-organisms

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 applica-
tion 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.................-. To cc.
Saturated alcoholic solution of gentian violet........... 1 “

(B)- —

APAMWICHACIC cine’: Graton 8a iy haerg: Soele war's Mie oo SIRE Row NSS I gram

Distilled: watercous isesee ss aoe 64 42 Oe 2 be sedis ts s'e 10 cc,

The solution should be made with cold water, and immediately
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 ate and
mounted. ;

Smith's Modification of Pitfield’s Method.t{—A boiling saturated solution of
bichlorid of mercury is poured into a bottle in which crystals of alam have
been placed in quantity more than sufficient to saturate the fluid. The
bottle is shaken and allowed to cool; 10 cc. of this solution are added to
the same volume of freshly prepared ‘tannic acid solution and 5 cc. of car-
bol fuchsin added. - Mix and filter. The filtrate, which is the mordant, is
caught 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.. ste HCC,
Saturated solution of ammonium alum............... To. é
Filter the stain 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 Ermengemt has devised a some-
what complicated method of staining flagella, which has given great
satisfaction. Three solutions, which he describes as the bain fixa-
teur, bain sensibilisateur, and bain reducteur et reinforcateur, are’ to
be used as follows:

1. Bain fixateur:

2 per cent. solution of osmic ACId se hard e ethene xn I part |
10-25 per cent. solution of tannin... seeeeeeeeees 2 Parts

The cover-glasses, which are very etal 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

* “Medical News,” Sept. 7, 1895. :

+ “Brit. Med. Jour.,” .»” IQOT, I, P. 205.

t “Travaux du Lab. d’ hygiene et des bact. de Gand.,” t. I, p. 3. Abstracted
in the “Centralbl. f. Bakt. u. Parasitenk.,” 1894, Bd. xv, p. 969.

Staining 163
next washed with distilled water, then with absolute alcohol, then
again with distilled water. All three washings must be very thorough.

2. Bain 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 reinfor¢ateur:

GaAllIG ACI ns 5. 0.s.aaives Sa Guebion h paaaane Oo MeN A date 5 grams
Tannin: fos agate sn aint hams 25 Weed yee ee ‘ a
Fused potassium acetate......................5 To “
. Distilled watefewn:;ivednns cou cea czades wense~ an B5O Cs

. 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 or two 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, of 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 attempted. 5

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 em-
ployed 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. :

Af favorable, a loopful of this dilution is added to 1 cc. 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 stirring, 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 1000 cc. 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)........0....20.55 2.5 grams
Absolute alcohol. .......0000. 000.00 e cece ee eeee 100.0 CC.
(C) Potassium hydrate....... 0.0.0.0 cece cece cee ee ee ees 1.0 gram

Distilled water........0 0.00.0 .c cece ee eeeeeeeeeeeeees 100.0 grams

*“Centralbl. f. Bakt. u. Parasitenk.,” Orig., 1903, XXXII, Pp. 572. .

164 ' Methods of Observing Micro-organisms

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
1§ to 20 cc. of the A B mixture into a glass-stoppered 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 precipitate has formed. When this has
occurred, a little experience will show when the proper moment arrives to throw
off the fluid and wash the 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 methodt
as being a simple and excellent method of staining flagella: The
material and cover-glasses are prepared with care as for the fore-
going methods, after which one proceeds as follows:

x. 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

A Tannie acids. ci2 va.anca-22cabe Soe ehd. Cen nne Ciaran I gram
Potassium: alutisc ye ce neuiy aedeu'hin tiga sane Ren wees <k Se Ere
Distilled watery ii doe-ac ss dd downs eyerd es mewenes cee ss 4o cc,

. Dissolve by. shaking or allow to stand overnight in the incubator.

Ts" Nieht: blue? a. ce cis wea cea Man Wha deka ws we deataates ©.5 gram

95 per cent. or absolute alcohol...................... 20.0 CC,

Mix Tand II thoroughly and remove the heavy precipitate by filtration.
Tf 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 min-
utes 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,
ona 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

* “Your. Med. Research,” 1901, VI, p. 341. :

t “Bacteria,”’ John Murray, London, 2d edition.
t James Strong & Son, Glasgow and Manchester.

‘Staining Protozoa 165

order that the formed elements may be separated sufficiently for
the individual 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 comparatively 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 formation 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 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 not widely separated.

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 illuminator.” The latter
serves better for the discovery of the very small transparent organ-
isms—Spirocheta 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—Spirocheta, Plasmodium,
etc.

As pointed out above, the protozoa, under such circumstances,
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.

1. 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 coin-

- cide. As soon as the drop is fairly well distributed the glasses are
gently slid apart.

2. 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.

166 _ Methods of Observing Micro-organisms

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,

<— »

‘Fig. 29.—Method of making dry film with two cover-glasses (from Daniels’
“Laboratory Studies in Tropical Medicine’’).

Jenner’s, Leishman’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.

Fig. 30.—Method of making dry films with two slides (from Daniels’ “Labora-’
. tory Studies in Tropical Medicine’’).

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 Roman-
owsky 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 1 gm. of the dye
to 100 cc. 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

* Mallory and Wright, “Pathological Technique,” rgrr, p. 364.

Staining Protozoa 167

thoroughly heated. The mixture is to be contained in a flask of such size
and shape that it forms a layer not more than 6cm.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 roo cc. of the filtered mixture add 500
cc. of a 0.1 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 0.1 gr. to 60 cc. 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 spontaneous 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 quantily of distilled
water by means of the medicine dropper, and allow it to remain for two
or three minutes, according to the intensity of the staining desired.
A longer period of staining may produce 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
pees parasites are present. It is an azur-eosin combination, prepared as
ollows: F

Solution I: :

Methylene-blue (medicinal)...........0....0..--.0.++. 0.5 gram

VAZUTE TDi = haar vg wetted Wisc guise to sate vase dona eperwen Sie aceien Ute as ous “*
: Water (distilled) ciisse sana ac ste bee dans toh @ bate an dohuaneeans 100.0 CC.
Solution II: ~~

Sodium carbonate....../.... 00000: cee eee ee eee eee ees = 0.5 gram

Water sc cag 34 ovine aiecn ues beege become Sees sma fsa s WOOHONCE:

Pour the two solutions together and stand the mixture in the thermostat
for forty-eight hours at 37°C.; then add o.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 maxi-
mum precipitation. The exact quantity can only be determined by
titration. A precipitate 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 cc. 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 pouring off the stain, twice the
- number of drops of a 1 : 100,000 aqueous eosin solution are added.t The

“*“ Ann. de PInst. Pasteur,” 1904, XVIU, 761.
Marino used a 1:20,000 aqueous solution of eosin, but the 1: 100,000
solution is less apt to cause objectionable precipitation of the dye and. gives
equally good results. +. 2 3 a ; mi

168 Methods of Observing Micro-organisms

two fluids gradually mix, transfusion currents are formed, and the speci-
men 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 distilled 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 hematoxylin and eosin,
thionin and eosin, methylene-blue and eosin, or any other dyes,
some of which sometimes bring out special details of structure.
The protozoa do not show the same reaction to Gram’s stain that
makes it so useful for differentiating the bacteria.

STAINING PROTOZOA IN TISSUE

For this purpose the sections should be embedded in paraffin,
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 modification of the time of staining.
and washing, be employed with good results.

Still better and more satisfactory for certain protozoa are the
jron-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 sections should be
ea vertically in the solution, so that no precipitate may form upon
them.

2. Wash quickly in water.

3. Stain in a o.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 decolorization.

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 1, Orange Geoususss sav axeseeckaRataka weds es 8 grams
WAL ED seis pes gpat seek seresee aco secede ave? duc vein oottnuseale te ives Too cc.
II. Ste ee eleagrowanames 20 grams
or Rubin S.
Walt 6 visvisician: o9 Rida a Seiad serale eee a wise eae I00 CC.
TIT. Methyl-green........... 00... ccc cue cena. 8 grams
Waters sic 3 sei adh aereadaacced acces Os aaa bale Too cc.

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 proportions:

* Mallory and Wright, ‘Pathological Technique,”’ rorz, p. 309.
+ Modified from Mallory and Wright, ‘Pathological Technique,” 1911, p. 289.

Staining Protozoa in Tissue 169

Ds Geiss asliiaherra asceese tare onseneiat aroun omen denis aeoatenlnudtenass Ioo parts
AUD aise arceavs seine) hestha-des oye Gi oxarplsm Sia Syed tisiencayerse 20
Thy, ssuascrositieasekoes eeeees ewdveteds katie 50

At the time of staining dilute the mixture 1:60 or 1: 100 with water.
To test the solution: (1) 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.

. Stain the sections from six to twenty-four hours.

. Wash out a little in 90 per cent. alcohol.

. Dehydrate in absolute alcohol.

. Xylol.

. Xylol balsam. :

t is important to place the sections directly from the staining fluid into the
alcohol, because water instantly washes out the methyl-green.

inp ON Hw

rat

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 magni-
fication and the method of manipulating the instruments must be
learned from the dealers’ catalogues.

Photographing Micro-organisms.—This requires special apparatus
and methods, for which it is necessary to refer to special text-books.*

* See the excellent chapter upon Photomicrography in Aschoff and Gaylord’s
“Pathological Histology,” Philadelphia, 1900.

CHAPTER VI
STERILIZATION AND DISINFECTION

BeForE considering the methods employed for the artificial cul-
tivation of micro-organisms and for 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 apply the methods to the elimination or destruction of micro-
organisms whose accidental presence might ruin the experiments.

The dust of the atmosphere, almost invariable in its micro-
organismal contaminations, constantly settles upon our glassware,
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 take use of our

knowledge of the conditions destructive to bacterial life, and sub-
ject 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 passing. 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 re-
moving all micro-organismal life, whether infectious or non-in-
fectious. 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 following table will serve to outline the methods used for
effecting sterilization or the complete destruction or removal of
living organisms:

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,
tubes, flasks, dishes, etc., 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 tem-
perature of 60°C., spore-bearers may withstand 100°C. for an hour;

170 :

171

Methods of Sterilization

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UOIPEZTMITS

172 Sterilization and Disinfection

it is therefore best to.employ a temperature high enough to kill
all with certainty. Theapparatusis known as a “hot-air sterilizer.”

Platinum wires used for inoculation are sterilized by being held
in the direct flame until they become incandescent. In sterilizing
the wires attention must be bestowed upon the glass handle, which
should be flamed for least half its length for a few moments.
Carelessness in this repect 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

Fig. 31.—Hot-air sterilizer. The gas jets are inclosed within the space be-
tween the outer and middle walls, C, and can be seen at F. The heat ascends,
warming the air between the two inner walls, K, then descends over the contents,
J, and escapes through to supply the draft at /’, perforations in the bottom, B,
and eventually escapes again at S; R, gas regulator; T, thermometer.

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 the hot-air sterilizer. 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

Sterilization and Protection of Culture-media 173

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 itis necessary to protect the sterilized objects and
media from future contamination.

To Schréder and Van Dusch belongs the credit of having first
shown that when 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 bacteriology. 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 instru-
ments 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 sometimes grow and allow their mycelia to
work their way through and drop into the culture-medium, Roux
has employed paper caps, with which the cotton stoppers can be pro-
‘tected from the dust. These are easily made by curling a small
square of paper into a “cornucopia,” and fastening by 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. 4

IL. 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 inappropriate for them, sterilization by stream-
ing steam being the only satisfactory method. The prepared
media are 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 streaming steam or
to some temperature judged to be sufficiently high, so that the adult
micro-organisms contained 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

174 Sterilization and Disinfection

third time, lest a few spores remain alive. When properly sterilized
in this way culture-media will remain free from contamination
indefinitely.

A prolonged single exposure to lower temperatures (60°-70°C.),
known as pasteurization, is employed for the destruction of bacteria
in milk and other fluids that are injured or coagulated by exposure
to 100°C. It is appropriate only when the organisms to be killed
are without spores and without marked resisting powers.

Fig. 32.—Arnold’s steam sterilizer (Boston Board of Health form). -

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 pres-
sure, sufficient heat being generated to immediately destroy 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 sterilization, to be complete, requires
that the exposure shall be for fifteen minutes at 110°C. (six pounds’
pressure).

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 minutes or longer if the media be in bulk in
flasks. The apparatus should be permitted to cool before.the valve is opened,
and the vacuum be slowly relieved. If the valve be opened suddenly the fluids
boil rapidly 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.

Sterilization by Filtration £75

Sterilization by Filtration.—Liquids that cannot be subjected
to heat without the loss of their most important qualities may be
sterilized by filtration—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 destroyed by heat. Various substances have been used for
filtration, as diatomaceous earth (Berkefeld filters), stone, sand,
powdered glass, etc., but experimentation’ has shown unglazed por-
celain to be the only reliable filtering material by which to remove
bacteria. Even the 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 porcelain bougies as well as their
attachments must be thoroughly sterilized
before use.

After having been used, a porcelain
filter must be disinfected, 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 por-
celain 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. Fig. 33—Modern

The filtration of water, peptone solu- autoclave.
tion, 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, 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 disinfection must be practised. Ever since Sir Joseph Lister
introduced antisepsis, or disinfection, into surgery there has been a
great struggle for the supremacy of this or that highly recommended

176 Sterilization and Disinfection .

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 surg-
ery of the present day, which strives to prevent the entrance of germs
into the wound rather than to destroy them afterwards.

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. Washing the hands with
soap, which has marked germicidal properties, will in many cases
suffice to destroy or remove bacteria from smooth skins. This

Fig. 34.—Different types of bacteriologic filters: a, Kitasato; b, Berkefeld; ¢,
Chamberland; d, Reichel.

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
overcome this, many surgeons prefer the use of sterilized gloves
of thin rubber to all other means of preventing manual infections.
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,

Disinfection of Sick-chambers, etc. 177

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 1: 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 1: 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; 1 part of
the biniodid in 500 of the spirit. Hands that cannot bear 1 : 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 1 : 2000 or 1 : 4000 biniodid of mercury solution.

It is a mistake to insist upon the employment of disinfecting
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 sterilized 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 preserved 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 168°-
178°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 Ber-
tarelli and Bocchia,} 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 contain, place it in a mixture of iodin 1,
iodid of potassium 1, and distilled water 100. After immersion for
eight days the catgut is removed, under aseptic precautions, to
alcohol or to 3 per cent. carbolic solution, in which it is indefinitely
preserved 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 1 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

* “Brit. Med. Jour.,” July 11, 1896.
t “Bull. of the Johns Hopkins Hospital,” Feb. and March, 1896.

t “Centralbl. fiir Bakt. u. Parasitenk.,”’ Orig. L, 620.
12

178 Sterilization and Disinfection

solution, or are dried with a sterile towel. Andrews makes special
mention of the fact that the instruments must be completely im-
mersed to prevent rusting.

Disinfection of the Wound. —Cleansing ivi (normal salt
solution) and disinfecting solutions (such as 1: 10,000 to 1: 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 carbolic acid or chlorinated lime. These
can serve no purpose 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.

DISINFECTANTS

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-organisms show marked variations
of their powers of endurance; different stages in the development of
the micro-organisms show different degrees of resisting power, and
the conditions urider which the germicide meets the micro-organism
effect marked variations in action. These factors make it necessary

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 oe

to the purpose for which they are intended. It kills cocci and non-
sporogenic bacilli in dilutions of 1:10,000 in from five minutes to
twenty-four hours, but to kill anthrax spores requires twenty-four
hours’ immersion in 1: 2000 solution. If albuminous substances are
present in the medium containing the micro-organisms they precipi-
tate the salt immediately, diminishing the strength of the solution
and so retarding or perhaps preventing the germicidal action.
Again, certain micro-organisms are defended from the action of de-
structive agents, and among them the germicides, through the pres-
‘ence of waxy matter in their substance. Such is the case with the
acid-fast organisms, and notably the tubercle bacillus. Antiformin,
a combination composed of equal parts of liquor sodz chlorinate and
a 15 per cent. solution of caustic soda, immediately dissolves the

great majority of micro-organisms, but has no destructive action _

upon the tubercle bacillus.

Inorganic Disinfectants 179

The most useful germicidal substances act destructively upon the
micro-organisms by forming chemical compounds with their cyto-
plasm. 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 de-
stroy the cells. In the 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 Krénig and
Paul* that ionization in solution plays an important 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. Dissociation, therfore, seems to have much to do with the
matter.

Inorganic Disinfectants.

Acrmws.—These agents are seldom employed, since the concentration required
makes them objectionable.

ALKALIS.—The same holds good with regard to these agents.

Satts.—In this group we find some of the most powerful and most useful
germicidal substances.

Copper Sulphate.—It is curious and interesting that while this salt is highly
destructive to alge 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 alge that.sometimes render the water of
reservoirs dirty and offensive. Some of the salt contained in a gunny-
sack and permitted 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 (HgClz).—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 exemplifying
the varying behavior of germicides under the varying conditions 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 solu-
tions of the germicide.

Thus, when the salt is dissolved in gelatin in a concentration of 1 :1,000,000,
anthrax bacilli cannot grow. If it is dissolved in blood-serum, the concentration
must be increased to 1 : 10,000 to prevent their growth.

When the anthrax spores are dropped in solutions of the salt, Krénig and Paul
found that they were killed in twelve to fourteen minutes by 1:65 solutions; in
eighty minutes by 1: 500 solutions, and in two hours by 1: 1000 solutions.
When the reaction takes place in albuminous media Behring and Nocht} found
that much more time was required. Thus, the destruction of the spores by a
1:200 solution required eighty minutes, and a 1:1000 solution twenty-four
hours to completely kill all of the spores.

Laplacet and Panfili§ found that the addition of 5 per cent. of tartaric or
hydrochloric acid facilitated the germicidal action through the prevention of
albuminate of mercury formation. Liibbert and Schneider and Behring have
used sodium chlorid and ammonium chlorid. Both of these salts diminish the

* “Zeitschrift fiir Hygiene,’’ 1897, xxv, I

t Ibid., rx, 432. ee
t “Deutsche med. Wochenschrift,” 1887, 866; 1888, 121.
§ “Ann. Ig. Roma,”’ 1893, II, 527.

180 Sterilization and Disinfection

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 roo per cent. alcohol dehydrates the
micro-organisms and prevents the diffusion currents by which the mercury is
carried into their substance. . er

For most purposes a 1: 2000 solution of the mercuric chlorid is to be recom-
mended.

Silver Nitrate (AgNO;).—The solutions of this salt are probably more
useful than the frequency of their employment might suggest. 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. ane

The germicidal power of the salt in aqueous solution is less than that
of the mercuric chlorid, but the power in albuminous fluids in greater.
Anthrax spores in blood-serum are killed in seventy hours ina 1 : 12,000
solution. The addition of other salts, as ammonium salts, interfere
with the germicidal activity by inhibiting ionization.

Combinations of the silver nitrate with albuminous compounds, and
variously known as argonin, argentum casein, argyrol, protargol, 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 pene-
tration, as in the treatment of gonorrhea.

Potassium Permanganate (KMnOs).—Solutions of this salt seem to act by ©
virtue of a strong oxidizing power. In 2 per cent. solutions anthrax
spores are killed in forty minutes; in 4 per cent. solutions, within
fifteen minutes. Koch’s experiments showed less activity of the germi-
cidal power against anthrax spores. In his hands a 5 per cent. solution
seemed to require about a day to effect complete destruction. A 1 per
cent. solution kills the pus cocci in ten minutes; a 1 : 10,000 solution
kills plague bacilli in five minutes.

The chief difficulty is that the salt is quickly reduced and its strength
destroyed by the organic substrata in which the bacteria are contained.

HaLocENs AND Compounps.—Those with the lowest atomic weight hav
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, CaOClz. The addition of any acid, including the atmospheric
CO, causes the evolution of Cl. The powder is readily soluble and
solutions of 1:500 kill vegetative forms of most bacteria in a few
minutes (not, however, resisting spores).

A proprietary compound known as ‘“‘electrozone,’’ made by electro-
lyzing sea-water in such a manner that magnesia and chlorin are liber-
ated and magnesium hypochlorite and magnesium 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 1:400 to 500
dilutions of it disinfected sewage in fifteen minutes; and Delépine, that
1:50 (equal to 0.66 per cent. of chlorin) rapidly killed the tubercle
bacillus and 1 : 10 (equal to 3.3 per cent. chlorin) killed anthrax spores.

Todin Terchlorid (ICls)—This compound, which is so unstable that it
only keeps in an atmosphere of Cl-gas, has great germicidal action,
that probably depends upon the readiness with which it decomposes.
In solutions of 1 : 1000 it kills vegetative bacteria in a few minutes,
and in 1: 100 it kills anthrax spores with equal rapidity. The presence
of organic and albuminous materials does not interfere with the germi-
cidal action, ;

Organic Disinfectants.

Carbolic Acid (CsHsOH) 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.

Organic Disinfectants 181

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 properties and cannot be touched
without destruction of the skin. In 2 to 3 or 5 per cent. solutions
carbolic acid destroys most bacteria within afew minutes. Anthrax
and other powerfully resisting spores, however, require prolonged ex-
posure. Tetanus spores are said not to be killed in Jess than fifteen
hours. There is no ionization; the reagent seems to act by coagulating
the bacterial 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 dis-
tillation are orthocresol, metacresol, and paracresol. ‘“‘Trikresol,” a
much used antiseptic, is a commercial product consisting 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 pre-
cipitate 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.
Tt has the advantage of forming a lather-like soap, so that it can be
employed both as a cleanser and disinfectant. In x 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 isfresh. When
exposed for long to the atmosphere it polymerizes into trioxmethylene
and paraformaldehyde and greatly loses its power. A 10 per cent. solu-
tion 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 in
ten to thirty minutes. Strong solutions are extremely irritating 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 aérial 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 (H,O:) 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:

Certain fundamental principles govern the rationale of disin-
fection, and must be kept in mind: (r) the reagent employed 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 con-
centrated form, and (4) it must be left in contact with the bacteria
long enough to accomplish the effect desired.

During the period of illness the chamber in which the patient is

182

Sterilization and Disinfection

confined should be freely ventilated. An abundance of fresh, pure
air is a comfort to the patient and a protection to the doctor and

nhurse.

After recovery or death one should rely less upon fumigation
than upon disinfection of the walls and floor, the similar disinfection

INHIBITION STRENGTHS OF VARIOUS ANTISEPTICS
; (Adapted from Fliigge, Leipzig, 1902)

Putrefactive
Anthrax Bacilli Other Bacteria Bacteria in
; Bouillon
AcIDs-
Sulphuries «+ ses sex gin d ou es 1: 3000 Chol. spir. 1: 6000
Hydrochloric.............. 1: 3000 B. diph. 1: 3000
; B. mallei 1: 700
B. typh. 1: 500
SuIPHUTOUS no uleadiced ena al emeouedacass Chol. spir. 1: 1000 1: 6000
ATSENOUSS ¢ 525 Bachan eecnee al ae ncdarislea wlll cla ate ds 'eueh See aa aes I: 200
BOLIC aavwmsciaustagen etaeeae lh TESOO. -eeeien aren eancece xd I: 100
ALKALIES
Potass. hydrox............. I:700 B.diph. 1: 600
Chol. spir. 1: 400
B. typh. 1: 400
Ammon hydrox............ I:700 Chol. spir. 1: 500
. B. typh. 1: 500
Calcium -hydrox............)........0008 Chol. spir. 1: 1100
we B. typh. 1: 1100
SALTS
Copper sulphate...........).0.0.00000..[0. ce cee eee eae ees I: 1000
Ferrie Sulphate civi4.ciecnuonnaa'| ans kuiehe abe allied eras ea webelacd aces 4 1:90
Mercuric chlorid........... I:100,000 | B. typh. 1:60,000 I: 20,000
Silver nitrate.............. 1: 60,000 Chol. spir.,
B. typhosus 1: 50,000
Potass. perman............ I: 1000 Eee ctaa metas meat I: 500
HALOGENS AND COMPOUNDS
Chlorin...... Weep aay a eso I:1500 Bee alandt Bale snes ae A dees 1: 4000
Bromin, ys. ca incs eee eae EU SOO.; Var Khalai dncdcmawel dics worcee 1: 2000
TOM eoesecctc ice ia Ueatae eas TR5COOr” nas INE nae dnzsnes seers 1: 5000
POtass: 1Odid ya s.9¢ ah dichcpo avedfssis avasd gdee chiles! eacharanonoaioes ode cee 1:7
Sodium chlor.............. 1:60
ORGANIC ComMpouNnDs , :
Ethyl alcohol.............. TO) | ieee oe ear I:10 |
Acetic and oxalic acids......|............ B. diph. 1: 500 1:40
Carbolic acid.............. 1: 800 B.typh. 1: 400
Chol. spir. 1: 600
_Benzoic acid............... I:1000
Salicylic acid... .. Bete tes 121500
Formalin (40% formaldehyd)|............ Chol. spir. 1: 20,000 | 1:1000
Staphylo. 1: 5000
Camphor. og5 jaev vey Saene 1: 1000
Phy: MOlse a ea 9 waesoeneed dics, a DPEIOjO00! isi avg oats eae need I:3500 |
Oil mentha pip.............| 123000
Oil of terebinth............ 1: 8000
Peroxid! of hydrogens iacc sc stvuees see ean [aasacsumuvewaneyars 122000

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 destruc-
tive to furnishings, so that they are rapidly giving way to the more

Comparison of Disinfectants

183

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

BACTERICIDAL STRENGTH OF COMMON DISINFECTANTS
(Adapted from Fliigge, Leipzig, 1902)

Peroxid of nySTOE EA

Streptococci | anthrax and Typhoid Bacilli,
Cee olera Syirilium | ;
Anthrax Spores
5 minutes 5 minutes 2 to 24 hours

Acips ‘

Sulphuric.......... I:I0 1: 100 I:1500 1:50 in 10 days

Hydrochloric....... I:ro I:I00 1:1§00 1:50 in 10 days

Sulphurousi... so ee feaive ses aiaive emunteee ee msc Typhoid

I: 700 |

SUIphurousisscceareiccssaccchors ard aaell coaapnae Grau 1:300 (Gas

. to vol. %)

BOriGiac vaxceecae a aay ates coe se eee sy sares 1230 Conc. sol. in-
complete disin-
fection.

ALKALIES :

Potass. hydrox... . Its I 1300

Ammon. hydrox....|............. 13300

"CAUM a. ncvee peaeaeteee tesa I: 1000 ;

SALTS . :
Copper sulphate....)...... 0... cc fecee cence ee efeee eee ee eens 1:20 (5 days)
Mercuric chlor... ..| 1: 10,000 to I: 2000 I:10,000 | 1:2000(26 hrs.)

1000

Silver nitraté...... ERTOOO || sais wares 1:4000

Potass: permang.::;) 2200) = |svewercevaasqsreszeaeaees 1:20 (1 day)

Cale, chlorid. soc leas ewdes sebr T2500° de wacenaeanins 1:20 (1 hr.)

HALOGENS AND Com-

POUNDS

-Chlorin........0.. 1% TOG | Thiceisun cen eae 2% (in 1 hr.)

Trichlorid of iodin.. 1: 200 TELOOO® ~.|iy'x'vvie "na sstabiays I:rooo (in 12

2 : hours)

_Orcanic ComPouNDs
Ethyl alcohol... ... 70%—15 70%—10 | 1:200 to 300} Alcol. 50% for 4
: minutes minutes months with-
Acetic and oxalic acids. out killing
5 spores (Kochf)
Carbolic acid. ...... 1:60 Cholera 1:300 I:20 (4 to 4§
I: 200 days) (at 40°
Typh. 1:50 in 3 hours)

Lysol. ...........0. 11300 I: 300
Creolin peisti aaehelaras otalag As I:I00 123000 (10% in 5 hrs.)
Salicylic acid.......| 1: 1000
Formalin (40% for- 1:10 1:20 I: 1000 1:20 (in 6 hrs.)

maldehyd).
Conc I: 200 12500 1:100 (in r hr.)

3: 100 (in x hr.)

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,”

* “Compte rendu de ]’Acad. des Sciences,”’ Paris, 1892.
+ Koch, Arb. a. d. kais. Gesundheitsamt, 1881, 1.

184 Sterilization and Disinfection

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
irritating to the mucous membrane of the eyes and nose.

The solution can be employed to spray the walls and floors of
rooms, though Rosenau* finds that unless the spray discharged
from a large atomizer be very fine, its action is uncertain.

The original method of disinfection, suggested by Robinson,f
consisted of the evolution of the gas by volatizing 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 4o 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. :

One of the best methods of evolving the gas for purposes of dis-
infection is that devised by Evans and Russell? who combine the
40 per cent. solution of formaldehyd with permanganate of
potassium, when an almost explosive liberation of the gas takes place.

Frankfortert found that a good method of escaping the un-
desirable 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 conditions unfavorable to the forma-
tion of oxids of manganese, such as otherwise tend to coat the grains
of permanganate and prevent further reaction between the formal-
dehyd solution and the permanganate.

The employment of calcium carbide for the same purpose is

suggested by Evans.§ The best results were obtained when the
calcium carbide wasin lumps about the size of a pea; when the formal-
dehyd solution was diluted with an equal volume of water, and when
the diluted formaldehyd was added to the carbide’in the propor-
tion of 5 cc. of the former to 3 grams of the latter. . In the perman-
ganate method the quantity of formalin (or 37-40 per cent.
formaldehyd in water) should equal 300 cc. to 1000 cubic feet of

space, but in the carbide method 500 cc. must be used to achieve —

the same result. Evans, therefore, prefers the permanganate
method.

* “Disinfection and Disinfectants,’’ P. Blakiston’s Son & Co., Philadelphia,
1902.
+ “Ninth Report of the State Board of Health of Maine,’’ 1896.

t‘‘Reports and Papers of the American Public Health Association,’’ 1906 +

vol. XXXII, part II, p. 114.
§ Ibid., p. 108.

Disinfection of Dejecta 185

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

‘js 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 undisturbed 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. If the permanganate of
potassium or calcium carbide methods are to be employed, the
cracks about the doors and windows are sealed with paper, a dish-
pan or wash-tub is placed in the center of the room, and in it the

_ can containing the permanganate or carbide and sand is stood. The
formaldehyd solution is poured into the can and the operator escapes,
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 formal-
dehyd leaves little to be desired. Care must, however, be exercised
to see that the required volume of gas is generated to disinfect
the apartment. A sufficient concentration of the gas is absolutely
necessary and the method selected should be one capable of discharg-
ing the gas in a short time, so that it immediately pervades the
atmosphere.

Gaseous disinfection of a room should always be followed by the
application of solutions of disinfectants to the woodwork, the baking
of the mattresses and pillows, the boiling of the linen, etc.

The Dejecta—In diphtheria the expectoration and nasal dis-

Fig. 35.—Pasteboard cup for receiving infectious sputum. When used the
pasteboard can be removed from the iron frame and burned.

charges are highly infectious and should be received in old rags or
in Japanese paper napkins—not handkerchiefs or 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, for the fastidious patients, cut-glass bot-
tles with tightly fitting lids may be used to collect the sputum, and

186 Sterilization and Disinfection

as these are not unsightly the patients make no objection to carrying
them with them. Tuberculous patients should be provided with
rice-paper instead of handkerchiefs, and should have their napkins,

towels, knives, forks, spoons, plates, etc., kept strictly apart from:

the others of the household and carefully sterilized by boiling after
using. Patients with sensitive dispositions need never be told of
these arrangements.

The excreta from cases of typhoid fever and cholera require
particular attention. These, and indeed all alvine matter the pos-
sible 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 com-
pound with albumin.

The Clothing, etc.—The bed-clothing, towels, napkins, handker-
chiefs, night-robes, underclothes, etc., used by a patient suffering
from an infectious disease, as well as the towels, napkins, handker-
chiefs, 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 asif made of cotton. |The
woolen outer clothing of the patient, if infective, requires special
treatment. Fortunately, the infection of the outer garments is un-

usual, The only reliable method for their sterilization is prolonged.

exposure to hot air at 110°C. In private practice 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 and baked.

The doctor visiting a case of dangerous infection or a hospital
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 precautions he will avoid spread-
ing infection among his patients or carrying it to his own family.

The Furniture, etc——The destruction of infective furniture is
unnecessary. The doctor treating a case of infectious disease, if
he properly perform his functions, will save much trouble and money

for his patient by ordering his immediate isolation in an uncarpeted,

Disinfection of the Patient 187

scantily and simply furnished room the nioment an infectious dis-
ease is suspected. If, however, the infectious disease can already be
recognized, it is best not to move him.

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 1:1000 bichlorid of mercury solution
before new paper is hung.

Strehl has demonstrated that when ro per cent. formalin solution
is sponged upon artificially infected curtains, etc., the bacteria are
killed. This is an important adjunct to our means of disinfecting
the furniture of the sick-chamber.

The floor should be scoured with 4o per cent. formaldehyd solu-
tion, 5 per cent. carbolic acid solution, or 1:1000 bichlorid 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. Ifa straw mattress
was used it should be burned and the cover’ boiled. If a hair mat-
tress 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 biniodid of mercury soap, bathed
with a weak bichlorid of mercury solution or with a 2 per cent. car-
bolic acid solution, or with 25-50 per cent. alcohol, before the pa-
tient is allowed to mingle with society, and the hair should either
be cut off or carefully washed with the disinfecting solution or an
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 atmos-
phere. 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’ 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
Mm a strong disinfectant solution, and given a strictly private funeral.
If this be impossible, the body should be embalmed, sealed in the
coffin, and the face viewed through a plate of glass; the body is

188 Sterilization and Disinfection

best disposed of by cremation, though it is not really necessary as
a dead body cannot remain a source of infection for an indefinite
period. Esmarch,* who made a series of laboratory experiments
to determine the fate of pathogenic bacteria in the dead body,
found that in septicemia, cholera, anthrax, malignant edema, tuber-
culosis, tetanus, and typhoid fever the pathogenic bacteria all die
sooner or later, more rapidly during active decomposition than dur-
ing preservation of the tissues.

* “Zeitschrift fiir Hygiene,’’ 1893.

CHAPTER VII

CULTURE-MEDIA AND THE CULTIVATION OF MICRO-
ORGANISMS

In order to observe them accurately micro-organisms must be
separated from their natural surroundings and artificially cul-
tivated upon certain prepared media of standard composition. 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
impossible it is to determine the natural history of any organism
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.

BACTERIA

Various organic and inorganic mixtures have been suggested for
the cultivation of bacteria, but few have met with particular favor
and become standards. At the present time a few well-known 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
behavior 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 composition which, for
the pathogenic forms at least, should approximate the juices of the
animal body. It might be added that transparency is a very desir-
able quality, and that the most generally useful culture-media are
those that can be liquefied and solidified at will.

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 knowledge is suggested by the pains taken
to arrive at it. The best bacteriologists of America have agreed
upon certain details that are explained in the following excerpts
from the Report of the Committee of Bacteriologists of the American
Public Health Association.*

* “Jour. Amer. Public Health Assoc.,” Jan., 1898, p. 72.
189

190 Cultivation of Micro-organisms

“The first thing to obtain is a standard ‘indicator’ which will give uniform
results. These requirements are best fulfilled by phenolphthalein.”

“The question of the proper reaction of media for the ‘cultivation of bacteria
and the method of obtaining this reaction have been discussed in @ valuable
paper by Mr. George W. Fuller, published in the ‘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—

Fig. 36.—Buret for titrating media. (From Hiss and Zinsser, ‘‘Text-Book of
~ Bacteriology,’ D. Appleton & Co., Publishers.)

“7, A o.5 per cent. solution of commercial phenolphthalein,.in 50 per cent.
alcohol.

“oA . 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 pre-
vent 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

Cultivation of Bacteria Igl

prescribed volume by the addition of distilled water to replace that lost by boiling,
and after being thoroughly stirred, 5 cc. are transferred to a 6-inch porcelain
evaporating-dish. To this 45 cc. of distilled water are added and the 50 cc. of
fluid are boiled for three minutes over a flame. One cubic centimeter of the
solution of phenolphthalein (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 developed, 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 addi-
tion 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 reaction always to be obtained. All titra-
tions 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 calcu-
lated 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 thoroughly 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 addition of sodium hydroxid, and the titrations and additions of alkali
must be repeated until the medium has the desired reaction (z.¢., 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.
nn. : sea

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 +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 +1.5 as the. provisional

192 Cultivation of Micro-organisms

standard reaction of media, but with the recommendation that the optimum
growth reaction be always recorded with the species.’

BOUILLON OR BROTH

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 1 per cent. of agar-agar makes it “agar-
agar.”

*T 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 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 cc. This
liquid is called the meat-infusion. To it 10 grams of Witte’s or
Fairchild’s dried beef-peptone or “Bacto-Peptone” made by the
Digestive Ferments Co., Detroit, Michigan, and 5 grams of sodium
chlorid are added, and the whole boiled until the albumins of the
meat-infusion coagulate, titrated or otherwise corrected 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 according to the directions
already given.

The filtered fluid is dispensed in previously sterilized tubes with
cotton plugs—about ro cc. to each—or in flasks, and is then sterilized
by steam three successive days for fifteen to twenty minutes each,
according to the directions already given for intermittent steriliza-
tion, 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 compara-
tively 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.

* “Trans. Assoc. Amer. Phys.,”” 1896.

To Prepare Bouillon from Meat Extract 193

II. To Prepare Bouillon from Meat Extract.—When desirable, the
bouillon may also be prepared from beef-extract, the method being
very simple: To tooo cc. of clean water 10 grams of 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 dispensed and
‘kept in small receptacles—test-tubes or flasks—in order that a single
contaminating organism, should it enter, may not spoil the entire

Fig, 37.—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, rubber tubing.

quantity. A convenient, simple apparatus for filling tubes with
liquid media consists of a funnel to which a short glass pipet is at-
tached by a bit of rubber tubing. A pinch-cock 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 plugged and sterilized by dry heat,
unless the subsequent sterilization is to be performed in the auto-
clave, when it may be unnecessary.
13

194 Cultivation of Micro-organisms

Sugar bouillon is bouillon containing in solution known percent-
ages of such sugars as glucose, lactose, saccharose, etc. As Smith*
has pointed out, if the quantity of sugar in the bouillon is to be ac-
curately known, it is necessary to first destroy the muscle sugars in
the meat-infusion. This can be done by adding a culture of the
colon bacillus to the meat-infusion and permitting fermentation to
continue overnight, then finishing the bouillon and adding the
known quantity of whatever sugar is desired. If the bouillon be
made from meat extract, fermentation may not be necessary. One
per cent. of dextrose, lactose, saccharose or galactose is the standard
addition.

The sugar bouillons should be sterilized in the Arnold apparatus
not in the autoclave, as the high temperatures chemically alter the |
sugars.

GELATIN

The culture-medium known as a gelatin is bouillon to which 10
per cent. of gelatin is added. It has the decided advantage 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 LaWall have examined commercial
gelatins and found that many of them contain sulphur dioxid in
quantities 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 prepared-as follows:

To 1000 cc. of meat-infusion or to 1000 cc. of water containing
2 grams of beef-extract in solution, ro 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 titra

tion, 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 solu-
‘tion, 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 vaporization during the process of

* “Jour. of Exp. Med.,” m1, No- 5. p. 546.

Agar-agar | 195

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 through gauze and
cotton until clear.

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 r10°-
115°C. for fifteen minutes, but this method is probably less well
adapted to the sterilization of gelatin than of the other media, as the -
high degree of heat lessens its subsequent solidifying power. To
overcome this evil it is recommended to plunge the freshly sterilized
media into ice water as soon as it has slightly cooled.

Gelatin becomes liquid at 37°C... It cannot, therefore, be ‘ised
with advantage for cultures that must be kept at body temperatures.

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, and recently in the form of powder.

The “Bacto-agar’”’ made by the Digestive Ferments Co. of Detroit,
Michigan, is a very satisfactory preparation. 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.
In addition to its ability to liquefy and solidify, it has the advantage
of remaining solid at comparatively high temperatures so as to
permit keeping the cultures grown upon it at the incubation tem-
perature—i.e., 37°C.,—at which temperature gelatin is always
liquid.

It is prepared as follows: To 1000 cc. of bouillon made as described
above, preferably of meat instead of beef-extract, 10 to 15 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-albumin is entirely coagulated, when it is filtered
through gauze and cotton.

Ravenel* prepares agar-agar by making two solutions, one con-
sisting of 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

*“Journal of Applied Microscopy,” June, 1898, vol. 1, No. 6, p. 106.

196 Cultivation of Micro-organisms

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,
but it must be filtered through gauze and cotton.

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 un-
sightly cloud.

The finished agar-agar should be a colorless, nearly transparent,
firm jelly. It is dispensed in tubes like the gelatin and bouillon,
sterilized by steam, either by the intermittent process or in the auto-
clave, and after the last sterilization, before cooling, each tube is
inclined against a slight elevation, so as to permit the jelly to solidify
obliquely and afford an extensive flat surface for the culture.

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.—Certain bacteria among which is the
tubercle bacillus, will not grow upon agar-agar prepared as described
above, but will do so if 3 to 7 per cent. of glycerin be added after
filtration. This fact was discovered by Roux and Nocard.

Blood agar-agar was recommended by R. Pfeiffer for the culti-
vation of the influenza bacillus and consisted of ordinary agar-agar
whose surface is coated with a little blood secured under aseptic
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. As powdered hemoglobin is
not sterile, the medium must be sterilized after its addition.

As employed at the present time for the cultivation of the meningo-
coccus, influenza bacillus and other fastidious micro-organisms, the
blood corpuscles are hemolyzed and added to the medium just
before use. For the micro-organisms mentioned a dextrose-hemo-
globin—agar-agar seems to be most appropriate and is prepared as
follows:

Ordinary meat juice agar-agar is prepared and sterilized. A one per cent.
aqueous solution of dextrose is made in distilled water in a flask and also sterilized.
Human blood is taken under aseptic precautions from a vein of the forearm

_ caught in a sterile flask, containing sterile glass beads with which it is defibrinated
by shaking and then decanted into a second flask containing twice the volume
of sterile distilled water. At the end of twenty-four hours the corpuscles are
usually laked and the hemoglobin in solution, the corpuscular bodies sedimenting.
The greatest pains must be taken to keep the blood and water mixture sterile.

The agar-agar is now melted and cooled to less than 50°C. The flask is
cautiously opened and one per cent. of the dextrose solution added (if the
sterilization of the agar-agar is to be made in the Arnold apparatus, the dextrose
addition may have been made beforehand) and then an addition of one per cent.
of the hemoglobin solution is made. When all are mixed, the medium may be
filled under aseptic precautions into tubes or Petri dishes as the work requires.

Blood-serum 197

But as the medium cannot be subsequently sterilized, the greatest dexterity
in filling the tubes or dishes is required to keep them from contamination, and it
is best to incubate them for 24 hours before using to make sure that they are
sterile. When the “blood-agar” is intended to be employed for the purpose of
determining the hemolyzing power of the micro-organismal products, the corpus-
cles are not laked, but the melted agar-agar receives an addition of one per cent.
of the defibrinated blood.

BLOOD-SERUM

The advantage possessed by this medium is that it is primarily a
constituent of the animal body, and hence offers conditions favor-
able for the development of the parasitic forms of bacteria. 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 liberated 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 1-quart fruit jars with tightly fitting lids. The jars
are sterilized by heat, closed, 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 con-
tamination is diminished. 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. i

As the demand for serum has been considerable during the last
few years, commercial houses dealing in biologic products now
market fresh horse serum, preserved with chloroform, in liter bottles.
This can be employed with great satisfaction, the chloroform being
driven off during coagulation 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 coagulation
occurs, and the tubes should be inclined, so as to offer an oblique

198 Cultivation of Micro-organisms

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 for coagulating blood-serum.
The bottom should be covered with wet 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 completely closed, for if the temperature of the serum be
raised too rapidly it is certain to bubble, so that the desirable smooth
surface, upon which the culture is to be made, is ruined.

Fig. 38.—Koch’s apparatus for coagulating and sterilizing blood-serum.

Like other culture-media, blood-serum and its combinations may
be sterilized in the autoclave and much time thus saved. The serum
should, however, first be coagulated, else bubbling is apt to occur
and ruin its surface. The autoclave temperature unfortunately
makes the preparation 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 coagulating 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.

Loéffler’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 containing 1 per cent. of glucose
and 3 parts of liquid blood-serum. After being well mixed the fluid

Potatoes 199

is distributed in tubes, and sterilized and coagulated like the blood-
serum itself. As prepared by Léffler it was soft, semi-gelatinous
and semi-transparent, not firm and white; therefore should be steril-
ized 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 diphtherie,
which grows 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 100 cc. of blood-serum add 1-1.5
cc. of a ro per cent. solution of sodium hydrate and shake it gently. Put suff-
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 toform. The result should be clear,
solid medium consisting chiefly of alkali-albumins. It is especially useful for
Bacillus diphtheriz. a :

Deycke’s Alkali-albuminate—One thousand grams of meat are macerated for
twenty-four hours with 1200 cc. 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 1 per cent. of peptone, 1 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.* a8

With the aid of a cork-borer or Ravenel potato cutter a little
smaller in diameter than the test-tube ordinarily used, a number
of cylinders are cut from potatoes. 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 removed 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 steam-
ing 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 cylin-
ders be allowed to stand for twelve hours in running water before be-
ing 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 Medical News,” 1887, vol. L. D. 138.

200 Cultivation of Micro-organisms

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 cleaning the tubes.

If the work to be done with potatoes is to be accurate, it is nec-
essary to correct their variable reaction, especially if the acids have
not been sufficiently removed by the washing in running water
already described. :

To do this the cut cylinders are placed in a measured quantity
‘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 corrected 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 cc. of water 100 grams of grated potato are
added, and allowed to stand on ice over night.
Of the pulp, 300 cc. are expressed through a
cloth and cooked for an hour on a water bath.
After cooking, the liquid is filtered, titrated if
desired, and receives an addition of 4 per cent.

bacillus grows well, especially when the reaction
of the medium is acid.

Milk.—Milk is a useful culture-medium. As

Fj = ,, the cream which rises to the top is a source
‘ig. 39.—Ravenel’s : 2 hice ‘
potato cutter. 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 sterilized by steam by the
intermittent method or in the autoclave. The opaque nature of
this culture-medium often permits the undetected development of
contaminating organisms. 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 pure reagent 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 sterilized
and treated like the culture media.

An excellent method of preparing reagent litmus from litmus cubes
is given by Prescott and Winslow* 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

* “Elements of Water Bacteriology,’ John Wiley & Sons, New York, 1904, P.
126.

of glycerin. Upon this medium the tubercle ~~

Peptone Solution 201

of water until 3 to 4 liters of water have been used and the cubes are well ex-
hausted of coloring matter. Pour the decantations together and allow them to
settle over night. Siphon off the clear solution. Concentrate to about 1 liter and
make the solution decidedly acid with glacial acetic acid. Boil down to about
44 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 =

HCI should turn it red, one drop of = NaOHO should turn it blue. Filter the

solution and sterilize at 110°C. This solution should be added to the media just
before use in the proportion of about }4 cc. to § cc. of medium.

If litmus bé added to the milk before sterilization, it is apt to be
browned or decolorized, so that it is better to sterilize the two sepa-
rately and pour them together subsequently. It is said that lac-
moid 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 1 liter of water is added. The
mixture is violently shaken. About 16 cc. 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, itisadded. 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 neutral liquid,
so that it has a violet color, changed to blue or red by alkalies or
acids.

Peptone Solution, or Dunham’s solution, is a perfectly clear,
colorless solution, made as follows:

Sodium chlorid.............0.0.0.000 000 cece eee eee 0.5
Witte’s dried peptone............-.... 0000 e eee 1.0
Wate. 6.c auschialecacmcummnaye reine nates aeinee ao Pepe E 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 bac-
teria known to produce it. He recommends testing the peptone to
be employed by the use of the biuret reaction. The reagent em-
ployed 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 successful tests.

The addition of 4 c.c. of the following solution—

*“Centralbl. f. Bakt. u. Parasitenk.,’’ xu, p. 790.

202 Cultivation of Micro-organisms

makes the peptone solution a reagent for the detection of acids and
alkalies. The solution is of a pale rose color. If the organisms cul-
tivated produce acids, the color fades; if alkalies, it intensifies. As
the color of rosolic acid is poate by glucose, it cannot be used in
culture-media containing it.

Theobald Smith* has called attention to the fact that many bac-
teria 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 six-
teen-hour-old cultures. His method of preparation - -is as follows:
Beef-infusion, prepared either by extracting in the cold or at 60°C,,
is inoculated in the evening with a rich fluid culture of some acid.
producing bacterium (Bacillus coli) and placed in the thermostat,
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 me-
dium of indol produced by the colon bacillus. This can be demon-
strated if the tests for indol be sensitive. Seltert 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 phos-
phate, o.5 part, and magnesium sulphate, o.1 part.

Other culture-media employed for special purposes will be men-
tioned as occasion arises.

* “Journal of Exp. Medicine,” Sept. 5, 1897, VI, p. 546.
t “Centralbl. f. Bakt. u. Parasitenk.,’’ Orig. 11, p. 465.

CHAPTER VIII
CULTURES, AND THEIR STUDY

Tue 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 considerable lengths of time, during
which their biologic peculiarities 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 difficult until he devised the
well-known ‘‘plate cultures.”

A growth of artificially planted micro-organisms is called a culture.
Tf such a growth contains but one kind of organism, it is known as a
pure culture.

It has t present become the custom to use the term “culture”
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, the typhoid bacillus is said to
occur in “pure culture” in the spleens of patients dead of typhoid
fever, because no other bacteria are associated with it; and some-
times, 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 recom-
mended, and a wire composed of platinum and iridium, which is
elastic in quality, is to be preferred. - The wires are about 5 cm.
i? length, of various thickness according to the use for which they
are employed, and are usually fused into a thin glass rod about
17cm. in length. The wires may be straight or provided with a
small loop at the end so as to conveniently take up small drops
of fluid. Heavy wires used for securing diseased tissue from ani-
mals 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.
Itconsists of the platinum wire fastened in a heavier aluminium wire
which in turn fits into a piece of glass tubing. When carried in

203

204 Cultures, and their Study

the pocket, the position of the platinum wire is reversed in the
glass tubing and protected by it. |

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.

¢ —— — —— |
a Ea

Fig. 40.—Platinum needles for transferring bacteria; made from No. 27 platinum
wire inserted in glass rods.

Capillary glass tubes are employed by the French for many of the
manipulations. They are made of 14- or 3¢-inch glass tubing cut
into 25 cm. lengths, heated at the center, and drawn out to capillary
ends about 5cm.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. They can be used for all the purposes for which the

———E——EEEEEE__
—SSSSSS__—SE SSS ——_

Fig. 41—Ravenel’s platinum wires for bacteriologic use.
& P

platinum wire is employed, and in addition can be used as con-
tainers 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.—Containers of stored culture-
media should be kept in an upright position, that the cotton stoppers

o Chez -
we,
Fig. 42.—Capillary glass tubes. @, Pipette for ordinary manipulations; },
constricted pipette in which small quantities of cultures, etc., can be sealed by

ene the glass; c, bulbous pipette in which larger quantities of fluids may be
sealed.

are not moistened or soiled. If moistened with the culture-media,
molds whose spores fall upon the surface of the stoppers may gradu-
ally work their mycelial threads between the fibers until they ap-
pear upon their inner surface and drop newly formed spores into the
contained media.

In handling tubes care must be taken to stand them up in tum-

Technic of Culture Manipulation 205

blers, racks, or other contrivances, and not lay them upon the table
so that the contents touch the stoppers.

When the cotton plugs are removed in order that the contents of
the tubes or flasks may be inoculated or otherwise manipulated, the
removal and replacement should be done as quickly as convenient,
and the mouth of the tube should be flamed before removal. The
plugs should be held between the fingers, by that part which projects
above the glass, not laid upon the table, from which dust, and in-
cidentally 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 con-
sideration lest they become the source of future contamination.

So soon as the cotton stopper is
removed, the medium is left with-
out protection from whatever
micro-organisms happen to be in
the air, so that it should be re-
placed as soon as possible, and
every manipulation requiring its
removal performed expeditiously.
During the time the 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. Some
bacteriologists make inoculations fig 43——Method of holding tubes
with the tubes reversed in all during inoculation.
cases in which solid media are
employed, but it is not necessary. If the tubes are held obliquely,
the danger of contamination is reduced toa minimum. It is well
to adopt some method of handling the tubes that has given satisfac-
tion 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 culture-tube, is, in detail, as follows:

In order that any bacteria loosely scattered over the surface of the
cotton stopper, and upon the glass near the mouth of the tube, may
be destroyed and prevented from entering the medium as the stopper
is withdrawn, both the tube containing 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 smoldering remains extinguished
by pinching with the fingers. The tubes are now placed side by side

206 Cultures, and their Study

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 re-
moved by a gentle twist and placed between the index and middle
fingers of the left hand; the stopper of the next tube similarly re-
moved and placed between the middle and ring fingers of the same
hand. 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 incandescence 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 touch-
ing 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 not being
touched. Each stopper is given a twist as it enters the mouth of the
tube. ,

Modifications of these directions can be made to suit the differ-
ent 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, form-
ing 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 observation of
Koch, that when bacteria are equally distributed throughout some
liquefied nutrient medium that is subsequently solidified in a thin
layer, they grow in scattered groups or families, called colonies, dis-
tinctly isolated from one another and susceptible of transplanta-
tion.

The plate cultures, as originally made by Koch, require con-
siderable apparatus, and of late years have given place to the more
simple and ready methods. So great is their historic interest, how-

Plate Cultures . 207

ever, 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 1o inches in diameter 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 1 : 1000 bichlorid of mercury solu-
tion 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. It consists of a wooden tripod with ad-
justable screws, and a glass dish covered by a flat plate of glass upon which a low
bell-jar stands. The 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 material to be examined,
a small quantity secured, and:stirred about
so as to distribute it evenly throughout
the contents of a tube of melted gelatin.
ee ay under examination be very
tich in bacteria, one loopful may contain a E
Tih le wie red?
a thin layer, would develop so many colonies
that it would be impossible to see any one
clearly; hence further dilation becomes nec- 2
essary. From the first tube, therefore, a _ Fig. 44.—Complete leveling ap-
loopful of gelatin is carried to asecond and Paratus for pouring plate cultures,
stirred well, so as to distribute the organ- 5 taught by Koch. .
isms evenly throughout its contents. In ;
this tube we may have no more than ten thousand organisms, and if the same
method of dilution be used again, the third tube may have only a few hun-
dred, 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. 1 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 cham-
ber prepared to receive it. As soon as plate
No. 1 is prepared, the contents of tube No. 2
are poured upon plate No. 2, allowed to spread

2 out and solidify, and then superimposed on ~
Fig. 45.—Glass bench. plate No. 1 in the moist chamber, being sepa-
rated from the plate already in the chamber by
small glass benches made for the purpose and previously sterilized. After the
contents of all the tubes are thus distributed, the moist chamber and its con-
tents are stood away to permit the bacterie 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

208 Cultures, and their Study

can be melted and solidified at will can be used for plate cultures—viz., gelatin,
agar-agar, and glycerin agar-agar. Blood-serum and Léffler’s mixture are en-
tirely inappropriate.

The chief drawbacks to this excellent method are the cumber-
some apparatus required and the comparative impossibility of
making plate cultures, as is oftea desirable, in the clinic, at the bed-
side, or elsewhere than in the laboratory. The method therefore
soon underwent modifications, 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 14 inch deep, with accurately fitting lids. They were first

Ce eet
ig ee TT

rt 0

Fig. 46.—Petri dish for making plate cultures.

recommended by Petri* and greatly simplify bacteriologic technic
by dispensing with. the plates and plate-boxes, the moist chambers
and benches, and usually with the leveling apparatus of Koch,
though this is still employed 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, closed and sterilized by hot air, care

Fig. 47.—Petri dish forceps.

being taken that they are placed in the hot-air closet right side up,
and after sterilization are kept covered and in that 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 protects the accidental entrance of dust between
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

*“Centralbl. f. Bakt. u. Parasitenk.,” 1887, 1, No. 1, p. 279.

Esmarch’s Tubes 209

flame, instead of in the hot-air closet, special forceps adapted to
holding them having been devised by Rosenberger.*

The dilution of the material under examination is made with gela-
tin or agar-agar tubes in the manner above described, the plug is
removed, the mouth of the tube cautiously held for a moment in the
flame, and the contents poured into one of the sterile dishes, whose
lid is just sufficiently 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 con-
densation may not drop from the lid upon the culture film and spoil
the cultures, and stood away for the colonies to develop.

Fig. 48.—Esmarch tube on block of ice (redrawn after Abbott).

To overcome the difficulty of excessive water of condensation 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 sudied 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 contamination, but to examine colonies in the Petri dish, one
simply examines through the thin glass of the bottom dish without
any exposure of contaminating organisms.

Esmarch’s Tubes.—This method, devised by Esmarch, converts the wall of
the test-tube into the plate and dispenses 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 inoculation 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 ina
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. Thus
the wall of the tube becomes the plate upon which the colonies develop.

In carrying out Esmarch’s method, the tube must not contain 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-
hess injured, and no water must be admitted from the melted ice.

*“Phila. Med. Jour.,”” Oct. 20, 1900, vol. vr, No. 16, p. 760.
I4

210 . Cultures, and their Study

Colonies.—The progeny of each bacterium form a mass which is
known. as a colony. When these are separated from one another,
each is spoken of as a single colony, and different characteristics
belonging to different micro-organisms enable us at times to recognize
by macroscopic aad microscopic study of the colony the particular
kind of micro-organism from which it has grown. The illustrations
show the various types of colonies and the legends the terms nutes
in describing them.

Growing colonies should be observed from day to day, as it not
infrequently happens that unexpected changes, such as pigmenta- —

Fig. 49.—Types of colonies: a, Cochleate (B. coli, abnormal form); }, conglom-
erate (B. zopfii); c, ameboid (B. vulgatus) ; d, filamentous (Frost).

tion and liquefaction, develop after the colony 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 bac- ,
teria 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.

Ob weezezzzezZZZZZZZEZZZA C LL, jf LilaLlldls
é

b LLL, = A LL 9 ttllllim,.

Fig. so = Sante elevations of growths: a, Flat; b, raised; c, convex; d, pulvinate;
e, capitate; f, umbilicate; g, umbonate (F rost).

Pure Cultures.—Single colonies alse subserve a second very
important purpose, that of enabling us to secure pure cultures of bac-
teria from a mixture. For 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 picked up with a sterile
platinum wire and transplanted to a tube of appropriate culture-
medium. If, however, the colonies are numerous, of small size, and _
close together, it may be necessary to do it under a dissecting

The Gelatin Puncture or “Stab” Culture 211

microscope or even a low power of the ordinary bacteriologic micro-
scope. This operation of transplantation is familiarly known as
fishing. | :

Fishing.—This is the transfer of a colony from the plate to a
fresh medium. It is done by touching the colony with the wire
and transferring. When the colony is large and well isolated no
particular skill is required, but when many small colonies are closely
associated it may be necessary to make the transfer while the colony
is under the microscope. A hand lens, a dissecting microscope
or the usual bacteriological microscope may be used. In the latter
case the low-power objective must be used. The colony to be trans-
planted, selected because of its isolation, its typical appearance,
and convenient position 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,
comfortably fixed upon the stage of the microscope, being used

. . . . 5
Fig. 51.—Microscopic structure of colonies: 1, Areolate; 2, grumose; 3,
moruloid; 4, clouded; 5, gyrose; 6, marmorated; 7, reticulate, 8, repand; 9, lobate;
10, erose; 11, auriculate; 12, lacerate; 13, fimbricate; 14, ciliate (Frost).

tosupport the hand. As the operator looks into the microscope the
point of the platiaum wire is carefully brought into the field of vis-
ion 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 focused 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 sur-
face of agar-agar or stabbed in gelatin or stirred in fluid medium, as
the case may be.

The Puncture or ‘‘Stab” Culture.—To make satisfactory punc-
ture cultures, the medium must be firm but not old or dry. Gela-

212 Cultures, and their Study

tin should not be soft and semi-fluid at the time the puncture is
made, or the bacteria diffuse themselves and the typical appearance
of the growth may be masked. On the other hand, if the gelatin
be old, dry, or retracted, it is very apt to crack after the culture has
been 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 appearances presented as the
growth progresses are subject to striking variations according to
the liquefying or non-liquefying tendency of the micro-organisms,

WU WW

“7

Fig. 52.—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, Crateri-

form (B. vulgare, 24 hours); 7, napiform (B. subtilis, 48 hours); 8, infundibuli-
le 8. prodigiosus); 9, saccate (Msp. finkleri); 10, stratiform (Ps. fluorescens)
rost).

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 incubating
oven, as the medium liquefies at such temperatures. On the other
hand, they must not be kept where the temperature is too low, else
the bacterial growth may be retarded. The temperature of a com-

Cultures upon Potatoes 213

fortably 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 care- ~
fully examined from day to day, as it not infrequently happens that
a growth 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 Stroke Culture.—In most cases, the culture is planted by
a simple stroke made from the bottom of the tube in which agar-
agar blood serum, or other solid medium has been obliquely solidified,
and where it is fresh and moist, to the upper part, where it is thin and
dry. Inaddition to this, it is advisable to make a puncture from the
center of the oblique surface to the bottom 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 par-

Ui 2 3 4 5

oT a
ae ca

Fig. §53—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).

ticular advantage except in cases where it is desired to scatter the
transplanted organisms as much as possible, in order that a large
bacterial mass may be secured. ens

Stroke cultures upon agar-agar have the advantage that the
cultures may be kept in the incubating oven. The colorless or
almost colorless condition of the preparation also aids in the detection
of chromogenesis.

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 (gonococcus).

Cultures upon Blood-serum.—Bacteria are planted upon coagu-
lated blood serum and blood-serum preparations as upon agar-agar.

Blood-serum is liquefied by some bacteria, but the majority of
organisms have no characteristic reaction upon it. A few, as the
bacillus of diphtheria, are, however, characterized, by rapid develop-
ment at given temperatures.

214 Cultures, and their Study

Cultures upon Potato.—These are made by simply stroking the
surface of the culture-medium, the density and opacity of the
' potato making it impracticable to puncture it.

Most bacteria producg smooth, shining, irregularly extending
growths upon potato, that may show characteristic colors.

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.

In milk and litmus milk one should observe change in color from
the occurrence of acid or alkali production, coagulation, gelatiniza-
tion, and digestion of the coagulum.

Adhesion Preparations.—Sometimes it is desirable to preserve
an entire colony as a permanent microscopic specimen. 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 integ-
rity of the colony. The cover is gently raised by one edge, and if
successful 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—
“ Klatschpréparat.”

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. Colo-
nies 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 media capable of alternate solidification 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.

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 better
to inject the fluid into the abdominal cavity of a guinea-pig, await
the development of tuberculosis in the animal, and then seek to

*“Centralbl. f. Bakt. u. Parasitenk.,”’ 1895, xvi, No. 16.

Microscopic Study of Cultures 215

secure pure cultures of the bacillus from the unmixed infectious
lesions.

In‘other cases, as when it is desired to isolate Micrococcus tetra-
genus, the pneumococcus, and other bacteria that pervade the blood,

Fig. 54.—Modern incubating oven.

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.

Microscopic Study of Cultures.—Some attention has been given
to the preparation of microtome sections of gelatin cultures, though

216 Cultures, and their Study

not much practical value has come of it. It can be done by warming
the glass of the tube sufficiently to permit the gelatin containing the
growth to be removed in a lump and placed in Miiller’s fluid (bichro-
mate of potassium 2-2.5, sulphate of sodium 1, 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.

Winkler* accomplishes the same end by boring'a hole in a block
of paraffin with the smallest size cork-borer, soaks the block in bi-
chlorid 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.

Museum Culture Preparations. —Neat museum specimens of plate
and puncture cultures in gelatin can be made by simultaneously
killing the micro-organisms and fixing the gelatin with formaldehyd,
which can either be sprayed upon the gelatin or applied in dilute
solution. As gelatin fixed in formaldehyd cannot subsequently be
liquefied, such preparations will last a long time.

Standardizing Freshly Isolated Cultures.—This is a matter of
some importance, as in bringing bacteria into the new environment
of artificial cultivation their biologic 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 successive generations upon two or
three of the most important culture media. Its saprophytic exist-
ence being thus established, the characteristics manifested become
the permanent peculiarities of the species.

*“Fortschritte der Medicin,’’? 1893, Bd. x1, No. 22.

CHAPTER IX
THE CULTIVATION OF ANAEROBIC ORGANISMS

THE presence of uncombined oxygen in ordinary cultures inhibits
the development of anaérobic bacteria. When such are to be culti-
vated, 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 excellent
review of which has been published by Hunziker,* who divides
them as follows, according to the principle by which the anaérobiosis
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 rarely em-
ployed. The appropriate container, whether a tube, flask, or some
special device such as the Novy jar, 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.

2. Displacement of the Air by Inert Gases.—This method is
decidedly preferable to the preceding, as it leaves no vacuum. It
is easier to displace the oxygen than to withdraw it, and any appa-
ratus permitting a combination of both features, as that designed by
Ravenel,f 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
who inoculated a culture-tube of melted gelatin or agar-agar, solidi-
fied it upon the wall of the tube, as suggested by Esmarch, sub-

* “Journal of Applied Microscopy and Laboratory Methods,’’ March, April —
and May, 1902; vol. v, nos. 3, 4, and 5.

t “Bacteria of the Soil,” “Memoirs of the National Academy of Sciences,”
First Memoir, 1896.

217

218 The Cultivation of Anaérobic Organisms

stituted for the cotton stopper a sterile rubber cork containing’;
long entrance and short exit tube of glass, passed hydrogen througt
the tube until the oxygen has been entirely removed, then sealed the
ends in a flame. In this tube the growth of superficial and deer
colonies can be observed. Hansen and Liborius constructed special

Fig. 56.—Frankel’s method of _ . Fig. 57.—Liborius’ tube for
making anaérobic cultures. anaérobic cultures.

tubes 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 inocu-
lation, hydrogen is passed into the small tube and permitted to es-
cape 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

The Absorption of the Atmospheric Oxygen 219

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 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 several hours, as the cotton stop-
pers retain the air in the test-tubes and prevent rapid diffusion.
When the air is all displaced, 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. It
consists of three parts—a deep
dish of glass (0), a stand to sup-
port the Petri dishes to be ex-
posed (c), and a bell-glass (a) to
cover the stand and fit inside of
the dish. The prepared dishes
are stood uncovered in the rack,
which is then placed in the dish
forming the bottom of the ap-
paratus, and into which liquid
paraffin is poured to a depth of
about 2 inches. The bell-glass
cover is now stood in place and 5 = ,
hydrogen gas is conducted “asking anaérobic’ cultures.
through previously arranged
rubber tubes (d, e). As soon as the air is displaced through tube d,
both tubes are withdrawn. It is well to place one Petri dish contain-
ing 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 atmos-
pheric oxygen by alkaline pyrogallic acid and permit the bacteria
to develop in the indifferent nitrogen. Various methods have been
Suggested for achieving this end,.Buchner’s own method consist-

_ ing in the use of two tubes, a small one to contain the culture and a
larger one to contain the absorbing fluid. A fresh solution of pyro-
gallic acid and sodium hydroxid were poured into the large tube,
the smaller tube placed within it, upon some appropriate support,
and the whole tightly corked.

Nichols and Schmitter, * at the suggestion of Carroll, have modified
the method by connecting the tube containing the inoculated

* “Jour. of Medical Research,” 1906, XV, Pp. 113.

220 The Cultivation of Anaérobic Organisms

culture medium with a U-shaped tube, to the other end of which
is attached a tube to contain the pyrogallic acid solution. The
apparatus will at once be understood by a glance at the cut.
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 Utube and the test-tubes are coated externally with
vaselin, the rubber tubes are adjusted on the U tube and a connec-
tion 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 ro per cent. solution of sodium hydroxid
are then poured into the tube and the
second connection made before the acid
_ and alkali react to any extent.”
Wright has suggested that the cotton
stopper of the ordinary culture-tube have

itself pushed down the tube for a short dis-

_tance. Some alkaline pyrogallic acid solu-
tion is poured upon the cotton, to saturate
it, and the tube tightly corked.

Zinsser* has recommended the follow-
ing method as satisfactory for use with
Petri dishes. The dishes selected should
be rather deeper than ordinary. They
are sterilized and inoculated in the
ordinary manner and then inverted. The
dish is cautiously raised, and some pyro-
gallic acid carefully poured into the lid
Fig. 59.—Spirillum ru- and the dish gently dropped into place

eo oe again. The alkaline solution is then
dant production of pigment Poured into the crevice between the edges
apap ee of the dish and the lid, and the remainder
fluid during handling by the Of the space filled with melted albolene.
photographer.) (Nichols When these dishes are carefully stood
and Schmitter.) away, the alkaline pyrogallic acid absorbs
all of ‘the contained oxygen and the
anaérobic cultures develop quite well. The growing colonies can
be examined 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 recom-
mended the cultivation of anaérobic bacteria in association with
aérobic bacteria by which the oxygen was to be absorbed. This

* “Journal of Experimental Medicine,” 1906, VIII, 542.

its projecting part cut off and the plug

Exclusion of Atmospheric Oxygen 221

method is too crude to be employed at the present time, as it destroys
the essential characteristics of the cultures by mixing the products
ofthe bacteria. ;

~ Chemic reduction of the oxygen has been attempted by the addi-
‘tion of 2 per cent. of glucose, as suggested by Liborius, 0.3-0.5 per
cent. of sodium formate, as suggested by Kitasato and Weil, 0.1
per cent. of sodium sulphate, suggested by the same authors, and
various other chemicals. None of these additions has been suffi-
ciently 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 accom-
plishing it have been tried with success.

Fig. 60—Buchner’s method of mak- . Fig. 61.—Hesse’s method of making
ing anaérobic cultures. anaérobic cultures.

The most simple plan is that of Hesse, who made a deep puncture
in recently boiled and rapidly cooled gelatin or agar-agar, then cov-
ered the surface of the medium with sterile oil. The so-called
“shake culture” is another 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 inoculated,
the inoculated bacteria being distributed by gently shaking. On
cooling, the medium “sets,” the organisms below the surface remain-
ing under anaérobic conditions.

Kitasato tirst used paraffin as a covering for the inoculated medium,
his recommendation having recently been revived by Park and made
successful for the cultivation of the tetanus bacillus. The paraffin

222 The Cultivation of Anaérobic Organisms

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 anaérobic organisms by cultivating =

them upon a film of gelatin covered by a thin sheet of sterilized

mica, by which the air was excluded.
Salamonsen has made use of a pipet for making anaérobic cultures,
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 out to a point at each end. The
inoculated gelatin or agar-agar is drawn
in while liquefied and the ends sealed.
The tube, of course, contains no air, and
perfect anaérobiosis results.

it excellently well adapted to the growth
of anaérobes, which, of course, grow only
in the closed limb.

Hens’ eggs have been used for anaérobic
cultures, and in them the tetanus bacillus
grows remarkably well. Conditions of
anaérobiosis are, however, not perfect, as
can be shown by the behavior of the egg
itself. If oxygen be completely shut out

egg will not develop.

A quite satisfactory and simple device
for routine work with anaérobic organisms
has been invented by Wright.* The es-
sential feature consists of a pipet, D, with
arubber tube, E, at the end, and one in-
a terruption connected by a rubber tube, C.
Figs. 62, 63.—Wright’s The device will be made clear at once by
method of making anaéro- a glance at the accompanying illustration.
bic cultures in fluid media The wethod of 1 + smal
(Mallory and Wright). 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.

*“TJour. Boston Soc. of Med. Sci.,” Jan., 1900.

Theobald Smith has found the fermen- “°° ~~
tation-tube and various modifications of _.

by oiling or varnishing the shell, a fertile

The Catalytic Action of Platinized Asbestos 223

In all cases where the presence of suspected micro-organisms is to
be demonstrated, it is necessary to make both aérobic and anaérobic
cultures. For routine work of this kind, this method of Wright is
probably the most convenient yet suggested.

6. The Catalytic Action of Platinized Asbestos upon Hydrogen
and Oxygen.—This method seems to have originated with Laidlaw
(British Medical Journal, March 20, 1915) who tried porous platinum,
colloidal platinum, and colloidal platinum and sodium formate in
various ways for the absorption and combination of the oxygen.
His preference was for the method in which porous platinum was
used as a catalysant, which is, briefly, as follows:

“Short pieces of platinum wire are fixed into glass holders at the blow-pipe
and the free ends are wrapped tightly round small pieces of gas carbon or other
porous material which will char readily and secured by twisting round the main
piece of wire. These pieces of carbon are then heated
in the flame from a Bunsen burner to expel the air, and
dipped while still hot into a strong solution of platinic
chloride. After soaking for some time, they are removed
and dried over the flame. They are then heated red
hot and redipped and the process repeated two or three
times. It will be found then that, on removal from the
flame, the reduced platinum on the surface of the carbon
will absorb sufficient oxygen from the air to keep the
mass a dull red until all the carbon is burned away...
The glass is cut short and pushed into the center ofa
cork. ... Suppose that an anaérobic culture is desired
on a blood agar slope ina test-tube. The tubeis infected
in the usual way. Itis turned upside down and the
cotton-wool plug removed. A sterile glass capillary tube
connected with a hydrogen apparatus (Kipp’s apparatus)

and with a cotton-wool plug in it is introduced from r—A
below, and a brisk stream of hydrogen run into the

_test-tube. No precautions are necessary to keep the D
hydrogen from diffusing out of the test-tube except that :

the remaining operations are carried through rather j E

_ quickly. The capillary is removed and the platinum

armed cork, which has been sterilized by passing through
a flame is pushed home. If an ordinary cork is used it is
advisable to paint the joint with melted paraffine wax. i
If the right amount of hydrogen has been introduced Fig. 64.—Sketch
the platinum will glow dull red as soon as it is intro- of anaérobic appara-
duced and continue to do so until all the residual oxygen tus for the cultivation
is used up in forming water. If two little hydrogen has of absolute anaérobes
been led into the tube, the platinum will burn white hot jn test-tubes (Smillie).
and an explosion will result. Using this method the
organisms of tetanus, botulismus and malignant edema grow with great freedom
and nearly the whole surface of the agar was covered with growth in forty-
eight hours. Transplants from these gave visible growths in fourteen hours.”

The method was somewhat modified and amplified by McIntosh
and Fildes (Lancet, Lond., April 8, 1916) who used asbestos wool
impregnated with palladium black, in the place of the platinized
carbon. It has also been amplified and considerably improved by
Smillie (Jour. Exp. Med., 1917, xxvi, No. 1, p. 59), who found that
Laidlaw’s tubes did not always prove to have had the oxygen com-
pletely removed. Smillie’s improvement is described as follows:

224 The Cultivation of Anaérobic Organisms

! “Two lengths of nichrome wire 6 cm. long, are separately fused into a glass
tube so thet they are insulated (A), and the glass tube (B), closed at each end
is passed through a one-hole rubber stopper AC). To the lower ends of the
nichrome wire is attached a coil of fine (No. 31) nichrome wire (D) thus completing

the circuit. In the coils of the fine wire is placed a small mass of platinized - - -

E). The apparatus is placed in a package and autoclaved. A large
oc x by 1.5 ana used, és which ro cc. of media are added, sterilized
and slanted. The water of condensation is removed and the tube inoculated.
The tube is then inverted, the cotton plug removed, and the tube filled with
hydrogen by means of a sterile capillary pipette. The platinized asbestos
mass is heated for a moment in a free flame and the rubber stopper is then firmly
inserted into the inverted tube and the end of the tube dipped in melted paraffine,

Glagsbulb conlaining
platinized asbestos

4 Ma 2m. perforations

Fig. 65.—Detail of the platinized asbestos bulb for the anaérobic jar (Smillie).

The tube may now be placed in an upright position and sufficient electric current
applied to the free ends of the wire to heat the fine nichrome wire wrapped
about the platinized asbestos to a red heat. ;

The catalyzer is then heated and the free oxygen and hydrogen unite to
form water. The tube is set aside for one-half hour to an hour, then the platin-
ized asbestos is reheated in order to ignite any residual oxygen. The tube
may now be incubated. The method is very useful for growing all anaérobes for
the oxygen is always removed, whereas the Laidlaw method frequently fails.
It is particularly useful for the cultivation of the stricter aérobes.” ‘The method
can be adapted to Blake bottles and flasks.

But the chief improvement made by Smillie was the adaptation
of the method to an anaérobic jar in which a considerable number of
tubes could be simultaneously placed for culture, and in which he
was successful in obtaining growths of the globoid bodies from the

The Catalytic Action of Platinized Asbestos 225

brains and infected tissues of cases of poliomyelitis. This was
prepared as follows:

“The jar used is an ordinary museum specimen jar about 30 cm. high, with an
inside diameter of 12.5 cm. Two holes 1.5 cm. are ground in the cover and
into each hole is firmly inserted a No. 4 one-hole rubber stopper carrying a
ground-glass ‘‘angle” stop-cock. To one of the stop-cocks is attached a rubber
tube, at the end of which is a short piece of glass tube which reaches to the
bottom of the jar. To the other stop-cock is attached by a short rubber tube a
glass bulb, 2 cm. in diameter which has been blown on the end of a capillary glass
tube. The glass bulb is perforated with 5-6 holes, 2 mm. in diameter and is
filled with platinized asbestos. The details are shown in the cut.

The cultures are placed after inoculation in a glass tumbler which is then

Meta! clamp

Stop-cock for so-

dium hydrate top-cock for

hydrogen
y Rubber tubing con-
necting bulb with
_. stop-cock
Perforated glass
bulb containing
platinized asbestos
~ Water of condensa-
tion formed from
hydrogen and
oxygen

Rubber ring

Rubber tube ex-
tending to pyro-
gallic acid

nsulated tubes in
glass container

Pyrogallic acid-
sodium hydrate |
mixture

Fig. 66.—Anaérobic jar with platinized asbestos bulb. Showing apparatus
complete (Smillie).

placed in the jar, to which roo c.c. of a 10 per cent. pyrogallic acid solution have
been added.

The glass bulb containing the platinized asbestos is heated over a free flame
for a few seconds and the cover is then cemented on. A rubber ring 0.5 cm.
which is placed between the jar and the cover, all surfaces are cemented with
Major’s glass cement, and the metal clamp is screwed down with thumb and
forefinger. The stop-cock to which the glass bulb is connected is placed on the
vacuum pump and gentle suction is applied for two or three seconds in order to
Secure a good initial flow of hydrogen and thus ignite the platinized asbestos at
once. The stop-cock is now closed and attached to the hydrogen apparatus,
and the gas allowed to enter. This should be done carefully at first, in order
that an excess of hydrogen does not enter at once; for the gas should be burned
as rapidly as it enters the jar. The platinized asbestos will soon be seen to
glow and from this time hydrogen and oxygen will slowly unite and the water
formed will be deposited on the sides of the jaw. When all of the oxygen has
united with the hydrogen the platinized asbestos will become cool but the hydro-

gen will continue to enter the jar until all the space formerly occupied by oxygen

226 The Cultivation of Anaérobic Organisms

is replaced by hydrogen. The result is a hydrogen-nitrogen jar under approzi-
mately atmospheric pressure. The whole process should take about 15 minutes,
In order to have an index of the completeness of anaérobiosis, the second stop-cock
is connected with a bottle of 20 per cent. sodium hydrate, freshly washed with
hydrogen. By means of slight suction throuph the first stop-cock, 25 ec of
the-sodium hydrate solution are drawn into the jar. Both stop-cocks are now
closed, the ends sealed with cement, and the jar is incubated. If the jar is
satisfactory the solution of sodium hydrate and pyrogallic acid will remain
colorless indefinitely. This solution should not be relied upon to absorb any
remaining traces of oxygen, but is simply an indicator of the pressure of oxygen
and if it becomes discolored shows that there has been a mistake in technic,
and the jar is unsatisfactory, therefore the cover should be removed and the

process repeated.

CHAPTER X
EXPERIMENTATION UPON ANIMALS

THE principal objects of medical bacteriology are to discover
the cause, explain the symptoms, and bring about the cure and
future prevention of disease. We cannot hope to achieve these ob-
jects 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 text-books of physiology, and whose “philanthropy has
ileneby been transformed to zoédlatry.”

Fig. 67.—1, Roux’s bacteriologic syringe; 2, Koch’s syringe; 3, Meyer’s
bacteriologic syringe. Such syringes, because of their complexity and the
destructible packings, give very unsatisfactory service and are no longer
employed.

It is largely through experimentation upon animals that we have
attained our knowledge of physiology, most of our important knowl-
edge of therapeutics, and most of our knowledge of the infectious
diseases. Without its aid we would still be without one of the great-
est achievements of medicine, the ‘‘blood-serum therapy.”

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 employed.

The bacteriologic methods are fortunately not cruel, the principal
modes of introducing bacteria into the body being by subcutaneous,
intraperitoneal, and intravenous injection.

Hypodermic syringes, expressly designed for bacteriologic work are

227

228 Experimentation upon Animals

shown in the illustration. Those of Meyer and Roux resemble
ordinary hypodermic syringes; that of Koch is supposed to possess

Fig. 68.—Altmann syringes for bacteriologic and hematologic work. These are
capable of sterilization without injury and are thoroughly satisfactory.

it,
Wi ‘S
mae |

see

Fig. 69.—Method of making an intravenous injection into a rabbit. Observe
that the needle enters the posterior vein from the hairy surface.

the decided advantage of not having a piston to come into contact
with the fluid to be injected. This is, however, really disadvanta-
geous, inasmuch as the cushion of compressed air that drives out the

Animal Inoculations 229

contents is elastic, and unless carefully watched will follow the injec-
tion into the body of the animal. In making subcutaneous injec-
tions 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 like those shown are to be
preferred. All syringes should be disinfected by boiling thoroughly,
before and after using. Syringes with packings to tighten the pistons
cannot be boiled with impunity, as it soon ruins them, and new pack-
ings may be difficult to obtain or fit. Syringes of such design should
be avoided.

. The intravenous injection is easy to achieve in a large animal, like
a horse, but is very difficult in animals smaller than a rabbit. Such
injections, when given to rabbits, are usually made into the ear-
veins, which are most conspicuous and accessible. A peculiar and
important fact to remember is that the less. conspicuous posterior
vein of the ear is much better adapted to the purpose than the an- -
terior. The introduction of the needle should be made from the

’ hairy external surface of the ear where 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 introduced, and the injection made as near the
root as possible. The fluid should be injected slowly.

By using very fine needles, similar injections may be made into the
ear veins of guinea-pigs. By dipping the tails of rats and even mice
into warm water so as to cause dilatation of the caudal veins, it may
be possible to effect intravenous injections of such animals. Kolmer
suggests that the tails be vigorously rubbed with xylol or alcohol,
and the epidermal cells softened and scraped off so as to expose the
veins better. As the first attempt to get the needle into the caudal
vein may fail, and new attempts be required, it is well to begin at a

_ point not too near the body.

Bacteria can be introduced into the lymphatics only by injecting
liquid cultures into some organ with comparatively few blood-vessels
and large numbers of lymphatics. The testicle is best adapted to
this purpose, the needle being introduced deeply into the organ.

Sometimes subcutaneous inoculations are made by introducing 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 sometimes made,
and in cases where it becomes necessary to determine the presence
or absence of the bacilli of tuberculosis or glanders in fragments of
tissue it may be necessary 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 abdomen near the groin, ’

230 Experimentation upon Animals

the skin picked up with forceps, a snip made through it, and the
points of.the scissors introduced for an inch or so and then separated.

Fig. 70.—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.

By this manceuver a subcutaneous pocket is formed, into which the °
tissue is easily forced. ‘The opening should not be large enough to
require subsequent stitching.

Fig. 71.—Guinea-pig confined in the. holder.

When tissue fragments or collodion capsules are to be introduced . -
into the abdominal cavity, the animal should be anesthetized and

Fig. 72.—Mouse-holder.

a formal laparotomy done, the wound being carefully stitched
together.

Securing Blood from Animals 231

When, in studying Pfeiffer’s phenomenon and similar conditions,
it is desirable occasionally to withdraw drops of fluid from the ab-
dominal cavity, a small opening can be burned through with a blunt
needle. This does not heal readily, and through it, from time to
time, a capillary pipet can be introduced and the fluids 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. Dogs, cats, sheep, and goats can
be tied and held in troughs. A convenient form
of mouse-holder, invented by Kitasato, is shown
in the figure.

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 com-
parison drawn by Frinkel, 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 Bloodfrom 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 re-
strain the animal, make a minute incision in Fig. 73.—Tube for
the skin over the jugular vein, which is easily Soe ee pe oe
found by compressing it at the root of the rabbit or guinea-pig.
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 canula 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 quanti-
ties 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 ex-
tended. Anesthesia can be used, but must be employed with great
care. The hair on the front of the neck is clipped and the neck
shaved, or, as is easier, the hair is pulled out, leaving a clean surface

232 Experimentation upon Animals

an inch square. The skin is then washed with a disinfecting solu-
tion, an incision one and a half inches long made through the skin
and superficial fascia in the middle line of the neck, the tissues care-
fully separated, the deep fascia cautiously opened, the tissues sepa-
rated with the point of the forceps and a grooved director, the
sheath of the vessels 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 ex-
posure, and a ligature placed in position and loosely looped ready to
tie about the proximal end. A tube with a sharp lateral tubulature,
as is shown in the illustration, is now made ready by breaking off

Fig. 74.—Showing the method of taking blood from the carotid artery ,
of a rabbit.

the closed tip, the moistened forefinger of the operator is placed
beneath 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 cc. may be collected in about as many
seconds. An assistant now ties the artery at its proximal end, the
tube is withdrawn, holding it so that the blood does not escape, and
_ the end sealed in a flame. 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.

Many experimenters now adopt a more simple method of obtaining
the blood from guinea-pigs, and that is by introducing a needle
through the chest wall into the heart and withdrawing the blood into

Post-mortems 233

a sterile syringe. The animal’s chest wall should have the hair
removed over a sufficient area and the skin should be disinfected.
Several cubic centimeters may thus be withdrawn without killing a
large guinea-pig.

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 into
the pleural cavities. After coagulation the serum can be secured
by carefully pipetting it from the cavities. ,

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 expert 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 bot iron, a puncture made into it with a
sterile knife, and the culture made by introducing a platinum wire.

lf the bacteriologic examination cannot be made at once, the or-
gans to be studied should be removed with aseptic precautions,
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 examination
should be cut into small cubes (of 1 cc.) and fixed in Zenker’s fluid
or absolute alcohol.

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 their virulence
is increased.

The capsules can be made of any size, though they are probably
most easily handled when of about 5-10 cc. capacity. The size is
always an objection, because of the disturbance 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 sufficiently thick,
homogeneous layer is formed. During the coating process the tube

234 Experimentation upon Animals

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 intro-
duction of culture-media in a fine pipet or
inoculation with a platinum loop, and that will
permit of ready sealing in a flame when
necessary; the rounded end is cut off, and the
edges are smoothed ina flame.’ The upper open
end of the collodion bag is carefully fittéd over
the end of the tube, shrunk on by a gentle
heating, and cemented fast with a little fresh
collodion applied to the line of union. Novy

recommends that a thread of silk be wound
Gece 1 —Prepare around the point of union, to hold the collodion
a, Test-tubeconstric- in place and to aid in handling the finished
eicler aa i a * sac. The sac is next filled with distilled 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
introduced with a pipet, the constricted portion is sealed in a flame,
and the capsule picked up with forceps is introduced into the
peritoneal cavity by an aseptic operation.

The collodion capsules may be made of any size. Those for rabbit
experiments should be of about ro cc. capacity, those for guinea-pig
experiments about 5 cc. By coating large glass tubes they can be
made of 500 cc. capacity, the large bags being useful for chemic
dialysis.

CHAPTER XI
THE IDENTIFICATION OF SPECIES

THE most difficult thing in bacteriology is the identification 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 difficulty. 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 characteristic agar-agar growths. The red
color of Bacillus prodigiosus 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 hun-
dreds of described species that lack any one distinct characteristic
that may be used for differential purposes, and require that for their
recognition we shall well-nigh exhaust the bacteriologic technic.

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 Grun-
driss der Bakteriologie und Lehrbuch der speziellen bakteriologischen
Diagnostik,” by Lehmann and Neumann,* and the “Manual of
Determinative Bacteriology,” by R. 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 thoroughly,
step by step, and compare it with the description and tables. In
this regard the differentiation of bacteria resembles the determina-
tion of the higher plants with the aid of a botanic key, or the qualita-
tive analysis for the detection of unknown chemic compounds.
Such a key for specific bacterial differentiation is really indispensa-
ble, even though it be imperfect, and every student engaged in re-
search 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 com-
paratively 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 suffi-
ciently described forms.” ‘That typical forms or species of bac-
teria do exist, no one can deny. These typical forms furthermore

* J. F. Lehmann, Miinchen, 1907.
235 :

236 The Identification of Species

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 pos-
sible, is outlined in the following tables:”

A PROPOSED SYNOPSIS OF GROUPS OF BACTERIA

‘BACTERIUM

I. Without endospores.
A. Aérobic and facultative anaérobic.
a. Gelatin not liquefied. :
* Decolorized by Gram’s method.
{ Obligate aérobic. AcETIc FERMENT Group.
tt Aérobic and facultative anaérobic.
Gas generated in glucose bouillon.
Gas generated in lactose bouillon. Bact. ARocEnes
Group. ;
Little or no gas generated in lactose bouillon. Frrep-
LANDER GROUP.
No gas generated in glucose bouillon.
Milk coagulated. Fowr CHOLERA Group.
Milk not coagulated. Swine PLacur Group.
** Stained by Gram’s method.
t Gas generated in glucose bouillon. Lactic FERMENT Group.
b. Gelatin liquefied.
* Colonies on gelatin ameboid or proteus-like. BACT. RADIATUM
Group. '
** Colonies on gelatin round, not ameboid. Bacr. ampicuum Grove.
II. Produce endospores.
1. No growth at room temperature, or below 22°25°C. THERMOPHILIC
Group.
2. Grow at room temperatures.
a. Gelatin liquefied. ANTHRAX Group.
b. Gelatin not liquefied. Bact. FacaLis Group.

BAcILLus

I. Without endospores.
A. Aérobic and facultative anaérobic.
a, Gelatin colonies roundish, not distinctly ameboid.
* Gelatin not liquefied.
} Decolorized by Gram’s method.
Gas generated in glucose bouillon.
Milk coagulated. Coton Group.
Milk not coagulated. Hoc CHoLERrA Group.

No gas generated in glucose bouillon. Typuoi Group.

tt Stained by Gram’s method. B. muriprstrreR GROUP.

** Gelatin liquefied. 2 :
} Gas generated in glucose bouillon. B. cLoac& Grouvp..

tt 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. PRoTrus vuLGARIS GROUP.
** Gelatin not liquefied. B. zoprt Group.
II. Produce endospores.
A, Aérobic and facultative anaérobic.
«. Rods not swollen at sporulation.
a, Gelatin liquefied. «

Chester’s Synopsis of Groups of Bacteria 237

* Liquefaction of the gelatin takes place slowly. Ferment
urea, with strong production of ammonia. Uro-BaciLLus
Group or MiquEL. y
** Gelatin liquefied rather quickly.
{ Potato cultures rugose. Potato Bacrttus Group.
Tf Potato cultures not distinctly rugose. B. susprizis Group.
b. Gelatin not liquefied. B. sorr Group.
2. Rods spindle-shaped at sporulation. B. LICHENIFORMIS GRouP.
3. Rods clavate at sporulation. B. suBLANATUS GRoUP.
B. Obligate anaérobic.
1. Rods not swollen at sporulation. MALIGNANT Eprema Group.
2. Rods spindle-shaped at sporulation. CLosTRrpIuM Group.
3. Rods clavate-capitate at sporulation. Trranus Grove.

Psrupomonas (Migula)

I. Cells colorless, without a red-colored plasma and without sulphur granules.
A. Grow in ordinary culture-media.
1. Without endospores.
a. Aérobic and facultative anaérobic.
*Without pigment.
¢ Gelatin not liquefied.
Gas generated in glucose bouillon. Ps. MONADIFORMIS
Group.
No gas generated in glucose bouillon. Ps. amBicuA Group.
TT Gelatin liquefied. :
Gas generated in glucose bouillon. Ps. coADUNATA GROUP.
No gas generated in glucose bouillon. Ps. FAIRMONTENSIS
Group.
*Produce pigment on gelatin or agar.
{ Pigment yellowish.
Gelatin liquefied. Ps. ocuracea Group.
Gelatin not liquefied. , Ps. ruRcosa Group.
tt Pigment blue-violet.
Gelatin liquefied. Ps. JANTHINA GROUP.
Gelatin not liquefied. Ps. BEROLINENSIS GROUP.
** Produce a greenish-bluish fluorescence in culture-media.
} Gelatin liquefied. Ps. pyocyaNrA Group.
t? Gelatin not liquefied. Ps. syNcyANEA GROUP.
2. With endospores, aérobic and facultative anaérobic.
a. Non-chromogenic.
* Rods not swollen at sporulation. Ps. RosEA Group.
** Rods swollen at one end at sporulation. Ps. TRomMMEL-
SCHLAGER GROUP.
b. Produce a greenish-bluish fluorescence in culture-media.
* Gelatin liquefied. Ps. virIDESCENS Group.
** Gelatin not liquefied. Ps. uNDULATA Group.
B. Be not grow in nutrient gelatin or other organic media. NITRIMONAS
ROUP. .
IT. Cell plasma with a reddish tint, also with sulphur granules. CHROMATIUM
Group.
Microspira (Migula)

I. Cultures show a bluish-silvery phosphorescence. PHOSPHORESCENT GROUP.
TI. Cultures not phosphorescent. ” :
A. Gelatin liquefied.
1, Cultures show the nitro-indol reaction. a
a. Very. pathogenic to pigeons. Msp. METCHNIKOVI 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.

Mycosacterium (Lehmann-Neumann)

I, Stain with basic anilin dyes, and easily decolorized by mineral acids when
stained with carbol-fuchsin.

238 The Identification of Species

A. Grow well on nutrient gelatin. Gelatin liquefied very slowly or merely
softened.

1. Stain by Gram’s method. Swine ErysipEtas 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—ir
regularly 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.
b. Do not stain by Gram’s method. INFLUENZA Grovp.
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 decolorizec: —

by acids. TUBERCLE GROUP.

CoccacE&

Cells in their free condition globular, becoming slightly elongated before division.
Cell division in one, two, or three directions of space.

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).

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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 contamination
in our bacteriologic researches and occasionally a menace to our
health.

Such ‘aérial organisms are neither ubiquitous nor uniformly
disseminated, but are much more numerous where the air is polluted
and dusty than where itis pure. The purity of the atmosphere bears

_adistinct relation to the purity of the surfaces over which its currents

blow.
The micro-organisms of the air are for the most part harmless
saprophytes taken up and carried about by the wind. They are

almost always taken up from dry materials, experiment 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 didia as well.

In some cases the bacteria are pathogenic, especially where dis-
charges 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. However, because of the expectora-
tion from cases of tuberculosis, influenza, and pneumonia, which is
often ejected upon the sidewalks and floors of public places, the

" presence of occasional pathogenic bacteria is far from uncommon in

street-dust.

Giinther 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 varieties 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 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 difficult.
Several methods have been suggested, of which the most important
may be briefly mentioned:

Hesse’s method is simple and good. It consists in making 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, the interior of which is coated with a film

239

240 The Bacteriology of the Air

of gelatin in the same manner as an Esmarch tube. The tube is clased 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
removed from the other end, and the air slowly passed through, the bacteria hav-
ing 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 represent pretty accurately the number of
bacteria in the volume of air that passed through the tube.

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 probably 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. 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-gauze coverings, are superimposed. One or both ends

Fig. 76.—Hesse’s apparatus for collecting bacteria from the air.

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 too 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 from the upper filter isthen

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 sedimenta-
tion. Sternberg 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. a

Sedgwick’s Method.—Sedgwick and Miquel have recommended the use of
a soluble material—granulated or pulverized 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, suggested by Sedgwick and Tucker,
has an expansion above the filter, so that as soon as the sugar is dissolved in the

aCe

Sedgwick’s Method 241

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 ro00 per cubic meter.

The bacteriologic examination of air is of very little importance
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

Fig. 77—Petri’s sand filter for air- Fig. 78.—Sedgwick and Tucker’s ex-
examination. panded tube for air-examination.

one walk across the floor so that dust rises, and the number of bac-
teria becomes considerably increased; if the room be swept, the in-
crease is enormous. From these and similar contingencies it be-
comes very difficult to know just when and how the air is to be ex-
amined, and the value of the results is correspondingly lessened.

The most sensible studies of the air aim rather at the discovery
of some definite organism or organisms than at the determination
of the total number per cubic meter. :

CHAPTER XIII
’ BACTERIOLOGY OF WATER

UnteEss water has been specially sterilized, and received and kept
in sterile vessels, it always contains some bacteria, the number
_ usually bearing a distinct, relationship to the quantity of organic
matter present.

The majority of the water bacteria are bacilli, and are as a rule
non-pathogenic. The bacteriological examination of water is directed |
toward two objectives, first, the determination of the number of
bacteria in a given quantity of the water; second, its purity or impurity
from the standpoint of sewage pollution and potability. A third
objective is sometimes added, namely, the demonstration of the
presence of the specific micro-organisms of typhoid and para-typhoid
fever, of dysentery and of cholera. The first two are comparatively
easy to perform and are regularly carried out in many municipalities

Fig. 79.—Wolthiigel’s apparatus for counting colonies of bacteria upon plates.

and water works; the third is so rarely successful that it is attempted
only under exceptional conditions.

I. The Determination of the Total Number of Bacteria in a Given
Sample of Water.—The method is very simple, and depends upon
the equal distribution of a measured quantity of the water to be
examined in some sterile liquefied medium, whose subsequent
solidification in a thin layer permits the colonies to be counted. It
is, however, of the utmost importance that the water to be examined
shall be in every respect unchanged by manipulation and the occur-
rence of artificial conditions before the examinations are made.

In the book upon “Standard Methods for the Examination of
Water and Sewage” published by the American Public Health
Association, Boston, 1917, the best suggestions and methods can be
found, and from that work the majority of our recommendations
have been selected. :

_ The samples for bacterial analysis should be collected in bottles
: 242

Determination of Bacteria in Water 243

that have been cleaned with great care, rinsed in clean water and
sterilized with dry heat for at least one hour and a half at 170°C., or
in an autoclave at 15 lbs. (120°C.) for fifteen minutes or longer after
the pressure reaches 15 lbs. Great care should be exercised to have
the samples representative of the water to be tested, and to see that
no contamination occurs at the time of filling the sample bottles.

The samples should be examined as promptly as possible after
collection as rapid and extensive changes take place in the bottled
samples even when stored at temperatures as low as 10°C.

The time allowed for storage and transportation of a bacterial
sample between the filling of the bottle and the beginning of the
analysis should not be more than six hours for impure waters and
not more than twelve hours for relatively pure waters. During the
period of storage the temperature should be kept as nearly at ro°C.,
as possible.

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 1:10 or 1:100 with sterile water,
mixing well. From the water sample, with or without dilution as
may be appropriate, 0.01; 0.1; and 1 cc. respectively are carefully
measured with a sterile pipet into a tube of melted agar-agar cooled
to a temperature that can be comfortably held in the hand.
After thorough mixing without shaking or forming bubbles, the con-
tents are poured into a sterile Petri dish which is inverted when the
medium solidifies, and is then stood in the incubating oven for
twenty-four hours.

It is best to count all the colonies developed upon the culture, if
‘possible; but when hundreds or thousands are scattered over it, an
estimate can be made by counting the number of colonies in each
of several of the divisions of some counting apparatus, such as have
been devised by Wolfhiigel, Esmarch, or Frost, and computing the
total number on the plate. In counting the colonies a lens is
indispensable.

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 sound an average of
398 colonies in a cubic centimeter.

244 Bacteriology of Water

II. The Determination of the Purity of the Water from the
Standpoint of Sewage Pollution.—The chief interest in ordinary
bacteriological examinations centers about sewage contamination as
indicated by the presence of numbers of the Bacillus coli group. It
is therefore recommended that the B. coli group be considered as
including all non-spore-bearing bacilli which ferment lactose with gas
production and grow anaérobically on standard solid media. The
formation of ro per cent. or more of gas in a standard lactose broth
fermentation tube within twenty-four hours at 37°C. is presumptive
evidence of the presence of members of the B. coli group, since the
majority of the bacteria which give such a reaction belong to this

Lf :
Hh Ah
ACEP EE
ANSZECNV
Se Th

SSaee a
ae

Fig. 80.—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.

group. To determine this one carries out the so-called presumptive
test.

A. Presumptive Test.—For this purpose one employes lactose
bouillon. lt is prepared by the addition of 3 grams of beef
extract and 5 grams of peptone to the 1ooo cc. of distilled water,
heating slowly on a steam bath to at least 65°C., until the ingredients
are dissolved. The lost weight is then made good and the reaction
adjusted by titration to + 1, when it is cooled to 25°C., and filtered
through filter-paper until lear, It then receives an addition of 1
. per cent. of chemically pure lactose, after which it is distributed in

Determination of Bacteria in Water 245

fermentation tubes, and sterilized in an autoclave at 15 lb. (120°C.)
for fifteen minutes after the pressure reaches 15 lb.

The tubes thus prepared are inoculated with appropriate quantities
of the water—say 10 cc., 1 cc. and }(9 cc.—and then incubated at
37°C. for forty-eight hours. If at the end of twenty-four hours more
than ro per cent. of gas occurs in the closed arm of the fermentation
tube the presumptive test is positive. If no gas is formed or if

‘ there be less than ro per cent. the incubation shall be continued to
forty-eight hours. The presence of gas in any amount in such a
tube, constitutes a doubtful test; the absence of gas after forty-eight
hours incubation constitutes a negative test.

B. The Partially Confirmed Test.—This consists in making one or
more Petri dish cultures, using either Endo’s medium (q.v.) or lactose-
litmus-agar (q.v.) and water from the fermentation tube showing gas
with the smallest amount of water tested.

The plates are incubated at 37°C. for eighteen to twenty-four
hours. If typical red colon-like colonies have developed upon the
plate within this period, the confirmed test may be considered to
be positive. If no typical colonies develop within twenty-four
hours the test cannot yet be considered negative as sometimes the
members of the B. coli group fail to form typical colonies in Endo’s
medium or litmus-lactose-agar within that time. In such cases it is
necessary to carry the test to a third step.

C. The Completed Test-—From the Petri dish cultures upon the
Endo’s medium or the litmus-lactose-agar, two typical colonies are
transferred, one to an agar slant, the other to a lactose broth fermen-
tation tube. If no typical colonies have developed at the end of the
twenty-four hour period, the dishes are reincubated for another
twenty-four hours, after which two, at least, of the colonies consid-
ered to be most like B. coli, are transplanted to agar slants and
lactose broth fermentation tubes.

' The transplants are incubated at 37°C. as usual, the formation of
gas being noted up to a period not exceeding forty-eight hours in the
fermentation tubes and microscopical examinations made of the agar
cultures. The formation of gas in the fermentation tubes and the
demonstration of non-spore-bearing bacilli on the agar may be con-
sidered a satisfactory completed test demonstrating the presence of
a number of the B. coli group. Absence of gas formation in lactose
broth or failure to demonstrate non-spore-forming bacilli in a gas-
forming culture constitutes a negative test.

' The potability of water is not determined by the bacteriological
analysis alone, but also by chemical analysis, optical condition, and
odor. So far as the bacteriology itself is concerned the standard
adopted in the Public Health Reports, Nov. 16, 1914, Vol. xlv, No. 29,
P. 2959, requires that the total number of bacteria shall not exceed
too per cubic centimeter when grown upon standard agar-agar plates
and counted after twenty-four hours incubation at 37°C. Further

Bacteriology of Water

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248 Bacteriology of Water

the presence of micro-organisms of the B. coli group shall not be shown
in more than one out of fivesamples of rocc.each. -Thereis, however,
no formal agreement as to how few or how many colon bacilli shall
make the water condemnable. It goes without saying that a water
without colon bacilli, such as can often be obtained from deep artesian
wells is better than surface waters containing afew. Howfewshallbe
regarded as consistent with safety remains, at present, a matter of
opinion among sanitarians, and varies widely in practice according to
the local conditions obtaining in different cities.

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 bac-
teria, 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
nourishment 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 dissemi-
nated 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. Frinkel, and others show that the
bacteria of the soil do not penetrate deeply, but gradually decrease
in number until the depth of a meter is reached, then rapidly di-
minish until at a meter and a quarter they rather abruptly disappear.

The bacteria of soil are, for the most part, harmless saprophytes,
though a few highly pathogenic organisms, such as the bacilli of
tetanus and malignant edema, occur. Many of them are anaérobic,
and it is interesting to speculate upon their biology. Whether they
develop and multiply in the soil in intimate association with strongly
aérobic organisms by which the free oxygen is absorbed, or whether
they remain 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, origi-
nated an accurate method of determining it. By means of a special
boring apparatus earth can be secured from any depth without
digging and without danger of mixing with that of the superficial
strata. A 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 alens. Fliigge found in virgin earth about
100,000 colonies in a cubic centimeter.

Samples of earth, like samples of water, should be examined as
soon as possible after being secured, for, as Giinther points out, the
number of bacteria changes because of the unusual dryness, warmth,
exposure to oxygen, etc.

249

250 Bacteriology of the Soil

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. Seen

2. They furnish conditions more suited to the multiplication of bacteria
than do virgin soils, unless the latter are contaminated 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 isolated and carefully studied
by Ravenel, there were two cocci, one sarcina, and five cladothrices;
all the others were bacilli.

*“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 con-
veyed to the body.

Il. Foods are chemnieallyy 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 diseased animals. Thus, milk
apparently normal in appearance has been found to contain danger-
ous pathogenic bacteria. The tubercle bacillus is one of the most
important of these, and at the present time the consensus of opinion
inclines toward the view that the great prevalence of tuberculosis
among human beings depends partly upon the ingestion of tubercle
bacilli in milk. It does not appear necessary that the udder of the
cow be diseased in order that the organisms 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,
imperative 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 centi-
meters 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 like cream. Such seriously in-
fected milk could not be used with safety to the consumer.

In market milk one occasionally finds pathogenic organisms, such
as the diphtheria bacillus, typhoid bacillus, streptococcus, etc., de-
rived 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

*“Maryland Medical Journal,’ Jan. 9, 1897.
251

252 The Bacteriology of Foods

always be treated with the greatest care to prevent such contamina-
tion, 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 infants 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 Hygienic 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. Dutton & 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 Bae with bacteria in a variety of ways
that will suggest themselves to the reader. The common 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 cleanli-
ness should prevail for the sake of the dealer, whose goods may be
spoiled by carelessness, and the consumer, who may be injured by
the food.

Shell-fish, especially oysters, seem to be common carriers of infec-
tion, especially of typhoid fever. The oysters seem to be contami-
nated 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 consump-
tion 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.—The nomenclature, suggested by Vaughan and
Novy,{ contains the following terms:

Bromatotoxism—food-poisoning;
- Galactotoxism—umilk-poisoning;

T yrotoxism—cheese-poisoning ;

Kreotoxism—meat-poisoning ;

* “Deutsche med. Wochenschrift,” 1900, No. 26; abstract in the “Centralbl.
f. Bakt.,” etc., 1901, XXIX, p. 300.
{Cellular Toxins,” Phila., 1902.

Food Poisons 253

Ichthyotoxism—fish-poisoning ;
M ytilotoxism—mussel-poisoning ;
Sitotoxism—cereal-poisoning.

The most important chemic alterations effected by bacteria occur
in milk and meat.

1. Milk-poisoning (Galactotoxism)-—Milk, even when freshly
drawn from the cow, always contains some bacteria, whose numbers
gradually diminish for a few hours, then rapidly increase until
almost beyond belief. These organisms are for the most part
harmless to the consumer, but ultimately 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 satis-
factory 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 presence of a ptomain
known as ¢yrotoxicon, formed by the growth of bacteria in the milk,
‘but whether by 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 bacteria 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 bac-
teria has led to the poisonous condition.

Cheese-poisoning (Tyrotoxism) 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 (Kreotoxism).—It was originally supposed
that the action of micro-organisms upon meats brought about
chemical changes resulting in the appearance of toxic ptomains
by which those eating it might be poisoned. It is now known that
such a change is rare. In 1888 Gartner* investigated a group of
fifty-seven persons who became ill, and some of whom died after
eating meat from a certain cow. From the flesh of this animal, and
also from the blood and spleen of one of the patients, he isolated a
bacillus which he named Bacillus enteritidis (g.v.). It has since
proved to be a member of a group of bacilli standing in an inter-
mediate position between the typhoid group and the colon group,
all of which are characterized by the production of an endotoxin

* Corrsp. BI. d. Aerzt. Vereins. Turingen, 1888.

254 The Bacteriology of Foods

that is highly toxic and not destroyed by heating. Human beings
consuming meat infected by these micro-organisms, and especially
by Bacillus enteritidis, may be seriously and perhaps fatally in-
fected if the bacilli are alive, or more or less seriously intoxicated
if the bacilli are dead as the result of the cooking.

One of the most important forms of meat poisoning is that known
as allantiasis (addas, a sausage) or botulism (botulus, a sausage).

, ALLANTIASIS, BOTULISM OR SAUSAGE POISONING
Bacillus Botulinus (von Ermengem)

General Characteristics—A large, motile, flagellated, anaérobic, aérogenic,
sporogenic, liquefying, non-chromogenic, pathogenic bacillus, staining by ordin-
ary solution of anilin dyes, and by Gram’s method.

In 1896 in the town of Ellezelles in Belgium a considerable number
of persons were taken with a peculiar illness, the nature of which
was obscure. Upon investigation it was found that they had all
eaten portions of a certain imperfectly cured ham. An investiga-

1 .

Fig. 82.—Bacillus botulinus (Kolle and Wassermann).

tion on the part of von Ermengem* resulted in the discovery, in the
ham, of the micro-organism to which he gave the name Bacillus
botulinus. It has since been much studied and has taken its place
as one of the most important micro-organisms of food-poisoning.

The organism is by nature a saprophyte of limited distribution.
Von Ermengem examined 52 samples of miscellaneous earths and
fecal matter and failed to find it. Kempner and Pollak} found it in
the dung of healthy hogs. Rémer{ found it in a remnant of ham
that had caused illness. The flesh was dotted with spots and per-
vaded by gas pockets. In both the original case studied by von
Ermengem and this later case studied by Rémer, the anaérobic
Bacillus botulinus grew in association with an aérobic coccus.

* Zeitschr. fiir Hygiene,” 1897, xxvi, p. 1.

t “Deutsche med. Wochenschrift,” 1897, No. 52.
t “Centralbl. f. Bakt.,” etc., 1900, xviI, 857.

Food Poisons 255

Morphology.—It is a large bacillary organism of a somewhat
quadilateral shape with rounded corners or sometimes with entirely
rounded ends, usually single or in pairs, and only in old cultures
with numerous involution forms occurring in the form of filaments.
It measures 4 to 64 X 0.9 to r.2u.

Motility —The motility is sluggish. Thereare petitrichial flagella,
but not usually more than eight.

Sporulation.—The bacillus forms good-sized oval spores that
are situated near one end and slightly increase the diameter of the
rod at that point. No distinct. drum-stick appearance occurs.
The spores are killed by exposure to 80°C. for an hour.

Staining.—The organism stains well by ordinary methods, and by
Gram’s method. It is not acid-fast.

Cultivation.—Bacillus botulinus is a strict anaérobe, and when
appropriate conditions are present, grows readily upon nearly any
culture-medium. The best development takes place at tempera-
tures ranging from 18° to 25°C. As it liquefies gelatin, it is recom-
mended that its isolation be undertaken upon slightly alkaline dex-
trose gelatin, kept at 25°C...

Colonies.—Colonies appear under anaérobic conditions and are
round, translucent, pale, yellow-brown and coarsely granular. After
some hours a liquid zone surrounds the colony, and as the lique-
faction of the gelatin continues, the coarse granules keep up a
constant slow streaming movement. When the maximum size is
attained, the colonies become brown and opaque.

Bouillon. —In plain bouillon a slight turbidity can be observed in
twenty-four hours. In glucose broth there is a dense turbidity.

Agar-agar.—The surface growth is rarely seen because of the
strict anaérobic conditions required. Colonies are in general round,
granular and yellowish-brown.

Gelatin.—Liquefaction begins to take place in a few hours and
progresses slowly.

Mica Slight acidulation occurs without conzulatien or pepton-
ization. Acid is produced.

Metabolic Products.—It liquefies gelatin through the formation
of a gelatinase that appears in bouillon and solid cultures. It

‘ ferments dextrose with the production of CHy,CO,and H. Butyric
acid is given off from appropriate substrata. It also forms a very
powerful exotoxin that saturates the fluids of the culture-media.

Toxin.—This is best prepared for study by cultivation in a sugar-
free bouillon at 25°C. for two weeks and then filtering through sterile
porcelain; o.cor' cc. of such a medium kills guinea-pigs in from
three to four days. Rabbits are also susceptible and not only die
when subcutaneously injected, but also if fed upon food to -which
0.1 to 0.5 cc. of the toxin has been added. The toxin is poisonous
to man, monkeys, kittens, rats, white rats, mice, and rabbits.
The toxin is destroyed by 80°C. in thirty minutes.

256 The Bacteriology of Foods

Pathogenesis.—The effects in man and animals are somewhat
similar. In the former, with or without any sign of gastro-
intestinal disturbance, the patient is seized with chilliness, vertigo,
tremor, prostration, faintness, feeble and accelerated pulse and
respiration, profuse salivation, muscular weakness and palsy,
protrusion of the eyeballs, dysphagia and various other nervous
disorders, ending fatally in bad cases. In some outbreaks of the
intoxication a mortality as high as 25 per cent. has been observed.

Bacteriological Diagnosis.—The suspected meat may furnish the
clue. As the patient is probably intoxicated but not necessarily
infected, not much can be learned by any examination of him.

A sample of the meat may show nothing abnormal to the naked
eye. A fragment of it may be macerated in sterile salt solution for
making the necessary tests. When the salt solution has taken up all
that it can extract, it is heated to 60°C. for half an hour to destroy all
but the spores of the B. botulinus, and then is planted on gelatin-
dextrose plates, and into fermentation tubes with dextrose broth.
These are placed under conditions of as perfect anaérobiosis as pos-
sible and kept at 25°C. to grow.

In cases of very bad infection of the meat, enough toxin may be
extracted by the salt solution to poison guinea-pigs. This may be
tried, the salt solution extract being sterilized by filtration through.
porcelain before being injected into the animals.

Prophylaxis.—Thorough cooking of meat is a very important
sanitary precaution in all cases, destroying all of the animal para-
sites, as well as most of the bacteria and their spores and the toxin of
B. botulinus. It does not, however, destroy the toxin of B. enterit-
idis (q.v.).

Treatment.—Kempner* has succeeded in preparing an antitoxin
that possesses both preventive and curative powers, but the manner
in which the intoxication occurs, makes it difficult to apply in
practice.

3. Fish-poisoning (Ichthyotoxism) sometimes follows the con-
sumption of canned and presumably spoiled fish, sometimes the
consumption of diseased fish. It is not known whether it depends
upon ptomains or upon toxicogenic germs, though probably the
latter as Silber has isolated a Bacillus piscicidus that is highly
toxicogenic.

4. Mussel-poisoning (M ytilotoxism) depends partly upon irri-
tating and nervous poisons in the mussel substance, in part upon
toxicogenic germs that they harbor.

5. Canned Goods.—Improperly preserved canned goods not in-
frequently spoil because of the growth of bacteria, but the occur-
rence of gas-formation, acidity, insipidity, etc., causes rejection of
the product, and but few cases of supposed poisoning from canned
goods can be authenticated.

* “Zeitschr. fiir Hygiene,” '1897, xxvi, 482.

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
fatal temperature 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 ordinary determinations
itis 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 ob-
served 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-poirit. If all the transplantations grow,
of course the maximum temperature was not reached, and the ex-
periment 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 tempera-
ture as 60°C. without losing its vitality, a dozen or more bouillon-
tubes may be inoculated with the organism to be studied, arid stood
in a water-bath kept at the temperature to be investigated. The
first can be removed as soon as it is heated through, another in five
minutes, 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, 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 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. Ap-
proximately 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

17 257

258 The Thermal Death-point

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 ngcessary is to stand them
aside for a day or two, and observe whether or not the bacteria
grow, determing the death-point exactly as in the other case. j,

CHAPTER XVII

DETERMINATION OF THE VALUE OF ANTISEPTICS,
GERMICIDES, AND DISINFECTANTS

. THE student must bear in mind that an antiseptic is a substance
capable of restraining the growth of bacteria; a germicide, one ca-
pable of killing them. All germicides are antiseptic in dilute solu-
tions, but not all antiseptics are germicides. Disinfectants must be
germicides.

Antiseptics are chiefly employed for purposes of preservation,
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 chem-
ically 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 obj ject desired might
be defeated.

The following methods of determining the antiseptic and germi-
cidal values of various agents can be elaborated according to the
extent and thoroughness of the investigation to be made.

I. The Antiseptic Value-—Remembering that an antiseptic is a
substance that inhibits bacterial growth, the determination of its
value can be made by adding varying quantities of the antiseptic
to be investigated to culture-media in which bacteria are subse-
quently planted. It is always well to use a considerable number of
tubes of bouillon containing varying strengths of the reagent to be
- investigated. 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 precaution

as regards asepsis, after sterilization and immediately before the test
is made. Control experiments—~.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 development, it will usually be
observed that those containing very small quantities develop al-
most as rapidly as the control tubes; those containing more, a little

250

‘260 , Value of Antiseptics

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 anti-
septic may be incompatible (as bichloride of mercury and albumin),
2. The nature of the test-organism, no two organisms being ex-
actly alike in their susceptibility.
“3. The temperature at which the experiment is conducted, a

Same rod immersed in broth after
exposure to disinfectant.

Fig. O35 —Glass rod in test-tube, for use in testing disinfectants. Tube
6 in. by 34 in.; rod 9 in. by 34 in. Ring marked with diamond 1 in. from lower
end, to show upper limit of area on which the organisms are dried. After ex-
posure the rod is placed in a similar tube containing broth, to test development.
a, Cotton plug wrapped around glass rod; b, broth; c, gummed label on handle
of rod. for identification; d, ring marked by diamond; e, dried organisms.

relatively greater amount of the antiseptic being necessary at tem-
peratures favorable to the organism than at temperatures un-
favorable.

4. The presence of spores which are always more resistant dine the
asporogenous forms.

II. The Germicidal Value.—Koch’s original method of determin-
ing 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 germi-
cidal solution. After the bath in the reagent the threads were

Testing Germicidal Value of Liquids 261

washed in clean, sterile water, transferred to fresh culture-media, and
their growth or failure to grow observed. This method also deter-
“mines the time in which a certain solution will kill micro-organisms, so
is advantageous.
Sternberg suggested a method by which the dilution necessary 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 quantity of a recent culture—usually 5 cc.—is
mixed with an equal quantity of a standard solution of the germi-
cidal agent, . . . and after two hours’ contact one or two loopfuls
are transferred to a suitable nutrient medium to test the question
of disinfection.”

A very simple and popular method of determining the germicidal
value is to make a series of dilutions of the reagent to be tested; add
to each a small quantity of a fresh liquid culture, and at varying in-
tervals of time transfer a loopful to fresh culture-media. By a little
ingenuity this method may be made to yield information as to both
lime and strength.

Hill* has suggested a convenient method of handling the cul-
tures, which are dried upon the ends of sterile glass rods and can then
be transferred from one solution to another or otherwise manipulated.

The Modern Method of Testing the Germicidal Value of Liquids.
—The methods of testing germicidal strength given above are uncer-
tain and inaccurate, and can only be looked upon as “rough and
ready”? methods, that should be willingly abandoned for anything
better. Three methods are now offered that hold out the promise of
scientific accuracy through an established standard of comparison.
In the order of their appearance, which is also, probably, the order of
their importance, these are the method of Rideal and Walker,{
“The Lancet Method,’’t and the method of Anderson and McClintic.§
The methods are similar in general principles, and have the same
object in view, i.e., the expression of the germicidal value of any sub-
stance as the carbolic acid or phenol ‘‘coefficient.” Experience
with the methods leads to the conviction that the Rideal and
Walker method is the more easy to execute, but that the Anderson-
McClintic method is the more accurate. As the latter in addition to
its accuracy has now become the standard method of the United
States Government, it is the method with which the student should
be acquainted and which will be given in detail.

I. The Apparatus, Reagents, etc., Required for the Test—1. A
Phenol Solution that shall act as the standard of comparison. In the .
preparation of this solution, pure phenol—as free from cresols, etc.,

* “Public Health,” vol. xx1v, p. 246.

{Journal of the Royal Sanitary Institute, London, 1903,'p. 424.

“The Standardization of Disinfectants’ (unsigned article), Lancet, London,

vol. Cixxvi, Nos. 4498, 4499, and 4500.
§ Bulletin No. 82 of the Hygienic Laboratory, Washington, D. C., 1912.

262 Value of Antiseptics

as possible—should be employed. Walker recommends that only
phenol with a melting point of 40.5°C., be used, as only such is en-

tirely free from impurities. The Eighth Revision of the U.S. Phar-- . -

macopceia declares phenol with a melting point of 40°C. to be pure
and that is the quality that may be accepted as the standard. |

The phenol used at the Hygienic Laboratory is Merck’s “Silver
Label.” The standard dilution, made by the U. S. P. method
(Koppeschaar), contains exactly 5 per cent. of pure phenol by weight,
in distilled water. From this stock solution, the higher dilutions are
made fresh each day for that day’s tests.

2. The Solution to be Tested—A 5 per cent. solution is made by
adding 5 cc. of the disinfectant to 95 cc. of sterile distilled water
with a standardized 5 cc. capacity pipet. After filling the pipet,
all excess of the disinfectant on its outside is wiped off with sterile

“gauze, The contents of the pipet are then delivered into a cyl-
inder containing 95 cc. of sterile distilled water and the pipet
washed out as clean as possible by aspiration and blowing out the
contents into the cylinder. The contents of the cylinder are then
thoroughly shaken.

3. The Test Organism selected is Bacillus typhosus. Before be-
ginning the tests, the organisms in bouillon culture should be trans-
planted to fresh media every twenty-four hours for at least three
successive days. In making the transfers one loopful of a 4-mm.
platinum loop is carried over. In exposing the culture to the dis-
infectant, 149 cc. of the culture is always added to 5 cc. of the
diluted disinfectant, the amount being measured by pipets graduated
in tenths of a cubic centimeter.

4. The Inoculating Loops.—These loops are made of No. 23 U.S.
standard gauge platinum wire, each loop being 4 mm. in diameter.
There should be four, and preferably six, such loops mounted in
the usual glass handles, ready for use. In order to facilitate their
sterilization, a special holder is used.

5. The Water-bath—As variations in the temperature of the
disinfecting solutions hasten or retard their destructive action, a
temperature of 20°C. has been arbitrarily adopted as the standard.
For its maintenance the following simply constructed water-bath
has been devised. It consists of a wooden box 20 inches deep, 21
inches long and 21 inches wide. Inside this box a 14-quart agate-
ware pail, 10 inches deep, is placed and saw-dust is well packed
around, sufficient being placed in the bottom of the box to bring
the rim of the pail on a level with the top of the box. A tightly
fitting wooden cover, so made that the edges project slightly over
the rim, is placed over the pail. .In the cover are a sufficient number
of holes for the seeding tubes, a thermometer, and the tube contain-
ing the culture. About 3 inches below the rim of the pail a false
bottom of wire gauze is placed; this is for the seeding tubes, etc.,
to rest on. Water is placed in the pail to within half an inch of

- Testing Germicidal Value of Liquids 263

the top. When an experiment is about to be made the tempera-
ture of the water in the pail is taken, and if above or below 20°C.,
4 ‘

Fig. 84.—Water-bath showing position of holes for seeding tubes and ther-
mometer in place (Anderson and McClintic, in Bulletin No. 82, Hygienic
Laboratory).

Fig 85.—Cross section of .water-bath showing seeding tubes in place (Ander-.
son and McClintic, in Bulletin No. 82; Hygienic Laboratory).

it is brought to the desired temperature by the addition of either
hot or cold water. When the proper temperature has thus been

264 Value of Antiseptics

adjusted, very little change takes place in an none time. The
apparatus is shown in the cut.

6. The Culture-media used for the primary cee and for the
subcultures, made after exposure of the micro-organism to the dis-
infectant, is nutrient bouillon made with Leibig’s beef extract in
the usual manner and given a reaction of exactly + 1.5. Anderson
and McClintic achieve this by so carrying out the titrating of the ©
medium that a distinctly perceptible pink color marks the point at
which the addition of the alkali stops (see directions for titrating
culture-media).

7. The Tubes for the culture and subcultures are ordinaty culture’
tubes, containing 5 cc. of the nutrient bouillon mentioned above.
_They are filled, plugged and sterilized in the usual manner.

The tubes for ‘‘seeding,” 7.e., exposing the bacteria to the ger-

* micide, are more convenient when shorter. At the time of transfer,
the platinum loop is to be introduced into the tube as it stands in
the water-bath and as this is not easy with tubes of standard length,
Anderson and McClintic recommend tubes 1 inch in diameter and
3 inches long. These are plugged and sterilized by dry heat,
or as recommended by the authors quoted, are sterilized mouth
down, without plugs in a paper-lined wire basket.

8. The Dilution of the Phenol and Test Solutions —This is done in
standardized graduates with standardized pipets, according to
the requirements of the particular case. Anderson and McClintic
give tables that are useful for making the dilutions, though with the
aid of a little arithmetic it is easy to calculate the proportions of
the 5 per cent. solutions already prepared, and sterile distilled water
necessary to make the test solutions required. As it is certain that
some of the dilutions will be below germicidal strength, and as

“weeds” may be more difficult to kill than the test organism (B.-
typhosus) it is important to see that the distilled water used for
dilution is sterile, and that the cylinders and bottles or pipets
used for making the dilutions are all sterile and that the dilutions
themselves are made with aseptic precautions.

Under the standard conditions recommended, the phenol solu-
tion that destroys all of the B. typhosus introduced, in two and one-
half minutes is 1 : 80, but it is always wise to make additional dilutions
to control the strength, as shown in the table below. When the
strength of the disinfectant or germicide to be tested is entirely
unknown, it is well to begin by making a number of tests with
widely separated dilutions, by one of the “rough and ready”
methods, so as to arrive at an approximate strength, before
commencing the more difficult technic required for the determination
of the phenol coefficient, which should be looked upon as the final
test for exact comparison.

9. Racks for Holding the tubes are indispensable. The ‘‘seeding
tubes,” that is, the tubes in which the actual exposure of the culture

Testing Germicidal Value of Liquids 265

to the germicidal solutions is to take place, have already been pro-
vided for in the construction of the water-bath.

For the “subculture” tubes, any test-tube rack will do, but it
is more convenient to have a special rack or stand made. That
recommended contains five rows of 14 holes each. Each tube of
culture-medium is carefully marked with a blue pencil to show three

Fy FN NONE ee 2
ole we “™ Foy ey (en
Fad tae

4.
‘i eM SG alta: Fi
h
t tel) | (2 i H
Hy |

Fig. 86.—Block for subculture tubes (Anderson and McClintic, in Bulletin
No. 82, Hygienic Laboratory). :

- Fig. 87. —Device for flaming inoculating loops (Anderson and McClintic,
in Bulletin No. 82, Hygienic Laboratory).

things, 1, the germicide; 2, the dilution; 3, the time of exposure, and
stood in its place in the rack as will be explained below.

The transplantations from the seeding tubes to the culture tubes
are to be made every two and one-half minutes up to fifteen minutes,
so that for each strength of dilution to be tested, there will be six
tubes. In addition to these test-tubes there will be four dilutions
of phenol to act as controls so that every two and one-half minutes
ten transplantations must be made.

As two and one-half minutes contain 150 seconds, and as the
picking up and opening of the subculture tube, the transfer of the
seed-culture to the medium, the replacement of the stopper and the

- 266 Value oi Antiseptics

_ return of the tube to the rack require about fifteen seconds at the
hands of an expert manipulator, the ten tubes in the series comprise
the maximum number that can be handled.

The illustration shows one of the racks, and indicates how the
tubes are placed in ten rows of six each, each row with an. empty
hole on the left. As the first tube, of each series is inoculated, it
is stood in the left-hand empty hole, the second stood in the hole
from which the first was taken, the. third in that from which the
second was taken, and so on, so that there is always an empty
hole to show the operator which tube to take up for the next inoculation:

The Technic of Determining the Phenol Coefficient.—Everything:

being ready as outlined above, one proceeds as follows: The twenty- =

four hour bouillon culture of B. typhosus is shaken, then poured
through a sterile filter-paper in a sterile glass funnel and caught in
a sterile tube. In this way clumps of bacteria are removed and
uniformly distributed bacteria secured for addition to the “seeding
tubes.”

Exactly 5 cc. of each dilution of the disinfectant to be tested is
now measured into a seeding tube. To economize glassware the
same pipet may be used for a whole series, by beginning at the lowest
dilution, measuring out the necessary 5 cc. into the first seeding tube,
with a 5-cc. delivery pipet. The contents of the pipet are then thor-
oughly blown out, and a pipetful of the next weaker dilution taken
up to wash out the pipet. After this has been thoroughly blown
out and thrown away, a pipetful of this second strength of diluted
disinfectant is carefully measured into a second seeding tube,
after which the same is done with each remaining dilution in turn.
The tubes are so marked and so arranged i in the rack of the water-
bath that no mistake can be made in transplanting from them in
regular order later. As each tube is filled, the stopper is replaced
and when all have been filled and stood in the rack, it is placed in
the water-bath and the temperature raised to 20°C. Anderson and
McClintic do not use cotton plugs for the seeding tubes but sterilize
them, open end down in a paper-lined wire basket. Some feel

safer, however, in using tubes with plugs. The culture now being =

filtered, and the seeding tubes each with the required 5 cc. of each
dilution of the disinfectant to be tested, all at 20°C. in the water-
bath, the subculture tubes marked and stood in their respective
places in the racks, sterilized pipets at hand, and four or six platinum
loops on the block ready sterilized, with the burner in place ready to
re-sterilize them, the technic is continued by the addition of the cul-
ture to the seeding tubes. At this point one should make aslight
calculation: if the culture is to be added to each of ten of the seeding
tubes, it must be done before the expiration of 150 seconds or two and
one-half minutes for at the conclusion of that time, the first trans-
plantation from each seeding tube to a culture tube must take
place. We have averaged fifteen seconds for each operation. If

Testing Germicidal Value of Liquids 267

each transfer takes an average of fifteen seconds, the operator must
have every detail of the technic so well in hand, and the materials
so conveniently placed, etc., that he can complete the entire per-
formance of the technic from the addition of the culture to the
seeding tubes to the last transplantation from seeding tubes to
subculture tube without a hesitation and without a distraction. It
is on account of the necessity of this “continuous performance”
that such care was taken to point out the exact details of
apparatus and materials needed, before describing the technic.

To return to the seeding of the tubes, a sterile pipet graduated
in Ko cc. is used. The cotton stoppers are removed from the
seeding tubes and thrown away as of no further use. One by
one as the time arrives, tubes are taken in one hand, inclined to
an angle of about 45 degrees, while the tips of the pipet are lightly
touched to that side of the tube from which the fluid has run away
on account of the slanting, and exactly o.1 cc. of the culture de-
livered. This may under no circumstances take longer to perform
than fifteen seconds, and if one succeed in finishing it in a shorter
time, he must wait until the calculated time arrives before delivering
the culture into the-next tube and so on until the end is reached.
Each tube is given three gentle shakes after being straightened up,
then returned to the water-bath:

With a ten-tube series, and a time allowance of fifteen séconds
for each tube, the entire series of tubes is no sooner completed
than the time (two and one-half minutes) for making the first
series of transplantations to the subculture tubes has arrived.
The operator therefore seizes at once the first of the culture tubes
in the two and one-half-minute series with one hand, and a sterile
platinum loop with the other. He cautiously removes the cotton
plug from the culture tube, and at the proper moment intro-
‘duces the platinum loop into the first seeding tube all the way
to its bottom, withdraws it, and carries one drop of the con-
tained fluid into the first subculture tube which he plugs and places
in the empty hole to the left of the row in the block, at once taking
up its neighbor on the right. As only fifteen seconds are allowed
for each such transfer, the operator must proceed without hesitation.

There is no time to sterilize the platinum loop, so he lays it on
the block, pushes the flame under it and takes up an already steril-
ized loop with which he performs the same act of transplantation
for the second tube that was done for the first, doing it on the
appropriate second of time, and so continuing through the whole
series.

Every test of the phenol coefficient of disinfection must em-
brace two such series, one made with the dilutions of the phenol
that is to act as the standard, the other made with the dilutions of
the disinfectant to be determined. If, however, a variety of dif-
ferent germicides are to be tested the same day, one phenol test

268 Value of Antiseptics

will answer the requirement of the whole group. The following
tabulation will make clear the details of a test (Table £7 from
Anderson.and McClintic’s paper).

TABLE 17

Name “A.”

Temperature of medication 20°C.

Culture used. B. typhosus, 24-hour extract broth-filtered.
Proportions of culture and disinfectant, 0.1 cc. X § cc.

Bea Time culture exposed to action of disin- Phenol
Sample Dilution fectant for minutes ~ coefficient

7% Io r2lg] 15

iS}
SX
mn

780
190
: 100
L110 ;
Hao. | ve Pade ves Meemetita clog gadia lanai’ 4.69 X 5.91
2375
1400
1425
1450
1500
1550
2600
2650
1700
2750

ws
~s
on
x
a
on
ra)

Phenol........

I+++ |
L++ 1
(apa
+1

Disinfectant,
“c A ” :

HHHHHH HH RH HHH

sessed
aie Eon mm | |
cs tal ea

+++4++4+44 11
4444410011

++4444444 |

To calculate the phenol coefficient, the figure representing the
degree of dilution of the weakest strength of the disinfectant that kills
within two and one-half minutes is divided by the figure representing
the degree of dilution of the weakest strength of the phenol control
that kills in the same time. The same is done for the weakest
strength that kills in 15 minutes. The mean of the two is the:
coefficient. The, coefficient of any disinfectant may, for practi-
cal purposes, be defined as the figure that represents the ratio of —
the germicidal power of the disinfectant to the germicidal power of
the phenol, both having been tested under the same conditions.

As many disinfectants and germicides are greatly modified through .
precipitation, combination or other transformation in the presence
of organic matter, in all of those whose coefficient is considerably
more than 1, it is wise to perform a second series of tests in which
the disinfectant is tested, and the control tests made in the presence
of organic matter and the coefficient calculated accordingly. It is
usually found that under these conditions the coefficient falls. In
a general way, those disinfectants are most valuable for general
employment, whose coefficients are highest in the presence of organic
matter in the test solutions.

The difference in the details of the test given and the new test to
be made are as follows:

Testing Germicidal Value of Liquids 269

1. The test dilutions are made 20 per cent. stronger to allow for
the dilution made by the addition of the solution of organic matter.

2, An organic matter solution is to be prepared. It consists of
water containing ro per cent. of peptone and 5 per cent. of gelatin.
The solids are dissolved and the solution sterilized. Titration is
not essential. :

The variations in technic are simple. Of the dilutions made
20 per cent. stronger than for the other experiment, 4 cc. (not 5 cc.)
are measured into each seeding tube. The culture after being fil-
tered is added to the organic matter in the proportion of 0.1 cc. to
each 1 cc. to be employed in seeding. The addition of 1.1 cc. of the
organic solution culture mixture to each seeding tube, gives a total
of 5 cc. of diluted disinfectant containing 0.1 coe. of culture and a
total of 2 per cent. of peptone and 1 per cent. of gelatin. Except
for the slight difference in the dilutions and the seeding with mixed
culture and organic fluid the method is the same, and the method
of calculating the results is the same.

Anderson and McClintic point out that it is manifestly cheaper
to purchase a disinfectant for 60 cents a gallon than to purchase one.
for 30 cents a gallon, providing the former has four times the effi-
ciency of the latter. The true cost of a disinfectant can only be
determined by taking into consideration the phenol coefficient and the
cost of the disinfectant per gallon. The cost of a disinfectant
-per 100 units of efficiency as compared with pure phenol is obtained
by first dividing the cost per gallon of the disinfectant by the cost
per gallon of pure phenol; the efficiency ratio is of course obtained by
dividing the coefficient of the disinfectant by the coefficient of phe-
nol, but as the coefficient is always 1, the efficiency ratio is repre-
sented by the phenol coefficient of the disinfectant.

The cost ratio divided by the efficiency ratio (the coefficient of the
disinfectant) gives the cost of the disinfectant per unit of efficiency as
compared with the cost per unit of efficiency of pure phenol = 1.
By multiplying by 100 the relative cost of roo units is obtained thus:

Cost of diingectant Coefficient of disinfec-

per gallon. 2 Be eer tant. ( = Efficiency
Cost of phenol per Nc Coefficient of phenol. ratio.)

gallon, (=1.)

= cost of the disinfectant per unit of efficiency as compared with
phenol = 1, and by multiplying by too the cost of 100 units is
obtained. For instance, the cost of disinfectant “Can” is $9.30
per gallon and it has a coefficient of 2.12; the cost of phenol is $2.67
and it has a coefficient of 1. Then,

0.30 , 2.12

mar eS O52
Therefore, the comparative cost per unit of efficiency of “Can” and
phenol respectively, is as 0.052:1; or, by multiplying by 100,
the relative cost per 100 units—s.2 : 100 is obtained.

270 | Value of Antiseptics

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 aclosed cham-
ber in which the gas is evolved. The germicidal 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
the cultures there are immense numbers of bacteria in the deeper
portions of the bacterial mass upon which the gas has no oppor-
tunity to act, and in the dust there are many sporogenous organisms
of extreme resisting power. Failure to kill all the germs exposed in
such manner is no indication that the vapor cannot destroy all
-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 ex-
periment. If, when transferred to fresh culture-media, the ex-
posed objects fail to give any growth, the disinfection has been
thorough so far as the particular test organism is concerned. It 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, allowed to dry, and then
exposed to the gas as long as required. 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 in-
sufficient amount of 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
BACTERIO-VACCINES

ABACTERIO-VACCINE is a culture of micro-organisms so modified as
to be no longer a source of dangerous infection, and so administered
as to stimulate the body defenses and thus assist either in prevent-
ing or overcoming more virulent infection.

The small amount of benefit that occurred from the employment
of the Oriental method of “inoculating against small-pox” was
based upon the theory that virus of low virulence, obtained from a
sporadic case of small-pox if introduced into the healthy body, must
result in a mild attack of the disease, by which the individual would
be left immune against the more virulent viruses by which epidemics
of the disease are brought about. The observation of Jenner, that
the virus of cow-pox would protect against small-pox, led to the
supposition that the essential causes of the two diseases had origin-
ally been the same, but had so diminished that the one became com-
paratively harmless for man after many generations of residence
in the cow. oot

The success of Pasteur’s preventive inoculation against chicken-
cholera depended upon the fact that the bacilli of the disease rapidly
lost their disease-producing power when grown artificially in cul-
ture-media, though they still retained the power of effecting a
change in the fowls which thereafter remained immune. His vac-
cination against anthrax was based upon the observation that the
spore-forming power and virulence of the anthrax bacillus could be
destroyed by cultivation at temperatures beyond a certain point,
and that animals infected with bacilli of this modified form subse-
quently resisted more virulent infections., His vaccination against
rabies was based upon the supposed diminution in virulence that
the unknown micro-organisms underwent when exposed to artificial
inspissation of the nervous tissue in which they were contained.
Such organisms of very low virulence protected against those of
higher virulence, and so on.

From the periods during which these early observations were
made, to the present time, when the term “bacterio-vaccine” is
in daily use, studies in immunity have been conducted in so great
a variety of ways by such a multitude of investigators, that it be-
comes tedious to endeavor to trace the logical and orderly steps
that lead to present knowledge, theory and practice. Two names,
however, stand out’ conspicuously in connection with the present
topic, because of the importance of their contributions, those of
Haffkine and Wright. The former used heated and killed cultures

271

272 Bacterio-vaccines

of the cholera spirillum as a prophylactic against cholera, and later
with equal success, heated and killed cultures of the plague bacillus
as a prophylactic against plague. Wright somewhat modified the
method, by using two or even three doses of modified cultures of
the typhoid bacillus at intervals of ten or even twenty days, to secure
complete prophylaxis against typhoid fever.

From prophylactic measures it was but a step to therapeutic
measures, and the endeavor to facilitate the cure of disease by the
administration of cultures of vaccine. The patient suffering from
an infectious. disease was already impressed by the toxic, enzymic
or other disease-producing substances in his body, and the adminis-
tration of cultures of micro-organisms seemed like adding so much
fuel to an already widespread conflagration. Indeed, experience
and experiment seemed to prove this to be the case, for when by any
mischance a patient in the early stages of plague received an in-
jection of the Haffkine plague prophylactic, he straightway became
much injured by the added culture and might even die quickly.

But there are certain infections in which conditions are different
both with regard to the bacteria and the disease. Thus, a certain
micro-organism with limited power of invasion and with difficultly
soluble toxic products (endo-toxins), whose injurious effects are
local and limited in extent, particularly when their effects are pro-
longed and the disturbances chronic, are essentially different from
actively invasive agents that quickly over-run the body, or those
with considerable soluble products by which it is generally disturbed.

In the former group it is not unreasonable to hope that through a
method of treatment by which the general body defences are stimu-
lated, the local infections may be overcome. Such cases of dis-
ease were, therefore, selected, especially by Wright, for investiga-
tion and treatment. Success of varying degree has followed, and
though it is difficult to calculate accurately the benefits obtained
in cases that are not susceptible of numerical expression, the almost
' uniform opinion of clinical and laboratory men is to the effect that
certain cases of sluggish infection, with little tendency to recover are
benefited and sometimes rapidly cured by treatment with bacterio-
vaccines.

From these preliminary considerations it should be clear to the
reader that the theoretical conditions necessary to success are the.
following:

1. That the disease should be of subacute or chronic duration.

2. That it should be fairly well localized. .

3. That it should be caused by a micro-organism incapable of ready: invasion
or much soluble toxin formation. :

4. That the micro-organism be known and capable of cultivation so that the
appropriate-specific vaccine can be made.

From these conditions certain lesions resulting from infection by
pus cocci, colon bacilli, acne bacilli, typhoid bacilli (post-typhoidal

Methods of Making the Vaccine 273

suppurations), tubercle bacilli, etc., etc., ought to be appropriate.
And, indeed, for them the treatment is highly recommended, and in
many cases remarkable success is claimed.

Remembering that the reactions of immunity are specific, it is
imperative that the essential organism of the lesion be found and
cultivated, and cultures of that organism used in the treatment.
So important is this that Wright insists that only ‘‘aztogenous
vaccines”’—that is, vaccines made of cultures of bacteria cultivated
from the very lesion to be treated—be used. This somewhat limits
the usefulness of the method for the rank and file of practitioners
can scarcely be supposed to have the knowledge, apparatus, or time
required for carrying out the technic, nor can all patients afford
to patronize the laboratory man. Commercial manufacturers are
therefore justified in the preparation and sale of what are known as
“stock vaccines” that can ‘be tried in lieu of autogenous vaccines,
though in checking up the results note should always be taken of
the fact that “autogenous” or “stock”? vaccines were used.

In spite of the general principles laid down above, there are re-
ports and observations to show that the theoretical considerations
may be faulty and that in some cases the method of treating by
‘vaccination may be beneficial in acute maladies, even when the
condition to be treated is toxic. It will be necessary, however, to
secure much more evidence with regard to the employment of the
method in such cases before it can be recommended as sound
practice. =

Should a case of appropriate kind, when investigated, yield more
than one species of micro-organism, of such kind as to make it un-
certain which is responsible for the injury done, both should be
cultivated, two vaccines made and mixed, and both infections
simultaneously antagonized: _ a

The Method of Making the Vaccine.—A pure culture of the
necessary micro-organisms is obtained from the lesion to be treated,
and cultivated on agar-agar.

One pint “Blake bottles,” pint or quart white glass whisky flasks, or other
good sized bottles with large flat sides, are selected and washed. Into each
enough melted agar-agar is filled to spread out over one of the flat surfaces to a
thickness of about 1 centimeter, after which a cotton plug is placed in the
mouth of the bottle, and it and its contents are sterilized in the autoclave. Upon
removal, after sterilization, the bottle is laid on its side so as to distribute the
agar-agar and permit it to solidify over the greatest surface, without flowing
into the neck and touching the cotton stopper. To the agar-agar culture of the
micro-organism to be used, about to cc. of sterile 0.85 per cent. sodium chloride
solution is added, the culture mass being detached with a platinum loop and
thoroughly mixed with the fluid. When the agar-agar is firm, each bottle
Tecelves by means of a carefully sterilized pipet, about 1 cc. of the culture
suspension which is thoroughly distributed over the entire flat surface of the
agar-agar by tilting the bottle this way and that until it has been completely
covered. The bottles are then placed in the incubating oven, lying upon the
side so as to permit the bacteria to vegetate undisturbed upon the moist flat
surface of the medium. After 24 hours, the growth having matured, the bottles
-are removed and about ro cc. of sterile distilled water containing 0.85 per cent.

274 Bacterio-vaccines

of sodium chloride and 0.5 per cent. of phenol is added to each, for the purpose
of washing off the bacteria that have grown. This is done by tilting the bottle
and permitting the solution to wash over and over the surface. If the culture
does not detach, it may be necessary to remove it with a sterilized glass rod, or
by means of a sterile swab made by fastening a small plgdget of cotton batting

upon the end of a wire. . ;
When the growth is detached and thoroughly mixed with the salt solution,

it is removed to a sterile receptacle by means of a sterile pipet.

What is next done. will depend upon the theory upon which the
treatment: is based. The culture washings contain: (A) sub-
stances derived from the culture-medium that certainly cannot be
regarded as useful or beneficial and may be harmful; i

(B) bacterial products, of soluble quality, eliminated from the
cells during the life activities, some of which may be useful;

(C) the bacteria themselves, which with their contained prod-
ucts—endo-toxins, etc.—are commonly -regarded as the essential
immunizing agents.

If one’s theory is that the bacterial cells are essential, and there
seems to be a growing tendency toward this view, further treatment
is necessary before actually preparing the vaccine for administra-
tion; if, however, the collected products of their growth are thought
to be of partial or equal value, and are to be preserved, this cannot .
be done without also retaining the less desirable matters from the
culture-medium.

Let us suppose that only the bacterial cells are to be employed.

The suspension of bacteria, under these circumstances, is transferred to
appropriate sterile tubes, plugged, and whirled in a powerful centrifuge until
the bacteria are thrown down to the end of the tube, leaving the supernatant
fluid fairly clear. The fluid is then removed by decantation or with a pipet,
and replaced by an equal volume of 0.5 per cent. phenol in 0.85 per cent. sodium
‘chloride solution in distilled water. In this the sediment is thoroughly mixed by
stirring. As the bacteria are often in masses, groups or chains, it is now necessary
to separate them. This is best done by adding a few small glas beads to the
contents of the tubes, changing the cotton stopper for a sterile rubber cork, and
shaking either in a shaking machine or by hand, until it can be supposed that the
micro-organisms are all separated. This is easily accomplished by the aid of the
shaking machine but is tedious to effect by hand. The tube is then returned to
the centrifuge and again whirled until the bacteria are again sedimented, after
which the fluid is again removed and again replaced and the bacteria again dis-
tributed. A few turns in the centrifuge now throw down particles of culture-
media and contained flakes of the culture and leave a uniformly clouded fluid
above.

If it be desired to conserve all of the bacterial products, the wash-
ings from the culture bottles are immediately transferred to the
appropriate tube, shaken with the glass beads, given a few turns
in the centrifuge to throw out flakes of culture and culture-media,
and we again arrive at the point of having a uniformly cloudy fluid -
with which to continue the preparation of the vaccine.

If the vaccine is to be of ‘scientific value, it should be made in
such manner that its composition represents what is desired—
bacterial cells only, or bacterial cells with their collected products—
and some means should be provided by which a reasonably accurate

Methods of Making the Vaccine 275

determination of its value can be estimated. This is done by cal-
culating the number of contained bacteria per cubic centimeter
of the fluid, and then either diluting or concentrating by means of
centrifugation until an appropriate result is reached. As the con-
centration by centrifugation is more difficult than dilution it is best
to take care at the very beginning of the process not to add too much
fluid to the culture bottles for the purpose of washing off the culture.
Whatever dilution of the final product may be necessary is made by
the use of the 0.5 per cent. phenol solution.

The most ready method of calculating the number of bacteria
in the fluid is that of A. E. Wright which will be found in the chapter
upon the ‘‘Calculation of the Opsonic Index.”

After having determined the number of bacterial cells per cubic
centimeter of fluid, dilution with the phenol solution is made until
single doses are contained in quantities easily injected into the
patient. As the doses vary with ‘the particular organism to be
injected, the operator must calculate from the number of bacteria
in the fluid, how much solution must be added to constitute a dose.
Several doses of each desired size should be prepared. Quantities
of the dilutions containing single doses or a number of doses as may
be preferred are now transferred, by means of a sterile pipet, into
previously sterilized, appropriate sized “ampules” or glass bulbs
made for the purpose, and the necks sealed in a flame.

The bacteria are, however, still alive, and though many of them
no doubt undergo autolysis in the phenol salt solution, it is necessary
to make certain that none remains alive to infect the patient.

The destruction of the vitality of the micro-organisms which is
the final step in the process of vaccine preparation is effected by ex-
posure to the lowest temperature that is known to be positively
destructive. As spore-producing micro-organisms may maintain
this vitality at temperatures beyond 100°C., at, which the micro-

- organismal substance as well as their products are altered by coagula-
tion and other destructive transformation, they are inappropriate
organisms to employ for purposes of vaccines, unless, through some
such ingenious means as was devised by Pasteur for the anthrax
bacillus, the production of spores can be prevented. :

With very few exceptions non-sporogenous bacteria are destroyed
by exposure for sixty minutes to a temperature of 60°C. Should any
escape destruction, they are probably so injured as to be incapable of
further injurious effect upon the human body.

The destruction of the bacteria is, then, effected by heat:

The ampules of vaccine are placed in some sufficiently commodious receptacle
filled with water, the heat being supplied by a flame below, and the temperature
determined by a thermometer whose bulb is at the center of the bath. When
small quantities of the vaccine are to be made for special cases, a large beaker
supported upon an asbestos plate upon a chemical tripod and heated by a

unsen’s flame answers very well. The burner is allowed to heat the bath until
the proper temperature is reached, when it is removed. As soon as the tem-

276 Bacterio-vaccines

perature begins to fall, it is replaced. Thus by alternately heating and re-
moving the source of heat for sixty minutes, the destruction is affected.

If there are many of the small ampules, containing different doses
or different cultures, each separate lot may be done up in a piece of
gauze, and labeled.

J. H. Small uses orange-colored “string tags”’ for this purpose,
writing upon them with either pen or pencil, and fastening them to
the gauze packages. In the water of the water-bath, the writing
does not wash off the tag, but the color comes out and gives the
water an orange tinge. This is found to be of the greatest use,
for as one or more of the factory-made ampules commonly cracks in
the water-bath, the color penetrates the contained fluid. Upon
removal from the water-bath, to glance at each ampule will inform
the observer whether it is cracked or not, through the change in the
the color of the contents. The tags, therefore, subserve a double
purpose.

After heating, one of the ampules can be opened and a drop of the
contents transferred to a tube of culture to make sure that the
bacteria are no longer alive.

The vaccine is now ready for use, but in what dose shall it be ad-
ministered? ‘There is no other information upon this subject than
that which is derived from the experience that certain doses seem to
accomplish good without producing ill effects. Thus experience
with doses at first selected arbitrarily has led to a fairly accurate
standard dosage. As the beginning dose for most vaccines 50-250
millions may be recommended, to be increased to 1000 millions or
more, the injections being given every four or six days or as con-
trolled by the opsonic index.

The benefit of the vaccine is commonly supposed to depend upon
the stimulation of the phagocytic cells of the body. This is very
probably the case, but when the bacterial bodies are administered,
their dissolution results in the liberation of the contained endo-toxin,
and when the entire culture is given, endo-toxins and perhaps exo-
toxins and other substances are also given so that the increased
phagocytosis is not likely to be the only effect of the treatment.

A. E. Wright who is a firm believer in the stimulating influence
upon the cells seeks to control the dosage and estimate the value of
the injections by such study of phagocytic activity, as is shown in
the next chapter. If after an injection of vaccine, the phagocytic
activity of the leukocytes is diminished (negative phase) harm is
supposed to have been done and the inference is drawn that the dose
was too large; if, on the other hand, the phagocytic activity is
increased for the respective organism, good is supposed to have been
done, and at the next injection the same or a larger dose may be given.

Besredka and Metchnikoff* have modified the vaccines by what
is called sensitization. This they accomplish by treating the bacteria

* Ann. d. l’Inst. Pasteur, to13, XXVII, 597.

Methods of Making the Vaccine 277

to be used with an antiserum, prepared by injecting animals with
such organisms as form the vaccine. In this manner the specific
bacteriolytic amboceptors are supposed to anchor themselves to the
bacterial cells, and so pave the way for immediate destructive treat-
ment in the body. To achieve such sensitization, some of the ap-
_ propriate serum is added to the bacterial suspension which need
not be subsequently killed, as the sensitized bacteria meet with
prompt destruction through the normal complement of the body
juices. However, if the bacteria are first killed by heat and then
sensitized, a similar result may be brought about, and one is re-
lieved of all anxiety as to the possibility of infection accidentally
resulting from the injections.

CHAPTER XIX

THE PHAGOCYTIC POWER OF THE BLOOD AND THE
OPSONIC INDEX

From the time that Metchnikoff connected the phenomena
of phagocytosis with those of immunity, there was no recognized
technic for the observation and comparison of the bacteria-con-
suming and bacteria-destroying power of the cells until 1902, when
Leishman* suggested the following simple method:

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 suffi-
ciently accurate observations could be made for controlling the spe-
cific 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 of washed leukocytes in ipeeiehe salt solution.

‘The serum to be tested.
A normal serum for comparison.

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 corpuscles 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 bacteria 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 sept

* “Brit. Med. Jour.,” Jan. rz, 1902, I, P- 73-
+Proc. Royal Soc. ‘of London, ” 1904, LXXXII, Pp. 357.

278

The Bacterial Suspension 279

rate 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, 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 hature that will not separate in

Fig. § 88. Tanne bacteria (Miller).

this manner (tubercle bacillus), it ‘ray be necessary to rub it between
two glass plates, or in a small agate mortar with a drop or two of
salt solution, other drops being added one at a time, until a homo-
Zeneous 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 standardization of the bacterio-
vaccines that the number of bacteria |
shall actually be counted. This he
does by mixing one part of the bac-
terial suspension with an equal volume |
of normal blood and three volumes
of physiological salt solution. After
thorough mixing a smear is made upon
a slide, the smear stained, and the _. Fig. 89.Diaphragm of eye-
number of bacteria and corpuscles in rai mal ss
successive fields. of the microscope -
counted until at least 200 red blood-corpuscles have been enu-
merated. As the number of red corpuscles per cubic millimeter
of blood is 5,000,000, the number of bacteria per cubic centimeter
can be determined from the results 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.
The prepared suspension must usually be greatly diluted before
using, but the reduction of bacteria is, of course, easily calculated.

280 The Phagocytic Power of the Blood

It requires experience to determine the appropriate number of
bacteria to be employed. When this is once determined, future
manipulations are made easy, because one first makes his suspension,

Fig. 90.—Photomicrograph showing cross-hairs, bacteria, and red blood-
corpuscles (Miller).

then enumerates the bacteria, and having determined their number,
immediately arrives at the appropriate concentration by dilution.

Fig. 91.—Collecting blood for corpuscles (Miller).

The Washed Leukocytes.—It is not necessary to have the leuko-
cytes 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 coagulation, and then separating the citrated
plasma from the corpuscles by centrifugalization.

The Washed Leukocytes 281

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 toa fine point in
the flame) a prick is made about a quarter inch
from the root of the nail. 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, balariced,
and centrifugalized until the corpuscles are
collected at the bottom of the tube. The
citrated plasma is now withdrawn and re-
placed with 0.85 per cent. sodium chloride
solution, through which the corpuscles are
distributed by shaking. The tubes are now
again centrifugalized until the corpuscles are
collected, when the saline is removed care-

Fig. g2.—Tube of
blood and citrate solu-
tion before and after
centrifugalizing (Miller).

fully, the last drop from the back of the meniscus. In the cor-
puscular mass that remains the leukocytes form a thin creamy

layer on the top.

Fig. 93.—Removing last drops of saline solution (Miller).

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.

282 The Phagocytic Power of the Blood

- The blood to furnish the serum is taken i in a small bent ube shown
in the illustration.

The blood from the puncture is allowed to flow into the Aiea end
of, the tube, into which it enters by
capillary attraction and from which it
descends to the body of the tube by
gravity. At least 1 cc. 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 im-

Fig. 94.—Special blood pipette (Miller).

mediately, and the serum usually sepa-
rates. quickly. If it does not do so,
Wright recommends hanging the curved
arm of the tube over the centrifuge tube
and whirling it for a moment or two, es
when the clot is driven into thestraight Fig. 95.—Opsenizing pipette a
arm of the tube and the clear serum containing biped cope es
appears above. The tube is then cut ues i ie
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

4
,

The Washed Leukocytes 283

by a rubber bulb. 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 1 centimeter from
the end of the capillary tube. He first draws up the leukocytic
layer of blood-corpuscles to this mark, then removing the tube,
permits the column to ascend 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; thus in the same capillary tube he has three
equal volumes of three different fluids, separated by bubbles of
air. It is next necessary to mix these, which is done by repeatedly
expelling them upon a clean glass slide, and redrawing them into the

Fig. 96. —Mixing liquids by repeatedly expelling on to slide and redrawing
into pipette (Miller).

tube. After thus being thoroughly mixed, the fluid is once more
permitted to enter the capillary tube and come to rest there. The
end is now sealed in 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 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 sedi-
mented, its 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 dis-
tribution 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

284 The Phagocytic Power of the Blood

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 counting, it is well to have the

Fig. 97.—A small incubator of special design for opsonic work (Miller).

smear terminate abruptly and not be drawn out into threads or
irregular forms.”

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. In order:
that the enumeration of the
bacteria taken up by the leu-
kocytes can be accomplished, it is

Pie 08 —The smear + (Miler). 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, Leishman’s stain, Jenner’s
stain, or J. H. Wright’s stains are appropriate. Marino’s stain,

* “Therapeutic Gazette,” March 15, 1907.

The Washed Leukocytes 285

recommended by Levaditi, * gives beautiful results. For the tubercle
bacillus the spreads may be stained with carbol-fuchsin and coun-
terstained with methylene-blue, or perhaps better with gentian
violet and counterstained 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 staphy-
lococcic cases, and separate averages made for polymorphonu-
clear and mononuclear cells in tubercle bacillus cases. It is best to
follow certain 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 polymorphonuclear cells and the elimina-
tion 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.

Leishman’s7 studies of the phagocytic power of the blood show
that in cases of furunculosis, etc., with each recrudescence of boils,
there is a marked diminution of the phagocytic power of the blood,
and with each improvement, a marked increase.

McFarland and Il'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 admin-
istration, 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 admin-
istration is 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 supposed, upon
theoretic grounds, and proved by practical experience, that the
increase of phagocytic activity brings about improvement. The

*“Ann. de l’Inst. Pasteur,” 1904, XVIII, p. 761.

} “Lancet,” 1902, 1, p. 73-
ft “ Medicine,” April, 1906.

286 The Phagocytic Power of the Blood

‘care of the operator should be to avoid giving so large a dose of the
vaccine that the negative 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 expressed—‘that the
estimation of the value of bacterio-vaccination by means of the
opsonic index was a very complicated way of finding out very little.” .

CHAPTER XX

THE WASSERMANN REACTION FOR THE DIAGNOSIS OF
SYPHILIS :

Tuts now popular and fairly reliable method for assisting in
the diagnosis of atypical syphilitic infections was devised by Wasser-
mann, 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 normal blood.

The test is twofold: (1) A combination of syphilitic antigen,
complement, and suspected serum. (2) A subsequent addition to
mixture of blood-corpuscles and hemolytic 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-corpuscles and hemolytic serum. If, on the other hand, the
suspected 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 hemo-
lysis 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.

(1) The Syphilitic Antigen.—It was supposed by Wassermann,
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 Treponema pallidum having at that
time been devised, cultures of the specific micro-organism could not
be employed. Histologists had, however, shown that greater num-
bers of the organisms were to be found in the livers of the congen-
itally syphilitic stillborn infants than anywhere else. With the

* “Deutsch. Med. Wochenschr.,” 1906, No. 19.. ;
287 ;

288 Wassermann Reaction for Diagnosis of Syphilis

purpose, therefore, of securing the greatest possible number 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 exsiccator over calcium chlorid if
_ placed where it is cool and dark. When the powder is to be used,
o.s gm. is extracted either at room temperature or in the ice-box
with 25 cc. 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 sedi-
mented liver tissué remnants, and the clear supernatant fluid
pipeted 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 substances 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 fragmented treponemata in the liver ex-
tract. The method scarcely began to meet with practical applica-
tion, 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. Porges and Meier,
indeed, found that lecithin could play the réle of syphilitic-antigen,
Leviditi and Yamanouchi place sodium glycocholate, sodium tau-
rocholate, protogon, and cholin among those bodies capable of
acting as syphilitic antigens, 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
and many now regularly employ human, beef or guinea-pig heart
muscle to which cholesterol is added. The method of making chol-
esterinized human heart muscle antigen recommended by Weston*
is as follows:

* Jour. Med. Research, 1914, XXX, p. 377.

The Serum to be Tested _ 289

Human heart muscle, free from pericardial fat was ground in a meat chopper
and covered with absolute alcohol in a wide-mouthed bottle. After forty-eight
hours the alcohol was decanted on to a dinner plate and the tissue expressed
between layers of cheese cloth. The expressed juice was added to the alcohol
and the tissue returned to the bottle. The alcohol was evaporated by means of
a current of air from an electric fan and the residue scraped up with a bone

=
=
=
=
=
S
=
x

Fig. 99.—The Kei-

del tube for collecting Fig. 100.—Parts of the Keidel tube. £

blood (Manufac- is the vacuum bulb which is attached to the

tured by the Steele needle by a piece of rubber tubing (D); the

Glass Co., of Phila- glass tube (B) covers the needle and the
- -delphia), whole is sterilized (Kolmer).

spatula and added to the tissue. Three volumes of absolute alcohol are then
added and the closed bottle kept at room temperature for two weeks. At the
end of this time the alcoholic extract was filtered off and to each 8 cubic centi-
meters was added 2 cubic centimeters of a 2 per cent. solution of cholesterol in
absolute alcohol.

290 | Wassermann Reaction for Diagnosis of Syphilis

(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. Noguchi, who works with very small quantities of
the reagents, secures the blood by obstructing the venous circulation
of the thumb or of a finger by means of a rubber band (see directions
for obtaining the blood for making the opsonic index) but the greater
number prefer to obtain it by introducing 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 momentary, spraying
with chlorid of ethyl will make the operation entirely . painless.
Some prefer to use the iodin without the preliminary washing, be-
lieving that soap makes it difficult for the iodin to effect satisfactory
disinfection of the skin. The sterilized needle is thrust into the
vein, care being taken that the vein is not too compressed and the
point of the needle thrust entirely through instead of into it. From '
15 to 25 cc. of blood may be withdrawn in a Keidel tube, or 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 pipette, or the clotted blood is placed i in a centrifuge
tube and whirled, 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 generally employed is

contained in the blood of a healthy adult guinea-pig. To obtain
it a piece of cotton moistened with ether or chloroform is held to
the guinea-pig’s nose until it becomes unconscious, 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 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-corpuscles 2g

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 se-
cures 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-four
hours. Should it be necessary to be economical with the guinea-
pigs, any serum not used may be preserved with a fair amount of
success by freezing. ‘This was first suggested by Morgenroth, and
improved by Weston* as follows: The guinea-pig serum, having been
three hours in contact with the clot, was placed in thick-walled test-
tubes 10 X Io0 mm., two cubic centimeters in each tube. The
tubes were closed with tightly fitting cork stoppers. A cake of arti-
ficial ice, left in the can and surrounded by brine was set aside for
the preservation of the serum. A hole was made in the ice, the
tubes placed in it so that the cork stoppers just reached the top
and ground ice was packed around them. One out of three tubes
kept its complementary value unchanged for three months.

Rhamyf found that chemically pure sodium acetate had no hemo-
lytic properties and could be used to preserve serum without loss of —
its complementary power. He used a to per cent. solution of the
sodium acetate in 0.9 per cent. sodium chloride solution for preparing

the usual 40 per cent.‘dilution of the complement, and found that
_ the complementary value of the serum was preserved until the solu-
tion was used up in routine work. In one month the deterioration
of complementary value was so slight that the dose of serum had
only increased to 0.125 instead of its original 0.1. The complement
- should, however, be titrated with each new batch of blood corpuscles
to make sure of the possible variation in value. The quantity of
the complement in the serum of the guinea-pig is fairly constant,
when the animal is regularly fed, and furnishes a fairly uniform
reagent that requires no titration.

II. For the second, or hemolytic, test two additional reagents
are required: 5

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 Morgenroth, in their
Ploneer experiments into the mechanism of hemolysis, used goat
corpuscles. Bordet used rabbit corpuscles; Wassermann, Neisser,

* Jour. Med. Research, 1915, XXXII, p. 391.
T Jour. Amer. Med. Asso., 1917, LXIX, Pp. 973-

292 Wassermann Reaction for Diagnosis of. Syphilis

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 ob-
tainable in any quantity, hence most investigators now follow
Wassermann and his collaborators and use sheep blood, which is
easily obtained ata 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 belonging 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 compact 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 throughout
the newly added fluid. The tubes are next returned to the cen-
trifuge and again whirled until the corpuscles are sedimented, when
the fluid resulting from this first washing 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
undisturbed. Some prefer to give the corpuscles a third washing,
but it does not seem to be necessary. Of the remaining corpuscular
mass, 5 cc. are added to 95 cc. 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 seventy-two hours to secure
satisfactory results, as they tend to soften when kept and so to lose
their standard value. If kept longer than twenty-four hours they
should be washed before using.

(5) The Hemolytic Amboceptor.—As the validity of the test de-
pends 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 stimulation 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 intra-
peritoneal injections of the 5 per cent. suspension of washed and

The Hemolytic Amboceptor 293

sedimented sheep corpuscles prepared as above described. These
injections are usually given about five days apart, and the dosage
is usually 5, 10, 15, 20 and 25 cc. respectively.

A serum of higher amboceptor content may be prepared by using
a greater number of corpuscles, and for this purpose the solid cor-
puscular mass thrown down by centrifugalization after the second
washing is employed. Of this, 2, 4, 8, and 12 cc., 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 injection. Much care must be taken to see that the injected
fluid is sterile and the operations performed under aseptic precau-
tions, as the rabbits are easily infected and not infrequently die.
They also seem prone to die after the last injection, 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 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 pipette. 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 em-
ployed in making the Wassermann test, the next step, that of titrat-
ing them, becomes essential. One of the first questions that pre-
sents 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 be least variable and
work up to those that are most so.

(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 in-
variable factor. One cubic centimeter of the 5 per cent. suspension
forms a good working quantity and constitutes the umit.

(2) The Normal Guinea-pig Serum Containing the Complement.—
As this also comes from a normal animal, is always treated in pre-
cisely 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, 0.1 cc. (1 cc. of a 1:10
dilution, made with physiological salt solution) forms the wnit, 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 treat-

294 Wassermann Reaction for Diagnosis of Syphilis

ment of the rabbit, and apparently, also, according to the ability
of thé individual rabbit to respond to the treatment by the forma-
tion of hemolytic amboceptors. It is, therefore, imperative to make.
a careful titration of it.

To do this we proceed as follows, the quantities recommended
being such as experience. has proved most satisfactory:

Into each of a series of common test-tubes or culture-tubes 1 cc.
of the 5 per cent. suspension of sheep corpuscles and 1 cc. of the
r:1o dilution of the normal guinea-pig serum (complement) are
measured with graduated pipettes, and then to each of these tubes
the rabbit serum (amboceptor), diluted with physiological salt solu-
tion so. as to make the correct measurement of the minute quantities
necessarily employed a matter of ease and convenience, is added in
diminishing quantities for the purpose of determining 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 solu-
tion 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

sion of corpuscles. serum. serum. after two hours).
I cc. 0.1 cc. o.or cc. Complete hemolysis.
I “ o.1 “ce 0.005 “ “ “
I (t4 oO.t “ce 0.002 “cc “ce “cc
I “ce O.1 ia O.00O1L cc “ “
I “cc O.1 ce ©.0005 “ “ce “
re o.r - 0.0003 “ Partial es
x o.r 0.0002 “ No ee
I “cc o.1 oe ©.0001 “ce “ it

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 ce.

From the results shown in the tubes it is evident that the hemolyz-
ing quantity of the rabbit sérum lies between 0.0005 and 0.0003
cc., and is probably 0.0004 cc. To be as accurate as possible, a
second series of experiments should be made with 0.0005, 0.00045,
and 0.0004 cc., so that the proportion of amboceptor serum neces- _
sary 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 (1 cc.), the unit of complement (0.1 cc.), and the unit
of hemolytic amboceptor is known as the hemolytic system.

As soon as this unit is known accurately, we are in position to

The Hemolytic Amboceptor a 295

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 conditions of age, feeding, health, etc., we
can now do so by determining whether, when 1 cc. of the corpuscles, »
t 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 manipulation, 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 Wassermann reaction.
In it are always employed 1 unit of sheep corpuscle suspension, 1
unit of complement, and 1 unit of amboceptor. Here, however, a
slight difference of opinion is reached, it being argued by many experi-
menters 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 comple-
ment fixation, it would result in positive readings where the final
result should be negative. To overcome this possibility, they dif-
ferentiate 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 hemo-
lysis is merely the relative 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 1 unit of com-
plement and by 1 unit of amboceptor as by 20 units of amboceptor |
and o.1 unit of complement or any other appropriate combination
of these two components.”

As in the performance of the test we work always with 1 unit of
complement, we do not want to unduly disturb its proper propor-
tional action by any excessive addition of amboceptor, but simply to
increase the latter sufficiently to provide for the accidental presence,
in the serum to be tested, of substances affecting hemolysis. Fortu-
nately, means are provided for controlling this action, as will be
shown below.

The amboceptor serum keeps alee ntel: 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 quan-

* “Serum Diagnosis and Syphilis,” 1910, p. 13 et seq.

. 296 Wassermann Reaction for Diagnosis of Syphilis

tity 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
to 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 pre-
liminary 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; 1 drop of serum usually covers
about 4 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 0.0001, 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 todry. 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, }4 cm., and a series of tubes prepared with cor-
puscle suspension and complement and the paper added 1 square, 2 squares,
24% 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 should be kept tightly closed in a dry, glass-
stoppered bottle or jar, the quantity for each test being cut off as
needed. The dry serum changes so little that the dose orice deter-
mined, the size of the square of paper needed for the test remains
about the same.

The method has the advantage that the amboceptor serum can-
not 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 complement
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 comple-
mentary action may be affected or set aside. Thus, temperature
affects it, and temperatures of o°C. suspend it. It is on this ac-
count that the test is always made at 37°C. Like most of the

The. Hemolytic Amboceptor 207

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,
cholesterol, 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 com-
plementary action.

Now, in all combinations of several serums and antigens it is
always possible that some of these complement-binding or comple-
ment-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 inhibit 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 com-
bination 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 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 cc., and this we regard as the unit of serum to be
tested.

To titrate the antigen we require (1) 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 labora-
tory in considerable 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 1 unit of complement. Each tube of
one group receives the addition of 0.2 cc. of the normal serum;
each tube of the other group, 0.2 cc. 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 accompanying tabulation.

From this we find that the unit of antigen is 0.09 cc., the largest

298 Wassermann Reaction for Diagnosis of Syphilis

quantity of the antigen that can be added without preventing hem-
olysis when the normal serum is used is probably 0.18 cc. At the
same time 0.09 cc. is the smallest quantity. that can be added, when
the syphilitic 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 leutic serum,
provided double the dose does not interfere with the complete hem-
olysis 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 nega-

TaBLe I.—Series with the Normal Serum

Tubes
1. t«unitof + tzunitof -+ antigeno.o1 8 | ga = Complete
complement normal serum B28 o hemolysis.
2. uu + oy + 0.03 gg@Ea =
; Fe Sa
3 “6 + 3 + ce 0.05 Cea aa ‘e
-BoO8s
cad ce ce esse e
4. + i+ / 0.07 BSyEom = .
a2 258
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: Of yg Ee
is) =
(a9 ec £3823
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; Een os
Brg &
cc ce ce Oe G — ce
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i ‘ oo
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OB8ue ‘:
: hemolysis,
or TaBLE II.—Series with the Syphilitic Serum
ubes
7 Pe Ska
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tr runitof + trunitof + antigeno.or °& og = Complete:
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“ ‘i su ees “
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a la
hb
moags

The Hemolytic Amboceptor 299

tive results, for in titrating the antigen 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 avoided.

A Wassermann reaction at the present time comprises not only
the test of the patient’s serum, but simultaneously includes a long
series of other tests by which the validity of every part of the test
and the correct titer of all the reagents employed can be simultane-
ously 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, un-
necessary to make more than one series of controls.

Of the complementary serum we add 1 cc. tog cc. of 0.85 per
cent. (physiologic salt solution, making each cubic centimeter of °
the dilution of the fluid equal 0.1 cc. This quantity, carefully
measured by the same volumetric pipette, is dropped into each
tube, and this pipette laid aside.

The serum to be tested is drawn into a second finely graduated
pipette, and 0.2 cc. added to tubes 1, 2, and g, and that pipette
laid aside. ;

The positive syphilitic serum used to control the test is similarly
drawn up in a fresh pipette and 0.2 cc. of it measured into tubes
3 and 4, and the pipette laid aside.

The normal serum used as a control is similarly drawn into still
another pipette and 0.2 cc. measured into tubes 5 and 6, and the
pipette laid aside.

The alcoholic extract composing the antigen is next added, either
by diluting it so that 1 cc. contains the unit, or measuring the unit
quantity directly into the tubes. The antigen is added to tubes
I, 3, 5, and 7, and the pipette 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 cc.

Each tube is now shaken carefully, so as not to cause frothing 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 1 unit of the sheep corpuscle suspension and, with
the exception of tube 9, receives one dose of amboceptor, either the
serum measured by diluting so that 1 cc. equals the dose, or the
necessary square of paper. ‘This, in the former case, brings the total

Wassermann Reaction for Diagnosis of Syphilis

300

I. The Fixation Test, the first part of the Wassermann Test.
Each tube receives the reagents indicated, and is then stood in the thermostat at 37°C. for one hour in order that the complement be fixed.

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Il. The Hemclytic Test, the second part of the Wassermann Test.—The same tubes as above.
Each tube receives the additions indicated below, and is then again stood in the thermostat at 37°C. for two hours in order that the occurrence or non-occur-
rence of hemolysis may show whether the complement had previously been fixed or not. If the complement be fixed there is no hemolysis in tubes 1 and 3,
and the patient has syphilis (within such limitations as the validity of the test necessitates).

Dose of 1 Dose of 1Dose of ‘1 Doseof 1 Dose of 1LDose of LDoseof 1 Dose of SaltSolution

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LO i ee ae ee a ee:

The Hemolytic Amboceptor 301

bulk of fluid to 5 cc., in the latter makes it necessary to add 1 more
cubic cemtimeter of salt solution to each tube. We aim to have
exactly 5 cc. of fluid in each tube.

The tubes are again stood in the thermostat, where they are per-
"mitted to remain for two hours, 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 tule, the readings taken after the second hour of incubation
and those taken after twenty-four hours correspond.

A valid test should show the following:

Tubes
1. No hemolysis in syphilis. Hemolysis in health.
2. Complete hemolysis.

3. No hemolysis (this is the standard of comparison).
| 4. Complete hemolysis. ;
Test Controls. { 5.
6. “ 6c
rE
8
9

_ No hemolysis, as a rule.

In the tubes in which hemolysis takes place the change is very
marked. The hemoglobin dissolves out of the corpuscular stroma
and saturates the fluid, transforming it from the opaque pale red
to a transparent Burgundy red. Sometimes the corpuscular
stroma dissolves, sometimes it sediments 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 corpus-
cles sediment to the bottom of the tube, leaving a colorless fluid
above.

When the complement fixation is complete there is no solution
of the hemoglobin. Such a result has been described by Citron as
+-+-+-+. 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 deposit, regardless of the state of the
supernatant fluid, and megative 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 examina-
tion contains, as is sometimes the case, in health as well asin syphilis,
sheep corpuscle amboceptors. If tube 9 shows such amboceptors
to be in the serum, it explains the redness of the fluid bathing the
_ corpuscles, and does not invalidate the test. Ifno such amboceptors
are present and the fluid is still red, it may indicate that a little of

302 Wassermann Reaction for Diagnosis of Syphilis

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 diagnosis, ,
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; Oppenheim, one in a case of tumor
of the cerebellopontine angle; Marburg, one in a similar case; New-
mark reports 2 cases of brain tumors with positive reactions; Cohn,
a positive in a patient with a cerebral tumor. The Wassermann

Fig. 1or.—A typical positive Wassermann reaction with the recommended

controls as it appears after standing twelve hours. Corpuscular sedimentation
without hemolysis is seen in tubes.1, 3, and 9; complete hemolysis in the others.

reaction is of no value for the differential diagnosis of syphilis and
framboesia or yaws. All cases of the latter give a positive reaction.
Positive reactions have been found in some cases of nodular leprosy,
in a few cases of malaria, in some cases of pellagra, and in a good
many cases of sleeping sickness. These seem to form the greater
part of positive reactions in non-syphilitics thus far recorded.

In active syphilis Wassermann had go per cent. of positive reac-
tions in 2990 cases; and most others report about the same. Basset-
Smith in 458 such cases found 94 per cent. positive reactions.

In latent syphilis Wassermann found 50 per cent. positive reac-
tions; Basset-Smith, 46 per cent.

In chronic, presumably syphilitic, disease of the nervous system,

Noguchi’s Modification 303

general paresis, and tabes dorsalis the positive reactions vary. In
the former disease some have found as high as go 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 treat-
ment, 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 considering
the nature of the reaction. It does not occur because of the pres-
_ ence in the blood of syphilitics of antibodies which combine with
the antigen and fix the complement. It is probably not comple-
ment fixation so much as complementary inhibition, through the
presence in the blood of syphilitics of certain metabolic products,
whose action interferes with the complement in some entirely
different manner.

NOGUCHI’S MODIFICATION OF THE WASSERMANN REACTION

Noguchi* has modified the Wassermann reaction, first by employ-
ing 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
Wassermann 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, pro-
vided it be recognized.

Noguchi also varies the technic. in such a manner that very sata
quantities of the various reagents are employed—a necessity that
arises from the relatively small quantity of the patient’s blood ob-
tainable according to the method he employs. The reagents
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 punc-
ture near the root of the nail with a Hagedorn needle, and collects
about 2 cc. 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 re-
moved ‘with a capillary pipet. The serum may or may not be in-
activated by heat, according to the option of the experimenter.
The dose of the unheated serum is 1 drop; of the inactivated serum,

* “Serum Diagnosis of Syphilis,” Philadelphia, rgro, J. B. Lippincott Co.

f Syphilis

jagnosis o

Wassermann Reaction for D

304

4 drops.

The same doses of the normal and syphilitic control serums

are used.

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hysiologic salt solution by

ion in p

Two units constitute the “dose.”

the unit.

(2) The Complement.—This consists of fresh guinea-pig serum,
cc. is

Of it he makes a 40 per cent. dilut
adding one part of the serum'to 1}4 parts of the salt solution

Noguchi’s Modification B05

(3) The Antigen.—The antigen is made, according to the direc-
tions 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 ac-
cording to the size of the square of paper needed, instead of the quan-
tity 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
a1 per cent. suspension is recommended. If normal corpuscles are
employed, it is necessary to wash them free of the normal serum or
plasma, which Noguchi accomplishes as follows: 8 cc. of normal salt
solution are placed in a large test-tube, and the blood flowing from:
a puncture (in the operator’s own finger, for example) permitted to
drop in, the proportion being 1 drop each 4 cc. 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, ina laboratory, the corpuscles can be washed
as usual with the aid of the centrifuge. If the patient’s own cor-

‘puscles are to be employed, some of them may be distributed,

through the serum without any washing, 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 cor-
puscles the eye becomes accustomed to the color, intensity and
density corresponding to the requirement.

(5) Lhe Antihuman Hemolytic Amboceptor—This is prepared
by injecting rabbits, according to the method already described,
with washed kuman corpuscles obtained from fresh human placente
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 cumber-
some than that recommended for the Wassermann test and includes
six tubes. It can best be understood by reference to the diagram.

The method recommends itself through its simplicity and con-
venience, 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.

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,

PART Il

THE INFECTIOUS DISEASES AND THE
SPECIFIC MICRO-ORGANISMS

CHAPTER I
SUPPURATION

SUPPURATION was at one time looked upon as a normal and in-
evitable outcome of the majority of wounds, and although bacteria
were early observed in the purulent discharges, the insufficiency of
information then at hand led to the belief that they were spon-
taneously developed there.

It is probable that the first contribution to the infectious nature
of pyemia and sepsis was made by Rindfleisch* who found micro-
organisms in the metastatic abscesses in the heart muscle of a patient
dead of pyemia. Similar observations were subsequently made by
numerous others, but the first to definitely connect them with the
process was Klebs.t In 1874 Billroth described a micro-organism
that he called Coccobacteria septica, but which he regarded as an
effect, not a cause of suppuration. In 1880 Pasteurt cultivated
streptococci from cases of puerperal fever and looked upon them as
the cause of the disease. It was only after Koch|| had broken the
way for the really scientific investigation of the subject, that Ogston§
was able to show that there were two principal micro-organisms con-
cerned in suppuration, one occurring in groups resembling bunches
of grapes or fish-roe, called Staphylococci, the other like strings of
beads, called Streptococci.

Other investigators followed and confirmed the work of Ogston,
and Fehleisen,** and Rosenbach}} finally settled the relation of the
common organisms to the process of suppuration.

Suppuration, is not a specific infectious process.

Being but the expression of tissue irritation accompanied by strong
chemotactic influences, as many bacteria may be, associated with it
as can bring about the essential conditions. Bacteria with which

* “Lehrbuch des Pathologischen Gewebslehre,” 1866.

+ Beitrage zur. path. Anat. d. Schusswunden, Leipzig, 1872.

Compt.-rendu. de la Soc. de Biol. de Paris, 1880, XC, p. 1035.

I Untersuchungen iiber die Aetiologie des Wundinfektionskrankheiten,”
1878 Leipzig,.

§ “Brit. Med. Jour.,” 1881, March, p. 369.

rN Aetiologie des Erysipels,” Berlin, 1883.
tt “Mikroorganismen bei der Wundinfektionskrankheiten,” Wiesbaden, 1884.

397

308 Suppuration

these qualities are exceptionally marked appear as the common
cause of the process; those with which it is less marked, as excep-
tional causes.

The relative frequency with which certain varieties of bacteria
are associated with suppuration is aes in the following table
from Karlinski:*

Suppuration in man— Streptococci, : 45 cases.
: Staphylococci, 144
Other bacteria, Is “
Suppuration in the lower animals—Streptococci, a3,
Staphylococci, ag
; Other bacteria, 15 “
Suppuration in birds— Streptococci, Tr
Staphylococci, 4o “
Other bacteria, 20 “
Andrewes and ene after the examination of large numbers
of staphylococci from lesions of the human skin and mucous mem-
branes, came to the conclusion that four varieties are differentiable.
Of these, the Staphylococcus pyogenes is the most common and
most important. When typical, it produces an orange-colored pig-
ment; when atypical, it may be lemon yellow or white. Staphy-
lococcus epidermidis albus is a distinct species. The differences

between these cocci are shown in the table.

SrapHyLococcus Epmermipis ALBUS (WELCH)

General Characteristics —A inon-motile, non-flagellate, non-sporogenous,
slowly liquefying, non-chromogenic, aérobic and optionally anaérobic, 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, Welcht has
described a micrococcus which seems to be habitually present upon
the skin, not only upon the surface, but also deep down in the Mal-
pighian 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 sup-
puration, the conclusions of Robb and Ghriskey, that sutures of
cat-gut when tightly drawn may be a cause of skin-abscesses by
predisposing to the development of this organism, are certainly
justifiable. As the morphologic and cultural characteristics of the
organism correspond fairly well to those of the following species, no
separate description of them seems necessary.

STAPHYLOcoccuUS PyoGENES ALBUS (ROSENBACH)§

General Characteristics.—A non-motile, _non-flagellate, non-sporogenous,
liquefying, non-chromogenic, aérobic and optionally anaérobic, mildly patho-
genic coccus, staining by the ordinary methods and by Gram’s method.

* “Centralbl. f. Bakt.,” etc., 1800, vir, S. 113.

t “Report of the Local Government Board of Great Britain,” Supplement;
“Report of the Medical Officers, ”” 1905-06, vol. XXXV, P. 543.

t “Amer. Jour. Med. Sci.,’ "1891, P. 439.

§ “Wundinfektionskrankheiten des Menschen,” Wiesbaden, 1884.

309

Staphylococcus Pyogenes Albus

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310 , Suppuration

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 injected into rabbits and guinea-pigs, abscesses
occasionally result. Injected into the circulation, the staphylococci
occasionally 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 Staphylococcus
albus is almost identical with the species next to be described, dif-
fering from it only in the absence of its characteristic golden pigment.

STAPHYLOCoccUS PYOGENES AUREUS (ROSENBACH*)

General Characteristics.—A non-motile, non-flagellate, non-sporogenous,
liquefying, chromogenic, pathogenic, aérobic and optionally anaérobic 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, or “golden staphylococ-
cus,” first observed by Ogston and cultivated by Rosenbach. As
the morphology and cultural characteristics of this organism are
identical with those of the preceding species, it seems convenient 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.

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 some-
times occur in the feces, especially of children.

Staphylococci are the most common micro-organisms in some ~*
acne pustules, in furuncles, in carbuncles, in superficial and deep
abscesses, and in the ordinary run of surgical infections. So com-
mon are they that one should never be satisfied that he has exhausted
the etiological possibilities of the case through their demonstration.
He should always seek for less evident though sometimes far more
important organisms. In the absence of such, and in their absence
only, should the case be referred to staphylococci.

* “ Mikroorganismen bei Wundinfektionskrankheiten des Menschen,” Wies-
baden, 1884.

Staphylococcus Pyogenes Aureus et Albus 311

Morphology.—The cocci are small spheres measuring about 0.7-
1.oin diameter. There is no definite grouping in either liquid
or solid cultures. It is only in pus or in the organs or tissues of dis-
eased animals that one can say that a true staphylococcus (bunch.
of grapes) grouping occurs.

The organisms are not motile and have no flagella. They do not
form spores.

Fig. 102.—Staphylococcus pyogenes aureus cone:

Staining.—They stain easily and baliantly with aqueous solutions
of the anilin dyes and by Gram’s method.

Fig. 103 Staphylococcus pyogenes aureus. Colony two days old, seen upon
an agar-agar plate. X 40 (Heim).

Isolation.—Staphylococci are easy organisms to isolate, and can
be secured by plating out a drop of pus in gelatin or in agar-agar.
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 absence of oxygen at

312 Suppuration

temperatures above 8°C. and below 45°C., the most rapid develop-.
ment being at about 37°C.

Colonies.—Upon the surface of gelatin plates the colonies appear
as small whitish points, after from twenty-four to forty-eight hours,
rapidly extending to the surface and causing extensive liquefaction
of the medium. The formation of the yellow 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 present in
Staphylococcus aureus. It is this precipitate
in particular that gives the organism its name,
“golden staphylococcus.”

Agar-Agar—The growth of the golden
staphylococcus upon agar-agar is subject to
considerable variation in the ‘quantity of pig-
ment produced. Sometimes, perhaps rarely, it
is golden; more commonly it is yellow, often

Fig. to4—Staph- cream color. Along the whole line of inocula-
ylococcus pyogenes tion a moist, shining, usually well-circum-
aureus. Puncture scribed growth occurs. When the develop-
ad i cen res ment occurs rapidly, as in the incubator, it
kel and Pfeiffer). 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, Staph-
ylococcus aureus producing an orange-yellow coating over 4
large part of the surface. The potato cultures may give off a
sour odor.

Bouillon.—When grown in bouillon the organism causes a diffuse
cloudiness, with a small quantity of slightly yellowish sediment.
The reaction of the medium becomes increasingly acid.

Milk.—In milk, coagulation takes place in about eight days, and
is followed by gradual digestion of the casein. In litmus milk slow
acid production is observed.

Blood-Serum.—Discrete and confluent yellow colonies appear on
the surface of the medium in twenty-four hours. Through softening

Staphylococcus Pyogenes Aureus et Albus 313

and evaporation of the medium they sink down into shallow ex-
cavations after a few days have passed.

Thermal Death Point.—Staphylococci are usually quite suscep-
tible 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 Lingelsheim 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.

Vital Resistance.—The staphylococci resist drying well and re-
main alive upon paper or cloth for as long as two or three
months. Daylight has no injurious effect; direct sunlight can
be endured for an unusually long time. In antiseptic solutions -
they show no unusual resisting power and are killed in about
fifteen minutes by 1: 1000 mercuric chlorid or 5. ad cent. phenol
solution.

Metabolic Products——Staphylococci can make use of free or
combined oxygen, hence are aérobic or anaérobic. In liberating
combined oxygen, no gas is generated in any culture-medium. They
produce ferments by which gelatin is liquefied, milk coagulated and
digested, blood-serum digested and slowly liquefied. Indol, phenol
skatol and trimethylamine result from the protein transformations,
according to Emmering.* The indol can easily be detected in Dun-
ham’s peptone medium. A yellow pigment is produced. Nitrates
are reduced to nitrites in cultures kept for three days at 37°C.
Staphylococci are capable of producing fatty acids from sugars,
hence acidity develops in media containing dextrose, saccharose,
lactose, maltose, mannite and glycerin. No gas is evolved from the
fermentation of the sugars. The acids most commonly produced
are acetic, valerianic, butyric and propionic.

Wells and Coopert have found small quantities of lipolytic ferment
in agar-agar cultures. Kraust first showed a hemolytic ferment in
the cultures.

Toxic Products.—Leber seems to have first conceived of suppura-
tion as a toxic process depending upon the soluble products of
parasitic fungi, and in 1888, through the action of alcohol upon
staphylococci, prepared an acicular crystalline 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 prop-
_ erties as sterilized cultures of the staphylococcus; that repeated in-
jections of sterilized pus induce chronic intoxication 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

**Berliner deutsche chemische Gesselschaft,”’ 1896, p. 2721.
+ “Jour. Inf. Diseases,” 1912, x1, 388.
t “Wiener Klin. Wochenschrift,” 1900, IIL

314 Suppuration

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 leuko-
cidin. 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
observations have been abundantly confirmed. Kraussf first ob-
served that certain products of the staphylococcus were hemolytic
_ and destroyed red blood-corpuscles. This hemolysin has been
carefully studied by Neisser and Wechsberg,t by whom it was called
staphylolysin.

Durme§ found staphylolysin produced most abundantly by viru-
lent staphylococci.

Ribbert|| found that both sterilized and unsterilized cultures 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.

To secure the endo-toxin, masses of culture, prepared as described
in the section upon “ Bacterio-vaccines,” are ground in a mortar, or
frozen by liquid air and then ground, or the culture masses are
treated by dilute acids and alkalies according to Vaughan, or the
culture masses are permitted to undergo autolysis in physiological
salt solution or in dilute serum containing amboceptor and comple-
ment (see Bacteriolysis).

Staphylococcus aureus is not only found in the great majority.
of furuncles, carbuncles, abscesses, and other inflammatory dis-
eases of the surface of the body, but also plays an important rdle 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, the staphylococcus be injected into the circu-
lation, osteomyelitis 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

* “La Cellule,” 1896, x1, p. 349.
} ‘Wiener. klin. Wochenschrift,” 111, 1900.
t ‘Zeitschrift fiir 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,” 1896, vol. 1, p. 613.

Staphylococcus Pyogenes Aureus et Albus S15

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.

Pathogenesis.—The Staphylococcus aureus is therefore a danger-
ous and sometimes a deadly organism. Its virulence is, however,
very variable both for the lower animals and for man. The most
susceptible laboratory animal is the rabbit. Guinea-pigs, rats, mice,
dogs and cats are much less susceptible.

Intravenous Injections —The classical test for virulence is to inject
4/9 cc. 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 which time
the animal suffers from fever and wasting, and the occurrence of
multiple widespread foci of colonization with minute abscesses in
many of the organs. The heart is sometimes the seat of purulent
myocarditis, less frequently of septic endocarditis. The kidneys

- show minute abscesses, with aggregations of cocci in the glomeruli

and in the tubules. Highly virulent cultures kill the animal in from
one to two days, without abscesses.

Subcutaneous Injection—If a few drops of a virulent culture
be injected beneath the skin of a rabbit, there is a local reaction, an
abscess forms, the temperature rises and the animal becomes ill.
In a few days the abscess points and empties, the temperature re-
turns to the normal and the animal recovers. In exceptional cases
a generalized injection occurs and the rabbit dies.

Intraperitoneal Injection.—If the injection be made into the peri-
toneal cavity, pleural cavity or into a joint, there is primarily a
localized suppuration, peritonitis, pleuritis or arthritis, which is
usually followed in a day or two by generalized infection and death.

Human Injection When the cocci enter human beings subcuta-
neously, furuncles, carbuncles and abscesses commonly result, ac-
cording to the virulence of the organism and the resisting power of
the individual. Garre* applied the organism in pure culture to the
uninjured skin of his arm, and in four days developed a large car-
buncle, 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 pro-
duced an abscess in every case. When conditions of invasion are
most favorable, fatal generalization of the organisms may occur.
In such cases they may be cultivated from the streaming blood,
though the greater number collect in, and frequently obstruct, the
capillaries. In the lungs and spleen, and still more frequently in

*“Fortschritte der Med.,” 1885, No. 6, p. 170.
t “Monatschrift fiir prakt. Dermatologie,” 1887, 1v, No. 10.

316 Suppuration

the kidneys, infarcts are formed by the bacterial emboli. The
Malpighian tufts of the kidneys are sometimes full of cocci, and be-
come the centers of small abscesses.

Virulence.—Experiments have shown that both Staphylococci
aureus and albus exist in attenuated and virulent forms, and there
is every reason to believe that in the majority of instances they in-
habit the surface of the body in a feebly virulent condition.

Agglutination.—Kolle and Otto* have found that immune anti-
staphylococcic serums agglutinate the staphylococci. The reaction.
is not specific and is peculiar. All pathogenic staphylococci are
agglutinated; non-pathogenic cocci are not agglutinated. The re-
action cannot, therefore, be used for specific differentiation.

Specific Therapy.—The treatment of staphylococcus infections
with immune serum has not met with encouraging success. Vi-
querat,} Denys and van de Velde, t and Neisser and Wechsberg§ and

others have experimented in this direction, but the literature con- .. - .

tains very little evidence that beneficial results have followed the
employment of antistaphylococcus serums.

Bacterio-vaccination.—Although specific serums have failed,. a
promising form of specific treatment for subacute and chronic
staphylococcic infections has been introduced by A. E. Wright,||
who first isolates from the lesion the particular strain of staphylococci
by which it is caused, cultivates this artificially, suspends the or-
ganisms in an indifferent fluid, of which a given quantity contains a
known (counted) 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
beginning dose is 100 million cocci. Doses are given every six or
eight days, increasing the dose each time, until, if necessary 1000
million are administered at a dose.

The treatment is controlled by studying the “opsonic index”
(q.v.), the objects being the avoidance of the “negative phase” or
condition of diminished resistance, and the progressive establish-
ment of the positive phase or stage of increased resistance. Asthe
resistance increases the patient rapidly improves, and many cases
of obstinate acne, furunculosis, and other pyogenic infections have
quickly recovered under this treatment.

STAPHYLOcoccus Cirreus (PASSET)

An organism similar to the preceding, except that its pathogenicity
for animals is doubtful, its growth on agar-agar and potato of a
brilliant lemon-yellow color and that it does not liquefy gelatin,

* “Zeitschrift fiir Hygiene,” etc., 1902, XLI.
Ibid., xvi, 1894, p. 483.
* “La Cellufe,” 1895, x1. ‘
“Zeitschrift fiir Hygiene,” 1901, XXXII.
|| “Lancet,” March 29, 1902, p. 874; “Brit. Med. Jour.,”? May 9, 1903, Pp. 1009.
** See Bacterio-vaccination.

Streptococcus Pyogenes 317

is Staphylococcus citreus of Passet.* As it is not common and is
doubtfully pathogenic, it is of much less importance than the pre-
viously described organisms.

StREPTOCOCCUS PYOGENES (ROSENBACH)

General Characteristics—The streptococcus is a non-motile, non-flagel-
late, non-sporogenous, non-liquefying, non-chromogenic, aérobic and optionally
anaérobic, spheric organism, infections for man and the lower animals. It
stains by ordinary methods and by Gram’s method.

In 1880 Pasteur} first cultivated streptococci from the blood of
patients suffering from puerperal fever. In 1881 Ogston{ called

attention to the fact that two distinct kinds of cocci were to be
found in pus, mentioning both staphylococci and streptococci. The

Fig. 105.—Streptococcus pyogenes, from the pus taken from an abscess.
X 1000 (Frankel and Pfeiffer).

beginning of real knowledge of the ‘streptococci, however, dates
from the time of their isolation and cultivation by Fehleisen§ and
by Rosenbach.||

Distribution.—Streptococci are parasitic pathogenic organisms,
not known apart from human and animal hosts. They seem to
occur not infrequently, in health, upon the surface of the body, in
its various openings and in the alimentary canal. Such organisms
are to be regarded as potentially virulent and pathogenic in all cases.

They are the primary infecting agents in many inflammatory,

* “Untersuchungen iiber die Aetiologie der eitrigen Phlegmone des Menschen,”
Berlin, 1885, p. 9. :

t Compt.-rendu. de la societe de biologie de Paris, 1880, xc, p. 1035. ©

“Brit. Med. Jour.,” March, 1881, p. 369.
§ “‘Aetiologie des Erysipels,” Berlin, Fischer, 1883.
; || “Mikroorganismen bei Wundinfektionskrankheiten des Menschen,” 1884,
1» 22, ?

318 . Suppuration

purulent and septicemic disturbances—erysipelas, cellulitis, phleg-
mons, osteomyelitis, puerperal infection, pseudo-membranous angina,
phlebitis, salpingitis, meningitis, endocarditis, etc.

Berson points out that they are secondary agents of importance
in all pathological conditions of the throat of whatever nature.

Hektoen found them to be the most frequent complicating or-
gainsm in scarlatina and Councilman the most frequent complicating
organism in variola.

The suppurative conditions for which streptococci are held to be
responsible, differ from those caused by staphylococci in being more
rapidly spreading, more locally destructive, and more prone to
generalized infection or septicemia.

: Morphology.—The organisms are spheric, of variable size (0.4-1
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)

———

Fig. 106.—Streptococcus colonies on serum agar (From Hiss and Zinsser,
“Text-Book of Bacteriology,” D. Appleton & Co., Publishers).

and Streptococcus brevis (chains of from four to ten), have been
described by v. Lingelsheim,* but do not hold as separate species.

The streptococcus is not motile has no flagella and does not form
spores. ;

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 contain-
ing 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 temperature
and that. of incubation, its best and most rapid development being
at about 37°C.

Colonies.—Upon gelatin plates very small, colorless, translucent
colonies appear in from twenty-four to forty-eight hours. When
superficial, they spread out to form flat disks about o.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

* “Zeitschrift fiir Hygiene,” 1891, Bd. x, p. 331; 1892, XII, p. 308.

Streptococcus Pyogenes 319

a frayed-out appearance 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 along the whole
length of the puncture and form a slightly opaque granular line.

Agar-agar.— Upon agar-agar a delicate transparent growth de-
velops slowly along the line of inoculation. It consists of small
colorless, or slightly grayish transparent colonies which do not read-
ily coalesce.

The addition of glycerin or of one per cent. of dextrose to the
agar-agar or other media greatly facilitates the growth of the cocci.

On agar-agar plates the colonies are small grayish, translucent
and do not coalesce. If blood corpuscles be disseminated throughout
the medium the majority of virulent cocci cause hemolysis in a wide
zone about the colonies.

Blood-serum.—The growth upon blood-serum and upon Léffler’s
blood-serum mixture, resembles that upon agar-agar. The colonies
are small, white, discrete, 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 ad-
hering to the sides of the tube, sometimes forming a sediment.
When the flocculi formation is distinct, the name Streptococcus con-
glomeratus (Kurth) is sometimes given to the organism; when the
medium is diffusely clouded, it is called Streptococcus diffusus.

In mixtures of bouillon and blood-serum or ascitic fluid the strep-
tococcus grows more luxuriantly, especially at incubation tempera-
tures, 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, 1 per cent. of sterile powdered CaCO;
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 along
time.

Milk.—The organism seems to grow well in milk, which is coagu-
lated in from three to five days because of the development of lactic
acid.

Reaction.—The streptococcus is sensitive to acids, and can only
grow well in media with a slightly alkaline reaction. All strepto-
cocci produce acids and eventually acidulate the media, thus check-
ing 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.

* “Text-book of Bacteriology,” p. 338.

320 Suppuration

Sternberg found that the streptococci succumb at temperatures of
52° to 54°C. if maintained for ten minutes. The ability to resist
heat depends somewhat upon the surroundings. In albuminous
media they resist more strongly and to kill streptococci in tuber-
culous sputum, heating to roo°C. for some minutes is necessary.
Their vitality in culture is slight, and unless frequently transplanted
they die. Bouillon 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
fairly well.

Differential Features.—It is not always easy to differentiate
Streptococcus pyogenes from the pneumococcus. One of the best
methods is to take advantage of the hemolytic activity of the or-
ganism first observed by Bordet* and Besredkat by the employment
of blood-agar plates, suggested by Schottmiiller.{ Such platesare
easily prepared by melting ordinary culture agar-agar, cooling to
about 45°C., and then adding about o.5 cc. 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 hemo-
lytic substance that destroys the blood-corpuscles in the vicinity of
the colony, thus surrounding each by a clear, pale halo that con-
trasts with the red agar. The colonies themselves appear gray.

The test is not specific. Colonies of the pneumococcus usually ©
appear dark and without hemolysis, but Ruediger§ finds that they
also sometimes cause solution of the hemoglobin. There are also
certain streptococci whose colonies are green and without hemolysis.
These were called Streptococcus viridans by Schottmiiller and were at
first regarded as practically non-pathogenic, though it is now known
that they cause endocarditis in rabbits and in man.

Pathogenesis.—The streptococcus has been found in erysipelas,

-malignant endocarditis, periostitis, otitis, meningitis, empyema,
pneumonia, lymphangitis, phlegmons, sepsis, puerperal endo-
metritis, and many other forms of inflammation and septic infection.
In man it is usually associated with active suppuration and sepsis.

The relation of the streptococcus to diphtheria is of interest, for,

_ though in all probability the great majority of cases of pseudo-

membranous angina are caused by the Klebs-Loffler bacillus, yet a

number are met with in which, as in Prudden’s 24 cases, no diph-
theria bacilli can be found, but which seem to be caused by the
streptococcus alone.

There are few clinical differences between the throat lesions pro-

duced by the two organisms, and the only positive method of dif-

*“ Ann. de l’Inst. Pasteur,” 1897, XI, 177-
ft “Ann. de l’Inst. Pasteur,” IQOT, XV, 880.
t“Miinch. med. Wochenschrift,”? 1903, L, p. 909.
§ “Jour. Amer. Med. Assoc.,’’ 1906, XLVI, p. 1171.

Streptococcus Pyogenes aot

ferentiating the one from the other is by means of a careful bacterio-
logic examination. Such an examination should always be made,
as it has much weight in connection with the treatment; in strepto-
coccus angina no benefit can be expected from the administration of
diphtheria antitoxic serum.

Hirsh* has shown that streptococci are by no means rare in the
intestines of infants, where they may occasion enteritis. In such
cases the organisms are found in large numbers 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.

Libmanf has reported 2 carefully studied cases of streptococcic
enteritis.

Flexner,t in a larger series of autopsies, found the bodies in-
vaded by numerous micro-organisms, causing what he has called
“terminal infection,” and hastening the fatal issue. Of 793 autop-
sies 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 requirements of a statistical
inquiry. Tuberculous infections were not included. Of the 255
cases, 213 gave positive bacteriologic results. ‘‘The micro-organ-
isms causing the infections, 38 in all, were Streptococcus pyogenes,
16 cases; Staphylococcus pyogenes aureus, 4 cases; Micrococcus

_lanceolatus, 6 cases; gas bacillus (Bacillus aérogenes capsulatus),
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 capsulated bacillus, and an unidentified
coccus.”

It is interesting to observe in how many cases the streptococcus
was present. All the streptococci found may not have been Strepto-
coccus pyogenes, but for convenience in his statistics they were re-
garded as such.

The presence of streptococci in the blood in scarlatina has been
observed in 30 cases by Crooke, by Frankel and Trendenburg,
Raskin, Leubarth, Kurth, and Babes. In 11 cases of scarlatina
studied by Wright§ a general streptococcus infection occurred in
4,4 pneumococcus infection in 1, and a mixed infection of pyogenic
cocci in 1,

Lemoine|| found streptococci in the blood during life in 2 out of
33 cases of'scarlet fever studied. Pearce** studied 17 cases of scarla-

*“Centralbl. f. Bakt. u. Parasit.,’’ 1897, Bd. xxu1, Nos. 14 and 15, p. 369.
{Centralbl, f. Bakt. u. Parasit.,” 1897, Bd., xxur, Nos. 14 and 15, p. 376.
_ Journal of Experimental Medicine,” 1896, vol. 1, No. 3.
ei Boston Med. and Surg. Jour.,” March 21, 1895.
‘Bull. et Mém. Soc. d’Hop. de Paris,” 1896, 3 s., XIII.
Jour. Boston Soc. of Med. Sci.,” March, 1898.
21

‘
i

322 Suppuration

tina 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 Staphy-
lococcus pyogenes aureus was associated with the streptococcus.

The streptococcus is the most common organism found in the
suppurative sequele of scarlatina, frequently occurring alone;
sometimes with the staphylococci; sometimes with the pneumococci.

Rosenow* cultivated streptococci from cases of arthritic, cholecys-
titic and ulcerative gastritis. He confirmed the observation of
Forsenert} that streptococci taken from an organ in which they have
successfully colonized in one animal and injected into a new animal,
colonize by preference in the organs corresponding to those from
which they were taken. -

Virulence.—Streptococci isolated from human beings vary greatly
in pathogenic action upon the laboratory experiment animals. In
many cases, although they have induced a fatal illness in human
beings, they are without effect upon the lower animals; in other
cases, although from a more simple lesion that recovered, they are
extremely fatal for the most susceptible animals, rabbits and mice.
Rats sometimes become ill when injected with virulent cultures in
large doses, but usually recover. Guinea-pigs, cats, and dogs are
but slightly susceptible even when the cultures are virulent. 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 diein from one to four days from generalinfection. If
the organisms 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. All streptococci seem to be most pathogenic
for that species of animal from which they have been isolated.

If the ear of a rabbit be carefully scarified, and cutaneously in-
oculated 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 may die 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.

When mildly virulent cultures of the variety called Streptococcus
viridans are intravenously injected into rabbits, some time elapses
before much disturbance is noted, then the animal becomes ill and
eventually dies of cardiac disease. Verrucose endocarditis with
marked calcification of the mitral valve, with secondary metastatic
subacute glomerulonephritis was observed in those cases which were
carefully studied by Libman.f

* Jour. Amer. Med. Asso.,”’ 1913, LX, 12233 LXI, 1947} 1914, LXII, 1835; “Jour.
Inf. Dis.,” 1915, xvi, No. 2, p. 240.

t Nordiskt diciniskt Archiv., 1902, xxxv, p. 1.

} Amer. Jour. Med. Sci., 1910, CxL, 516; 1912, CLXIV, 313; Trans. Asso. Amer.
‘Phys., 1912, XXVII, 157.

Streptococcus Pyogenes 323

According to Marmorek,* the virulence of the streptococcus can be
increased to a remarkable degree by rapid passage through rabbits,
and maintained by the use of a culture-medium consisting of 3 parts
of human blood-serum and 1 of bouillon. The blood of the ass or
ascitic or pleuritic exudates may be used instead of the human
blood-serum if the latter be unobtainable. By these means he
succeeded in intensifying the virulence of a culture to such a degree
that one hundred-thousand millionth (un cent milliardiéme) of a
cubic centimeter injected into the ear vein was fatal.

Petruschky{ found the virulence of the culture to be well re-
tained when the organisms were planted in gelatin, transplanted
every five days, and when grown, kept on ice.

Holst{ observed a virulent Streptococcus brevis that remained
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.

Metabolic Products.—The streptococcus produces a ferment by
which milk is coagulated. A few streptococci (S. faecalis of And-
rewes and Horder) are said to produce gelatine softening ferments,
but this Streptococcus pyogenes never does.

The organisms derive O from the atmosphere or from compounds,
but no gas is ever evolved in the process, though acids are always
produced in the presence of saccharose, lactose, rhamnose (isodulcite)
-Yaffnose, inulin, amygdalin, arbutin, coniferin, digitalin, helicin,
populin, salicin, glycerin, sorbite and mannite (Gordon). No acids
are formed from starch, glycogen, arabin, convolvulin, huperidin,
jalapin, methyl glucoside, saponin, glycol, erythrite or dulcite
(Gordon).

Marmorek|| and Lubenau** found that cultures of the strep-
tococcus when grown in bouillon containing glucose, produced a
hemolytic substance—streptokolysin—not seemingly present in
cultures grown in ordinary bouillon. Besredkat{ found that strep-
tokolysin was produced only by highly virulent cultures of the
streptococcus and not by saprophytic organisms that have been
for some time under cultivation in the laboratory.

Levintt investigated the subject thoroughly and found that
different strains of streptococci produced streptokolysin 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

*“Ann. de l’Inst. Pasteur,” July 25, 1895, p. x, No. 7, 593-
t“Centralbl. f. Bakt. u. Parasitenk.,” May 4, 1895, Bd. xvii, No. 16, p. §51.
{ Ibid., March 21, 1896, Bd. xx, No. 11.
Fliigge’s “Die Mikroorganismen.”
“Annales de l’Inst. Pasteur,’’ 1895, 593.

Centralbl. f. Bakt.,” etc.. 1901, Bd. xxx, Nos. 9 and 10.

{ “Ann, de I’Inst. Pasteur,” 1901, p. 880.
Th“Nord. Med. Ark.,”’ 1903, 11, No. 15, p. 20.

|
7K

324 ’ Suppuration

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 filtra-
tion or by centrifugation there is nearly one thousand times as
much as in the filtered fluid culture. The streptokolysin is not
destroyed by the death of the bacteria. Avntistreptokolysin is pres-
ent in antistreptococcus serum.

Varieties and Types of Streptococci.—The discussion of the meta-
bolic products of the streptococcus brings up the subject of the
unity or plurality varieties, which has not yet been settled. Scho-
telius* thought that definite varieties could be differentiated through
the hemolytic test and described 1. Streptococcus longus sur hemo-
lyticus, and 2. Streptococcus metior sur viridans. The former was
hemolytic, the latter not. Gordont believed that it was better ac-
complished through attention to the fermenting powers of the organ-
ism. His results were carefully investigated by Andrewes and
Horder,t who give us the following tabulation.

= 13) is} a
2/z/8/ Sle) sla/ dl] ios] sas
Streptococcus pyogenes..... | — | — +|+ Sey eat [ie ae) be sete +
Streptococcus salivarius..... |+|+|+|]+]+/—|]—-|-|-/+/;-|]4+]-
Streptococcus anginosus....|+/+!+/+/—|—-|—]—!—l4}+ +
Streptococcus fecalis....... +Hyei ty 4+ i —)—} +} +l 4+]4+)-)4+i-
Pneumococcus.,........... shy feeb ee fetes) ae pe bee ef et ae

Endeavors by Buerger§ to improve upon this plan were not con-
clusive, and attempts by Kinsella and Swift|| to make type separa-
tions by complement-fixation tests have failed.

Toxic Products.—The toxic products of the streptococcus are
not well known. Cultures from different sources vary greatly in
the effects produced by hypodermic or intravenous 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 strepto-

* Miinchen med. Wochenschrift, 1903, No. 21-and 22. ;
t Report of the Medical Officer of the Local Government Board, 1903-4}
Lancet, London, 1905, Nov. 11, p. 1400.
tLancet, Lond., 1906, 1, pp. 708, 775, 852.
§ “Jour. of Exp. Med.,” 1907, ix, p. 428.
| “Jour. of Exp. Med.,” 1917, xxi, p. 877.
** “Centralbl. f. Bakt.,” Dec. 18, 1903, XXxv, No. 3, p. 308.

Streptococcus Pyogenes 325

cocci of different stocks had nothing to do with their virulence.
Simon* also found that the toxic products of the streptococcus were
diverse and peculiar. The bodies of the cocci contained an intra-
cellular toxin the activity of which was independent of 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 in-
dependent 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 un-
less 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 occasional ac-
cidental erysipelatous infection of malignant tumors is followed
by sloughing and the subsequent disappearance of the tumor,
suggested the experimental inoculation of such tumors with Strep-
tococcus 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 Coleyf has recommended that the
toxin instead of the living coccus be used for injection.

A virulent culture of the streptococcus is obtained, by preference from a fatal
case of erysipelas, inoculated into small flasks of bouillon, and allowed to grow for
three weeks. The flask is then reinoculated with Bacillus prodigiosus, allowed
to grow for ten or twelve days at the room temperature, well shaken up, poured
into bottle of about £3ss 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

had been most efficacious; but, although Coley still recommends it and Czerny
upholds it, the majority of surgeons have failed to secure the desired results.

Antistreptococcus Serum.—Since 1895 considerable attention
has been bestowed upon the antistreptococcus serum of Marmorekt
and Gromakowsky,§ which is said to act specifically upon strepto-
coccus 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.

The serum is prepared by the injection of cultures of living viru-
lent streptococci into horses, unti! a high degree of immunity is

*“Centralbl. f. Bakt.,” Jan. 16, 1904, xxxv, No. 4, p. 350.

+ “Amer. Jour. Med. Sci.,”’ July, 1894.

aa de l’Inst. Pasteur,” July 25, 1895, Ix, No. 7, Pp. 593-
id.

326 Suppuration

ane: The serum is probably both antitoxic and buctenicaal
in action.

The success following the serums of some acimcntes 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 streptococci. To obviate this -
inequality Van de Velde* 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 Tavelt and Moser,{ is made by using cultures from dif-
ferent 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 thera-
peutic application.

Streptococcus Vaccine.—Vaccines made by the method given in
the chapter on “ Bacterio-vaccines”’ are now used in all streptococcus
infections with varying succes. As, however, there is no knowledge
by which one can foretell exactly what course a streptococcus in-
fection will pursue, it is impossible to determine with accuracy
what advantage results from the treatment. Judged upon its
clinical merits, streptococcus vaccine does good, especially when
the vaccine is homologous. When homologous vaccine cannot be
prepared, preference might next be given the so-called “polyvalent”
vaccines made by combining cultures from many sources. Such
especially when “sensitized”? by admixture with antistreptococcus
serum according to the method of Besredka, give promise of benefit
upon theoretical grounds. :

Bacteriological Diagnosis and Differentiation —The micro-organ-
isms sometimes appear in the original tissue juices as diplococci or
in such short chains as to be mistaken for diplococci. Under such
conditions mistakes are easily made and Boston and Pfahler§ were
led to believe that erysipelas was caused by a diplococcus. Pairs
and short chains also sometimes occur in clumps and can be mis-
taken for staphylococci. Cultures upon solid media also appear in
such form as to make it difficult to tell the correct grouping. Under
such circumstances cultures in liquid media usually offer the char-
acteristic rosary-like chains.

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

* “ Archiv. de. méd. Expér.,” 1897.

t ‘Deutsche med. Wochenschrift,” 1903, No. 50.

{ ‘Berliner klin. Wochenschrift,”’ 1902, 13.

§ “Phila. Med. Jour.,” Jan. 13, 1900.

|| “Journal of Medical Research,” 1901, N. S. 1, 163.

Streptococcus Mucosus 327

again later by Schottmiiller* from a case of parametritis, peri-
tonitis, meningitis, and phlebitis.

It occurs as a rounded coccus in pairs and in short chains, though
sometimes long chains of a hundred have been observed. The pairs
resemble gonococci. They measure 1.25 to 1.75 uw in length and
0.5 to 0.75 win breadth. Each is surrounded by a halo that varies
in width from 1.5 to 3.0 wu, 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 the
organism ferments inulin, which made Hiss think it related to the

Fig. 107.—Streptococcus mucosus, from peritoneal exudate. < 1200
(Howard and Perkins, in “ Journal of Medical Research”’).

pneumococcus. It is now generally believed to correspond to type
1 of the pneumococci (q.v.).

STREPTOCOCCUS ERYSIPELATIS (FEHLEISEN)

The streptococcus of Rosenbach is generally thought to be iden-
tical with a streptococcus described by Fehleisent 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 individuals, but sometimes reach-

- Ing a hundred or more in number. Occasionally the chains occur
in tangled masses.

* “Minch. med. Wochenschrift,’”’ 1903, XXI. ;
} “Verhandlungen der Wurzburger med. Gesellschaft,” 1881.

328 _ Suppuration

They can be cultivated at the room temperature, but grow much
better at 30° to 37°C. They are not particularly sensitive to'the
presence or absence of oxygen, but perhaps develop 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. 5

Micrococcus TETRAGENUS (GAFFKY)

General Characteristics.—Large, round, encapsulated cocci, regularly asso-
ciated in groups of four, forming tetrads. They are non-motile, non-flagellated,
non-sporogenous, non-liquefying, non- -chromogenic, non-aérogenic, aérobic and
optionally aérobic, pathogenic for mice and other small animals, and stain well
by all methods, including that of Gram.

A large micrococcus surrounded by a broad capsule, ea in
fours and hence known as Micrococcus tetragenus can sometimes

Fig. 108.—Micrococcus tetragenus in spleen of infected mouse. (From Hiss
and Zinsser, ‘“‘Text-Book of Bacteriology,” D. Appleton & Co., Publishers.)

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 im-
portance in connection with the pulmonary abscesses which compli-
cate tuberculosis. It was discovered by Gaffky.*
Morphology.—The cocci are rather large, measuring about 1
* “Archiv. f. Chirurgie,” xxvuiz, 3.

Micrococcus Tetragenus 329

in diameter. In cultures they do not show the regular arrange-
ment in tetrads as constantly as in the blood and tissues of animals,
where they occur in groups of four surrounded by a transparent
gelatinous capsule.

Staining —The organisms stain well by ordinary methods and
beautifully by Gram’s method, by which they can best be demon-
strated in tissues.

Isolation.—The organism can be isolated by inoculating a white
mouse with sputum or pus containing it, and after death recovering
it from the blood.

Cultivation.—It grows readily upon artificial media. At temper-
atures ranging from 12°C. to 45°C., the optimum being 37°C.

Colonies.—Upon gelatin plates small white colonies are produced
in from twenty-four to forty-eight hours. Under the microscope

Fig. 109.—Micrococcus tetragenus; colony twenty-four hours old upon the -
surface of an agar-agar plate. X 100 (Heim).

they appear spheric or elongate (lemon shaped), finely granular, and
lobulated like a raspberry or mulberry. When superficial they are
white and elevated, 1 to 2 mm. in diameter.

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.

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.

Bouillon.—A uniform clouding of the medium takes place. Acid
but no gas is produced when dextrose, lactose, saccharose and
mannite are added.

Milk.—The milk is not changed in appearance and is not coagu-
lated. Litmus milk is slightly acidulated.

Blood-serum.—The growth upon blood-serum is also abundant,

330 Suppuration

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 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 ab-
scesses result from subcutaneous inoculation.
The tetracocci, when present, probably hasten the tissue-necrosis

Fig. 110.—Bacillus pyocyaneus, from an agar-agar culture. XX 1000
(Itzerott and Niemann).

in tuberculous cavities, aid in the formation of abscesses 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 demostrating that the tetracoccus may be the cause of a pseudo-
membranous angina, 3 cases of which came under his observation.
Bezangonf has isolated this organism from a case of meningitis.
Forneaca} has reported a case of generalized tetragenous septicemia.

Bacittus PyocyAaNEeus (GESSARD)

General Characteristics.—A minute, slender, actively motile, flagellated, non-
sporogenous, chromogenic and feebly pathogenic, aérobic or facultative anaérobic,
liquefying bacillus, staining by ordinary methods, but not by Gram’s method.

* “Phila. Med. Jour.,’’ April 22, 1899.
t “Semaine Medicale,” 1898.
} “Riforma Medica,” 1903.

Bacillus Pyocyaneus 331

In some cases pus has a peculiar bluish or greenish color, which
depends upon the presence of Bacillus pyocyaneus of Gessard.*

Distribution.—The bacillus appears to be a rather common
saprophyte, being found in feces, manure, and water. It easily
takes up its. residence upon the skin and mucous membranes, and
has been found in the perspiration. It sometimes occurs as a sapro-
phyte upon the surgical dressings applied to wounds, and some-
times invades the tissues through wounds, to occasion dangerous
infections.

Morphology.—It is a short, slender organism with rounded ends,
measuring 0.3 X 1 to 2 mw, according to Fliigge; 0.6 X 2 to 6 u, ac-
cording to Ernst, and 0.6 X 1 yu, according to Charrin. It is quite
pleomorphous, which probably accounts for the difference in measure-
ments. It is occasionally united in chains of four or six. It is

actively motile, has one terminal flagellum, and does not form spores.

It closely resembles a harmless bacillus found in water, and
known as Bacillus flourescens liquefaciens, from which RuzickaT
thinks it has probably descended.

Staining.—It stains well with the ordinary staining solutions, 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.—The organism grows readily upon all ordinary
culture media, under aérobic and anaérobic conditions and at tem-
peratures ranging from 18° to 45°C., the optimum temperature
being 37°C.

Colonies.—The superficial colonies upon gelatin plates are small,
irregular, slightly greenish, ill-defined, and produce a distinct
fluorescence of the neighboring medium.

*“De la Pyocyanine et de son Microbe,’”’ Thése de Paris, 1882.
1 “Centralbl. f. Bakt. u. Parasitenk.,’”’ July 15, 1898, p. 11.

332 Suppuration

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 the liquefaction pro-
gresses. Four or five days must elapse before the medium is all
fluid.
Gelatin Punctures.—In gelatin puncture cultures the chief de-
velopment 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 (fluorescin) which soon
saturates the culture-medium and gives it the characteristic floures-
cent appearance. As the culture ages, or if the medium upon which
it grows contains much peptone, 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 Jordan.* Its
formula, according to Ledderhose, tf is CuH4N20.

A well-known feature of the growth upon fresh agar-agar, upon
which much stress has recently been laid by Martin,t is the forma-
tion of crystals in fresh cultures. Crystal formation in cultures of
other bacteria usually takes place in old, partially dried agar-agar,
but Bacillus pyocyaneus often produces crystals in a few days upon
fresh media. 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 flourescence, 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 formation of a,third pigment,
but the work of Boland§ has shown this to be a transformation prod-
uct 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

* “Journal of Experimental Medicine,” 1899, vol. rv.

{ ‘Deutsche Zeitschr. f. Chirurgie,” 1888, Bd. xxvut. ’
I “Centralbl. £. Bakt.,”’ April 6, 1897, XXI, Pp. 473.

§ “Centralbl. f. Bakt.,”’ 1899, Bd. xxv, p. 879.

Bacillus Pyocyaneus 333

ferment, the pyocyanase of Emmerich and Liéw. It produces no
diastatic ferments, so does not ferment carbohydrates.

It also produces, under favorable conditions, a toxin which has
been studied by Wassermann, who found it fatal in doses of 0.2
to o.5 cc. when intraperitoneally injected into guinea-pigs. The
animals show peritonitis and punctiform hemorrhages on the serous
membranes.

Bullock and Hunter* found that Bacillus pyocyaneous also pro-
duces a hemolytic substance, pyocyanolysin, by which corpuscles of
man, oxen, sheep, apes, rabbits, cats, rats, dogs, and mice are dis-
solved. The peculiar substance was produced in greatest quantity
in virulent cultures three or four weeks old. Jordanf believes that
this hemolytic property depends solely upon the intense alkali
formed in old cultures. Gheorghewskit found a leukocyte-destroy-
ing substance in the cultures.

In addition to the metabolic pigments mentioned, the organism
produces toxins. Wassermann§ found that filtrates of old cultures
were more toxic for guinea-pigs than the endotoxins 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
cc. of a virulent bouillon culture, injected into the subcutaneous
tissue of a guinea-pig, causes rapid edema, suppurative inflamma-
tion, 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.
Rats and mice behave similarly to guinea-pigs when inoculated
subcutaneously. ;

Rabbits are less susceptible and subcutaneous injections rarely
cause death. Intraperitoneal injection may be followed by fatal
infection if the bacillus be highly virulent or if it be not virulent,
recovery may occur. Intravenous inoculation causes fever, al-
buminuria, diarrhea and death in a day or two. If the dose be
smaller or the virulence of the culture less, a subacute disturbance ~
characterized by wasting, palsy and convulsions may occur. If
the animal dies, nephritis can usually be found, and perhaps explains
the symptoms.

Dogs are susceptible to infection by B. pyocyaneus, the symptoms
bearing a considerable resemblance to rabies.

Blum|| reports a case of pyocyaneus infection with endocarditis
‘in a child.

*“ Centralbl. f. Bakt.,” xxVIII, 1900, p. 865.
. {Ibid., Bd. xxxrm, Ref. 1903.
t “Ann. de l’Inst. Pasteur,” 1899, XIII.
§ “Zeitschrift fiir Hygiene, 1896, XXII. f
|| “Centralbl. £. Bakt. u. Parasitenk.,’’ Feb. 10, 1899, xxv, No. 4.

3 34 Suppuration

Lartigau,* in his study of “The Bacillus Pyocyaneus as a Factor
in Human Pathology,” sums up what is known about this réle of
the organism as follows:

“The Bacillus pyocyaneus, like many pathogenic micro-organisms, is occasion-
ally found in a purely saprophytic réle 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 Eberth and Audanard. Abelous demonstrated its presence
in the stomach as a saprophyte. Its existence in suppurating wounds has
long been known, and Koch early detected its presence in tuberculous cavities,
regarding it as an organism incapable of playing any pathologic réle. The
etiologic relation of the organism to certain cases of purulent otitis media in
children was pointed out by Martha, Maggiora 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 cultures 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, Ehlers, and Barker are interesting instances of its réle 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 bronchopneu-
monia, by Monnier; diarrhea of infants,by Neumann, Williams, Thiercelin and
Lesage, and other observers; dysentery, by Calmette and by Lartigau; and
general infection, by Ehlers, Neumann, Oettinger Karlinski, Monnier, Krannhals,
Calmette, Finkelstein, and L. F. Barker.”

Nine additional cases of human infection are reported by Perkins. t

Immunity.—Immunity against pyocyaneus infection develops
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 injection of either the endo-
toxin or the exotoxin. In the immunity resulting from the treat-
ment with bacterio-vaccines the serum of the animal becomes
agglutinative and bactericidal; in the immunity resulting from
treatment with the exotoxin, antitoxin is produced.

Bacittus Proteus Vuicaris (Hauser)

Synonym.—Proteus vulgaris

General Characteristics.—An actively motile, flagellated, non-sporogenous,
non-chromogenic, liquefying, aérobic and optionally anaérobic, doubtfully
pathogenic, aérogenic 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 Hausert 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 wher-

* «Phila. Med. Jour.,” Sept. 17, 1898.

+ “Jour. of Med. Research,’’ rgo1, vol. vI, p. 281.
{ “Ueber Faulnissbakterien,” Leipzig, 1885.

Bacillus Proteus Vulgaris 335

ever 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—pleo-
morphic—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 occasional 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 yw, but the length varies from 1.2
por less to 4 zor more. No spores are formed. The organisms are
actively motile. The long filaments frequently form loops and
tangles. Flagella are present in large numbers. Upon one of the

Fic. 112.—Bacillus proteus, showing flagella (Migula).

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 but not
by Gram’s method.

Cultivation.—The proteus is easily cultivated and grows well in
all the artificial media. -

Colonies.— Upon gelatin plates a typical phenomenon is 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.

_Krusef 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 magnification the center of each is seen to be surrounded by radiations ex-
tending in all directions into the solid gelatin, and made up of chains of bacilli.
Between the radiations and the granular center bacteria are seen in active motion.

pon the surface the colony extends as a thin patch, consisting of a layer of
bacilli arranged in threads, sending numerous projections from the periphery.

* Fliigge’s “‘ Die Mikroorganismen.”

436° Suppuration

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 extension and circular movement, detach themselves from the colony and
wander about upon the plate. From the radiated 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 have been
frequently transplanted, the phenomenon is less marked or 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. ,

A
= ~ iN “
oe
eg

EEN, 4

Fig. 113.—Swarming islands of proteus bacilli on the surface of gelatin; X 650
(Hauser).

Gelatin Punctures.—Puncture cultures in gelatin are not char-
acteristic. A stocking-like liquefaction occurs and extends so rap-
idly that the entire medium is liquefied in a few days. Anaérobic —
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 ameboid movement of the
colonies sometimes occurs.

Potato.— Upon potato the growth occurs in the form of a smeary
patch of soiled appearance. os

Milk is coagulated and peptonized. .

Metabolic Products.—The bacillus rapidly decomposes pro-
teins, albumen, fibrin, blood-serum and gelatin. According to

Amebe and Suppuration 337

Emmerling* in,so doing it gives off as cleavage products, trimethyl-
amine, betain, phenol and hydrogen sulphide. Taylor} found that
casein was transformed with deuteroalbumose, peptone, histidin,
lysin, tyrosin, indol and skatol as cleavage products. Ammonia is
liberally formed so that sugar-free cultures become alkaline.

In bouillon containing sugars, dextrose and saccharose are fer-
mented with the evolution of H:CO, = 24, and the formation of
some lactic and formic acid. Lactose is untouched. Nitrates are
reduced to nitrites, and then partly reduced to ammonia.

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 cul-
ture 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 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. Tsiklinsky{
in studying the diarrheas of nursing infants found Bacillus proteus
vulgaris i in 65 per cent. and believed it to be the important patho-
genic agent concerned in the etiology of the trouble. °

Bordoni-Uffreduzzi has shown that the proteus quite regularly
invades the tissues after death, though it appears unable to main-
tain 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 pro-
duce 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 Wesenberg,§ who
cultivated the organism from the putrid meat by which 63 persons
were made ill. Silverschmidt|| and Pfuhl** have made similar in-
vestigations with similar results.

AMEBZ AND SUPPURATION

The process of suppuration is not confined to bacterial micro- ©
organisms, but is shared to a limited extent by the protozoa. Thus,

*“Berliner Chemische Gesellschaft,” 1896, p. 2711.

} ‘Zeitschrift £. Phys. Chem., 1902, XXXVI.

f “Ann. de I’Inst. Pasteur,” 1917, XXXI, Pp. 517.

} “Zeitschrift fiir Hygiene, ”” etc., 1898, XXVIII.

|| Ibid., 1899, xxx. ** Thid., 1900, XXXV.
22

338 - Suppuration

Entameeba histolytica (g.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.

Entameeba buccalis (Prowazek*) is a small ameba that has been
found in purulent exudates in the oral tissues of persons with carious
teeth. It isat present thought to be the cause of Riggs’ disease or
pyorrhea alveolaris.

Ameeba kartulisi (Dofleint) appears to be capable of exciting
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 operated upon for the removal of a
piece of bone. It is 30 to 38 w in diameter, is actively motile. Its
coarse protoplasm contains red and white blood-corpuscles. Kar-
tulist found the same organism five times in other cases, and
Flexner§ found it also.

Amceba 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 DeEscrIBED 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.

* “Arbeiten a. d. Kaiserl. Gesundh. Amt.,” 1904, XxI, 1, Bull. p. 42.
t ‘Die Protozoa als Krankheitserreger,”’ Jena., 1901, p. 30.
Coon. f. Bakt. u. Parasitenk.,” 1903, XXXII, p. 471.

“Bulletin of the Johns Hopkins Hospital,’ 1892, xxv.
|| ‘University of Pennsylvania Medical Bulletin,” Sept., 1910.

CHAPTER II
MALIGNANT EDEMA

BaciLLus CEpEMATIS Maticni (Kocn)
Synonym.—Vibrion septique.

General Characteristics—A motile, flagellated, sporogenous, anaérobic,
liquefying, aérogenic, 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 putrescent
animal infusions and called by him (1875) Vibrion septique. It was
later more carefully studied and described by Koch. ft

It is supposed that this bacillus was among the organisms whose
introduction into wounds in the days of pre-antiseptic surgery,
commonly occasioned the then prevalent ‘Hospital gangrene.”
' Distribution.—The organism is widely distributed in nature,
being commonly present in garden earth. It is also found in dusts,
in waste water from houses, and sometimes in the intestinal content
of animals. “

Morphology.—The bacillus of malignant edema is a large rod-
shaped organism with rounded ends, measuring 2 to 10 uw by 0.8
to1.ou. It is usually motile, and possesses many flagella. It pro-
duces 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 as a rule.

Cultivation.—The organism is a strict anaérobe, but under con-
ditions 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 pire culture, being most easily obtained
from the edematous tissues of guinea-pigs and rabbits inoculated
with garden. earth.

Colonies.—The colonies which develop upon the surface of
gelatin kept under anaérobic 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 fila-
ments which under a high power exhibit active movement. The
edges of the colony have a fringed appearance, much like the colonies
of the hay or potato bacillus.

Gelatin—In gelatin tube cultures the characteristic growth
cannot be observed unless the tube be placed under anaérobic

*“Bull. Acad. Med.,” 1877 and 188r.
t “Mittheilungen aus dem kaiser]. Gesundheitsamte,” 1, 53.
339

340 Malignant Edema

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
which contain a srnall amount of gas. In gelatin to which a little
grape-sugar has been added the gas production is marked.
Agar-agar.—The growth takes place in the form of a cloudy
stream, in the lower’part of deep punctures in recently heated
agar-agar, from which the air has been expelled. If the agar-agar
contains 1 per cent. of glucose, it is soon split up by the gas for-
mation. Such cultures give off a very disagreeable odor.

Fig. 114.—Bacillus of malignant edema, from the body-juice of a guinea-pig
inoculated with garden earth. XX 1000 (Frankel and Pfeiffer).

Bouillon.—In deep tubes of recently heated bouillon a diffuse
turbidity occurs in about twenty-four hours. After the third day
the upper half.clears, the bacilli and spores sedimenting or moving .
away from the oxygen. The culture gives off a very disagreeable
odor. ‘

Milk.—Milk is slowly coagulated, and later digested.

Potato.—The bacillus grows upon the surface of potato if kept
under anaérobic conditions.

Blood-serum.—Upon coagulated blood-serum, growth occurs
under anaérobic conditions, the medium being slowly digested
liquefied.

Vital Resistance.—The bacilli themselves soon succumb when ex
posed to the air. They are destroyed in a few moments by heating
to 60°C. The spores, on the other hand, resist drying and exposure
to the atmosphere well and can be kept alive for years in garden
earth. The complete destruction of the spores requires exposure to

Lesions 341

go°C. for a half hour. Moist heat at 100°C. kills them in a few
minutes.

Metabolic Products.—The organism decomposes albumin, forming
indol, HS, fatty acids, leucin, hydroparacumaric acid, and an oil
with an offensive odor. It liquefies gelatin and digests blood-serum.
It ferments dextrose with the evolution of carbonic acid, hydrogen,
and marsh gas.

Pathogenesis.—When introduced heneadh 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

Fig. 115.—Bacillus ceedematis, dextrose gelatin culture (Giinther).

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.

‘Washed spores of the bacillus are quickly taken up by phagocytes
and destroyed without producing infection. Salt-solution suspen-
sions of such spores quickly infect, however, if mixed with some
tissue-injuring agent such as lactic acid, or if combined with
a harmless micro-organism such as Bacillus prodigiosus by which
the phagocytic activity of the leukocytes is distracted through
preference, ;

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

Teach all the organs.

342 Gaseous Edema

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.

Grigorjeff and Ukket 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 transforma-
tion 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 bacil-
lus of malignant edema, the general impression one receives from the
description of the lesions suggests that it was Welch’s Bacillus
aérogenes capsulatus.

Immunity.—Cornevin found that the passage of the bacillus
through white rats diminished its virulence, and that the animals
of various species that recovered were immune against subsequent
infection with the virulent organisms. Roux and Chamberlandt
found that the filtered cultures were toxic and that animals could be
immunized by injection with this toxic filtrate.

GASEOUS EDEMA

Bacittus AROGENES CAPSULATUS (WELCH)

Synonym.—Bacillus Welchii; Bacillus enteritidis sporogenes; Bacillus phleg-
mone emphysematose; Bacillus perfringens; Bacillus emphysematis vagine;
Granulobacillus saccharobutyricus immobilis liquefaciens; Welch’s gas bacillus.

General Characteristics.—A large, stout, non-motile, non-flagellate, sporogen-
ous, non-chromogenic, purely anaérobic, markedly aérogenic, doubtfully patho-..
genic 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 organism to be identical with Kline’s Bacillus

‘enteritidis sporogenes,** and that it belongs to the butyric acid
group. It is probably also identical with Bacillus phlegmone em-
physematose of Frankel.t{t In many systematic writings the organ-
ism is now called Bacillus welchii. English writers identify it

* “Berliner klin. Wochenschrift,” 1882, No. 44.
{ “ Militdér-medizin. Jour.,” 1898, p. 323.
{ “Ann. de |’Inst. Pasteur.,” 1887.
§ Bull. of the Johns Hopkins Hospital,’’ July and Aug., 1892, vol. vii, No. 24.
|| ‘Jour. of Experimental Medicine,’ Jan., 1896, vol. 1, No. 1, p. 6.
** “Centralbl. f. Bakt. u Parasitenk,”’ 1895, xvi, 737.
Tt ‘“Centralbl. f. Bakt.,” etc., Bd. xm, p. 13.

Morphology 343

with Bacillus pefringens of Veillon and Zuber,* and Besson describes
itunder thisname. Pending final decision upon the identity of these
organisms, it is here called by the name originally given it by Welch
who first secured it from the body of a man dying suddenly of
aortic aneurysm with a peculiar gaseous emphysema of the sub-
cutaneous tissues and internal 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.—It is believed that the natural habitat of the ba-
cillus is the soil, but there is reason to think that it commonly occurs
in the intestine, and may occasionally be found upon the skin.

Fig. 116.—Bacillus aérogenes capsulatus (from photograph by
Prof. Simon Flexner).

Morphology.—The bacillus is a large organism, measuring 3-5
in length, about the thickness of the anthrax bacillus, with ends
slightly rounded, or, when joined, square. It occurs chiefly in pairs
and in irregular groups, but may also occur in chains. 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 somewhat in size, especially in length, in different
culture-media. 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. In the blood and
tissues of animals and in albuminous media it forms rather broad
_ capsules.

. Dunhamf found that spores were produced upon blood-serum, and

* Archiv de méd. expér. et d’anat. path., 1898, x, 517.
t “Bull. of the Johns Hopkins Hospital,” April, 1897, p. 68.

344 Gaseous Edema

especially upon Léffler’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 moderate exposure to the action of 3 per cent. solu-

tion 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 appearance is sometimes
observed, due to the presence of unstained dots in the cytoplasm.

Usually in the body-fluids and often in cultures the bacilli are
surrounded by distinct capsules—clear, unstained zones. To dem-
onstrate this capsule to the best advantage, Welch and Nuttall de-
vised 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 solu-
tion, after soaking up the excess with filter-paper, the thin layer of
coloring fluid not interfering with a clear view of the bacteria 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.

Cultivation.—The bacillus is anaérobic. It grows upon all cul-
ture media at the room temperature, though better at the tem-
perature of incubation, 37°C. Growth does not occur below 20°C,
or above 45°C.

Gelatin.—It grows in ordinary neutral or alkaline gelatin, but
better in gelatin containing glucose, in which the characteristic gas
production is marked. Soft media, made with 5 instead of 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 demonstrated by tilting the
tube and observing 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 anaérobic pre-
cautions 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 un-
less such tubes are placed in a Buchner’s jar or other anaérobic
device. The deeper colonies are the largest. Sometimes the growth

Cultivation 345

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 cul-
ture-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 0.5-1.0 mm. in diameter, though they
may subsequently attain a diameter of 2-3
mm. or more. Their first appearance is as
little spheres or ovals, more or less flattened,
with irregular contours, due to the presence
of small projecting prongs, which are quite
distinct under a lens. The colonies may ap-
pear as little irregular masses with pro-
jections.

- 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
branchings—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 appearance 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 abundant at 37°C.
than at the room temperature. . Fig. 117.—Bacillus

The agar-agar is not liquefied bythe growth aérogenes capsulatus,
2 the bacillus, but is often broken up into ae crotdeap by

gments and forced into the upper part of Prof. Simon Flexner).
the tube by the excessive gas-production.

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 anaérobic conditions, it occurs with abundant gas-formation,
especially in glucose-bouillon, and the formation of a frothy layer
on the surface. The growth is rapid in development, the bouillon

346 Gaseous Edema

becoming clouded in two to three hours. After a few days the ba-
cilli sediment and the bouillon again becomes clear. The reaction
of the bouillon becomes strongly acid.

Milk.—In milk the growth is rapid and luxuriant under anaérobic
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 be-
comes decolorized when the culture is kept without oxygen, but
turns pink when it is exposed to the air. Butyric acid is formed in
the milk.

Potato.—The bacillus will also grow upon potato when the tubes
are inclosed in an anaérobic 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 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 hy-
drogen 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 absorp-
tion 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 to three days’
growth.

Metabolic Products.—The bacillus is unable to make use of the
uncombined oxygen of the atmosphere, and derives its oxygen sup-
ply entirely from carbohydrates in the medium in which it grows.
It causes fermentation of dextrose, saccharose and lactose with the
evolution of ni) asians and hydrogen gases in the approximate
proportion H: CO, :: 1:214, and the production of lactic and
butyric acids. It acti milk and softens gelatin.

Simonds* divides the organisms known as B. aérogenes capsulatus
or B. welchii into four groups according to their metabolic activities
as follows:

1. Organisms that ferment inulin and glycerin with production
of gas and increase of acidity. Do not form spores in media con-
taining either substance. Produce strong hemolysins, and are
pathogenic for guinea-pigs, even after many months cultivation

“upon artificial media.

2. Organisms that produce acid and gas from glycerin but not
from inulin. Form spores in inulin but not in glycerin broth.
Hemolytic and pathogenic powers variable.

* Jour. Infectious Diseases, 1915, XVI, 32.

Pathogenesis 347

3. Organisms that produce acid and gas from inulin but not from
glycerin. Form spores in glycerin but not in inulin broth. Hem-
olysis and pathogenicity variable.

4. Organisms that do not produce acid or gas from either
inulin or glycerin and from spores in both inulin and glycerin
broths.

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 predis-
posing to its growth.

Being anaérobic, the bacilli are unable to live in the circulating
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 2.5
cc. of a fresh sugar-bouillon culture are injected into the ear-vein of
a healty rabbit, it usually recovers. After similar injection with
but 1 cc. 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 embryos, and there multiplied suffi-
ciently 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 die in from seven to twenty-four hours, but may recover.
Gas-production causes the tissues to become emphysematous.

Intraperitoneal inoculation sometimes causes fatal purulent peri-

tonitis of laboratory animals.
. 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, commjnuted fracture of the humerus, with fatal infec-
tion, reported by Dunham, or in wounds and injuries in the neigh-
borhood 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 in-
fection occurred.

Dobbin, t P. Ernst, Graham, Stewart and Baldwin,§ and Krénig

* “Journal of Experimental Medicine,’” Jan., 1896, vol. 1, No1.
+ “Bull. Johns Hopkins Hospital,” Feb., 1897, No. 71, p. 24.

t “Virchow’s Archiv.,’’ Bd. cxxxi, Heft 2.

§ “Columbus ‘Med. Jour.,” Aug., 1893.

348 Gaseous Edema

and Menge* have studied cases of puerperal sepsis and sepsis follow-
ing abortion either caused by the bacillus or in which it played an
important réle. :

Williams} has found the bacillus in a case of suppurative pyelitis.

The symptoms following infection are quite uniform, consisting
of redness and swelling of the wound, with rapid elevation of tem-
perature and rapid pulse. The wound usually becomes more or
less emphysematous, and discharges a thin, dirty, brownish, offensive
fluid that contains gas bubbles and is sometimes frothy. The pa-
tients 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, may attain twice its
normal size and be unrecognizable. Upon palpation a peculiar crepi-

Fig. 118.—“Frothy liver” from Bacillus aérogenes capsulatus infection
(Aschoff).

tation can be felt in the subcutaneous tissue nearly everywhere,
and the presence of gas in the blood-vessels is easy of demonstra-
tion. 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 German term
“Schaumorgane”’ (frothy organs). The liver is especially apt to
show this coridition. When such tissues are hardened and ex-
amined 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

* “Bakteriologie des weiblichen Genitalkanals,” Leipzig, 1897.
{ “Bull. Johns Hopkins Hospital,” April, 1896, p: 66.

Pathogenesis 349

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 bacteria. On this
account it is difficult to estimate just how much of the damage be-
fore 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 distribution of the bacilli throughout the circulation,
and then killing the rabbit. In a few hours the animal 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 aérogenes capsulatus.

An excellent review of the early literature of the-subject is to be
found in “A Contribution to the Knowledge of the Bacillus Aéro-.
genes Capsulatus,” by W. T. Howard, Jr.* »

Immunity and Specific Therapy.—Few experiments along these
lines have resulted in anything worthy of much attention until
Bull and Pritchett} found that under certain conditions Bacillus
aérogenes capsulatus gives off a soluble toxin that can be utilized
to make an antitoxic serum by administration to animals, and later}
that when such a serum was prepared by the immunization of horses,
it was capable of conferring an immunity upon animals that was of
about two weeks’ duration. It is believed by the investigators that
such serum could be used to prevent gaseous wound infection in
man.

Other Micro-organisms of Gaseous Infections.—Elliott and
Henry§ in studying cases of infection of hemothorax by anaérobic
gas-producing bacilli, found the following: ,

1. Bacillus perfringens group.—These include:
Bacillus perfringens (Veillon and Zuber).
Bacillus aérogenes capsulatus (Welch).
Bacillus enteritidis sporogenes (Klein).
Bacillus of articular rheumatism (Achaline).
Bacillus phlegmonis emphysematose (Fraenkel).
Bacillus saccharobutyricus immobilis (Schattenfiroh and Grossburger).

: 45 , coniations to the Science of Medicine by the Pupils of W. H. Welch,”
900, p. 401.
1 Jour. Exp. Med.,” 1917, XXVI, p. 119.
t'Jour. Exp. Med.,” 1917, XXVI, Pp. 603
Brit. Med. Jour.,” 1917, I, p. 413.

350 Gaseous Edema

They are all characterized as stout, Gram-negative organisms, non-motile,
causing explosive disruption of casein through fermentation of carbohydrates.
They do not form spores in milk. Minced meat turns red. Cultures tend to
die out in a few days. .

2. Bacillus sporogenes group.—These micro-organisms endure heating to 80°C.
for twenty minutes. They sporulate in milk. They slowly digest and clear
milk from the cream downwards. They blacken minced meat. They are the
same motile organisms that have been described by recent English authors as
Bacillus cedematis maligni.

3. Bacillus von Hibler IX.

4. A small diphtheroid bacillus.

Henry* found it convenient to divide the anaérobic bacilli into
two groups:

J. The Saccharolytic group.—z. Bacillus welchii (B. perfringens).
2. Bacillus tertius (B. von Hibler IX).
3. Bacillus fallix (Weinberg).
4. Bacillus aérofcetidus (Weinberg).
5. Bacillus cedematicus (Weinberg).
6. Vibrion septique.

II. The Proteolytic group.—z. Bacillus sporogenes (Metchnikoff).
2. Bacillus histolyticus (Weinberg).
3. Bacillus putrificus coli (Bienstock).
4. Bacillus cadaveris sporogenes (Klein).
5. Bacillus tetani (Nicolani).

Weinberg and Seguin{ give the following tabulation of the different
anaérobic bacilli in infected gun-shot wounds:

Cases Per cent.
Bacillus perfringens (Veillon and Zuber).......... 70 17
Bacillus cedematicus (n. sp.)..........00.0 0000 31 34
Bacillus sporogenes (Metchnikoff)................ 25 27
Bacillus fallax (0. sp.) vices, doa eiee sete coe wane en eet I5 16.5
Vibrion septique (Pasteur). .............00 0 eee 12 13
Bacillus tetanhevccc cc casieg sas eed ees den ees eames 9 5)
Bacillus histolyticus (n. sp.).........0000e eee eens 8 9
Bacillus aérofcetidus (n. sp.)........0 cece eee 5 5.5
Bacillus putrificus........ 2.2.0... 0c cece eee eee 2 2
Bacillus bifermentous................00000 seve 2 2
- Bacillus Gohn-Sachs IT.................00 00s eae I I
Bacillus Certius acco: 24 ees seus Mos s-gug maleate I -

Pure infections by these organisms are less frequent than mixed.
Not only are the infections caused by combinations of the anaérobes,
but aérobes are also frequently associated with them.

They found the relations of the different organisms as follows:—

A single species Two or more
of anaérobic earls Total
organisms ane TOpI.
organisms
Anaérobic organisms only... Io 14 24
Anaérobes and aérobes,.... 27 40 67
Ota oe siec os hictn ee os 37 54 gr

*“Brit. Med. Jour.,” 1917, I, p. 806.
t ‘‘Annales de l’Inst. Pasteur,” 1917, XXXI, p. 444.

Other Micro-organisms of Gaseous Infections 351

All of the investigators cited found the aérobic organisms most
commonly occurring in the mixed gas infections to be streptococci,
staphylococci, pneumococci, Bacillus proteus, Bacillus subtilis, and
Bacilluscoli. In the thoracic infections Bacillus pneumoniz, Bacillus
influenze, Micrococcus tetragenus, Micrococcus catarrhalis, Bacillus
pyocyaneus and Sarcina were also sometimes found.

CHAPTER III
TETANUS

Bacittus TETANI (FLUGGE)

General Characteristics——A motile, flagellated, sporogenous, liquefying,
obligatory anaérobic, non-chromogenic, aérogenic, 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 tetanus or “lock-jaw.”’

Distribution.—The tetanus bacillus is a common saprophyte in
garden earth, dust, and manure, and is a constant parasite in the in- -
testinal contents 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 occur-
rence of the baccilli 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 anaérobic
conditions. Verneuil has observed that tetanus rarely occurs at sea
except upon cattle transports.

Le Dantect has shown that the tetanus bacillus is a common or-
ganism in New Hebrides, where the natives poison their arrows
by dipping them into a clay rich in its spores.

Morphology.—The tetanus bacillus is a long, slender organism
measuring 0.3 to 0.5 X 2 to 4 mw (Fliigge). Its most striking char-
acteristic 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 solu-
tions of the anilin dyes and by Gram’s method.

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

* “Deutsche med. Mechpnschaitt,” 1884, 42.

} Ibid., 1889, No. 3
t See abstracts i in the “Centralbl. f. Bakt. u. Parasitenk.,” rx, 286; XII, 351.

352

Isolation 353

Fig. 120.—Bacillus tetani; six-day- Fig. 121.>-Bacillus tetani; culture
old puncture culture in glucose-gelatin four days old in glucose-gelatin (Frankel
(Frankel and Pfeiffer). "and Pfeiffer).

354 Tetanus

twenty-four hours, during which time all of the contained micro-

organisms, 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.

A method more certain in its results has been suggested by Theobald
Smith* and depends upon increasing the number of tetanus spores
in the inoculation material before the actual isolation of the organ-
ism is attempted. Fermentation tubes are employed for the pur-
pose, and are filled with dextrose-free culture bouillon. Into each
tube a small bit of sterile rabbit- or guinea-pig liver is introduced so
as to occupy the constriction between the bulb and upright arm,
After sterilization—or after incubation long enough to show that
the introduced tissue has caused no contamination— the suspected
material is introduced. The tube is kept at 37°C. for forty-eight
hours, when it will be found that a great increase of tetanus spores
has obtained about the implanted tissue. When examination of
stained drops, removed with a sterile pipet, shows plenty of spores,
the culture is heated to 80°C. for thirty minutes to kill any non-
sporulating contaminating organisms in the originally implanted -
media, and’ transfers made with a pipet from the neighborhood of
the bit of tissue, to fresh appropriate media for tubing or plating. ©

Cultivation.—The tetanus bacillus is difficult to cultivate because
‘it will not grow where the smallest amount of free oxygen is present.
It is hence a typical obligatory anaérobe. Farranf and Grixoni
believe it to have originally been an optional anaérobe, and it is said
by these writers that the organism can gradually be accustomed to
oxygen so as to grow inits presence. When this is achieved, it loses
its virulence. These observations have not been confirmed.

The general methods for the cultivation of anaérobic organisms,
are given under the appropriate heading (Anaérobic Cultures), and
need not be repeated here.

The cultures grow best at 37°C., and at this temperature the spore-
formation is at a maximum. Growth takes place between the
extremes of 15° and 45°C.

The cultures usually give off a disagreeable odor resulting from
H.S and mercaptan.

The colonies of the tetanus bacillus, when grown upon gelatin
plates in an atmosphere of hydrogen, resemble those of the well-
known hay bacillus. There is a rather dense, opaque central mass
surrounded by a more transparent zone, the margins of which con-
sist of a fringe of radially projecting bacilli. Liquefaction occurs
slowly.

cue Med. Research, 1905, XIV, 193. _
‘Centrabl. f. Bakt. u. Parasitenk.,” July 15, 1898, p. 28.

Vital Resistance 355

The colonies upon agar-agar are similar but effect no liquefaction.
If blood corpuscles be distributed throughout the medium, a rather
broad zone of hemolysis is occasioned by the tetanolysin .

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 liquefaction spreads slowly, but may
involve the entire mass of gelatin and resolve
itinto a grayish-white syrupy liquid, at the
bottom of which the bacilli accumulate.
The growth in gelatin containing glucose is
rapid and the medium becomes filled with
bubbles of gas before it melts.

Agar-agar—The growth in agar-agar
punctures is slower, but similar to .the
gelatin cultures except for the absence of
liquefaction. In agar-agar containing glu-
cose the gas production may break up the
medium.

Bouillon—The organism can be grown
in bouillon without difficulty, when once
habituated to the medium. The bouillon
should be heated to drive off the air, then
rapidly cooled and the transplantation
made. If there be a depth of 10 cm. the
bacilli grow readily in the lower half of the
fluid. If the surface be covered with liquid
paraffin before the final sterilization and
inoculation, they grow throughout the
entire medium. The organism attains its
maximum development at a temperature
of 37°C. No change except a well-
marked cloudiness appears in the bouillon.
After about two weeks growth ceases and
the bacilli settle to the bottom leaving the
medium fairly clear again. ,

Milk is favorable for the development
of the tetanus bacillus. There is no
coagulation. Litmus milk is acidified. Me: set ea nus

Potato.—Upon potatoes under strict bacillus; glucose-agar
anaérobic conditions the bacilli grow but Culture, five months old.
slightly. (Curtis).

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

356 , Tetanus

which 0.5 per cent. of hydrochloric acid has been added, destroys
them in two hours. They are destroyed in three hours by 1: 1000
bichlorid of mercury solution, 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,*
exposure to streaming steam for from five to eight minutes is cer-
tain to kill tetanus spores, and this statement has found its way
into most of the text-books without discussion. Theobald Smith,t
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 indol, hydrogen sulphide, mercaptan and proteolytic fer-
ments. In sugar-bouillons the tetanus bacillus ferments dextrose
and maltose, giving off lactic and other acids, carbon dioxid and
hydrogen gases. Maltose and polysaccharides are not fermented.

Toxic Products.—The most ready method of preparing the toxins
for experimental study is to cultivate the bacilli in freshly prepared
neutral or slightly alkaline sugar-free bouillon under conditions of
most strict anaérobiosis, at a temperature of 37°C., and then filter
the culture through porcelain. Fieldt found the highest degree of
toxicity about the’sixth or seventh day. It may attain a toxicity
so great that 0.c00005 cc. will cause the death of amouse. The aver-
age culture has such toxicity that o.oor cc. is fatal to a guinea-pig.
Knorr§ 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............. 13x toxin
i I gram of rabbit is destroyed by............. 2,000x% toxin
: 1 gram of hen is destroyed by.............:.200,000% toxin

The toxin is very unstable, and is easily destroyed by heat above
60°C. It is also quickly destroyed by light, especially direct sun-
light. Flexner and Noguchi|| found that 5 per cent. of eosin added
to the toxin destroyed it through the photodynamic .power of the
stain. It is also easily destroyed by electric currents. 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 con-
ditions.

To keep it for experimental purposes it is advisable to precipitate

* “Zeitschrift ftir Hygiene,” x1, p. 225.

Lae feter sled is March 21, 1908, vol. L, No. 12, p. 931.
“Proc. N. Y. Path. Soc.,”” March, 1904, p. 18.

§ “ Miinch. med. Wochenschrift,” 1898, D. 321.

|| ‘Studies from the Rockefeller Institute,’’ 1905, v.

Metabolic Products 357

the toxin from the bouillon by supersaturation with ammonium sul-.
phate, which causes it to float upon the liquid in the form of a sticky
brown scum that can be skimmed off and dried. Such dry precipi-
tate retains its activity for months.

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,” “tetanotoxin,” tetanospasmin,”
and a fourth substance to which no name is given. All were very
poisonous and productive of tonic convulsions. Later Brieger and
Frinkel isolated an extremely poisonous toxalbumin from sugar-
bouillon cultures of the bacillus. :

The purified toxin of Brieger and Cohn was fatal to mice in doses
of o.coo00005 gram. Lambert* considers the tetanus toxin to be
the most poisonous substance that has ever been discovered.

_ Fermi and Pernossif found most toxin produced in agar-agar
cultures, less in gelatin cultures, and least in bouillon cultures.

Ehrlicht found two poisons in the tetanus toxin, one of which
was convulsive and was in consequence called fetanospasmin, the
other hemolytic and called ¢efenolysin. When tetanus toxin is
added to defibrinated blood, the tetanolysin is absorbed by the
corpuscles, many of which are dissolved, while the tetanospasmin
remains unchanged.

Dénitz§ and Wassermann and Takaki|| have found that the
tetanus toxin has a specific affinity for the central nervous system,
with whose cells it combines im vitro and becomes inert.

Roux and Borrel** have found that when tetanus toxin is injected
into the brain substance a very much 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 effect-
ive when produced in or injected into the tissues and absorbed into

the circulation. It is harmless when given by the digestive tract,

Ramon{t having administered by the mouth 300,000 times the fatal
hypodermic dose without producing any symptoms..

One of the most interesting peculiarities about the toxin is the com-
parative uniformity of the period intervening between its administra-
tion 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:

*“New York Med. Jour.,” June 5, 1897.
7 “Centralbl. f. Bakt.,” etc., xv, p. 303.
T “Berliner klin. Wochenschrift,” 1898, No. 12, p. 273.
§ “Deutsche med. Wochenschrift,” 1897, p. 428.
|| “Berliner klin. Wochenschrift,”’ 1898, 35.
** “ Ann, de Inst. Pasteur,” 1898, x1.
Tt “Deutsche med. Wochenschrift,’’ Feb. 24, 1898.

358 Tetanus

13 lethal doses........... 00. ceeees symptoms in 36 hours
tro lethal doses...... Ssh Wusissinae gee eters symptoms in 24 hours
333 lethal doses.............200000- symptoms in 20 hours

1300 lethal doses....>........0000005 symptoms in 14 hours
3000 lethal doses............000ee eee symptoms in 12 hours

The local action of the toxin is very painful and associated with
spasm of the muscular fibers with which it comes in contact. Pit-
field,* 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 causes 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
the axis cylinder substance; the latter found the toxin carried to the
central nervous system solely by the motor nerves, the action de-
pending upon the integrity of the axis cylinder. They believe that
the toxin is absorbed by the axis cylinder endings, and reaching the
corresponding spinal nerve center by that route spreads to the corre-
sponding center in the other half of the cord and outward, 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 fefanus ascendens
and tetanus descendens. The former always follows the intramus-
cular 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 distribution in the blood.
Under these conditions Zupnik believes it acts upon the central

* “Therapeutic Gazette,’ March 15, 1897.

7 “Ann de V'Inst. Pasteur,’ ? 1902, XVI, p. 818; and “Bull. de I’Inst. Past.,”

1903, I, p. 4
t “Arch. . exper. Path. u. Pharmak.,” 103, xLIx.
§ “ Wiener klin. Wochenschrift,”’ Jan. 23, 1902.

Pathogenesis 359

nervous system, especially upon the spinal cord, manifesting itself
in extreme reflex excitability with irregular motor discharges result-
ing 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, depending upon the centric action.
The latter are the more dangerous for the sufferer.

The lockjaw or trismus and the opisthotonos that are so charac-
teristic 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 muscles without
equally powerful opposing groups. 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 Ran-
som,* 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. In-
jection 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 dolorosus because the transportation of the poison along
these trunks is so slow.

The fefanolysin is a hemolytic component of the toxic bouillon,
and is entirely separate and distinct from the tetanospasmin or con-
_vulsive 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 infinitesimal
amount of tetanus culture, or with garden earth containing the tet-
anus bacillus, the first symptoms come on in from one to two days,
when the mouse develops typical tetanic convulsions, first beginning
in the neighborhood 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 unfavorable to

* “Archiv. £. exper. Path. u. Pharmak.,” 1903, Bd. xLrx, p. 396.

360 ‘Tetanus

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 most cases of tetanus are mixed infections in
which the bacillus enters with aérobic bacteria, that aid its growth
by absorbing the oxygen in the neighborhood. The amount of
poison produced must be exceedingly small and its power tremen-
dous, 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 un-
less 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 suc-
cumb to the disease without showing any sensible difference.”

The circulating blood of diseased animals is fatal when injected
into susceptible animals because of the toxin it contains; and the
fact that the urine is also toxic to mice proves that the toxin is ex-
creted by the kidneys.

Two classes of infected wounds are particularly apt 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 aérobic and other inappropriate conditions of growth. It -
reaches 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,t
who found that if the tetanus spores were introduced 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

* Quoted before the Académie de Médicine, Oct. 22, 1895.
} See “Centralbl. f. Bakt. Infekt., u. Parasitenk.,” vol. xvi, p. 208.

Antitoxin of Tetanus 361

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 pre-
vious 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 imucosa exist.

Montesano and Montesson,t unexpectedly found the tetanus
bacillus in pure culture in the cerebro-spinal fluid of a case of para-
lytic 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 castration, accouchement,
or abortion. Most birds are scarcely at all susceptible either to the
bacilli or to their toxin. Amphibians and reptiles are immune,
though it is said that frogs can be made susceptible by elevation of
their body-temperature.

The injection of the toxic bouillon or of the redissolved ammonium
sulphate precipitate, in progressively increasing doses, into animals,
causes the formation of antibodies (antitoxin) by which the effects of
both the tetanospasmin 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 preparing 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.—Welcht early pointed out that the antitoxin of tetanus
is a disappointment in the treatment of the disease. Moschowitz,§
in an excellent review of the subject, came to the conclusion 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.

Trons|| analyzed 225 cases of tetanus treated with antitoxic serum
and found the mortality 20 per cent. lower than in cases otherwise
treated. On the other hand, Gessner,** in an analysis of cases

» treated in the Charity Hospital of Louisiana, located in New Orleans, :
found that the percentage of deaths from tetanus in the decade from
1840-1849 was 68.7, and that in the decade from 1900-1909 was
68.7 and that for the year 1910-1917, 68.5: A comparison of all
the cases in the years 1840-1889, the pre-antitoxin period, with those
in the years 1890-1917, shows the former group to have a death-
tate of 79.1 per cent., the latter 70.7 per cent., a gain of 8.4 per cent.

*“Centralbl. £. Bakt u. Parasitenk.,”’ 1895, XVIII, p. 513.
+ “Centralbl. f. Bakt. u. Parasitenk.,” Dec., 1897, Bd. xxu, Nos. 22, 23, Pp. 663.
aoe ae the Johns Hopkins Hospital,” July Ee August, 1895.
nals of Surgery,” 1900, XXXII, 2, Pp. 219, 416, 567.
(Jour. Am. Med. Asso.,” 1914, LXIL, 2025. ;
Jour. Am. Med. Asso.,”’ Sept. 14, 1918, LxxI, No. 11, p. 867.

362 Tetanus

But too much emphasis must not be placed upon these latter figures
as there were variations quite as great in the various decades making
up the totals. Thus between 1840-1849 the deaths numbered 68.7
per cent.; in 1880-1889, 83.9 per cent. Irons says that it is im-
portant that the full effect of the antitoxin be immediately obtained,
the best method of using it being that outlined by Park in which
3000 units are given intraspinously at the earliest possible moment
after the symptoms appear, and 10,000 to 20,000 units given intra-
venously at the same time. On the following day the intraspinous
injection of 3000 should be repeated. On the fourth or fifth day,
10,000 units should be given subcutaneously. By these means a
high antitoxic content of the blood and juices is maintained.

The use of antitoxic serums must not replace other non-specific
modes of treatment such as local treatment of the wound and the
administration of sedatives, etc. The result of its experimental in-
jection, in combination with the toxin, into mice, guinea-pigs, rab-
bits, and other animals is perfectly satisfactory, and affords protec-
tion 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 anti-
toxin 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 incep-
tion or to begin the treatment until it may be 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 symptoms of tetanus is
probably found in a ready fixation of the toxin in the bodies of the
infected animals. This is well shown by the experiments of Dénitz,*
who found that if a mixture of toxin and antitoxin were made before
injection into an animal, twelve minimum fatal doses were neutralized
by 1 cc. of a 1:2000 dilution of an antitoxin. If, however, the
antitoxin was administered four minutes after the toxin, 1 cc. of a
1 :600 dilution was required; if eight minutes after, 1 cc. of a 1:200
dilution; if fifteen minutes after, 1 cc. of a r:100 dilution. He found
that similar but slower fixation occurred with diphtheria toxin.

It was found by Roux and Borrel} that doses of tetanus antitoxin
absolutely powerless to affect the progress of the disease, when ad-
ministered in the ordinary manner by subcutaneous injection, read-

* Reference 18, in “Jour. of Hygiene,” vol. 1, No. 2, in Ritchie’ s article.
{ ‘Ann. del’ Inst. Pasteur,” 1898, No. 4.

Bacilli Resembling the Tetanus Bacillus 363

ily saved the animal if the antitoxin were injected into the brain
substance.

Chauffard and Quénu,* 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, however, 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 to be recommended at
present is the intraspinous, intravenous and subcutaneous injection
of large and frequently repeated doses of the antitoxic serum. There
can be little doubt but that the administration must be so free as to
load up the patient’s blood with the antitoxin in hopes that its pres-
ence there may detach the toxic molecules from their anchorage to
the nerve cells.

Prophylactic Treatment.—While tetanus antitoxin is extremely
disapppointing, in practice, for the cure of tetanus, it is most satis-
factory for its prevention. ‘An ounce of prevention is better than a
pound of cure,” and if the surgeon 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.

BAcILLI RESEMBLING THE TETANUS BACILLUS

Tavelf has called attention to a bacillus commonly found in the intestine,
sometimes in large numbers in the appendix in cases of appendicitis, and locked
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 measures 0.5 by 5~7u,.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 Léffler’s
method without the addition of acid or alkali. The organism does not stain so

. well by Gram’s method as the true tetanus bacillus. The bacillus isa pureanaérobe.

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 encompassed by a narrow, clear
zone, which is often notched. The spores are killed at 80°C.

The organism produced no symptoms in mice, guinea-pigs, and rabbits even
when 2-5 cc. of a culture were subcutaneously introduced.

Sanfelicet and Lubinski§ have observed a bacillus in earth and meat-infusions
that is morphologically and culturally like the tetanus bacillus, but differs from
it in not possessing any pathogenic powers.

Boucle also described a bacillus much like the tetanus micro-organism that
grows aérobically. It is not pathogenic. :

*“Ta Presse méd.,” No. 5, 1898.

+ “Centralbl. f. Bakt.,” etc., March 31, 1898, xx, No. 13, p. 538.
{ “Zeitschrift fiir Hygiene,” vol. x1v.

eer: f. Bakt. u. Parasitenk.,’’ xx1, 19.

|| Fligge, “Die Mikroorganismen,” vol. 1, p. 267.

CHAPTER IV
ANTHRAX

Bacittus ANTHRACIS (Koc)

General Characteristics.—A non-motile, non-flagellated, sporogenous, liquefy-
ing, non-chromogenic, non-aérogenic, pathogenic, aérobic and optionally anaéro-
bic bacillus staining by. the ordinary methods and by Gram’s method.

The disease of herbivora known as anthrax, “splenic fever,”
“ Milzbrand,” and “charbon,” is a dreaded and common malady in _
France, Germany, Hungary, Russia, Persia, and the East Indian |
countries. In Siberia the disease is so common and malignant as to

Fig. 123.—Bacillus anthracis; colony three days old upon a [gelatin plate;
adhesive preparation. X 1000 (Frankel and Pfeiffer).

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. ;

Herbivorous animals are most frequently affected, especially
cows and sheep. Carnivorous animals are less often affected,
though notimmune. Among laboratory animals, white mice, house-
mice, guinea-pigs, and rabbits are highly susceptible; rats, scarcely
; ; 364

Sporulation 365

susceptible; birds, reptiles and amphibians usually immune. Man
is susceptible in varying degree.

Anthrax was one of the first infectious diseases proved to depend
upon a specific micro-organism. As early as 1849 Pollender* dis-
covered 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 in-
teresting 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,{ who, observing

Fig. 124.—Bacillus anthracis; showing the capsules. From a case of human
infection. Magnified 1000 diameters (Schwalbe).

that the bacilli bore spores, cultivated them successfully outside
the body, and produced the disease by the inoculation of pure
cultures.

Morphology.—The anthrax bacillus is a large rod-shaped organ-
ism, of rectangular form, with slightly rounded corners. It meas-
ures 5 to 20 uw in length and from 1 to 1.25 win breadth. It hasa
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 appearance of a
bamboo rod. In preparations made by staining blood or other
animal juices the bacilli often appear surrounded by transparent
capsules. Such are not found in specimens made from artificial
cultures.

Sporulation.—The formation of endospores is prolific in the pres-

* “Vierteljahrsschr. fiir ger., Med.,” 1855, Bd. vit.

“Compte-rendu,” 1863, LVIL.
t “Beitrige zur Biol. d. Pflanzen,” 1876, It.

366 Anthrax

ence of oxygen. When oxygen is withheld spore-formation does
not occur. In the bodies of experiment animals spore-formation
is unusual and its occurrence signifies the local presence of abundant
oxygen. On account of this peculiarity of the organism, the dead
body of an animal is less dangerous as a source of infection than the
discharges from living animals, As, however, the wool, hair and
hides of infected animals are always soiled by the discharges, these
are a menace to all that handle them and ought not-be used. 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. When a spore is placed under conditions favorable
to its development, it increases in length and ruptures at the end,

Fig. 125.—Bacillus anthracis, stained to show the spores. X 1000 (Franke
and Pfeiffer).

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.

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 beautiful, clear picture. The spores can
be stained by any of the special methods (q.2.).

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 tis-

Cultivation 367

‘

sues, by the plate method, or by the inoculation into animals and
recovery of the micro-organisms from their blood.
Cultivation.—Colonies.— Upon the surface of a gelatin plate the
bacillus forms beautiful and highly characteristic colonies. To the
naked eye they appear first as minute round, grayish-white dots.
Under the microscope they are egg-shaped, slightly brown and granu-
lar. Upon the surface of the medium, they spread out into flat,
irregular, transparent tufts like curled wool, and from a tangled cen-
ter large numbers of curls, made up of parallel threads of bacilli,
extend upon the gelatin. Before the colony attains to any consider-
able size liquefaction sets in. Beautiful adhesion preparations can

Fig. 126.—Bacillus anthracis; colony upon a gelatin plate. X 100 (Frankel
and Pfeiffer).

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 surrounding gelatin, with a beautiful
arborescent effect. :

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

368

eat huess

grayish-white, pamiens ekely wrinkled layer with irregular

Fig. 127.—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).

edges, from which curls of bacillary threads
extend upon the medium. When the cul-
ture is old, the agar-agar usually becomes
brown in color. Spore-formation is lux-
uriant.

Bouillon.—In bouillon the anthrax bacil-
lus grows chiefly upon the surface, where
a thick felt-like pellicle forms. From this,
fuzzy extensions descend into the clear
bouillon’ below. .After a few days some
wooly aggregations can be seen in the
bottom of thetube. 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. Sporula-
tion is marked. ‘

Blood-serum.—Blood-serum cultures
lack characteristic peculiarities; the cul-
ture-medium is slowly liquefied.

Milk.—The anthrax bacillus grows well
in milk, which it coagulates and acidulates.
Later the coagulum is peptonized and dis-
solved, leaving a clear whey.

Vital Resistance.—The bacillus grows
between the extremes of 12° and 45°C.,
best at 37°C. The exposure of the organ-
ism to the temperature of 42° to 43°C.
slowly diminishes its virulence.

When dried upon threads, the spores
retain their vitality for years, and are
highly resistant to heat and disinfectants.
The spores of anthrax are killed by five
minutes’ exposure to 100°C. It is said by
some that spores subjected to 5 per cent.
carbolic acid can subsequently germinate
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 two hours by ex-

posure to 1:1000 bichlorid of mercury solution.
Metabolic Products.—The anthrax bacillus produces a curdling

Pathogenesis

369

ferment. Iwanow* found that the organism forms acetic, formic,
and caproic acids, but it produces no important change of reaction in

themediumin whichit grows. It generates
noindol. Its proteolytic enzyme is active,
digesting both casein and fibrin. No acid
or gas is formed through the change of any
carbohydrate.

It is doubtful whether the anthrax bacil-
lus produces any important toxic sub-
stance. Hoffat isolated a basic substance
from anthrax cultures and called it an-
thracin; Hankin and Wesbrook,t an albu-
mose fatal in large doses and immunizing
in small ones. Brieger and Friinkel§
isolated a tox-albumin from the tissues of
animals dead of anthrax. Martin|| sepa-
rated 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. Conradift in an
elaborate research failed to find that the
anthrax bacillus produced any soluble ex-
tracellular or intracellular poison capable of
affecting susceptible animals, and concluded
that it was highly improbable that the an-
thrax bacillus produced any toxic sub-
stances at all.

Pathogenesis.—Avenues of Infection.—
Infection usually takes place through the
respiratory tract, through the alimentary
canal, or through the skin. It may take
place through the placenta.

I. The Respiratory Tract.—The inhala-
tion of the spores of the anthrax bacillus
is possible whenever they are present in
the atmosphere. The effect produced will
depend upon the number of spores inhaled

*“ Ann, de Inst. Pasteur,” 1892.

Fig. 128.—Bacillus an-
thracis; glycerin agar-agar
culture (Curtis).

t “Ueber die Natur. des Milzbrandgifts,” Wiesbaden, 1886.

t “Ann. de. l’Inst. Pasteur,” 1892, No. 9.

§ “Ueber Ptomaine,” Berlin, 1885-1886.

|| ‘Proceedings of the Royal Society,” May 22, 1890.

** “Ann. de V’Inst. Pasteur,”’ 1895, p. 533-
it “Zeitschrift fiir Hygiene.” June 14, 1899.

370 Anthrax

and the resistance or susceptibility of the animal. In man, a
resisting animal, anthrax is rarely so caused except the number of
spores be great, when it results in a disturbance at first localized in
the lungs, and much resembling pneumonia. From the lungs
generalized infection may later occur and destroy life. This form
of infection is of occasional occurrence among men whose occupation
occasionally brings them into contact with the hair or hides of
animals dead of anthrax, and is often spoken of as “wool-sorters’
disease.” :

Anthrax in cattle probably results from the inhalation or ingestion
of the spores of the bacilli from the pasture. Interesting discussions
arose concerning the infection of the pastures. It was argued that,
the bacilli being inclosed in the tissues of the diseased animals, in-
fection 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 moisture in the
soil. Koch seems, however, to have demonstrated 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

Fig. 129.—Anthrax carbuncle or malignant carbuncle (Lexer).

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.

ITI. The Alimentary Tract.—When the bacilli are taken into the
stomach they are probably destroyed by the acid gastric juice.
The spores, however, are able to endure the acid, and pass uninjured
into the intestine, where the suitable alkalinity enables them to
develop into bacilli, surround the villi with thick networks of bacil-
lary threads, separate the covering epithelial cells, enter the lym-
phatics, and then the blood, and effect general infection.

Pathogenesis - 371

IIT. The Skin.—The bacillus frequently enters the body through
wounds, cuts, scratches, and perhaps occasionally fly-bites, though
from the work of Nuttall* it is pretty clear that flies play little part
in the transmission of the disease. Under these conditions the organ-
isms at once find themselves in the lymphatics or capillaries, and
may cause immediate general infection. In human beings a “malig-
nant pustule” is apt to follow local infection, and may recover or
ultimately cause death by general infection.

The malignant pustule usually makes its appearance upon the face,
hands or arms. The first symptom is a reddish papule that extends
and becomes vesicular. At the point of infection necrosis is rapid,

Fig. 130.—Anthrax bacilli in glomeruli of kidney.

and within forty-eight hours there may be a brownish eschar sur-
rounded by a crop of secondary vesicles, beyond which there is edema
or brawny swelling. According to the susceptibility of the patient
the disease may soon localize, the slough detach and recovery set in,
or the edema and swelling may continue, blood invasion occur and
death ensue. Heinemann,t in compiling statistics of the fatality of
malignant pustule, shows that the danger of the lesion is greatly miti-
gated by complete excision. Koch found the death-rate among 1473 |
cases to be 38.8 per cent., but Heinemann’s statistics upon 2255
cases show the deaths to be only 5.8 per cent.

Lesions.—The disease as seen in the laboratory is accompanied
by few marked lesions. The ordinary experimental inoculation is

”

* “Johns Hopkins Hospital Reports,” 1899.
t “Deutsche Zeitschrift fiir Chirurgie,” 1912, CXIX, 201.

372 Anthrax

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 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 subcutane-
ous 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 somewhat enlarged.
The lungs are usually slightly congested.

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 capil-
laries 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 few, so that the burden of
bacillary obstruction is borne by the minute vessels.

Death from anthrax seems to depend more upon the obstruction.
of the circulation by the multitudes of bacilli in the capillaries, and
upon the appropriation of the oxygen destined to support the tissues,
by the bacilli, than upon intoxication by the metabolic products
of bacillary growth.

Virulence.—The anthrax bacillus maintains its virulence almost
without modification because of the prolific formation of spores and
their remarkable resisting powers. By artificial means, however,
the formation of spores can be inhibited and the bacilli attenuated.
This was first achieved by Pasteur* by cultivation at temperatures
above the optimum, at which no spores were formed. Toussaintt
found that the addition of 1 per cent. of carbolic acid to blood of
_ animals dead of anthrax destroyed the virulence of the bacilli;
Chamberland{ and Roux found the virulence destroyed when 0.1-0.2
per cent. of bichromate 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 an immune animal, such as the frog,
and their subsequent recovery from its blood, diminishes the virulence.

Vaccination.— Pasteur|| early realized the importance of some prac-

* “Rec, de Méd vet.,”’ Paris, 1879, p. 193.
+ ‘“Compte-rendu Acad. des Sci. de Paris,” xct, 1880, p. 135.
{ “Ann. de l’Inst. Pasteur,” 1894, p. 161.

§ “Compte-rendu de l’Acad. des Sci.,”’ Paris, 180%, CXIV, P. 152i.
|| ‘Rec. de Méd. vet.,” Paris, 1879, D. 193.

Bacteriologic Diagnosis 408

tical measure for the protective vaccination of cattle against the
disease, and devoted himself to investigating the problem. He
found that the inoculation of attenuated bacilli into cows and sheep,
and their subsequent reinoculation with mildly virulent bacilli,
afforded them immunity against highly virulent organisms.

The protective inoculations prepared by Pasteur consisted of
two cultures of diminished virulence, to be employed one after the
other, each rendering the vaccinated animals more immune. The
cultures were prepared, that is, attenuated 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 tem-
perature of 42°C. for from ten to twelve days and killed mice,
guinea-pigs and occasionally rabbits.

The vaccine is administered by hypodermic injection into the tis-
sues of the neck or flank, the second being given from two to three
weeks after the first. Of each broth culture about 1 cc. is admin-
istered. The animals frequently become 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
1881 and 1894 there were 1,788,677 sheep vaccinated, with a mor-
tality 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.

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 albu-
minose, that exerted a protective influence. Rettgert prepared
“prodigiosus powder” from potato cultures of B. prodigiosus, which
when injected into guinea pigs during experimental anthrax infection
prolonged life or induced recovery.

Serum Therapy.—In 1890 Ogata and Jasuhara showed that the
blood of experiment animals convalescent from anthrax possessed
an antitoxic substance of such strength that 1 : 800 parts per body-
weight would protect a mouse. Similar results have been attained
by Marchoux.t Serum therapy in anthrax is, however, of no prac-
tical 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 bacterio-
logic diagnosis of anthrax made where no laboratory facilities are at
hand, an ear of the dead animal can be inclosed in a bottle or fruit

*Les Maladies Infectieuse, 11, p. 1489.
t “Jour. of Infectious Diseases,” Nov. 25, 1905, vol. m1, No. 4, p. 562.
t “Ann. de l’Inst. Pasteur,” November, 1895, 1x, No. 1, pp. 50-75.

374 Anthrax

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 that 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 114-2 meters, so that the sporulation
of the bacilli is made impossible. The dejecta should also be care-
fully disinfected with 5 per cent. carbolic acid solution. As the
pastures and barnyards are certainly infected wherever an animal
has been the victim of anthrax, all other susceptible animals upon
the farm, and all such upon neighboring farms, should at once be
vaccinated.

Cases of human anthrax must be treated by isolation, careful
dressing of the lesions when external, the dressings being burned
as soon as removed. The expectoration, urine and feces should be
disinfected with care. The patient should be defended from flies,
and the nurse and others who come into contact with the patient
should be warned of the dangerous character of the infection.

Bacittr RESEMBLING THE ANTHRAX BACILLUS

Bacilli presenting the morphologic and cultural characteristics
of the anthrax bacillus, but devoid of any disease-producing power,
are occasionally observed. Of these, Bacillus anthracoides of Hiippe
and Wood,* Bacillus anthracis similis of McFarland,t and Bacillus
pseudoanthracist have been given special names. What relation-
ship they bear to the anthrax bacillus is uncertain. They may be
entirely different organisms, or they may be individuals whose viru-
lence has been lost through unfavorable environment.

* “Berliner klin. Wochenschrift,”’ 1889, 16.

+ “Centralbl. {. Bakt.,” vol. xxrv, No. 26, p. 556.
t Hygienisehe Rundschau,’ ” 1894, No 8.

CHAPTER V
HYDROPHOBIA, LYSSA, OR RABIES.

NEURORRHYCTES HyDROPHOBI& (CALKINS)

Hypropuosia, 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 infection must be a in the

saliva.
The infected animals manifest no symptoms during a varying in-
cubation period in which the wound heals kindly. For human be-
ings this period-may be of twelve months’ duration; in rare cases
may be only a few days; its average duration is about six weeks.
Toward the close of the incubation period an observable altera-
tion occurs in the wound, which becomes reddened, may suppurate,
_ andis painful. The victim has a sensation of horrible dread, which
passes into wild excitement, with paralysis of the pharyngeal mus-

_Cles and inability to swallow. The wild delirium ends in a final stage
of convulsion or palsy. The convulsions are tonic, rarely een and
finally cause death by interfering with respiration.

During the convulsive period much difficulty is odes in
swallowing liquids, and it is supposed that the popular term “ hydro-
phobia ” arose from the reluctance of the diseased to take water be-
cause of painful spasms brought on by the attempt.

The infectious nature of rabies seems to have been first demon-
strated by Galtier.* Pasteur, Chamberland and Rouxf continued
the investigation and found that in animals that die of rabies the
salivary glands, pancreas and the nervous system contain the
infection, and are more appropriate for the 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 de-
velopment of typical rabies in the rabbit in about six days.

Specific Organism.—It is not yet generally conceded that the
pathogenic micro-organism of rabies has been discovered, though
there is continually accumulating evidence in favor of the “bodies of
Negri.”{ Believing that the evidence at hand is strongly in favor

an Compte-rendu de l’Acad. des Sciences de Paris,” 1879, LXXXIX, 444.

} Ibid., 1881, XCII, 159. °
re ‘Zeitschrift fiir Hygiene,” 1903, XLII, 507}; XLIV, 520; 1909, LXIT, 421.

375

376 Hydrophobia, Lyssa, or Rabies

of the protozoan nature and etiological importance of these bodies,
they are tentatively accepted as the cause of the disease and treated
accordingly in the text. To these bodies Calkins has given the
name Neurorrhyctes hydrophobiez. :

Morphology.—By appropriately staining sections of the cerebrum,
cerebellum, pons, basal ganglia, spinal ganglia, and salivary glands,
of human beings or animals dead of rabies, it was possible to demon-
strate small rounded bodies measuring 4 to 10 yw as a rule, though
varying from 1 to 20 p, in the interior of the protoplasmic process of
the cells. In experimental infections they are most numerous in
the hippocampal convolution. The bodies, when stained by the
methods given below, usually appear red in color. They are ovoid
in shape, well-circumscribed, and vary in size from invisibility to
20 w in length. The smaller of them do not show any structural
differentiation, but the larger show central condensations that may
be nuclear material. The greater number of them lie in the cyto-
plasm of the nerve cells; some are free. These are the Negri bodies.

Williams and Lowden* are convinced that they are protozoan
organisms, that they are the cause of rabies, and that their presence
is pathognomonic of rabies. They believe:

1. The smear method of examining the Negri bodies (vide infra) is superior
to any other method so far published for the following reasons: (a) It is simpler,
shorter and less expensive; (b) the Negri bodies appear much more distinct
and characteristic. For this reason and the preceding one its value in diagnostic
work is great; (c) the minute structure of the Negri bodies can be demonstrated
more clearly; (d) characteristic staining reactions are brought out.

2. The Negri bodies as shown by the smears, as well as by the sections, are
specific to hydrophobia.

3- Numerous “bodies” are found in fixed virus. ;

4. “Bodies” are found before the beginning of visible symptoms, 2.¢., on the
fourth day in fixed virus, on the seventh day in street virus, and evidence is given
that they may be found early enough to account for the appearance of infectivity
of the host tissues.

5. Forms similar in structure and staining qualities to the others, but just
within the limits of visible structure (at 1500 diameter magnification), have been
seen; such tiny forms, considering the evidence they give of plasticity, might be
able to pass the coarser Berkefeld filters.

6. The Negri bodies are organisms belonging to the class Protozoa. The
reasons for this conclusion are: (¢) They have a definite characteristic morphology;
(b) this morphology is constantly cyclic, z.e., certain forms always preponderate .
in certain stages of the disease, and a definite series of forms indicating growth
and multiplication can be demonstrated; (c) the structure and staining quali-
ties, as shown especially by the smear method of examination, resemble those
of certain known Protozoa, notably of those belonging to the sub-order
Microsporidia. ;

7. The proof that the Negri bodies are living organisms is sufficient proof
that they are the cause of hydrophobia; a single variety of living organisms found
in such large numbers in every case of a disease, and only in that disease, appear-
ing at the time that the host tissue becomes infective, in regions that are infect-
ive, and increasing in those infective areas with the course of the disease can
be no other, according to our present views, than the cause of that disease.

One of the objections urged against the bodies of Negri as the
specific cause of the disease was the failure of the organism to

*“Jour. of Infectious Diseases,” 1906, I, 452.

PLATE |

e Nerve-cells containing Negri eae Hippocampus impression preparation,
Sie Van Gieson stain; 1000. , Negri bodies; 2, capillary; 3, free red
ood-corpuscles. (Courtesy of LaneaGn Frothingham.)

Morphology a7%

appear elsewhere than in the central nervous system, when the
saliva, the salivary glands and the pancreas were known to harbor
it. This has now been overcome by the demonstration of the
bodies in the salivary glands in precisely the same form as that
seen in the nervous system by Manuelian.*

Steinhardt, Poor and Lambertt have endeavored to determine

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Journal of Experimental Medicine).

graphs were taken from the film preparation stained with Giemsa solut
X 1100 (Noguchi,

Fig. 131.—Nucleated bodies in culture (Giemsa stain)
different stages of development in a culture (second an

pared from the brain of a rabbit exper
material there were no such forms to be seen either

whether Negri bodies are parasitic micro-organisms or degeneration
products of the nervous system, and have shown that when cells
of the normal guinea-pig brain are incubated in blood plasma, their
cytoplasm, when stained by Van Gieson’s stain, show small pink-

*“Ann. de VInst. Pasteur,” 1914, XXVII, 233.
ft Jour. of Infectious Diseases, 1912, XI, 459.

378 Hydrophobia, Lyssa, or Rabies

staining bodies surrounded by a blue granular ring, indistinguishable
from the unstructured Negri bodies observed with great frequency
in the rabid guinea-pig brain. In a few instances these forms con-
tained a blue-staining central ring or point, and closely. resembled
the structured forms of Negri bodies. The normal guinea-pig
brain inoculated with rabid material, street or fixed virus, incubated
in the same manner, showed the same structures. The brains of
guinea-pigs dying of street virus and rabbits dying of fixed virus,
incubated in small fragments, gave no development of Negri bodies
in blood plasma, beyond the small structured and unstructured
forms, although in one preparation the ganglion cells appeared to be
living at the end of twenty-one days’ incubation.
Cultivation.—Attempts to cultivate Negri bodies were made by
Moon,* but the success of his attempts seemed doubtful. The first

Fig. 132.—From rabbit “‘fixed-virus” brain; a, b, c, d, f, and z, types of Negri
bodies seen at death of rabbit; e, g, 4, andj, apparent multiplication and segmen-
tation of the bodies after three days at 24°C. Drawing made from smears
stained by Giemsa’s method and magnified about 2000 diameters (Williams, in
Jour. Am. Med. Assoc.). ;

claim to successful cultivation of the Negri bodies was made by
Noguchi.t The cultivation was done according to his already suc-
cessful method for Spirocheta of various kinds. Large, small and
dividing bodies appeared in the culture fluid, after inoculation with a
fragment of nervous tissue from various animals with infection fol-
lowing inoculation with street virus and “fixed” virus. But Wil-
liamst at once pointed out that there is no certainty that the bodies
increased in numbers in the cultures, though Noguchi says that they

* “Jour. of Infectious Diseases,’’ 1913, XIII, 213.

{ “Jour. of Experimental Medicine,” 1913, XVIII, 314.
t “Jour. Amer. Med. Assoc.,’’ 1913, LXI, 1509. -

Staining 379

reappear in new cultures “through many generations.”” Noguchi’s
paper seems more like a preliminary report than a finished work, and
future publication on the subject is promised. Two methods of
obtaining the virus of rabies freed from the cells of the host and free
from contaminating organisms, published by Poor and Steinhardt,*
give some promise of permitting the introduction of the bodies of
rabies into artificial culture media in a measured quantity of fluid,
perhaps containing a known number of organisms, and thus per-
mitting better methods of estimating the growth in artificial culture.

ee 7 >

IAT Ee

Fig. 133—From dog “street-virus,” brain; a, b, c, and f, types of Negri
bodies seen at death of dog; d, ¢, g, and 4, apparent multiplication and segmenta-
a ss the bodies after three days at 24°C. (Williams, in Jour. Am. Med.

SSOC.

Staining. —The Negri bodies are not difficult to stain and find
when one is familiar with them or when they are present in the
hervous tissue in considerable numbers. To find a few, to find them
quickly, and to recognize them unmistakably is, however, a different
matter. They stain by all of the Romanowsky modifications, by all
of the eosin-methylene blue combinations, and by various other
methods.

*“Jour. of Infectious Diseases,’’ 1913, XII, 202.

380 Hydrophobia, Lyssa, or Rabies

Williams and Lowden* stained Negri bodies by one of the follow-
ing methods:

(a) Giemsa’s Solution—The smears are fixed in methyl alcohol for about
five minutes. The staining solution recommended is that last used by Giemsa:

Agut- I.) FOS s 034 Sdovcaie 4.5 Sab anes sc terene Be AAO tee 3.0
ABE TD seiscis ace cient adage aoa wres 6 gape layer benetectaleusi 0.8
Glycerin (Merck’s chemically pure)................- 250.0
Methyl alcohol (chemically pure)............... Sakae 250.0

Both the glycerin and methyl alcohol are heated to 60°C. The dyes are
put into the alcohol and the glycerin is added slowly, stirring. The mixture is
allowed to stand at even temperature over night, and after filtration is ready
for use. At the time of use one drop of the stain is added for every cubic centi-
meter of distilled water made alkaline by the addition of one drop of a 1 per
cent. solution of potassium carbonate to ro cc. of the water.

The stain is poured on the slide and allowed to stand for from one-half to three
hours. The longer time brings out the structure better and in twenty-four hours
well-made smears are not overstained. After the stain is poured off, the smear
is washed in running tap water for from one to three minutes and dried with
filter-paper.

By this method the “bodies” are stained blue and the central bodies and
chromatoid granules blue, red or azure. The cytoplasm of the nerve cells stains
blue also, but the bodies can be seen distinctly within it. For diagnostic purposes
the method may be shortened thus:

Methyl alcohol...............-05 Sis ee eae ee we geen 5 minutes.
Equal parts of Giemsa solution and distilled water........ Io minutes.

(b) The eosin-methylene blue of Mallory (g.v.).

The smears are fixed in Zenker’s solution for one-half hour; after being rinsed
in tap water they are placed successively in 95 per cent. alcohol and iodine
for one-quarter hour, 95 per cent. alcohol for one-half hour, absolute alcohol
one-half hour, eosin solution twenty minutes, rinsed in tap water, methylene blue
solution fifteen minutes; differentiated in 95 per cent. alcohol, lasting one to five
minutes and dried with filter-paper.

With this method the cytoplasm of the “bodies”’ is magenta, light in the small
bodies, darker in the larger; the center bodies and chromatoid granules are a
very dark blue, the nerve-cell cytoplasm a light blue, the nucleus a darker blue
and the red blood-cells a brilliant eosin pink.

Harris} uses the following method of staining Negri bodies that
seems to have the advantages of coloring them so as to bring out their
structure, and to do away with the granular precipitate that occurs
in most methods.

Smears of the appropriate material are made upon slides and fixed by the
application of methyl alcohol for one minute, are then washed with water to
remove the alcohol, placed for from one to three minutes in an old saturated
solution of eosin in 96 per cent. alcohol, after which they are washed for two or
three seconds with water to remove the excess of eosin. This stains the Negri
bodies. Counterstaining is effected by immersing for five to fifteen seconds
in a fresh solution of Unna’s alkaline methylene blue, after which there is a brief
washing in water, decolorization in 95 per cent. alcohol and then the usual treat-
ment with absolute alcohol, xylol and balsam if the preparation is to be covered
and preserved, or the spread is blotted and dried if to be examined without a
cover. The whole process requires less than five minutes.

Smears that have been dried for several days or weeks cannot be thus stained
with satisfaction. The older the eosin solution the more rapidly and intensely
it stains. To secure the best results it should not be less than two months old.
The methylene blue should not be more than a week or two old, else it will yield
an objectionable precipitate. .

a . .
* “Tour. of Infectious Diseases,’? 1906, II, 452.
? , 2

Tt “Jour. of Infectious Diseases,” 1908, v, 566.

Pathology 381

Reichel and Engle* stain Negri bodies with the following:

Sat. alc. sol. methylene violet.................0005 Io cc.
Sat. alc. sol. fuchsin... 6... eee 7 drops.
Sterile? Water edisies.sicccess va aidaus, a baton travel aoa ace emus Wai 40 CC.

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.

Luzzanif gives the following method of staining Negri bodies.

The tissue to be stained should be fixed in Zenker’s solution, imbedded in
' paraffine and cut into very thin slices. Mann’s stain is used:

I :zoo aqueous solution of eosin. .... 2... eee ee eee 45 cc.
I:100 aqueous solution of methylene blue........... 35 cc.
Distilled: water's::.oscaiessau es soy eauewecse nae vasa 100 CC.

" (The solution of eosin and of methylene blue should be kept separately,
and only mixed and diluted at the time of using. The diluted mixture does
not keep longer than some days, or at best, a few weeks.)

After the sections are cut, they are fixed to the slides with Mayer’s glycerin
albumen, the paraffine removed with xylol, the xylol with alcohol, and the
alcohol with water. The stain is then applied for some minutes after which the
section is rapidly washed in tap water, then in absolute alcohol; when dehydrated

‘in the absolute alcohol, they are washed in a solution of

Absolute alcohol... ..... ccc cee e eee e eee eaee 30 cc.
Saturated solution of caustic soda in absolute alcohol 5 drops.

until they lose the blue color and become entirely red. They are then given a
washing in absolute alcohol, plunged into tap water and then washed with
distilled water slightly acidified with acetic acid until they turn blue again. The

* final steps are absolute alcohol, xylol and Canada balsam. The Negri bodies are
red, the cells blue.

The method should be as applicable for smears or contact spreads
as for sections, and for purposes of diagnosis the hippocampal con-
volution can be cut across, a clean side touched to the cut surface and
removed. Nerve-cells adhere to the glass which is dried and treated
as though it had an adhering section of tissue. The Negri bodies are
best seen in the processes of the nerve-cells.

Pathology.—It is generally supposed that the activity of the rabic
virus is largely confined to the nervous system, and that from the
point of admission to the body it ascends the peripheral nerves to
effect its final and fatal influence upon the central nervous system.
The seat of inoculation has, therefore, much to do with the facility
and rapidity with which the symptoms and termination come on.

When the virus enters through the skin of the forearm or lower
limb, it has a long way to travel, and the period of incubation is long;
when it enters about the face, a correspondingly short distance to go,
and a correspondingly brief period of incubation. The occurrence

* Personal communication.
; “Ann. de. l’Inst., Pasteur,” 1913, XXXVII, 1039.

382 Hydrophobia, Lyssa, or Rabies

of symptoms is accepted as evidence that the central nervous
system has been reached.

When as in experimental inoculation the virus is at once placed in
the central nervous system, symptoms do not at once develop, hence
it is concluded that not only must the essential parasites reach the
central nervous system, but they must do so in sufficient numbers
before enough damage can be done to produce the symptoms.
Under the most favorable conditions of infection, this requires about
six days.

The virus is, however, not confined to the nervous system for the
saliva is infective, and the salivary glands, pancreas, and perhaps
other glands harbor the infective agent. How-it reaches these
structures has not yet been determined. In them Negri bodies are
present but whether they reach the glands through the blood or by
way of their nervous connections is not known.

There is no morbid anatomy of rabies. Carefully made autopsies
upon the bodies of rabid human beings and animals show nothing
by which the nature of the disease can be determined. Most inter-
est naturally centers about the brain and spinal cord as being the
chief sources of disturbance and chief seats of the virus. There are,
however, so few changes as scarcely to merit description. In some
cases the meninges are distinctly congested, but in uncomplicated
cases there is no meningitis and therefore no inflammatory exudation.

In a few cases there may be scattered minute hemorrhages. In
many cases there are no lesions.

The pathologic histology of rabies reveals certain fairly constant
lesions described in the next section, but they are not now regarded
as characteristic of the disease.

Diagnosis of Rabies.—There are three means of arriving at a
diagnosis of rabies in cases of suspected “mad dogs.”

The animal having been killed, its head is cut off by an incision
through the neck at some distance from the skull, and immediately
taken to an appropriate laboratory or carefully packed. in plenty of
ice and sent to the laboratory by express. The fresher the tissue
received by the laboratory worker, the more cértain his results
can be.

Carefully opening the skull of the dog, the bwin isremoved toa

sterile dish. Good sized bits of tissue are taken from the appropriate
portions of the brain and placed in glycerin for future inoculation
operations if necessary, small bits of the same tissue are spread upon
slides according to the “smear” method of Williams and Lowden,
or slides may be spread by the “adhesion” method of Frothingham*
who makes an incision into the brain, lays it open in the appropriate
areas, and then applies the flat surface of a perfectly clean slide to
the flat cut surface of the brain. When the slide is lifted up (not
slid off), nerve-cells adhere to it, in which the Negri bodies may

* Jour. Med. Research, 1916, XIV, 477.

Inoculation of Rabbits 383

later be found. Other parts cut from the appropriate areas of the:
brain tissue are placed in fixative to prepare for sectioning should
that later become desirable.

Williams and Lowden* devised a new technic of examination for
Negri bodies. that has been of considerable advantage to those en-
gaged in looking for them for assisting in the diagnosis of rabies, as
well as in studying the bodies themselves. It may be called the
“smear method” to differentiate it from the older and less certain
“section method.” Briefly, the method is as follows:

Glass slides and cover-glasses are washed thoroughly with soap and
water and heated in a flame to get rid of oily substances. A small
bit of the gray substance of the brain chosen for examination is
placed upon one end of a slide, a cover-glass placed upon it and
pressed down so as to spread out the nervous tissue in a thin layer,
when the cover is slowly moved to the opposite end of the slide
spreading out the nerve-cells and distributing them over the surface.
The tissues selected for examination should come from at least three
different parts of the gray matter of the central nervous system, first,
from the cortex of the brain in the neighborhood of the fissure of
Rolando, or in the region corresponding to it; second, from Ammon’s
horn; third, from the cerebellum.

The smears are dried in the air and them stained as stated above.

Formerly an examination of the spinal sympathetic ganglia was
made, and the diagnosis made from what was found in them. This
constitutes the least important and most rarely pursued form of
diagnostic procedure at the present time. However, we will sup-
pose some sympathetic ganglia secured. The remainder of the ani-
mal’s head can then be destroyed. With the material thus secured
we make the following diagnostic tests:

1. Examination for the Negri bodies.
2. Inoculation of rabbits.
3. Examination for histological changes in the ganglia.

1. The Negri Bodies.—As now generally conceded, the discovery.
of these bodies in the cells of the central nervous system may be
taken as positive evidence of the existence of rabies in its transmis-
sible stage..

2, The Inoculation of Rabbits.—This is only necessary in highly
suspicious cases in which no Negri bodies are found, or in which the
investigator is not satisfied that such bodies are specie indications
of the disease.

The glycerinated or fresh nervous tissue can be employed. A bit
of the tissue is made into a creamy suspension, under aseptic precau-

tions, by adding physiological salt solution, crushing and grinding
ina small agate mortar. When it is ready a rabbit is anesthetized,

* Jour. of Infectious Diseases,” i906, It, 452.

384 Hydrophobia, Lyssa, or Rabies

the hair is pulled out over one side of the skull (or if it be preferred,
‘the skin can be shaved), the scalp is washed with an antiseptic
solution and an incision about an inch long is made and the skull
exposed. With a small trephine a button of bone is cut out and the
dura exposed. The suspension of nervous tissue is drawn up in a
sterile hypodermic syringe, and one or two drops of it injected be-
neath the dura mater or deeply into the brain tissue. If the opera-
tion be successful the wound heals and no meningitis follows, but at
the end of about six days the rabbit becomes paralyzed, “dumb
rabies.” Several rabbits should be simultaneously inoculated as
should a single rabbit develop meningitis, through accident or bad
technic, no information is gained, and no diagnosis is possible. The
rabid rabbits die in a day or two after the onset of the palsy, and
Negri bodies can be found in the brain tissue, which is infectious for
other rabbits in endless series.

3. The Histological Changes in the Nervous System.—These are
now rarely looked for, as experience has shown them to be the least
reliable means of making the diagnosis. The chief changes are the
“tubercles of Babes,’’* which consist of perivascular collections of
cells, and collections of newly formed cells about the ganglionic
nerve-cells of the brain and cord.

Van Gehuchten and Nelis,+ and Ravenel and McCarthy{ have
studied these lesions. Ravenel and McCarthy think that Babes
gave undue prominence to the rabid tubercle, which consists of an
aggregation of embryonal cells about the central canal of the cord,
about the ganglionic nerve-cells, and about the capillary blood-
vessels. They think, however, that the lesions of the nerve-ganglion
cells are pathognomonic if taken in connection with the clinical
manifestations of the disease. The specific changes consist of de-
generation, chromatolysis and even total disappearance of the nuclei
of the ganglion cells, dilatation of the pericellular space, and 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, brownish and in part metachromatic. Spiller§ refused to
regard these lesions as pathognomonic of rabies and it is now gen-
erally conceded that they are not to be looked upon as of more than
confirmatory evidence of the disease.

Virulence.—The virus of rabies is variable in virulence to a marked
degree. ‘Street virus,” or that obtained from rabid dogs, is so
variable that before scientific study with it is possible, it must
be standardized. This is done by passage through rabbits, the tech-
nic of the inoculation being the same as that given in the section
on “Diagnosis.” After being passed successively from rabbit to

* Ann. de l’Inst., Pasteur, 1896, VI, 209.

+ “Univ. med. Mag. »” Jan., s00ts

{ ‘Archiv. de Biologie,” 1900, xv

§ “Pathological Society of Philadelphia, ”? March, 1901.

Prophylaxis 385

rabbit from twenty to thirty times, a maximum virulence is attained
and the virus is said to be “ fixed.”

Pasteur found that the virulence of the nervous tissue was dimin-
ished by inspissation, by drying under aseptic precautions in a sterile
jar over calcium chloride. There is some doubt whether this results
in actual diminution in the virulence of the organisms as Pasteur
thought, or whether the virulence is diminished by dilution, i.e., by
effecting the destruction of many of the organisms. There seems
to be no means of determining this at present. The diminution of
virulence is in proportion to the length of time the nervous tissue is
dried.

Prophylaxis.—To prevent rabies, means must be devised for
preventing dog-bites. In an island community like England, rabies
may be successfully eliminated by destroying all animals suspected
‘of having the disease, muzzling the dogs for a time, and denying
admission to new dogs until they have spent a long enough period
in quarantine to exclude the possibility of their being infected with
the disease.

Upon continents it seems unlikely that rabies can ever be com-
pletely eradicated as it is not only a disease of dogs, but also of
wolves, foxes, skunks and other wild animals by which dogs may be
bitten.

However, it is the dog that is the common distributor and to
which attention must be directed.

All rabid animals should at once be killed, and all others known
to have been bitten by them also killed so soon as the diagnosis of
rabies in the first animal is confirmed. If the bitten animals cannot
for any reason be killed, they should be carefully confined until the
incubation period is long past. All stray dogs and cats should be
destroyed because not being under any observation, their condition
isnot known. Dogs in general should be muzzled when abroad.

Immunity to rabies may be brought about in human beings by
the method of active immunization given below, but as rabies is a
somewhat rare disease of human beings, it does not seem worth
while to advise immunization except when there is some particular
danger of its occurrence. Such danger obtains when human beings
have been attacked and bitten by rabid animals or by dogs running
at large, whose health is a matter of doubt. Recovery from rabies
in human beings is practically unknown. Any individual, therefore,
that is bitten under suspicious circumstances may be in danger of
developing an almost certainly fatal malady. This is not to be con-
Strued to mean that every person bitten by a certainly rabid dog
must necessarily contract rabies, for there are accidents and cir-
cumstances attending the transmission of diseases of infectious na-
ture, but whether certain or not, the danger of rabies is great in
such cases and they ought to receive immediate care and attention.
Many content themselves with an attempted destruction of the

386 Hydrophobia, Lyssa, or Rabies

introduced virus by applying the actual cautery, or caustics, or
powerful germicides to the wounds made by the dog’s teeth, and
Lambert who worked upon this matter experimentally came to the
conclusion that though a few cases might thus be saved, the method
was too unreliable to be recommended. The long period of incuba-
tion of human rabies (from 15 to 250 days and averaging 4o days)
is the source of salvation for many infected persons, for it makes it
’ possible to effect immunization during that period and so inhibit
the development of the disease itself.

Immunization against Rabies.—Pasteur* observed that the viru-
lence of the virus was less in animals that had been dead for some
time than in those just killed, and by experiment found that when
the nervous system of an infected rabbit was dried in a sterile atmos-
phere its virulence attenuated in proportion to the length of time
it was kept. A method of attenuating the virulence was thus sug-
gested to Pasteur, and the idea of using attenuated virus as a pro-
tective vaccine soon followed. After careful experimentation he
found that by inoculating a dog with much attenuated, then with
less attenuated, then with moderately 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 method, based upon limited accurate
biologic knowledge, and upon experience with very few micro-organ-
isms, should find absolute confirmation as our knowledge of im-
munity, toxins, and antitoxins progressed. Pasteur introduced the
unknown poison-producers, attenuated by drying and capable of
generating only a little poison, accustomed 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.

The Attenuation 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 viru-
lence. This can be prepared, according to the directions of Hégyes,f
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 emulsion made-
with a fragment of it, and a second rabbit inoculated, and so on through the

*“Compt.-rend. de Acad. de Sciences de Paris,’ xc, 1259; XCV, 1187,
XCVIII, 457, 1229; CI, 765; CI, 459, 835; CII, 777. —
t See Kraus and Levaditi, “‘Handbuch der Immunititsforschung,” 1.

The Attenuation Method 387

series until a standard virulence is attained and the virus is said to be “fixed.”
Tt 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 laboratories and kept passing through rabbits.
In this manner uniformity of dosage and virulence is most easily maintained.

The technic of obtaining the rabbit’s cord given by Oshida* is the one now
generally employed. As given by Stimson,} 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 (x 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.r inch wide is seared with
a hot iron around the occiput and nuchal region and ear openings. The skull

Fig. 134.—Removal of the spinal cord from a rabbit (Stimson, Bull. No. 65,
Hygienic Laboratory).

is then transversely divided in the center 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 beneathit. 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.

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 over sticks of caustic potash or calcium chloride.

The longer the cord dries, the more the virulence of the micro-
. organisms attenuates.

When the cord has reached the necessary attenuation, 1 cm. of

*“Centralbl. f. Bakt. u. Parasitenk.,”” 1901, XxIx, Orig., 988.
+ “Facts and Problems of: Rabies,” Hygienic Laboratory Bulletin No. 65,
June, 1910, Washington, D. C. °

388 Hydrophobia, Lyssa, or Rabies

it is emulsified with 3 cc. of sterile 0.8 per cent. salt solution and is
ready for use. There can be no absolute accuracy of dosage. The
injection material made in the laboratory. under strict aseptic pre-
cautions can be used with perfect safety for many hours subsequently
if kept cold, and can be packed in ice and sent by express to the phy-
sician to use at the home of his patients.

Fig. 135.—Method of drying the spinal cord of a rabbit for the purpose of
attenuation (Stimson, Bull. No. 65, Hygienic Laboratory).

As the transfer of the cord to glycerin preserves the virulence for
some time 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, always available for furnishing vaccines of all re-
quired strengths, independently of new experimental rabbits, and
also makes it possible for one rabbit cord to furnish material for
several cases. The treatment of a patient bitten by a rabid
animal, and in danger of acquiring rabies, requires numerous injec-
tions with material of varying virulence, as shown in the following
tabulations:

Scheme for Mild Treatment 389
PASTEUR’S ORIGINAL SCHEME (Marx)
Light schema |! Intense schema
Age of ‘| Amount of Age of Amount of
Day of treatment dried injected Day of treatment dried injected
cord emulsion cord emulsion
Days ce. Days ce.

” 14 3 F 14 3

EUR gue se 13 3 Firsts: seiesaev eee 13 3
12 3 ‘12 3

Second......... ne 3 ct 3
“ 10 3 Io 3
Third......... 5 z er ee i ;
Fourth . : ‘3
6 2 | Third é :

: a le Dhnrd cilcag saad 2
Wifthiescisce cia seesinn 6 j 6 ;
Sixthizg. 40 watee 5 2 Fourth. <.4 45000 5 2
Seventh........ 5 2 Pifthyses sn gerk eles is 2
Fighth......... 4 2 Sixths. vscemay ans x 4 2
Ninth.......... 3 I Seventh.......... 3 I
Tenthsinss:scicsesce 5 2 EFighth........... 4 2
Eleventh 5 2 Ninth. 3 I
Twelfth........ 4 2 Tenth 5 2
Thirteenth...... 4 2 Eleventh......... 5 2
Fourteenth... . 3 2 TPwelfth +3 s<s02s< 4 2
Fifteenth....... 3 2 Thirteenth........ 4 2
Sixteenth....... 5 2 Fourtcenth....... 3 2
Seventeenth. ... 4 2 Fifteenth......... 3 2

. Eighteenth. .... 3 2 Sixteenth......... 5 2
Seventeenth....... 4 2
Eighteenth........ 3 2
Nineteenth....... 5 2
Twentieth........ 4 2
Twenty-first...... 3 2

(From Bulletin No. 65, Hygienic Laboratory, June, 1910, U. S. Public Health
and Marine-Hospital Service.)

The system of treatment at present used at the Hygienic Labora-
tory is shown in the following tables:

SCHEME FOR MILD TREATMENT

Amount injected Amount injected

mai Home + Five | One Day a Five One

Adult | to ten | to five Adult| to ten | to five

years | years years | years
Injections ce. ce. ce. Injections| cc. ce. ce.

Tew... 8—7-—6=3] 2.5 | 2.5 | 2.0 |lr2....) g=r | 2.5 | 2.5 | 2.5
Qe, G42) 2.5 | Sas | us WESee S| aE | bh [26§. | 2S
See, 4—-3=2| 2.5 2.5 2.0 |[14....] 3=2 | 2.5 2.5 2.0
fesse. S=1/ 2.8 || age | Busi. [lPgecca| FEL | os | ees | eve
Sees, 4=1/ 2.5 2.5 2.5 |\t0....| 2=2 | 2.5 2.0 I.5
Oo se 3=1] 2.5 205 220! WOFicc | =p an§ 2.0 2.5
Tee 3=1| 2.5 2.5 2.0 |/18....] 4=1] 2.5 2.5 2.5
Bike 2=1/ 2.5 1.5 1.0 j/f9....] 3=1 | 2.5 2.5 2.5
Qe, 2=1| 2.5 | 2.0 | 1.5 |l20....] 2=1 | 2.5 | 2.5 | 2.0
10s, 5=1| 2.5 |] 2.5 225 ||2ka.) PET | 2.5 Be 2.0

IL, 5=1] 2.5 | 2.5 | 2.5

390 Hydrophobia, Lyssa, or Rabies

SCHEME FOR INTENSIVE TREATMENT

Amount injected Amount injected
a
Day cae Five |: Gna || Per |. eet Five | One
Adult | to ten | to five | ‘ Adult | to ten | to five
years | years years years
| ; :
| Injections ce. ce. ce. Injeclions| ce. ce. ce
I....../8-7-6=3] 2.5 | 2.5 7-2.5 || 12....] g=1 | 2.5 | 2.5 | 2.0
Dicidewela AS32215 2.5 | 2.0 13....] 3=2 | 2.5 | 2.5 |.2.0
ee 5—4=2! 2.5 25, 2.5 14...| 2524 |] 2.5 | 1.5 | 2.0
a a aiate 3=1| 2.5 2.5 2.0 Pee Ve aE. ||) Bag. | 220
Bie ZH Tz) 205 2.5 2.0 16....] 4=1 | 2.5 2.5 | 2.5
Gscv as 2=1} 2.5 2:0 | Bus Pixel BEE) 2.5 | a8 | a8
Tassel 2=1/ 2.5 2.5 2.0 18....] 2=1 | 2.5 2.5 | 2.0
c: eeee I=I] 2.5 1.5 I.0 TQss5| Z=E 7 2.5 | 205 | 220°
Oye 5=1| 2.5 2.5 2.5 2060) 2 | a5 2.5 | 2.5
Io.. 4=1) 2.5 2.5 2.5 2000] FE | B58 Din 2.0
Ekim as 4=1/ 2.5 2.5 2.5 ; .

(From Bulletin No. 65, Hygienic Laboratory, June, 1910, U. S. Public Health
and Marine-Hospital Service.) . : ;

The Dilution Method.—Higyes,* of Budapest, believes that Pas-.
teur 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 (1 gram of medulla to 10
cc. of sterile-broth) as a stock solution, to be prepared freshly every
day, and uses it for treatment, the first dilution used being 1: 10,000;
then on succeeding days 1:8000, 1:6000, I:5000, 1: 2000, I: 1000,
I:500, 1:250, 1:200, 1:100, and finally the full strength, 1:10.

Cabott found the dilution method attended with danger to the
animal immunized, which was not true of the dried-cord method of
Pasteur.

The Inspissation Method.—A new method of carrying out the
dilution method, suggested by Harris:and Shackell,t seems to be
devoid of danger to the patient and bids fair to recommend itself
on the ground of greater accuracy than former methods. It depends
upon Shackell’s method of desiccation:t

The material to be dried is placed in the bottom of a Schubler’s vacuum
desiccating jar, in the upper part of which is a separate dish containing sulphuric
acid. The temperature is reduced by placing the jar, half submerged, in a salt
and ice mixture, and after thorough solidification of the material has resulted,
a rapid vacuum is produced by a Geryk ‘pump to less than 2 mm. of mercury.
During the process of-desiccation, the temperature in the lower half should be
kept several degrees below o°C. Unless the sulphuric acid be repeatedly shaken
to prevent saturation with water, the time required for complete desiccation
will be unduly prolonged.

By this method brains and cords may be desiccated in toto, with-

*“ Acad. des'Sciences de Buda-Pest,” Oct. 17, 1897; ‘‘Centralbl. f. Bakt. u.
Parasitenk.,”’ 1887, 11, 579.

t “Journal of Experimental Medicine,” 1899, vol. 1v, No. 2.

{Lab. Sec. Amer. Pub. Health Asso., Sept. 6, 1910.

The Inspissation Method 391

out destruction of virulence, in from twenty-four to thirty-six hours.
The material thus dried is like chalk and easily pulverized. It
is, however, highly hygroscopic and if permitted to absorb water
becomes leathery and loses virulence rapidly.

‘In a later paper Harris* found that the more thoroughly and
rapidly the material is frozen, the greater will be the amount of
virulence remaining after desiccation. A new method suggested
is as follows:

The brain or cord is ground in a porcelain mortar, with the addition of
water drop by drop until a thick smooth paste is formed. Carbon dioxide snow
is then collected from a tank in the ordinary manner and is added in small
amounts to the paste which should be stirred thoroughly meanwhile to prevent
the material freezing in 3 solid mass. Freezing occurs rapidly and when complete
the material is very brittle and easily reducible to a fine powder. During the
pulverization more snow is added from time to time to prevent thawing. When
the material is thoroughly pulverized, it is transferred to a small beaker with an
excess of snow and placed in the bottom of a Schubler’s vacuum jar which has
previously been half immersed in a mixture of salt and ice and become thoroughly
cold. A beaker of sulphuric acid is then placed on wire gauze in the upper part
of the jar in such manner that there is free access of air between the frozen material
and the sulphuric acid. The acid is placed in the upper part because if placed
below, it soon freezes at the low temperature. The vacuum should measure
less than 2 mm. of mercury. During desiccation the temperatures should not
be allowed to rise above — x 5°C. The jar should be rotated gently several
times daily to mix the water and theacid. Asingle brain will become thoroughly
dry in from thirty-six to forty-eight hours.

The object in thoroughly pulverizing the virus is two-fold. It
results in a more complete mixture, so that all parts contain an equal
amount of virulence. Secondly, it permits of more rapid drying
and an easy transfer into smaller containers for subsequent tests.
To avoid any absorption of moisture, the dry powder is transferred .
from the beaker to small glass tubes the ends of which are sealed in
aflame. The transfer is effected in a moisture-free atmosphere by
covering the top of the beaker with rubber dam held in place by ad-
hesive strips. A small puncture is made in the rubber large enough
to admit the tube, and through this the tubes are inserted and filled.
From 20 to 100 mg. is a convenient amount put into each tube. If
the tube has a diameter of 4 mm., each millimeter of powder will
weigh approximately 2 mg.

Harris believes that the use of desiccated virus in anti-rabic im-
munization of animals and persons offers many advantages over
other methods.

Harrist reports that 182 patients have been injected with the
virus thus prepared for the purpose of immunizing them against
hydrophobia. No deaths have occurred and no complications de-
-Veloped. It is thus to all appearances a safe and efficient method
and is especially economical to the laboratory in time,.labor and
money.’ Material can be prepared two or three times a year and
put aside in the cold to be used only when needed and as one rabbit

*“TJour. of Infectious Diseases,” 1912, X, 369.
1 “Jour. of Infectious Diseases,” 1913, XIII, 155.

392 Hydrophobia, Lyssa, or Rabies

furnishes enough material to immunize 20-25 patients, the initial
cost is negligible. The work can be undertaken in any hospital or
municipal laboratory without increasing the staff or the expense.
To be able to prepare at one time enough material for from six to
twelve months’ use and to have this always ready for any number of
patients is such a lessening of labor and anxiety as only those who
have followed the classic method of drying cords can appreciate.

If the conclusion of Harvey and McKendrick* be correct, and
“the immunizing power of any given portion of a rabies cord is a
function of the unkilled remnant of the rabies virus which is con-
tained in that cord,” one should be able to find out with mathe-
matical certainty how many minimum infective doses will produce
a definite degree of immunity. For this purpose they suggest that
the virulence of the virus is expressed in “units,’”’ one unit being
the smallest amount which, when injected intracerebrally into a
full-grown rabbit, will produce paresis on the seventh day.

Specific Treatment.—Babes and Leppf thought that the serum
of animals that had received repeated injections of the crushed
nervous tissue of rabid animals was neutralizing or destructive to
the rabies virus in vitro, called it “antirabic serum,” and believed
that it conferred a defensive power upon other animals. Marie,t
however, found it to be a simple neurotoxic serum and inert in its
action upon the virus. It is never used in the treatment of rabies,
at present.

* “Theory and Practice of Anti-rabic Immunization,” Calcutta, 1907.

t “Ann. de l’Inst. Pasteur,” 1889, 11.
t “Compt.-rendu Soc. Biol.,” June 18, 1904, LVI, p. 1030.

CHAPTER VI
ACUTE ANTERIOR POLIOMYELITIS

ACUTE anterior poliomyelitis, atrophic spinal paralysis, infantile
palsy, “spinale Kinderlahmung,” is an acute infectious disease,
largely confined to the first three years of life, and characterized by
fever, destruction of cells in the gray matter of the central nervous
system, palsy and rapid atrophy of the palsied muscles. It is of
sporadic and occasionally of epidemic occurrence in all parts of the
world. Although infectious, its transmissibility is so slight as to
make contagiousness a matter of doubt. .

The essential cause is in doubt, though it is probable that it is a
minute coccoid organism capable of artificial cultivation. It is
certain that there is an infectious agent and that it is filterable
through the Berkefeld filters. Probably the best account of the
history and epidemiology of the disease has been compiled by
Wickman.*

The disease was investigated bacteriologically by various workers,
and it went through the usual experience of having various micro-
organisms isolated and described, to be afterward abandoned as
accidental and unimportant agents. The modern studies of the sub-
ject, by modern methods of investigation, were begun by Landsteiner
and Popper.| Their method of procedure was to emulsify the

_ Spinal cord of a fatal case of the disease, in a nine-year-old child, in

physiological salt solution, and inject it into the peritoneal cavities
of monkeys. One monkey became ill and died on the eighth day;
the other became paralyzed on the seventeenth day after the inocu-
lation. A similar emulsion of the cord of the paralyzed monkey
failed to infect other monkeys into which it was injected. Knépfel-
macher,t and Strauss and Huntoon§ were also able to infect one
monkey with human virus, but could carry the infection no further.

Flexner and Lewis|| made careful experiments upon 81 monkeys
inoculated with the disease. They found the incubation period to
vary from 4 to 33 days, the average being 9.82 days. During this
period there were prodromal symptoms such as nervousness and
excitability, fatigue, tremor of the face and limbs, shifting gaze

*“Beitrige zur Kentniss der Heine-Medinischen Krankheit,” Berlin, 1907.

t “Zeitschrift fiir Immunitatsforschung,” 1909, II, 377.

“Med. Kiin.,” 109, v, 1671.
Phat York Med. Jour.,” 1910, XCt, 64.

“Journal of the Amer. Med. Asso¢.,” 1909, LIZ, 1639, and “Jour. Medical
esearch,” 1910, XII, 227.

393

394 Acute Anterior Poli omyelitis

when the attention was attracted, and a wrinkled and mobile rather
than smooth and placid face. The onset of the disease is sudden,
with or without the given signs, and consists of paralysis. In gen-
eral, any of the larger voluntary muscle groups may be affected;
other groups may be weak or partially paralyzed. The paralysis
may be of all grades of completeness. There may be some anes-
thesia; occasionally there was evidence of pain. The animals may
die or they may recover. In the latter case the paralysis sometimes
entirely disappears; more frequently it persists and the paralyzed
member gradually stiffens and is deformed by contractures.
. In the dead monkeys, or those that were killed for study, the
chief lesions were in the gray matter of the céntral nervous sys-
tem and consisted of edema, diffuse livid injections of the blood-
vessels and punctiform and pin-head-sized hemorrhages. When
healing sets in, the lesions are firmer, paler, non-circumscribed, and
raised somewhat above the level of the surrounding gray and white
matter.

The chief histological changes were also in the gray matter es-
pecially in the cord, where they occurred in either the anterior or
posterior horns, but more frequently and more extensively in the
anterior horns. There was a high degree of cellular infiltration of
the perivascular spaces, edema of the spaces, and hemorrhage into
the spaces. From the spaces the cells often passed into the ground
substance. But independent foci of small cells, edema and hemor-
rhage also existed in the nervous tissue. The nerve cells often
showed degerieration which consisted of hyaline transformation
and necrosis leading to loss of the tigroid substance, cell-processes,
nuclei, etc. Often the cell was surrounded by lymphocytes or in-

vaded by polymorphonuclear leukocytes. Sometimes the nerve- |.

cells had disappeared and the leukocytes taken their places. UIti-
mately, a part of the nervous elements would be removed and re-
placed by an indefinite cellular tissue, containing many compound
granular corpuscles.

The monkeys were infected by various methods, the first being
the direct inoculation of the brain by a needle introduced through
_ the opening made by a small trephine. They found, however,
that the virus readily finds its way to the nervous system when
introduced subcutaneously, and less readily when introduced in-
traperitoneally. The blood of the infected animal contains the
virus at the beginning of the attack but how richly was not deter-
mined. The cerebro-spinal fluid also contains it at the time the
palsy appears. The vaso-pharyngeal mucosa also contains it, and
_ can convey it to other animals.

The virus readily passed through Berkefeld filters, and the clear
filtrate thus obtained, when injected into monkeys by the intra-
‘cerebral or subcutaneous routes, regularly produced the disease
in an infectious form so that it was clear that the lesions were in-

Bacteriology 396

fectious and not toxic in character though brought about by filtered
fluid.

The virus resists freezing but is readily destroyed by heating to
45°-s0°C. for half an hour.

Various attempts were made by Kraus and Werhicke,* Lentz
and Huntemiillert and Marksf{ to infect rabbits with the virus, but

’ Fig. 136.—Micro-organism causing epidemic poliomyelitis. 3, Separate glo-
boid bodies, X 1000; 4, aggregated masses of globoid bodies, X 1000; 5, chains
and pairs of globoid bodies, X 1000; 6, chains of globoid bodies compared with

' Streptococcus pyogenes, X 1000; 7, agar fragment showing pairs of globoid bodies
compared with Streptococcus pyogenes, X 1000 (Flexner and Noguchi, in Journal
of Experimental Medicine).

though some successes were reported, there seems to be no develop-

ment in the rabbit of lesions or disturbances resembling the char-

' acteristic lesions and symptoms of acute anterior poliomyelitis in
man and the monkey. a
**Deutsche med. Wochenschrift,” 1909, XXXV, 1825; 910, XXXVI, 693.

} “Zeitschrift fiir Hygiene,” 1910, LXVI, 481.
{*Jour. Exp. Med.,” 1911, XIV, 116.

396 Acute Anterior Poliomyelitis

In 1912, Rosenau and Brues* reported that in 50 per cent. of their
experiments, the virus of acute anterior poliomyelitis was trans-
mitted from monkey to monkey by the bite of the stable fly Stomoxys
calcitrans, and expressed the belief that it was a biological and not a
mechanical transfer, and that the virus underwent some change and
development in the flies. These results were confirmed by Ander-
son and Forst,t+ but failed to be confirmed by other workers and
later could not be successfully repeated by the same investigators.

Howard and Clarkt worked over the subject of transmission of
the disease by insects, and investigated the house-fly Musca domes-
tica; the bed-bug, Cimex lectularius; the lice, Pediculus capitis and
Pediculus vestimenti; various mosquitoes, Culex pipiens, Culex
solicitans and Culex cantator, and found that only one of these
insects, the common house-fly, Musca domestica, can carry the virus
in an active state for several days both upon the surface of its
body and in its gastro-intestinal tract. None of the suctorial -
insects withdrew the virus with the blood of the infected monkeys
to which they were applied.

Flexner and Noguchi§ made experiments upon the cultivation
of the micro-organism supposed to be the infective agent. The
technic employed was much like that employed for the cultivation
of Treponema pallidum (q.v.), and resulted in an undoubted quan-
titative increase in the infectiveness of the virus. Further, they
were now able, for the first time, to describe an organism that
might be the specific infectious agent. It is a globoid body meas-
uring from 0.15—0.3 «, arranged in pairs, chains and indefinite masses.
Its small size makes it barely visible and able to penetrate the pores
of the Berkefeld filters.

This organism they were able to stain both by the methods of
Giemsa and Gram. Having come to recognize it in the culture,
they were subsequently able to find it in sections of tissue from the
lesions of poliomyelitis, and conclude that “The micro-organism
exists in the infectious and diseased organs; it is not, so far as is
known, a common saprophyte, or associated with any other patho-
logical condition; it is capable of reproducing, on inoculation, the
experimental disease in monkeys, from which animals it can be re-
covered in pure culture. And besides these classical requirements,
the micro-organism withstands preservation and glycerination as
does the ordinary virus of poliomyelitis within the nervous organs.
Finally, the anaérobic nature of the micro-organism interposes no
obstacle to its acceptance as the causative agent, since the living
tissues are devoid of free oxygen and the virus of poliomyelitis has
not yet been detected in the circulating blood or cerebro-spinal fluid

* “Monthly Bull. of the State Board of Health of Massachusetts,’1912, VII,

14.

: + «Public Health Reports,” 1913, XXVIII, 833.

t “Jour. Exp. Med.,” 1912, xvi, 850.
§ “Jour. Exp. Med.,” 1913, XVIII, 461.

Bacteriology 397

of human beings, in’ which the oxygen is less firmly bound; nor need
it, even should the micro-organism be found sometimes to survive
in these fluids.”

From these discoveries it is now certainly well established that
acute anterior poliomyelitis is an infectious disease, occasioned by
a minute anaérobic organism, of globoid form, capable of resisting the
bactericidal effects of glycerin for months, and capable of passing
through the pores of a Berkefeld filter. When nervous or other
tissue containing it, or pure cultures of it, are introduced into the
nervous tissue or into the subcutaneous tissues of certain animals,
of which the monkey is the chief one, the disease is readily induced.

The mode of transmission remains to be discussed. From the
failure of those who continued the insect experiments to achieve
continued success, and because of the short time the infectious agents
are in the blood—only the first few days—and the small number that
seem to be there, it is well to assume that insects play a doubtful
réle, unless it be the common house-fly, Musca domestica.

Flexner and Clark* have shown that when the virus is introduced
into the upper nasal mucosa in monkeys its propagation can be
followed from the olfactory lobes of the brain to the medulla ob-
longata and spinal cord. Since the virus can thus find its way from
the nasal mucosa to the deeper nervous tissues, they hold the opinion
that it is through this avenue that infection commonly takes place.

During the disease, the infectious agents are upon the nasal
mucosa, they may be discharged from the surface into the atmos-
phere, and inhalation by others may be the means of infection. It
is also not impossible that house-flies first visiting the nose of an
infected sleeping child, and then some other sleeping child, may carry
the organisms.

One attack of the disease confers immunity, and experimental
immunization can be effected by a succession of doses beginning
with great dilutions and ascending to greater concentrations like
the Hégyes method in rabies, but as the disease comes on without
a preliminary dog-bite, and as the period of incubation is short, and
as our first knowledge of it coincides with the appearance of the
paralysis when the damage is already done, no practical utilization
can be made of our knowledge of the facts of immunity to the dis-
ease at the present time.

*“Proc. Soc. Exper. Biol. and Med.,” 1912-13, X, I.

CHAPTER VII
CEREBRO-SPINAL MENINGITIS

DipLococcus INTRACELLULARIS MENINGITIDIS
(WEICHSELBAUM)

Synonyms.—Meningococcus; Micrococcus meningitidis.

General Characteristics—A minute non-motile, non-flagellate, non-sporog-
enous, non-chromogenic, non-liquefying, aérobic, pathogenic: coccus, staining
by ordinary methods, but not by Gram’s method.

Acute cerebro-spinal 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 pneumococci, staphylococci, strep-
tococci, Bacillus influenze, B. typhosus, B. coli, B. mallei, B. pestis
and others.

In addition to these, however, are numerous cases of seemingly
primary infection of the membranes, either sporadic or epidemic in
occurrence and constituting the disease known as cerebro-spinal
fever, epidemic cerebro-spinal meningitis, or “spotted fever.” It isa
very dangerous febrile malady, characterized by sudden onset, high
temperature, an irregular exanthem, early meningitis with or without
coma, and a high mortality. The disease is transmissible, though
but slightly contagious, and is caused by the meningococcus, or
Diplococcus intracellularis meningitidis. This micro-organism seems
to have been first seen and described by Marchiafava and Celli*
and by Leichtenstern.t It was, however, confused with the pneu-
mococcus and not much attention was paid to it until Weichselbaum}
reported that he had found it, isolated it and cultivated it from six
cases of epidemic cerebro-spinal meningitis.

Later careful studies by Jager,§ Scherer,||:Councilman, and Mal-
lory and Wright** (embracing 55 cases, in which the cocci were
found by culture or by microscopic examination in 38), and of Flat-
ten,tt Schneider,{ + Rieger,} {+ Schmidt,+{ Gdppert,}t Fligge,tt von
Lingelsheim,{+ Besredka,tt Flexner§§ and others have shown the

* “Gaz. degli Ospedali,” 1884, vir.

t “Deutsche med. Wochenschrift,” 1885.

{t“Fortschritte der Med.,” 1887, No. 18 and 10.

§ “Zeitschrift fiir Hygiene,” xrx, 2, 351.

| Centralbl. f. Bakt. u. Parasitenk.,” 1895, xvu1, 13 and 14.
** “ Amer. Jour. Med. Sci.,” March, 1898, vol. cxv, No. 5.
tt “Klinisches Jahrbuch,” 1906. ;
tt‘ Annales de l’Inst. Pasteur,” 1906, xx, 4.
§§ “Jour. Exp. Med.,” 1906-07.

398

Identification 399

diplococcus of Weichselbaum to be, without doubt, the specific
organism.

Distribution.—The distribution of Diplococcus intracellularis
in nature is as yet not fully known. It can be found in nearly all
cases of epidemic cerebro-spinal meningitis. What seems to be the
same organism has been found in the nose, in coryza, by Scherer,
on the conjunctiva by Carl Frinkel* and Axenfeld,f and in the puru-
lent discharges or rhinitis and otitis by Jager.t During epidemics of
the disease, apparently identical organisms can be cultivated from
the naso-pharynx of many healthy persons. Such are called
“carriers,” and though apparently immune to the disease themselves,
are supposed to aid in disseminating the cocci among others whose
susceptibility causes them to fall victims to it. A careful study
of the distribution of the organism in the respiratory passages of.
carriers has been made by Herrold.§

Fic. 137—Meningococcus in spinal fluid (from Hiss and Zinsser, ‘“Text-Book
of Bacteriology,’ D. Appleton & Co., Publishers).

Morphology.—The micro-organism is a biscuit-shaped diplococcus
_ having a great resemblance to the gonococcus. It measures

about 1 w in diameter, but is variable in size according to its age.
Cocci in young growing cultures are much the same size; in old and
dying cultures many are large. They are not motile, have no flag-
ella and no spores. No capsules occur about the organisms.

Like the gonococcus the cocci are usually to be found in the cy-

, toplasm of polymorphonuclear cells in the exudate of the inflamed

membranes. Occasional free cocci are also found. It was the com-
‘mon occurrence of the cocci in the phagocytes that led Weichselbaum
toname the organism Diplococcus intracellularis. Many of the cocci
inclosed in the cells are apparently dead and degenerated, as they

* “Zeitschrift fiir Hygiene,” June 14, 1899.
Lubarsch and Oestertag, ‘Ergebnisse der allg. Path. u. path. Anat.,” m1,

+ 573.
1" Deutsche med. Wochenschrift,” 1894, S. 407.
§ “Jour. Amer. Med. Asso.,” Jan. 12, 1918, LXx, 82.

400 Cerebro-spinai Meningitis

stain badly and do not grow when the pus is transferred to culture-
media. It has recently been claimed by Hort* that we are mistaken
in regarding the meningococcus as an organism belonging to the
coccacee. His observations lead him to believe that it is not a
bacterium at all, but one of the ascomycetes. Certain large organ-
isms in the cultures, to which he applies the term “giant meningo-
‘cocci’ are the fully developed organisms, the meningococci as usu-
ally observed, being regarded by him ascospores of the higher
organism.

Identification.—There should be no difficulty in identifying the
organism under what may be spoken of as normal conditions. Thus,
a Gram-negative diplococcus inclosed in the polymorphonuclear leu-
kocytes of a cloudy fluid drawn from the spinal canal of a case of
suspected cerebrospinal fever, can scarcely be anything else, and is
sufficient not only to make certain that the organism is the menin-
gococcus, but also that the patient is suffering from the disease.

The difficulty arises when the micro-organisms are to be iden-
tified under abnormal conditions, as, for example, when a search
is being made for “meningococcus carriers” by an examination of
the nasal secretions. Should nasal or pharyngeal mucus from an
apparently normal man be found to contain Gram-negative diplococci,
the question at once arises whether these may notbe Micrococcus
catarrhalis, Micrococcus flavus, Diplococcus pharyngis siccus
or some other similar but comparatively harmless organism. This
question cannot be settled by microscopic examination alone, but
must be achieved through cultivation and specific serum agglutination
of the organisms.

Staining.—The organism is stained with the usual aqueous ~~~

solutions of the anilin dyes. The effect of ‘staining is not, however,
always uniform. Some may stain uniformly and intensely, others
unequally and palely, some may not stain at all. Large cocci
usually show the greatest irregularity. It is supposed that the young
actively growing cocci stain well, the old dying cocci, irregularly
or not at all. It does not stain by Gram’s method..

For staining the meningococcus the method of Pick and Jacob-
sohnj was highly praised by Carl Frankel, who modified it by adding
three times as much carbol-fuchsin as was recommended in the
original instructions, which were as follows: Mix 20 cc. 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. Car-
bol-thionin also stains meningococci well.

Isolation.—The organism can be secured for cultivation either
from the purulent matter of the exudate found at autopsy, or from
the fluid obtained during life by lumbar puncture. To obtain this

* “Brit. Med. Jour.,” 1917, 1, p. 377-
t “Berliner klin. Wochenschrift,” 1896, 811.

Morphology 401

fluid Park* gives the following directions: “The patient should lie on
the right side with the knees drawn up and the left shoulder de-
pressed. 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 1 mm. for children,
and larger for adults. The puncture is generally made between
the third and fourth lumbar vertebre. The thumb of the left
hand is pressed between the spinous processes, and the point of the
needle is entered about 1 cm. to the right of the median line and

Fig. 138.—Technic of spinal puncture. The patient is sitting on the edge
ofa chair and is bent forward; the crests of the ilia are indicated by black lines,
and are on a level with the spinous process of the fourth lumbar vertebra;:
the “soft spot” is found just above. The first tube receives the first few drops of
fluid, which are usually blood tinged (Kolmer).

on a level with the thumb-nail, and directed slightly upward and
inward toward the median line. Ata 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 ina stream. If the needle meets
a bony obstruction, withdraw and thrust again rather than make
lateral movements. Any blood obscures microscopic examination.
Adults, not too ill, may sit upon a chair or upon the edge of the bed

* “Bacteriology in Medicine and Surgery,” Philadelphia, 1899, p. 364. —
26.

402 Cerebro-spinal Meningitis

while the spinal puncture is made, as shown in Kolmer’s illustration.
The fluid is allowed to drop into sterile test-tubes or vials with sterile
stoppers. From 5 to 15 cc. should be withdrawn. No ill effects
have been observed from the operation.”

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 1 cc.) must be used.

The cocci can also be cultivated from the nasal discharges as
was shown in 6 cases of cerebro-spinal meningitis studied by Weich-
selbaum, and in 18 studied by Scherer. Elser* isolated the organism
from the circulating blood of patients suffering from that form of
epidemic cerebro-spinal fever known as “spotted fever” in which
there is a purpuric exanthema.

Cultivation.—The meningococcus though successfully cultivated
by. Weichselbaum is not easy to cultivate and disdains most of the
usually employed media. It is aérobic. Growth takes place within.
a narrow temperature range 25°-40°C., the optimum temperature
being 37°C. In handling cultures of any kind, great care should
be taken to prevent them from becoming chilled. Cultures that
have been growing well, sometimes fail to continue when taken from
an incubation oven to a cold room for a short time for transplanta-
tion. It develops very slight growths upon agar-agar and glycerin
agar-agar. Growth is better upon agar-agar containing ascitic fluid
containing 1 per cent. of dextrose and upon Léffler’s blood-serum
mixture. According to Goldschmidt, it can grow upon potato,
though most investigators fail to find any development upon this me-
dium. It does not grow in gelatin. It does not grow in plain bouil-
lon but when a little calcium carbonate is added to neutralize any
acid formed, and 1 per cent. of dextrose and some ascitic fluid or
sheep-serum added, the broth becomes an excellent medium. The
cultures are usually scanty and without characteristic features.

Flexnert found that the difficulties of cultivation were greatly
reduced by the employment of sheep-serum water prepared according
to the method of Hiss (sheep-serum 1 part, water 2 parts, sterilized
in the autoclave) and mixed with a beef-infusion agar-agar contain-
ing 2 per cent. of glucose. The quantity of sheep-serum need not
exceed 149 to {0 of the volume of the agar-agar. It is added to the
sterile melted agar, which is afterward slanted in test-tubes and
allowed to congeal. But by far the best medium for the isolation and
cultivation of the meningococcus is slightly alkaline (-+o.5) agar-
agar, containing 1 per cent. of dextrose and 1 per cent. of laked
human blood. (See directions for making the medium in the chap-
ter upon Culture Media.) Upon this medium the cocci rarely
fail to grow, whether taken from the nasal secretions, the cerebro-

* “Tour. Medical Research,” 1906, xiv, 89.
+ “Centralbl. f. Bakt. u. Parasitenk.,”’ 11, 22, 23.
t “Jour. Experimental Med.,” 1907, Ix, p. 105.

Cultivation 403

spinal fluid, the blood of the patient or the purulent exudation upon
or in the removed brain or spinal cord.

As has been remarked, the life of the culture is brief so that daily
transplantation may be required to keep the culture growing. But
once the culture has been isolated, the blood medium may no longer
be necessary, sheep-serum dextrose agar-agar being easier to pre-
pare and just as satisfactory. Experience indicates that the greatest
longevity of a generation of cocci may be secured by the employment
of a medium suggested by. Bordet and Gengou for the cultivation
of Bacillus pertussis, which Roos (“Jour. of Bacteriology,” 1916,
vol. 1, No. 1, p. 67) makes as follows:

1. a. Potato peeled, cut into small pieces and washed for two

hours in running water..........0....00..000 0 eee Loo grams.
b. Water containing 4 per cent. double distilled glycerin free ,
: from acidec'ssccauieo nes eas $40 OOH Ete Kee our edn eae ee 200 C.C.

c. Mix and autoclave for 40 minutes.
d. Allow to stand over-night and strain through cheese-cloth.
2. a. Mix in an Erlenmeyer flask.

Potato extract as made above................ 50 ce.
0.65 per cent. sodium chloride solution......... 150 cc.
Agar-apatixvoacy gue veo aue bee Puy beeen gee 5 grams.

b. Heat in an Arnold sterilizer until the agar-agar is melted. This requires
from one-half to one hour.
. Tube without filtering and sterilize in an autoclave for about 40 minutes.
. When wanted for use, melt the medium, cool to about 45°C., and then add
5 per cent. of sterile, defibrinated horse’s blood.

Upon this medium Roos succeeded in keeping the meningococcus
alive for as long as four weeks.

Colonies——When grown upon blood agar-agar plates, the col-
onies develop only upon the surface appearing larger or smaller
according to circumstances. When the culture is pure and the
colonies not crowded, they may attain a diameter of two or three
millimeters. They are uniformly creamy white and look soft. Col-
onies close together become confluent When touched they are
found to be viscid. Small colonies viewed under a low-power lens
appear regularly rounded, finély granular or transparent. The
colonies are usually colorless by transmitted light, but may be uni-
formly slightly yellowish.

Vital Resistance.—The organism is soon killed by drying, by
exposure to the sun, and by quite moderate variations of tempera-
ture. It succumbs to very high dilutions of most germicides in a
very short time. i

The thermal endurance of the organism is very slight. It is
lulled in five minutes at 60°C.

-Agglutination.—When animals are immunized by repeated in-
jections of the Diplococcus intracellularis, their blood-serum and
body-juices become agglutinative. Such serums carefully titrated
and kept in the laboratory are indispensible for the identification
of the coccus in fresh culture. The serums have an agglutinating
power that varies from 1:500 to 1: 3000.

404 Cerebro-spinal Meningitis

Metabolic Products.—The meningococcus breaks up dextrose and
maltose with the production of acids, but has no similar action upon
levulose, saccharose, or inulin. Acid production is unaccompanied
by gas evolution. To determine the acid the coccus may be grown
upon acetic-fluid agar containing the sugar under examination, and
a little litmus or neutral red. ©

No indol is produced, no gelatin-softening, coagulating or other
ferments are formed.

Elser and Huntoon (‘‘Jour. Med. Research,” xv, 1909) give the
following table showing the fermentative peculiarities of the
Gram-negative diplococci.

Strains tested Strains |Dextrose | Maltose Tere oe oe Lactose
Meningococci.......... 200 + + ° ° ° °
Pseudomeningococci.... 6 + + ° ° fo) °
Gonococcus........-... I5 + ° ° Ze) ° °
Micrococcus catarrhalis... 64 ° ° ° ° ° °
Micrococcus pharyngis

SICCUS: aoc aged oa db ared. els 2 + + + + ° °
Chromogenic Group I...| 28 + + + + ° °
Chromogenic Group II. .| 11 + + + ° ° °
Chromogenic Group III. + + ° ° ° °
Jaeger Meningococcus : :

Ritalin sae ores I op + ae an ee
Diplococcus crassus . ;
BWP ale ou idine de neta # odes I + + + + + +

The meningococcus produces an endotoxin. Albrech and Ghon*
were able to kill white mice with dead cultures. Lepierret obtained
a toxin from bouillon cultures by precipitating them with alcohol.

Pathogenesis.—Flexner{ found that in large doses the coccus

was always capable of killing small guinea-pigs and mice when in-

jected intraperitoneally. To achieve this, however, the organisms
should be suspended in sheep-serum water, not in salt solution,
which is an active poison to them.

Bettencourt and Franca§ 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 Lingel-
sheim and Leuchs]|| and Flexner** were more successful. Flexner’s
method was to introduce a hypodermic needle into the spinal canal,
wait until a few drops of cerebro-spinal 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

* “Wiener klin. Wochenschrift, ” IgoI.

} “Jour. de phys. et de path. gén. ’ v, No. 3.
tLoc. cit.

fine Ja f. Hyg. u. Infekt.,” eres Pp. 463.

“Klin. Jahrbuch,” 1906, xv, Pp. 489.
** Loc. cit.

Mode of Infection 405

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 preparations from
the nasal mucosa show many polymorphonuclear leukocytes con-
taining the cocci in a degenerated form. The cocci were not culti-
vated from the nasal exudates.

Mode of Infection.—It is not known by what channels infection
with Diplococcus intracellularis meningitidis takes place. Weich-
selbaum 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 organ-
isms 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 dis-
ease, and that of another child with suspicious symptoms.

Steelf has found what may be a variety of the meningococcus
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.§

Bacteriological Diagnosis.—In cases with the clinical symptoms
of meningitis, the bacteriological diagnosis is of great assistance in
determining the correctness of the diagnosis and the nature of the
infection. It is accomplished by means of the lumbar puncture
(vide supra) and the study of the cerebro-spinal fluid thus secured.
Normal cerebro-spinal fluid is clear, that in meningitis is cloudy. A
few cubic centimeters of the fluid can be used for culture and in-
oculation experiments of as many kinds as are deemed advisable.
The remainder is placed in a tube and whirled in a centrifuge. From
the sediment, smears are made upon slides and stained by various
methods, including Gram’s method. The occurrence of polymor-
phorfuclear leukocytes containing Gram-negative diplococci is diag-
nostic of cerebro-spinal meningitis. The occurrence of poly-
morphonuclear leukocytes and Gram-positive diplococci may mean
Pneumococcus or streptococcus infection. If the chief cells

* “Journal of Infectious Diseases,” 1906, Supplement No. 2, p. 21.
t “Klinisches Jahrbuch,” xv, 1906.

t “Pediatrics,” Nov. rs, 1898.

§ “Journal of Medical Research,” 1909, XX, 377.

406 Cerebro-spinal Meningitis

appearing in the sediment are lymphocytes, tuberculous meningitis
should be thought of and smears stained for tubercle bacilli, and
guinea-pigs inoculated.

Considerable difficulty may be experienced in the identification
of the meningococcus when it is encountered in other locations,
especially the naso-pharynx, because of the presence of other Gram-
negative diplococci with which it may be confused. It is, however,
precisely under these conditions that its identification becomes of
the greatest importance when it becomes necessary to stamp out
an epidemic of the disease. : :

Sanitation. Discovery and Treatment of Carriers.—Epidemics of
cerebro-spinal meningitis occur not infrequently in civil life, but
are much more common and more destructive in military life where
large numbers of young and susceptible individuals from many
different centers of population are suddenly brought together.
Under such circumstances a certain number of sporadic cases may
always be expected, and epidemic outbreaks feared. Although it is
the common experience of those that treat the disease that direct
transmission is rare, and some mystery still surrounds the exact.
mode of infection, the undoubted infectivity of the patients and the
probably infectivity of the “carriers” makes it incumbent upon the
sanitarian to isolate the former at once, and to discover and segre-
gate the latter.

The first consideration in regard to the carriers must be bestowed
upon those that have been in contact with the patients. As, how-
ever, there seem to be sporadic carriers just as there are sporadic
cases, it may be necessary to go farther than merely to consider
the contacts, and even become necessary to examine entire military
organizations that the sporadic carriers may be found and segregated.

I. The detection of carriers is accomplished by swabbing the
naso-pharynx, cultivating the secretions upon appropriate media -
and identifying the meningococci.

1. Swabbing.—West* has invented a simple apparatus for swabbing the naso-
pharynx that has met with favor at many hands. It consists of a glass tube
about 1.5 cm. in diameter and rs or 16 cm. long, bent at the end so as to have its
opening at right angles. Enclosed in this is a cotton swab attached to a wire. —
The whole is sterilized by dry heat. When used, the tube is passed over the
tongue and into the pharynx and by pushing on the wire the swab is thrust out
into contact with the mucous membrane, twisted about and then drawn into the
tube again. For this purpose we prefer a slender stick six inches long, with a
pledget of cotton fastened to one slightly roughened end. Bundles of these,
tied up in paper are sterilized by dry heat, ready for use at anytime. Petri
dishes are prepared arid into each is poured about to cc. of dextrose-blood-agar-
agar, which is permitted to congeal and then kept at the body temperature.

The swab is passed into the nasal passage through the anterior nares, care
being taken not to touch the skin, and thrust back until it touches the posterior
pharyngeal wall, when it is withdrawn with a rapid rotary motion, gathering
up the naso-pharyngeal secretions upon the cotton. The Petri dish is cautiously
opened and the swab applied so as to draw a number of horizontal lines from side

to side, not too close together. The dish is then stood in an incubating oven for
12-18 hours when the colonies will have developed.

*“Jour. Amer. Med. Asso.,” Aug. 25, 1917, vol. LXIX, p. 640.

Sanitation, Discovery and Treatment of Carriers 407

2, Finding the Colonies.—In most cases a limited variety of colonies appears.
It is best to look for those of meningococci with a hand lens along these lines last
traversed by the swab where they are most isolated and least numerous. Any
perfectly round colorless or creamy colony that is transparent and pearly by
transmitted light should be marked for further study.

3. Transplanting the Colonies—With a platum wire each marked colony is
touched. If it prove to be slightly viscid, it is transplanted to sheep-serum
dextrose agar-agar in tubes for further study, and placed in the incubator for
12-18 hours to grow.

4. Identification of the Cultures—When grown many of the cultures can be
rejected on the naked eye appearance as not meningococci. Suspicious growths
are next spread upon slides and stained by Gram’s method. All not proving to
be Gram-negative diplococci are rejected. : :

s. Final Identification of the Organism.—The few cultures remaining may be
meningococci or they may be other Gram-negative cocci—Micrococcus catarr-
halis, Micrococcus flavus, Micrococcus pharyngis siccus, etc. So it now becomes
necessary to apply the final test which is the application of the agglutinating
serum. For this purpose the investigator must be provided with the serum

r “>

Fig. 139.—West tube.

either making it himself or obtainig it from some laboratory where it is made, and
must be acquainted with its agglutinating value for previous titration with known
meningococci.

One cubic centimeter of sterile physiological salt solution is placed in a small
test-tube. With a platinum loop a small quantity of the suspected culture is
picked up, and gently rubbed upon the inner wall of the test-tube just at the
ney of the salt-solution until a uniform suspension of the micro-organism is
made.

With a sterile pipette, the agglutinating serum is removed from the cautiously
opened container, and enough transferred either directly by measurement, or in-
directly after dilution, to each tube to give the appropriate dilution for effecting
the agglutination. One known culture of meningococcus similarly suspended in
salt solution, two tubes being prepared. One receives the agglutinating serum
and acts as the serum control, the other receives none and acts as the salt-solution
control. When all have received the necessary additions, they are stood in an
incubating oven and kept at a high temperature (55°C.) for two hours, when the
results are read. :

The salt-solution meningococcus control tube should still contain a uniform
suspension; the serum meningococcus control tube and such of the others as are

“Meningococci should show fine agglutinations like tiny snow flakes. These
commonly sediment so that the tube should first be observed for its clarity and
then shaken to show the flocculi. Only the tubes showing fine agglutinations
should be regarded as meningococci.

This method requires 48 hours for its completion and requires
two cultivations. As time is an important consideration, and/as
culture media are not always available in large quantities, a method
devised by Olitsky* may be used to advantage. The suspected

* “Tour. Am. Med. Asso.,” 1918, Lxx, No. 8, p. 153.

408 Cerebro-spinal Meningitis

marked colonies are transplanted to tubes containing 1 cc. each of
a medium made as follows:

To z per cent. glucose broth (made from veal infusion and having an acidity
of from o.5-0.7+ phenolphthalein) is added 5 per cent. of unheated, sterile,
clean, normal horse-serum. The medium is then ‘distributed in small tubes
(from 8-ro mm. in diameter, and 9 cm. in length), one cc. being placed in each
tube.

The tubes are then incubated for twelve hours at 37°C. Owing to
the presence of the normal horse-serum in the medium, Micrococcus
flavus, Micrococcus crassus, Micrococcus pharyngis siccus, and an
unclassified Gram-positive bacillus will show firm agglutinations.
As hemoglobin is absent, Bacillus influenze fails to grow. Micro-
coccus catarrhalis grows with a dense turbidity and often shows a
pellicle on the surface. The meningococci on the other hand
grows in a characteristic manner. The fluid becomes slightly
turbid and a slight sediment forms which emulsifies uniformly
when the tube is shaken. The final test, however, must be made by
the agglutinating serum which is added as o.1 cc. of a 1:10 dilution
of a high titre serum. The tubes are then stood in a water-bath
at 37-38°C. for two hours when the characteristic agglutinations
will appear. :

II. The treatment of the carriers is an equally important matter
sometimes fraught with difficulty as they are well and therefore
capable of performing the daily duties and anxious to doso. They
should, however, be carefully segregated, and their naso-pharynges
sprayed with some appropriate disinfecting solution several times
daily, for several days. Then, after an interval of a day when no
spraying is done, the nasal passages should be swabbed and meningo-
cocci sought for. Three negative examinations should suffice to
release them, but an occasional swabbing should be done to see that
they do not relapse into carriers again.

Specific Therapy.—Kolle and Wassermann* carefully studied
antimeningococcus sera for specific opsonins, for bacteriotropic
substances, and for other evidences of favorable therapeutic action,
but came to no definite conclusions. Flexnert 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 re-
place it by the antiserum, of which not more than 30 cc. must be
injected. Several such injections should be made. Tabulations
of the results following the employment of Flexner’s serum show a
large percentage of recoveries.

*Loc. cit.
} “Jour. Experimental Medicine,” 1907, rx, p. 168, and 1908, X, Pp. 141.

Specific Therapy — 409

. According to the investigation of Gordon* there are four types,
I, II, III, IV, of the meningococcus, capable of identification through
their behavior toward their respective agglutinating specific sera.
To treat a case therefore it becomes necessary either to use a serum
specific for that type, as in pneumonia, and make sure that a power-
ful polyvalent serum potent against all the types be employed. As
the determination of the type is more difficult than the determina-
tion of the types of the pneumococci, the polyvalent serum treatment
is that given preference at the present time.

* “Medical Research Committee, Special Reports,” Series No. 3, 1917, 19.

CHAPTER VIII
GONORRHEA

Micrococcus GONORRHG (NEISSER)
Synonyms.—Gonococcus; Diplococcus gonorrhcea; Neisseria gonorrhoea,

General Characteristics—A minute, biscuit-shaped, non-montile, non-spro-
genous, non-liquefying, non-chromogenic, non-flagellate, aérobic, strictly para-
sitic coccus, not stained by Gram’s method, cultivable 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 con-
junctival secretions of gonorrhea and purulent ophthalmia by
Neisser* in 1879.

Bummyf found other cocci closely resembling the gonococcus
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 discharges of gonorrhea
from the beginning until the end of the disease, and often for many
months and even years after recovery from it. After the period
of creamy pus has passed, its numbers are usually outweighed by
other pyogenic organisms. Wertheimt 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, in the acute stage of- the disease.
They become less numerous as the sub-acute stage is reached, and
are much less numerous, and largely extra-cellular in the chronic or
“gleet” stage. They should always be sought for as diagnostic
of gonorrhea, as purulent urethritis is sometimes caused by other
organisms, as Bacillus coli communis§ and Staphylococcus pyogenes.

Morphology.—The organisins occur in pairs. Each pair of young
cocci is composed of two spherical organisms, but as they grow older

*“Centralbl. f. d. med. Wissenschaft,’’ 1879, No. 28.

t‘‘Der Mikroorganismus der gonorrhoischen Schleimhauterkrankungen,”
““Gonococcus Neisser,”’ second edition, 1887.

t “Archiv. f. Gynikologie,’’ 1892, Bd. xu, Heft. 1.

§ Van der Pluyn and Loag, “Centralbl. f. Bakt. u Parasitenk.,’’ Feb. 28, 1895,
Bd. xvu1, Nos. 7, 8, p. 233.

410

Isolation and Cultivation __ “art

the inner surfaces become flattened and separated from one another
by anarrow interval. A pair of the cocci resembles a coffee-bean or
a German biscuit, and is described by the Germans as semmelformig.

The gonococci are small, the length of one of the coffee-bean cocci
being about 1.6 yw, its breadth about 0.8 uw. They are not motile,
nor provided with flagella, and are without spores.

Quite as characteristic as the form of the organism is its rela-
tion to the cells. In most of the inflammatory exudates the gono-
cocci 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. Itmay
not obtain in old lesions.

Staining.—They stain readily with all the aqueous solutions of
the anilin dyes—best with rather weak solutions, but not by Gram’s
method.

Fig. 140.—Gonococci in urethral pus.

The organisms contained in pus can be beautifully shown by
first treating the prepared film with alcoholic eosin, and then with
Léffler’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. Ordinary pus cocci, taking the
Gram’s stain, appear blue-black; the gonococci, taking the counter-
stain, are brown in the former, purplish red in the latter case.

Isolation and Cultivation.—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

”

* “ Archiv. fiir Gynakologie,’’ 1892.

412 Gonorrhea

observed. 'Those upon the surface showed a dark center, sur-
rounded by a delicate granular zone. ana

Glycerin agar-agar stroked with defibrinated human blood,
heated to 55°C. for one-half hour to destroy any bacteriolytic sub-
stances the blood may contain, and to aid in ensuring its sterility,
answers quite well as a medium for starting a culture from an acute
case of gonorrhea, and ascitic-fluid bouillon (x part ascitic fluid and
2 parts bouillon) is an excellent medium for maintaining it and
growing large numbers of the cocci. Cultures grow only at 37°C.

Young* had excellent success with a hydrocele-agar prepared as
follows: , ;

“©The fluid (hydrocele or ascitic) is obtained 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 re-
placed. Collecting the fluid in this way we have very rarely had it contaminated,
often keeping it several months before using it. The fluid is mixed with ordi-
nary 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 hav-
ing 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 canbe 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 mixtures obliquely
coagulated, isolated little gray colonies occur, later becoming con-
fluent 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 occurs in the
water of condensation.

Heimanj found that the gonococcus grows best in a mixture of
1 part of pleuritic fluid and 2 parts of 2 per cent. agar. Wrightt
prefers a. mixture of urine, blood-serum, peptone, and agar-agar.

Wassermann§ used a mixture of 15 cc. of pig-serum, 35 cc. of
water, 3 cc. 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 containing 1 per cent.
of peptone and o.5 per cent. of sodium chlorid, mixed with one-
third to one-half its volume of cyst or ascitic fluid, very satisfactory.

* “Contributions to the Science of Medicine by the Pupils of William M.
Welch,” Baltimore, 1900, p. 677.
t “Medical Record,” Dec. 19, 1688.
t “Amer. Jour. Med. Sci.,”’ Feb., 1895.
§ “Berliner klin. Wochenschrift,” 1897.
|| See “Text-Book of Bacteriology,” by Hiss and Zinsser, 1910, p. 383.
** “Centralbl. f. Bakt. u. Parasitenk.,” June 1, 1898, vol. x11, No. 20, p. 874s

Toxic Products 413

-The gonococcus could be kept alive upon these media for two
months.

Metabolic Products.—Laitinen found that the gonococcus pro-
duces acids in the early days of its development, and alkalies sub-
sequently. The acids are produced only in dextrose broth, no other
sugars being fermented.

Vital Resistance.— Authorities agree that the gonococcus 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 killed in a few minutes at 55°C.

Gonococci are very delicate organisms, unable to resist external
conditions. ‘They cease to grow and soon die out if the temperature
becomes low. They die quickly if dried. They are extremely
susceptible to the action of germicides.

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, 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 urethral pus and epithelial cells is a very simple matter. The
identification of the micro-organisms under other conditions is by
no means easy. ‘Thus, when gonorrhea becomes chronic and the
cocci are no longer taken up by the phagocytes, one may be in
doubt whether Gram-negative diplococci found in a urethral
discharge are gonococci or not. A patient getting over gleet and
wanting to marry desires to know definitely whether gonococci are any
longer present in his urethra or not. Again, when the gonococcus- .
like organisms occur upon the conjunctiva, in the pus taken from
joints, upon the valves of the heart, or in 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 confusion of the gonococcus, the pneumococcus, and Micrococcus
catarrhalis (g.v.) which only careful staining and culture experiments
can solve. The pneumococcus may be readily separated by its
Gram-positive staining, its lanceolate form and capsules, 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 ae
nosis can be made.

The complement fixation test is probably the court of final resort,
but is attended with such great technical difficulty that it can
scarcely be recommended at present.

Toxic Products.—The toxic metabolic products of the gonococcus
appear to be contained within the bodies of the bacteria and dis-
seminated but slightly throughout the culture-media. Christmas,*

* “Ann. de l’Inst. Pasteur,” 1897.

AIA . Gonorrhea

Nicolaysen,* and Wassermann{ 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.1
cc. would kill mice; others, of which 1.0 cc. 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.

When the cocci are injected into the peritoneal cavity of mice, a
purulent form of peritonitis is produced. Injected into the joints
of young rabbits results in purulent arthritis. Applied to the con-
junctiva, conjunctivitis is produced. From all these lesions the
gonococci rapidly die out, and Kendall thinks that it is the toxin and
not the cocci that produces the inflammatory reaction.

There is no doubt but that the gonococcus causes gonorrhea.
Bumm{ and Finger, Gohn and Schlaugenhaufer§ have several times
intentionally and experimentally inoculated gonococci into the
human urethra with resulting typical disease. It is constantly pres-
ent in the disease, and very frequently in its sequela, though it not
infrequently happens that the lesions secondary to gonorrhea are
caused by the more common organisms of suppuration that have -
entered through the surface denudations caused by the gonococcus.

Opinions differ as to whether the gonococci can, with equal facility,
penetrate squamous and columnar epithelium. Their attacks are
usually made upon surfaces covered with squamous epithelium.

Gonococci rarely enter the circulation of human beings and occa-
sion a peculiar septic condition with irregular temperature, aptto
be followed by invasion of the cardiac valves, joints, or other
tissues. P. Kraus|| has twice succeeded in cultivating the gono-
coccus from the blood of patients in the stage of septic
infection.

The deep lesions caused by the gonococcus are, however, numer-
ous, and in Young’s paper (loc. cit.) its widespread powers of pyo-
genic infection are well shown in a collection of the cases recorded
in the literature, and some original observations showing the un-
doubted occurrence of the gonococcus in gonorrhea, ophthalmia
neonatorum, arthritis, tendosynovitis, perichondritis, subcutaneous
abscess, intramuscular abscess, salpingitis, pelvic peritonitis, adenitis,

* “Centralbl. f. Bakt. u. Parasitenk.,’’ 1897, Bd. xxu, Nos. 12 and 13, p. 305.

t “Zeitschrift fiir Hygiene,’ 1898, and “Berliner klin. Wochenschrift,” 1897,
No. 32, P- 685.

t“Die Mikroorganisum des gonorrheischen Schleinhautkrankungen Gono-
coccus,” Neisser, Weisbaden, 1885.

§ “Centralbl. f. Bakt. u., Parasitenk.,” 1894, XVI, 350.

| Berliner klin. Wochenschrift,”’ May 9, 1904, No. 19, p. 494.

Immunization 415

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 irri-
tation as the superficial cells desquamate.

All urethral inflammations, and in gonorrhea all of the inflam-
matory symptoms, do not depend upon the gonococcus. The
periurethral abscess, salpingitis, etc., not infrequently depend upon
ordinary pus cocci, anid the author has seen a case of gonorrhea with '
double orchitis, general septic infection, and endocarditis, in which
the gonococci had no réle in the sepsis, which was caused by a large
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 success-
fully achieved. Wassermann failed altogether; Christmas 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 said that the exact reaction was its most
important feature, as otherwise 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’ incubation 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 rabbits were used for making the
serum, and were intraperitoneally inoculated with 10 cc. 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 ob-
tained when cultures from six to fifteen days old were employed.

*“Yournal Amer. Med. Assoc.,’’ Jan. 27, 1906, XLVI, p. 261.

416 _ Gonorrhea

' 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 pedanen with appar-
ently good results.

Good results in gonorrheal arthritis and in gleet are an claimed
for treatment with gonococcus-vaccines.

* «Tour. Amer. Med. Assoc.,’”’ Jan. 27, 1609, XLVI, p. 261.

CHAPTER IX
CATARRHAL INFLAMMATION

Micrococcus CATARRHALIS (SEIFERT)

General Characteristics.—A small, slightly ovoid, non-motile, non-sporulating,
non-flagellated, non-liquefying aérobic and optionally anaérobic, non-chromo-
genic 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 cultivated in 1890 by .
Kirchner f from 1o cases of an influenza-like affection. It has since
been frequently demonstrated in the exudates from various in-

Mig dete Cae Y P
Bae igi Ke sa : rr (eee

Fig. 141.—Micrococcus catarrhalis in smear from sputum (F. T. Lord; photo by
L. S. Brown).

flammatory conditions of the respiratory tract and conjunctiva,
and seems to be a not uncommon organism of superficial inflamma-
tions. 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

* ¢Volkmann’s klin. Vortr.,”’ Nr. 240.
t ‘‘Zeitschr. f. Hyg.,” Bd. 9.

27 417

418 Catarrhal Inflammation

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. It may
occur singly, though it usually appears in pairs or clusters. Large
numbers are enclosed in the leukocytes or other cells. The spheric
organisms have a diameter of about 1 yw; the ovoid organisms may
measure as much as 1.5 by 2 wu. The relation of the cocci to the
cells seems to have something to do with the course of the inflam-
matory conditions with which they are asso-
ciated. 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.

The organisms are not motile, have no

flagella and do not form spores.

Staining.—The cocci stain by ordinary meth-
ods, but not by Gram’s method.

Cultivation.—The organism can be easily
cultivated, both inthe incubator and at room
temperature, 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 transplantation made after from
four to six days fail to grow; and in the cul-
tures one usually finds many deeply staining,
supposedly living cocci, and many poorly stain-

Fig. 142.—Micrococ- ing, supposedly dead organisms.

Se eae cog, — AGar-Agar.—The culture in general re-
photo by L. S. Brown). sembles that of Staphylococcus albus. When
blood is added to the agar-agar, the growth is
more luxuriant, whitish, and usually consists of closely approxi-
mated colonies which do not become confluent.

Gelatin.—This medium is not liquefied.

Bouillon.—At the end of the first day no growth seems to have
taken place, but at the end of the second day there is a slight cloud-
ing and a meager precipitate. The organism seems to maintain
its vitality somewhat longer in bouillon than in other culture-
media.

Metabolic Products.—No enzymes, no acids, no gases and no toxic
products are known to be formed. Blood corpuscles in the media
are not hemolyzed. (

Pathogenesis 419

Pathogenesis.—The organism seems to be scarcely pathogenic
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, only once succeeded in producinga
fatal infection, by the intraperitoneal injection of 0.4 cc. 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 whooping-cough, and in
pneumonia.

CHAPTER X °
CHANCROID

Bacittus DucREYI

General Characteristics.—A small, ovoid streptobacillus, with rounded, deeply
staining ends, non-motile, non-flagellate, non-sporogenous; aérobic and optionally
anaérobic, 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 cleanliness. It is characterized
by the appearance of a soft reddish papule, which makes its appear-
ance usually upon the genital organs, .rarely upon other parts of the
body, soon after the infection, and soon becomes transformed to an
ugly ulceration 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 organ-
ism whose presence he was able to demonstrate with great con-
stancy, sometimes in pure culture, in the lesions of chancroid, and
which he believed to be the specific organism of the affection. Unnat
later described an organism resembling that of Ducrey, and the later
observations of Krefting,t Peterson,§ Nicolle,|| Cheinisse,** and
Davistt 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 “strepto-
bacillus.” It is very small, short, and ovoid in shape, and occurs
habitually in longer or shorter chains. Each organism measures
about 1.5 X 0.5 w. 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

* “Congres. Inter. de Dermatol. et de Syphilog.,”’ Paris, 1889; “‘Compt.-
rendu,” p. 229.

t“‘Monatschr. f. praktische Dermatologie,”’ 1892, Bd. xiv, p. 485.
t “Archiv. f. Dermatol. u. Syphilol.,’’ 1897, p. 263; 1897, p. 41.
; “Centralbl. f. Bakt.,” etc., 1893, XIII, p. 743. ;
|| “Med. Moderne,” Paris, 1893, Iv, p.-735.
** “Ann. de Dermat. et de Syphil.,”’ Par., 1894, p. 272.
TT ‘Jour. Med. Research,” 1893, Ix, p. 401.
420

Pathogenesis 421

no relation between the cells and the bacilli. As a rule, they are
free, sometimes they are inclosed in leukocytes. The bacilli are not
motile, have no flagella and do not form spores.

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 cultivation of the
organism seems to have been by Benzangon, Griffon and Le Sourd*
upon a culture-medium consisting of rabbits’ blood 1 part, and agar-
agar 2 parts. Davist 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 and
mixed with fresh rabbits’ blood drawn under aseptic precautions,

Fig. 143.—Smear of pus of chancroid of penis stained with carbol-fuchsin
and briefly decolorized by alcohol. XX 1500 (Davis). (Photomicrograph by
Mr. L. S. Brown.)

in the proportion of two-thirds agar to one-third blood, and slanted
while in a fluid state. Ata later period tubes of rabbits’ blood-serum
_ uncoagulated, also rabbits’ blood bouillon, one-third blood to two-
thirds bouillon, were used, and gave equally satisfactory results.
By employing small tubes of freshly drawn human blood, pure cul-
tures 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 blood apparently inhibiting to a certain
extent the growth of extraneous organisms.’

No growth takes place upon ordinary culture-media under either
» aérobic or anaérobic conditions.

Cultures are best obtained by puncturing an unopened bubo iat
a sterile needle and planting the pus directly and immediately upon
the special medium which should have been warmed in the incubator

* «Ann. de Dermat. et de Syphilog.,”’ 1901, TI, p. 1.
Loc. cit. ‘

422 Chancroid

so that the pus is not chilled. In this way pure cultures which are
difficult to get from the soft sore itself, may be secured.

Colonies.—The colonies appear upon the appropriate media in
about twenty-four hours, and attain their complete development in
about forty-eight hours. They are at first round bright globules,
and later become grayish and opaque. They measure 1 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.

Fig. 144.—Culture from ulceration on monkey resulting from inoculation of
culture from a case of chancroid of finger, first generation. Stained with carbol-
fuchsin and briefly decolorized by alcohol. Culture of twenty-four hours’
lah rabbit’s bouillon. XX 1500 (Davis). (Photomicrograph by Mr. L.S.
Brown.

Vital Resistance.—The organisms seem to possess little vitality,
their life in artificial culture being limited to a few days. Fre-
quent 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 chancroidal 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,
II, p. 209. :

’

CHAPTER XI
ACUTE CONTAGIOUS CONJUNCTIVITIS

Tue Kocu-WEEKS BAcILLuUs

General Characteristics——A minute, slender bacillus, non-motile, non-flagel-
lated, non-sporogenous, non-liquefying, non-chromogenic, aérobic, and optionally
anaérobic, staining by the ordinary methods but not by Gram’s method, sus-
ceptible 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 characteristics, and es-
pecially 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 investigation
of acute contagious conjunctivitis was made by Robert Koch,*
when in Egypt investigating a cholera epidemic. While in Alex-
andria he examined the secretions from 50 cases of conjunctivitis,
finding the gonococcus, or an organism closely resembling it. Ina
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 experiments
are mentioned.

The organism was observed from time to time, but no serious
consideration seems to have been devoted to it until Weeks+ pub-
lished an account of what seemed to be the identical organism, which
he not only observed, but also cultivated, and eventually success-
fully inoculated into the human conjunctiva. In the same year
Kartulist in Alexandria succeeded in cultivating the same organ-
ism. In 1894 Morax published a brochure in Paris in which he
says that “the disease [which he describes under the name of acute
conjunctivitis] 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 Atiological
Factor in Acute Contagious Conjunctivitis.” §

*Wiener klin. Wochenschrift,” 1883, p. 1550.

tN. Y. Med. Record,’”? May 21, 1887.
t“Centralbl. £. Bakt. u. Parasitenk.,” 1887, p. 289.

§“New York Eye and Ear Infirmary Reports,” Jan., 1895, vol. 1, Part 1,
P. 24,

423

424 Acute Contagious Conjunctivitis

Morphology.—The organism is very tiny and is said to bear
some resemblance to the bacillus of mouse-septicemia. It measures
1 to2 X 0.25 (Weeks). The length is more constant in individuals
found in the pus than those taken from cultures. In cultures the
organisms are longer and more slender. Involution forms of con-
siderable 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.

Fig. 145.—The Koch-Weeks bacillus in conjunctival secretion. Magnified 1000
diameters (Rymowitsch and Matschinsky).

Cultivation.—The organism refuses to grow upon any of the
ordinary culture-media. Weeks found, however, that if the per-
centage of agar-agar used was reduced to 0.5 per cent., growths

' could be secured by incubation at 37°C., and successful transplanta-

tions carried on to the sixteenth generation. 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 agar 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

The Morax-Axenfeld Bacillus 425

patch is noticeable; this is of a pearly color and possesses a glisten-
ing 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 organ-
ism 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 organism in
intimate association with a larger club-shaped bacillus, which
was regarded as the pseudo-diphtheria 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 Axenfeld{ pre-
sented to a congress in Heidelberg cultures of the same bacillus that
he had isolated from 51 cases of what he called ‘ Diplobacillen-
conjunctivitis” that occurred a few months before as an epidemic
in Marburg. De Schweinitz and Veasy,t Alt§ 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 dis-
tributed 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 secre-
tion, and to secure the micro-organism either for microscopic ex-
amination or for cultivation recourse must be had to minute flakes
of grayish mucus that collect upon the caruncle. :

Morphology.—The bacillus is small, commonly occurs in pairs
or chains. It measures approximately 2 uw in length. It is not
motile, has no flagella, and forms no spores. It is somewhat pleo-
morphous. 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 Léffler’s blood-serum mixture.
The colonies appear in twenty-four hours at 37°C. The blood-
serum is almost immediately liquefied, s0 that the growing colonies
*Ann. de l’Inst. Pasteur,” June, 1896; “Ann. d’Oculist,” Jan., 1897.
} “Heidelberg Congress,” 1896; “‘Centralbl. f. Bakt.,” etc., 1897, XXI.

{t “Ophthalmological Record,”’ 1899.
_ § “Amer. Jour. of Ophthalmology,” 1898, p. 171.

426 Acute Contagious Conjunctivitis

appear to be sinking into the medium after thirty-six hours. The
entire tube of medium may eventually be liquefied.

Fig. 146.—Morax-Axenfeld .diplobacillus. Smear taken from conjunctiva
(Brown Pusey). ‘

Upon agar-agar containing serum, grayish-white colonies. of
small size, resembling colonies of gonococci, are formed. Growth
is slow. Bouillon is slowly clouded.

Fig. 147—The Morax-Axenfeld diplobacillus of conjunctivitis. Magnified
ooo diameters (Rymowitsch and Matschinsky). .

Pathogenesis.—The pathogenic and specific nature of the diplo-
bacillus was made clear by Morax, who produced the disease in
man by placing a pure culture upon the human conjunctiva.

Zur Nedden’s Bacillus 427

ZUR NEDDEN’sS BACILLUS

This bacillus was the only organism that Haupt* was able to
isolate from a neuroparalytic with confluent peripheral ulcera-
tions of the cornea. It seemed to be identical with an organism
that zur Nedden had found previously in a case of corneal ulcera-
tion in the clinic at Bonn.

Morphology.—It is a tiny bacillus, less than 1 uw 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 yellowish 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 infrequently
make their appearance as excitants of conjunctivitis. The most
frequent of these being 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, meningococct, colon bacilli, Bacillus pneumoniae
(Friedlander), and other organisms have occasionally been found
and appear to be responsible for a few cases of conjunctivitis.

*“Tnaugural Dissertation,’ Bonn, 1902.

_ CHAPTER XII
DIPHTHERIA

Bacittus DreHTHERIe (KLEBS-LOFFLER)

Synonyms.—Bacterium diphtherie; Corynebacterium diphtherie, Klebs-
Loffler bacillus. ;

General Characteristics—A non-motile, non-flagellate, non-sporogenous,
non-chromogenic, non-aérogenic, non-liquefying, aérobic, 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 Léfflert succeeded in
isolating and cultivating it. The organism is now known by both
their names, and called the Klebs-Léffler bacillus.

Morphology.—The bacillus is about the length of the tubercle
bacillus (1.5—-6.5 4), but about twice its diameter (0.4-1.0 mu), hasa
slight curve similar to that which characterizes the tubercle bacillus,

Fig. 148.—Westbrook’s types of Bacillus diphtheria: a, c, d, Granular types;
a}, c1, d', barred types; a”, c?, d®, solid types. XX 1500.

and has rounded and usually clubbed ends. It does not form |
chains, though two, three, and rarely four individuals may be found
conjoined; usually the individuals are separate from one another.
The bacillus has no flagella, it is non-motile, and does not form spores.
* “Verhandlungen des Congresses fiir innere Med.,” 1883.
tT “Mittheilungen aus dem kaiserlichen Gesundheitsamte,” 2.
428

Cultivation 429

Fig. 149.—Bacillus diphtherie, five Fig. 150.—Bacillus diphtherie, same
hours at 36°C. This shows only solid culture, eight hours at 36°C. This also
‘staining forms. shows solid forms, many’ of them with

parallel arrangement.

Fig. 151.—Bacillus diphtherie, same Fig. 152.—Bacillus diphtherie, same
culture, twelve hours at 36°C. Thé culture, fifteen hours at 36°C. The
bacilli stain faintly at their ends, and in bacilli stain more unevenly and the gran-
some small granules are seen at the tip of ules are larger.

the faintly stained portions.

Fig. 153.—Bacillus diphtherie, same Fig. 154.—Bacillus diphtheriz, forty-

culture, twenty-four hours at 36°C. This eight hours at 36°C. This is the same
shows clubbed and barred forms as well as bacillus as in the preceding figures, but
franular forms. - At the lower part of the from a culture where the colonies were
eld is a paired form which shows the discrete. It shows long filamentous forms.

characteristic clubbing of the distal ends. ook
(Photomicrographs by Mr. Louis Brown. The magnification is the same in all—X 2000.

ie, Of the preparations were] made from growth on blood-serum.) (Francis P. Denny, in
Jour. of Med, Research."’) :

430 Diphtheria

Distinct polar granules, Babes-Ernst granules, can be defined at the
ends of the bacilli by special methods of staining. Occasional
branched forms are observed, though Abbott and Gildersleeve*
do not regard branching as a ‘phase of the normal development
of the organism and do not find it common upon the standard
culture media. 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 enlarged and club shaped, some
greatly elongated, with both ends similarly and irregularly expanded.
Westbrook, Wilson and McDaniel} have found it convenient
to describe three chief types of the diphtheria bacillus as it occurs
in twenty-four-hour-old cultures on Léffler’s blood-serum, sent to
the laboratory for diagnosis.. The classification places all types in
three general groups: (a) granular, (b) barred, and (c) solid or’
evenly staining forms. Each group is subdivided into types based
on the 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; (b) 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 obsevers called ‘pseudo-diphtheria bacilli,” may cause
severe clinical diphtheria. Solid types may sometimes be re-
placed by granular types when convalescence is established and
just before the throat is cleared of diphtheria-like bacilli.
Staining.—The bacillus can most readily and most character-
istically be stained with Léffler’s alkaline methylene blue:

Saturated alcoholic solution of methylene blue......... 30°
I :I0,000 aqueous solution of caustic potash............ 100

Emery prefers Manson’s borax methylene blue. A stock solu-
tion which keeps well is prepared by dissolving 2 grams of meth-
ylene blue and 5 grams of borax in roo cc. of water. This is diluted
with from five to ten times its volume of water for ordinary use.

The small dark-staining dots at the poles of the bacilli, the so-
called metachromatic or Babes-Ernst{ granules were at first thought
to be sporogenic granules. Later, when it became definitely known
that the organism produced no spores, their presence was thought
to be significant of virulence and special pains were taken to define
them by special methods of staining. An aqueous solution of dahlia
recommended by Roux for the purpose is made and used as follows:
Two solutions are prepared:

*“Centralbl. f. Bakt.,”’ etc., Dec. 18, 1903, Bd. xxxv, No. 3. - :

1 ‘Trans. Assoc. Amer. Phys.,’’ 1900; Trans. Amer. Public Health Asso.,
1900; Jour. Boston Society of the Medical Sciences, 1900 Iv, 75.

t “Zeitschrift fiir Hygiene,” 1880, v.

Cultivation 431

J, Dahliaviolet................. t II. Methylgreen................ I
Alcohol (90 per cent.)........ Io Alcohol (90 per cent.)....... Io
Water to..... ccc eee eee 100 Water to......... 0.0.0.0 0 ee 100

Of the solutions, one part of I is mixed with three of II and the fixed spread
stained for two minutes without warming.

The Neisser* method of staining the diphtheria bacillus, to show
the metachromatic granules, is as follows: -

The prepared cover-glass is immersed for from two to three
seconds in

Alcohol (96 per cent.)..............4. Larner tine 20 parts
Methylene blue........0...000000 000.0 I part
Distilled wateric.c. ain nan vou senshyxecawee ve oe 950 parts
Acetic acid (glacial)...........0...0..... 0000000 ee 50 parts

Bismarck brown...........00.0.. 00000 cece eee I part
LRERRLER GET OREEREL ERGO BES 500 parts

Park} 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.

The bacilli stain well by Gram’s method, which is excellent for
their definition in sections of tissue, though Welch and Abbott found
that Weigert’s fibrin method and picrocarmin gave the most beau-
tiful 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. It is almost purely aérobic. It grows at tem- .

peratures ranging from 20°C. to 40°C., the optimum being 37°C.
To secure it a sterile swab or a platinum loop is introduced into
the mouth of a patient suffering from diphtheria, and brought into
contact with the false membrane, after which it is immediately rub-
bed over the surface of a tube of Léffler’s blood-serum mixture.
After twelve to eighteen hours in the incubator, the diphtheria bacilli
will usually be found to have outgrown all other micro-organisms, and
appears in scattered, rounded, cream-colored colonies or as a conflu-
ent surface growth. Transplantation to other media for further
study in pure culture can usually be effected by transplanting a
colony.

Colonies.—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. Upon agar-agar and glycerin agar-agar the
colonies are slower to develop, larger, more translucent, without the

* “Zeitschrift fiir Hygiene,” 1897, XxIv, 443.

{ “Bacteriology in Medicine and Surgery,’ 1900.

432 Diphtheria

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. If blood corpuscles be suspended in the agar-agar,
a narrow zone of hemolysis occurs about each colony. The colonies
that develop upon Léffler’s blood-serum mixture are rounded,
yellowish-white, good sized and more or less confluent when closely
approximated. They are smooth, moist and shining on the surface.

a b : Cc

Fig. 155.—Diphtheria bacilli (from photographs taken by Prof. E. K. Dun-
ham, Carnegie Laboratory, New York): a, Pseudobacillus; 5, true bacillus; c,
pseudobacillus.

They are with difficulty differentiated from those of Bacillus hof-
manni, the pseudo- diphtheria bacillus.

Gelatin.—The growth in gelatin puncture is scanty, not char-
acteristic, and consists of small spheric colonies along the line of
inoculation. The gelatin is not liquefied.

Agar-Agar.—Cultures upon the surface of agar-agar slants are
usually meager when contrasted with those upon Léffler’s blood-
serum mixture, and may be whitish in color. They consist of dis-
crete and confluent whitish colonies devoid of differential qualities.
The oftener the organism is transplanted to fresh agar-agar, the
more luxuriant its growth becomes. The growth is rapid and lux-
uriant upon glycerin agar-agar.

Bouillon 433

Bouillon.—When planted in bouillon a distinct, whitish, granular
pellicle forms upon the surface of the clear medium. ‘The pellicle
appears quite uniform when the tube or flask is undisturbed, but it
is so brittle that it at once falls to pieces if disturbed, the minute
fragments slowly sedimenting and forming a miniature snow-storm
in the flask or tube. When dextrose is added to the bouillon the
organism causes a diffuse cloudiness of the medium, but, not being
motile, soon settles to the bottom in the form of a flocculent precipi-

Fig. 156.—Bacillus diphtheria; colony fenentaetour hours old, upon agar-agar
X100 (Frankel and Pfeiffer).

tate which has a tendency to cling to the sides of the glass, and leave
the bouillon clear.

No fermentation occurs in bouillon to which sugar is added, though
acids are soon formed by which the growth is checked. If, how-
ever, the quantity of sugar be too small to check the growth, the
acidity gives place to increasing alkalinity at a later period.

Blood-serum.—Thé bacillus grows upon blood-serum.

Léffler* 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—Léffler’s blood-serum
mixture:

Blood-serum (of sheep or calf).................000005 rage 33
Ordinary bouillon + 1 per cent. of glucose............... I

This mixture is filled into tubes, coagulated, and sterilized like
blood-serum, and is one of the best known media to be used in con-
nection with the study of diphtheria.

_ Material from the infected throat can be taken with a er or
platinum loop and spread upon the surface of several successive
tubes of Léffler’s blood-serum media. Upon the first a confluent

* “Mitt. a. d. Kais.-gesundheitsamt,”’ 1884, IL
28

434 Diphtheria

growth of the bacillus usually occurs; but upon the third, scattered
cream-white colonies suitable for transplantation can usually be
found. .

' 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.

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.
Duboist 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 pure culture upon the blood-serum mixture to which it is added.
The bload-serum is not liquefied or otherwise visibly changed.

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
diphtherie. The milk is not coagulated. Litmus milk is useful
for detecting the changes of reaction brought about. Alkalinity,
which at first favors the development of the bacillus, is soon replaced
by acidity that checks it. When the culture becomes old, the reac-
tion may again become strongly alkaline. This variation in reac-
tion seems to depend entirely on the transformation of sugar in the
media.

Vital Resistance.—As the diphtheria bacillus does not form spores,
it possesses very little vital resistance and is delicate in its thermic
sensitivity. It grows slowly at 20°C., rapidly at 37°C., and ceases
to grow at about 40°C. It is killed when exposed to 58°C. for a
few minutes. Besson states that when dried in fragments of false
membrane it resists high temperatures and has been found alive
after exposure to 100°C. for an hour. Drying quickly destroys it,
but if organic matter be present it may remain alive a long time.
Roux and Yersin were.able to keep the bacilli alive in a piece of dry
pseudo-membrane, kept in the dark, for five months.

Reyes 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 alive seventy-five to one hundred and five
days in dry air, and one hundred and twenty days in moist air.

* “Centralbl. f. Bakt. u. Parasitenk.,’’ Sept. 24,1897, Bd. xx11, Nos. 10 and 11
+ “Seventeenth Annual Report of the Department of Health and Charities,”’
Indianapolis, Ind., 1907.

Metabolic Products 435

The organism is highly susceptible to disinfectants except when
dried in false membrane.

Metabolic Products.—The diphtheria bacillus forms acids (lactic
acid?) in the presence of dextrose, galactose, levulose, maltose,
dextrin and glycerin. It also forms acids in meat-infusion bouillon,
probably because of the muscle sugars it contains. In the absence
of sugars it produces alkalies. It is unable to evolve gas from any
carbohydrates. It does not coagulate milk; does not liquefy
gelatin or blood-serum.

Palmirski and Orlowski* assert that the bacillus produces indol,
but only after the third week. Smith,t however, found that when
the diphtheria bacillus grew in dextrose-free bouillon no indol was
produced.

Toxin.—The earliest researches upon the nature of the poisonous
products of the diphtheria bacillus seem to have been made in 1887
by Loffler,f 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 belong 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 de-
generation 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 hind quarters. The effect of the poison
was slow and death took place days or weeks after injection, some-
times 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 mechan-
ically carried down by calcium chlorid. Brieger and Frinkelll|
confirmed the work of Roux and Yersin, and concluded that the
poison was a toxalbumin. Tangl** 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 Ehrlichff
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 harmless for animals—or at least not fatal
tothem. 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 toxins or toxoids and

*“Centralbl. f. Bakt. u. Parasitenk.,’”? March, 1895.
dcGntait Macken pos
§ “Ann. de l’Inst. Pasteur,’’ 1888-1889.
|| Berliner klin. Wochenschrift,”’ 1890, 11-12.

** “Centralbl. f. Bakt.,’’ etc., Bd. xI, p. 379.

TT “Klinisches Jahrbuch,”’ 1897.

436 Diphtheria

the poisonous quality is lost though the power of combining with
antitoxin remains.

The toxin is extremely poisonous, and a filtered bouillon con-
taining it may be fatal to a 300-gram guinea-pig in doses of only
0.0005 cc. 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 Uschinsky, 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.

Pathogenesis.—The Bacillus diphtherie is pathogenic for man,
monkeys, guinea-pigs, rabbits, dogs, cats, cows, and horses. Spar-
rows, pigeons and fowls are susceptible to experimental infection;
ratsandmiceareimmune. Spontaneous or natural infection is pretty
well limited toman. The effects of artificial experimental infection
vary with the avenue of infection, the quantity of culture and its
virulence.

1. Subcutaneous inoculation in rabbits and guinea-pigs is usually
fatal in from seventy-two hours to five days. The animal suffers
some rise of temperature in twelve to twenty-four hours, soon is
depressed, weak, loses flesh, remains quiet and dies. At the seat
of infection there is a swelling caused by combined edema, hemor-
rhage and fibrinous exudation. If the culture be of feeble viru-
lence so that death does not occur, this area sloughs, and then
heals slowly.

2. Intraperitoneal and Intrapleural Infection.—This is not so
serious in its results as might be supposed. Some animals recover
from doses that might be fatal under the skin. Death does not
occur until after a week or twelve days. Fluid of slightly turbid
character with flakes of fibrin is found in the peritoneum.

3. Mucous Membrane Inoculations.—When implanted upon the
scarified surface of the mucous membranes, the bacillus causes the
formation of a fibrinous and necrotic pseudo-membrane. Such con-
ditions may recover or death may follow after some days.

In all cases the bacilli remain fairly well-localized at or near the
seat of inoculation and only rarely invade the blood. Death and
‘ness result from toxemia, not from bacteremia.

When examined post-mortem, the liver is found to be enlarged and

sometimes shows minute whitish points, which upon microscopic ... .

examination prove to be necrotic areas in which the cells are com-

pletely degenerated, and the chromatin of their nuclei scattered

about in granular form. Similar necrotic foci, to which attention
* “Soc. de Biol. Paris,’” 1903 No. 25.

Pathogenesis , 437

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 adrenals
enlarged and hemorrhagic. The kidneys show parenchymatous
degeneration. :

Roux and Yersin found that when the bacilli were introduced
into the trachea of animals, a typical pseudo-membrane was
formed, and that diphtheritic palsy sometimes followed.

Diphtheria in man is characterized by a pseudo-membranous in-
flammation of the mucous membranes, particularly of the fauces,
though it may occur in the nose, in the mouth, upon the genital
organs, or upon wounds. Williamst has reported a case of, diph-
theria of the vulva, and Nisot and Bummf{ 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 pres-
ence and development of the bacilli upon the diseased mucous mem-
brane, but is accompanied by a serious intoxication resulting from
the absorption from the local lesions of a poisonous metabolic product
of the bacilli. The bacilli are found only in the membranous exuda-
tion, 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. Diphtheria bacilli were first .
found in the heart’s blood, liver, spleen, and kidney, by Frosch.§
Kolisko and Paltauf|| had already found them in the spleen, and
other observers in various lesions of the deeper tissues and oc-
casionally in the organs. In the blood and organs it is commonly
associated with Streptococcus pyogenes and sometimes with other
_ bacteria. While present in nearly all of the inflammatory sequele
of diphtheria, the Klebs-Léffler bacillus probably has very little in-
fluence in producing them, the conditions being almost invariably
associated with the pyogenic cocci, either the streptococci or staph-
ylococci. Howard** studied a case of ulcerative endocarditis
caused by the diphtheria bacillus, and Pearce}} has observed it in
1 case of malignant endocarditis, 19 out of 24 cases of broncho-
pneumonia, 1 case of empyema, 16 cases of middle-ear disease, 8
cases of inflammation of the antrum of Highmore, 1 case of in-

’ *“Bull. of the Johns Hopkins Hospital,’” Aug., t901.
{ “Amer. Jour. of Obstet. and Dis. of Women and Children,’’ Aug., 1898.
t “Zeitschrift fiir Geburtshiilfe u. Gynakologie,” 1895, XXXII. ‘<
“Zeitschrift fiir Hygiene,’ etc., 1893, x1, Heft 1. °

.! “Wiener klin. Wochenschrift,”’ 1889.
*“ Amer. Jour. Med. Sci.,”? Dec., 1894.

Tt Jour. Boston Soc. of Med. Sci.,”’ March, 1898.

438 Diphtheria

flammation of the sphenoidal sinuses, 1 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 associated with diphtheria.

A case of septic invasion by the diphtheria bacillus is reported
by Ucke,* who gives a synopsis of the literature of similar cases.
The writer has recently seen a case of double otitis media from the
pus of which pure cultures of the diphtheria bacillus were obtained,
and in which they persisted for many weeks, descending the Eustach-
ian tube with the pus, and scattering over the pharynx. The
patient was thus a dangerous “carrier” of the disease, though not
at all ill.

The disease pursues a variable course. In favorable cases the
patient recovers gradually, the pseudo-membrane first disappearing,
leaving an inflamed mucous membrane, upon which virulent diph-
theria bacilli persist for weeks and sometimes for months. Smith
describes the bacteriologic condition of the throat in diphtheria
as follows: ‘“‘The microscope informs us that during the earli-
est local manifestations the usual scant miscellaneous bacterial
flora of the mucosa is quite suddenly replaced by a rich vege-
tation of the easily distinguishable diphtheria bacillus. Frequently
no other bacteria are found in the culture-tube. This vegeta-
tion continues for a few days, then gradually gives way to another
flora of cocci and bacilli, and finally the normal condition is
reéstablished.

Associated Bacteria.—Streptococcus pyogenes and Staphylococci
pyogenes aureus and albus are, in many cases, found in associa-
tion 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,} it was found that in 26 cases of pseudo-
membranous angina due to streptococci, staphylococci, colon bacilli,
and pneumococci, 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 staph-
ylococci; of these, 25, or 32.89 per cent., died. Twenty cases
showed the diphtheria bacilli and Streptococcus pyogenes, with 6
deaths—3o 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,t there were 94 cases of diphtheria, 46

*Centralbl. f. Bakt. u. Parasitenk.,”’ Original, xtv1, Heft 4, March 10, 1908,
Pp. 292.

+ “Ann. de I’Inst. Pasteur,’’ 1896, p. 387.

t “Jour. Boston Soc. of Med. Sci.,’’ March, 1808.

Pathogenesis 439

cases of complicated diphtheria (29 with scarlet fever, 11 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-Léffler
bacilli were present in the heart’s blood in 4, twice alone and twice
with streptococci. In g cases the streptococcus occurred alone; in
1 case the pneumococcus occurred alone. In the /iver the bacillus
was found in 24 cases, alone in 12 and together with the strepto-
coccus in 12; the streptococcus occurred in 27 cases, alone in 14,
with the Klebs-Léffler bacillus in 12, and with Staphylococcus
pyogenes aureus in 1. Staphylococcus pyogenes aureus occurred
in 4 cases, alone in 3 and associated with the streptococcus in 1.
The pneumococcus occurred alone in 1 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-Léffler bacillus twice, and with Staphylococcus pyogenes
aureus once. Staphylococcus pyogenes occurred twice, once alone
and once with the streptococcus. The pneumococcus occurred
twice alone.

In the kidney the Klebs-Léffler 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. Staphylo-
coccus 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-Léffler 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-Léffler bacillus once. The diphtheria
bacillus occurred alone once.

In the diver, in 10 cases streptococcus occurred alone, in 7 cases
associated with the Klebs-Léffler 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 Klebs-
Léffler bacillus is not apparent. It occurred generally, but not
always, in the gravest cases, or those known as ‘septic’ cases. It

440 Diphtheria

is probable that it. may be due to a diminished resistance of 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-Léffler bacillus was about even, though slightly in
favor of the streptococcus.”

The mixed infections add to the clinical diphtheria the patho-
genic effects of the associated bacteria. The diphtheria bacillus
probably begins the process, growing upon the mucous membrane,
devitalizing it by its toxin, and producing coagulation-necrosis.
Whatever pyogenic germs happen to be present are thus afforded
an opportunity to enter the tissues and add suppuration, gangrene,
and remote metastatic lesions to the already existing ulceration.

Diphtheritic inflammations of the throat are not always accom-
panied 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 suffocation, only a bacteriologic
examination revealing the true nature of the disease.

Lesions.—The pseudo-membrane characterizing diphtheria con-
sists 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 are embedded
in coagulated exudate—fibrin—and show a peculiar hyaline degen-
eration. 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 superficial layer the proc-
ess may descend to the deepest layers, all of the cells being included
in the coagulated fibrin and showing more or less hyaline degenera-
tion. 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 differences 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 with difficulty, leaving an abraded
surface. When the membrane is divulsed during the course of the
disease, it immediately forms anew by the coagulation of the in-
flammatory 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. ,

Flexner} has made a study of the minute lesions caused by bac-

* Ann. de l’Inst. Pasteur,” 1898, p. 210.
t “‘Johns Hopkins Hospital Reports,” v1, 259.

Specificity A4I

terial toxins and especially of the diphtheria toxin, and Council-
man, Mallory, and Pearce,* of both gross and minute lesions, that
the thorough student should read.

Specificity Herman Biggs,} in an interesting discussion of the
occurrence of the diphtheria bacillus and its relation to diphtheria,
came to the following conclusions: ,

1. “When the diphtheria bacillus is found in healthy throats,
investigation almost always shows that the individuals 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 diphtheria bacilli are
found are to be regarded from a sanitary standpoint in exactly
the same way as the cases of true diphtheria.

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
clinica] standpoint as diphtheria, but bacteriological examination
shows that some other organism than the Klebs-Léffler bacillus is
the cause of the process.”

Any skepticism of the specificity of the diphtheria bacillus on the
author’s part was dispelled by a somewhat unique experience.
Without having been previously exposed to diphtheria while ex-
perimenting in the laboratory the author accidentally drew a living
virulent culture of the diphtheria bacillus through a pipet into his
mouth. Through carelessness no precautions were taken to prevent
serious consequences and two days later the throat was filled with
typical pseudo-membrane which private and Health Board bacterio-
logic examinations showed to contain pure cultures of the Klebs-
Léffler bacilli.

Some have been led to doubt the specificity of the diphtheria
bacillus because of the existence of what is called the pseudo-diph-
theria 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§ also found that the morphology of the two organ-
isms was continually different when they were grown upon the
same medium for the same length of time, and that the short pseudo-
diphtheria 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 pseudo-

*“Diphtheria: A Study of the Bacteriology and Pathology of Two Hundred
and Twenty Fatal Cases,” 901.

{ “Amer. Jour. Med. Sci.,” Oct., 1896, vol. xm, No. 4, p. 411.

i“ Archiv Russes de Path.,’’ etc., Aug. 31, 1902.
§ American Public Health Association, 1902.

442 Diphtheria

diphtheria 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 pseudo-bacillus is not pathogenic for
animals.

Contagion.—The diphtheria bacilli, being always present in the
throats of patients suffering from diphtheria, constitute the element
of contagion.

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 dis-
charge 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 from
the sick-room to the outer world. Still more extraordinary 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 ob-
servation must be apparent to all readers, and serves as further
evidence why thorough isolation should be practised during conva-
lescence and after it so long as the patient can be shown to bea
“‘carrier”’ of the infectious agents.

Neumannf 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. Such cases, not, being
segregated, may easily serve to spread the contagion of the disease.

The occasional occurrence of true diphtheria bacilli in the throat
of healthy persons who have been exposed to diphtheria, has been a
stumbling-block to many practitioners uninformed upon bacterio-
logic subjects, who are unable to account for its presence, fail to
realize how rare its appearance under such circumstances really is, .
and hesitate to concede that persons so harboring it are “carriers”
and may spread infection.

Park{ found virulent diphtheria bacilli in about 1 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,

* “Brit. Med. Jour.,”’ Feb. 1, 1896.

+ “Centralbl. f. Bakt. u. Parasitenk.,’’ Jan. 25, 1902, Bd. xxxi, No. 2, p. 41.

{ “Report on Bacteriological Investigations and Diagnosis of Diphtheria, from

May 4, 1893, to May 4, 1894.” “Scientific Bulletin No. 1,”” Health Department,
City of New York. : ;

Bacteriologic Diagnosis 443

until cultures made from their throats show that the bacilli have
disappeared.

Bacteriologic Diagnosis.—It is impossible to make an accurate
diagnosis of diphtheria without a bacteriologic examination.

Such an examination is now within the power of every physician.
All that is required is 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, and a tube of Léffler’s blood-serum-medium. These
are now commonly provided
free of charge by state and
municipal health boards or if
desired, can be bought from
almost any modern druggist.
The swab is introduced into
the throat and applied to the
false membrane, after which
it is carefully smeared over
the surface of the blood-
serum. The tube thus in-
oculated is stood away in an
incubating oven or other-
wise kept at the temper-
ature of 37°C. for twelve
hours, then examined. If
the diphtheria bacillus be
present, a smeary, creamy-
white layer with outlying
colonies will be present.
These colonies, if found by
microscopic examination to be
made up of diphtheria bacilli,
will confirm the diagnosis
of diphtheria. There are Fig. 157—The Providence Health De-
very few other bacilli that partment outfit for diphtheria diagnosis,
oy 0 tapidly upom Punune Ss perce oy omnes

Loffler’s mixture and etched surface on which to write the name
scarcely any other is found and address of patients, etc.

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 required, 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 ismixed with distilled
water, spread on a cover-glass, dried, fixed, stained with methylene
blue, and examined. An abundance of the organisms is usually

444 Diphtheria

found and valuable time is saved preparatory to the use of the
antitoxin.

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-kérper) capable of annulling the effects
of the bacilli or the toxin when simultaneously or subsequently
inoculated into susceptible animals. This substance, held in solu-
tion in the blood-serum of the immunized
animals, is the diphtheria antitoxin. For
the method of preparing see Antitoxins.
The serum may be employed for purposes
of prophylaxis or for treatment.

Prophylaxis.—The serum can be relied
upon for prophylaxis in cases of exposure
to diphtheria infection. In most cases a
single dose of 1000 units is sufficient for
the purpose. The protection thus afforded
does not continue longer than about six’
weeks. The transitory nature of the im-
munity afforded by prophylactic injec-
tions of antitoxin is probably dependent
| upon the fact that the antitoxin is slowly
} eliminated.
| Treatment.—Diphtheria antitoxin is

preferably administered by the hypodermic
| method at some point where the skin

is loose. Some clinicians prefer to inject
into the abdominal wall; some, into the
| tissues of the back. A slightly painful
| swelling is formed, which usually disap-

] pears in a short time.
Ehrlich asserts that a dose of 500 units
is valueless for the treatment of diphtheria,
4 2000 units being probably an average
Fig. 158—Sterilized dose for an adult and roo units for a child.
test-tube and swab for {t is far better to err on the side of admin-
collecting pus and fluids - :
for bacteriologic examina- istering too much than*on that of not
tion (Warren). enough. Forty thousand units have been
administered to a moribund child with
resultingcure. Theadministration 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—

**Deutsche med. Wochenschrift,” 1890, Nos. 49 and 50; “Zeitschrift fiir
Hygiene,” 1892, xt, 1.

Treatment 445

rarely general urticaria. Sometimes considerable local erythema
results. Fever and pain in the joints (serum disease of von Pirquet)
also occur, especially if the patients have been previously treated

_ with horse-serum. In a few cases sudden death, with symptoms

suggesting anaphylaxis (g.v.), has followed the injection.

. Inmoribund cases or those in which for any reason the treatment
is begun late, the first doses should be large and the injection made
into a vein.

Diphtheria paralysis is said to be more frequent after the use of

0 ~ : = :
i 80 85 a0} | 95 oo ros

ago

«LPN A

in LAY M W« A
NED Y AS |
«SRD OPO ANEIOSD
4 Z Pca WAN AN :
» SWAP LETTS

MRS Fig. 159.—Deaths from diphtheria and croup per 100,000 in 19 large cities,
1878-1905 (Park and Bolduan i in Nuttall and Graham-Smith’s ‘* Bacteriology
of Diphtheria”).

antitoxin than in cases treated without it. McFarland* has 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 the early statistics upon the antitoxic
treatment of diphtheria in the hospitals of the world was published
by Welch,t who, excluding every possible error in the calculations,
“showed an apparent reduction of case-mortality of 55.8 per cent.”

A more recent statistical study of diphtheria mortality, with care-

* “Medical Record,” New York, 1897.

t Bulletin No. 38 of the Hygienic Eaporeorys U.S. Public Health and Marine

Hospital Service,” Washington, D. C.; 1907.
t “Bull. of the Johns Hopkins Hospital, ” July and Aug., 1895.

446 Diphtheria

ful comparisons of the pre-antitoxin period with the present anti-:
toxin period, by Park and Bolduan is found in “The Bacteriology of.
Diphtheria” edited by G. H. Nuttall and G. S. Graham-Smith,
London, 1908. The paper is ill adapted to the purpose of quotation
and should be read by those interested in the subject. The chart
shows the diminishing death-rate in 19 large cities between the years
1878 and i905.

Nothing should so impress the clinician as the necessity of begin-
ning 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 dis-
ease yielded a fatality of 27.8 per cent.

On the other hand, it can scarcely be said that any time is foo 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, ap-
parently 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 ee and
in such cases antitoxin must fail.

One disadvantage under which the diphtheria antitoxic serum is
administered both for purposes of prophylaxis and treatment, is
the inability of the operator to find out what may be the already
existing antitoxin content of the patient’s blood. Though it is cer-
tain that existing diphtheria is proof that the patient needs the
remedy, it is by no means certain that all normal persons exposed
to diphtheria in institutions, etc., require it for prophylactic purposes.
Some may already possess enough to defend them and the promiscu-
ous administration of the serum to every child in an asylum, may re-
sult in sensitizing some to the allergizing effect of the horse-serum
without just reason. A means by which some knowledge of the nor-
mal diphtheria-toxin neutralizing quality of the blood of a healthy
individual can be arrived at, has been devised by Schick,* and is now
known as Schick’s reaction. It consists in the intracutaneous ad-
ministration of a minute dose of diphtheria toxin. If the patient’s
blood contains the neutralizing substance, no reaction takes place;
if it contain none, a reddened and tumefied circumscribed area ap-
pears, persists for from seven to ten days and then disappears with
desquamation. W. H. Park uses one-fiftieth of the L+ dose of diph-
theria toxin, injecting it into the skin with a very fine hypodermic
needle. Kolmer prefers to use one-fortieth of the L+ dose.t The

* “Miinchener. med. Wochenschrift,”’ 1913, p. 2605.
t The L-+ dose is the least quantity of diphtheria-toxin that will kill a 250-
gram guinea- pig on the fourth day. For the method of computing it, see
‘Antitoxins.”

ve yy

Bacilli Resembling the Diphtheria Bacillus 447

presence of one-thirtieth of a unit of antitoxin in 1 cc. of the patient’s
blood prevents the reaction. Kolmer*has also made use of the Schick
reaction for the important purpose of determining how long the anti-
toxin serum injected into the patient remains and confers immunity.
When the reaction reappears, the immunity can be supposed to
have disappeared, and the patient again becontes susceptible to the
infection.

A very interesting paper by Park { shows the effect of the intro-
duction of antitoxin upon the death-rate from diphtheria and the
advantages of itsemployment. From it the following table is taken:

“Combined statistics of deaths and death-rates from diphtheria and croup in
New York, Brooklyn, Boston, Pittsburgh, Philadelphia, Berlin, Cologne, Bres-

lau, Dresden, Hamburg, Kénigsberg, Munich, Vienna, London, Liverpool,
Glasgow, Paris, and Frankfort:

Year Population Deaths from diphtheria Deaths per
and croup 100,000
TBQO sacra, weeded 16,526,135 11,059 66.9
TOOT retinal dnwok we teas 17,689,146 12,389 70.0
1802: cacewe Ses dee eres 18,330,787 . 14,200 VTS
1803 vacandonuys dave oe 18,467,970 15,726 80.4
TBOA Ss ilesisnsurond o-oo ESE one 19,033,902 15,125 79.9
BBQ SH savaluenanicssscncs acids 19,143,188 10,657 55.6
T8Q0!s scasscctnanes oe esa taste 19,489,682 ; 9,651 40.5
ISO Fccreciteaes avaewicna Batu ie 19,800,629 8,942 45.2
1809 oe iceeitd nude ease 20,037,918 7,170 35-7
1800; uewaaaweee sane ss 20,358,837 7,256 35.6
TQOOS seaside ood queers 20,764,614 6,791 32.7
LOOTS 12. anaewned Ae ahr 20,874,572 6,104 29.2
TQO2) scsaicavsieaee ssw noasd Se 21,552,398 5,630 26.1
TQO Sic atseectann/ ate had anne 21,865,299 5,117 23.4
1004: fountain ew ht ee 22,532,848 4,917 21.8
TOOSs Sela artettiarmte- x yun ace aa 22,790,000 4,323 19.0

BACILLI RESEMBLING THE DIPHTHERIA BACILLUS—
DIPHTHEROID BACILLI
BacitLus HormManni

The pseudo-diphtheria bacillus, bacillus of Hofmann-Wellenhof,§
Bacillus. pseudo-diphthericus, or as it is now called Baccillus hof-
manni, was first found by Léffler|| in diphtheria pseudo-membranes
and in the healthy mouth and pharynx. Ohlmacher has found it
with other bacteria in pneumonia; Babes, in gangrene of the lung;
and Howard,** in a case of ulcerative endocarditis not secondary to
diphtheria.

Park tt found that all bacilli with the typical morphology of the
diphtheria bacillus, found in the human throat, are virulent Klebs-

* “Phila. Pathological Society,’ Feb. 11, 1915.

t “Journal of the Amer. Med. Assoc.,”’ Feb. 17, 1912, Lvim, No. 7, p. 453.

Introduction of antitoxin treatment. :

§ “Wiener klin. Woch.,”’ 1888, No. 3.

| “Centralbl. f. Bakt. u. Parasitenk.,”’ 11, 105.

* “Bull. of the Johns Hopkins Hospital,’’ 1893, 30.
tt “Scientific Bulletin No. 1,’’ Health Department, city of New York, 1895.

448 Diphtheria

Léffler bacilli, while forms closely resembling them, but more
uniform in size and shape, shorter in length, and of more homo-
geneous staining properties, with Loffler’s alkaline methylene-
blue solution, can with reasonable safety be regarded as pseudo-
diphtheria bacilli, especially if it be found that they produce an alka-
line rather than an acid reaction by their growth in bouillon. The
pseudo-diphtheria bacilli were found in about 1 per cent. of throats
examined in New York; they seem to have no ep to dpb:
theria, and are never sarnlents

Morphology.—This micro-organism bears a more or less marked
resemblance to Bacillus diphtheriz, but differs in certain particulars
that usually make it possible to recognize and identify it. It is
shorter and stouter, is straight, and usually slightly clubbed. It
usually stains intensely, and commonly shows but one unstained
transverse band. There are no flagella and no spores.

* Se “4 f ee
yf ee ‘So cme i
< W 2! ; S. wt S
Fig. 160.—Pseudo-diphtheria bacilli.

Staining.—The organism stains intensely and more uniformly
than Bacillus diphtherie. 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 similarities and morphologic resem-
blances to Bacillus diphtherie. It grows more luxuriantly upon the
ordinary culture-media than B. diphtheriz. The colonies are larger,

less transparent and whiter, as seen upon agar-agar. In bouillon

there is more marked clouding and less marked pellicle formation.
Upon Léffler’s blood-serum the cultures are too much alike to be
easily differentiated.

G. F. Petri* found no substances i in filtrates of cultures of Hof-

a “Jour. of Hygiene,” April, 1905, vol. v, No. 2, p. 134.

Bacilli Resembling the Diphtheria Bacillus 449

mann’s bacillus capable of neutralizing diphtheria antitoxin; he also
found that horses immunized with large quantities of filtrates of the
Hofmann bacillus did not produceany antitoxin to diphtheria toxin.

Cobbett* and Knappt show that there is a chemicobiologic dif-_
ference between the true and pseudo-diphtheria bacilli, in that the
latter 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 independent micro-or-
ganism. Under no circumstances does it produce or can it be
made to produce toxin. Under no circumstances can it be made
to produce acid through the decomposition of sugars.

Pathogenesis.— Dr. Alice Hamilton{ carefully studied 29 organ-
isms, of which 26 corresponded fully with the pseudo-diphtheria
bacilli. They were divisible into three groups: I, Those non-patho-
genic 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, or-
ganisms which form gas in glucose media, produce bacteremia in
guinea-pigs, and are neutralized neither by diphtheria nor by pseudo-
diphtheria 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 irremedi-
able by or deleteriously affected by antitoxic serum may depend
upon one of these organisms. It is also probably one of them that
Councilmdn found in his case of “general infection by Bacillus
diphtherie,” and that Howard encountered in his case of acute
ulcerative endocarditis without diphtheria, from the valves of whose
heart cultures of a diphtheria-like organism not pathogenic for
guinea-pigs was 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. diphtherie 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, | who regarded it as the cause of xerosis conjunctive, having
found it upon the conjunctiva in that disease. It has, however, been

*“Centralbl. f. Bakt. u. Parasitenk.,’’ 1898, XXIII, 395.
{ Jour. of Med. Research,” 1904, XII (N. S., vol. vit), p. 475.
{ * Jour. Infectious Diseases,” 1904, I, p. 690.
§ “Jour. Infectious Diseases,” VII, 1910, 335-
ll “Deutsche med. Wochenschrift,” 1884, Nos. 21, 24.
2 o]
29

450 Diphtheria

so frequently found upon the normal conjunctiva that it can no
longer be looked upon as pathogenic. It is also found upon other
mucous membranes than the conjunctiva; 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, Baumgarten, and:others it appears that Bacillus xerosis is a
harmless saprophyte that is occasionally found upon the conjunctiva.
Happening to be found in xerosis it was accorded undue distinction.
Morphology.—It resembles Bacillus diphtheriz very closely, but
is probably a little shorter. The ends are clubbed, and in them meta-
chromatic bodies are stained by Neisser’s and Roux’s methods.
There is no motility; there are no flagella and no spores.
Cultivation.—Upon Léffler’s medium and other media commonly
used for the diagnosis of diphtheria, the organism grows with so close
resemblance to the Bacillus diphtheriz as to make the differentiation
difficult. Transplanted to other media, it continues to resemble
B. diphtheriz. ,
Chemistry.—The organism is incapable of forming any toxin.
It ferments dextrose and saccharose with the production of acids
and thus differs from Bacillus hofmanni which does not ferment
either, and from Bacillus diphtheria which ferments only dextrose.
Pathogenesis.—The organism is not pathogenic for man and is ©
certainly not the cause of xerosis. It is not toxicogenic and is not
known to be pathogenic for any animal.

CHAPTER XIIT
VINCENT’S ANGINA

VINCENT’S angina is an acute, specific, infectious, pseudo-mem-
branous form of pharyngitis or tonsillitis characterized by the forma-
tion of a soft yellowish-green exudate upon the mucous membranes,
which, when removed, leaves a bleeding surface which becomes an ul-
cer. 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 pseudo-membrane is sometimes quite
distinct, the disease is apt to be mistaken for diphtheria, and may be
differentiated from it only by a bacteriologic examination. When
such an examination is made two apparently different micro-or-
ganisms may be found. The first is the Bacillus fusiformis; the sec-
ond, Spirocheta vincenti.

BaciLtLus Fusirormis (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 Babesf also
described a fusiform bacillus which seems to be somewhat different,
that occurred in a necrotic exudation from a pseudo-membranous—
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 Matzenauer observed similar
organisms in zoma. Fusiform bacilli are, therefore, not infrequently
associated with necrotic processes of various kinds. Similar but

not identical bacilli were found by Babes in the gums of scorbutic
patients.

SPIROCHETA VINCENTI (PLAUT-VINCENT)

Plaut{ and Vincent§ observed 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,

*“Micro-organisms of the Human Mouth.” Philadelphia, 1890.
t “Les Bactéries,” 1884.

t “Deutsche med. Wochenschrift,” 1894, XLIX.
§“Ann. de l’Inst. Pasteur,’’ 1896, 488.

451

452 - Vincent’s Angina

always took faint but uniform coloring, varied in length, and showed
such irregular and non-uniform undulations as to appear more ser-
pentine than ‘‘corkscrew-like.”” They seem never to occur without
associated fusiform bacilli. The writers believe these organisms 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 it.

RELATION OF THE ORGANISMS TO ONE ANOTHER

- We have, in Vincent’s angina, to do with two micro-organisms that

occur in habitual association. Neither was found to be cultivable
by the earlier writers. The spirocheta 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 sus-
ceptible of cultivation. Later, however, reports were made of the
growth of the organisms 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 dif-
ferent stages of the same organism. Tunnicliff* found that in pure
cultures of Bacillus fusiformis, after forty-eight hours, spiral organ-
isms resembling those seen in smear preparations from the original
source were found. From Tunnicliff’s results it would seem as
though Bacillus fusiformis and Spirocheta vincenti are identical
organisms in different stages of their life-history. But the matter
is not yet settled for Krumwiede and Prattt by a different method of
cultivation have apparently obtained B. fusiformis pure—i.e., free
from the spirocheta—have not found any apparent transformation
of the bacilli into spirocheta, and insist that the two are essentially —
different organisms.

Morphology.—The Bacillus fusiformis presents the same appear-
ances, no matter what medium it grows upon. It measures 3 to lou
in length, 0.3 to 0.8 win 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 uniform, the substance usually
being interrupted by vacuoles or fractures, reminding one of those
seen in the diphtheria and tubercle bacilli. According to Tunnicliff,
the bacilli form endospores sometimes situated at the center, but
more frequently toward one end. Krumwiede and Pratt never ob-
served spores. In twenty-four to forty-eight hours filaments are

* “Tour. of Infectious Diseases,’’ 1906, 111, 148.
t“‘Jour. of Infectious Diseases,’’ 1913, XIII, 438.

Relation of the Organisms to One Another 453

seen. These are of the same diameter throughout, and usually con-
tain deeply staining bodies, sometimes round, oftener in bands.
Most of the filaments are made in the strings of bacilli, but some

Fig. 161.—Bacillus fusiformis. Smear from gum in normal mouth. (Ruth
Tunnicliff in ‘Journal of Infectious Diseases.’’)

stain uniformly. Tunnicliff found that about the fourth or fifth
day the spirals made their appearance, sometimes in enormous
numbers. As a rule, they stained uniformly, some showed the

Fig. 162—Bacillus fusiformis. Pure culture grown forty-eight hours anaé-
Rhecally Léffler’s blood-serum. (Ruth Tunnicliff in ‘Journal of Infectious
iseases,””

dark bodies seen in the bacilli and filaments. They had from one
to twenty turns, which were not uniform. The spirals were flexible,
the ends pointed. The spirals persisted in the cultures, at times
for fifty-five days. Krumwiede and Pratt never found spirals in
their cultures.

454 Vincent’s Angina

Neither the bacilli nor the spicals showed any progressive move-
ment, though with the dark-field illuminator they showed a slight
vibratile and rotary movement. No flagella were observed.

Fig. 163.—Bacillus fusiformis. Pure culture grown forty-eight hours anaé-
robically in the fluid of condensation of Léffler’s blood-serum. (Ruth Tunnicliff
in ‘Journal of Infectious Diseases.’’) :

Cultivation.—The organisms were cultivated by Tunnicliff upon
the surface of ascitic fluid agar-agar (1:3) under strictly anaérobic
conditions at 37°C., and by Krumwiede and Pratt, also under strictly

Fig. 164.—Bacillus fusiformis. Pure culture grown four days in ascites broth
(Ruth Tunnicliff in “Journal of Infectious Diseases.’’)

anaérobic conditions, upon ascitic fluid agar-agar and serum agar-
agar. The latter investigators isolated and studied cultures from
fifteen different sources. After two or three days the fusiform bacil-

Pathogenesis 455

lus 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.

Liffler’s Blood-serum Mixture. Agia 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 regains its lost color.

Potato.—No growth.

Bouillon and Dextrin-free Bouillon —No growth.

Glucose-bouillon No growth when more than 1 per cent. of glu-
cose is present. The medium is clouded with some sediment. No
gas was produced in dextrose, galactose or levulose, but gas is some-
times produced in saccharose. All of the carbohydrates gave rise to
acidity of the culture media.

From all of the cultures a somewhat offensive odor is given
off.

Pathogenesis.—Pure cultures of the organisms were inoculated
into guinea-pigs without result. As in Vincent’s angina the throat
always contains staphylococci and streptococci, 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 inflammations 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.

Bacillus fusiformis, with the associated spirals are not confined to
Vincent’s angina, but are found in a variety of other necrotic and
gangrenous affections. Vincent* himself found them in all cases of
hospital gangrene; Veillon and Zuber, } found them in certain cases
of appendicitis; Bernheim and Popischellt in gangrenous laryngitis;
Silberschmidt§ in fetid brochitis; Freejmuth and Petruschky,||

* “Ann. del’ Inst. Pasteur,” 1896, x, 488.
t “Archiv. de med. Exp.,’’ 1898, p. 517.
{ “Jahresb. fiir Kinderheilkunde,” 1898, XLv.

§ “Centralbl. f. Bakt., etc.,’’ 1901, Orig., xxx, 159.
|| “Deutsche med. Wochenschrift,” 1898, Be 232.

456 Vincent’s Angina

Seiner* and others i in noma; way in certain chronic ulcers of

the legs in Gambia.
The complete literature of the subject collected by Beitzke, is

published in the Centralbl. fiir Bakt. u. Parasitenk. (Referata) 1904,
XXXV, p. I.

* “Wiener klin. Wochenschrift,’’ 1899, No. 2.
t “Journal of Medical Research,’’ 1912~13, XXVII, 27.

CHAPTER XIV
THRUSH

Ofprum 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 substance proves to be composed of masses of mycelia with en-
larged epithelial cells and leukocytes. The affection is far more fre-
quent 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 childcen, 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.t 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, Oidium
albicans. Many systematic writers have exercised themselves con-
cerning the exact place in the botanical system in which the organ-
isms should be placed. Thus, Gruby and Heim regarded it as a
sporotrichum; Robin, as an oidium; Quinquaud, as a syringospora;
Hallein called it Stemphylium polymorpha; Grawitz, as Myco-
derma 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 budding, of hyphe and

*See Kehrer, “Ueber den Soorpilz,” etc., Heidelberg, 1883.

} See Behrend, “Deutsche med. Wochenschrift,’”’ 1890.

i “Histoire naturelle des vegetaux parasites qui croissent sur l’homme et
sur les animaux vivants,” Paris, 1853.

457

458 Thrush

mycelial threads into which these grow, and of chalmydospores and
conidia.

The yeast-like elements measure 5 to 6 uw in length and 4 yp in
breadth. They have an oval form and cannot be distinguished from
yeast cells. The mycelia are formed by elongation of these elements,
some of which appear slightly elongate, 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
granules. In the interior of the hyphe conidia-like organs often
appear, and chalmydospores are found. 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 regarding the dif-
ferent elements.

Fig. 165——Oidium (Kolle and Wassermann).

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 for-
mation 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.

Fermentation 459

Potato.—Various in different cases. Often floury.

Milk.—The organism grows very poorly in milk, which is not coagu-
lated or fermented. ;

Fermentation.—The organism utilizes dextrin, mannite, alcohol,
lactose, and glycerin without fermentation.
Saccharose is destroyed without invertin forma-
tion. Glucose, levulose, and maltose are fer-
mented very slowly.

Metabolic Products.—In addition to the
ferments that act upon the sugars, etc., and
soften the gelatin, the organism 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 in-
oculated the scarified vaginas of rabbits with
mixed cultures of pyogenic cocci and oidium
and obtained thrush plaques. The oidium
alone was unable to secure a foothold. Déder-
lein, Grosset, and Stooss all succeeded in pro-
ducing abscesses, sometimes by subcutaneous
injection of the oidium, but usually only when it
was combined with pus cocci. Insuchabscesses
the cocci are killed off by phagocytes, and when
cultures are made only the oidium grows.
Plaut points out that this is exactly the reverse ; -”

: ar Fig. 166.—Oidium
of what happens in artificial cultures of the two ajpicans. Culture in
organisms where the cocci outgrow and kill off gelatin (Hansen).
the oidium.

Intravenous injection sometimes causes generalized oidium infec-
tion, with colonies of the micro-organism in the kidneys, heart-
muscle, peritoneum, liver, spleen, stomach, and intestines. The
central nervous system may also show small foci of the infection.

Immunity.—Roger* and Noissettet were able to immunize ani-
mals against oidium.

* “Compt.-rendu de la Société de Biologie,’ Paris, 1896.
{ “Thése de Paris,” 1898.

CHAPTER XV
WHOOPING-COUGH

BACILLUS PERTUSSIS
Tur BorDET-GENGOU BACILLUS

General Characteristics——A minute, non-motile, non-flagellate, non-sporo-
genic, non-liquefying, non-chromogenic, non-aérogenic, strictly aérobic, patho-
genic bacillus, capable of cultivation upon special media, and pathogenic for
man.

The subacute, contagious, undoubtedly infectious disease of
childhood, characterized by periodic attacks of spasmodic cough and
largyngeal spasm, terminating in a prolonged crowing inspiration ©
and frequently followed by vomiting and prostration, known as
pertussis, or whooping-cough, ‘“Keuchhusten” (German) and
“coqueleuch” (French), has long been subject to bacteriologic
investigation. Deichler, Kurloff, Szemetzchenko, Cohn, Neumann,
Ritter, and Afanassiew have all written upon bacteria which they
supposed to be the causal factors of the disease, but which time has
consigned to oblivion. Koplik* and Czaplewski and Henself de-.
scribed micro-organisms that for some years attracted 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 observations lacked sufficient confirmation to
carry conviction of their importance, and they, too, fell into oblivion.
A still different organism was described by Vincenzi,t 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 fre-
quent 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 Gengoutt described a new organism whose
importance was supported by such weighty evidence as the forma-
tion of an endotoxin sufficiently active to éxplain the symptoms, and
the fixation of complement by the serum of the infected animal.

* “Centralbl. f. Bakt.,” etc., Sept. 15, 1897, xx1, 8 and 9, p. 222.
‘ Tt caw Seas dar meee ee 57, p. 586; “Centralbl. f. Bakt.,”
etc., Dec. 22, 1897, xx, Nos. 22 and 23, p. 641. F
. 1 “Atti ams Accademia di Medicina in Torino,” x1, 5-7; “Centralbl. f.
akt.,”’ etc., Jan. 19, 1898, XXIII, p. 273.
§ “Deutsch. med. ‘Wochengchrift,” 1897, 830.
|| “Zeitschrift fiir Hygiene,” etc., 1901, XXXVI, 193.
** “Tour. Infectious Diseases,’’ 1906, III, 1.
tt “Ann. de l’Inst. Pasteur,” 1906, xx, 731.
460

Morphology 461

This organism, therefore, presents itself as sufficiently meritorious
to maintain the field for the present.

Morphology.—The organisms, as found in the sputum, occur as
very minute ovoid rods of about the same size as the influenza
bacillus. They measure approximately 1.5 » in length by 0.3 win
breadth. They do not remain united as chains or rods, but separate
as individuals. They are somewhat pleomorphous, yet the varia-
tions are not considerable. 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

' Fig. 167—The Bordet-Gengou bacillus of whooping-cough. Twenty-four
hour-old culture upon solid media containing blood (Bordet-Gengou).

the middle. They do not stain by Gram’s method. The discoverers
recommend that the organism be stained with—

eed gener Dissolve and add 500 of 5 per cent. aqueous
Wateti.s snc... gon) carbeliencid. “Atter two days flter,

Isolation—The organisms occur in almost pure cultures in the
whitish expectoration which escapes from the bronchi in the begin-
ning 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. Potato chips............... ep¥ vs aL ‘i .
4 per cent. aqueous glycerin........ Wiad off the fluid.

II. Potato extract (made as above).. 50 cc. Boil, dissolve,’ filter, and
0.6 per cent. aqueous NaCl...... I50 cc. tube; 2 to 3 cc. to a
Agar-agar.... 0... eee eee 5 gm. tube.

human) blood before cooling to the point of coagulation. Permit the tubes
to solidify in the oblique position.

462 Whooping-cough

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 aérobe. 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.——The organism is incapable of producing
either acids or gases from carbohydrates. It produces no indol and
has no recognized enzymes.- 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 im 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 1 to 2 cc. usu-
ally killed a rabbit about twenty-four hours after intravenous injec-
tion. Subcutaneous injection caused a necrosis without suppuration
and without constitutionalsymptoms. Small quantities of the toxin
placed in the rabbit’s eye caused local necrosis with little inflam-
matory 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 ba-
cillus failed to produce the disease. Klimenko, t however, succeeded
in infecting monkeys and pups by intratracheal introduction of
pure cultures. After a period of 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 contact with
those suffering from the infection. Frankelt obtained similar results.

The differences between the Bordet-Gengou bacillus and the in-
fluenza bacillus are not great. In size, mode of occurrence, grouping
and staining there is much resemblance between the two. Cultur-
ally, however, they differ because the influenza bacillus grows best
upon hemoglobin or blood agar-agar, which is less adapted for the
isolation of the Bordet-Gengou bacillus than the culture-medium
recommended above, upon which the influenza bacillus does not
grow well. Further, we have as differential features 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.

* Bordet, “Bull. de la Soc. Roy. de Bruxelles,’ 1907.
t “Centralbl. f. Bakt.,” etc. (Orig.), x~vm, 64.
t ‘“Miinchener med. Wochenschrift,’ 1908. p. 1683.

Pathogenesis 463

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 com-
plement when applied to the bacilli; Friénkel and Wollstein,* 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 performing the complement fixation tests.

* “Journal of Exp. Med.,’’ 1909, XI, 41.

CHAPTER XVI
PNEUMONIA
LOBAR OR CROUPOUS PNEUMONIA

Drietococcus PNEUMONIZ (WEICHSELBAUM)

Synonyms.—Micrococcus pasteuri, Diplococcus lanceolatus, Streptococcus
lanceolatus, Streptococcus mucosus, Bacterium pneumoniz, Bacillus septicus
sputigenus. ;

General Characteristics—A minute, spheric, slightly elongate or lancet-
shaped, non-motile, non-flagellate, non-sporogenous; aérobic and optionally
anaérobic, non-chromogenic, non-liquefying diplococcus, pathogenic for man and
the lower animals, staining by ordinary methods and by Gram’s method.

‘The micro-organism, that can be demonstrated in at least 90 per
cent. of cases of lobar pneumonia, which is almost universally ac-
cepted to be the cause of the disease, and about whose specificity
very few doubts can now be raised, is the Diplococcus pneumoniz
or, as it is most commonly called, the puewmococcus, 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. Pasteurt seems to
have cultivated the same micro-organism, also from saliva, in the
same year. Telamon,{ Frankel,§ and Weichselbaum,|] however,
discovered the relation which the organism bears to pneumonia.

Distribution.—The pneumococcus is present in the lungs, sputum,
and-blood in croupous pneumonia. It is also found in the saliva
of a large number of healthy persons (Parke and Williams**), espe-
cially during the winter months (Longcope and Fox ff), and the inocu-
lation 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 Fliigge to describe it as
Bacillus septicus sputigenus. It is occasionally found in inflamma-
tory lesions other than pneumonia, as will be pointed out below.

. Morphology.—The organism is variable in morphology according
to the conditions under which it is examined. In the fibrinous
exudate from croupous pneumonia, in the rusty sputum, in the

* “National Board of Health Bulletin,’’ 1881, vol. 11.
t “‘Compte-rendus Acad. des Sciences,’’ 1881, XCII, p. 159.
t“Compte-rendus de la Société d’ anatom. de Paris,’ Nov. 30, 1883.
§ “Deutsche med. Wochenschrift,”’ 1885, 31. :
|| ‘Wiener med. Jahrbuch,”’ 1886, p. 483.

** “Tour, Exp. Med.,” Aug. 7, 1905, VII, p. 403.

tt Ibid., p. 430.

464

_ Staining 465

blood of rabbits and mice, and in albuminous liquids generally,
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 fluid culture media
without albumin, it appears more rounded, lacks the capsule and
though it has a pronounced disposition to occur in pairs, not in-
frequently forms chains of five to six members, so that some have
been disposed to look upon it as a streptococcus (Gamaléia). When
grown upon solid media, the capsules are not apparent. The
lanceolate form led Migula* to describe it under the name Bacterium
pnheumonie. .

The organism measures about 1 yw in greatest diameter, is without
motility, has no flagella and forms no spores. _

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. Dead pneumococci are
commonly Gram-negative.

To demonstrate the capsules, the glacial acetic acid method of
Welcht 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 for an instant, poured (not
washed) off, and at once followed by anilin-water gentian violet, in
which the staining continues 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 1 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.

Hiss{ recommended the following as an excellent method of stain-
ing the capsules of the pneumococcis: The organism is first culti-
vated upon ascites serum-agar to which x 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 gentian 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 employed a 5 or ro per cent. solution
of fuchsin or gentian violet (5 cc. saturated alcoholic solution of

*“System der Bakterien,” Jena, 1909, p. 347.

. { “Bull. of the Johns Hopkins Hospital,” Dec., 1892, p. 128.

T Abstract, “Centralbl. f. Bakt. u. Parasitenk.,’? Bd. xxx1, No. 10, p. 302,

arch 24,.1902.. More complete details appear in a later paper in the “Journal
of Experimental Medicine,” v1, p. 338.

466 Pneumonia

dye in 95 cc. 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 preparation is then dried and mounted in balsam.

Hiss found this stain a useful aid in differentiating the pneumo-
coccus from the streptococcus, with which it is easily confounded if
the capsules are not distinct.

Isolation.— When desired for purposes of study, the pneumococcus
may be obtained by inoculating beneath the skin or into the peri-
toneal cavity pneumonic sputum of white mice and recovering the
organisms from the heart’s blood, or peritoneal fluid. Or it may be
obtained from the rusty sputum of pneumonia by the method em-

[ae . = |
k o
i

Fig. 168.—Capsulated pneumococci in blood from the heart of a rabbit; carbol-
fuchsin, partly decolorized. XX 1000.

ployed 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, care-
fully 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,
used a 2 per cent. glucose-agar of a neutral, or, at most, 0.5 per cent.
phenolphthalein acid titer.

“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. . . +

“Thefplan finally adopted [for inoculating the plates] was as follows¥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

* “Jour. Exp. Med.,” Aug. 25, 1905, vit, No. 5.

Cultivation 467

may be made. A small portion of the dilute mixture was poured at a point near
the periphery of the prepared plates. By a slight tilting motion. the fluid was
carefully distributed 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 colonies.”

Cultivation—The organism grows upon all the culture-media ex-
cept 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 because of the metabolic formation of lac-
tic and formic acids. The addition of alkali to the culture-medium
favors the growth of the pneumococcus by neutralizing this acid.
Hiss and Zinsser* advise that the culture-media used for the pneu-
mococcus 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, circum-
scribed, 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 sur-
rounded 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.

Gelatin Punctures.—In gelatin puncture cultures, made with
15 instead of the usual xo per cent. of gelatin, the growth takes
place along the entire puncture in the form of minute whitish gran-
ules distinctly separated from one another. The growth in gelatin
is always meager. The medium is not liquefied.

Agar-agar and Blood-serum.—Upon agar-agar and blood-serum
the growth consists of minute, transparent, semi-confluent, colorless,
dewdrop-like colonies. The medium is not liquefied. Upon
glycerin agar-agar the growth is more luxuriant. The addition of a
very small percentage of blood-serum facilitates growth.

Bouillon.—In bouillon the organisms grow well, slightly clouding
the medium. With the death of the organisms and their sedimenta-
tion, 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.

*“Text-book of Bacteriology,” 1910, p. 356.
Ortmann asserts that the pneumococcus can be grown on potato at 37°C.,
but this is not generally admitted. The usual acid reaction of potato makes
it an unsuitable culture-medium. ‘

468 Pneumonia

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 some-
times be kept alive for several weeks. Hiss and Zinsser* 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-Uffreduzzit 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 nine-
teen 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 temperature
of 52°C. It is highly sensitive to all disinfectants, weak solutions
quickly killing it.

Neufeld§ found that the pneumococci was extremely susceptible
to the action of bile, and that when 0.5 per centro per cent. of
rabbits’ bile was added to cultures, the organisms began to disappear
‘at once and all disappeared in twenty minutes leaving the culture
sterile.

Cole|| found that when fresh culture of pneumococci, not having
an acid reaction, received an addition of 10 per cent. of a freshly
prepared 2 per cent. solution of sodium chlorate, the organisms dis-
solved when kept at incubation temperature.

Metabolic Products.—Hiss** found that the pneumococcus pro-
duces acid from monosaccharids, disaccharids, and such complex
saccharids as dextrin, glycogen, starch, and inulin. The fermenta-
tion of inulin by the pneumococcus is a most important means of
differentiating it from the streptococcus. Butterfieldand Peabody tt
found that when pneumococci were grown upon blood-agar, the
colonies became surrounded by a greenish zone of what they deter-
mined to be methemoglobin.

Toxic Products.— As early as 1891 Klemperertt found that culture
filtrates of pneumococci were toxic for the small laboratory animals.
This was confirmed by Isaeff$§ and by Washburn.||||

Auld*** 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

* Loc. cit. :
{ “Arch. p. l. Sc. Med.,” 1981, xv. * :
tT“ Atti della R. Acad. Med. di Roma,” 1888, rv.
§ “Zeitschrift ftir Hygiene,’ 1900, XXXIV, 454.
|| “Jour. Exp. Med.,” 1912, xvi, 658.
** «Tour. Exp. Med.,” va, No. 5, Aug. 25, 1905.
tt “Jour. Exp. Med.,” 1913, xvi, 587. i
tt “Zeitschrift fiir klin. Med.,” 1891, xx, 165.
§§ “Annales de I’ Inst. Pasteur,” 1892, vil, 250.

\\|| “Jour. of Path.'and Bact.,” 1897, III, 214.
*** “Brit. Med. Jour.,”? Jan. 20, 1900.

Pathogenesis 469

produce much stronger toxin. Macfadyen* found that by freezing
cultures of the pneumococcus with liquid air, destroying them by
trituration in the frozen state and then extracting the frag-
ments with 1:1o00o 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 cc.

Neufeld and Dodd followed by Rosenowt obtained a toxic fluid by

. permitting the pneumococci to undergo autolysis either in lecithin-
ized or plain physiological salt solution. Cole§ found that the
toxic values of such extracts was not uniform, but that autolysates
of pneumococci in dilute solutions of bile salts were very uniform in
strength, and possessed hemolytic effects upon the blood-corpuscles
of human beings, rabbits, sheep and guinea-pigs. This hematoxic
substance produced immunity reactions when repeatedly injected
into animals in increasing doses, antihematoxin being produced.
The toxin liberated by autolysis was found by Rosenow,]|| to be soluble
in ether. Heating the clear autolysate to 60°C. for twenty minutes
destroys it, though toxic pneumococcus suspensions remain toxic even
after boiling. Hydrochloric acidin weak solutions destroys the toxic-
ity of pneumococcus autolysates. The toxic substance is absorbed
by blood charcoal from which it can again be obtained by shaking with
ether. The toxic substance is probably a base which contains amino
groups of nitrogen. Indications have been obtained showing that
during pneumococcus infections toxic substances are produced that
do not call forth any immunizing response.” Rosenow** found
that the autolysate contained a proteolyticenzyme. He alsofoundt}f
thatit was capable of producing, in dogs, symptoms strikingly like ana-
phylaxis, with a striking drop in the blood pressure, pronounced
hemorrhages, marked depression of respiration, extreme cyanosis and
the presence of CO. in the stomach.

Pathogenesis.—If a small quantity of a pure culture of the viru-
lent organism be introduced into a mouse, rabbit, or guinea-pig, the
animal dies in one or two days. Exactly the same result can be ob-
tained by the introduction of a piece of the lung-tissue from croupous
pneumonia, by the introduction of-some of the rusty sputum, and
frequently by the introduction of human saliva. Postmortem ex-
amination 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.

*“Centralbl. f. Bakt. u. Parasitenk.,”’ 1907, Orig. xLuII, p. 30.
t “Berl. klin. Wochenschrift,” 1911, XLVIII, 1069.
t “Journal of Infectious Diseases,’’ 1911, Ix, 190.
§ “Jour. Exp. Med.,’? 1912, XVI, 644.
"|| “Journal of Infectious Diseases,” 1912, XI, 94, 235.

4 Mt i of Infectious Diseases,” 1912, XI, 286.
id., p. 480.

470 ’ Pneumonia

The spleen is enlarged, firm, and 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.

In such cases the lungs show no consolidation. Even if the in-
oculation be made by a hypodermic needle plunged through the
chest-wall into the pulmonary tissue, pneumonia rarely results.
Gamaléia* reported that pneumonic consolidation of the lungs of

Fig. 169.—Lung of a child, showing the appearance of the organ in the stage
of red hepatization of croupous pneumonia. The pneumonia has been preceded
by chronic pleuritis, which accounts for the thickened fibrous trabecule extend-
ing into the tissue, and which may have had something to do with the peculiarly
prominent appearance of the bronchioles through the lung.

dogs and sheep could be brought about by injecting the pneumo-
coccus through the chest-wall into the lung. Tchistowitschf stated
that by intratracheal injections of cultures into dogs he succeeded in
producing in 7 out of 19 experiments typical pneumonic lesions.

*“ Ann. de l’Inst. Pasteur,’’ 1888, 11, 440.
t Ibid., 1890, m1, 285.

Pathogenesis 471

Monti* claimed to have found that a characteristic croupous pneu-
monia results from the injection of cultures into the trachea of sus-
ceptible animals. A very interesting review of the literature of the
experimental aspects of the subject, embracing 108 references, will
be found in Wadsworth’s paper upon ‘‘Experimental Studies on the
Etiology of Acute Pneumonitis.” +

The final proof that true pulmonary consolidation, z.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 for-
ward so that the posterior aspect of the epiglottis presented an in-
clined 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 pipette containing a liquid culture of the pneumococcus was next
attached to the external end of the tube, and by means of a syringe
the culture (about 6 cc.) was injected into the bronchus. The
syringe was then removed, the piston withdrawn, and the syringe
again attached to the pipette. 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 consolidation and lobar distribution,
was produced in 42 successive cases. The course of the inflamma-
tory disturbance thus produced was rapid, and in one case nearly
complete consolidation had occurred in seven hours.

Lesions.—The lesions of croupous pneumonia of man are almost
too well known to need description. 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 invaded portion of the lung is supposed to
pass through a succession of stages clinically described as (1) con-
gestion, (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 approxi-
mately like liver tissue in appearance. The third stage is charac-
terized by dissolution of the erythrocytes and invasion of the diseased
ar-cells by leukocytes, so that the color of the tissue changes from
ted to gray. At the same time the coagulated exudate begins to

* “Zeitschrift fiir Hygiene,” etc., 1892, XI, 387.
“Amer. Jour. Med. Sciences,’’ 1904, CXXVI, ps 851.
t “Jour. Exp. Med.,” 1912, xv, No. 2, p. 133.

472 Pneumonia

soften and leave the air-cells by the natural passages, and the stage
of resolution begins.

The pneumococci, though present in enormous numbers in the
pulmonary lesions, are not confined to them. In practically all
cases pneumonia is a blood infection (bacteremia) as well as a pul-
monary infection. It is through the blood infection that many of
the complications and sequele of the disease are brought about.
Not only are the pneumococci found in this typical lobar form of pneu-
monia, but also in the atypical scattered consolidations of the lung
known as broncho-pneumonia, catarrhal pneumonia and inspiration
pneumonia. Here, however, they are by no means so constant in
occurrence.

The pneumococcus is not infrequently discovered in diseased con-
ditions other than croupous pneumonia; thus, Foa, Bordoni-Uffre-
duzzi, and others found it in cerebro-spinal meningitis; Frankel, in
pleuritis; Weichselbaum, in peritonitis; Banti, in pericarditis; numer-
ous observers, in acute abscesses; Gabbi isolated it from a case of
suppurative tonsillitis; Axenfeld observed an epidemic of conjunc-
tivitis caused by it; Zaufal, Levy, and Schréder and Netter have
been able to demonstrate it in the pus of otitis media, and Foulerton
and Bonney* isolated it from a case of primary infection of the
puerperal uterus. It has also been found in arthritis following pneu-
monia, and in primary arthritis without previous pneumonia by
Howard. f

Interesting statistics concerning the relative frequency of pneumo-
coccus infections in adults given by Netter{ are as follows:

Pneumonia...... eid daen sa A aha'e en Taare IN FINN
Broncho-pneumonia

Meningitis.................. Paaticoa sani tant t hans
FMP VOM sys 456d 5-HS ad WA ADORE etukoe APE

Endocarditis............... M isciiacysilaloone tile dlgin aaglidags wll g
Hepatic AbsCess ici; inc eu gihaeecee nad veh aun dines od

In 46 consecutive pneumococcus infection of children he found:

OTIS MEM fies aida adres Go race chee BAAS 29°
Broncho-pneumonia.......... 0.0.00 cece cece ene e eee 12
Meningitisigis's cp.ancon e4 Gail 3 £4906 NS KS BOE OS CH BOD Aes 2
PHEUMOD ale ie ais Gexciay 6:6 BaEN IIH Sanne AE nerd ONE KIS SRE I
PlCULIS Y= tad a tsnosisaies arid veGes Rasemnce dea a Gade ba maunea AS I
POriGarditis.s cies ic sete s dual 4 NE Seca oMlacen A awl R ae 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 inoculation of large doses. .

Specificity.—The etiologic relationship of the pneumococcus to
pneumonia is based chiefly upon the frequency of its presence in

*“Trans. Obstet. Soc. of London,”’ 1903, part 1, p. 128.
+ “Johns Hopkins Hospital Bulletin,’? Nov., 1903.
t “Compte-rendu,” 1889.

Specificity — 473

croupous pneumonia. Netter* found it 82 times in 82 autopsies
upon such cases; Klemperer, 21 times out of 21 cases studied by
puncturing the lung with a hypodermic syringe. Weichselbaum ob-
tained it in 94 out of 129 cases; Wolf, in 66 out of 70; and Pierce,
in 110 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 pneu-
monia are not known. It is probable that some systemic 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

Fig. 170.—Diplococcus: pneumoniz. Colony twenty-four hours old upon
gelatin. X 100 (Frankel and Pfeiffer).

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 itis 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 niné
rabbits developed a permanent virulence when kept on agar-agar
so made that it was not heated beyond 100°C., and alkalinized 4 cc.
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

* “Compte-rendu,’’ 1889.

T “Jour. Infectious Disedsés,!*

1904, I, p. 280.

474 Pneumonia

37.5°C. Ordinarily pneumococci seem unable to accommodate
themselves to a purely saprophytic life, and unless continually trans-
planted to new media die in a week or two, sometimes sooner.
Lambert found, however, that in Marmorek’s mixture (bouillon 2
parts and ascitic or pleuritic fluid 1 part) the organisms would some-
times 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 of pneumonia.
If, for any reason it be considered necessary, three means are avail-
able: 1, the blood culture; 2, the inoculation of animals with
the expectoration; 3, the cultivation of the organism from the
expectoration.

1. To make the blood culture, the elbow is encircled with aband,
the skin washed and after an application of iodine has been made,
a hollow needle is introduced into one of the distended veins, and
from five to ten cubic centimeters of the blood permitted to drop
into a small flask containing about too cc. of appropriate media.

2. To inoculate an animal with the sputum, or with fluid drawn
from the lung or pleura, a white mouse is usually selected as suitable,
the inoculation of a drop of the sputum, diluted with salt solution
if necessary, being made beneath the skin or into the abdominal
cavity. It is usually fatal in twenty-four hours.

3. The recovery of the organism from the sputum can be accom-
plished by stroking appropriate media with a platinum wire dipped
in the sputum. The characteristic colonies can be picked up and
transplanted as soon as they appear.

Identification of the Organism.—The identification of the pneu:
mococcus may be a matter fraught with considerable difficulty
when it makes its appearance in an unusual localization and espe-
cially when it lacks its characteristic lanceolate shape, its surround-
ing capsules and its occurrence in pairs. Under such circumstances
it is easily confused with the streptococcus and for the differentia-
tion recourse must be had to certain characters upon which much em-
phasis is laid by some authors, though others regard them lightly.
Here one should recall that a few authors even express the opinion
that the pneumococcus may be but the streptococcus temporarily or
permanently modified in small particulars by the mode of its occur-
rence. Rosenow* indeed, believes that he has succeeded in bring-
ing about such a mutation, between streptococci and pneumococci.

To make the identification, therefore, advantage must be taken

of such peculiarities as are commonly supposed to belong to each
organism:
* “Centralbl. f. Bakt. u. Parasitenk.,” Orig. 1914, LXxI, 284.

Types of Pneumococci 475

1. The morphology of the two organisms, the pneumococcus tending to be
elongate or lanceolate, occur in pairs and have a capsule.

2. The more luxuriant growth of the streptococcus in artificial culture.

3. The ability of the pneumococcus to ferment inulin, which the streptococcus
ordinarily fails to do.

4. The solution of pneumococci within 15-20 minutes by the addition of 5-10
per cent. of bile to a fluid culture.

s. The solution of the pneumococci in 0.2 per cent. sodium chlorate solution.

6. The production of a reddish hemolytic zone by streptococci and of a
greenish zone by pneumococci, about colonies upon blood-agar.

7. The agglutination of the organisms by antipneumococcus and antistrepto-
coccus serums respectively.

Wadsworth* has shown that agglutination reactions can be ob-
tained by concentrating the pneumococci by centrifugalization
in isotonic salt solution and adding the serum. Neufeld} and
Wadswortht have also found that when rabbit’s bile is added to
a pneumococcus culture so as to produce 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 value for purposes of diagnosis, but is useful in assisting
in the recognition of the pneumococcus and differentiating it
from the streptococcus, for when the latter organisms are similarly
treated no precipitate takes place.

Buerger§ found that all pneumococci, irrespective of source,were
agglutinating by pneumococcus-immune serum, that such serum
was capable of agglutinating various pyogenic streptococci, certain
atypical organisms, and certain strains of Streptococcus capsulatus.
_ Types of Pneumococci.—In the first studies of the comparative viru-
lence of pneumococci, Kruse and Pansini|| observed striking differ-
ences in cultural, morphological and pathogenesis among these
micro-organisms. The matter was puzzling and was not greatly
clarified by the studies of Neufeld and Hindel** Itseemedcurious.
that some pneumococci in an avirulent state should be found in the
mouths of well persons, that some slightly virulent organisms should
be found in the lesions of pneumonia, and that other cultures ob-
tained from pneumonia should appear somewhat differently and
possess great virulence. The solution of the problem begun with
the work of Docheztt and was continued by Dochez and Avery,tt
Cole,§§ Pisek and Pease, |\|| Mitchell, *** Cole,tt+ Stillman,ttt

* “Jour. Med. Research,” 1904, x, p. 228.
{ “Zeitschrift fiir Hygiene,’ 1902, x1.
T Loc. cit.
§ “Jour. Exp. Med.,’’ Aug. 25, 1905, vu, No. 5.
|| “Zeitschrift fiir Hygiene,” 1892, x1, 279.
** “Zeitschrift fir Immunitétsforschung,” 1909, III, 159.
tt “Jour. Exp. Med.,” 1912, XVI, 680.
tt “Jour. Amer. Med. Asso.,” 1913, LXI, 727.
§§ “New York Medical Journal,” rors, cl, p. 1."
Ih “ Amer. Jour. of the Med. Sci.,”#1916, CLI, 14.
*** “Denna. State Medical Journal,” 1917, Xx, p. 343-
ttt Jour. Exp. Med.,” 1917, xxiv, No. 4, 56.
tit “Jour. Exp. Med.,” 1917, XXVI, p. 513.

476 Pneumonia

Blake* and others. The result has been the acceptance of a system
devised by Dochez, by which the pneumococci are divided into four
groups, known as I, I]. III and IV respectively. Of these, groups I
and I] constitute the organisms found in the more benign forms of
clinical pneumonia, group III (supposed to be identified with the
Streptococcus mucosus of Schottmiiller g.v.) those found in the rapidly
fatal variety of ¢linical pneumonia, and group IV made up of mis-
cellaneous mildly virulent organisms found in the human mouth 7
in a variety of pathological lesions other than pneumonia.

Groups I, II and IV have much the same morphological and sil
tural appearances, but type III is characterized by a ach more
‘broad and distinct capsule.

The chief criterion for the identification of the types, inca is
the phenomenon of agglutination, for each organism reacts strikingly
to the agglutinating effects, of antiserums produced by organisms of
its own type and very slightly to others. Moreover, the reactions of
immunity among the organisms are dissimilar among the organisms,
the pneumococci of type III being unable to induce the production of
any considerable amount of antibody formation (antitoxin). The
result is that in endeavoring to treat clinical pneumonia with anti-
serum and draw scientific conclusions regarding the efficiency of
the treatment, it is necessary to learn which type of organism is pres-
ent and which type of antiserum to administer. Types I and II-
pneumococci, producing pneumonia that is amenable to treatment
by the antiserum, while type III causes a form of the disease that
cannot be influenced favorably, the organisms of this type not excit-
ing immunity reactions in experiment seals to the (horses) pro-
duction of potent antiserum.

It then becomes important to know hee to. differentiate the types
of pneumococci. To do this requires that serums be prepared by
injecting cultures of the respective strains into animals until strong.
agglutinating values are obtained. These serums then become the
. standards of differentiation.

It is necessary to have on hand agglutinating serum for the various
types. These must, at first, be obtained from some laboratory where
they are already recognized standards, or one must have for their
preparation, cultures of the known organisms of the various types,
especially types I and II. If the cultures are at hand and it is de-
sired to make the serums, that is done by repeatedly injecting a
rabbit with first killed cultures, then living cultures, in increasing:
doses until a drawn sample of the blood shows a sufficient agglutinat-
ing value, when brought into contact with the cocci, to give satis-
factory agglutination in considerable dilutions.

One then proceeds as follows: A portion of the freshly coughed up expectora-
tion of the patient, is diluted with physiological salt solution and injected into the
abdominal cavity of a white mouse. In from five to twenty-four hours, as deter-

* “Jour, Exp. Med.,” 1917, XxvI, p. 67.

Types of Pneumococci 477

mined by puncture of the abdominal wall by a capillary pipet, the cocci will have
increased sufficiently and the mouse can be chloroformed to death. The abdomi-
nal cavity is then carefully opened and the fluid in the peritoneal cavity trans-
ferred with a pipit to 4-5 cc. of physiological salt solution in a centrifuge tube.
After mixing, the contents are centrifuged for a minute or two at low speed to
throw down leukocytes, shreds of fibrin and any clumps of bacteria, and the
supernatant fluid which is a fairly uniform, slightly opalescent suspension of
pneumococcic removed with a pipet to a second tube and centrifuged at high
speed to thrown down the cocci. The supernatant fluid is thrown away, and the
bacterial sediment distributed throughout fresh salt solution and placed in
several small tubes. To one of these, asmall quantity of the serum agglutinating
type I, to the other a small quantity of that of type II, to a third that of type III
is added, and the tubes stood in an incubating oven for eighteen to twenty-four
hours. If one agglutinates, the type is at once known; if none agglutinates, the
disease affecting the patient is caused by type IV.

The type organism now being known, the therapeutic antipneumo-
coccic serum appropriate in antagonizing type I or type IT should
be administered. If the disease is caused by type III, the patient
cannot be benefited, as no serum has thus far been produced that
is of use in treating cases of pneumonia occasioned by type III.

A second method of determining the type of pneumococci in any
case has been suggested by Blake* and depends upon specific
precipitation. Blake points out that the method by agglutination
is sometimes delayed by the occurrence of complicating infections .
—influenza bacillus, mouse typhoid bacillus—and is always delayed
by the time of incubation necessary for the occurrence of the
agglutination.

To obviate these, he proceeds according to the method given above, inoculating
the mouse with the sputum, making occasional punctures with capillary pipets
to determine when the organisms have multiplied sufficiently, kills the mouse
at the appropriate time, collects the fluid from its abdominal cavity, dilutes it
with 4-5 cc. of salt solution and immediately centrifuges at high speed to throw
out the leukocytes, fibrin and bacteria, leaving aclear fluid. This issaved and

constitutes the fluid susceptible of specific precipitation by the immune serums.
Five-tenths cubic centimeter of the fluid is placed in each of a series of small

‘ test-tubes, and to each o.5 cc. of the immune serums of types I, II, ITI, and IV are

added. An immediate precipitate takes place in that tube containing the homo-
logous immune serum, the other tubes remaining clear. The association of the
micro-organisms mentioned above does not interfere with the success of the
specific precipitation test.

Blake, and also Stillman} both found that the organisms of type
II were not always uniform in behavior and have therefore divided

them into several sub-types Ia, Id and IIx.
Stillman observed and studied 454 cases of pneumonia and found

the organisms as follows:

Typel................. PS Baik aarann Geeta 33.26 per cent.
Type Ls geccca waves vn eee 13 Gis ace havo ele Suveoes 29.29 per cent.
Type Qe «ss sissies eevee Ospina ee 1.32 per cent.
VDE Lb ssi’ s esac wise y daenen't Ariste nee sann ese ight 0.88 per cent.
Dype tive eves 2k seg eee Obaeas ora nes wewe I.98 per cent.
Type IIL... 0.0.00. 00 0. 5Q wie osGremtanee ene 12.99 per cent.
Pype- LV es ssucs aoease aan 2 Bie ROM aad eet 20.26 per cent.

* “Tour. Exp. Med.,” 1917, xxvi, No. 1, p. 67.
tT “Jour. Exp. Med.,” 1917, xxv1, No. 4, p. 513.

478 Pneumonia

As much valuable time is lost in the treatment of the case through
the delays in making a diagnosis of the type of organism concerned
by this method, Mitchell and Muns* recommend a new and
quicker method, tried upon 69 sputa from as many cases of pneu-
monia, and controlled by the mouse method. The method depends
upon obtaining material for specific precipitation from the sputum
itself. By this means the identification of the type of pneumococcus
may be made in an hour or two. The method is as follows:

About 5 cc. of sputum, collected in a sterile container, is pipetted into a small
mortar and sufficient relatively fine sand added to make a rather stiff mixture.
This is ground with the pestle and in about three minutes becomes a thick,
tenacious gritty paste. Ten cubic centimeters of normal saline solution are now
added, two at a time, the stirring being continued. _ After thorough mixing, the
whole is permitted to stand for a few minutes, that the sand may settle. The
fluid is then pipetted into a clean centrifuge tube. Another ro cc. of saline solu-
tion is then added to the sandy precipitate and stirred for a few minutes, allowed
to sediment and then pipetted into a second centrifuge tube, held in reserve,
The tubes are now centrifuged at high speed until the fluid is perfectly clear.

Into each of three small test-tubes 0.2 cc. of immune sera, type I, type II
and type III, respectively are placed, and to each 1 cc. of the centrifugated
sputum fluid is added, carefully mixed by shaking and stood in a water bath at
37°C. Specific precipitation soon appears in the tube containing the serum
corresponding to the type of pneumococcus occasioning the pneumonia from
which the serum was obtained. It sometimes appears at once, sometimes only
after an hour or two and is distinctly feathery in appearance. ~The precipitate
sediments after standing in the water-bath. ;

Avery} recommends a simple method in which the mouse is elimi-
nated and advantage taken of the accelerating influence of glucose
and blood proteins upon the growth of pneumococcus, and the lytic
action of ox bile for pneumococci alone. The method is carried out
as follows:

A kernel of sputum is selected with care, as coming from the deeper portions
of the air passages and being as free as possible from admixture of naso-pharyn-
geal mucus and saliva. It should be caught in a sterile container and have a
bulk about equal to that ofa bean. If it cannot be examined at once it should be .
kept on ice. Such a mass of material should be placed in a centrifuge tube con-
taining 4 cc. of a medium specially adapted to the cultural requirements of the
Pneumococcus and made as follows: A meat infusion broth is made and titrated
with phenolphthalein to 0.3-0.5 acid. It is then sterilized on three successive
days in the Arnold sterilizer, twenty minutes each time. To each 100 cc. of
the broth one adds 5 cc. of a sterile 20 per cent. solution of glucose and 5 cc. of
sterile defibrinated rabbits’ blood. The medium, thus containing 1 per cent. of
glucose and 5 per cent. of blood is distributed in small centrifuge tubes, about
4 cc. in each tube.

The tube of medium inoculated as directed above is placed in a water-bath
and kept at 37°C. for five hours. A blood-agar plate is then streaked witha -
platinum loop full of the culture and set aside for the isolation of the pneumo-
coccus, should that prove to be desirable.

The type determination may now be made either by specific precipitation or by
agglutination. .

1. The Method by Specific Precipitation—The tube is placed in the centrifuge
and whirled at low speed to throw out the blood-corpuscles, after which 3 cc. of
the supernatant bacterial suspension are pipetted into a second centrifuge tube
containing about 1 cc. of sterile ox bile and stood in the water-bath at 37°C. for

*" * “Tour, Med. Research,” 1917, Xxx1I, No. 2, p. 339.
t “Jour. Amer. Med. Assoc.,”’ 1918, LXX, p. 17.

Immunity 479

about twenty minutes, by which time the bodies of the pneumococci are usually
dissolved. It may be well at this point to test the reaction of the fluid. If acid, it
should be neutralized by the addition of a drop or two of an alkaline solution.
One-half of a cubic centimeter of the bile solution is mixed with an equal volume
of each of the immune serums corresponding to the types of pneumococci known,
te corresponding to the coccus tested, causes a delicate precipitate in the
medium.

2. The Method by A gglutination—When no ox bile is available, the fluid culture
cleared of blood-corpuscles by centrifugation is pipetted into small tubes, 0.5
cc. each, and each receives 0.5 cc. of the type immune serums. As the pneu-
mococci have not been dissolved, they agglutinate with the serum of their own
type.

Krumwiede and Valentine* recommend the following method also
based upon the specific precipitation of the pneumococcic antigen
by the homologous type serum:

From 3 to 10 cc. of the sputum, depending upon the amount available, is
poured from the sputum container into a test-tube. This is placed in boiling
water for several minutes or longer until a more. or less firm coagulum results,
which will occur if the specimen bea suitable one. The coagulum is then broken
up with a heavy platinum wire or glass rod, and saline is added. Just enough
saline should be added so that, on subsequent centrifuging, there will be sufficient
fluid to carry out the test. If too much saline is added the resulting antigen
may be too dilute.

In some instances little or no saline is necessary as sufficient fluid separates
from the coagulum. :

After the addition of saline, the tube is again placed in boiling water for a
few moments to extract the soluble antigen from the coagulum, the tube being
shaken several times during heating.

The broken clot is then thrown down by the centrifuge and the clear superna-
tant fluid used for the test. To hasten the appearance of the reaction and to
obtain a reaction even should the antigen be dilute, the antigen is layered over
the “type” serums, using the latter undiluted.

Two-tenths cubic centimeter of the three “type” serums are placed in narrow
test-tubes and the antigen added froma capillary pipet with a rubber teat. If
the tubes containing the serums are tilted, and the antigen dropped slowly on the
side of the tube just above the serum, no difficulty will be encountered in obtaining
sharp layers as the undiluted serum is sufficiently high in its specific gravity. The
tubes are then placed in a water-bath at 50-55°C. and observed after several
minutes. If a fixed type was present in the sputum, and should the sputum
have been rich in antigen, a “definite contact ring is seen in the tube contain-
ing the homologous serum. With sputum less rich in antigen, the ring may
develop more slowly, and it will be less marked. Some experience is necessary
in detecting the less marked contact rings and in differentiating these from an
apparent ring which may be confusing if one of the serums is darker in color giv-
ing them a sharper contrast with the supernatant antigen. The true ring is
more or less opaque, and this quality can be seen by tilting the tubes and looking
at the area of contact against a dark back-ground.

Immunity.—Pneumonia in human beings terminates by crisisas
though from a supply of antitoxin or other immunizing agent sud-
denly liberated. Ordinarily the pneumococci are not taken up by
the leukocytes, but immediately following the crisis the leukocytes
become and seem to remain actively phagocytic and distend them-
selves with the organisms. Recovery is followed by immunity from
further infection by pneumococci of that particular type by which
the patient was infected, but not from the other types. Thus_are
explained the frequent “relapses” of the disease.

* “Jour. Amer. Med. Assoc.,”’ 1918, LXX, P. 513.

480 _ Pneumonia

Immune Serum.—The early experiments were remarkably con-
tradictory in the hands of different investigators. G. and F. Klemp-
erer* showed that the serum of rabbits immunized against the pneu-
mococcus protected animals infected with virulent cultures.. When
applied in human medicine, the serum failed to do good.

The treatment of pneumonia by the injection of blood-serum‘

from convalescent patients, tried by Hughes and Cartert, was at
once abandoned as useless and dangerous. ~

Antipneumococcic sera were experimentally investigated by De
‘Renzi,{ Washbourn,§ and Pane.||
~ McFarland and Lincoln** succeeded in immunizing a horse against
large doses of a virulent culture of the pneumococcus, and obtained a
serum of which 0.5 to 0.25 cc. protected rabbits from many times the
fatal dose. In‘ the treatment of pneumonia it gave excellent results
in some cases but failed in others.

The experiments by Passler{{ showed some gain over ‘the earlier

work. :
_ With the differentiation of the various types of pneumococci
described above, and the discovery that the immunity reactions in-
duced by each were specific in character, the varying successes and
failures attending the employment of the earlier antipneumococcic
serums became intelligible. They succeeded when the serum was
homologous with the type organism, and failed when it was not.

There has been, therefore, a return to the therapeutic employment
of antipneumococcic serums, but with restrictions. It is essential
first to determine the type of pneumococcus infecting, and then to
employ the appropriate serum in treatment. The most complete
investigations along this line have been and are being conducted by
Cole and his associates. tf
- Good results follow the usé of the serum for type I, fairly satis-
factory results that for type II, but for type III a satisfactory serum
remains to be found, and the cases resulting from the organisms of
this type are extremely serious and highly fatal.

A leukocytic extract prepared by Hiss arid Zinsser§§ from an
aleuronat exudation in the rabbit’s pleura was tried in the treat-
ment of pneumonia in man.

Rosenow]||| found that the injection of autolyzed pneumococci into
25 patients with lobar pneumonia seemed to have a beneficial effect.

* “Berliner klin. Wochenschrift,” 1891, Nos. 34 and 35.

+ “Therapeutic Gazette,” Oct. 15, 1892.

t ‘Tl Policlinico,” Oct. 31, 1986, Supplement.

§ ‘Brit. Med. Jour.,” Feb. 27, 1897, p. 510.

| <‘Centralbl. f. Bakt. u. Parasitenk.,”” May 29, 1897, Xxt, 17 and 18, p. 664.

** “Tour, Amer. Med. Assoc.,” Dec. 16, 1899, Pp. 1534.

tt ‘Deutsches Archiv fiir klin. Med.,” Bd. 1905; txxxu, Nos. 3, 4, “Jour.
Amer. Med. Assoc.,” May 13, 1905, p. 1538.

tt ‘Jour. Amer. Med. Assoc.,” 1913, LXI, 663; ‘“N. Y. Med. Jour.,” 1915, Cl,
No. 1, p. 1 and p. 59. :

§§ “Jour. Med. Research,” 1908, XIX, 323.
Ill ‘Jour. Amer. Med. Assoc.,’? June 20, L1v, No. 24, p. 1943.

Bacillus Capsulatus Mucosus 481

Sanitation.—Pneumonia is undoubtedly a transmissible 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. Pneumonia cases should be segre-
‘gated and treated apart from the general run of hospital cases. As
cases of pneumonia in neighboring beds may be occasioned by
pneumococci of different types, which to alJ intents and purposes
are different organisms, they may infect one another, thus bringing
about what seem to be relapses. To prevent this it is well to sepa-
rate the patients by sheets hung as curtains between the beds in
such manner as to make it impossible that drops of moisture from
the respiratory passages of one can find their way to another.
Wood* has shown that “the organisms in the sputum die off rapidly
under the action of light and desiccation. In sunlight or diffuse
daylight the bacteria die within an hour, and in about four hours if
kept in the dark. The danger of infection from powdered sputum
may, therefore, 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.

Bacittus Capsutatus Mucosus (FascHinct)—PNEUMococcus
(FRIEDLANDER)—BACTERIUM PNEUMONIE (ZorrFf)

General Characteristics——An encapsulated, non-motile, non-flagellated,

hon-sporogenous, non-liquefying, aérobic and optionally anaérobic, non-chromo-

genic, aérogenic and pathogenic organism, 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 pneumonia, and, being
thought by its discoverer to be the cause of that disease, was called
the pneumococcus, and later the pueumobacillus. 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
present looked upon as one whose presence in the lung is, in most
cases, unimportant, though it is sometimes associated with and is
probably the cause of a special form of pneumonia, which, ac-
cording to Stuhlern,|| is clinically atypical and commonly 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
ee

Jour. Exp. Med.,” Aug. 25, 1905, vil, No. 5, p. 624.

{ “Spaltpilze,” 1885, p. 66.

t ,Centralbl. f. Bakt.,” etc., 1892, XII, p. 304..

f. Rortshritte der Medizin,” 1883, 22, 715. :

I'“Centralbl. f. Bakt.,” etc. (Originale), July 21, 1904, Bd. xxxvz, No. 4, p. 493-

482 Pneumonia

growing and more delicate pneumococcus. In the light of present
knowledge Friedlinder’s bacillus must be looked upon as the type
of @ group of organisms (the “mucosus-capsulatus group’’) varying
among themselves in many minor particulars. The other members
of the group are Bacillus rhinoscleromatis (g.v.), and Bacillus
lactis aérogenes.

Distribution.—The organism is sometimes found in normal saliva;
it is a common parasite of the respiratory apparatus; not infrequently
occurs in purulent accumulations; is occasionally found in feces, and
sometimes occurs under external saprophytic conditions.

Morphology.—Though usully distinctly bacillary in form, the
organism is of variable length and when paired sometimes bears a
close resemblance to the pneumococcus of Frankel and Weichsel-

Fig. 171.—Bacterium pneumonie (modified after Migula).

baum. It measures 0.5 to 1.5 w 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 commonly surrounded by a distinct
transparent capsule, hence its name “capsule 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 pneumonic exudate be mixed with
gelatin and poured upon plates, small white spheric colonies aj pear
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. The gelatin is not liquefied.

Bacillus Capsulatus Mucosus 483.

Bouillon.—There is nothing characteristic about the bouillon

cultures of Friedlinder’s bacillus. The
medium 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 elevated, rounded white
mass is formed, 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 oc-
curs. 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 sur-
face with a thick yellowish-white layer,
which sometimes contains bubbles of
gas.

Milk is not coagulated asarule. Litmus
milk is reddened.

Vital Resistance.—The bacillus grows at.

a temperature as low as 16°C., and, ac-
cording to Sternberg has a thermal death-
point of 56°C.

Metabolic Products.—Friedlinder’s ba-
cillus ferments nearly all the sugars, with
the evolution of much gas. It generates
alcohol, acetic and other acids, and both
CO, and H. According to the best author-
ities 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 carbohydrates:

Fig. 172.—Friedlin-
der’s pneumobacillus;
gelatin stab culture,
showing the typical
nail-head appearance
and the formation of
gas bubbles, not always
present (Curtis).

I. Bacillus aérogenes type which ferment all carbohydrates,

with the formation of gas.

* “Jour. of Infect. Dis.,” 1904, 1, No. 2, p 241.

484 Pneumonia

II. Bacillus pneumonie (Friedlander) type which ferment all
carbohydrates except lactose, with formation of gas.
III. Bacillus lactis aérogenes type which ferment all carbohy-
drates 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 pathogenic effects that Friedlander
observed occurred in mice, into whose lungs and pleura he injected
the cultures, with resulting inflammation.

That Friedlinder’s bacillus may be the cause of true lobar pneu-
monia there can be no room for doubt after the demonstrations of
Lamar and Meltzer,* who found that its experimental 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.

Curry{ found Friedlinder’s bacillus in association with the
pheumococcus in acute lobar pneumonia; in association with
the diphtheria bacillus in otitis media associated with croupous
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 inflamed tonsils in children.

Abelf cultivated it from the discharges of fetid ozena, and sup-
posed it to be the specific cause (Bacillus ozenz).

Occasionally Friedlinder’s bacillus bears an important pelations
ship 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 contained 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- ies preparations from the

* “Your. Exp. Med.,” 1912, xv, 13
t “Jour. Boston Soc. of Med. Sci. a March, 1808, vol. 1, No. 8, P. 137.
t “Zeitschrift fiir Hygiene,” xxz.
§ “Jour. Boston Soc. of Med. Sci.,” May, 1898, vol. 1, No. ro. p. 174.

Mixed Pneumonias 485

heart, blood, spleen, and peritoneal cavity showed large numbers of
the capsule bacilli.

Howard* has also called attention to the importance of this bacil-
lus in connection with numerous acute and chronic infectious proc-
esses, among which’ may be mentioned croupous pneumonia, sup-
puration of the antrum of Highmore 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 pro-
duces local inflammatory lesions, and sometimes death from septic
invasion. |

CATARRHAL PNEUMONIA OR BRONCHO-PNEUMONIA

This form of pulmonary inflammation occurs in local areas, commonly situated
about thé distribution of a bronchiole. 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 staphylococci and streptococci of suppuration, Friedlander’s bacillus, the
bacillus of influenza, and other well-known organisms.

TUBERCULOUS 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, or the tubercle bacillus
together with staphylococci, streptococci, tetragenococci, pneumococci, pneu-
mobacilli, and other organisms accidentally present in a lung in which ulceration
and cavity formation are advanced. _

PLAGUE PNEUMONIA

The pneumonic form of plague is characterized by consolidation of the lung
histologically and anatomically, indistinguishable from pneumococcic and
other extensive pulmonary infections.

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 pneumococci is commonly found. Sometimes pneumococci
and staphylococci simultaneously affect. the lung, purulent pneumonia with
abscess formation being the conspicuous feature. Almost any combination
of bacteria may occur in the lungs, so that it must be left for the student to work
out what the particular effects of each may be. ; :

Among the mixed forms of pneumonia may be mentioned those called by
Klemperer and Levy “é¢omplicating pneumonias,” occurring in the course of
typhoid fever, etc.

* “Phila. Med. Jour.,” Feb. 19, 1898, vol. 1, No. 8, p. 336.

CHAPTER XVII
INFLUENZA

BacitLus INFLUENZ& (R. PFEIFFER)

General Characteristics —A minute, non-motile, non-flagellated, non-sporog-
enous, non-liquefying, non-chromogenic, aérobic, pathogenic bacillus, staining
by the ordinary methods, but 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 de-
termine 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 conformed, in large part, to the require-
ments of specificity.

Morphology.—The bacilli are very small, having about the same
diameter as the bacillus of mouse septicemia, but only half its length
(o.2 by 0.5 w). They are usually solitary, 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 Léffler’s alkaline meth-
ylene blue, and even with these more deeply at the ends than inthe
middle, so that they appear not a little like diplococci. They do not
stain by Gram’s method.

Canonf recommends a rather complicated method for the demon-
stration 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 Waterecs.cinge i gare dats canada vedas 84 lewd waa 40

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 leukocytes blue; and the bacilli, also
blue, appear as short rods or as dumb-bells.

Large numbers of bacilli may be present, though sometimes only a
few can be found after prolonged search, as they are prone to occur

*“Deutsche med. Wochenschrift,” 1892, 2; “Zeitschrift fiir Hygiene,” 1893,

XIII, 357-
} “Centralbl. f. Bakt.,” etc., Bd. x1v, p. 860.

486

Cultivation 487

in widely scattered but dense clusters. They are frequently inclosed
within the leukocytes. It is scarcely necessary to pursue so tedious
a staining method for demonstrating the bacilli, for they stain well
enough for recognition by ordinary methods.

Isolation.—The influenza bacillus grows poorly upon artificial
culture-media, and is not easy to isolate, because the associated bac-
teria tend to outgrow it. When isolated it is difficult to keep, as it
soon dies in artificial cultures.

Pfeiffer found that the organism grew when he spread pus from
the bronchial secretions upon serum-agar. Subcultures made from
the original colonies did not “take.” 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,

Fig. 173.—Bacillus of influenza. Smear from sputum (after Heim).

with precautions as to sterilty, from the finger tip. Later it was
found that the addition of hemoglobin to the culture-medium was
equally efficacious. The most ready means of cultivation is by
the use of 1 per cent. dextrose agar-agar containing 1 per cent. of
laked, defibrinated human blood. 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. :

Cultivation.— After twenty-four hours in the incubator, minute
colorless, transparent, dewdrop-like colonies may be seen. They
look like condensed moisture, and Kitasato 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 organisms die

488 : Influenza

quickly and must be transplanted every three or four days if they are
to be kept alive.

The organism is aérobic 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 luxu-
riantly upon culture-media containing hemoglobin or blood, and can be
transferred from culture toculture many times before losing vitality.

Vital Resistance.—Its resisting powers are very restricted, as it
speedily succumbs to drying, and is certainly killed by an exposure to

Fig. 174.—Bacillus of influenza; colonies on blood agar-agar. Low magnifying
power (Pfeiffer).

a temperature of 60°C. for five minutes. It will not grow at any
temperature below 28°C. M

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 conclusion 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, meningitis, etc.
Horder* has cultivated it from the valvular vegetations of 2 cases of
endocarditis following influenza.

Davis found the influenza bacillus in the respiratory passages of a
large number of patients suffering from whooping-cough.

* “Path. Soc. of London,” “Brit. Med. Jour.,” April 22, 1905.
} “Jour. Infectious Diseases,” 1906, 1, 1.

Immunity 489

Pathogenesis.—The bacillus is pathogenic for very few of the
laboratory animals. The guinea-pig is susceptible of fatal infec-
tion, the dose required to cause death varying considerably.

Pfeiffer and Beck* 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 relapses, and
from which one rarely seems to acquire protection against future
attacks, there must be scarcely any immunity induced through ordi-
nary infection. Moreover, the organism once finding its way into
the body seems to remain almost indefinitely, especially when, as in

Fig. 175.—Bacillus of influenza; cover-glass preparation of sputum from a case
of influenza, showing the bacilli in leukocytes. Highly magnified (Pfeiffer).

pulmonary tuberculosis, there is already present an abnormal con-
dition furnishing discharges or exudates in which it can thrive.

Delius and Kollet found that the toxicity of the culture does not
depend upon a soluble toxin, but upon an intracellular toxin. The
outcome of the researches, which were made most painstakingly, was
total failure to produce experimental immunity.

Increasing doses of the cultures, injected into the peritoneal
cavity, enabled the animals to resist more than a fatal dose, but
never enabled them to recover when large doses of living cultures
were administered.

A. Catanni, Jr.,t trephined rabbits and injected influenza toxin

* “Deutsche med. Wochenschrift,’’ 1893, XXI.
t “Zeitschrift fiir Hygiene,” etc., 'Bd. 1897, xxiv, Heft 2.
tIbid., Bd., 1896, xx.

490 : Influenza

into their brains, at the same time trephining control animals, into
some of whose brains he injected water. The animals receiving
o.5 to 1 mg. of the living culture died in twenty-four hours with all
the nervous symptoms of the disease, dyspnea, paralysis beginning
in the posterior extremities and extending over the whole body,
clonic convulsions, stiffness of the neck, etc. Control animals in-
jected 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 diagnosticating
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
coexists 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

Pfeiffert 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 Wollsteint believes that they are influenza bacilli.

*“Bureau and Division Reports,” Department of Health, city of Chicago,

‘Jan., 1899.
Tt “Zeitschrift fiir Hygiene,” etc., 1892, XIII.
t “Jour. Exp. Med.,” 1906, vit.

CHAPTER XVIII
MALTA OR MEDITERRANEAN FEVER

Micrococcus MELITENSIS (BRUCE); BAcILLUS MELITENSIS (BABES)

General Characteristics—A non-motile, non-flagellate, non-sporogenous,
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 read-
ily on artificial media, and which, when injected into monkeys, pro-
duced the disease.

Morphology.— Micrococcus melitensis, as Bruce called it, is a
round or slightly oval organism measuring about 0.3 uw 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 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{
at 57.5°C.

Cultivation——The best medium for its cultivation is said to be
ordinary agar-agar. After inoculating, by a puncture, 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 scattered 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 becomes 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.

When the sloping surface of inoculated agar-agar is examined by
transmitted light, the appearance of the colonies is somewhat dif-
"ferent. At the end of nine or ten days, if kept at 37°C., some of the
the colonies have a diameter of 2 to3 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

*“Practitioner,” xxxIv, p. 16

p. 161. :
Kolle and Wassermann, “Die Pathogene Mikroérganismen,” Il, p. 443.
Jour. of Hygiene,” 1904, IV, p. 157.

491

492 Malta or Mediterranean Fever

thecolonies by transmitted light, the center of eachis seen to be yellow-
ish, while the periphery is bluish-white in color. The same colonies
by reflected light appear milky-white. 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 naked eye
before the seventh day; at 37°C., before the third or fourth day.

In bouillon culture kept at 37°C., diffuse clouding of the medium
occurs in three or four days. There is noscumonthesurface. No
indol is formed. In sugar bouillon there is no fermentation.

In milk the orgainsm grows slowly without coagulation and with-
out acid production.

The growth in gelatin takes place at room temperature with

ME. ae!
Mg

Fig. 176.—Micrococcus melitensis.

great slowness, first appearing in about a month, and no liquefac-
tion of the medium occurs.

No growth takes place on boiled potato.

Plate cultures are not adapted to the study of the organism be-
cause of its extreme slowness of growth.

Bacteriologic Diagnosis.—The specific agglutinative effect of the
serum can be made use of for the purpose of diagnosis. This has
been studied by Wright,* Birt and Lamb,f{ and later by Bassett-
Smith.

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 1:30, never greater than 1:50, must be used. Birt and
Lamb also arrive at certain conclusions regarding the prognosis

* “Lancet,” 1897, March 6; “Brit. Med. Jour.,” 1897, May 15.

{ Ibid., 1899, u, p. 7or.
{ “Brit. Med. Jour.,” 1902, 11, p. 861.

treet

Treatment 493

based upon a study of the agglutinative phenomena. Their conclu-
sions are:

1, Prognosis is unfavorable if the agglutinating reaction is persistently 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 con-’
valescence is favorable.

4. A long illness may be anticipated if the agglutination figure, at first high,
decreases considerably.

The agglutination reaction appears early, is available by the
end of the first week, and often persists 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, goats, dogs, horses,
asses, and mules, when agar-agar cultures are injected beneath the
skin.

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 followed by an
attack of the disease. Six cases of this kind in human beings have
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 means of
bacterio-vaccines has been attempted with what seems to be glit-
tering results by Bassett-Smith. ¢

Sanitation.—The report of “British Government Commission for
the Investigation of Mediterranean Fever,’ published by the Royal
Society, April, 1907, has greatly elucidated our knowledge of the

_ pathogeny 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 specific 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 percentage of the
animals were infected with the specific cocci. The commission has,
therefore, concluded that it is by goats’ milk that the disease is com-
monly 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 4o per cent. of the goats of Malta gave the serum
teaction, showing that they had had the disease, while 10 per cent.

*See Wright and Windsor, “Jour. of Hygiene,” 1902, 1, p. 413.
t+ “Journal of Hygiene,” 1907, VII, p. 115.

494 Malta or Mediterranean Fever

of them were actually secreting the cocci in their milk. The au-
thorities 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 emana-
tions, 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 directed 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 go per cent. :

‘For example, in the second half of 1905 there were 363 cases of Mediter-
ranean 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.” :

CHAPTER XIX
MALARIA

PiasmoDIUM MALARI@ (LAVERAN); PLASMODIUM Vivax (GRASSI
AND FELETTI); PLasmoprum Fatcrparum (WELCH)

MatariA, 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 de-
rived from the Latin, malus and aer, coming from the Greek djp,
air from dev, to blow. The other name, “paludism,” from the
Latin palus, 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 be-
tween 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, es-
pecially 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 remit-
tent 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 fol-
lowed by pronounced rigors in which the patient shivers violently,
his teeth chattering. The temperature soon begins to rise and at-
tains a height of 102°, 104°, or even 106°F., according to the severity
of the case. As the temperature rises the sense of chilliness disap-
pears 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 fever and head-
ache 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 in-
capacitate 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 per-

*D. Appleton & Co., New York, 1892. :
495

496 Malaria

nicious, are rapidly fatal, others develop into a chronic cachexia,
with profound anemia and complete incapacitation for physical
or.mental effort. The discovery of Peruvian or Jesuits’ bark, and
its introduction into Europe by the Countess del Cinchén, 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 im-
portant 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 thename
Plasmodium malarie, in the blood of patients suffering from the
disease. His observations were immediately confirmed, Biitschli
recognizing the parasitic nature of the bodies observed. For the
discovery he was awarded the Bréant prize.

Laveran, however, threw no light upon the source of infection, and
malaria continued to be described as a miasmatic disease.

It was, however, recognized that there were different types of
parasites corresponding to the different clinical forms of the disease,
and Golgift succeeded in correlating the various appearances of the
parasites so as to express their life cycles. But in spite of the in-
teresting and important work of Golgi, Celli, Bignami and Marchia-
fava, 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 detail, proved in the endto — -

open the door to the next important discovery. Finding that the

malarial parasites could not be shown to leave the body in any of

its eliminations, 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 mos-
quitoes that had been permitted to distend themselves with the
blood containing the parasites, Manson found that in the stomach
of the insect the peculiar phenomenon known as “flagellation,”
long before observed by Laveran, 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 in-
fection 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 destina-
tion. Ross{ accepted the opportunity that soon presented itself,

* “Acad. d. Méd.,” Paris, Nov. 28 and Dec. 28, 1880.
t “R. Accad. di Medicina di Torino,” 1885, x1, 20.
{ ‘Indian Medical Gazette,” xxxim, 14, 133, 401, 448.

Malarial Parasites 497

and, after a most painstaking investigation, the details of which are
given in a paper which can be found in the International Medical
Annual,* 1895, made the second great discovery in the parasitology
of malarial fever. 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 organisms taken up by the mos-
quito 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 mosquito 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 in-
fectious disease whose parasites divide their life cycle between man
and the mosquito, each becoming infected by the other. The only
réle of the swamp is to furnish the mosquitoes, and since these are
only more numerous where swamps are numerous, but may occur
without swamps, the not infrequent occurrence of malarial fevers
apart from swamps is also explained. Ross further discovered that
all mosquitoes are not equally susceptible of infection, and, therefore,
not all able to spread the infection. Grassi, in Italy, quickly
followed with the demonstration that human paludism was
transmitted in the same manner, and only by mosquitoes of the
genus Anopheles.

There remained, however, dne more important fact to be eluci-
dated, and one more mysterious body to be accounted for, viz., the
“flagellated’”’ body that had misled Manson. This was found by
MacCallum f to be but the spermatozoit of the male parasite. While
observing one of the malarial parasites of birds—Plasmodium dan-
liewskyi—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. In 1900 two demonstra-
tions of momentous importance were made. First, Sambon and
Low went to Italy, to one of the most pestilential parts of the
Campagna Romana, and lived there during three months of the
most malarious time of the year in a mosquito-proof house, taking
every precaution to avoid mosquitoes, and escaped infection;
second, anopheles mosquitoes infected in Italy, by biting malarial
patients, were taken to England, where they were permitted to
bite Dr. P. J. Manson and Mr. George Warren, both of whom, after
4 period of incubation suffered from malarial paroxysms and showed

*E. B. Treat & Co., New York.
t “Journal of Exper. Med.,” 1898, U1, 117.
32 :

498 Malaria

plasmodia in their bloods. What may perhaps be regarded as the
final step in the perfection of the knowledge of the parasite was
reached in 1911, when C. C. Bass* devised a method of cultivating
the parasite in its asexual stage, in vitro.

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 malariz by Laveran.

1890—Discovery of its human developmental cycle by Golgi.

1895—Discovery of the mosquito cycle by Ross.
1895—Discovery of the transmission of the human parasite by
Anopheles by Grassi.

1898—Discovery of the sexual fertilization of the parasite by
~ MacCallum.

1911—Discovery of the method of cultivation in vitro by Bass.

The interest aroused by Laveran’s original discovery gave a great
impetus to the study of hematology with special reference to para-
sites, and it soon became evident that the plasmodium was but one
of a group of similar parasites. Of these we have now become ac-
quainted with the following:

Parasite Disease Host Insect Host
Plasmodium Quartan fever. Man. Anopheles, My-
malarie. zorrhynchus,
ee Cel-
Plasmodium Tertian fever. Man. Anopheles, My-
vivax. zorrh yin|chus,
M yzomyia,
Cellia.
Plasmodium: Aestivo-autumnal Man. Anopheles, My-
falciparum. fever. zorrhynchus,
Myzomyia,
Cellia.
Plasmodium Cercopithicus. Unknown.
kochi. ;
Plasmodium Macacus (Inuus Unknown.
inui. cynomolgus).
Plasmodium Orang-outang Unknown.
pitheci. (Pithecus satyrus).
Plasmodium Brachyrus calores. Unknown
brazilianum.
Plasmodium Inuus cynomolgus Unknown.
cynomolgi. and Inuus nem-
istrinus.
Plasmodium Sparrows, canary Culex pipens.
grassii (Proteo- birds, and other
soma grassi). small birds.

Plasmodium Owls, hawks, crows, Unknown.
danliewskyi and other large
(Halteridium birds.
danliewskyi).

* “Journal of the American Medical Association ”

IQII, XLVI, 1534.

Malarial Parasites 499

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 intermediate hosts vary and may be birds or

Fig. 177.—Plasmodium falciparum. Oédkinetes in the stomach. of Anopheles
(Grassi).

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 become infected by
being bitten by infected mosquitoes, and so on, in endlesscycles.

The parasites differ but little in the details of structure and de-
velopment, 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 mw in length and
0.2 win breadth, and known 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 irreg-
ularly spherical. There is some differ-
ence of opinion whether the little bodies
are simply upon the corpuscles, as Koch . :
believed, or in the corpuscles, as the _, Fis: oe dasmodiom, fel:

é.7 48) 7 : art ae clparum. Transverse .section
Majority of writers believe, but itis an of the stomach of Anopheles,
immaterial difference, for the parasite snewine the a one
soon makes clear thatitisconsuming the Feyclopment attached to the
corpuscle. This little body is known outer surface (Grassi).
as a schizont. When stained with poly-
chrome methylene-blue, and examined under a high power of the
microscope, it appears 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 granules of a
bacillary or rounded form, presumably melanin. In a length of

500

Fig. 179.—Developmental cycle of Plasmodium vivax, the tertian malarial
parasite. Figures 1 to 17 are magnified 1200 diameters; 18 to 27, only 600
diameters: 1, Sporozoit; 2, penetration of a sporozoit into a red blood-corpuscle;
3 and 4, schizont developing in the red blood-corpuscles; 5 and 6, nuclear divi-
sion of the schizont; 7, free merozoits; 8 (following the arrows to the left to
3), merozoits entering red blood-corpuscles, and multiplying by schizogony 3
to 7; after longer continuance of the disease the sexual forms arise; 9a to 124,
macrogametocytes; 9b to r2b, microgametocytes still in the circulatory blood of
man. If the macrogametocytes (aaah are not-taken into the alimentary canal of
the mosquito, they multiply parthenogenetically (12a, 13c to 17c) and the
resulting merozoits (17c) become schizonts (3 to 7). The figures below the
dotted line represent what takes place in the alimentary canal of anopheles
(13 to 17); 13b and 14b the formation of microgametocytes; 13a and 13b, matura-
tion of the macrogametes; 15b a microgamete; 16, fertilization; 17, odkinete;

Malarial Parasites 501

time that varies—twenty-four to forty-eight hours (Plasmodium
falciparum), forty-eight hours (Plasmodium vivax), seventy-two
hours (Plasmodium malariz)—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, commonly known as spores.
Of these there are usually eight in the meroblasts of Plasmodium
malarie, from fifteen to twenty-five in those of Plasmodium vivax, _
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 parox-
ysms have occurred it may be observed that not all of the schizonts
change to meroblasts andform spores. Someremain large spheroidal
bodies or, as in Plasmodium falciparum, assume a peculiar crescentic
form and remain unchanged in the blood. These are the sexual
parasites. The female is usually the larger and is known as the
macrogametocyte, the male, the smaller, the microgametocyte. These
are the bodies which, when removed by the mosquito, lay the foun-
dation of its infection. When they are withdrawn for microscopic
examination or exposed to the intestinal juices of the mosquito, the
microgametocyte becomes tumultuous, its granules are observed to
be in a state of active cytoplasmic streaming, and suddenly there
burst forth long slender filaments, the microgametes or spermatozoits.
These correspond with the flageila 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 by “reduction division” and polar body
formation, thus fertilizing them. As the result of this fertilization a

'. gygote or odkineteisformed. 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, project-
ing into the body cavity. It grows larger and rounder, divides into
several segments, and eventually forms an odcyst 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 salivary glands, entering into the epithelial cells and taking
radial positions about the nuclei, where they remainforatime. Later,

18, odkinete on the wall of the mosquito’s stomach; 19, penetration of the gastric
epithelium by the odkinetes; 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; 1, 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).

502 Malaria

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 re-
main 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. Itis
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 in estivo-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 estivo-autumnal fever, but sometimes also those of
tertian and quartan fever undergo regressive schizogony, by par-
thenogenesis, in the patient’s blood, and without fertilization suddenly
break up into spores which enter the red blood-corpuscles and occa-
sion a relapse of the infection that had apparently spent itself.

Diagnosis of Malarial Fever.—Prior to the discovery of the mala-
rial parasites and their relation to paludism any vague febrile
process was regarded as “malarial,” but with an understanding of the
disease based upon its parasitology, they have all been ascribed to
other causes, and at present only those accompanied by the presence
of malarial parasites in the blood are called “malarial fever.”
The diagnosis is therefore clinical in that the symptoms point to
the infection, and microscopic in that the discovery of the parasite
in the blood clinches it. ;

In all suspected cases, therefore, the diagnosis hinges upon the
discovery of the parasite in the blood, and to find and recognize it
is the problem. There are various ways of accomplishing this:

1. The Examination of Freshly Drawn Blood.—A drop from the lob-
ule of the ear or from the finger is placed upon a slide, covered, and
examined directly with an oil-immersion lens. If rouleaux formation
prevents the observation of individual cells, the cover should be
pressed upon a few times with a needle, to distribute the corpuscles.
The film should be thin enough to enable individual corpuscles to be
distinctly seen. The parasites are in the red corpuscles, and accord-
ing to their ages will present the appearances later to be described.
By this means the living motile parasites can be observed to
advantage.

2. The Examination of Stained Blood Films.—For purposes of diag-
nosis this method is to be preferred as the colored parasites are more
quickly found than the live and uncolored ones. The method of

Diagnosis of Malarial Fever 503

staining is given in detail in the section of this work dealing with
“The Staining of Protozoa,” q.v.

3. Ross’s Method of Finding the Parasites When Presets in Small .
Numbers.—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 with-
‘ out 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 completion. When all of the hemoglobin has been
removed, the slide is taken out, dried, and then fixed and stained.

i
i ———i

Fig. 180.—A, large capillary tube (a) indicating place to cut; B, manner of draw-
ing out the cells and plasmodia (Bass).

There now being no red corpuscles to distract the attention or obscure
the vis‘on, the stained parasites can quickly be found.

4. Bass’ Ss Method of Concentrating the Parasites by C entrifugation.— _
Bass and Johns} withdraw 10 cc. of blood from a vein of the forearm
.and mix it with 0.2 cc. of a solution of 50 grams of sodium citrate
and 50 grams of dextrose in sufficient water to make a volume of
too cc. The blood thus prepared is placed in two centrifuge tubes
and whirled at a speed of 2500 revolutions per minute for one min-
ute. All of the plasmodia, except the small estivo-autumnal rings,
and leukocytes rise to the top of the cell sediment and are found in
the firsto.rcm. Witha large capillary pipet this “cream” is taken

* “Lancet,” Jan. 10, 1903.
\. t “Amer. Jour. of Tropical Diseases and Preventive Medicine,” 1915, 11, 298.

504 Malaria

up, the column being not more than 5 cm. in length. The tip of
the tube is sealed, the excess of glass cut off, and then the remainder,
containing the blood is placed in the centrifuge and whirled again.
A small grayish mass of leukocytes and parasites rises to the top.
The capillary tube is cut just above this layer, and the grayish mass:
removed with a fine capillary pipet is spread upon a slide and stained.
The parasites are so concentrated as to be easily found.

THE Human MaAtariAL PARASITES

There are three known forms of human malarial parasites: Plas-
modium malarie, Plasmodium vivax, and Plasmodium falciparum.
I. Plasmodium Malariz (Laveran,* 1880).—This is the smallest

Synonyms.—Oscillaria malarie pro parte, Laveran, 1881. Plasmodium var-
quartana, Golgi, 1890. Hemamceba malaria, Grassi et Feletti, 1892.
Hemamceba laverani var. quartana, Labbé, 1894. Plasmodium malarie quart.
anum, Labbé, 1899. Hzmomenas malariz, Ross, 1900. Plasmodium golgii,
Sambon, 1902. Plasmodium quartane, Billet, 1904; Celli, 1904.

of the human malarial parasites. Its occurrence is relatively infre-
quent, as is that of the quartan fever that it occasions. The schiz-
ogonic period is seventy-two hours.long, and as each is completed,
a paroxysm of the disease occurs.

The parasite, in the red blood-corpuscles, 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 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 depres-
sions 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 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 nor 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

* “Acad. de Med.,” Nov. 23, Dec. 28, 1880.

The Human Malarial Parasites 505

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
frequency of the paroxysms.

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, the larger, the macrogametocyte or
female, the other, the microgametocyte or male. Each has proto-
plasm, with a tendency to take a blue-gray color and appear uni-
formly granular, 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.

J
Fig. 181.—Parasite of quartan malarial fever: a, 6, c, d, ‘enlarging intracellular
parasites; e, f, g, h, segmenting parasites forming a distinct rosette from which
the spores separate; 4, macrogametocyte; j, microgametocyte; k, sporozoit.

II. Plasmodium Vivax (Grassi and Feletti,* 1890).—This is the

Synonyms —Oscillaria malarize pro parte, Laveran, 1891. Plasmodium var.
tertiana, Golgi, 1889. Hemameeba vivax, Grassi et Fe eletti, 1890. Hzemamceba
laverani var. tertiana, Labbé, 1894. Plasmodium malarie tertianum, Labbé,
1899. Hzmamceba malariae var. magna, Laveran, 1900. Haemamoeba malari«
var. tertiane, Laveran, 1904. Plasmodium tertiane pro parte, Billet, 1904.

most common of the malarial parasites of man, and occasions the
“benign” tertian fever. It is a large parasite, the full-grown schiz-
ont (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 aay, para-
Sites 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
ting (wher the blood is stained with polychrome methyene blue)
is outlined with blue, at one side there is a distinct blue dot, and the
center appears colorless and like a vacuole.. The dot is usually

*“Centralbl. f. Bakt. u. Parasitenk.,’’: 1890, v1I, 396; 1891, X, 449, 481, 517.

506 Malaria

on the side of the vacuole that has the thinner protoplasmic outline.
The smallest such rings usually have a diameter equal to about 14
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 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 be-
coming pale in color. It seems also as though the disk shape of

Anil 65

sat BAe _— : i ey ee
Fig. 182. : Fig. 183.

Figs. 182, 183.—Gametocytes of Plasmodium malariz: 85, The macrogametocyte;
86, the microgametocyte (Kolle and Wassermann).

the corpuscle was lost, and it had become swollen into a more spher-
ical—sometimes irregular—form. The parasite, which may still
show a relic of its original ring-form, now shows plentifully through-
out its protoplasm exceedingly fine granules of yellow-brown pig-
ment. When from thirty-six to forty hours old, all trace of the
“seal-ring”’ form disappears, the ameboid 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 forma-
tions 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 lobulated surface,
and with its pigment granules collected to form one or two rounded
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 even-

DESCRIPTION OF PLATES II AND III.

Various forms of malarial parasites: Figs. 1 to 10 inclusive, tertian
parasites; Figs. 11 to 19 inclusive, quartan parasites; Figs. 20 to 26
inclusive, estivo-autumnal parasites.

1.—Normal red blood-cell. 2.—Young tertian ring. 3.—Largeter- -
tian ring. 4.—Half-grown tertian parasite. 5.—Infected cell showing
Schiiffner’s dots. 6.—Adult tertian parasite. 7.—Beginning sporula-
tion. - 8.—Sporulation completed. 9.—Tertian microgametocyte. 10.
—Tertian macrogamete. 11.—Young quartan ring. 12.—Older quar-
tan ring. 13.—Quartan band. 14.—Older quartan band. 15.—Full-
grown quartan parasite. 16.—Mature parasite with divided chromatin.
17.—Sporulation completed. 18.—Quartan microgametocyte. 19.—
Quartan macrocyte. 20.—Young estivo-autumnal ring. 21.—Large
estivo-autumnal ring. 22.—Mature parasite. 23.—Sporulation com-
pleted. 24.—Estivo-autumnal microgametocyte. 25.—Estivo-autum-
nal macrogamete. 26.—Estivo-autumnal ovoid.

(From Deaderick, ‘‘A Practical Study of Malaria.”)

PLATE II

10 11 12

PLATE IIT

REE pow
%. ene 2 a
_ weedy oe a
13 14 15

20 21 22

23 24 25 26

The Human Malarial Parasites 507

tuates 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 peripherally or centrally situated, and colorless.
Except in this area the pigment is distributed throughout the parasite.
The larger or macrogametocyte, the female parasite, measures 10
to 14 u in diameter. It has a greenish or grayish-green or almost

Fig. 184.—Parasite of tertian malarial fever: a, b, c, d, e, f, g, Growing pig-
mented parasite in the red blood-corpuscles; #, spores formed by segmentation
of the parasite—no rosette is formed, but concentric rings of the cytoplasm divide;
4, Macrogametocyte; 1, microgametocyte with spermatozoits.

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 sparingly scattered throughout the
substance elsewhere.

The microgametocyte or male form is approximately the size
ofa red blood-corpuscle—8 to 9 win diameter. Itstains more deeply
than its mate and contains more and coarser pigment granules.

Il. Plasmodium Falciparum (Welch,t 1897).—This is the

Synonyms—Oscillaria malarie pro parte, Laveran, 1881. Hemameéba
precox, Grassi et Feletti, 1890. Laverania malarie, Grassi et Feletti, 1890.

mamozba malaria precox, Grassi et Feletti, 1892. Hamomenas precox,
Ross, 1899. Plasmodium malarie precox, Labbé, 1899. Plasmodium pre-

* Bass asserts that Plasmodium vivax produces 32 merozoits.
Article “Malaria” in “A System of Practical Medicine by American
Authors,” 1897, p. 138. ;

508 Malaria

cox, R. Blanchard, 1900. Haemamoeba. malaria var. parva, Laveran, 1900.
Plasmodium immaculatum, Schaudinn, 1902. Laverania precox, Nocard et
Leclainche, 1903.

parasite of estivo-autumnal or malignant tertian malarial fever.
It is a very small parasite, whose occurrence, even multiple occur-
rence, in the corpuscles does not change their size or shape. It
does, however, quickly change the appearance of the corpuscles,
which become polychromatophilic, and frequently show numerous
small dots—the granulations of Schiiffner—in the corpuscular
substance. a

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.

Fig. 185. Fig. 186.
Figs. 185, 186 ee of Plasmodium vivax: 87, The microgametocyte;
88, the macrogametocyte (Kolle and Wassermann).

and sometimes seem to be incompletely loka, 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 differentiated from the “tiny rings” of the tertian parasite.
As 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

* Bass asserts that Plasmodium falciparum, like Plasmodium vivax produces
32 merozoits.

The Human Malarial Parasites 509

there are no regularly occurring paroxysms. The duration of the
asexual cycle is from twenty-four to forty-eight hours.

The gametocytes are striking and characteristic oviod and cres-
centic bodies—crescents—1}4 times the diameter of a red blood-
corpuscle in length, and about half the diameter of the corpuscle

ow 3

e al :
min - oe ™ Ci
Ss 3. Tae: tf

Fig. 187.—Parasite of estivo-autumnal fever: a, b, c, Ring-like and cross-
like hyaline forms; d, e, pigmented forms; /f, g, segmentary forms; /, 7,7, crescents.

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.

SS ia

Fig. 188. : Fig. 189.

Figs. 188, 189.—Gametocytes of plasmodium falciparum: 91, The microga-
metocyte; 92, the macrogametocyte (Kolle and Wassermann).

The macrogametocytes are broader, not curved, and sometimes
are ovoidal or prolate spheriodal in shape. The pigment granules
are more widely scattered throughout the substance. The cres-
cents are most numerous after the fever has lasted for some time or
in recurrences of the fever. The life duration of a crescent is about
three weeks.

The'fever in this form of malarial infection may beintermittent

510 Malaria

with daily—quotidian—paroxysms, or with irregular paroxysms, or
the fever may be remittent. The infection 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 been successfully
cultivated in blood, prevented from coagulation, by Bass.

In the first paper, Bass* announced that the cultivation of these
parasites was made possible by the maintenance of the culture at
40°C., the selection of such an elevated temperature being based
upon the theory that in the bloods of infected human beings, there
were specific amboceptors directed against the invading organisms,
but unable to effect their destruction until complement is formed.
Complement soon appears in the drawn blood, according to Bass,
unless the temperature be sufficiently elevated to prevent it, and he
finds 40°C. sufficient for the purpose. A later paper by Bass and
Johnsf gives the details of cultivation as follows:

When blood is to be taken from a malarial patient for the purpose of cultivat-
ing the parasites, one prepares a sterile 50 per cent. solution of Merck’s dex-
trose, in distilled water, and measured into a sterilized test-tube, 1 inch in
diameter o.1 cc. for each 10 cc. of blood to be collected. The tube, which is called
the “‘defibrinating tube” is provided with a glass rod that passes through the
cotton plug to the bottom of the ube. A needle is plunged into the arm vein
of the patient, and the infected blood is permitted to flow into the defibrinating
tube until the requisite quantity has been collected. The needle is then with-
drawn, the arm dressed, and the blood gently stirred or whipped until defibri-
nated. In the process of collecting and whipping, the admixture of air with the
blood is to be avoided.

If only one generation of parasites is to be cultivated, the culture may be
grown in the defibrination tube, provided that the contained column of blood be
not greater than 1-2 inches. There is no advantage in having a deeper column
of blood, but there is danger in having less depth as under such circumstances
the parasites die before the stage of segmentation is reached. In case the column
is more than the required depth, some of the blood can be pipetted to other tubes
and several cultures made. The plasmodia grow in the top layer of the sedi-
mented cells, near the clear supernatant serum above. The thickness of the
layer of cells in which they live is said to be not more than %o of an inch.

If the cultures are to be continued for numerous generations, precautions
must be taken to exempt the parasites from the destructive activities of the
leukocytes. The method is therefore varied in this manner: The defibrinated
blood is centrifugalized until three layers are formed, clear serum above, leu-
kocytes in a thin layer below, and red corpuscles at the bottom. The clear serum
is pipetted off and filled into small culture tubes to make a column not deeper
than 1}¢ inches. Red blood-corpuscles and plasmodia are then drawn up from
the deeper part of the corpuscular layer, thus escaping the leukocytes at the top,
and planted at the bottom of each tube of serum. It is thought to be advan-
tageous to use cultures tubes with flat bottoms. A still better method is the
~ introduction of a paper disk into a half-inch tube, about half an inch below the
surface of the serum, and then place one- or two-tenths of a cubic centimeter
of corpuscles upon it. Under these circumstances all of the plasmodia are
said to grow and segment. Two or three generations of parasites grow in such
cultures, then the plasmodia begin to die out, so that if the culture is to be
perpetuated, they must be transplanted to freshly prepared blood-corpuscle

* Jour. Amer. Med. Asso., 1911, LVII, 1534.
} “Jour. Exp. Med.,” ro12, xvi, 567.

Cultivation of the Parasites 511

tubes of the same kind. The method of transplantation recommended is
very simple: a drop of the culture is drawn into a fine (not capillary) glass
pipet and then followed by about five times the volume of the fresh corpuscle
suspension. These are mixed in the pipet, care being taken not to mix air
with the blood, and are then transferred to the new media in the same manner
asin making the original inoculation. The transplantation should be done within
five hours of the time of maximum segmentation, and therefore every forty-
eight hours for the tertian and estivo-autumnal parasites. All species of the
plasmodia have been successfully cultivated by these means. The parasites
have also been grown in red blood-cells in Lock’s solution, free of calcium chlorid
and in the presence of ascitic fluid.

According to Bass and Johns, the parasites grow in the corpuscles,
not upon them as believed by Koch. They are destroyed in a few
minutes 7” vitro by normal human serum or by all the modifications
of it that they have tested. This fact, together with numerous ob-
servations of parasites in all stages of development apparently
within the corpuscles render untenable the idea of extra-corpuscular
development. Leukocytes phagocytize and destroy malarial para-
sites growing 7 vitro only when they escape from their red-corpuscle
capsule or when the latter is perforated or becomes permeable.

The substance of the malarial plasmodium is very different in con-
sistency from that of the blood-cells, and therefore they cannot pass
through the smallest capillaries like the more yielding fluid-like red
blood-cells. That the consistency of the protoplasm of the parasite
is less yielding than that of the red blood-cell is shown by the fact
that when a small quantity of a culture containing large parasites
is spread over a slide with the end of another slide, the parasites are
dragged to the end of the spread though the red blood-cells are left
behind. Large estivo-autumnal plasmodia are round or oval; the
tertian variety are more or less flattened. As a result of their un-
yielding consistency, malarial parasites lodge in the capillaries of
the body, especially where the current is weakest, and remain and
segment. In the meantime other red corpuscles are forced against
them and if in a favorable situation, one or more merozoites pass
directly into the other cells. When the segmented parasite has be-
come sufficiently broken up it can pass through the capillary into
the circulating blood where the remaining merozoites are almost
instantly destroyed.

They further observed that calcium salts added to cultures of
estivo-autumnal parasites caused hemolysis of the infected, possibly
also of non-infected red blood-cells. Such salts have no effect on
the corpuscles of normal blood, possibly because of the precipitation
of other substances from the serum. The amount of calcium neces-
sary to cause hemolysis of malarial blood is only slightly in excess
of the quantity present in normal blood and possibly might be
reached by the ingestion of considerable quantities of calcium in.
drinking water or food. They speculate that malarial hemoglobi-
nuria may be the result of the presence of an excess of calcium in
drinking water.

512 Malaria

Bass and Johns believe that quinin has no direct effect upon the
malarial parasites, but effects its curative influence by rendering the
substance of the corpuscles more permeable to the all-sufficient de-
structive influence of the serum. The quinin would then affect only
the parasites in the circulation, and not those lodged in the capil-
laries, which would not be reached until they had segmented. The
effect of quinin isesaid to be defeated by influences such as diet,
exertion, etc., which increase the dextrose content of the blood,
whereby the permeability of the red blood-cells seems to be de-
creased. It is hoped that a better understanding of the principles
involved in the treatment of malaria may result from the study of
the organism in culture by which empiricism may be exchanged for
rationalism.

Animal Inoculation.—The human malarial parasites cannot be
successfully transmitted by experimental inoculation to any of the
lower animals.

Human Inoculation.—The blood. of one human being contain-
ing schizonts, when experimentally introduced into another human
being in doses of 1 to 1.5 cc. transmits the disease. 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 synchrony of the seg-
mentation of the parasite with the occurrence of the paroxysms
seems to indicate that a toxic substance 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 destruc-
tion 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 enor-
mous, 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 activity 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 hypertrophy. The tis-
sue is pigmented. The liver and kidneys are also enlarged and
pigmented.

Prophylaxis. —_With the knowledge of the réle of the mosquito

Pathogenesis 513

in the transmission of malaria, its prophylaxis becomes a matter
of simplicity when certain measures can be systematically carried
out. There are two equally important factors to be considered—
the human being and the mosquito. The measures must be di-
rected toward preventing each from infecting the other.

1. The Human Beings—In districts where malarial fever pre-
vails, 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 aunt to sleep in mosquito-

y |
Fig. 190.—Anopheles maculipennis: Adult male at left, female at right (Howard)

proof houses under nets, and as the mosquitoes are nocturnal and
begin to fly at dusk, the patients should shut themselves in before

‘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 development of the
parasites when exposure cannot be avoided.

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

33 ;

514 Malaria

making the house mosquito-proof most of the insects can be kept
out, while those that get in can be caught and killed.

oO ¢c

Fig. 191.—Various mosquitoes in attitudes of repose: a, Culex pipiens; d, Myzor-
rhynehus pseudo-pictus; c, Anopheles maculipennis (Manson).

~.Prothorax
Mesothorax
Scutellum
Metathorax

wt
Halteres ,,

First abdominal segment + /f «Abdomen

“s+.,Basal lobes

£ / First tarsal segment
/ e ff ;

Fig. 192.—External morphology of a female mosquito (Manson).

By draining the swamps and destroying all the breeding places
in and near human habitations, the number of mosquitoes can be
greatly diminished. Fortunately this is particularly true with

Mosquitoes and Malarial Fever 515

reference to the mosquitoes most concerned—the anopheles—which
fly but short distances. 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 anopheles mos-
quitoes 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.

MosQuliITOES 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 correctly use which, however,
the student should have some familiarity with insect structure and
the general principles of entomology. The best works of reference
for this purpose, that have come under observation to the present
time are ‘‘A Text-book of Medical Entomology” by Patton and
Cragg, published by the Christian Literature Society for India,
London, Madras and Calcutta, 1913, and the ‘‘ Handbook of Medical
Entomology” by Riley and Johannsen, the Comstock Publishing
Co., Ithaca, New York, rors.

The mosquitoes comprise a family of dipterous or two-winged

‘' insects, included in the family Culicide. They can be recognized,

first by their well-known general form, and second by the presence
of scales upon some part of the head, thorax, abdomen, and wings.
For the rough and ready identification of the larger groups and
principal genera, the following table compiled from various authors
may answer. For more precise information and for the identi-
fication of the species, of which hundreds are now described, refer-
ence must be made to the large works recommended above.

CLASSIFICATION (Stitt)

al are four subfamilies of CULICIDA, differentiated according to the
pal mi
I. Palpi as long or longer than the proboscis i in the male.
1. Palpi as long as the proboscis in the female;

proboscis straight. . . . ANOPHELINE.
2. Palpi as long or shorter than the proboscis;
proboscis curved. ...22s arenes ne nas aarners MEGARRHININE
3- Palpi shorter than the proboscis............... CULICINE.
II. Paipi shorter than the gies in the male and
female.. os . EDINE.

Of these the Anophelinze i is the one “family concerned in the transmission of
malarial fever, so that it is important to be able to differentiate the genera in-
cluded in the family.

ANOPHELINE
1. Scales on head only; hairs on thorax and abdomen.

I. Scales on wings rs and lanceolate. wel oaly
slightly scaled.. Hie F . Anopheles.

516 * Malaria

2. Wing scales small, narrow, and lanceolate. Only
a few scales on palpi.............-...02.2...Myzomyia.
3. Large inflated wing scales.............0ee ee eeeee Cyclolepteron.
2. Scales on head and thorax. Scales narrow and
curved. Abdomen with hairs, not scales. :
1. Wing scales small and lanceolate................. Pyreto phorus.
3. Scales on head, thorax, and abdomen. Palpi covered
‘with thick’scales.
1. Abdominal scales on ventral surface only. Thoracic
scales like hairs. Palpi rather heavily scaled... . Myzorrhynchus.
2. Abdominal scales narrow, curved or spindle :

shaped, in tufts and dorsal patches............Myssorrhynchus.
3. Abdomen almost completely covered with scales

and also having lateral tufts..................Cellia.
4. Abdomen completely scaled............0.0000000- Aldrichia.

Species of the genera Anopheles, Myzomyia, and Myzorrhynchus, are known

to transmit malarial parasites. The Culicine include Stegomyia and Culex,

. which have some medical interest, as the former transmits yellow fever and the
latter, filarial worms.

CULICINE
I. Posterior cross-vein nearer the base of the iis than the mid-cross-vein.
1. Proboscis curved in the female.. dene ala . .Psorophora.
2. Proboscis straight in the female: \

A. Palpi with three segments in the female.
a. Third segment somewhat longer than
the first twos: dsaesssivesess oacan se 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 pro-
boscis. Spotted wings.............. Theobdaldia.
b.. Palpi longer than the third of the Brakes :
cis. Irregular scales on the wings. ...Mansonia.
C. Palpi with fine segments in the female....... Teniorrhynchus.
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. . 5 abe wyate . Mucidus.

Male mosquitoes can at once be recognized by the pennate
antenne which appear like plumes on each side of the head. They
commonly “swarm” in flocks, do not suck blood, and are not com-
monly found in or about human habitations. Comparatively little
is known of their habits. Cohabitation of the sexes occurs but once
after which the males commonly die. The females after fecunda-
tion require a meal of blood before they become gravid and ready to
oviposit. Oviposition takes place in water. During the winter
many gravid females hibernate in cellars in a very inactive condition,
but are immediately ready to fly to appropriate places and lay their
eggs with the return of warm weather. In hot climates some of
them estivate—i.e., become similarly inactive during the dry
period, but are ready to fly to the water and oviposit as soon as the
rains begin again. The breeding places vary with the species.
Fresh water is the usual preference, but a few select pools of brack-
ish water, and one or two species prefer salt water. Most of the
malaria-bearing species of anopheles prefer pools of fresh clear
water, some prefer running water in small streams with a slow cur-
rent. A few breed in large rivers. Some species aré notably domes-

Mosquitoes and Malarial Fever . 517

tic and oviposit in wells, cisterns, water-butts, cans and any other
available collection of water.

The eggs are laid as the female hovers upon the surface, touch-
ing the water from time to time, with the tip of the abdomen, each

Fig. 193.—Pupa of Anopheles maculipennis (Brumpt).

es
,, Brushes

“s Silky bristles

Abdomen

= . Chitinous combs
| ~ .. Stigmata .
y Anal papille
la \% ger _ Large bristles

Fig. 194.—Larva of Anopheles maculipennis (Brumpt).

time depositing an egg. Culex eggs are fastened together side by
side to form a kind of minute raft, but anopheles eggs are laid singly:
and float away independently of one another. If at the time the
eggs are laid the waters are receding, they may catch upon the leaves

518 Malaria

and stems of plants, and remain alive until the waters rise again, be-
fore hatching. Dry eggs are sometimes able to remain alive for long
periods, and may even be frozen without being killed. Cazeneuve
hatched eight larve from eggs obtained by thawing a block of
ice taken from a swamp in North China, where the temperature

Fig. 195.—Method of withdrawing the digestive tube of the mosquito for
study (Blanchard.)

had gone as low as —32°C. When conditions are favorable the
eggs hatch in two or three weeks. The anopheles larve feed at
the surface of the water along the banks where they are protected by
thevegetation. They are voracious feeders and satisfy their appetites

Fig. 196.—Method of withdrawing the salivary glands of the mosquito for
study (Blanchard).

with all kinds of minute vegetable and animal organisms or rem-
nants. In a day or two the larve molt for the first time. In
five or six days, having grown larger, they molt a second time and
pupate. The appearances of the larve and pupe are shown in the
accompanying diagrams. The pupa floatsat the surface of the water,
is comparatively inactive and does not feed. If disturbed, it
is capable of swimming vigorously to escape. In about three days
the imago issues and is ready to fly. Anopheles do not fly great
distances; a few hundred yards is the common range of their activi-
ties. They do not always return to the same pools from which they
issued, any similar pool or stream is good enough for oviposition.

Mosquitoes and Malarial Fever 519

After having deposited the first lot of eggs, the female is ready to feed
again and produce a new lot. This can go on for a number of broods.
How long the insects can live, probably depends upon their activities.
When actively engaged in reproductive activities they probably
live a shorter time than when hibernating or estivating. It is known
that some of them can live the greater part of a year.

The mosquitoes used for study and for classification should be
mounted dry in the usual way well known to all entomologists.

eis 2 ‘ ones

Fig. 197.—Imago of Anopheles maculipennis escaping from the pupa case upon
the surface of the water (Brumpt).

Fine entomologic pins (00-000) 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 sawdust, 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 entomologic forceps, touching the pins
only, Every insect thus mounted should have placed upon the pin, at the junc-
tion of the middle and lower thirds, a small bit of card or 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, and
the mode of pulling the insect apart to show them can be learned from the dia-
gtam. 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 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, embedding the insect in paraffin
and cutting serial longitudinal vertical sections.

To iafect 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 smail 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, kept as many days as may be desired, then killed and
sectioned. In this way, remembering that the entire mosquito cycle of develop-
ment takes about a fortnight, any stage of the cycle may be observed. :

CHAPTER XX
RELAPSING FEVER

SPIROCHZTA RECURRENTIS (LEBERT); SprrocH#zTA DuTTONI,
Novy AND Knapp; SprrocHzZTA Novyi, SCHELLAK;
SPIROCHZTA CARTERI, MACKIE

General Characteristics.—An elongate, flexible, flagellated, non-sporogenous,
actively motile spiral organism, pathogenic for man and monkeys, susceptible
of cultivation in special media, stained by ordinary methods, but not by Gram’s
method.

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 spirocheta rarely played any
pathogenic réle. Millerf had, indeed, called attention to the con-
stant presence of Spirocheta dentinum in the human mouth, but

it had not been connected with any morbid condition. In 1890
Sacharoff{ discovered a spirillary infection of geese in the Caucasus,
caused by an organism much resembling Spirocheta obermeieri
and called Spirocheta anserinum. In 1903 Marchoux and Salim-
beni§ found a third disease, fatal to chickens, caused by Spirocheta
gallinarum, and found that the spread of the disease was determined
by the bites of a tick, Argas miniatus. In 1902 Theiler,|| in the Trans-
vaal, observed a spiral organism in a cattle plague. This has been
named after him by Laveran, Spirocheta 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 spirochetal infections were fairly well dis-
seminated among the lower animals and that the spirocheta were
of different species with different hosts and intermediate hosts.

In 1904 Ross and Milneff and Dutton and Toddtt studied a
peculiar African fever which they were able to refer to a spirocheta

*“Centralbl. f. d. med. Wissenschaft,’ 1873.
t Micro-organisms of the Human Mouth, Phila., 1890, p. 44 et seq.
t “Ann. de l’Inst. Pasteur,” 1891, XvI, No. 9, Pp. 564.
Tbid., 1903, XVII, p. 569.
\| Jour. Comp. Path. and Therap.,”’ 1903, XLVII., 1903, XLVI, p. 55.
** “ Compt.-rendu de la Soc. de Biol. de Paris,” July 22, 1905, LIX, p. 200.
tt “British Med. Jour.,” Nov. 26, 1904, p. 1453.

tf“ Memoir xvu, Liverpool School of Tropical Medicine,” “Brit. Med. Jour.,”
Nov. 11, 1905, p. 1259.

525

Relapsing Fever 521

for which Novy* has proposed the name Spirocheta duttoni in
memory of Dutton, who lost his life while studying it. In 1905
Koch} while working in Africa discovered a spirocheta that he re-
garded as identical with that already described by Ross and Milne
and Dutton and Todd. Later studies of the organism convinced
C. Frankel{ that it was a separate species. For it Novy later sug-
gested the’ name Spirocheta kochi. In 1906 Norris, Pappenheimer
and Flournoy§ found a spirocheta in the blood of a patient suffering
from relapsing fever in New York. This having been extensively
studied by Novy, has since been called Spirocheta novyi.

With the work of Schaudinn and his associate, Hoffman,|| the
spirocheta 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, the rdéle of an intermediate
host (ticks, etc.) in transmitting them, and the longitudinal mode of
division.

Fevers characterized by relapses and by the presence of spirocheta

Fig. 198.—Spirocheta recurrentis from human blood (Kolle and Wassermann).

in the blood have been found in northern and northeastern Europe
(true relapsing fever with Spirocheta recurrentis), in various parts
of equatorial Africa (African relapsing fever with Spirocheta dut-
toni); in North Africa (Spirochzta berbera); in Bombay and in other
parts of India (Spirochzta carteri); in Persia (Spirocheta persica);
and in America (Spirocheta novyi). The question, therefore, arises
whether these similar diseases are slight modifications of the same

* “Tour. Infectious Diseases,’ 1906, Il, p. 295.

Deutsche med. Wochenschrift,” 1905, xxxI, p. 1865; “Berliner klinische

ochenschrift,” 1906, XLII, 185.

1 “Med. klin.,” 1907, m1, 928; “Miinchener med. Wochenschrift,” 1907, LIV,
201,

i “Jour. Infectious Diseases,” 1906, 111, 266.

| “Deutsche med, Wochenschrift,” Oct., 1905, XXXI, p. 1665; “Arbeiten aus
dem kaiserlichen Gesundheitsamte,”’ 1904, XX, pp. 387-439-

522 Relapsing Fever

thing caused by the same parasite, or whether they are different
diseases caused by slightly different parasites.

If Nuttall be correct, there are no adequate grounds upon which
to conclude that the spirochetes are really different species. On
this account, and as the differences between the organisms are
minute, it scarcely seems well to devote space to the consideration
of each, but better to select the oldest and the best known—Spiro-
cheta recurrentis—as the type, describe it, and then point out such
variations as are shown by its close relations.

Morphology.—The Spirocheta recurrentis is extremely slender,
flexible, spirally coiled, like a corkscrew, and pointed at the ends.

* Fig. 199.—Spirocheta recurrentis (Novy). Rat blood No. 321a. X 1500.

It measures approximately 1 » in breadth and 10, 20, or even 40 4
in length. The number of spiral coils varies from 6 to 20; the di-
ameter of the coils varies so greatly that scarcely any two are uni-
form. 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, consisting of
rotation about the long axis and serpentine flexions. No structure
can be made out by our present methods of staining and examining
the spirocheta. No spores are found. Multiplication is thought
to take place by longitudinal division, though some believe the di-
vision to be transverse.

*“Kolle and Wassermann’s Handbuch der pathogene Mikroérganismen,”
1903, Il, p. 82.

Cultivation 523

The Spirocheta duttoni is said by Koch,* in his interesting
studies of “African Relapsing Fever,” to resemble the Spirocheta
recurrentis in all particulars.

The Spirocheta novyi with which Novy and Knappt experi-
mented, and which they believed to be identical with Spirocheta
obermeieri, measured 0.25 to 0.3 win breadth by 7 to 19 win length.
The number of coils varies from three to six. The shorter forms are
pointed, with a long flagellum at one end and a short one at the
other.

Staining —The spirocheta can be stained with ordinary anilin
dye solutions, by the Romanowsky and Giemsa methods, and by
the silver methods (see Treponema pallidum). It does not stain
by Gram’s method.

Fig. 200.—Spirocheta duttoni (Novy). Tick fever, No. 520. Rat blood.
X 1500.

Cultivation.—Following the suggestion of Levaditi, Novy and
Knappt cultivated Spirocheta obermeieri in collodion sacs in the
abdominal cavity of rats, and succeded 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 cor-
puscles had been removed by centrifugation and so proved that
no intercellular developmental stage of the organism takes place.
Organisms thus cultivated attenuate in virulence.

_ Norris, Pappenheimer, and Flournoy§ believe that they succeeded
M securing multiplication of the spirocheta by placing several drops
* “Berliner klin. Wochenschrift,” Feb. 12, 1906, xxxrv, No. 7, p. 185.
t “Jour. Infectious Diseases,” 1906, III, p. 291.

1 “Jour. Amer. Med. Assoc.,” Dec. 29, 1906, XLVII, p. 2152.
§“Journal of Infectious Diseases,” 1906, 111, 266.

524 _ Relapsing Fever

of blood containing them in 3 to 5 cc. of citrated rat or human
blood. A third generation always failed.

Noguchi* was the first to achieve the successful cultivation of
the spirocheta in artificial culture-media. The best success was
obtained as follows: Into each of a number of sterile test-tubes
2 X 20 cm. in size is placed a fragment of fresh sterile rabbit kidney
and then a few drops of citrated blood from the heart of an infected
mouse or rat. Following this, about 15 cm. of sterile ascitic or
hydrocele fluid are quickly poured into the tubes and the contents
of some of the tubes are covered with a layer of sterile paraffin

oil, while the rest are left without the oil.. The tubes are placed in _

the incubating oven at 37°C. By these means cultures of Spirocheta
duttoni, Spirocheta kochi, Spirocheta recurrentis and Spirocheta
novyi were secured. The maximum growth was obtained in 7, 8 or
g daysat37°C. The presence of some oxygen seemed to be essential.
By transplantations to fresh media of the same kind they were
all kept growing for many generations during which they did not lose
their virulence.

Mode of Infection.—The means by which Spirocheta recurrentis
is transmitted from individual to individual is not definitely known.
Tictint 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 prede-
cessors, were unable to infect monkeys by permitting infected bed-
bugs to bite them.

Mackie,§ Graham-Smith,|| Bousfield,** Ed. Sergent and H.
Foley, tT studied the louse and found that it was undoubtedly capa-
ble of acting as a transmitting agent, and possibly was the only de-
finitive host of the parasite. Nicolle, Blaizot and Conseil{{ studied
the North African relapsing fever of Tunis and Algeria, and proved
that the body and head lice are undoubtedly the common definition
hosts of its spirochaete. When the lice were fed upon blood of in-
fected patients, the spirochetes rapidly disappear in their bodies, but
after eight days reappear and remain for almost twelve days during
which time the insects can transmit thedisease. They alsofound that

* “Journal of Experimental. Medicine,” 1912, XVI, 199.
+ “Centralbl. f, Bakt. u. Parasitenk., av 1894, 1 Abt. , XV, p. 840.
Tt Ibid., Oct., 1906, xxu, Heft 6, p. 537.
§ “Brit, Med. Jout., > Dec. 14, 1907.
| Ann. de l’Inst. Pasteur, ”” TOIO, p. 63.
** Report of the Wellcome Tropical Research Laboratories, 1911, p. 63.
tt ‘Ann. de l’Inst. Pasteur,” 1910, p. 337.
ti“ Ann. de l’Inst. Pasteur,” Mar. 25, 1913, vol. xxvu1, No. 3, p. 204.

Mode of Infection Cea

‘the infectious agent passes to a new generation of the lice, which are
also infective. They also studied a tick, Ornithodorus savignyi,
found in those countries, thinking that it might behave like Ornith-

-odorus moubata toward Spirocheta duttoni, and found that it
could transmit the spirochete of the Tripolitan relapsing fever,
though apparently not that of the Tunisian fever.

When we come to consider Spirocheta duttoni, however, we find
our knowledge much further advanced. On Nov. 26, 1904, Dutton
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. Dutton
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 Dutton and Todd, then
working on the Congo: “Spirilla cause human tick fever; naturally
infected ornithodorus infect monkey.” It was not until Nov. 11,
‘1905, that the paper upon the subject was read and published in
the same journal by Dutton and Todd, and the etiology of the dis-
ease made clear. These observers found the horse-tick, Ornith-
odorus moubata (Murray) is the intermediate host of the spirilla
or spirocheta 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, who examined the ticks, observed the distribution of the
micro-organisms in their bodies, and found that they collected in
large numbers in the ovaries, so that the eggs were commonly in-
fected and the embryo hexapod ticks hatched from them were in- —
fective. Not only is this second generation of ticks infected, but
Mller has found the third generation also infected by the spiro-
cheta, and it is not improbable that the infection is kept on passing
from female to offspring through many generations. Leishman,
who followed the spirocheta throughout the body of the tick,
observed that it entered the ovaries and appeared in the ova in the
spiral form, but that in the ova it not infrequently became trans-
formed to “coccoid” granules which held together more or less
closely like tiny streptococci. He supposed that it was in the
granular form that the micro-organism found its way into the embryo
and so infected the developing nymph. There is reason to believe
that this was an error and that the spirals alone are the sources of

. transmission and infection. What is true of the tick seems to be

equally true of the lice, the infective micro-organisms being passed

down from generation to generation. How the ticks and lice effect
the transmission of micro-parasites is to a certain extent in dispute.

It was at first supposed that the spirochetes entered the human

* «Brit. Med. Jour,”’ Nov. 26, 1904..
ft “Berliner klin. Wochenschrift,”’ Feb. 12, 1906.

526 _ Relapsing Fever

hosts with the saliva of the respective arthropods, but there is some
reason to think that this is a mistake, and that the scratching of the
itching bite conveys the spirocheta deposited upon the skin in the
excrement of the arthropod, into the deeper layers and lymphatics
through which it reaches the blood.

Pathogenesis.—The spirocheta of relapsing fever are pathogenic
for man and monkeys, some of them for smaller animals. Novy
and Knapp* found their organism and Spirocheta duttoni to be
infectious for mice and rats, and attribute the failure of others to
discover this to their failure to examine the blood during the first
and second days. Fulleborn and Meyer, and Nartin} were able
successfuly to transmit the spirocheta 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 of the disease 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 characteristic.
After a short incubation period the invasion comes on with chill,
fever, headache, pain in the back, nausea and vomiting, and some-
times convulsions. The temperature rises rapidly and there are
frequent sweats. The pulse is rapid. By the second day the tem-
perature may be 104° to 105°F. and the pulse r1o to 130. There
is enlargement of the spleen. Icteroid discoloration of the conjunc-
tiva 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 fourteenth day the relapse characteristic
of the affection makes its appearance as an exact repetition of what
has gone before. This is followed by another apyretic interval,
and then by another relapse, and so on. The patient usually re-
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 second or third relapse,
the African not until after a greater number.

Lesions.—There are no lesions characteristic of pelandine fever.

Bacteriologic Diagnosis.—This should be quite easily made by
an examination of either the fresh or stained blood, provided the

* Loc. cit. Tt Loc. cit.

The Vectors of Relapsing Fever 527

blood be secured during a febrile paroxysm. 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 im-
portant. At the moment of decline of the fever a powerful bacterio-
lytic substance appears in the blood and dissolves 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 in-
jecting the animal with blood containing spirocheta. Serum con-
taining 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.

THE VECTORS OF RELAPSING FEVER :
I. Ticks

The ticks thus far known to act as vectors of relapsing fever are two species of
the genus Ornithodorus. Thirteen species of this genus are described in “A
Text-book of Medical Entomology,’’ by Patton and Cragg, who give excellent
tables for their identification and additional valuable information is to be found
in the excellent ‘“‘ Monograph of the Ixodoidea,’’ by Nuttall. Ornithodorus ticks
of various species are to be found pretty widely distributed throughout tropical
and semitropical regions of both hemispheres. In general, they are most numer-
ous where the temperature is highest and the soil driest.

The genus Ornithodorus was described by C. L. Koch and characterized as
follows: ‘‘The body is flat when starving and convex when replete, and may be
nearly as broad anteriorly as posteriorly, or pointed and beak-like anteriorly.
The margin of the body is not distinct but is of a similar structure to the rest of
the integument which is generally mamillated. On.the ventral surface there are
two well-marked folds, one internal to the coxe, the coxal fold, and the other
above the coxz, the supracoxal fold; there is also a transverse pre-anal groove,
as well as a transverse postanal groove. Eyes are either absent or present in
pairs on the supracoxal fold; one pair between coxe I and II, and the other
between coxe III and IV.

The Ornithodorus savignyi is the transmitting agent of Spirocheta berbera;
Ornithodorus moubata of Spirochzta duttoni.

Ornithodorus savignyii—The description given by Patton and Cragg (“A
Text-book of Medical Entomology,” 1913 p. 586) is as follows: Integument
leathery and covered by distinct non-contiguous mammille and numerous short
hairs interspersed. Supracoxal folds well marked, with two eyes one each side.
Coxal folds less well marked. Pre-anal groove distinct. The basis capituli
broader than long and shorter than the rest of the rostrum. Hypostome with
six principal rows of teeth, the external the stoutest. Palps with first and second
Segments of equal length, third segment the shortest. Cox contiguous; pro-
tarsus and tarsus of legs I, II, and III with three well-marked humps; the|two
proximal humps on tarsus of leg IV are close to each other, while the thirdis
separated by an interval of about two and a half times the distance between the
first and second. :

Length 5-12 mm. Width 4-8.5 mm. The female and male resemble each
other except that the latter are smaller. Its genital orifice is markedly smaller.
In the female the genital orifice is a broad transverse slit which can be made to
gape and is guarded by two flaps like valves; in the male the orifice is oval and

528 Relapsing Fever

the valves are absent. The eggs number 50-100, measure 1.3-1.5 mm. in length
and 0.8-1 mm. in breadth. They are oval, smooth and of a dark brown or black
color.

Habitat.—Arabia, Nubia, Egypt, Somaliland, Abyssinia, German East Africa,
Cape Colony, Rhodesia, Bechuanaland and Portuguese East Africa. In India it
is common in the Madras Presidency, in Gujarat, and in many parts of the Bom-
bay Presidency. In Aden it is widely distributed throughout the Hinterland,
where its principal host is the camel.

Ornithodorus moubata.—Patton and Cragg describe this tick as follows: Body
almost as broad anteriorly as posteriorly; covered with non-contiguous mamille‘

Fig. 201.—Ornithodorus moubata. Tick that transmits African relapsing fever:
a, Viewed from above; 6, viewed from below (Murray from Doflein).

an f

Fig. 202.—Ornithodorus savignyi. An, anus; cam, camerostome; cz.I., coxa
I; cx, coxa II; cw.JII, coxa IIT; cx.IV, coxa IV; cx.f., coxal fold; e, eye; g.c.,
genital aperture; g.g., genital groove. :

but with fewer hairs than savignyi.’ Basis capituli broader than long and shorter
than the palps; hypostome with six principal rows of teeth. Tarsi of legs I, II
and XII with three humps as in savignyi; those on the pro-tarsus are subequal,
more pointed and about equidistant, while those of savignyi are unequal, less
pointed and not equidistant. The tarsus of leg IV in moubata is shorter and
thicker than in savignyi, and its humps are nearly equidistant. Eyes absent.
Length 8-12 mm.; breadth 6-1o mm. The eggs are ovoid, measure 0.8-0.9 mm.
in length, are smooth on the surface and dark yellow in color.

Habitat.—Africa: from British East Africa to the Transvaal, and across to the
Congo; southward to German East Africa and Cape Colony. It is common in
Egypt, Abyssinia and in parts of Somaliland and in Portuguese East Africa.

The Vectors of Relapsing Fever 529

Female Male

Ovum or nit Embryo
Fig. 203.—Pediculus capitis, or head-louse. X10. a, Female; b, male; c, egg
cemented toa hair; d, nymph. (From Beattie and Dickson’s ‘“‘A Text-book of
General Pathology,” by kind permission of William Heinemann, Publisher.)

Male ' Female

Embryo Ovum

Fig. 204.—Pediculus vestimenti, the clothes or body louse. X 10. a, Male;
}, female; c, nymph; d, egg. (From Beattie and Dickson’s “(A Text-book of
General Pathology,” by kind permission of William Heinemann, Publisher.)

530 Relapsing Fever

Ornithodorus savignyi is chiefly a parasite of the camel and only occasionally
bites man; Ornithodorus moubata is essentially a human pest.

The eggs of these ticks hatch in eight to fourteen days. The larval stage which
has six legs is spent in the eggs and the creature that emerges is usually a first
nymphalinston, which has eight legs. After hatching it remains inactive for
several days, then becomes very active and ready to suck blood. As it grows it
becomes voracious, distending itself with blood, then dropping off, hiding itself
for a time, molting, then being ready to feed again. This continues for a number
of months, the ticks molting four times before passing from the nymph to the
adult stage. ;

Ornithodorus moubata is a common inhabitant of the native African huts
along the caravan routes. To avoid it and escape relapsing fever R. Koch in his
African expedition camped near but not in the villages, and avoided the native
houses. It lives in the cracks in the mud walls, in the thatch, in the mats and
sometimes simply upon the ground where its small size and dull color make it diffi-
cult to see. From these hiding places it crawls at night and like a bed-bug

on a

|
Bee PAW Secs | | ise lata oN Fle ist 4

Male Female
Fig. 205.—Pediculus pubis, Phthirius inguinalis or crab-louse. X17. (From
Beattie and Dickson’s “A Text-book of General Pathology,’ by kind permission
of William Heinemann, Publisher.) ‘

attacks the sleeping host. When handled it feigns death, remaining quiet for
so long a time that it is hard to believe it alive.

The Ornithodorus savignyi is less adapted to the requirements of the spiro-
cheta than. its relative. Brumpt* found that the spirocheta did not pass
through the eggs of O. savignyi to subsequent generations, and that the infectivity
of the tick itself soon was lost. The spirochete remain indefinitely in O. mou-
bata, and are passed through their eggs to at least three generations. It is,
therefore, difficult to be certain that any particular tick is uninfected unless its
progenitors be known.

The spirochzta pass from female to the ovum and infect the young nymphs as
such. The granules observed in the eggs of infected ticks, also occur in those of
non-infected ticks and have nothing to do with the spirocheta.

II. Lice

Lice are apterous insects formerly classed in the order Hemiptera, but now
placed in a separate order, the Anoplura. Two genera, and three species are
common upon human beings. a

I. Pediculus (Linn, 1758). In this genus there are two species:

1. Pediculus capitis (de Geer, 1778). This is the head-louse. It is of a
gray color. The abdomen is composed of eight and not of seven seg-
ments as was stated by Piaget, and is blackened along the edges. The

* “Precis de Parasitologie,”’ 1910, 538.

The Vectors of Relapsing Fever 531

males and females look much alike, but the male measures 1.8 mm. in
length and o.7 mm. in breadth, while the female measures 2.7 mm, in
length by 1 mm. in breadth. ;

These parasites live in the hair, close to the scalp. Rarely they pass
from the scalp to the beard. Still more rarely do they occur upon other
hair-covered surfaces. The female produces large eggs, one at a time,
which are firmly anchored to the hairs by a mucilaginous secretion.
In them the embryo develops in about sixteen to eighteen days then
escapes as a nymph with proportionally smaller body and larger legs
than the adult. There are three molts before the insect reaches matur-
ity. The full and empty eggs occur in great numbers upon the hairs
and are known as “nits.”

The insects are sometimes present on the head in great numbers and
cause intolerable itching.

2. Pediculus vestimenti (Nitzsch, 1818). This is a larger louse of much the
same appearance and structure as P. capitis. Indeed there are such
minute differences between the two that there is some dispute as to
whether they should not form subspecies of the same insect instead of
different species of insects.

The size is, however, larger. The male theasures 3 mm. in length
and 1 mm. in breadth; the female 3.3 mm. in length and 1.14 in
breadth. ;

The “body louse” as this is commonly called, lives in the clothing and
passes to the skin to feed, then returns again to the seams of the garments.
Its eggs are fastened to the fabric of the clothing, not to the skin or hairs. It is
sometimes present in great numbers and its bites cause much annoying itching.

Both of these lice have been found to be capable of effecting the transmission
of the spirocheta of relapsing fever. The infection in the lice is transmitted to
their offspring. .

II. Phthirius (Leach, 1815). In this genus there is only one human parasite.
Phthirius inguinalis (Ridi, 1668). This pubic louse or ‘‘crab louse” is
often incorrectly called Pediculus pubis. It is a shorter, stouter-
bodied creature with more powerful legs terminating in large tarsal
hooks that give it a crab-like appearance. The thorax and abdomen
are compressed and shortened to a heart-like body. The abdomen is
composed of six segments, each of which has a pair of stigmata, but
the stigmata of the first, second, third, fourth, and fifth segments ap-
pear to be in one broad segment. The males measure 1 mm. in length,
the females 1.5mm. These lice live chiefly in the pubic hair and that
of the perineum. Rarely they are found in the axilla, the beard, the
eyebrows and even upon the eye-lashes. The eggs are fixed to the
bases of the hairs as in P. capitis. They hatch in about seven days and
the nymphs grow to maturity fifteen days later.

The bites of these lice are very irritating and cause severe itching and
the eruption cf pink papules that sometimes become bluish spots nearly
a centimeter in diameter. Such spots known as “taches ombrées” are
frequent in tyhoid fever when lice are present.

It is not known that this louse can harbor spirocheta or any patho-
genic bacteria or protozoa. :

CHAPTER XXI
INFECTIVE JAUNDICE; WEIL’S DISEASE;
SPIROCHATOSIS ICTEROHEMORRHAGICA

SPIROCHATA IcTEROHEMORRHAGIE (INADA AND Ino)

General Characteristics.—A minute, slightly bent, irregularly coiled, inactively
motile, flagellated spiral organism, capable of cultivation by special means in
special media. It is aérobic, non-chromogenic, non-aérogenic, non-sporogenic, and
is pathogenic for guinea-pigs, ratsand man. It can be stained by certain methods
only and not by Gram’s method.

Occasional epidemic outbreaks of jaundice have been noted since
the time of Hippocrates, by whom they were mentioned, and have
received mention from the pens of many eminent writers. Lance-
reaux spoke of the disease as ‘‘Ictére grave essentiel;”’ Landouzy as
“Fiévre bileuse;”’ Mathieu as “Ictére febrile 4 rechutes” on account
of the frequency of relapses. In 1886 Mathieu described cases and
pointed out that “catarrhal jaundice” was an inadequate name for
the affection as the severity of the fever, the severe constitutional
symptoms, the enlargement of the spleen and the occurrence of
albuminuria justified the name infectious jaundice. In the same
year Weil* described four cases, two of which suffered from relapses,
and from the time of the appearance of his contribution the disease
has been known as Weil’s disease. :

_The disease is characterized by a sudden onset with occasional
vomiting, malaise sometimes amounting to severe prostration,
muscular pains sometimes of great severity, fever ranging from 103°
to 105°F. and lasting for a number of days during which the tongue
becomes dry and brown and herpes hemorrhagica appear about the
lips in about half of the cases. About the fourth day jaundice
appears, deepening until its greatest intensity is reached by the ninth
day, and continuing until the twelfth day. There is usually consti-
pation. Bile-pigments, albumen and tube casts appear in the urine.
In severe cases epistaxis, hemoptysis, hematemesis, melena, and
subcutaneous hemorrhages occur. In nearly all cases lymphatic
enlargements are present. Hume and Bedsonf point out that jaun-
dice is not always present in otherwise typical Weil’s disease. Under
these conditions it is difficult to recognize and may figure as “Fever
of unknown origin,” and be one form of what is sometimes called
“Trench fever.”

* “Deutsches Archiv. f. Klin. Med.,” 1886, xxxIx, 209.
t “Brit. Med. Jour.,” Sept. 15, 1917, p. 345.
532

Morphology . 533

In 1914 Inada and Ido began the study of Weil’s disease in Japan
and in 1915 made the first report upon the discovery of the causal
organism.* By means of intraperitoneal injections, they succeeded
in transmitting the disease to guinea-pigs in whose blood and tissues
it wasfound. Uhlenhuth and Fromme,} Hiibener and Reiter,t and
Stokes and Ryle§ confirmed the observations and achieved similar
results. Inada, Ido, Kaneko and Itolj injected the blood of eighteen
cases into the abdominal cavities of guinea-pigs and had thirteen
successes—i.e., in that number of cases the guinea-pigs showed con-
junctival hemorrhage, jaundice, fever and albuminuria. In all of
these animals they found in the blood, various organs and urine, a
spiral organism for which they suggested the name Spirocheta
icterohemorrhagice.

Morphology.—The organism is a closely wound. cylindrical
thread with gradually tapering ends. Its measurements vary.

Fig. 206.—Weil’s disease. Spirochete excreted in the urine of patients (Inada
Ido, Hoki, Kaneko and Ito).

According to Noguchi** it averages 9u X 0.25 u. In cultures, how-
ever, it may vary from 3 4 to 4o in length. The number of coils
Is greater in a given length than in any other known spirocheta,
there being to~r2 coils in 5 » of length. The coils are closest near
the ends. They are never deep, and not seen very distinctly, the
appearance being somewhat like a transversely barred chain of
streptococci.

Motility —When viewed under a dark field illuminator the move-
ment and position are characteristic. Active specimens show a
. *“Tokyo Ijishiushi,” 1915, No. 1908. A full bibliography of their writings
is to be found in the Journal of Experimental Medicine, 1916, xxu1I, p. 400.

t “Med. Klinik,” 1915, XI, 1202. '
Deutsche med. Wochenschrift,” 1915, XL1, 1275.
§ “Brit. Med. Jour.,”’ Sept. 25, 1916, 1, 413.
.! “Jour. Exp. Med.,” 1916, xxi, 377.
‘Jour. Exp. Med.,” 1917, XXV, 758. ~

534 Infective Jaundice

straight body with one orboth ends curved in the form of a semicircle,
the length of the hook at the end varying from 3-5 wu. In motion the
organism, without relaxing its elementary windings, rotates upon its
axis two to four times per second, giving the impression of a drawn
out figure eight. The movement is bipolar and the direction al-
ternates at short intervals. When passing through a semi-solid
medium such as fibrin or soft: agar the body of the spirochata
assumes a wavy spiral not unlike S. refringens.

Noguchi who has studied the morphology of the organism with
great care finds the body to be absolutely flexible. There is a dis-
tinct halo about it but no membrane has been demonstrated.
The part of the body which forms the hook terminates in a fine
point, but no minute flagellum-like projection can be demonstrated
by staining or by dark field illumination. Noguchi found it to be
devoid of a terminal filament such as characterizes Treponema and
is resistant to 10 per cent. saponin solutions, in which it is unlike
all other spirochete, and believes that it should be placed in a new
genus for which he suggests the name Lepiospira. Martin, Pettit
and Vaudremer* describe flagella, terminally placed and varying in
number and length and terminating in tiny knobs.

The hooked ends form one of the most characteristic poses of the
organism while rotating on its axis in a free space, but as soon as it
meets a solidor semi-solid obstacle, it begins to penetrate into it.
Its habitat seems to be a porous gelatinous substance, the organisms
swarming in and out of it.

Staining.—The organism may be colored by Giemsa’s or Roman-
owsky’s stains and appears pinkish purple, not taking the color
intensely. Burri’s Indian ink method gives good results on smear
preparations, but Hume and Bedsonj{ think that smears are best
stained by Fontana’s method which consists of treating the fixed
smear with a mordant consisting of 5 per cent. aqueous solution of
tannic acid and then applying an ammoniated silver nitrate solution.

For staining the organism in sections of tissue, the older method
suggested by Levaditi for staining Treponema pallidum (q.v.) is said
to be most satisfactory. .

Isolation and Cultivation.—The organism was first obtained in
pure culture by Inada, Ido, Hoki, Kaneko and Ito,t the method
employed being that of Noguchi for Treponema pallidum. They
employed guinea-pig instead of rabbit kidney and always used liquid
paraffines. The most important part was the temperature. The
usual incubation temperature of 37°C. was found to be inappropriate,
the organisms becoming sluggish in two or three days and then de-
generating and disappearing from the culture media. The best
results were obtained between 22° and 25°C.

*C.R. de la Soc. de Biol., 1916, LXxIx, 1053.
1B. M. J., Sept. 15, 1917, Il, p. 345.
TJour. Exp. Med., 1916, xx1v, p. 387.

Pathogenesis 535

The culture fluid remains perfectly clear although the spiro-
chete distribute themselves throughout the medium. The slightest
cloudiness indicates contamination. No odor is given off, and
the ascitic fluid is not coagulated. The multiplication of the spiro-
chete does not begin at once, but only after several days and some-
times not for a week or two. The cultivated organism does not
differ from that obtained directly from the animal body except that
when multiplication is at its height the organism is very short
and has brisk movements. Sometimes two are connected and some-
times from 8 to 15 may be collected about some granular substance
forming a kind of rosette, but maintaining very brisk movements.

Noguchi* thinks that the most reliable procedure for securing
initial growth is to produce strands of loose fibrin in the fluid culture
media by using a small quantity of citrate plasma in combination
with the diluted or undiluted serum of a suitable animal. The dilu-
tion of the serum made in any proportion above 1 : 10 by adding
sterile (0.9 per cent.) saline Ringer solution or even plain water.
For obtaining the spirochetal material for inoculation, the citrated
blood derived from the heart of a guinea-pig having the disease is
best although an emulsion of kidney or liver may also be used. He
obtained a good growth from infected guinea-pigs’ blood in dilution
as high as 1 : 100,000.

Noguchi believes that blood cultures for diagnostic purposes in
human cases are feasible and for the purpose recommends two media.
The first is rabbit serum 1 part + Ringer’s solution or 0.9 per cent.
sodium chloride solution 3 parts + citrated rabbits’ plasma 0.5
part, covered with a thin layer of sterile paraffine oil. The second
is the same except that o.5-1 0 parts of neutral or slightly alkaline
2 per cent. agar-agar are added while liquid and at about 65°C.
and mixed well. These media, because of the paraffine oil layer, can
be preserved at room temperature for many months in a cool place.

In the case of an infected guinea-pig, the detecting of the spiro-
cheta in the blood can be made in forty-eight to seventy-two hours
if the culture tubes are kept at 30°-37°C. The search for the organ-
isms should be made within the aérobic zone immediately below the
surface (1.0-1.5 cm.), because, according to Noguchi’s experience the
organism is an obligatory aérobe unable to grow in the absence of
oxygen. The growth is visible as a distinct haze.

Pathogenesis.—The spirocheta is pathogenic for man, guinea-
pigs,rats and mice. The guinea-pig seems to be the most susceptible
animal—even more so than man. Rabbits are almost insuscept ble.

Distribution in the Animal Body.—This has been made the subject
of a careful investigation by Kaneko and Akuda,t who follow Inada
and Ido in dividing the disease into three stages (1) the febrile, (2)
the icteric, (3) the convalescent. In the febrile or first stage, lasting

* Jour. Exp. Med., 1917, xxv, 758.
+ Jour. Exp. Med., 1917, xxvi, No. 3, p. 325.

536 Infective Jaundice

about a week, the micro-organisms live-in the blood. In whatever
organ they may be found they are either in the capillaries, the lymph
spaces or the intercellular spaces. In unusual instances they may
enter into epithelial cells, especially those of the liver. Presumably
dead organisms are sometimes seen in phagocytic cells of the blood.
In animals, as a rule, the greatest number of micro-organisms is to
be found in the liver, the next greatest in the adrenals and kidneys.
In the liver large numbers surround the individual liver cells like a
garland; in the kidneys they occur inside the interstitial tissues and
also in the walls and lamina of the uriniferous tubules, then entering
the urine in large numbers. There are few in the spleen, bone mar-

Fig. 207.—Weil’s disease. Spirochete in the liver of a patient autopsied on the
sixth day. (Inada, Ido, Hoki, Kaneko and Ito.)

tow or lymph nodes. Diffuse hemorrhages in any organ always con-
tain them. In the icteric or second stage they disappear from the
blood and are destroyed in most of the organs, though they can be
constantly found in the kidneys and cardiac muscle. In the third or
convalescent stage they are abundantly excreted in the urine and die
out in all the organs. The varying occurrence and distribution
depend upon the development and destructive effects of immune
bodies. In man the post-mortem examination of organs showed the
greatest number of spirochete in the kidneys. They escaped in the
lumen where they were caught in tube casts in those dying before
the oth day and were free in the tubules in those succumbing about

Bacteriological Diagnosis 537.

the 14thday. The distribution differs in guinea-pigs and men in that
in the latter the number is smaller, degenerative forms more numer-
ous and a greater number inclosed within the cells.

Lesions.—In animals the chief pathological changes are marked
jaundice, hemorrhages into or from the lungs, intestinal walls,.
retroperitoneal tissues and the fatty tissues of the inguinal region,
and cloudy swelling of the organs. The liver shows cloudy swelling
of the parenchyma, while the color varies according to the degree
of jaundice and the quantity of blood present. - Microscopically
the precipitation of bile is not marked in spite of the presence of jaun-
dice and there is no congestion of bile in the biliary tract. The
kidneys show parenchymatous nephritis; the lungs large or small
hemorrhagic spots; the intestines hemorrhages in their walls.

Escape of the Spirochete from the Body.—In seven cases studied by
Inada, Ido, Hoki,.Kaneko and Ito, many spirochete were found in
the urine from the roth to the 30th day of the illness. The num-
ber was sometimes countless. They were chiefly present in cylin-
droids and nubeculz and in small numbers in the cylinders. At
the time that the urine contains the micro-organisms it is highly in-
fectious. A number of the spirochetz also seem to leave the body
in the feces. Bloody sputum contains such as have escaped in the
blood.

Bacteriological Diagnosis.—This can sometimes be made by
microscopic examination with dark field illumination but best by the
injection of 4 to 5 cc. of the blood of the patient into the abdominal
cavity of a guinea-pig. Inada, Ido, Hoki, Kaneko and Ito, however,
found that the spirochete are present in the blood only during
what'may be described as the first stage of the lesion. Thus, all blood
gave positive results on the 4th and 5th days of the disease; on
the 7th day one blood gave a positive, the other a negative result;
one blood gave a positive result on the 9th day; no blood gave a
positive result on the 12th day. Guinea-pigs thus inoculated
_ show jaundice by the 7th or 8th day.

During the second stage of the disease, from the 7th to the 14th
day, no spirochete are in the blood, the development of immune
bodies having destroyed them, but they remain in heart-muscle
and kidneys. During this period, though the clinical signs are
striking and characteristic, bacteriological confirmation may be diffi-
cult. In the third stage of the disease, from the 14th day to conva-
lescence, the spirochzete appear in large numbers in the urine,
and bacteriological confirmation of the clinical findings again be-
comes possible, by microscopic examination of the urine, by dark
field illumination, India ink staining and by guinea-pig inoculation
with the urine. Garnier and Reilly* centrifugalize 50 to 15occ. of
the urine and inject the sediment suspended in 5 cc. NaCl
solution.

*C. R. de la Soc. de Biol., 1917, LXXx, p. 38.

538 Infective Jaundice

As suggested by Noguchi, blood cultures may be possible and
of use in making the diagnosis.

The finding of spirochzte in urine subjected to dark field exami-
nation should not be relied upon in suspected cases unless supported
by guinea-pig inoculation, for Stoddart* has found that spirochete
of various species are not uncommon organisms in the urethra, and
could be found in 44 out of too cases examined.

Sources of Infection.— Where the spirocheta comes from and how
it finds its way into the body are matters of great practical interest.
The escape of the organisms in such great numbers in the urine,
at once causes suspicion to center about that excretion as the chief
agent. The Japanese writers, so often quoted, observed that epi-
demics sometimes occurred in mines and always in wet mines.
Stokes, Ryle and Tatler observed that when the disease occurred
among soldiers in the trenches, it was always in particular trenches.
When the soldiers were removed from there they ceased to have new
cases; when new soldiers were placed there, the disease appeared
among them. Such trenches were always wet.

In 1915, Miyajima called the attention of Inada, Ido, Hoki, Ito
and Wanif to the fact that he had observed spirochetz, similar to
those of infective jaundice, in the kidney of a field mouse. This
led them to begin a study of various rodents, but being occupied
with the Spirocheta icterohemorrhagiz, the problem was set aside for
future solution. In 1916, Miyajima made additional mention of
having found similar spirochete that infected guinea-pigs, and that
he believed to be identical with S. icterohemorrhagie, not only on
account of the symptoms produced, but because the immune serum
of Spirocheta icterohemorrhagie was capable of destroying them.
It was then remembered that cooks working in kitchens frequented
by rats frequently suffered from infective jaundice, and at the be-
ginning of 1917 they observed two typical cases following the bites
of rats. They were led to the conclusion that rats play an important
part in the spread of the disease and therefore undertook an investi-
gation of the rats in the city of Fuknaka and its vicinity. They
were able to find S. icterohemorrhagie in the kidneys of 4o.2 per cent.
of 149 Mus decumanus specimens examined. Their results were
soon confirmed by Stokes, Ryle and Tatlert of the British Army in
Flanders and by Noguchi.§ This occurrence of the spirochzta in
the kidney of the rat compared with that of the convalescent cases in
man. “The behavior of the spirocheta within the rat is open for
further study, but we know that the rats harboring spirochete
always excrete them in the urine. The organisms thus find their
way to the ground where they may infect other rats if opportunity
offers. In all probability they are disseminated by means of the

* Brit. Med. Jour., Sept. 20, 1917, p. 416.

t Jour. Med. Research, 1917, XXV, No. 3, Pe e4ts
t Lancet, 1917, 1, 142.

§ Jour. Exp. Med., 1917, Xxv, 755.

Prophylaxis 539

rats, the soil and the animals forming a circle of habitation for the
spirochete. It happens rarely that human. beings are infected
directly through the bites of rats, the infection being usually trans-
mitted from the soil where the excreted spirochete lodge and thrive.
On these grounds we can explain the epidemics of spirochztosis
icterohemorrhagica which occur in coal mines and among farmers in
the vicinity. Rats are constant tenants of the mines, and it is
known that miners go barefooted (in Japan). A similar statement
may be made concerning the transmission of Weil’s disease on the
battlefields of Europe. There the rats living in the trenches infect
the soldiers.”

Spirochetosis icterohemorrhagica is therfore one form of ‘“rat-
bite fever,” though as will be shown below, not the common form.
Inada, Ido, Hoki, Kaneko and Ito, found that when the abdomen
of a guinea-pig was cleanly shaven, washed with soap and water,
then with alcohol and then dried, 10 out of 13 animals became
infected when the spirochzte were applied to the uninjured skin.
They conclude, therefore, that traumatic injury may be unnecessary
to bring about infection, the micro-organisms finding their way into
the body through the skins of persons exposed to wet soil con-
taining them.

Prophylaxis.—The necessity of exterminating rats through whose
urine the chief dissemination of the spirochztz takes place is an indis-
pensable factor in preventing the infection. In mines and military
trenches, drainage and drying of the soil are eminently desirable
as a secondary procedure, tending to destroy the micro-organisms.
Where this is not possible and the localities must be occupied,
advantage might be taken of the observations of Inada, Ido, Hoki,
Ito and Wani that the spirochete die in soil that is acid, to apply
a chemical effecting the necessary change of reaction to the earth
of the trench or mine concerned.

Ido, Hoki, Ito, and Wani* have attempted to produce active im-
munity in guinea-pigs and in men by vaccination with a preparation
made as follows: To a liver emulsion or a pure culture, which con-
tained 10 to 15 spirochete ina single field (172 oil immersion, ocular
tm under dark field illumination) carbolic acid was added in the pro-
portion of 0.5 per cent., after which the mixture was left in the ice-
box for a week. The clear supernatant fluid was employed for the
injections and administered to guinea-pigs intraperitoneally in doses
of 2-4 cc., three times at 7-9 day intervals. An uncertain degree
of immunity was developed.

They also undertook the immunization of a horse through in-
oculation with the vaccine and after having demonstrated the appear-
ance of immune bodies in the blood of the horse, proceeded to the
active immunization of man. They found that by the employment of
a vaccine ten times as strong as that originally employed for guinea-

* Jour. Exp. Med., 1916, XXIV, p. 471.

540 _ Infective Jaundice

pigs, an uncertain degree of immunity, as shown by Pfeiffer’s test or
guinea-pigs with the human serum, could be established.

Treatment.—The blood of horses immunized with the vaccines
described above was tried upon 24 cases of infectious jaundice
with a mortality of 17.3 per cent., as against the untreated cases
of which Oguro gives the mortality as 40 per cent., Nishi as 48 per
cent. and Inada as 30.6 per cent. Thus, with the employment
of the horse serum the-mortality seems to be reduced almost to
two-thirds. ; :

In a later paper Inada, Hoki, Ito and Wani* again report upon
the use of intravenous injections of immune horse serum in the treat-
ment of Weil’s disease. A total of 41 patients had been treated, the
total death-rate being 23.7 per cent., as contrasted with 30.6 per
cent. in untreated cases. A thorough analysis of the cases and
results are given.

RAT-BITE FEVER

SPIROCHZTA Morsus Muris (Futaki, TAKAKI, TANIGUCHI
AND OsuMI)

It has already been pointed out that spirochetosis icterohemorrhag-
ica is one form of disease that may be caused by the bites of rats. -
The “‘rat-bite fever” is, however, a disease that seems to be suffi-
ciently different in its clinical manifestations to constitute a sepa-
rate'entity. The affection, known in Japan as “sodoku,” seems to
have been first described by Miyakit who reported eleven cases of
his own and added others collected from the Japanese literature.
Prior to his time, occasional cases were reported in the literature of
various countries—in 1840 in America, by Wilcox;{ in France in
1884, by Millot-Carpentier;§ scattered cases have since appeared in
the literature of most countries of the world, and in 1916 eighty.
odd cases were on record.

According to the description of Blakel| rat-bite fever is a paroxys-
mal febrile disease of the relapsing type following the bite of a rat.
The wound heals readily, but after an incubation period varying from
a few days to a month it becomes inflamed and painful. Lymphan-
gitis and adenitis set in and are quickly followed by symptoms of
systemic infection ushered in by a chill and a rapid rise in tempera-
ture. There is extreme postration, severe generalized muscular pain,
headache, weakness and loss of appetite. Stupor, delirium and even
coma may supervene. There is muscular pain and rigidity and the
tendon reflexes are frequently exaggerated. A characteristic exan-
them of bluish red, erythematous, sharply marginated macules ap-

* Jour. Exp. Med., 1918, xxvu, No. 2, p. 283.

+ Mitt. aus der Grenzgebete d. Med. u. Chir., 1900, Vv, 231.
t Amer. Jour. Med. Sci., 1840, xxvI, 245.

§ L’Union, Med., 1884, xxxVIII, 1069.

|| Jour. Exp. Med., 1916, xx1II, 39.

Rat-bite Fever 1 AT

pears, varying in size from 1-10 cm. in diameter and of general dis-
tribution. After 5 to 9 days the temperature falls by crisis accom-
panied by a drenching sweat and all symptoms subside. The disease
then assumes the relapsing type with paroxysms occurring at fairly

Fig. 208.—Section of the lung of a mouse inoculated with venous blood from
a patient with rat-bite fever. The length of the body of the spirocheta is 2.24;
including the flagella it is 64. Silverimpregnation. X 1500. (Futaki, Takaki,
Taniguchi, and Osumi.)

regular intervals, usually about once a week. The course may vary
from two to three months or even longer. Gradually the relapses
become less frequent and less severe and the disease often terminates
with an abortive paroxysm. The more important complications

Fig. 209.—Spirochete froma guinea-pig with experimental rat-bite fever.
The length of the bodies varies from 2.2 to 4u. Giemsa’s stain. X 1250.
(Futaki, Takaki, Taniguchi,.and Osumi.) .

are nephritis, severe anemia and‘emaciation. About ro per cent.
of the cases terminate fatally, usually during the first febrile period,
occasionally later from nephritis or exhaustion.

542 Infective Jaundice

Ogata* thought the disease to be caused by a sporozoan which he
carefully described. Schottmiillert thought it was caused by a
Streptothrix muris ratti that he found. Middletont and Proeschi§
cultivated rod-like organisms and Blakel| a streptothrix that he
identified with that of Schottmiiller.

Futaki, Takaki, Taniguchi and Osumi** have, however, come toa
different conclusion and appear to have shown that the true etiolog-
ical factor is a spirochzta which ina later contribution tf they describe
carefully under the name Spirocheta morsus muris. This they found
in nine cases of the affection that they were fortunate enough to ob-
tain for study. Simultaneously appeared a contribution by Ishiwara,
Ohtawara and Tamura{t in which a slightly different appearing
spirocheta was described and almost the same results remained.

Morphology.—Generally speaking these spirochete present thick
and short forms of about 2-5u and have flagella at both ends.
Including the flagella they measure 6-10 in length. Some forms
in the cultures reach a length of 12-19u excluding the flagella.
The curves are regular and the majority have one curve in tp.
Smaller ones are found in the blood and in the tissues.

Staining.—These spirochete stain easily, taking a deep violet
red color with Giemsa’s stain, which also colors the flagella. They
also stain with ordinary anilin dyes.

. Movements—The movements are very rapid, resembling a vibrio
and distinguishing them from all other spirochete.

Cultivation.—The spirocheta can be cultivated upon a’ medium
devised by Shimamine prepared as follows: o.5-0.75 gram of
sodium nucleate and 100 cc. of horse serum are shaken until the
former is completely dissolved, after which carbon dioxide is
passed through the solution for 3-4 minutes until the serum becomes
transparent. The liquid is heated on three successive days, for about
an hour at6o°C. On the 4th day it is heated to 65°C. for about thirty
minutes, when it separates into a fluid and a coagulated protein.
It can also be cultivated by Noguchi’s method for T. pallidum.

_ The inoculations are made by thrusting a capillary tube filled with |
the blood of an animal containing the spirochaete deeply into the
medium. No liquid paraffine is added. The tubes are kept at
37°C. for two weeks. No change is apparent in the medium but
the micro-organisms can be detected by the dark field illumination
er by staining. Ishiwara, Ohtawara and Tamura were unable to
cultivate their spirochete.

* Mitt a. d. med. Fakult. d. k. Univ. z. Tokyo, 1909, vmI, 287; I91T, IX, 343
t Dermat. Woch., 1914, Lvim, Suppl. 77.
. ELancet, toro, 1, 1618.
§ Internat. Clinics, 1911, Series 21, Iv, 77.
|| Jour. Exp. Med., 1916, Xx1mt, 39.
** Jour. Exp. Med., 1916, XXIII, 249.
tt Jour. Exp. Med., 1917, xxv, 33.
Tt Jour. Exp. Med., 1917, xxv, p. 45.

Distribution in Nature 543

Distribution in Nature.—The spirocheta of rat-bite fever seems
to be a pathogenic parasite of rats. It, like other parasites is not
present in all rats, but only in those suffering from or convales-
cent from infection, and seems to be spread from animal to animal
through bites. In no case has the spirocheta been found in healthy
guinea-pigs or mice, yet guinea-pigs readily contract the infection
when bitten by a diseased rat, and men when bitten sometimes
do the same. The spirocheta is not found in the saliva of the rat
and probably accidentally enters it through admixture of blood from
the gums.

Pathogenesis.—The organisms are pathogenic for rats, white-
rats, mice, guinea-pigs, monkeys and man, but not for rabbits.

Distribution in the Animal Body—Always present in the blood of
infected animals, though not always in numbers permitting dis-
covery by direct observation, they pervade the body but collect
chiefly in the liver and kidneys.

Lesions —In general the lesions consist of swelling and congestion
of the lymph nodes, congestion of the liver. and lungs, congestion,
swelling and sub-capsular as well as interstitial hemorrhages in the
kidneys, congestion and hemorrhage of the adrenals.

Treatment.—The spirochete quickly disappear from the blood
of guinea-pigs when salvarsan is administered, in which particular
they differ from those of infective jaundice which were much less
susceptible to the effects of the arsenic compounds.

CHAPTER XXII
SLEEPING SICKNESS

TRYPANOSOMA GAMBIENSE (DUTTON) TRYPANOSOMA RHODE-
SIENSI (STEPHENS AND FANTHAM)

SLEEPING sickness, African lethargy, Maladie du sommeil,
Schlafkrankheit, or human trypanosomiasis is a specific, infectious,
endemic disease of equatorial Africa characterized by fever, lassi-
tude, 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 pro-
duced by a chronic meningo-encephalitis. Slow irregular febrile
disturbance, headache, lassitude, deepening into profound physical
and mental lethargy, muscular tremor, spasm, paresis, sopor, ulti-
mately wasting, bed-sores, and death by epileptiform seizure, or by
exhaustion, or by some intercurrent infection.

“In every case the lymphatic glands, especially the cervical,
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 decided symp-
toms, 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 faculties 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 con-
dition and of the miserable fate that is in store for him; and he looks
as if he knew it.”’

*“An Account of Native Africans in the Neighborhood of Sierra Leone,” 1803.

} “ The Lane Lectures for 1g05,”” Chicago, 1905.

544

Specific Organism 545

Specific Organism.—The discovery of the specific organisms
was foreshadowed by Nepveu,* who recorded the existence of try-
yanosomes in the blood.of several patients coming from Algeria,
by Barron, and by Brault.t

In 1901 Forde received under his care at the hospital in Bathurst
(Gambia), a European, the captain of a steamer on the River Gam-
bia, who had navigated the river for six years, and who had suffered
several attacks of fever that were looked upon as malarial. The ex-
amination of his blood revealed the presence not of malarial para-
sites, but of small worm-like bodies, concerning the nature of which
Forde was undecided.§ Later, Dutton, in conjunction with Forde,
examined this patient, whose condition had become more serious,
and recognized that the worm-like bodies seen by Forde were try-
panosomes. Of these parasites he has written an excellent descrip- -

Fig. 210.—Trypanosoma gambiense (Todd).

tion, calling them Trypanosoma gambiense.|| The patient thus
studied by Forde and Dutton died in England, January 1, 1903. In
1903 Dutton and Todd** examined 1000 persons in Gambia and found
similar trypanosomes in the bloods of 6 natives and 1 quadroon.
In the same year Manson{f discovered 2 cases of trypanosomiasis in
Europeans that had become infected upon the Congo. Brumpt{t
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

* “Memoirs, Soc. de Biol. de Paris,” 1891, p. 40.
1 “Transactions of the Liverpool Medical Institute,” Dec. 6, 1894.
{ “Janus,” July to August, 1898, p. 41.
“Trypanosomes and Trypanosomiasis,’’ Laveran and Mesnil, 1907.
|| See Forde, “Jour. Trop. Med.,” Sept. 1, 1902; Dutton, Ibid., Dec. 1, 1902;
utton, “Thompson-Yates Laboratory Reports,’’ 1902, V, 4, part Il, p. 455.
."*«First Report of the Trypanosomiasis Expedition to Senegambia,” 1902,
Liverpool, 1903. i
‘tt “Jour. Trop. Med.,”” Nov. 1, 1902, and March 16, 1903; “ Brit. Med. Jour. ””
@Y 30, 1903. -
It“ Acad. de Med.,”” March 17, 1903.
§§ “Brit. Med. Jour.,”” May 30, 1903.
35 :

546 Sleeping Sickness

micro-organisms and African lethargy, and much interest was being
taken in a coccus—the hypnococcus—-that was being studied by
Castellani in Uganda. As Castellani* was prosecuting the investi-
gation of this organism, he chanced to examine the cerebro-spinal
fluid of several negroes in Uganda who were suffering from sleeping
sickness, and in it found trypanosomes. Even then, though Cas-
tellani realized that these organisms were connected with sleeping
sickness, he did not identify them in his mind with the Trypano-

Fig. 211.—Various species of trypanosomes: 1, Trypanosoma lewisi of the rat;
2, Trypanosoma lewisi, multiplication rosette; 3, Trypanosoma 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 gambiense, undergoing division; 8, Trypanosoma theileri, a harm-
less trypanosome of cattle; 9, Trypanosoma transvaliense, a variation of T. theileri;
10, Trypanosoma avium, a bird trypanosome; 11, Trypanosoma damonie ofa
tortoise; 12, Trypanosoma solee of the flat fish; 13, Trypanosoma~granulosum of
the eel; 14, Trypanosoma raje of the skate; 15, Trypanosoma rotatorium of frogs;
16, Cryptobia borreli of the red-eye (a fish). (from Laveran and Mesnil.)

* The Lancet, London; June 20, 1903.

Morphology 547

soma gambiense discovered in the blood by Forde and Dutton, and
described the newly discovered organism as Trypanosoma ugan-
dense. Kruse,* thinking to honor the discoverer, called it Try-
panosoma castellani. Bruce and Nabarrot found the new try-
panosome in each of 38 cases of sleeping sickness in the cerebro-
spinal 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
negative in every case. They also declared that, contrary to what
had been stated, there were no appreciable morphologic differences
between Trypanosoma gambiense and Trypanosoma ugandense.
Dutton, Todd, and Christy{ arrived at the same conclusion. The
matter was finally settled by Thomas and Linton§ and Laveran,]||
who, by means of animal experiments, determined not only the
complete identity of the organisms, but their uniform virulence.
Early in 1910 J. W. W. Stephens** studied the blood of a rat in-
oculated with blood from a patient suffering from sleeping sickness,
with which he had become infected in North Eastern Rhodesia,
.and observed certain definite morphological differences between the
trypanosomes in it, and Trypanosoma gambiense. Later he and
Fantham{f studied this organism with great care and came to the
conclusion that it was a new and separate species, and gave it the
name Trypanosoma rhodesiense. In this they received the support
of Mesnil.tt
Morphology.—(1) Trypanosoma gambiense is a long, slender,
spindle-shaped, flagellate micro-organism that measures 17 to 28 wu
in length and 1.4 to 2 win breadth. From the anterior 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 undulat-
ing membrane the organism swims rapidly with a wriggling and
rotary movement that gives it the name Trypanosome, which means
“boring body.”

* “Gesell. f. natur. Heilkunde,’’ 1903.
+ “Brit. Med. Jour.,” Nov. 21, 1903.
TIbid., Jan. 23, 1904, also “Thompson-Yates and Johnson Lab. Reports,”’
a v, 6, part I, Pp. 1-45.
“Lancet,” May 14, 1904, pp. 1337-1340.
.! “Compt.-rendu de l’Acad. des Sciences,” 1906, V, 142, p. 1056.
* “British Medical Journal,” 1912, 11, 1182.
' “Proceedings of the Royal Society,”’ 1910, LXXXIII, 28, 31; 1912, LXXXV, 223;
ulletin of the Sleeping-sickness Bureau,”’ 1911-1912, Nos. 33, 38.
tt “Brit. Med. Jour.,” 1912, 1, 1185.

548 Sleeping Sickness |

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 that is always well in advance of the centro-
some or blepharoplast, and not infrequently is near the center
of the organism. There is also a centrosome or blepharoplast,
which appears as a distinct, deeply staining dot near the posterior
blunt end and from which the flagellum appears to arise. Near
this a vacuole is sometimes situated.

(2) Trypanosoma rhodesiense differs from Trypanosoma gam-
biense in that the nucleus is never near the center, rarely far in ad-
vance of the blepharoplast, and not infrequently is posterior to the
blepharoplast.

Staining—The organisms are best observed when stained with
one of the polychrome methylene-blue combinations—Leishman’s,
Wright’s, Jenner’s, Romanowsky’s, or Marino’s. To stain them a
spread of the blood or cerebro-spinal fluid is made and treated pre-
cisely as though staining the blood for the differential leukocyte
count or for the malarial parasite.

Cultivation. -sheypanesoma lewisi of the rat and Trypanosoma
brucei of ‘‘nagana” or “‘tsetse-fly” disease of Africa have been culti-
vated by Novy and.McNeal* in mixtures composed of ordinary
culture agar-agar and defibrinated rabbit-blood, combined as nec-
essary, I:1, 2:1, 1:2, or 2:3, etc. - The 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 Try-
panosoma 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.

Bayont has foienil it easy to cultivate Trypanosoma rhodesiense
in Clegg’s ameba-agar (q.v.) and in blood agar-agar containing
dextrose. The organisms thus cultivated retain their virulence for
rats for a long time.

Reproduction.—Multiplication takes place by binary division,
the line of cleavage being longitudinal and beginning at the posterior
end. The centrosome and nucleus divide, then the flagellum and |
undulating membrane divide longitudinally, and finally the proto-
plasm divides, the two organisms hanging together for some time by
the undivided tip of the flagellum..

In addition to this simple longitudinal fission, the trypanosomes
seem to possess a sexual mode of reproduction. When the well-

* “Contributions to Medical Research dedicated to Victor Clarence Vaughan,’’
Ann Arbor, Michigan, 1903, p. 549; “ Journal of Infectious Diseases,” 1994, I, p. I.
} “Proc. Royal Society, Series B,’” 1912, LXxxv, 482.

Transmission — 549

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 reproduc-
‘tion from Trypanosoma lewisi. Prowazek found that when rat
blood containing the latter organism was taken into the stomach
of the rat louse, Hematopinus spinulosus, the male trypanosome
enters the female near the micronucleus and the various parts of
the two individuals become fused. A non-flagellate odkinete re-
sults, 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 transmitted to a fresh host..

Another form of multiplication consists in the “shedding” of
infective granules. This has been studied by Ranken.* The organ-.
isms from which this is about to take place are observed to contain
three or four, sometimes five or six granules of small size, highly
refractile and spherical in shape. They are distinctly within the
protoplasm of the trypanosome and swing backward and forward.
as it makes its lashing movements. When these are closely watched
a time comes when one of the granules shoots out. At first the:
granule is carried about by whatever currents of fluid it happens to:
meet, having no motility of its own, but soon a dot appears, then a
flagellum, and provided with means of locomotion, and now hav-
ing a pyriform shape, the new embryo parasite swims away. Ran-
ken thinks these granular forms develop in the internal organs
ee found them of pyriform shape in the liver, spleen, and.

ungs.

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 and those in the country
less than a year frequently died of it, there was no spread of the
affection to those that were acclimated. 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 fellow countrymen. A case from the Congo that died in a
hospital in Philadelphia and came to autopsy at the hands of the

_ author, did not spread the disease in this city.

* “Brit. Med. Jour.,”’ 1912, 11, 408.

550 Sleeping Sickness

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 withit. 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
suggested 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

Fig. 212. —Glossina palpalis. A Fig. 213.—Glossina palpalis before
perfect insect just escaped from the and after feeding (Brumpt).
pupa (Brumpt). Showing how the
wings close over one another like the
blades of a pair of scissors.

that inoculations with the proboscis of the tsetse-flies were harmless.
The exact connection between 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 imme-
diately afterward on a healthy dog, conveyed the disease to the
latter. The flies were infectious for twelve, twenty-four, and even
for 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.
* “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 551

So soon as African lethargy was shown to be a form of trypano-
somiasis, the question arose, Was it spread by tsetse-flies? Sambon*
and Brumptf both suggested it, but it was soon discovered that the
_ geographic distribution of the tsetse-fly, Glossina morsitans, that

distributes nagana, does not coincide with the geographic distribu-
tion of sleeping sickness. There are, however, different kinds. of
tsetse-flies, and Bruce and Nabarrot first showed that it was not
Glossina morsitans, but a different tsetse-fly, Glossina palpalis, that
is the most important source of the spread of human trypano-
somiasis. They submitted a black-faced monkey (Cercopithicus)
to the bites of numerous tsetse-flies caught in Entebbe, Uganda, and
found trypanosomes in its blood. 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 geographic
distribution of African lethargy and of Glossina palpalis, which
-were found perfectly to correspond.

But the natural history of sleeping sickness is less simple than these
facts make it appear. Kinghorn and Yorkel| observed that in
the Luangwa Valley where tsetse-flies (Glossina morsitans) abound,
there is much game but few domestic animals. This led them to
study the bloods of all the game animals in an attempt to discover
how many harbored trypanosomes and what kind they were. The
results are interesting, but two are of great importance in the present
connection. They discovered that antelopes harbored Trypano-
soma rhodesiense, and that it could be transmitted by Glossina
morsitans. As Trypanosoma rhodesiense is the more virulent
parasite, and as the antelope regularly harbors it and the widely
distributed Glossina morsitans distributes it, the likelihood of
an early and successful outcome of the campaign against sleeping
sickness becomes improbable.

The flies are found to become infective in from eleven to twenty-
five days after consuming infected blood, and to remain so as long
as they continue to live.

Bruce, Hamerton, Bateman and Mackie, the members of the
“Royal Society Sleeping-sickness Commission”’ for 1908-9** have
found that under experimental conditions the development of the
parasites takes place only in about 5 per cent. of infected flies.
The shortest time in which their flies became infective was 18 days,

. the longest 53 days, the average 34 days. An infected fly was kept

*“ Your. Trop. Med.,”’ July 1, 1903.
t“C. R. Soc. de Biol.,”’ Jan. 27, 1903.
{ “Reports of the Sleeping Sickness Commission of the Royal Society,” 1903,
Ae ee em
. ¥Ibid., 1903, No. 4, VIII, 3.
|| “Brit. Med. Jour.,” 1912, 11, 1186.
** “Brit. Med. Jour.,” 1910, 1, 1312.

552 Sleeping Sickness

alive in the laboratory for 75 days and aiinlned infective all that

time. Experiments directed toward finding out how long the flies ©

might remain infective in nature indicate that they may be able to
transmit the parasites for at least two years.

It is, of course, not impossible that other flies, generally other
species of tsetse-flies, may act as distributing hosts of the trypano-
somes, but there is no doubt about the chief agents being Glossina
palpalis and .Glossina morsitans. With increased entomologic
and geographic information it has been found that there are certain
districts where these flies abound though the disease is unknown,
but that only shows that in those districts the flies are not infected.
Tsetse-flies are not, as was formerly 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. disease. The inability of the disease to spread in
England and America depends upon the absence of tsetse-flies from
those countries.

It is possible for the disease to be transmitted from human deities
to human being through such personal contacts as may afford oppor-

tunity 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, probably through sexual intercourse, a prob-
able 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.—Trypanosoma 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, however, so far as is known, subject to
natural infection. ‘
Trypanosoma rhodesiense, being a more virulent parasite than its
close relative, probably infects a greater variety of animals. Among
these, in nature, antelopes seem to be commonly infected.
Pathogenesis.—The first effect of human trypanosomiasis seems
to be fever of an irregular and atypical type, occurring in irregular
paroxysms. It was in this early febrile stage of the disease that
Forde and Dutton first found the trypanosomes in the circulating

blood. The number of organisms in the peripheral circulation is,

however, usually so small that it is tedious to look for them. The
search may be made in thick smears stained by any blood stain, but
it is better to proceed by washing the corpuscles in citrated blood
as in preparing to calculate the opsonic index, and to collect the
“leukocyte cream” for staining and examination. The trypano-

Transmission to Lower Animals 553

somes, which seem to have much the same specific gravity of the

leukocytes, appear in greatest numbers where the leukocytes collect.

In African natives the trypanosomes may be present in the blood

for a long time before any symptoms are discovered, but in Euro-

peans their presence is soon followed by fever. As the infection

progresses, the micro-organisms increase in great numbers in the.
organs, and almost entirely disappear from the blood. The lymph

nodes swell and Winterbottom, who first described the disease, called

particular attention to the enlargement of those of the posterior

cervical triangle, which he regarded as of diagnostic significance.

When the blood examination fails to reveal trypanosomes, they
may frequently be found by puncturing an enlarged lymph node
with a dry needle and examining the drop of fluid obtained.

Wolbach and Binger* found that the trypanosomes invade the
connective-tissue structure of all organs, the reticular tissue of lymph
nodes and spleen, and the substance of the brain. The lesions are
due to the presence of the flagellated form of the parasite in the
tissues. They found the initial cell reaction to be the proliferation
of endothelial cells. They believe the discovery of numerous
intravascular mitoses of endothelial cells in the lung, liver, spleen
and kidney to indicate the source of the increase of the large mono-
nuclear leukocytes of the blood in human trypanosomiasis.

Lymphocytosis is the rule in trp aoson aes but is of no diag-
nostic importance.

’ As the invasion of the body continues, the trypanosomes dis-
appear in large measure from the blood to multiply in the organs.
In the spleen, in particular, the parasites assume a different form:
a deep band makes its appearance between the nucleus and the ~
blepharoplast. The former becomes surrounded by a large vacuole,
and the trypanosome becomes disintegrated and reduced to a
nucleus, which represents the latent form of the organism. The
nucleus later divides, giving rise to a new blepharoplast from which a
new flagellum arises, an undulating membrane later forms, and the
usual appearance of a trypanosome again develops. When perfected,
this new trypanosome enters the circulating blood. At the time that
the first indications of somnolence appear, the parasites are present
in the cerebro-spinal fluid. The fluid is collected by the technic
given in the chapter upon cerebro-spinal meningitis. To find the
trypanosomes in the fluid, it should be rapidly centrifugalized for a
few minutes and the whitish sediment collected, and examined imme-
diately, when the micro- organisms may be studied alive, or the fluid
may be spread upon slides and stained according to the technic
for blood spreads, when, the trypanosomes being killed, fixed and
stained, their structure can be studied to advantage. In studying
the morbid anatomy of sleeping sickness, Mott came to the con-

* “Your. Med. Research,” 1912-1913, XXVII, 83.
t “Brit. Med. Jour.,’”? Dec. 16, 1899, 1.

554 Sleeping Sickness

clusion that the essential lesion is an extensive meningo-encephalitis.
To the naked eye, there are scarcely any lesions in sleeping sickness,
except the enlargement of the lymph nodes, and even in the nervous
system when one looks with care, there is but little to be seen. The

Fig. 214.—Photomicrograph of an eosin-methylene-blue-stained section; -

1000 diameters. Shows trypanosomes about a small vessel of the cortex of the
brain (Wolbach and Binger, in “Jour. of Med. Research’’).

histological examination of the nervous tissues, on the contrary,

shows that in both the brain and spinal cord there is proliferation
_and overgrowth of neuroglia cells, especially those connected with

' Fig. 215.—Photomicrograph of Fig. 216.—Trypanosoma gambiense.
a Giemsa-stained section; rooo Formation of the latent stage and
diameters, showing a trypano- transformation of the latent stage into

some deep in the cortex of the a trypanosome (after Guiart).
brain (Wolbach and Binger, in
“Jour. of Med. Research”’).

the subarachnoid space and the perivascular space, with accumula-
tion and probably proliferation of lymphocytes in, the meshwork.
Wolbach and Binger found that the trypanosomes actually escape
from the blood-vessels and make their way into the nervous tissue.

Prophylaxis nae

The period of lethargy seems to coincide with that at which the
parasites are invading and injuring the nervous tissue.

Prophylaxis.—Reasoning from knowledge of the successful cam-
paigns that have waged against yellow fever and paludism, it at
first appeared as though the prophylaxis of sleeping sickness ought
to be based partly upon measures taken to prevent the infection
of men by tsetse-flies, and partly upon those taken to prevent the
infection of the flies by men.

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 bath-
ing, 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 intro-
duction of chickens, to pick up the larve and pupez, have been
recommended. ,

To prevent infection of the flies with Trypanosoma gambiense is
impossible where, as in some sections of Africa, 50 per cent. of the
population of some of the villages already harbor the parasites,
and still more impossible when, as is the case with Trypanosoma
thodesiense, the wild animals, especially antelopes which are ex-
tremely numerous, continually harbor the parasites and act as
reservoirs from which the flies receive a continuous supply.

The importance of undertaking radical measures for the prevention
of the disease may be imagined when it is understood that in the
last few years no less than a half-million of the natives of the infected
districts have died of sleeping sickness.

TsETSE FLIES

_ The Tsetse flies are dipterous insects belonging to the family Glossinie, and
included. in a single genus Glossina. With one exception, G. tachinoides, the
entire family lives in tropical and subtropical Africa. About sixteen species of
Glossina are now described, for the rough and ready identification of which the
following table from Brumpt (‘‘Précis de Parasitologie,”’ 1910, p. 630) will be found
useful. For those who desire more accurate information, Austin’s ‘Handbook of
the Tsetse Flies,’ the “Sleeping Sickness Bulletin,” and Patton and Cragg’s
Text-book of Medical Entomology” will prove useful books of reference.

Tsetse flies are easily recognized by their fly-like appearance, by their hori-
zontal proboscis, slender but swollen at the base, and by their habit of resting with
the wings crossed like the blades of a closed pair of scissors.

The greater number of the flies occupy sections of country, spoken of as “fly
belts” or “‘fly districts,’ some of which are permanently infected, others tem-
Porarily infected. Such “belts” are usually deep forests along the banks of
Streams or on the shores of lakes. The adult flies seem to love the shade, though
they fly from it into the hot sun to seek their prey. The large game animals seem
to be the natural prey of the flies, though a number of them bite human_beings,
and one, Glossina palpalis, seems to prefer human blood to all others. The flies
seem to attack moving animals by preference. So long as the creature moves
they pursue. When it stands, many of them fly away to the shade again.

Both males and females bite. The latter distend themselves with blood until

556 Sleeping Sickness

they are so heavy that they can scarcely fly and drop off to the ground. Biting
is almost entirely confined to bright sunny weather. On dull or cloudy days the
flies remain in the brush. Exceptions are found among the few species that live
in arid sections. Such may bite at night. Few of the flies fly far from their
native haunts where they seem to prefer to await the coming of their prey, rather
than to make excursions after it. Clouds of the flies often arise at the same time
and attack the animals in swarms.

The flies are larviparous and do not lay eggs. Copulation of the sexes takes
place but once, the sperm being retained in a spermatotheca. The eggs are:
fertilized as they descend from the oviduct to the uterus where they hatch into a
larva on the fifth day. The larva grows rapidly, molts three times and attains
its full size by the tenth day, when it is born. The larva at the time of birth is
cylindrical in shape, consists of thirteen segments and measures 6-7 mm. in length.
It is nearly white but has a black head which is small and inconspicuous. The
larve are usually deposited on the sand of the banks of streams or lakes, and at
once burrow into the ground to a depth of an inch or so. Ina half hour or an
hour the larva changes to a pupa in which state it continues for about a month.
The imago or fly then emerges. The average duration of life of the imago fly is
about three months, during which time each female bears an average of ten new
larve.

Glossina palpalis is commonly infested by a flagellate called Crithidia grayi,
that seems in some way to pass from fly to fly, and to have nothing to do with the
bloods upon which it feeds. It is to be regarded as a parasite of the fly, and
should be known lest it be confused with the Trypanosoma of which the fly is
the vector.

TABLE FOR THE IDENTIFICATION OF THE COMMON TSETSE FLIES

Large Species; body measuring more than 12 mm. in length.
Pattern on thorax faint; four very distinct black spots. .G. longipennis.
Pattern on thorax sharp and distinct, no black spots. .G. fusca.
Small species; body in general measuring less than 12 mm. in length. |
All five tarsal joints of the third pair of legs black.
Colors dark; antenn@ black; last two tarsal joints of
the first pair of legs black... .G. palpalis.
All of the tarsal joints of the first ] pair of legs yellow. G. bocagei.
Very small species; markings like those of G. morsi-
tans on abdomen: ss evr es shaw ede sdeiesscdaes G. tachinoides.
Colors dark; antennz yellow. . .G, pallicera.
Only the last two tarsal joints of the third pair “of legs
black; all the others yellow.
The fifth tarsal joint of the first and second pairs of
legsits:-yelloW.s. ws ¢2 sce ah, pe duines at odan Bee Saas < G. pallidipes.
The last two joints of the tarsi of the first and second pairs of legs are black.
. The yellow band on the abdominal segments takes up

one-third of the segment.............. 0000 eee G. morsitans.
The yellow band on the abdominal segments takes up
one-sixth of the segment. . .G. longipalpis.

Full information and beautiful colored illustrations of the Tsetse flies can‘be
found in E. E. Austin’s “A Monograph of the Tsetse Flies,” 1903 and in his
“Handbook of the Tsetse Flies,” ro1r.

AMERICAN TRYPANOSOMIASIS
SCHIZOTRYPANUM CruzI (CHAGAS)

No sleeping sickness has thus far been found to occur upon either
of the American continents, though human trypanosomiasis in
another form has been observed in Brazil where it has been studied
by Chagas.* ie

* “Archives fiir schiffs u. tropen Hygiene,” 1909, Heft 4; abstract “Centralbl.
f. Bacteriologie, etc. Ref.,”’ 1999, XLIV, 639; “‘ Bull. de Inst. Pasteur,’’ 1910, VIH,
373-

American Trypanosomiasis 557

The disease, which in Minas Gaeras often attacks the entire popu-
lation, chiefly affects the children and goes by the local name of

ss salen Cc 2 »
Fig. 217.—1. Glossina palpalis, d' X 6. 2. Glossina morsitans, 2 X 6
(Patton and Cragg). :

Opilacao. In childhood it usually assumes the form of an acute
malady characterized by an incubation period of ten days, and by
high continued fever, puffiness of the face, enlargement of the

558 y Sleeping Sickness

thyroid gland, of the lymph nodes and spleen. In some cases men-
ingitis occurs. It is extremely fatal.

In adults it is apt to take a more chronic course in which the
chief symptoms are enlargement of the thyroid gland, and a myx-
edematous condition of the skin. The lymph nodes usually enlarge.
If the adrenal glands become affected, symptoms resembling Ad-
dison’s disease make their appearance. If the heart muscle be
invaded by the parasites, its power is diminished and the pulse
becomes feeble and irregular. If the nerve-cells or neuroglia cells
of the central nervous system be affected through parasitic invasion,
symptoms occur according to the extent and localization of the
disturbance. There is always irregular fever and marked
anemia.

Chagas found a trypanosome in the peripheral blood of patients
suffering from Opilacao, and gave it the name Trypanosoma cruzi.
Later studies of the micro-parasite have, however, shown that its
method of reproduction differs so strikingly from that of the trypano-
somes, that it was necessary to make a generic distinction between
the two, and it is now called Schizotrypanum cruzi.

Morphology.—The Schizotrypanum is present in the peripheral
circulation only during the febrile stages of the disease, when it
may be found by the usual methods of staining for trypanosomes.
It is a long slender trypanosome-like organism, with the char-
acteristic fusiform shape, with a nucleus, a large blepharoplast, a
flagellum and an undulating membrane. No measurements are
given, but the parasite is rather small. No dividing forms are
observed in the circulating blood. The trypariosomes may be-free,
may be attached to the erythrocytes or may be partly or entirely
in the red corpuscles. They show sexual dimorphism, the males
being long and slender, the females shorter and stouter.

Reproduction.—Gametogony takes place in the lungs. Such of
the trypanosome forms as are caught and retained there, lose the
undulating membranes, the two ends curve toward one another
forming first a crescent, then unite and form a ring. The female
parasites shed the blepharoplasts, and in both male and female
parasites the nucleus breaks up into eight secondary nuclei, giving
rise to eight merozoits. The merozoits derived from the female
parasites have a single nucleus, those derived from the male para-
sites, a nucleus and a blepharoplast connected by a fine thread of .
chromatin. The merozoits thus formed enter into erythrocytes
where they eventually develop into the trypanosome forms. Hence
is explained the peculiar relation of the trypanosomes to the eryth-
rocytes mentioned above.

The chief multiplication of the parasites, however, takes place in
the cells of the voluntary muscles, the heart muscle, the central nerv-
ous system, the thyroid, the adrenal glands and the bone marrow.
In these situations, according to Chagas, the parasites take on a

American Trypanosomiasis 559

rounded form, and by schizogony give rise to a great number of
daughter parasites, each having a nucleus and a blepharoplast.
For a time the schizonts are quiescent, then develop flagella and
undulating membranes. The infected cells are destroyed. Chagas
thinks that gametes are formed only in the lungs.

In the definitive host, the Lamus (or Conorhinus) megistis, the
sexual conjugation occurs in the mid-gut. The blepharoplast
approaches and seems to blend with the nucleus, the undulating
membrane disappears and the parasites assume a spherical form.
Actual conjugation does not seem to have been observed. Multi-
plication takes place by division of these rounded organisms, the
daughter parasites becoming flagellated, the flagellum originating
from the blepharoplast. Numerous flagellated trypanosome and
crithidia forms of the parasite are observed in the hind-gut of the
insect. Chagas observed trypanosome forms in the body cavity
and in the salivary glands of the insect, and it is probable that it is
through these that the infection is transmitted when the insect
bites a susceptible animal, though Brumpt thinks the infection may
take place through the feces of the bug, especially when these are in
some way brought to the conjunctiva.

Transmission.—Chagas was able to show that a large bug, Lamus
(Conorhinus) megistis, common in the neighborhood in which Op-
liagao occurs, is the principal definitive host of the parasite. Both
males and females of this flying bug are vicious biters and both
_ live upon human blood as well as upon the bloods of other warm-

blooded vertebrates. The bugs are common in the thatch and in

the cracks between the timbers of the native houses. Whether
other species of Lamus may also harbor the parasites is not known.

Brumpt* found that Cimex lectularius, Cimex boneti and Ornitho-

dorus moubata could also act as definitive hosts. A study of

Cimex lectularius, the common bed-bug, as a definitive host of the

parasite, was made by Blacklockt who found that only a very
occasional bug becomes so infected as to be able to effect the
transmission.

‘Cultivation—The parasites are easily cultivated in vitro in the
medium recommended for trypanosomes by Novy and McNeal.
In culture the organisms resemble those found in the bugs, i.e.,

_Tound and crithidial forms, or pear-shaped rapidly dividing forms.
re than two subcultures can rarely be made before the organisms

e out.

Pathogenesis.—The Schizotrypanum is pathogenic for certain
monkeys (Callithrix), dogs, rabbits and guinea-pigs. Guinea-pigs
usually die in five to ten days, though the trypanosome forms are not
usually found in the peripheral blood. They are, however, present
in larger numbers in the lungs. Monkeys live longer. Trypano-

* “Centralbl. f. Bakt. etc. Ref.,”’ Lv, No. 3, p. 75.
t “Brit. Med. Jour.,” 1914, I, 912.

560 . Sleeping Sickness

some forms of the parasite appear in the blood in about a week,
then may disappear. The animals live a month or two.
Diagnosis.—As the trypanosomes are present in the circulating

Fig. 218.—Schizotrypanum cruzi developing in the tissues of the guinea-pig. 1.
Cross-section of a striated muscle fiber containing Schizotrypanum cruzi: Note
dividing forms. 2. Section of brain showing a Schizotrypanum cyst within a
neuroglia cell, containing chiefly flagellated forms. 3. Section through the
suprarenal capsule, fascicular zone. 4. Section of brain showing a neuroglia cell
filled with round forms of Schizotrypanum (From Low, in Sleeping Sickness
Bulletin, after Vianna).

blood of human beings in somewhat small numbers, and only at
certain times, it is unwise to rely upon them as a means of making

American Trypanosomiasis 561

the diagnosis, though if they be found the diagnosis is certain. It
is usually much better to inoculate 1 or 2 cc. of the blood of the
suspected case into a guinea-pig and then make frequent exami-
nations of its blood. Here, again, the common absence of try-
panosome forms from the blood complicates matters. If none can
at any time be found, the muscles of the guinea-pig must be exam-
ined for the dividing forms of the parasites, which are usually quite
numerous.

Prophylaxis.—As the bugs fly it is somewhat difficult to defend
the sleeping patient against them, so long as he lives in a carelessly
built and thatched country house. Sulphur fumigation and white-
washing may help. Well-built habitations with screened windows
and the use of mosquito bars should constitute the best defense.

Lamus (ConorHINUS) MeEcisTIs (BURN)

Patton and Cragg* describe this bug as follows: ‘‘Dark brown to black. Pro-
notum broadly expanded, with two broad raised red lines extending from the

Fig. 219—Lamus (Conorhinus) megistis (female), the insect host and dis-
tributing agent of Schizotrypanum cruzi (Chagas). X2.

middle of the posterior border, and a red spot on the postero-lateral angles of pro-
notum, At the anterior border of the pronotum there are six short spines, three
on each side; the most anterior are the longest and project on each side of the.
eyes; two are situated further back, one on each side of the middle line at the
‘origin of the two admedial ridges; the third spine is situated on a ridge at the
Junction of the middle and anterior third of the pronotum just above the first pair
of legs. Scutellum dark brown with two short red lines converging toward the
apex, where they meet; apex red, turning upward and bluntly rounded off. Cor-
lum and membrane fuscous, the former with one or more red streaks. Connexi-
vum with six well-marked bright red lines, broader in the male; in both sexes the
es extend round to the ventral border. In the male the last segment, except
for a central black mark, is entirely red. Length 30 to 32 mm.”

*“A Text-book of Medical Entomology,” 1913, p. 492.
36

552 Sleeping Sickness

The L. megistis ‘‘is almost entirely a domestic insect.’’ ‘‘The adults enter
inhabited houses but never those that have been abandoned. In houses which are
old or badly kept they are to be found in cracks and holes in the walls, where
they lay their eggs; the early stages, which are wingless, crawl out of their resting
places in the walls so soon as the lights are put out and make their way to the beds
of the occupants of the house. The adults behave in the same manner, but as
they are powerful fliers, they can reach the people. who sleep in hammocks.
The bite is said to be painless and to leave no mark.”

“The eggs of L. megistis are of a creamy white color and are laid in batches of
from eight to twelve, and as many as forty-five such batches may be laid. Ac-
cording to Neiva they hatch in twenty-five to forty days. The larva is of a uni-
form light color when it emerges, becoming darker later; it takes its first feed
from five to eight days after emerging from the egg, and the second from the
fifteenth to the twentieth day; it changes its skin (first nymphal stage) after
about forty-five days. The second molt takes place during the second or third
month, and the third during the fourth or sixth month. The fourth molt occurs
about’the 190th day after the larva has hatched out from the egg; this stage lasts
at least forty-two days. Neiva states that this time is the most critical period in
its life, and that large numbers of them die. After the next molt the adult stage
is reached, and eight days later they are ready to suck blood; egg-laying com-
mences about the fifty-fifth day after the first feed. One female kept under
observation by Neiva for about three and a half months laid 218 eggs in thirty-
eight batches. Under favorable conditions of food supply the cycle from egg to
egg is completed in about 324 days.’

This bug, when experimentally infected with Schizotrypanum cruzi, trans-
mitted the infection to monkeys, guinea-pigs, rabbits and dogs. Both males and
females bite and may transmit the parasites. ‘

CHAPTER XXIII
KALA-AZAR (BLACK SICKNESS)

LeIsHMANIA DonovaniI (LAVERAN AND MESNIL)

“Kara-azar,” ‘““Dumdum fever,” “ Febrile tropical splenomegaly,”
“Non-malarial remittent fever,” is a peculiar, fatal, infectious
disease of India, Assam, certain parts of China, the Malay Ar-
chipelago, North Africa, the Soudan and Arabia, caused by a
protozoan micro-organism known as Leishmania donovani, and -
characterized by irregular fever, great enlargement of the spleen,
anemia, emaciation, prostration, not infrequent dysentery, occa-
sional 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.

In 1900 Leishman* noticed in the spleen of a soldier returned
from India and suffering from ‘“ Dumdum fever”’—a fever acquired
at Dumdum, an unhealthy military cantonment not far from Cal-
cutta—certain peculiar bodies. He reserved publishing the observa-
tion until 1903, so that it appeared almost simultaneously with a
paper upon the same subject by Donovan.t 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,t Ross,§ and others, and its de-
velopmental forms observed, it was agreed that it belonged in a new
genus of micro-organisms, not far removed from the trypanosomes,
and eventually Ross, and then Laveran and Mesnil, honored both
of its discoverers by calling it Leishmania donovani, which name
has been generally accepted.

Morphology.—As seen in a drop of splenic pulp the organism is a
Minute round or oval intracellular body measuring 2.5 by 3.5 yu.
When properly stained with polychrome methylene blue (Wright's, '
[Biases 2 2S |

1 “Quarterly Jour. Microscopical Society,” xtvi, 367; “Brit. Med. Jour.,”
1904, I, 1249; 1, 645; “ Proceedings of the Royal Society,” Lxxvi, 284.

i “Brit. Med. Jour.,”’ 1903, 1, 1401.

563

564 Kala-Azar

Leishman’s, or Jenner’s stains) and examined under a high magnifi-
cation, 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

rr =
&

Fig. 220.—Evolution of the parasite of kala-azar: 1 to 5. Parasites of kala-
azar. 1. 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, phagocytosis of a parasite by a polynuclear
leukocyte. 6to15. Parasites from cultures. 6, First changes in the parasites.
The protoplasm has increased in bulk and the nucleus has become larger; 7,
further increase in size; vacuolization of the protoplasm; 8, division of the en-
larged parasite; 9, evolution of the flagella; 10, small pyriform parasite showing
flagellum; 11, 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).

the long diameter of the organism. This is regarded as the nucleus.
The second body is smaller, of bacillary shape, usually lies with its
long diameter transverse to the nucleus, stains more intensely than
the nucleus, and is looked upon as the blepharoplast. In addition
to these bodies the protoplasm may contain one or two vacuoles.

Cultivation 565

All of the bodies are intracellular, as can easily be determined
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.

The bodies divide by binary and multiple fission, without rec-

a. oO", as:
eyes a
es

Fig. 221.—Leishman-Donovan bodies from the spleen of a case of Kala-azar.
X about 1000 (from Beattie and Dickson’s ‘‘A Text-book of General Path-
ology,” by kind permission of Rebman, Limited, publishers).

ognizable 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, McNeal,
and Hall for trypanosomes, and in the N. N. N. medium of Nicolle,
which has the following composition:

Watetisvinnonguleerecuaeenrep use ess puscenr sees te ReOOO Co
Salt(NaC)) es evi sccuadeviaed ne Pedic eed ean cee OTM:
PA aT BAT oid cos au tnarcane tevnlevinee hesaiovesed Gooubtsooss 4 Sdeuseasada Atriene LOS EM

_ Dissolve, distribute in tubes, sterilize, and add to the medium in each tube after

liquefying and cooling to 40°-50°C., one-third of its volume of rabbit’s blood
obtained by cardiac puncture. Slope the tubes for twelve hours, incubate at
37°C. for five days to test the sterility of the medium, then keep at the ordinary
temperature of the laboratory, sealed to prevent evaporation.

It is imperative that the material planted be sterile so far.as bacteria

* “Brit. Med. Jour.,”’ 1904, 11, 645.

366 Kala-Azar

are concerned as any associated growing bacteria quickly destroy
them.

Under conditions of cultivation the appearance of the organism
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 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 pw in length and 3 to 4 yu in breadth, its
whip or flagellum measuring about 3 uw 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 organ-
ism. It multiplies by a peculiar mode of division first observed by

Fig. 222.—Leishmania donovani. Flagellated forms obtained in pure cultures
(Leishman).

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 later, as they grew larger, a 1 flagellum was
thrust out at one end,

Distribution.— The Leishman-Donovan bodies are widely distrib-
uted throughout the body of the patients suffering from kala-azar.
They occur 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 leuko-
cytes. They are always intracellular, or when in the circulating —
blood may be found in indefinite albuminous masses, probably de-
stroyed 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 specific. The

Transmission 567

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.—Rogers’ observation, that the round bodies grew
into flagellate bodies at temperatures much below that of the human
body, led Manson to conjecture that the extrahuman 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 distribut-
ing host. Later, Patton} reported a very thorough study of insects
in relation to kala-azar, in which after a long series of experimental
investigation, he came to the conclusion that the Indian bed-bug,
Cimex rotundatus, is the specific invertebrate host of Indian kala-
azar. It seems that in order that the parasites shall mature in the
bed-bug, and undergo those changes that shall result in the insect’s
infectivity, the bug must receive one full meal of the infected blood.
Ifa second meal is taken, the digestive condition in the bug’s alimen-
tary canal is changed, and instead of continuing to develop, the
parasites die out. When the conditions are all favorable, Patton
found that the flagellates continued to multiply actively from the
fifth to the eighth day. By the twelfth day practically all had.
reached the postflagellate stage and were only found in the stomach
of the bed-bug. These results convince Patton that Cimex rotund-
atus is the definitive host, but the proof is lacking. No animal
is known. to be sufficiently susceptible to Leishmania donovani, to
acquire anything resembling kala-azar, therefore there is none
that the bug can successfully infect. Human experiment with
so fatal a disease being out of the question, the case rests at this
point. Rowt has however, shown that when a monkey, Macacus
sinicus, is inoculated cutaneously or subcutaneously with a three-
weeks-old culture of Leishmania donovani, a cutaneous or sub-
cutaneous lesion may result. This may facilitate future studies
with biting insects.

It may be, however, that Patton and others are wrong in thinking
that the flagellate stage at which the parasites arrive in the bed-bug
is the infective stage, and have, therefore, gone astray. Bayon§ ©
points out that Leishmania infantum is infective for dogs and
Monkeys in the rounded or oval stages, not in’ the elongate: or
cultural stages, and that the same may be true of Leishmania dono-

* “Scientific Memoirs of the Government in India,” 1907, No. 27.
+ “Brit. Med. Jour.,” 1912, 1, 1194.
{ “Brit. Med. Jour.,” 1912, 11 1106.
§ “Brit. Med. Jour. ” 1912, 11, 1197.

568 | Kala-Azar

vani. The fleas, which are the vectors of infantile kala-azar among
dogs, show only the rounded and oval forms of the parasites, never
the flagellated forms.

Quite recently Patton and Donovan have been successful in infect-
ing puppies with Leishmania donovani, though the mature dogs seem
never to be infected, the examination of 2000 street dogs in Madras
and other cities failing to reveal any of the parasites in either the
liver or spleen. Patton inoculated a white rat with 3 cc. of an emul-
sion of human spleen containing the oval forms of Leishmania dono-
vani from a case of Indian kala-azar, and fifteen days later found the
spleen several times the normal size and containing large numbers of
the parasites.

Diagnosis.—The anemia of kala-azar is usually not profound.
The erythrocytes number about 3,000,000 in ordinary cases and
the hemoglobin is correspondingly diminished. As in malaria,
there is leukopenia, but it is usually more severe, the white
corpuscle sometimes being as few as 600 to 650 per cubic milli-
meter 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 hepatic or splenic
puncture. A large hypodermic needle should be used, and it should
be carefully sterilized. It should by preference be thrust into the
liver and a drop of fluid secured for examination. If nothing be
found it may later be necessary to puncture the spleen, though it
is dangerous because of the probability of subsequent hemorrhage.
If decided upon as a justifiable method of examination, the needle
is thrust into the spleen, and a bit of splenic pulp secured by firmly
withdrawing the piston of the attached syringe.

Before making such a puncture, leukemia should be excluded, lest’

hemorrhage occur.

INFANTILE KALA-AZAR

LEISHMANIA INFANTUM (NICOLLE)

Pianese* found infantile kala-azar in Italy, and in the children
suffering from it he was able to find Leishmania.

Nicolle, f 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. Méesnil 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 distinguishable either by
microscopic examination or by cultivation from Leishmania dono-

* “Gaz. Intern. di Medicin,” 1905, vit, 8.
{ “Ann. de l’Inst. Pasteur,” 1909, XXIII, 361, 441.

Tropical Ulcer | 569

vani, but, finding that it was infectious for dogs, he came to
the conclusion that it was a separate species, and called it
Leishmania infantum. He also found that the dogs in Tunis
frequently suffered from spontaneous infection from this parasite,
and it is possible that it is from the dogs that the children become
infected.

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 cul-
tivating the organism and infecting dogs with artificial cultures
containing its flagellate forms.

It is now thought by many that infantile kala~azar and Indian
kala-azar are identical diseases, caused by identical parasites. In
considering the probable source of the disease Stitt* says: “It has
been suggested that the Mediterranean basin may have been the
original focus of visceral kala-azar and that it spread thence to India
by way of Greece and the Russian Caucasus, cases having been re-
ported from districts which would join the two foci. Just as chil-
dren bear the brunt of malaria in old malarial districts and adults
suffer in places in which the disease has been more recently imported,
so by analogy we may consider the disease as of more recent intro-
duction in India. . . . In the Mediterranean basin there is a
natural canine Leishmaniasis and some think the human form may
be contracted from the dog through the medium of the flea.”

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 occasionally observed,.
and is variously known as Tropical ulcer, Oriental sore, Biscra boil,
Biscra button, Aleppo boil, Delhi boil, Bagdad boil, Jericho boil,
and Buton d’Orient. It has long been known as a specific ulcerat-
ing 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—sometimes a year or more—and gradually cicatrizes,
forming a contracting scar that is quite disfiguring when upon the
face. 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.

* Diagnosis and Treatment of Tropical Diseases, 1914, Pp 73-

57° KalJa-Azar

Fig. 224.—Helcosoma tropicum, from a case of tropical ulcer (‘Delhi sore”’)
smear preparation from the lesion stained with Wright’s Romanowsky 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. XX 1500 approx. (Wright) (From photograph
by Mr. L. S. Brown).

Tropical Ulcer 571

Organism.—In 1885 Cunningham* described a protozoan organ-
ism found in the tropical ulcer, the observation being confirmed
by Firth,t who called the podies Sporozoa furunculosa. . Later,
J. H. Wright} studied a case of tropical ulcer and found bodies pre-
cisely like the Leishmania donovani. He gave it the name Hel-
cosoma 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 infection like kala-azar, it is now
known as Leishmania furunculosa.

Cultivation.—The organism has been cultivated by Nicolle and
Manceaux§ upon the same media and in the same manner as Leish-
mania donovani and Leishmania infantum with which these in-

‘Fig. 225—Oriental sore (Wellcome Research Laboratory).

vestigators believe it to be identical. Cultivation was also success-
fully achieved by Row. |

Pathogenesis.—The virus is pathogenic for man, monkeys such
as Macacus simius, M. cynomolgus, M. rhesus and M. inuus, and for
dogs. The same effects are produced whether fresh virus from a
human ulcer, or from an artificial culture be employed. In dogs
the inoculations produce only nodular formations; in monkeys,
nodules like those in human beings that go on to ulceration. Intra-
Peritoneal inoculations usually fail. The most successful inocula-
tions are made beneath the skin in the neighborhood of the nose.

* “Scientific Memoirs by Medical Officers of the Army in India,” 1884, 1.

+ “Brit. Med. Jour.,”” Jan. 10, 1891, p. 60.

t Jour. of Med. Research,” 1904, X, 472. i

“Ann. de I’Inst. Pasteur,’’ 1910, XXIv, 683.
| “Brit. Med. Jour.,” 1912, 1, 540.

572 Kala-Azar

One successful infection with the parasite usually confers immunity;
unsuccessful intraperitoneal introduction of large quantities of
culture produce no immunity. ;

Transmission.—The disease can be transmitted by inoculation
- from human being to human being.

The usual 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 act as vectors of the parasites.

Preventive Inoculation.—Jackson* is authority for the statement
that “the Jews of Bagdad recognized that tropical ulcer is inoculable
and autoprotective years ago, and practised 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.”’ Nicollet sought
to vaccinate according to modern methods with killed and liv-
ing cultures of the organism, and was successful when he first
used a killed culture, then after a year a live culture, and then three
months later another live culture.

Treatment.—Rowt has endeavored to cure already existing
lesions by vaccination, and has met with what seems to be encour-
aging success. Cultures of the organism were permitted to grow for
seven days, then sterilized with glycerin. Patients can bear 0.25
cc. at a dose, there is little febrile reaction, and the lesions proceed
to heal nicely.

HISTOPLASMOSIS
HistoptasMA CapsuLatum (DARLING)

In 1906 Darling,§ working at the Isthmus of Panama, observed
certain cases presenting pyrexia, anemia, leukopenia, splenomegaly,
and emaciation, and bearing a close resemblance to kala-azar. The
disease was quite chronic, and it tetminated fatally. When ex-
amined at autopsy, these cases showed necrosis with cirrhosis of
the liver, splenomegaly, pseudo-granulomata of. the lungs, small
and large intestines, ulceration of the intestines, and necrosis of
the lymph nodes draining the infected 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 encap-
sulated micro-organism.

The organism is small, round or oval in shape, and measures 1
to4min diameter. It possesses a polymorphous, chromatin nucleus,
basophilic cytoplasm, and achromatic spaces all enclosed within an
achromatic refractile capsule.

* “Tropical Medicine,” Phila., P. Blakiston’s Son & Co., 1907, p. 478.

ft “Annales de l’Inst. Pasteur,” Tunis, 1908.

t “Brit. Med. Jour.,” 1912, 1, 540.

§ “Jour. Amer. Med. Assoc.,” 1906, XLVI, 1283; “Archiv. of Int. Med.,” 1908,
mr, 107; “Jour. Exp. Med.,” 1909, XI, 515.

Histoplasmosis 573

The micro-organism differs from the Leishman-Donovan body
of kala-azar in the form and arrangement of its chromatin 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 blood- and lymph-vessels and cap-
illaries, and by the infection of distant regions by the dislodgment of
infected endothelial cells and their transportation thither by the

Fig. 226.—Histoplasma capsulatum. Mononuclear cells from the time con-
taining many parasites (Samuel T. Darling in “Journal of Experimental
Medicine”).

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
in 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 XXIV
YELLOW FEVER

TuE bacteriology of yellow fever has been studied by Domingos .
Freire,* Carmona y Valle,t Sternberg,{ Havelburg,§ and Sanarelli,||
but all of their work has been shown to be incorrect by the interest-
ing researches and very conclusive results of Finlay,** Carter,tt
Reed, Carroll, Lazear, and Agramonte,{{ and Reed and Carroll,§§
which have proved the mosquito to be the definitive host of an in-
visible micro-organism.

Reed, Carroll, Lazear, and Agramonte,|||| constituting a Board
of Medical Officers “for the purpose of pursuing scientific investiga-
tions with reference to the acute infectious diseases 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 technic they withdrew and examined the
blood from the veins of the elbow of 18 cases of yellow fever, mak-
ing 48 separate examinations 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. They
were entirely unable to confirm the findings of Wasdin and Ged-
dings,*** that Bacillus icteroides was present in blood obtained
from the ear in 13 out of 14 cases, and concluded that both Sanarelli,
and Wasdin 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 infection, the
commissioners, remembering the theory of Beaupertius, who in 1854

* “Doctrine microbienne de la fievre jaune et ses inoculation preventives,”
Rio Janeiro, 1885.
{ “Legons sur l’étiologie et la prophylaxie de la fievre jaune,’’ Mexico, 1885.
I “Report on the Etiology and Prevention of Yellow Fever,”? Washington,
Bes ; “Report on the Prevention of Yellow Fever by Inoculation,’”’ Washington,
1888. ;
; ‘Ann. de V’Inst. Pasteur,” 1897.
|| Brit. Med. Jour.,”’ July 3, 1897; “Ann. de l’Inst. Pasteur,” June, Sept.,
and Oct., 1897. ; ‘
__** “Amer, Jour. Med. Sci.,” 1891, vol. cur, p. 264; “Ann. dela Real Academia,”
1881, vol. XVIII, pp. 147-169; “Jour. Amer. Med. Assoc.,’’ vol, xxxvum, April 19,
1902, P. 993.
tt “New Orleans Med. Jour.,’’ May, 1890.
tt “Phila. Med. Jour.,”’ Oct. 27, 1900; “Public Health,” vol. xxv1, 1900, p. 23.
§§ “Public Health,” 1901, vol. xxvm, p. 113.
\||| “Phila. Med. Jour.,”’ Oct. 27, 1900. .
*** “Report of the Commission of Medical Officers Detailed by the Authority
a the President to Investigate the Cause of Yellow Fever,’’ Washington, D. C.,
1899.
574

Mosquitoes and Yellow Fever 575

ascribed yellow fever to the bites of mosquitoes, the work of Fin-
lay,* who in 1881 published an experimental research showing that
mosquitoes spread the infection of yellow fever, and the interesting °
and valuable observations of Carter f upon the interval between in-

Fig. 227.—Stegomyia fasciata (Stegomyia calopus): a, male; b, female (after
Carroll). ;

fecting and secondary cases of yellow fever, turned their attention

to the mosquito. Securing mosquitoes from Finlay and continuing

: the work where he had left it, they found that when mosquitoes

_ Stegomyia fasciata seu calopus) were permitted to bite patients suf-

ering from yellow fever, after an interval of about twelve days they

** Annales dé la Real Academia,” 1881, vol. XVIII, pp. 147-169.
t “New Orleans Med. Jour.,” May, 1900.

aye. Yellow Fever

became able to impart yellow fever through their bites. This in-
_ fectious character, having once developed, seemed to remain through-

out 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 mos-
quito and in man must explain the symptomatology of yellow fever.

In order to establish these observations, experimental inocula-
tions were made upon human beings in sufficient number to prove
their accuracy. Unfortunately, Dr. Lazear 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 individual by means of the
bite of the mosquito that has previously fed on the blood of those sick with the
disease.

3. An interval of about twelve days or more after contamination appears to be
necessary before the mosquito is capable of conveying the infection.

4. The bite of the mosquito at an earlier period after contamination does not
appear to confer any immunity against a subsequent attack.

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. f
6. An attack of yellow fever produced by the bite of a mosquito confers im-

munity against the subsequent injection of the blood of an individual suffering
from the non-experimental 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. ;

g. 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 para-
site of this disease.

to. The spread of yellow fever can be most effectually controlled by measures
directed to the destruction of mosquitoes 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 place of mosquitoes
should be destroyed by drainage. Cisterns 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. ‘

* Pan-American Medical Congress, Havana, Cuba, Feb. 4-7, 1901; Sanitary
Department, Cuba, series 3, 1902.

Prophylaxis » Key

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 convalescent.

4. The premises where such a case has occurred should be fumigated by burning
pyrethrum powder (1 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 disin-
fection and without regard for fomites, 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.
Tf coarser than this the stegomyia mosquitoes can pass through.

Reed and Carrollt were the first to filter the blood of yellow
fever patients and prove that after it had passed through a Berke-
feld 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 intentionally
permitting non-immunes who are to be exposed to the disease to
be experimentally infected by being bitten by infected mosquitoes,
after which they are at once carefully treated. His first con-
clusion was that “the intentional inoculation gives the patient a
better chance of recovery,” but the danger of death from the ex-
perimental infection 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.

t Report of Working Party No. 2, Yellow Fever Institute, Bull. 14, May, 1904.

t“Am. Med.,” Feb. 22, 1902.

§ “Bull. No. 14, U. S. Public Health and Marine Hospital Service,’’ Washing-
ton, D. C., May, 1904.

|| “Revista de Medicina Tropical,” Havana, Cuba, 1902.

CHAPTER XXV
TYPHUS FEVER

Typuus fever, also known as jail-fever, ship-fever, army-fever,
and by a large number of other names, of which about a hundred
have been collected by Murchison,* has long been known, but was
probably not recognized as a definite disease before 1760, when
Gaultier de Sauvage endeavored to give it individuality, or 1769
when Cullum of Edinburgh defined it. Its eventual separation

from typhoid fever, with which it continued to be confused, was:

the result of the studies of Gerhard “On the Typhus Fever which
occurred in Philadelphia in the Spring and Summer of 1836, etc.” t
The Germans still speak of typhus abdominalis, meaning typhoid
or enteric fever, and typhus exanthematicus, meaning the typhus
fever of the present day. The Spanish and Mexicans call it
tabardillo.

The disorder is largely a disease of poverty, filth and crowding, and
is of frequent occurrence both in sporadic and epidemic form where
such conditions occur permanently or temporarily. Its most
common epidemic occurrence is therefore among the slums, in jails,
’ in ships, in asylums, in hospitals and in armies. With the improved
hygienic conditions of the present time its occurrence in consider-
able epidemics is much diminished, and it is not to be expected in
sanitary dwellings, among cleanly people or in well-regulated
institutions.

It is undoubtedly transmissible and therefore infectious, but
it early became clear that the infection was not air-borne and did
not readily pass from individual to individual. Further, it seems
clear that the survival of an attack confers immunity against future
infection.

In 1876 Moczutkowskif inoculated himself with the blood of a
patient suffering from typhus fever, and developed the disease
eighteen days later. In 1907 Otero§ endeavored to induce the dis-
ease in human beings by inoculation. In one out of four attempts
he was successful.

Experiments with a not infrequently fatal malady made upon
human beings being immoral and inexpedient, it became necessary
to find some animal susceptible to the disease, with which further
experiments could be prosecuted.

Se ‘A Treatise on the Continued Fevers of Great Britain,’’ 3d edition, 1884, p.
IO1r.
+ Amer. Jour. of the Med. Sciences, 1836, XIX, p. 283; 1837, XX, p. 289.

t “Allgemeine Med. Central Zeitung,” 1900, Lxvutt, 1055.
- § “Mem. pres. a l’Acad. de Med. de Mex.,” 1907.

578

Transmission 579

In 1909 Nicolle* succeeded in producing the disease in a chim-
panzee by inoculating it with human blood. Later he was able to
transmit the disease from the chimpanzee, and still later from human
beings, to Macacus sinicus by inoculating with infected blood. In
1909 Anderson and Goldbergert were successful in transmitting
the disease to monkeys, by inoculating them with human blood.
Other workers corroborated these results, and thus it became clear
that the suspicion that the disease was infectious was correct,
and that the infectious agent was in the blood with which it could
be carried over to new men and animals and reproduce the disease:
Later Nicolle, Couer and Conseil§ were able to transmit the disease
to guinea-pigs.

In Mexico, Gavefio and Girard|| were able to carry the infection
through 11 transplantations from guinea-pig to guinea-pig, and still
find it infective for monkeys.

Still, however, the micro-organism could not be found. Two
additional problems therefore became -important for solution.
First, what was the nature of this virus that could not be found,
second, how did it naturally pass from patient to patient?

In October, 1916, Nicolle, Couer and Conseil** instead of working
with artificially defibrinated blood, permitted the blood to coagulate
spontaneously, then passed it through the most porous kind of a
Berkefeld filter, and successfully infected one out of two monkeys
injected with the filtrate. After other series of experiments, these
investigators came to the conclusion that the serum of artificially
defibrinated blood, when filtered, was always without infective
power, and that of spontaneously coagulated blood, commonly
so, and that hence, though the virus of the disease is a filterable virus,
it consists of organisms so large as to be commonly held back by the
coarsest Berkefeld filters.

In regard to the transmission of the disease the investigators had
before them the usual exemption of physicians, nurses, attendants
and others who cared for patients suffering from the disease, as
contrasted with its persistent spread to new patients at the foci of
‘infection. They also had the recently gained knowledge of the part
played by insects and arthropods in the transmission of malaria,
relapsing fever, African lethargy, etc., the whole matter being of
such nature as to make the conclusion that the infection was trans-
mitted by an insect host, a justifiable one. .

The first to work upon this problem were Nicolle, ,Couer and
Conseil, tt the selected insects being pediculi. They permitted lice

* Ann. de l’Inst. Pasteur,’ 1910, XXIV.
| “Compt.-rendu Acad. d. Sciences de Paris,” 1910.
{ “Public Health Reports,’’ 1909, XXIV, p. I94I.
§ “Ann. de l’Inst. Pasteur,”’ 1910, XXV, 97.
| “Publ. de l’Inst. Bact. Noc. Mex.,” 1910, Nov. 9.
** “Ann. de |’Inst. Pasteur,” 1911, XXV, 97-
tt “Compt.-rendu de l’Acad. des Sciences de Paris,” 1909, CxLIx, 486.

580 Typhus Fever

to feed upon the blood of an infected monkey, and then upon a
healthy monkey. The healthy monkey contracted typhus fever.
In the same year, and working independently, Goldberger and
Anderson* made two attempts ot infect healthy monkeys by per-
mitting lice fed upon cases of typhus fever in men, to bite them.
They had partial success—the monkeys became diseased but no
immunity tests were made for confirmation of the nature of the
disease.

Ricketts and Wilder working in Mexico succeeded in transmitting
typhus fever from man to monkeys by means of lice—Pediculus
vestimenti. They also succeeded in transmitting the disease to a
monkey by scarifying its skin and applying the abdominal contents
of some infected lice, so that it was proved by them that the cause
of infection was in the lice. In the course of these experiments
Ricketts contracted typhus fever and unfortunately died. Later
Nicolle and Conseilt also succeeded in infecting a monkey by the
bites of infected lice. :

Wilder§ further found that the infectious agent passes from the
infected lice to a second generation of insects, as does the spiro-
cheta of relapsing fever to subsequent generations of ornithodorus
ticks. Wilder failed in experiments directed toward infecting
monkeys by fleas or bed-bugs.

In the experiments recorded by Wilder, the transmission of typhus
fever to monkeys, by lice, was successful in 7 out of 10 attempts.
It required 17 lice to infect a monkey. In one case a monkey
seemed to be immunized by being bitten by very young lice.

Goldberger and Anderson|| also experimented with the head
louse Pediculus capitis and succeeded in showing that it too takes

up the typhus fever virus and may pass it on from human being to

monkey, and hence probably from man to man.
A description of the lice will be found in the chapter upon “Re-
lapsing Fever.”

Bacittus TyPHI-EXANTHEMATICUS (PLATZ)

Ricketts and Wilder had observed a small bacillus both in the blood of some of
their patients and in the intestinal contents of some of the lice that they investi-
gated, but saw no reason for believing it to bejthe cause of the disease. What may
be the same organism was rediscovered by Platz (“Jour. Amer. Med. Asso.,”’ 1914,
LXII, p. 1556) in the blood of a series of caselet the variety of typhus fever called
Brill’s disease. It is too early to accept this bacillus as the cause of the disease,
but it is certainly worthy of careful consideration. According to Platz it is
characterized as follows: :

Mor phology.—It is a small straight, short, coccoid pleomorphus bacillus measur-
ing 0.9-1.93 u in length by 0.3-0.6u in breadth. Polar granules can be demon-
strated by appropriate staining. It has no capsule. It is not motile and has no
flagella. It forms no spores.

* “Public Health Reports,” 1910, Xxv.

tT “Jour. Amer. Med. Asso.,’’ 1910, LIV, 1304.

t “Compt.-rendu. de l’Acad. des Sciences de Paris,’ 1911, CLII, 1522.
§ “Journal of Infectious Diseases,’ 1911, IXI.

|| “Public Health Reports,” 1912, xxvil.

Bacillus Typhi-exanthematicus 581

Staining. —It stains ordinarily and is Gram-positive. It is not acid-fast.

Cultivation. —The organism is an obligatory anaérobe. It grows only at 37°C.,
better on solid than in fluid media, the best growth resulting from the use
of 0.5 per cent. glucose serum-agar. Upon plates under anaérobic conditions
in a Novy jar, colonies developed after seven days. They attained a size of 1.5—
2mm., were round or oval, were cream-colored by reflected light, opaque by trans-
mitted light.

The bacillus produces acid from glucose, maltose, galactose and inulin; no
acid from raffinose, mannite, arabinose, saccharose, dextrin or lactose. No vis-
ible gas results from the transformation of any of these carbohydrates.

No growth occurs in gelatin; an “invisible growth” occurs on potato.

Thermal Death Point—This was found to be 550°C. maintained for ten minutes.

Pathogenesis——When inoculated into guinea pigs the same symptoms were
observed as followed inoculation with typhus blood—.e., fever terminating by
crisis. ;

Complement-fixation—Complement-fixation tests were positive in six out of
eight cases tried, but only after the crisis of the disease had taken place.
There was no complement-fixation with any of thirty-six bloods used as controls.

CHAPTER XXVI
PLAGUE

Bacittus Prestis (YERSIN, KITASATO)

General Characteristics.—A minute, pleomorphous, diplococcoid and elongate,
sometimes branched, non-motile, non-flagellated, non-sporogenous, non-liquefy-
ing, non-chromogenic, non-aérogenic, aérobic, and optionally anaérobic, patho-
genic organism, 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 nodes, marked pul-
monary, cerebral and vascular disturbance, and the presence of the
specific bacillus in the lymphatic nodes and blood.

The history of plague is so full of interest that many references
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 Boccaccio 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 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 Em-
pire. 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 eighteenth centuries. In 1894 an
epidemic broke out in the western Chinese province of Yunnan
and reached Canton in January, 1894, thus escaping from its en-
demic center and began to spread. It can be traced from. Canton
to Hongkong. In 1895 it appeared also in Amoy, Macao, and
Foochoo. In 1896 it had reached Bombay and reappeared in Hong-
kong. In 1897 Bombay, the Madras Presidency, the Punjab, and
Madras were visited. In 1898 the disease spread greatly through-
out 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 Para-
guay on the south. In 1900 it had spread to nearly every part of
the world. In those places in which sanitary measures could not be
carried into effect the people died in great numbers—thus in India

582

Bacillus Pestis 583

in 1901 there were 362,000 cases and 278,000 deaths. In the first
‘six months of the epidemic of 1907, the deaths in India were much
more numerous, reaching a total of 1,062,908. Where sanitary
precautions are possible and co-operation between the people and
the authorities can be brought about, as in New York, San Fran-
cisco, and other North American and European ports, the disease
remains confined pretty well within limits and does 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 observa-
tions, carried off 95 per cent. of the cases. The death-rate varies in
different epidemics from 50 to 90 per cent. In the epidemic at

|
|
eg
ie
A
&

‘Fig. 228.—Axillary bubo (Reproduced’ from Simpson’s “A Treatise on Plague,”
1905, by kind permission of the Cambridge University Press).

Hongkong in 1894 the death-rate was 03. 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, pros-
tration, delirium, and the occurrence of exceedingly painful lym-
phatic swellings—buboes—affecting chiefly the inguinal nodes,
though not infrequently the axillary, and sometimes the cervical,
i nodes, 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 re-
E veals the characteristic enlargement of the lymphatic nodes, whose

contents are soft and sometimes purulent.
Wyman* in his very instructive pamphlet, ‘““The Bubonic Plague,”

* “Government Printing Office, Washington, D. C., 1900.

584 Plague

divided plague into (2) bubonic or ganglionic, (6) septicemic, and
(c) pneumonic forms. Of these, the bubonic form is most frequent
and the pneumonic form most fatal.

Specific Organism.—The bacillus of bubonic plague was inde-
pendently discovered by Yersin* and Kitasato 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 de-
scribed by the two observers are identical.

Ogatat 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 nodes; 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,

Fig. 229.—Bacillus of bubonic plague (Yersin). ‘

grayish white, with a bluish-tint, and resemble glass-wool when
slightly magnified; those of Yersin’s bacillus, white and transparent,
with iridescent edges. Ogata, in his investigations, found that
the bacillus corresponded with the description of Yersin rather than
that of Kitasato, and it is certain that of the two the description given
by Yersin is the more correct.

In the “Japan Times,” Tokio, November 28, 1899, Kitasato
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 Yersin’s observation
that the bacilli first multiply in the lymphatics.

Both Kitasato and Yersin showed that in blood drawn from tlie
finger-tips and in the softened contents of the buboes the bacillus
may be demonstrable.

*“ Ann. de l’Inst. Pasteur,’ 1894, 9

} Preliminary notice of the bacillus at bubonic plague, ee aie July 7, 1894.
t “Centralbl. f. Bakt. u. Parasitenk.,”’ 1897, Bd. xxt, p. 769.

Morphology 585

Morphology.—The bacillus is quite variable. Usually it. is
short and thick—a ‘‘cocco-bacillus,”’ as some call it—with rounded
ends. Its size is small (1.5 to 2 w in length) and 0.5 to 0.75 uw in
breadth. It not infrequently occurs in chains of four or six or even
. more, and is occasionally encapsulated. It shows active Brownian
movements, which probably led Kitasato to consider it motile.
Yersin did not regard it as motile, and was correct. It has no’
flagella. No spores are formed.

Staining.—It stains by the usual methods; not by Gram’s method.
When stained, the organism rarely appears uniformly colored, be-
ing darker at the ends than at the center, so as to resemble a dumb-
bell or diplococcus. The bacilli sometimes appear vacuolated,
and nearly all cultures show a variety of involution forms. Kitasato

Fig. 230,—Bacilli of plague and phagocytes, from human lymphatic gland X 800
(Aoyama). :

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 characteristic. The microbes
swell and form large, round, oval, pea-shaped, spindle-shaped or
biscuit-like bodies which may attain twenty times the normal
size, and gradually lose the ability to take the strain. Such involu-
tion forms are not seen in liquid culture.

Cultivation.—Pure cultures may be obtained 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 observed by
Kitasato, though Versin observed that the cultures resembled ery-

586 Plague

sipelas cocci, and contained zodglea attached to the sides and at
the bottom of the tube of nearly clear fluid.

Haffkine* found that when an inoculated bouillon culture is
allowed to stand perfectly at rest, on a firm shelf or table, a char-

Fig. 231.—Bacillus pestis. Highly virulent culture forty-eight hours old, from
the spleen of arat. Unstained preparation (Kolle and Wassermann).

acteristic appearance develops. In from twenty-four to forty-
eight hours, the liquid remaining limpid, flakes appear underneath
the surface, forming little islands of growth, which in the next
twenty-four to forty-eight hours grow into a jungle of long stalactite

Fig. 232.—Bacillus pestis. Involution forms from a pure culture on 3 per

cent. sodium chlorid agar-agar. Methylene-blue (Kolle and Wassermann). ~

like masses, the 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

* “Brit. Med. Jour.,” June 12, 1897, p. 1461.

Gelatine Punctures 587

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 microscopic examination the borders
of the colonies are found to be sharply defined. The contents be-
come 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. Magnifica-
tion shows the colonies to be serrated at the edges and made up of
short, oval, sometimes 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 colonies of Proteus

Fig. 233.—Stalactite growth of bacillus pestis in bouillon (Reproduced from
Simpson’s “A Treatise on Plague,” 1905, by kind permission of the Cambridge
University Press). - .

vulgaris. At first these were regarded as contaminations, but later
their occurrence was regarded as characteristic of the plague bacillus.
The peculiarities of these colonies cannot be recognized after forty-
eight hours.

Gelatin Punctures.—In gelatin puncture cultures the develop-
Ment isscant. 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. Sometimes fine filaments project into
the gelatin from the central puncture.

Abel found the best culture-medium to be 2 per cent. alkaline
peptone solution containing 1 or 2 per cent. of gelatin, as recom-
mended 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

fn % ‘
*“Centralbl. f. Bakt. u. Parasitenk.,” July 10, 1897, xxi, Nos. 24 and 25

588 Plague

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 microscope 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 diganosis
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 in-
volution forms so characteristic of the bacillus occur with ex-
ceptional rapidity. In bouillon containing this high percentage of
salt the stalactite formation is beautiful and characteristic.

Blood-serum.—Upon blood-serum, growth, at the temperature
of the incubator, is luxuriant and forms a moist layer of yellowish-
gray color, unaccompanied by liquefaction 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.

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’ ex-
posure to c.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 o.5 per cent. carbolic acid. In o.1 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 sunlight for three or four
hours; and Bowhillf found that they are killed by drying at ordinary
room temperatures in about four days.

Wilson§ 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 influence of sunlight and desicca-
tion cannot be relied upon to destroy it.

: “Centralbl. f. Bakt. u. Parasitenk.,’’ Oct., 1897, Bd. xxu, Nos. 16 and 17, p.
w + Quoted by Wyman.

t “Manual of Bacteriological Technique and Special Bacteriology,”’ 1899, p. 197.
§ “Journal of Medical Research,” July, rgo1, vol. vi, No. 1, p. 53.

Metabolism 589

Rosenau* 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 tem-
perature of 70°C. is invariably fatal in a short time.

Metabolism.—The bacillus develops best under aérobic con-
ditions though it develops to a slight extent also under anaérobic
conditions. In sugar-containing media it does not form gas. Acids
are formed from dextrose, lactose, galactose, mannite and maltose
but not from saccharose, sorbite, dulciteorinulin. Noindolis formed.
Ordinarily the culture-medium is acidified, the acid reaction persist-
ing for three weeks or more. .

Ghon,{ Wernicke,{ and others who have studied the toxic prod-
ucts 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.

Bielonovsky || finds that broth, agar, and serum cultures of the
plague bacillus possess the property of hemolyzing the blood of
normalanimals. The hemolytic power of filtrates of plague cultures
increase up to the thirteenth or fourteenth day, then gradually di-
minishes, but without completely disappearing. The hemolysins
are notably resistant to heat, not being destroyed below 100°C.

_ Experimental Infection—Mice, rats, guinea-pigs, rabbits, and
“monkeys are all susceptible to experimental inoculation. When
_blood, lymphatic pulp, or pure cultures are inoculated into them,
the animals become 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 suffer from both an acute septicemic and a chronic form of
the disease. In the former 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 yellowish gelatinous exuda-
tion. The spleen and liver are enlarged, the former often pre-
senting an appearance similar to that observed in miliary tuber-
culosis. Sometimes there is universal enlargement of the lymphatic

* Bulletin No. 4 of the Hygienic Laboratory of the U. S. Marine Hospital

ervice, T9OT,

t Wien, 1898.

{“Centralbl. f. Bakt.,” etc., 1898, xxiv.

“Arbeiten aus d. kaiserl. Gesundheitsamte,” 1901, XVII.
|| “Arch, des Sci. Biol.,” Petersb., 1904. St. Tome x, No. 4.

590 Plague

glands. Bacilli are found in the blood and in all the internal organs.
Skin eruptions may occur during life, and upon the inner abdominal
walls petechiz and occasional hemorrhages may be found. The
intestine is hyperemic, the adrenals congested. Serosanguinolent
effusions may occur into the serous cavities.

Sometimes encapsulated caseous nodules in the submaxillary
glands, caseous bronchial glands, and fibroid pneumonia, are found
months after infection. In all such cases virulent plague bacilli are
present. ;

In and about San Francisco the extermination of rats for the
eradicaton 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* 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 necessitated continued observation of these
animals and the extermination of diseased colonies, as well as their
complete extermination in the neighborhood of human habitations.

Devell} has found frogs susceptible to the disease.

Mode of Infection.—The plague bacillus may enter the body by
inhalation, from an atmosphere through ‘which it is disseminated, |
under which circumstances it usually causes the pneumonic type
of the disease which 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.

Plague pneumonia does not necessarily imply infection through
inhalation of the bacilli, however, for it occasionally occurs as a
complication in the bubonic form of the disease.

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. Simond has confirmed his results and
it is not improbable that the disease is sometimes acquired by
rats through feeding upon their companions that have died of it.
The micro-organisms seem able to penetrate any of the mucous mem-
branes, so that infection usually follows their application to the un-
injured conjunctiva, nasal, buccal, vaginal or gastro-intestinal
surfaces.

Cutaneous and Subcutaneous Inoculation.—All susceptible ani-
mals quickly become infected if a needle infected with a culture
of the bacilli or with material from a bubo or other infective lesion
be used to puncture or scratch the skin. Wyssokowitsch and
Zabolotny{t found monkeys highly susceptible to plague, especially
when subcutaneously inoculated. 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

* “Journal of Infectious Diseases,’ 1910, VII, p. 374.
t “Centralbl. f. Bakt. u. Parasitenk.,’ Oct. 12, 1897.

Mode of Infection ‘ 591

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. Weichselbaum, Albrecht and Gohn have found that
rats may be infected by rubbing the infective material upon the
surface of the shaved skin, the method being employed for making
a diagnosis of the disease in suspected cases. Rats and mice in-
fected through the skin usually die in two or three days, guinea-
pigs in two to five days, rabbits in three to eight days.

The facility with which dermal infection could be brought about,

Antepygidial bristle Abdomen Thorax Head Antenna
fa Eye
Pygidium sat OF iS > “. Ocular bristle

—

_. Oral bristle

Mi See
Stigmata -4\I\¥3 f

Penis , iN Maxillary palp

" Maxilla

Vth... Hip or coxa
S---- Trochanter

j A. Femur
Se, Tarsi
nh ;
¢
‘a
ry

Sr
7,

Fig. 234.—Xenopsylla cheopis (male) (from Rothschild).

quickly suggested that the skin might be the common route, and
that biting insects might act as vectors. :

Yersin showed that flies taking up the bacilli may die of the in-
fection. Macerating 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 observa-
tion correct, and showed that flies fed with the cadavers of plague-
infected mice die in a variable length of time. Large numbers of
plague bacilli were found in their intestines. 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

*“Centralbl. f. Bakt. u. Parasitenk.,”’ xxr, No. 24, Aug. 13, 1897.

592 Plague

of his experiments upon this point was great enough to prove that
plague cannot be thus spread. Vergbitski,* however, was more
successful and a bed-bug that he caused to bite a patient suffering
from plague, subsequently transmitted the disease to a rat. It is
quite possible that mosquitoes and biting flies may transmit it.

M. Herzog} has shown that pediculi may harbor plague bacilli
and act as carriers of the disease.

As epidemics of human plague are commonly preceded by epi-
demics among the rats which die in great numbers, it early became
a question whether the plague among them was not caused by the
bites of fleas, and whether it might not also be fleas that infected
man.

Ogatat 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 spontaneously of the disease, presumably because of
flea infection.

Galli-Valerio§ and others thought that the fleas of the mouse and
rat were incapable of living upon man and did not bite him, and
that it was only the Pulex irritans, or human flea, that could transmit
the disease from man to man. Tidswell||, however, found that
of roo fleas collected from rats—there were four species, of which
three—the most common kinds—bit men as well as rats. Lisbon**
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 Investiga-
tions 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 1906 onward. {Tt
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

* “Tour. of Hygiene,” 1904, vit, 185.
t “Amer. Jour. Med. Sci.,”” March, 1895.
t“ Centralbl. of Bakt. in Parasitenk., 1897, XxI, p. 769.
§ Ibid., xxvu, No. 1, p. 1, Jan. 6, 1900.
| “Brit. Med. Jour.,” June 27, 1903.
** «Times of India,” Nov. 26, 1904.
tt “Journal of Hygiene,” Sept., 1906, vol. v1, p. 421; July, 1907, vol. vil, p.
324; Dec., 1907, vol. vil, p. 693; May, 1908, vol. vit, p. 162; 1909, vol. 1x; 1910,
vol. X; 1911, vol. x1. *

Mode of Infection 593

of the infected houses, the fleas of all kinds quickly attack them
with resulting 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 fleas. When the rats die, the fleas being hungry,
jump upon any convenient warm-blooded animal to satisfy their
appetites, and when human beings become their victims, infection
may follow the bites. It is now clearly demonstrated that though
Pulex irritans, the human flea, prefers to bite human beings, and
Xenopsylla cheopis, the rat flea, prefers to bite rats, under stress of
necessity preferences are set aside and miscellaneous feeding prac-
tised by these and probably all other fleas.

A peculiar circumstance attending flea infection has been dis-
covered by Bacot and Martin* who find that when Xenopsylla cheo-
pis and Ceratophyllus fasciatus are fed upon septicemic plague blood,
the respective fleas suffer from a temporary obstruction at the en-
trance of the stomach, caused by a massive growth of the plague
bacilli. This culture appears to start in the intercellular recesses of
the proventriculus and grows so abundantly as to choke this organ
and extend into the esophagus. Fleas in this condition are not
prevented from sucking blood, as the pump is in the pharynx, but
they only succeed in distending an already contaminated esophagus,
and on the cessation of the pumping act, some of the blood is forced
back into the wound. Such fleas are persistent in their endeavors to
feed and this renders them particularly dangerous.

Bacott found that infected fleas remained infectious when starved
for forty-seven days, and that when they were subsequently per-
mitted to feed upon mice, another period of twenty days might
supervene before the mice became infected.

The cutaneous and subcutaneous inoculation in man is followed
by lymphatic invasion with bubo formation. Beyond this lymphatic
barrier but few bacilli get so that in the greater number of cases
with buboes there is little blood infection. However, should the
bacilli be highly virulent or the patient exceptionally susceptible,
the septicemic form of the disease may supervene, and the case
Progress to a rapidly fatal termination.

Intravenous and Intraperitoneal Inoculations produce rapidly fatal
Septicemic forms of plague. .

Kleint 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

* «The Journal of Hygiene,” Plague Supplement, 1, 1914, p. 423.

t “Journal of Hygiene,”’ Plague Supplement, No. rv, Jan., 1915, p. 770.

}“Centralbl, f. Bakt. u. Parsitenk.,” 200, No. 24, July 10, 1897, p. 849.
38

594 Plague

chains of the plague bacillus, occurring in from twenty-four to
forty-eight hours.

Diagnosis.—It seems possible to make a diagnosis of the disease
in doubtful cases by examining the blood, but it is admitted that a
good deal of bacteriologic practice is necessary for the purpose.

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

ae ca

Fig. 235.—Fig. 1 is a diagrammatic representation of a longitudinal section
through the esophagus (@), proventriculus (p), and stomach (s) of a heavily
infected specimen of Ceratophyllus fasciatus. The light shaded portion shows
where fresh blood, impregnated with free individuals of B. pestis, is present in
the specimen, the darker shading indicates the solid mass of bacteria which has
so far become disintegrated at its center as to be ruptured by the force of the
blood pumped into the esophagus, thus allowing the passage of blood to the
stomach. ‘The action of the valve is, however, inoperative, owing to the solidity
of the mass of bacteria in which the spines of the proventriculus are embedded.
X_ about 180 reduced to one-fourth the size.

Fig. 2 shows a similar representation of a section through the dissected pro-
ventriculus and esophagus of a specimen of C. fasciatus. It differs from Fig. 1
in that the lumen of the valve is still obstructed by the disintegrating mass of
an old plug and that the growth of bacteria surrounding this, which is of more
recent growth, though yielding to the pressure of the fresh blood pumped into
the esophagus, has not yet been ruptured. X about 180 reduced to one-fourth
the size (Bacot, in Journal of Hygiene).

injury of the skin. The infective material is carefully rubbed into
the shaved skin. Important, 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 pro-
duction 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.”

i Bee Havelburg, “‘Public Health Reports,” Aug. 15, 1902, vol. xvi, No. 33,
p. 1863.

Virulence 595

The postmortem appearance of the body of a plague-infected
rat is as follows:* Subcutaneous hemorrhages occur in about 4o
per cent. of the animals and are most frequently to be seen in the
submaxillary region. Buboes are present in the majority of cases,
usually in some one locality, and commonly about the neck. The
liver may show necrotic changes which have the appearance of an
excessive deposit of fat, and a condition of the greatest importance
in diagnosis is the occurrence of small necrotic foci scattered over
its surface and throughout its substance. The spleen is firm and
does not collapse like a soft normal spleen; granules or nodules may
be well marked in it and may be confluent. The kidneys and
suprarenal capsules are often congested. Hemorrhages are fairly
common in the lungs and visceral pleura. The presence of pleural
effusion is very characteristic and of great value in diagnosis. In
naturally infected plague rats, the most important features for

_ purposes of diagnosis are:

1. A typical bubo—most commonly in the neck.

2. Granular liver—not seen except in plague rats.

3. Hemorrhages beneath the skin and in the internal organs are

"very suggestive.

4. Pleural effusion. :

In putrid rats, bubo, granular liver and pleural effusion may persist
and are of great significance. A microscopical examination of
scrapings from buboes and spleen and inoculation tests will clinch
the diagnosis (Besson).

Virulence.—It was formerly supposed that by frequent passage
through animals of the same species the bacillus could be much in-
creased in virulence. Kolle recommended rats for this purpose, and,
indeed, declared that without the use of rats it is impossible to keep
cultures at a high grade of virulence. Yersin thought 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.

Knorr, Yersin, Calmette, and Borrelft found that the bacillus
made virulent by frequent passage through mice is not increased in
virulence for rabbits. According to the researches of the Advisory
Committee for the study of plague in India, this is an error. The
virulence of plague bacilli for rats is subject to very little change.
Their members in investigating the question made twenty-six
passages from rat to rat, by subcutaneous inoculation, during
eighty-nine days, and found the original virulence of the Sree
unchanged.

Sanitation—A. disease that may be transmitted from man to
man in by,atmospheric infection and inhalation, that can be transported

. _ * See “Journal of Hygiene,” 1907, VII, 324. |
_ t “Ann. de Inst. Pasteur.,’ ~” July, 1895.

596 . ‘Plague

from place to place by fomites, that occurs in epidemic form among
the lower animals as well as among men, and that can be trans-
mitted 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 a plague-stricken
port, means must be taken to see that all passengers are healthy

Fig, 236.—Plague-infected rat. A composite picture, illustrating some of the
common naked-eye pathological changes found in various organs and tissues in a
plague-infected rat. Note (a) marked subcutaneous congestion causing a
peculiar pink appearance of the tissues; (b) submaxillary bubo; (c) subcutaneous
punctate hemorrhages most frequently found in the neck; (d) marked congestion
and hemorrhages in the thoracic cavity, especially in the lungs; (e) advanced
stage of mottled granular liver; (f) enlarged and congested spleen.

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 goods are landed. ‘ But the rats must be
given special consideration, for, so soon as the vessel reaches port
some of them jump overboard and swim to the shore, carrying the
disease with them. When a vessel visits a plague port, every pre-

Sanitation 597

caution should be taken to prevent the entrance of rats, first by
anchoring in the stream instead of tying to the dock; by carefully
scrutinizing 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 swim-
ming at night. Arrangements should also be made for rat destruc-
tion on board the ship by means of sulphurous oxid or other poi-
sonous vapors to rid the ship of rats before the next port is reached.

Fal

Fig. 237—Healthy rat to be contrasted with plague-infected rat, Fig. 236.

Passengers and crew should also be kept in quarantine before ming-
ling with society. It is much more easy to keep plague out ofa
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 immediate warfare upon the rats and
other small animals of the neighborhood. To emphasize how
dificult the latter may be it is only necessary to point out that
plague reached San Francisco in May, 1907, during which year

598 Plague

there were 156 cases and 76 deaths. Every precaution was taken
to prevent its spread, and though the extermination of rats was
practised at great expense and with the utmost thoroughness, the
disease spread to the ground squirrels and other small rodents, and
in 1914 plague-infected rodents were still to be found in the cuisines
of the city.

Immunity.—An attack of plague usually exempts from farts
attacks. Artificial immunity may therefore be induced in both man
and the lower animals by a variety of methods.

I. Active _Immunity—Haffkine* followed his plan of oie
inoculation as employed against cholera, and has devised a method

of prophylaxis based upon the use of devitalized cultures. Bouillon

cultures are grown in flasks for six weeks; small floating drops of
butter being employed to make the “islands’’ of plague bacilli
float. Successive crops of the island-stalacite growth are ‘pre-
cipitated by agitating the flasks. In this manner an “intense extra-
cellular toxin,’”’ containing large numbers of the bacilli is prepared.
After testing the purity of the culture by transplantation to agar-
agar, it was killed by exposure to 65°C. for one hour and received
an addition of 0.5 per cent. of phenol. The preparation was used in
doses of 2 to 3 cc. as a preventive inoculation. A more thorough
and prolonged immunity resulted from the administration of a
second dose ten days after the first.

An interesting collection of statistics, showing in a convincing
manner the value of the Haffkine prophylactic, is published by
Leumann, of Hubli. The figures, together with a great deal of
interesting information upon the subject, can be found in the
paper upon “A Visit to the Plague Districts in ae by Barker
and Flint.t

The German Plague Commission{ believed that an important
improvement in the vaccine could be brought about by the use of
the method now generally employed in making bacterio-vaccines
(q.v.). They therefore caused the bacilli to grow in Roux bottles
upon the surface of agar-agar for forty-eight hours, washed off the
bacteria with bouillon or physiological salt solution so that 1 cc. of
the suspension contained about 2.5 mg. of bacilli, and then heated
the suspension for an hour or so at 65°C. After heating, 0.5 per
cent. of phenol was added. This mode of preparation has the ad-
vantage of excluding the possibility of the accidental growth of
tetanus bacilli and other micro-organisms in the culture. The
vaccine appeared to give excellent results in Brazil where it was ex-
tensively used. Haffkine, however, considers his method preferable
because of the greater quantity of immunizing metabolic products
of the bacilli contained in the fluid cultures on account of their pro-
longed growth.

* “Brit. Med. Jour.,”’ June 12, 1897; ‘‘India Medical Gazette,” 1897.

+ “New York Med. Jour.,’’ Feb. 3, 1900.
t“Arbeiten aus dem Kaiserl. Gesundheitsamte,’’ 1899, XVI.

Immunity 599

: The immunity conferred by the Haffkine prophylacticis supposed
to last about a year. The preparation must never be used if the
person has already been exposed to infection, and is in the incuba-
tion 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.

Kolle and Otto* from experimental studies of plague immunity
in rats, came to the conclusion that a prophylactic injection con-
sisting of a culture of attenuated plague bacilli would have a much
more powerful and lasting effect than one consisting of killed bacilli.
The same conclusion was reached by Kolle and Strongt and the
first use of living cultures for preventive inoculation in human beings
was by Strong{ who found them to be devoid of danger, and is
hopeful regarding their efficacy. By

Besredka§ advises the use of a killed culture sensitized by the

. application of immune serum. Such vaccine seems to be productive
of long enduring immunity when tried upon experimental animals.

Rowland|| is under the impression that the essential immunizing
antigen is in the bacterial nucleoproteins. These he extracts from
the bacterial cells by treating them while moist with anhydrous
sodium sulphate, freezing, permitting the water to be absorbed by
the chemical, thawing, and then filtering off the fluid at 37°C. The
filtrate thus obtained is highly toxic, fatal to rats in minute doses and
capable of effecting immunization.

IL. Passive Immunity against plague, through the employment
of the serums of experimentally immunized animals for hypo-
dermatic injection into man was tried soon after the discovery of
the plague bacillus. 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 pre-
venting the disease, but also of curing it in mice and guinea-pigs
and probably in man.

Study of plague serums has been conducted by Yersin, Calmette
and Borrel,** but their value as a prophylactic lacks demonstration.

Wyssokowitsch and Zabolotny,}{ used 96monkeys in the study
of the value of the “plague serums,” and found that when treatment

* “Deutsche med. Wochenschrift,” 1903, p. 493; ‘Zeitschrift fiir Hygiene,”
a XLV, 507.
“Deutsche med. Wochenschrift,”” 1906, XXXII, 413.
i “Jour. Medical Research,” N. S., 1908, XVIII, 325.
§ “Bull. de l’Inst. Pasteur,’’ 1910, VIII, 241.
al Jour. of Hygiene,” 1912, x1, 344.
“Ann. de Inst. Pasteur,” 1895, IX, 589.
tt Loe. cit.

600 Plague

was begun within two days from the time of inoculation the animals
could be saved, even though symptoms of the disease were marked.
After the second day the treatment could be relied upon. The dose
necessary was 20 cc. of a serum having a potency of 1:10. If too
little serum was given, the course of the disease was retarded and the
animal impoved for a time, then suffered a relapse, and died in ~
from thirteen to seventeen days. The serum also produced im-
munity, but of only ten to fourteen days’ duration. Immunity
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.
THE PLAGUE FLEAS

Fleas were formerly classed as a suborder of the Diptera, or two-winged insects,
and because they had no wings, were known as Aphaniptera. At the present
time they constitute an order by themselves, the Siphonaptera.

Fig. 238.—Various fleas, magnified about 30 diameters. The specimens are
treated with hot 20 per cent. caustic potash for a few minutes, dehydrated in
alcohol, cleared in xylol and mounted in balsam. a, Ceratophyllus fasciatus, 3’;
b, Ceratophyllus fasciatus, ? ; c, Leptopsylla musculi, ; d, Leptopsylla musculi, ?
(Bacot, in Journal of Hygiene, “Plague Supplement 11, 1914’).

Every flea undergoes a complete metamorphosis. It begins its life history as
a minute, oval pearly white egg measuring about 0.6 mm. in length, that falls
from the body of the female to the floor or ground. The eggs of fleas are not
cemented to the hairs like those of lice, but drop to the ground where the larva
lives. More or less eggs are therefore always scattered about where dogs, cats,
Tats, Mice or other animals that harbor fleas are to be found, and more or less
larvee and pupz are likewise to be found in such places. In the course, of from

The Plague Fleas 601

five to ten days, a minute, active, caterpillar-like larva emerges from the
egg to feed upon such organic matter as it may find for the six to eight weeks of
this stage. During the larval period the skin is shed three or four times. When
full grown, the larva empties its alimentary canal, spins itself a tiny silken cocoon,
sometimes including minute bits of rubbish or grains of sand in its structure,
sheds its skin for the last time, and becomes a pupa. As such it is inactive for
from two to eight weeks, according to external conditions of temperature and
moisture, then opens the cocoon and emerges from the pupa shell, a perfect. in-
sect—the flea proper.

The adult fleas, both males and females, have soft exoskeletons at first, but
soon they harden, through the formation of chitin, to the well-known tough and
brittle armor. ;

Fig. 239.—Various fleas, magnified about 30 diameters. The specimens are
treated with hot 20 per cent. caustic potash for a few minutes, dehydrated in
alcohol, cleared in xylol and mounted in balsam. a, Ctenocephalus canis, 07; b,
Ctenocephalus canis,?; c, Ctenocephalus felis, o’; d, Ctenocephalus felis, ?
(Bacot, in Journal of Hygiene, “‘ Plague Supplement 11, 1914”).

The male differs from the female in being smaller and in its shorter abdomen.
Both insects hop about in search of the appropriate warm-blooded hosts upon
whose blood they are to live. Each kind of flea has a preferred host, but the
tastes of all are more or less cosmopolitan, so that in the absence of the preferred
host, another kind of warm-blooded creature will do. Adult fleas live solely by
sucking blood.

The longevity of a flea varies according to conditions of temperature and mois-
ture. Life is longest when the temperature is high and the ground not too dry.
' They may. live for months without feeding; when regularly fed they can live at
least a year and a half. The longevity of the fleas in the adult stage, the long
periods of abstention from food that they may suffer without dying, and the ac-
cessions to their numbers that may occur through the maturation of their embry-
onal fellows in the same place, explain why families returning to their closed city

uses, or going to their closed country houses, sometimes find them after months

602 - Plague

of desertion, occupied by a welcoming host of fleas. They are the progeny of the
fleas of the former dog, cat, rat or mouse tenants, that have matured or survived
the interval and are now hungry because the removal of the family months before,
was probably followed by the withdrawal of the rats and mice no longer able to
find food in the deserted habitation. 4

To get rid of such fleas is often a perplexing question. A way to accomplish
it is to place a cage containing a cat or a guinea-pig, or a trap containing living rats
or mice on the floor of a room and surround it by sticky fly-paper. Fleas. when
empty and hungry, were found by Strickland* to be able to jump 4 inches;
those recently fed only 3 inches. In their endeavors to reach the caged animals
the fleas jump upon the fly-paper and are caught. This can be done in several
rooms of the house and soon cleans up the fleas.

Fig. 240.—Various fleas, magnified about 30 diameters. The specimens are
treated with hot 20 per cent. caustic potash for a few minutes, dehydrated in
alcohol, cleared in xylol and mounted in balsam. a, Pulex irritans, 0°; b, Pulex
irritans, 9 ; c, Xenopsylla cheopis,o"; ¢, Xenopsylla cheopis, @ (Bacot, in Jour-
nal of Hygiene, “Plague Supplement 1, 1914’).

During such periods of fasting the sexes do not copulate and no ova are pro
duced. As soon as blood is taken, copulation takes place; and if the blood be
that of the preferred host, ovulation follows in about twenty-four hours. The
eggs are relatively large, and small numbers are produced.

In the case of Sarcopsylla penetrans, a flea that has no known interest in con-
nection with plague transmission, the female after copulation imbeds itself in the
skin of the host and suffers an enormous saccular distension of the abdomen where
many ova are produced. Ordinary fleas never imbed themselves but simply bite
and suck blood, leaping off the host when satisfied. ;

Epidemics of plague among men are commonly preceded by epizootics of plague
among rats. The mortality of the rats being high and their number diminishing,
many fleas are unprovided for and seek human hosts upon whom to satisfy their
appetites. In this way, the plague which was at first transmitted by the fleas to

* “Journal of Hygiene,” 1914, XIV, p. 129.

The Plague Fleas 603

the rats, is now transmitted to men. Human fleas may also transmit the infec-
tion from man to man, but the bulk of the transmission probably takes place
through rat fleas.

When the plague spreads from the rat to ground squirrels or to marmots, rare
fleas may engage in the transmission of the disease from animal to animal and
from man to man, but ordinarily it is the common rat fleas that are responsible
for it.

. Both rats and fleas vary in prevalence and in relative frequency in different
parts of the world. Thus there are three common rats: Mus decumanus, the
_ brown or sewer rat, Mus rattus, the black or house rat and Mus norvegius, the
Norway rat. In Northern Europe, the Mediterranean coast, Egypt and North
America, the Norway rat has colonized more or less successfully. Where it
preponderates Ceratophyllus fasciatus is a common flea. Where Mus decumanus
and Mus rattus alone are found, or are preponderant, Xenopsylla cheopis is the
common flea. In the Orient, Xenopsylla cheopis is the chief flea that is to be
taken into account in plague transmission. The dog flea Ctenocephalus canis
is common everywhere as is Pulex irritans, the human flea. It is likely that any
or all of these engage in plague transmission when once an epidemic has started,
but the most active vector of the disease, the world over, and the most important
agent in starting human epidemics of plague is Xenopsylla cheopis.

Much interesting and valuable information concerning the biology, bionomics
and relation of rats and fleas to plague, will be found in the “Reports of the
India Plague Commission”? many of which are to be found in the “Journal of
Hygiene,” vols. 1-xIv. : :

The following illustrations and tabulations. will enable the student to identify
the common genera of fleas. For more intimate systematic study he must be
teferred to “A Text-book of Medical Entomology,” by Patton and Cragg.*

* “Christian Literature Society of India, London, Madras and Calcutta,”’ 1913.

604 Plague

TABLE FOR THE IDENTIFICATION OF THE FLEAS CONCERNED IN
PLAGUE TRANSMISSION ;

Family—PULICID.
Subfamily—PULICIN A.
All have eyes.

A. Have no combs or spines on
head, thorax or abdomen.

a. The meso-sternite is narrow
and has no rod-like incrassa-
tion from the insertion of the
coxa upward.........+-00- Pulex,

b. The meso-sternite has a rod-

like incrassation from the

. insertion of the coxa up-
ward....... Foss etapmitohee ads ‘ NX enoteila:

B. With‘combs.

c. Combs on the _ prothorax
ON Yiice ves new cay nee oe Ceratophyllus,

d. Combs on the prothorax and
on the gena or lower margin
of the face.......... Ctenocephalus.

OTHER MICRO-ORGANISMS OF THE PLAGUE GROUP

The Bacillus pestis is a member of a group of organisms col-
lectively known as the bacilli of hemorrhagic septicemia. Two,of
these organisms are of sufficient interest to deserve special mention.

Chicken-cholera 605

Bacittus CHOLERA GALLINARUM (PERRONCITO); BactLLus CHOL-
ER; Bacittus AviciIpumM; BAcILLus AVISEPTICUS;
BACILLUS OF RABBIT SEPTICEMIA; BACILLUS

CUNICULICIDA

General Characteristics—A non-motile, non-flagellated, non-sporogenous -
non-liquefying, non-chromogenic, aérobic and optionally anaérobic bacillus,
pathogenic for birds and mammals, staining by the ordinary methods, but not by
Gram’s method, producing acids, indol, and phenol, and coagulating milk.

The barnyards of both Europe and America are occasionally visited by an
epidemic disease known as ‘‘ chicken-cholera,’’? Huhnercholera, or cholera de poule,
which rapidly destroys pigeons, turkeys, chickens, ducks, and geese. Rabbit-
watrens 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, measur-
ing 1 X 0.4 to 0.6 wu, sometimes joined to produce chains. Pasteur at first re-
garded them as diplococci, because the poles stain intensely, a narrow space
between them remaining almost uncolored. This peculiarity 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.

{Thoinot and Masselin assert that the organism is motile. ‘‘Précis de
Microbie,”’ 2d ed., 1893. : :

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 the bacilli be
planted upon the surface of obliquely solidified gelatin, 4 much more pronounced
growth takes place, and along the line of inoculation a dry, granular coating is
formed. There is no liquefaction of the medium.

Bouillon.—The growth in bouillon is accompanied by a slight cloudiness.

Agar.—This growth, like that upon agar-agar and blood-serum, is white,
shining, rather luxuriant, and devoid of characteristics.

Potato.—Upon potato no growth occurs except at 37°C. It is a very insignifi-
cant, yellowish-gray, translucent film. :

Milk is acidulated and slowly coagulated. :

Vital Resistance.—The bacillus readily succumbs to the action of heat and
dryness. The organism is an obligatory aérobe.

Metabolic Products.—Indol and phenol are formed. Acids are produced in
sugar-containing media, without gas formation.

Pathogenesis.—The introduction of cultures of this bacillus into chickens,
geese, pigeons, sparrows, mice, and rabbits is sufficient to produce fatal septice-
Mia. 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
tufledup. The eyes are closed shortly after the illness begins, and the birds grad-
ually fall into a stupor, from which they do not awaken. The disease is fatal in

tom twenty-four to forty-eight hours. During its course there is profuse
diarrhea, with very frequent fluid, slimy, grayish-white discharges.

Lesions.—The autopsy shows that when the bacilli are introduced subcuta-
neously a true septicemia results, with the formation of a hemorrhagic exudate and

* “ Archiv, f. wissenschaftliche und praktische Thierheilkunde,” 1879.
t “Compte-rendu de I’Acad. de Sci. de Paris,” vol. xc.

606 Micro-organisms of the Plague Group

gelatinous infiltration at the seat of inoculation. The liver and spleen are en-
larged; circumscribed, hemorrhagic, and infiltrated areas occur in the lungs; the
intestines show an intense inflammation with red and swollen mucosa, and occa~
sional ulcers following small hemorrhages. Pericarditis is frequent. 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 the bacilli were inoculated into the pectoral muscles of pigeons, if
death did not come on rapidly, portions of the muscle (sequesira) underwent
degeneration and appeared anemic, indurated, and of a yellowish color. _
Immunity.—Pasteur* discovered that when cultures are allowed to remain un-
disturbed for several months, their virulence becomes greatly lessened, and new
cultures transplanted from them are also attenuated. If chickens be inoculated

Fig. 241.—Bacillus of chicken-cholera, from the heart’s blood of a pigeon.
X 1000 (Frinkel and Pfeiffer).

with such attenuated cultures, no other change occurs than a local inflammatory
reaction that soon disappears and leaves the birds protected against future
infection with virulent bacilli. From these observations Pasteur 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 Australia, where they are
pests. It is estimated that two gallons of bouillon culture will destroy 20,000
rabbits, irrespective of infection by contagion. 7
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 ““Wildseuche” (Hiippe), bacillus of ‘

Biiffelseuche”’ (Oriste-
Armanni), etc. :

* An interesting account of Pasteur’s experiments upon chicken-cholera can be
found in. the “Life of Pasteur,” by Vallery-Radot; translated by Mrs. R. S.
Devonshire, 1909. Popular Edition, New. York, Doubleday, Page and Co.

Swine-plague 607

BaAcILLUS SUISEPTICUS (LOFFLER AND Scutirz)

General Characteristics—A non-motile, non-flagellated, non-sporogenous,
non-liquefying, non-chromogenic, non-aérogenic, aérobic and optionally anaéro-
bic bacillus,, pathogenic for hogs and many other animals, staining by the ordi-
nary methods, but not by Gram’s method.

The bacillus of swine-plague, or Bacillus suisepticus of Léffler and Schiitz* and
Salmon and Smith, but slightly resembles the bacillus of hog-cholera (q.v.),
though it was formerly confounded with it and at one time thought to beiden-
tical 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 diag-
nosis makes it difficult to determine exactly how fatal either may be in uncom-
plicated cases.

Morphology.—The bacillus of swine-plague much resembles that of chicken-
cholera. It is a short organism, rather more slender than the related species,
not possessed of flagella, incapable of movement, and producing no spores.

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,t however, points out that when the bacillus
grows in bouillon the liquid remains clear, the bacteria gathering to form a floc-
culent, stringy sediment if dextrose or saccharose be added to the bouillon, a
strongly acid reaction develops, but no gas is formed. If lactose be added neither
acidity nor gas appears. Indol is sometimes but not always formed. The organ-
ism does not grow upon ordinary acid potato, but if the reaction of the medium be
alkaline, a grayish-yellow patch is formed. In milk a slight acidity is produced,
but the milk is not coagulated.

Vital Resistance.—The vitality of the organism is low, and it is easily de-
stroyed. Salmon says that it soon dies in water or when dried, and that the tem-
perature for its growth must be more constant and every condition of life more
favorable than for the hog-cholera bacillus. 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.

Rabbits, mice, and small birds are very susceptible to the infection, 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 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, intravenous, and intraperito-
neal inoculation, not by feeding.

’ As seen in hogs, the symptoms of swine-plague closely resemble those of hog-
cholera, but differ in the occurrence of cough, swine-plague being prone to affect

e lungs and oppress the breathing, which becomes frequent, labored, and pain-
ful, 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 td be inflamed, and to contain nu-
merous small, pale, necrotic areas, and sometimes large cheesy masses 1 or 2 inches
in diameter. Inflammations of the serous membranes affecting the pleura, peri-
cardium, and peritoneum, and associated with fibrinous inflammatory deposits on

esurfaces,arecommon. ‘There may be congestion of the mucous membrane of
the intestines, 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.

' * “Arbeiten aus den kaiserlichen Gesundheitsamte,”’ 1.
[t “Zeitschrift f£. Hygiene,” x.
t Fliigge’s ““Die Mikroorganismen, 1896,” p. 419.

CHAPTER XXVII
ASIATIC CHOLERA

SPIRILLUM CHOLERZ ASIATICe (Kocu*)

Synonyms.—Vibrio cholere asiatice; Microspira comma; comma bacillus;
cholera spirillum; cholero vibrio.

General Characteristics——A motile, flagellated, non-sporogenous, liquefying,
non-chromogenic, non-aérogenic, parasitic, and saprophytic, pathogenic, aérobic
and optionally anaérobic spiral organism, staining by ordinary methods, but
not by Gram’s method.

Cholera is a disease endemic in cee parts of India and prob-
ably 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 century has made scientific observation of the disease
possible and has 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 most 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 quan-
tity 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 must be great, for there are few months
in the year when the city is exempt from the disease.

The pilgrimages and great festivals of both Hindoos and Woden:
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 distribu-
tion of the disease when the festivals break up. Probably no
more favorable conditions 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

* “Deutsche med. Wochenschrift,” 1884-1885, Nos. 19, 20, 37, 38, and 39.

608

Distribution 609

Europe, and by steamships plying foreign waters has several times
been carried to our own continent. Many cases are on record which
show conclusively 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 Ham-.,
burg 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 ro1z the
disease again appeared in Europe and invaded the countries along
the Mediterranean coasts.

ne ac

tone te oD ee. .

Fig. 242—Cholera spirilla.

Specific Organism.—The discovery of the spirillum of cholera
was made by Koch while serving as a member of a German com-
mission 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 immense amount of literature, a large part of
which was stimulated 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

39 ;

610 Asiatic Cholera

water from polluted gutters; how they enter milk with water used
to dilute it; how they appear to be carried 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 mention even the most instructive examples, A bacteri-
ologist 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,

Fig. 243.—Spirilla of Asiatic cholera, from a bouillon culture three weeks old,
showing long spirals. Xt1o000 (Frankel and Pfeiffer).

and can be found alive after months have passed. Frankel, how-
ever, points out that this ability to grow and remain vital for long
periods in sterilized water does not guarantee the same power of
growth in unsterilized water, for in the latter the simultaneous
growth of other bacteria serves to extinguish the cholera spirilla
in a few days.
Morphology.—The micro-organism is a short rod 1 to 2 mw in
length and o.s yw 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-shape.
When the conditions of nutrition are good, multiplication by fission
progresses with rapidity; but when adverse conditions arise, long
spiral threads—unmistakable spirilla—develop. Frankel found

*Deutsche. med. Wochenschrift, 1885, No. 37, a, 7.

Staining 611

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 a single flagellum attached to one end can be
demonstrated without difficulty.
Involution-forms of bizarre appearance are common in old
and sometimes in fresh cultures. Many individuals show by

. Aree
SON Sete
Sy boas ‘
* x
ws ‘st & hae we
Ren =
ats es ~
<< Ss eS
x ~ s
~ .
< = eS
~ » aw 8
.
s ~
“
~ ~ es
_ ~
= =
. ~. ~
= ~ ve
A x x
eo ~
2 ~
x e oo
~
s . =

Fig. 244.—Cover-glass preparation of a mucous floccule in Asiatic cholera.
X 650 (Vierordt).

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
es observed, by examination in the “‘hanging-drop,” certain large
spheric bodies which he described as spores (arthrospores). Koch
and, indeed, all other observers fail 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 se-
cure homogeneity. The organism does not stain by Gram’s method.
It may be colored and examined while alive; thus, Cornil and

612 | Asiatic Cholera

Babes, in demonstrating 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 weak solu-
tion 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, though they take up enough stain
to color them, they still retain the power of vigorous movement,
which would be entirely lost if the specimen were dried, stained, and
mounted in the ordinary fashion.”

Fig. 245.—Spirillum of Asiatic cholera; colonies two days old upon a gelatin
plate. X 35 (Heim).

Isolation of the Organism.—One of the best methods of securing
a pure culture of the cholera spirillum, and also of making.a bacterio-
logic diagnosis of the disease in a suspected case, is probably that
of Schottelius,*
_ Asmall 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, at temperatures
between 10° and 45°C., the optimum being 37°C.

* Deutsche med. Wochenschrift, 1885, No. 14.

Cultivation 613

Colonies.—The colonies grown upon gelatin plates are character-
istic and appear in the lower strata of the gelatin as small white
dots, which gradually grow out to the surface, effect a slow lique-
faction of the medium, and then appear to be situated in little pits
with sloping sides. The 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 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

Fig. 246.—Spirillum cholere asiatice; gelatin puncture cultures aged forty-eight
and sixty hours (Shakespeare).

granules do the same and attain a peculiar transparent appearance
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 also
quite characteristic. It occurs along the entire puncture, but best
at the surface, where it is in contact with the atmosphere. Lique-
faction 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 is the formation of a short, rather wide funnel
at the top of the puncture. As the growth continues, evapora-
tion of the medium takes place slowly, so that the liquefied gelatin

614 Asiatic Cholera

is lower than the surrounding solid portions, and the growth ap-
pears to be surmounted by an air-bubble.

The luxuriant development of the spirilla in the liquefying 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, translucent growth along
the entire line of inoculation. It is in no way peculiar or char-
acteristic. 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 organ-
isms 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 usually
alter its appearance. The life of cholera organisms in milk is, how-
ever, rather short-lived, for when the acidity that invariably develops
reaches a certain point, they die out.

Vital Resistance.—Although an organism that multiplies with
great rapidity under proper conditions, the cholera spirillum does
not possess much resisting power. Sternberg found that it was
killed by exposure of 52°C. for four minutes, but Kitasato found that
ten or fifteen minutes’ exposure to 55°C. was not always fatal to
it; 60°C. maintained for thirty minutes is certainly fatal. 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 disap-
peared in a few hours. Kitasato found that upon silk threads.the
vital ty might be retained longer. Abel and Claussen* have shown
that it does not live longer than twenty or thirty days in fecal matter,
and often disappears in from one to three days. Zeatogorofft
found that if air was excluded from the fecal matter, the organisms
might remain alive for nine months. The organism is very suscep-
tible to the influence of carbolic acid, bichlorid of mercury, and other

* “Centralbl. f. Bakt. u. Parasitenk.,” Jan. 31, 1895, vol. xvi, No. 4.
{ “Centralbl. f. Bakt. u. Parasitenk.,” 1911, LVI, 14.

Pathogenesis 615

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, the organisms in the liquefied cultures all
die in eight weeks, and cannot be transplanted. Kitasato, how-
ever, has found them living and active on agar-agar after from
ten to thirty days, and Koch occasionally found some alive after
two years.

This low vital resistance of the microbe is very fortunate, for
it enables us to establish satisfactory precautions.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 innocuous 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. All that is necessary to demon-
strate 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.

This reaction depends upon the fact that the organism produces
nitrites, so that their addition is not necessary to bring out the red
color. On this account, the product is known as wiiroso-indol.

The organism also produces acid in milk and other media. Bitter
has also shown that the cholera organism produces a peptonizin
and probably also a diastatic ferment.

Toxic Products.—Rietsch thinks the intestinal changes depend
upon the action of the peptonizing ferment. Cantani, 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,t Gamaléia,§ Sobernheim,|| and Villiers have
studied more or less similar toxic products. The real toxic substance
- is, however, not known.

Pathogenesis.—Through what activity the cholera organism
provokes its pathogenic action is not yet determined. The organ-
isms, however, abound in the intestinal contents, penetrate spar-
ingly into the tissues, but slightly invade the lymphatics, and
almost never enter the circulation; hence it is but natural to conclude
that the first action must be an irritative one depending upon toxin-
formation ‘in the intestine.

In the beginning of the disease the small and large intestines

* “Kwai Med. Jour.,” Tokyo, 1893.
+ “Berliner klin. Wochenschrift,’’ 1887, p. 817.
t “Untersuchungen iiber die Bakteriengifte,” etc., Berlin, 1890.

§ “Archiv de méd. exp.,” Iv, No. 2.
|| “Zeitschrift fiir Hygiene,’ 1893, XIV, 145.

616 Asiatic Cholera

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 become extremely thin from the ad-
‘mixture 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 macer-
ated, and necrotic, its epithelium separating in small shreds or
flakes. The evacuations of watery exudate rich in these shreds con-
stitute the characteristic ‘‘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 ulcera-
tions 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
become markedly degenerated, 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.

Zeatogoroff* found that the cholera spirilla could remain alive in
the intestinal canals of those recovered from the disease as long as
93 days. Their duration he supposed to depend upon associated.
organisms by which it might or might not be extinguished. In lieu
of the observations of Kuleschaf that disease of the biliary passages
were occasioned sequel of cholera, and that the spirilla could long
remain in the gall-bladder, it seems as though a new supply of the
spirilla might at frequent intervals enter the intestinal contents.

This makes it not improbable that there are ‘“‘cholera carriers’

just as there are “typhoid carriers’”’ among convalescents.

So far as is known, cholera is a disease of human beings only,
and never occurs spontaneously in the lower animals.

Supposing that the lower animals were immune against cholera
because of the acidity of the gastric juice, Nicati and Rietsch,t
Van Ermengem, and Koch§ have suggested methods by which
the micro-organisms can be introduced 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 hypo-
dermic 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

* “Centralbl. f. Bakt. u. Parasitenk,” Originale, 1911, LVI, 14.
{ “Centralbl. £. Bakt. u. Parasitenk,” Originale, 1909, L, Pp. 417.

t “Deutsch. med. Wochenschrift,”’ 1884.
§ Ibid., 1885.

Pathogenesis 617

the peristaltic movements of the intestine can be checked. The
amount necessary for the purpose is large and amounts to about
1 gram for each 200 grams of body-weight. It completely nar-
cotizes the animals for a short time (one to two hours), but they re-
cover without injury. The contents of the stomach are neutralized
after administering the opium, by introducing 5 cc. of a 5 per cent.
aqueous solution of sodium carbonate through a pharyngeal catheter.
With the gastric contents thus alkalinized and the peristalsis -para-
lyzed, 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 paralyezd, 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 thé
blood or tissues, but only in the intestine, where they frequently
enter between the basement membrane and the epithelial cells, and
aid in the detachment of the latter.

Issaéff 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 susceptible to intraperitoneal injections of
the spirilum, and speedily succumb. The symptoms are rapid
fall of temperature, tenderness over the abdomen, and collapse.
The aytopsy shows an abundant fluid exudate containing the micro-
organisms, and injection and redness of the peritoneum and viscera.

Specificity—The cholera spirillum is present in the dejecta
of cholera with great regularity, and as regularly absent from the
dejecta of healthy ,individuals and those suffering from other
diseases. No satisfactory proof of the specific nature of the organ-
isms can be obtained by experimentation upon animals. Ani-
mals 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 in-
oculations do not produce cholera.

A sufficient number of laboratory infections have occurred among
human beings, however, to be convincing. Such have been reported
by Koch,* Metchnikoff,t Hasterlik,t Renners,§ Kolle,|| Voges,**
Zeatogorofft{ and others.

* “Deutsche med. Wochenschrift,” 1885, 37a, p. 7.

¢ “Ann. de l’Inst.,” Pasteur, 1893, VII, 403; 562.

t “Wiener klin. Wochenschrift,” 1893, p. 167.

“§ “Deutsche med. Wochenschrift,” 1894, 52.

|| “Zeitschrift fiir Hygiene,” 1894, Xvi, 17. ;
** “Centralbl. f. Bakt. u. Parasitenk.,” 1895, XVIII, 629.
tt “Berliner klin. Wochenschrift,” 1909, No. 44.

618 _ Asiatic Cholera

Detection of the Organism.—It often becomes a matter of im-
portance 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 ordi-
nary methods. One of the most expeditious methods is that
recommended by Léffler, who adds 200 cc. of the water to be
examined to 10 cc. 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.

Castellani* recommends that a few drops of agglutinating sera for

such associated bacteria as may be expected to contaminate the
culture, be added to the medium. By this means they will grow
slowly and settle to the bottom of the tubes in clumps, leaving the
cholera organisms relatively more numerous and more easily obtained
from the surface.

Gordont employs a medium composed of lemco 1 gram, peptone
I gram, sodium bicarbonate o.1 gram, starch 1 gram, and distilled
water 100 cc. 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:
(1) 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
pathogenic for guinea-pigs, and harmless to pigeons. (3) Vegeta-
tive: The organism should liquefy to per cent. gelatin and should
not coagulate milk. (4) Metabolic: the 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 introduced 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 reciprocally. Both organ-

* “Brit. Med. Jour.,” Oct. 13 al p- 476.
+ “Brit. Med. Jour., f July 23, 1906. :

Immunity 619 .

isms 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 agglutination tests alone, as will be made clear by a perusal
of the paper upon Bacteriological Diagnosis of Cholera by Ruffer.*
(7) The complement fixation test should be position.

Immunity.—One attack of cholera usually leaves the victim
immune from further attacks of the disease. Gruber and Wiener,t+
Haffkine,t Pawlowsky,§ and Pfeiffer) have immunized animals
against toxic substances from cholera cultures and against living
cultures.

Sobernheim** found the Pfeiffer reaction specific against 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 60°70°C., and its bactericidal
power thus destroyed, 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 temperatures.

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 the serum to confer immunity is lost much sooner.

Prophylaxis.—Of the numerous attempts to produce immunity
against cholera in man, or to cure cholera when once established
in the human organism, nothing very favorable can be said. Experi-
ments in this field are not new. As early as 1885 Ferran, in Spain,
administered hypodermic injections of pure virulent cultures of the
cholera spirillum, in the hope of bringing about immunity. The
work of Haffkine, t+ however, is the chief important contribution, and
his method seems to be followed by a positive diminution of mortal-
ity in protected individuals. Haffkine uses two vaccines—one mild,
the other so virulent 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;

* “Brit. Med. Jour.,”” March 30, 1907, I, p. 735-
+“ Centralbl. £. Bakt.,”’ 1892, XIV, p. 76.
{“Le Bull. méd.,” 1892, p. 1113, and “Brit. Med. Jour.,” 1893, p. 278.
§ “Deutsche med. Wochenschrift,”’ 1893, No. 22.
al ‘Zeitschrift fiir Hygiene,” Bd. xvii and xx.
“Zeitschrift fiir Hygiene, xx, p. 438.
tt “Le Bull. méd.,”’ 1892, p. 1113; ‘Indian Med. Gazette,’’ 1893, p. 97; “Brit.
Med. Jour.,” 1893, p. 278.

. 620 Asiatic Cholera

in the coolies of the Assam-Burmah survey party, where, as far as I can gather
from my preliminary 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 in-
oculated fourteen to fifteen months after vaccination in an epidemic of exceptional

virulence. This makes it probable that a protective effect could be obtained

even for long periods of 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. .

“sy. 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.”

Serum Therapy.—Pawlowsky and others have found the dog
susceptible to cholera, and have utilized it in the preparation of an
antitoxic 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 1: 130,000 upon
experimental animals.

Freymuth* and others have endeavored to secure favorable
results from the injection of blood-serum from convalescent patients
into the diseased. One recovery out of three cases treated is
recorded,

In all these preliminaries the foreshadowing of a future thera-
peusis must be evident, but as yet nothing satisfactory has been
achieved.

One of the chief errors made in the experimental preparation of
anticholera serums is that efforts have been directed toward endow-
ing 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 neutralize the depressing effects of the toxalbumin,
and some means of destroying the bacteria in the iritestine.

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.

So soon as cholera asserts itself, the chief danger lies in the prob-
able contamination of the water-supply. To prevent this the
utmost effort must be made to locate all cases and see that the de-
jecta 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 discharged 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
uncooked vegetables or fruits eaten. Foods should be carefully

* “Deutsche med Wochenschrift,” 1893, No. 43.

Cultivation 621

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.

Possible “carriers” of the disease among convalescents should be
looked for and detained in the hospital until the spirilla are no longer
to be found in their intestines. es

SPIRILLA RESEMBLING THE CHOLERA SPIRILLUM

THE FINKLER AND PRIoR SpPrRILLUM (SPIRILLUM PRoTEvs)

Synonym.—Vibrio proteus; Vibrio of Finkler and Prior.

This spirillum was obtained from the feces of a case of cholera nostras by Finkler
and Prior.*

Fic. 247.—Spirillum of Finkler and Prior, from an agar-agar culture. XX 1000
o (Itzerott and Niemann).

Morphology.—It is shorter and stouter than the “‘comma bacillus,” has a more
Pronounced curve, and rarely forms long spirals. The central portion is also
somewhat thinner than the ends, which are a little pointed and give the organ-
ism a less uniform appearance. Involution forms are common in cultures,
and appear as spheres, spindles, clubs, etc. Like the cholera spirillum, each
a 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
Secure few enough organisms to enable one to observe the growth of a single
colony. To the naked eye the deep colonies appear as small white points. They
Tapidly reach the surface, begin liquefaction of the gelatin, and by the second 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 a
zone of sharply circumscribed liquefied gelatin. Careful examination with a
high-power lens shows rapid movement of the granules in the colony.

*“Centralbl. fiir allg. Gesundheitspflege,” Bonn, 1885, Bd. 1; “Deutsche med.
Wochenschrift,” 1884, p. 632.

622, The Finkler and Prior Spirillum

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
liquefaction of the medium, the wider top to the funnel, the absence of the air-

Fig. 248.—Spirillum of Finkler and Prior; colony twenty-four hours old, upon a
gelatin plate. > 100 (Frankel and Pfeiffer).

Fig. 249.—Spirillum of Finkler and Prior; gelatin puncture cultures aged forty-
eight and sixty hours (Shakespeare).

bubble, and the clouded nature of the liquefied material, all serve to differentiate
the culture from the cholera spirillum. i

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.

Morphology 623

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 fermentation 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 tempera-~
ture, and produce 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.

Milk.—The spirillum does not grow well in milk, and speedily dies in water.

Metabolic Products.—The organism does not produce nitroso-indol, nor does
any indol make its appearance before 24 hours, and then only in small quanti-
ties. 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

Fig. 250.—Spirillum of Denecke, from an agar-agar culture. X 1000 (Itzerott
and Niemann).

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 39 per cent. of the animals die, but the
intestinal lesions produced are not identical with those produced by the cholera
spitillum. The intestines in such cases are pale and filled with watery material
having a strong putrefactive odor. This fluid teems with the spirilla. '

Tt seems unlikely, from the evidence thus far collected, that the Finkler and
Prior spirillum is pathogenic for the human species. As Frinkel points out, it 1s
probably a frequent and harmless inhabitant of the human intestine.

Tue SprRItLUM OF DENECKE (SPIRILLUM TYROGENUM)

Another organism with a partial resemblance to the cholera spirillum was found
y 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 Finkler and Prior spirillum, and are more tightly coiled than
those of the cholera spirillum.

*Deutsche med. Wochenschrift,” 1885.

624 The Spirillum of Denecke

Like the related species, this micro-organism is actively motile and possesses a
terminal flagellum.

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 Finkler and
Prior spirillum. The colonies appear as small whitish, round points, which soon
reach the surface of the gelatin and commence liquefaction. 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 granu-
lar, 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 from those of cholera in the prompt liquefaction of the
gelatin, the rapid growth, yellow color, irregular form, and distinct line of
circumscription.

Fig. 251.—Spirillum of Denecke; gelatin puncture cultures aged forty-eight and
sixty hours (Shakespeare).

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 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 surface.

Bouillon.—In bouillon the growth of the organism is characterized by a diffuse
turbidity. No gas-formation occurs in sugar-containing rhedia.

Potatoes.—The culture upon potato is luxuriant if grown in’ the incubating
oven. It appears as a distinct yellowish, moist film, and when examined micro-
scopically 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.

Pathogenesis 625

THE SPIRILLUM OF GAMALETIA* (SPIRILLUM METCHNIKOVI)

Resembling the cholera spirillum in morphology and vegetation, and possibly,
as has been suggested, a descendant of the same original stock, is a spirillum
which Gamaléia 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 curved, and has similar rounded ends. It forms
long spirals in appropriate media, and is actively motile. Each spirillum 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 comparatively easy to differentiate between a plate of pure cholera spirillum
and a plate of pure Spirillum metchnikovi, yet it is almost impossible to pick out
a few colonies of the latter if mixed upon a plate with the former.”’

Frinkel regards this organism as a species intermediate between the cholera.
and the Finkler-Prior spiriJlum.

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 spirillum occur.
The liquefaction of the gelatin is quite rapid, the resulting fluid being turbid.
Usually, upon a plate of Vibrio metchnikovi some colonies are present which
closely resemble those of the cholera spirillum, being deeply situated in conical
depressions in the gelatin. Under the microscope the contents of the colonies,
which appear of a brownish color; are observed 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 yellowish-brown growth takes place.
Sometimes the color is quite dark, and chocolate-colored potato cultures are not
uncommon. g

Bouillon.—In bouillon the growth which occurs at the temperature of the incu-

bator 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
ina 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 nitroso-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@. When guinea-pigs are treated by Koch’s method of narco-

* “Ann, de l’Inst. Pasteur,” 1888.
40

626 Spirillum of Gamaléia

tization and cholera infection, the temperature of the animal rises for a short time,
then abruptly falls to 33°C. or less. Death follows in from twenty to twenty-four
hours. A distinct inflammation of the intestine, with exudate and numerous
spirilla, may be found. The spirilla can also be found in the heart’s blood and in

Fig. 252.—Spirillum metchnikovi, from an agar-agar culture. 1000 (ltzerott
and Niemann).

the organs of such guinea-pigs. When the bacilli are introduced by subcutane-
ous 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 Vibrio-

Fig. 253.—Spirillum metchnikovi; puncture culture in gelatin forty-eight hours —
old (Frankel and Pfeiffer).

nensepticemia. In the intestines very few alterations are noticeable, and very
few spirilla can be found.

Immunity.—Gamaléia 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.

Spirillum Schuylkiliensis 627

SPIRILLUM SCHUYLKILIENSIS (ABBO1T)

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 metchnikovi.

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.—Léffler’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 anaérobe.

Vital Resistance.—The thermal death point is 50°C. maintained for five
minutes. :

Pathogenesis.—The organism is pathogenic for pigeons, guinea-pigs, and mice,

_ behaving much like Spirillum metchnikovi. 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 Spirillum schuylkiliensis and Spirillum metch-
nikovi, the immunity thus produced being of about ten days’ duration.

In a second paper by Abbott and Bergey} it was shown that the spirilla oc-
curred 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 iden-
tical in all points. There seems to be, therefore, a family of river spirilla, closely
telated 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 water Bergeyt 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 bacteriologists 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 organism, and that the deviations in the morpho-
logic and biologic characters from those of the cholera organism were brought
about by their prolonged existence in water. No such explanation of the occur-
tence of the organisms in Philadelphia 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 origi-
nal descriptions, references to which are given in each case.

*“Tournal of Experimental Medicine,” July, 1896, vol. 1, No. 3, p. 419.
{Journal of Experimental Medicine,” vol. 11, No. 5, p. 535.
{Journal Amer. Med. Assoc.,” Oct. 23, 1897.

Table of Spirilla

628

: ‘E°0
“beir -d ‘z6gr ,,{yLIyDsusYyIOMA “pau aqosynaq_ ,, a
"iz ‘d ‘b6gr ‘Ar ,,‘neyospuny oyostuar3A zy ,, 4h
“gb -d ‘f6gr ‘xrx ,,‘auaIBAH Any AIWOIY ,, 82
“rhe -d ‘Arx “999 IAT “J 1qQLe1WaD ,, ae
“Segt .IluyosusysoM “pour ayosynaq ,, t

‘1 ‘JOA ‘6rb -d ‘g6gr ‘Ain ,,S9UlDIpay] [eyUsuITIedxy Jo yeurnof,, ff]

“gtr ‘d ‘Sger ,,{yyryosuayIO AA “paul ayIsyNIq ,, xx

“621 “d ‘v6gr ‘Ixx ,,“aUdsISAP In aryoiy ,, tt

664 ‘d ‘C6g1 ,,“ Wy IsuayIO MA “paw sysjneq va)
‘zl -d ‘bggi ,,{WIIySsuayIOAA “pawl ayosjnaq ,,

“f6gr ,,‘neqospuny syosuar3Ay ,, ¢
“Gob -d ‘/ggt ‘1 “pg “979. IAPT "J “IGIETIUID ,,
*zlr *d ‘pOgt ‘1xx ,,‘aUaIZAP Inj AIWIIY ,,

segh-d ‘ir ‘ggg ,,‘anaqseg “jsuy,[ ap ‘uuy ,,

“Ze pur 1 ‘son ‘Pggt ,, WYLYIsusyIOAA "UITY JOUTLIEg ,, ¥

AaZiag pue yoqqy) sisuar[ryyAnyos wmypiaids
scores (98g azayyund) sipyenbe wmy[taids

cross soe + (ftfaassrayy) sisusurjossq wnypiids
DER a 8 * ({}pseyjund) snuasiia} wnypids

“Gees TOTAL) Poy UL umy[itids

rash See Aaa stots + (i PqreAA) Taq wnyptatds
cy (22 yoyuog) suarovyenbiy wny[ids
SP ee en ee (ff eyoruz9 AA) TT wnypds
aie, Ge ta Sel Geb lair. e, oe s+ (Lbayoruiaa) [ wmypiaids

"1 + (4g J@ploH) snoiqnuep wumypitds

PDR ID ER aS ce (|| sequnq) stsuatequnp winyyiaidg

*dnosp J3a1e¥AA

Trees + + (Qergqeureg) taosuypsyour winyTEds
Be fe ah ar Be see ge 8% ({ ay9auIeq]) wNnusgo14] winds
a Ses foe Soli Sos Ne eS re song pue sopqurg)

yoid winyidg) seisou ‘seiajoyds uiny[iuids
Pr Sep os (x YOY) BoyeIse setapoyD winy[iids
: *dnosp [eulsazuy

-1989}0] Ul punoy

ojo + [+pOj+|+] |o] + jol+/+j+jololo}]+}o]+] o Jo] +] o | + |+/+Jolsol+}4+] o |4] o
D\|o fe) ° +]o]o +/o] o Jo} + ° oO |+/+}/o;/o}+/4+] o |/+] 0
ojo + ° co) Sm ie Os +/o] o J+} 0 | o | + J4}+4fojo]+]+4] o |+]} o
o}o ° oj+ a/oj;0 +]/o] o }o| o | ¢ é |+/+]/o]o}+j4+}] 0 }o} o
oloj}o] o Jolo ° +]o}ololo}o o|+] + Jo} o | o | + J4+J+}olo}+/+] o J}o] o
° ° + +]o}o +/o}] + Jo] o | o | + J+}4+}o}o}/+]/+] o |4] 0
ojo + ° fo} o|+/+}o]o}+ ojo} o Jol o o | + J+}/4+j)o}/o}+]/+] o j+] 0
o}oj+) + J+ + o jo} +] o | + |+]+]/o}ol+/+] o ;+] 0
a ae ; ea) oe) | ae ee Poe] a Lb)
o|o + ol+]+ ° +]o]o}ojolo/+/+}o} o J+] o | o | + J+]+]o}+}o}+] o J+] o
Sr + ) +\|+/+j)o}ofoj+] Jo} 4+ Jo} o | o | + J+/+}ofo}o}+] o |4] o
o}o}o) + i+)0]+/+]}o0]o +/+]4+]o}o}+] Jo/+t} o Jo] + | + | o J+/+}oJo]+}+] o Jo
olo + ° ° +]/o}o}olo}oj+/o]+] o |+}] o | o | + |+/+)o]o}+]4+] o Jo
o|o + fo} o; + +)0/0;0/+]/o/+]/o]+] + |o] o o | + |+}+}o}o}j+]4+] o Jo
O}o/O) + /O/Ol}oj;+lolo} + joj+]+)t}oi+jo] J+fo} o |+] o | o | + J+{+}o}oj+/+] o J+
y > wn Cte)
g/g Ble |B 8/3 2/2/0)8)2/8/2/8) 5/2) ..2)33| 33/8/32] 3 /a/F 525 |3/2
ia i . a is oO
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CHAPTER XXVIII
TYPHOID FEVER .

BacitLus TypHosus (EBERTH-GAFFKY)

Synonyms.—Bacterium typhosus; Bacillus typhi abdominalis.

General Characteristics.—A motile, flagellated, non-sporogenous, non-lique-
fying, non-chromogenic, non-aérogenic, aérobic, and optionally anaérobic,
pathogenic bacillus, staining by ordinary methods, but not by Gram’s method,
not forming indol, not forming acids from sugars, nor coagulating milk.

TypHow fever, “typhus abdominalis,” enteric fever, “la fiévre
typhique,” is a disease so well known and of such universal distribu-
* tion, that no introductory remarks concerning it are necessary.

The bacillus of typhoid fever (Bacillus typhosus) 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.t

iE SRN
i “i! Ae) Py, *¥y/
OOK; Korres
oe
NN EP aI
DP EM ey.
wes) fys=
apes 2 “aes Slew 0
RY a

a ‘ BAS tN A

Fig. 254.—Bacillus typhosus, from twenty-four-hour culture on agar (From
aes ae Zinsser, “Text-book ‘of Bacteriology,” D. Appleton & Co., pub-
ishers).

Distribution.—The typhoid bacillus is rarely found in nature apart
from the human beings that are suffering from typhoid fever or
have suffered from it, and when it is otherwise encountered, it
can in almost all cases be traced to them. Leaving the human body
in the feces and urine, it naturally finds its way to the soil and to the
water. As a saprophyte it appears to survive but a short time,
though just how long will depend upon its numbers and the nature of

*©Virchow’s Archiv,” 1881 and 1883.

; } “Mittheilungen aus dem kaiserl. Gesundheitsamte,” 1, 45.
: t Ibid., 2.

629

630 ' Typhoid Fever

its environment. Levy and Kayser* found it still alive in soil that
two weeks previously had been manured with the five months old
contents of a privy vault. From privy vaults and from infected
soils it may easily find its way into wells and streams. Gé§rtnert
‘found that it lived long enough to be transported a distance of 140
kilometers in running water. Jordon, Russell and Zeit{ found it
alive and retaining its virulence for five days in natural bodies of
water.

Morphology.—The typhoid bacillus measures about 1 to 3 u (2 to
4 u—Chantemesse, Widal) in length and 0.5 to 0.8 mu in breadth
(Sternberg). The ends are rounded, and it js 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. Thoi-

Fig. 255.—Bacillus typhosus.

not and Masselin,§ in describing these morphologic variations, point
out that 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 filaments. It produces
no spores.

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
Léffler’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 meth-
ods, but not by Gram’s method. © As it gives up its color in the pres-
ence of almost any solvent, it is difficult to stain in tissue.

*“Centralbl. f. Bakt. u. Parasitenk,” 1903, XxIII, 480.
} Klinisches Jahrbuch, 1902, 335.

t “Jour. Infectious Diseases,” 1904, 1, 641.
§ “Précis de Microbie,” Paris, 1893.

Isolation 631

When sections of tissue are to be stained for the demonstration of
the typhoid bacilli, the best method is to allow them to remain in
Léffler’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 1, and phenol 5. After
staining they are washed in distilled water containing 1 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 microscope, as
reddish specks, and readily resolved into bacilli with the oil-im-
mersion lens.

Fig. 256.—Bacillus typhi abdominalis; superficial colony two days old, as seen
upon the surface of a gelatin plate. X 20 (Heim).

In bacilli stained with the alkaline methylene-blue solution,
dark-colored dots (Babes-Ernst or metachromatic granules) may
sometimes be observed near the ends of the rods.

' Isolation —The bacillus can be secured in pure culture from an
enlarged lymphatic gland or from the splenic pulp of a case of
typhoid.

As the groups of bacilli are sometimes widely scattered through-
out the spleen, E. 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 ma-
terial for cultures obtained by introducing a platinum loop into the
substance of the organ through the sterilized surface.

Cultures may be more easily obtained from the blood of the
living patients. (See “Blood culture,” under the section “ Bacterio-
logic Diagnosis.”’)

' The bacilli can also be secured from the alvine discharges of

632 Typhoid Fever

typhoid patients during the second and third weeks of the
disease.

Cultivation.—The bacillus grows well upon all culture-media
under both aérobic and anaérobic 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, iridescent 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 puncture cul-
tures, the typhoid bacilli develop along the entire 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 liquefied, but is sometimes slightly clouded in the neigh-
borhood 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 potato is inoculated and stood in the incubating
oven, no growth can be seen even at the end of the second day,
but if the surface of the medium be touched with a platinum wire,
it is found entirely covered with a rather thick, invisible layer of
sticky vegetation which the microscope 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.—In 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 sugais. No indol
is formed.

Milk.—In milk containing litmus a very slight and slow acidity
is produced, which later gives place to distinct alkalinity. The
milk is not coagulated.

Vital Resistance.—The organisms grow well at all ordinary tem-
peratures. The thermal death-point is given by Sternberg at 56°C.,
destruction being effected in ten minutes. Upon ordinary culture-
media, the organisms remain alive for several months if drying is
prevented. In carefully sealed agar-agar tubes Hiss found the or-

Metabolic Products 633

ganism still living after thirteen years. According to Klemperer and
Levy,* the bacilli can remain vital for three months in distilled water,
though in ordinary water the commoner and more vigorous sapro-
phytes outgrow them and cause their disappearance in a few days.
There seems to be some doubt, however, on this point, as Tavelt
found that it lived for six months in the blind terminal of a water-
supply pipe, and Hofmann, after planting it in an aquarium con-
taining fish, snails, water-plants, and protozoa, was able to recover
it from the water after thirty-six days, and from the mud in the bot-
tom after two months. In elaborate experimental studies of this
question Jordan, Russel, and Zeit§ found its longevity to be only
three or four days under conditions resembling 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. i

Cold has little effect upon typhoid bacilli, for some can withstand
freezing and thawing several times. Observing that epidemics of
typhoid fever have never been traced to polluted ice, Sedgwick and
Winslow** made some investigations to determine what quantitative
reduction might be brought about by freezing, and accordingly ex-
perimentally froze a large number of samples of water intentionally
infected with large numbers of typhoid bacilli from different sources.
It was found that the 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 bacilli per thousand
appeared very resistant and sometimes remained alive after twelve
weeks.

The typhoid bacillus resists the action of chemic agents rather
better than most non-sporogenous organisms. The addition of
from.o.1 to 0.2 per cent. of carbolic acid to the culture-media is
without effect uponits 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 1 : 500 bi-
chlorid 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 lactic and formic acids when grown in
sugar-containing media, but its regular tendency is to form alkalies

*“ Clinical Bacteriology.” Translated by A. A. Eshner, Phila., W. B. Saun-
ders Co., r900. :
t“Centralbl. f. Bakt. u. Parasitenk.,” 1903, XXXVIII, p. 166.
i“ Archiv. f. Hyg.,”? 1905, LI, 2, 208.
§ “Journal of Infectious Diseases,’ 1904, 1, p. 641.
|| “Brit. Med. Jour.,” Jan. 8, 1898. ‘

**“ Jour, Boston Soc. of Med. Sci.,”? March 20, 1900, vol. 1v, No. 7, p. 181.

634 Typhoid Fever

of which the chief is probably ammonia. It forms no coagulating
or proteolytic enzymes.

Toxic Products.—The disproportion of local to constitutional dis-
turbance in typhoid fever and the irritative and necrotic charac-
ter of its lesions suggest that we have 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 diphtheria and tetanus,
dissolved in the culture-medium. This was an obstacle to the immu-
nization experiments of both Pfeiffer and Kolle and Léffler and Abel, ¢
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 intracellular. Macfadyen and Rowlandf liberated
an intracellular toxin from cultures of the typhoid bacilli by freezing
them with liquid air and grinding them in an agate mortar. Animals
immunized with this poison produced an antiserum active against it,
but useless against infection with typhoid bacilli. Wright, of Net-
ley,§ observes that Macfadyen’s method of securing this intracel-
lular toxin was unnecessarily 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 Macfayden think** that exotoxin is also formed.
Vaughanff has obtained poisonous and non-poisonous fractions by
extracting massive cultures of typhoid bacilli with 2 per cent. solu-
tions of sodium hydrate in absolute alcohol at 78°C.

Mode of Infection—The typhoid bacillus enters the body by
way of the alimentary tract with infected foods and water. It is
commonly believed that the great majority of typhoid epidemics,
and the sporadic cases as well are caused by infected drinking water.

* “Deutsche med. Wochenschrift,’’ Nov. 12, 1896.

+ “Centralbl. f. Bakt. u. Parasitenk., ” Jan. 23, 1896, Bd. xIx, No. 23, D. SI.
t “Brit. Med. Jour.,”’ 1963.

§ Ibid., April 4, 1903, 1, p. 786.

i] “Ann. de Inst. Pasteur,” 1895, x, 1896, XI.

** “Centralbl. f. Bakt.,” etc., 1906, I.

tt “Amer. Jour. Med. Sci.,’ 3 "1908, CXXXVI.

Pathogenesis 635

Opposed to this view is the rarity with which the bacilli are found in
the water, in favor of it the almost invariable decline in the incidence
of the disease when the water supply is purified or filtered, and the ©
continued low incidence thereafter.

Next to water, milk is probably the most frequent vehicle through
which it is admitted to the body. Schiider* found that r10, out of
460 epidemics that he studied, could be referred to milk.

Rosenau, Lumsden, and Kastlef were able to connect ro per cent.
of the cases of typhoid fever 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
may occasionally enter milk in water used to dilute it or to wash the
cans, but may also be directly introduced by the hands of careless
milkers who are carriers, or be conveyed from infected fecal matter
by flies. :

. 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{ 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 vegetables
grown in soil fertilized with infected human excrement or sprinkled
with polluted water. Conn§ investigated an epidemic of typhoid
fever at Wesleyan College, and believed that he traced it to the
eating of raw oysters that had been “fattened” in sewage-polluted
water. Broadbent|| believed an outbreak of the disease in England
to be traceable to the same cause. Newsholme** found that in 56
cases of typhoid fever about one-third were attributable to eating
raw shell-fish from sewage-polluted beds. Footef} found that when
typhoid bacilli were placed in water containing oysters, they could
be found alive in the mollusks for three weeks after they had dis-
appeared from the water.

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 intesine, where local dis-
turbances are set up. Whether during 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

* Zeitschrift fiir Hygiene, 1901, xxxvili, 343.
} “Hygienic Laboratory Bulletin No. 33,” Washington, D. C., 1907.

1 { “Report on Typhoid Fever in the U. S. Military Camps in the Spanish War,”
vol. 3.
§ Medical Record, Dec. 15, 1894.
ll Brit. Med. Jour., Jan. 12, 1895.
Brit. Med. Jour., Jan., 1895.
» tf Med. News, 1895.

636 Typhoid Fever

intestinal and mesenteric lymphatics at the time we are able to
recognize the disease.

It is quite certain that the chief operations of the typhoid 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 exist-
ence 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 present. 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 es-
capes 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 in-
dividuals have become known as “typhoid carriers” and are a men-
ace to the public.

In a case studied by Miller* bacilli were found in the gall-bladder
seven years after recovery from typhoid fever; in a case studied by
Drobat they were found in both the gall-bladder and a gall-stone
seventeen years after recovery from the disease; Humer{ found
them in the gall-bladder of a patient suffering from cholecystitis,
eighteen years after recovery from an attack of typhoid fever, and in a
case studied by Dean,§ they were present in the stools of a man
twenty-nine years after he had had an attack of typhoid fever.

Cushing|| 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 cholecystitis. Cushing collected
6 cases of operation for cholecystitis with calculi in which typhoid
bacilli were present, and 5 in which Bacillus-coli was present in
the gall-bladder. ;

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 arule. The greatest number was obtained when there
was much blood in the stool.

* “Bull. of the Johns Hopkins Hospital,’ May, 1898.
. ~ “Wiener klin. Wochenschrift,” 1899, xl, p. 1141.
1 “Bull. of the Johns Hopkins Hospital,” Aug. and Sept., 1809.
; “Brit. Med. Jour.,”” March 7, 1908, 1, p. 562.
|| “Bull. of the Johns Hopkins Hospital,”’ rx, No. 86.
“ e ‘Journal of the American Medical Association,’’ Sept. 7, 1907, XLIX, p-
46.

Pathogenesis 637

There is always well-marked blood-infection during the first
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 disease—one to
three weeks. The immediate result of their activity in the lymphatic
structures is an increase in the number of cells, the ultimate effect
is necrosis and sloughing of the Peyer’s patches and solitary glands.
From the intestinal lymphatics the bacilli pass, in all probability,
to the mesenteric nodes, which become enlarged, softened, and
sometimes rupture. They also invade the spleen, liver and some-

_ Fig. 257.—Intestinal perforation in typhoid fever. Observe the threads of
tissue obstructing the opening. (Museum of the Pennsylvania Hospital.)
(Keen, “Surgical Complications and Sequels of Typhoid Fever’’).

times the kidneys, and other organs where they may be found
in small clusters in properly stained specimens.

Mallory* found the histologic lesions of typhoid fever to be wide-
spread throughout the body and not limited to the Peyer’s patches of
the intestine, where they are most evident. His conclusions regard-
ing the pathology 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, ec-

* “Journal of Experimental Medicine,” 1898, vol. 111, p. 611.

638 Typhoid Fever

centrically situated nuclei, abundant, sharply defined, acidophilic
protoplasm, and are characterized by marked phagocytic properties.
These phagocytic cells are produced most abundantly along the line
of absorption from the intestinal tract, both in the lymphatic ap-

paratus and in the blood-vessels. They are also produced by dis-

tribution of the toxin through the general circulation, in greatest
numbers where the circulation is slowest. Finally, they are pro-
duced all over the body in the lymphatic spaces and vessels by ab-
sorption 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 degen-
eration of phagocytic cells beneath the lining endothelium 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 ab-
sorbed by the lymphatics; the other is due. to obstruction of liver
capillaries by phagocytic cells derived in small part from the lining
endothelium of the liver capillaries, but chiefly by embolism through
the portal circulation of cells originating from the endothelium 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 polymorphonuclear leukocytes is rare.”

“. -, . Histologically the typhoid process is proliferative 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 albuminuria, 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 investi-
gated; 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. Petru-
schky{ 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

* “Brit. Med. Jour.,” Feb. 13, 1897.

t “Journal of Experimental Medicine,’ May, 1898.

t “Centralbl. f. Bakt. u..Parasitenk.,” May 13, 1898, No. 13, p. 577.
§ “Phila. Med. Jour.,”’ March 3, 1900.

Pathogenesis 639

centimeter of urine, and mentions a case of Cushing’s in which
the bacilli persisted in the urine for 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. Their persistence in the urine after recovery from typhoid
fever, depends upon continued growth in the bladder and not upon
continuous 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.

Bacilli can be found in the circulating blood. The eruption
may be regarded as one of the local irritative manifestations of the
bacillus, as the blood from the roseole contains them. Richardson,*

however, found it necessary to examine a number of spots in each
case. He carefully disinfected the skin, freezing it with chlorid of
ethyl, made a crucial incision, and cultivated from the blood thus
obtained. He was able to secure the typhoid bacillus in 13 out of
14 cases examined. ;

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 expectoration,
especially when pulmonary complications arise in the course of the
disease. Cases of this kind have been reported by Chantemesse and
Widalt and Frankel. t

The pyogenic power of the typhoid bacillus was first pointed
out by A. Frinkel, 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 common results of the
lodgment of the bacilli in bony tissue. Ohlmacher|| has found the
bacilli in suppurations of the membranes of the brain. The ‘bacilli
are also encountered 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 widespread to
constitute a real septicemia.

* Phila. Med. Jour.,”? March 3, 1900.
t “Archiv. de physiol. norm. et. path.,” 1887.
{Deutsche med. Wochenschrift,”’ 1899, XV, XVI.
, ; = der k. k. Gesellschaft d. Aerzt. in Wien.,’’ “Aerztl. Central-Anz.,”
: I 96, NO. 3.
_ || “Jour. Amer. Med. Assoc.,”’ Aug. 28, 1897.
~ ** “Bull, Johns Hopkins Hospital,” Dec., 1897.

640 Typhoid Fever

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 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 pol-
luted with typhoid bacilli for a considerable time, yet without
symptoms. Griinbaum* produced typhoid fever in chimpanzees
by inoculating them with the bacillus. The introduction of viru-
lent cultures into the abdominal cavity of animals is followed by
peritonitis.

Germano and Maurea} found that mice succumbed in from one to
three days after intraperitoneal injection of 1 or 2 cc. of a twenty-
four-hour-old bouillon culture. Subcutaneous injections in rabbits
and dogs caused abscesses.

Lésener 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 subcutaneous
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 infective character of the discharges, 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 would 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 infancy,
might be checked.

In large cities where typhoid fever hag 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.

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 progress, green vegetables
and oysters that may be polluted by infected water must be guarded
against.

* “Brit. Med. Jour.,” April 9, 1904.
t “Ziegler’s Beitrige,” Bd. xu, Heft 3, p. 494.
t “Zeitschrift fiir Hygiene,” 1892, Bd. xu, p. 261.

Prophylaxis 641

Typhoid Carriers.—The persistence of typhoid bacilli in the gall-
bladder for years after an attack of typhoid fever is commonly at-
tended with the regular or occasional appearance of the bacilli in
the intestinal contents of the individuals concerned, who then be-
come “‘carriers,” and as such are a menace to the health of those
about them. In military cantonments, in institutions, and in as
many other places when people are congregated together as prac-
ticable, examinations should be made, from time to time, of all those
whose occupation brings them into contact with food subsequently
eaten by the rest, to see that there are no “carriers” among them.

The method is comparatively simple. The suspect is furnished with a small
bottle containing about § cc. of sterilized ox-bile, and instructed to introduce a
; re a He his feces about the size of a soup bean, and bring it to the laboratory

e next day.

The bottle is then thoroughly shaken so as to mix the bile and feces thoroughly
together and distribute the contained bacteria with fair uniformity, after which
a platinum loopful is stroked upon Conradi-Drigalski plates, or mixed with
melted Endo’s medium and poured into Petri dishes. Bluish white, thin, leaf-
like colonies, should be picked and tested for fermentation and milk coagulation
and in the absence of either, if composed of motile, gram-negative, non-sporulating
bacilli, further tested by means of an agglutinating serum.

Prophylactic Vaccination.—Following the principle of Haffkine’s
anticholera inoculations Pfeiffer, and Kolle,* Wright, t and Semplet
have used subcutaneous injections of sterilized cultures as a prophy-
lactic measure. One cubic centimeter of a bouillon culture sterilized
by heat was used.

The “Indian Medical Gazette” gives the following important
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 to00. There were 4502 inoculations, and among them
there were only 9 deaths from typhoid fever—o.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 675 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 prophylactic
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.

*“Teutsche med. Wochenschrift,’’ 1896, XXII; 1898, XXIV.
+ “Lancet,”’ Sept., 1896. :

t “Brit. Med. Jour.,”’ 1897, I, p. 256.

§ “Phila. Med. Jour.,’”’ Oct. 13, 1900, p. 688.

|| “The Lancet,”’ Sept. 6, 1902.

642 Typhoid Fever

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 ro11, the United States Govern-
ment began, on March to, 1911, the mobilization of regiments of the
United States Army on the Mexican frontier near San Antonio,
Texas. In order to prevent repetition of 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 immunized against the disease. Many of the
soldiers arriving on the ground had already been immunized, the
remainder were at once given the necessary injections. The mean
strength of the command at San Antonio was 12,000 up to June 30,
IQII, a period approximating four months. During all that time
there were only 2 cases of typhoid fever in the encampment, 1 in-
an uninoculated civilian teamster and 1 in an inoculated soldier.
Both cases recovered. The soldier suffered from so mild an attack
that it would not have been diagnosed had not a blood-culture been
made. During the four months from March roth to June 3oth the
typhoid fever was prevalent among the civilians of San Antonio,
there being 4o cases with 19 deaths.

The prophylactic used was prepared from a specially selected
strain of Bacillus typhosus 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, standard-
ized by counting the bacteria according to the method of Wright,
and after being diluted with salt solution, 0.25 per cent. of trikresol
was added. One cubic centimeter of the finished prophylactic con-
tained 1,000,000,000 bacilli. The first dose injected contained
500,000,000 bacilli, the second and third, given after ten and twenty
days, contained 1,000,000,000 each. The injections caused little
inconvenience either locally or constitutionally. Only 1 case had
fever, chills, and sweats, and this was the only case requiring treat-
ment in the hospital. It subsequently developed that this soldier
was suffering from early tuberculosis, which may explain the unto-
ward symptoms from which he suffered.

Specific Therapy.—Animals can be immunized to this bacillus, and
then, according to Chantemesse and Widal, develop antitoxic blood
capable of protecting other animals. 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

* “Brit. Med. Jour.,”’ Oct. 10, 1903.

t “Report of the Surgeon-General of the United States Army to the Secretary
of War,” 1911, Washington, D. C.

t “Zeitschrift fiir Hygiene,’ 1894, Xv1, p. 458.

Specific Therapy 643

previous one by Chantemesse and Widal, and has recently been
abundantly confirmed.

The immunization of dogs and goats by the meaacnas of
increasing doses of virulent cultures has been achieved by Pfeiffer
and Kolle* and by Léffler and Abel.} From these animals immune
serums were secured.

Walgert reported 4 cases treated successfully with a serum ob- |
tained from convalescent patients. Ten cubic centimeters were.
given at a dose, and the injection was repeated in 1 case with relapse.

Rumpf§ and Kraus and Buswell|| report a number of cases of
typhoid favorably influenced by hypodermic injections of small doses
of sterilized cultures of Bacillus pyocyaneus.

Fig. 258.—Typhoid bacilli, unaggluti- Fig. 259.—Typhoid bacilli, showing

nated (Jordan). typical clumping by typhoid serum
(Jordan).

Jez** believes that the antitoxic principle in typhoid fever is con-
tained in some of the internal organs instead of the blood, and claims
to have obtained remarkable results in 18 cases treated with extracts
of the bone-marrow, spleen, and thymus of rabbits previously in-
jected with the typhoid bacillus.

Chantemesse,{t Pope, {tt and Steele§§ have all used serums from
animals immunized against typhoid cultures for the treatment of
typhoid fever, with more or less success, but an analysis of the results
shows them to be very inconclusive.

The serum prepared by Macfadyen,|||| by crushing cultures
frozen with liquid air and injecting animals with the thus liberated
intracellular toxin, seems to be no improvement upon others.

cm ae f. Bakt. u. Parasitenk.,’’ Jan. 23, 1896, Bd. xrx, No. 23, p. 51.
id., 1896
“Miinchener med. Wochenschrift,’’ Sept. 27, 1898.
‘: Deutsche med. Wochenschrift,’’ 1893, No. 41.
“Wiener klin. Wochenschrift,”’ July 12, 1894.
** “Méd. moderne,” March 25, 1899.
tt “Gaz. des Hopitaux, ” 1898, LXXI, p. 397.
tt “Brit. Med. Jour.,” 1897, 1, 259.
§§ Ibid., April 17, 1897.
Ill “Brit. Med. Jour.,”’ April 3, 1903.

644 Typhoid Fever ©

‘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 Leyden 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 certain value in
therapeutics.

Bacteriologic Disgrod= nee are four bavtadblogis methods
that may assist the clinician in completing 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. The 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 Griin-
baum,{ working independently, observed 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—agglutina-
tion. 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 fairly accurate
results.

The Blood-culture—The technic of this operation is simple.
The skin of the fold of the elbow is thoroughly cleansed, a fillet put
about the arm, and as the veins become prominent, a sterile hypo-
dermic needle is introduced into one and about 10 cc. of blood
drawn into a syringe. Before clotting can take place, this is dis-
charged into a small flask containing 100 cc. of bouillon, mixed,
and stood away to incubate. 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 sepa-
rated from contaminating organisms by plating.

* “Med. Klinik,” m1, No. 31, p. 917, Aug. A 1907.
t “Berl. klin. Wochenschrift, ” 1907, No. 18
} “La Semaine Médicale,” 1896, p- 295.

Bacteriologic Methods _ 645

The Isolation of the Bacillus from the Feces——This method of
making the diagnosis has practically been abandoned because of its
uncertainty, its cumbersomeness, its tediousness, and because the

‘ preceding methods suffice in all cases.

An excellent résumé 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 diagnosis of
typhoid, in doubtful cases, based upon the Wolff-Eisner-Calmette
reaction in tuberculosis, is the “ocular typhoid reaction” of Chan-
temesse.{ This test consists in the instillation into the eye of a
solution made by extracting the typhoid bacillus as follows: ‘“ Gela-
tin plates covered with an eighteen to twenty-four 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 coagulum. This was subsequently
dried, powdered and dissolved in sterile water in the proportion
of 0.02 mg. to a drop.” ft

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 mucopuru-
lent exudate in some cases. The reaction persists about ten hours
and then declines, usually disappearing in twenty-four hours. Ham-
burger§ 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 bacteriologic 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

*“Boston Medical and Surgical Journal,” 1907.

+ “Deutsche med. Wochenschrift,” 1907, No. 31, p. 1264.

ie Hamburger, “Jour. Amer. Med. Assoc.,” L, 17, p. 1344, April 25, 1908.
Cc. cit. :

646 Typhoid Fever

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 differentiation when the cultures have
already been isolated. The method is described under the heading
“ Agglutination,” in the section devoted to the “Special Phenomena
of Infection and Immunity.”

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 o.s5 cc. 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. Inasecond mention of this methodf
he has found its use satisfactory in practice and the paper serviceable after four-

teen 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., Elsnert suggested the employment
of a special potato medium, and Rémy§ an artificial medium ap-
proximating a potato in composition, but without dextrine or glu-
cose. These media have ceased to be used.

Wiirtz|] and Kashida** make the differential diagnosis by observ-
ing the acid production of Bacillus coli in a medium consisting of
bouillon containing 1.5 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. Ifa glass rod dipped in hydrochloric acid be held over the dish,
vapor of ammonium chlorid is given off. The typhoid bacillus pro-
duces 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.

* “Centralbl. f. Bakt. u. Parasitenk.,” 1897, p. 445.

{ “Journal of Experimental Medicine,” May, 1808, p. 353, note.

t “Zeitschrift fiir Hygiene,” 1895, xx, Heft 1; Dec. 6, 1896.

Taare de l’Inst. Pasteur,” Aug., 1900. ~ :

|| “Archiv. de med. Experimentale,” 1892, rv, p. 85.
**“Centralbl. f. Bakt. u. Parasitenk,”’ June 24, 1897, Bd. xx1, Nos. 20 and 21.

Differentiation of Typhoid and Colon Bacilli 647

Rothberger* first employed neutral red for the differentiation of the
typhoid and colon bacilli. When grown in fluid media containing
it, the colon bacillus produces a yellowish flourescence, while the
typhoid bacillus does not destroy the port-wine color. Savage} and
Irons{t 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 propor-
tion of o.1 gram per liter, and uses the medium in the fermentation
tube. The colon bacillus always ferments the sugars and produces a
typical color reaction.

Hiss|| recommends the use of two special media.

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 cc. 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 a few minutes’ :boiling. The me-
dium is then titrated to determine its reaction, phenolphthalein being used as the
indicator, and enough HCl or NaOH added to bring it to the desired reaction—
ie., a reaction indicating 1.5 per cent. of normal acid. To the clear medium add
one or two eggs, well beaten in 25 cc. 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 ro grams of agar, 25 grams
of gelatin, 5 grams of beef-extract, 5 grams of sodium chlorid, and ro 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 a 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 con-
sists 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 yel-
lowish-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 urine
two days old, to which o.5 per cent. of peptone and 3.3 per cent. of
gelatin have been added. Colonies of the typhoid bacillus appear
radiated and filamentous; those of the colon bacillus, round, yellow-
ish, and sharply defined at the edges. The cultures should be kept
at 22°C., and the colonies should appear in twenty-four hours.

*“Centralbl. f. Bakt.,”” 1893, p. 187. ;
t “Journal of Hygiene,” rgor, 1, p. 437-

t Ibid., 1902, 1, p. 437.

§ “Jour. of Infectious Diseases,’ 1904, I, p. 341.

|| ‘Jour. of Experimental Medicine,” Nov., 1897, vol. 11, No. 6.

648 Typhoid Fever

Adami and Chapin* have suggested a method for the isolation of
typhoid bacilli from water, in which use is made 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 cc. 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, ro cc. 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 oneend. About 1 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 1 : 60, 1 : 100, 1:150, and
1s'200. 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 agglu-
tinated bacilli is complete, 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 loopful of the washed sediment is transferred to a tube of
nutrient broth, and finally from this tube plate cultures are made upon Elsner’s or
Hiss’ media.

A culture-medium for isolating the typhoid bacillus from feces is

recommended by Drigalski-Conradif and by Petkowitsch.t It is
made as follows:

Horse-meat infusion (3 pounds of horse meat to 2 liters

of water)......... oer 2 liters
Witte’s peptone....................000.000.2205+.+ 20 Tams
INMLIOSEs. cr aciacanscy wien ed eS aaeetiee sen lvaye ened y @ 20 eras
Sodium chlorid..................0................ IO grams
Agar-agaticcccccieeatiee stan See ea lite ae erase rans OO Brams
Litmus solution (Kubel and Tiemann)............... 260 cc.
TACOS Crectr crete wea Madan c.g sal iecse ese tie Ah Am oO 30 grams
Crystal-violet solution (0.01 per cent.)............... 20 €¢.

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 1 liter of water to 680 grams of finely chopped lean beef and place in
the cold for twenty-four hours. Express the juiceand make upto rliter. Coagu-
late 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 chlorid. 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 autoclave
at a temperature of 120°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 cc. of litmus solution (Kubel and Tiemann’s) add 15 grams of chem-
ically pure lactose. Boil for ten minutes.

* “Berliner klin. Wochenschrift,” Feb. 13, 1899.
t “Zeitschrift f. Hygiene,” Bd. xxrx. ;
{ “Centralbl. f. Bakt.,” etc., May 28, 1904, Bd. xxxvt, No. 2, p. 304.
se See F. F. Wesbrook, “Jour. Infectious Diseases,’’ May, 1905, Supplement,
0. I, Pp. 310. - ;

Differentiation of Typhoid and Colon Bacilli 649

(c) Mix (a) and (6) while hot. Render slightly alkaline to litmus, if necessary.
To the mixture add 2 cc. of hot sterile solution of 10 per cent. sodium hydrate in
distilled water and ro cc. of a fresh solution of Héchst’s crystal violet (0.1 gram of
crystal violet to 100 cc. of sterile water).

The medium is now poured into Petri dishes and is of a deep purple 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 dries up rapidly.

A very ingenious method of isolating the typhoid and colon bacilli
from drinking water has been suggested by Starkey,* who uses a
tubular labyrinth of glass filled with ordinary bouillon containing
0.05 per cent. of carbolic acid, or, as recommended by Somers,f
Pariette’s bouillon. The original for-
mula for the latter medium is as
follows:

1. Measure out pure hydrochloric acid, 4
cc., and add it to carbolic acid solu-
tion (5 per cent.), roo cc. Allow the
solution to stand at least a few days
before use.

2. This solution is added in quantities of
0.1, 0.2, and 0.3 cc. (delivered by
means of a sterile graduated pipet to
tubes, each containing ro cc. of pre-
viously sterilized nutrient bouillon).

3. Incubate at 37°C. for forty-eight hours
to eliminate contaminated tubes.

The restraining medium prevents the
ready growth of most organisms ex-
cept colon and typhoid bacilli. The
anaérobic conditions prevent the de-
velopment of aérobic organisms
which form the majority of bacteria pig 260.—Starkey’s labyrinth
with which one comes in contact in as modified by Somers.
ordinary bacteriological examinations. &

- The typhoid bacillus, being more motile than the colon, travels
more quickly through the coils of the labyrinth 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 labyrinth 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 anaérobic conditions can easily be attained.

Hesset has recommended the following medium:

Agar apa... ssccdaniui ak eer aut 5 grams (4.5 grams absolutely dry).
Witte’s peptone............... Io
Liebig’s beef-extract........... 5
Sodium chlorid............... 8.5
Distilled water..............-. 1000

* © Amer. Jour. Med. Sci.,” July, 1906, cxxxu, No. 1, No. 412, p. 109.
{ “Trans. Phila. Path. Soc.,” 1906.
t “Zeitschrift fiir Hygiene,’ 1908, LVI, 441.

650 Typhoid Fever

Dissolve the agar-agar in 500 cc. of the water over a free flame, making up the
loss by evaporation. Dissolve the other ingredient, in the remaining 500 cc.
of water, heat until dissolved, replacing the loss by evaporation., Pour the two
solutions together, 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—rocc.toatube. 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......... 0... cece eee ee ee eeeeeeees 20 gfams
Gelatin 5 si.3. 5h demir aig 4:4 Mara aiid aaah nh koew ds chan tain Io grams
Agar-Agan. .vcimucaccyseousntet cee spstemeese ane eeaeve ZO: BRATS
Dextrose or mannite................-.e000000.-.+.8.. TO grams
Sodium chlorid vcs oc csdeies ad ed host Sh yriee SR ED EES 5 grams
Potassium: chlotid <2 .scosvi eee: sedis es ans a Se4 weed eee 5 grams

Distilled ‘water!...»3s0542.<desawassgse aw ute amen: . 1000 grams
Dissolve the agar in 500 cc. of water, the other ingredients in the other 500 cc.
of water. Pour together, add 10 cc. of NaOH, filter, and tube.

Upon this medium the typhoid colonies are small, glistening
bluish, and translucent. Colon colonies are larger, opaque, and
brownish.

Endof recommends the employment of the following medium upon
which colonies of the typhoid bacillus grow large and remain colorless
while those of the colon bacillus remain small and red:

tooo cc. of meat infusion.
30 grams of agar-agar.
ro grams of peptone (Witte’s).
5 grams of sodium chlorid.

Neutralize to +1, and clear by filtration. Distribute the medium in 150 cc.
flasks and sterilize in the autoclave. When the medium is to be used, have ready a
Io per cent. solution of basic fuchsin in 95 per cent. alcohol that has stood for 20
hours and been decanted, and also a 10 per cent. solution of anhydrous sodium sul-

_ phite. To rocc. of the latter solution add 2 cc. of the former and steam the mix-
ture in an Arnold sterilizer for five minutes. To each too cc. of the agar-agar add
1 gram of pure lactose, dissolve in streaming steam or on a water-bath and then
add }4 cc. of the fuchsin-sulphite solution. The medium is then ready for use
in Petri dishes into which it can be poured as soon as mixed with the water, or
without admixture, to be inoculated later by stroking with a platinum wire. The
ae of the fuchsin by the sulphite should result in a nearly colorless
medium.

Léffler{ has found malachite green a very useful adjunct to our
means of differentiating the typhoid from other similar bacilli.
For the purpose, 2}4 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 1
pound of meat macerated in 1 liter of water, neutralized with potassium, with

* “Zeitschrift fiir Hygiene,” 1896, xxin, 47 Se
+ “Centralbl. f. Bakt.,”’ etc., 1904, Xxxv.
¢ “Boston Med. and Surg. Journal,” Feb. 13, 1908, CLVIII, p. 213.

Differentiation of Typhoid and Colon Bacilli 651

the addition of 2 per cent, of peptone, 5 per cent. of lactose, 1 per cent. of glucose,
0.5 per cent. of sodium sulphate, 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, Gart-
ner’s bacillus and the paratyphoid bacillus 6 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 sur-
rounded 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,* who found
great differences in the quality and reactions of different malachite
greens in the market. That with which Loffler worked was com-
mercially 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 concentra-
tion of the dye to the reaction of the medium. When this is done,
malachite green becomes a valuable adjunct to specific differentia-
tion. Their studies of the media led Peabody and Pratt to the inven-
tion 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 “enrich-
ing” 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 Limbourgf and
have been more or less popular ever since, though for differentiation
of typhoid and colon bacilli they cause occasional disappointment.

Buxton and Colemant prepare a medium composed of:

Ox- bile 32 a arde agers gaara nes a alie Agee es 900 cc
Glycerinies occa cence eae ee Bae eR 100 CC
PePtone eic.a: sen diss Ad scanel eon cosueien os a dade ack Oinanece aa a 20 grams

This was placed in a number of roo cc. flasks, sterilized in the Arnold
sterilizer, and employed chiefly for blood-culture. ° The typhoid
bacillus grows well in it.

Jackson§ prepares a medium for water examination when typhoid
and colon bacilli are suspected that consists of undiluted ox-bile;

if fresh ox-bile cannot be secured, an 11 per cent. solution of dry

* “Zeitschrift f. physiol. Chemie,” 1889, TI, p. 196.

1 “Inst. hyg. Univers. Griefswald,’’ see ‘‘Bull. Inst. Past.,’’ 1v, No.9, May
T5, 1906, P. 393.

t “Journal of Infectious Diseases,’’ 1900, v1, No. 2, p. 194. . ;

§ “Biological Studies of the Pupils of W. T. Sedgwick,” 1906, University of
Chicago Press

652 Typhoid Fever

ox-bile can be used to which 1 per cent. of peptone and 1 per cent.
of lactose are added. It is filled into fermentation-tubes of 40 cc.
capacity and sterilized in the Arnold apparatus ro cc. of suspected
water or milk are planted in the tubes of this medium. The con-
tained 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 MacConkey* has the following
composition:

Agar.. Janeen teeees dp beehineee &— ie Bierams
Sodiunitaurosholate.. Bias iain nema bed aetna OG Orany
Peptone.. acini went la ache ere rauataaetiegta) 2xOseTams
Water.. a et . 100.0 CC.

It is boiled, clarified “andl Gleered « as a wie, then receives an addition of ro,
gram of lactose, i is tubed, and then sterilized three times on successive days.

For determining fermentation by colon bacilli the same investiga-
tor advises a broth composed of:

Sodium taurocholate @ (pare): Jeeeneeieecpeere: OxSi pram
Pep iane = DiniGiioneedaresevacs —2.6'gTams
Glucose. . Gash Crisis hls Dron eAgres MACAU IAI Soeeeie epee dotkiads ie, Ogi BrOUy
Water. . es .anane 206.0-66.

Boil, filter, ada Mmificlent siduital ifentig; “fil i into 5 fennientation-tubes. and steril-
ize at 100°C. Colon colonies appear red; typhoid, blue.

In a careful study of the bile-salt media MacConkeyf 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 zed preferable to litmus, and makes the medium as
follows:

1. A stock solution is made:
Sodium taurocholate (commercial from ei tile and

neutral to neutral red) ...... 00. eee o.5 per cent.
Peptone (Witte’s).. peigacsdeanagnegeesecea, 200 per cents
Water (distilled or tap)... . 100.0 CC.

(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, o.5 per cent.; lactose, 1 per cent.;
cane-sugar, I per cent.; dulcit, o.5 per cent.; adonit, 0.5 per cent., or inulin, 1
per cent.; and neutral red (x per cent. solution), 0.25 per cent., distributing into
fermentation-tubes and sterilizing in the steamer for fifteen minutes on each of
three successive days.

* “The Thompson-Yates Laboratory Reports,” 111, p. 151.
t “Journal of Hygiene,”’ 1908, vit, p. 322.

Bacilli Resembling the Typhoid Bacillus 653

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 cc. When required for use, the fer-
mentable 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 loopful 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.

Castellani* recommends the following method to facilitate the
isolation of the bacilli of the typhoid-paratyphoid groups:

1. Inoculate with the fecal matter to be investigated several tubes of tauro-
cholate of soda peptone water, or Browning, Gilmore and Mackie’s telluric acid
peptone water might be used.

2. Immediately after, or better immediately before, the inoculation, add 5
drops of polyvalent lactose fermented intestinal bacteria serum (B. proteus
group, etc.), taking care to use serums containing only a very small amount of
typhoid coagglutinin; or serum can be used from which the typhoid coagglutinin
has been removed by absorption.

3. Incubate for twelve or twenty-four hours, then make plates on MacConkey,
Conradi-Drigalsky or similar media, from the most superficial portion of the
liquid medium, and further investigate any suspicious colonies that may develop
with typhoid, paratyphoid A and paratyphoid B serums, etc. When there are
many flocculi of agglutinated bacilli also in the upper part of the tube, these
may be got rid of by a short centrifugation with an ordinary electric centrifuge
which causes the agglutinated bacilli to fall to the bottom, while it has practi-
cally no effect upon the non-agglutinated organisms in young cultures.

BACILLI RESEMBLING THE TYPHOID BACILLUS

Bacillus typhosus is one of a group of organisms possessing a con-

- siderable number of common characteristics, each member of which,

however, can be differentiated by some one fairly well-marked pecu-
liarity. 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 formacids. At the other
extreme stands Bacillus coli, an organism whose typical representa-
tives coagulate milk, form indol, ferment dextrose, lactose, saccha-
rose, and maltose with the formation of hydrogen and carbon dioxid
: ‘ H 2 '
in the proportion of co." a

Between these extremes are numerous organisms known as “ inter-
mediates.” It is usually a simple matter to differentiate 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 atten-
tion to them is, as yet, uncertain; and, indeed, we do not know

* Brit. Med. Jour., 1917, II, p- 477.

654 . Typhoid Fever —

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.
Buxton* summarizes the main points of difference as follows:

B. coli com- '
munis Intermediates B. typhosus
Coagulation of milk.. ive + - ao
Production of indol.. + - -
F ermentation of lactose with ;
AAS a ycavenstatstityau 6 oh ae sgraeks avas ve + — =
Fermentation of glucose with
gas.. Kee Hees AAG se + + -
Agglutination by typhoid 2
SOLU 0 agit esa eek ha eects = - +

The characteristics of the three groupe as shown by the fermenta-
tion-test stand thus:t

Gas upon Gas upon Gas upon

dextrose lactose saccharose
Bacillus typhosus. . iin =. | = 1
Intermediates................. + = -
Bacillus coli communis......... + + =
Bacillus coli communior. Zn + + +

Buxton finds those en 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.

(6) The Pneumonic or Psittacosis Group—Psittacosis is an epi-
demic infectious disease with pneumonic symptoms and a high
mortality. Its origin has been traced to diseased 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—tThe organisms to be included in this
group occasion symptoms closely resembling typhoid fever, though
they differ biologically from the typhoid bacillus, and do not agglu-
tinate with typhoid serums.

It is thus evident that some of the intermediates occasion symp-
toms 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.

Achard and Bensaude,t Johnson, Hewlett, and Longcope,§

* “Journal of Medical Research,” vol. vit, No. 1, June, 1902, p. 201.
+ Hiss and Zinsser, “‘Text-book ‘of Bacteriology,” IQIO, Pp. 429.

¥ “Soc. Med.,”’ Nov., 1896.
§ “Amer. Jour. Med. Sci. ,»” Aug., 1902.

655

Bacilli Resembling the Typhoid Bacillus

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656 - Bacillus Coli

Gwyn,* Libman,} Cushing,{ Durham,§ Savage and Read,|| Rus-
sell,** Krumwiede, Pratt and McWilliamstt and many others have
studied these organisms from various points of view. The impor-
tant points are to recognize their presence in cases of suspected in-
fection and to differentiate them quickly from the typhoid and
dysentery bacilli. 2

Russell{t first plated the material to be examined on Endo’s plated -
medium, and then transplanted the suspicious colonies to a tube of
culture-media so arranged as to contain two sugars that were incon-
siderably mixed. About 5 cc. of glucose litmus agar-agar were put
into each tube, and after sterilizing and cooling, enough sterilized
lactose litmus agar-agar was added to make a good slant. The
tubes were then incubated over night to permit any contaminating
organism to grow. The glucose-agar is at the bottom, the lactose
agar forms the surface, the tube is inoculated by stroking the surface
and stabbing the agar. In this way both media are brought into
use. Typhoid cultures thus inoculated after eighteen hours show
the usual non-spreading colorless growth on the surface of a blue
background of unchanged medium. In the depth of the tube, how-
ever, the medium is changed to a bright uniform red color. Later
Russell found that it was not necessary to keep the media separated,
but that the sugar could be added to the agar-agar containing litmus
and enough sodium hydrate to make the mixture just neutral to the
litmus. Last of all o.1 per cent. of glucose and 1.0 per cent. of lac-_
tose are added and the mixture sterilized. The autoclave should
not be employed for the sterilization because the high temperature
tends to break down the lactose.. In such a tube the typhoid bacil-
lus causes a colorless surface growth upon a blue background with
a red color in the bottom of the tube when punctured.

There is no gas. The colon bacillus, on the other hand, produces
abundant gas, and the medium is reddened throughout. The
dysentery bacillus behaves like the typhoid. The paratyphoids ap-
pear like the typhoid on the surface, but in the lower part of the punc-
ture made in the medium, a few gas bubbles appear.

Krumwiede and Kohn§§ have found that if Andrade’s indicator is
employed instead of litmus, three sugars, glucose, lactose and sac-
charose may all be added to the same agar-agar and the separation
of the “intermediates” facilitated by observing the fermentation
produced. To this end they use a stock agar made as follows:

*“ Johns Hopkins Bulletin,” 1898, vol. xx.
t “Journal of Medical Research,” 1902, vit, p. 168.
t “Johns Hopkins Bulletin,” 1900, vol. xt.
§ “Journal of Experimental Medicine,” 1901, vol. v, p. 353. .
|| Journal of Hygiene, 1913, XII, 343.
** Tour. Med. Research, 1911, XXV, 217.
tt Jour. Infectious Diseases, 1916, XVIII, I. -
tt Jour. Med. Research, 1912, xxv, p. 217.
§§ Jour. Med. Research, 1917, XXXII, p. 225.

Bacilli Resembling the Typhoid Bacillus 657

Liebig’s extract of beef......... 0.0.0... ce eee 3 grams
Witte’s peptone................ ce cece ee ee esse eee = TO grams
Salton ctemmay Sea aes ehere teem se eae ae vanende 5 grams
TAD AT are.s:savspsaavamivusneete ay ana acm wh aust elena Adcsecwanstioe: fk Sp SbAINS
Wate caine angsk ated tnaundtad aie Sane Att ae aegnanyiondaseanss MOOOTEC,

issolve in the autoclave. Tuitrateas nearly as possible to theslightly
kaline reaction desired and then add 1 per cent. of Andrade’s indica-
r which consists of 100 cc. of a 0.5 per cent. solution of acid fuchsin
scolorized by the addition of 16 cc. of a normal solution of sodium
ydrate. After titration to the final reaction, add the sugars, and
nally o.1 per cent. solution of brilliant green.

Fig. 261.—Bacillus coli (Migula).

Bacittus Cort (ESCHERICH)

Synonyms.—Bacillus coli communis; Bacterium coli; Bacillus neapolitanus.

General Characteristics.—A motile, flagellated, non-sporogenous, aérobic and
ptionally anaérobic, non-chromogenic, non-liquefying, aérogenic, saprophytic,
iccasionally pathogenic bacillus, staining by the ordinary methods, but not by
jam’s method. It produces indol, coagulates milk, and produces acids and
sases from dextrose, lactose, and sucrose. :

This micro-organism was first isolated from human feces by
Emmerich,* in 1885, who thought it to be the specific cause of
Asiatic cholera, and called it Bacillus neapolitanus. It was isolated
tom the feces of infants and thoroughly studied culturally and mor-
dhologically by Escherich,t and has since frequently been described
is “Escherich’s bacillus.” Weissner{ showed that it was to be
found in the normal human intestine. Many have since studied it
until it has now become one of the best known bacteria, and one

* “Deutsche med. Wochenschrift,”’ 1885, No 2.
t Die Darmbakterium des Saiiglings, Stuttgart, 1886.
t Zeitschrift fiir Hygiene, 1886, 1, 315.

658 Bacillus Coli

that is almost universally prevalent in the intestines and fecal evacu-
ations of man and the higher animals.

Distribution.—It is habitually present in the feces of animals, and
in water and soil contaminated by them. Soon after birth the or-
ganism finds its way into the alimentary canal and permanently
establishes itself in the intestine, 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* believes them to be
identical. It may also be identical with Bacillus lactis aérogenes,
Bacillus cavicida, and other separately described species.

Morphology.—The bacillus is rather variable, both size and form
depending to a certain extent upon the culture medium on which it
grows. It measures about 1-3 X0.4-0.7u. It usually occurs in the
form of short rods, with rounded ends but coccus-like and elongate
individuals may be found in thesame culture. The bacilli are usually

Fig. 262.—Bacillus coli; superficial colony two days old upon a gelatin
plate. XX 21 (Heim).

separate from one another, though occasionally joined in pairs, are
sluggishly motile, and provided with flagella, which are variable in
number, usually from four to a dozen. The organisms from some
cultures swim actively, even when the culture is some days old;
others are sluggish even when young 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,
at temperatures varying from 10° to 45°C., the optimum being about
37 C.

Colonies.—Upon gelatin plates the colonies are visible in twenty-
four hours. Those situated below the surface appear round, yellow-

* Society of American Bacteriologists, Dec. 31, 1902.

Bacilli Resembling the Typhoid Bacillus 659

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 col-
onies rapidly increase in size and become more and more opaque.
The gelatin is not liquefied.

Gelatin Punctures.—Development in gelatin punctures occurs
upon the surface, and also in the needle’s track, causing the forma-
tion 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, sufficient gas-production may occur to
break up the medium. The gelatin may become slightly clouded
but is not liquefied.

Agar-agar.— Upon agar-agar, along the line of inoculation, a gray-
ish-white, translucent, smeary growth, devoid of any characteristics,
takes place. The entire surface of the culture-medium is never cov-
ered, the growth remaining confined 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 bac-
teria, a delicate pellicle at times forming upon the surface. 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 in-
oculation over about one-half to two-thirds of the potato. The
color varies considerably, sometimes being pale, sometimes quite
brown, sometimes greenish. It cannot, therefore, be taken as a
characteristic of much importance. The growth on potato may be
almost invisible.

Milk.—In milk coagulation and acidulation occur, with varying
rapidity. 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 germi-
cides, and grows in culture-media containing from 0.1-0.2 per cent.
of carbolic acid. It i is, however, easily killed by heat, and is de-
stroyed by exposure to 60°C. for 120 minutes (Frankel) or 75°C.
for fifteen minutes (Kendall).

Metabolic Products.—Wiirtz found that Bacillus coli produced
ammonia in culture-media free from sugar, and thus caused an in-
tense alkaline reaction in the culture-media. The cultures usually
give off an unpleasant odor.

Nitrates are reduced to nitrites by the growth of the bacillus.

In bouillon containing 1 per cent. of dextrose, lactose, levulose,
galactose, and mannite, the colon bacillus ape up the sugar, lib-

2
erating CO, and H, the gas formula being ~~ C i =T This gas for-

660 . Bacillus Coli

mula is very constant for the micro-organisms of the colon group
and forms one of their most important differential characteristics.
In calculating the gas formula Winslow has shown that some care
ought to be taken to doit at the appropriate time. According to his
observations the given formula only obtains between the twenty-
fourth and forty-eighth hours. Before this period the H, which is
first formed, preponderates; after it the CO. may preponderate.
In sugar-containing bouillon, acetic, lactic, and formic acids are
produced. Thecolon bacillus does not, as a rule, fermentsaccharose.
When a similar bacillus is found regularly to ferment saccharose, it
is best to regard it as a subspecies or separate type, for which Dun-
ham has introduced the name Bacillus coli communior.

The bacillus requires very little nutriment. It grows,in Uschin-
sky’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 Sal-
kowski’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 pulver-
ized. Ofsucha preparation 0.0002 gram will kill a 200-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 continuity of tissue, or via the blood-vessels.

Although under normal conditions a saprophyte, the colon bacillus
is not infrequently found in the pus in suppurations connected with
the intestines—as, for example, appendicitiss-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 suppurative lesions in which it is the essential factor.
When absorbed from the intestine, it frequently enters the kidney
and is excreted with the urine, causing, incidentally, local inflamma-
tory areas in the kidney, and occasionally cystitis. A case of ure-
thritis is reported to have been caused by it.

The bile-ducts are sometimes invaded by the bacillus, which may
lead to inflammation, obstruction, suppuration, or calculus formation.

* «Tour. Amer. Med. Assoc.,” 1901; “American Medicine,” 1901.
f “Trans. Assoc. Amer. Phys.,” ro9o1.

Bacilli Resembling the Typhoid Bacillus 661

The colon bacillus has also been met with in puerperal fever,
Winckel’s disease of the newborn,* endocarditis, meningitis, 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 Medical Journal”’
for Nov. 4, 1911, p. 1186.

In a certain number of cases general hemic infection may be caused
by Bacillus coli. In 1909 Jacobt published an analysis of 39 such
cases, and in 1910 Draper{ increased the number to 43. Wiens§
also reported 6 cases and Maher|| x 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 strangulated portion of intestine. Dreyfustt 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
¢ases of cholera, cholera nostras, and other intestinal diseases are
more pathogenic than those obtained from normal feces or from pus.

For the laboratory animals the colon bacillus is pathogenic 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 intraperitoneal and intravenous in-
jection. Subcutaneous injections are of less effect, and in rabbits
produce abscesses only.

_ When injected into the abdominal cavity; the bacilli set up a sero-
- fibrinous or purulent peritonitis, and are numerous in the abdominal.
fluids.

Cumston, tt from a careful study of 13 cases of summer infantile
diarrheas, came to the conclusion that Bacillus coli seemed to be the
pathogenic agent of the greater number of cases.

Lesage,§§ in studying the enteritis of infants, found that in 4o out
of 50 cases depending upon Bacillus coli the blood of the patient

* “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.

+x “Ann. de l’Inst. Pasteur,’’ 1895, No. 9.

tt “Centralbl. f. Bakt.,”’ etc., XvI, p. 581.

{ “International Medical Magazine,” Feb., 1897.
§§ “La Semaine Médicale,” Oct. 20, 1897-

662 Bacillus Coli

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 colonbacillus. Léffler and Abel immunized dogs by progressively
increased subcutaneous doses of live bacteria, grown in solid culture
and suspended in water. The injections at first produced hard swell-
ings. 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 similar 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 similar to be differen-
tiated into groups, types, or families by our present methods.

In order to establish a type species of Bacillus coli communis,
Smith* says:

“T 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 margins; 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 approximately 14;
H : CO, = approximately 2:1; reaction strongly acid. ;

. ae per cent. lactose-bouillon: as in dextrose-bouillon (with slight varia-
tions).

“One per cent. saccharose-bouillon; gas-production slower than the preceding,
lasting from seven to fourteen days. Total gas about 24; H: CO. = 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 B:

*“ Amer. Jour. Med. Sci.,”’ 1895, 110, p. 287.

Bacilli Resembling the Typhoid Bacillus

663

“The same in all respects, excepting as to its behavior in saccharose-bouillon;

neither gas nor acids are formed in it.”

DIFFERENTIAL CHARACTERISTICS

TypHoIp BacILLus

Bacilli usually slender.

Flagella numerous (10-20), long, and
wavy (peritricha).

Growth not very rapid, not particu-
larly luxuriant.

Upon Elsner’s, Hiss’, Piorkowski’s, and
other media gives characteristic ap-
pearances.

Upon fresh acid potato the so-called
“invisible growth” formerly thought
to be differential.

Acid-production in whey not exceeding
3 percent. Sometimes slight in or-
dinary media, and succeeded by
alkali-production.

Grows in media containing sugars with-
out producing any gas.

Produces no indol.
Growth in milk unaccompanied by

Coton BaciLius

Bacilli a little thicker and shorter.
Flagella fewer (8-10) (peritricha).

Growth rapid and luxuriant. This
character is by no means constant.
Upon Elsner’s, Hiss,’ Piorkowski’s, and
other media gives characteristic
appearances.

Upon potato a brownish-yellow distinct
pellicle.

Acid-production well marked through-
out.

Fermentation with ga -production well
marked in solutions containing dex-
trose, lactose, etc., the usual for-
mula being H: CO. = 2:1.

Indol-production marked.

Milk coagulated.

coagulation.

Gives the Widal reaction with the ser-
um of typhoid blood. -

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 con-
genial 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 1,000,000 colon bacilli per cubic
centimeter and pure water very often o per cubic centimeter (only
1 cc. 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

Does not react with typhoid blood.

664 Bacillus Suipestifer

the repeated examination of 1 cc. samples shows the presence of
numerous colon bacilli, the water is seriously polluted and doubtfully
potable, but when samples of 1 cc. 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 intestinal parasitism and taken up
a saprophytic life. The chief of these characters is the ability to fer-
ment lactose. Only recently isolated organisms manifest this fer-
mentative 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 detection of the
colon bacilli in water, the favorite at the present time probably be-
ing the litmus-lactose-agar (g.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
elsewhere blue plate. These red colonies are then fished up and
transplanted to appropriate media for further study.

Other media and methods useful in studying the colon bacilli are
also discussed in the chapter upon Typhoid Fever (q.v.).

Bacititus ENTERITIDIS (GARTNER)

General Characteristics.—A motile, flagellated, non-sporogenous, non-chro-
mogenic, non-liquefying, aérogenic, aérobic and optionally anaérobic, pathogenic
bacillus staining by the ordinary methods, but not by Gram’s method.

This bacillus was first cultivated by A. Gartnert 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
oe and thick, is surrounded by a slight capsule, is actively motile, and has

agella. a

Staining.—It stains irregularly with the ordinary solutions, but not by Gram’s
method. It has no spores.

Cultivation.—Upon gelatin plates it forms round, pale gray, translucent col-
onies. 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. It ferments
dextrose, mannite, dulcite and sorbite with the production of acid and the evolu-
tion of gas. It does not ferment saccharose, adonite, inosite or inulin. Upon
potato it forms a yellowish-white, shining layer.

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, intraperitoneally,
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 hemorrhages, are found. The bacilli occur in the intes-
tinal contents. The spleen is somewhat enlarged. :

The bacillus is differentiated from the colon bacillus chiefly by its inability
to ferment lactose and saccharose, by the absence of indol-production, by its

*“ Archiv, de méd. Experimentale,” 1892, IV, p. 85.
{ “Korrespond. d. allg. arztl. Ver. von Thiiring,” 1888, 9. °

Bacilli Resembling the Typhoid Bacillus 665

ability to produce infection when ingested, and by the fact’that it elaborates a
ne substance capable of producing symptoms similar to those seen in the
infection.

BACILLUS Fa&cALIS ALKALIGENES (PETRUSCHKY)

General Characteristics.—A motile, flagellated, non-sporogenous, non-lique-
fying, non-chromogenic, non-aérogenic, aérobic and optionally anaérobic, 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 animals 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 cul-
tures are not agglutinated by the typhoid serums.

Baciiius PsitTacosis (NocARD)

General Characteristics—A motile, flagellated, non-sporogenous, aérobic,
optionally anaérobic, non-chromogenic, aérogenic, pathogenic, non-liquefying
bacillus, staining by the ordinary methods, but not by Gram’s method.

This micro-organism was discovered by Nocard,t 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 spe-
cific organism. :

The subsequent work of Gilbert and Fourniert 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 cultures, closely resemble
those of the typhoid fever organism. Upon potato it more closely resembles the
colon bacillus. Bouillon becomes clouded. Doms,

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
eat-vein of a rabbit kill it in from twelve to eighteen hours. Guinea-pigs are
more resistant. Subcutaneous injection of dogs produces a hard, painful swell-
ing, which persists for a short time and then disappears without suppuration. It
is also infectious for man, a number of epidemics of peculiar pneumonia, charac-
terized by the presence of the bacillus in the blood, traceable to diseased parrots,
having been reported.

Differentiation Bacillus psittacosis can best be differentiated from the ty-
phoid and the colon bacilli and others of the same group by its pathogenesis and

*“Centralbl. f. Bakt. u. Parasitenk.,’”’ x1x, 187.
{“Séance du Conseil d’hygiene publique et Salubrite du Department de la
Seine,” March 24, 1893. . :

t“Comptes rendu de la Société de Biologie,’ 1896; ‘‘La Presse médicale,”’
Jan. 16, 1897.

666 Bacillus Suipestifer

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, invad-
ing their tissues when they become ill, or whether it is a purely pathogenic micro-
organism found only in psittacosis.

BACILLUS SUIPESTIFER (SALMON AND SMITH)*

Synonym.—Bacillus cholere suis. ;

_ General Characteristics.—An actively motile, flagellated, non-sporogenous,
non-chromogenic, non-liquefying, aérobic and optionally anaérobic, aérogenic
bacillus pathogenic for hogs and other animals. It stains by the ordinary
methods, but not by Gram’s method. It ferments dextrose, 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 go 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 DeSchwein-
itz 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 cultivation to be free of bacilli. It therefore
depends upon a filterable and unknown virus.

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
rhe Bolton, and McBrydet and by Dorset, McBryde, and Niles§ are worth
treading.

These investigations entirely changed our ideas of the importance 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 Salnion and Smith,|| but was for
a long time confused with the bacillus of “‘swine-plague,”’ which it closely resem-
bles, and in association 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 soo hogs. It
occurs in the blood and in all the organs, and has also been cultivated from the
urine. ;

Morphology.—The organisms appear as short rods with rounded ends, 1.2 to
1.54 long and 0.6 to 0.7m in breadth. They are actively motile and possess long
flagella (peritrichia), easily demonstrable by the usual methods of staining. No
spore production has been observed. In general the bacillus resembles that of
sea 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 aérobic and anaérobic 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.

* Annual Report of the United States Bureau of Animal Industry, 1885, Vol. ii.

t ‘Circular No. 41 of Bureau of Animal Industry,’ U. S. Dept. of Agriculture,
Washington, D. C.

t “Bull. No. 72 of Bureau of Animal Industry,’ U. S. Dept. Agriculture,
Washington, D. C., 1905.

§“ Bull. No. 102 of Bureau of Animal Industry,” U. S. Dept. Agriculture,
Washington, D. C., Jan. 18, 1908.

|| ‘Reports of the Bureau of Animal Industry,’ 1885-91; and “Centralbl. f.
Bakt. u. Parasitenk.,’’ March 2, 1891. Bd. rx, Nos. 8, 9, and 10.

Bacilli Resembling the Typhoid Bacillus 667

They are rarely larger than 0.5 mm. in diameter and are homogencous through-
out. The superficial colonies have little tendency to spread upon the gelatin.
They rarely reach a greater diameter than 2mm. 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. Upon
Conradi-Drigalski agar-agar plates the colonies are blue. On Léffler’s malachite-
green plates, transparent colonies appear surrounded by a yellowish change in the
agar.

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, circum-
scribed, grayish, smeary layer without characteristic appearances.

Potato.— Upon potato a yellowish coating is formed, especially when the cul-
ture is kept in the thermostat. No growth occurs upon acid potato.

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; after a slight initial acidity it becomes and
remains alkaline in reaction.

Vital Resistance.—Smith found the bacillus vital after being dry for four
months. It ordinarily dies sooner, however, and difficulty may be experienced in
keeping it in the laboratory for any length of time unless frequently transplanted.
The thermal death-point is 58°C., maintained for ten minutes.

Metabolic Products.—Gas Production.—The hog-cholera bacillus is capable of
breaking up dextrose, arabinose, xylose, fructose, galactose, mannose, maltose,
dulcite, mannite and sorbite into CO», H, and an acid, which, formed late, even-
tually checks its further development. It does not ferment saccharose or lactose,
nor does it decompose glycogen, inulin, adonite, starch, erythrite or raffinose.

Indol.—No indol and no phenol are formed in the culture-media, but H2S is
formed from peptone.

Toxin.—In pure cultures of the hog-cholera bacillus Novy* found a poisonous
base with the probable composition CisH2sN2, 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 white rats. He has
also obtained a poisonous protein of which 50 mg. were fatal for white rats, and
which immunized them against highly virulent hog-cholera organisms, when
administered by repeated subcutaneous injection.

DeSchweinitz+ 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 ix vacuo, forms needle-like crystals with
platinic chlorid, and was classed among the albumoses.

_ Pathogenesis.—The bacillus is disappointing in its effects upomhogs. When
it is subcutaneously or intravenously 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
hog cholera to others.

_Smith found that 0.75 cc. of a bouillon culture injected into the breast muscles
of pigeons would kill them. a

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 temperature abruptly rises 2° to 3°C., and remains high until death. Sub-
cutaneous injection of larger quantities may kill in five days. Injected intra-
venously in small doses the bacillus may kill rabbits in forty-eight hours. __

Agglutination.—Pitfieldt found that after a single injection of a killed bouillon
culture of the bacillus into a horse, the serum, which originally had very slight
agglutinative 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 dilu-
tion of 1 : 10,000 a typical agglutination occurs in sixty minutes.

McClintock, Boxmeyer and Siffer§ found that the serum of normal hogs
agglutinates strains of ordinary hog-cholera bacilli in dilutions occasionally as

* “Medical News,” 1900, p. 231.

t “Medical News,’’ 1900, p. 237.

1“ Microscopical Bulletin,” 1897, p. 35.

§ “Jour. of Infectious Diseases,”’ March 1, 1905, vol. 11, No. 2, p. 351.

668 Bacillus Icteroides

high as 1:250 and consider reaction in a dilution of less than 1 : 300 without
diagnostic value.

Bacittus IctERomwES (SANARELLI)

General Characteristics—An actively motile flagellated, non-sporogenous
non-liquefying, non-chromogenic, aérogenic, aérobic and optionally anaérobic,
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 rz autopsies upon yellow fever cases, but al-
ways in association with streptococci, colon bacilli, pro-
teus, 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 accomplished
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 ex-
amination.

Morphology.—The bacillus presents nothing morpho-

bacillus with rounded ends, usually joined in pairs. ‘It is
2 to 4m 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.

263-—Cul: Agar-agar.—The culture upon agar-agar is said to be
3: 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 cultures are kept for the first twelve to sixteen
hours at 37°C., and afterward at the room temperature, when the colonies will
show a flat central nucleus transparent and bluish, surrounded by a prominent
and opaque zone, the whole resembling a drop of sealing-wax. Sanarellirefers to
this appearance as constituting the chief diagnostic feature of Bacillus icteroi-
dgs. It can be observed in twenty-four hours. :

Blood-serum.—Upon blood-serum the growth is very meager. :

Potato.—The growth upon potato corresponds with that of the bacillus of
typhoid fever. ; :

Fig.
ture of Bacillus
icteroides on agar
(Sanarelli).

* Il Policlinico, 1897, 1v, Nos. 8-9, p. 1.

logically characteristic. It is a small pleomorphous. . .

Bacilli Resembling the Typhoid Bacillus 669

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
solarrays. It can live for a considerable time in sea-water.

Metabolism.—The bacillus is an optional anaérobe. It slowly ferments dex-
trose, forming gas. It does not coagulate milk. In the cultures a small amount
of indol is formed.

Pathogenesis.—The bacillus is pathogenic for the domestic animals, all mam-
mals seeming to be more or less sensitive to it. Birds areoftenimmune. 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 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 it is injected
intravenously, symptoms appear almost immediately and recall the clinical and
anatomic features of yellow fever in man. The most prominent symptom 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.

BacitLus TypHt Murium (LOFFLER)

General Characteristics.—A motile, flagellated, non-sporogenous, non-liquefy-
ing, non-chromogenic, non-aérogenic, aérobic and optionally anaérobic bacillus,
pathogenic for mice and other small animals, staining by the ordinary methods,
but not by Gram’s method. It acidulates but does not coagulate milk. ;
_ Bacillus typhi murium was discovered by Léffler* in 1889, when it created
havoc among the mice in his laboratory at Greifswald.

Morphology.—The organism bears a close resemblance to that of typhoid fever,
sometimes appearing short, sometimes 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 Léffler’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. Superficial 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 devoid of peculiarities
occurs,

he a potato a rather thin whitish growth may be observed after
a few days.

i Milk.—The bacillus grows well in milk, causing acid reaction, without coagu-
ation.

Bouillon.—In bouillon it produces clouding. There is no fermentation of
saccharose, dextrose, lactose, or levulose. ci

Pathogenesis.—The organism is pathogenic for mice of all kinds, which suc-
cumb in from one to two days when inoculated subcutaneously, and in from eight
to twelve days when fed upon material containing the bacillus. The bacilli
multiply rapidly in the blood- and lymph-channels, and cause death from
septicemia, So

Léffler expressed the opinion that this bacillus might be of use in ridding in-
fested 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

*“Centralbl. f. Bakt. u. Parasitenk.,’’ x1, p. 129.

670 Bacillus Murium

mice that had eaten the bread, but also infected others that ate their dead bodies,
the extermination progressing until scarcely a mouse remained.

In discussing the practical employment of this bacillus for the satisfactory de-
struction of field-mice, Brunner* calls attention to certain conditions that are
requisite: (1) It is necessary, first of all, to attack extensive areas of the invaded
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 preparation 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

Fig. 264.—Bacillus typhi murium (Migula).

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 Laboratory 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
Rosenauf 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.

+ “Ann. de l’Inst. Pasteur,’ April, 1900.

t ‘Bulletin No. 5 of the Hygienic Laboratory of the U. S. Marine Hospital
Service,’ Washington, D. C., rgor. : :

CHAPTER XXIX
DYSENTERY

DysENTERY is an acute, subacute or chronic, infectious colitis,
usually characterized by an acute onset, mild fever, pain in the abdo-
men, 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 dysen-
tery that is meant by “emerods” in describing an epidemic that took
place among the people of Israel during the time of the Judges. Hip-
pocrates 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. Inaddition
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 great-
est sufferers. The incidence of dysentery in the Federal Army dur-
ing the War of the Rebellion was appalling. Woodward* states
that there were 259,071 cases of acute and 28,451 cases of chronic
dysentery.

Endemics also occur from time to time and assume devastating
proportions, as in Japan, where between 1878 and 18099 there were
1,136,096 cases, with 275,308 deaths—a mortality of 25.23 per cent.f
Osler 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 bacteriolo-
gists, and Klebs, Ziegler, Ogata, Grigorjeff, de Silvestri, Maggiora,
Arnaud, Celli and Fiocca, Galli-Valerio, Valagussa, Deycke, and
others published descriptions of various micro-organisms isolated
from dysenteric stools, and looked upon by their discoverers as its
cause. The results were, however, so discordant that none of
the described micro-organisms could be agreed upon as the excitant
of the disease.

In 1860 Lamblt 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.

* “Medical and Surgical History of the War of the Rebellion,” Medical, 11.

} “Public Health Reports,” Jan. 5, 1900, xv, No. r.
t“Aus. d. Franz Joseph Kinderspital zur Prague,’’ 1860,1, 326.

671

672 Dysentery

In 1875 Lésch* described an ameba which he found in great num-
bers 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 obser-
vation until eight years later, when Koch and Gaffky,f in studying
the cholera in Egypt, also observed amebas in the intestinal dis-
charges in certain cases, and Kartulis{ wrote upon the “Etiology of
the Dysentery in Egypt,’”’ which he referred to them. In America
the study of these amebas was quickly taken up. Osler§ dis-
covered the organisms in the evacuations of a case of dysentery
contracted by a patient during a visit to Panama. Councilman and
Lafleur|| wrote a fine monograph upon ‘ Amebic Dysentery,” while
Quincke and Roos** and Kruse and Pasqualett confirmed the ob-
servations and results in Europe.

' Thus it came to be recognized that an ameba might be the causeof _
dysentery. It was soon pointed out, however, that there were cases
of dysentery in which no amebas could be found in the intestinal dis-
charges, or in which they were so few that it seemed impossible that
they could be the cause of the disease. This was particularly im-
pressive throughout the years of the endemic dysentery in Japan,
already referred to. Great numbers of cases occurred, great num-
bers 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 a difficult
one, and might not have been solved had he not made use of a then
new means of investigation, viz., the phenomenon of agglutination.
By studying such bacteria as could be cultivated from the intes-
tinal discharges, with particular reference to the agglutinating
effect of the blood of dysenteric patients upon them, Shigatt suc-
ceeded in discovering a new micro-organism which he called Bacillus
dysenteriae. Two years afterward Kruse§§ investigated an outbreak
of dysentery in an industrial section of Westphalia and found the
same bacillus and Flexner|||| showed it to be present in the epidemic
dysentery of the Philippine Islands.

Thus through the discovery of Shiga it became evident that
there are two forms of dysentery, one amebic the other bacillary.
Both occur sporadically and endemically in the tropics and in tem-
perate climates, and both may occur epidemically, though of the
two the bacillary form is the more liable to do so. Of the chronic
cases of dysentery 9o per cent. are amebic.

* “Virchow’s Archives,” 1875, Bd. Lxv.

} “Bericht tiber die Erforschung der Cholera,’’ 1883; “Arbeiten aus dem
kaiserl. Gesundheitsamte.,” 111, 65.

{ “Virchow’s Archives,” 1886, cv.

§ “Centralbl. f. Bakt. u. Parasitenk.,”’ 1890, vir, 736.

|| ‘Johns Hopkins Hospital Reports,” 1891, 11. .

** “Berliner klin. Wochenschrift,” 1893.

tt “Zeitschrift £. Hygiene,” etc., 1894, xvI.

tt “Centralbl. f. Bakt. u. Parasitenk.,” 1898, xxrv, 817.

i “Deutsche med. Wochenschrift,’? 1900, No. 40.
\|Il ‘Centralbl. f. Bakt. u. Parasitenk.,” 1900, xxvut, No. 19.

Amebic Dysentery 673

I. AMEBIC DYSENTERY

Amasa Cort (Léscu, 1875); AM@pa DysEnTERI& (Coun-
CILMAN AND LAFLEUR, 1893); ENTAMG@BA HistTo-
LYTICA (SCHAUDINN, 1903)

As has been shown, amebas were first found in the human in-
testine by Lambl. Their presence and probable importance in dys-
entery was subsequently worked out by Lésch, Koch, Gaffky, Kar-
tulis, Osler, Councilman and Lafleur, and many others.

Celli and Fiocca* were the first to study the amebas system-
atically 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 Amceba dysenterie. The
former was supposed to be a harmless commensal, the latter a

Fig. 265.—Ameeba coli in intestinal mucus with blood-corpuscles and bacteria
é (Lisch).

pathogenic organism and the cause of dysentery. As, however,
Lésch had called the organism found in dysentery the Amoeba
coli, Stiles declared the nomenclature faulty, and pointed out that
Ameeba coli, variety dysenteriz, must be the name of the pathogenic
form. Schaudinnt reviewed the subject and grouped all of the
intestinal amebas under the following: ,
I. Chlamydophrys stercorea (Cienkowsky).
II. Ameeba coli rhizopodia.
1. Entamoeba coli (Lésch) (Schaudinn).
2. Entameeba histolytica (Schaudinn).
To these has been since added in 1907:
Entameeba tetragena (Viereck).
1. Entameeba Coli (Lisch, 1875)——This organism seems to be a
harmless commensal, living in the intestines of man, many domestic,
*“Centralbl. f. Bakt. u. Parasitenk.,’’ 1894, xv, 470.
t “Arbeiten aus d. kaiserl. Gesundheitsamte.,” 1903, XIx, No. 3:
43

674 Dysentery

and many wild animals. It may be abundant when the reaction
of the intestinal contents is neutral or alkaline. It usually measures
between ro and 20 uw in diameter when free, but when encysted from
15 to 50 uw. It is spheroidal 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 methy-
lene-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 organism first be-
coming 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
intestines of experiment animals have failed.

2. Entameeba Histolytica (Schaudinn*).—This is now recognized

as the organism seen by Lésch, 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. It is usually present in great numbers
so that its discovery in the evacuations is easy.

Morphology. —It is usually considerably larger than Entameeba
coli and varies in diameter up to 504. When at rest it is spherical,
when active it is very irregular. Its movement 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. In the intestinal evacuations of
dysentery its protoplasm commonly contains many red _ blood-
corpuscles, upon which the organism seems to feed.

Staining.—When stained with polychrome methylene-blue the
- ectoplasm stains more deeply than the endoplasm. ht nucleus
contains relatively little chromatin.

Reproduction.—Multiplication takes place by binary division
after karyokinesis and by encystment and sporulation. The sporu-
lation is quite different from that seen in Entamceba coli, and only
takes place when conditions are unfavorable to continued division.

, * “Arbeiten a. d. k. k. Gesundheitsamt.,”’ 1903, XIX, 547.

Amebic Dysentery 675

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 u in diameter, and are

Fig. 266.—Reproductive cycle of parasitic ameba (Wenyon). ‘The small
circle indicated by 1, 2, 3, 3’ and 3” indicated multiplication by schizogony, or
binary division. The large circle indicated by 1-12, the sporogeny or sexual
cycle. The ameba having arrived at its full size (3) becomes enycsted (4). The
nucleus then divides into two ( 5), each half expels a small fragment of nuclear

material (6), and when this has been effected, they conjugate (7) forming a

synkaryon. The synkaryon then divides into two, into four, and then generally
into eight (8-9-10-11-12) when the cyst ruptures, ‘the spores are liberated
(t) and both cycles are again started.

surrounded by a yellowish envelope, which resists drying and the

“+ penetration of stains and chemicals.

Craig gives a tabulation of the differential features of Entamceba
coli, Entamoeba histolytica, and Entamceba tetragena (vide infra).

3. Entameeba Tetragena (Viereck*)—This organism resembles
Entamoeba histolytica more than Amceba coli, but differs from it
in the mode of reproduction, the sporocysts containing four instead
of eight spores.

* “ Archiv. f. Schiffs. u. Tropenhygiene,” 1907, It, 1.

676 Dysentery

Relationship of the Organisms.—In recent years (1910-1915)
much morphological and experimental study of these amcebas has
been conducted with results that are given in full, together with the
literature, in a paper ‘“‘The Identity of Entamceba Histolytica and
Entameeba Tetragena, with Observations upon the Morphology and
Life Cycle of Entamceba Histolytica”’ by Charles F. Craig.* The
results of his studies, as set forth in the paper, go to show that
Schaudinn was in error in regard to the developmental cycle of
Entameeba histolytica, that what he supposed to be its sole method
of reproduction, is only that means that preponderates during the
period of its greatest activity; that as the acme of the dysenteric
disease is passed and the process of repair sets in, the other mode of
reproduction characteristic of Entamoeba tetragena is observed,
and that the two species Entamceba histolytica and Entamceba
tetragena are one. There is, therefore, to all appearances, and
according to the best information available at present, only one
pathogenic intestinal: amceba, the Entamceba histolytica. The
same conclusions have also been arrived at by Darling.f

With regard to Entomeeba coli, opinion as to its non-pathogenic
disposition is much less certain than a few years ago. Williams and
Calkinst close their excellent paper upon “Cultural Amoeba; a
Study in Variation’’ with the statement that “it is unwise for any-
one at present to be too positive in regard to the distinctive features
of Entameeba coli, E. tetragena and E. histolytica, or any of the
Entamceba groups. There may be in man, three or more, or two
(as Hartmann, Whitman, Walker and Craig now think) or possibly
only one species of ameba manifesting different forms under different
conditions.”

Isolation and Cultivation—Many experimenters have made
more or less successful attempts to cultivate amebas. Musgrave 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: ,

PABA ow g wiarlodrte cetyeee ote Gace bade Reyes eh aaeeeane ee -ROeO grams
SOM UM CHIGHG wich sn sauieddn ao awh ae wba ole Ao 0.3-0.5 *
Extract OF D€€ivcinscw vcs ceiaae ane sad sewers 0.3-0.5 “
WALERS cc issets oe ei ron ie dra acre Ape iicers aise sees 1000.0 ce.

Prepare as ordinary culture agar, and render 1 per cent. alkaline to phenol-
phthalein. 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, naturally, not pure; they contain
various amebas and numerous bacteria.

* “Tour. Infectious Diseases,” 1913, XIII, 30.

tT ‘Trans. of the Fifteenth International Congress on Hygiene and Dermo-
graphy,” Washington, D. C., Sept., 1912.

t “Jour Med. Research,” 1913-1914, XXIV, 43.

§ ‘Department of the Interior, Bureau of Government Laboratories, Biological
Laboratory,” Manila, Oct., 1904, No. 8.

Amebic Dysentery 677

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 separated from
one another that not more than one is in a microscopic field of a low-power objec-
tive. 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 D D ) 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 organ-
ism. 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 surface
of the culture-medium, when, upon elevating it again, it usually leaves the
ameba behind. Observation 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 useful for the nourishment of the ameba, which is holophagous, and cannot
live by the absorption of nutritious fluids.

Later it was shown by Tsugitani* that killed cultures of bacteria
could supply the necessary nourishment. All cultures of amebas
must contain some kind of cells upon which the amebas can feed.
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 deter-
mined 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 bacteria, and 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.
lose 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 transplant-
ing amebas from plate to plate with suitable bacteria or other cells
for them to feed upon, the cultures may be kept growing almost
indefinitely.

Anna Williamst has been able to grow ameba in pure culture
without bacteria, either dead or alive, by smearing the surface of
a freshly prepared agar-agar plate with a fragment of freshly re-
moved 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 discharges
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 penetration of germicides. Direct sunlight
inhibits the activities of the organisms, but does not kill them.

*“Centralbl. f. Bakt. u. Parasitenk.,” Abt. 1, xxiv, 666.
t “ Journal of Medical Research,” Dec., 1911, xxv, No. 2, p. 263.

Dysentery

678

,OWER GROUP

GROUP

UPPER

Fig. 267.

Amebic Dysentery 679
EXPLANATION OF FIG. 267.

(All figures drawn by Charles F. Craig, M. D.)

J. Upper Group.—Entameba coli stained with Giemsa stain.

A,B, and C. Vegetative organisms showing nuclear membrane, karyosome,
and collections of chromatin upon the nuclear membrane and within the
hyaloplasm. Vacuoles are also present. =

D, An organism containing a protozoan parasite which might be mistaken
for spores. :

H. Division of nucleus (primitive mitosis).

E. Partially divided ameba containing two nuclei.

F,G. Ameba resulting from simple division.

M. eee of Entameeba coli. Eight daughter nuclei in vegetative

orm.

N. Ameba resulting from schizogony.

I, Earliest stage in cyst formation. Cytoplasm clear of foreign bodies and
nucleus showing collection of chromidial masses upon the inner side of
the nuclear membrane.

K,L, O, P. Two- and four-nucleated stage of reproduction within the cyst.

Q. Encysted form containing two large nuclei and a mass of chromatin.

R. Fully developed cyst of Entamceba coli containing eight nuclei.

Lower Group.—Entameba coli, fixed in sublimate alcohol and stained with Dela-
field’s hematoxylin. Note the more delicate staining of the nucleus and
the greater detail obtained with this method of staining.

A, B, C. Vegetative amebe showing variations in the structure of the
nucleus.

D. An organism during schizogony, containing eight nuclei.

E. Mitotic division of the nucleus as observed in this species. ;

FLA me developed cyst of Entamceba coli containing eight daughter
nuclei.

G. The four-nucleated cystic stage of Entamceba coli sometimes mistaken
for the cyst of Entamceba tetragena.

H. Two-nucleated cyst of Entameeba coli.

I. Young amebe originating from the cysts of Entameba coli.

K. Fully developed cyst in which the cystic membrane is apparently absent.

L. Degenerated cyst of Entameeba coli, filled with vacuoles, and containing
masses of chromatin. No nucleus is visible.

Il. Entameba histolytica stained with Giemsa stain.

A. Organism showing distinction between the ectoplasm and endoplasm,
nucleus and vacuole.

B. Organism showing vacuole and red blood corpuscle and nucleus contain-
ing minute karyosome and chromatin dots in the hyaloplasm.

C. Organism showing nucleus and numerous red blood corpuscles.

D. Organism in first stage of nuclear division, showing division of the karyo-
some and minute dots of chromatin in hyaloplasm. :

E. Organism showing later stage of nuclear division, the polar bodies being
connected by a filament of chromatic substance. Bark

F, First stage of formation of spore cysts; the nucleus distributing chro-
matin to the cytoplasm. eee

G to I. Stages in the process of formation of spore cysts, the chromatin being
distributed to the cytoplasm and collected in threads or masses,
while the nucleus is observed as a flattened body crowded against the
periphery of the parasite. :

L. Degenerated parasite containing vacuoles and free chromatin.

K, M, N. Entameeba histolytica in the final stage of the formation of spore
cysts. The free chromatin has collected at the periphery, and sur-
rounded by a small amount of cytoplasm, is being budded off from the
parent organism. .

O. Degenerated organism filled with vacuoles and free from chromatin.
The nucleus stains abnormally and there is no distinction between the
ectoplasm and endoplasm. : :

P. Entameeba histolytica filled with erthyrocytes, the nucleus being crowded
to the periphery and staining abnormally (Charles F. Craig, M. D.,
in Journal of Medical Research, vol. xxv1, No. 1, April, 191 2).

680 Dysentery

LOWER GROUP

UPPER GROUP

Bor.
fwd
ie j

UPPER GROUP

Fig. 268.

Amebic Dysentery 681

EXPLANATION OF FIG. 268.

(All figures drawn by Charles F. Craig, M. D. )

Ill. Upper Group.—Entameba tetragena fixed in sublimate alcohol and stained
with Delafield’s hematoxylin. Note the great delicacy of the stain-
ing when compared with the staining with the Giemsa method.

A. A vegetative parasite showing three erythrocytes in the cytoplasm'and a
nucleus in which the nuclear membrane, and the karyosome with its
centriole are shown. ;

B. A vegetative organism showing thick nuclear membrane and karyosome
containing a centriole. ;

C. A vegetative parasite containing vacuoles and nucleus showing karyo-
some containing a centriole surrounded by an unstained area.

D. A degenerative form filled with vacuoles and showing abnormal appear-
ance of the nucleus.

E. Precystic form of Entamceba tetragena.:

G. Another precystic form which is more typical in the free chromatin in the
cytoplasm is visible. The form E would probably degenerate before

; the cyst wall was fully formed.

F. A cystic form of Entamceba tetragena showing two chromatin spindles in
the cytoplasm and a nucleus having a centriole surrounded by an
unstained area and a definite network upon which are arranged dots of
chromatin.

H. An encysted form showing a very large mass of chromatin and a nucleus
containing a karyosome and centriole.

I. Two-nucleated cyst of Entamceba tetragena showing mass of free chroma-
tin and the morphology of the nuclei after division.

K. Fully developed cyst of Entamceba tetragena containing four daughter
nuclei and a mass of chromatin. a

L. Degenerated form of Entamoeba tetragena containing some free
chromatin and a nucleus in which the karyosome stains deeply and
nearly fills the nucleus. This form might be mistaken for a free living

* ameba. ,

M. Mlustrating the typical nuclear structure of Entamoeba tetragena.
Note the large karyosome containing a centriole surrounded by an
unstained area. ,

Lower Group.—Entameba histolytica fixed in sublimate alcohol and stained with
Delafield’s hematoxylin. -

Aand B. Vegetative organisms showing vacuoles and typical morphology of
the nucleus. No distinction between the endoplasm and ectoplasm. —

C. Vegetative form of Entameeba histolytica showing the type of mitosis
during simple division. .

D. First step in the formation of spore cysts. The distribution of the chro-

"matin by the nucleus to the cytoplasm. ’
E, F and H. Organisms showing chromidia in the cytoplasm arranged in
rods, threads, and masses, the nucleus being flattened out against the
a periphery and staining poorly. :
% G. A degenerative form of Entamceba histolytica filled with vacuoles and
with an atypical nucleus. :

I and K. Budding of the spore cysts from the periphery of Entameba
histolytica. . :

L. Illustrating the typical nuclear structure of Entamceba histolytica.

IV. Upper Group.—Entameba tetragena stained with Giemsa stain.

A, B, C. Vegetative organisms. Note that the nuclear membrane and
karyosome stain very heavily and are not as well differentiated as in
‘specimens stained with hematoxylin. .

D. Precystic form containing masses of chromatin in the cytoplasm.

E. Degenerative form containing vacuoles, masses of chromatin, and an
atypically stained nucleus. ;

F, Two-nucleated stage of the cyst of Entamceba tetragena, showing heavy
staining of the nuclear membrane and karyosome. Two masses of

Ez. chromatin are present. : ;

682 . Dysentery

Lésch was the first to observe that quinin was destructive to in-
testinal 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 1:25000 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.

Vedder found that emetin would kill ameba in dilutions up to
1:100,000, and Rogers has shown that this drug is the most de-
structive agent we possess as an amebicide. Unfortunately it does
not kill the encysted forms.

Exposure to 1:1000 solution of formalin did not kill encysted
amebas in twenty-four hours. Acetozone did not kill amebas in
1:1000 dilutions. If, however, the acetozone was made 1 per cent.
acid to phenolphthalein the amebas were all kil'ed by 1 ; 5000 solu-
tions in ten minutes.

Metabolic Products.—It seems as though Entamceeba histolytica
must produce some metabolic product that exerts an enzymic ac-
tion upon the human tissues and thus accounts for the destructive
nature of the lesions. This has not, however, been demonstrated
as yet. :

G. Fully developed cyst of Entamceba tetragena containing four nuclei and
one mass of chromatin.

H. Illustrating the type of nucleus as observed in Entamceba tetragena in
specimens stained with Giemsa stain.

Lower Group.—Ameba lobospinosa stained with Delafield’s hematoxylin after
fixation with sublimate alcohol.

1, 2, and 3. Vegetative organisms showing the large contractile vacuole and
"the typical nucleus containing a deeply stained karyosome almost
filling the nucleus.

4. A vegetative ameba in which the nucleus has divided.

5, 6. Vegetative amebe in which the nucleus is dividing. Polar bodies
are present connected by filaments and a well-marked equatorial
plate is apparent.

7. Degenerated vegetative ameba filled with vacuoles and with atypically
staining nucleus.

8. Amceba lobospinosa containing a protozoan organism. These forms
have been mistaken for sporulating amebe.

g and 10. Encysted forms of Amceba lobospinosa during the first few

days in cultures.

11 to 18 (except 14). Various cystic forms of Amceba lobospinosa show-
ing the character of the cyst wall in the older cysts. At 12 the cyst
contains two vacuoles and the cyst membrane is folded in, an appear-
ance frequently observed in cultures which have become dry; 15 and
17 represent cysts in which the cyst wall is cracked and a nucleus can-
not be distinguished; 16 represents a cyst filled with deeply staining
granules of chromatin derived from the degenerated nucleus; 18 is a
cyst in which only the cystic membrane is visible, the ameba having
escaped from the cyst.

14. A fragmenting ameba frequently mistaken for a budding organism before
the separation of the fragments (Charles F. Craig, M. D., in Journal
of Medical Research, vol. xxv1, No. 1, April, 1912).

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Amebic Dysentery

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684 Dysentery

Pathogenesis.—Schaudinn was the first to prove the pathogenic
action of the organism. He inspissated the evacuations of a case
suffering from dysentery, so that it contained considerable numbers
of encysted amebas. When this was fed to kittens they died in two
weeks with the typical lesions of dysentery. Musgrave and Clegg
had less satisfactory results with cats, dogs, and other laboratory
animals, but were quite satisfied with 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 in-
vestigators, and was strongly opposed by Craig,* who found both

Secondary abscesses

Falciform ligament

. - Secondary abscess in
Main abscess Lymphatic spigelian lobe
gland

Fig. 269.—Multiple amebic abscesses of the liver (J. E. Thompson, in Interna-
tional Clinics, vol. 11, r4th Series, J. B. Lippincott Co., Publishers).

varieties, and though he was never able to infect animals with
Entameeba 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 differentiate 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
almost never occurs in bacillary dysentery.

The distinct and somewhat rigid ectoplasm of the Entameba
histolytica is supposed to make it easy for the organisms, which it

* “Journal of Infectious Diseases,” 1908, V, p. 324.

Amebic Dysentery

Figs. 270, 271.—Colon: A, Tropical or amebic dysentery;
B, bacillary dysentery.

685

686. Dysentery

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 tissues with resulting exfoliation and
the production of round, oval, or ragged ulcerations with markedly
infiltrated and undermined edges. As the amebas continue to in-
crease 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

Fig. 272.—Entameeba histolytica. Section of the human intestinal wall
showing the amebas at the base of a dysenteric ulcer: A, A, A, Amebas, some of
which are in blood-vessels, Gf (Harris).

destroyed, leaving the entire large intestine denuded, except for
occasional islands of much congested, inflamed, and partly necrotic
mucous membrane. The diseased intestinal 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 lymphatics, 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

Bacillary Dysentery 687

material which may be so extensive as to involve the entire thick-
ness of the organ. There is usually one large abscess, but there may
be several small ones, or the liver may be riddled with minute ab-
scesses. The contents of the abscesses is pinkish necrotic material
in which amebas are few. The walls are of semi-necrotic material, in
which great numbers of amebas abound. The liver sometimes be-
comes adherent to the diaphragm, may perforate it, and after adhesion
of the lung to the diaphragm may evacuate through the lung, the
pinkish abscess contents with amebas being expectorated.
Sections of the intestinal wall and of the liver near the border
of the abscess show the amebas well when stained with iron-hema-
toxylon, 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.

#, 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 brown-
ish red; the nuclei of the mast-cells and of all other cells are stained blue.

II. BACILLARY DYSENTERY

BacitLus DysENTERI# (SHIGA)

General Characteristics.—A non-motile, non-flagellated, non-sporogenous,
non-liquefying, aérobic and optionally anaérobic, non-chromogenic, non-aéro-
genic, pathogenic bacillus of the intestine, staining by ordinary methods, but not
by Gram’s method. It does not produceindol It first acidifies, then alkalinizes
milk, but does not coagulate it.

After considerable investigation of the epidemic dysentery pre-
valent in Japan, Shigat came to the conclusion that a bacillus
which he called Bacillus dysenteriae was its specific cause.

It is not improbable that the bacillus of Shiga is identical with
Bacterium coli, variety dysenteri@, of Celli, Fiocca, and Scala,t
a view that has been further confirmed by Flexner.§ It may also
be identical with an organism described in 1888 by Chantemasse
and Widal.||

In 1899 Flexner,** while visiting the Philippine Islands, isolated
a bacillus from the epidemic dysentery prevailing there, which he
regarded as identical with Shiga’s organism. In 1890 Strong and

*“Pathological Technic,” 1911, p. 434.
+ “Centralbl. f. Bakt. u. Parasitenk., we 1808, xxiv, Nos. 22-24
: az “Hygien. Institut. Rom. Univ.,’ ” 1895, and ‘“Centralbl. f. pak u. Parasi-
en. ’ 1899.
§ “Univ. of Penna. Med. Bulletin,’ Aug., 1901.
|| “Deutsche med. Wochenschrift,’”? 1903, No. 12.
** “Bulletin of the deta Hopkins Hospital,” 1900, 1x

688 Dysentery

Musgrave* isolated what appeared to be the same organism, also
from cases of dysentery in the Philippines. Almost at the same
time Krusef was investigating an epidemic of dysentery in Germany,
and succeeded in isolating a bacillus that also bore fair correspond-
ence to that of Shiga. In 1901 Spronck{ found a bacillus in cases
of dysentery occurring in Utrecht, Holland, that corresponded with
a slightly different organism first found.and described by Kruse§
as a ‘‘pseudodysentery bacillus.”

In 1902 Park and Dunham] investigated a small epidemic of
dysentery in Maine, and there found a bacillus similar to those al-
ready described. In 1903 Hiss and Russell described a bacillus
“VY” from a case of fatal diarrhea in a child.

Bacillus dysenteriz was also found by Vedder and Duval** in
the epidemic and sporadic dysentery of the United States. Duval
and Bassetttt and Martha Wollsteintt found Bacillus dysenteriz in
cases of the summer diarrheas of infants, especially when such diar-
rheas were epidemic. ;

Lentz§§ has shown that dysentery and pseudodysentery bacilli
present differences in their behavior toward sugars. Numerous ob-
servers found differences in the behavior of the various bacilli to
the agglutinating effects of artificially prepared immune serum.
The outcome of these investigations is the discovery that Bacillus
dysenteriz 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 bacteri-
ology of dysentery. .

Varieties of the Dysentery Bacillus——Three varieties of the
dysentery bacillus may now be described:

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
their varying agglutinability by artificially prepared immune serums,
each of which exerts a far more pronounced effect upon its own
variety than upon the others, and in the behavior toward sugars
with reference to acid formation and gas production. It seems
not improbable that the future will have much to say about the

* “Report Surg. Gen. U. S. Army,” Washington, 1900.
I eee med. Pe dinaeda 1900, XXVI.
ef. Baumgarten’s Jahresberichte,” 1901.
§ ‘Deutsche med. Wochenschrift,” 1901, Nos. 23 and 24.
|| “New York Bull. of Med. Sciences,” 1902.
. ey Journal of Experimental Medicine,” 1902; vol. v1, No. 2, “American Medi-
cine,’ 1902. ;

tt “American Medicine,” Sept. 13, 1902, vol. 1v, No. 11, p. 417.
tt “Jour. Med. Research,” 1904, X, p. It. ‘ ;

§§ ‘Zeitschrift {. Hygiene,” etc., 1902, XLI.

Bacillary Dysentery 689

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 table taken from Muir and Ritchie’s ‘ Manual
of Bacteriology.”

Morphology.—The organism is a short rod with rounded ends,
generally similar to the typhoid bacillus. It measures 1.5-3 mu in
length by 0.8 —1 w in breadth. It usually occurs singly, but may
occur in pairs and rarely in short chains. It forms no spores, is not
motile and is 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 irregularities. It stains with or-
dinary solutions, but not by Gram’s method.

Isolation.—The bacillus may be obtained in greatest numbers
from the flakes of mucus in the dysenteric discharges. To free these
from the numerous bacteria of the feces, it has been recommended
that they be washed in salt-solution, before being smeared over
the surface of plates of such media as are used for the isolation of
the typhoid bacillus. As the general cultural difficulties experienced
in regard to the typhoid and dysentery bacilli are much the same,
the recommendations concerning the former apply equally to the lat-
ter. When the colonies supposed to be those of the dysentery bacilli
have been isolated and transplanted, the final identification must
be made by comparison with the table showing the general require-
ments, and by the application of the agglutination test by appropriate

. Serums.

Cultivation.—The organism grows well in slightly alkaline media,
at temperatures between 10° and 42°C. The most vigorous growth
takes place at about 37°C. It is an aérobe and optional anaérobe.

Colonies.—The colonies upon gelatin plates are small and dew-
drop-like in appearance. Upon microscopic examination they are
seen to be regular and of spheric form. By transmitted 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
medium.

Agar-agar.—Upon the surfate 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
alas it is composed cease to stain evenly, presenting involution
orms,

Dysentery

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Bacillary Dysentery 691

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 increas-
ing 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 indol, does
not ferment dextrose, lactose, saccharose, or other carbohydrates.
Acids are produced in moderate quantiteis after twenty-four hours
in dextrose media. Milk is not coagulated. Gelatin and blood-
serum are 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, which is removed by dialysis and
the residuum dried iz vacuo. Of the powder thus obtained 0.0025-
0.005 gr. is fatal, when intravenously administered toa rabbit. The
most interesting effect of the toxin is seen when rabbits are given
large enough doses to induce death in about twelve hours. In the
course of a few hours they develop diarrhea and when examined
postmortem are found to have, among other lesions, an enterocolitis
of varying severity, sometimes with the formation of a pseudomem-
brane upon the mucosa of the intestine. Small increasing doses of .
the toxin, administered in succession, produce immunity against its
effects, but the antibody thus formed is not antagonistic to the
living bacilli.

Vital Resistance.—The thermal death-point is 65°C. maintained:
for fifteen minutes. It grows slowly at ordinary temperatures
rapidly at the temperature of the body.

Pathogenesis.—Shiga and Flexner found that infection of young
cats and dogs could be effected by bacilli introduced into the stom-
ach, and that lesions suggestive of human dysentery were present im
the intestines. Kazarinow* found that when guinea-pigs and
young rabbits were narcotized with opium, the gastric contents
alkalinized with 10 cc. of a ro per-cent. NaOH solution, and a
’ quantity of Shiga bacilli introduced into the stomach with an

* “Archiv. f. Hyg.,” Bd. 1, Heft 1, p. 66; see also “Bull. de l’Inst. Pasteur,” 15
Aout, 1904, p. 634.

692 Dysentery

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 cc. of a virulent culture intro-
duced into the peritoneal cavity would cause fatal infection, to
produce infection by the mouth as above stated required the en-
tire 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 infection without
intestinal lesions.

Lesions.—The lesions found in human dysentery are usually
fairly destructive. They consist of a severe catarrhal and pseudo-
membranous colitis, which later passes into a stage of marked
ulceration. There is great thickening of the submiicosa and the
whole of the intestinal lining is corrugated. For the most part the
ulcerations are more superficial 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 agglutination was first care-
fully studied by Flexner, and is peculiar in that the serums pre-
pared 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 prepa-
ration. 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.

Some experience with the serum employed is necessary for identi-
fying supposed dysentery bacilli, or with the bacilli employed when
diagnosticating the supposed disease. Normal serums sometimes
agglutinate in dilutions as low as-1:10, hence dilutions as high as
1:20, 1:50 or even 1:100 should be compared.

Serum Therapy.—By the progressive immiunization of horses
to an immunizing fluid, the basis of which is a twenty-four-hour-
old agar-agar culture dried in vacuo, Shiga 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 Labor-
atory Hospital, 91 cases, with a death-rate of 8 per cent.; in 1899,
in the Hirowo Hospital, 110 cases, with a death-rate af 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,

Monilia Psilosis 693

furnishing a total of 1,136,096 cases, with 275,308 deaths (a total
mortality for the entire period of 24.23 per cent.).*

The serums prepared according to Shiga’s plan have since been
found to be specific in activity against the particular variety of
dysentery bacillus used in the immunization. Todd, Kraus and
Doerrt have shown that serum prepared from Shiga’s bacillus con-
tained antitoxin. Shiga and later Kruse found the serums to be
bacteriolytic. They are agglutinating according to the height of
immunity attained, 1 : 5000 being observed in some cases. They
also contain specific precipitins and opsonins. ,

Prophylaxis.—The prophylaxis of bacillary dysentery by the use
of killed cultures as vaccines has been attempted by Shigaft and by
Kruse.§ Monovalent, polyvalent and sensitized vaccines, have been
tried, but not upon a sufficient scale to enable a satisfactory judg-
ment to be formed.

SPRUE OR PSILOSIS
MoNILIA PSILOSIS (ASHFORD)

PARASACCHAROMYCES ASHFORDI (ANDERSON)

Sprue is an interesting form of catarrhal inflammation of the
mucous membrane of the whole or part of the alimentary canal. It
is common to most hot and moist climates, and chiefly affects
immigrants from the temperate zones. According to Manson it
“is characterized by irregularly alternating periods of exacerbation
and of comparative quiescence; by an inflamed, bare and eroded
condition of the mucous membrane of the tongue and mouth; by
flatulent dyspepsia, by pale, copious and generally loose, fermenting
stools; by wasting and anemia; and by a tendency to relapse. It
may occur as a primary disease or it may supervene on other affec-
tions of the bowels. It is very slow in its progress; and unless prop-
erly treated, tends to terminate in atrophy of the intestinal mucosa,
which sooner or later, proves fatal.”

It is variously known as sprue, “tropical diarrhea,” ‘*diarrhea
alba,” “aphthe tropica,” “Ceylon sore mouth,”. “psilosis lingue”’
“Cochin China diarrhea,”’ etc.

Ashford|| studied 379 cases and deduced the following from the
data collected:

1. Sprue is usually a mild disease with a veiled picture, a tendency to spon-
taneous cure and a usually ready submissiveness to a non-carbohydrate diet.

2. Tongue lesions are often clinically and histopathologically indistinguishable
from ordinary thrush, a disease, as a rule, due to Monilia albicans.

3. Clinically and histopathologically the picture of the tongue is projected on
through stomach and intestine.

* “Public Health Reports,’’ Jan. 5, 1900, vol. xv, No. r.

} “Zeitschrift fiir Hygiene,” 1902, XLI, 355.

t “Deutsche med. Wochenschrift,” 1901.

fener med. Wochenschrift,” 1903. : a

| American Journal of Tropical Diseases and Preventive Medicine,’
ti, No. 1, p..32.

1895,

604 . Sprue

4. Chronic intoxication supervenes after well-developed sprue and the liver
atrophies without cirrhotic changes, secondary anemia making its appearance.

5. The intestinal lesions produce large, acid, frothy, white stools with excessive
gas accumulation. The character of these stools does not warrant the belief that

serious ulcerative changes take place. . : : :
6. There is a tendency to chronicity and to periods of latency in which decided

betterment and apparent cure may take place. ; ;
7. Drugs are of little avail save when used symptomatically for definite critical

crisis and no specific has yet been found.
From the epidemological studies made, he comes to the following

conclusions:

1. Sprue is a disease of towns and cities where bread is a staple food.

2. Sprue is rare in the country districts where bread is not at least a daily food
and where often it is not eaten at all or only at long intervals.

3. Family endemics are noticeable.

4. There seems to pe a racial predisposition to sprue in persons of northern
birth.

In the light of these facts he recalled that Bahr* in his “ Researches
on Sprue”’ had given the following account:

““The whole of the intestinal canal was covered with a layer of ropy mucus,
the tongue with a film of thrush. The esophagus was coated with a yellowish
substance, resembling a diphtheritic membrane, composed almost entirely of
yeast fungi; microscopically complete desquamation of the epithelial covering of
the tongue and of the esophagus, and deep infiltration with yeast cells and
mycelial threads were found. In smears from the liver from one postmortem a
few yeast cells were seen and in preparations of the intestinal mucus, stained by ©
Gram’s method, from every part of the intestinal tube great numbers of these
cells and branching mycelium were found; in fact, they were by far the most abun-
dant organisms. Yeasts were grown in glucose broth from every part of the
intestinal canal, also in one case from the liver and spleen and from the kidneys
in the other.”’

Ashford, therefore, made cultures from the tongue, the stools or
both of 197 persons. Forty-nine were distinctly cases of sprue; and
in them he found Monilia psilosis (not Monilia (Oidium) albicans) of
an undescribed species in 100 per cent. Ninety-two were cases of
gastro-intestinal disturbances ranging from mere vagaries to serious
disease, chiefly accompanied by excessive gas production; 17 per cent.
were found to harbor the same organism. Sixty-six were apparently
normal as far as their intestinal tract was concerned, and but 3
per cent. harbored it.

Investigation of the Monilia showed it to be pathogenic for ani-
mals. Feeding it to them caused diarrhea and excess of intestinal
gas. Complement fixation tests showed positive in a few sprue
cases and negative in normal cases.

In his excellent studies of the “ Yeast-like Fungi of the Human In-
testinal Tract” Anderson} describes the yeast-like organism isolated
by Ashford, giving it the name Parasaccharomyces ashfordi. It is
perhaps too soon to declare this organism to be specific, but it seems
to be sufficiently well incriminated in the pathology of the disease
to justify a brief mention. Anderson describes it thus:

* “Transactions of the Society of Tropical Medicine and Hygiene,” 1914, I,

0. 5.
} “Jour. Inf. Diseases,” 1917, XxI, p. 380.

Balantidium Diarrhea 695

Morphology.—In young cultures the cells are round or slightly oval; in old cul-
tures cells are of many forms; oval, elongate, elliptical, round, or irregular; giant-
cells are common. Septate mycelium develops in gelatin hanging-drop and in
old cultures. Budding occurs from any point on the young cells, but usually near
the ends of articles in old cultures. Thesizeis 4.5 x 5 yu.

Cultural Characters —On glucose agar the streak is filiform, raised, glistening,
chalk-white and smooth; later the central portion may become rugose or ribbed;
the edge of the streak may remain entire or may become decidedly filamentous,
due to the outward growing hyphal elements under the surface of the medium.
There is a growth in gelatine stab, at first filiform, later it develops scattered,
bushy clusters of filaments. In liquid sugar medium, and beer-wort a very evi-
dent ring formation occurs; no pellicle is present. Giant colonies occur.

Physiological Properties —It ferments glucose, maltose and levulose; occasion-
ally sucrose and galactose arefermented. Yeast-water sugar mediums, with an
initial acidity of +1, become more alkaline. Litmus milk is rendered alkaline
in two weeks, but is not clotted. Gelatin is rarely liquefied.

This species strongly resembles the fungus variously called Oidium albicans,
Monilia albicans, and Endomyces albicans. Castellani has, however, reserved
the name Monilia albicans for a species which always clots milk and liquefies gela-
tine. Monilia albicans, Monilia psilosis, Oidium albicans and Endomyces
albicans are synonyms.

Specific Therapy. — Michel* has treated a number of cases of sprue
with a monilia vaccine and claims good results.

‘A
BALANTIDIUM DIARRHEA

BaLantipium Coir (MALMSTEN)

In certain rare cases a severe form of diarrhea, or a mild form of dysentery
appears to depend neither upon Entameeba histolytica nor Bacillus dysenteria,
but upon a protozoan parasite known as Balantidium coli. This organism was
first observed by Malmstenft 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
his original observation and up to 1908, Braun{ had been able to collect 142 cases
of human infection. In all of these cases the presence of the balantidium was
accompanied by obstinate diarrhea with bloody discharges (dysentery) in some,
and many of the cases ended in death.

Morphology.—The Balantidium coli is a ciliate protozoan micro-organism of
ovoid or ellipsoidal form, measuring from 30 to 200 » in length and from 20 to 70 #
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 infun-
dibuliform 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 opening in the ectosarc, the anus. The mouth
is the simple termination of the infundibuliform extension of 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
ab eis 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.

* “Jour. Inf. Diseases,” 1918, xxtt, No. 1, p. 53.
+ “Archiv f. pathologische Anatomie,” etc., x11, 1857, p. 302.
t “Tierische Parasiten des Menschen,” Wiirzburg, 1908.

696 Balantidium Coli

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.

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 satisfactorily studied whild
alive. To stain it a drop of the fluid containing the balantidia is spread upon a
slide and permitted to dry. Just before the moisture disappears from the film,
methy] 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-hema-
toxylon. The cilia usually do not show.

Reproduction.—This commonly takes place by karyokinesis, followed by trans-
verse division, and in cases of experimental! infection so rapidly that the organ-

_ Fig. 273.—Reproduction of Balantidium coli: 1-5, Asexual reproduction by
division; 6, encysted form of single individuals; 7, conjugation of-two individu-
als; 8, reproductive cyst; 9, cyst with peculiar contents whose further devel-
opment has not been followed (Brumpt). :

isms have not time to grow to the full size before dividing again. The result is
that many appear that are no more than 30 win length. In addition to multi- -'
plication by division, there is a sexual cycle of development with conjugation.
This was first pointed out by Gourvitsch,* studied by Leger and Duboscq,{ and
further confirmed by Brumpt.{ In the process of conjugation two individuals
come together, become attached lengthwise, 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
determined. :

Habitat.—The balantidium is unknown except as a parasite of thecolon. Itis
very common in hogs and has been found in the orang-outang, in certain lower
monkeys (Macacus cynomolgus), and in man.

" * “Russ. Archiv. f. Path. klin. Med. u. Bact. St. Petersb.,”’ 1896, quoted by
raun.

t “Archiv. de Zoé]. Exper.,”? 1904, 11, No. 4.

t ‘Compt.-rendu de la Soc. de Biol.,”’ July 10, r909.

Balantidium Diarrhea 697

Cultivation.—The organism quickly dies when transplanted 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
ulcerative. Some doubt has been expressed as to the exact réle 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 importance.

Animal Inoculation.— Experiments made by Casagrandi and Barbagallo,*
Klimenko, t and others upon kittens and pups have failed to produce the disease
even when the colon was already inflamed. Brumpt,t on the contrary, suc-
ceeded in reproducing it in monkeys and pigs by introducing the encysted organ-
isms into the already inflamed intestine via the anus.

Fig. 274.—Balantidium coli deeply situated in the interglandular tissue of the
intestinal mucosa (Brumpt).

Lesions.—In the majority of fatal cases postmortem examination of the colon
shows it to be in a state of catarrhal inflammation with numerous superficial
ulcerations with considerable surrounding infiltration of the mucosa. ‘Twenty-
four hours from the time of the death of the patient the balantidia are all dead.
Strong and Musgrave, § Solowiew,|| _Klimenko,** and others have shown that in
microscopic sections 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 beer reported by

* “Bal. coli,” etc., Catania, 1896, quoted by Braun.
t “Beitrige zur. path. Anat. u. allg. Path.,”’ 1903, XxxI1, 281.
{‘‘Précis de Parasitology,”’ 1910, 152.
§ “Bulletin of the Johns Hopkins Hospital,” 1901, XII, 31.
|| “‘Centralbl. f. Bakt.,” etc., 1 Abl., r90z, XxIx, 821, 840.
** Loc. cit.

698 Balantidium Coli

Manson, * and a case of abscess of the lung caused by the organism by Winogradow
and Stokvis. ft :

Transmission.—The transmission of the disease can only come about through
the encysted form of the parasites. Great numbers are passed in the feces of the
infected animals, but except the encysted forms all die very quickly as the fecal
matter dries. Unfortunately the further life-history of the encysted forms is
unknown.

CRAIGIOSIS
Craicia Hominis (CALKINS) t

Craigia hominis is an ameboid and flagellated intestinal protozoan parasite of
man, described in 1906 by Craig§ and recently carefully and elaborately studied
by Barlow.|| It is a minute organism and has an amebic stage during which it
reproduces by simple division like a typical ameba for several generations or
as long as conditions are favorable. It then encysts, and within the cysts numer-

Fig. 275.—Craigia hominis (Barlow, in American Journal of Tropical Diseases).

ous small bodies called ‘‘swarmers” develop and escape. Each of these has a
long single protoplasmic flagellum and is actively motile. The swarmers multiply
by longitudinal division for several generations after which the flagella disappear
and the amebic stage begins again.

In 56 cases of infection by this parasite studied by Barlow, diarrhea was the
most invariable symptom. Enterrhagia is less frequent and less severe in
craigiosis than in amebiasis. Of the 56 cases, 11 developed abscess of the liver,
one a pulmonary abscess, two appendicitis, one arthritis, two duodenal ulcer,
while others had more vague complications and sequel. It seems, from Barlow’s
studies, that the parasite deserves considerable attention. The discovery of the
parasite was made in the Philippine Islands, but Barlow’s cases were in Honduras.
One case has been reported in Texas, another in Tennessee.

Barlow recognizes two species, Craigia hominis and Craigia migrans.

HARMLESS FLAGELLATES OF THE HUMAN INTESTINES

In certain cases of diarrhea, flagellates—Trichomonas intestinalis, Cercomonas
intestinalis, and Lamblia (Megastomum) 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.

* “Tropical Diseases,”’ 1900, p. 394.

t “ Niederl. Tijdschr. v. Geneeskde.,’’ 1884, xx, No. 2, quoted by Braun.

{ Trans. xvth Internat. Congress of Hygiene and Demography, 1912, I, 287.

§ Amer. Jour. Med. Sciences, 1906, CXXXII,214.

|| The American Journal of Tropical Diseases, etc., 1915, II, 680.

CHAPTER XXX
TUBERCULOSIS

BacitLus TuBERCULOsIS (KocH)

Synonyms.—Bacterium tuberculosis; Mycobacterium tuberculosis.

General Characteristics.—A non-motile, non-flagellate, non-sporogenous, non-
liquefying, non-chromogenic, non-aérogenic, distinctly aérobic, acid-proof,
purely parasitic, highly pathogenic organism, staining by special methods and by
Gram’s method. Commonly occurring 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 destructive and, unfortunately,
one of the most common diseases. It is no respecter 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 fre-
quency 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 dis-
ease, but when caged and kept in zodlogic 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 some-
what modified form in birds, and it is said occasionally 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 im-
printed 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.

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
Kocht to demonstrate and isolate the Bacillus tuberculosis, the

* “Virchow’s Archives,” Bd. LXXXII, p. 397.
+ “Berliner klin. Wochenschrift,” 1882, 15.
699

FOO. Tuberculosis

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 except in the
bodies and discharges of animals affected with tuberculosis, and
in dusts of which these are component parts.

The widespread distribution of tuberculosis at one time sug-
gested that tubercle bacilli were ubiquitous in the atmosphere, that
we all inhaled them, and that it was only our vital resistance that
prevented us all Gam becoming its victims. Cornet,* however,
showed the bacilli to be present only in dusts with which pulverized
sputum was mixed, and to be most common where the greatest
uncleanliness prevailed.

Fig. 276.—Tubercle bacillus in sputum (Frankel and Pfeiffer).

Morphology.—The tubercle bacillus is a slender, rod-shaped
organism with slightly rounded ends and a slight curve. It meas-
ures from 1.5 to 3.5 w in length and from o.2 to 0.5 uw in breadth.
It commonly occurs in pairs, which may be associated end to end,
but generally overlap somewhat and are not attached to each
other, or in small groups in which most of the individuals have their
long axes in the same general direction, though one frequently
crosses the other at an angle. Organisms found in old pus and
sputum show a peculiar beaded appearance caused by fragmentation
of the protoplasm and the presence of metachromatic granules.

These were thought by Koch to be spores, but are irregular in
shape, have ragged surfaces, are without the high refraction peculiar
to spores, and do not resist heat.

The organism not infrequently presents projecting processes or

* Zeitschrift fiir Hygiene,” 1888, v, pp. 191-331.

Staining 701

true branches, a circumstance that has modified the present opinion
regarding its classification. It is probable that it has been errone-
- ously placed among the bacilli, and really belongs among the higher
bacteria.
The organism is not motile, does not possess flagella, and has
no spores. '
Staining.—The tubercle bacillus belongs to a group of organisms
which, because of their peculiar behavior toward stains, are known
as “sdurefest” or acid-proof. Young organisms may stain quite
easily with ordinary solution of anilin dyes, but 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).

Fig. 277.—Bacillus of tuberculosis, showing branched forms with involution
(Migula).

Koch* first stained the bacillus with a solution consisting of 1 cc.
of a concentrated solution of methylene blue mixed with 20 cc.
of distilled water, well shaken, and then, before using, receiving an
addition of 2 cc. of a 10 per cent. solution of caustic potash. Cover-
glasses were allowed to remain in this.for twenty-four hours and sub-
sequently counterstained with vesuvin. Ehrlich subsequently modi-
fied 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 by mineral acids which

* “Mittheilungen aus dem Kaiserlichen Gesundheitsamte,”’ 1884, 11.

702, _ Tuberculosis
characterizes the acid-fast or acid-proof (sdurefest) micro-organisms.
But it is not improbable that we have been led into error by the
assumption, upon inadequate grounds, that this is a constant and
uniform quality of the tubercle bacillus and similar micro-organisms.
The interesting observations of Much* have shown that many of
the paradoxes of tuberculosis can be accounted for by the fact that
during certain stages, or under certain conditions, the bacilli are not
acid-proof at all. Thus, caseous masses from the lungs of cattle
show complete 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 organ-
isms can be found. This shows that Gram’s method is really a
more useful method for demonstrating the bacillus than those in
which acids are employed. 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 con-
dition 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 ,t who claims to be able,
by appropriate methods of cultivation, to make many of the ordi.
nary saprophytic bacteria (Bacillus coli, B. subtilis, etc.) thor-
oughly acid-proof, must be mentioned.

In all cases where the detection of tubercle bacilli in pus or secre-
tions is a matter of clinical importance, it must be remembered that
the quantity of material examined by the staining method is ex-
tremely small, so that a few bacilli in a relatively large quantity of
matter can easily escape discovery.

As the purpose for which the staining is most frequently performed
is the differential diagnosis of the disease through the demonstra-
tion of the bacilli in sputum, the method by which this can be
accomplished will be first described.

» Staining the Bacillus in Sputum.—When the sputum is muco-
purulent and nummular, any portion of it may suffice for exami-
nation, 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.

* “Berliner klin. Wochenschrift, ” April 6, 1908, p
Tt “International Conference on Tuberculosis,” Philo delphia, 1907.

Staining 703

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 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 principally of caseous material from the
tuberculous tissue, and are the most valuable part of the sputum
for examination. 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 become opaque
in staining, but should the layer be spread, as is. often advised,

“as thin as possible,” there may be so few bacilli upon the glass
that they are found with difficulty.

The film is allowed to dry thoroughly, is passed three times
through the flame for fixation, and is then stained and examined.

Where examination by these 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, anti-
formin (g.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 sediment at the bottom of the tubes is then spread upon the
glasses and stained and will often reveal the bacilli which, having
been freed from the viscid materials in the sputum, are thrown
down in masses by the centrifuge.

The purpose of the staining being the discovery of the tubercle
bacillus, success is only possible when the method employed en-
ables that particular micro-organism to be recognized, as such,
so soon as itisseen. This can be accomplished by taking advantage
of the “acid-proof’’ quality of the micro-organism, which permits
it to take up the penetrating stains employed, but does not permit
it to let them go again in the bleaching agents, and assume the
counter stain. It is owing to this peculiarity that the tubercle
bacillus alone is colored blue by the Koch-Ehrlich method, and the
tubercle bacillus alone red by the Ziehl method, and it is because
no advantage is taken of the acid-proof peculiarity in using Gram’s
method, that the latter, which colors all micro-organisms stained,
the same blue-black color, and hence is not differential, is never
used for diagnostic purposes.

704 Tuberculosis

Ehrlich’s Method, or the Koch-Ehrlich M ethod.—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:

AMID oe 5 8 aagadisnd sade tara ee eid tata ae he 4
Saturated alcoholic solution of gentian violet............... 42
Water........ LUE CASED Yee ee EERE ReRO ER GES . 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. ;

This method, requiring twenty-four hours for its completion, is rarely used.

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, 1; 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 nowand then be -
added. The staining 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 hydro-
chloric acid in 70 per cent. alcohol, 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 examined or mounted in Canada balsam. Nothing will be colored ex-
cept the tubercle bacilli, which appear red. _

Gabbet’s Method—Gabbet modified the method by adding a little methylene-
blue to the acid solution, which he makes according to this formula:

Methylene: blueivem: s04 oad duu bcecivnn Go os baal adameeeecsn 2
SUIPHUTIC ACId ys esse casas nel Racutelnk Fe Evneeenean aya BS
Water nt os i iictcadey gaualanae be eine - 495

In Gabbet’s method, after staining with carbol-fuchsin, 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 examined or
finally mounted in Canada balsam. The tubercle bacilli are colored red; the
pus-corpuscles, epithelial cells, and unimportant bacteria, blue.

Pappenheim, * having found bacilli stained red by Ziehl’s method in the sputum
of a case which subsequent postmortem examination showed to be one of gan-
grene of the lung without tuberculosis, recommends the following as superior to
methods in which the mineral acids are employed:

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: naiiaat accede nine aeety aeekleteag eee aeiass. DoE
Absolute alcohol... 0... ... 0... cc cece cece eee ees I0o CC.
Methylene-blue.............0 0.00.00. cc cece esses ad Sat,
GLY COLIN se yee usta Gt iy Biectp sence bob wiz dteuii'a aa aye ae Gaiden 2 ees 20 CC.

* “Berl. klin. Wochenschrift,” 1898, No. 37, p. 809.

tid

Staining 705

5. Wash quickly in water.

6. Dry.

7. Mount.

The entire process takes about three minutes. The tubercle bacilli alone
remain red.

The possible relation that the number of bacilli in the expectora-
tion of consumptives might bear to the progress of the disease was
investigated by Nuttall.*

But a glance down the columns of figures in the original article
is sufficient to show that accident may cause wide variations in the
quality of the sputum and the number of bacilli it contains.

Staining the Bacillus in Urine.—The detection of tubercle bacilli
in the urine is sometimes easy, sometimes difficult. The centrifuge

Fig. 278.—Bacillus tuberculosis in sputum, stained with carbolic fuchsin and
aqueous methylene-blue. >< 1000 (Ohlmacher).

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 adda
little white of egg to secure good fixation of the urinary sediment
to the glass. The method of staining is the same as that for sputum
but as the smegma bacillus (q.v.) is apt to be present in the urine,
the precaution should be taken to use Pappenheim’s solution for
differentiation or to wash the stained film with absolute alcohol,
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.

* “Bull. of the Johns Hopkins Hospital,’ May and June, 1891, 11, 13.
45

4706 Tuberculosis

Staining the Bacillus in Sections of Tissue.—Ehrlich’s Method
for Sections —Ehrlich’s method must be recommended as the most
certain and best. The sections of tissue should be cemented to the
slide and then freed from the paraffin or other 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 absolute 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 become 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 colorless, and then removed to the slide by méans
of a section-lifter. The water is absorbed with filter-paper, and then the slide is
heated over a Bunsen burner until the section becomes 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 not adapted for differentia-
tion. They should not be neglected when no tubercle bacilli are demonstrable
by the other methods, as they are particularly well adapted to the demonstration
of such of the organisms as may not be acid-proof.

Isolation.—Piatkowski* has suggested that the cultivation of the
tubercle bacillus and other “acid-proof”’ organisms may be achieved
by taking advantage of their ability to resist the action of formal-
dehyd. The material containing the acid-proof organism is mixed
thoroughly with 10 cc. of water or bouillon, which receives an ad-
dition of 2 or 3 drops of 4o 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 formaldehyd. __

Still further improvement in the means by which the tubercle
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 Sjéo and Tornell, consists of Javelle water
to which sodium hydrate is added. To make it in the laboratory
one first makes the Javelle water as follows:

Cadi. A edanth sive esd wide hencehech sc tng TENA news Sea a 80
Water. . bes sees accitrokad oats va Nam eee

and after irene the salts add an equal volume of 15 per cent.
aqueous solution of caustic soda.

* “Deutsche med.’ Wochenschrift,”’ June 9, 1904, No. 23, p. 878.

Isolation 407

Uhlenhuth and Xylander* investigated its usefulness and recom-
mend it highly for assisting in manipulating the tubercle bacillus.
The sputum or tissue supposed to contain these organisms receives
an addition of antiformin, by which the tissue elements, the pus cells,
the mucus and other objectionable substances, and bacteria are
quickly dissolved, leaving the tubercle bacilli uninjured. It is
then centrifugalized, 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 sedi-
ment, rich in bacilli, may be immediately transferred to appropriate
culture-media, where the organisms frequently 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 first to inoculate a guinea-pig,
allow artificial tuberculosis to develop, and then make eltures 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 advised the following method,
with which he never experienced an unfavorable result.

With a sharp-pointed pair of scissors a snip about 14 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 1 cm., and
the scissors opened and closed so as to make a broad subcutaneous
pocket.- Into this pocket the needle of the hypodermic syringe
containing 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 instrument is with-
drawn, the mouth of the pocket is left open. A slight suppuration
usually occurs and carries out the organisms of wound infection,
while the tubercle bacilli are detained and carried to the inguinal
nodes, which usually enlarge 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 1 : 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 ab-
dominal 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 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

* “ Arbeiten a. d. kaiserlichen Gesundheilsamte,” 7G05: Xxx1, 158; ‘‘Centralbl.
{. Bakt. u. Parasitenk.,” Referata, 1910, XLV, 686.

708 Tuberculosis

and immediately dropped upon the surface of appropriate culture-
media in appropriate tubes: The fragments of tissue from the
spleen of the tuberculous guinea-pig are not crushed or comminuted,
but are simply laid upon the undisturbed surface of the culture
medium 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 almost immediately
begins from bacilli scattered over the surface as the bit of tissue was
moved. As the appropriate medium, blood-serum was recom-
mended by Koch; glycerin agar-agar,
by Roux and Nocard; glycerinized po-
tato, by Nocard; coagulated dogs’
blood-serum, by Smith, or coagulated
egg, by Dorset, may be mentioned.
The most certain results seem to follow
the employment of the dogs’ serum and
egg media.

Cultivation.—Blood-serum—Koch
first achieved artificial cultivation of the
tubercle bacillus upon blood-serum,
upon which the bacilli are first ap-
parent to the naked eye in about two
weeks, in the form of small, dry,
whitish flakes, not unlike fragments
of chalk. These slowly increase in size
at the edges, and gradually form small
scale-like masses, which under the
microscope are found to consist of
tangled masses of bacilli, many of
which are in,a condition of involution.
The medium is so ill adapted to the
Fig. 279.—Bacillus tuberculosis requirements of the tubercle bacillus

on “glycerin agar-agar, 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 discovery that
the addition of from 4 to 8 per cent. of glycerin to bouillon and agar-
agar made them suitable for the development of the bacillus, and
that a much more luxuriant development could be obtained upon
such media than upon blood-serum. The growth upon “glycerin
agar-agar” resembles that upon blood-serum. A critical study of
the relationship of massive development and glycerin was made
by Kimla, Poupé, and Vesley,t who found that the most luxuriant
growth occurred when the culture-media contained from 5 to 7 per
cent. of glycerin.

* “Ann. de l’Inst. Pasteur,” 1887, No. 1.
t ‘Revue de la Tuberculose,”’ 1898, v1, p. 25.

Cultivation

7°9

Dogs’ Blood-serum.—A very successful method of isolating the

tubercle bacillus has been published by Smith.*

A dog is bled from the femoral artery, the blood being caught in a sterile
flask, where it is allowed to coagulate. The serum is removed with a sterile

pipette, placed in sterile tubes, and coagulated at 75° to 76°C.
Reichel has found it advantageous to add to each 100 cc. of
the dogs’ serum 25 cc. of a mixture of glycerin 1 part, and dis-
tilled water 4 parts. The whole is then carefully shaken with-
out making a froth, and dispensed in tubes, 10 cc. 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 this medium was employed with thorough satis-
faction for the isolation of many different tubercle bacilli.
Smith prefers 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 tubulature,
to permit the air in the tubes to enter and exit during the con-
traction and expansion resulting from the heating incidental to
sterilization.

To the same end the ventilators of the incubator are closed,
and a large evaporating dish filled with water is stood inside,
so that the atmosphere may be constantly saturated with
moisture.

Egg Media—Dorset{ recommends an egg medium,
which has the advantage of being cheap and easily
prepared. 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 egg
makes them particularly adapted for the purpose.

The medium is prepared by carefully opening the egg and
droppping 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 mixture is then poured

into sterile tubes, about 10 cc. ineach, inclined in a blood-
serum sterilizer, and sterilized and coagulated at 70°C. on two

each day. The medium appears yellowish and is usually dry,
so that before using it is well to add a few drops of water.

Potato —Pawlowski{ was able to isolate the ba-
cillus upon potato. Sander found that it could be
readily grown upon various vegetable compounds,
especially upon acid potato mixed with glycerin.
Rosenau§ has shown that it can grow upon almost any
cooked and glycerinized vegetable tissue.

Fig. 280.—
Glass-capped
culture -tube
used by Theo-
bald Smith for
the isolation of
the _ tubercle
bacillus.

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

*“Transactions of the Association of American Physicians,’’ 1898, vol. xm,

p. 417.
+ “American Medicine,” 1902, vol. II, p. 555.
ee de l’Inst. Pasteur,” 1888, t. vz.

“Jour. Amer. Med. Assoc.,” 1902.

|| “Centralbl. £. Bakt. u. Parasitenk.,” I. Abl. Orig., 1910, LI, 553.

710 Tuberculosis

lung for the purpose. The tissues are first cooked in a steam ster-
ilizer, 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 tissue and keep it moist, and the

whole then sterilized in the autoclave.

The organisms are planted upon the tis-
sue, 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 aérobic grows
only at the surface, where a much
wrinkled, creamy white, brittle pellicle
forms. .

’ Non-albuminous Media.—Instead of re-
quiring 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 mix-
ture of commercial ammonium carbonate,
0.35 per cent.; primary potassium phos-
phate, 0.15 per cent.; magnesium sulphate,
0.25 per cent.; glycerin, 1.5 per cent.
Tuberculin was produced 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.

Appearance of the Cultures.—Irre-
spective of the media upon which they
are grown, cultures of the tubercle bacillus
present certain characteristics which serve
Fig. 281.—Bacillus tuber- to separate them from the majority of

culosis; glycerin agar-agar other organisms, though insufficient to
fou) ania months old enable one to identify them with certainty.
POEs The bacterial masses make their ap-
pearance very slowly. As a rule very little growth can be ob-
served at the end of a week, and sometimes a month must elapse
before the growth is distinct.

* “Zeitschrift fiir Hygiene,’ Aug. 10, 1894, xvi, No. 1.

Cultivation 711

They usually develop more rapidly upon fluid than solid media. '
The organism is purely aérobic, and the surface growth formed
upon liquids closely resembles that upon solids.

It 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 in-
stances be shining in spots 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

Fig. 282.—Bacillus tuberculosis; adhesion cover-glass preparation from a four-
teen-day-old blood-serum culture. X 100 (Frankel and Pfeiffer.)

to nine months) without transplantation, but may be transplanted
indefinitely.

Reaction.—The tubercle bacillus will grow upon otherwise ap-
propriate media whether the reaction be feebly acid or feebly alkaline.
The human bacillus has been shown by Theobald Smith* to produce
acid, the bovine bacillus to produce alkali in artificial cultures. .

Relation to Oxygen.—The tubercle bacillus is a strict aérobe and
grows only upon the surface of the culture-media.

Temperature Sensitivity—The bacillus is sensitive to tempera-
ture variations, not growing below 29°C. or above 42°C. Rosenaut
found that an exposure to 60°C. for twenty minutes destroyed 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.

* “Jour. Med. Research,” 1905, XIII, 253, 495.
t ‘Hygienic Laboratory,” Bulletin No. 24, Jan., 1908.

712 Tuberculosis

’ 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 lesions, but coming from a tuberculous woman, caused
fatal tuberculosis in guinea-pigs into which they were inoculated.
Baumgarten* has expressed the opinion that tubercle bacilli entering
the body of the child through the placenta, may remain dormant
for months or years, to begin an invasion at any time that the vital

Fig. 283.—Bacillus tuberculosis: a, Source, human; b, source, bovine. Mature
colonies on glycerin-agar. Actual size (Swithinbank and Newman).

resistance was sufficiently diminished to permit them to do so. It
seems unlikely, however, that transmission through the placenta
takes place sufficiently often to make this more than occasional.
The most frequent channel of infection is the respiratory tract,
into which the finely pulverized pulmonary discharges of consump-
tives and the dusts of infected rooms and streets enter. Fliigge,
Laschtschenko, Heyman-Sticher, and Benindet found that the
greatest danger of infection was from the atomized secretions, dis-
charged 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 con-
sumptives, these atoms of moisture are not infectious except when
there are open lesions in the lungs, etc. Experiments show that
they usually do not pass farther than o.5 meter from the patient,
though occasionally they may be driven 1.5 meters. A knowledge

* “Deutsche med. Wochenschrift,” 1882, No. 22. ;
{ ‘Zeitschrift fiir Hygiene,” etc., Bd. xxx, pp. 107, 125, 139, 163, 193-

Pathogenesis 713

of these facts teaches us that visits to consumptives should not be
prolonged; 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 consumptives should also be frequently washed
with a disinfecting solution.

Probably all of us at some time in our lives inhale living virulent
tubercle bacilli, yet not all suffer from tuberculosis. 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. Present evidence points to danger from
tubercle bacilli in the milk of cattle affected with tuberculosis.

The ingested bacilli may enter the tonsils and be carried to the
cervical lymph-glands, but seem more commonly to reach the in-
testine, from which they enter the lymphatics, sometimes to produce
lesions immediately beneath the mucous membrane, sometimes
to invade the more distant mesenteric lymphatic glands, but more
frequently to enter the thoracic duct and then through the venous
system find their way to the lungs. Passing this barrier they may
distribute through the arterial systemic circulation. The entrance
of tubercle bacilli into the systemic circulation with subsequent
deposition in the brain, bones, joints, etc., explains primary lesions
of these tissues.

Koch* believed 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 bovine bacilli to be equally
infectious for man. Behringt believes that nearly all children be-
come infected by ingesting tubercle bacilli in milk, though a certain
predisposition is necessary before the disease can develop.

Infection also occasionally takes place through the sexual appara-
tus. In sexual intercourse tubercle bacilli from tuberculous testicles
can enter the female organs, with resulting bacillary implantation.
Sexual infections are 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

an enatenal Congress on Tuberculosis,’’ London, 1901, and Washington,
190. 4
"| “Deutsche med. Wochenschrift,” 1903, No. 39.

714 Tuberculosis

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 retrogressive 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. Ex-
amination 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 ex-
amination usually shows a cluster of tubercles at the point of inocu-
lation, tuberculous enlargement of lymphatic glands both near and
remote from the primary lesion, and a widespread tuberculous in-
vasion of the lungs, liver, spleen, 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 disease, 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 alimen-
tary 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 common. The development of the lesions in what-
evet tissue or animal always depends upon the distribution of the
bacilli by the lymph or the blood.

The experiments of Koch, Prudden, and Hodenpyl,* and others
have shown that when dead tubercle bacilli are injected into the
subcutaneous tissues of rabbits, small local abscesses develop in the
course of a couple of weeks, showing 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 tuberculosis are caused. When the dead tubercle bacilli, instead
of being injected en masse into the areolar tissue, are introduced 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 organisms.

Baumgarten, whose researches were made upon the iris, found

* “New York Med. Jour.,” June 6-20, 1891.

Lesions 715

that the first irritation caused by the bacillus is followed by multi-
plication of the fixed connective-tissue cells of the part. The cells
increase in number by karyokinesis, and form a minute cellular
collection or primitive tubercle.

The group of epithelioid cells and lymphocytes constituting .the
primitive tubercle scarcely reaches visible proportions before central
coagulation-necrosis begins. The cytoplasm of the cells takes on a
hyaline character; the chromatin of the nuclei becomes dissolved in

Fig. 284.—Miliary tubercle of the testicle: a, Zone of epithelioid cells and
leucocytes; b, area of coagulation-necrosis; c, giant cell with its processes; per-
ipherally arranged nuclei and necrotic center; d, seminiferous tubule (Cameron, in
“International Text-book of Surgery’’).

the nuclear juice and gives a pale but homogeneous appearance to the
stained nuclei. As the tubercle grows, large protoplasmic masses—
giant cells—which contain many nuclei are formed. They some-
times occur near the center, more frequently near the periphery of
the lesion.

Giant cells are not always formed in tubercles, as the necrotic
changes are sometimes too rapid and widespread.
_ Tubercles are constantly avascular—.e., in them no new capillary
blood-vessels form—and the coagulation-necrosis soon destroys pre-
existing capillaries. Avascularity may be a factor in the necrosis
of the larger tuberculous masses, though probably playing no im-

716 Tuberculosis

portant 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.

Fig. 285.—Tuberculosis of the lung: the upper lobe shows advanced cheesy
consolidation with cavity-formation, bronchiectasis, and fibroid changes;
hae lowes lobe retains its spongy texture, but is occupied by numerous miliary
tubercles.

As almost all tissues contain a supporting connective-tissue frame-
work whose fibers are more resistant to necrosis than the cells, after
the cells of a tubercle have been destroyed, fibers may still be visible
among the granules, and give the tubercle a reticulated appearance.

‘Lesions 717

As a rule, tubercles progressively increase in size by the invasion
of fresh tissue. The tubercle bacilli are usually observed in greatest
number at the edges, among the healthy cells, where the nutrition
is good. From this position they are swept along by currents of
lymph or occasionally are picked up by leukocytes and transported
through the lymph-spaces, until the phagocyte falls a prey to its
prisoner, dies, and sows the seed of a new tubercle. It is by such
continuous invasion of new tissue, the formation of necrotic areas
in the lungs, and evacuation through the air-tubes that cavities
are formed. In pulmonary tuberculosis the process of destruction
is greatly accelerated by inspired saprophytic bacteria that live in
the necrotic tissue. The patient also suffers from secondary infec-
tions, especially by the streptococcus and pneumococcus.

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 cicatrize, and becomes surrounded by
a zone of newly formed contracting fibrillar tissue, by which it is
’ circumscribed and isolated. This constitutes recovery from tuber-
culosis. 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 infection.

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 “Variation 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 employing
cultures only for inoculation, and taking of each bacillary mass
exactly 5 mg. by weight, suspending it in 5 cc. of an indifferent fluid
until the density was uniform and the microscope showed no clumps,
and injecting into rabbits 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
induced varying degrees of tubercuolsis in animals; that the in-
jection of the same culture in different amounts produces different
results; that the extent and rapidity of development usually cor-
respond to the virulence of the culture; that doses of 1 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 produce any lesion in rabbits or guinea-pigs; that no mor-

*“Journal of Medical Research,” July, rgor, vol. vi, No. 1; N. S., vol. 1,
No. 1, p. 156.

718 Tuberculosis

phologic 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 viru-
lence; the bacilli in pulmonary tuberculosis with ulceration are
of feeble virulence, those of miliary tuberculosis of very great viru-
lence; that the so-called “healed tubercles” of the lung may con-
tain 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 tub-
erculosis of the bones may contain bacilli of high or low virulence,
and that variations in virulence among human tubercle bacilli.
may possibly sometimes depend, like many other qualities among
tubercle bacilli, on peculiarities inherited through serial transmis-
sions in other than human hosts.

Metabolism.—Tubercle bacilli require a plentiful supply of oxygen
and therefore grow only upon the surface of culture-media. They
produce no diastatic enzyme and give off no gas from cultures con-
taining carbohydrates. Carrier* and Wells and Cooper have shown
that they produce some lipase, and Kendall, Walker, and Dayt that
they produce some esterase.

They disintegrate protein with the production of amino-acids and
ammonia. In doing so no indol is formed. They do not produce
enzymes by which gelatin is softened, blood-serum digested or
milk coagulated or digested. ;

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 insoluble 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 Schweinitz|| showed that it was possible to extract from
the tubercle bacillus an acid closely resembling, if not identical with, .
teraconic acid. It melted at 161° to 164°C. and was soluble in ether,
water, and alcohol. He thought the necrotic changes caused by the
organism depended upon it..

Ruppel** believed that three different fatty substances were present

* “Compt.-rendu de la Soc. de Biol. de Paris,” 1901, LIT, 320.

t Jour. Infectious Diseases, 1912, x1, 388.

t ‘Jour. Infectious Diseases,” 1914, XV, 443.

§ “Centralbl. f. Bakt.,’”’ 1896, xx, p. 488. :

|| “Trans. Assoc. of Amer. Phys.,”’ 1897; “‘Centralbl. f. Bakt.,” etc., Sept. 15,
1897, Bd. xxu, p. 200.

* “Zeitschrift fiir physiol. Chemie,” 1899, xxvI.

Toxic Products 719

in the tubercle bacillus, making up from 8 to 26 per cent. by weight.
The first could be extracted with cold alcohol, the second with hot
alcohol, the third with ether. In addition to the fatty substance
Ruppel also found what he believed to be a protamin, and called
tuberculosamin. It seemed to be combined with nucleinic acid, and,
indeed, from it he isolated an acid for which he proposed the nam

tuberculinic acid.

Behring* found that this acid contained a histon-like body whose

removal left chemically pure tuberculinic acid. One gram of this
acid was capable of killing a 600-gram guinea-pig when administered
beneath the skin. One gram was fatal to 90,000 grams of guinea-
pig when introduced into the brain. If injected into tuberculous
guinea-pigs it was much more fatal, 1 gram destroying 60,000 when
injected subcutaneously and 40,000,000 when injected into the
brain.
- Levenet 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{ announced some observations
upon the toxic products of the tubercle bacillus and their relation
to the diagnosis and treatment of tuberculosis, which at once afoused
an enormous though transitory enthusiasm. The observations were,
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 enlarge-
ment of the lymphatic glands of the neighborhood disappear. In
about two weeks, however, there appears, at the point of inocula-
‘tion a slight induration, which develops into a hard nodule, ulcer-
ates, 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
ultimately slough away, leaving an ulcer which rapidly and per-
manently 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 also occurred 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

* “Berliner klin. Wochenschrift,” xxxv1.
t “Jour. of Med. Research,” 1, 190r.
t ‘Deutsche med. Wochenschrift,” 1891, No. 343.

720 Tuberculosis

extract of cultures of the tubercle bacillus—tuberculin—produced
the same effect as the dead cultures originally 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 al-
buminous 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 ani-
mals, its injection into them being followed either by a violent
febrile reaction or by death, according to the extent of the disease
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 surface-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 cultures 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 sometimes 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 cc. to 1 liter, are more convenient. The con-
tents 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. Superfi-
cial tuberculous lesions (lupus) sometimes ulcerate and slough away. The febrile
reaction is sufficiently characteristic to be of diagnostic value, though tuberculin
can aly be used with perfect safety asa diagnostic agent upon the lower
animals. ‘

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 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
zomg. It is soluble in water and glycerin and gives the protein 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 1 per cent. aqueous carbolic
acid solution that 1 cc. equals a dose. The dose of the concentrated tuberculin
is 0.4 to 0.5 cc.; that of the diluted tuberculin, x cc.

Tuberculin does not exert the slightest influence upon the tubercle
bacillus, but acts upon the tuberculous tissue, augmenting the
poisonous influence upon the cells surrounding the bacilli, destroy-
ing their vitality, and removing the conditions favorable to bacillary
growth, which for a time is checked. This action is accompanied

Toxic Products 721

by marked hyperemia of the perituberculous tissue, with tran-
sudation of serum, softening of the tuberculous mass, and absorp-
tionlinto 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

iin

Fig. 286.—Massive culture of the tubercle bacillus upon the surface of glycerin-
bouillon, used in the manufacture of tuberculin.

areas, could occasion a widespread metastatic invasion of the disease.
Old tuberculous 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 recep-
tion, 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 tuber-

722 Tuberculosis

culosis 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 characteristic reaction and a
positive diagnosis can be obtained without danger.

von Pirquett found that if a drop or two of Koch’s (old) tuberculin
is placed upon the skin of a tuberculous child, and a small scarifica-
tion made, through the drop, with a sterile lancet, a small papule
develops at the point of inoculation 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 re-
actions 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,” “cutaneous tubercuiin reaction” or
“cutaneous test.”

Detre,{ desiring to kill two birds with one stone, modified the von
Pirquet test by applying tuberculin made from cultures of human
bacilli to one arm of the patient, and tuberculin made from cultures
of bovine bacilli to the other. Accordingly as the reaction took
place upon one or the other arm he divined that the infection was
caused by the one or the other bacillus. The method has not
proved to be a satisfactory means of differentiation.

A modification of this method by Lign-:éres§ is called by him the
“ cutituberculin reaction.” Ligniéres 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 un-
diluted tuberculin, rubbing the reagent in with a pledget of cotton.
The reaction obtained is purely local and without fever.

Moro|| has modified the 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. This method is now known as
the percutaneous test.

Hiss** says that “it is more simple and equally efficient to massage
into the skin a drop of undiluted ‘old tuberculin.’”

* “Berliner klin. Wochenschrift,’”’ 1899, Dec. 18-25.
+ “Ibid., May 20, 1907.
t “Wiener klin. Wochenschrift, 1908,” No. 41.
F§ “Centralbl. f Bakt. u. Parasitenk.,” Orig., x~v1, Hft. 4, March 10, 1908, p.

73.
|| “‘Mitinch. med. Wochenschrift,” 1906, p. 216.
** “Text-book of Bacteriology,” 1901, p. 489.

Toxic Products 723

Calmette* suggested the “‘ophthalmo-tuberculin reaction,” which
consists of instilling 1 drop of a solution of prepared tuberculin into
the eye of the suspect. If no tuberculosis exists, no reaction follows,
but if the patient be infected with tuberculosis, the eye becomes red-
dened in a few hours and soon shows all of the appearances of a more
or less pronounced acute mucopurulent inflammation of the con-
junctiva. This attains its maximum in six or seven hours, and en-
tirely recovers in three days. It usually causes the patient very
little discomfort, but a number of patients have been unfortunate
enough to suffer from supervening corneal ulceration and other de-
structive 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 Calmette
is to precipitate the stuberculin with alcohol, dry the precipitate
and dissolve it in roo 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 reactions
seems to belong to Wolff-Eisner,t who was the first to point out
that the injection of all albuminous substances resulted in hyper-
sensitivity instead of immunity unless certain precautions were
observed. Upon this ground Levyt gives him credit as the founder of
the method. The reaction is undoubtedly an allergic phenomenon.

Klebs§ made strong claims for his own modifications of tuber-
culin, known as antiphthisin and tuberculocidin, but according
to the experimental studies of Trudeau and Baldwin, antiphthisin
is only much diluted tuberculin, and exerts no demonstrable in-
fluence upon the tubercle bacillus iz 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 “bouillon-filtrate” (bouillon filtré), of Denys|| is a porcelain
filtrate of bouillon culture of the tubercle bacillus and corresponds
to Koch’s original tuberculin before concentration, except in that
it has not been subjected to heat.

Tuberculin-R.—TR or tuberculin-R appears to be an important
addition to the immunology of tuberculosis, made by Koch.**
TR signifies “tuberkelbacillen resten” or bacillary fragments.
Pursuing the idea of fragmenting the bacilli, or treating them chemically to
increase their solubility, Koch found that a ro 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,

*“Ta Presse Médicale,” June 19, 1907.
t “Centralbl. f. Bakt. u. Parasitenk.,”’ 1904, Orig., XxxvI..
t “Verein fiir innere Medizin zu Berlin,” Dec. 16, 1907.
§ “Die Behandlung der Tuberculose mit Tuberculocidin,”’ 1892.
| “Acad. royale de med. de Belgique,” Feb. 22, 1902; abst. ‘‘Centralbl. f. Bakt.
u. Parasitenk.,”” Ref., 1902, XXXI, p. 563.
** “Deutsche med. Wochenschrift,” 1897, No. 14.

724 Tuberculosis

except that they were more brief in duration and more constant in result; but
the disadvantage of abscess formation following the injections remained. The
fluid, when filtered, possessed the properties of tuberculin. sees :

Mechanical fragmentation of bacilli had been employed by Klebs in his studies
of antiphthisin and tuberculocidin, 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 envelope of fatty acid, triturat-
ing only very small quantities of the bacteria at a time.

Having thus reduced the bacilli to fragments, he removed them from the mor-
tar, placed them in distilled water, washed them, and collected them by cen-
trifugation, as a muddy residuum at the bottom of an opalescent, clear fluid.
For convenience he named the clear fluid TO; the sediment, TR. TO was found
to contain tuberculin. In ordér 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 recentrifugated. 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. i

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. i

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
occur only 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 undesirable 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 injec-
tions are made with a hypodermic syringe into the tissues of the back. The
pesoning dose is }490 mg., rapidly increased to 20 mg., the injections being made

aily.

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 inocula-
tion with virulent bacilli. The point of inoculation presents no
change when the remedy is administered; 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:

. “Lhave, in general, got the impression in these experiments that full immuni-
zation sets in two or three weeks after the use of large doses. A cure in tubercu-

Toxic Products _ 725

lous 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 treatment 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 pa-
tients who suffer chiefly from secondary infection, especially with the streptococ-
cus, and in whom the septic process has put the tuberculosis entirely in the
background.”

One very serious objection, first urged against commercially pre-
pared 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 TR-tuberculin, and, like Trudeau, found living, virulent
bacilli in the preparation secured from Héchst. Many others have
since discovered 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:failure to crush
every bacillus could be overcome, Koch having specially depre-
cated such additions as producing destructive changes in the TR.
Until this possibility of danger can be removed, and our confidence
that attempts to cure patients may not result in their infection
’ be restored, it becomes a question whether TR can find a place in

human medicine, or must remain an interesting laboratory product..

Baumgarten and Walzt find that the administration of tuber-
culin-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, Koch§ 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 emulsion” Bazillenemulsion, be investigated for its im-
munizing and curative properties.

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{y or

‘disadvantages of any preparation, therefore, depend upon

the personal opinions of those employing them rather than upon
any demonstration regarding them—a very unscientific state of
knowledge.

* “Medical News,”’ Aug. 28, 1897.

t “Centralbl. f. Bakt.,”’ etc., July 5, 1902, xxx, No. 1, p. 28.

t“Centralbl. f. Bakt. und Parasitenk.,” April 12, 1898, xx, No. 14, p. 593.
§ “Deutsche med. Wochenschrift,”’ 1901, No. 48, p. 829.

726 Tuberculosis

The suggestion of A. E. Wright that the administration of all such
products should be controlled 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 “nega-
tive 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
expense of the examinations makes them impracticable.

Agglutination—Arloing* and Courmontt found it possible to
prepare homogenized cultures of the tubercle bacillus, and saw them
agglutinated by the serum of immunized animals and by the serum
of tuberculous patients. The subject was investigated by Koch,{
who carefully reviewed the details of technic and investigated the
method, which, he concluded, was valueless for the diagnosis of
human infection, though a good guide to the extent of immunization
achieved by the therapeutic administration of tuberculin-R. Thel-
ling§ has also shown the reaction to be too irregular to be of practical
diagnostic importance.

The technic of the agglutination test as given by Kochll is as
‘follows: ;

Any culture of the tubercle bacillus can be made useful by the following treat-
ment: 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
tub it in an agate mortar, adding, drop by drop, a 149 normal sodium hydroxid
ee until the proportion of 1 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 pipetted off, and rendered
feebly alkaline by adding diluted hydrochloric acid solution. The fluid thus
obtained is too concentrated 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 1 : 10, 1:25, 1 :50,1:75,1 :100
I: 200, I : 300, etc., and is to stand for twenty-four hours. By inclining the tube
a ene through a thin stratum of the fluid the agglutinations can be at once

etected.

Complement-fixation.—The complement-fixation test of Bordet
and Gengou was first applied to the study of the tubercle bacillus
by Wassermann and Bruch** and investigated by a long line of
clinicians and laboratory workers. It has, however, been abandoned

*“Congress de méd. int. Montpellier,’ 1898; ‘‘Compt.-rendu Acad. de
Sciences de Paris,’”’ 1898, T. CxxvI, pp. 1319-1321. ‘

t “Compt. rend. Soc. de Biol. de Paris,” 1898, No. 28, v; ‘Congr. pour
Vetude de la Tuberculose,”’ Paris, 1898.

¢ ‘Deutsche med. Wochenschrift,” 1901, No. 48, p. 829.

Loc. cit.
bPeutethe med. Wochenschrift, ” 1901, No. 48, p. 829.
** “Tyeutsch med. Wochenschrift,”’ 1906, p. 449.

Antitubercle Serums 727

because the amount of immune body in the blood of tuberculous
patients is generally too small to enable the test to be successfully
applied.

Antitubercle Serums.—Tizzoni and Centanni,* Bernheim,t
Paquin,{ Viquerat§ and others have experimented in various ways,
hoping that the principles of serum therapy might apply to tuber-
culosis. Nothing has, however, been achieved. Maragliano’s||
antitubercle serum has been used in a very large number of cases in
human medicine, but the glittering results reported by its author
have not been confirmed. Behring** comments upon it by saying
that “ Maragliano’s tubercle antitoxin contains no antitoxin.”

Babes and Proca,t} Mafucci and di Vestea,tt McFarland,§§ De
Schweinitz,|||| Fisch,*** and Patterson{{T{ have all endeavored to ob-
tain 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 experi-
ments, 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 collect the expectoration in a suitable receptacle,
that is sterilized 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 physician 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 disinfection
practised after scarlatina, diphtheria, and smallpox.

*“Centralbl. f. Bakt.,”” etc., 1892, Bd. x1, p. 82.
{ “Ibid., 1894, Bd. xv, p. 654.

t“New Vork Med. Record,” 1895.

§ “Zur Gewinnung von Antituberkulin, Centralbl. f. Bakt.,” etc., Nov. a
1896, xx, Nos. 18, 19, p. 674

| “Berliner klin. Wocheuacheift; ” 1895, No. 32.

** “Fortschritte der Med.,” 1897.

tt “La Med. Moderne,” 1896, p. 3
+ ot “Centralbl. f. Bakt.,” etc., Boe. Bd. xix, p. 208.

“Jour. Amer. Med. ’Assoc., "» Aug. 21, 1897.

lll “Centralbl. £. Bakt. und ’Parasitenk.,’ Sept. 15, 1897, Bd. xx, Nos. 8
and 9.
*** “Tour. Amer. Med. Assoc.,’’ Oct. 30, 1897.
ttt “Amer. Medico-Surg. Bull.,” Jan. 25, 1898.

728 Tuberculosis

Boards of health are now becoming more and more interested in
tuberculosis, and, though exceedingly slow and conservative in their
movements, are disseminating literature 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 registra-
tion of tuberculous cases as a means of collecting accurate data con-
cerning their origin; insist upon the careful domestic sterilization
and disinfection of all articles used by the patients; recommend pub-
lic disinfection 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
BacitLtus TUBERCULOSIS Bovis

The tuberculous diseases of the lower animals and especially cattle
have lesions closely resembling those of human tuberculosis, and
containing bacilli similar both in morphology and in staining reac-
tion to those found in human tuberculosis. The conclusion that
they are identical seems inevitable, but in his monograph upon
tuberculosis Koch called attention to certain morphologic and cul-
tural differences that obtain between bacilli obtained from human
and from animal tuberculosis. Unfortunately, very little attention
was paid to the subject until Theobald Smith* carefully 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 4). 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.

Staining.—The bovine bacillus usually stains homogeneously; 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.—Smith} observed that cultures of the two
organisms in glycerin bouillon differ in the induced reaction of the

* “Trans. Assoc. Amer. Phys.,” 1896, XI, p. 75, and 1898, xim, p. 417; “Jour.

of Experimental Medicine,” 1898, 111, 495.
{“ Trans. Assoc. Amer. Phys.,” 1903, vol. xvIII, p. 109. _

Bovine Tuberculosis 729

media. The cultures of the bovine bacillus tend toward alkalinity,
those of the human bacillus toward acidity.

Pathogenesis—(a) Guinea-pigs.—The bovine bacilli are more
virulent than those of human tuberculosis, intraperitoneal inocula-
tion of the former producing death in adult animals in from seven
to sixteen days; of the latter, in from ten to thirty-eight days. Sub-
cutaneous 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 imogulatad 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 inocu-
lated experimentally with the bovine bacillus die.

Lesions.—In general the lesions produced by the bovine bacillus
are 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
resembled those observed by Prudden and Hodenpyl* after the
intravenous injection of boiled, washed tubercle bacilli.

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 im-
portant 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,t
Ravenel, and Salmon§ contain evidence showing 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 veterinary surgeons who
became infected through wounds accidentally inflicted during the

*“New York Med. Jour.,’’ June 6-20, 1891.

t “Lyon Méd.,” Dec. 1, 1901.

t “Univ. of Pa. Bulletin,” x1v, p. 238, 1901; ‘‘Lancet,”’ Ave. 17 and 19, 1901;
“Medicine,” July and Aug., 1902, vol. viz1.

fe ‘Bull. No. 33, Bureau of Animal Industry,” U. S. Dept. of Agriculture, rgor.
|| ‘Phila. Med. Jour.,”’ July 21, 1900.

730 Tuberculosis

performance of necropsies upon tuberculous cattle. The tubercle
-bacilli were demonstrated 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 Kocht analyzed the cases usually selected 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 investiga-
tion and demonstration before it will be possible to express any posi-
tive 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 that of the Royal Commission on
Tuberculosis of Great Britain} The general tenor of this report
is contrary to Koch’s views, and many believed it settled the ques-
tion. Atthe International Congress on Tuberculosis in Washington,
1908, Koch reviewed the subject and stated his continued belief
in the principle he had enunciated 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 thesubject, 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 dis-

eases 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 collected dur-
ing seven years, showed how errors had crept into the investi-
gations, 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 supposed
modifiability of the tubercle bacillus in different environments.
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,
Moriyal|| seems to have shown that such changes are either purely

*“ Amer. Jour. Med. Sciences,” Aug., 1904, vol. cxxvi, No. 389, p. 216.
t Eleventh International Congress for Tuberculosis, Berlin, 1902.

t See the “British Medical Journal,” 1907 and 1908. °

§ “Jour. Amer Med. Assoc.,” Oct. 10, 1908, 11, No. 15, p. 1256.

|| ‘‘Centralbl. #. Bakt. y. Parasitenk.,” 1909, 1, Abt. Orig., LI, 460.

Bovine Tuberculosis 731

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 codperation of many laboratories
throughout different parts of the world. Unfortunately he died
before the results could be compiled, but much work had been done
and much support thereby given 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,
come to the following conclusions:

CoMBINED TABULATION CASES REPORTED AND Own SERIES OF CASES

PUN eis wic sues we ey oa emits 2 - 4 - 2 ~
‘Tuberculous adenitis, cervical...... 27 I 36 2 15 2i
Abdominal tuberculosis............ 14 4 8 o) 9 13
Generalized tuberculosis, alimentary

OTE eye vcore aeanse route smecmiies 6 I 2 3 13 12
Generalized tuberculosis............ 29 - 4 1 | 43 5
Generalized . tuberculosis including

meninges, alimentary origin...... - - I _ 3 8
Generalized tuberculosis including :

MENINGES: c5:coyee vraag ee eeews + s - 7 — 52 I
Tubercular meningitis.......... eee I - 3 - 27 4
Tuberculosis of bones and joints.....; 27 I 38 3 26 -
Genito-urinary tuberculosis......... 17 I 2 - - -
Tuberculosis of skin............--- 3 - I - I -
Miscellaneous cases:

Tuberculosis of tonsils........... = = = I - =

Tuberculosis of mouth and cervical

MOD ES i crscagenceddie crea tulyh diebaar nase - I - - - -
Tuberculous sinus or abscess...... 2 _ - - - -
Sepsis, latent bacilli............. = -_ = - I

Total Sica. nccavoadauinmn eto ee 777 Io | I17 36 | 215 65

Mixed or double infections, 4 cases.
° Total cases, 1224.

Conclusions —Bovine tuberculosis is practically a negligible
factor in adults. It very rarely causes pulmonary tuberculosis
or phthisis which causes the vast majority of deaths from tuber-
culosis in man, and is the type of disease responsible for the spread
of the virus from man to man.

* “Journal of Medical Research,”’ 1910, xxl, No. 2, p. 205; 1911, XXV, No. 2,
P. 313.

732 Tuberculosis

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 disfigurement.
Tt causes a large percentage of the rarer types of alimentary tuber-
culosis requiring operative interference 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
614 to ro 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, therefore, becomes imperative, and the utmost
efforts should be made to bring it ‘about. Several separate meas-
ures must be considered: , ;

i. 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 increasing information
regarding the use and abuse of tuberculin, etc.

2. Means by which infected animals shall be destroyed. Here
the municipal and state governments furnish inadequate 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 animals. In
many places this is being achieved with brilliant success by sepa-
ration of the herd, healthy and newly born animals constitut-
ing 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 bacteriotbxins made by
comminuting them.

Experiments of this kind have been conducted by McFadyen,*
on a large scale by von Behring, by Pearson and Gilliland,t Cal-
mette and Guérin,§ and by Theobald 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 reac-
tion caused by the injection of tuberculin into tuberculous animals
is an important adjunct to our means of diagnosticating the disease.

*“Jour. Comp. Path. and Therap.,” June, rgor.

{ “Beitriige zur experimentellen Therapie,” 1902, Hit. 5. :

i “Jour. of Comp. Med. Vet. Archiv.,’”” Nov., 1902, “Univ. of Penna. Med.
Bull.,” April, 1905.

§ “Ann. de l’Inst. Pasteur.,” Oct., 1905, May, 1906, and July, 1907; and
‘International Congress on Tuberculosis,’ Washington, 1908.

|| ‘Journal of Medical Research,” June, 1908, xvi, No. 3, p. 451.

Fowl Tuberculosis 733

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 normal diurnal and
nocturnal variations of temperature shall be known. The tuber-
culin is then administered by hypodermic injection into the shoulder
or flank, and the temperature 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
BacitLus TUBERCULOSIS AVIUM

The occasional spontaneous occurrence of tuberculosis in chickens,
parrots, ducks, and other birds, observed as early as 1868 by, Roloff*

Fig. 287——Bacillus tuberculosis avium.

and Paulicki,t 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 pe-
culiarities which make it a different variety, if not a separate species.
Cadio, Gilbert, and Roger succeeded in infecting fowls by feeding

* “Mag, f. d. ges Tierheilkunde,” 1868.

+ “Beitr. zur vergl. Anat.,” Berlin, 1872.

+‘ Giorn. anat. fisiol. e. path.,” Pisa, 1883.

§ “Zeitschrift fiir Hygiene,” Bd. x1. -
|| “La Semaine medicale,” 1890, p. 45

734 Tuberculosis

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 temperature of the birds with
antipyrin and lessening their vitality by starvation.

- Morphologic Peculiarities.—Morphologically, the organism 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 ordinary
agar-agar and bouillon prepared without glycerin.

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 ‘heraig
sensitivity and will grow at 42° to 45°C. quite as well as at 37°C.,
while the growth of the human and mammalian bacilli ceases at
42°C. Moreover, growth at 43°C. does not attenuate its virulence.

The thermal deathpoint is 70°C. Upon culture-media it is said to.

retain its virulence as long as two years.

Pathogenesis.—Birds are the most susceptible annie for ex-
perimental inoculation, the embryos and young being more sus-
ceptible 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 dis-
tribution 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 experiments 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.

*Centralbl. f. Bakt.,” etc., x11, 750.

Bacilli Resembling the Tubercle Bacillus 735

FISH TUBERCULOSIS

Dubarre and Terre* 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 the tubercle bacillus,
growing readily upon simple culture-media at 15° to 30°C., and not at 37°C.

Weber and Taube 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 morphology and staining peculiarities, have a common ancestry
and are descended from the same original stock. The most important of these
fal organisms are Bacillus lepre (q.v.), B. smegmatis, and Moeller’s grass

acillus.

BAcILLUS SMEGMATIS

Alvarez and Tavel,t Matterstock,§ Klemperer and Bittu,|| 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 axille, and the anus. They are also sometimes found in urine, and oc-
casionally in the saliva and sputum.

Morphology and Staining.—The organisms are of somewhat variable mor-
phology, but in general resemble the tubercle 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 declares the smegma
bacillus to be absolutely alcohol-proof as well as acid-proof, and admits no tinc-
torial difference between it and the tubercle bacillus. The bacillus, being about
the size and shape of the tubercle bacillus, is very readily mistaken for it, and its
presence in cases of suspected tuberculosis of the genito-urinary apparatus, and
in urine and other secretions in which it is likely to be present, may lead to con-
siderable 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.{t Doutrelepont and Matterstock cultivated it upon
coagulated hydrocele fluid, but were unable to transplant the growth successfully.

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 cc. 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 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 pur-
pose of investigating 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 remained
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

* “Compt. rendu de la Soc. de Biol. de Paris,” 1897, 446. ie
ee pa Arbeiten aus ‘—o eros ar aaa 1905.
“ Archiv de Physiol. norm. et Path.,’”’ 1885, No. 7.
fi ae ee as med. Klin. d. Univ. zu. Wiirzburg,” 1885, Bd. vi.
“Virchow’s Archives,” V, 103.
** “ Tournal of Experimental Medicine,” 1900-01, vol. v, p. 205.
tt “Miinchener med. Wochenschrift,” 1897.
{tf “Laboratory Work in Bacteriology,” 1899. -
§§ “Centralbl. f. Bakt. u. Parasitenk,” March 12, 1902 (Originale), Bd. xxx1,

(0. 7, p. 278.4

736 Tuberculosis

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. da

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 tempera-
ture the dry appearance of the growth was retained. The water of condensation
remained clear. ae :

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 coagulation. .

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 sapro-
phyte.

MOoELLER’s GRASS BACILLUS
BaAcILLus PHLEI

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.*
The organisms so closely resemble the tubercle bacillus that guinea-pig inocu-
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 instructive study of these organisms has been made by Abbott and Gilder-
sleeve,t 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 complete references to the
literature of the subject, come to the following conclusions:

1. That the majority of the acid-resisting bacteria may be distinguished from
true tubercle bacilli by their inability to resist decolorization 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 histologic structure, may nevertheless usually be
distinguished from them by the following peculiarities:

(a) When occurring as a result of intravenous inoculation, they are always
seén in the kidneys, only occasionally in the lungs, and practically not at all in
the other organs.

(b) They constitute a localized lesion, having no tendency to dissemination,
metastasis, or progressive destruction of tissue by caseation.

(c) They tend to terminate in suppuration or organization rather than in pro-
gressive caseation, as is the case with true tubercles.

(d) They are more commonly and conspicuously marked by the actinomyces
type of development of the organisms than is the case with true tubercles, and
these actinomycetes are less resistant to decolorization by strong acid solutions
than are those occasionally seen in tubercles.

3. That by subcutaneous, intravenous, and intrapulmonary inoculation 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 resulting 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 considered as accidental

contaminations from the surroundings, and not as evidence of disease in the

animals.

* “Deutsche med Zeitung,” 1898, p. 135; ‘Deutsche med. Wochenschrift,”
1898, p. 376, etc. ; /
“Univ. of Penna. Bulletin,’’ June, 1902.

Bacilli Resembling the Tubercle Bacillus 737

5. That the designation “bacillus” as applied to this group of bacteria and to
the exciter of tuberculosis is a misnomer; they are more correctly classified as
actinomyces.

THE BUTTER BACILLUS

Petri,* Rabinowitsch,t and Korn} 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 mor-
phologic 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.

Fig. 288.—Bacillus pseudotuberculosis from agar-agar. X 1000 °
(Itzerott and Niemann).

PSEUDOTUBERCULOSIS

BACILLUS PSEUDOTUBERCULOSIS

Pfeiffer,§ Malassez and Vignal,|| Eberth,** Chantemesse,{{ Charrin, and
Rogertt have all reported cases of so-called pseudotuberculosis occurring in
guinea-pigs, and characterized 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 disease
was reproduced. oo .

Morphology and Cultivation.—Bacillus pseudotuberculosis is characterized by
Pfeiffer as follows: The organisms are rod-shaped, the rods varying in length. (0.4
to 1.2 4) and sometimes united in chains. They may be almost round, and then
resemble diplococci. They stain by ordinary methods, but not by Gram’s

* “ Arbeiten aus dem kaiselichen Gesundheitsamte,” 1897.
+ “Zeitschrift fiir Hygiene,” etc., 1897.
t “Centralbl, i. Bakt.,” etc., 1899.
§ “Bacillire tuberculose, u. s. w.,” Leipzig, 1889.
|| “Archiv de Physiol. norm. et. Path.,” 1883 and 1884.
** “Virchow’s Archiv.,” Bd. ci.
tf Ann. de V’Inst. Pasteur,” 1887. | ; ;
tt “Compte-rendu de l’Acad. des Sci.,” Paris, t. CvI. F

738 Tuberculosis

method. They are motile and have flagella like the typhoid and colon bacilli.
They form 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 concen-
tric markings. The gelatin is not liquefied. The bacilli grow readily upon agar
and on potato, but without characteristic 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 XXXI
LEPROSY

Bacittus Lepra (HANSEN) *
Synonyms.—Bacterium lepre; Mycobacterium lepre.

General Characteristics——A non-motile, non-flagellate, non-sporogenous,
chromogenic, non-liquefying, non-aérogenic, distinctly aérobic, parasitic and
highly pathogenic, acid-resisting bacillus, staining by Gram’s method, and culti-
vable upon specially prepared artificial media. It does not form indol, or acidu-
late or coagulate milk

Leprosy very early received attention and study. Moses in-
cluded in the laws to the people of Israel rules for its diagnosis, 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 people. 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 malig-
nant 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 dis-
ease and exists much the same as it did several thousand years ago
in Palestine, Syria, Egypt, and the adjacent countries, and is com-
mon in China, Japan, and India. South Africa has many cases,
and Europe, especially Norway, Sweden, and parts of the Mediter-
ranean 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 re-
ported 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. :

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 frac-

*“Virchow’s Archives,” 1879. .
739°

740 . Leprosy

tures, giving it a beaded appearance, like the tubercle bacillus. It
occurs singly or in irregular groups. There is no characteristic
grouping and filaments are unknown. It is not motile and has no

flagella and no spores.
Staining.—It stains in very much the same way as the tubercle

bacillus, but permits of a more ready penetration of the stain, so .

that the ordinary aqueous solutions 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. 289.—Lepra bacilli. Smear from a lepra node stained with carbol-fuchsin
: (Kolle and Wassermann).

Cultivation—Many endeavors have been made to cultivate
this bacillus upon artificially prepared media, but in 1903 Hansen,*
who discovered the organism, declared that no one had yet culti-
vated it. 7

Bordoni-Uffreduzzit was able to cultivate a bacillus which par-
took of the staining peculiarities of the lepra bacillus as it appears
in the tissues, but differed in morphology.

Czaplewskit confirmed the work of Bordoni-Uffreduzzi, 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 consisting 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 intermittent method.

* Kolle and Wassermann’s ‘“‘Handbuch der pathogenen Mikroérganismen,”

XI, p. 184, 1903.
t “Zeitschrift f. Hygiene,” etc., 1884, III.
f“Centralbl. f. Bakt. und Parasitenk.,” Jan. 31, 1898, vol. xxim1, Nos. 3 and

4, P. 97-

Cultivation 741

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 aureus and the
bacillus of Friedlander, developed, and in addition a number of
colonies, composed of slender bacilli about the size and form of
the lepra bacillus.

These colonies were grayish yellow, humped in the middle, 1 to

Fig. 290.—Section of one of the nodules from the patient shown in Fig. 292,
stained by the Weigert-Gram method to show the lepra bacilli scattered through
the tissue and inclosed in the large vacuolated “‘lepra-cells.’’ Magnified 1000
diameters.

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 formed upon agar-agar were much like those
described by Bordoni-Uffreduzzi, and appeared as isolated, grayish,
rounded flakes, thicker in the center than at the edges, and char-
acterized by an irregular serrated border from which a fine irregular

742 Leprosy

network extended upon the medium. These projections consisted
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 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
Léffler mixture was also luxuriant. :

Upon agar-agar the growth was more meager; it was 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 extended to the water of condensation
to form a floating layer.

The bacillus developed well upon gelatin after it had grown arti-
ficially for a number of generations and become accustomed to a
saprophytic existence. Upon the surface of gelatin the growth was
in general, similar to that upon agar-agar. In puncture cultures
most of the growth occurred upon the surface to form a whitish,
grayish, or yellowish wrinkled layer. Below the surface of the
gelatin the growth occurred as a thick, granular column. The
medium was not liquefied.

Tn bouillon, growth occurred only at the bottom of the tube in the .

form of a powdery sediment.

Spronck* believed that he had successfully cultivated the organ-
ism upon glycerinized, neutralized potatoes, first seeing the growth
after the lapse of ten days. Cultures thus prepared were found to
be agglutinated by the blood-serum of lepra cases, and he recom-
mended the agglutination test for the diagnosis of obscure cases of
the disease.

Ducrey claimed to have cultivated the lepra bacillus in grape-
sugar, agar, and in bouillon im vacuo. His results need confirmation.

Rostf claimed to have isolated and cultivated the lepra bacillus
upon media free from sodium chlorid. The technic of his method
is thus described by Rudolph:t

‘Small lumps of pumice stone are washed and then dried in the sun, and then
allowed to absorb a mixture of 1 ounce of meat-extract and 2 ounces of water.
This pumice stone is then placed in wide-mouthed bottles and placed in the auto-
clave. Each bottle is provided with a stopper through which pass two tubes, the

* “Weekblad van het Nederlandsch Tijdschrift voor geneeskunde,” Deel u,
1898, No. 14; abstract ‘‘Centralbl. f. Bakt.,’? etc., 1899, XXV, DP. 257.

t “Brit. Med. Jour.,” Feb. 22, 1905, and “‘Indian Med. Gazette,”’ 1905.

t “Medicine,” March, 1905, p. 175. ;

Cultivation 743

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 passed 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.”

Clegg* 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 re-
search by Duval,t who, after confirming 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 grew, 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 some symbiotic organism,
such as the colon, typhoid, proteus, or other bacilli, added. Or
if the tissues were already contaminated 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 was as follows:

“Egg-albumin or human 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 1 mm. in thickness, which are dis-
tributed over the surface of the coagulated albumin. By means of a pipet the
medium thus seeded with bits of tissue is bathed in a 1 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 gray-
ish 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-

* “Philippine Journal of Science,” 1909, Iv, 403.
+ ‘Journal of Experimental Medicine,” 1910, XII, 649; 1911, XIII, 365.

744 Leprosy

trypsin medium. After the third or fourth generation the bacilli may be grown
without difficulty upon glycerinated serum agar prepared in the following
manner: d

“Twenty grams of agar, 3 gm. of sodium chlorid, 30 cc. of glycerin, and 500
cc. of distilled water are thoroughly mixed, clarified, and sterilized in the usual
way. To tubes containing ro cc. of this material is added in proper proportion 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 cc. of distilled water.
This is 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 cc. 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 collect 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 multiplication 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.

“Tn using an agar medium it is well to leave out the peptone 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
multiplication of the lepra ba¢illus.

“Bacillus leprz 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 lepre is very moist, and in this respect
unlike that of Bacillus tuberculosis, except possibly the avian strain. Sometimes
when the medium is devoid of water of condensation, the growth is dry and occa-
sionally 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.”

Although each of the workers upon leprosy has begun by asserting
that he had certainly cultivated the specific organism, a time comes
when a more extended acquaintance with the bacteriology of the
disease seems to cause him to doubt the results of his own work.
This is particularly true of this work of Duval, which was prosecuted
with enthusiasm, carried conviction with it, and then was partially
repudiated by its author, for in the discussion before the 17th Inter-
national Medical Congress in London in 1913, Duval is reported as
saying that “he knew less of the bacteriology of leprosy now than he
did some four years ago. He had made several mistakes, had

Pathogenesis 745

stated openly that he had cultivated the leprosy bacillus, but now
admitted frankly that he was mistaken.”

The interesting question that awaits settlement now seems to be, if
these bacilli, and specially the bacillus of Duval, are not Bacillus
lepre, what. are they? What relation do they bear to leprosy?

Pathogenesis.—Melcher and Ortmann* introduced fragments
_ of lepra nodules into the anterior chambers of the eyes of rabbits,
and observed the death of the animals aftec some months, with what

they considered to be typical leprous lesions of all the viscera,
_ especially the cecum; but the later careful experiments of Tashirof
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.

Nicollet 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 Sugail| found Japanese dancing mice susceptible
to infection with leprous material, the micro-organisms not remain-
ing localized at the seat of inoculation, but disseminating through-
out the animal’s body. Their observation has been confirmed by
Duval,** who later{{ was also able to infect: monkeys—Macacus
rliesus—with pure cultures of the organism and produce the typical
disease.

Very few instances are recorded in which actual inoculation has
produced leprosy in man. Arningtt was able to experiment upona
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 observa-
tions 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.

* “Berliner klin. Wochenschrift,’”’ 1885-1880.
t “Centralbl. f. Bakt. u. Parasitenk” (Originale), March 12, 1902, Xxx1, No.
71 P. 276.
t “Semaine medicale,” 1905, No. 39 P. I10.
§ Philippine Journal of Science,” 1909, IV, 403.
\| “Lepra,” 1909, VIII, 203.
+e “Journal of Experimental “Medicine,” IQIo, XH, 649.
if Tbid., 1911, XM, 374
it “Centralbl. f. Bakt.,” etc., 1889, VI, p. 201.
§§ “ Mittheilungen und Verhandlungen der internationalen wissenschaftlichen
Lepra-Konferenz zu aia Oct., 1897, 2, Theil.

740. Leprosy

2. The nasal lesion is peculiar—i. ey 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 tuberculous
lesions, but are superficial, affecting the skin and subcutaneous
tissues. Rarely they may also occur in the organs. Virchow*
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 con-
nective 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 grecorum) and lepra anesthetica, 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 re-
sembling new growths. The 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-sheaths.

Lepra nodules do not degenerate so readily as 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 éxternal
agencies upon the feebly vital pathologic tissue.

According to the studies of Johnston and Jamieson,} the scutes

logic 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. In such preparations the bacilli are pres-
ent in enormous numbers, forming a marked contrast to tuber-
culous skin diseases, in which they are very few.

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 anesthesia following the peripheral
nervous lesions predisposes to the formation of ulcers, etc., by allow-

*“Mittheilungen und Verhandlungen der internationalen wissenschaftlichen

Lepra-Konferenz zu Berlin,’”’ Oct., 1897, 2, Theil.
Montreal Med. Journal, ‘Jan. 1897.

jee pe ee

oe ee

Lesions 747

ing injuries to occur without detection and to progress without
observation. 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 the lymphatics and ex-
tends to the internal viscera. Death ultimately occurs from ex-
haustion, if not from the frequent intercurrent affections, especially
pneumonia and tuberculosis, to which the patients seem predisposed.

Specific Therapy.—Carrasquilla’s* “leprosy serum” was pre

Fig. 291.—Lepra anesthetica (McConnell).

pared by injecting the serum separated from blood withdrawn from
lepers, into horses, mules, and asses, and, after a number of in-
jections, bleeding the animals and separating the serum. There is
no reason for thinking that such a product could have therapeutic
value. In practice it proved worthless. ;

Rostt prepared massive cultures of the lepra bacillus, filtered
them through porcelain, concentrated the filtrate to one-tenth of its
volume, and mixed the filtrate with an equal.volume of glycerin.
The resulting preparation was called Jeprolin and was supposed to be

* “Wiener med. Wochenschrift,” No. 41, 1897.
ft “Brit. Med. Jour.,”’ Feb. 11, 1905.

748 ' Leprosy

analogous to tuberculin. With it he treated a number of lepers ©
at the Leper Hospital at Rangoon, Burmah, many of whom greatly
improved and some of whom seemed to be cured. Confirmation of
the work by others is greatly desired.

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.

= et ‘)

Fig. 292.—A case of lepra nodosa treated in the Medico-Chirurgical Hospital of
Philadelphia.

Rat LEPROSY

In 1903 Stefansky* reported the occurrence of a disease of rats that bore a
striking resemblance to lepra of man, and was caused by a very similar acid-fast
bacillus. Many others have since confirmed his observations. ‘The disease ap-
pears to be wide-spread among rats, its distribution seeming to bear no reference
to the presence or absence of human leprosy, so that no connection between the
epidemiology of the two can be traced. That the two depend upon similar '
micro-organisms is not only shown by the morphological and tinctorial resem-
blances between the two, but also by the fact that the serum of each will give
complement fixation reactions with the organisms from the other.

*Centralbl. f. Bakt. u. Parasitenk, 1903, Orig., XxxIm, p. 481.

CHAPTER XXXII
GLANDERS

Bacittus MALLer (LOFFLER AND ScHiTz)*

General Characteristics.—A non-motile, non-flagellate, non-sporogenous, non-
liquefying, non-chromogenic, non-aérogenic, aérobic and optionally anaérobic,
acid-forming and milk coagulating bacillus, pathogenic for man and the lower
animals, staining by ordinary methods, but not by Gram’s method.

GLANDERS, “Rotz” (German) or “‘morve” (French), is an infectious
mycotic disease which, fortunately, 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 suc-
cumbed 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
are formed by the breaking down of inflammatory nodules which
can be detected in all stages upon the diseased membranes. Hav-
ing once formed, they 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 sub-
maxillary lymphatic glands begin. to enlarge, soften, and ulcerate.
The lungs may also become infected by inspiration of the infectious
material from the nose and throat, and contain small foci of broncho-
pneumonia not unlike tubercles in their early appearance. The
animals ultimately die of exhaustion.

Specific Organism.—In 1882, shortly after the discovery of the
tubercle bacillus, Léffler and Schiitz discovered in the discharges
and tissues of the disease the specific micro-organism, the glanders
bacillus (Bacillus mallei).

Distribution—The glanders bacillus does not seem to find con-
ditions outside the animal body suitable for its growth, and prob-
ably 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 m, and is slightly bent.
Coccoid and branched forms sometimes occur. It usually occurs
_Singly, though upon blood-serum, and especially upon potato,

* “Deutsche med. Wochenschrift,” 1882, 52.
749

750 | Glanders

conjoined individuals may occasionally be found. Long threads
are never formed. |

When stained with ordinary aqueous solutions of the aniline
dyes, or with Léffler’s alkaline methylene-blue, the bacillary sub-
stance does not usually appear homogeneous, but, like that of the
diphtheria bacillus, shows marked inequalities, some areas 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 bacillus readily
gives up the stain in the presence of decolorizing agents, so is dif-

Fig. 293.—Bacillus mallei, from a culture upon glycerin agar-agar. XX 1000
: (Frinkel and Pfeiffer).

ficult to stain in tissues. Léffler accomplished the staining by
allowing the sections to lie for some time (five minutes) in the alka-
line methylene-blue solution, then transferring them to a solution of
sulphuric and oxalic acids:

Concentrated sulphuric acid...............0.00005 2 drops
Five per cent. oxalic acid solution................. 1 drop
Distilled ‘watetics «is caesceewoess ey saesesa pee ners Io cc.

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 car-
bolmethylene-blue.

VCO ROME e532 oss ayes Seated aoc Sepia ost Ach Bee ea Mtoe 10.0

Vital Resistance 751

Kiihne stains the section for about half an hour, washes it in water,
decolorizes it carefully in hydrochloric acid (zo drops to 500 cc. of
water), immerses it at once in a solution of lithium carbonate (8 drops
of a saturated solution of lithium carbonate in 10 cc. of water),
places it in a bath of distilled water for a few minutes, dips it into
absolute alcohol colored 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.—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 1
per cent. carbolic acid destroys it in about half an hour; 1: 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 to isolate the glanders bacillus from infec-
tious discharges, by the usual plate method, are apt to fail, on ac-
count of the presence of other more rapidly growing organisms.

A better method seems to be by infecting an animal and recov-
ering the bacillus from its tissues. For this purpose the guinea-
pig, being a highly susceptible as well as a readily procurable
animal, is appropriate. ;

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, .
however, it be killed and its testicles examined, the tunica vaginalis testis will be
found to contain pus, and sometimes to be partially obliterated by inflammatory
eco 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,t that a new bacillus, which he has classed
among the pseudo-tubercle bacilli, produces a similar testicular
swelling when injected into the abdominal cavity; also by Levy and
Steinmetz,{ who found that Staphylococcus pyogenes aureus was
also capable of provoking suppurative orchitis. However, the

* “Compt. rendu Acad. d. Sciences,”’ Paris, CVIH, 530.

} “Zeitschrift fiir Hygiene,’ Bd. xx1, Heft 1, Dec. 6, 1895.
t “Berliner klin. Wochenschrift,” March 18, 1895, No. 11.

752 Glanders

diagnosis is certain if a culture of the glanders bacillus be secured
from the pus in the scrotum.

For the diagnosis of the disease in living animals, subcutaneous
injections or ophthalmic instillations of mallein (g. 0.) are also em-
ployed.

McFadyen* 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 govern-
ment. Moore and Taylor, 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 1:500, but occurs in very few cases. The maxi-
mum agglutinative power of the serum of diseased horses not suffer-
ing 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 sometimes unexpectedly lose their
ability to agglutinate.

The diagnosis of glanders by means of the complement-fixation
method has been tried with success by Mohler and Eichhorn.{

Cultivation.—The bacillus is an aérobic and optionally anaérobic
organism, and can be grown in bouillon, upon agar-agar, better upon
glycerin agar-agar, very well upon blood-serum, and quite character
istically upon potato.

Temperature Reactions.—The optimum temperature is 37.5°C.
Growth,scarcely occurs at less than 25°C. and ceases entirely at
43°C. The thermal death-point is between 55° and 60°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 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 scanty moist, shining, viscid, grayish or slightly yellow-
ish layer, confined to the path of the inoculating wire.

Blood-serum.—Upon blood-serum the growth is rather character-
istic, the colonies along the line of inoculation appearing as cir-
cumscribed, clear, transparent drops, which later become confluent
and form a transparent layer unaccompanied by liquefaction.

* “Tour. Comp. Path. and Therap.,” 1896, p. 322.
{ “Jour. Infectious Diseases,’ 1907, Iv, p. 85, supplement.
t ‘Report of the Bureau of Animal taddatry, IgIo

Cultivation 753

Potato.—The most characteristic growth is upon alkaline 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 proteolytic ferments.

; Fig. 294.—Culture of glanders upon cooked potato (Léffler).

There is no exotoxin. All the poisonous substances seem to be
endotoxins. i “4
Mallein.—Babes,* Bonome,} Pearson, { and others have: prepared
asubstance, mallein, from cultures of the glanders bacillus, and have
employed it for diagnostic purposes. It seems to be useful in ‘veteri-
nary medicine, the reaction following its injection into glandered
animals being similar ‘to that caused by the injection of tuberculin
into tuberculous animals. The preparation of mallein is simple.
- Cultures of the glanders bacillus are grown for several weeks and
‘killed by heat. The culture is then filtered through porcelain, to
temove the dead bacteria, and evaporated 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 agent is employed
*“ 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.
¢ “Jour. of Comp. Med. and Vet. Archiv,” Phila., 1891, XII, pp. 411-415.

7154 . ‘Glanders

exactly like tuberculin, the dose for diagnostic purposes is 0.25 cc.
for the horse, the temperature being taken before and after its
hypodermic injection. A febrile reaction of more than 1.5°C. is
said to be indicative of the disease. If instilled into the eye of a
glandered horse it excites intense redness of the conjunctiva, quickly
followed by purulent discharge. Normal horses show only a slight
reddening of the conjunctiva.

Pathogenesis.—That the bacillus is the cause of glanders there is
no room to doubt, as Léffler and Schiitz have succeeded, by the
inoculation 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.

In field-mice the disease is rapidly fatal. 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 prelimina-
ries, tumbles over dead.

Infection may take place through the mucous membranes of the
nose, mouth, or alimentary tract, and apparently without preéxist-
ing demonstrable lesions.

The disease assumes either an acute form, characterized by de-
structive 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-organisms throughout the body,
with resulting more or less widespread nodular formations (farcy-
buds) in the skin. The acute form is quickly fatal, death some-
times 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 arefound -
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 leuko-
cytes, but also contain numerous epithelioid cells. As is the case
with tubercles, 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 ulcerations
-depend upon retrogressive changes occuring upon mucous surfaces,
the breaking down of the nodules permitting the softened material .
to escape. At times the lesions heal with. the formation of stellate
scars.

Baumgarten* regarded the histologic lesions of glanders as sitaeli
like those of the tubercle. He first saw epithelioid cells accumulate,
followed by the i invasion of leukocytes. Tedeschi was not able to

oe *'Pathdloviache, Mykologie,” Braunschweig, 1890.
‘ }.‘Zeigler’s Beitrage z. path. Anat.,” Bd. xm, 1893.

755

Lesions

Fig. 295.—Pustular eruption of acute glanders as exhibited on the day of the
patient’s death, twenty-eight days after initial chill (Zeit).

Fig. 296.—Lesions of glanders in the skin of a horse (Kitt)..

756 Glanders

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 Tede-
schi. 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 in-
fected. The disease has, however, occurred among those in frequent
contact with horses and among bacteriologists. It occurs either
in an acute form in which, from whatever primary focus may have

Fig. 297.—Farcy affecting the skin of the shoulder (Mohler and Eichhorn, in
Twenty-seventh Annual Report of the Bureau of Animal Industry, U. S. De-
partment of Agriculture, 1910). ;

been its starting-point, the distribution of micro-organisms may
be so rapid as to induce an affection with skin lesions resembling
smallpox and terminating fatally in eight or ten days.

The chronic form in man is chiefly confined to the nasal and laryn-
geal mucosa. It is commonly mistaken for more simple infections,
and though it sometimes shows its character by generalizing, it not
infrequently recovers.

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 im-
mune to the disease, may be made susceptible by feeding with
phloridzin and causing glycosuria.

} “Journal of Experimental Medicine,” vol. 1, No. 4, p. 577.

Glanders in Human Beings 757

Babes has asserted that the injection of mallein into susceptible
animals will immunize them against glanders. Some observers
claim to have seen good therapeutic results follow the repeated in-
jection of mallein in small doses. Others, as Chenot and Picq,*
find blood-serum from immune animals like the ox to be curative
when injected into guinea-pigs infected with glanders.

t pies sits

Fig. 298.—Lesions of glanders in the nasal septum of a horse (Mohler and
Eichhorn, in Twenty-seventh Annual Report of the Bureau of Animal Industry,
U. S. Department of Agriculture, 1910). ‘

Pseudo-glanders Bacillus.—Bacilli similar to the glanders bacillus
in tinctorial and cultural peculiarities, but not pathogenic for mice,
guinea-pigs, or rabbits, have been isolated by Babes, f and by Selter,{
and called the pseudo-glanders bacillus. oe

* “ Compte-rendu de la Soc. de Biol.,”” March 26, 1892.

+ “Archiv de med. exp. et d’anat. path.,” 1891.
t“Centralbl. f. Bakt.,” etc., Feb. 18, 1902, XXXV, 5, P- 529.

CHAPTER XXXIII
RHINOSCLEROMA

BaciILius RHINOSCLEROMATIS (von FRIScH*)

General Charactéristics.-—A non-motile, non-flagellate, non-sporogenous, non-
chromogenic, non-dérogenic, aérobic and optionally anaérobic, capsulated ba-
cillus, pathogenic for man and identical with Bacillus pneumoniz of Pee
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

Fig. 299.—Rhinoscleroma (Courtesy of Mr. Owen Richards, Cairo, Egypt).

Germany. A few cases have been observed in Egypt and a few
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 membrane and the adjoining skin of the nose, and
spreads to the skin in the immediate neighborhood by aslow invasion,
involving the upper lip, jaw, hard palate, and sometimes even the

* «Wiener med. Wochenschrift,”’ 1882, 32.
758

Bacillus Rhinoscleromatis 759

pharynx. The growths are without evidence of acute inflammation,
do not usually 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 hyaline degeneration.

In the nodes, von Frisch discovered bacilli closely resembling the
pneumobacillus of Friedlander, both in morphology and vegetation,
and, like it, surrounded by a capsule. The only differences between

Fig. 300.—Rhinoscleroma (Courtesy of Mr. Owen Richards, Cairo, Egypt).

the bacillus of rhinoscleroma and Bacillus pneumoniz 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 resemble
those of the bacillus of Friedlander (g.v.) so closely as to be almost
indistinguishable from it. The chief difference lies in its inability

to endure acid media and to ferment carbohydrates. Even when
inoculated into animals the bacillus behaves much like Friedlander’s
bacillus.

760 Rhinoscleroma

Inoculation has, so far, failed to reproduce the disease either in
man or in the lower animals. i

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

Fig. 301.—Bacillus rhinoscleromatis. Pure culture on glycerin agar-agar.
Magnified tooo diameters (Migila), 3

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 numbers. The older lesions consist of a
firm sclerotic cicatricial tissue.

CHAPTER XXXIV
SYPHILIS

TREPONEMA PALLIDUM (SCHAUDINN AND HoFFMANN)
Synonym.—Spirocheta pallida.

General Characteristics—A non-chromogenic, non-aérogenic, anaérobic,
minute, slender, closely coiled, flexible, motile,’ flagellated, non-sporogenous,
non-liquefying, spiral organism, cultivable upon specially prepared media, patho-
genic for man and certain of the lower animals, staining by certain methods only
and not by Gram’s method, ;

ALTHOUGH syphilis has been well known for centuries, its specific
cause has but recently been discovered. The supposition that the
disease could not be successfully communicated to any of the lower
animals was supposed to explain the delay, but has not proved
to be the case, for in spite of the discovery by Metchnikoff and
Roux* that chimpanzees could be successfully inoculated with virus
from a human lesion, the confirmation of their work by Lassart and
others, and the additional discovery by Metchnikoff and Roux,t
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. )

It has long been known that preputial smegma and various
ulcerative lesions of the generative organs contain spiral organisms.
Bordet and Gengou§ studied them with care, expecting to find that
they’ were concerned with the etiology of syphilis, but it was to
Schaudinn and Hoffmann|| that the discovery of the specific micro-
organism is to be credited. These investigators studied chancres,
syphilitic bubos and mucous patches, both by examination of matter
collected from the surfaces of the living tissue, and by sections of
excised tissue. The almost uniform result was the discovery of spiral
organisms of which two chief varieties were particularly studied.
One of these was very slender, uniformly and relatively tightly
coiled, and seen with difficulty because of its tenuity and because it
could scarcely be induced to stain by any method tried. This organ-
ism was found only in lesions from cases of syphilis, never in control
cases. Because of its pallor when stained they called it Spirocheta
pallida and because it was apparently constant in syphilis and absent

*« Ann. de l’Inst. Pasteur,’’ Dec., 1903, p. 809.
+ “Berliner klin. Wochenschrift,” 1903, p. 1189.
t “Annales de l’Inst. Pasteur,” Jan., 1904.

§ “Bulletin de l’acad. de med. Paris,” May 16, 1905.
|| “Deutsche med. Wochenschrift,” May 4, 1905.

761

762 Syphilis

from all other lesions, they looked upon it as the specific organism
of the disease. The other spiral organism being readily stained was
easy to find and was present in both syphilitic and non-syphilitic
cases. They called it Spirocheta refringens (q.v.) and looked upon
it as an accidentally present complicating organism.

The discovery was quickly confirmed by Metschinkoff* and Rouxt
who upon examining the secretions from the lesions of experi-
mental syphilis in apes and monkeys, always found the Spirdcheta
pallida, though they did not always find any other micro-organism.

A voluminous confirmatory literature quickly sprung up, and the
Spirocheta pallida became universally accepted as the cause of
syphilis.

For various reasons, chief among which are the relative rigidity
of this organism, as compared with the spirocheta, and the absence
of any vestige of an undulating membrane, the organism is now trans-
ferred by most writers from the genus Spirocheta to a new genus
Treponema. However, as Schaudinn and Hoffmann first placed it
among the spirocheta, no inconsiderable number of writers con-
tinue to adhere to the original nomenclature.

Morphology.—The organism is a slender, closely coiled spiral,
usually showing from eight to ten uniform undulations, but occa-
sionally being so short as to show only two or three, or so long as to
show as many as twenty. It is flexible, but does not bend itself.

It is very slender, measuring from 0.33 to 0.5 w in breadth to 3.5
to 15.5 w in length (Levaditi and McIntosh).

It forms no spores. Mulapheshin seems to ike place by longi-
tudinal division.

It is motile, and when ebeewal alive with a ne field illuminator,
can be seen to rotate slowly about its longitudinal axis at the same
time that it slowly sways from side to side.. The organism is, pro-
vided with a flagellum at one end, sometimes one at each end.

Noguchit observed two types of treponema, one slender, one
stouter. When carried through culture and used to inoculate rabbits
their differences were found to be fairly constant. The lesions pro-
duced in rabbit’s testicles varied with the variety of organism in-
oculated, one causing a diffuse, the other a nodular, orchitis. He
conjectured that the distinction may be of value in explaining certain
obscure points in human syphilis.

Staining.—I. Films.—The original discovery of the organism
was achieved through the employment of Giemsa’s stain—a modifi-
cation of the Romanowsky. method. But by this method the organ-
isms appeared very pale and not very numerous. Goldhorn§ im-
proved it as follows:

* “Bull. Acad. de med. de Paris,” May 16, 1905.
lane de l’Inst. Pasteur,’’ 1905, XIX, 673.

‘Journal of Experimental Medicine,” 1912, XV, No; 2, p. 201.
tra 1906, VIII, p. 451.

Staining 763

In 200 cc. of water, 2 grams of lithium carbonate are dissolved and 2 grams of
Merck’s medicinal, Griibler’s BX, or Koch’s rectified methylene 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 cottoninafunnel. 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 remaining half of the
solution'is added, so as to carry the reaction back to a low degree of alkalinity.. A
weak eosin solution is now prepared, approximately o.5 per cent. French eosin,
and this is added gradually while the mixture is being stirred until a filtered sam-

‘ ple 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 tem-
perature (heating spoils it). When completely dried it can easily be removed
from the paper and may then be dissolved without further washing in commercial

Fig. 302.—Treponema pallidum in the periosteum near an epiphysis (Bertarelli).

{not pure) wood alcohol. The solution should be allowed to stand a day, then
filtered. The strength of this alcoholic solution is approximately 1 percent. 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 mounting in balsam. The treponemas
appear violet in color. :

Ghoreyeb* recomniends 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 x per cent. aqueous solution of osmic acid, and per-

mit 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 1 : 100 dilution of Liquor plumbi subacetatis (freshly

* “Jour. Amer. Med. Assoc.,’’ May 7, 1910, Lv, No. 19, p. 1498.

764 Syphilis
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 oath ‘ulptiid
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 i in running water.

The whole process is to be repeated in exactly the same manner
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.

- Fig. 303.—Treponema pallidion impregnated with silver. Film prepared from
the skin of a macerated, congenitally syphilitic fetus. XX 750 diameters (Flex-
ner). The dense aggregation of organisms may indicate agglutination.

When serum from a primary sore or other syphilitic Jesion is.
treated by these methods, a number of the treponemas appear well
stained and a number very palely stained, so that one is in doubt
whether there may be many others unstained, and this seems to be
the case, for when similar smears are treated by other methods many
more can be found.

Stern* has applied the method of silver incrustation to, the ex-
amination of films 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 ina 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 brown-

* “Berliner klin. Wochenschrift,” 1907, No. I4.-

Staining 765

ish metallic in appearance, when they are thoroughly washed in water. The
treponemas appear black, the background brownish.

Burri* has recommended a simple and rapid method of demonstrat-
ing the treponema and other similar organisms by the use of Indiaink.

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.

IT. Section.—Staining the organism in the tissues is a more
difficult matter, for the Giemsa stain scarcely shows it at all. Bert-
arelli and Volpinof tried a modification of the van Ermengem method
for flagella with some success, but there was no real success until
Levaditif devised his methods of silver impregnation.

This consists in hardening pieces of tissue about 1 mm. in thickness in 10 per
cent: formol for twenty-four hours, rinsing in water, and immersing in.gs per cent.
alcohol for twenty-four hours. The block is then placed in diluted alcohol 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, 5 cc.;
distilled water, 100 cc., and kept in the dark, at room temperature, from twenty-
four to seventy-two hours, then 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-
Seas mixture, cleared in oil of bergamot, then in xylol, and mounted in Canada
balsam.

This method was later improved by Levaditi and Manouelian§
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 1 to 2 mm. in thick-
ness 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 impreg-
nated by immersion in a bath composed of a'1 per cent. solution of nitrate of
silver, to which, at the moment of employment, ro per cent. of pyridin is added.
Keep the blocks immersed in this solution at room temperature for two or three
hours, and at 50°C. for four or six hours, then wash rapidly in a 10 per cent. solu-
tion of pyridin, and reduce ina bath composed of 4 per cent. pyrogallic 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 toluidin blue, differentiated with glycerin-ether, and finally mounted in
Canada balsam. J :
* “Wiener klin. Wochenschrift,” July 1, 1909.
+ “Centralbl. f. Bakt. u. Parasitenk.,” Orig., 1905, XI, p. 56.
t “Compt.-rendu de la Soc. de Biol. de Paris,” 1905, LIX, p. 326.
§ ‘“Compt.-rendu de la Soc. de Biol. de Paris,’’ 1906, LVIM, p. 134. |

766 Syphilis

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 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 at-
_ tempted by Levaditi and McIntosh,* 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, how-
ever, 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 maintain it for generations
and study its morphology and behavior. During cultivation its
virulence was lost.

Schereschewsky{ endeavored to cultivate the treponema by
placing a fragment of human tissue, containing it, deep down into
gelatinized horse-serum. The treponema grew together with the
contaminating organism and no pure culture was secured. Muh-
lenst 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 Bruckner
and Galasesco** and Sowade,fft that an enormous multiplication
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
culture-medium used in these experiments was a “serum water,”
composed of 1 part of the serum of the sheep, horse, or rabbit
and 3 parts of distilled water; 16 cc. 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 eal of a series of such tubes a carefully removed fragment 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 anaérobiosis
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 solution for ten minutes. He then uses a

* “Ann. de l’Inst. Pasteur,” 1907, p. 784.
+ “Deutsche med. Wochenschrift,” 1909, Xxxv, 835, 1260, 1652.
Tt Ibid., 1909, xxxv, 1261.
§ “Zeitschrift fiir Hygiene und Infektionsk.,” 1911, LXVIII, 27.
Fl “Journal of Experimental Medicine,” 1911, xIv, 99. :
“Compt.-rendu de la Soc. de Biol. de Paris,” 1910, LXVIIT, 648.
tt ‘Deutsche med. Wochenschrift,” 1911, XxxvII, 682... <

Cultivation . 767

vacuum pump to exhaust the atmosphere in the jar, and lastly permits the alka-
aon (KOH) to flow down one of the tubes and mix with the pyrogallic

In these cultures the organism 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 serumi-agar-tissue
medium, but that the bacteria grow only in the stab or puncture,
whereas the treponemata grow out into the medium as a hazy
cloud. By cautiously breaking the tube and securing material for
transplantation from the scarcely visible cloud, the organisms may
be transplanted to new media and pure cultures obtained.

In a later paper, Noguchi* details the cultivation of the trep-
onema 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 1 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 addi-
tion 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 determine sterility.
The bits of human tissue are snipped up with sterile scissors in
salt solution containing 1 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. A bit of the tissue
should be emulsified in a mortar with citrate solution and exam-
ined 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 culti-
vation, each of the remaining bits of tissue is taken up by a thin
blunt glass rod and pushed to the bottom of a culture-tube and
into each tube several drops of the emulsion examined are intro-
duced by means of a capillary pipet, also inserted deeply into the
medium. ‘The tubes are next incubated 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 opal-
escence of the agar-agar caused by the extension into it of the grow-
ing treponemata. A capillary tube cautiously inserted into the opal-
escent medium withdraws a particle that can be examined with
the dark field illuminator. When such observation shows the cause
of the opalescence to be, in fact, the treponema, the tube can be

* “Journal of Experimental Medicine,” 1912, XV, I, p. 90.

768 Syphilis

cautiously broken at some appropriate part and the transplanta-
tion made from the opalescent part of the medium to fresh appro-
priate culture-media. By these means, after a few trials, pure
cultures of Treponema were secured.

The colonies were never sharp, but always faintly visible. There
is no color and no odor.

By inoculating the organisms recently secured from ere
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. aoe of the
organisms was not lost in the cultivation.

Zinsser, Hopkins and: Gilvert* found it possible to grow Tre-
ponema pallidum in massive cultures in fluid media. They em-
ployed a flask with a long slender neck like a “specific gravity flask.”
The flask was filled with slightly acid (0.2 to 0.8 per cent. acidity)
broth containing sheep-serum, ascitic fluid, horse-serum or rabbit-
‘serum, with an addition of autoclaved and hence thoroughly steril-
ized tissue (kidney, liver, brain, lung or heart muscle) and covered
with sterile neutral paraffine oil. The culture contains the greatest
number of organisms after three weeks. To collect them for mak-
ing luetin, etc., the fluid in the flasks was poured into tubes and
centrifugated for a short time to throw down scraps of the nutrient
tissue, the fluid then decanted and recentrifugated rapidly and for
a longer time to throw down the micro-organisms.

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 experi-
ment animals. As syphilis is, however, unknown under natural
conditions, except in man, it may be looked upon asa human disease.

The organism enters the body through a local breach of con-
‘tinuity of the superficial tissues, except in experimental and con-
‘genital infections, where it may immediately reach the blood.

In ordinary acquired syphilis the point of entrance shows the
first manifestations of the disease after a period of primary incuba-
tion about three weeks long, in what is known as the primary lesion
or chancre. This appears as a papule, grows larger, undergoes super-
ficial indolent ulceration, and eventually heals with the formation
of an indurated cicatrix. It is in the chancre 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

* “Journal of Experimental Medicine,” 1915, xx1, No. 3, p. 213.

Lesions 769

numbers in juice secured from a puncture 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 invasion suggests that those first enter-
ing are destroyed, and that it is only when their numbers are great
and their virulence increased that they suddenly become able to
overcome the defenses and permit the development of the secondary
symptoms. The 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., probably due to intoxication, and
local symptoms, such as alopecia, exanthemata, etc., due to local
colonization of the organisms. 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 mul-
.tiplication of the parasites, or there may be after many years a
‘period of tertiary symptoms characterized by the sudden appear-
ance of severe lesions in which the parasites are very few in number.

The specific organisms are present in juice expressed from the
primary lesion, in juice from the buboes and enlarged 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 the ovum, the
spermatozoén, or 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 demonstration 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 granulomata, 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 diag-

770 Syphilis

nosis 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 organisms 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 test-
ing the complement fixing power of the patient’s serum. This
method, now known as the “ Wassermann reaction” (g.v.) is given
elsewhere in complete detail.

The success of the von Pirquet cutaneous tuberculin reaction in
assisting the diagnosis of tuberculosis led to experiments on the
part of a number of investigators—Meirowsky, Wolff-Eisner,

Tedeschi, Nobe, Ciuffo, Nicholas, Favre, and Gauthier and Jodas- ,

sohn—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 Treponema
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 diagnosis. To this end he pre-
pared two cultures, one in ascitic fluid containing a piece of sterile
placenta, the other in ascitic fluid agar also containing a piece cf
placenta. After permitting them to grow under strictly anaérobic
conditions at 37°C. until luxuriant development occurred, the lower
part of the solid culture was carefully cut off, the tissue fragment
removed, and the rich culture carefully ground i in a sterile mortar,
the thick paste being diluted from time to time by adding a little
of the fluid culture. The grinding was continued until the emulsion
became perfectly clear, when it was heated to 60°C. for one hour upona
' water-bath and o.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 remained
sterile and inoculation into rabbits’ testicles was without result.

This extract of the treponema culture he called luetin. When it.

was applied to the ear of a normal rabbit, by means of an endermic
* “Journal of Experimental Medicine,” ror1, xu, p. 557.

Diagnosis 771

' injection with a fine needle, an erythema appeared, 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 redness developed into an induration the
size of a pea and persisted 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 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 1o 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 forpid, the erythema cleared
away and a negative result was supposed to have resulted, when
. suddenly the spots lighted up again and progressed to vesiculation
or pustulation. In 3 cases there were constitutional symptoms—
malaise, loss of appetite, and diarrhea. 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.

SPIROCHZETA 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.Donné 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 genitalia because of the
smegma upon which it customarily lives.

Pathogenesis—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 has also been found in the mouth and on the tonsils.
According to Hoffmann and Prowazek* it is not entirely harmless, 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.

Mor phology.—It is much broader than Treponema pallidum, its spiral waves
are much coarser and less regular.

' Staining. —It is easy to stain by all methods and is hence easily found. It
does not stain by Gram’s method. 5

Cultivation.—It has been cultivated by Noguchi,f in acetic fluid agar-agar in
which it grows but when a small fragment of sterile tissue is added. No mul-
tiplication of the organisms takes place except under anaérobic conditions. The
isolation of the organism in pure culture is not easy but can be effected by the
means employed for Treponema pallidum.

*“Centralbl. f. Bakt.,”’ etc., 1906, XLI.
{ ‘Journal of Experimental Medicine,” May 1, 1912, xv.

CHAPTER XXXV
: FRAMBESIA TROPICA (YAWS)

TREPONEMA PERTENUE (CASTELLANI)

Synonyms.—Treponema pallidulum; Spirocheta pallidula; Spirillum pertenue;
Spirocheta pertenuls.

Tuts peculiar, specific, infectious, contagious, chronic, febrile
disease of the tropics is characterized by the appearance upon the
skin of one or more primary papular lesions—the yaws—bearing
some resemblance to raspberries, and by subsequent 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 recovers 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 constitutional dis-
turbances occur only at the period preceding the development of
the eruptions and for a short time afterward. Little children
frequently die; older children and adults may die of exhaustion in
case extensive lesions with marked ulcerations 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 resemblances to syphilis; but the
peculiar raspberry-like character of the primary lesion, its dispo-
sition to occur upon the face, mouth, nose, eyes, neck, limbs, fingers,
and toes, as well as upon the genitals, seem to point in another di-
rection, 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 Malabar, Assam, Ceylon, Bur-
mah, 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 disease rarely makes it appearance in the United States, and

Specific Organism 773

it is of interest to know that Wood* has been able to collect nine
such cases from the literature.

Specific Organism.—The cause of the disease was unknown until
the discovery of Treponema pallidum, which opened a way for its
investigation. 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 Spirocheta pertenuis
and Spirocheta pallidula, but it is now recognized as a treponema
and is called Treponema pertenue.

Morphology.—The organism so closely resembles Treponema
pallidum that it is rather by knowing the source from which the
organism was derived than by any morphologic distinctions that

Fig. 304.—Yaws (photograph by P. B. Cousland, M. B., Swatow, China).

the two are separated. It is said to be a little shorter than T.
pallidum, measures 7 to 20 u 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 appropriate, both for
demonstrating it in smears from the lesions or in sections of tissue.

Cultivation.—The organism seems not yet to have been cultivated.

Pathogenesis.—Castellanit has succeeded in infecting monkeys
with the scrapings from yaws papules. The infection usually re-
sulted 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 re-

*“Brit. Med. Jour.,” 1905, I, 282, 1280, 1330.
+ “Jour. of Hygiene,” 1907, VII, p. 558.

774 Frambesia Tropica

moved, 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
successfully inoculated with yaws are afterward infected with syph-
ilitic 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* differ from Castellani
in this particular, and found that monkeys infected with syphilis
are refractory to yaws. Castellani was able, by means of com-
plement-fixation tests, to ‘detect different specific antibodies for
syphilis and yaws. MHalberstadtert has successfully infected
orang-outangs.

Human beings have been successfully inoculated with the disease,
the initial lesions appearing at the seat of introduction. How the
transmission naturally takes place is not known. Some think the .
micro- organisms may be carried from man to man by insects.

There is no doubt but that in their clinical manifestations and in
their etiology frambesia and syphilis are closely related. _

Diagnosis.—In addition to the clinical manifestations which are
usually quite sufficient for diagnosis, the discovery of the Treponema
pertenue is of assistance. It can usually be found without difficulty
by expressing the serum from a lesion and staining it by any of the
methods recommended for Treponema pallidum, the India-ink
method being the most simple.

. The Wassermann reaction is always positive in yaws, hence is of
no use for purposes of differential diagnosis. _

‘CHAPTER XXXVI
ACTINOMYCOSIS

AcTINoMycEs Bovis (BOLLINGER)

Synonyms.—Discomyces bovis: Streptothrix actinomyces: Streptothrix israeli:

Nocardia actinomyces: Odspora bovis: Nocardia bovis: Cladothrix actinomyces:
Bacterium actinocladothrix.

General Characteristics.—A parasitic, anaérobic, non-motile, non-flagellate,
non-sporogenous (?), branched micro-organism, belonging to the higher bacteria,

staining by ordinary methods and by Gram’s method, and pathogenic for
man and the lower animals. :

ACTINOMYCOSIS is a specific, infectious, granulomatous disease of:
man and the lower animals, characterized by a chronic course, and
by lesions that are partly purulent and partly productive.

The disease is fairly common among cattle, sheep and hogs, rare

in deer, horses, dogs and cats. One case has been observed in an
elephant. The disease is comparatively rare among men.

Fig. 305.—Bovine actinomycosis.

On account of its common occurrence about the face and mandibles,
it has long been known as “lumpy jaw” and “big head’ or “swelled
head.”” When it affects the tongue and interior of the mouth, itis
frequently spoken of as “ wooden tongue” or “ Holzzunge.”

The peculiar micro-organism by which it is caused was carefully
studied by a German botanist named Harz* and called by him
actinomyces, from the Greek, axris, a star and puxas a fungus.
From this botanical name the disease has become known as Actino-
mycosis. The French modify the technical name slightly —actino-
mycose, and the Germans translate it ‘Strahlenpitzkrankheit.”

Though the disease has long been known to agriculturists and
drovers it has only attracted the attention of the medical profession

*“Jahresberichte der kaiserlichen Central-Tierarzneischule in Miinchen,”
1877-1878, p. 125.

775

776 Actinomycosis

since 1847 when von Langenbeck observed a case of spinal caries in a
human being, in which he discovered “drusen” (as the parasitic
organismal masses are called), and identified as the same disease as
that occurring in the lumpy jaw of cattle. The “drusen” he
sketched and after examining them came to the conclusion that they
were masses of fungi.

Lebert* undoubtedly saw the same fungus masses but failed to
appreciate their nature. Rivolta,} Perroncitot and Hahn§ all rec-
ognized the bodies and regarded them as fungi, but it remained for
Bollinger|| to carefully describe them and point out their peculiar
radiating club-shaped formations.

J. Israel** studied the lesions of the disease and described it as a
new form of mycosis. Ponfickft proved the identity of the disease
described by Israe] with the well-known disease of cattle.

Important biological and cultural studies of the fungus were made
by Bostromft and by M. Wolff and J. Israel.$§

Following these came a long series of publications by many
authors in different countries, all generally confirmatory of the
main facts but filled with contradictions and paradoxes. Thus,
the parasitic fungus isolated by Bostrém grew easily and was an
aérobe of pathogenic properties, while those isolated by Wolff and
Israel were extremely difficult to cultivate, anaérobic for the most
part and irregularly pathogenic for animals. Most of the contribu-
tions favored the parasite of Bostrém.

It was not until J.H. Wright|||| made a critical study of the litera-
ture and supplemented it by a careful study of newly isolated organ-
isms from 13 cases of human and 2 cases of bovine actinomycosis,
that the discrepancies began to disappear. Even then there seemed
to be some doubt as to whether the results were entirely conclusive,
but no one has yet controverted Wright and it seems that the time
has come for a more widespread acceptance of his ideas. He be-
lieved that the error in the work of his predecessors depended upon
the circumstance that two separate and distinct organisms had been
isolated by Bostrém and his followers, and by Wolff and Israel and
himself.

Bostrém’s organism was a branched fungus of which all was prob-
ably true that was claimed for it, that is, it was isolated from cases

*Traité d’Anat. pathologique générale et specielle, Paris, 1857.

+ “Tl med. veter.,” 1868. ‘

t “Encyclop. agraria italiano, di Catani,” 1875.

§ “Jahresberichte der k. Central-Tierarzneischule in Miinchen,” 1877-81, 132.

|| “Deutsche Zeitschrift fiir Tiermedizin,” 1877, m1; and ‘“Centralblatt fiir
Medizinische Wiesenschaft,” 1877, xv.

** “Virchow’s Archiv.,” 1878, No. 74. :

tt “Breslauer drzl. Zeitschrift,” 1879, and 1885, p. 30.

tt “Berliner klin. Wochenschrift,” 1885; “Beitrige zur pathol. Anat. u. zur
allg. Path.,” 1890, rx, Heft 1.

§§ “Virchow’s Archives,” 1891, cxxv1; ‘Deutsche med. Wochenschrift,” 1890

and 1894; ‘‘Virchow’s Archives Bd.” CLI, p. 471.
\|ll “Jour. of Med. Research,” 1904, XIII, p. 340.

Morphology 777

of actinomycosis, was widely distributed in nature upon vegetation
generally and grains particularly, and had the cultural and biological
peculiarities ascribed to it, but was not the cause of actinomycosis,
but an accidentally and commonly present contaminating organism.

The true cause of the disease, the real actinomyces was the diffi-
cultly: cultivable organism of Wolff and Israel.

The organism of Bostrém is placed by Wright in the genus Nocar-
dia. A careful perusal of Wright’s paper will convince most readers
of the probable correctness of his views which are followed in the
succeeding text.

Fig. 306.—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). :

Distribution——The actinomyces is known only as a parasitic
organism associated with actinomycosis.

Morphology.—When an actinomyces grain or ray-fungus is ex-
amined in a section of tissue it is found to consist of several distinct
zones composed of differentelements. Thecenter consists of a granu-
lar mass containing numerous bodies resembling micrococci or spores.
Extending from this center into the neighboring tissue is a
branched, tangled mass of mycelial threads. In an outer zone these
threads are seen to terminate in conspicuous, club-shaped, radi-
ating forms which give the colonies their rosette-like appearance.

778 Actinomycosis

The clubs are commonly so inconspicuous in the lesions of the hu-
man form of the disease as to lead some to suppose that the parasite
is of a different species.
When clumps formed in artificial cultivations of the parasite
‘are properly crushed, spread out, and stained, the long mycelial
threads, 0.3—0.5 yw in thickness, occasionally show flask- or bottle-
like expansions—the clubs—at the ends. These probably depend |
upon gelatinization of the cell-membrane of the degenerating para-
site. 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 and closely packed together,
making the whole mass form a rounded little body often spoken of
as an “actinomyces grain.” When tissues are stained first with -

Fig. 307.—Actinomyces granule crushed beneath a cover-glass, showing radial
striations in the hyaline masses. Preparation not stained; low magnifying
power (Wright and Brown).

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.

The fungus is of sufficient size to be detected in pus, etc., by the
naked eye. As it usually has a bright yellow color it is not in-
frequently spoken of as a “sulphur grain.”

Cultivation—The actinomyces fungus may be grown upon arti-
ficial culture media, as was first shown by Wolff and Israel, and
later by Wright. The method of isolating given by the latter, is
as follows:

“The granules, preferably obtained from closed lesions, are first
thoroughly washed in sterile water or bouillon and then crushed and
disintegrated between two sterile slides. If one is working with a

Cultivation 779
bovine case it is well to examine microscopically the disintegrated
material, after mixing it with a drop or two of bouillon under a cover-
glass, to see if filamentous masses are present. If they are not, or
if they are very few, proceed no further, but begin again with another
granule, because the granules in bovine lesions sometimes contain
no living filaments at all, but may be composed entirely of de-
generated structures from which no growth of micro-organisms can
be generated. If filaments and filamentous masses are found to be
present in the granule, then the disintegrated products of the gran-
ule are to be transferred by means of the platinum loop to melted
1 per cent. dextrose agar-agar contained in test-tubes filled to a
depth of 7 or 8 centimeters which have been cooled to about 40°C.
The material is to be thoroughly distributed throughout the melted
agar-agar by means of the loop, and the tube then placed in the in-
cubator. Several tubes should be prepared. At the same time a
number of granules, after washing in sterile water or bouillon, should
be placed on the sides of sterile test-tubes plugged with cotton and
kept at room temperature in the dark. The sugar-agar tubes in-
oculated as above described should be examined from day to day for
the presence of the characteristic colonies in the depths of the agar-
agar. If very many colonies of contaminating bacteria have devel-
oped in the tubes, it will probably be very difficult or impossible to
isolate the specific micro-organism. If there are few or no contami-
nating colonies, then the colonies of the specific organism should be
expected to develop in the course of two or three days to a week.
If a'good number of living filaments of the micro-organism have
been distributed throughout the agar, the specific colonies that
develop will be very numerous in the depths of the agar, especially
throughout a shallow zone situated about 5 to 12 mm. below the
surface of the agar-agar. When the presence of the characteristic
colonies has been determined, slices or pieces of the agar containing
colonies are to be cut out of the tube by means of a stiff plati-
num wire with a flattened and bent extremity. A piece of the agar-
agar is to be placed upon a clean slide and covered with a clean
cover-glass. It is to be examined under a low power of the micro-
scope, and an isolated colony selected for transplantation. By
obvious manipulations, under continuous control of microscopic
observation, the selected colony, together with a small amount of
the surrounding agar-agar is to be cut out, care being taken to
be sure that no other colony is present in the small piece of agar-
agar containing the colony. The small piece of agar-agar thus cut
out should not have a greatest dimension of more than2 mm. The
piece of agar-agar is then transferred from the slide by means of a
platinum loop to a tube of sterile bouillon where it is thoroughly
shaken up to free it from any adherent bacteria. If there be any
reason to believe that the small piece of agar has been very much
contaminated with bacteria, it should be washed in a second tube of

Actinomycosis

780

Fig. 308.—Colony of actinomyces with well-developed ‘‘clubs’’ at the periph-

ery in a nodule in the peritoneal cavity of a guinea-pig inoculated with a. cul-
ture from another guinea-pig. Paraffin section. Low magnification (Wright)

(Photograph by Mr. L. S. Brown).

Fig. 309.—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 developed and show some indications
of stratification. Paraffin section. XX 750 approx. (Wright) (Photograph by

Mr. L. S. Brown).

Cultivation 781

bouillon, then the piece of agar-agar is to be transferred by means of
the platinum loop to a tube of melted sugar-agar cooled to 40°C.

Fig. 310.—Actinomycosis: Surface colonies oh agar and on coagulated Léffler’s
blood serum (J. H. Wright; photograph by L. S. Brown).

It should be immersed deeply in the agar and the tube placed in the
incubator. If the colony thus transferred to the agar-agar is
capable of growth, in the course of some days it will have formed

Fig. 311.—Actinomycosis: Gross appearances of sugar agar suspension cultures
(J. H. Wright; photograph by L. S. Brown).

a good-sized colony from which transplants in various culture-
media may be made.”

782 Actinomycosis

The characteristic rosettes so constantly found in the tissues are
never seen in artificial cultures.

Agar-agar.—The best medium for cultivation seems to be agar-
agar containing 1 per cent. of dextrose or glucose. The trans-
plantations should be made deeply and the material inoculated
distributed. The culture should be kept at incubation temperature.
Colonies appear scattered through the media in the course of a few
days, very few appearing close to the surface, and those a’short
distance below being very small in size. The colonies will be found
most numerous in a zone about 5-10 mm. below the surface, where
they may meet with a gentle stimulation by small amounts of oxygen
absorbed from the air. Lower down, the colonies, though less num-
erous, grow much larger and form irregularly spherical or nodular
opaque areas, composed of branching filaments radiating from the
center. The filaments show true branching and tend after a time to
break up into segments and form a compact mass.

When a puncture-culture is made in glucose agar, the organism
grows as an anaérobe in the line of inoculation, but never upon
or near the surface.

Under anaérobic conditions the agar spread culture gave poor
success.

Bouillon.—The growth takes place only at the bottom of the tube
in the form of solid whitish masses commonly of nodular integer
character. The bouillon remains clear.

Potato.—Potato cultures made under anaérobic conditions give
poor results.

Peptone Solution.—Furnished poor cultures.

Milk and Litmus Milk.—There are apparently poor media for
the cultivation of actinomyces.

Eggs.—Wright tried ten strains of his organisms in eggs and ege
media but found very little growth. In one case there were me
filaments that appeared to be degenerated.

Staining —The organism stains easily and retains. the stain ‘in
Gram’s method. It is not acid-fast.

Metabolism.—Actinomyces grows only under sialon conditions.

It does not ferment sugar, and does not evolve gas.

Temperature.—The cultures grow only at 37°C. Wright found
the organism killed after fifteen minutes at 60°C.

Pathogenesis.—There is little evidence that the cultivated organ-
isms are pathogenic for laboratory animals. Wolff and Israel fre-
quently found nodular masses with communicating suppurating
sinuses containing the fungi, in the peritoneal cavities of inoculated
animals, but it is doubtful whether the organisms had multiplied
after inoculation or whether they would long have remained alive.
In the abdominal cavities of experimentally inoculated rabbits the
peritoneum, mesentery and omentum may show typical nodules
containing the actinomyces rays in cases of successful inoculation,

Mode of Infection 783

but there is little evidence that the introduced micro-organisms have
multiplied.

Mode of Infection—The manner by which the organism enters
the body is not known. There are some who believe that the organ-
ism occurs in nature as a saprophyte, or as an epiphyte upon the hulls
of certain grains, especially barley. Woodhead has reported a case

Fig. 312.—Section of liver from a case of actinomycosis in man
(Crookshank). ; :

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

784 Actinomycosis

the lower animals. The fungi may enter the organism through
the mouth and pharynx, through the respiratory tract, toroueh 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.

In some way, the organisms sometimes enter the lung and cause
a suppurative bronchopneumonia with adhesive inflammation of
the contiguous pleura. After the formation of the pleuritic'adhesions
the disease may 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.—The lesions of actinomycosis vary under circumstances
not well understood. They fall into the groups of granulomas, but
how they shall comport themselves has some reference to the tissue
in which they occur. A primitive lesion may be described as con-
sisting of the ray-fungus at the center and a reaction zone round
aboutit. In close approximation to the fungus it is not uncommon to
find a number of foreign-body giant-cells in a zone of lymphocytes,
plasma cells, and endothelial cells. Beyond these are endothelial
cells and fibroblasts. As the lesions enlarge the cellular collections
die at the center while growing at the periphery.

The disease in the tongue eventuates in dense indurations, at the
centers of which dead and calcified actinomyces or small collections
of pus or necrotic matter can be found.

In the maxillary bones, the cell collections result in absorption
and redeposition of the bone in a rarefied form, and instead of the
induration seen in the tongue, necrosis and suppuration are apt to
eventuate in the formation of sinuses through which the pus and acti-
nomyces are evacuated. These sinuses commonly become seconda-
rily infected by a miscellany of bacteria from the surface and further
suppuration, necrosis and destruction quickly follow. It is such
secondarily admitted organisms that make the isolation of the
actinomyces difficult, and that so commonly lead to the appearance,
in the culture, of the aérobic easily cultivated Nocardia, confused
by so many investigators with the true cause of the disease.

As the complicating infection and suppuration leads to the en-
larging sinuses and fistule, the actinomyces are liberated and escape
.in the pus. They are large enough to be seen by the naked eye

.* Centralbl. f. Bakt. u. Parasitenk., xvi, p. 7.

Lesions 785

and have a bright yellow color. The discovery of these “ driisen,”’
“grains,” “sulphur grains” or “yellow granules” is sufficient to
base a suspicion of the disease upon. The only confirmation re-
quired is a microscopic examination of a granule crushed between
glasses, by which the radiating filaments with their clavate expansions
may be found.

CHAPTER XXXVII-
MYCETOMA, OR MADURA-FOOT

A curious disease of not infrequent occurrence in the Indian prov--

ince 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 disease 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
almost 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 com-
mon in men than in women, individuals between twenty and forty
years of age suffering most frequently, though persons of any age
may suffer from the disease. It is insidious in onset, no symptoms
being observed 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, thick-
ened, indurated, and adherent. The ball of the great toe and the
pads of the fingers and toes are the points most frequently invaded.
The lesions progress 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 mate-
rial, occasioning the formation of ulcers and sinuses. The matter
discharged from the lesions at this stage of the disease is a thinserum,
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 detec-
tion of these particles that the diagnosis rests. According 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 enlargement of the
affected part. The malady is usually painless.

The micro-organismal nature of the disease was early 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 Surveyor found that the black

particles of the melanoid variety consisted of a large branching

septate fungus.
786

The Pale or Ochroid Variety 787

THE PALE OR OCHROID VARIETY

ACTINOMYCES Mapur& (VINCENT)

Synonyms.—Streptothrix madure; Dyscomyces madure.

General Characteristics. A non-motile, non-flagellate, sporogenous (?),
non-liquefying, non-aérogenic, chromogenic, aérobic and optionally anaérobic,
branched, parasitic organism belonging to the higher bacteria, staining by ordi-
nary methods and by Gram’s method, and pathogenic for man.

Kanthack* was the first to show the presence of fungus threads
in microscopical sections of the tissues. He considered them to be

ids

Fig. 313——Mycetoma. Dorsum of foot showing sinuses, some of which are
covered by hard brownish crusts (courtesy of Dr. John W. Perkins). |

a form of streptothrix, probably closely related to the well-known
actinomyces. Vincent} also studied lesions of the ochroid variety of
the disease and found a streptothrix. Later Vincent and Gérnyt
isolated this streptothrix from the lesions of a second case of the
disease. Hewlett§ studied the same organism and found it like the

* Trans. Path. Soc.. London, 1892.

t ‘Ann. de l’Inst. Pasteur,’’ 1894, vIII, 30.
t Ann. de dermat. et syph., Paris,” 1896, v1.
§ Trans. Path. Soc., London, 1893.

788 Mycetoma, or Madura-foot

actinomyces in structure. Boyce and Surveyor,* after studying
eighteen cases of the disease, concluded that the fungi present pre-
sented many of the characteristics of the fungus actinomyces.
Boycet later cultivated a streptothrix, from a case of Madura foot
that differed somewhat from fungi previously cultivated. Adami

and Kirkpatrick} believe that the fungus observed by them was

identical with the actinomyces.

It seems probable, therefore, that Madura disease is a form of
actinomycosis, caused by a parasite
best called Actinomyces madura.
It differs from Actinomyces bovis
in the greater ease with which it
is cultivated—if, indeed the organ-
isms cultivated are the same as
those observed in the sections.

Morphology.—Under the micro-
scope the organism which belongs
to the higher bacteria, is found to
consist of long, branched threads
forming a tangled mass. The
peripheral filaments radiate from

like those of Actinomyces. In
some cases it is said that no clubs
were observed, in other cases that
they were longer and more slender
than in Actinomyces.
Staining.—The organism stains
easily and holds the stain well
after Gram’s method. It is not
Fig. 314.—Actinomyces mad- aeld-ias t F :
ure an Sy Cee on of diseased tis- Cultivation.—Vincent succeeded
sue (Vincent). in isolating the specific micro-
organism by puncturing one of the
nodes with a sterile pipette, 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 Actinomyces. They cling to the
glass, 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.

* Trans. Royal Soc., London, 1894.
t+ “ Hygienische Rundschau,” 1894, Iv, p. 529.
{ Montreal Med. Jour., Jan., 1896.

the center, and form clubs much >

Lesions 789

Gelatin.—The growth in gelatin is not very abundant, and forms
dense, slightly reddish, rounded clumps. Sometimes 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 be-
come rose-colored or bright red. The majority of the clusters re-

Fig. 315.—Melanoid form of mycetoma. Section showing black granules
and general features of the lesions as they appear under a lo w-magnifying power.
Zeiss dz (James H. Wright).

Fig. 316.—Melanoid form of mycetoma, showing structure and appearance
of the hyphz of the mycelium obtained from the granules. Zeiss apochromat;
4mm. (James H. Wright).. :

main 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 it becomes dull white or
downy from the formation of aerial hyphe. The colonies are very
adherent to the surface of the medium, and are of almost cartilagi-
nous consistence.

790 Mycetoma, or Madura-foot

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 forma-
tion of spores or of aerial hyphe.

Lesions.—Microscopic study of the diseased tissues in myce-
toma is not without interest. The healthy tissue is sharply sepa-
rated from the diseased areas, which appear like large degenerated
tubercles, except that they are extremely vascular. The mycelial
or filamentous mass occupies 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 the pur-
pose. The tissue surrounding the nodes is infiltrated with small
round cells. The youngest nodules consist of granulation-tissue,
whose development is checked by early coagulation-necrosis. Giant-
cells are few. ;

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. These
may partly explain the occurrence of considerable iron in granular
detritus about the fungi.

The general tendency is for the older lesions to heal with the for-
mation of much connective-tissue induration, as new lesions form on.
the outskirts.

B. THE MELANOID VARIETY

ASPERGILLUS BOUFFARDI?

General Characteristics.—A pathogenic hyphomycete composed of branching
septate hyphe 3-8y in diameter. It is non-motile, non-flagellate, non-sporogen-
ous, liquefying (?) aérobic or optionally anaérobic, and is pathogenic for man.

This form of mycetoma depends upon an entirely different micro-
organism from that causing the ochroid form of the disease, and
properly classed among the hyphomycetes. It was probably first
seen by Carter.* Bristowe,t Hogg,t Barsini,§ Kanthack,|] Boyce
and Surveyor** and Wrightff have all observed and studied it.

Wright found it in the diseased tissues, taken from the pads of a
toe of a patient who came for treatment in the Massachusetts Gen-
eral Hospital. It occurred in the form of black granules that were
embedded in the tissue and appeared mulberry-like and less than
I mm. in diameter. They were firm, and when enucleated and

* “On Mycetoma or the Fungus Disease of India,” London, 1874.
} Trans. Path. Soc., London, 1871.

{ Trans. Path. Soc., Lond., 1872.

§ Arch. per le scienza mediche, 1888, x11, 309.

| Jour. Path. and Bact., 1893, 1, 140.

i" Trans. Royal Soc., London, 1894.
Tt Jour. Exp. Med., 1898, 111, 421.

The Melanoid Form 791

pressed between cover and slide did not crush. Only after digestion
with a solution of caustic potash and careful teasing could the gran-
ules be resolved into the hyphe of the mold. The central part of the -
granule formed a reticulum, with radiating, somewhat clavate ele-
ments projecting from it.

In sections of tissue it was found possible to stain the fungus with

Fig. 317.—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. 318. —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).

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

792 Mycetoma, or Madura-foot

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 sup-
ports drops of brownish fluid. The long branched hyphe thus
formed were from 3 to 8 uw in diameter, with transverse septa in the
younger ones. The older hyphe 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.

Pathogenesis.—No results followed the introduction of the gran-
ules from the original lesions, or of the cultures made from them,
into experiment animals.

Lesions.—These are not essentially different from those of the
other form of the disease and consist of granulation tissue, more or
less atypical in structure, with numerous small foci of suppuration.
The granules lie at the center and are surrounded by giant and epi-
thelioid cells with many lymphocytes and plasma cells. The occur-
rence of suppuration causes this structure to be disrupted. -

3 CHAPTER XXXVIII
BLASTOMYCOSIS
Biastomyces DERMATITIDIS (GILCHRIST AND STOKES)

THE first case in which yeasts or blastomycetes were definitely
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. Post-
mortem examination showed numbers of broken-down nodular for-
mations 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 introduced in pure culture into animals—mice and
rats—proved infective for them. He called the organism Saccharo-
myces hominis, and the affection in which it was found “Saccharo-
mycosis hominis.”

In May, 1904, three months before the appearance of Busse’s
paper, Gilchrist exhibited to the American Dermatological Associa-
tion 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, Gilchristt 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,f Rabinowitsch,§
and others, the chief and almost the sole form in which these infec-
tions make their appearance is a dermal infection known as “blasto-
mycetic 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 and begins to
heal, but at the periphery new and usually minute foci of suppura-
tion occur, so that while the original lesion tends to heal very slowly,
with much cicatricial formation, it is always spreading. The prog-
ress is usually slow, and Gilchrist’s first case spread only 2 inches
in four years.

Though the progress is slow, it is sure, and there is no tendency
to spontaneous recovery in most cases, nor is the condition modified
by treatment. The patients may die from intercurrent disease or

*“Centralbl. f. Bakt. u. Parasitenk.,’”’ 1894, XVI, 175.

t Johns Hopkins Hospital Reports,’’ 1, 269, 291.

t“Centralbl. f. Bakt. u. Parasitenk.,’”’ 1895, XVII, 113, 625; XVII, 521; XX, 219.
§ “Zeitschrift fiir Hygiene,” etc., 1896, XXI, 11.

793

7904 Blastomycosis

from a generalization of the blastomycetic infection, which not in-
frequently happens.

After the work of Gilchrist had made clear the symptomatology
and parasitology of the disease, a number of other cases were re-
ported 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,t published in Igo2,
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 granuloma.

It seems to have. been first observed by Posadas and Wernicket:

and has been carefully studied by Ophiils.§ In this form of the
disease the lesions are in the internal organs, macroscopically and

Fig. 319.—Cutaneous blastomycosis (Montgomery).

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.
According to Evans** the disease seems 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 blastomycetic
dermatitis and the blastomycetic granuloma, or that they are caused
by different micro-organisms. Regarding the organisms, however,
we are by no means sure that there are not several species.

*“Tour. Med. Research,” 1901, I, 373.

t Jour. Amer. Med. Assoc. Cd June 7, 1902, 1, 1486.
t “Jour. de Micro-organismen,’ ” 1891, XV, 14.

§ “Jour. _ Experimental Medicine,” 1905, VI, 443. Ophiils and Mofit, “‘ Phila.

Med. Jour.,’’ 1900, v, 1471.
{| “Jour. Amer. Med. Assoc.,” 1902, XXXVIII, 867.
** “Your. of Infectious Diseases,’’ 1909, VI, 535-

Cultivation 795

Specific Organism.—The organism presents a variety of appear-
ances which may be thus brought together: F irst, 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 mw in greatest
diameter, are distinctly doubly contoured, highly refracting, and,
though sometimes clear and transparent, are frequently granular and
vacuolated. From them buds may grow, as in the yeasts, or hypha
may form, as in oidium. In artificial cultivations the hypha may
form a tangled mycelium. — :

Staining.—The organisms are usually better found without
staining. They do not stain with aqueous anilin dyes, but are pene-
trated by warm thionin, alkaline methylene-blue, and polychrome
methylene-blue. In sections of tissue stained with hematoxylon

Fig. 320.—Giant-cell from a cutaneous lesion in blastomycosis, showing a group
of blastomyces (Montgomery). :

and eosin they show as uncolored 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 from the
dermal lesions. Hiss and Zinsser recommended that this be done

* “Journal of Experimental Medicine,” 1898, 11, 53.

796 Blastomycosis

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 1 per cent. of dextrose being excellent. When
once isolated the organism is easily kept growing by transplanting
every month or two.

The colonies appear in a few days as small, round, hemispheric
dots with numerous prickles about the surfaces. Later they havea
moldy appearance from the development of aérial hypha. They are
almost purely aérobic, 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

Fig. 321.—Blastomyces dermatitidis. Budding forms and mycelial growths
from glucose agar (Irons and Graham, in “Journal of Infectious Diseases”).

a few weeks the agar-agar begins to turn yellow and later may be-
come brown, though the growth itself remains white and unchanged.
The growth is firmly attached 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 aérial hypha. Liquefaction
does not occur or is very slow.

Potato.—Abundant growth with aérial hypha.

Milk.—Not coagulated, not acidified, slowly digested.

There is some difficulty in describing the cultures, as different
authors describe them quite differently, evidently having different
organisms or different strains under observation.

;

Transmission 7197

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 vulgaris, other cases present an exaggeration
of the ulcerative element. Cases have also been mistaken for

Fig. 322.—Cultures of Blastomyces dermatitidis upon solid culture-media
(Montgomery).

syphilis. The intractable character of the lesions is suggestive, and
the finding of the micro-organisms in the viscid pus is pathogno-
monic.

Upon section the lesions still resemble ]upus and other tuberculous
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 in-
fection is not known.

CHAPTER XXXIX
RINGWORM
TRICHOPHYTON TONSURANS (MALMSTEN)

TINEA trichophytina, ringworm of the scalp, herpes tonsurans,
tinea circinata, ringworm of the body, herpes Circinatus, tinea
unguium, onychomycosis, tinea imbricata, herpes desquamans, tinea
vesicolor, pityriasis versicolor, erythrasma, etc., are diseases: with
well-marked clinical manifestations and differences, all of which .
may be comprehended under the general term dermatomycosis, and
are caused by closely related forms of parasitic fungi, whose generic
and specific differences are matters of considerable uncertainty.

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 saprophytic, while others pene-

_ trate more deeply and are undoubtedly 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 deplor-
able confusion of ideas, anda feeling that the synonomy is too com-
plicated and the. use of terms too loose to pent of ‘systematic
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,{ between the epi-
thelial cells. The organism appears to have been called Trichophy-
ton tonsurans in 1845 by Malmsten. The parasitology of all of the
trichophyton infections was thoroughly studied by Sabouraud,t
and the old species, Trichophyton tonsurans, divided into eleven new
species, to 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. ful-
matum, T. umbilicatum, T. regulare, T. pilosum, T. glabrum, T.
sulphureum, T. polygonum, T. exsiccatum, T. circonvulatum, 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
small, the latter large, spores.

* “ Compt.-rendu,”’ Paris, 1842, xv.

+ “Handbuch der speziellen Path. u. ,_ Therapie von Virchow,” m1, 1860.

t“‘Ann. de dermat. et de syphilis,” 1892, 11; 1893, IV; 1804, v; “‘ Monats-
hefte,” 1896, 576; ‘‘La euiaus dermatologique. Trichophytie,” Igoo.

798

Cultivation 709

Morphology.—The trichophyton parasites form delicate mycelia
composed of somewhat slender septate hypha. They can best be
observed by extracting one of the hairs, including its root, from the
diseased area, or if the affection be upon a hairless 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 zodglea upon the outside of the hair. In
some cases, however, especially in Trichophyton megalosporon, the
hypha may be observed with thespores
inside. The hypha measure from 2 to
8 » in diameter, are usually simple, and
rarely divide. The spores are from 2
to 3 win diameter in the Trichophyton
microsporon and 7 to 8 win T. mega-
losporon. The former is the more
common 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 annoy-
ing and almost constant presence of
the associated: bacteria of the skin.
By crushing the hair and scales in a
mortar with some dilute KOH solu-
tion, and then thoroughly distributing

Fig. 323.—Invasion of a hu-
: man hair by trichophyton: A,
the spores through the alkaline Points at which the parasitic

medium which dissolves many of the’ fungi coming from the epider-

BEA fae . * mis are elevating the cuticle
bacteria, plates can be made with high o¢ "the hair and entering ‘into

dilutions, or drops of the fluid be its substance. Magnified 200
spread over potato, which is an ex- ‘diameters (Sabouraud).
cellent 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 aérial hypha, looking like a tiny white
powder-puff. Upon the surface of liquid culture-media the growth
appears as a thick wrinkled pellicle with aérial hypha of velvety
appearance. As the cultures grow older the lower mycelial growth
becomes yellowish and wrinkled, but the aérial hypha maintain
the velvety white appearance. Some of the colonies are mammil-
lated, 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

800 Ringworm

for the lower animals. They spread from animal to animal by con-
tact and by inoculation. Men, dogs, cats, horses, sheep, goats, and
swine all suffer from the infection. The growth ‘of the hypha be-
tween the epidermal layers causes a chronic inflammation, with
hyperemia, desquamation, the formation of some papules, and oc-

Fig. 324.—Trichophyton tonsurans. Primary cultures twenty days old on
maltose agar-agar. Natural size (Sabouraud).

casional 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 transmission
of the infection by the barber’s razors. The disease is easily trans-
missible and precautions should always be taken to prevent its
dissemination. ,

CHAPTER XL
FAVUS
ACHORION SCHONLEINIT (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 Schénlein,* and called in his honor Achorion
schénleinii 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 separates, forming
the “scutulum,” or characteristic lesion of the disease. The reac-
tion 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 inflammatory reaction takes place, and leukocytic
invasion and ulceration cause the scutulum to separate.

Although usually confined to the skin, the favus infection may ex-
tend to the mucous membranes, and Kaposi and Kundrat{ have
reported a case in which fuvas fungi were found to have invaded
the stomach and intestines.

The disease runs a course sometimes extending over many years.
Crocker{ mentions a case that recovered after thirteen years. It
may remain localized upon the scalp or may spread 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 scarring, and atrophy of hair-
follicles, sweat, and sebaceous glands is inevitable.

The Specific Organism.—The Achorion schénleinii is probably
better regarded as a group of closely related organisms than as a
single one. Quincke has described three species, though they are
not yet generally accepted.

* Miiller’s ‘ Archiv,” 1839.

ft ‘‘Ann. de Dermat. et de Syph.,’”’ 1895, p. 104.

t ‘Diseases of the Skin,” Phila. - 1903, p. 1276.
cE, 801

802 Favus

The organism can be studied by extracting a hair and examining »
‘it in KOH (40 per cent.) or NaOH (20 per cent.), solution 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

A . B
Fig. 325—Favus. Hairs of a child infected with Achorion schénleinii. A,
Magnified 260 diameters; B, 75 diameters. The large rounded bodies are drop-
lets of air which always appear after treatment with 4o per cent. potash solution.
The linear threads are the fungi. Some are without spores, others contain rows
of spores (Sabouraud).

scutulum consists of masses of spores at the center and about the hair,
with mycelia containing spores at the edges. From the mycelium,
hypha are given off, the ends being knobbed or clavate.

The mycelial threads are highly refractile, contain granular proto-
plasm, and are of varying thickness. Sometimes the terminal

Cultivation 803

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
win length and 3 to 4 uw 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 of each scutulum contains pure
cultures of the organism. The best method is probably that of
Kral,* 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

Fig. 326.—Achorion schonleinii. Fig. 327.—Achorion — schénleinii.

Four weeks old culture upon beer- Pure culture, four wéeks old, on
wort agar-agar (Kolle and Wasser- beerwort agar-agar (Kolle and
mann). Wassermann).

with two or three loopfuls of the crushed material and poured into _
Petri dishes. Greater dilution can be made if desired, The plates
are examined 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 recommended
that, instead of tubes, they be made in Erlenmeyer flasks, the trans-
planted little colonies being placed at the center of the medium con-
gealed upon the bottom of the flask.

The appearance of the cultures varies considerably. Plaut gives

*See Plaut, in Kolle and Wassermann’s ‘‘Pathogene Mikroérganismen,” 1,
p. 608. :

804 Favus

two principal varieties: (1) The waxy type—a yellowish mass of a
waxy character with radiating folds and a central elevation. As a
rule no aérial hyphe, but occasionally short aérial hypha.

(2) The downy type—this forms a white disk with a velvety or
plush-like covering of white aérial hypha. Sometimes instead 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. foe

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 simi-
lar objects. On account of its chronicity, its obstinacy, its disfig-
urement, and its transmissibility, it is a dangerous disease, and one
that requires prompt isolation of the patient and the utmost care for
the prevention of contagion.

CHAPTER XLI
SPOROTRICHOSIS

SPOROTRICHOSIS is a somewhat rare disease of man, caused by
various members of a genus of fungi known as S porotrichum (Link-
Saccardo). The first occurrence of human sporotrichosis seems to
have been reported by B. R. Schenck.* The isolated micro-organ-
ism in this case was carefully studied and later was found to be
identical with a micro-organism isolated from another case of some-
what similar character studied by Hektoen and Perkins,t who de-
scribed it as Sporotrichum schencki. In 1903 de Beurmannt and
his associates took up the subject in France, and Lutz and Splendore§
in Brazil, and newcases werereported. On Aug. 8, 1908, the writer of
an editorial in the Journal of the American Medical Association was
able to give references to 14 cases of the disease. In 1912 Ruediger||
was able to collect 57 cases that had occurred in the United States.
In 1912 de Beurmann** reported that more than 200 cases had been:
put on record since the beginning of his work in 1903. It will thus.
be seen that the recognition of the cause of the disease and the im-
provement in diagnosis that followed it have made possible the
detection of many cases of a disease not recognized until 1900.

According to de Beurmann who has shown great interest in the
affection and prosecuted its study with much industry, the known
organisms of the Sporotrichum group comprise the following:

Sporotrichum schencki.

Sporotrichum beurmanni.

Sporotrichum beurmanni var. asteroides Gplentene
Sporotrichum beurmanni var. indicum (Castellani).
Sportrichum jeanselmei.

Sporotrichum gougerati.

Specific Organism.—The Sporotrichum is characterized by a
filamentous spore-bearing mycelium. The filaments are fine, meas-
uring about 2 u in diameter, partitioned, colorless, much branched
and tangled. The chief feature is the occurrence of the spores which
are situated along the length of the recumbent filaments either ‘on

* “Bulletin of the Johns Hopkins Hospital,” Dec., 1898.
t “Journal of Experimental Medicine,” 1900, 1901, V, 77.
t Ann. de Dermatologie et pupbllopraphic, 1906, 538.
§ “Centralbl. f. Bakt., etc.,” xiv, Orig., 632.
|| “Jour. of Infectious Diseases,” 1912, XI, 193.
** Brit. Med. Jour.,”” Aug. 10, 1912, Il, 2900.
805

806 Sporotrichosis

their extremities or on branches. They are arranged in cylindrical
cuffs about ro w in size and in glomeruli. As a matter of fact the
spores are readily isolated from one another. They arise one by one
in variable numbers along the mycelium, but as a rule in very large
quantity in each segment of the thallus. There is no apparent order
in their arrangement. So long as it remains on the filament the spore
appears pear-shaped. It is attached by a very fine sterigma, from
1~2 winlength and about 0.5 w in width. When shed, the spore is
oval. Its dimensions vary from 3-5-6 yw in length and from 2-3-4 u
in breadth. The form, the distribution and the brown color of the
spores and their fructification in the form of cylindrical cuffs, ar-
ranged in branches at the extremities of the filaments, constitute

Fig. 328.—Sporothrix schenckii. Fig. 329.—Sporothrix schenckii.
Margin of living hanging-drop cul- Slant culture on glucose agar, eight
ture (gelatin) X about 150 (Hektoen days old (Hektoen and Perkins, in
a Naas in ‘Jour. of Exper. ‘Jour. of Exper. Med.’’).

Med.”’). :

together with the original substratum of the fungus, a group of
characters which differentiates Sporotrichum beurmanni sharply
from all other sporotrichs (Matruchat). .

Hektoen and Perkins thus describe Sporotrichum schenckii: The
threads of the mycelium are seen to be doubly contoured; the proto-
plasm is somewhat granular and interrupted at fairly regular inter-
vals by transverse septa; the diameter of the threads varies some-
what, the average being about 2 4; the branches are not frequent and
do not bear any fixed relations to the septa. In the hanging-drop
cultures the relations of the conidia to the mycelium are very nicely
shown. The spore-bearing branches which grow out in a radiating
manner from the central feltwork, are commonly tipped by a cluster

Staining 807

of from three to six or more conidia, which, in the case of the larger
cluster, are attached by the smaller end to the slightly expanded
extremity of the branch. Similar ovate buds also arise from the
sides of the hyphz at shorter or longer intervals. The spores are also
doubly contoured and granular, resembling yeast cells. These
various features are well shown in the
photographs on the accompanying plate.
The attachment, by means of the short
pedicles of the spores to the threads, is-very
easily severed as shown by the difficulty in
obtaining stained preparations with the
spores i situ. When placed in the
hanging drop, the conidia grow out into
one or more straight germ tubes which
spring from either or both ends or from
the side. These embryonal threads
again give rise to lateral .or terminal
buds, which in all particulars resemble
the spores and some of which form branch-
ing spore-producing threads, so that in the
early stages very peculiar-looking bodies
are produced.

In the tissues and in the pus from the
lesions of the disease the parasites have
quite a different appearance, assuming
a short oblong form like a thick short
bacillus 3-5 w in length and 2-3 u broad,
basophilic, finely granular and surrounded
by a very delicate, ‘colorless membrane.
de Beurmann has watched the growth of
this degraded form of the parasite into the
filamentous and spore-bearing form, in
artificial culture. Meee = Ss Tae

Staining.—The micro-organism ismuch q,,.64 “hy Sporothrix
better examined in the fresh and living schenckii. “Arm of patient
condition than dried and stained, as it showing ulcers and scars,

° at a latestage of the lesions
greatly changes in appearance through (Hektoen and Perkins, in
shrinking. It does stain, however, with “Jour. of Exper. Med.”).
the usual dyes, andretains Gram’s stain ;
except when the alcohol washing is unduly prolonged. ;

Cultivation. Colonies——Upon agar-agar, at the end of about

‘ forty-eight hours, the colonies appear elevated, whitish, with feathery
fringes and some filamentous downgrowths into the medium. Upon
gelatin the downward growth results in liquefaction and the grow-
ing colonies sink below the surface.

Agar-Agar.—Along the needle track made by a stroke culture, a
grayish granular slightly elevated line with feathery edges forms in

'
i

808 Sporotrichosis

forty-eight hours and in seventy-two hours assumes the form of a
band with numerous transverse wrinkles; in a couple of days more
“the surface becomes more markedly corrugated and looks like a
chain of mountains on a map.” About the seventh day, the
growth, which has increased in thickness, becomes light brownish in
color, the margins being smooth and wavy and marked by shallow
transverse grooves. Still later the growth becomes dark brown,
wrinkled and covered by a delicate fuzz. The agar-agar becomes
brown.

Gelatin.—In gelatin punctures the growth is confined to the upper
strata. Lateral branches are sent out from the needle track. A sur-
face felt-like mass of mycelial threads forms beneath which the gela-
tin liquefies. The surface growth sinks into the liquid medium.

Blood-serum.—The growth is somewhat like that on agar-agar
but not so massive. It is apt to be covered by a white down.

Bouillon.—The growth which is fairly abundant, is in flakes and
tufts, shreds and filaments that settle to the bottom or cling to the
sides. A white surface film is apt to cover the liquid. No fermenta-
tion occurs in sugar bouillon.

Potato.—Upon potato, tufts form in twenty-four hours. These
have a brownish-gray color and soon become raised, wrinkled, and
frosted. The potato is darkened.

Milk.—The growth is scanty and owing to the opacity of the
medium, difficult to see. Litmus milk is not acidified. There is
no coagulation. .

Vital Resistance.—The optimum temperature is about 37°C.
The organism grows slowly at room temperature but in the end at-
tains pretty much the same magnitude as those kept in the thermo-
stat. The death point is 55°C. for one hour. Hektoen and Perkins
found S. schenckii killed in four and one-half minutes at 60°C.

Metabolic Products.—The organism produces no curdling or
proteolytic ferments for milk or blood-serum. It does, however,
liquefy gelatin. It grows aérobically or anaérobically, but under the
latter conditions it does not produce acid or ferment sugars, or
evolve gas. No indol is formed. It has a remarkable tolerance
for acid media. Page, Frothingham and Paige* found that it grew
well in media at least six times as acid as those ordinarily employed
for bacteria. They also found that the organism does produce acid
in media containing dextrose.

‘Distribution in Nature.—According to de Beurmann, the Sporo-
trichum is a widely distributed micro-organism in nature. It has
been found on green: vegetables, upon bark, thorns, potatoes, various
implements, in the soil, and in infected insects.

Pathogenesis.—The ‘Sporotrichum is pathogenic for men, horses,
rats, dogs, and white mice.

*“ Jour. Med. Research,” 1910, XXII, p. 129.

Pathogenesis 809

It would seem as though the rarity of its occurrence as a patho-
genic agent signified that it was by no means easy for it to effect the
invasion of the animal body. However, de Beurmann mentions a
man wounded in the forehead by a coster’s awl whom he believed
to have been infected by a cap, used to conceal the untreated wound,
that usually lay on the fruit and vegetables that filled his barrow;
a market woman infected by the salad that she was in the habit of
handling all day. Dominici and Duval report a case following a cut
inflicted while peeling a potato; Saint-Girons, a case following the
prick of a thorn of a barberry bush. A patient of Lutz’s was inocu-
lated through the bite of a cat; one of Wyse-Lauzun’s through the
bite of a parrot. Perkins’ case was that of a child that had abraded
a finger with a hammer. de Beurmann found the organism in the
pharynx of healthy persons “carriers,” whose saliva might, therefore,

*beinfectious. He believes that infection may take place through the
hair-follicles; that the healthy skin may be penetrated, and that the
healthy gastro-intestinal mucosa may be invaded.

Lesions.— The primary disturbance is a chronic and destructive
ulceration from which the disease spreads to numerous secondary |
foci chiefly by lymphatic metastasis. Hektoen and Perkins describe
the appearance of the primary lesion in Perkins’ case of infection
by S. schenckii, thus: “the finger from the first to the third joints
is swollen to twice its original size, presenting in the center a deep,
well-defined, sharp, undermined ulceration, the size of a ten-cent
piece. The base of the ulceration is rough and covered with gray-
ish-looking pus. This, when sponged away, leaves a bright red
surface; the ulcer extends through the whole thickness of the skin.
Surrounding the ulcer over about one-half of the infiltrated area, are
a large number of vesicles and a few pustules. The dorsal surface
of the hand and the extensor surface of the forearm present a chain
of swollen lymphatics along which are about twenty nodules from
the size of asmall pea toa Jarge hazel nut. . . This little patient
does not complain of much pain.” In the course of two months
Perkins opened and treated more than twenty abscesses resulting
from the enlargement and softening of the nodes.

De Beurmann and Gougerot found that the most characteristic
lesion of the skin is a nodule in which three processes are found, some-
times mixed up in an irregular manner, but most frequently arranged
concentrically. ‘In the center an abscess containing polymorpho-
nuclear leukocytes and macrophages; in the intermediate zone an
area of degenerated epithelioid giant-cells and tuberculous follicles
and at the periphery a proliferation of basophile lymph and con-

‘ nective-tissue cells.or a fibro-cellular infiltration.” ‘The structure
of the sporotrichoma is, therefore, very closely allied to that of the
lesions caused by syphilis, tuberculosis, and by the agents of chronic
suppuration, and it resembles sometimes the one, sometimes the

other.”

810 Sporotrichosis

De Beurmann and Raymond, 1903, and de Beurmann and
Gougerot, 1906, describe three clinical varieties of the disease.

1. Disseminated Gummatous Sporotrichosis.—The onset is insidious. An

accident usually leads to the development of the first gummata. The number of
.gummata may vary upto too. The first takes origin from any point in the sub-

cutaneous tissue. Others disseminate themselves over the whole body. Each’
gumma has an autonomous evolution which is the same for all. At first itisa -
little rounded mass, hard, elastic, painless and invariably in the subcutaneous
tissue. ‘The mass evolves rapidly in the direction of softening and in four or
six weeks terminates in a characteristic cold abscess. When it undergoes lique-
faction, it contains a fluid which is at first transparent, viscid, gummy, and with
purulent streaks and later becomes opaque, thick and purulent. It does not
undergo complete softening, and when it becomes fluctuating we find a central
cup-shaped depression surrounded by a firm and resisting zone, and when its
contents are evacuated, there remains round the empty pocket a persistent and
indurated ring.

2. Disseminated Subcutaneous, Gummatous Sporotrichosis with Ulceration.
—In this variety, the subcutaneous gummata after having passed through the,
phases described above, become hypodermo-dermic and destroy the skin by
ulceration more or less rapidly, sometimes in twenty days, sometimes in two or
three months. As a rule the ulcers are tuberculous in appearance. Frequently
the ulceration is at first no more than a narrow fistula from which oozes a viscid,
colorless and sometimes reddish pus or a yellowish serous fluid. :

3. Mixed forms are frequent. When the disease has existed for a long |
time it presents a complete clinical picture. Side by side are lesions of different
age with different tendencies and different appearances; tuberculous looking,
syphilitic looking, ecthymous, rupial and furuncular. There may be associated
lesions of the lymphatics, and lesions of the dermis, epidermis, mucous mem-
branes, muscles, osseous tissues, synovial membranes, eyes, epididymis, etc.

4. Localized Sporotrichosis.—The Sporotrichum penetrates by a cutaneous
lesion at the site of which it produces an initial lesion, which may be called the
‘“sporotrichotic chancre.”? Then it gradually invades the lymphatics and a hard
lymphatic cord studded with gummata—centripetal gummatous sporotrichosis—
makes its appearance. Sometimes the lymph-nodes of the region react, but this
is not constant. The disease remains localized to the region primarily affected.

Sporotrichosis of the mucous membranes, of the muscles, of the bones and
joints, of the synovial membranes, of the eye, of the epididymis, of the kidney,
and of the lung are described by de Beurmann.*

Bacteriologic Diagnosis.—Diagnosis by immediate and direct
examination of the pus either stained or unstained is difficult be-
cause the parasites are few in number, and are present in the bacil-
lary form that is so difficult to recognize.

_ The approved method is to carefully cleanse the skin over one
of the closed lesions, disinfect it with iodine, and then puncture the
abscess with a hollow needle. The pus obtained is spread plenti-
fully over the surface of culture-media in a number of tubes and stood
in the incubating oven. The characteristic colonies should appear in
from four to twelve days.

_ Should cultures be on hand in the laboratory at the time.a case
presents itself for diagnosis, two other methods may be employed.

1. The Agglutination Test.—A suspension of the spores from cul-
tures of the Sporotrichum will be agglutinated by the patient’s
serum in dilutions of 1-400 to 1-500 on the average.

* “Brit. Med. Jour.,”’ 1912, 1, 293.

Bacteriologic Diagnosis 811

2. The Complement-fixation Test.—The entire culture is used as
‘an antigen, the serum of the patient and guinea-pig complement em-
ployed as usual. As, however, Oidium, Actinomyces, Discomyces
and other fungi give the same degree of fixation, the method lacks
precision.

Bloch has also employed an intra-dermic injection of a sterilized
emulsion of the Sporotrichum for purposes of diagnosis. In twenty-
four hours, patients with sporotrichosis give.a marked reaction in
the form of an indurated nodule with a broad reddish surrounding
areola.

Bape siete, pe Re ee Eg,

BIBLIOGRAPHIC INDEX

AEDETT OF 92, 159, 160, 199, 209, 331,

Abbott and Bergey, 627, 628

Abbott and Gildersleeve, 430, 736

Abbott and Welch, 431

Abderhalden, 143, 144, 145, 146

Abderhalden and Freund, 146

Abel, 116, 484

Abel and Claussen, 614

Abel and Léffler, 634, 643, 662

Abelous, 334

Achaline, 349

Achard and Bensaude, 654

Adami, 69, 77 n

Adami and Chapin, 648

Adami and Kirkpatrick, 788 -

Afanassiew, 460

Agramonte, Carrol, Lazear and Reed,
574

Agramonte, Reed and Carrol, 576

Akuda and Kaneko, 535

Alav, 457 |

Albrecht and Ghon, 404

Albrecht, Weichselbaum and Gohn
591

Alessi, gt

Alt, 425

Altmann, 228 ©

Alvarez and Tavel, 735

Anaximander, 17

Anderson, 41, 75, 693, 694

Anderson.and Forst, 396 :

Anderson and Goldberger, 579, 580

Anderson and McClintic, 261, 263,
264, 265, 266, 268, 269

Anderson and Rosenau, 109, 110, 135

Andrade, 656, 657

Andrewes and Gordon, 308

Andrewes and Horder, 323, 324

Andrews, 178 .

Anjeszky, 160

Aoyama, 585

Aristotle, 17

Arloing, 102, 372, 726, 729

Arnaud, 671

Arning, 745

Arnold, 171, 174, 194, 478, 651, 652

Arrhenius, 24

Arustamoff, 38

Aschoff, 116, 348

Aschoff and Gaylord, 169

Ashford, 40, 693, 694

Audanard, 334

Auld, 468

Austin, 556

Avery, 478

Avery and Dochez, 475

Axenfeld, 399, 425, 426, 472

BABES, 31, 321, 334, 384, 430, 447,

451, 452, 491, 631, 753, 757
Babes and Cornil, 4515 ‘6r2
Babes and Ernst, 149

. Babes and Lepp, 104, 392

Babes and Proca, 727 ;
Bacot, 593, 594, 600, 601, Soy:
Bacot and Martin, 593

Bahr, 604

Bail, 123, 127

Baker, 545

Baldwin and Trudeau, 723, 725

. Baldwin, Graham and Stewart, 347,

348
Banti, 214, 472
Barbagallo and Casagrandi, 697
Barker, 334
Barker and Flint, 598

' Barlow, 698
_ Barron, 545
- Barsini, 790

Bass, 498, 503, 507, 508, 510

Bass and Johns, 503, 510, 511, 512

Bassett and Duval, 688

Bassett-Smith, 302, 492, 403

Bateman, Bruce, Hamerton and
Mackie, 551

. Baumgarten, 79, 450, 688, 699, 712,

714, 754, 756
Baumgarten and Walz, 725

- Bauzhaf and Steinhardt, 134

Bayon, 548, 567

. Beattie and Dickson, 529, 565

Beaupertius, 574

Beck and Pfeiffer, 489

Beck and Proskauer, 54, 710
Becker, 314

Beckman, 648

Bedson and Hume, 532, 534
Beebe, 442

' Behrer, 457

Behring, 24, 104, 130, 132, 357 444,
713) 719, 732

Behring and Kitasato, 105, 134

Behring and Nissen, 115

Behring and Nocht, 179

Beitzke, 456

Belfanti and Carbone, 137

Beninde, 712

Bensaude and Achard, 654

Benzangon, Griffon and Le Sourd, 421

Berestneff, 39

Berg, 457

Bergell and Meyer, 644

Bergey, 627

Bergey heel Abbott, 627, 628

Bergholm, 75

Berkefeld, 175, 176, 744, 767

813

814 Bibliographic Index

Bernheim, 72, 727

Bernheim and Popischell, 455
Berson, 318, 343, 434, 595
Bertarelli, 763

Bertarelli and Bocchia, 177
Bertarelli and Volpino, 765
Bertrand and Phisalix, 105, 135

Besredka, 320, 323, 326, 398, 462, 599.

Besredka and Metchnikoff, 276

Besredka and Steinhardt, 110

Bettencourt and Franca, 404

Beurmann, 805, 807, 808, 809, 810

Beurmann and Gougerot, 809, 810

Beurmann and Raymond, 810

Beyer, Rosenau, Parker and Francis,
577

Beyerinck, 67

Bezancon, 330

Bielonovsky, 589

Bienstock, 74, 350

Biggs, 441, 442

Bignami, 496

Billet, 504, 505

Billroth, 23, 35, 238, 307

Binger and Wolbach, 553, 554

Biondi, 73, 168

Biondi and Heidenhain, 168

Birch-Hirschfeld, 712

Birt and Lamb, 492

Bitter, 63

Bittu and Klemperer, 735

Blacklock, 559

Blaizot, Nicolle and Conseil, 524

Blake, 224, 273, 476, 477, 540, 542

Blanchard, 25, 508, 518

Blasi, 438

Block, 811

Blum, 333

Blumer, 334

Boa, 472

Boas and Oppler, 74

Bockhart, 76, 315

Boehm, 22

Boland, 332

Bolduan and Park, 446

Bollinger, 39, 775, 776

Bolton, 115, 141

Bolton and Globig, 199

Bolton and Pease, 56

Bolton, Dorset and McBryde, 666

Bomstein, 441

Bonhoff, 628

Bonney and Foulerton, 472

Bonome, 104, 753

Bonome and Gros, 57

Bonome and Viola, 56

Bordet, 24, 107, 122, 124, 137, 139, 201,

. 320, 460, 461, 462

Bordet and Gay, 141

Bordet and Gengou, 106, 142, 403, 726,
761

ee Uffreduzzi, 337, 468, 472, 740,

Hoel and Roux, 357, 362
Borrel, Knorr, Yersin and Calmette, 595

Borrel, Yersin and Calmette, 599

Boston and Pfahler, 326

Bostrém, 776, 777

Botkin, 219

Bousfield, 524

Bowhill, 588

Boxmeyer, McClintock and Siffer, 667

Boyce, 786, 788

Boyce and Surveyor, 786, 788, 790

Brault, 545

Braun, 695, 696, 697

Brebeck-Fischer, 457 —

Brefeld, 43, 45

Breinl, Kinghorn and Todd, 524

Brieger, 615

Brieger and Ehrlich, 114, 342, 357,

Brieger and Frankel, 82, 357, 369, 435,
615, 634

Bristowe, 790

Brown, 417, 418, 421, 429, 570, 780,
781

Brown and Wright, 777, 778

Browning, Gilmore and Mackie, 653

Bruce, 491, 493, 550

Bruce and Nabarro, 547, 551

Bruce, Hamerton, Bateman and
Mackie, 551

Bruce, Nabarro and Greig, 551

Bruck, 292

Bruck and Wassermann, 726, 770

Bruck, Wassermann and Neisser, 287,

290
Bruckner and Galasesco, 766
Brues and Rosenan, 396
Brumpt, 517, 519, 530, 545, 550, 551,
555, 559, 096, 607
Brunner, 670
Buchner, 114, 115, 139, 219, 221, 344
Buchner and Metchnikoff, 114
Buerger, 324, 466, 467, 475
Bujwid, 133
Bullock and Hunter, 333
Bumm, 315, 410, 414
Bumm and Nisot, 437
Bunsen, 150, 160, 163, 175, 207
Burn, 561
Burri, 534, 765
Burroughs and McCollum, 446
Burse,.40
Busse, 713
Buswell and Kraus, 643
Biitschli, 496
Butterfield and Peabody, 468
Buxton, 654
Buxton and Coleman, 651
Buxton and Torry, 112
Buxton and Vaughan, 127

CABOT, 390

Cadio, 733

Cadio, Gilbert and Roger, 733
Calkins, 27, 375, 376, 698

Calkins and Williams, 676

Calmette, 105, 135, 136, 334, 645, 723 |
Calmette and Guérin, 732

Bibliographic Index

Calmette, Borrel and Yersin, 599
Calmette, Knorr, Yersin and Borrel,
595 ves
Cameron, 715
Canon, 24, 486
Cantani, 615
Capaldi, 650
Carbone and Belfanti, 137
Cardan, 17
Carmona y Valle, 574
Carrasquilla, 747
Carrier, 718 :
Carroll, 219, 575
Carroll and Reed, 577
Carroll, Reed and Agramonte, 576
Carroll, Reed, Lazear and Agramonte,
574
Carter, 574, 575, 786, 790
Carter and Hughes, 480
Casagrandi and Barbagallo, 697
Castellani, 25, 546, 618, 653, 772, 773,
774, 805
Catanni (A.), Jr., 489
Cazeneuve, 518
Celli, 496, 504
Celli and Fiocca, 671, 673
Celli and Marchiafava, 398 '
Celli, Fiocca and Scala, 687
Celli-Shiga, 655
Centanni and Tizzoni, 727
Chagas, 556, 558, 559, 561
Chamberland, 176, 372, 720
Chamberland and Roux, 104, 342
Chamberland, Roux and Pasteur, 375
Chantemesse, 630, 643, 645, 737
Chantemesse and Widal, 639, 642, 643,
687
Chapin and Adami, 648
Charin, 56
Charrin, 104, 331, 737
Charrin and Roger, 91, 125
Chauffard and Quénu, 363
Chauveau, 104, 111, 372
Cheinisse, 420
Chenot and Picq, 757
‘Chester, 235!
Chester and Migula, 37
Chevreul, 20
Cheyne, 88
Christmas, 413, 415
Christy, Dutton and Todd, 547
Cienkowsky, 673
Citron, 297, 301
Ciuffo, 770
Clark, 449 ;
Clark and Flexner, 397
Clark and Howard, 396
Clarke and Miller, 177
Claudius, 177.
Claussen and Abel, 614
Clegg, 548, 743, 745
Clegg and Musgrave, 676, 677, 682,
684
Cobbett, 95, 115, 449
Cohn, 302, 460

815

Cohn and Brieger, 357

Cohnheim, 699

Colbach, 22

Coleman and Buxton, 651

Cole, 468, 469, 475, 480

Coley, 57, 325

Colla; 92 ©

Comte and Nicolle, 520, 569

Comus and Gley, 137, 141

Conn, 86

Conradi, 369, 641

Conradi-Drigalski, 653, 667

Conseil, Nicolle and Blaizot, 524

Conseil, Nicolle and Couer, 579

Cooley, 81 :

Cooley and Vaughan, 660

Cooper and Wells, 313, 718

Coplin, 154, 704

Cornet, 700

Cornevin, 342

Cornevin and Thomas, 102

Couer, Nicolle and Conseil, 579

Councilman, 25, 318, 449 .

Councilman and Lafleur, 672, 673, 674

Councilman, Mallory and Pearce, 441

Councilman, Mallory and Wright, 398

Courmont, 726

Cousland, 773

Coventon and Pelletier, 496

Cowie, 735

Cragg and Patton, 515, 527, 528, 555;
557, 561, 603

Craig, 675, 676, 679, 682, 683, 684, 698

Craig and Walker, 676

Creite, 136

Crocker, 801

~Crooke, 321

Crookshank, 783
Cruveilhier, 134

Cullum, 578

Cumston, 661

Cunningham, 571

Curry, 484

Curtis, 40, 355, 368, 369, 483, 710
Cushing, 636, 639, 655, 656
Czaplewski, 735, 740
Czaplewski and Hensel, 460
Czenzynke, 486

Czerny, 325

Datton and Eyre, 491

Daniels, 166

Danliewskyi, 497, 498

Dantec, 352

Danysz, 670

Darling, 572, 573) 676

d’Arsonval, 56

Davaine, 22, 24, 365

Davidson, 495

Davis, 420, 421, 422, 460, 488

Day, Kendall and Walker, 718

Dean, 636

de Beurmann, 805, 807, 808, 809, 810
de Beurmann and Gougerot, 809, 810.

816 Bibliographic Index

de Beurmann and Raymond, 810
de Geer, 530

Deichler, 460

Delafield, 679, 682

Delage, 27

Delépine, 180

Delezene, 108, 138

Delius and Kolle, 489

de Mondeville, 21

Denecke, 623, 624, 628

Denny, 429, 441

Denys, 723

Denys and van de Velde, 316
De Renzi, 480 '

de Sauvage, 578

de Silvestri, 671

DeSchweinitz, 425, 667, 718, 727
DeSchweinitz and Dorset, 666 .
Descos and Nicholas, 77

Detre, 292, 722

Detweiler, 81

Deutsch, 103

Deutsch’ and Feustmantel, 103
Devell, 590

Deycke, 199, 671

Dickson and Beattie, 565
Dineur, 127

Distaso and Douglas, 30

Di Vestea and Maffucci, 727
Dixon and Beattie, 529
Dobbin, 347

Dochez, 475, 476

Dochez and Avery, 475

Dodd and Neufeld, 469
Déderlein, 459

Déderlein and Winternitz, 76
Doerr, Todd and Kraus, 693
Doflein, 338

Dominici and Duval, 809
Dénitz, 357, 362
Donné, 771

Donovan, 563, 565, 566, 573
Donovan and Leishman, 25
Donovan and Patton, 568
Dopter and Vaillard, 692
Dorset, 708, 709

Dorset and DeSchweinitz, 666
Dorset, Bolton and McBryde, 666
Dorset, McBryde and Niles, 666
Douglas and Distaso, 30
Douglas and Wright, 113, 278, 285
Doutrelepont and Matterstock, 735
Draper, 661

Dreyfus, 661
Drigalski-Conradi, 648, 651
Droba, 636

Drysdale, 550 4

Dubarre and Terre, 735
duBary, 46

Dubois, 434

Duboscq and Leger, 696
Ducrey; 78, 420, 742

Dujardin, 19, 26

Dunbar, 628

‘Dungern, 106, 107, 108, 138

Dunham, 201, 202, 343, 347,:432, 6555

660, 662
Dunham and Park, 688
Durham, 656
Durham and Gruber, 126
Durme, 314
Dusch, 173

Dutton, 25, 521, 544, 545 i

Dutton and Forde, 547, 552

Dutton and Todd, 520, 521; 525, 545
Dutton, Todd and Christy, 547
Duval, 743, 744, 745

Duval. and Bassett, 688

Duval and Dominici, 809

Duval and Vedder, 688

EAGER, 583

Eberth, 24, 247, 334; 629, 655, 737

Effront, 61

Ehlers, 334

Ehrenberg, 19, 26, 246, 247 -

Ehrlich, 24, 97, 105, 106, 107, 116, 117,
118, I19Q, 120, 121, 122, 126, 128,
132, 133, 142, 155, 156, 157, 159,
357) 435) 444, 701, 703; 704, 706, 740

Ehrlich and Brieger, 114, 342

Ehrlich and. Marshall, 124

Ehrlich and Morgenroth, 116, 137, 139,

291
Eichhorn and Mohler, 752, 756, 757
Eisenberg, 235, 246, 247
Eisenberg and Voll, 127
Elders and Matzenauer, 451
Ellermann, 452 ‘

Elliott and Henry, 349 ~

Elmassian and Morax, 440

Elsching, 784

Elser, 402, 646, 648, 663

Elser and Huntoon, 404, 405

Emery, 430

Emmerich, 657

Emmerich and Léw, 68, 333

Emmerling, 313, 337

Emory, 74

Empedocles, 17

Endo, 245, 641, 650, 656

Engle and McFarland, 285

Engle and Reichel, 381

Eppinger, 39

Erlenmeyer, 720, 803

Ermengem, 254 ~

ge 31, 133) 331, 334, 347, 349, 430,
31

’ Ernst and Babes, 149

Ernst and Robey, 127

Escherich, 33, 75, 247, 655, 057

Esmarch, 188, 199, 209, 217, 240, 241,
243, 249

Evans, 184, 794

Evans and Russell, 184

Eyre, 46

Eyre and Dalton, 49x

FAIRCHILD, 192
Fantham, 547

Bibliographic Index

Fantham and Stephens, 544

Farran, 354

Fasching, 481

melee
ehleisen, 307, 317, 327, 328

Fehling, 201, 719 dala

Feletti and Grassi, 504, 505, 507

Fermi, 63

Fermi and Pernossi, 357

Fermi and Salsano, 734

Ferran, 619 ;

Feustmantel and Deutsch, 103

Fick, 450

Field, 356

Fildes and McIntosh, 223

Finger, Gohn and Schlaugenhaufer,
44

Finkelstein, 334

Finkler, 618

Finkler and Prior, 621, 624, 625

Finlay, 525; 574

Fiocca, 160

Fiocca and Celli, 671, 673

Fiocca, Celli and Scala, 687

Firth, 569, 571

Fisch, 727

Fischel, 116

Fischel and Wunschheim, 116

Fish, 124 .

Fitzpatrick, 599

Flatten, 398

Flexner, 25, 39, 321, 338; 343, 345, 398,
402, 404, 408, 440, 672, 687, 688,
690, 692, 764

Flexner and Clark, 397

Flexner and Harris, 639

Flexner and Lewis, 393

Flexner and Noguchi, 136, 137, 356,
395; 3906

Flexner and Shiga, 691

Flexner and Welch, 342, 347, 437

Flint and Barker, 598

Flournoy, Pappenheimer and Norris,
521, 523

Fliigge, 58, 113, 115, 154, 181, 182, 183,

246, 247, 249, 331, 352, 398, 464,

606, 712
Fodor, 113
Foley and Sergent, 524
Fontana, 534
Foote, 635
Forde, 25, 545
Forde and Dutton, 547, 552
Forneaca, 330
Forssner, 123, 322
Foulerton, 39
Foulerton and Bonney, 472
Fournier and Gilbert, 665
Fox and Longcope, 464
Fracastorius, 21
Franca and Bettencourt, 404
Francis and Grubs, 65 4
Francis, Rosenau, Parker and Beyer,

577
Frank and Heiman, 146
52

817

Franke and Frankel, 450

Frankel, 58, 95, 217, 218, 231, 249, 250,
342, 349, 370, 374, 399, 400, 450,
462, 464, 472, 481, 482, 521, 610,
ait 615, 617, 623, 625, 631, 639,

Frankel and Brieger, 357, 360, 435,
615, 634

Frankel and Franke, 450

Frankel and Pfeiffer, 312, 317, 340,
353, 364, 366, 367, 433, 473, 606,
610, 622, 626, 700, 711, 750

Frankel and Trendenburg, 321

Frankel and Weichselbaum, 78, 338

Frankel and Wollstein, 463

Frankforter, 184

Frankland, 246, 247

Fredericq, 113

Freejmuth and Petruschky, 455

Freire, 574

Freund and Abderhalden, 146

Freymuth, 620 :

Friedlander, 31, 59, 76, 155, 236, 427,
481, 482, 483, 484, 485, 741, 758, 759

Frisch, 334, 758, 759

Fromme and Uhlenhuth, 533

Frosch, 437

Frosch and Kolle, 323 »

Frost, 58, 210, 211, 212, 213, 243, 244

Frothingham, 382

Frothingham, Page and Paige, 808

Frugoni, 709

Fulleborn and Meyer, 526

Fuller, 190

Funck, 108

Futaki, Takaki, Taniguchi and Osumi,

540, 541, 542

GABBET, 704, 740, 746

Gabbi, 472

Gaffky, 328, 629, 640, 673

Gafftky and Koch, 672

Galasesco and Bruckner, 766
Galeotti, 64

Galli-Valerio, 57, 592, 671

Galtier, 375 ;
Gamaléia, 465, 470, 615, 625, 626, 628
Garini, 201 ;

‘Garnier and Reilly, 537

Garr, 315

Garré, 76

Gartner,253,630, 651, 654, 655, 664, 690

Gaspard, 20

Gauss, 31 :

Gauthier and Jodassohn, 770

Gay, 109

Gay and Bordet, 141

Gay and Southard, 109, 110

Gaylord and Aschoff, 169

Geddings and Wasdin, 574

Gelston, 81

Gelston and Marshall, 81

Gengou, 460, 461, 462, 463

Gengou and Bordet, 106, 142, 403, 726,
761

818 Bibliographic Index

Gerhard, 578 are

Germano and Maurea, 640

Gérny and Vincent, 787

Gessard, 64, 246, 330

Gessner, 361

Gheorghiewski, 106, 333

Ghon, 589

Ghon and Albrech, 404

Ghoreyeb, 763

Ghriskey and Robb, 72, 308

Gibier, 91

: Gibson, 134

Giemsa, 166, 378, 380, 396, 452, 523,
534, 542, 554, 679, 682, 696, 762,
765, 770 .

Gilbert and Fournier, 665

Gilbert, Cadio and Roger, 733

Gilchrist, 40, 793, 794, 797

Gilchrist and Stokes, 793, 795

Gildersleeve and Abbott, 430, 736 -

Gilliland and Pearson, 732

Gilmore, Browning and Mackie, 653

Gilvert, Zinsser and Hopkins, 768

Gley and Comus, 137, 141

Globig and Bolton, 199

Gohn, Finger and Schlaugenhaufer, 414

Gohn, Weichselbaum and Albrecht, sor

Goldberger and Anderson, 579, 580

Goldhorn, 762

Goldschmidt, 402

Golgi, 496, 498, 504

Gomez, 496

Goodby, 73

Goodsir, 238

Goodwin and Sholly, 405

Géppert, 398 ~

Gorden, 163:

Gordon, 323, 324, 409, 618

Gordon and Andrewes, 308

Gorgas, 577

Gorhan, 65

Gottschalk and Immerwahr, 76

Gottstein, 109

Gougerot and de Beurmann, 809, 810

Gourvitsch, 696

Gradenigo, 334

Graham and Irons, 40, 796

Graham-Smith, 524

Graham, Stewart and Baldwin, 347,
348

Gram, 155, 156, 157, 158, 168, 236,
238, 308, 310, 311, 317, 318, 319,
327, 328, 320, 330, 331, 334, 335,
339, 342, 344, 350, 352, 363, 364,
366, 396, 398, 400, 405, 406, 407,
408, 410, 411, 413, 415, 417, 418,
420, 421, 423, 424, 425,427, 428,

431, 452, 462, 464, 465, 481, 482, |

486, 490, 491, 520, 523, 532, 582,
584, 585, 605, 608, 611, 621, 625,
629, 630, 657, 658, 664, 665, 666,
668, 669, 687, 689, 694, 699, 702,
703, 706, 737, 739; 74°, 741, 749,
750, 758, 759,761, 771, 775, 778,
782, 787, 788, 790, 791, 807

Gram and Weigert, 156.

Grassi, 497, 498, 499

Grassi and Feletti, 495, 504, 505,
507

Grawitz, 42, 457, 459

Greig, Bruce and Nabarro, 551

Griffon, Benzangon and _Le Sourd,
421

Grigorjeff, 671

Grigorjeff and Ukke, 342

Grimme, 149

Grixoni, 354

Grohman, 113

Grohn and Sachs, 350

Gromakowsky, 325

Gros and Bonome, 57

Grossburger and Schattenfiroh, 349

Grosset, 459

Gruber, 217

Gruber and Durham, 126

Gruber and Wiener, 619

Griibler, 763

Grubs and Francis, 65

Gruby, 798

Gruby and Heim, 457

Griinbaum, 640

Griinbaum and Widal, 644

Grysez and van Steenberghe, 77

Gscheidel, 113

Gscheidel and Traube,. 139

Guarniere, 468

Guérin and Calmette, 732

Guiart, 554

Guidi, 457

Guiteras, 577

Giinther, 194, 239, 243, 249, 311, 341,
628

Giinther and Wagner, 765

Gwyn, 638, 656

HAECKEL, 26

Haffkine, 102, 271, 272, eae 586, 598,
619, 641

Hagedorn, 303

Hahn, 776

Halberstadter, 774

Hall, 565

Hallein, 457

Hallier, 238

Hamburger, 645

Hamerton, Bruce, Bateman} and
Mackie, 551

Hamilton, 449

Handel and Neufeld, 475

Hankin, 58, 91, 114, 373

Hankin and Leumann, 588

Hankin and Wesbrook, 369

Hansen, 24, 61, 218, 459, 739, 740

Harris, 380, 391, 686

Harris and Flexner, 639

Harris and Shackell, 390

Hartmann, 676

Harvey, 18

Harvey and McKendrick, 392

Harz, 775

Fe ee en eee

Bibliographic Index

Hashimoto, 615

Hasslauer, 76

Hasterlik, 617

Haupt, 427

Hauser, 334, 336

Havelburg, 574, 594

Hebra, 798

Heidenhain, 168

Heidenhain and Biondi, 168

Heider, 628

Heim, 311, 487, 612, 631, 658

Heim and Gruby, 457

Heiman, 412

Heinemann, 371

Hektoen, 318

Hektoen and Perkins, 805, 806, 807,
808, 809

Henle, 22

Henry, 350

Henry and Elliott, 349

Hensel and Czaplewski, 460

Herman, 88

Herrold, 399

Herzog, 592

Hesse, 221, 239, 240, 649

Hesse and Liborius, 221 |

Hewlett, 787 :

Hewlett and Nolen, 442

Heyman-Sticher, 712

Hibler, 350

Higgins, 765

Hildebrand, 72, 106

Hill, 148, 149, 209, 261, 649

Hippocrates, 532, 671

Hirschwald, 116

Hirsh, 321 ;

Hiss, 46, 181, 319, 327, 402, 465, 466,
468, 632, 647, 648, 663, 722, 765

Hiss and Russell, 688 ;

Hiss and Zinsser, 38, 190, 318, 328,
399, 412, 467, 468, 480, 629, 751,

795

Hiéchst, 145, 649, 725

Hodenpyl, 714 :

Hodenpyl and Prudden, 729

Hoffa, 369

Hoffmann, 25, 432, 766

Hoffmann and Prowazek, 771

Hoffmann and Schaudinn, 72, 521, 761,
762, 771

Hofmann, 441, 447, 449, 633

Hogg, 790

Hogyes, 386, 390, 307

Hoki, Ido, Ito and Wani, 539

Hoki, Inada, Ido, Kaneko and Ito, 533,
534, 536, 537, 539

Hoki, Ito, Wani and Inada, 540

Hoki, Ito, Wani, Inada and Ido, 538,
539 ' :

Holmes, 22

Holst, 86, 323 ; ,

Hopkins, Zinsser and Gilvert, 768

Horder, 488 a, ha

Horder and Andrewes, 323, 324

Hort, 400 :

819

Howard, 349, 437, 447; 449,472,485,513
Howard and Clark, 396

Howard and Perkins, 326, 327
Hiibener and Reiter, 533

Hughes and Carter, 480

: Hume and Bedson, 532, 534
' Humer, 636

Huntemiiller and Lentz, 395
Hunter and Bullock, 333
Huntoon and Elser, 404, 405
Huntoon and Strauss, 393
Hunziker, 217

Hiippe, 247, 373, 606, 611, 690
Hiippe and Wood, 374
Huxley, 27

Ivo and Inada, 532, 533, 535

Ido, Hoki, Inada, Kaneko and Ito, 533,
534) 536, 537, 539

Ido, Hoki, Ito and Wani, 539

Immerwahr and Gottschalk, 76

Inada, 540

Inada and Ido, 532, 533, 535

Inada, Hoki, Ito and Wani, 540

Inada, Ido, Hoki, Ito and Wani, 538,

539 :
Inada, Ido, Hoki, Kaneko and Ito, 533,
534, 536, 537, 53
Trons, 361, 647.
Irons and Graham, 40, 796
Ishiwara, Ohtawara and Tamura, 542
Israel, 776, 784
Israel and Wolff, 776, 777, 778, 782
Issaéff, 126, 468
Issaéff and Kolle, 617 ;
Ito, Hoki, Ido, Wani and Inada, 538,

539

Ito, Inada, Kaneko, Ido and Hoki, 533,
534, 536, 537, 539

Ito, Wani, Hoki and Ido, 539

Ito, Wani, Inada and Hoki, 540

Itzerott and Niemann, 330, 621, 623.
626, 737

Iwanow, 369

JACKSON, 572, 651

Jacob, 661

Jacobsohn and Pick, 400
Jacoby, 124

Jadkewitsch, 334

Jager, 247, 398, 399, 404
Jamieson and Johnston, 746

’ Jasuhara and Ogata, 104, 373

Jenner, 99, 101, 166, 271, 284,564
Jez, 643

Jobling, 408

Jodassohn and Gauthier, 770
Johannsen and Riley, 515

Johns and Bass, 503, 510, 511, 512
Johnson, Hewlett and Longcope, 654
Johnston and Jamieson, 746

Joos, 127

Jordan, 247, 332, 333, 643

Jordan, Russell and Zeit, 630, 633
Jorgensen, 61

820

KAENSCHE, 62
Kamen, 361
Kaneko and Akuda, 535
Kaneko, Inada, Ido, Hoki and Ito, 533,
534, 536, 537, 539
Kanthack, 787, 790
Kaplan, 298 |
Kaposi and Kundrat, 801
‘Karlinski, 308, 334, 664
Karlinski and Lubarsch, 664
Kartulis, 338, 423, 672, 673, 674
‘Kashida, 646
Kastle, Lumsden and Rosenau, 635
Kayser and Levy, 630
Kazarinow, 691
Keen, 637
Kehrer, 457
Keidel, 145, 289, 290, 644
Kempner, 256
Kempner and Pollak, 254
Kendall, 74, 414, 659
Kendall, Walker and Day, 718
Kerr, MacNeal and Latzer, 75
Kimla, 708
Kinghorn and Yorke, 551
Kinghorn, Breinl and Todd, 524
Kinsella and Swift, 324
Kipp, 223
Kircher, 17, 21
Kirchner, 417, 419
Kirkpatrick and Adami, 788
Kitasato, 24, 104, 105, 176, 221, 231,
352, 356, 359, 360, 466, 487, 582,
584, 585, 588, 599, 614, 615, 672,
I

Kitasato and Behring, 105

Kitasato and Weil, 221

Kitt, 102, 755

Klebs, 23, 111, 307, 320, 428, 437, 439,
440, 441, 447, 615, 671, 699, 718,
723, 724

Klein, 349, 350, 587, 590, 593

Klemperer, 468, 473

Klemperer (G. and F.), 480

Klemperer and Bittu, 735

Klemperer and Levy, 485, 633, 634

Klencki, 661

Klimenko, 462, 697

Kline, 342

Knapp, 449

Knapp and Novy, 520, 523, 526, 527

Knapp, Levaditi and Novy, 523

Knisl, 623

Knépfelmacher, 393

Knorr, 356

Knorr, Yersin, Calmette and Borrel,
595

Kny, 44

Koch, 22, 24, 53, III, 171, 180, 183,
198, 203, 206, 207, 208, 209, 222,
227, 228, 260, 307, 334, 339, 364,
365, 370, 371, 423, 424, 499, 511,
521, 523, 525, 530, 552, 606, 608,
609, 610, 611, 614, 615, 616, 623,
625, 628, 673, 674, 699, 700, 701,

Bibliographic Index

703, 704, 707, 708, 73, 714, 719,
720, 722, 723, 724, 725, 720, 728,
729, 730 731, 763, 708

Koch (C. L.), 527

Koch and Gaffky, 672

Koch and Van Ermengem, 616

Kohlbrugge, 74

Kohn and Krumwiede, 656

Kolisko and Paltauf, 437

Kolle, 154, 594, 595, 617, 642

Kolle and Delius, 489

Kolle and Frosch, 323

Kolle and Issaéff, 617

Kolle and Otto, 316, 599

Kolle and Pfeiffer, 634, 641

Kolle and Strong, 599

Kolle and Wassermann, 31, 33, 35, 36,
37,38, 42, 44, 116, 254, 405, 408,
458, 506, 508, 509, 521, 522, 586,
740, 803

Kolmer, 144, 229, 289, 401, 402, 446,

447
Koplik, 460
Korn, 737
Kossee and Overbeck, 589
Kossel, 105, 106, 137, 141, 334
Kral, 404, 803 ;
Krannhals, 334
Kraus, 106, 123, 124, 313, 414
Kraus and Buswell, 643
Kraus and Jochmann, 460
Kraus and Levaditi, 386
Kraus and Wernicke, 395
Kraus, Todd and Doerr, 693
Krefting, 420
Kronig, 177
Krénig and Menge, 348
Krénig and Paul, 179 .
Krumwiede and Kohn, 656
Krumwiede and Park, 731
Krumwiede and Pratt, 452, 453, 454
Krumwiede and Valentine, 479
Krumwiede, Pratt and McWilliams,
656
Kruse, 85, 334, 335, 363, 547, mol 659.
672, 688, 693
Kruse and. Pansini, 475
Kruse and Pasquale, 672
Kruse and Shiga, 688
Kubel and Tiemann, 648
Kiihne, 750, 751 :
Kulescha, 616
Kundrat and Kaposi, 801
Kurloff, 460
Kurth, 319, 321
Kutcher, 751
Kutschbert, 449

Lazpt, 504, sos, 507

Laennec, 716

Lafleur, 2

Lafleur and Councilman, 672, 673, 674
Laidlaw, 223, 224

Laitenen, 412, 413 :

Lamar and Meltzer, 471, on

Bibliographic Index

Lamb and Birt, 492

Lambert, 357, 386, 474

Lambert, Steinhardt and Poor, 377

Lambl, 671, 673

Lammershirt, 451

Lancereaux, 532

Landois, 136

Landouzy, 532

Landsteiner, 108

Landsteiner and Popper, 393

Langenbeck, 457, 776

Laplace, 179

Larkin, 39

Lartigau, 330, 334, 717

Laschtschenko, 712

Lassar, 761

Latapie, 138, 230, 231

Latour, 18

Latour and Schwann, 19

Latzer, MacNeal and Kerr, 75

Laurent, 457

Laveran, 25, 495, 496, 498, 501, 504,
505, 507, 508, 520, 547

Laveran and Mesnil, 546, 548, 563

LaWall, 194

Lazear, 576 :

Lazear, Read, Carroll and Agramonte,

574
Leach, 81, 531
Leber, 45, 313, 450
Lebert, 520, 776
Leclainche and Nocard, 508
Le Dantec, 352
Ledderhose, 332
Leeuwenhoek, 18, 26
Leffmann, 194
~ Leger and Duboscq, 696
Lehman and Neumann, 235, 237
Leichtenstern, 398
Leidy, 19, 26
Leishman, 166, 284, 285, 525, 548, 563,
564, 565, 506, 573
Leishman and Donovan, 25
Lemoine, 321
Engle and McFarland, 285
Lenglet, 422
Lenholm, 61
Le Noir, 334
Lenthold, 109
Lentz, 688
Lentz and Huntemiiller, 395
Leo, 92, 756
Lepierre, 404
Lepp and Babes, 104, 392
Lesage, 246, 334, 661, 662
Le Sourd, Benzangon and Griffon, 421
Leubarth, 321
Leuchs and von Lingelsheim, 404
Leumann, 598
Leumann and Hankin, 588
Levaditi, 285, 288; 534, 765
Levaditi and Kraus, 386
Levaditi and Manouelian, 765
Levaditi and McIntosh, 762, 766
Levaditi and Nattan-Larrier, 774

821

Levaditi, Novy and Knapp, 523 |

Levene, 719

Levin, 323

Levy, 472, 723

Levy and Kayser, 630

Levy and Klemperer, 485, 633, 634

Levy and Steinmetz, 751

Lewis, 25

Lewis and Flexner, 393

Lexer, 370

Libman, 321, 322, 656

Liborius, 218, 221

Liborius and Hesse, 221

Lichtowitz, 451

Liebig, 20, 264, 647, 657

Ligniéres, 722

Limbourg, 651

Lincoln and McFarland, 480

Lindemann, 108

Lindt, 44

Lingelsheim, 313, 318, 398

Lingelsheim and Leuchs, 404

Link-Saccardo, 805

Linn, 530

Linossier, 457

Linossier and Roux, 457

Linton and Thomas, 547

Lisbon, 592

Lister, 23, 175, 592

Livingstone, 550

Lockwood, 177

Léffler, 24, 154, 161, 198, 199, 208, 320,
344, 303, 402, 411, 425, 428, 430,
431, 432, 433) 435) 437, 439, 440
441, 443, 447, 448, 450, 452, 454,
455, 486, 618, 627, 630, 631, 650,
651, 655; 667, 669, 742; 759; 753

754
Loffler and Abel, 634, 643, 662
Léffler and Schiitz, 607, 749
Longcope and Fox, 464
Longcope, Johnson and Howlett, 654
Lord, 417, 418
Lésch, 25, 672, 673, 674, 682
Lésener, 640
Low, 560, 639
Low and Emmerich, 68, 333
Low and Sambon, 497 ;
Lowden and Williams, 376, 380, 382,

383
Lubarsch, 115, 372
Lubarsch and Karlinski, 664
Liibbert, 179
Lubenau, 323
Lubinski, 363
Lugol, 155
Liihe, 501
Lumsden, Kastle and Rosenau, 635
Lutz, 809 ;
Lutz and Splendore, 805
Luzzani, 381

MacCa.tvuy, 497, 498, 501
MacConkey, 652, 653
Macfadyen, 82, 469, 634, 643

822

Macfadyen and Rowland, 634

Macgregor, 671

Mackie, 520, 524

Mackie; Browning and Gilmore, 653

Mackie, Bruce, Hamerton and Bate-
man, 551

MacNeal, Latzer and Kerr, 75

Madsen, 24, 108, 134

Madsen and Noguchi, 136

Mafucci, 733

Mafucci and di Vestea, 727

Magendie, 109

Maggiora, 71, 334, 671

Maher, 661, 702

Malassez and Vignal, 737

Mallory, 158, 380, 637, 687

Mallory and Wright, 166, 168, 222

Mallory, Pearce and Councilman, 441

Mallory, Wright and Councilman, 398

Malmsten, 695, 798

Malvoz, 126, 127

Manceaux and Nicolle, 571

Mann, 381

Mannatti, 313

Manouelian and Levaditi, 765.

Manson, 430, 496, 497, 501, 514, 544,
545, 567, 693, 698

Manuelian, 377

Maragliano, 727

Marburg, 302

Marchiafava, 496

Marchiafava and Celli, 398

Marchoux, 373

Marchoux and Salimbeni, 520

Marcot and Bacot, 593

Marie, 392

Marie and Morax, 358

Marino, 166, 167, 168, 284, 548

Marks, 395

Marmier, 369

Marmorek, 88, 323, 325, 474

Marshall and Ehrlich, 124

Marshall and Gelston, 81

Martha, 334

Martin, 131, 136, 332, 360, 526

Martin and Roux, 115 .

Martin, Pettit and Vaudremer, 534

Marx, 149, 3890

Masselin and Thoinot, 605, 630

Masterman, 570

Mathieu, 532

Matruchat, 806

Matschinsky and Rymowitsch, 424,
426

Matterstock, 735

Matterstock and Doutrelepont, 735

Mattson, 109

Matzenauer and Elders, 451

Maurea and Germano, 640

Mayer, 363, 381

McBryde, Bolton and Dorset, 666

McBryde, Dorset and Niles, 666

McCarthy and Ravenel, 384

McClintic and Anderson, 261, 263,
264, 265, 266, 268, 269

Bibliographic Index

McClintock, Boxmeyer and Siffer, 667

McCollum and Burroughs, 446

McConkey, 690

McConnell, 747

McCoy and Smith, 590

McDaniel, Westbrook and Wilson, 430

McFadyen, 732, 752

McFarland, 374, 445, 727

McFarland and |’Engle, 285

McFarland and Lincoln, 480

McFarland and Small, 65

McIntosh and Fildes, 223

McIntosh and Levaditi, 762, 766

McIntyre, 81 ;

McKendrick and Harvey, 392

McNeal, 565

McNeal and Novy, 548, 559, 744

McWilliams, Krumwiede and Pratt,
656

Megnin, 550

Meier and Porges, 288

Meirowsky, 770

Melcher and Ortmann, 745

Meltzer and Lamar, 471, 484

Menge and Krénig, 348

Mense, 564

Merck, 167, 763

Mesnil, 113, 547, 568 ;

Mesnil and Laveran, 546, 548, 563

Messea, 32

Metalnikoff, 108, 138

Metchnikoff, 24, 94, 96, 107, 108, 111,
II2, 113, 114, 116, 119, 122, 126,
142, 237, 278, 350, 617, 762

Metchnikoff and Besredka, 276

Metchnikoff and.Buchner, 114

Metchnikoff and Roux, 761

Meunier, 57

Meyer, 152, 153, 227, 228, 519

Meyer and Bergell, 644

Meyer and Fulleborn, 526

Meyer and Ransom, 358

Michel, 434, 695

Middleton, 542

Migula, 32, 35, 36, 235, 237, 238, 465,
482, 657, 670, 701, 760

Migula and Chester, 37

Mikulicz, 760

Miller, 61, 72, 73, 279, 280, 281, 282,
283, 284, 520, 636

Miller and Clarke, 177

Millot-Carpentier, 540

Milne and Ross, 520, 521

Miquel, 237, 240

Mitchell, 22, 475

Mitchell and Muns, 478

Mitchell and Stewart, 136

‘Mittman, 71

Miyajima, 538

Miyaki, 540

Moczutkowski, 573

Moeller, 735, 736

Moffitt and Ophiils, 794

Mohler and Eichhorn, Bey, 756, 757
Moller, 160, 525

Bibliographic Index

Mondeville, 21

Monnier, 334 ;

Montesano and Montesson, 361

Montesson and Montesano, 361

Montgomery, 77, 794, 795, 797

Montgomery and Walker, 794

Monti, 471 :

Moon, 378

Moore and Taylor, 752

Morax, 423, 424, 425, 426

Morax and Elmassian, 440

Morax and Marie, 358

Morgan, 690

Morgenroth, 106, 107, 108, 124, 291

Morgenroth and Ehrlich, 116, 137, 139,
291

Moriya, 730

Moro, 114, 722

Morse, 314

Moschowitz, 361

Moser, 326

Mosso, 137

Mott, 553

Motz, 334

Mouton, 113

Much, 702

Muhlens, 766 :

Muir and Ritchie, 156, 159, 163, 680,
690

Miiller, 152, 159, 216, 623

Muns and Mitchell, 478

Murchison, 578

Murphy, 784

Murray, 164, 525

Musgrave and Clegg, 676, 677, 682,
684

Musgrave and Strong, 687, 697

Myers, 105, 107, 108, 124

Naparro and Bruce, 547, 551

Nabarro, Bruce and Greig, 551

Nattan-Larrier and Levaditi, 774

Neélow, 78

Negri, 375, 376, 377, 378, 380, 381, 382,
383; 384

Neisser, 24, 201, 410, 419, 431, 448,
449, 450, 628

- Neisser and Sachs, 143

Neisser and Wechsberg, 140, 141, 314,
316 ‘

Neisser, Bruck and Wassermann, 287,
290

Nelis and van Gehuchten, 384

Nepven, 545

Nessler, 67

Netter, 472, 473

Neufeld, 468, 475

Neufeld and Dodd, 469

Neufeld and Handel, 475

Neumann, 334, 442, 460

Neumann and Lehman, 235, 237

Newman, 164

Newman and Swithinbank, 712

Newmark, 302 j

Newsholme, 635

823

Nicati, 615

Nicati and Rietsch, 615

Nicholas, 770

Nicholas and Descos, 77

Nicholls, 77 ‘

Nichols and Schmitter, 219, 220

Nicolaier, 24, 352

Nicolani, 350 |

Nicolaysen, 414

Nicolle, 158, 420, 451, 565, 568, 572,
579, 745

Nicolle and Comte, 520, 569

Nicolle and Manceaux, 571

Nicolle, Blaizot and Conseil, 524 |

Nicolle, Couer and Conseil, 579

Niemann and Itzerott, 330, 621, 623,

._ 626, 737

Niles, McBryde and Dorset, 666

Nishi, 540

Nisot and Bumm, 437

Nissen, 180

Nissen and Behring, 115°

Nitzsch, 531

Nobe, 770

Nocard, 30, 360, 362, 508, 654, 665, 708

Nocard and Leclainche, 508

Nocard and Raillet, 550

Nocard and Roux, 196, 708

Nocht and Behring, 179

Noguchi, 25, 136, 288, 290, 202, 295,
296, 303, 305, 377, 378, 379, 524,
533) 534, 535, 538, 542, 762, 766,
767, 768, 770, 771

Noguchi and Fletcher, 137

Noguchi and Flexner, 136, 356, 395,

396

Noguchi and Madsen, 136

Noissette and Roger, 459

Nolf, 124

Norris, 39

Norris and Oliver, 64

Norris, Pappenheimer and Flournoy,
521, 523 ;

Nothnagel, 45

Novy, 151, 217, 218, 219, 234, 521,
522, 523, 565, 569, 649, 667, 735, 766

Novy and Knapp, 520, 523, 526, 527

Novy and McNeal, 548, 559, 744

Novy and Vaughan, 61, 252

Novy, Knapp and Levaditi, 523.

Nowlen and Hewlett, 442

Nuttall, 113, 125, 149, 150, 371, 522,
527, 591, 705 eee

Nuttall and Graham-Smith,. 446'

Nuttall and Inchley, 125

Nuttall and Welch, 342, 344, 347

OBERMEIER, 23, 24, 520
Oertel, 437

Oettinger, 334

Ogata, 542, 584, 588, 592, 671
Ogata and Jasuhara, 104, 373
Ogston, 307, 310, 317

Oguro, 540

Ohlmacher, 443, 447, 639, 705

824

Ohtawara, Ishiwara and Tamura, 542
Olitsky, 407

Oliver and Norris, 64

Olsen, 457

Ophiils, 40, 794

Ophiils and Moffitt, 794
Oppenheim, 302

Oppler and Boas, 74
Oriste-Armanni, 606
Orlowski and Palmirski, 435
Orth, 158

Ortmann, 467

Ortmann and Melcher, 745
Oshida, 387 5
Osler, 671, 672, 673

Osumi, Futaki, Takaki, and Taniguchi

540, 541; 542

Otero, 578

Otto, 109

Otto and Kolle, 316, 599
Overbeck and Kossee, 589
Ovid, 17 :

Oviedo, 772

Pacez, Frothingham and Paige, 808

Paige, Frothingham and Page, 808

Palmirski and Orlowski, 435

Paltauf, 44

Paltauf and Kolisko, 437

Pane, 480

Panfili, 179

Pansini, 334

Pansini and Kruse, 475

Pappenheim, 704, 705

Pappenheimer, Flournoy and Norris,
521, 523

Paquin, 727

Pariette, 649

Park, 88, 221, 362, 401, 402, 431, 442,
446, 447

Park and Bolduan, 446

Park and Dunham, 688

Park and Krumwiede, 731

Parke and Williams, 464

Parker, Rosenau, Francis and Beyer,

577

Pasquale and Kruse, 672

Passet, 310, 316, 317, 658

Passler, 480

Pasteur, 19, 20, 24, 91, 101, 102, III,
220, 230, 231, 271, 275, 307, 317,
339, 350, 370, 372, 373, 385, 386,
389, 390, 464, 605, 606, 607, 709,
720, 743, 761

_ Pasteur and Toussaint, 605

Pasteur, Chamberland and Roux, 375.

Patterson, 727

Patton, 567

Patton and Cragg, 515, 527, 528, 555,
557, 561, 603

Patton and Donovan, 568

Paul and Krénig, 179

Paulicki, 733 ;

Pawlowski, 57, 709

Pawlowsky, 619, 620

Bibliographic Index

Peabody and Butterfield, 468

Peabody and Pratt, 636, 645, 651

Pearce, 321, 437, 438

Pearce, Councilman and Mallory, 441

Pearson, 753 ,

Pearson and Gilliland, 732

Pease and Bolton, 56

Pelletier and Coventou, 496

Perkins, 334, 483, 787, 809

Perkins and Hektoen, 805, 806, 807,
808, 809

Perkins and Howard, 326, 327

Pernossi and Fermi, 357

Perroncito, 605, 655, 776

Peterson, 420 .

Petkowitsch, 648

Petri, 148, 196, 208, 209, 219, 220, 240,
241, 243, 244, 245, 249, 270, 288,
290, 321, 406, 448, 641, 646, 649,
653, 676, 735, 7371 740, 743, 803

Petruschky, 38, 39, 201, 323, 638, 640,
655, 665, 722

Petruschky and Freejmuth, 455

Pettit, Vandremer and Martin, 534

Peyer, 637

Pfahler and Boston, 326

Pfeiffer, 24, 57, 107, 139, 154, 196, 231,
486, 487, 488, 489, 490, 540, 618,
619, 625, 626, 737, 738

Pfeiffer and Beck, 489

Pfeiffer and Frankel, 312, 317, 340,
353, 364, 366, 367, 433, 473, 606, 610,
622, 626, 700, 711, 750

Pfeiffer and Kolle, 634, 641

Pfuhl, 337, 719

Phisalix and Bertrand, 105, 106, 135

Piaget, 530

Pianese, 568

Piatkowski, 706

Pick and Jacobsohn, 400

Picq and Chenot, 757

Pictet, 58

Pierce, 473

Piorkowski, 647, 663

Pirquet, 445, 722, 770

Pirquet and Schick, 109

Pisek and Pease, 475

Pitfield, 162, 358, 667

Plant, 44, 451, 457, 458, 459, 803

Platania, 92 .

Platz, 580

Plencig, 22

Pohl, 247

Pollak and Kempner, 754 |

Pollender, 22, 24, 365 i

Ponfick, 776

Poor and Steinhardt, 379 .

Poor, Lambert and Steinhardt, 377

Pope, 643

Popischell and Bernheim, 45

Popper and Landsteiner, 393

Porges and Meier, 288

Portier and Richet, tog

Posadas and Wernicke, 793

Pott, 57

Bibliographic Index

orig) is

ratt and Krumwiede, 452,

ae McWilliams and ed a
5

Pratt and Peabody, 636, 645, 651

Preindelsberger, 72 ?

Prescott, 61, 658

Prescott and Winslow, 200

Prior, 618

Prior and Finkler, 621, 624, 625

Proca and Babes, 727

Proeschi, 542

Proskauer and Beck, 54, 710

Proskauer and Voges, 600

Prowazek, 338, 549

Prowazek and Hoffmann, 771

Prudden, 243, 320, 714

Prudden and Hodenpyl, 729

Pusey, 426

Quénv and Chauffard, 363
Quincke, 801

Quincke and Roos, 672
Quinquaud, 457

RABINOWITSCH, 252, 737, 793

Railliet, 550

Railliet and Nocard, 550

Ramon, 357

Ransom and Meyer, 358

Rappaport, 588

Raskin, 321

Ravenel, 58, 77, 195, 199, 200, 203,
204, 217, 246, 247, 250, 729

Ravenel and McCarthy, 384

Raymond and de Beurmann, 810

Read and Savage, 656

Redi, 17 :

Reed and Carroll, 577

Reed, Carrol and Agramonte, 576

Reed, Carroll, Lazear and Agramonte,

574

Reed, Vaughan and Shakespeare, 635
Reichel, 176, 709
Reichel and Engle, 381
Reilly and Garnier, 537
Reiter and Hiibener, 533
Remak, 801

Rémy, 646

Renners, 617

Ress, 457

Rettger, 75, 373

Reyes, 434

Rhamy, 291
_Ribbert, 314, 315
Richards, 758, 759 ;
Richardson, 638, 639, 64
Richet and Portier, 109
Ricketts, 794

Ricketts and Wilder, 580
Rideal, 180

Rideal and Walker, 261
Ridi, 531

Riedel and Wolffhiigel, 610
Rieger, 398

825

Rietsch and Nicati, 615 -

Riggs, 338

Riley and Johannsen, 515

Rindfleisch, 307, 625

Ringer, 535

Rist, 436

Ritchie and Muir, 156, 159, 163, 680,
690 :

Mites 460 :
ivolta, 25, 733, 776

Robb, 176.

-| Robb and Ghriskey, 72, 308

Robertson, 633

Robey and Ernst, 127

Robin, 457

Robinson, 184

Rodet, 314

Roger, 88, 90, 373,737

Roger and Charrin, 91, 125

Roger and Noissette, 459

Roger, Cadio and Gilbert, 733 |

Rogers, 416, 563, 565, 567, 682

Rogone, 177

Rolleston, 661

Roloff, 733

Romanowsky, 166, 168, 379, 523, 534,
548, 570, 762

Romer, 254 -

Roos, 403

Roos and Quincke, 672

Rosenau, 110, 133, 184, 445, 589, 670,
qII

Rosenau and Anderson, 109, I10, 135

Rosenau and Brues, 396.

Rosenau, Lumsden and Kastle, 635

Rosenau, Parker, Francis and Beyer,

$77

Rosenbach, 307, 308, 310, 314, 317

Rosenberger, 209

Rosenow, 322, 469, 473, 474

Roser, 111

Ross, 495, 497, 498, 503, 504, 507; 525,
503,574

Ross and Milne, 520, 521

Rossi, 163

Rost, 742, 747

Rothberger, 647

Rothschild, 591

Rouget and Vaillard, 360

Roux, 24, 104, 131, 173, 217, 220, 227,
228, 372, 430, 431, 448, 450, 508,
710, 762

Roux and Borrel, 357, 362

Roux and Chamberland, 104, 342

Roux and Linossier, 457

Roux and Martin, 115

Roux and Metchnikoff, 761

Roux and Nocard, 196, 708

Roux and Yersin,; 82, 104, 434, 435,
436, 437

Rone Pasteur and Chamberland, 375

Row, 567, 571, 572

Rowland, 82, 599

Rowland and Macfadyen, 634

Rudolph, 742

826

Ruediger, 320, 805
'Ruffer, 619

Rumpf, 643

Ruppel, 718, 719

Russell, 656

Russell and Evans, 184

Russell and Hiss, 688

Russell, Jordan and Zeit, 630, 633
Russo-Travali, 438

Ruzicka, 331

Ryle and Stokes, 533

Ryle, Tatler and Stokes, 538
Rymowitsch and Matschinsky, 424,

426

SABOURAUD, 798, 799, 800, 802, 803
Sabrazes, 451

Sacharoff, 520

Sachs and Gohn, 350

Sachs and Neisser, 143
Saint-Girons, 809

Salamonsen, 222

Salant, 92

Salimbeni and Marchoux, 520
Salkowski, 65, 660

Salmon, 607, 655, 729 -

Salmon and Smith, 104, 607, 655, 666
Salsano and Fermi, 734 -
Sambon, 504, 551

Sambon and Low, 497

Sanarelli, 57, 574, 655, 668, 669
Sander, 709

Sanfelice, 334, 363, 793

Sattler, 450.

Savage, 647

Savage and Read, 656

Scala, Celli and Fiocca, 687
Schattenfiroh and Grossburger, 349
peas 25, 508, 673, 674, 676, 683,

684

Schaudinn and Hoffmann, 72, 521, 761,
762, 771, 773 :

Schellak, 520

Schenck, 805

Scherer, 398, 399, 402

Schereschewsky, 766

Schering, 153, 181

Schick, 446, 447

Schick and von Pirquet, 109

pelleigenliauies Finger and Gohn,

414
Schleich, 450
Schmidt, 398
Schmitter and Nichols, 219, 220
Schneider, 179, 398
Schénlein, 801
Schottelius, 324, 612
Schottmiiller, 320, 327, 4 476, 542
Schréder, 24, 173, 472
Schroter, 247
Schubler, 390, 392
Schiider, 635
Schiiffner, 508
Schulze, 18
Schumburg, 31

Bibliographic Index

Schiitz, 24, 754
Schiitz and Léffler, 607, 749
Schiitze and Wassermann, 106, 125
Schwalbe, 365
Schwann, 18
Schwann and Latour, 19
Sedgwick, 240
Sedgwick and Tucker, 240, 241
Sedgwick and Winslow, 58, 63 3
‘Seifert, 417
Seiner, 456
-Selter, 202, 757
Semmelweiss, 22
Semple, 641
Sergent and Foley, 524
Shackell and Harris, 390
Shakespeare, 613, 622, 624
Shakespeare, Vaughan and Reed, 635
Shaw, 8
Shiga, 24, 672, 687, 688, 692
Shiga and Flexner, 691
Shiga and Kruse, 688
Shimamine, 542
Sholly and Goodwin, 405
Sicard and Widal, 126
Sievenmann, 47
Siffer, McClintock and Boxmeyer, 667
Silber, 256
Silverschmidt, 337) 455
Simon, 324, 325
Simond, 590
Simonds, 346
Simpson, 587
Sjéo, 706
Small, 276
Small and McFarland, 65
Smillie, 223, 224, 225 .
Smirnow, 56
Smith, 62, 109, 127, 131, 162, 164, 192,
etl 435, 484, 638, 652, 662, 667,
70
Smith and McCoy, 590
Smith and Salmon, 104, 607, 666
Smith and Weidman, 338
Smith (Lorrain), 199
Smith (Theobald), 202, 222, 354. 356.
709, 711, 728, 730, 732
Sobernheim, 615, 619
Solowiew, 697
Somers, 649
Southard and Gay, 109, 110
Sowade, 766
Soyka, 53
Spallanzani, 18
Spengler, 460
Spiller, 384
Splendore, 805
Splendore and Lutz, 805
Spronck, 688, 742
Starkey, 649
Steele, 75, 405, 643
Steenberghe and Grysez, 77
Stefansky, 748
Steinhardt and Bauzhaf, 134
Steinhardt and Besredka, IIo

Bibliographic Index

Steinhardt and Poor, 379

Steinhardt, Poor and Lambert, 377

Stephens, 547

Stephens and Fantham, 544

Stern, 116, 642, 764, 770

Sternberg, 63, 92, 111, 240, 258, 260,
261, 313, 320, 334, 355, 464, 483,
574, 614, 630, 632

Stewart, 152, 166

Steve Baldwin and Graham, 347,
34

Stewart and Mitchell, 136

Stic er, 45, 745

Stiles, 673

Stillman, 475, 477

Stimson, 387, 388

Stitt, 515, 560

Stoddart, 538

Stokes, 251, 647

Stokes and Gilchrist, 793, 795

Stokes and Ryle, 533

Stokes, Ryle and Tatler, 538

Stokvis and Winogradow, 698

Stooss, 459

Strasburger, 75

Straus, 751

Strauss and Huntoon, 393

Strehl, 187

Strickland, 602

Strong, 599

Strong and Kolle, 599

Strong and Musgrave, 687, 697

Stuhlern, 481

Sugai, 745

Surveyor and Boyce, 786, 788, 790

Swift and Kinsella, 324

Swithinbank and Neuman, 252

Swithinbank and Newman, 712

Szekely, 34

Szemetzchenko, 460

Takaki and Wassermann, 105, 357

Takaki, Futaki, Taniguchi and Osumi,
540, 541, 542

Tamura, Ishiwara and Ohtawara, 542

Tanaguchi, Osumi, Futaki and Takaki,

| 540, 541, 542

Tangl, 435

Tashiro, 745

Tatler, Stokes and Ryle, 538

Taube and Weber, 735

Tavel, 44, 326, 363, 633

Tavel and Alvarez, 735

Taylor, 337

Taylor and Moore, 752

Tchistowitch, 107, 124

Tedeschi, 754, 756, 770

Telamon, 464

Terre and Dubarre, 735

Theiter, 520

Thelling, 725, 726

Theodoric, 21 ;

Thiercelin, 334

Thoinot and Masselin, 605, 630

Thomas and Cornevin, 102

827

Thomas and Linton, 547
Thompson, 652, 684
Thiiring, 664 :
Tictin, 524
Tidswell, 592
Tissier, 75
Tizzoni and Centanni, 727
Hee 545

odd and Dutton, 520, 521, 525, 54
Todd, Christy and Dutton, 547 ial
Todd, Kinghorn and Breinl, 524
Todd, Kraus and Doerr, 693°
Tornell, 706
Torrey, 415, 416
Torry and Buxton, 112
Toussaint, 372
Toussaint and Pasteur, 605
Trambusti, 59
Traube, 113
Traube and Gscheidel, 139
Trendenburg and Frankel, 321
Treskinskaja, 55
Triboulet, 334
Trudeau, 725
Trudeau and Baldwin, 723, 725
Tschistowitsch, 470
Tsiklinsky, 58, 337
Tsugitani, 677
Tucker, 240
Tucker and Sedgwick, 240, 241
Tunnicliff, 452, 453, 454
Tyndall, 19, 53

UckE, 438

Uhlenhuth, 124

Uhlenhuth and Fromme, 533
Uhlenhuth and Xylander, 707

Ukke and Grigorjeff, 342

Unna, 71, 158, 380, 420, 706, 740, 765
Uschinsky, 436, 660 .

VAILLARD and Dopter, 692
Vaillard and Rouget, 360
Valagussa, 671

Valentine and Krumwiede, 479
Valle y Carmona, 574

Van de Velde, 314, 326

Van de Velde and Denys, 316

van Dusch, 24, 173:

van Ermengem, 162, 254, 615, 765
van Ermengem and Koch, 616

van Gehuchten and Nélis, 384

van Gieson, 377.

van Helmont, 17

van Steenberghe and Grysez, 77
Varro, 20 h
Vaudremer, Martin and Pettit, 534
Vaughan, 81, 110, 253, 314, 548, 634
Vaughan and Buxton, 127
Vaughan and Cooley, 660

’ Vaughan and Novy, 61, 252

Vaughan, Reed and Shakespeare, 635
Veasy, 425

Vedder, 682

Vedder and Duval, 688

828 Bibliographic Index

Veillon and Zuber, 343, 349, 350, 452,
455.
Vergbitski, 592
Verneuil, 352
Vesley, 708
Vianna, 560
Viereck, 673, 675, 683
Vierordt, 611
Vignal, 38
Vignal and Malassez, 737
Villemin, 699
Villiers, 615
Vincent, 451, 452, 455, 787, 788
Vincent and Gérny, 787
Vincentini, 73
_ Vincenzi, 460
Viola and Bonome, 56
Viquerat, 316, 727
Virchow, 629, 672, 721, 739,746,776,7908
Virgil, 17
Voges, 617
Voges and Proskauer, 690
Volkmann, 417
Voll and Eisenberg, 127
Volpino and Bertarelli, 765
von Behring, 24, 104, 130, 132, 357,
444, 713, 719, 732
von Diingern, 106, 107, 108, 138
von Fodor, 113
von Frisch, 334, 758, 759
von Hibler, 350
von Langenbeck, 457, 776
von Lenthold, 109
von Lindt, 44
von Lingelsheim, 313, 318, 398
von Lingelsheim and Leuchs, 404
von Mayer, 363, 381
von Pirquet, 445, 722, 770
von Pirquet and Schick, 109
von Szekely, 34
von Thiiring, 664
Vuillemin, 457

WandsworTH, 471, 475
Wagner, 92

Wagner and Giinther, 765
Walger, 643

Walker, 123, 262

Walker and Craig, 676
Walker and Montgomery, 794
Walker and Rideal, 261
Walker, Kendall and Day, 718
Walz and Baumgarten, 725
Wani, Ido, Hoki and Ito, 539
Waal Inada, Ido, Hoki ‘and Ito, 538,

Want Inada, Hoki and Ito, 540

Warren; 193, 444, 407

Wasdin and Geddings, 574

Washbourn, 473, 480

Washburn, 468

Wassermann, 24, 105, 124, 125, 143,
288, 291, 292, 293, 295, 296, 297,
298, 299, 300, 302, 303, 305, 333,
412, 414, 415, 770, 771, 774

Wassermann and Bruck, 726, 770

Wassermann and Kolle, 31, 33, 35, 36,
37, 38, 42, 44, 116, 254, 405, 408,
458, 506, 508, 509, 521, 522, 586,
740, 893

Wassermann and Schiitze, 106, 125

Wassermann and Takaki, 105, 357

Wassermann, Neisser and Bruck, 287,
290

Weaver, 452

Weber and Taube, 735

Wechsberg and Neisser, 140, 141, 314,
316

Weeks, 423, 424, 425, 450

Weibel, 628

Weichselbaum, 247, 398, 399, 402, 405,
464, 472, 473, 482, 639

Weichselbaum, Albrecht and Gohn,

591

Weichselbaum and Fraenkel, 78, 338

Weidman and Smith, 338

Weigert, 24, 116, 118, 147, 149, 366,431,
465, 706, 740, 741, 790, 791

Weigert and Gram, 156

Weil, 337, 532, 533, 536; 539, 540

Weil and Kitasato, 221

Weinberg, 350

Weinzirl, 55

Weissner, 657

Welch, 72, 116, 123, 176, 308, 342, 343,
349, 361, 412, 445, 440, 495, 507

Welch and Abbott, 431

Welch and Flexner, 342, 347, 437

Welch and Nuttall, 342, 344, 347

Wellenhoff, 447

Wells and Cooper, 313, 718

Wenyon, 675 ;

Wernich, 111

Wernicke, 24, 589, 628

Wernicke and Kraus, 395

Wernicke and Posadas, 794

Wertheim, 410, 411, 413

Wesbrook and Hankin, 369

Wesenburg, 337

West, 406, 407

Westbrook, 428, 434

Westbrook, Wilson and McDaniel, 430

Weston, 288, 291, 644

Wheeler, 81

Whitman, 676

Wickman, 3

Widal, 126, 630, 636, 663, 644

Widal and ‘Chantemesse, 639, 642, 643,
687

Widal and Griinbaum, 644

Widal and Sicard, 126

Wiener and Gruber, 619

Wiens, 661

Wigura, 72

Wilcox, 540

| Wilder and Ricketts, 580

Whee: 334; 348, 378, 379, 437; 677;

795
Williams and Calkins, 676 =
Williams and Lowden, 376, 380, 382,383

ii: Serre, eel me

Bibliographic Index -

Williams and Parke, 464

Wilson, 587, 588

Wilson, McDaniel and Westbrook, 430

Winckel, 661

Windsor and Wright, 493

Winkler, 216

Winogradow and Stokvis, 698

Winogradsky, 66

Winslow, 72, 660 -

Winslow and Prescott, 200

Winslow and Sedgwick, 58, 633

Winterbottom, 553

Winternitz and Déderlein, 76

Witte, 192, 201, 202, 648 649, 650,
652, 657

Wladimiroff, 522

Wolbach, 456

Wolbach and Binger, 553, 554

Wolf, 473

Wolff and Israel, 776, 777, 778, 782

Wolff-Eisner, 645, 723, 770

Wolffhiigel and Riedel, 610

Wolfhiigel, 242, 243

Wollstein, 462, 490, 688

Wollstein and Frankel, 463

Wood, 151, 481, 773

Wood and Hiippe, 374

Woodhead, 33, 783

Woodward, 671

Wright, 103, 166, 168, 220, 222, 223,
246, 247, 271, 272, 273, 275, 276,
279, 281, 282, 283, 284, 286, 303,
316, 412, 492, 548, 503, 570, 571,
634, 641, 642, 683, 726, 756, 776,
777, 778, 780, 781, 782, 789, 790,
791, 792

Wright and Brown, 777, 778

Wright and Douglas, 113, 278, 285

Wright and Mallory, 166, 168, 222

Wright and Windsor, 493

Wright, Mallory and Councilman, 398

Wunschheim, 116

829
Wiirtz, 646, 650, 664

Wyman, 583, 588

Wynekoop, 490

Wyssokowitsch, soo

Wyssokowitsch and Zabolotny, 599’
Wyze-Lauzun, 809

XYVLANDER and Uhlenhuth, 707

YAMANOUCHI, 288
Yates, 652
Yersin, 24, 582, 583, 584, 585, 587, 591,

599
Yersin and Roux, 82, 104, 434, 435, 436
Yersin, Calmette and Borrel, so9
Yersin, Calmette, Borrel and Knorr,

595
Yorke and Kinghorn, 551
Young, 412, 413, 414
Yung, 58

ZABOLOTNY, 590

Zabolotny and Wyssokowitsch, 599
Zaufal, 472

Zeatogoroff, 614, 616, 617

Zeit, 56, 755

Zeit, Jordan and Russell, 630, 633

Zenker, 152, 158, 168, 233, 380, 381

Ziegler, 671

Ziehl, 154, 160, 631,°703, 704

Zieler, 158

Zimmermann, 246, 247

Zinsser, 220, 484

Zinsser and Hiss, 38, 190, 318, 328, 399,
412, 467, 468, 480, 629, 751, 795

Zinsser, Hopkins and Gilvert, 768

Zopf, 33, 247, 481

Zuber and Veillon, 343, 349, 350, 452,
455

Zupinski, 217, 358

Zur Nedden, 427

INDEX OF SUBJECTS

ne method of staining spores,
160
Abderhalden reaction, 143
dialysis test, 144
dialyzing shells for, 144
for pregnancy, 145
optical test, 144
aes of liver in amebic dysentery,
86 '
Accidental infection, 97
Acetic acid fermentation, 60
Achorion, 42
schénleinii, 801
cultivation, 803
Kral’s method, 803 -
pathogenesis, 804
varieties, 804
Acids as disinfectants, 179
production of, by bacteria, 63
Acquired activity, active, 97
immunity, 97
Passive, 104
Actinodiastase, 113
Actinomyces, 40
bovis, 775
cultivation, 778
Wright’s method, 778
distribution, 777
lesions from, 784
metabolism, 782
morphology, 777
pathogenesis, 782
staining, 782
temperature, 782
grain, 778
madure, 787
cultivation, 788
general characteristics, 787
lesions from, 799
morphology, 788
staining, 788
Actinomycosis, 775
Bostrém’s organism in, 776
history, 776
lesions, 784
mode of infection in, 783
Active immunity, 95
Adami and Chapin’s method of iso-
lating Bacillus typhosus, 648
Addiment, 107, 122
Adhesion cultures, 214
Aérobes, 54
Aérogens, 60 3
African lethargy. See Sleeping sick-
ness.

831

Agar-agar as culture medium, 195
blood, as culture medium, 196
glycerin, as culture medium, 196

Agglutination, 125
test for sporotrichosis, 810

technic, 128
Widal’s, in typhoid fever, 644

Agglutinins, 120,126 ~

Aggressins, 99, 123

Ague-cake, 512

Air, bacteria in, 239

quantitative estimation, Hesse’s
method, 239
Petri’s method, 240
Sedgwick’s method, 240
bacteriology of, 2309 :
Air-examination, Petri’s sand filter for,

24r ;
Sedgwick’s and Tucker’s expanded
tube for, 241 .
Alcoholic fermentation, 60
Aleppo boil, 569
Alexins, 114, 122, 139
Algid malaria, 510
Alkali-albuminate, Deycke’s, 199
Alkalies as disinfectants, 179
production of, by bacteria, 63
Alkaline blood-serum, 199
Allantiasis, 254
Allergia, 109
Altmann syringes, bacteriologic, 228
Amboceptor dose in Wassermann reac-
tion, 295 :
hemolytic, for Wassermann reaction,
292
unit in Wassermann reaction, 294
Amboceptors, 120, 121
Amebadiastase, 113
Amebe, parasitic, reproductive cycle,
675
suppuration and, 337
Amebic dysentery, 672, 673
American trypanosomiasis, 556.
also Sleeping sickness, American.
Ameeba coli, 673
thizopodia, 673
dysenteriz, 673
kartulisi, 338
mortinatalium, 338
Anaérobes, 54
facultative, 54
optional, 54
Anaérobic bacilli of infected gun-shot
- wounds, 352
bacteria, cultivation of, 217

See

832 Index of
Anaérobic bacteria, cultivation of, by
absorption of atmospheric oxy-
gen, 219
by catalytic action of platinized
asbestos on hydrogen and
oxygen, 223
by displacement of air with
inert gases, 217
by exclusion of atmospheric
oxygen, 221
by formation of vacuum, 217
by reduction of oxygen, 220
cultures, Botkin’s apparatus
making, 219
Buchner’s method of making, 219
Frinkel’s method of making, 218
Hesse’s method of making, 221
Koch’s method of making, 222
Laidlaw’s method of making, 223
Smillie’s modification, 223
Liborius’ tubes for, 218
Nichols and Schmitter’s method
of making, 219
Novy’s jars for, 218
Salamonsen’s tube for, 222
use of hen’s eggs for, 222
Wright’s method of making, 222
Zinsser’s method of making, 220
Anaphylactin, 110
Anaphylaxis, 109
passive, Iz
symptomatology, 110
Anderson-McClintic method of testing
germicidal value of liquids, 261-269
Anesthetic leprosy, 746
Angina, Vincent’s, 451
Animal holder, guinea-pig, 230
Latapie’s, 230
mouse, 230
fluids, increase of virulence by addi-
tion to culture-media, 87
Animalcule, 26
of Ehrenberg, 19
Animals, experimentation on, 227
immunization of, for diphtheria
antitoxin, 131
method of making injections into,
229
of securing blood from, 231
postmortems on, 233
typhoid fever in, 640
ca ee method of staining spores,
160
Anopheles maculipennis, 513, 514, 517
Anterior poliomyelitis, acute, 393.
See also Poliomyelitis.
Anthracin, 369
Anthrax, 364
alimentary tract infection in, 370
avenues of infection, 369
bacillus of, 364.° See Bacillus an-
thracis.
bacteriologic diagnosis, 373
in cattle, 370
lesions in, 371

for

Subjects

Anthrax, pathogenesis, 369
respiratory tract infection in, 15 369
sanitation, 374
serum therapy, 373
skin infection in, 371
vaccination in, 372
Anti-amboceptor, 121
Antianapbylactin, 110
Antibiosis, effects of, on growth of bac-
teria, 57
Antibody, 103
Anticomplement, 121
Anti-enzymes, 119
Antiformin, 178
for isolating tubercle bacillus, 706
Antigen, 103
syphilitic, 287
titration of, in Wassermann reaction,
296
iAuiRieanwedue serum, 415
Anti-immune bodies, 141
Antikérper, 444
Antiphthisin, 723
Antipneumococcus serum, 480
Antirabic serum, 392
Antisepsis, 23
Antiseptics, 170 i
action of, results, 259 ,
determination of value, 259
inhibition strengths of, 182
Antistreptococcus serum, 325
Antistreptokolysin, 324
Antitoxin, 116, 119, 128
diphtheria, 130, 444.
Diphtheria antitoxin.
tetanus, 134, 361
Antitubercle serums, 727
Antivenene, 106
Aphthe tropica, 693
Argas miniatus, 520
Army fever, 578 ,
Arnold’s steam sterilizer, 174
Aromatics, production of, by bacteria,
65
Arthrospores, 34
Ascococcus, 35
Asiatic cholera, 608
carriers, 616
clinical picture, 616 4
discovery of specific organism, 609 ’
Haffkine’s vaccines in, 619
history, 608
immunity, 619
prophylaxis, 619
rice-water discharges in, 616
sanitation in, 620
serum therapy, 620
specific organism, 609
Aspergillus, 44
bouffardi, 790
cultural characteristics, 791

See: also

general characteristics, 790 b
lesions from, 792 b
pathogenesis, 792 a

flavus, 45 -

Index of Subjects

Aspergillus fumigatus, 45
malignum, 44
nidulans, 45
niger, 45
subfuscus, 45
Association, effects of, on growth of
bacteria, 57
Atrophic spinal paralysis, 393
Auditory meatus, external, bacteria in,
72 3
Autoclave, modern, 175
sterilization in, 174
Autogenous vaccines, 273
Avery’s method of identifying types of
pneumococcus, 478

BaBeEs, tubercles of, 384
Babes-Ernst granules, 31
Bacillary dysentery, 672, 687
emulsion, 725
Bacilli, Gram-negative, of coli-typhoid
group, 690
Bacillus, 36
acidi lactici, 658
aérogenes capsulatus, 342
cultivation, 344
distribution, 343
general characteristics, 342
immunity, 349
metabolic products, 346
morphology, 343
pathogenesis, 347
sources of infection, 347
specific therapy, 349
staining, 344
vital resistance, 346
anthracis, 364
bacilli resembling, 374
cultivation, 367
general characteristics, 364
isolation, 366
metabolic products, 368
morphology, 365
motility, 366
sporulation, 365
staining, 366
virulence, 372
vital resistance, 368
avicidum, 605
avisepticus, 605
botulinus, 254
colonies, 255
cultivation, 255
general characteristics, 254
metabolic products, 255
morphology, 255
motility, 255
pathogenesis, 256
sporulation, 255
staining, 255
toxin, 255
butter, 737
butyricus, 737
capsulatus mucosus, 481
general characteristics,-481

833

Bacillus cholerz, 605
gallinarum, 605
cultivation, 605
general characteristics, 605
immunity against, 606
lesions from, 605
metabolic products, 605
morphology, 605
pathogenesis, 605
staining, 605
vital resistance, 605
suis, 666
coli communis, 657
Bacillus typhosus and, differ-
entiation, 645, 662, 663
cultural, 646
serum, 645
cultivation, 658
distribution, 658
general characteristics, 657
immunization against, 662 ©
in drinking water, 663
isolation, Léffler’s method, 650
Starkey’s method, 649
metabolic products, 659
morphology, 658
Fi pathogenesis, 660
‘ staining, 658 -
toxic products, 660
virulence, 661
vital resistance, 659
cuniculicida, 605
diphtheriz, 428
bacilli resembling, 447
bacteria associated with, 438
bacteriologic diagnosis, 443
carriers, 442
contagion from, 442
cultivation, 431
entrance into internal organs, 437
general characteristics, 428
infection with, 439
habitat, 437, 439 .
infection, intraperitoneal, 436
intrapleural, 436
lesions from, 440
metabolic products, 435
morphology, 428 .
mucous membrane inoculations,
436 :
pathogenesis, 436
specificity, 441
staining, 430
Léffler’s method, 430
Neisser’s method, 431
subcutaneous inoculation, 436
toxin, 435
types, 430
vital resistance, 434
diphtheroid, 447
ducreyi, 420
cultivation, 421
Davis’ method, 421
general characteristics, 420
morphology, 420

834

Bacillus ducreyi, pathogencs, 422

staining, 421
vital resistance, 422
dysenteriz, 672, 687
cultivation, 689
Flexner variety, 688
general characteristics, 687
Hiss- Russel variety, 688
isolation, 589
lesions from, 692
metabolic products, 691
morphology, 689
pathogenesis, 691
Shiga-Kruse variety, 688
staining, 689
varieties, 688
vital resistance, 691
.emphysematis vagine, 342
enteritidis, 664
cultivation, 664
‘general characteristics, 664
lesions, 664
morphology, 664.
pathogenesis, 664
sporogenes, 342
staining, 664
fusiformis, 451
cultivation, 454
morphology, 452
pathogenesis, 455
Spirocheta vincenti and, relation,

452
gas, of Welch, 342
hofmanni, 430, 441, 447
chemistry, 449
cultivation, 448
morphology, 448
pathogenesis, 449
staining, 448
icteroides, 668
cultivation, 668
distribution, 668
general characteristics, 668
metabolism, 669
morphology, 668
pathogenesis, 669
staining, 668
vital resistance, 669
yellow fever and, 576
influenze, 486
Bordet-Gengou bacillus and, dif-
ferentiation, 462
cultivation, 487
general characteristics, 486
immunity, 489
isolation, 487 _
morphology, 486
pathogenesis, 489
specificity, 488
staining, 486
vital resistance, 488
Klebs-Loffler, 428. See also Bacillus
diphtheria.
Koch-Weeks, 423
association, 425

Index of Subjects

Bacillus, Koch-Weeks, cultivation, 424
general characteristics, 423
morphology, 424
pathogenesis, 425
staining, 424

lactis aérogenes, 482
lepre, 739,
cultivation, 740
Clegg’s method, 743
Duval’s method, 743
Rost’s method, 742
general characteristics, 739
lesions from, 746
morphology, 739
pathogenesis, 745 — .
staining, 740
mallei, 749
cultivation, 752
distribution, 749
immunity to, 756
isolation, 751
lesions from, 754
metabolic products, 753
morphology, 749
pathogenesis, 754
staining, 750
Kithne’s method, 750
Léffler’s method, 750
temperature reactions in, 752
virulence, 756
vital resistance, 751
melitensis, 491. See also Micrococ-
cus melitensis.
Moeller’s grass, 736
Morax-Axenfeld, 425
cultivation, 425
morphology, 425
pathogenesis, 426
staining, 425
mucosus capsulatus, wildvation, 482
distribution, 482
metabolic-products, 483
. morphology, 482
pathogenesis, 484
virulence, 485
vital resistance, 483
neapolitanus, 657. Seealso Bacillus
coli communis.
cedematis maligni, 339
cultivation, 339
distribution, 339
general characteristics, 339
immunity, 342
lesions, 341
metabolic products, 41
morphology, 339
pathogenesis, 341
staining, 339
vital resistance, 340
of Bordet-Gengou, 460. See also
Bacillus pertussis.
of Biiffelseuche, 606
of diphtheria, 428
of Ducrey, 420. See also Bacillus
ducreyi.

‘Index of Subjects

Bacillus of Hofmann, 441
of plague, 582. See also Bacillus
pestis.
of rabbit septicemia, 605
of rhinoscleroma, 750
of swine-plague, 607.
illus stisepticus.
of tuberculosis, 699.
lus tuberculosis.
of typhoid fever, 629.
illus typhosus.
of Weeks, 423.
Koch-Weeks.
of whooping-cough, 460.
Bacillus pertussis.
of Wildseuche, 606
of Zur Nedden, 427
Oppler-Boas, 74
paracolon, 654
paratyphoid, 654
perfringens, 342
group, 349
pertussis, 460
Bacillus influenze and, differen-
tiation, 462
cultivation, 461
general characteristics, 460
isolation, 461
metabolic products, 462
morphology, 461
pathogenesis, 462
staining, 461
pestis, 582
colonies, 586
cultivation, 585
metabolism, 589
mode of infection with, 590
by cutaneous inoculation, 590
by inhalation, 590
by intraperitoneal inocula-
tion, 593
_by intravenous inoculation,

See also Bac-
See also Bacil-
See also Bac-
See also Bacillus,

See also

93
by subcutaneous inoculation,

590
morphology, 585
staining, 585
virulence, 595
vital resistance, 588
phlegmone emphysematose, 342
phlei, 736
piscicidus, 256
proteus vulgaris, 334
cultivation, 335
distribution, 334
general characteristics, 334
metabolic products, 336
morphology, 335
pathogenesis, 337
staining, 335
pseudo-diphtheria, 430, 441,
See also Bacillus Hofmanni.
pseudodysentery, 688
pseudoglanders, 757
pseudo-influenza, 490 .

447.

835

Bacillus pseudotuberculosis, 737
cultivation, 737
morphology, 737
pathogenesis, 738

psittacosis, 665
cultivation, 665
differentiation, 665
general characteristics, 665
isolation, 665
metabolic products, 665
morphology, 665
pathogenesis, 665
pyocyaneus, 330
cultivation, 331
distribution, 331
general characteristics, 330
immunity, 334
isolation, 331
metabolic products, 332
morphology, 331
pathogenesis, 333
staining, 331
pyogenes foetidus, 658
resembling anthrax bacillus, 374
rhinoscleromatis, 482
general characteristics, 758
pathogenesis, 750
septicus sputigenus, 464
smegmatis, 735°
cultivation, 735
morphology, 735
pathogenesis, 736
staining, 735
sporogenes group, 35°
suipestifer, 666
agglutination, 667,
cultivation, 666
general characteristics, 666
metabolic products, 667
morphology, 666
pathogenesis, 667
vital resistance, 667
suisepticus, 607
cultivation, 607
general characteristics, 607
lesions from, 608 —
morphology, 607
staining, ‘607
vital resistance, 607
tetani, 352
bacilli resembling, 363
cultivation, 354
distribution, 352
general characteristics, 352
metabolic products, 355
morphology, 352
staining, 352
toxic products, 356
vital resistance, 355
tuberculosis, 699
agglutination, 726 e
appearance of cultures, 710
avlum, 733
cultivation, 734
morphology, 734 -

836

Bacillus tuberculosis avium, patho-
genesis, 734
staining, 734
thermic sensitivity, 734
bacilli resembling, 735
bovis, 728
lesions of, 729
metabolic products, 728
morphology, 728
pathogenesis, 729
staining, 728
vegetation, 728
channels of infection for, 712
gastro-intestinal tract, 712
placenta, 712
respiratory tract, 712
sexual apparatus, 713
wounds, 713
chemistry, 718
cultivation, 708
Beck and Proskauer’s method,

710
Dorset’s method, 709
Frugoni’s method, -709
Koch’s method, 708
Nocard and Roux’s method, 708
Pawlowski’s method, 709
Smith’s method, 709

distribution, 700

effect of light on 711
tuberculin on, 720

general characteristics, 699

isolation, 706
antiformin for, 706

Koch’s method of obtaining cul-
tures, 707

lesions from, 714

metabolism, 718

morphology, 700

pathogenesis, 712

reaction, 711

relation to oxygen, 711

staining, 7o1
Ehrlich’s method for sections, 706
Gabbet’s method, 704

Gram’s method for sections, 706

in feces, 705
in sputum, 702
in tissue sections, 706

' in urine, 705
Koch-Ehrlich method, 701, 704
Pappenheim’s method, 704
Unna’s method for sections, 706
Ziehl’s method, 704

temperature sensitivity, 711

toxic products, 719

virulence, 717

typhi murium, 669
cultivation, 669
for extermination of field-mice,
670

general characteristics, 669
isolation, 669
morphology, 669
pathogenesis, 669

Index of Subjects

Bacillus typhi murium, staining, 669
typhi-exanthematicus, 580
typhosus, 629

bacilli resembling, 653
meat- -poisoning group, 654
pneumonic or psittacosis
group, 654
table for differentiation, 655
typhoidal group, 654
Bacillus coli and, differentiation,
645, 662, 663
cultural, 646
serum, 645
Capaldi’s medium for plating, 650
cultivation, 632
Hiss’ method, 647
Piorkowski’s method, 647
Rothberger’s method, 647
Wiirtz and Kashida’s method,
646
distribution, 629
effect of chemic agents on, 633
of cold on, 633
general characteristics, 629
Hesse’s medium for plating, 644
histologic lesions from, 637
in blood, 639
in drinking water, 634
in green vegetables, 635
in milk, 635
in raw oysters, 635
in urine, 638
invisible growth, 632
isolation, 631
Adami and ae s method,
641
Beckman’s methnd, 648
Buxton and Coleman’s method,
651
Castellani’s method, 653
Conradi-Drigalski’s method, 648 .
Endo’s method, 650
from feces, 645
Jackson’s method, 651
- MacConkey’s method, 652
Petkowitsch’s method, 648
Starkey’s method, 649
metabolic products, 633
mode of infection with, 634
morphology, 630
staining, 630
Ziehl’s method, 631
: toxic products, 634
vital resistance, 632
Welchii, 342
xerosis, 449
chemistry, 450
cultivation, 450
morphology, 450
. pathogenesis, 450
Y, 688

Bacteremia, 85

Bacteria, 26
anaérobic, culture of, 217. See also

Anaérobic bacteria, cultivation of.

Index of Subjects

ar associated with suppurations,
30
binary division of, 32
Brownian movement, 32
capsule, 31
chromogenic, 64
classification, 29
colonies, 206, 210
combination ‘of nitrogen by, 67
cultivation of, 189
cytoplasm, 31
discovery of, 18
effects of antibiosis on growth, 57
of association on growth, 57
of chemic agents on growth, 59
of metabolism on growth, 60
of movement on growth, 57
of symbiosis on growth, 57
of temperature on growth, 58
fission of, 32
flagella, 31
formation of nitrates by, 65
grinding, 279
groups of, proposed synopsis, 236
higher, 30, 37
in air, 239
quantitative estimation, Hesse’s
method, 239
Petri’s method, 240
Sedgwick’s method, 240
in bladder, 76
in butter, 252
in conjunctiva, .72
in external auditory meatus, 72
in foods, 251
in intestine, 74
in larynx, 76
in lungs, 76
in meat, 252
in milk, 251
in mouth, 72
in mucous membranes adjacent to
skin, 71
in nose, 76 -
in oysters, 252
in shell-fish, 2 52
in skin, 71
in soil, 249
in stomach, 93
in trachea, 76
in urethra, 76
in uterus, 75
in vagina, 75
in water, determination of total
number in given sample, 242
influences of electricity on growth,

56
of food on growth, 54
of light on growth, 55
of moisture on growth, 55
of oxygen on growth, 53
of reaction on growth, 55
of x-rays on growth, 56
invasive power, 81
isolation of, 206

837

Bacteria, liquefaction of gelatin by, 63
living, study of, 147
lower, 29
morphology, 34
motility, 32
non-chromogenic, 64
non-pathogenic, 67
nucleus, 30 |
number of, causing infection, 87
observation of, in sections of tissue,
152
Parasitic, 69
pathogenic, 67
in healthy body, 79
peptonization of milk by, 67
polar granules, 31
preparations for general examina-
tion, 149
production of acids by, 63
of alkalies by, 63
of aromatics by, 65
of disease by, 67
of enzymes by, 68
of gases by, 62
of odors by, 65
of phosphorescence by, 65
reduction of nitrates by, 66
reproduction, 32
saprophytic, 69
size, 27
species of, identification, 235
sporulation, 33
staining of, 149.
bacteria.
structure, 30
sulphur, 30
thermal death-point, determination
of, 257
transfer of platinum wires for, 203,
204
transplantation of, from culture-
tube to culture- tube technic, 205
true, 29
Bacterial suspension, 278
standardization of, 279
Bactericidal strength of disinfectants,
183
Bacterination, ror
Bacteriology, evolution of, 17
of air, 239
of foods, 251
of soil, 249
of water, 242
Bacteriolysins, 120
Bacteriolysis, 139
technic, 139
Bacteriolytic serums, therapeutic se-
rums, 141
Bacterio-toxins, 103,
Bacterio-vaccinations, conditions nec-
essary to success in, 272
Bacterio-vaccines, 271
Bacterium, 36__ ;
actinocladothrix, 775
coli dysenteriz, 687

See also

FACULTATIVE anaérobes, 54
Farcin du beeuf, 39
Farcy, 754
Farcy-buds, 754
Fatigue, susceptibility from, 91
Faulnisszymoid, 23
Favus, 801
scutulum of, 801
specific organism, 801
Febrile tropical splenomegaly, See:
See also Kala-azar.
Feces, isolation of Bacillus typhosus
from, 645
staining tubercle bacillus from, 70
Ferment, inflammatory, 23
Fermentation, 19, 60
alcoholic, 60
acetic, 60

Subjects

Fermentation, butyric acid, 61
lactic acid, 61
Fermentation-tube. Smith’s, 62
Ferments, defensive, 143
Fever, army-, 578
enteric, 629
jail-, 578
Malta, 491
Mediterranean, 491
non-malarial, remittent, 563.
also Kala-azar.
relapsing, 520
ship-, 578
splenic, 364
spotted, 398
typhoid, 629 :
typhus, 578
‘yellow, 574
Fiévre bileuse, 532
Filter, Berkefeld, 176
Chamberland, 176
Kitasato’s, 176
Reichel, 176
Filterable viruses, 27 |
Filtration, sterilization by, 175
Finkler and Prior spirillum, 621
Fiocca’s method of staining spores,
160
Fish tuberculosis, 735
Fish-poisoning, 256
Fishing, 211
Fixateur,, 121
Fixation of complement, 142
test of Wassermann reaction, 300
Flagella of bacteria, 31
methods of staining, 161.
also Staining flagella.
Flagellates, harmless, of human intes-
tines, 698
Flagellation of malarial parasites, 496
Fleas, plague, 600. See also Plague
fleas.
Fleischvergiftung, 61
Flexner variety of Bacillus dysenteriz,
688
Fluorescin, 64
Fomites, 70
foods as, 251
Food as fomites, 251
bacteria in, 251
influences of, on growth of bacteria,
54
poisons, 252
Foot, madura-, 786
Forceps, cover-glass, Stewart’s, 152
- Petri dish, 208
sterilization of, 172
Formaldehyd, 183
Formalin, 183
as disinfectant, 181
Fowl tuberculosis, 733
Frambesia tropica, 772
diagnosis, 774
history, 772
specific organism, 773

See

Index of

Frankel’s instrument for obtaining
earth for bacteriologic study, 250
method of making anaérobic cul-
tures, 218
Friedlander’s pneumococcus, 481. See
also Bacillus capsulatus mucosus.
Frost’s plate counter for counting col-
onies of bacteria, 244
Frugoni’s method of cultivating tuber-
cle bacillus, 709
Fungi, branched, 42
budding, key to genera of, 41
imperfect, 42, 46
Furniture, disinfection of, 186

GasBBET’s method of staining tubercle
bacillus, 704
Galactotoxism, 253
Gangrene, hospital, 339
Gas bacillus of Welch, 342
Gaseous disinfection, 270]
edema, bacillus of, 342. See also
Bacillus aérogenes capsulatus.
infections, micro-organisms of, 349
Gases, production of, 62
Gastro-intestinal tract, infection with
tubercle bacillus through, 712
Gelatin as culture-medium, 194
liquefaction of, by bacteria, 63
Generation, spontaneous, doctrine of,
17
Genital apparatus, infection through,
78
Germ theory of disease, 22
Germicidal value of liquids, modern
method of testing, 261
Germicides, 170
determination of value, 259, 260
apparatus for, 261 ;
culture-media for, 264
dilution of phenol and test solu-
tions for, 264
Hill’s method, 261
inoculating loops, 262
Koch’s method, 260
racks for holding tubes in, 264
solution to be tested, 262
Sternberg’s method, 261
technic of determining phenol
coefficient, 266, 268
test organism, 262
tubes for, 264
water bath for, 262
Germination of spores, 34
Ghoreyeb’s method of staining Tre-
ponema pallidum in films, 763,
Giant meningococci, 400
Giant-cells in tubercles, 715
Gilvert, Zinsser and Hopkins’ method
of cultivating Treponema pallidum,
768 | :
Glanders, 749
bacillus of, 749.
mallet.
diagnosis, 751

See also Bacillus

Subjects 843

Glanders, diagnosis, .
fixation test, 752

mallein in, 753
McFadyen’s agglutination test,
2

complement-

75
Straus’ method, 751

general characteristics, 749

immunity to, 756

in human beings, 756

‘lesions, 754

specific organism, 749
Glassware, sterilization of, 170, 172
Globulin precipitation for concentra-

tion of diphtheria antitoxin, 134
Glossina morsitans, 551, 557

palpalis, 550, 557
Ghasin agar-agar, as culture medium,

19
Glycerin-gelatin, embedding, 154
Golden staphylococcus, 310

-Goldhorn’s method of staining Trep-

onema pallidum in films, 762
Gonococcus, 410
Gonotoxin, 414
Gonorrhea, 410
complement-fixation test in, 413
diagnosis, 413
pathogenesis, 414
serum therapy, 415
Gordon’s method of detecting Spiril-
lum cholere asiatice, 618
Grain actinomyces, 778
‘sulphur, 778
Gram-negative bacilli of coli-typhoid
group, 690
Gram’s method of staining, 155
as aid to identification of spe-
cies, 156 :
Nicolle’s modification, 158
sections for tubercle bacillus,
706
solution for staining, 155
Gram-Weigert stain, 156
Granules, metachromatic, 31
polar, of bacteria, 31 .
Granulobacillus saccharobutyricus im-
mobilis liquefaciens, 342
Granuloma, coccidioidal, 794
Grass bacillus, Moeller’s 736
Grinding bacteria, 279
Guinea-pig holder, 230
serum, titration of, for Wassermann
réaction, 293 .
Gun-shot wounds, infected, anaérobic
bacilli of, 350

HarrkineE prophylactic against plague,

saceines in Asiatic cholera, 619
Halogens and compounds as disinfec-
tants, 180
Hands, disinfection of, 176
Hanging block, Hill’s, 148
drop, 148
Haptines, 119

844 Index of

Haptophiles, 117
Haptophore group, 120
Haptophores, 117
Hardening, 152
Harris and Shackell’s
treatment of rabies, 390
Harris method of staining Negri bodies,
380
Head, big, 775
swelled, 775
Head- louse, 529, 530
Heidenhain’s iron- -hematoxylin stain
for protozoa, 168
Heimann’s method of cultivating Mi-
crococcus gonorrheee, 412
Helcosoma tropicum, 570, 571
Hematopinus spinulosus, 549
Hemolysins,. 120
Hemolysis, 107, 136
technic, 137 ;
Hemolytic amboceptor for Wasser-
mann reaction, 292
serum, titration of, for Wassermann
reaction, 293
system in Wassermann reaction, 294
Hemorrhagin, 136
Hen’s eggs, use of, for anaérobic cul-
tures, 222
Herpes circinatus, 798
desquamans, 798
tonsurans, 798
Hesse’s apparatus for collecting bac-
teria in air, 240
medium for plating Bacillus typho-
sus, 649
method of making anaérobic cul-
tures, 221
quantitative method for estimation
of bacteria in air, 239
Higher bacteria, 37
Hill’s hanging block, 148
method of determining germicidal
value of disinfectants, 261
Hiss’ method of cultivating Bacillus
typhosus, 647
method of staining Diplococcus
pneumonia, 465
Hiss-Russell variety of Bacillus dysen-
teriz, 688
Histoplasma capsulatum, 572, 573
Histoplasmosis, 572
Historical introduction, 17
Hofmann’s bacillus, 441
Hog-cholera, 666
Hégyes’ attenuation treatment of
rabies, 386
dilution treatment of rabies, 390
Holzzunge, 775
Hopkins’, Gilvert’s and Zinsser’s
method of eallivaning: Treponema
pallidum, 768
Hospital gangrene, 339
Host, definition, 69
susceptibility of go.
ceplibility.

inspissation

See also

532. See also

See also

Subjects 845

Jacxson’s method of isolating Bacillus
typhosus, 651
Jactationstetanus, 359
Jail-fever, 578
Jaundice, infective,
Weil’s disease.
Jaw, lumpy, 775
Jennerian vaccination, 99
Jericho boil, 569, 570

532. See also

KALA-AZAR, 563
diagnosis, 568
infantile, 568
lesions, 566
transmission, 567
Keidel tube, 289
Keuchhusten, 460
K tasato’s filter, 176
Klatschpraparat, 214
Klebs-Léffler bacillus, 428.
Bacillus diphtheria.
Knives, sterilization of, 172
Koch-Ehrlich method of staining tu-
bercle bacillus, 701, 704
Koch’s apparatus for
blood-serum, 198
bacteriologic syringe, 227
law of specificity o bacteria, 22
method of cultivating tubercle bacil-
lus, 708
of determining germicidal value of
disinfectants, 260
of making anaérobic cultures, 222
of obtaining cultures of tubercle
bacillus, 707
plate cultures, 206
technic for agglutination test of tu-
bercle bacillus, 726
tuberculin, 720
Koch-Weeks bacillus, 423
Kolle’s method for diagnosis of plague,

See also

coagulating

594
Kolmer’s method of testing dialyzing
shells for Abderhalden reaction,

144 .

Kral’s method of cultivating Achorion
schinleinii, 803

Kreotoxism, 253

Krumwiede and Valentine’s method of
identifying types of pneumococcus, .

479 ;
Kiihne’s method of staining Bacillus
mallei, 750 .

LaByrintH, Starkey’s, Somers’ modi-
fication, 649

Lactic acid fermentation, 61

La fiévre typhique, 629

Laidlaw’s method of making anaérobic

cultures, 223
Smillie’s modification, 223

Laitenen’s method of cultivating Mi-
crococcus gonorrheee, 412

Lamblia intestinalis, 698

846

Lamus megistis, 559, 561
appearance, 561
breeding habits, 562
habitat, 562
habits, 562
Larynx, bacteria in, 76
Latapie’s animal holder, 230
instrument for preparing tissue pulp,
138 ©
Latent tuberculosis, 717
Lateral-chain theory of immunity, 116
Leeuwenhoek’s discovery of bacteria,

18 ;
Leishman-Donovan body, 563
Leishmania donovani, 563 .

cultivation, 565
distribution, 566
evolution, 564
lesions from, 566
morphology, 563

furunculosa, 569, 571

cultivation, 571
pathogenesis, 571

infantum, 568
Leistenkern, 117
Lepra anesthetica, 746

cells, 746

nodosa, 746
Leprolin, 747
Leprosy, 739

anesthetic, 746

bacillus of, 739.

leprae.

Carrasquilla’s serum for, 747

distribution, 739

etiology, 739

history, 739

lesions, 746

nasal lesions in, 745

nodular, 746

pathogenesis, 745

rat, 748

sanitation in, 748

specific therapy, 747

transmission of, 745
Leptopsylla musculi, 600
Leptospira, 534
Leptothrix, 38
Lethargy, African.

ness.
Leuconostoc, 36
Leukocidin, 314°
Leukocytes, washed, opsonic value of

blood in, 280
Levaditi’s method of staining Prepon

ema pallidum in sections, 765
aaa tube for anaérobic cultures,

21
Lice, 530
Ligatures, disinfection of, 175

sterilization of, 177
Light, influences of, on growth of bac-

terla, 55
Ligniéres cutituberculin reaction, 722

See also Bacillus

See Sleeping sick-

Index of Subjects

Listerism, 24
Litmus milk, 200
Liver abscess i in amebic dysentery, 686
postmortem appearance, in diph-
theria, 436
Lobar pneumonia, 464
Lockjaw, 359
Lockwood’s method of hand disinfec-
tion, 177
Léffler’s blood-serum mixture, 198
method of detecting Spirillum chol-
ere asiatice, 618
of isolating Bacillus typhosus and
Bacillus coli, 650
of staining, 154
Bacillus diphtheriz, 430
mallei, 750
flagella, 161
Louse, body, 529, 531
crab, 531
head-, 529, 530
Luetin, 770
Lumbar puncture, technic, 401
Lumpy jaw, 775.
Lungs, bacteria in, 76
Lupus, avenue of infection in, 89
Luzzani’s stain for Negri bodies, 385
Lysin, 107
Lysol as disinfectant, 181
Lyssa, 375. See also H ydro phobia.

MacConkeEy’s method of
Bacillus typhosus, 652
Macrocytase, 113, 122
Macrogametocyte, 501
Macrophages, 112
Madura-foot, 786

Maladie du sommeil. See Sleeping

sickness.
Malaria, 495.
ague-cake in, 512
algid, 510
congestive chills in, 510
diagnosis, 502
fever of, 495
geographic distribution, 49 5
history, 495
parasites, animal inoculation, 512
Bass’ method of concentrating,
503
classification, 498
cultivation, 510
Bass’ and Johns’ method, 510
estivo-autumnal, 507
examination of fresh blood for, 502
of stained blood films for, 502
flagellation in, 496
human, 504
inoculation, 512
life cycles of, 499
method of infecting mosquitoes
with, 519
parthenogenesis of, 502
pathogenesis, 512

isolating

Index of Subjects

Malaria, parasites, quartan, 504
Ross’ method of ee, 503
tertian, 505

paroxysms, 495 :
prophylaxis, 512
human beings, 513
mosquitoes, 513
relation of mosquitoes to, 496

Malarial fever. See Malaria.

Malignant edema, 339
bacillus of, 339. See also Bacillus

edematis maligni.
polyadenitis, 582
pustule, 371 |
Mallein, 753 |
Mallory’s eosin and methylene-blue
stain, 158

Malta fever, 491
bacteriologic diagnosis, 492
sanitation in, 493
treatment, 493

Manouelian’s method of staining Tre-

ponema pallidum in sections, 765

Marino’s stain for protozoa, 167

Mastigophora, 47

McClintic-Anderson method of testing

germicidal value of liquids, 261-

269
McFadyen’s agglutination test for
glanders, 752
Measurement of micro-organisms, 169
Meat, bacteria in, 252

extract, Prepaneticin of bouillon
from, 1

fresh, preparation ‘of bouillon from,
192

Meat-infusion, 192
Meat-poisoning, 61, 253
Medical contributions, 20
Mediterranean fever, 491
Megastomum intestinalis, 698
Melanoid mycetoma, 790
Meningitis, cerebro-spinal, 398.
also Cerebro-spinal meningitis.
Meningococcus, 398
carriers, 400
giant, 400
Mercuric chlorid as disinfectant, 179
Merismopedia, 34
Merozoits, 501
Messea’s classification of bacteria, 32
Metabolism, effects of, on growth of
bacteria, 60
Metachromatic granules, 31
Metazoa, 47
Metchnikoff’s phagocytosis theory of
immunity, 112
Meyer’s bacteriologic syringe, 227
Micrococcus, 34
catarrhalis, 417
cultivation, 418
metabolic products, 418
morphology, 418
pathogenesis, 419

See

staining, 418

847

Micrococcus gonorrheee, 410
cultivation, 411
Heiman’s method, 412
Laitenen’s method, 412
Wassermann’s method, 412
Wertheim’s method, 411
Young’s method, 412
diagnosis of gonorrhea from, 413
distribution, 410
general characteristics, 410
immunization against, 415
isolation, 411
metabolic products, 413
morphology, 410
pathogenesis, 414
staining, 411
toxic products, 413
vital resistance, 413
melitensis, 491
cultivation, 491
general characteristics, 491
morphology, 491
pathogenesis, 493
staining, 491
thermal death point, 491
meningitidis, 398
pasteuri, 464
tetragenus, 328
cultivation, 329
general characteristics, 328
isolation, 329
morphology, 328 .
pathogenesis, 330
staining, 329
Microcytase, 113, 122
Microgametes, 501
Microgametocyte, 501
Micron, 27
Micro-organismal tenants of human
body, 71
Micro-organisms, biology of, 53
classification, 26
cultivation of, 189
invasion of body by, 85
measurement of, 169
methods of observing, 147
of gaseous infections, 349
photographing, 169
structure, 26
Microphages, 112
Microspira, 37
comma, 608
Microsporon, 43
Miliary tubercles, 716
Milk, as culture- medium, 200
Bacillus typhosus i in, 635
bacteria in, 251
peptonization of, by bacteria, 67
Milk-poisoning, 253
Milzbrand, 364
Mitchell and Muns’ method of iden-
tifying types of pneumococcus, 478
Mixed infections, 92
Mixture, Coley’s, 325
Moeller’s grass bacillus, 736

848 Index of
Moisture, effects of, on growth of bac-
teria, 55
Molds, 42
black, 43

Miller’s method of staining spores, 160
Monilia psilosis, 693
Morax-Axenfeld bacillus, 425.
also Bacillus, Morax-Axenfeld.
Morbid conditions, susceptibility from,

See

92 z
Moro’s percutaneous tuberculin reac-
tion, 722
Morve, 749. See also Glanders.

Mosquitoes, 515
breeding habits, 516
classification, 515
destruction of, in prevention of
malaria, 513
development of larve, 518
of pupe, 518
habitat, 516
habits of pups, 518
in malaria, 513, 515
method of dissection, 519
of infecting with malarial para-
sites, 519
relation of, to malaria, 496
table for identification, 515
yellow fever and, 575
Motility of bacteria, 32
Mouse-holder, 230
Mouth, bacteria in, 72
Movement, effects of, on growth of
bacteria, 57
Mucor, 43
conoides, 44
corymbifer, 44
mucedo, 43
pusillus, 44
ramosus, 44
rhizopodiformis, 44
septatus, 44
Mucous membranes adjacent to mouth,
bacteria in, 71
Muguet, 457
Muir and Ritchie’s method of staining
spores, 159
Museum culture preparations, 216
Musgrave and Clegg’s method of culti-
vating amebe, 676
Mussel-poisoning, 256
Mutilation of body,
from, 92
Mycetoma, 786
lesions, 790, 792
melanoid, 790
ochroid, 787
pale, 787 Zi
pathogenesis, 792
Mycobacterium lepre, 730.
Bacillus lepre.
Mycophylaxins, 114
Mycosozins, 114
Mytilotoxism, 256
Myzorrhynchus pseudo-pictus, 514

susceptibility

See also

Subjects

NaGAna, 350 :
Nasopharynx, swabbing of, West’s ap-
paratus for, 406
Natural immunity, 95 ;
Negative phase of opsonic index, 285
Negri bodies, 376. See also Neuror-
rhyctes hydrophobie.
Neisseria gonorrhoea, 410 é
Neisser’s stain for Bacillus diphtheriz,
431
Neisser-Wechsberg phenomenon, 140
Nephrotoxins, 108
Neurorrhyctes hydrophobiz, 375
cultivation, 378
examination for, 383
morphology, 376
staining, 370
Harris’ method, 380
Reichel and Engle’s method
381
Williams and Lowden’s method,

380
Nichols and Schmitter’s method of
making anaérobic cultures, 219 _
Nicolle’s modification of Gram’s stain,

158
Nitrate broth, 66 ,
Nitrates, formation of, by bacteria, 65
reduction of, by bacteria, 66
Nitrogen, combination of, by bacteria,

7
Nitrosoindol reaction, 65
Nits, 531
Nocard and Roux’s method of culti-
vating tubercle bacillus, 708
Nocardia actinomyces, 775
bovis, 775
Nodular leprosy, 746
Noguchi’s method for diagnosing syph-
ilis, 770 :
of cultivating Spirocheta ober-
meieri, 524
Treponema pallidum, 766
modification of Wassermann reac-
tion, 303 ©

‘Non-malarial remittent fever, 563.

See also Kala-azar. :
Non-pathogenic bacteria, 67
Nose, bacteria in, 76
Novy’s jars for anaérobic cultures, 218
Noxious vapors, inhalation of, suscep-
tibility from, 91
Nucleus of bacteria, 30
of protozoa, 50

OcuRorp mycetoma, 787
Odors, production of, by bacteria, 65
Oidia, 41 :
Oidium albicans, 457
fermentation, 459
immunity against, 459
metabolic products, 459
morphology, 457
pathogenesis, 459
Onychomycosis, 798

Index of

Oscyst, sor
Odkinete, 501
Oéspora bovis, 775
Ophidiomonas, 37
Opilacao, 557
Opisthotonos of tetanus, 359
Oppler-Boas bacillus, 74
Opsonic index, 27
determination of, 285
negative phase, 285
value of blood, requirements for test
of, 278
serum to be tested, 281
washed leukocytes in, 280
Opsonins, 113
Opsonizing pipette, 282
Optical test in Abderhalden reaction,
144
Optional anaérobes, 54
Organic disinfectants, 180°
Oriental sore, 569
Ornithodorus moubata, 525, 528, 559
habitat, 528
savignyi, 525, 527
habitat, 528
Oven, incubating, 215
Oxygen, influences of, on growth of
bacteria, 53
Oysters, Bacillus typhosus in, 635
bacteria in, 252

PALUDISM, 495
Pappenheim’s method of staining tu-
bercle bacillus, 704
Paracolon bacilli, 654
Paraffin embedding, 153
Paralysis from diphtheria antitoxin,
_ 445.
infantile, 393.
elitis. =
Parasaccharomyces ashfordi, 693, 694
cultural characters, 695
morphology, 695
physiological properties, 695 |
Parasite, definition, 69
malarial, 496. See Malaria, para-
Site. ;
Parasitic ameba, reproductive cycle,
675
bacteria, 69
stomatitis, 457
Paratyphoid bacilli, 654
Parthenogenesis of malarial parasites,
502 j
Partially confirmed test for purity of
water, 245
Passive anaphylaxis, 111
immunity, 95
Pasteur treatment of rabies, 102, 389
Pasteurian vaccination, 101
Pasteurization, 174
Pathogenesis, 8: _
Pathogenic bacteria, 67
"in healthy body, 79
_ Pathogens, 60
54

See also Bacillus

850 Index of
Plague, death-rate, 583
diagnosis, 594
experimental infection with, 589
fleas, 600
breeding habits, 602
life history, 600
longevity, 601
method of extermination, 602
table for identification, 604
varieties, 600, 601, 602
group of micro-organisms, 604
history, 578, 582
immunity against, 598
active, 598
Haffkine prophylactic in, 599
passive, 599
mode of infection with, 590
mortality, 583
pneumonia, 485, 590
postmortem appearance in, 595
quarantine in, 597
rat extermination in, 597
sanitation in, 595
specific organism, 584
Planococcus, 35
Planosarcina, 35
Plasmodium falciparum, 495, 507
gametocytes in, 508
malaria, 495, 504
gametocytes of, 505
meroblasts of, 504
quartanz, 504
vivax, 495, 505
developmental cycle of, 500
gametocytes of, 501
Plasmolysis, 31
Plate cultures, 206
disadvantages, 208
Koch’s method for, 207
leveling apparatus for making, 207
Petri dish for, 208
Platinum wires for transferring bac-
teria, 203, 204
sterilization of, 172
Pneumobacillus, 481
Pneumococcus of Frinkel and Weich-
selbaum, 464. See also Diplo-
coccus pneumonia.
of Friedlander, 481. See also Bacil-
lus capsulatus mucosus.
types of,475
methods of identifying, 476
Avery’s, 478
Blake’s, 477
Krumwiede’s and Valentine’s
method, 479
Mitchell and Muna? >» 478
Pneumonia, 464
bacteriologic diagnosis, 474
broncho-, 485 -
catarrhal, 485
complicating, 485
croupous, 464
lesions, 471
lobar, 464

Subjects

Pneumonia, mixed, 485
plague, 485, 59°
’ sanitation in, 481
tuberculous, 485
Poisoning, cereal-, 253
cheese-, 253
fish-, 256
from canned goods, 256
ice-cream, 253
meat-, 253
milk-, 253
mussel-, 256
sausage, 254
Poisons, food, 252
Polar granules of bacteria, 3I
Poliomyelitis, acute anterior, 393
avenues of infection, 397
cause, 393
characteristics, 394
histological changes, 394
immunization against, 397
possible infective agent, 396
transmission, 396, 397
virus, 394
Polyadenitis, malignant, 582
Ponos, 568
Positive phase of opsonic index, 285
Post-mortems on animals, 233
Potability of water, 245
Potassium permanganate as disinfec-
tant, 180
Potato as culture-medium, 199
cultures on, 214
cutter, Ravenel’s, 200 ‘
Potato-juice as culture-medium, 200
Precipitate, specific, 106 ~
Precipitation, specific, 123
Precipitins, 120, 124
Predisposition to infection, 90

Pregnancy, diagnosis of, by Abder-

halden reaction, 145
Prescott and Winslow’s method of pre-
paring reagent litmus, 200.
eraaame test for purity of water

Brophoiacue vaccination in typhoid
fever, 641 :
Proskauer and Beck’s method of culti-
vating tubercle bacillus, 710
Proteins, defensive, 114
Proteolytic group of bacilli, 350
Protista, 26
Protozoa, 47, 49
classification, 47
cytoplasm, 49
encystment, 52
living, observation of, 164
movement, 50
nucleus, 50
reproduction, 51
size, 51 ’
structure, 49
Pseudo-diphtheria bacillus, 430, 441,
447. See also Bacillus hofmanni.
Pseudodysentery bacillus, 688

Index of Subjects

Pseudo-glanders bacillus, 757

Pseudo-influenza bacillus, 490

Pseudomonas, 36

Pseudotuberculosis, 737

Psilosis, 693

lingue, 693

Ptomains, 61

Pulex irritans, 592, 602

Puncture cultures, 211

lumbar, technic, gor.

Pure culture, 203, 210

Purity of water, determination of, 244
completed test, 245
partially confirmed test, 245
presumptive test, 244

Pustule, malignant, 371

Putrefaction, 19, 61

Putrefactive ferment, 23

Pyemia, 85

Pyocyanase, 68, 333

Pyocyanin, 64

Pyocyanolysin, 333

QuaRTAN malaria, parasites of, 504

Rassir septicemia, bacillus of, 605
Rabies, 375. See also Hydrophobia.
Rat extermination in plague, 597
leprosy, 748
Rat-bite fever, 540
course, 540
lesions, 543
spirochete of, 540. See
Spirocheta morsus muris.
symptoms, 540
treatment, 543
Ravenel’s platinum wires for bacteri-
ologic use, 203
potato cutter, 200
Reaction, Abderhalden, 143.
Abderhalden reaction.
Calmette’s ophthalmo - tuberculin,
723
complement-fixation 142
influences of, on growth of bacteria,

also

See also

55
Ligniére’s cutituberculin, 722
Moro’s percutaneous tuberculin, 722
tuberculin, in bovine tuberculosis,
732
von Pirquet’s cutaneous tuberculin,
722
Wassermann, 287
Reagents employed in Wassermann
reaction, 287
Receptors, 117, 121
free, 119
of first order, 119
of second order, 120
of third order, 120
Refined tuberculin, 720
Regressive schizogony, 502 :
Reichel and Engle’s stain for Negri
bodies, 381
filter, 176

851

| Relapsing fever, 520
carriers, 527
method of infection, 524
Relative immunity, 95
Remittent fever, non-malarial, 563.
See also Kala-azar.
Respiratory apparatus, infection
through, 78
tract, infection with tubercle bacillus
through, 712
Retention theory of immunity, 111
Rhinoscleroma, 758
bacillus of, 758
Rhipicephalus decoloratus, 520
Rhizopoda, 47
Richardson’s method of differentiating
a typhosus and Bacillus coli,
4
Ringworm, 798
Robinson’s method of disinfection, 184
Ross’ method of finding malarial para-
sites, 503
Rossi’s method of staining flagella, 163
Rost’s method of cultivating Bacillus
lepre, 742 .
Rothberger’s method of cultivating
Bacillus typhosus, 647
Rotz, 749. See also Glanders.
Roux and Nocard’s method of. culti-
vating tubercle bacillus, 708
bacteriologic syringe, 227
Rubber stoppers, _ sterilization
172

“of

SACCHAROLYTIC group of bacilli, 350
Saccharomyces hominis, 793
Saccharomycosis haminis, 793
Sacs, collodion, increase of virulence
by use of, 87
Salamonsen’s tube for anaérobic cul-
tures, 222
Salkowski’s test for indol, 65
Salts as disinfectants, 179
Sand filter, Petri’s, for air-examination,
241
Sapremia, 85
Saprogens, 60
Saprophytic bacteria, 69
Sarcina, 35
Sausage poisoning, 254
Scalp, ringworm of, 798°
Schaumorgane, 348
Schering’s method of embedding, 153
Schick’s reaction in diphtheria, 446
Schizogony, regressive, 502
Schizonts, 499
Schizotrypanum cruzi, 556, 560
morphology, 558
pathogenesis, 559
reproduction, 558
transmission, 559
Schlafkrankheit, 544. See also Sleep-
ing-sickness.
Schmitter andNichols’ method of mak-
ing anaérobic cultures, 219

852 Index of

Schottelius’ method of securing pure
culture of cholera spirillum, 612
Scissors, sterilization of, 172
Scutulum of favus, 801
Sedgwick and Tucker’s expanded tube
for air-examination, 241°
Sedgwick’s method of quantitative
estimation of bacteria in air, 240
Seitenketten, 117
Seitenkettentheorie, 116
Seminaria contagionum, 21
Sensitization of vaccines, 276
Septicemia, 85
rabbit, bacillus of, 605
Serum, antigonococcus, 415
antipneumococcus, 480
antirabic, 392 ;
antistreptococcus, 325
antitubercle, 727
antivenomous, 135
bacteriolytic, therapeutic uses, 141
Coley’s, 325
‘determining potency of, in diph-
theria antitoxin, 132
disease, 109
in Wassermann reaction, 290
guinea-pig, titration of, 293
hemolytic, titration of, 293
leprosy, Carrasquilla’s, 747
preparation of, for diphtheria anti-
toxin, 132
Sexual apparatus, infection with tu-
bercle bacillus through, 713
Shake culture, 221
Sheep corpuscles, titration of, for
Wassermann reaction, 293
Shell-fish, bacteria in, 252
Shiga-Kruse variety of Bacillus dysen-
teriz, 688
Ship-fever, 578
Siberian pest, 364
Sickness, black, 563. See also Kalaazar.
Sick-room, disinfection of, 178
Side-chain theory of immunity, 116
Silver nitrate as disinfectant, 180
Sitotoxism, 253
Skin, bacteria in, 71
disinfection of, 176
infection through, 76
Sleeping sickness, American, 556
clinical picture, 558
diagnosis, 560
prophylaxis, 561
transmission, 559
clinical picture, 544
distributign, 551
lesions, 554
natural history, 551
prophylaxis, 555
réle of tsetse fly in, sso.
specific organism, 545
transmission, 549
to lower animals, 552
trypanosome of, 534. See also
Trypanosoma gambiense.

Subjects

Smallpox, early inoculation for, 98
Smillie’s modification of Laidlaw’s
method of making anaérobic cul-
tures, 223
Smith’s method of cultivating tubercle
bacillus, 709
modification of Newman’s method
of staining flagella, 164
of Pitfield’s stain for flagella,
162
Smith’s (T.) fermentation tube, 62
phenomenon, 109
Sodoku, 540
Soil, bacteria in, 249

Somers’ modification of Starkey’s laby-

rinth, 649 rs
Soor, 457
Sore mouth, Ceylon, 693
Oriental, 569
Sozins, 114 és
Species of bacteria, identification, 235
Gram’s method for, 156
Specific precipitate, 106
precipitation, 123
Spermatoxin, 108
Spermatozoits, 501
Spinal paralysis, atrophic, 393
Spinale kinderlahmung, 393
Spirillum, 37
cholere asiatice, 608
cultivation, 612
detection, 618
Castellani’s method, 618
Gordon’s method, 618
Léffler’s method, 618
distribution, 609
general characteristics, 608
immunity against, 619
isolation, 612
metabolic products, 615
morphology, 610 ~
pathogenesis, 615
Schottelius’ method of making
pure cultures, 612
specificity, 617
spirilla resembling, 621
staining, 611
table for separating organisms
resembling, 628
toxic products, 615
vital resistance, 614
Metchnikovi, 625
obermeieri, 520.
cheta obermeieri.
of Denecke, 623
cultivation, 624
metabolic products, 624
morphology, 623
pathogenesis, 624
of Finkler and Prior, 621
cultivation, 621
metabolic products, 623
morphology, 621
pathogenesis, 623
staining, 621

See also

with

854 Index of
Staining flagella, Smith’s modification
of Newman’s method, 164
Van Ermengem’s method, 162
jar, Coplin’s, 154
Loffler’s method, 154
protozoa in tissue, 168
Biondi-Heidenhain method, 168
Heidenhain’s method, 168
Marino’s method, 167
Wrigat’s method, 166
spores, 159
Abbott’s method, 160
Anjeszky’s method, 160
Fiocca’s method, 160
Miller’s method, 160
Muir and Ritchie’s method, 159
Staphylococci in man, chief types,
309
Staphylococcus, 35
citreus, 316 -
epidermidis albus, 308
golden, 310
pyogenes albus, 308
aureus, 310
et albus, 310
agglutination, 316
bacterio-vaccination, 316
colonies, 312
cultivation, 311, 312
distribution, 310
isolation, 311
metabolic products, 313
morphology, 311
pathogenesis, 315
specific therapy, 316
staining, 311
thermal death-point, 313
toxic products, 313
virulence, 316
vital resistance, 313
Stanhylolysin, 314
Starkey’s labyrinth, Somers’ modifica-
tion, 649 :
method of isolating Bacillus coli and
Bacillus typhosus, 649
Steam sterilizer, Arnold’s, 174 .
Stegomyia calopus, 575
fasciata, 575
Stein’s method of staining Treponema
pallidum in films, 764
Sterilization, 170
by filtration, 175
in autoclave, 174
intermittent, 173
of corks, 172
of culture-media, 173
of forceps, 172
of glassware, 170, 172
of instruments, 170
of knives, 172
of ligatures, 177
of platinum wires, 172
of rubber stoppers, 172
of scissors, 172
of surgical instruments, 177

Subjects

Sterilization of wooden apparatus, 172
Sterilizer, steam, Arnold’s, 174 :
Sternberg’s method of determining
germicidal value of disinfectants,
260
Stewart’s cover-glass forceps, 152
Stock vaccines, 273
Stomach, bacteria in, 73
Stomatitis, parasitic, 457
Strahlenpitzkrankheit, 775
Straus’ method of diagnosis of glan-
ders, 751 :
Streptococcus, 35
conglomeratus, 319
differential diagnosis, 326
diffusus, 319
erysipelatis, 327
lanceolatus, 464
mucosus, 326, 464.
pyogenes, 317
cultivation, 318
differential features, 320
distribution, 317
general characteristics, 317
isolation, 318
metabolic products, 323
morphology, 318
pathogenesis, 320
reaction, 319
staining, 318
virulence, 322
vital resistance, 319
toxic products, 324
vaccine, 326
varieties and types, 324
viridans, 320
Streptokolysin, 323
Streptothrix, 39
actinomyces, 775
israeli, 775
madure, 787
Stroke culture, 213
Subinfection, 69
Substance sensibilisatrice, 121
Sucholotoxin, 667
Suctorial insects, infection by, 71
Sugar bouillon as culture-medium, 194
Sulphur bacteria, 30
grain, 778
Suppuration, 307
amebe and, 337
bacteria associated with, 308
Surgical contributions, 20
instruments, sterilization of, 177
Susceptibility from diet, 91
from exposure, 91
from fatigue, 91
from inhalation of noxious vapors,
gt
from intoxication, 92
from morbid conditions, 92
from mutilation of body, 92
from traumatic injury, 92
of host in infection, 90
Susotoxin, 667

Index of

Suspension, bacterial, 278
standardization of, 279
Sutures, disinfection of, 175
Swelled head, 775
Swine-plague, bacillus of, 607.
also Bacillus suisepticus.
Symbiosis, 57
effects of, on growth

57
Syphilis, 761
bacillus of, 76x.
ema pallidum.
chancre, 768
diagnosis, 769
by dark-field illumination, 770
by Noguchi’s method, 770
by serum, 770
by staining, 770
_ by Wassermann reaction, 287
lesions, 769
primary incubation, 768
secondary incubation, 769
tertiary symptoms, 769
Syphilitic antigen, 287
Syringe, bacteriologic, Altmann, 228
Koch’s, 227
Meyer’s, 227
Roux’s, 227

See
of bacteria,

See also Bacillus

Subjects 855

Toxemia, 85
Toxins, chemic nature, 83
Coley’s, 325
extracellular, 82
intracellular, 81
soluble, 82
specific action, 83
affinity of cells for, 84
Toxophylaxins, 114
Toxosozins, 114
Trachea, bacteria in, 76
Traumatic injury, susceptibility from
92
Trench fever, 532
Treponema, 37
pallidulum, 772
pallidum, 761
cultivation, 766
Noguchi’s method, 766
Zinsser, Hopkins and Gilvert’s
method, 768
distribution, 766
film staining, 762
Ghoreyeb’s method, 763
Goldhorn’s method, 762
Stein’s method, 764
general characteristics, 761
identifying, Burri’s India-ink
method, 765
lesions from, 769
morphology, 762
section staining, 765
Levaditi’s method, 765
Manouelian’s method, 765
specificity, 768
staining, 762
pertenue, 772 ;
cultivation, 773
morphology, 773
pathogenesis, 773
staining, 773
Trichomonas intestinalis, 698
Trichophyton, 43
acuminatum, 798
circonvulatum, 798
crateriform, 798
effractum, 798
exsiccatum, 798
flavum, 798
fulmatum, 798
glabrum, 798
microsporon, 798
pilosum, 798
plicatili, 798
polygonum, 798
regulare, 798
sulphureum, 798
tonsurans, 798
cultivation, 799
morphology, 799
pathogenesis, 799
umbilicatum, 798
violaceum, 798
Trikresol as disinfectant, 181
Trismus of tetanus, 359

856 Index of Subjects

Tropical diarrhea, 693
ulcer, 569 ‘
organism, 571
preventive inoculation, 572
transmission, 572
treatment, 572
Trypanosoma avium, 546
brucei, 546
castellani, 547
damonia, 546
equinum, 546
gambiense, 546
cultivation, 548
morphology, 547
pathogenesis, 552
reproduction, 548
staining, 548
transmission, 549
granulosum, 546
lewisi, 546
raja, 546
thodesiensi, 544, 547
morphology, 548
rotatorium, 546
solez, 546
theileri, 546
transvaliense, 546
ugandense, 547
Trypanosomiasis, American, 556. See
also Sleeping sickness, American.
human, 544
Tryptic enzymes, 63
Tsetse fly, 555
appearance, 555
breeding habits, 556
disease, 550
habitat, 555
habits, 555
larve of, 556
table for identification, 556
Tube, expanded, Sedgwick and Tuck-
er’s, for air-examination, 241
Keidel, 289
Tubercle bacillus, 699. See also Bacil-
lus tuberculosis.
crude, 716
giant-cells in, 715
miliary, 716
of Babes, 384
Tuberculin, 719
concentrated, 720
crude, 720
dangers from, 721
Denys’, 723
diluted, 720 ;
effect on tubercle bacillus, 720
Koch’s, 720
preparation, 720
refined, 720
test for bovine tuberculosis, 732
Tuberculin-R, 723
Tuberculin-TO, 724
Tuberculin-TR, 723
Tuberculinic acid, 719
Tuberculocidin, 723

Tuberculosamin, 719
Tuberculosis, bacillus of, 699. See
also Bacillus tuberculosis.
bovine, 728
communicability to man, 729
in young children, 732
lesions in, 729
prophylaxis, 732
tuberculin test for, 732.
complement-fixation test in, 726
diagnosis, Calmette’s ophthalmo-
tuberculin reaction, 723
von Pirquet’s cutaneous method,-
722
Ligniéres’ modification, 722
Moro’s modification, 722
distribution, 699
fish, 735
fowl, 733
latent, 717
lesions in, 714
prophylaxis, 727
pseudo-, 737
specific organism, 699
Tuberculous pneumonia, 485 ,
Tubes, Esmarch, 209
Tucker and Sedgwick’s expanded tube
for air-examination, 241
Typhoid fever, bacillus of, 629. See
also Bacillus typhosus.
bacteriologic diagnosis, 644
blood-culture diagnosis in, 644
carriers, 636, 641
Chantemesse’s ocular reaction in,
645
conjunctival reaction in, 645
histologic lesions in, 639
in lower animals, 640
isolation of bacillus from feces in,
645
method of detecting carriers, 641
mode of infection in, 634
pathogenesis, 635
prophylactic vaccination in, 641
prophylaxis, 640
specific therapy, 642
Widal reaction in, 644
pig, 666
Typhus abdominalis, 578, 629
exanthematicus, 578
fever, 578
inoculation into animals, 579
transmission by lice, 580
Tyrotoxicon, 61, 253
Tyrotoxism, 253

UBLENHUTH’S test, 124

Ulcer, tropical, 569 :

Unna’s method of staining sections for
tubercle bacillus, 706

Urethra, bacteria in, 76

Urine, Bacillus typhosus in, 638
staining tubercle bacillus from,

795 ae
Uterus, bacteria in, 75

Index of Subjects

VACCINATION, accidents, ror
efficient, 100 :
in anthrax, 372
inefficient, 100
Jennerian, 99
Pasteurian, ror
prophylactic, in typhoid fever, 641
Vaccines, 99
autogenous, 273
bacterio-, 271
dosage, 276
Haffkine’s, in Asiatic cholera, 619
method of making, 273
sensitization, 276
stock, 273
streptococcus, 326
Vaccinia, rox
Vacuoles, contractile, 49
Vagina, bacteria in, 75
Van Ermengem’s method of staining
flagella, 162
Vegetables, green, Bacillus typhosus
in, 635 :
Vibrio, 36
cholerz asiatice, 608
of Finkler and Prior, 621
proteus, 621
Vibrion septique, 339
Vibrionensepticemia, 626
Vincent’s angina, 451
Virulence, 85
decrease, 86
increase, 86
by addition of animal fluids to
culture-media, 87
by passage through animals, 86
by use of collodion sacs, 87
variation in, 86
Virus of acute anterior poliomyelitis,

394
street, in hydrophobia, 384
Viruses, filterable, 27
invisible, 27
von Pirquet’s cutaneous tuberculin
reaction, 722

WASSERMANN method of cultivating
Micrococcus gonorrhee, 412
reaction, 287
amboceptor dose in, 295
unit in, 294
antigen in, 287
blood-corpuscles for, 291
complement for, 290
fixation test, 300
hemolytic amboceptor for, 292
‘system in, 294
nature, 303
Noguchi’s reaction, 303
reagents employed, 287
serum to be tested in, 290
technic for, 287, 288
theoretic basis, 288
titration of guinea-pig serum for,
293

857

Wassermann reaction, titration of
hemolytic serum for, 293
of sheep corpuscles for, 293
validity of, 302
Water, bacteria in, 242
determination of purity, 244
completed test, 245
partially confirmed test, 245
presumptive test, 244
of total number of bacteria in
given sample, 242
cries Bacillus coli communis in,
3
potability of, 245
Weil’s disease, 532
bacteriological disease, 537
lesions, 537
prophylaxis, 539
sources of infection in, 538
stages, 535
treatment, 540
Welch’s gas bacillus, 342
method of staining Diplococcus
pneumoniz, 465
Wertheim’s method of cultivating
Micrococcus gonorrhcee, 411

| West’s apparatus for swabbing naso-

pharynx, 406
method of discovering cerebro-spinal
meningitis carriers, 406
Whey, Petruschky’s, as culture-me-
dium, 201 ;
Whooping-cough, bacillus of, 460.
See also Bacillus pertussis.
Widal reaction, 126
in typhoid fever, 644
Wildseuche, bacillus of, 606
Williams and Lowden’s method of
staining Neurorrhyctes hydropho-
biz, 380

Williams’ method of cultivating ame-_

be, 677
Wiston’s method of making syphilitic
antigen, 288
Wolfhugel’s apparatus for counting
colonies of bacteria on plates, 242
Wooden apparatus, sterilization of, 172 .
tongue, 775
Wool-sorters’ disease, 370
Wounds, disinfection of, 178
infection with tubercle
through, 713
Wright’s blood-stain for protozoa, 166
method of cultivating actinomyces
bovis, 778
of making anaérobic cultures, 222
Wurtz and Kashida’s method of cul-
tivating Bacillus typhosus, 646

X-Rays, influences of, on growth of
bacteria, 56
Xenopsylla cheopis, 591, 593, 602

Yaws, 772. See also Frambesia trop-
tea.
Yeasts, 40

bacillus

858 Index of

Yellow fever, 574
Bacillus icteroides and, 576
historical study, 574
mosquitoes and, 575
prophylaxis, 577
tules for prevention, 576
Young’s method of cultivating Micro-
coccus gonorrheee, 412

ZENKER’S fluid, 152
Ziehl’s method of staining Bacillus
typhosus, 631

Subjects

Ziehl’s method of staining tubercle
bacillus, 704

Zieler’s method of staining, 158

Zinsser’s method of making anaérobic
cultures, 219

Zinsser, Hopkins and Gilvert’s method
of cultivating Treponema pallidum, |

768

Zur Nedden’s bacillus, 427

Zygote, 501

Zymogens, 60

Zymophore group, 120

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