Gould's Pocket Pronouncing
Medical Dictionary
Eij
nounced
Thin,
$2.00, \
Wi
Bacilli,
and Me
both Ei
"The (
accuracy
nearly e
British 2
By GEORGE M. GOULD, A.M., M.D.
PO(
By
Dictiona
by R. J.
Gilt Ed
Eve
and tho
be given
thorough v
BIOLOQY
LIBRARY
G
Medical
n, Edited
I Corners,
e a brief
lich may
d treated
BLuuent desiring TX> refresh his
rds Pro-
30 Pages.
I Corners,
, Nerves,
, Weights
larians in
3 in which
to include
srature."
memory concerning any medical or surgical theme the book will
prove invaluable.
"In small and convenient size, with the text clearly printed and the
subject-matter condensed, this concentrated form of the same author's
larger work on medicine and surgery commends itself to the profession
for its handiness. The text is well arranged ... the definitions are
well written." Detroit Medical Journal.
5-8-22 2KM A
Hughes'
Practice of Medicine
ELEVENTH EDITION REVISED
GIVING THE SYNONYMS, DEFINITIONS, CAUSES, SYMPTOMS, PATHOLOGY,
DIAGNOSIS, PROGNOSIS, AND TREATMENT OF EACH DISEASE.
By DANIEL E. HUGHES, M.D., Late Chief Resident Physician,
Philadelphia Hospital; formerly Demonstrator of Clinical Medicine,
Jefferson Medical College. Edited by R. J. E. S( OTT, M.A.,B.C.L.,
M.D., Formerly Attending Physician to the Demilt Dispensary, New
York; Editor of Gould and Pyle's Cyclopedia of Medicine and
Surgery, etc.
63 Illustrations. 12mo. xix -f 785 Pages. Cloth, $4.25 postpaid.
The Treatment is specially full. 406
valuable prescriptions have been included.
"This popularity is due to the fact that the book gives practical
discussions of diseases as briefly as is consistent with the subject,
theories being eliminated." Journal American Medical Association.
"This book is intended as a working manual in which the
physician may find that which he needs for a rapid restudy of any
disease without having to go over the controversial matter usually
found in treatises on practice. * * * For case reference and rapid
study, there is no better book." Medical Council.
"The work is thoroughly modern and is particularly valuable
for its discussion of diagnosis and treatment, an immense number
of excellent prescriptions being incorporated under the latter head.
It is, we believe, unique in including sections on mental diseases and
diseases of the skin." New York Medical Journal.
The Practitioner's Medical
Dictionary
- Third Edition, Revised and Enlarged by R. J. E. SCOTT, M.A.,
B.C.L., M.D., Fellow of New York Academy of Medicine, etc. Many
thousands of new medical words are included in this edition. The
work contains over 70,000 terms, 962 pages and weighs only 2^ Ibs.
Bound in Handsome Flexible Cloth, Marbled Edges, Round Corners,
$4.00; with Thumb Index, $4.50.
Morris Human Anatomy
A Complete Systematic Treatise
6th Edition Revised and Largely Rewritten. With 1164 Illus-
trations. Cloth, $10.00..
CONTRIBUTORS Charles R. Bardeen, A.B., M.D., Professor of
Anatomy, University of Wisconsin, formerly Associate Professor
Johns Hopkins; Eliot R. Clark, A.B., M.D., Professor of Anatomy,
University of Missouri, formerly Associate in Anatomy, Johns
Hopkins University; Albert C. Eycleshymer, Ph.D., M.D., Professor
of Anatomy, University of Illinois; J. F. Gudernatsch, Ph.D., As-
sistant Professor of Anatomy, Cornell University Medical College,
New York; Irving Hardesty, A.B., Ph.D., Professor of Anatomy,
Tulane University of Louisiana; C. M. Jackson, M.S., M.D., the
Editor, Professor of Anatomy, University of Minnesota; Dean D.
Lewis, M.D., Associate Professor of Surgery in the Rush Medical
College, Chicago, Ills.; Richard E. Scammon, Ph.D., Assistant Pro-
fessor of Anatomy, University of Minnesota; J. Parsons Schaeffer,
Ph.D., M.D., Professor of Anatomy, Jefferson Medrcal College, Phila-
delphia; H. D. Senior, M.B., F.R.C.S., Professor of Anatomy, Uni-
versity and Bellevue Hospital Medical College, New York; G. Elliot
Smith, M.A., M.D., F.R.C.P., F.R.S., Professor of Anatomy, Uni-
versity of London; Charles R. Stockard, Ph.D., D.Sc., Professor of
Anatomy, Cornell University Medical College, New York; R. J. Terry,
A.B., M.D., Professor of Anatomy, Washington University, St. Louis,
Mo.
A very important feature of any textbook of anatomy is that
of the illustrations. They should be very well executed arid clear to
give the student a proper notion of the part under discussion. In
the new Morris, the illustrations have been carefully selected and
prepared. Many of them are printed in colors. This edition in-
cludes many new pictures redrawn to supplant the older ones. The
drawings are a distinguishing feature of Morris's Anatomy.
Microbiology and Microanalyses of Foods
131 Illustrations. 8vo. Cloth, $3.50. By ALBERT SCHNEIDER,
M.D., Formerly Microanalyst, U. 8. Bureau of Chemistry.
BACTERIOLOGY
P ITF I E LD
BLAKISTON'S COM PEN DS
The Best Series of Manuals for the Use of Students
Price of each, Cloth, $2.00 net
CSTThese Compends are based on the most popular text-books and the lectures of
prominent professors, and are kept constantly revised, so that they may thoroughly
represent the present state of the subjects upon which they treat.
t^~The authors have had large experience as Quiz- Masters and attaches of
colleges, and are well acquainted with the wants of students.
GP^They are arranged in the most approved form, thorough and concise,
containing over 900 fine illustrations, inserted wherever they could be used to
advantage.
t^"Can be used by students of any college.
t^They contain information nowhere else collected in such a condensed, practical
shape.
Illustrated Circular Free
POTTER'S ANATOMY. Eighth Revised and Enlarged Edition. Including
Visceral Anatomy. Can be used with either Morris' or Gray's Anatomy.
139 Illustrations and 16 Plates of Nerves and Arteries, with Explanatory Tables,
etc.
BRUBAKER. PHYSIOLOGY. Fifteenth Edition, with 26 Illustrations. Enlarged
and Revised.
LANDIS. OBSTETRICS. Ninth Edition. Revised and Edited by WM. H.
WELLS, M. D., Late Associate Professor of Obstetrics, Jefferson Medical College,
Philadelphia. 80 Illustrations.
POTTER. MATERIA MEDICA, THERAPEUTICS AND PRESCRIPTION
WRITING. Eighth Revised Edition.
WELLS. GYNECOLOGY. Fourth Edition. With 153 Illustrations.
GOULD and PYLE. DISEASES OF THE EYE AND REFRACTION. Including
Treatment and Operations and a Section on Local Therapeutics. With Formulae
and 109 Illustrations, several of which are in colors. Fourth Edition.
LIPSHUTZ. COMPEND OF SURGERY. 185 Illustrations.
This volume replaces the Compend of Surgery formerly written by the late
Orville Horwitz, M. D.
LEFFMANN. CHEMISTRY, Inorganic and Organic. Sixth Edition. Including
Urinalysis, Animal Chemistry, Chemistry of Milk, Blood, Tissues, the Secretions,
etc.
STEWART. PHARMACY. Ninth Edition. Based upon Prof. Remington's
Text-book of Pharmacy.
ST. CLAIR. MEDICAL LATIN. Second Edition.
SCHAMBERG. DISEASES OF THE SKIN. Sixth Edition. Revised and
Enlarged. 119 Illustrations.
PITFIELD. BACTERIOLOGY. Fourth Edition. 82 Illustrations.
HIRSCH. GENITO -URINARY AND VENEREAL DISEASES, AND SYPHILIS.
Third Edition. With 59 Illustrations.
BJL AKISTCM'S C O NX F K 1SL P S
A COMPEND
ON
BACTERIOLOGY
INCLUDING
PATHOGENIC PROTOZOA
BY
ROBERT L. P1TFIELD, M. D.
PATHOLOGIST TO THE GERMANTOWN HOSPITAL; LATE DEMONSTRATOR OF
BACTERIOLOGY AT THE MEDICO-CHIRURGICAL COLLEGE, PHILA-
DELPHIA; VISITING PHYSICIAN TO ST. TIMOTHY'S HOS-
PITAL AND CHESTNUT HILL HOSPITAL, PHILA.
FOURTH EDITION
WITH 4 PLATES AND 82 OTHER
ILLUSTRATIONS
PHILADELPHIA
P. BLAKISTON'S SON & CO
1012 WALNUT STREET
v n
COPYRIGHT, 1922, BY P. BLAKISTON'S SON & Co.
PRINTED IN U. S. A.
IY THE MAPLE PRESS YORK PA
PREFACE
This little book was designed by the writer to serve the needs
of the medical student preparing for examination, and for the
prac itioner of medicine who desires to acquaint himself with the
principle facts of the rapidly growing science of bacteriology. An
effort has been made to reduce the subject matter to as concrete
a form as possible.
While the literature of the subject of immunity is as vast almost
as the rest of bacteriology, yet it is hoped that the chapter in this
book on immunity gives in outline the essential accepted teachings
on the subject.
Minute details of cultures and technic are not given. They
must be sought for in books on descriptive bacteriology.
The author has drawn very freely from many standard text-
books. Many illustrations are from Kolle & Wassermann's
Atlas, Park and Williams, Williams, McFarland, Tyson's Prac-
tice and Abbott.
The writer's best thanks are tendered to Dr. Herbert Fox of
the University of Pennsylvania (Pepper Laboratory) to whom
entire credit is due for the chapters on filterable viruses; the re-
arrangement of the chapters, and the new matter that has been
added throughout the book.
ROBERT L. PITFIELD.
4 8 885 3
TABLE OF CONTENTS
CHAPTER I
PAGE
THE CLASSIFICATION, MORPHOLOGY, AND THE BIOLOGY OF BACTERIA. . . i
CHAPTER II
PRODUCTS OF BACTERIAL ENERGY 23
CHAPTER III
INFECTION 30
CHAPTER IV
IMMUNITY 46
CHAPTER V
STUDY OF BACTERIA 95
CHAPTER VI
BACTERIOLOGICAL LABORATORY TECHNIC 108
CHAPTER VII
ANTISEPTICS AND DISINFECTANTS 135
CHAPTER VIII
BACTERIA 143
CHAPTER IX
ANIMAL PARASITES '. 232
CHAPTER X
THE FILTERABLE VIRUSES 255
CHAPTER XI
BACTERIOLOGY OF WATER, SOIL, AIR AND MILK 278
INDEX 287
vii
COMPEND OF BACTERIOLOGY
CHAPTER I
THE CLASSIFICATION, MORPHOLOGY, AND THE
BIOLOGY OF BACTERIA
BACTERIA (fission fungi or schizomycetes) may be defined
as very minute unicellular vegetable organisms, almost always
devoid of chlorophyll, and generally unbranched, that reproduce
themselves asexually by means of direct division or fission, spores,
or gonidia. They are allied closely on the one hand to the higher
fungi, such as the moulds, and on the other to the algae. Many
forms in one phase of development closely resemble members of
other groups, and it has always been difficult to classify them.
Various botanical classifications have been employed by different
bacteriologists. The following one is based somewhat upon
Migula's, and that adopted by Lehmann and Neumann, which was
compiled from the systems of Flugge, Fischer, Ldffler, and Migula.
CLASSIFICATION. Bacteria may be conveniently divided
into six families, according to their morphology or shape.
I. COCC AC EJE. Spherical or spheroidal bacteria. Globular
in free state but usually seen with one axis slightly larger.
They do not have parallel sides like the bacilli. To mul-
tiply, the cell divides into halves, quarters, or eighths, each
of which grow again into perfect spheres. Endospores
and flagella are very rare (Lehmann and Neumann). If
mobile they are called Planococcus or Planosarcina.
2 . . . BACTERIA
(a) Streptococcus. Cells that divic^e in one direction only
and grow in chains.
(b) Micrococcus. Cells that divide in two directions, or
irregularly; with this group staphylococcus may be
classed. Also tetrads, which form into fours by division
in two directions.
(c) Sarcina. Cells that divide in three directions so that
bale-like packages, or blocks of eight are formed. At
least one variety (Sarcina agilis) is motile, having fla-
gella. Plates of cocci, one thick in the plane, are called
" merismopedia""
II. BACTERIACE^:. ROD bacteria are straight or slightly
curved. Each cell is from two to six times as long as broad.
Division takes place in one direction only, and at right angles
to the long axis. Spores may be produced or may not.
They may have flagella, or may not.
(a) Bacterium.- Neumann Have no endospores. Migula
no flagella.
(b) Bacillus. Neumann Have endospores, and often grow
in long threads. Migula Flagella present at any part
of cell, peritrichic in arrangement.
(c) Pseudomonas. Have endospores very rarely. Flagella
only at ends.
in. SPIRILLACE^:. -Spiral bacteria. Unicellular, more or less
elongated. Twisted more or less like a corkscrew. Cells
are sometimes united in short chains. Generally very
motile. Spores are known in two varieties only.
(a) Spirosoma. Rigidly bent. No flagella.
(b) Vibrio or Microspira. Cells that are rigidly bent like
a comma, and have always one, occasionally two polar
flagella.
(c) Spirillum. Are long and spiral, like a corkscrew, are
rigid, and have a bunch of polar flagella.
CLASSIFICATION 3
(d) Spirochaeta. Cells with long flexible spiral threads,
without flagella. Some move by means of an undulating
membrane. These have been thought to belong to the
bacteria but those that move by an undulating membrane
should be. classified with the protozoa.
IV. MYCOBACTERIACE^S. Cells as short or long filaments,
which are often cylindrical, clavate (club-shaped), cuneate
or irregular in outline, and display true or false branching.
Spores are not formed, but gonidia are. They have no fla-
gella, and division takes place at right angles to the long
axis. There is no surrounding sheath as in the next family (V) .
(a) Mycobacterium, Cells are short cylindrical rods, some-
times wedge-like, bent, or Y-shaped : long and filamentous.
They exhibit true branching, and perhaps produce coccoid
elements and gonidia, but no flagella. The Corynebac-
terium of Lehmann and Neumann belongs to this group.
Many are acid-fast.
(b) Streptothrix or Actinomyces (ray fungus) are long
mycelial threads, that radiate in indian-club, or loop-like
forms, with true branching and delicate sheaths, devoid of
gonidia and flagella. Growth coherent, mould-like and
dry. Often powdery on the surface in culture media,
frequently emitting a musty odor. A few species are
weakly acid-fast.
V. CHLAMYDOBACTERIACE^:. Sheathed bacteria. Cells
are characterized by an enveloping sheath about branched
and unbranched threads. Division takes place at right
angles to the long axis of the cells.
(a) Cladothrix are distinguished by false dichotomous
branching. Multiplication is affected by separation of
whole branches, and by swarm spores or motile gonidia
having flagella.
4 BACTERIA
(b) Crenothrix. Filaments are fixed to a nutrient base.
Are usually thinner at the base than at the apex, formed
of unbranched threads that divide in three directions of
space, and produce in the end two kinds of gonidia,
probably of bisexual nature.
(c) Phragmidiothrix. Cells are first united into unbranched
threads by means of delicate sheaths, branching threads
are then formed. Division takes place in three directions
of space, producing sarcina-like groups of gonidia,
which, when free, are spherical.
(d) Thiothrix. Are unbranched cells, sheathed, without
flagella, divided only in one direction^ and contain sulphur
granules.
VI. BEGGIATOACE^:. Cells united to form threads that are not
sheathed: have scarcely visible septa; divide in one direction,
and motile only by an undulating membrane, not by flagella.
(a) Beggiatoa. Cells containing sulphur granules.
Bacteria may furthermore be classified according to their biolog-
ical characteristics, which may be wonderfully different. The ulti-
mate differentiation of one species from another depends not only
on the morphology, which may be precisely similar, but on its bio-
logical behavior in culture media and in the tissues of animals un-
der identical conditions. Again, different individuals of a given
species may vary extraordinarily one from another in form and
size, yet the chemical behavior is invariably the same. Hence it
is only by observation of the development of bacteria in culture
media, and the reactions produced in it, and in the bodies of ex-
periment animals, that we can identify them positively from others
of a foreign species. No bacteriologist is able by a simple micro-
scopical examination of a given bacterium, to identify it absolutely
at all times.
The higher groups of fungi may be classified conveniently as
follows :
CLASSIFICATION 5
BLASTOMYCETES-YEASTS. Budding fungi. Character-
istic lies in predominant round or elliptical unit; some few form
mycelia; division by endospores or budding; important in fer-
mentation and in disease. Divided into:
Saccharomyces. Endospores and budding, fermenters. No
mycelia.
Monilia. Budding. No spores mycelia fermenters.
Oidia. Budding. No spores mycelia non-fermenters.
Coccidioides. Spores. No budding mycelia non-fermen-
ters.
HYPHOMYCETES-MOULDS. Mycelium-forming fungus;
division by spores, branching, budding, or intercalary division;
some bisexual. Divided into:
Phycomycetes. Mucorinae sometimes bisexual division by
grouped spores or segmentation of mycelium. Not important
pathologically. Example Mucor.
Mycomycetes. Asexual forms dividing by spores in a sac
or by end organs sexual forms dividing by specially developed
cells. Mycelia predominate. Example Aspergillus.
These are the principal groups of yeasts which can be reasonably
well classified. There are others, Microsporon, Trichophy ton, and
Sporothrix, that have a decided pathogenic importance but for
which a systematic position is not easy to give. They belong
probably between the two above classes in that mycelial growth
with lateral budding and spore formation are their characters.
Bacteria that are globular in form are called cocci.
Cocci that divide in one direction of space and grow in chains
are called streptococci (Fig. i).
Cocci that divide irregularly and form parrs of fours, or irregular
groups, are called micrococci. Those of this class that form pairs
are frequently called diplococci. 1 When they form fours by divi-
sion in two directions, they are called tetrads. But when they
1 This word is frequently used as if it were a biological term indicating some
species identity, e.g., Diplococcus pneumoniae. There is no biological
group called Diplococci and the term should be used in a descriptive sense.
The cause of pneumonia is now called Streptococcus pneumoniae.
BACTERIA
divide irregularly and form masses resembling bunches of grapes,
they are spoken of a staphylococci (Fig. 2).
Coeci that divide in three directions are called sarcina. One
single coccus, by division in three directions, forms cubes of eight
FIG. i. Large and very large streptococci. (Kolle and Wassermann.)
or more, each of which becomes globular and equal in size to the
parent.
Motile micrococd are those that divide in two directions of space
and have flagella. They are known as planococci.
FIG. 2. Staphylococci. Streptococci. Diplococci. Tedrads. Sarcinae.
(Williams.)
Micrococci that divide in three directions, and are motile, are
called planosarcina (Fig. 3).
Bacteria that resemble straight rods are called bacilli. These
may be short and thick, or long and thread-like; are never curved,
but may be slightly bent.
CLASSIFICATION
Bacilli may grow singly or in chains; may be flagellated; contain
spores and gonidia; or, may be devoid of flagella.
Members of the spirillaceae that resemble a curved rod, or are
FIG. 3. Planosarcina ureae, showing very long flagella.
(Kolle and Wassermann.)
comma-shaped, are known as vibrios (Fig. 4). Those of the same
family that resemble a corkscrew, are called spirilla. When they
are like long spiral threads they are called SpirocJUttd (Fig. 5) .
Any of these different members of the
family of Spirillaceae may grow in chains.
In clinical medicine it is common to speak
of the streptococcus pneumonia as the pneu-
mococcus. As the organism appears in the
diseased lung, or in the sputum, one diameter
of the coccus is invariably longer than
another, and the rule of equal diameters
cannot be applied to it. But in culture
media, the organism resembles a true coccus,
being globular and growing in chains. It is then called the
Streptococcus pneumoniae. It is common also to speak of mem-
bers of the family of Mycobacteriacea as bacilli, as they are more
FIG. 4. Cholera
vibrios.
(Greene's Medical
Diagnosis.)
8 BACTERIA
commonly met with in this form in clinical examinations, and in
cultures. Hence, we frequently hear of the bacillus of tubercu-
losis, and not the Mycobacterium tuberculosis.
Among the higher bacteria, the differentiation of those belong-
ing to the sheathed group, or Chlamydobacteriacea, is difficult, as it
depends largely upon the formation of the false branching and
the gonidia. When bacteria exhibit many, or various forms,
in the same culture, as does the typhoid bacillus, we speak of
FIG. 5. Spirochaeta of relapsing fever. (Kolle and Wassermann.)
them as pleomorphic, or as showing pleomorphism. To eluci-
date: Man is pleomorphic, because among adult individuals
some are tall or short, fat or thin.
Involution or Degeneration Forms. When the best or opti-
mum conditions for bacterial life (see page 18) are not found,
bacteria present appearances quite different from those of the
young, active or perfect adult type. These changes are called
involutionary if temporary, or degenerative if permanent.
For example: the diphtheria bacillus under good conditions for
life is a straight or slightly bent rod staining in a granular manner.
CLASSIFICATION Q
If living under unsuitable conditions it becomes quite short,
and stains solidly. Again, bacilli that are accustomed to appear
as short elements may grow to long threads without dividing,
or swell into unrecognizable form. Branching is sometimes
seen in rods and spirals, a condition due in certain cases to
involution, in others naturally among the higher bacteria.
To measure bacteria, we use the thousandth part of a milli-
meter, called the micromillimeter, or micron, as the unit. The
Greek letter ju is the symbol for this unit. A micron is about
M5>ooo of an inch, yet a bacterium i n long, and % fj. in width,
is very large in comparison to some things that scientists measure,
such as the thickness of oil films, soap bubbles, or light-wave
lengths, in which the unit is a micromicron, and is symbolized
by /*/*. The shortest light-wave lengths are about 400 /*/*> or
.4 ju, while chromatic threads in cells of bacteria are often 100 w
in width. Then again there are many things smaller than these
threads. The thinnest part of a bursting soap bubble is but
7 juju in thickness. There are certain infectious agents that are
submicroscopic; that is, invisible even by the aid of Siedentopf's
ultraviolet microscope, which shows objects smaller by half a
light- wave length (.2 AIJU).
The structure of the bacterial cell is very simple, consisting of
a delicate poorly staining limiting membrane or wall enclosing
a mass of substance with strong affinity for basic dyes like
methylene blue. Just what part of the bacterial interior is
cytoplasm and what is nucleus is not definitely known. Some
observers believe that all that is stained is chromatin, or nuclear
matter diffusely distributed through the bacterial cell, while
others think that a delicate cytoplasm exists under the wall and
that it is overshadowed by relatively great proportional bulk
of the nucleus.
In the course of the rod we often see metachromatic bodies,
called the Babes-Ernst granules, and unstained spaces called
vacuoles, both of which are common to many bacteria. They
10 BACTERIA
may be ingested substances but some are lipoidal or carbohy-
drate in nature. These bodies are demonstrated by staining
with basic dyes and may be of importance in determining the
mycobacteria. It is thought that they play a role in reproduc-
tion (Fig. 65).
The food of the bacterium passes through the cell wall by
osmosis. The cell wall of certain organisms, for example the
FIG. 6. Zooglea formation. (Leuconostoc.) (Kolle and Wassermann.)
pneumococcus, undergoes a change whereby a mucilaginous or
gelatinous capsule is formed outside the cell wall. Its use is
not known. The cell wall is generally the first portion of the cell
to be attacked by certain specific substances (ferment) found
in the blood of immunized animals, called bacteriolysins and
agglutinins. Where great masses of bacteria are clumped in
excessive mucilaginous material we speak of this condition as
zooglea (Fig. 6).
We sometimes find, as a prolongation of the cell wall, filament-
ous organs of locomotion known as flagella. Bacteria without
flagella are sometimes called gymnobacteria, those possessing
CLASSIFICATION
II
them, trichobacteria but these terms are falling into disuse
because the latter is now-a-days applied to higher groups that grow
in hair like forms. However the following may be described:
When they have one flagellum we call them monotrichous bacteria,
and amphitrichous when there are two flagella, one at each pole
(Fig. 7). When the cell is surrounded by flagella, it is known as
a peritrichous bacterium, and lopho-
trichous when the flagella are ar-
ranged in tufts of two or more.
These are simple adjectives and not
FIG. 7. Spirillum undula with polar
flagella. (Kolle and Wassermann.)
FIG. 8. Bacillus proteus vul-
garis, showing peritrichous fla-
gella. (Kolle and Wassermann.)
now used as terms of classification. The tetanus bacillus is an
example of a peritrichous organism, while the bacillus of green
pus is called monotrichous, because of its single flagellum.
Flagella are not pseudopods, but distinct organs of locomo-
tion.
In certain bacteria of the Beggiatoa, locomotion is accom-
plished by a peculiar amoeboid motion, or by an undulating
membrane. On looking at bacteria known to have no powers
12 BACTERIA
t
of voluntary motion, they are seen to oscillate, tremble or move
slightly. Suspensions of india-ink in water are seen to do the
same thing, as are other inanimate suspensions. This molecular
movement is known as the Brownian motion. By ordinary
staining methods, and in preparations of living bacteria known
to be flagellated, these organs of locomotion cannot be seen,
as a rule. Occasionally, however, one may be seen under either
condition. Generally, strong solutions of aniline dyes, to which
powerful mordants have been added, are necessary to stain the
capsule of bacteria and the attached flagella. The motion of
bacteria varies from a simple rotatory, on one axis, to a swing-
ing, shaking, boring or serpentine action. The location of the
flagella has some influence upon the motion they impart.
Flagella may be broken off from the cell body by agitation,
but when separated may still be clumped by agglutinating sera.
Flagella may have other functions than locomotion. It is
possible that they serve as organs for the absorption of nour-
ishment from the surrounding media. The presence of very long
or very numerous flagella does not necessarily presage very
active motion. At times, under certain conditions, an organism
ordinarily motile and flagellated will appear immobile and non-
flagellated (Lehmann and Ziferler), but this is rare. Certain
flagella have in their continuity little round granules, or bodies,
which apparently have nothing to do with the functions of
locomotion but may have something to do with the nutrition
of the cell. The test of motility of a bacterium is to see it pro-
gress by itself completely across the field of the microscope.
REPRODUCTION. The process of direct cell division is
the commonest way by which bacteria multiply; hence comes
the name of fission fungi. The ways of reproduction of the
bacteria high in the scale are by direct division, branching, and
by means of spores, and by other granules called gonidia. The
spores appearing in the lower bacteria, bacilli for example, are
not reproduction forms but states of high resistance.
SPORULATION 13
The process of direct or binary division is very simple, and
may be a matter of twenty minutes, or as long as six hours.
Division is almost always across the cell in the direction of the
short axis, though it may in some bacteria be in a direction
parallel to the long axis, but this is uncommon.
By means of the hanging-drop or the block-culture method,
on an inverted cover-glass the process may be observed easily.
The phenomena of division begin by an elongation of the cell,
soon followed by a constriction of pinching in of the cell on both
sides, at an equatorial point. The process begins to be apparent
in the cell wall and extends inward.
Division may occur in one, two, or three directions, or planes.
By cell division bacteria multiply by geometrical progression.
One cell at the end of a period becomes two, and at the end of a
second period these two become four; at the end of another
period these four become eight; after twenty-four hours they
may number many millions.
It is well that the food supply soon gives out and that the
products of bacterial metabolism, such as acids and ferments,
inhibit their growth. By this rapid bacterial multiplication,
carcasses of animals are disintegrated and the higher nitrogenous
compounds are reduced to simple gases that are quickly dissi-
pated in the air.
SPORULATION. Sporulation is of two kinds: the first and
most important for hygiene is that into which some pathogenic
bacteria go when they meet unfavorable conditions and it affords
protection against all but the most vigorous disinfection; the
second kind is a specialized function of the higher bacteria and
moulds by which reproduction occurs (vegetative). In the
latter case it is not impossible that some sexual specialization
occurs. The first mentioned are called Endospores.
Vegetative sporulation corresponds to the flowering of the
higher plants, and is observed under the most favorable vital
conditions. Endospores are produced under stress of circum-
BACTERIA
stances, when certain agencies or conditions, such as absence of
food, drying, and heat, threaten the extinguishment of the organ-
ism. Spores are bright, shining, oval, or round bodies, which do
PIG. 9. The formation of
spores. (After Fischer from
Frost and McCampbell.)
FIG. 10. Spores and their location in bac-
terial cells. (After Frost and McCampbell.)
not take aniline dyes readily, and which, when they are stained,
retain the color more tenaciously than the adult cells. They
resist heat, often withstanding a temperature of i5oC. dry heat
for an hour. Steam under pressure at a temperature of i5oC.
will invariably kill them after a short exposure.
fl 8
FIG. ii. Spore germination, a, direct conversion of a spore into a bacillus
without the shedding of a spore-wall (B. leptosporus) ; b, polar germination of
Bad. anthracis; c, equatorial germination of B. suUilis; d, same of B. mega-
terium; e, same with "horse-shoe" presentation. (After Novy.)
Spores are situated either in the ends of the adult organism
(polar) or in the middle (equatorial), and the spore is discharged
(sporulation) either from the end or through the side.
SPORULATION . 15
The spore is developed in the bacterial cell as follows: If
the organism is a mobile one it becomes quiet before sporulation,
during which the flagella are retained. The position of the
spore is early marked by a granularity of the bacterial body at
one point, an area soon assuming a clear glistening character,
often with a double contour, which may or may not increase the
thickness of the cell. If unfavorable conditions continue the
cell body disintegrates and disappears leaving the spore bare.
?IG. 12. Capsules. Bad. pneumonia (Friedlander;. (After Weichselbaum
from Frost and McCampbell.)
Certain spore bearing bacteria grown for a week at 42C. lose
the power to form spores; likewise their progeny. As a rule the
anthrax bacillus does not form spores in the bodies of animals.
Free oxygen is required for sporulation by some bacteria. One
spore only is produced by an adult cell. Some forms of bacteria
can be differentiated from each other only by the way in which
they sporulate, whether from the poles or the equator.
Spores are formed chiefly by the rod-shaped bacteria especially
the anaerobic and saprophytic organisms and these varieties
always have a high thermal death-point. Certain round bodies
I 6 . BACTERIA
found in bacteria of high thermal death-point, are called by
Heuppe arthrospores. It is believed that they are without
significance. Arthrospores are common among the micrococci
and may be associated with capsule formation and cell enlarge-
ment. The whole cell may stain more intensely. They are also
to be sought among the Streptothrix genus.
Spores resist chemicals for a long period, and withstand drying,
even in lime plaster, for years. It is believed that the thick
capsule enables them to resist these deleterious agents.
FIG. 13. Pest bacilli showing capsules. (Kolle and Wassermann.)
Sporulation is more apt to occur under poor nutritive conditions.
The anthrax bacillus thrives at i3C. but cannot sporulate
below i8C. Anthrax spores have been known to resist the
germicidal action of a 5 percent carbolic acid solution for forty
days.
Capsules. Certain well-known pathogenic bacteria have
thick well-marked capsules. The pneumococcus, pneumobacillus,
and Bacillus aerogenes capsulatus, are well-known examples of
such capsulated organisms. The capsule is not always constant.
It often disappears when the organism is grown in culture
media (Figs. 12 and 13).
THE CHEMICAL COMPOSITION OF BACTERIA 17
The higher bacteria are those from the Mycobacteriacea up to
the yeasts and moulds. They are higher than the Bacteriacea
because they tend to form truly or falsely branching filaments
and specialized segments, gonidia, which may behave as sex
organs. Few of them are pathogenic, except in the genera
Mycobacterium and Streptothrix. To the former belongs the
diphtheria and tubercle bacillus, both of which are said to have
branching involution forms, while to the latter belong the organ-
isms of actinomycosis and Madura foot. The Chlamydo-
bacteriacea and Beggiatoa are Saprophytes. These require special
technique for the laboratory culture.
The Yeasts or Blastomycetes or budding fungi are next in order.
They consist of sharply and doubly outlined, refractive, oval
bodies which may grow out into short stalks called mycelia. They
grow well in the laboratory and may produce pigments. They
are much larger than the bacteria (10-25 /x long). They multi-
ply by budding with a separation and removed growth of the
young form. They may produce a local or general infection in
man, Blastomycosis. They are used in beer making. The
commonest genus is Saccharomyces.
The Moulds or Uyphomycetes represent the next highest group
of the plant algae. They are characterized by a greater promi-
nence of the mycelium over simple segments or bodies. They
are widespread in nature and many are pathogenic. They
multiply by segmentation of the mycelia into gonidia or by the
development of special spore masses called sporangia. Fur-
ther refinements of the spores into sexual elements is known.
They are chiefly of interest to the physician on account of the
skin diseases that they occasion.
THE CHEMICAL COMPOSITION OF BACTERIA
Bodies of bacteria contain water, salts, certain albumins, and
bodies that may be extracted with ether. Among the latter are
lecithin, cholesterin, and triolein. In acid-fast organisms, fatty
1 8 BACTERIA
acids and wax have been found. In others, xanthin bases, cellu-
lose, starch, chitin, iron salts, and sulphur grains have been dis-
covered. The essential protein of the cell body is highly nitrog-
enous and is usually combined with some carbohydrate as a
glyconucleo-protein. The salts in the ash are mostly composed
of various phosphates. Intracellular toxins in combination
with the cytoplasm are found in certain groups of bacteria,
e.g., B. typhosus.
BIOLOGICAL CONDITIONS
Bacteria are arbitrarily classes as parasites, or saprophytes.
They may be so dependent upon the tissues of the infected
organism as to be a strict parasite and incapable of growth under
any other condition (Mycobact. leprce), or they may be capable
of life on artifical culture media (tubercle bacillus), or of life in
the body, on culture media containing organic matter (influenza
bacillus), or in the soil (B. tetani).
Saprophytes are bacteria capable of living upon dead organic
matter, in soil, in water, in air; they are not parasitic and do not
resist the defenses of the living body.
Certain biological conditions are essential for the growth of
bacteria: water, oxygen, carbon ; nitrogen, and salts are neces-
sary. For certain parasitic bacteria, highly complex substances
are indispensable: meat albumins, peptones, milk, egg albumin,
blood serum, and sugars are the ingredients of various culture
media.
The chemical reaction of such media is important: it should
either 'be faintly acid or faintly alkaline. The greatest number
of water bacteria grow in media that are slightly acid, while
diphtheria produces its strongest toxins and grows best in
alkaline media. Salt-free media is required for a number of
pathogenic bacteria, e.g., the gonococcus, B. leprae.
All bacteria require for their growth either free oxygen, as in
air, or combined oxygen, as in albumin, water, etc. Those that
BIOLOGICAL CONDITIONS 1 9
only grow when deprived of free oxygen are known as obligate
anaerobes, while those that require the presence of oxygen are
called obligate aerobes. Those that grow under either conditions
are named facultative anaerobes. Free oxygen is needed for spore
formation by certain bacteria. Anaerobes obtain oxygen as
they need it by breaking up their foodstuffs.
Nutriment is most important for the growth of bacteria,
nitrogenous compounds (albumins) particularly being required.
Simple aquatic forms of bacteria can live and grow in distilled
water. The addition of the various sugars is of advantage in
the cultivation of many bacteria, and glycerine for the growth
of some members of the Mycobacteriacea. Blood serum or whole
blood is required by some pathogenic organisms. The foodstuffs
must be in a form that can diffuse through the cell wall.
The temperature of the medium in which various bacteria
grow is most important. Bacterial growth is possible between
oC. and 7oC., some varieties thrive at the one extreme, and
others at the other.
Psychrophilic bacteria, are those that grow at i5C., with a
maximum of 3oC. and a minimum of oC. Water bacteria of
the polar seas belong to this group.
Mesophilic grow best at 37C. the temperature of the body
and thrive from ioC. (minimum) to 45C. (maximum). All
pathogenic bacteria belong to this group.
Thermophilic (min. temp. 4oC., max. 6o-7oC.) are most
prolific at 50-5 5 C. To this class belong bacteria of the soil.
All of this class are spore-bearing.
Darkness favors bacterial growth.
Association of different kinds of bacteria is of some importance
in their growth and welfare and when thus associated, they some-
times benefit each other. Such combination is called symbiosis.
Antibiosis is the condition when one or more of a mixture of
organisms suffers by the presence of others, e.g., the destruction
of putrefactive germs in the intestinal tract by lactic acid bacilli.
20 BACTERIA
Certain anaerobic bacteria grow in the presence of oxygen if
other particular varieties of aerobic bacteria are present.
Attenuated tetanus bacilli become virulent if cultivated with
Bacterium vulgae. Again, complicated chemical changes, as the
decomposition of nitrites with the evolution of nitrogen cannot
be accomplished by certain bacteria severally, but jointly, this
is quickly brought about.
Pfeiffer has shown that certain chemical substances (foods,
albumins, etc.), attract bacteria (positive chemotaxis) , while
other substances, as turpentine, repel them (negative chemotaxis).
Oxygen repels anaerobes and is particularly attractive to aerobes.
FREE AGENTS PREJUDICIAL TO THE LIFE OF
BACTERIA
High temperatures are surely germicidal: 6oC. coagulates
mycoprotein of bacteria and other common albumins. The degree
of temperature at which bacteria are killed is called the thermal
death-point. Most vegetative forms die after a short exposure at
6oC., though some require a higher temperature, e.g., tubercle
bacillus.
Spores resist boiling, often for hours. Spore-bearing bacilli
from the soil often survive a temperature of ii5C. moist heat
(steam), from thirty to sixty minutes. Bacteria resist dry heat
of i75C. from five to ten minutes.
Cold inhibits bacteria; destroys some; but is not a safe germi-
cidal agent, as typhoid bacilli have been isolated from melted ice
in which they had been frozen for months.
Ravenel exposed bacteria to the extreme cold of liquid air
( 3i2F.) and found that typhoid bacilli survived an exposure
of sixty minutes; diphtheria, thirty minutes, and anthrax spores,
three hours; during this exposure, however, many were destroyed.
Light is inimical to the life of bacteria, direct sunlight being the
most germicidal, as it destroys some, reduces the virulence of
AGENTS PREJUDICIAL TO BACTERIAL LITE 21
others, or interferes with the chromogenic properties. Typhoid,
cholera, diphtheria, and many other organisms are killed after an
hour or two's exposure to bright sunlight. The ultraviolet or
actinic rays are the efficient ones. If free oxygen is excluded, the
germicidal action is very materially reduced. Sunlight acting
on culture media (free oxygen and water being present) produces
after ten minutes, peroxide of hydrogen. This action of light on
bacteria has been extensively used, notably by Hansen, as a
therapeutic measure for the cure of bacterial skin diseases, espe-
cially lupus. Diffuse sunlight, electric light, Rcentgen-rays, con-
tinuous and alternating currents of electricity, are also more or
less germicidal. Antiseptics, such as metallic salts, formalin,
carbolic acid, cresol, mineral acids, and essential oils, are powerful
germicides; some even in high dilution.
According to Koch, absolute alcohol, glycerine, distilled water,
and concentrated sodium chloride solution do not affect anthrax
spores, even after acting on them for months. Halogen elements
(iodine, bromine, chlorine) are the most powerful germicides.
Free acids and alkalies must be very strong to act as disin-
fectants. Excessive amounts of sugar, salt, glycerine, and the
pyroligneous acids act as destroyers, or inhibitors to bacterial
growth in foodstuffs.
Metals act as lethal agents in the presence of light and water, by
forming metallic peroxides, which either destroy the vitality of
bacteria or hinder their growth. Silver, zinc, cadmium, bismuth,
and copper, have this action. Consequently silver wire and foil,
are used in surgery because of their antiseptic action. Metallic
fillings in teeth prevent the growth of bacteria that cause caries.
Certain cells in the bodies of animals (leucocytes) and some ele-
ments of the blood serum, being bactericidal, are a powerful means
of internal defense against infection.
If the water of the cytoplasm of bacterial cells is dried out, the
vitality of the organism suffers. The length of time required for
drying varies, anthrax spores resisting the process for over ten
22 BACTERIA
years. Ancient methods of preserving foods from putrefying, and
which are still in vogue, depend upon the employment of some of
these agents, which are prejudicial to bacterial life. Meats are
salted, pickled, dried, or smoked. Fruits are dried, pickled, or
immersed in strong saccharine solution, in order to preserve them
from decay, in every instance, the absence of moisture, the excess
of salt, sugar, or vinegar, or the pyroligneous acid from the smok-
ing, prevents bacterial growth, and consequently, decay of the
foodstuff. The products of bacterial growths often inhibit, or
destroy, the cells that made them, as well as other bacteria.
B. pyocyaneus and S. cholera, have this property of secreting
autolytic ferments.
CHAPTER II
PRODUCTS OF BACTERIAL ENERGY
According to their chemical activities, bacteria are arbitrarily
divided into the following classes:
Photogens Chromogens Zymogens
Saprogens Aero gens Pathogens
Photogens are those bacteria of the sea, putrefying flesh, and
damp rotten wood, that produce a faint phosphorescence.
Chromogens are bacteria that produce colors as they grow, nota-
ble among which may be mentioned the Staphylococcus aureus,
that are golden in hue; B. pyocyaneus, of a greenish-blue; and
B. prodigiosus which appears a brilliant red.
Zymogens are the bacteria of fermentation, which is the
chemical transformation of carbohydrates by the action of bac-
teria, with the evolution of CO2 CO & H. Such bacteria are use-
ful in the industries for the production of alcoholic beverages,
wine, beer, etc. Through the actions of these organisms grape
sugar is converted into alcohol, lactic acid, and acetic acid.
C 6 Hi 2 O 6 = 2 C 2 H 6 O + 2 CO 2
glucose 2 alcohol 2 carbonic acid
or
C 6 Hi 2 O 6 = 2 C 3 H 6 O 6
2 lactic acid
or
C 6 H 12 6 = 3C 2 H 4 2
2 acetic acid
23
24 PRODUCTS OF BACTERIAL ENERGY
From the bodies of ground yeast cells a soluble ferment,
Zymase, has been expressed, which causes alcoholic fermentation
of cane and grape sugars. This fact proves that fermentation
is not necessarily a vital process. The fermentations of bacterial
enzymes may give acids, and also aldehydes, ketones, CC>2,
CO, H, N, NH 3 , marsh gas and H 2 S. The carbohydrate splitting
powers are used in determinative bacteriology.
Fermentation and putrefaction are bacterial enzymic processes
of indispensible importance to life. Bacteria reduce excrementi-
tious matters to their elements and then others build up these
elements into conditions favorable for plants. This process
affects the cycle of utility of carbon, sulphur and particularly
nitrogen in the air and soil. Some soil bacteria can fix nitrogen
from the air for the use of plants. Because of the importance of
these processes, culture of appropriate bacteria may be spread
upon exhausted soil. These are chiefly nitrifying bacteria.
Manure contains the denitrifying organisms. Bacterial fermenta-
tions produce the flavor of tobacco, opium and butter.
Enzyme Production by Bacteria. These products are difficult
to define because few have been obtained in an entirely pure
state. They may be described as soluble, but non-dialyzable
products, precipitable by salts of heavy metals or by alcohol,
destroyed by 70 but resisting drying and decomposition.
They are restrained by excess of alkali, of acid, and by an accum-
mulation of their own products. Ferments of great variety
and power are formed by the zymogens, as proteolytic, which dis-
solve proteids, such as casein; tryptic, gelatine liquefying; diastase,
which converts starch into sugar; invertase, which changes cane
sugar into grape sugar; ferments that curde the casein of milk;
and it may well be that the activity of pathogenic bacteria in the
body is due to ferments of some kind. The hemolytic action of
the golden staphylococcus or the tetanus bacillus is thought, by
some, to be of enzymic nature.
Organized ferments (bacteria, yeasts) differ from the unorganized
SAPROGENS AND PATHOGENS 2$
(pepsin, diastase). The latter " exercise solely a hydrolytic
action" (Fischer), causing the molecules of insoluble compounds
to take up water and to separate into less complex molecules of a
different constitution, which are soluble in water. The organized
ones act differently. Highly complex molecules are split up, and
numerous substances of a totally different character are formed
with the evolution of gases and by-products (Fischer). The
reason for this is, perhaps, to be found in the supposition that
the bacteria abstract oxygen for their own use, and thus cause the
atoms to unite into an entirely different substance. According
to the above-named investigator, it is not possible to express
such chemical changes by a simple equation. Experiments have
shown that B. typhosus and pyocyaneus are able to split up olive
oil or fat, and produce glycerine and fatty acids, thus making
them accessible to fermentation (Fischer). The action of the
buttermilk organisms, while usually very complex, may be
represented by the following :
Ci 2 H 22 On + H 2 O = C 6 H 12 O 6 + C 6 Hi 2 O 6
lactose galactose dextrose
C 6 Hi 2 O 6 = 2C 3 H 6 O 3
galactose lactic acid
Saprogens produce putrefaction which is the chemical trans-
formation of albuminous bodies with the evolution of nitrogen,
and of alkaloidal substances, known as ptomaines. Aromatic
elements are also produced, such as indol, phenol, kresol, etc.
It is therefore obvious that fermentation and putrefaction are
separate processes, the former an action upon carbohydrates,
the latter a splitting up of proteins. If has been found that when
organisms can attack both substances, the sugars and starches
are first broken up; this is what is meant when it is stated that
carbohydrates have a "sparing action" upon proteins.
Pathogens. If the tissues are receptive to bacteria, and if the
latter, in any way, injure the tissues, then the invading organism
is called pathogenic. Theoretically the tissues of the body are
26 PRODUCTS OF BACTERIAL ENERGY
sterile, but as a matter of fact, isolated pathogenic bacteria
such as colon and diphtheria bacilli, streptococci, and pneu-
mococci, have been found in the tissues and cavities of the body
in the absence of pathological evidence of their presence.
Sixteen hours after death the blood and tissues teem with
bacteria that have wandered in from the intestines. It has been
shown that bacteria, even non-motile ones, can migrate through
the body during the agonal period.
Bacteria may cause disease in the following ways : (a) mechan-
ically, a clump of bacteria may plug a capillary; (b) simply over-
whelm the tissues and absorb the oxygen (anthrax); (c) they may
cause new growths (tubercle) ; or false membranes to form in the
larynx causing suffocation (diphtheria); (d) ulceration of heart
valves causing cardiac insufficiency; (e) thrombosis in the veins
and arteries; (J) pus formation; (g) by generating toxins that
cause anaemias, or degeneration of important elements of the
nervous system, parenchymatous organs and the walls of the
blood-vessels.
The tissues of certain animals are receptive for particular
bacteria, and the latter are therefore pathogenic to that animal.
B. of swine plague is pathogenic to swine, but not to man. B.
typhosus is pathogenic for man, but not to swine.
As emphasized above, the activities of bacteria are due to the
enzymes they produce. In the course of their life, bodies, called
toxins, are formed that have the power of producing illness in
higher plants and animals. These bodies are similar to the
enzymes. Both are produced in minute quantities. Their exact
chemistry is not known, and pure toxins, at least, have probably
never been isolated. We test for them by animal experiments
while the presence of enzymes may be observed upon artificial
culture media. Toxins of bacteria are not the only ones formed.
Castor bean produces a body classed among the toxins as does
the rattlesnake in its venom. These bodies differ from ptomaines,
also poisons, by being less resistant to heat, in causing a peculiar
TOXINS 27
blood reaction and by refusing isolation. The toxins are not
essential to the life of pathogenic bacteria and some of the usually
virulent organisms may grow without toxin development. Toxin
productions may be lost and regained. The real object of the
toxins is not known, as it is not thought that bacteria gain any-
thing by producing disease. They are separate from the other
chemical bacterial products. Toxins may be divided into those
which are secreted through the bacterial cell wall and diffuse
through the median in which organisms are growing, the extra-
cellular or soluble toxins, and those which remain within the
bacterial cells and are only liberated upon their death and dis-
integration, the endotoxins. Closely related to the second class
are the so-called toxic bacterial proteins or plasmins. These do
not separate from the structures since bacteria which produce
them furnish a toxic mass if thoroughly washed, ground and
rewashed.
Examples and Characters. Soluble or Extracellular Toxins.
The best examples are those of the tetanus and diphtheria bacilli.
In diseases caused by these germs, bacteria do not enter the body
i fluids but the general manifestations are due to absorbed soluble
I poisons. Such toxins are soluble in water; they are rendered inert
i by heating, sunlight and some chemicals. They dialyze very
i slowly and are not crystallizable. They may be precipitated with
j the albumen fraction of the medium. They may be precipitated
and dried, in which state they keep much longer than when in
solution, and then are more resistant to heat. Curiously enough
I the toxins may be destroyed by proteoly tic enzymes. Some toxins
! are complex; the tetanus toxin for example, contains two elements,
; one a dissolving power on red blood cells, the other a stimulator
i of the motor system. They are specific for each organism.
Endotoxins. These are exemplified by the poisons of the ty-
phoid and plague organisms. We know little of their chemistry
but we may assume that it is of protein material and similar to
that of the bacterial cell. These toxins are less rigidly specific
28 PRODUCTS OF BACTERIAL ENERGY
than the extracellular poisons. They are probably quite complex
in activity as they give rise to various anti-poisons when in the
animal body. These poisons are resistant to heating at 8oC. and
keep under artifical conditions much longer than soluble toxins.
The toxic bacterial proteins are best exemplified by tuberculin.
This is complex mixture of the proximal principles of the tubercle
bacillus and is probably albuminose in character. These sub-
stances are almost as specific for their own germs as the toxins and
much more so than the endotoxins. They are capable of produc-
ing a reaction in animals similar to that which might be produced
by the organisms themselves. For example tuberculin, wholly
free from tubercle bacilli, will produce a reddening of the skin or a
rise of temperature if injected into a tuberculous individual. The
dead tubercle bacillary mass if placed beneath the skin of a healthy
guinea pig will set up a local limited miliary tubercle. The
reactions from mallein and luetin (q.v.) injection are due to toxic
proteins. The proteins are usually thermostable, that is not
destroyed at iooC.; this is also called coctostabile.
In practice it may not be so simple to separate bacteria that
produce the various poisonous elements as the above descriptions
would indicate. Toxins are all in a sense specific, that is they
are for the most part selective in action, and are harmless if
swallowed. The diphtheria toxin is absorbed from a raw inflamed
surface under cover of an exudate composed of fibrin and bacteria.
The tetanus toxin is absorbed from its seal of manufacture in the
depths of a punctured wound. The endotoxin of typhoid bacilli
has no pathogenic effect if swallowed or rubbed in skin or mucous
membrane. If it be injected under the skin in the absence of
bacteria it will call forth reactions on the part of the body similar
to those expressed when living typhoid germs are circulating.
Toxins are again relative in their affinities. Tetanus toxin is fatal
for man and horses while rats and birds are resistant to it. We
use this expression of specificity for determining the nature of
certain germs. We may speak of failures to react as failures
TOXINS 29
of receptivity on the part both of the microbe and the injected
animal.
Other characters of toxins are that they act in dilute suspen-
sions, are destroyed by heat, and produce, when injected in small
doses into animals, a specific anti-substance.
CHAPTER III
INFECTION
Infection means the successful invasion of the tissues of the
body by either animal (protozoa, vermes) or vegetable (bacteria
and moulds) organisms with the evidences of their action. To
successfully infect the body, bacteria must enter the tissues, be
of sufficient number, find the tissues receptive, and continue
to multiply.
The skin, mucous membranes, and the various cavities of the
body connected with the outside air, teem with countless bacteria
at all times, many of which are pathogenic, yet there is no infec-
tion, because the tissues are not invaded. Again, there can be no
doubt that highly pathogenic bacteria enter the tissues of healthy
people at times, in small numbers, and yet no disease is produced,
because of their scarcity, or by reason of the tissues not being
receptive. Infection implies not only invasion of the body, but
injury to the tissue. Certain bacteria may invade a body, and
yet create no harm. These bacteria may enter dead or dying
body tissues, and secrete poisonous substances (toxins) which
may be absorbed, and produce pathologic symptoms known as
Saprcemia. Clots of blood in the parturient uterus, and gan-
grenous limbs may be invaded by strict saprophytes incapable of
life in living tissues, and yet cause much harm by the absorption
of their products.
Infestation is when organisms, even pathogenic, are present in
a place without exciting a reaction; the term is best used however
to imply the presence and action of animal parasites. Matter
carrying pathogenic germs is called infective.
30
Depending upon the ability to grow in the body, bacteria may
be divided into: (i) purely saprophytic ; (2) occasionally para-
sitic; <ind (3) purely parasitic. A host harbors a parasite.
Purely saprophytic germs cannot live in tissues at all; those
that are occasionally parasitic lead a saprophytic existence in
the soil or water, and yet may invade the body, and produce
disease: the tetanus and malignant oedema bacilli are examples
of this group. Those bacteria that are purely parasitic are only
known as they exist in the tissues of the infected host, and have
no outside existence at all.
Koch's Postulates
In order to prove that a certain organism is the infectious agent
of a given disease, Koch has devised four postulates which the
given organism must fulfill before it can be considered the cause
of the disease.
1. The organism must be found microscopically in the tissues
of the animal having the disease, and its position in the lesion
should explain the latter.
2. It must be isolated in pure state from bodies of the diseased
animals.
3. And then it must be grown for successive generations in
culture media.
4. If injected into a healthy animal, or animals, it must produce
the same disease, and be found in the lesions of the disease in
the animal's tissues.
Some of the many organisms that certainly fulfill these condi-
tions, are as follows :
Streptococcus Pyogenes (Sepsis). Actinomyces.
B. of Tuberculosis. B. of Diphtheria.
B. of Anthrax. B. of Tetanus.
B. of Glanders. B. of Malignant (Edema
B. of Bubonic Plague. B. of Malta Fever.
32 INFECTION
B. of Typhoid. B. of Dysentery.
Spirillum Choleras. Meningococcus.
Pneumococcus (Pneumonia) .
Spiroch&ta of Relapsing Fever and of Syphilis
There are several other organisms that are considered to be
the cause of specific disease, but they do not fulfill the postulates.
Among these are :
The Protozoa of Malarial Fever
Amoeba Dysenteries.
While the specifications outlined by Koch as indicating the
etiological role of an organism were sufficient for the period at
which they were laid down, advances in immunology have added
so much information about antigens and antibodies that it is
but right today to expect that a virus should behave as an
antigen by calling forth certain immunity reaction under spon-
taneous and experimental conditions. Such as expectation is
fulfilled in practically all cases, and indeed has been, even in
a few instances where all Koch's postulates could not be com-
pleted, typhoid fever being a notable example.
In rheumatic fever, measles, mumps, yellow fever, chicken-
pox, rabies, and dengue, the specific cause has, thus far, eluded
discovery. In the case of measles, hog cholera, and some of
the eruptive diseases, it has been found that the cause of these
diseases resides in the blood, and if the serum of the latter is
carefully filtered through a Berkefeld filter, it is still capable of
producing the disease in susceptible animals. Careful micro-
scopic search fails to show any bodies in the serum that might be
considered the agents of infection, and it is thought that these
organisms are submicroscopic (see chapter on Filterable Viruses).
If the invading organism is a pure saprophyte the various"
forces for internal defense immediately act upon and destroy it.
Bacteria are disposed of in diverse ways. By means of the
ATTENUATION OF BACTERIA 33
lymph channels they are carried to the various mucous surfaces
of the body, intestinal and bronchial. The liver, according to
Adami, destroys at once bacteria absorbed from the intestines.
During typhoid fever, the typhoid bacilli are often found in
the urine, the organisms escaping from the blood or from the
lymphoid foci in the kidney. Pathogenic bacteria are discharged
from the body in feces, pus, sputum, and in scales in the des-
quamating skin diseases.
To successfully inoculate a guinea pig with tuberculosis, the
tubercle bacilli should be injected beneath the skin.
It has been said that successful invasion demands a sufficient
number of organism; it is equally true that the number admitted
will determine the character of disease to arise, as is indicated
by the following observation of Cheyne.
In experimenting with the staphylococcus aureus, it was found
that 250,000,000 were required to cause an abscess; and 1,000,000,-
ooo were needed to cause death. The internal powers of defense
were able to cope with or limit the action of a few million to a
certain locality, but could not withstand the injection of over-
whelming numbers, which caused the animal's death.
There are three attributes which make successful the invasion
of pathogenic germs into the body: virulence, toxicity and
pathogenicity. These factors shade into each other sometimes
very confu singly and are of course capable of varying proportions
in the same organism. In order to combat successfully the pri-
mary defenses of the body, a virus requires virulence, the degree
and permanency with this is accomplished being due to the amount
of poison the invader can elaborate to keep the safety devices
of the economy from conquering. The physical damage done
is attributable to the pathogenicity of the germ. It is like a
fight where the man has strength, and staying powers and does
physical damage to his adversary.
Ehrlich's explanation of virulence assumes that bacteria have
binding posts or receptors and the more of these a germ has, the
34 INFECTION
more of the natural defenses it can anchor and remove from the
field.
Their virulence can be lessened by cultivation at a higher
temperature than the body, 42.5-47C.; by drying; the exposure
to light; the action of chemicals; compressed oxygen; and by
passing the organism through the bodies of non-susceptible
animals. The attenuation or weakening of the pathogenic powers
of bacteria is useful for the production of various vaccines which
are valuable in preventive medicine.
By growing the anthrax bacillus atahigh temperature, 42.5C.,
it becomes so avirulent that it is incapable of destroying sheep or
rabbits. It is then used as a vaccine to prevent infection with
virulent bacilli. By exposing the spinal cords of animals dead
from hydrophobia to the action of drying for various periods,
Pasteur was able to attenuate the virus, so that it would not
produce hydrophobia, but on the contrary, it, by repeated
inoculation, caused immunity. The inoculation of monkeys
(which are non-susceptible) with hydrophobia virus attenuates
it. The growth of the small-pox organism in the cow, causing
cow-pox, so reduces the virulence of the germ that it is incapable
of producing small-pox in man, but only vaccinia; infection with
this gives immunity against small-pox. The flesh of animals
that have died from quarter-evil is so changed by heat and desic -
cation that if it is injected into susceptible animals, they do not
succumb but are vaccinated against infection with the virulent
organism.
When we speak of attenuation of virulence we usually refer to
the effects on experimental animals and specify what attenuation
is meant when they are to be used as vaccine. A very interesting
pathogenic, yet attenuated, form of streptococcus is to be met
in subacute endocarditis. These organisms produce serious or
even fatal valvulitis, and yet have no effect upon lower animals.
They are extremely hard to remove from the body. They
have accustomed themselves to residence in the body, have estab-
AVENUES OF INFECTION 35
lished a balance of poise between their offenses and the bodily
defenses and practically cannot be rapidly dislodged. These are
called fixed or fast strains. Such strains may be seen under other
conditions such as the typhoid bacillus in the gall-bladder. These
fast strains usually are found at places remote from intimate con-
tact with the defenses of the body, the leucocytes and blood serum
as in the cases cited.
The malignancy of bacteria may be heightened in various ways :
(i) By passing them repeatedly through the bodies of susceptible
animals; (2) by cultivation in culture media in collodion sacs
placed in the abdominal cavities of animals; (3) by injections
mixed with other injurious substances, such as lactic acid, and the
metabolic products of foreign bacteria. Cultures of pneumococci
may be made so virulent by the first means that only one pneu-
mococcus is capable of setting up a fatal septicaemia in a rabbit.
By injecting attenuated diphtheria bacilli with streptococci into a
rabbit, the virulence of the bacilli can be raised, as mixed infection
often adds to the virulence of an organism. Malignant strepto-
coccic infection added to virulent diphtheria infection, greatly
increases the severity of the disease. The transference of infec-
tive agents from one person to another during an epidemic
increases the virulent action of the organism by reason of the
rapid passage from individual to individual.
Mixed infections are those in which more than one kind of
virus is active. It is of course possible that two kinds may
originate a disease, but it is usual for one germ to initiate a process
and another to be superimposed upon it, usually intensifying the
lesions. The active ulcerative inflammation in tuberculous lungs
is usually due to be secondary effect of streptococci.
The secondary streptococcic infection in small-pox and in
phthisis complicates the primary infection and frequently causes
death of the individual affected.
The avenue of infection and the tissues infected alter the type of
the disease exceedingly. Streptococci invading the tonsils cause
36 INFECTION
tonsillitis, but the same organisms entering the skin cause erysipe-
las of phlegmons; or if the uterus is infected after the birth of a
child the disease is still different and more serious. If the tubercle
bacilli enter the skin they produce lupus; if swallowed they cause
ulceration of the bowels, and subsequently invade the peritoneum;
if inhaled, tuberculosis of the air passages, phthisis, or tuberculous
laryngitis may follow. If cholera spirilla be injected into a vein
of a guinea pig, it may develop choleraic septicaemia; if they are
injected into the peritoneal cavity, a choleraic inflammation of the
peritoneum is produced, and not a septicaemia. Pneumococci if
injected into a vein cause a rapid septicaemia, or they may give
rise to abscesses anywhere in the body. Like streptococci, they
may be the cause of inflammation in any tissue, particularly
serous membranes, and show different clinical entities, according
to the organs involved, and the morbid anatomy and physiology
produced. The fatality of a bacterial infection varies with the
avenue of inoculation: it is safer to have a skin infection than a
meningeal, or endocardial one, not only from the likelihood of
rapid toxin absorption, but from purely mechanical damage, as
pressure and interference with vital functions by inflammatory
products such as fibrin, tubercles, serum and pus.
How Bacteria Are Brought to the Body. Air-borne infection
may occur by the direct transference of the bare organisms, a very
rare occurrence, or by dust or by droplets of fluid usually sputum.
Organisms settle on objects of our environment when leaving the
sick and can be stirred up with the dust. This is important for
diphtheria, the acute exanthemata and tuberculosis although
the 'most dangerous source for the last is the coughing con-
sumptive. The transmission of pertussis and pneumonia is almost
surely always a droplet convection.
Water-borne infection, including typhoid, cholera and dysen-
tery occurs by the contamination of water courses with the dis-
charges of the respective diseases.
Milk-borne diseases, tuberculosis, diphtheria, epidemic sore
SOURCES OF INFECTION 37
throat and some others, occur because persons or animals suffering
with the disease have handled or supplied the milk. This direct
contamination also applies to food like meat and oysters (typhoid
and meat poisoning).
Soil-borne diseases are chiefly those arising by direct implanta-
tion of earth into the body. Of course the ground may be soiled
by discharges from infectious diseases and contamination of
hands and clothing.
Animal carriers of disease include those acting as intermediate
hosts (anopheles mosquito in malaria) ; as mechanical conveyances
of a direct or indirect nature, in the former case like transmission
of organisms from a sick man or animal to a well one, in the latter
case transferring the germs to food consumed by a healthy being;
or acting as a passive host for the germ as is the case in the
transmission of plague bacilli by the rat flea.
Human transmission of infective matter is the most important
of all methods as it is an axiom that a person suffering with a
disease is most capable of transmitting it. This occurs by direct
contact, by the carrier state and by passing the contagium to the
embryo.
Carriers. After recovery from certain diseases, notably ty-
phoid fever, diphtheria and cholera, convalescents may carry
in themselves fully virulent germs with no outward evidences
thereof. Such persons are called " carriers" and are of the highest
importance in hygiene. The reasons for this condition are several.
These germs may be removed from the bodily defenses or the
body may be immune to them; again they may be fixed or fast
strains. Wherever they are they may escape and infect another
person. After typhoid fever bacilli remain in the gall-passages
and bladder; after cholera in the deep mucous membranes and
after diphtheria the crypts of the tonsils or the nasopharynx may
hold them. Vaccination or operation may be needed to remove
them. Persons never known to have had enteric fever have
been known to harbor bacilli in their gall-bladder. One typhoid
38 INFECTION
carrier, " Typhoid Mary" a cook, is known to have infected 26
persons. Such persons because of their apparent innocence
might be called " hidden carriers." They have been found trans-
mitting dysentery and poliomyelitis as well as the above typhoid
fever, and, judging from the continued existence of the exanthe-
mata in cities, it may be that we shall find such persons harboring
the virus of varicella, mumps and pertussis.
Local Immunity to Infection. There is evidently more resistance
offered by the liver against invasion than by the peritoneum. It
is not likely that a man would contract typhoid through skin
infection, nor is it probable that he would contract tetanus by
swallowing tetanus bacilli, but the reverse of these conditions
certainly produces infection.
Infection may be caused from without the body, or from within.
Lockjaw, sepsis, hydrophobia, or anthrax may follow injuries from
rusty nails, splinters, weapons, unsterile fingers, or instruments.
Personal intercourse, bites, kisses, sexual intercourse, association
with persons suffering from exanthematous or contagious diseases
may transmit disease.
Winslow has found colon bacilli upon 9 percent of the hands he
examined. Tubercle bacilli have been found on the hands of the
non-tuberculous. Some organisms, notably the smegma bacillus,
pyocyaneus bacilli and cocci resembling the white pus former, may
be said to be normal inhabitants of the skin.
The bites of insects that are intermediate hosts of infectious
agents (plague bacilli, malarial organisms, etc.) are sources of
infection from without, as is also the ingestion of infected food or
water.
Infection from within may be caused by the migration of bac-
teria from the skin inwards, or from any of the mucous membranes,
on which, and in which many pathogenic bacteria at all times may
be found.
Bacteria from the mouth, stomach intestines and the rectum
may invade the tissues and the blood under certain conditions.
SOURCES OF INFECTION 39
This is particularly the case during the last stages of diseases, not
necessarily infectious, such as chronic heart disease, kidney
disease, or diabetes. Vital resistance is much lowered, and intes-
tinal bacteria, invading the tissues in enormous numbers, set up
what is known as terminal infection, which is often the immediate
cause of death.
The stomach with its gastric juice, containing during digestion
.2 percent to .3 percent of hydrochloric acid, guards the lower ali-
mentary tract against infection. A great many bacteria are in-
gested with foods, particularly with milk, cheese, and overripe
fruit. These in the most part are quickly destroyed by the hy-
drochloric acid. When the stomach is diseased and the contents
become stagnant, as in stenosis of the pylorus, and in carcinoma,
when HC1 is diminished, or absent, fermentative bacteria give rise
to great amount of gas, and lactic acid, to the great discomfort of
the patient. The normal acidity of the stomach is a great
safeguard against infection with cholera. If tubercle bacilli are
swallowed, and if infection occurs, the lesion is not always localized
to the alimentary tract. Lesions of the lymph glands, peritoneum,
bones, and nervous tissues often follow the ingestion of these or-
ganisms. Dogs fed on soup containing great numbers of tubercle
bacilli, and then killed three hours after, were found to have bacilli
in the thoracic duct. Chyle from the duct, injected into guinea
pigs, caused tuberculosis in them (Nicolas and Descos).
The interior of the uterus, the bladder, urine, deep urethra, and
lungs are generally sterile in health. With the exceptions noted
where germs are not usually found, all tissues, especially the inlets
and outlets of the body, may be said to have a normal bacterial
flora. Bile has a distinct antibacterial power, which indeed is one
of its functions in the intestines.
The placenta is an avenue of infection in several diseases: not-
ably small-pox, anthrax, glanders, typhoid fever, and sometimes
tuberculosis pass through the placenta from mother to foetus.
Streptococci may pass through the placenta of a woman with
40 INFECTION
ante-delivery sepsis and cause peritonitis in the child. Recurrent
fever has been transmitted from mother to foetus, and the specific
spirillum has been detected in the latter's blood.
A case has been recorded in which a woman suffering from
pneumonia gave birth to a child, which died thirty-six hours after-
ward, and autopsy revealed a consolidation of the lower left lung,
and microscopic examination discovered pneumococci. A hydro-
phobic cow was delivered of a calf that developed rabies three days
after birth.
McFarland divides microbic infection in three heads :
Phlogistic. Characterized by restricted growth and local
irritation.
Toxic. Characterized by restricted growth and toxin dissemi-
nation.
Septic. Characterized by unrestricted growth in the blood and
lymph. In the three groups, the damage is done ultimately, by
metabolic products acting on the tissues. If the product be not
soluble the harm done is purely local, as in the formation of tuber-
cles by the toxin of the tubercle bacilli.
If the growth be restricted, as in tetanus and diphtheria, the
toxin being soluble and diffusible, harm is done to tissues remote
from the infected area.
Anthrax and streptococci and other pus organisms by rapid
increase in the blood eventually infect all the tissues.
Combinations of these forms of infection may be at first confined
to some particular area; the pneumococcus, which is generally
restricted to the lungs at the outset, may ultimately infect the
blood, causing septicaemia and localized lesions in more or less
remote parts, such as the veins of the leg, or inflammation of the
meninges. In a topographical sense infection may be local, focal
and general. Local disease is limited in extent and at most gives
only trifling general manifestations by absorption of products of
inflammation, a boil. When an infection becomes well established
in a small locality but without active general evidences, it may
SOURCES OF INFECTION 41
still send out a few organisms or small quantities of poison which
can attack other parts. Thus from a root abscess, germs may
sneak into the blood stream and settle in the kidneys or joint
membranes; this is focal infection and is usually subacute or
chronic in character. General inffection is self explanatory.
Bacteria may become accustomed to the fluids of the body by a
similar process and may elaborate free receptors or their own
protection, i.e., anti-bacteriolysins (Welch's theory).
In the aged, and in chronic disease of the liver and kidneys,
the complement existing in the blood may become reduced in
quantity, and the individual may succumb to an infection, which
ordinarily would be mild.
Soluble products of bacterial activity which are alkaloidal
(basic), crystalline in character, and mostly poisonous, are known
as ptomaines, or putrefaction alkaloids. They are highly com-
plex in chemical structure, and are difficult to isolate.
The foregoing processes are due to the toxic products of bac-
teria during their growth in the tissues, substances mentioned
before but now deserving a more detailed study from the stand-
point of the diseased tissue.
Bacterial endotoxins are poisonous substances liberated only
upon the death and disintegration of the germ cells. They are
moderately active in attacking wandering and special tissue
cells; they are more resistant to heat and ferments than toxins.
The identity of these toxins has given rise to considerable dispute
and there will be given the two principle ideas concerning them.
The older theory considered them integral parts of the cell, peculiar
to each virus and calling forth specific responses because of the
individuality of each toxin. Recent work has shown that the
chemical and immunological response to bacterial injection is
similar to that obtained by the use of serum, or egg white or
animal cells. For this and more minute reasons it is believed by
some that there is in every protein (bacterium, cell, serum) a
toxic part with a common construction. Another and peculiar
42 INFECTION
moiety for each is non-toxic but represents the part which calls
forth specific antibodies. The characteristic infectious phenomena
of each disease are therefore due to the non-toxic part of its
molecular construction. It has been shown that protein
cleavage products can cause fever if injected into animals.
Cholera and typhoid organisms do not produce soluble toxins
in the body, but when they are disintegrated therein, soluble
poisons (intracellular) are liberated.
Bacterioprotein or plasmins are albuminous bodies produced
by bacteria, are not altered by heat, and produce fever and in-
flammation. The best examples of these are mallein a product
obtained from old cultures of glanders bacilli, and the original,
or old tuberculin of Koch.
Toxins or toxalbumins are soluble, non-crsytallizable, non-
dialyzable bacterial products which are removable by filtration
from the bacteria, and which are thermolabile.
These various poisons produce many of the clinical pathological
entities and symptoms, known to physicians. Their highly
complex molecular structure enables a group of atoms in the
toxic molecule to unite with a certain other group of atoms in
the protoplasmic molecule of a body cell. The latter is either
killed outright, or else is stimulated to produce other free groups
of combining atoms (lateral chains) which may unite with other
toxic groups.
Various kinds of cells are attacked in infective processes.
Leucocytes may be degenerated, forming pus; red blood cells may
be dissolved, causing anaemia; important nerves may be degener-
ated; or muscle fibers of the heart may undergo fatty degeneration
and die. Again, mechanically important serous cavities may be
filled with serum, interfering with normal functions of the en-
veloped organs. The heart orifices may be closed partially or
emboli may form, or false membranes block the air passages, and
a hundred other pathological changes may be wrought by these
toxins.
TOXINS OR TOXALBUMINS 43
Most toxins are easily decomposed by sunlight, air, and heat.
Absolute alcohol separates the active principles from the bouillon
in which it grows. Ammonium sulphate also precipitates the
toxins from cultures of tetanus and diphtheria bacilli, from which
they may be collected, dried and powdered, and in this state
may be kept much longer without deteriorating into inert sub-
stances. Small quantities of bile and pancreatic juice destroy
the toxic properties of diphtheria and tetanus toxin.
Since the toxins cannot be isolated in a chemically pure form,
their exact composition cannot be known, except by studying
their effects upon animals and animal tissues. Hence, when anti-
toxin, added to toxin in a test-tube is injected into an animal,
and no harm results, it is rightly assumed that the toxin is
neutralized, and both are chemically bound; yet if fresh toxin is
added to the mixture, it is no longer neutral.
If the toxin of the pyocyaneus and the anti-toxin be mixed so
that they neutralize each other, and if the mixture is heated, the
neutralization disappears, and the mixture becomes toxic again.
That the union is a chemical one, may be inferred from the fact
that it is more rapid in concentrated solution than in weak, and
is much quicker when warmed than when cold, and it follows the
law of multiples, i part toxin neutralizing i part of anti- toxin,
and 10 parts of toxin neutralizing 10 parts of anti- toxin. All
this is in accord with chemical laws. Toxins sometimes degener-
ate into what Ehrlich has called toxoids, substances that bind
(unite with) anti-toxin just as effectively as toxins, while they
are not poisonous, yet may stimulate healthy cells to secrete
anti- toxins if they are injected into the body of experiment
animals.
More is known about the toxins of diphtheria and tetanus bacilli
than of any other. Diphtheria toxin has numerous component
substances, one of which is the toxin that causes the acute phe-
nomena of diphtheria intoxication. Another, toxon, causes
cachexia and paralysis some time after infection.
44 INFECTION
Tetanus toxin is composed of two substances: tetanospasmin
and tetanolysin. The first unites chemically with the motor ele-
ments of the nervous system, producing degeneration and causing
tremendous contractions of the muscles governed by the nerves
involved. The second has the property of dissolving tissue, such
as blood cells.
Tetanus toxin travels from the infected site to the cord by
way of the nerves; it is exceedingly poisonous; a single prick of
the finger with a needle moistened with toxin, has induced tetanic
symptoms.
If tetanus toxin of known strength is mixed in a test-tube with
fresh brain substance of a guinea pig, the toxin is no longer toxic
for guinea pigs. This shows that there is a chemical union of
the toxin and the cells of the brain. Cells of other organs have
no such effect. This explains specific action of tetanus upon
nervous tissue.
Aggressins. If tubercle bacilli are injected into the abdominal
cavity of a guinea pig, tuberculosis is produced. If the exudate
produced in the peritoneum, consisting of fluid and cells, be
sterilized and injected into another guinea pig, together with
some virulent tubercle bacilli, the animal will succumb in twenty-
four hours. If the exudate alone be injected no effect will follow;
if bacilli alone are injected, a tuberculous peritonitis will be
produced in a few weeks. It is the exudate plus bacilli that
does the harm. The exudate is, in this instance, the aggressin.
Bail, who originated the doctrine of aggressins, believes that a
bacteriolysin is produced, which, acting on the bacilli, liberates
an endotoxin, which paralyzes the polynuclear leucocytes,
inhibiting their action as phagocytes.
By heating the exudate to 6oC. the aggressins are increased in
activity, and it has been found that small amounts are relatively
stronger than larger ones.
This phenomenon has been explained by Bail in this way. He
assumes that there are two substances in the exudate, one is
AGGRESSINS 45
thermolabile, which prevents rapid death, the other is thermo-
stabile and this is favorable to rapid death.
Bail assumes that a tuberculous cavity in an animal contains
a great amount of the aggressin, which prevents chemotaxis of
the poly nuclear leucocytes, but no.t of the mononuclears or
lymphocytes.
In the peritoneal cavity into which tubercle bacilli without
aggressins, have been injected, an active phagocytosis at once is
begun by the polynuclears, and the injected bacilli are in a great
measure destroyed, and those left develop more slowly, producing
a tuberculosis in normal course of time. It is possible to im-
munize animals against this aggressin producing an anti-ag-
gressin, which substance will not only neutralize the aggressin
but also stimulate the leucocytes to phagocytosis.
This aggressin theory has been applied to other infections with
like results, notably in pneumococcus, typhoid, dysentery, and
plague infection.
CHAPTER IV
IMMUNITY
By immunity is understood the inherent power of a living body
to successfully withstand the invasion of infective agents, e.g.,
bacteria, or such deleterious and toxic substances as toxins, drugs,
complex poisonous albumins, snake venom, foreign blood sera, etc.
The following tables will, perhaps, be helpful in the study of the
subject.
Racial immunity
I. Immunity
2. Inmiuiiity
Natural
Acquired
Inherited immunity
Active immunity
Passive immunity
f Anti-toxic
I Anti-bacterial
It is a well known fact that one attack of an infectious disease
generally protects an individual against a subsequent attack. It
has also been known for centuries that the human system, by first
taking very small doses, and gradually increasing them, can be so
accustomed to poison, that large, and otherwise deadly quantities
may be taken at one time with impunity. Among the poisonous
substances to which men can accustom themselves are: tobacco,
morphia,' arsenic, and alcohol. Animals treated in a like manner
also become immunized to powerful toxins, snake venom, etc.
Natural Immunity. The hog is immune to snake venom; the
chicken to tetanus. Man is immune to hog, or chicken cholera.
The negro is not so susceptible to yellow fever as is the white.
Animals cannot be infected with scarlet fever, malaria, and
measles. Young adults are more susceptible to typhoid fever
than are elderly ones. Infants are exceedingly prone, to suffer
from milk infection while older children are not. Again, one
4 6
PHAGOCYTOSIS 47
individual may contract a disease, while another exposed at the
same time will not. Inherited immunity is exemplified by the
history of races into which a new disease was introduced at first
with high mortality but later with great reduction in the severity
of the infection.
Acquired Immunity. Actively acquired by infection. One
attack of yellow fever immunizes the individual against subse-
quent attacks. Vaccination actively immunizes against small-pox.
Passively Acquired. Actually injecting protective substances
(anti-toxic sera) into the blood. The immunity againsta given
disease (diphtheria) resides in the anti-toxic sera.
Immunity is nearly always relative. A small quantity of toxin
may be innocuous, while a large quantity may cause a fatal
toxaemia.
There have been several theories advanced to account for the
various phenomena of immunity, the oldest ones, beginning with
Pasteur, being that some substances vitally necessary to the
virus was used up or that something was retained to affect new
microbial attacks.
The modern conception of immunity deals with two theories,
the theory of phagocytosis of Metchnikoff, which may be termed
the cellular or biologic one, and the lateral-chain, or the humoral
or chemical theory of Ehrlich. Both of these are extremely
ingenious and explain satisfactorily why certain bacteria are
unable to infect the body, and why, the body once infected, cannot,
in many diseases, be again infected. Furthermore these theories
make it clear to us why the body tissues during life do not fall
an easy prey to many putrefactive bacteria, as after death.
Phagocytosis is essentially a theory of cell-devouring. Leuco-
cytes which are white mobile cells of the blood, and other fixed
cells, defend the body against infection by devouring the in-
vading agents of disease (Fig. 14).
Metchnikoff considers the subject of phagocytosis under three
aspects; (i) nutritional; (2) resorptive; (3) protective.
48 IMMUNITY
Amoeba and certain other unicellular vegetable organisms
belonging to the myxomycetes possessing amoeboid properties and
having the faculty of throwing out pseudopodia or protoplasmic
arms, acquire their food by enveloping smaller organisms and
other nutritious matter which they absorb. Certain intracellular
ferments, which they possess, digest fibrin and gelatine, and con-
vert starch into sugar. These cells protect themselves against
inimical microorganisms by enveloping and digesting them.
FIG. 14. Phagocytosis. Gonococci in leucocytes in pus from gonorrhoea.
(Kolle and Wassermann.)
They are attracted by food and moisture (called positive chemo-
taxis) and repelled by strong solution of salt, poisons, etc. (nega-
tive chemotaxis) .
Higher in the animal scale among the multicellular organisms,
the cells of the intestines have the property of absorbing and di-
gesting food. These fixed cells are called sessile phagocytes.
Still higher in the scale (man) certain digesting cells are present
in the digestive tract, which are incapable of absorbing food.
They, however, secrete ferments which digest gelatine and fibrin,
and convert starch into sugar. But other cells of the animal '
body, the leucocytes, large mononuclears and certain fixed tissues-
cells, have the property of engulfing foreign bodies like bacteria
PHAGOCYTOSIS 49
and of digesting them; it is to these that Metchnikoff ascribed
the protective power of phagocytosis. Cells of a protecting
character in man are either microphages or macrophages. The
microphages are the polynuclear leucocytes, which are concerned
in the protection of the organism against acute infections, the
bacteria of which they take up and devour. The macrophages
consist of the large lymphocytes, the endothelial cells, and some
connective tissue cells, which take up foreign bodies. Both of
these classes contain ferments; microcytase being found in the
microphages; and macrocytase in the macrophages. The latter
absorb connective tissue cells through their particular ferments,
and are active in immunizing against tuberculosis. These cells
perform various functions in the body. When the tissues are
invaded with bacteria, the blood shows an increase in the number
of these microphages, which have been called the "hygienic
police." Summoned to repel invasion, they leave the lymph
stream for that of the blood. All the phenomena of leucocytic
emigration in inflammation is a manifestation of positive
chemo taxis. During practically all the infections, the peripheral
blood contains an excess of leucocytes over the normal amount
per cubic millimeter (7,600). In exceptional infections, typhoid
fever, influenza, measles, and tuberculosis, there is no such
increase, or leucocytosis. In malaria (not a bacterial infection)
there is also no leucocytosis.
Metchnikoff has described a process in which the phagocytes
undergo what he calls phagolysis. The ferment, cytase, is dis-
charged and acts extracellularly, digesting the cell body and
freeing ferment which will act against the invaders. Metchnikoff
further claims that both phagocytosis and phagolysis, either
severally, or in combination, are responsible for natural or ac-
quired immunity.
In the case of acquired immunity, it is supposed that the leuco-
cytes become educated. Regarding the toxins against which
animals can be immunized by gradually increased doses, it is held
50 IMMUNITY
by him that the educated leucocytes neutralize the poison by
their secretions.
In the case of anthrax infection, animals infected with virulent
cultures of this organism quickly succumb, without exhibiting
any leucocytosis (negative chemo taxis).
If the animal has been previously immunized with attenuated
culture the injection of a virulent culture is followed by an enor-
mous outpouring of leucocytes at the site (positive chemo taxis),
while if the site of the inoculation in the non-immune animal is
examined, only a few leucocytes, and some clear serum will be
found.
Toxins, if injected, cause a negative chemotaxis. If tetanus
spores are injected into an animal, together with some toxin, the
animal rapidly succumbs to tetanus, without evincing any leuco-
cytosis. If the spores are washed free from toxin, and injected,
active leucocytosis occurs and the animal survives.
A mixed infection of a highly virulent culture, and a non-
virulent one, often hastens the action of the virulent one. It
is supposed that the non-virulent bacteria engage the leucocytes,
so that these cells cannot cope with the virulent ones.
Phagocytosis thus plays an important part in a protective role
in natural immunity, but no satisfactory theory has yet been
offered in explanation of the protective process in acquired
immunity, at least against toxins and other soluble and unorgan-
ized poisons.
In order to meet the criticisms arising after Ehrlich's theories.
Metchnikoff added to his theory by stating that complement
and-anti-body are enzymic substances derived from phagocytes.
The cellulo-humoral theory claims the attention of most bac-
teriologists, as the probable explanation of the phenomena of
immunity. It is certain that cells, either sessile or mobile, and
fluids, are important means of internal defense. In order that '
this theory may be comprehended, certain well-known properties
on formal and artificially immunized serum must be understood.
NATURAL IMMUNE BODIES 51
Alexins. It has been found by numerous observers, that nor-
mal blood serum is germicidal for many bacteria, and pecu-
liarly active substance that is contained in the serum, was called
by Buchner Alexin. This dissolves bacteria and destroys them.
It also destroys the red blood cells of other animals. The alexin
of a dog's serum dissolves the red cells of a rabbit; it is therefore
hamolytic. It also is thermolabile, that is, its properties are
destroyed by heat (55C.). It is identical with the complement
of Ehrlich, and the cytase of Metchnikoff.
The complement, as it will hereafter be called, takes, as already
stated, an active part in bacteriolysis, or bacteria-dissolving, and
in haemolysis, or blood-dissolving, it is present hi normal non-
immune sera. R. Pfeiffer found that if some serum from a guinea
pig immunized against cholera spirilla is injected into the peri-
toneal cavity of a healthy non-immune guinea pig, with some
cholera spirilla, that the latter are agglutinated, and ultimately
dissolved, having undergone bacteriolysis (Pfeiffer's reaction).
The immune serum alone in a test-tube, with the cholera spirilla
does not have this action, but if some normal guinea-pig serum
is added to the mixture, an immediate solution takes place,
showing that the presence of both the normal serum containing
the complement, and the immune serum, containing the immune
body, or amboceptor, are necessary to complete the solution of
the bacteria. If the complement is heated above 55C. for an
hour, solution does not take place, even if the immune serum is
present, but, after heating the mixture, it may be reactivated by
adding some fresh unheated complement. The complement is
thermolabile, i.e., destroyed by heat.
The immune serum is not affected by heat, and is therefore
called thermostabile.
These various reactions may be expressed concretely thus:
Bacteria + immune body = no solution.
Bacteria + complement = no solution.
52 IMMUNITY
Bacteria +inimune body + complement = solution (Pfeiffer's
reaction).
Bacteria + immune body + complement (heated) = no solu-
tion.
Bacteria + immune body (heated) + complement = solution.
The same phenomena have been observed in the blood of
animals immunized against the red blood corpuscles of another
animal of foreign species.
If a rabbit is immunized with the blood of a dog by repeated
and increasing doses, the serum of that rabbit will become h&mo-
lytic to the corpuscles of the dog's blood if they are mixed, pro-
vided some normal rabbit's blood complement is added to the
mixture.
Dog's erythrocytes + immune rabbit serum = no solution.
Dog's erythrocytes + immune rabbit serum + complement =
solution.
Dog's erythrocytes + immune rabbit serum + complement,
heated = no solution.
The immune body acts as a preparer of the corpuscles, or
bacteria, so that the complement can act upon the cells. The
reaction is very like the action of pepsin on fibrin. Hydrochloric
acid must be present.
(1) Pepsin + fibrin = no solution or lysis.
(2) HC1 + fibrin = no solution or lysis.
(3) Pepsin + HC1 + fibrin = solution or lysis.
The HC1 corresponds to the immune body.
In the case of haemolysis, or bacteriolysis the action of the im-
mune body is specific. The immune body of cholera spirilla will
not prepare, or fix typhoid bacilli, so that they can be acted upon by
the complement. Nor will the immune body of dog's erythrocytes
prepare those of a pig, so that the complement may act on themu
A loose chemical union takes place between the bacteria and
the immune body, but no such union occurs between the comple-
AGGLUTININS 53
ment and the bacteria. The same chemical union occurs between
the red cells and the immune body in haemolysis, but not between
the cells and the complement.
Ehrlich holds that there are many complements, each one dif-
ferent from the other, and that their action is specific for the
different kinds of bacteria or cells with which an animal may be
immunized. Bordet and Buchner, on the other hand, maintain
that there is but one complement.
The solution of any cells by immune bodies, or anti-bodies, as
they have been called, is known as cytolysis. And cytolysins
may be produced by making anti-bodies of nerve cells, leucocytes,
epithelial cells, liver cells, as well as blood cells, by immunizing
an animal against these different cells with repeated injections
of the cells or emulsions of them.
Agglutinins are peculiar bodies which have the property of
causing certain cells to agglutinate. One of the earliest manifes-
tations of immunity of a certain serum to bacteria, or to blood
cells, is this peculiar action of the serum causing either the bac-
teria or blood cells to clump together in masses. Part of Pfeiffer's
reaction is the agglutination of the cholera spirilla in clumps
before they are dissolved by the complement and immune body.
Recent studies accord to agglutinins one of the most impor-
tant places in the defense against disease. Their action is
believed to facilitate that of lysins and opsonins, helping the
latter by fixing a group of bacteria so that they may be the
better prepared for phagocytosis.
If the serum of a typhoid fever patient is mixed, even in high
dilutions with some typhoid bacilli, the latter are clumped in
isolated groups. Clinically this is known as the Widal reaction,
and is the most reliable single sign of typhoid fever.
These agglutinins may be produced artificially by injecting
large and increasing doses of bacteria into animals. After a time,
in the serum of the rabbit, there develops a peculiar body which
agglutinates typhoid bacilli, if they are brought in contact with
54 IMMUNITY
it. Sera can be rendered so highly agglutinative as to produce
this reaction even if diluted 100,000 times or more.
If an animal is immunized against spermatozoa, or the red
blood cells of a foreign species, its serum becomes agglutinative
to these cells.
Precipitins. If a rabbit, or any other animal in fact, is immun-
ized by repeated injections of foreign protein (blood, bacterial
culture, etc.) peculiar bodies develop in its blood serum called
precipitins, and these can be demonstrated by adding to the
serum of the immunized animal in a test-tube a minute portion
of the material against which the animal was immunized. As
soon as the immunized serum and the specific substance are
mixed, a precipitate forms. This is another phenomenon of
immunity, and is of more than theoretical importance in medicine.
The reaction is strictly specific; thus, if the serum of a goat is
injected into a rabbit repeatedly the rabbit's blood will form a
precipitate with normal goat's serum if the two are mixed in
a test-tube. Old dried blood, semi-putrid blood, blood on white-
wash, or rusty steel, even in minute quantities, if dissolved in salt
solution, may be used to produce this reaction. In medico-legal
matters, this test is of use for the identification of human blood.
By some, the phenomenon of agglutination is supposed to be due
to the formation of a precipitin, in the meshes of which bacteria or
blood cells are caught and agglutinated, and that agglutination is
but a modification of the formation of precipitins.
Anti-toxin formation is also another phenomenon of immunity.
If an animal, such as a horse, receives numerous increasing doses
of a given toxin, say that of tetanus, it, in a short time, becomes so
accustomed to the poison, that it can withstand the administration
of immense doses. (If these large doses had been given at first,
they would have proved fatal.) If the horse is then bled, and its
serum injected into rabbits or guinea pigs, they may receive
shortly after, at one dose, enough toxin to kill ten such animals.
The horse serum thus protected these animals against the toxin,
as it was anti-dotal, or in other words anti-toxic. A chemical
LATERAL CHAIN THEORY 55
union occurs between the toxin and the anti-toxin, since, according
to the law of multiples, a definite amount of anti-toxin unites
with a definite amount of toxin. If ten times the amount of
anti-toxin is used it will exactly neutralize ten times the amount
of toxin, and the mixture becomes inert. Again, the union of
the two substances follows well-known chemical laws, whereby
chemical union takes place more rapidly in concentrated than in
dilute solutions, and when the solutions are warm. If the mixture
of toxin and anti-toxin is heated, it, instead of being neutral,
becomes toxic again. This toxicity can be neutralized again by
the addition of fresh unheated an ti- toxic serum (reactivation).
The production of bacteriolysins, cytolysins, agglutinins, pre-
cipitins, and anti-toxins are manifestations of the activity of the
immunized organisms. To further understand this activity,
Ehrlich's side-chain theory of immunity must be comprehended.
This is known as the chemical theory. To understand it fully
some consideration must be given to the study of the toxin mole-
cule. Ehrlich believes that each molecule of toxin is made up of
two groups of atoms, constituting what is known in chemical
nomenclature as lateral chains. Many molecules are made up
of a central body and lateral chain of atoms which are free to
combine with other groups of atoms without disturbing the central
body.
The benzol ring is very suitable for the demonstration of the
relationship of the side chain to the central body.
H
I
/ C \
H C ^C H
II I
H (
H
BENZOL.
56 IMMUNITY
The benzol molecule CeH 6 is here represented graphically as a
ring with a central nucleus of C 6 with lateral chains of H. con-
necting each atom of C.
If one of these lateral chains H. is supplanted by the acid radical
COOH. the benzol is converted into benzoic acid and its formula is
represented thus:
O
y
C OH
H C ^C H
H C C H
H
BENZOIC ACID.
If to this acid radical of the benzoic ring, sodium hydroxid
unites, supplanting an H in the OH of this radical, we have,
instead of benzoic acid, benzoate of soda. .
O
y
C O Na
/\
H C C H
II I
H C C H
\y
C
I
H
BENZOATE OF SODA.
RECEPTORS 57
It is thought that as the soda is brought in contact with the
central nucleus of the benzol ring, so foodstuffs unite with the
central body of the cell molecule in the organism and nourish it.
In the case of toxin, the two lateral chains of its molecule are
called haptophores and toxophores. The haptophores seize the
lateral chains of the cell and the toxophores poison it.
Ehrlich conceived that cells were nourished by their lateral
chains, each having a central nucleus with many lateral chains
called receptors bristling all over it. Complex albumins, food-
stuffs or poisons (as the case may be) unite with it. This means
a chemical union of a part of a cell with all or part of a group of
atoms. But certain body cells are only capable of uniting with
certain toxins. It is known that the toxin of tetanus has a chem-
ical affinity for the nervous system and for its neural elements
and not for liver or spleen cells. The poisons of snake venom
seem incapable of uniting with any cells of the pig; therefore, this
animal is immune to snake venom.
Now, as these toxins unite with the cells by means of the recep-
tors, the cell is stimulated to produce an excessive number of these
receptors, which are cast off and become free. Nature is very
prodigal and whenever any of the tissues of the body have been
injured, or there is a deficiency, an enormous excess of reparative
cells is produced. Weigert first called attention to this phenome-
non, which has been called Weigert's overproduction theory. So
when the haptophores of the toxin molecule combine with the
receptors of the cell, the latter are incapable of any further union
and are useless to the cell. Accordingly a great number of free
receptors are generated, and floating in the blood, engage the
haptophorous portion of the toxin. Thus the toxophore is neu-
tralized and rendered innocuous before it can reach the cell.
These free overproduced receptors constitute the anti-toxin.
This is the essence of Ehrlich's theory (Fig. 15).
Through the process of time and oxygenation the toxophorous
group in the toxin becomes innocuous, and only the haptophorous
IMMUNITY
group remains active; nevertheless the haptophorous group is able
to combine with the receptors and to stimulate the cell into
generating free receptors. This attenuated toxin is called by
Ehrlich toxoid. The receptors have been compared to a lightning
rod, which if placed within a building would, if struck, cause
disaster, while the same rod placed outside of the building, is a
means of protection to the structure against lightning. This
FIG. 15. a, receptor on cell; b, toxin molecule; c, haptophorous portion of
the molecule; d y toxophorous portion; e, receptor. (Williams.)
theory can be applied to the production of other anti-bodies.
If blood cell, bacterial cell, or any animal fluid possessing a hapto-
phore is capable of combining with side chains (receptors) of the
cells of the immunized, just as a key fits a lock, then the cells are
stimulated to produce excessive numbers of receptors, and these
constitute the anti, or immune body. It is possible to produce
from rennet, egg-albumin, cow's milk, and from many other-
albuminous substances, immune bodies by injecting these sub-
stances into animals (Figs. 16, 17).
IMMUNE BODIES
59
FIG. 16. EHRLICH'S LATERAL-CHAIN THEORY. Cell with num-
>us receptors of various kinds and shapes to which are united the toxin
jlecule. Note the free receptors.
FIG. 17. EHRLICH'S LATERAL-CHAIN THEORY. In one figure the
free receptors (anti-bodies) are united with the toxin molecule, the attached
receptors have no haptophores united to cell.
6o
IMMUNITY
List of immune bodies and their anti-bodies (Ricketts)
Antigens or Products of
Immunizing Immunization
Substances
Toxins
Complements
Ferments
Precipitogenous
Substances
A gglutinogenous
Substances
Opsinogenous
Substances
Cytotoxin Produc
ing Substances
Complement
Alexin.
Cytase
Anti-toxins
Anti-comple-
ments
Anti-ferments
Precipitins
Agglutinins
Opsinins
Cytotoxins . . .
Uemolysins
Bacteriolysins
Special cytotox-
ins Such as
Spermotoxin
Nephrotoxin
Hepatotoxin, etc.
Synonyms
Amboceptor.
Immunkb'rper.
Zwischenkb'rper.
Intermediary body.
Fixateur.
Preparateur.
Consisting of
two bodies
Complement
Amboceptor
Desmon.
Substance sensibilisatrice.
It is well known that rennet coagulates milk, but if some of the
serum of an animal immunized against rennet is added to the milk,
the latter cannot be coagulated because the anti-rennin combines
with the rennet and renders it inert.
IMMUNE BODIES 6 1
The production of bacteriolysins is explained by Ehrlich's lat-
eral-chain hypothesis. Immunization against bacteria which do
not produce soluble toxins is easily secured by repeated injection
of either dead or living bacteria into the organism. It is not easy,
however, to confer passive immunity, as in the case of diphtheria,
by the injection of the serum of the immunized animal. The
immune body is alone present in the serum generally and some
complement must be added to effect bacteriolysis. The serums
which aid in the solution of bacteria are known as anti-bacterial
serums, which, though not anti-toxic, may check invasions and
aid in recovery by destroying bacteria. It is possible to effect
an in corpore bacteriolysis in the case of typhoid fever if the
immune body and complement are injected in sufficient amounts
and proportions. As yet the results are not satisfactory from a
clinical standpoint.
A study of Fig. 18 will show clearly the exact combinations of
various substances engaged in the immunity process. Some of
the terms must be defined.
Antigen, the body, bacterium, red blood cell, etc., used for
stimulating the production of thermostabile anti-bodies, which
latter are then the substances formed against antigens; inciting
substance-antigen.
Toxins, ferments, see above.
Toxophore, the poison-carrying fraction of the antigen.
Haptophore, the binding fraction of antigen or anti-body.
Complement, alexin the normal thermolabile anti-body sub-
stance in serum.
Zymophore, toxophore for agglutinins and precipitins.
Cytophile fraction is that part of anti-body which combines
with cell, while complement phile fraction joins with complement.
Immune body, the thermostabile anti-body against bacterial or
other cells.
By immunizing with complement or anti-body we obtain
respectively anti-complement and anti-immune body which
62
IMMUNITY
u
. ^
H
!
N
TJ a
|:
& ?
| c
{
, E
'I
tea
3 1
-5
3
V 5
n 1
til
|i
-1 .
1- 4
fl : .i
3i
ANAPHYLAXIS 63
i-
experimentally will neutralize the action of these two substances.
The complement being the really responsible potent factor in all
these reactions it may be assumed to have two binding affinities,
one to the cells which it designs to help and another effect upon
antigen. If the former be absorbed in any abnormal manner
the latter is valueless.
Cell Receptor and Immune Bodies (follow Fig. 18). First Order:
I Simple union of toxins (soluble) and fixed or free receptors or antil
! toxins; no complement needed.
Second Order : Concerns agglutination and precipitation. Anti-
gen has two affinities, one for the haptophore of anti-body, another
for the agglutinin of the anti-body. The anti-body must therefore
have reversed corresponding fractions. The zymophore of anti-
body acts when the two haptophores have united and produces
the agglutination or precipitation. No complement is needed.
Third Order : Concerns bacteriolysins, hemolysins or bacterioly-
sins, etc. ; have haptophore for anti-body, and a toxophore. Anti-
body has haptophore for antigen and for the haptophore of the
complement. The union of the three must occur. Complement
is necessary for the destruction of the bacteria which it accom-
plishes through its zymophore.
Anaphylaxis. Against protection, the opposite of prophylaxis;
also called Hypersusceptibility. This phenomenon, first de-
scribed by Theobald Smith, Portier and Richet, consists in a
condition of extreme sensitiveness of animals against foreign pro-
teins. If a guinea pig be injected into the peritoneum with a
minute quantity, say M>000 g ram > of horses' serum and eight to
ten days later receive a quantity of Ko g ram > the animal will be-
come uneasy, then depressed, have dyspnea, scratch itself vio-
lently about the face and finally die after an intensification of
these symptoms. Similar symptoms have been observed in per-
sons receiving diphtheria anti-toxin therapeutically. The condi-
tion of high sensitivity to this anti-toxin is called allergie and
upon its degree depends the reaction following anti-toxin admin-
64 IMMUNITY
istration. The skin eruptions, joint pains and edema of serum
sickness are also evidences of this condition. It is said that those
persons who suffer after anti-toxin are susceptible to the emana-
tions from horses and the physician should make inquiries in this
direction when contemplating the injection of all sera.
In experimentally induced hypersusceptibility the reaction is
specific. The condition is transmissible from mother to foetus
and it can be transferred from adult to adult passively by injecting
the blood of a sensitive animal into a normal one. The first dose
is called the sensitizing one, the second the intoxicating. The
incubation period of the sensitization varies with the nature of the
protein; for horse serum it is from eight to twelve days, for bac- |
terial proteins from five to eight days. The sensitive period may j
last for several years. In searching for the cause of this reaction
it was found that there are (i) a spastic distention of the pul-
monary alveoli probably both of central and local nature, (2)
scattered hemorrhages in the organs and (3) hemorrhages with
ulcerations in the gastric mucosa. There have been many theories
for this phenomenon, but those of Vaughan, Friedberger and Wolff
Eisner may be condensed and compounded about as follows. The
body is unprepared to care for parenterally (otherwise than gastro-
intestinal tract) introduced protein and must develop anti-body
or enzyme to care for it. This enzyme or anti-body works slowly
and carefully disposes of the foreign protein, the products of which
are slowly absorbed and removed. In accord with the overpro-
duction theory this anti-substance is in large quantity when an-
other introduction of protein occurs, and goes to its work with
avidity so that it rapidly breaks the protein up into toxic elements
which cannot suddenly be cared for by the body. It is also
thought by some workers that the body protein of the animal in
question is attacked and split, liberating toxic fractions, since the
injected protein would not be adequate in amount to accomplish
poisoning. These protein toxins attack nervous and parenchy-
matous tissues. Another very plausible theory would have it
ANAPHYLAXIS 65
that the first injection directly sensitizes important cells which are
destroyed when an intoxicating dose arrives.
The similarity of characters between serum shock and immedi-
ate traumatic shock was thought to shed some light on the sub-
ject. The latter is believed to be due to protein degradation
products, due to trauma or low blood pressure, which have a
paralyzing effect upon unstriped muscle or upon the vasomotor
centers.
It has been shown that an anti-anaphylactic state can be pro-
duced by repeated small injections of protein at intervals too
short to allow incubation of an intoxicating dose. Use is made
of this knowledge in the case of persons who need anti-serum but
who show sensitivity to it. Repeated small but increasing quan-
tities are put into or under the skin or into the vein until the
patient can receive without reaction the full dose. This is called
desensitization.
Friedberger has used these facts to elaborate a theory of infec-
tion. He believes that bacteria circulating in the body stimulate
anti-bodies, combine with them and that when complement acts
upon this union toxic substances are set free.
In explaining all infectious diseases on this basis one assumes
that sometime in life a person has been sensitized by bacteria or
their proteins so that he is receptive for a virulent germ when
this has overcome the primary external bodily defenses. It is also
to be considered the modern explanation of diathesis.
McKail divides anaphylaxis as follows:
Natural Anaphylaxis, depending upon
(a) Species of animal, for example cholera in man, anthrax
in cattle, glanders in horses.
() Age diphtheria in children, erysipelas in the elderly.
(c) Individual to white of egg, or blood serum, even by in-
gestion ("one man's meat is another man's poison"),
5
66 IMMUNITY
Acquired Anaphylaxis, depending upon
(a) An attack of disease, erysipelas, diphtheria.
(b) The injection of dead cells, tuberculin.
(c) Injection of nitrogenous matter, blood serum and egg-
white.
Direct practical application of theoretical speculation about
hypersensitivity may be made in explaining, treating and prevent-
ing certain states in man. Mention has already been made of the
possibility of intoxicating persons susceptible to the presence of
horses by the injecting of horse serum. The only treatment for
such a "Schock" is an injection of epinephrin or atropin.
Serum sickness is explained as a digestion of foreign serum still in
the body as such when sufficient ferment has accumulated to
digest it, a peroid of three to twelve days. Whether this be cor-
rect or not, symptoms can be made milder or prevented by dividing
the doses by some hours. A state of hypersensitivity or allergic
may also exist to pollen of plants, certain foods and drugs, dusts
of feathers and hair or dandruff from cats and dogs. The evi- j
dences of this state take the form of asthma, gastrointestinal dis- '
turbances, and skin eruptions of which urticaria and eczema are i
the commonest. Detection of this hypersensitivity may be sub- |
jective at times but usually it has to be established by technical
means. It so happens that allergic persons have either a definite
anti-body reaction or the local sessile receptors in the cutaneous
cells are stimulated, for the application of the responsible protein
to an abraded area on the skin will produce at the point a swollen
red areola. In the cases of infection of a bacterial or toxic mix-
ture this reaction usually requires twenty-four hours to develop,
a delay suggesting that anti-bodies are involved. In the case of
serum or pollen allergic, the reaction is almost immediate, suggest-
ing local cellular preparation. This is the basis of skin tests,
except with tuberculin and the S chick test. Prevention of such
allergic poisonings is best accomplished by the avoidance of
ANAPHYLAXIS
6 7
the offending material. Treatment and prophylaxis take the
form of injecting solutions of the responsible protein under
the skin. For example ragweed is the most common cause
of hay fever. Solutions of its pollen, made in standard dilu-
tions of 1-10,000 to 1-500 are injected in rising quantities,
beginning with the weakest. Treatment should be given during
the winter and spring so that as high a degree of disensiti-
zation as possible will be accomplished. Little can be expected
in the hay fever season.
FIG. 19. Illustrating the conception of deviation of complement.
Amboceptor; b, antigen; k, complement. (MacNeal.)
. Complement Fixation. Hemolysis occurs when the serum of a
rabbit immunized against washed sheep's red blood cells is mixed
with fresh washed sheep's corpuscles in the presence of comple-
ment. If, however, complement be absorbed in any way, a solu-
tion of the coloring matter of the red cells will not occur in this
mixture. Complement will combine with anti-body in the pres-
ence of antigen. This fact has been taken advantage of in deter-
mining both the nature of antigen and the presence of anti-body.
Its most important practical use is in syphilis, to the diagnosis of
which Wassermann applied it, and the Wassermann test is for the
presence of syphilitic anti-body in the blood serum of syphilitics.
68 IMMUNITY
This test is positive from the initial lesions all during life unless
the patient has been successfully treated. Indeed the para-
syphilitic states also give it. The principles of the test are also
used for determining the presence of tuberculous, leprous, ty-
phoid and other anti-bodies.
The materials necessary in the Wassermann test are as follows:
i . Syphilitic antigen, extract from the syphilitic liver of a foetus,
in alcohol, ether or water; lipoids like lecithin or extracts from
guinea pig's heart will act as antigen.
2 a. Serum from a known case of syphilis and containing there-
fore syphilitic anti-body.
2b. Known non-syphilitic serum without anti-body.
3. The suspected serum.
4. Fresh serum from a guinea pig, rich in complement.
5. Serum from a rabbit that has been immunized against
washed red cells from a sheep; called amboceptor.
6. Fresh sheep's red blood cells, washed in saline and made into
5 percent supension.
The solutions are all standardized so that only sufficient of each
is added to complete the absorption or produce the hemolysis.
The serum known to be syphilitic and the suspected serum are
heated to 56C. for thirty minutes to destroy the native and in-
herent complement. The rabbit anti-sheep cell serum is also
heated to this degree.
The hemolytic series, i.e., sheep's cells, rabbit's anti-sheep's
cells serum and complement are standardized to find out what
quantities will exactly complete hemolysis. These quantities
are the units. It is necessary to control tests to find out what
quantity of the antigen and known syphilitic anti-body will unite
to bind the determined quantity of complement. The tests are
performed in small tubes so as to have a long column of fluid easier 1
to observe. Tubes are set as follows:
A.
unit #i +i unit #2a -f- I unit
unit #i +i unit #3+1 unit
unit #1+1 unit #20 + I unit #4.
unit #2a + i unit #4.
unit #ab + i unit #4.
unit #3+i unit #4.
unit #4.
unit #i.
unit #2a.
unit #2b.
unit #3.
FACTION 69
unit #5 +
unit #6 = No hemolysis.
unit #5 +
unit #6 =
if #3 be
syphilitic, no hemolysis.
if #3 be
^on-syphilitic hemolysis.
unit #5 +
unit #6 = Hemolysis.
unit #5 +
unit jj'6 = Hemolysis.
unit #5 +
unit #6 = Hemolysis.
unit #5 +
unit #6 = Hemolysis.
unit #5 +
unit #6 = Hemolysis.
unit #5 +
unit #6 = No hemolysis.
unit #5 +
unit #6 = No hemolysis.
unit #5 +
unit #6 = No hemolysis.
unit #5 +
unit #6 = No hemolysis.
The tubes receive first the solutions on the left and are placed
in the 37C. incubator for two hours to allow union of their various
parts, particularly the complement with others. They then re-
ceive the solutions on the right, are placed in the incubator for
half an hour and in the ice-box overnight, when they are ex-
amined for a solution of the red coloring matter. If it occurs, the
column is perfectly clear red with some residue of extracted cells.
If no hemolysis has occurred, the red cells form a layer at the bot-
tom, and the column is clear and colorless.
A and B are the tests of syphilitic sera while the remaining are
to find out if the other solutions affect the results of A and B. Of
course tube G represents simply the complete hemolytic system.
The extra tests are to exclude the possibility of interference on
the part of any single member with the complement No. 4. The
character of the test is found in tube A where syphilitic antigen and
serum have bound or fixed the complement so that it cannot unite with
the rabbit serum and sheep's corpuscles to hemolyze the latter. This
is a positive test. A negative test is when hemolysis occurs, since no
anti-body is present to unite with complement in the presence of
antigen.
Complement Deviation. This is a condition arising when there
is too much amboceptor and too little complement. The free
amboceptors adsorb complement and there is none left for cell
needs or renewed demands. It is to be distinguished from com-
plement fixation. The terms are not interchangeable.
70 IMMUNITY
ANTI-TOXINS, VACCINES, AND TOXINS
The following is Wassermann's list of anti-toxins:
Anti-toxins for bacterial toxins :
Diphtheria
Tetanus
Botulism
Pyocyaneus
Symptomatic Anthrax
Anti-leucocidin, an anti-toxin against the leucolytic poinson of
staphylococcus
Anti-toxins for the blood dissolving toxins of certain bacteria.
Anti-toxin for animal toxins :
Anti-venene for snake venom
Anti-toxin for spider poison
Anti-toxin for scorpion poison
Anti-toxins for certain poisons in fish, eel, salamander, turtle, and wasp
sera.
Anti-toxins for plant poisons :
Anti-ricin for castor-oil poison
Anti-abrinforjequerity bean poison
Anti-robin for locust bean poison
Anti-croton for crotin-oil bean poison
Anti-pollen for pollen of plants that produce hay-fever.
There may be added to this list a number of anti-sera developed
to a practical value in recent years whose activity is dependent
not on anti-toxin content, but upon their ability to agglutinate,
precipitate, and opsonify their respective microorganisms. These
sera are made against infections with:
Pneumococcus
Meningitis coccus
Streptococcus
Plague
Dysentery (bacillary)
Staphylococcus
Typhoid fever
MANUFACTURE OF ANTI-TOXINS 71
The first two have proven of more definite value than the rest.
Manufacture of Anti-toxins. If small doses of a given poison,
such as diphtheria toxin, be repeatedly injected into a susceptible
animal, and if the dose is gradually increased, there appears, after
a time, in the blood serum, an anti-body, or anti-toxin. This
substance in the serum is secreted by the cells and corresponds to
the free receptors in Ehrlich's lateral-chain theory. If an animal
be injected with the anti- toxin, and then with a large dose of toxin
say ten times the amount necessary to kill it if it had not
received the anti-toxin it will not be harmed. Here the free
receptors artifically supplied to the animal unite with the hapto-
phorous chains in the toxin molecule, and naturalize, or bind, the
toxophorous or poisonous chains in the molecule, and prevent
toxophore from attacking important vital cells belonging to the
animal. And if the anti- toxin and toxin, after being mixed in a
test-tube, are injected into a susceptible animal, no harm results,
if they are in proper proportions, since the same thing has hap-
pened in vitro that happened in the animal, the receptors and
haptophores have united; the toxophores are bound, and the
animal is unharmed.
The manner of making the diphtheria anti-toxin can be taken
as a type.
Diphtheria bacilli are grown for seven to ten days in . i percent
dextrose bouillon at 37C.; as the bacilli grow they elaborate a
very powerful poison or toxin, which is highly complex in composi-
tion. Strains that habitually grow on the surface of the bouillon
are used, the access of air enhancing the production of toxin. It
is easily decomposed by heat, light and oxygen, and should be
used soon after it is prepared. After the cultures have grown for
several days, the bouillon is filtered through a porcelain filter, and
is then stored in sterile bottles in an ice chest. Horses are gen-
erally immunized, since they are susceptible to the action of the
toxin, and are easily managed. Before being used they are care-
fully tested with tuberculin for tuberculosis and with mallein for
72 IMMUNITY
glanders. Being very susceptible to infection with tetanus while
undergoing treatment, a prophylactic injection of tetanus anti-
toxin is given each animal. McFarland found that the death
rate from tetanus, in a large stable, was greatly reduced after
using tetanus anti-toxin as a prophylactic measure.
Immunization is started by injecting into a previously examined
healthy horse a mixture of toxin and an ti- toxin in which the
former is not quite neutralized by the latter. These quantities are
determined by guinea-pig .tests. Such a mixture is safer for the
horse and begins the immunity reaction more promptly. A
few doses like this are given after which pure toxin is used.
This is followed by a rise of temperature, local reaction and
systemic disturbance. After waiting for all reactions to disappear
injections are continued by slow increases, until, after a few
weeks or months, 1,000 c.c. of toxin are injected at one time
(enough to have killed a dozen horses that had not received the
smaller doses previously). The injection of the toxin is followed
by an immediate fall in the anti-toxic power of the serum, only
to be followed by a quick rise. The horse will not produce anti-
toxin indefinitely. After the animal has been immunized suffi-
ciently, his blood is drawn from the jugular vein, and after the
clot has formed the serum is drawn off and stored.
Anti-toxins are found to lie in the pseudoglobulin fraction of
the serum. Even though an anti-serum be strong, a large quan-
tity would have to be injected to obtain high unit value. The
globulins of the whole blood are now precipitated by 30 percent
ammonium sulphate and the pseudoglobulins are dissolved by
normal salt solution. This concentrates high unit values into
small bulk. When removed from the horse, serum may contain
300-800 units per cubic centimeter. After concentration a value
of 3-10,000 units may be obtained.
McFarland found that a horse was capable of producing enough
an ti- toxin to protect 806 other horses against doses of toxin, each
one of which was equivalent to the total amount of toxin that the
ANTI-TOXINS 73
immunized horse received. Thus there is evidently a tremendous
overproduction of anti-toxin far above the needs of the animal.
The various component parts of the toxin stimulate the cells of
the horse to produce the receptors, or anti-toxin. The toxoids,
themselves not poisonous, have the property of stimulating the
production of anti-toxin. We measure the anti-toxic powers of
the anti- toxin with units arbitrarily devised. An anti-toxic unit
is that amount of horse serum just necessary to protect a 2$o-gram
guinea pig against 100 times the minimum lethal dose of toxin.
To standardize anti- toxin, we must employ animals, into the
bodies of which toxins and anti-toxins are injected. If a certain
amount of anti-toxin is necessary to protect a guinea pig against
ten times the minimum fatal dose of toxin per 100 grams of guinea-
pig weight, then we know that the anti-toxin contains so many
units. The minimum lethal dose of toxin is the smallest quantity
that will kill a guinea pig of 250 grams in four days.
A standard anti-toxin is kept by governments to be used as a
control of the products of biological chemists.
Against this standard anti-toxin a toxin of unknown strength is
measured by means of guinea pigs. The toxin unit thus found is
then used to determine the anti-toxic unit of anti-toxins of un-
known power.
Anti- toxic serum is preserved by the addition of .5 percent of
tri-cresol or phenol. It remains practically unchanged in strength
for a year or more.
It is not only of value as a curative agent, neutralizing the toxins
already formed, but is valuable as an immunizing one against in-
fection. Persons exposed to diphtheria and giving a positive
Schick test should be given an immunizing dose of 1,000 to 1,500
units. If injected early in a case of diphtheria, it is much more
likely to do good, than if used later. Some desperate cases have
received 100,000 units and have recovered. The following is the
very good guide given by Park:
74 IMMUNITY
SINGLE DOSE ONLY
Infant, 10 to 30 pounds (under 2 years)
Mild Moderate Severe Malignant
2,000 3,000 5,000
3,000 5,ooo 10,000 10,000
Child, 30 to 90 pounds (under 15 years)
3,000 4,000 10,000 10,000
4,000 10,000 15,000 20,000
Adults, 90 pounds and over
3,000 S,ooo 10,000 15,000
5,000 10,000 20,000 40,000
Method of Administration
Intramuscular
or
^ Intravenous
Subcutaneous
Intramuscular
% Intravenous
and
or
or
and
3^ Intramuscular
Intramuscular
Subcutaneous
% Intramuscular
or
or
Subcutaneous
Subcutaneous
Tetanus Anti-toxin. Tetanus anti-toxin is produced in a man-
ner similar to that of diphtheria anti-toxin. As the horse is ex-
ceedingly sensitive to tetanus toxin, before the immunizing process
is begun, the toxin is attenuated by heat or iodine.
The an ti- toxin is standardized, as in diphtheria, by testing its
potency against the toxin. A guinea pig of 500 grams weight is
used, and test toxin is employed of such strength that .01 c.c. will
kill the guinea pig in about four days. The United States unit
of tetanus an ti- toxin is now the least quantity of an ti- tetanic
serum necessary to save the life of a 350-gram guinea pig for
ninety-six hours against the official test dose of standard toxin
furnished by the Hygienic Laboratory of the Public Health
Service.
The toxin of the tetanus bacillus has such an affinity for nervous
tissue that, once it becomes attached, separation is difficult, or
even impossible if union has existed for some time. More than
this the toxin has a greater affinity for nervous tissue than for its
ANTI-TOXINS 75
own anti-toxin. If an animal be injected intravenously with
anti-toxin and intracerebrally with toxin it will die of tetanus.
This explains why the use of anti-toxin in the treatment of the
diesase must be vigorous, early and direct. Dosages of 10-50,000
units are necessary before symptoms have become well estab-
lished. Subcutaneous methods are too slow and indirect. Anti-
toxin should be given into the spinal canal, into the vein and
around the wound. Prophylactically anti-serum should be given
in every case of penetrating, lacerated wound especially if soiled
with earth, street dirt, rust or gun waddings. Tetanus antitoxin
is more efficient as a prophylactic than as a remedy.
Streptococcus Antiserum. The three principal groups of
streptococci, hemolytic, non-hemolytic and viridans, have differ-
ent immunity factors and each kind produces it own anti-bodies.
These however are never in great amount and this seems
due to the rather feeble antigenic power of the cocci them-
selves. The only useful anti-sera are those prepared against a
large number of strains of each of the varieties by repeated
injections over a long period. Their antigenic fractions seem to
be hemolytic, leucocytolytic and neurolytic. The anti-bodies
formed in horse's serum seem to decrease in value after removal
from the body. They are chiefly agglutinative and anti-toxic.
For clinical use the serum should be as fresh as possible and used
intravenously in doses of 50 to 200 c.c. It has been employed
in endocarditis, osteomyelitic and puerperal septicemia with some
promise.
The Anti-pneumococcus serum is prepared in the same way.
Horses are immunized by the injection of first autolysates then
living cultures, and the horse's blood, after a period of treatment
by cultures, is drawn off, preserved with tri-cresol. It is used in
the crude form of serum as the anti-bacterial powers are not easily
concentrated. It is standardized so that .2 c.c. shall protect a
mouse against 100,000 times the amount of a culture of pneumo-
cocci that would kill a control mouse. It has been found that
76 IMMUNITY
there are in this country four groups of pneumococci in a serolog-
ical sense, a discovery confirmed by therapeutic results. It is
possible to prepare curative anti-sera against two of the groups
while the other two fail to call forth useful anti-bodies. In any
given case of pneumonia the type of infecting organism is deter-
mined by isolating it from the sputum and performing the agglu-
tination test with the individual sera of Types I and II, whereupon
should one of these react the appropriate anti-serum can be in-
jected. The organisms are obtained for the agglutination test
by injecting the sputum into a mouse's peritoneum, killing the
animal after six to eight hours and using the rich growth of cocci
in the peritoneal fluid as the bacterial suspension. With Type I
the therapeutic results are very promising; with II helpful at times.
Type III is the Pneumococcus mucosus yielding no useful anti-
serum, while Type IV is a heterogeneous group possessing no
serological uniformity and unable to call forth any valuable anti-
body in the injected horse. The serum is given intravenously in
dose of 50-100 c.c. and repeated when the temperature rises
again, sometimes every eight hours.
Meningitis Anti-serum. Epidemic meningitis, caused by the
Micrococcus meningitidis intracellularis or meningococcus, can
be treated by anti-serum. The cocci owe their power to endo-
toxins and pus formation, exerted chiefly in the coverings of the
central nervous system. They are present in the spinal fluid,
only being found in the blood early in an ordinary attack or
in highly septicemic cases. Anti-serum is made by injecting
horses with first dead then living bacteria until its serum shall
have acquired high agglutinative, opsonic and bactericidal
power. The horse's serum is separated, preserved as usual, and -
used by injecting into the arachnoid space by lumbar or cranial
puncture. This route is selected to bring the anti-serum into
close relation with the cocci since by the subcutaneous or intra- *
venous routes insufficient anti-bodies pass from the blood to the
meningeal fluid. It should be given also into the blood stream
VACCINATION 77
in order to prevent septicemia. Its effect is to increase phagocy-
tosis and cause bacteriolysis. No satisfactory standard has been
devised. Dosage varies but a safe guide is to inject under the
meninges 75 percent as much as the fluid removed at lumbar
puncture. Intravenous dosage should be 20-50 c.c. The anti-
serum probably has no prophylactic value.
Anti-plague Serum. Yersin, a French bacteriologist, treated
horses with living cultures of plague bacilli, and after a long period
of immunization used a serum which either effectually vaccinated
an individual against the plague, or greatly modified the disease
after it had once begun.
The action of the serum is bactericidal, as well as anti-toxic.
The dose varies with the stage of the disease; 20 c.c. is an effective
prophylactic dose, while from 20 to 300 c.c. have been used often
as curative doses.
VACCINATION
By the use of attenuated, or killed microorganisms, it is possible
to effectively vaccinate men and animals against many diseases,
notably, small-pox, hydrophobia, plague, cholera, typhoid fever,
anthrax and quarter-evil.
Any of the bacterial products used as prophylactics are some-
times called vaccines, the word being borrowed from small-pox
vaccine. It is better to use the word bacterin for the purpose,
even when they are given prophylactically. Bacterin is employed
for the dead bacterial masses used therapeutically. Sensitized
bacterins are vaccines that have been exposed to the action of the
respective anti-sera before their use. It is the purpose of this
procedure to prepare the organisms by combining them with the
homologous anti-bodies so that when injected they have only to
combine with complement to begin their immunizing effect.
Theoretically this is correct but the practical value has not been
fully confirmed.
78 IMMUNITY
Vaccination Against Small-pox
Jenner's observations in 1794 established the etiological rela-
tionship between human variola and cow-pox. Human small-
pox virus passed through a cow will lose its power to produce
typical variola but will produce in man a local condition called
vaccinia which, upon recovery, leaves behind immunity to
variola.
By the term vaccination, in its strict sense, we mean the applica-
tion of attenuated small-pox virus, weakened by passage through
kine, to human beings and infecting them with the modified
disease. The disease is localized at first at the site of inoculation,
and a bleb or vesicle forms. As a rule the disease does not become
generalized. It creates, in the vaccinated individual, an active
immunity against small-pox. The toxins diffused through the
blood-stream stimulate the cells of the body into forming either
anti-toxic or other anti-bodies. These various substances, as
yet unknown, remain for a long period within the body of the
vaccinated person and may protect it for years against invasion
and infection with the cytorcytes in virulent form. A person who
has variola cannot be vaccinated, subsequently he is immunized
against vaccinia by this attack of variola, just as he can^be immun-
ized against variola by vaccinia infection.
Since Jenner first discovered that cow-pox introduced into the
body prevented small-pox, it has been the world- wide custom to
use either the dried virus or liquid glycerinized virus from the cow
or human beings in the process of vaccination. It has been found
that human virus generally used was likely in rare instances to
transmit syphilis, so it is now the universal custom to use cow
virus. This virus is collected from fresh vesicles in calves or
young heifers, as clean as possible, as it is used as seed to inoculate
the animals and the operation is done under strict antiseptic
precaution. After a week the virus is collected under similar
antiseptic precautions by scraping the base of the vesicle with a
VACCINATION AGAINST CHOLERA 79
sterile curette. The pulpy substance thus obtained is mixed
with glycerine and stored for a month or more. The action of the
glycerine is to rid the virus of many of the bacteria, through, it is
supposed, a hydroly tic action. This virus is then rubbed into the
skin of the individual to be vaccinated under strict aseptic pre-
cautions. At the end of a week, a pearly white vesicle is formed,
and it is then considered that vaccination has "taken" and that
the individual is protected against variola. This action of
immunization is supposed to be complete on the fourth day after
the virus has been introduced. This is a matter that is difficult
to decide, but the immunization process is, no doubt, a very slow
one, like every other immunizing process where the immunity is
autogenous and active, and not passive, as in the case of diph-
theria anti-toxin.
Persons who are entirely immune to variola will show twenty-
four hours after vaccination a distinct red areola without vesicles
or pustules; this is the reaction of immunity. Recent successful
vaccination leaves the patient in a condition of resistance and
four to six days after revaccination an areola surmounted by a
reddish papule is seen; this is vaccinoid.
Vaccination Against Cholera
By the attenuations of cholera spirilla, Haffkine has produced
vaccines which effectively protect individuals against infection
with cholera, or if they become infected with the disease, it is so
modified that they can, and do, more easily recover. He employs
two vaccines, a weak one and a stronger one. The weak one is
used to prepare for the stronger one, which is the effective vaccine.
The weak, or first virus, is prepared by growing the cholera
vibrios at a high temperature, 39C., in a current of air. The
stronger is prepared by passing the vibrios through a series of
guinea pigs, so increasing the virulence that the virus is invariably
fatal to the guinea pigs in eight hours. After cultivating this
8o
IMMUNITY
virus on agar, the surface growth is washed off with sterile water
(8 c.c.) and % part of this is used as a dose. As the virus rapidly
attenuates it must be reactivated by passing it through guinea
pigs from time to time.
The first injection is given in the flank, and the second follows
in five days. Accordingly as the symptoms are severe, so will the
resulting protection be strong. Haffkine has given 70,000 injec-
tions without an accident. The following results were obtained
by Haffkine who worked in India for the British Government:
Population
Case Cholera
Deaths
Total
Percent
Total
Percent
Non-inoculated, 1,735
171
21
10.63
4.20
ii. 3
19.0
6.51
3-80
Inoculated, 500
The immunity conferred by this mode of vaccination is not
complete until ten days after treatment. It is possible to vac-
cinate with these relatively virulent bacteria because they are
given under the skin, a place where life of the vibrios soon ceases.
During an attack of cholera the vibrios do not enter the blood
but remain in the deep layers of the intestinal mucosa.
Vaccination Against Typhoid
By the injection of sterilized cultures of typhoid bacilli, it is
possible to create an immunity of a moderate kind against enteric
fever. The method was perfected by Wright, and his mode
of procedure is to secure a culture of typhoid both virulent and
able to call forth a large amount of anti-body in the injected
person, which is tested on guinea pigs, and the minimum lethal
dose for a loo-gram guinea pig is used as the dose for man. This
dose varies from .5 c.c. to 1.5 c.c. of a culture sterilized by heat
ANTI-TYPHOID VACCINATION 8 1
at 6oC., and preserved with lysol. After the injection there is
often redness and pain at the site of inoculation, some fever and
lymphangitis. The method at present in use in the United
States is to employ a twenty-four hour agar slant or bouillon
culture, killed by heating as above; these are suspended in
saline with trikresol or phenol and counted in a hemocytometer.
In order to control the toxicity of this suspension a mouse is in-
jected subcutaneously with a billion; it should live at least five
days. The doses are 500 million, 1,000 million and 1,000 million
eight to ten days apart.
The results of Col. F. F. Russell, U. S. A., a man who has had
much experience, since he was in charge of the army vaccinations,
are interesting and instructive. He says :
1. " Anti- typhoid vaccinations in healthy persons is a harmless
procedure.
2. It confers almost absolute immunity against infection.
3. It is the principal cause of the immunity of our troops
against typhoid in the recent Texas maneuvers.
4. The duration of the immunity is not yet determined, but
is assuredly two and one-half years and probably longer.
5. Only in exceptional cases does its administration cause an
appreciable degree of personal discomfort.
6. It apparently protects against the chronic bacillus carriers,
and is at present the only means by which a person can be pro-
tected against typhoid under all conditions.
7. All persons whose profession or duty involves contact with
the sick should be immunized.
8. The general vaccination of an entire community is feasible
and could be done without interfering with general sanitary
improvements and should be urged wherever the typhoid rate
is high."
The wisdom of these conclusions has been abundantly proven
in our army during the World War when practically no typhoid
fever occurred in vaccinated men. It might be added that no
82 IMMUNITY
immunizing procedure is perfect and this one does not justify
the drinking of water known to be polluted.
Vaccination against all the typhoid fevers, due to the typhoid
bacillus, the paratyphoid bacillus A and B, can be accomplished
by combining these organisms in one vaccine. The form now in
common use, as employed by our armed forces, is one suspension
in each cubic centimeter of which are contained 1,000 million
typhoid bacillus, 750 million of paratyphoid A and 750 million
of paratyphoid B. Three doses, .5 c.c., i c.c. and i c.c are given
ten days apart. This makes a total of 6,250 million organisms
injected.
Vaccination Against Pneumococcus Infections
Experiences in South Africa by Lister and in the American
Army by Cecil seem to hold out encouragement for the prophy-
laxis of pneumonia by vaccines of the respective coccus. The
types of pneumococci prevalent in a district must be determined
and used in the suspension. The preparations employed by
Cecil contained the three fixed American types and were given
in doses of 6,000 million four times at six to eight day intervals.
Under war conditions some advantage was observed but available
data are too few to form a general evaluation. Experimenta-
tion and human experience indicate the harmlessness of this
procedure and use is recommended. The action of the vaccine
seems to be stimulation of opsonins and of bacteriolysins.
Vaccination Against Diphtheria
According to the experiments of Behring, Theobald Smith and
W. H. Park it is perfectly feasible and without danger to immun-
ize children against diphtheria. As already mentioned immuni-
zation of horses is begun with nearly neutral toxin-anti-toxin
mixtures. For immunization of children only such are used.
Solutions of the toxin and anti- toxin are made to contain i unit
VACCINATION AGAINST ANTHRAX 83
of the latter and 60 percent and 80 percent of a unit of the former.
Two or three injections are given, the first or the first and second
of the former strength, the last having the higher value of toxin.
A local reaction occurs and not infrequently a general one
fever, malaise. Immunity requires a week to start and about a
month to be fully developed. It is believed to last at least
four years. Immunization need only be given to those who have
a positive Schick test.
Vaccination Against Plague
Haffkine, in India, has vaccinated natives and others against
plague by somewhat the same methods employed in anti-cholera
vaccination. The B. pestis is cultivated in flasks of bouillon; as
it grows, the stalactite-like scum on top is shaken from time to
time to the bottom of the flask. After growing for six weeks in
the bouillon, the culture is killed at 7oC. for three hours. It is
then used as vaccine, 3 c.c. is the usual dose for man, 2 c.c. for
woman, and children still less. After the inoculation, heat and
redness appear at the site of inoculation, and the patient feels ill
and has some fever. Haffkine holds that immunity against the
plague is complete in twenty-four hours after vaccination. His
results are at times really very good.
The Indian Plague Commission reported that the measure was
valuable as a means of preventing infection; while it was not an
absolutely certain 'means, yet it sensibly diminished the death
rate. The immunity lasts about a month. Such vaccines are
not to be used after attack has started.
Vaccination Against Anthrax
Of all forms of vaccination against disease with attenuated
bacteria this is the most successful. Its use is confined to domes-
tic animals, sheep, cattle, and horses, and has reduced the
mortality in the country where it is used from 10 percent to .5
84 IMMUNITY
percent. The method requires the employment of two vac-
cines made of attenuated anthrax bacilli. No. i is a culture of
bacilli attenuated by growing them at a high temperature, 42.5C.,
in a current of air for twenty-four days. No. 2 is grown at the
same temperature for only twelve days. The first vaccine is
used to immunize the animal against the second, which causes a
marked local reaction, and which is the real immunization agent
against infection with virulent anthrax bacilli. The injections
are given about one week apart. Many State Governments as
well as the Federal Government of the United States supply
the vaccine gratis to stock raisers and others.
Vaccination Against Black-leg or Quarter-evil
Quarter-evil, or Rauschbrarid, is due to a specific bacillus.
Vaccination against this disease may be accomplished by inocu-
lating with a powder consisting of dried muscle from the affected
part of infected animal. There are two vaccines, No. i, and No.
2. The first is prepared by heating (and thus attenuating) the
bacilli up to 103 C. The second is prepared by raising the tem-
perature up to Q3C. These vaccines are given at a short time
apart, and the immunity is effective. The method is valuable
to stockmen.
Vaccination Against Tuberculosis
There is at present no safe and satisfactory prophylactic meas-
ure for the production of increased resistance in man to tubercu-
losis. In cattle the repeated injection of bacilli of the human type
has been found capable of raising the animal's resisting power to
a sort of immunity. The therapeutic use of the various tubercu-
lines has on the other hand, met with better success, and it seems
that for human medicine at least that they have won a place in
the treatment of surgical tuberculosis. They may be also of
value in pulmonary disease.
THE TUBERCULINS 85
The Tuberculins
The toxin of the tubercle bacilli (old tuberculin) is prepared by
growing the organism for a long period in glycerinized veal broth,
after which the flasks are steamed in a sterilizer for an hour or
more, and then the bacilli are filtered out through porcelain filters.
The filtrate is reduced by boiling to one-tenth of its bulk, and to
this .5 percent of carbolic acid is added as a preservative. If
this toxin, even in minute doses, is injected under the skin of a
tuberculous animal, it acts as a powerful poison. In a few hours,
it causes a rapid rise of body temperature, accompanied by nausea
and, perhaps, vomiting. About the localized foci of tuberculosis,
a vigorous reaction occurs. Around indolent old sores and other
lesions there is a tendency to heal by the casting off of necrosed
tissues, and the infiltration of the perituberculous area with leu-
cocytes. In lupus (tuberculosis of the skin) redness and heat
occur about the lesion. This febrile phenomenon following the
injection of tuberculin into tuberculous animals is a valuable
diagnostic feature toward the recognition of tuberculosis in
animals and in man. In 90 percent of cases the reaction is
trustworthy.
Its use in man has been much questioned, as it is thought by
some to disseminate the disease from original and confined foci.
This, however, has been denied. Many able clinicians use it and
recommend it (Osier, Trudeau, Musser).
Koch's new, or T.R. tuberculin was, like the old, designed by
him as a therapeutic agent for the cure of tuberculosis. It is
made by pulverizing the bodies of living tubercle bacilli and dis-
solving the residuum in an indifferent fluid, centrifuging this and
collecting the sediment which is Tuberculin Rest, T.R. The
solution above this sediment containing soluble substances from
the bacillary bodies is Tuberculin Obers, T.O. It produces a
more intense reaction than the old tuberculin. Like the old, it
is used in the treatment of lung, bone, laryngeal, and skin tuber-
86 IMMUNITY
culosis. It certainly causes a local reaction about tuberculous
foci, and no doubt aids in the building up of healthy tissue.
The dose of tuberculin for testing purposes should be .5-2. mg.
for a child, 2-6. mg. for a young or weak person, and 5--io. mg.
for a larger person. It is well to give the highest dose that it is
believed the patient will stand in order to get a prompt and
definite result thus avoiding the necessity of a repetition. Re-
peating the injection of such amounts is not without danger as
it might light up a latent lesion. Any focus that is at the bottom
of a clinical condition requiring such a test, will give a positive
reaction with the quantities mentioned. For therapeutic pur-
poses one begins with an injection of .000000 1 gram or smaller
and increases .slowly according to the patient's condition. Tuber-
culin should only be administered by experts.
Ma lie in
Mallein is a preparation made from the toxin of the glanders
bacilli, and is prepared precisely as the old tuberculin. By in-
creasing the virulence of the glanders bacilli, by passage through
a series of guinea pigs, a highly virulent bacillus is obtained. It
is then grown in glycerinized bouillon for a month at 37C. The
resulting fluid is sterilized by heat and filtered through a Pasteur
filter. The filtrate is evaporated to one-tenth its quantity
when intended for conjunctival use or left in its natural state
when for subcutaneous use. A small amount of carbolic acid
is added in order to preserve it.
In a horse with glanders, the injection of mallein is followed by
a large painful swelling at the injection site. With this there
is a rise of temperature, which is the diagnostic reaction that
indicates infection with glanders. In this respect the reaction
is like tuberculin. In healthy horses no rise of temperature
follows the injection, and the resulting swelling more quickly sub-
sides. A convenient test is the introduction of a drop of concen-
trated mallein into the conjunctival sac. Positive reaction is
IMMUNIZATION AGAINST HYDROPHOBIA 87
indicated by lacrimation and purulent collections within twenty-
four hours. Mallein has been used as a prophylactic agent
against glanders with some success, and treatment can be carried
on with it in valuable animals.
Immunization Against Hydrophobia
While the actual causal agent of hydrophobia has thus far
eluded bacteriologists, certain well-marked histologic lesions have
been discovered in the ganglia of the central nervous system, and
in the medulla, which are not found in any other disease. This
dispels all doubt as to the fact that hydrophobia is a real clinical
entity.
It is possible to immunize animals and man against this disease
by the use of attenuated virus. In common with many other
viruses, that of hydrophobia can be weakened through the action
of either heat, drying, light, or chemicals. Pasteur found that by
drying the spinal cords of rabid animals for two weeks, they be-
come totally avirulent. If the cord is dried but three or four days,
the virulence is but slightly reduced. Immunity to rabies can be
produced by injecting minute quantities of the poison, and then
gradually increasing the dose until virulent virus can be employed.
Recent work seems to indicate that simple dilutions of the
virus so that minute quantities are used, can be employed
prophylactically instead of dried material. . '
Modification of the amount of poison used may be affected by
employing equal quantities of spinal cords from rabid animals that
have dried varying lengths of time. The vaccine consists of pieces
of cord, i cm. in length, from rabbits that have been killed by
inoculation with fixed virus. This is emulsified with sterile salt
solution. Cord that has dried for fourteen days is first injected,
after which cords that have dried fewer and fewer days, until,
finally, one that has dried only three days is injected.
In cases of bites by rabid dogs on the face or head, the vaccina-
tion must be rapid, so two injections per diem are given. In
88 IMMUNITY
Berlin the weakest injection used (the first) is made from a cord
that has dried but eight days, and the course is much quicker.
It was at first thought that short drying might carry over too
much virus but in order to treat certain serious head bites, cords
of 3 and 4 days drying were tried not only without damage but
with promising results. Now in threatening cases treatment
may be begun with 3 day cords, then 2 day cords. The effect
of this mode of inoculation is to produce in the bitten individual
a very rapid active immunity, quicker in its action than the
infection. The treatment is solely prophylactic and in no way
curative. If symptoms of rabies have set in, the treatment
is of no avail. In rabies the incubation period is about six weeks,
so that there is plenty of time to immunize the patient by injec-
tion with attenuated virus.
Since the immunizing process is always begun after the bite of a
rabid, or supposedly rabid dog, it differs from other vaccinations,
which are resorted to before infection.
Results of Treatment. Among those bitten by rabid animals
the total mortality before the introduction of vaccination was not
less than 10 percent. Among the same class of patients in the
Pasteur institutes, the death rate of all cases, early and late, has
been reduced to a fraction of i percent. Those cases in which
the bites are on the head, are always more serious, and the mortal-
ity is higher. Like tetanus the virus travels, it is supposed, from
the site of injury to the central nervous system by way of the
nerves. If the bite was on the toe, it would take longer for
infection to reach the brain, than if it was on the upper lip. This
is a very plausible explanation of the varying incubation periods
in both tetanus and hydrophobia.
Coley's Fluid in the Treatment of Tumors
This method of treatment is in no wise a prophylactic one, but
strictly a curative one. It consists in the injection of the toxins
of streptococci, in the hope that they will cause a shrinking, or dis-
OPSONINS AND OPSONIC INDEX 89
appearance of malignant sarcomata. An attack of erysipelas (it
has long been observed) occurring in a patient with some malig-
nant disease, has the effect of causing a disappearance, or retro-
gression, of the tumors. Artificial infection with streptococci
was then practiced with the idea that it might produce the same
effect. But this was found to be dangerous. Coley prepared
toxins of streptococci by allowing them to grow with the B. Pro-
idgiosus. The mixture after a Jong period of incubation was steril-
ized by heat, and the fluid thus obtained was injected into the
tissues. Virulent strains of streptococci are used and the dose
of the dead culture is about half a drop given under strict anti-
septic precautions. The best results are obtained in spindle-
cell sarcoma, and the poorest in the melanotic variety. The
method by no means should be employed where the tumor can
be removed by operation. It cannot supplant the knife, and
only in inoperable cases or as a supplementary treatment where
other forms of treatment are employed, should it be used.
Opsonins and Opsonic Index
Peculiar substances in blood serum have been called by Wright and Doug-
lass opsonins (Greek: prepare food for) . If fresh blood is mixed with an emul-
sion of some bacteria and then incubated for half an hour, it will then be
found that many of the bacteria are within the polymorphonuclear leucocytes.
If the serum is washed away from the leucocytes before adding bacteria, none
of the latter will be found within the leucocytes. This proves that the serum
has some influence on phagocytosis. In order to show that this effect is on
the bacteria rather than on the leucocytes, the bacterial suspension may be
treated with some serum for half an hour and then washed free from this
serum by means of a salt solution in a centrifuge, and then mixed with some
serum-free leucocytes; then it will be found that phagocytosis occurs as before.
The bacteria have been "sensitized." According to Wright this action is
comparable to cooking or peptonizing.
Phagocytosis then depends upon the action of serum upon bacteria,
which are coped with in the body, first by the action of the serum, and then
by the leucocytes. It is thermostabile.
The quantitative action of phagocytosis may be estimated by Leishman's
method. He mixed blood and an emulsion of bacteria in salt solution in equal
QO IMMUNITY
quantities, and allowed them to stand for thirty minutes in the incubator.
After this the mixture was stained and the average number of bacteria per
leucocyte was obtained. The result was known as the phagocytic index.
Wright has devised the following technique. Young cultures, a few hours
old, are employed. These are scraped off agar tubes and mixed with salt
solution. After this has sedimented, the supernatant fluid is separated from
the bacterial masses by a centrifuge; is pipetted off, and preserved.
Washed leucocytes are obtained by collecting 2 c.c. of blood in 30 c.c. of
salt solution containing i percent citrate of soda to prevent blood coagula-
tion. The serum and citrate of soda are separated from corpuscles by
washing twice in a centrifuge. The upper layer of the sediment is rich in
washed leucocytes, and is used in the experiments.
To obtain the opsonic index, blood serum from various cases is collected.
In the case of staphylococcus infection say furuncle the blood serum is
drawn from the patient and, with equal portions of an emulsion of staphylo-
cocci (young culture), and a suspension of washed corpuscles, is thoroughly
mixed in a pipette, which after the ends are sealed, is placed in an incubator
for fifteen minutes. A drop of the mixture is then spread upon a slide; fixed,
and stained with Jenner's stain. The number of staphylococci in 50 poly-
nuclear leucocytes is determined and divided by 50 to obtain the average.
At the same time that this experiment is being performed, some normal
serum should be used in another experiment; an emulsion of staphylococci
and washed leucocytes being used as above. After pursuing the same steps
in this experiment as in the first, the average number of staphylococci per
leucocyte is determined.
Tq obtain the opsonic index, it is necessary to know the ratio of staphylo-
cocci in the leucocytes treated with furuncular serum and with normal serum.
If the normal serum leucocytes contained 10 staphylococci, and the furun-
cular serum contained 15, the index would be 1.5.
In the case of tubercle bacilli, the latter must be heated to iooC. to kill
them, otherwise they will be agglutinated by the serum, and a homogeneous
emulsion will not be obtained. After heating, the clumps must be broken up
by grinding the masses in an agate mortar, adding a little salt solution from
time to time until the mass is thoroughly broken up. The bacilli must then,
after phagocytosis, be stained by carbol fuchsin and decolorized with acid
alcohol. If the leucocytes are left too long in contact with the organisms
they may become so engorged as to prevent counting, the number increasing
from 5.7 percent after five minutes to 28.5 percent in two hours.
Highly immunized anti-bacterial serums have much greater opsonic powers
than have normal ones, anti-streptococcus and anti-pneumococcus sera
being especially pwerful toward streptococci and pneumococci. It is pos-
OPSONINS AND OPSONIC INDEX 91
sible to increase the opsonic powers of the blood of an individual suffering
from an infection, by vaccinating him with killed cultures of the organism
with which he was infected.
The determination of the opsonic index is a long and tedious affair so that
in practice it is only used when it is necessary to estimate the value of the
vaccine treatment or to control dosage. Under ordinary circumstances
clinical phenomena will indicate the correctness of dosage and interval
but certain cases that fail to do well should be checked by opsonin indicators.
The value rises slowly and steadily with appropriate vaccine, dosage and
interval, falls with too large quantity, shows no change with inadequate
quantities.
The Local Reactions or Tests. We have learned in the past few years
that the skin and mucous membranes will react more or less specifically to
the bacterial proteins. It is a form of allergic (see page 66). There have
been developed local tests for tuberculosis, syphilis, typhoid, glanders and
other diseases. The first two being the most important are considered below.
The others are of similar nature.
Tuberculosis. If tuberculin of any form be rubbed into an abraded skin
area (Von Pirquet's cutaneous) or injected between the layers (Moro's'per-
cutaneous) of the skin a red maculopapule or even vesicle upon an inflamed
base will appear within twenty-four hours. There may be a mild general
reaction of fever and malaise. A positive reaction to such an installation
simply indicates the presence of a tuberculous lesion and that an allergic
fstate of the skin exists but does not show whether or not the lesion is active.
For this reason it is only of value in children since three-fourths of adults
are believed to have a healed lesion within them. Not only upon the skin
but upon the conjunctiva can this reaction be obtained.
Syphilis. The poison of the Treponema pallidum is called luetin. It is
made by grinding up in salt solution a culture of the germ, heating the result-
ing mass to 6oC. for an hour and preserving it with phenol. If this be in-
stilled into an abraded skin area a maculopapule or nodular eruption occurs
in a syphilitic. This positive outcome, however, appears only in late cases,
those of tertiary stages and in treated cases. It therefore complements the
Wassermann reaction, being positive where this is apt to fail.
Schick Test. It has been found by Schick and others that if Ko minimum
lethal dose of diphtheria toxin in .2 c.c. of saline be injected into the skin of
a person, a swollen, pink, tender area will appear in persons susceptible to
infection with Klebs-LofHer bacilli. If no such reaction occurs, the person
is not susceptible. This depends upon the fact that if anti-toxin be present
in the blood it will combine with the toxin and no reaction will appear, while
if no anti-toxin be present the toxin is free to exert its effect. It has been
Q2 IMMUNITY
determined that persons giving a negative reaction need not receive immuniz-
ing doses of anti-toxin. About 80 percent of adults and 30 to 40 percent
of children are immune. A negative reaction seems to indicate that the blood
contains Ko c.c. more unit of anti-toxin per cubic centimeter.
Isoagglutinin and Isohemolysin
There are frequently in the blood of animals, ly tic and agglutina-
tive anti-bodies for the red cells of other members of the same
species. By this is meant that if the blood of a man be mixed with
that of another the cells of one of them may be clumped or dis-
solved. This is of considerable importance to the surgeon who
wishes to transfuse blood, for were he to use a donor whose blood
was unlike that of the recipient the latter would have a serious
chill, indications of blood destruction and shock, and he might die.
These two substances are probably independent in action but
are so commonly found acting in harmony that they may be con-
sidered inter-dependent. Landsteiner and Jansky divided persons
into four groups according to their agglutinin and agglutinogen.
Moss made the same observation but classified them differently.
JANSKY'S GROUPING
Serum
Cells I II III IV Percent
I 42.84
II + + 10.36
HI + + 41-38
IV + + + 5-42
This means that the cells of Type I are not agglutinated by an]
of the serums, whereas the cells of Type IV are clumped by
serums but their own.
Agglutination is accomplished according to Landsteiner by tw<
agglutinins a and b which are both contained in serums clumping
all type of corpuscles, whereas both are absent in serums clumpii
no cells. Agglutinable bodies, represented by a and b, are suppos
to be present in the respective cells which can be clumped, absent
in the others. Group I therefore contains agglutinins A and B
but does not contain agglutinable bodies a and b. Other groups
ISOAGGLUTININ AND ISOHEMOLYSIN 93
would be explained on the same basis. While Jansky's methods
have priority in time of publication the American surgeon has
during the great war become accustomed to the Moss grouping
which is as follows :
Serum
Cells I II III IV
I + + +
II + +
HI + +
IV
It will be seen that this is merely the reverse of the first one and
that Type IV has agglutinins but no agglutinable bodies.
Agglutinins and agglutinable bodies may be absorbed from a
given blood by saturation with their respective antigen.
In transfusion, homologous bloods should be mixed, but groups
I or IV respectively may be considered as universal donors since
experience shows it safe to use a blood which agglutinates the
recipient's cells. It is however, incorrect to use a blood whose
corpuscles are clumped by the recipient's serum. The agglutina-
tive titer of human blood is not very high, i-io to 1-25, and as
the entering serum is diluted the agglutination titer would be
exceeded.
Further experiments on the independence of the two anti-bodies
would indicate that usually agglutination precedes hemolysis but
this need not be so, in a small percentage of cases. For practical
purposes agglutination tests are sufficient indications of the com-
patibility of bloods. Tests for compatibility take the form 'of
(i) direct mixture of the blood of recipient and of donor and (2)
the testing of donor's blood by standard serums.
i. Patient's blood A serum separated from the clot. B
cells from defibrinated blood washed in saline and resuspended in
saline. Donor's blood a serum separated from the clot, b cells
from defibrinated blood washed in saline and resuspended in saline.
Test A 4 parts b i pt.
a 4 parts B i pt.
Incubate at 37 for one hour.
94 IMMUNITY
If bloods be compa table no change will occur in the blood cells.
If donor's corpuscles be agglutinated by recipient's serum there
will be clumping in the first tube; if the reverse, a change will
occur in the second tube. This method while often used is more
trouble and no more reliable than the following :
2. It is necessary to have for the group determinations, known
sera of the various types, but for practical purposes types II and
III will reveal any type corpuscles suspended in them. The blood
to be tested is caught from a prick in the finger into a few drops of
sodium citrate solution. One drop of this cell suspension is mixed
with one drop of type II and of type III sera separately on a slide.
After a period of five to thirty minutes clumping may occur.
If it occur in both, the blood is by the Moss scale group I; if in
neither it is group IV; if clumping occur in serum III and not in
serum II, the blood is group II; if clumping occur in group II
and not in group III the blood is group III.
CHAPTER V
STUDY OF BACTERIA
Bacteria are studied in the following various ways:
1. Morphological characteristics, form, size, motility, presence
of spores, granules, capsules, and flagella. Reaction of proto-
plasm to dyes and reagents.
2. Characteristics of growth in culture media; appearances of
culture; chemical activities; production of acid, gases, toxins,
colors, etc.; reactions to heat, disinfectants, light, etc.
3. Study of the action of bacteria on the tissues of man and
animals, and of the toxins on the tissues and functions of the
various organisms.
The simplest way to study bacteria is to make a hanging drop of
a fluid containing bacteria, and observing the organisms under a
microscope. To do this, a cover-slip, and a slide with a concavity
ground in it are used. A drop of bacteria laden fluid is placed on
the cover-glass, and after the edges have been smeared with vase-
line, the cover-slip is inverted over the concavity in the slide, and
the bacteria can then be examined with either the dry % inch, or
the one-twelfth oil immersion objective. If the preparation is
kept warm for some time, various vital phenomena may be noted.
Direct division, sporulation, motility, agglutination, and bacterio-
lysis can be studied by this means. Instead of using a fluid, a
block of nutrient agar may be cemented to the cover-glass; after
the bacteria have been planted on the agar, the various vital
phenomena may be noted.
All minute bodies, whether they be bacteria, dust particles or
granules of india ink in suspension, exhibit a trembling vibrating
motion called the Brownian motion. Motile bacteria either move
95
96 STUDY OF BACTERIA
so swiftly that the eye can hardly follow them, or they may merely
roll or wiggle across the field slowly. Direct division, if pro-
ceeding under the best conditions, requires but fifteen to forty
minutes. It is best observed in a warm stage or when working in
a room kept at a temperature of 35C. Sporulation occurs differ-
ently in different species. In some it will be found soon after the
culture has been removed from the incubator, while in others
several hours are required. Sporulation, it must be remembered
is a resistant stage when unfavorable conditions are met.
The Gruber-Widal reaction is thus studied. A drop of the
serum and bullion culture, mixed in proper proportions, is dropped
on a cover-slip, which is then placed, drop downward, over the
cavity of the slide (hanging drop, Fig. 20) (see Agglutination) .
FIG. 20. Hanging drop, over hollow ground slide. (Williams.)
Staining bacteria is a matter that is easily accomplished, and
very many staining solutions and methods have been invented for
this purpose.
The simplest procedure is to take a drop of pus, blood or culture,
and spread it upon a very clean slide with a sterilized platinum
needle. The matter must be spread thinly and evenly. After the
water has evaporated and the preparation has become dry without
the use of Jieat, it must be fixed. To do this various agents are
used. The object of the fixing is to coagulate the protoplasm of
the cells, and to fasten all the smeared matter fast to the glass,
so that the staining fluid and water will not wash them off. This
is accomplished, for bacteria usually, by holding the smeared slide
in the^apex of a bunsen flame until quite warm to the hand. Great
care must be used not to char the film. Experience is need(
to fix slide smears correctly. The beginner would do well t(
use cover-slips. If a cover-slip is used it must be passed througl
the flame three times rapidly. After fixing and thorough cooling,
STAINING BACTERIA 97
the staining fluid is poured on, and after remaining a few minutes
is poured off and the slide is washed, dried by blotting paper,
and examined. If a cover-slip has been used a drop of balsam is
put upon a clean slide and the cover, smeared with stained
bacteria, is inverted on the balsam. Upon the stained bacteria
themselves (if a cover-glass has not been used) or upon the cover-
slip a drop of cedar oil may be placed, and the preparation
examined with a one-twelfth objective. This is one of the
simplest staining procedures practised in bacteriology. Other
more complicated methods will now be described.
Besides heat, absolute alcohol, methyl alcohol, or formalin may
be used as fixatives. Some stains are made up with methyl
alcohol, and instead of fixing by heat, the stain is merely dropped
upon the dried film, and the bacteria are fixed and stained by the
same solution at the same time, water being added for differentia-
tion at the end.
Aniline dyes are almost entirely used as stains in bacteriology
and these are divided into two classes, the basic and acid stains,
according as their staining properties depend upon the basic or
acid part of the molecule. Basic dyes stain nuclear tissues of cells
and bacteria. The acid are used as contrast stains and do not
color bacteria, but tissues in which they may be imbedded.
The common basic stains are methyl violet, and gentian violet,
methyl green, methyl blue, and methylene blue, thionin blue, Bis-
marck brown, fuchsin, and saffranin. These are used for staining
different bacteria under different conditions. The most useful
stain is methylene blue, since it is difficult to overstain with it, and
it is very easily applied. It has been found that certain physical
and chemical conditions are necessary for successful staining with
aniline dyes. Alcoholic solution of dyes entirely devoid of water
do not stain; absolute alcohol does not decolorize bacteria after
staining with aniline colors, while diluted alcohol decolorizes
readily. The more completely a dye is dissolved, the weaker is its
staining power. A dyestuff unites, as a whole, with the bacterial
7
98 STUDY OF BACTERIA
plasm, forming, as it were, a double salt between the two. Cer-
tain substances, alkalies, carbolic acid, iron and copper sulphate,
tannic acid, alum, and aniline oil, are added to a solution of aniline
dyes, and they act as mordants, or fixatives, making the dye bite
into the protoplasm of the bacterial cells. Spores, capsules, and
flagella, are hard to stain, and special heavily mordanted stains are
used to demonstrate them. Chemical reaction occurring in the
cell protoplasm is of great value in differentiating bacteria. The
presence of granules in bacterial cells is often only shown by
the use of special stains, which deeply color them. Bacteria of the
tubercle group are called "acid-fast," because, after being stained,
it is difficult to decolorize them with acid solutions. These bac-
teria are hard to stain and resist decolorizing agents after they
are stained.
1 . Loffler's alkaline methylene blue solution consists of
Saturated alcoholic solution of methylene blue 30 c.c.
Ho, ooo solution caustic soda solution in water 100 c.c.
Mix.
This is the most useful of all the staining mixtures employed.
2. ZeihFs solution carbol-fuchsin consists of
Fuchsin i gram.
Carbolic acid crystals 5 grams.
Dissolved in 100 c.c. of water, to which is added 10 c.c. of absolute alcohol.
This can also be made by taking a 5 percent solution of carbolic
acid in water and adding sufficient saturated solution of fuchsin in
water until a bronze scum persists upon the top. This is used for
staining tubercle bacilli in sputum and sections. It must be
heated when used for rapid staining. Tubercle bacilli can be
stained in cold solution, if immersed over night in it.
3. Fuchsin solution.
Saturated alcoholic solution of basic fuchsin i c.c.
Water. . . . 100 c.c.
STAINING BACTERIA 99
4. Bismarck brown solution.
Water 100 c.c.
Bismarck brown sufficient to saturate.
Filter and use a contrast stain.
5. Weigert's aniline gentian violet stain.
Gentian violet i gram.
Dissolve in absolute alcohol 15 c.c.
Distilled water 80 c.c.
Then add to this
Aniline oil 3 c.c.
Mix, shake and filter.
This stain can also be prepared by taking a
Sat. watery solution of aniline oil 100 c.c.
Filter, then add
Sat. alcoholic solution gentian violet 10 c.c.
Sterling's permanent solution is made by mixing 2 c.c. of aniline oil with
10 c.c. of 95 percent alcohol; the mixture is shaken and 88 c.c. of distilled
water added; 5 grams of gentian violet powdered in a mortar receives the
above fluid, added slowly while grinding; filter. This solution while expen-
sive to make, requires only a sm,all quantity, stains rapidly and keeps well.
This is a very intense bacterial stain used for demonstrating
bacteria by the Gram method.
Gram's method of staining.
A cover-glass is spread with a smear of bacteria, or pus to be
examined. After air-drying it, and fixing it in the flame, the ani-
line gentian violet is poured on, allowed to stand for three minutes,
then poured off and the preparation treated with
Iodine crystals i gram.
Potassium iodide 2 grams.
Water 100 c.c.
for two minutes. This renders the purplish preparation grayish
in appearance. Alcohol is now poured upon the preparation re-
peatedly until the alcohol does not dissolve any more color. A
contrast stain of Bismarck brown or dilute fuchsin or safranin is
100 STUDY OF BACTERIA
now used. If the bacteria on examination remain a dark violet-
blue they are then said to stain by Gram's method, or are " Gram-
positive." If they are decolorized they take the contrast stain and
are said not to stain by this method, and are "Gram-negative. "
Many bacteria stain in this way, and many do not. Important
bacteria often may be differentiated in this manner.
Examples of Gram's stain are as follows:
Gram-positive Bact. aero genus capsulatus, Bact. anthracis,
Bact. diphtheria, B. tetani, Bact. tuberculosis, Streptococcus
pneumonia, Staph. pyogenes, Strep, pyogenes. Gram-negative
B. coli, B. dysenteries, Bact. influenza, Bact. mallei, Bact. pestis,
B. pyocyaneus, B. typhosus, Diplococcus intracellularis menin-
gitidis, Micr. catarrhalis, Micr. gonorrhoea, Spirillum cholera.
Thionin Blue, or Carbol Thionin
This is a useful stain, prepared thus:
Thionin blue i gram.
Carbolic acid ! 2.5 grams.
Water 100 c.c.
Filter. Good for staining bacteria in tissues.
Special Stains
Wright's Stain. This not only stains, but fixes. It has a wide
range of usefulness in a bacteriological laboratory for the staining
of blood, pus, malarial parasites, trypanosomes, as well as many
bacteria, and is prepared as follows :
.5 percent solution of sodium bicarbonate 100 c.c.
Methylene blue i gram.
Mix and heat in sterilizer one hour at iooC. Cool, filter, then mix Ko per-
cent yellowish eosin in water until the mixture loses its blue color and becomes
purplish. Of the eosin solution add 500 c.c. to each 100 c.c. of the methylene
blue mixture. Mix and collect the abundant precipitate which immediately
forms on a filter. Dry this and dissolve in methyl alcohol in the proportion
of i gram of powder to 600 c.c. of the alcohol. This is the staining fluid.
Keep well stoppered. Fresh alcohol may be added for that which evaporates.
SPECIAL STAINS ' IOI
This complex stain represents -a type *of v which Jenner's, Leish-
man's, and Romanowsky's are members. To use this stain, a
blood or pus film is spread and air-dried. The stain is then run
on the slip, or slide, for one minute. After this time slowly drop
distilled water in quantity similar to that of stain used. This is
when the true staining takes place. After three minutes wash in
distilled water, dry and mount. Nuclei, malarial parasites, try-
panosomes, and bacteria are stained blue; red cells are stained
pinkish-orange, while the granules of the leucocytes are stained
pink, lilac, or blue, depending upon their character.
Giemsa's Stain
This stain is used for demonstrating the organism of syphilis,
trypanosomata, granules and the like, and is prepared as follows :
Azur II Eosin 3 grams.
Azur II 8 grams.
Glycerine C. P 250 c.c.
Methyl alcohol 250 c.c.
Bacteria are often covered with capsules that are difficult to
stain, and special methods have been devised to demonstrate
them.
1. Air-dry the specimen.
2. Harden and fix in absolute methyl alcohol.
3. Dilute stain with distilled water, using one drop of stain to each cubic
centimeter of water.
4. Cover preparation with dilute stain fifteen minutes to three hours
(longer period for spirochaetesj.
5. Wash in running water.
6. Blot and mount.
Capsule Staining
Welch's Method.
1. Cover-glass preparations are made in the usual manner, and over the
film after fixing, glacial acetic acid is poured.
2. Without washing off the acid, aniline water gentian violet is poured on.
102 ST?JE\ OF BACTERIA
Charge the stain four or five times to remove the acid. Stain four
minutes, and wash with 2 percent NaCl solution, not water. This
demonstrates the capsule very well.
His's Method.
"A."
1. Make cover-glass preparation mixing specimen with blood serum. Fix
in flame.
2. Stain for a few seconds with a half concentrated water solution of gentian
violet.
3. Wash in weak potassium carbonate solution for a few minutes.
4. Dry and mount.
"B."
. 2. Dry and fix.
3. Heat and pour on the following stain, steaming thirty seconds:
(a) Saturated alcoholic solution of gentian violet 5 c.c.
(b) Water 95 c.c.
4. Wash in a 20 percent solution cupric sulphate.
5. Dry and mount.
Spore Staining
Spores resist stains, and when stained are hard to decolorize.
1. Dry and fix in the usual way.
2. Flood cover-glass with hot carbol-fuchsin; heat until it steams; repeat
this once or twice. This stains bacteria and spores.
3. Wash in water.
4. Decolorize with
Alcohol 2 parts.
i percent acetic acid i part.
5. Wash.
6. Counterstain with methylene blue.
7. Wash, dry and mount.
By this method, which is a simple and satisfactory one, the
spores are stained a brifliantjred, while_the body of the bacilli
are stained blue.
Flagella Staining
To a beginner flagella staining is difficult; there have been many
well-known methods devised. The simpler are as effective as the
more complicated but do not always make as pretty preparations.
FLAGELLA STAINING 103
Flagella, being processes extending from the capsule, are, like
the latter, hard to demonstrate. They are not stained by the
common bacterial stains. In general a powerful stain mixed with
a strong mordant must be employed. Some methods appear
to be not so much a staining method in the ordinary sense but
either a precipitaing of the stain in the substance of the flagella
or else a decomposition of silver salts in the flagella substance.
To stain flagella, a young culture grown on agar must be employed ;
glycerine agar must never be used. A mass of the organism is
gently mixed with a drop of distilled water until a uniform emul-
sion is made. A dozen cover-slips carefully washed and cleaned
by alcohol are thoroughly flamed in order to remove the slightest
trace of grease. The watery emulsion of bacteria is then spread
over the cover-slips evenly and thinly. After they are dry the
bacteria are fixed by holding them for a minute just above the
apex of the flame with the fingers. The following methods may be
pursued :
Pitfield's Method Modified by Muir.
Two solutions are necessary for this method.
A. Mordant.
10 percent watery solution tannic acid '. . . 10 c.c.
Corrosive sublimate saturated water solution 5 c.c.
Carbol-fuchsin solution 5 c.c.
This forms a dense precipitate which must be removed by the centrifuge,
or sedimentation, and the clear fluid, or mordant, is stored in a bottle. It
keeps for two weeks.
B. Stain.
Saturated watery solution of alum 10 c.c.
Saturated alcoholic solution gentian violet 2 c.c.
This keeps but two or three days.
Flood the cover-slip with the mordant and gently steam for one
minute, then wash and dry thoroughly, pour the stain on and
steam for one minute more. Wash, dry and mount.
This method yields very good results.
104 STUDY OF BACTERIA
Pitfield's Method.
This is the simplest stain and the easiest to use, but does not
give the good results that the previous one does. But one solution
is needed, this is made in two parts and mixed.
A. Tannic acid i gram.
Water 10 c.c.
B. Saturated watery solution alum (old) 10 c.c.
Saturated alcoholic solution gentian violet i c.c.
Mix.
A heavy precipitate is formed by this process which is useful in the stain-
ing. The stain is almost a saturated solution of alum and tannic acid, and
when it becomes supersaturated by evaporation and heat, staining takes place.
After this the process is very simple. The cover-slip is carefully flooded with
the stain and warmed for a minute over the flame of a bunsen burner, turned
very low, until steam arises. Not too much stain should be run over the
cover-slip. After steaming occurs, the stain should remain for a minute, then
the preparation is washed, dried and mounted. It will be found that the best
stained flagella are on those bacteria nearest to the edges where the evapora-
tion has been most intense. If the preparation is not equally stained, Wei-
gert's aniline gentian violet can be run on for a minute to deepen the color.
Loffler's Method.
This is the original flagella stain and is a very good one.
It is made as follows :
A. Mordant
20 percent watery solution tannic acid 10 c.c.
Sat. solution ferrous sulphate 5 c.c.
Fuchsin sat. alcoholic solution i c.c.
Mix.
B. Stain
Carbol-fuchsin.
Proceed as in the previous methods.
The most important steps in flagella staining are to clean the
cover-slips thoroughly, to mix the culture with water and have no
culture media with it, to fix gently, and not to overheat the stain.
Even in expert practised hands it is not always easy to demon-
strate flagella readily.
STAINING DIPHTHERIA BACILLI 10$
Neisser's method of staining the diphtheria bacillus.
Two stains are needed (Fig. 21):
FIG. 2i. B. Diphtheria stained by Neisser's method. (Williams.)
A Methylene blue
i gram.
95 percent alcohol
Water
20 c.c.
o^o c.c.
Mix and add
Glacial acetic acid
50 c.c.
B Vesuvin
2 grams.
Distilled water. . .
. 1000 C.C.
The staining steps are as follows:
1. Prepare film, fix and dry.
2. Pour on "A" for thirty seconds.
3. Wash well in water.
4. Dry and pour on "B" for thirty seconds.
5. Wash, dry and mount.
The protoplasm of the bacilli will be stained brown, and the
characteristic (diagnostic) chromatin points will be stained a
deep blue black.
106 STUDY OF BACTERIA
Tubercle Bacillus Stain
1. Spread the sputum, pus or culture, over the surface of the cover-slip.
Allow the preparation to thoroughly dry.
2. Fix in flame and cool.
3. Pour carbol-fuchsin over the slide and heat with steaming for five min-
utes. Young bacilli in tubercles and other fluids are very difficult to stain
in this way. The preparation containing them should be stood in cold carbol-
fuchsin for twenty-four hours. This method stains everything on the slide.
4. Wash in water.
5. Decolorize the preparation with a 25 percent solution of sulphuric acid
in water until the red color is lost. Repeat this once or twice.
6. Wash and counterstain with Loffler's methylene blue.
7. Dry and mount.
Gabbet's solution, methylene blue 2 grams, H 2 SO4 25 c.c.,
water 75 c.c., is a very useful, convenient decolorizer and counter-
stain for sputum.
In such a preparation, if tubercle or other acid-fast bacilli are
present, the bacilli will be colored a brilliant red, while the pus
cells, epithelial cells, and other bacteria will be stained blue.
The microscope dark field illumination enables one to see
flagella and capsules. This illumination is obtained by blocking
out the central portion of the Abbe condenser in the substage of
the microscope. Light is admitted only from the sides and objects
in the field at the point of crossing of the rays reflect these from
their sides. India ink may be used as a background for bacteria
that stain poorly and have low refractive index.
Protozoa are stained by Wright's or Giemsa's method in one of
its various forms. Spirochetes, particularly that of syphilis,
may be stained by Giemsa's methocl but show up more clearly
in the following technic of Stern.
1. Dry film in incubator for several hours.
2. Immerse in 10 percent aqueous silver nitrate in diffuse
daylight, six hours to three days, depending on thickness of smear
and need for haste.
TUBERCLE BACILLUS STAIN 107
3. Correct color for smear is a dull gray brown with metallic
sheen wash in water, dry and examine. Spirochetes are deep
brown or black, cells delicate brown.
Microscopic objects are measured by viewing with an ocular
fitted with a graduated glass disc. Their values are indicated
on the apparatus.
CHAPTER VI
BACTERIOLOGICAL LABORATORY TECHNIC
In order to study bacteria by other methods than the simple
examination of their morphology by means of stains, and by the
hang-drop, or block method, they must be cultivated either in
the bodies of experiment animals, or in culture media artificially
prepared. The latter method is the most widely used in labora-
tories. It is necessary, in order to study bacteria, that the media
shall not contain any extraneous bacteria to begin with, and
that they shall be cultivated under such conditions that such
bacteria cannot reach the media at any time. To accomplish all
this, the culture media must be kept in glass vessels, such as test-
tubes and flasks that have been sterilized. And, since all animal
and vegetable substances, not actually alive, are teeming with
a multitude of bacteria, these substances must be sterilized too,
in order that the media shall be free from any living organisms.
Glassware is cleaned by boiling with soap suds or powder or if
very dirty by immersion in saturated watery solution of bichro-
mate of potash plus an equal part of sulphuric acid. This latter
must be very carefully washed away in running water.
Glassware, such as pipettes, Petri dishes, flasks and test-tubes,
are sterilized best by dry heat in hot-air sterilizers. The appara-
tus is subjected to a temperature of i5oC. for one hour, or until
the cotton plugs are slightly brown. The glassware should be
put in wire baskets and the test-tubes should be kept erect.
Petri dishes are best sterilized in a wrapping of paper. Flasks
and test-tubes* are always plugged with raw cotton, which pre-
vents the ingress of bacteria, while air can reach the media
through it freely.
108
STERILIZATION
I0 9
Sterilization of culture media is accomplished in steam steril-
izers of two patterns; of these, the autoclave, using steam under
pressure, is the most satisfactory and is most generally used at
present.
The baskets containing the culture media are placed in the
autoclave after sufficient water has been put in it. The instru-
FIG. 22. Autoclave.
ment must never be allowed to run without water. The lid is
screwed down and the flame started; free flowing steam should
escape from the valve before the latter is shut. When the
pressure has risen to i atmosphere (15 pounds) or i2oC. and
held there for twenty minutes, all bacteria are destroyed, and
the media can be safely assumed to be sterilized. If media
containing sugar or gelatine are to be sterilized, the temperature
should not run above noC., since, if this is done the gelatine will
no
BACTERIOLOGICAL LABORATORY TECHNIC
not solidify when cold, the sugar is caramelized and the media
blackened.
Potato tubes are harder to sterilize at times, and it is safer
to repeat the operation in twenty-four hours.
Fractional method of sterilization, or Tyndallization, is accom-
plished by heating the media to iooC. on three successive days
FIG. 23. Arnold sterilizer.
in a lyoch or Arnold sterilizer. By heating culture media to this
temperature, all the vegetative, or adult, forms are killed, while
the spores are not affected; after the first sterilization, at room
temperature, the spores vegetate and become adult bacteria,
when on the second sterilization they are non-resistant to iooC.
and are killed. Spores remaining after this develop into adult
forms again and are killed on the third day, at the third steriliza-
tion. This fractional sterilization is employed under many
BACTERIA CULTIVATION
III
circumstances, and is certainly the best for media containing
carbohydrates of any kind. To be effective, the media must be
exposed to a temperature of iooC. for thirty minutes, that is,
thirty minutes after the steam has begun to form. Overheating
of sugars causes them to caramelize and turn black.
FIG. 24. Incubator.
Bacteria that grow best at a temperature of 37C. (most of the
pathogenic ones do) develop more rapidly and luxuriantly in an
incubator, or thermostat. Indeed some organisms, like the
tubercle bacillus, cannot be cultivated without it. An incubator
comprises an air chamber surrounded by a water chamber, and
this, in turn, is surrounded by another air chamber. It is essen-
tial that the interior of the incubator be kept at an even, unvary-
ing temperature. This is accomplished by using a small bunsen
flame under the incubator. The heat from the flame warms the
outer air chamber or jacket, and it in turn warms the water
112 BACTERIOLOGICAL LABORATORY TECHNIC
jacket, and the interior air chamber, where the cultures are kept,
is thus heated to the required temperature. The amount of heat
is automatically regulated by a thermo-regulator, which dimin-
ishes the gas supply if the temperature runs too high, or increases
it if it runs too low. The Roux regulator is the simplest and most
efficient one.
FIG. 25. Blood serum coagulating apparatus.
A serum coagulating apparatus is needed in laboratories in
order to coagulate the tubes of blood serum (Fig. 25).
Serum tubes are coagulated in it at a temperature of about 7oC.
They are then sterilized by heating them either by the fractional
method or in the autoclave.
The separation of bacteria from the bouillon in which they
grow for the preparation of toxins requires the use of a bacteria
or germ proof filter, the best type of which is the Chamberland or
Pasteur unglazed porcelain filter. These filters are of varying
grades of fineness, and are so made as to be easily sterilized. The
common pathogenic bacteria cannot pass through the pores of
the ordinary filter, but toxic agents are known to pass through the
finest filters, though they cannot be discovered, as they are
submicroscopic.
To operate the porcelain filter it must fit into the neck of a
NUTRIENT MEDIA
vessel very tightly, so that a vacuum may be maintained in the
latter by means of an air pump.
Collodion sacs are sometimes used in animal experiments.
Bouillon cultures are placed within the
sacs, which are then inserted in the ab-
domen of an animal and left there. The
sac is made of coUodion because it is
non-absorbent and allows the bacterial
juices and products to osmose outward
and be absorbed by the animal, while the
animal fluids percolate into the sac.
There are several very ingenious ways of
making these sacs, but the details are too
elaborate to be described here.
BOUILLON
Bouillon or broth is the most useful of
all the nutrient media, since it is not only
used as a liquid medium, but by the addi-
tion of gelatine, or agar, it is converted
into solid media.
There are two methods of making
bouillon:
Method i.
Take 500 grams of lean beef free from all fat, chop it fine and
cover with 1,000 c.c. of water, shake and place on the ice over-
night. Then squeeze the fluid out of the met by means of a
cloth, and supply enough water to make a litre. Inoculate this
meat juice with a fluid culture of the colon bacillus for the pur-
pose of fermenting the meat sugar. For this purpose the inocu-
lated juice is allowed to stand at room temperature overnight.
Bring to a boil and add
10 grams of Witte's peptone.
5 grams common salt.
FIG. 26. Kitasato
filter for filtering toxins.
(Williams.)
114 BACTERIOLOGICAL LABORATORY TECHNIC
Weight the saucepan and contents and heat to 6oC. Supply
the water lost by evaporation. Neutralize either by adding
sufficient sodium hydrate, 10 percent solution, until red litmus
paper is colored a faint blue, or else titrate 10 c.c. of the mixture
with a decinormal solution of sodium hydrate, using phenol-
phthalein as an indicator, and after finding how much of a normal
solution is required to neutralize 990 c.c. (1,000 c.c. 10 c.c.
used for titration) this normal solution is added. The mixture
thus neutralized is then boiled for five minutes and the weight
restored. After boiling, from .5 percent to 1.5 percent normal
hydrochloric acid solution is added and the acidity thus produced
is spoken of as + .5 percent or + 1.5 percent as the case may be.
Upon boiling, the albumins are coagulated by heat, and the
phosphates are thrown down. The acid re-dissolves the latter.
The former must be removed by filtration. The filtrate is a clear
straw-colored fluid of an acid reaction which should not become
cloudy upon boiling. This is then run into flasks or test-tubes
and sterilized.
The second method is much more convenient, and is prepared
by adding 3 grams of Liebig's beef extract to a litre of water, and
adding the peptone and salt, as in the previous method, and pro-
ceeding as before. To filter the bouillon, the filter paper must
be folded many times, and the funnel must be carefully cleaned.
Newer methods for the titration of media have been constructed
on a physico-chemical basis, attempt being made to estimate
exact reaction in terms of ionic dissociation and hydrogen concen-
tration. Distilled water contains ionizable hydrogen according
to the following formula i + 10 7 = log. 7, a mathematical
statement abbreviated for convenience to 7., or the symbol of
strict neutrality and called the Ph or hydrogen concentration.
As the ionization of hydrogen increases, acidity becomes greater
and the numerical factor drops to 6.8 or lower; as the hydrogen
value decreases the figure rises. Substances in solution, such as
salts or organic matter act as buffers or agents which attempt to
NUTRIENT MEDIA
keep the hydrogen ion concentration fixed. Dilution does not
change the concentration. Mixtures of acid and alkaline salts
which will give hydrogen ion concentration ranging from Ph
values of 5. (acid) to 9. (alkali) have been prepared for use in
bacteriology. To these solutions may be added dye indicators.
Different dyes have different ranges of color changes. The most
useful are the following:
Range] of r Ph)
5.2 6.8
6.0 7.6
6.8 8.4
Brom cresol purple yellow-purple
Brom thymol blue yellow-blue
Phenol red yellow-red
Litmus which has so long served a useful purpose in the laboratory
has a Ph of about 6.8. A convenient method for practical use in
the laboratory is as follows :
Materials :
1. Chemically clean test tubes.
2. Freshly distilled water.
3. N/i5 KH2PO4 (primary phosphate) solution. (1.078 gm. to litre of
water.)
4. N/i5 Na 2 HPO 4 (secondary phosphate) solution (11.996 gm. per litre
of water.)
5. Phenolsulphonphthalein .01 percent water solution (or phenol red).
6. N/20 NaOH, N NaOH.
7. N/20 HC1, N HC1.
Phosphate mixture for Ph values 7.0 to 8 o.
Ph value
Amt. of primary
phos. in c.c.
19.4
14.2
9-7
6-5
4-3
2-5
Procedure.
Add 20 c.c. freshly distilled water to chemically clean test tube. Add
10 drops phenol red and 5 c.c. of medium to be titrated. Compare with
standard.
7.0 -
7-8
8.0
Amt. of secondary phos.
in c.c.
Quantities to be
mixed to get in-
dicated Ph value.
5 drops indicator
to each.
30.6
35-8
40.3
43-5
45-7
47-5
Il6 BACTERIOLOGICAL LABORATORY TECHNIC
Titrate with N/20 NaOH or N/2o HC1 to match tint of stand-
ard tube corresponding to the Ph desired. Calculate the amount
of normal alkali or acid to be added to the medium to give the
proper reaction.
If the natural color of the medium makes it difficult to match
with the standard solutions, a tube of 20 percent solution of the
medium in water may be placed back of the standard solution.
The best generally useful reaction is 7.4, a figure suitable for
most pathogenic organisms.
GELATINE
To make gelatine, bouillon is made to which gelatine is added
in order to render it solid. The following steps are taken:
(a) Take a litre of water in a saucepan and add chopped beef or
beef extract as in bouillon. After standing overnight
squeeze the beef and extract the juice.
(b) Add i percent peptone, .5 percent salt, 10 percent to 15
percent best gelatine and weigh.
(c) Heat until ingredients are all dissolved.
(d) Neutralize, gelatine is highly acid and requires much alkali.
(e) Boil five minutes and restore weight, boil till albumin
coagulates.
(/) Cool to 6oC. and add an egg well beaten up in water.
(g) Boil slowly till all the egg is coagulated. This clears the
jnedium of fine particles that are not removed by filtration.
Add .5 percent normal hydrochloric acid.
(h) Filter through absorbent cotton on a funnel previously wet
with boiling water.
(i) Tube and sterilize in autoclave for fifteen minutes at i ioC.
Litmus, or lacmoid, or neutral red may be added to the
gelatine as an indicator.
AGAR-AGAR 117
AGAR-AGAR
To make agar :
(a) Take 20 grams of powdered or chopped agar.
(b) Add to 500 c.c. of water, place in a can in autoclave and
heat to 1 2oC. Then cool.
(c) Add this to 500 c.c. of bouillon of double strength, making
1,000 c.c.
(d) Neutralize.
(e) Cool to 6oC.
(/) Add the egg to the mixture, stir.
(g) Boil till egg is coagulated thoroughly.
(h) Titrate and adjust to desired acidity as given under bouil-
lon, and while boiling hot, filter through absorbent cotton
wet with boiling water.
(i) Run into tubes. Sterilize. Slope the tubes for twelve
hours and store in dark place.
To make glycerine agar add 5 percent of glycerine to the agar
before neutralizing. To make agar for tubercle bacilli, veal
bouillon may be employed, and glycerine must be added.
Litmus Milk
Carefully skimmed milk, to which litmus tincture has been
added, is run into tubes and sterilized. This is a valuable culture
medium. It is also a reagent.
Potato Tubes
i. Wash some large potatoes and with a Ravenel potato cutter,
cut out semi-cylinders of potato. Immerse in running water over-
night, in order to prevent them from turning black. It is well
to wash these bits of potato with i- 10,000 bichloride of mercury
six hours and running water over night. Some laboratories soak
their slices in sodium carbonate solution. It is desirable to know
n8
BACTERIOLOGICAL LABORATORY TECHNIC
the reaction of the medium and each batch should be tested, then
marked whether faintly or strongly acid or alkaline.
Thrust absorbent cotton to the bottom of the tube and wet
with distilled water; place the potato upon the
cotton, then plug the tube and sterilize in auto-
clave twice. The tubes should be sealed.
PEPTONE SOLUTION Dunham
Take Peptone 10 grams.
Salt 5 grams.
Water 1,000 c.c.
Mix. Boil. Filter and store in tubes and sterilize.
This is used to demonstrate the production of
indol. Reaction should be neutral.
SUGAR MEDIA
One of the most important parts of determinative
bacteriology is the discovery of the different fer-
mentative powers upon carbohydrates of otherwise
similar germs. Monosaccharides (dextrose and
galactose), disaccharides (lactose, saccharose),
alcohols (glycerine mannite) and some starches
(dextrin, inulin) are in common use daily in the
laboratory to show the enzyme action of various
species, indicated by acidity and gas production.
One percent solutions of these carbohydrates are made in neutral
broth or agar. The best method is to prepare a 20 percent
solution of the material, sterilize it in the steam sterilizer and add
from this to the stock medium sufficient to make the required
percentage. This avoids repeated heating. The media should
be neutral. Addition of litmus tincture or Andrade's indicator
(100 c.c. of .5 percent watery acid fuchsin decolorized by the
addition of 16 c.c. N/i NOOH) will supply an index for acid
production.
FIG. 27. Po-
tato in culture
tube. (Wil-
liams.)
LOFFLER'S BLOOD SERUM MIXTURE 119
BLOOD AGAR
Is prepared by adding to melted agar sterile defibrinated blood
of any animal in the proportion of i of blood to 5 of agar. The
mixture may then be allowed to harden in a slanting position or
poured into plates.
BLOOD SERUM
The blood of a dog, sheep or cow drawn under strictly aseptic
precautions is collected in a sterile jar and after the serum has
separated, it is run into tubes by sterile pipettes and simply
coagulated by heat. Sterilization is not necessary, and is harm-
ful for the growth of the tubercle bacilli, because salts are formed
which interfere with the growth of the bacteria. If this serum
be mixed with 3 or 4 parts of distilled water and sterilized five
days in the Arnold at 7oC. it will be a slightly turbid, opalescent
liquid very suitable for many organisms, particularly cocci.
This medium with the addition of bits of animal tissue is a good
medium for spirochaetes.
LOFFLER'S BLOOD SERUM MIXTURE
Blood serum of an ox, a sheep or a horse is employed, mixed
with bouillon containing i percent of grape sugar.
Seventy-five percent of blood serum is mixed with 25 percent
bouillon. This is run into sterilized tubes and the latter are
placed in a blood serum coagulator and coagulated in a sloping
position at a temperature of 65C. or thereabouts.
After they are coagulated they are sterilized by heating an
hour each day at 65C. five successive days, or at 95C. for an
hour on three successive days. After sterilization the tubes
should be sealed carefully.
Ascitic and hydrocele fluids may be used for this medium or
in the liquid form combined with plain broth; they need no
addition of sugar as they contain a small percentage.
120 BACTERIOLOGICAL LABORATORY TECHNIC
SPECIAL MEDIA
Endo medium for the Typhoid series. Add i percent lactose to
plain neutral agar and i percent of the following solution: 10 c.c.
of 10 percent watery sodium sulphite and i c.c. of saturated
alcoholic solution of fuchsin, heated in the Arnold twenty minutes.
This should be mixed freshly each time since the medium is
properly colorless but becomes a pale pink on standing, being
then useless. It is poured into plates and allowed to cool and
harden thoroughly before use.
Russell's Medium. Plain neutral agar receives i percent
lactose, .1 percent glucose and an indicator, either litmus or
Andrade. It is used as slants with a deep butt.
The first medium is to distinguish typhoid colonies from colon
bacillus; pale bluish round colonies versus irregular red ones.
The Russell medium gives with typhoid, a colorless surface
growth and acid in the butt; paratyphoids give bubbles of fermen-
tation in the butt; colon makes distinct red slant growth and
much gas.
Eggs are employed as culture media. The yolks and whites of
a number of eggs are shaken together in a flask and then strained
through a towel to remove the froth. The mixture is then run
into tubes and coagulated and sterilized like blood serum. On
this mixture the tubercle bacillus grows very well.
These are the common culture media used in laboratories. For
a more technical description of the manufacture of these and
other media, the student is referred to books devoted to labora-
tory technique.
Litmus tincture is made by adding a large handful of litmus
cubes to a pint of water and boiling down to one-fourth its volume.
This is then filtered through paper and stored after sterilization.
The Study of the Growth of Bacteria Cultures
Bacteria growing in groups on culture media are spoken of as
colonies. Aerobic bacteria may be made to grow on culture
THE STUDY OF THE GROWTH OF BACTERIA 121
media by simply inoculating the media with some pus or blood
containing them, by means of a sterile pipette or platinum needle.
But such cultures are made up of colonies of different sorts of
bacteria some pathogenic, some non-pathogenic, etc. To sepa-
rate the various bacteria so that they will grow in isolated groups,
is a comparatively easy matter, and is accomplished in several
ways. The simplest is to employ several tubes of agar or blood
FIG. 28. Colonies in gelatine plate showing how they may be separated and
the organisms isolated. (Williams.,)
serum. Over the surface of each of these, a platinum loop con-
taining pus, or other matter, is rubbed successively. These tubes
are then incubated. After a few hours, the first one exhibits a
copious growth of many different kinds of bacteria growing con-
fluently together, from which it is impossible to isolate any pure
cultures. The second tube is less covered with bacteria while,
the third, instead of containing a mass of bacteria, exhibits tiny
little dots, or colonies (pure cultures) growing discretely isolated.
By means of a sterilized platinum needle these little colonies
122 BACTERIOLOGICAL LABORATORY TECHNIC
may be fished out and transplanted to fresh culture tubes, and
after a few hours' growth they become pure cultures. An old
method employed in many laboratories, in breweries and orig-
inated by Pasteur was what is known as the dilution method.
FIG. 29. Series of stab cultures in gelatine, showing modes of growth of
different species of bacteria. (Abbott.)
Numerous flasks are inoculated by matter containing bacteria
very highly diluted in bouillon and by means of a sterile pipette
drops of this highly attenuated mixture are dropped into flasks
of sterilized bouillon or wort. Most of the flasks will show a
mixed growth but a few will show only one kind of organism.
THE STUDY OF THE GROWTH OF BACTERIA
123
Another method is to inject some matter containing patho-
genic bacteria into a rabbit of guinea pig. The various juices
and the leucocytes of the animal destroy the non-pathogenic
bacteria and a pure culture, of a pathogenic form, may be isolated
from the blood or pathological lesions at
autopsy and transferred to culture media.
By far the most useful and ingenious
method of procedure is the Koch, or plate
method. Koch was the first to employ solid
culture media for this purpose, and his
method depends upon the principle that a
liquid culture media may be inoculated with
bacteria and then spread out on sterile glass
plates or dishes where it quickly hardens, the
bacteria being uniformly separated from each
other, and for a time at least kept isolated
by means of the solid media, and after they
have developed into isolated colonies they
may be transplanted to tubes of media in
which they may be stored. In another way
if a man wanted to secure a pure lot of seed
of a single variety from a multitude of many
kinds, it would perhaps be impossible to
pick out by hand the seed wanted because
of their fewness and smallness, but if he
sowed them and waited until the plants
developed they could then be identified and gathered (Abbott).
Thus it is with plate cultures.
To isolate a pure culture of bacteria, say the Bacillus pyocya-
neus from pus, the following procedure is adopted in this method.
Three sterilized petri dishes, and three tubes of agar or gelatine
melted at 4oC. are used. A loopful of pus is taken up by a ster-
ilized platinum loop and mixed with the gelatine of the first tube.
To do this the tube is held across the left hand in a horizontal
FIG. 30. Needles
used for inoculating
media.
124 BACTERIOLOGICAL LABORATORY TECHNIC
position and the cotton plug is removed, and held by its outside
end between the fingers of the left hand, care being taken to pre-
vent the tubal part of the plug touching anything and being con-
taminated. The platinum loop is then slowly and carefully
introduced into the median, and stirred around so that the tube
walls are not touched. The needle is again sterilized and tube
number two is held in the palm of the left hand parallel to
the first one and its plug is removed also; then with a carefully
sterilized needle, three loops of the inoculated gelatine are re-
moved from number one and mixed with number two tube.
FIG. 31. Method of inoculating culture media. (Williams.) '
The needle is then again carefully sterilized in the flame, the plug
of number one is carefully replaced and another tube, number
three, is held in the palm of the left hand and its plug is carefully
removed and held as the previous ones were. With the sterilized
loop three loopfuls of the gelatine from number two are carefully
introduced into number three and the needle is then sterilized
and p'ut aside. The petri dishes should now be laid on a cold level
slab, and the contents of the tubes run into the different dishes.
Tube number one is taken first; the lip of the tube is wiped with
the cotton plug and then held in the flame to destroy all bacteria
clinging to it. The lid of a petri dish is carefully and partially
lifted and the contents of the tube 'rapidly and evenly poured
over the bottom of the plate, and the lid quickly replaced.
THE STUDY OF THE GROWTH OF BACTERIA 125
FIG. 32. Dilution method of making cultures, i, Is first tube containing
great number of colonies; 2, contains less number; 3, relatively few.
(Williams.)
126 BACTERIOLOGICAL LABORATORY TECHNIC
This procedure is followed with the other tubes, and then the
plates or dishes are put in a cool dark place, and the tubes are put
into a solution of bichloride of mercury, or into boiling water.
The plates should be examined from time to time. After several
days a perfect cloud of round colonies are seen in number one; a
large number in No. 2 and a much fewer number, say fifty, in No. 3.
It is an easy matter then to pick out a colony that is surrounded
by a bluish-green halo and transfer it to a tube of agar or bouillon.
In the case of pus it is more than probable that the colony is that
of the pyocyaneus bacillus, and that it contains nothing but these
bacilli. It must be studied in a dozen ways, before it is certain
that it is this bacillus, but the preceding method is a necessary
primary step to secure this organism in pure culture and may
be taken as a pattern for all plate methods.
Agar plates are usually used since they have this advantage
they do not melt at 37C. incubator temperature. When agar is
used it must be melted at iooC. and cooled below 48C. and
above 43C. Above 48C. bacteria may be killed. Below 43C.
the agar begins to harden, so this method must be performed
quickly; the plates should be slightly warmed, the culture poured
on and the agar hardened, they must be invented in the incubator,
since the water of condensation forming in the lids of the plates
often falls and washes one colony into another. When gelatine
plates are made, they must be kept at 2o-25C. It is often of
advantage to cool the plates by means of ice, before they are
filled.
The so-called "Stroke plates" are extremely useful for hospital
bacteriology. The agar is softened, poured into plates, allowed to
harden and the material to be examined is smeared upon the firm
surface by a flattened platinum rod, a "Spatula." The separate
colonies develop along the lines of spread and can be isolated to
individual tubes as given above.
Roll Citllure. Instead of pouring out the contents of the in-
oculated tubes the gelatine may be made to harden on the walls of
ROLL CULTURE 127
the tubes by quickly rotating the tube in a groove melted in a
block of ice. The centrifugal force distributes the gelatine over
the glass, and the ice hardens it rapidly while in contact with the
glass. Such tubes are veritable plates, and in them colonies of
bacteria often grow as well as on the plates and may be fished
out.
The various characteristics of bacterial growth may be studied
in cultures. Some organismal cultures grow rapidly and luxuri-
antly; some discretely and slowly; colors and odors are produced
by some; gelatine is liquefied by many, while others do not liquefy
gelatine. Milk is curdled and digested by some; gas and acids
produced by others. These various characteristics enable us to
identify and differentiate bacteria.
The cultivation of bacteria in the laboratory has for its purpose
a demonstration of their vital activities. This may indicate only
their botanical character or it may show their relation to disease.
In order that we may classify germs systematically certain criteria
have been established which when added together permit us to
identify and name the organisms. This is called determinative
bacteriology. The principal characters to be noted are com-
plete morphology, staining characters, particularly with Gram's
method, colonial growth on agar and gelatine, potato, blood
serum, milk, sometimes inorganic salt solutions, the enzymic
products as indicated by fermentation of carbohydrates and
solution of proteins like milk curd and gelatine. With this last
comes ammonia and nitrite productions. The optimum tempera-
ture and media, and resistance to physical and chemical agencies
must be taken into consideration. For pathogenic bacteria we
establish as far as possible the relations with lower animals.
This includes, of course, the production of soluble toxins and
endotoxins.
The chemical activities of many bacteria are well displayed in
litmus milk culture in which decolorization, acid or alkali forma-
tion, coagulation and clot digestion are the important ones.
128 BACTERIOLOGICAL LABORATORY TECHNIC
The property of converting sugar into acids and gases is best
studied in fermentation tubes.
Into sterile fermentation tubes bouillon containing sugar is run,
these are plugged and sterilized. They may be inoculated with
bacteria and if gas production occurs it is quickly manifested in
the closed arm. The component gases may be studied and the
various properties determined. This gas ratio is of use in identi-
FIG. 33. Fermentation tube. (Williams.)
fying various bacteria and differentiating them. The closed arm
of the tube being shut off from free air by the amount of bouillon
in the open arm is practically an anaerobic tube and is employed
for this purpose. Bacteria that grow only in the closed arm are
considered anaerobes. By inoculating a gelatine tube with bac-
teria while it is melted and then letting it solidify, previously
shaking the tube vigorously, gas formation will be speedily mani-
FERMENTATION METHODS 1 29
fested by the presence of bubbles. Acids are detected in cultures
by the employment of various indicators in the culture media.
Litmus, lacmoid, andrade, and neutral red are used for this pur-
pose. By titrating bouillon of previous known acidity with a
decinormal soda solution, the amount of acid produced by differ-
ent bacteria can be estimated.
Various sugars are fermented by bacteria, and lactic, acetic,
and butyric acids are produced. Indol is also produced by many
bacteria (colon bacillus, cholera bacillus), and its presence in cul-
ture is an important means of identifying different bacteria. The
organism to be studied must be grown in culture media known to
be free from indol. For this purpose, all meat extracts must be
excluded and a simple solution of peptone and salt, run into tubes
and sterilized, is used. After bacteria have grown in this media
for several days the indol produced, if it is produced, is detected
by adding a few drops of pure sulphuric acid. If a red color
(nitroso-indol) is not produced, a few drops of sodium nitrite solu-
tion (.02 gram to 100 c.c. of water) must be added, and if a pink
to deep red color does appear it may be safely assumed that indol
is present.
Ammonia is detected in culture by suspending a piece of paper
wet with Nessler's reagent above a bouillon culture of a given or-
ganism. If a yellow to brown color is produced ammonia is
present.
Nitrites are detected by growing the organism in a solution of a
nitrate (.02 gram potassium nitrate, 10 grams peptone in 1,000
c.c. of water).
Incubate for a week and then add i c.c. each of the following
solutions :
.
(a) Sulphuric acid .5 gram.
Acetic acid 150 c.c.
(6) Amido naphthaline . i gram.
Water . 20 c.c.
Boil, filter, and add 180 c.c. of dilute acetic acid.
9
130
BACTERIOLOGICAL LABORATORY TECHNIC
If nitrites are present a pink color is produced by these reagents.
Enzymes may be detected by noting whether gelatine is liquefied,
or milk curd digested. Both these actions are evidences of the
presence of enzymes.
Bacteria growing exclusively in the absence of oxygen are
known as anaerobes; to cultivate these successively various forms
of apparatus are necessary.
FIG. 34. A streak made in agar by a needle inoculated with aerobic bacilli
and then covered at one spot with cover-glass. The aerobic organisms will
not grow in the anaerobic conditions under the glass. (Williams.;
The following methods are pursued in ordinary laboratory
manipulations :
1. Exclusion of oxygen.
2. Exhaustion of oxygen by means of an air-pump.
3. Absorption of oxygen by means of chemicals that absorb
oxygen from the air. A mixture of pyrogallic acid and sodium
hydrate absorbs oxygen rapidly, leaving nitrogen only in the
chamber.
ANAEROBIC METHODS 131
4. Displacement of air by means of an air-pump and allowing
hydrogen to enter the vacuum.
Under \hzfirst method we may either exclude oxygen by laying
sheets of sterile mica or a cover-glass on the surface of the agar
or gelatine plates (Fig. 34), thus excluding air, or deep punc-
tures may be made in tubes half filled with gelatine or agar, for
growths often occur in the depths of the medium, especially if
FIG. 35. Novyjar.
the latter has been boiled previously to expel the oxygen; or,
instead of mica, sterile paraffine may be poured over the top of
the tube. The layer of paraffine excludes the air. Flasks filled
with bouillon, or tubes filled with bouillon, or melted agar may be
inoculated with an anaerobic culture, but the filling of the vessel
with the medium must be absolute so that no space is left for air,
otherwise the organisms may not grow. Roux employs a long
sterile glass tube, which he completely fills with melted agar
132 BACTERIOLOGICAL LABORATORY TECHNIC
inoculated with the organism he wishes to grow. The ends of
the tube are then sealed in a bunsen flame and there being no air,
anaerobic conditions are fulfilled, and organisms grow. After
colonies appear the tube is broken at a file-mark near the colony
and tubes inoculated therefrom.
Under other methods large Novy jars are used for the reception
of petri dishes and test-tubes. From these jars the air is with-
drawn, and hydrogen allowed to flow into it. A solution of
pyrogallic and sodium hydrate is placed in the bottom of the jar
to absorb any remaining oxygen. There are many other ingenious
mechanical ways of growing bacteria under anaerobic conditions
and the student is referred to works devoted entirely to technique.
Animal Experiments
To determine the pathogenicity of bacteria; to measure the
strength of toxins and anti-toxins, to standardize anti-toxins, and
to recover bacteria in pure culture, it is often imperative that
small laboratory animals be used. Guinea pigs, rabbits, and
mice are oftenest employed. Strong young animals are the best.
Culture toxins and pathological material are introduced into their
bodies in various ways. A favorite one is to' shave the abdomen,
scour it with soap and water, and then bichloride of mercury, and
finally sterile water. With a pair of sterile scissors a small hole
is cut in the abdominal parieties and through it a loop containing
a drop of culture is run into the peritoneal cavity, or under the
skin. With ordinary fluid material, a syringe may be used to
inject it directly through the abdominal wall. The animal is
carefully weighed, and it is watched from day to day. If it dies
an autospy is made on it.
Other methods consist in injecting fluid culture into the veins
of the ear, or into the peritoneum, by means of sterile hypoder-
mic syringe. The autopsy should be made carefully, the animal
should be thoroughly wet with a solution of bichloride of mercury,
HISTOLOGICAL METHODS 133
then it should be stretched over a pan, especially devised for the
purpose, or nailed to a board. The skin over the abdomen and
thorax must then be shaved and sterilized with a solution of
bichloride of mercury. The walls should then be seared in a line
from the throat to the pubes with a hot knife, and through this
line a cut should be made opening up the thoracic and abdominal
cavities.
By means of a hot knife spots must be seared on the various
organs, and with another sterile knife cuts should be made into the
organs, then through these cuts sterile platinum needles are
thrust, and then culture media are inoculated with them. Some-
times it is necessary to remove bits of tissue from various organs
and place them in culture media. In the recovery of the tubercle
bacillus from animals this procedure is necessary. Great care
must be taken in making the culture and all tubes should be
carefully stored. It is of great importance to make smears on
cover-slips as well as cultures, from the heart cavities, liver,
kidneys, peritoneal cavity, etc., and stain them directly with
Gram stain. It is sometimes necessary to inject cultures, or
bits of nerve tissue from a rabies case into the brain. To do
this, remove under strict aseptic precautions, a button of bone
from the skull by means of a trephine. It should not be forgotten
that animals inoculated and killed or dying after infection may
present dangerous material to the laboratory personnel. After
an autopsy, a strong disinfectant should be generously spread
over all parts of the animal and autopsy tray.
Histological Methods
Sections of tissues from infected animals are often examined and
stained by appropriate methods. To demonstrate bacteria, the
tissues should be hardened in alcohol or formaldehyde solution
(4 percent), and imbedded in celloidin, then cut into sections and
mounted in the following different ways :
134 BACTERIOLOGICAL LABORATORY TECHNIC
I. Loffler's Method.
(a) Float section in water.
(6) Remove with section lifter to Loffler's methylene blue from five to
thirty minutes.
(c) Decolorize in i percent solution of acetic acid for ten seconds.
(d) Dehydrate in absolute alcohol for a few minutes.
(e) Clear in xylol.
(/) Mount in balsam.
II. Weigert's Method.
(a) Place in lithium carmine five minutes.
(6) Then in acid alcohol fifteen seconds.
(c) Wash in water.
(d) Transfer to slide and dry with blotting paper.
(e) Apply Ehrlich's gentian violet for three minutes.
(/) Blot and place in Gram's solution for two minutes,
(g) Wash and dehydrate in aniline oil,
(h} Wash with xylol.
(i) Dry, mount in balsam and examine.
In Loffler's method all the tissues, especially the nuclei and the
bacteria, appear blue.
In Weigert's method, if the bacteria stain by Gram's method,
the tissues appear pink, the bacteria a deep blue-black. Tubercle
bacilli are to be stained in tissues, best fixed in formaldehyde solu-
tion, by heating with carbol-fuchsin as given on page 106; the sec-
tion is decolorized by 3-5 percent hydrochloric acid and cleared
by passing it through 95 percent alcohol, absolute alcohol and
finally xylol. It is then mounted in balsam.
Parafnne embedding methods may be employed, but for these
and -other methods of staining the student is referred to works
solely devoted to technique. The staining methods are the
same for paraffine and in experienced hands give better results.
CHAPTER VII
ANTISEPTICS AND DISINFECTANTS
Many chemical substances have the power of entering into
chemical union with the protoplasm of bacterial cells and so
forming new compounds, and coagulating the protoplasm;
other chemicals dissolve the bacterial bodies.
Bacteria differ in their powers to resist these agencies; the
anthrax spore is much more difficult to kill than the typhoid
bacillus; these chemical substances act better at a high than a low
temperature.
A chemical disinfectant, such as copper sulphate, acts more
rapidly and effectively in a watery solution than in a complex
albuminous one.
Park's division of the change of viruses under the influence of
chemicals is convenient and instructive. Attenuation is the tem-
porary restriction of growth, but especially of virulence and
pathogenicity; these are resumed upon cessation of action of the
chemical. Antisepsis is a definite restriction of growth but there
is no destruction. Incomplete sterilization is the destruction of
vegetative forms of bacteria but not spores. Disinfection or
sterilization is the destruction of all disease producing virus.
It is often necessary to determine the exact minimum amount of
an antiseptic that will destroy a given organism or produce a
complete inhibition of growth; for this purpose small amounts of
a disinfectant are added to gelatine in test-tubes and these are
poured into plates and the result noted.
Previous to pouring the plates each tube is inoculated with a
loopful of culture and thoroughly mixed with the medium.
136 ANTISEPTICS AND DISINFECTANTS
Another method is to make bouillon cultures of an organism and
add to each a certain percentage of the solution of the antiseptic,
and abstract every few minutes after the addition of the chemical
one loopful of the mixture and inoculate fresh media.
Pieces of thread sterilized, and then put in fluid cultures may
be used in experiments; they are dipped into solutions of chemicals
for varying lengths of time and then placed in culture media
and growth noted.
It will be found in the case of most antiseptics in dilute solution
that an interval of time must elapse before the organisms are
killed. This is determined by observing the cultures made from
the mixture. After five minutes, growth may occur, but after
one hour, all may be dead, or it may take two or three hours.
The student should refer to works on hygiene for standard
methods of controlling disinfectants, for example the Hygienic
Laboratory and the Rideal- Walker methods.
The most valuable chemical disinfectants are those that kill in
highly dilute solution in a short time.
Bichloride of mercury is a highly efficient germicide in watery
solutions; if, however, albuminous matter is present its action is
inhibited very much.
CHEMICAL DISINFECTANTS
Mercury Salts. Bichloride of mercury in highly dilute solution
is a very valuable antiseptic. It dissolves in 16 parts of tepid
water. It requires an acid reaction for most favorable action
and the tablets now on the market are made up with some acid
having no effect upon the mercury salt. In i-ioo water solution
this salt will kill anthrax spores in twenty minutes. In blood,
the anthrax bacillus is killed by a 1-2,000 solution in a few minutes.
In bouillon the same organism is killed in a dilution of 1-40,000;
in water, 1-500,000; all in the same interval of time. The pres-
ence of the albumins in the blood or bouillon, no doubt acts as a
CHEMICAL DISINFECTANTS 137
protecting envelope about the bodies of the bacteria, being there-
fore unreliable for disinfecting sputum and pus. It is also more
useful and powerful when it is acidulated with a 5. percent of
HC1, or when it is mixed with common salt or ammonium chloride.
In culture 1^1,000,000 solution prevents the growth of most
pathogenic bacteria. Biniodide of mercury is said by some ob-
servers to be more powerful than the bichloride. It is certainly
less likely to be interfered with by albumins.
Sulphate of copper in water is a powerful germicide. It is more
potent in watery solution than in bouillon. It has a remarkable
affinity for algae and for moulds. The author found that if
moulds are put into alkaline solution of copper sulphate and
heated, the copper enters into chemical union with the protoplasm
of the mycelia, hyphae, and the spores; 1-400,000 of copper sul-
phate in water destroys the typhoid bacilli. Even nascent copper
kills the typhoid bacilli, so that copper foil in drinking water has
the power, after a few hours' contact, of destroying bacteria in
the water.
The silver salts are useful in medicine as disinfectants, especially
on mucous surfaces. The nitrate of silver is one of the most valu-
able of all preparations; it is about a fourth as efficient as
bichloride of mercury and is not nearly so toxic. Some of the
albuminates of silver are useful because of their non-irritating
action.
Acids, especially the mineral ones, are valuable disinfectants in
not too dilute solutions. They act chiefly as inhibitors of growth
rather than destroyers of bacterial cells. In the healthy stomach,
hydrochloric acid acts as a normal disinfectant, and in disease,
where it is absent, it must be added in order to prevent decom-
position of food. Boric acid is useful in medicine on mucous
membranes.
The halogens, iodine, bromine and chlorine, are active agents
for the destruction of bacteria. The cheapest of these is chlorine.
It acts best in contact with moisture, since it decomposes the
ANTISEPTICS AND DISINFECTANTS
molecule of water combining with the hydrogen to form free HC1
and setting free oxygen.
Dry chlorine gas (45 percent) failed to kill dry anthrax spores
in one hour, but when moisture was introduced 4 percent chlorine
killed the spores.
" Chloride of lime ," chlorinated lime, in i percent solution kills
most bacteria in one to five minutes. Iodine preparations like
chlorine ones are very powerful. They are of great use in medi-
cine; ordinary tincture of iodine painted over infected areas acts
as a powerful germicidal agent. It is too expensive to use in
house disinfection and it is exceedingly destructive to all metallic
objects. A 5 percent solution in 50 percent alcohol acts as a
splendid disinfectant for intrauterine injection in puerperal sepsis.
It is now said that 10 percent iodine tincture in 70 percent alcohol
is the most efficacious, practical, medical disinfectant. Many
claim it to have the highest penetrating powers.
Dakin's solution is a mixture of chlorinated lime and sodium
carbonate in water and sodium bicarbonate, with an alkalinity of
.45 percent. This mixture when in the presence of organic
matter decomposes with the formation of hypochlorous acid
which may further change into chlorimido (NCI) to which
changes the antiseptic action is due. The solution is used for
infected wounds, being introduced by methods of infiltration and
drainage. It has an irritant effect upon the skin which tissue
must be protected. The germ-killing power of this solution is
very high. Its use was followed in the great European war, by a
marked reduction in spreading and fatal infections from lacerated
wounds.
Chloramin is another product of chlorinated lime, depending
on chlorin for its action. Being less toxic it may be applied to
tissues or even mucous membranes in 2 percent solution but it is
irritating and surrounding surfaces must be protected.
DicUoramin T. still a chlorine disinfectant, is less irritating than
the foregoing but being unstable, must be suspended in an oil like
CHEMICAL DISINFECTANTS 139
eucalyptoe, saturated with chlorine gas. It can be sprayed on a
surface or pressed into a wound.
Carbolic acid is valuable as a disinfectant because of its stability.
A 1-1,000 solution inhibits bacterial growth; a 5 percent solution
is a thoroughly reliable disinfectant for morbid discharges; this
strength is not injurious to metals or fabrics. A thorough solu-
tion should be made, and to be very efficient, 5 percent HC1
should be added to it.
Cresol, lysol and creolin are useful as disinfectants, but are
sometimes unreliable since perfect solution cannot always be
made. The mixture of one of these substances with water is
more of an emulsion than solution. Anthrax spores have been
known to live for hours in creolin solutions. The value of these
cresols is that when applied to a surface the water may evaporate
but the germicide sticks and continues its effects. Glycerine is
sometimes added to lighter phenol solutions to assist this action.
Peroxide of Hydrogen has a great reputation in medicine as an
antiseptic. It kills bacteria, especially the pus cocci, in a few
minutes in a 15 percent solution. A 40 percent solution will kill
anthrax spores in a few hours. It is a powerful agent when fresh,
and is not poisonous. It combines with organic matter and
becomes inert. It degenerates if exposed to atmosphere and if it
comes in contact with the ferments of the blood (haemase).
Formaldehyde gas, CH^O, is, by all means, the most useful, as
well as the most powerful disinfecting agent that we have. In
solution 40 percent in water, it is known as formaline. It has a
marked affinity for organic substances and forms chemical com-
binations with many organic bodies. When it unites with am-
monia it becomes inert until some acid frees it. It unites with
iron, but other metals are unaffected. Its use in medicine is wide
and varied. It is a deodorizer; renders gelatine glass-like and
insoluble in boiling water. It may be liberated as a gas in apart-
ments and ships, actively destroying all bacteria. One percent
of the vapor in the air of a closed room, if the air is moist, destroys
140 ANTISEPTICS AND DISINFECTANTS
bacteria after twelve hours. It is best to keep the room closed
for twenty-four hours. It may be thrown into the room in many
ways; by generators which decompose the vapor of wood alcohol,
when they reach hot platinum sponges, salt, or hot copper; by
vaporizing a solution by means of heat; by adding permanganate
of potash to a solution of formaline; by spraying a concentrated
solution over bedding, floors, and walls, then closing the apart-
ment. It is very much more active in warm air than in cold, and
when the air is moist. It has been known to destroy anthrax
spores wrapped up in paper and placed under blankets. All of
the pathogenic bacteria are killed by it, the Staphylococcus
aureus and anthrax spores being more resistant than anything
else. It will not kill moulds unless highly concentrated. As di-
lute watery and alcoholic solutions decompose they should only
be used when freshly made.
Sulphur Dioxide Gas. An old and rather unreliable form of
disinfectant. It does not kill anthrax spores very readily, as
it requires an exposure of twenty-four hours to a 40 percent vapor
in a room. It is generated by burning sulphur in a room tightly
closed, and it is much more efficient if weter is vaporized in
the room. It is not very penetrating, is poisonous to breathe,
speedily bleaches fabrics, and attacks metal objects. It is much
superior to formaline as an agent for the destruction of insects,
especially mosquitoes, also to kill rats infected with plague bacilli.
Lime. Ordinary quick lime, or whitewash, is highly germicidal.
It is especially efficacious in disinfecting feces from typhoid cases.
Typhoid bacilli are killed after one hour's exposure to a 20 percent
mixture.
Potassium permanganate in 3 percent solution is said by Koch
to kill anthrax spores in twenty-four hours. It is not so efficient
a germicidal agent as supposed.
Turpentine and essential oils are efficient germicides in con-
centration. Common mustard rubbed in the hands is said to
make them sterile.
CHEMICAL DISINFECTANTS 141
Alcohol. Ninety-five percent and absolute alcohols are not
antiseptic for the anthrax spores, since they will live for many
hours in contact with absolute alcohol. In general it is unreliable.
Seventy percent alcohol is the most efficient strength.
Zinc chloride in concentration is a powerful germicide. A 2 per-
cent solution will kill the ordinary pyogenic bacteria in two hours.
Sputum, urine and dejecta are best disinfected by heat. Chem-
icals often are inert because they cannot penetrate the albumi-
nous masses of the sputum or feces. Long contact with carbolic
acid acidulated with HC1 is very efficient. Concentrated forma-
line and solutions of chloride of lime may be used, also a heavy
mush of lime in water.
Boiling or heating instruments and dressings by high moist heat,
as in an autoclave, is the most reliable method of rendering them
sterile. The exposure of dressings to i5oC. for one hour, or
boiling instruments, thoroughly cleaned mechanically, for twenty
to thirty minutes makes them certainly sterile.
Disinfection of the skin is a difficult undertaking from a
bacteriological standpoint. In the deep layers of the skin, and in
the sweat glands and hair follicles, bacteria often exist, even after
the most thorough and prolonged disinfection. The application
of soap and water with a stiff brush is by all means the most
valuable part of the process, since with the removal of the dirt
most of the bacteria are removed. Thorough scrubbing with soap
and sterile water, followed by scrubbing with a 1-1,000 bichloride
solution, cleansing the nails with a sterile brush, and prolonged
immersion in bichloride or permanganate of potash solution,
complete the process. Modern methods, even after all this
preparation, require the use of rubber gloves that have been
sterilized by boiling. The faultiest part of the preparation for an
aseptic operation from a bacteriological standpoint, has always
been considered to be the sterilization of the hands, and if these
can be covered by rubber gloves that are sterile, the fault can be
surely eliminated.
142
ANTISEPTICS AND DISINFECTANTS
Antiseptic Values (after Park).
The figures refer to the relative antiseptic powers of various
agents for fluids containing organic matter.
Alum i to 222
Aluminium acetate i to 6,000
Ammonium chloride to 9
Boric acid to 143
Calcium chloride : to 25
Calcium hypochlorite to 1,000
Carbolic acid to 333
Chloral hydrate to 107
Copper sulphate to 2,000
Ferrous sulphate to 200
Formaldehyde, 40 percent to 10,000
Hydrogen peroxide to 20,000
Mercuric iodide to 25,000
Mercuric chloride to 40,000
Quinine sulphate to 800
Silver nitrate to 12,500
Zinc chloride to 500
Zinc sulphate to 20
CHAPTER VII
BACTERIA
STREPTOCOCCUS PYOGENES
Streptococcus Pyogenes.
Streptococcus Erysipelatis. Chain Coccus (Fig. 36).
Morphology and Stains. Cocci grow in catenate form of from
4 to 40 individuals to a chain. The cocci divide in a single plane,
FIG. 36. Streptococcus pyogenes. (Kolle and Wassermann.)
by transverse fission thus giving rise to chains. The cocci are
not motile, and do not have spores. They can be stained with
all basic stains, and retain the Gram's stain.
Relation to Oxygen. They grow either in the presence or
absence of oxygen, and are, therefore, facultative aerobes.
Temperature and Food Requirements.
Develop best at 37C. Will not grow at 47C. Never vege-
tate luxuriantly on any culture media, but are most prolific on
143
144 BACTERIA
one that is faintly acid and contains animal juices like serum.
They must be transplanted frequently. On gelatine they grow
scantily without liquefaction, the growth consists of discrete little
masses, while on agar they appear translucent colonies of very
small grayish granula. In bouillon cultures some varieties either
cloud the medium uniformly, or else sedimentate in the form of
little balls, the supernatant fluid remaining clear. It ferments
some simple sugars but does not form gas. In milk the growth is
more luxuriant, and becoming acid, may be coagulated in twenty-
four hours. On potato the growth is invisible and scanty, or
absent. On blood agar plates colonies appear as tiny gray
points with a zone of hemolysis about them. Sugar media
of the simpler carbohydrates, show acidification.
Vital Resistance. Thermal death-point is 54C. in five min-
utes. Virulence in dried albuminous matter (pus) is retained for
months. If kept on ice, vitality and virulence are retained for
months also.
Chemical Activities. Lactic acid and sulphuretted hydrogen are
produced, also ferments which have the property of dissolving
fibrin under anaerobic conditions. They are also capable of dis-
solving red blood corpuscles, either in culture media or in the
body and about cultures on blood agar plates there is a clear halo
of hemolysis, streptocolysin. They produce a strong soluble
toxin, which can be filtered from the bouillon and precipitated
with alcohol. This causes necrosis, anaemia and death.
Habitat. In sewage, dwellings, dust, on the healthy human
body, and in the cavities of the respiratory tract, vagina, rectum,
and in the faeces. It is the cause of many diseases, i.e., erysipelas,
puerperal fever, meningitis, pneumonia, endocarditis, peritonitis,
tonsillitis, osteomyelitis, and the diarrhoea of children.
In general septicaemia streptococcus is found in the blood, and
plays an important role in secondary infection, causing an aggra-
vation of the original infection, and often death. It is especially
active in phthisis, scarlatina, small-pox, and diphtheria, in which
STREPTOCOCCUS PYOGENES 145
diseases it is often the cause of death. Many of the symptoms
of phthisis are due to the toxins of the streptococcus; cavity for-
mation and hectic fever for example. Its virulence can be in-
tensified by passing it through a series of animals, until, finally,
M>ooo cu. mm. kills in one day all the mice injected with this
dose. The toxin contains a peculiar haemolytic substance, which,
as before remarked, dissolves red cells of the blood, hence the
anaemia in septicaemia and in suppuration. The toxin of the
streptococcus, if injected under the skin, causes redness like ery-
sipelas. Coley's fluid containing this toxin is used to treat
sarcomata, since infection with the streptococcus has been known
to cause a disappearance of these tumors. Practically all animals
are susceptible to the streptococcus.
Agglutinations. The serum from an animal injected with strep-
tococci, or immunized against it, will agglutinate streptococci.
Anti-toxic sera have been prepared by injecting horses with
highly virulent living culture of streptococci. The serum protects
to a limited degree, and has some curative properties. Cultures
of cocci from human sources have been found to produce the
best toxins; there are, however, many strains.
The foregoing description represents the principal characters of
the most important member of a large group of closely related
streptococci. Distinctions in the group are based upon the
solution of red blood cells, the fermentation of lactose, of maltose,
of salicin and the coagulation of milk. The relative value of
each of the members is not yet settled but investigations have
enabled laboratory workers to elaborate certain techniques
which may be expected to clear up the matter. The varieties
now recognized are Str. hemolyticus, epidemicus, anginosus,
fecalis, salivarius, equinus, and mitis. Some of these organisms
produce no hemolysis; they come under the term non-hemolyticus
and are of importance in certain respiratory and allied diseases.
There is a group of organisms, a sort of connecting link with
pneumococci, which produce green colonies on blood agar and
10
146 BACTERIA
are called Str. viridans. These are to be distinguished from
true pneumococci by the formation of a tiny rim of blood
clearing in blood plates and by their insolubility in bile. These
and the group of non-hemolytic streptococci are held responsible
for some cases of arthritis, endocarditis, sinusitis, nephritis, etc
PNEUMOCOCCUS
Streptococcus pneumonias commonly known as the pneumo-
coccus, or Diplococcus lanceolatus (Fig. 39). (For types see
page 76.)
Morphology and Stains. This organism is usually found in the
tissues and sputum, in the form of lance-shaped cocci, surrounded
by a capsule. Is almost always associated in pairs, though some-
times in chains of five or six members. In albuminous fluids, or
blood serum, and in milk, the organism exhibits a well-defined
capsule; in bouillon and other media, it loses the capsule and the
lanceolate shape, and often appears spherical, in pairs, or chains.
It is not motile, has no flagella or spores, is easily stained by all
the basic aniline dyes, and keeps the color by Gram's method.
Under certain conditions it strongly resembles the streptococcus
pyogenes, and may be differentiated therefrom by growing it on
agar smeared with blood. The streptococcus causes a haemolysis
of the corpuscles, while the pneumococcus does not and the
colonies are greenish.
Oxygen Relations. It is a facultative aerobe.
Grows rapidly, but never luxuriantly at 37.5C.; at 22C. much
more -slowly, often not at all. Grows better in the presence of
serum or haemoglobin.
Vital Resistance. Easily killed at a temperature of 52C., ex-
posed for ten minutes. Direct sunlight also kills it in twelve
hours. While it quickly dies on ordinary culture media, it may
live in dried sputum or pus exposed to diffuse light and desiccation,
for four months.
PNEUMOCOCCUS
147
Cultures. On gelatine plate it produces very minute colonies
after quite a length of time. On glycerine agar it grows better,
but the colonies are small and difficult to see. In both, the colo-
nies are whitish, with a pearly lustre. On blood serum it grows
in transparent colonies. On blood agar the colonies are tiny and
FIG. 37. Diplococcus pneumonias, from the heart's blood of a rabbit. X
1,000. (Frankel and Pfeiffer.)
of a greenish color, lying on a brown base due to production of
methemoglobin. In bouillon it grows feebly, with a whitish
sediment, and in the form of chains. Here the growth is inhibited
by the products of its own metabolism, i.e., lactic acid. If this
is neutralized by putting chalk into the bouillon the growth
becomes luxuriant and the bouillon becomes thick. On potato it
148 BACTERIA
will not grow. It ferments some of the sugars, the most impor-
tant and characteristic being inulin. No gas is formed. Pneu-
mococcic are soluble in bile or a solution of its salts, a distinguishing
determinative character.
Habitat. Outside the human body it has not been found, but
is normally present in the mouth of about 30 percent of all people.
In apparent health cultures from the throat or the sputum in-
into animals often causes pneumococcic septicaemia because
Type IV (see page 76) may be frequently found in them. The
so-called "fixed types" I, II, and III occur in the throat during
pneumonia but disappear shortly after recovery. It also may be
found on the conjunctiva and nose in health.
Chemical Activities. No soluble toxin has been discovered.
The toxic properties are due to an endo-toxin. This organism is
a pyogenic one, and causes dense fibrinous exu dates on serous
membranes. All tissues of the body may be attacked. Some
strains of pneumococci are more neurotoxic than others.
In rabbits an intravenous injection of pneumococci will cause
a septicaemia with at times areas of lobular pneumonia. The
only successful reproduction of pneumonia in the lower animals
is accomplished by tracheal insufflation of pure virulent cultures.
In human infection the organisms are forcibly inhaled into the
deepest recesses of the lungs. Pneumonia may be haematogenous
in origin also.
Besides pneumonia, any serous membrane may be attacked and
pleuritis, peritonitis, pericarditis, or meningitis may be caused.
Abscesses anywhere may be due to the pneumococcus. Mucous
membranes of the throat often are affected; middle ear abscesses
also may be caused by this organism. Pneumococcic septicaemias
are common.
During pneumonia, pneumococci may be recovered from the
blood before the crisis by means of blood cultures; 10 c.c. of blood
abstracted from veins is mixed with 500 c.c. of milk or bouillon
and incubated. In twenty-four hours pneumococci, if present,
COCCUS OF MENINGITIS 149
grow luxuriantly. Just before the crisis the organisms will not
grow.
Immunity and Susceptibility. The susceptibility of man varies
greatly. Exposure to cold and hardships of various kinds predis-
pose to pneumonia. One attack does not prevent another. It
has been observed that normal leucocytes only become phagocytic
toward the pneumococcus when lying in anti-pneumococci serum.
It has even been noticed that these organisms grow better in the
anti-serum, rather than in the normal serum. Animals have been
immunized by injecting cultures and toxin. The immune serum
thus produced protects small animals against infection, and stimu-
lates phagocytosis. It has been used therapeutically in man for
the cure of pneumonia with hopeful results. Oleate of soda aids
in bacteriolysis of pneumococci by sera, if added to the various
varieties of immune sera (see page 76). Most mammals, but few
if any birds are susceptible to the pneumococci; mice, being
very easily infected, are used for isolation purposes.
Agglutination of pneumococci is caused by the blood of infected
individuals, even diluted at 1-60. Immune serum also has the
same action.
Opsonins increase during the course of pneumonia and are at
their height at or just after crisis.
Pneumococcus mucosus, also called Type III is distinguished
by its large size, long chains, capsule, more generous growth
in large moist colonies of not such a distinct green color and its
ability to produce a serious form of pneumonia.
COCCUS OF MENINGITIS
Micrococcus Meningitidis.
Diplococcus intracellularis meningitidis.
Meningococcus (Fig. 38).
This organism is the cause of epidemic cerebro-spinal meningitis.
Morphology and Stains. Resembles the gonococcus closely,
150
BACTERIA
because it grows in biscuit-shaped pairs; is nearly always within
pus cells, and like the gonococcus it is decolorized by Gram's
stain. It has no spores or flagella; is not motile; grows in short
chains at times, and on ordinary media best at 37C.
Q
V
FIG. 38. Meningococcus in spinal fluid. (From Hiss and Zinsser's Bacteri-
ology, Copyright by D. Appleton & Co.)
Relation to Oxygen. It can be cultivated from the meninges
at first best under conditions of low oxygen tension, as in a Novy
jar or in an atmosphere of carbon dioxide and moisture; once
growing in the laboratory it is more luxuriant in aerobic tubes.
Vital Resistance. It is killed after ten-minutes' exposure to
COCCUS OF MENINGITIS I$I
65C. and is easily destroyed by drying, and by light. It dies
out rapidly on artificial culture media.
Cultures. Best isolated on neutral semi-solid ascitic fluid or
serum agar in a moist chamber. Growth is pale gray white
translucent moist separate colonies. On glycerine agar it grows,
sparingly as white viscid colonies; occasionally it develops on
potato ; thrives on blood serum, especially if smeared with blood,
and does not liquefy the serom.
Habitat. It is found in the pus from the meninges, sputum,
and nasal mucus of persons afflicted with epidemic meningitis, or
spotted fever. It has been found in the mucous membranes of
healthy individuals, and these persons may be "carriers" of infec-
tion. After spinal puncture, it may be seen in the pus cells, and
the diagnosis of the disease can be made in this way.
Virulence. It is scarcely virulent for lower animals. If given
by hypodermics into the pleura, or peritoneum, it produces death
in mice. Meningitis may be, in monkeys, produced by subdural
injection.
Chemical Activities. Produces an endo-toxin but no soluble
toxin. It is not chromogenic.
Agglutination is caused by immune serum and it is upon their
property that anti-sera are standardized. It has been discovered,
by means of this anti-body and by the failure of certain sera to do
good, that there are several types of meningococci of different
antigenic qualities. Anti-sera are made now with all available
varieties.
Method of Infection. The infection atrium of the coccus is
not certainly known but most of the evidence points to the nasal
passages and cribriform plate to the subdural space.
Specific Therapy is practicable; it has been discussed just above
and on page 76.
There is another important Gram-negative diplococcus in the
nose called Micrococcus catarrhalis. It is differentiated from
the meningitis organism by its free growth on agar, its sugar reac-
152 BACTERIA
tions and absence of active pathogenic properties. It will appear
on nearly all plates made from sputum and throat cultures, in
crowded city life of temperate zones. It seems not to be able to
incite an infection but to continue or aggravate one already under
way.
STAPHYLOCOCCUS PYOGENES AUREUS
Staphylococcus Pyogenes Aureus (Fig. 39).
Micrococcus Pyogenes.
Staphylococcus pyogenes aureus, albus, and 'citreus are known
commonly as Staphylococcus, or grape coccus. They differ only
in color production on artificial media.
FIG. 39. Staphylococcus aureus. (Williams.)
The Micrococcus pyogenes aureus only is here described; the
other varieties have similar but much feebler pathogenic powers.
Morphology and Stains. Round cocci, often growing in
bunches like grapes. Individual cocci dividing in two planes.
They stain very well with all basic dyes, and are not decolorized
STAPHYLOCOCCUS PYOGENES AUREUS
153
by Gram's method. They are not motile; have neither flagella
nor spores.
Oxygen Requirements. The coccus grows well in oxygen, and
poorly without it.
Temperature and Vital Resistance.
Thrives best at body temperature, but
grows well at room temperature. Resists
drying for over one hundred days in pus.
Dry thermal death-point is 8oC. for one
hour. Moist heat 7oC., kills in ten to
twenty minutes. Resists freezing tempera-
ture for many months.
Exceedingly resistant to formaldehyde,
more so than some spore-bearing organisms.
Resists light also. It is killed by corrosive
sublimate i-iooo in fifteen minutes; i per-
cent H2C>2 in thirty minutes.
Chemical Activities. Produces a golden-
yellow pigment only under oxygen. Gener-
ates acids, but no free gases. Creates indol
and sulphuretted hydrogen; ferments urea,
and produces ferments that dissolve gelatine,
and the coagulated proteids of milk. The
toxin is soluble in water, and acts intensely,
causing violent local reaction. If in the ab-
dominal cavity, it causes peritonitis. Subcu-
taneously it may produce sterile abscess, or
local necrosis. There is produced in cultures
a toxin having a destructive action upon leucocytes and red
blood cells.
Cultures. In gelatine it rapidly forms golden-yellow colonies,
that quickly liquefy the gelatine (Fig. 40). Sterile products
of the growth also liquefy gelatine. On gelatine plate, yellowish
to orange colonies are formed. On agar streak a luxuriant orange
FIG. 40. Gelatine
culture staphylococ-
cus aureus one week
old. (Williams.)
154 BACTERIA
growth develops. In bouillon there is a marked even cloudiness,
with a fine pellicle on surface; moderate sediment, which upon
shaking is broken up. Milk is rendered acid and curdles very
soon, the curd being digested finally.
Potato cultures are dry, whitish then yellow, and finally deep
orange.
Habitat. Widely distributed; found in dirty water, sewage, air,
dust of streets and houses; also upon the skin; normally present in
the mouth, nose, rectum, anterior urethra, vagina, and external ears.
Pathogenesis. In man it is the cause of carbuncles, abscesses,
osteomyelitis, septicaemia, puerperal infection, and any inflamma-
tion of the serous membranes. It causes acne and boils; can, and
does attack any tissue of the body. Endocarditis is a very grave
affection that is caused by this organism. It also plays an
important role in secondary infection, causing necrosis of pre-
viously infected tissues (tubercles) and is active in small-pox and
diphtheria. Experimental endocarditis has been produced in
animals by injecting it into the veins. By passage through
animals it is rendered highly virulent. In young, diabetic and
anaemic subjects, its action is often rapidly fatal. Its pathogenic
action is often wide and disastrous. By growing it under an ae-
robic conditions its virulence may be intensified, and the ac-
tivity with which it liquefies gelatine is an index of its malignancy.
In man acne, boils, and carbuncles have followed the rubbing of
culture into the skin.
Immunity. Careful injections may result in the immunization
of the lower animals. An anti-serum with opsonic, agglutinative,
lytic and anti-toxic properties has been produced and, if used
fresh, seems to have a slight beneficial effect upon staphylococcus
septicemia. Too little is known for definite statements. Bac-
terins made from this germ have been used with excellent results
in all but the very aggravated and fulminating affections caused
by it. Bacterin treatment of acne and furunculosis has estab-
lished itself as most efficacious.
GONOCOCCUS
155
There is a member of this group infesting the deep layers of the
skin called Micro, epidermidis albus. It is of feeble pathogenic
power, but may delay the healing of surgical wounds.
GONOCOCCUS
Micrococcus Gonorrhoeas (Neisser).
Diplococcus Gonorrhoea, commonly called the gonococcus (Fig. 41).
FIG. 41. Gonococci and pus cells. X 1000. (MacNeal.)
Morphology and Stains. The morphology of this organism is
peculiar and characteristic. Always found in pairs which are
cemented by an invisible substance. These pairs resemble coffee
beans with the concave sides opposite each other and slightly apart;
or kidneys placed with the hilums facing each other.
In pus it is generally found within the protoplasm of the leuco-
cytes, about, though never within, the nuclei. It is non-motile;
has no flagella, or spores, and stains readily with all the basic stains;
but best with Loffler's blue. It is decolorized by Gram's stain.
156 BACTERIA
This point is most important in differentiating it from other
diplococci, except the meningococcus. A diplococcus is said to
exist normally in some urethras that resembles the gonococcus,
but is Gram-positive.
Oxygen Requirements. It is a facultative anaerobe.
Vital Conditions. It is cultivated with difficulty in culture
media. Grows best at about 37C. As it dies quickly in usual
culture media, a special one must be employed; that containing as-
citic or hydrocele fluid, blood or urine is best. It does not with-
stand high temperature, drying, or light, very long, and is very
easily killed in culture by silver salts. In tissues of the urethra it
may live many months.
Cultures. On agar, containing ascites fluid, it grows very spar-
ingly. The colonies are exceedingly delicate, and gray, turning
to yellowish, and are scarcely above the culture media. It will not
grow in gelatine, milk, or ordinary bouillon, but in one made of
nutrose, serum, beef-extract, and peptone.
Habitat. Never found outside the human organism, except on
linen, towels, instruments, etc. It is in all senses a strict parasite.
Bacterial Activities. Apparently does not produce a soluble
toxin, but an endo-toxin (gonotoxin) , which is highly resistant to
heat.
Pathogenic Virulence. This organism does not infect any of
the lower animals. The "gonotoxin," if injected into small ani-
mals, produces a doughy infiltrated area, which undergoes necrosis.
It has been found that filtrates of old cultures (sterile), if placed on
urethral mucous membranes, can produce suppuration. In man,
the organism causes a distressing disease (gonorrhoea), which may
become a dangerous one, ending even in death. It may produce
violent inflammation of the urethra vagina, uterus fallopian tubes,
and the peritoneum. It frequently affects the conjunctive, and
sometimes causes a pan-ophthalmia, which destroys the sight. It
may be a cause of plastic arthritis gonorrhceal rheumatism, endo-
carditis, pleuritis. In fact, any serous membranes may be infected,
MICROCOCCUS TETRAGENUS 1 57
and very serious results follow. Cystitis caused by the gonococcus
is sometimes followed by infection of the kidneys. In the urethra,
the cocci may burrow deep beneath the epithelial cells, and set up
a metaplasia, or abscess formation. The purulent exudate is rich
in phagocytes gorged with cocci, often as many as 40 being found
within a cell.
Immunity . One infection does not confer immunity against
further infection. There is no reliable means of producing artifi-
cial immunity. However, gonococcus bacterins are of some value
for chronic gonorrhoea. Torrey has been able to obtain from
rabbits an anti-serum of therapeutic value in gonorrhoeal arthritis.
MICROCOCCUS TETRAGENUS
Micrococcus Tetragenus.
Morphology and Stains. Round or oval cocci; found in pairs;
more commonly in fours differing in size. In culture this form of
growth is apt to vary, and not to be characteristic. In sections of
human or animal tissues, tetrads only are found that are always
surrounded by a capsule which is stained easily by eosin. The
cocci are stained by Gram's method. It is not motile, and does
not form spores.
Oxygen Requirements. It grows very well in the presence of
oxygen, and poorly without it.
Cultures. Grows well on all common culture media. On gela-
tine plates its growth is characterized by small white colonies,
elevated, with sharp outlines. It does not liquefy the gelatine.
On agar it grows even more luxuriantly than on galatine. In
bouillon it thrives well, depositing a heavy precipitate. In milk
it causes acidity but no coagulation. On potato it also grows,
leaving a silvery streak where the inoculating needle was drawn.
Chemical Activities. It produces acid in sugar bouillon, but
does not form gas, indol, or H 2 S.
Habitat. Has never been found outside the human body; is
158 BACTERIA
normally present in the saliva, sputum of tuberculous subjects, in
the cavities of phthisical lungs, and in abscesses.
Pathogenesis. While causing a fatal septicaemia in mice, and
abscesses in rabbits, it is not of much moment from a pathological
standpoint, though it plays an important role in secondary infec-
tion in phthisis and bronchiectasis.
COCCUS OF MALTA FEVER
Micrococcus Melitensis.
Bacterium Melitensis.
Bacillus of Malta Fever.
Coccus of Malta Fever.
An organism belonging somewhere between the Coccacae and
Bacteriacae. It is small, oval-shaped, and of about .$n diameter,
occurring in culture singly, in pairs, or in chains. In the latter
form, the organism elongates and resembles, more strongly, bacilli.
It is non-motile and it has no spores. Stain faintly with the com-
mon basic dyes, but not by Gram's method. It has been found in
the blood during life, and by splenic puncture.
Cultures. On gelatine its growth is slow, without liquefaction.
On agar the growth, at 37C., is more rapid. The colonies are
pearly white, becoming yellow. In bouillon it produces turbidity,
with a flocculent deposit. No pellicle is formed. On potato an
invisible growth occurs. Milk is not coagulated, nor are acids or
gases produced.
Pathogenesis. It causes in man, Malta fever. Rabbits,
guinea pigs, and mice are not susceptible to inoculation, but the
disease can be produced in monkeys.
Agglutination. The serum from an individual suffering from
Malta fever agglutinates the bacilli, even in dilutions as high as j
i-ioo.
INFLUENZA BACILLUS 159
Diagnosis of the disease can be effected by the agglutination
test, and by splenic puncture, and blood cultures.
It is present in the blood and is excreted via the urine and milk.
The goat while not suffering with Malta fever can carry the germs
in its body and excrete them in the milk. Goats' milk is a general
food in Malta. The inference is obvious. Flies may transmit the
bacilli.
INFLUENZA BACILLUS
Bacterium Influenzse.
Influenza bacillus.
Morphology and Stains. Very small short rods which are
often in pairs, found within epithelial and pus cells, and in sputum;
from 40 to 80 in a cell. May grow out into short mycelia. No
flagella or spores are formed. Stains weakly. Carbol-fuchsin, di-
luted, gives the best result. The ends of the bacillus stain more
deeply than do the rest of the cell. It is decolorized by Gram's
stain.
Oxygen Requirements. It is a strict aerobe.
Cultures grow best on blood-smeared agar, or in blood bouillon
between 27 and 4iC.; best at 37C. Blood or haemoglobin is
demanded for all cultures. In bouillon it grows in thin white
flocculi. On agar in small transparent "dewdrop" colonies,
never luxuriantly. Grown in the same culture with Staphylo-
coccus aureus, it increases more luxuriantly (symbiosis). It is
probable that the cocci, in some way, alter the blood of the culture
media. Very satisfactory media may be made by heating blood
agar or by the addition of sodium oleate to it.
Vitality. It is easily killed by light, heat and drying. Lives
but a day in distilled water, and from eight to twenty-four hours
in dried sputum.
Habitat. Never outside the body; always a strict parasite. It
is found in the mucous membranes of the upper respiratory tract,
and in the mucous secretions.
l6o BACTERIA
Pathogenesis. Catarrhal symptoms follow the smearing of a
culture upon the nasal mucosa of monkeys. Pure cultures,
injected into the peritoneum of guinea pigs cause fatal peritonitis.
This bacillus was isolated by Pf eiffer during the influenza epidemic
of 1889 and by him believed to be the cause of the disease. Be-
tween that time and the pandemic of 1918 it has been found in
acute respiratory infections, chronic bronchitis, sinusitis, otitis and
meningitis, all attacks being characterized by great depression.
Conviction has never been obtained that it was the principal cause
of acute disease except for meningitis, but the bronchitis of tuber-
culosis has been ascribed to it on many occasions. The pandemic
cases of 1918 showed a high percentage of positive findings in the
sputum and lungs at autopsy. There was however no definite in-
crease in agglutinins, lysins or complement fixing anti-bodies so that
many have doubted its etiological relation in epidemic influenza. It
was present in a large percentage of cases and certainly aggravated
pneumonitis and sinusitis in association with streptococci. Its
effect seems due to an endotoxin, possibly also to some exotoxin,
having a definite affinity for the nervous system. It circulates
in the blood rarely, principally in the meningitic form and in
early stages of the intense general cases.
Influenzal meningitis is more frequent than formerly or
at least is more often diagnosed. It can be reproduced in
monkeys.
By immunizing a goat with influenza bacilli Wollstein obtained
a serum which has a pronouncedly favorable effect upon the
experimental disease in monkeys and promises some therapeutic
power for human beings. Its most important effect is to stimu-
late phagocytosis in the cerebro-spinal fluid.
A short immunity remains after a spontaneous attack in man
but attempts at production of immunity by vaccines have been
disappointing. By the use of a mixed vaccine of influenza
bacilli, streptococci and pneumococci, the chance of pneumonic
complications seems reduced, but influenza may not be prevented.
INFLUENZA BACILLUS l6l
Therapeutic use of vaccines is useful only to increase leucocytes
which are characteristically low during the disease.
Bordet-Gengou Bacillus of Whooping Cough. This is a very
minute ovoid rod lying separately, varying from .8-1.5^ long and
being .^fj, wide. No spores, no motility or flagella. Stains poorly,
best at ends; Gram-negative. It may be cultivated from expec-
toration early in the disease upon media containing glycerine,
potato, blood and agar. Aerobe, and grows best at 37C. There
is an endo-toxin. Infective for monkeys. The discoverers claim
this to be the cause of pertussis, because it will act as an antigen
and fix complement away from the hemolytic series.
Its relation to pertussis has been explained by the report that it
is found lying between the cilia of the respiratory epithelium, an
embarrassment of the movements of which causes the coughing
attack. The only bacteriological diagnosis is by growing the
microbes on the medium given above, and by the complement
fixation test. Vaccines are said to have a prophylactic value,
and some relief of paroxysms certainly seems to follow their use.
The last two microorganisms are types of the so-called hemo-
globinophilic bacteria because of the requirement of blood
coloring matter in laboratory culture media. Other forms have
been found in trachoma and in the spinal fluid.
Conjunctivitis. There are two specific germs for conjunctivitis
separate from the gonococcus. They are the bacillus of Koch-
Weeks and that of Morax and Axenfeld.
Koch-Weeks Bacillus. The organism of pink eye. This is
a minute, i.5juX.2/-i non-motile, Gram-negative, sporeless, poorly
staining rod, very like the influenza bacillus. It is aerobic and
non-liquefying. It grows as minute, pearly, glistening, discrete
colonies only upon agar of 5 percent strength plus serum.
The Bacillus of Morax and Axenfeld. A non-motile, sporeless
diplo-rod; negative to Gram stain. Grows only in the presence of
serum or blood and liquefies the former. It is larger than the
Koch- Weeks bacillus, measuring up to 2/*.
11
l62
BACTERIA
PLAGUE BACILLUS
Bacterium Pestis.
Plague Bacillus (Fig. 42).
Morphology and Stains. Short plump rods with rounded ends,
containing no spores and non-motile. It is said by some that a
capsule is formed. Organisms from exudates, or blood, exhibit
characteristically peculiar polar staining. They are often found
FIG. 42. Pest Bacilli from spleen of rat. (Kolle and Wassermann.)
within the leucocytes. In bouillon the organism grows in long
chains; is stained with all the common basic dyes, but is not
colored by Gram's method in cultures. It exhibits a great variety
of involution forms when grown in salty culture media (3^
percent salt).
Relation to Oxygen. Strict aerobe, the growth is stopped by
the exclusion of oxygen.
Vital Requirements. Grows well at 22C., but best at 35C.;
is killed after a short exposure to 55-6oC., stands drying from
four to eight days, and dies in water after a week. In the buried
bodies of man and animals it lives from twenty-two to thirty-eight
clays. Withstands freezing for months, but does not stand light
or chemicals very long.
PLAGUE BACILLUS 163
Cultures. Grows very well on culture media. In bouillon it
thrives abundantly, with a heavy pellicle which produces dependent
stalactites that drop to the bottom of the vessel. On gelatine
plates it grows in small flat colonies, which are gray and trans-
parent, and which do not liquefy the gelatine (Fig. 43). In gela-
tine tubes it forms a faint thread-like line, without liquefying
the media. On agar the growth is whitish and abundant, and
resembles the colon bacillus. Old cultures are luxuriant. Milk
FIG. 43. Colonies of plague bacilli forty-eight hours old. (Kolle and
(Wassermann.)
is not coagulated, but a faint acidity appears. Potato yields a
slow whitish-yellow growth that is sharply outlined.
Chemical Activities. Does not produce H^S, enzyme, colors,
or odors, indol or nitrites. The toxin produced is not soluble and
the nitrate is non-poisonous. Old killed bouillon cultures can
be extracted and a highly poisonous substance precipitated there-
from with alcohol, or ammonium sulphate, that is lethal for mice.
Habitat. Never found in healthy human bodies. In persons
afflicted with plague, the organism is widely distributed in buboes
and in the cutaneous pustules, lymphatics and in the lungs in
plague pneumonia; more rarely in the blood and other organs.
164 BACTERIA
In animals, plague occurs in rats. It is supposed that some
tropical soil bacilli infect rats, and becoming accustomed to the
rodent's body, are eventually transmitted to man. The bacilli
may be transmitted from rat to rat in India by the rat fleas
which also can bite man. The organisms remain in the flea for
some time. Rats are also infected from dead rats. In epidemic
times the soil becomes infected and persons going barefoot may
be infected.
FIG. 44. B. Pestis in pus of bubo. . (Jackson.)
Pathogenesis. Highly pathogenic for man. Is the cause of
the bubonic or Oriental plague; bacilli gain entrance by way of
the skin, causing localized foci of infection from which buboes
develop, followed by pest-sepsis and death. The lungs may be
the original site of invasion, and plague pneumonia (worst form
of the disease) may result. The typical bacilli. can be found in
the sputum of the patient thus affected but not in quietly expired
air. The mortality from this plague is from 50 percent to 80
percent.
Almost all domestic animals rats, mice, guinea pigs, rabbits
and squirrels are susceptible; horses and swine are very suscep-
tible; cows and dogs less so. Rats seem to be affected with a
PLAGUE BACILLUS 165
chronic form of the malady, and by inhabiting ships and ware-
houses in foreign countries, spread the disease. Post mortems
on infected animals reveal haemorrhagic petechia and serous
infiltration into serous cavities. Death is generally due to a
profound toxaemia and exhaustion.
The virulence of the organism can be raised by passing it
through a series of animals.
Serum from infected animals agglutinates plague bacilli.
FIG. 45. Pest bacillus involution forms produced by growing on 3 percent
salt agar. (Kolle and Wassermann.)
The diagnosis of the plague bacilli is made by rubbing the sus-
pected culture upon the freshly shaven skin of a guinea pig; if
the animal develops buboes and dies, and polar staining bacilli
are found, it is probable that the organism is the plague bacillus.
Further, if curious involution forms develop an heavily salted
agar (3 percent) the diagnosis is confirmed (Fig. 45).
Immunity. It is possible to immunize against the disease.
Kitasato and Yersin produced an anti-toxic serum, which has,
not only a prophylactic, but a curative action. By the use of
killed culture Haffkine vaccinated many people against the plague
very successfully (see page 83).
I 66 BACTERIA
MUCOSUS CAPSULATUS GROUP
There is a large group of organisms of moderate pathogenic
powers and importance called variously, Bacterium aerogenes,
Bacterium mucosus or Aerogenes mucosus group of which the
Friedlander bacillus is the most important. They all have a
luxuriant growth on media; are negative to Gram stain; ferment
most of the carbohydrates; are non-motile and most of them
show a capsule when in the animal body.
Perkins divides them as follows:
I. Bacterium aerogenes type ferments all carbohydrates with
gas.
II. Bacterium pneumoniae group ferment all carbohydrates but
lactose, with gas.
III. Bacterium lactis aerogenes group ferment all carbohydrates
except saccharose, with gas.
These organisms are important members of the intestinal flora.
Bacterium Pneumoniae.
Friedlander 's Pneumonia Bacillus.
Morphology and Stains. Short plump rods with rounded ends,
surrounded by a thick gelatinous capsule .in animal fluids, and
when grown in milk; is not motile, and has no spores; does not
stain by Gram's method, but easily by the common basic dyes.
Oxygen Requirements. Grows in and without oxygen, upon
all culture media.
Chemical Activities. Produces abundant acids, COz and H,
gas, alcohol, indol, ferment and H 2 S.
Habitat. Has been found in soil; sometimes in healthy saliva.
Culture Media. Grows luxuriantly on all culture media.
On gelatine it grows in roundish elevated colonies that are
yellowish-white with a slimy lustre, and never liquefies the gela-
tine. In agar it multiplies even more abundantly with a moister
growth. The border of streak cultures is smooth and wavy, and
the water of condensation is cloudy. In bouillon the growth is
MUCOSUS CAPSULATUS GROUP 167
very cloudy with a silvery deposit at the bottom. The bouillon
becomes thickened. Milk is not coagulated, and potato yields
a luxuriant yellowish, moist shining growth.
Pathogenesis. It is possible to cause pneumonia in mice, also
septicaemia. Guinea pigs and dogs are susceptible. It may be
found in normal mouths. Friedlander's pneumonia is much less
frequent than that due to the pneumococcus, but it is very fatal.
Members of this group may also be responsible for cystitis,
pyelitis, sinusitis and in children pneumonia and pleuritis. The
exudate produced by all of them is mucoid, stringy. Anti-bodies
are not readily formed against any of the mucosum group so that
anti-sera and vaccines are not of great value.
The Bacterium lactis aerogenes group is a very large one and
includes nearly all the forms engaged in milk souring. The
ordinary B. lactici is very like the colon bacillus, but is non-motile.
It forms lactic acid among its principal products. The most
important lactic acid producer related to but not belonging
directly in this group, is Bact. bulgaricum of Massol. This is the
principal ferment of the eastern sour milks, Kumyss and Yoghurt.
Because of the large amount of lactic acid formed by this germ,
Metchnikoff has advocated cultures of it and sour milk made
by it in the treatment of intestinal putrefaction and fermentation.
The Bacterium bulgari^um produces a soft milk curd and an excess
of lactic acid and alcohol. The bacteria are non-motile, non-
spore-forming, Gram-positive and vary from 21* to 5oju in length.
They grow with difficulty in the laboratory, best on milk and
whey. Optimum temperature 44C. They form branching fila-
mentous colonies. Milk is coagulated in eighteen hours at 44C.
and in thirty-six hours at 37C. The clot is not dissolved. Gela-
tine is not liquefied. Congeners with this organism are Bac.
acidophilus and Bac. acidophil-aerogenes differing from it in fer-
mentative powers.
1
1 68 BACTERIA
TYPHOID BACILLUS
Bacterium Typhi. Eberth.
Bacillus Typhosus
Typhoid Bacillus (Fig. 46).
A most important pathogenic organism which causes typhoid
fever.
Morphology and Stains. Generally short plump rods i to 3/4
long, and .6 to .8/x broad. Forms long threads in cultures,
especially on potatoes. Polar metachromatic
bodies are sometimes seen as are unstained
areas when alkaline methylene blue is used.
The rod is flagellated (peritrichous) ; con-
tains no spores; exhibits pleomorphic and
involution forms; is actively motile, and
stains with all the basic aniline dyes, but
ella. (Kolle and Vital Resistance. The thermal death-
Wassermann.) point ig 6Q o c ^ ten tQ fifteen minutes> Re _
mains alive in ice for three months; even the temperature of
liquid air does not destroy it at once. In distilled water it lives
for months, but if other saprophytic bacteria are associated
with it, however, it quickly dies. Does not resist drying or
chemicals, except carbolic acid, towards which it exhibits a
tolerance. Sunlight kills it in an hour.
Habitat. It never exists in nature, except where water or soil
has been contaminated by fasces or urine. It may multiply in
potable waters, in milk, and the juices of oysters.
Chemical Activities. Does not produce proteolytic enzymes;
forms H 2 S, but will not ferment the sugars with gas formation.
Does not yield indol or nitrites. Produces levorotatory lactic
acid. Its toxin is all contained within the bacterial cell (endo-
toxins) and is not water-soluble. This toxin is manifested by
injecting washed and killed bacilli into animals, or by freezing the
TYPHOID BACILLUS
169
bacilli with liquid air, and then crushing them. This injected
into guinea pigs causes diarrhoea, mydriasis and death.
Oxygen Requirements. It is a facultative aerobe.
Cultural Characteristics. It grows upon all media at the tem-
perature of the body, 37C. and more slowly at 2oC. On gela-
tine plate it produces at first small colonies, yellowish and punctate,
which become whitish, delicately notched and ridged (Fig.
47). In gelatine stab culture it grows in a thread-like granular
FIG. 47. Seventy-two hour old culture of typhoid bacillus on gelatine.
(Kolle and Wassermann.)
line, without producing gas. In neither case is the gelatine lique-
fied. On agar plates the colonies are not so characteristic, being
round, grayish-white, and shining. In milk it grows well, not
coagulating it even after boiling, and only a very little acid is
produced. On acid potato the growth is characterized by its
invisibility, and this fact is used to differentiate it from other
kindred bacteria. The growth is detected only by scratching
with a needle. In bouillon it grows uniformly, producing very
little acid, and no gas. In special media (Hiss's semi-solid media)
thread-like colonies are produced, which are characteristic. On
Eisner's potato media it produces small granular, glistening
1 70 BACTERIA
points. It also grows characteristically in Endo and the Drigalski
and Conradi media. In sugar media no gas is formed but there
is acidification in dextrose, galactose, mannit, maltose, xylose and
levulose. The addition of sterile bile to culture medium, 10-50
percent, increases the chance of isolating the germ in blood, faeces
or urine cultures; it acts by inhibiting other organisms and by
supplying salts favorable to the typhoid bacillus.
Invasion of Body. This organism generally invades the body
by way of the alimentary tract, in food and water. Flies may
infect milk and other foods. Oysters may become infected and
cause disease. Personal contact, by hand to hand for example,
is a very potent method of transmission.
Pathogenesis. It is certainly the cause of typhoid fever.
During the attack the germs circulate in the blood-stream during
the entire fastigium but are obtained with ease only in the first
two weeks. They are constantly in the faeces for varying periods
even after clinical recovery and may be frequently found in the
urine. Rose spots also contain them. Also found in the spleen
and gall-bladder. It produces well-marked histological changes
in the lymphoid structures, particularly in Peyer's patches,
solitary foUicles, and other lymph-glands. There is, according
to Mallory, a massive endothelial proliferation in the lymph-
glands. This causes occlusion of the lymph-vessels, and is fol-
lowed by necrosis (ulceration) of the Peyer's patches. The in-
tense phagocytic action of the fixed lymphatic cells in the glands
is manifest toward the red blood cells, which are devoured in
great , numbers. The toxin causes degeneration of other organs,
particularly in the liver. Bacilli are found in the spleen and
blood. The rose-colored spots are found to be full of them.
The disease is certainly not a merely localized infection of the
lymph structures, but is a bacteriaemia. There is often a mixed
infection in which streptococcus pyogenes in the blood plays an
active role. In the necrosis of bone and in subphrenic abscess
the typhoid bacilli may act as a pus former. Commonly it
TYPHOID BACILLUS
171
produces death by (i) profound toxaemia; (2) ulceration of the
Peyer's patches, causing perforation and peritonitis; (3) by the
destruction of a blood-vessel in the floor of an ulcer producing a
haemorrhage.
In animals, as a rule, typhoid bacilli if injected, produce no
disease, and the bacilli rapidly die. In chimpanzees, how-
ever, it is possible to produce atypical typhoid lesions and
symptoms.
Natural and Acquired Immunity. Human blood serum is
strongly bactericidal toward the typhoid bacillus. Normal gas-
tric juice, with its hydrochloric acid, destroys
the bacillus when ingested and this forms the
natural means of protection. Immunity fol-
lowing an attack of typhoid is generally of
long duration. If bacilli do reach the blood-
stream of an immune individual, the ambo-
ceptors originated by a previous infection,
together with the complement normally pres-
ent, effect a solution of the invading organism.
For vaccination against typhoid fever see
page 80. Anti-serum for typhoid has been
prepared by injecting horses with killed
culture of typhoid bacilli, but it has not
proved to be effective.
Agglutinations. One of the most important means of diagnos-
ing typhoid fever is by the so-called Widal test, really the Gruber
and Durham agglutination reaction. This consists in applying
the serum of the blood of a person, supposedly ill with typhoid,
to a fresh bouillon culture of typhoid bacilli. If the person has
the disease, and it has lasted for five or more days, the bacilli are
promptly agglutinated in clumps. Undiluted normal serum, and
serum from people suffering other diseases, will bring about the
same reaction at times; it is therefore best to dilute the serum
with water 1-50, and if the reaction comes within an hour the
FIG. 48 Widal
reaction. One-half
of the field shows
typhoid bacilli un-
clumped, other half
shows clumping.
(Greene's Medical
Diagnosis.)
172 BACTERIA
disease is considered typhoid fever. The test may be either with
a hanging drop and examined microscopically, or macroscopically
by adding a drop of diluted serum to fresh bouillon culture of
typhoid bacilli, when, if the case is typhoid, large clumps of the
bacilli will form and drop to the bottom of the tube. Animals
immunized against typhoid exhibit this reaction to a high degree.
Serum diluted with 10,000 parts of water has caused the reaction
in less than one hour's time. This reaction with a known culture
of typhoid bacilli is used clinically to identify serum from a doubt-
ful case of typhoid, and establish a diagnosis. On the other hand,
a known serum prepared artificially by immunizing rabbits with
bacilli is used to identify typhoid bacilli when found in water,
or elsewhere. There are two stages to the reaction; immediately
after mixing the serum and culture, the bacilli will be seen to
become less motile, and then still. After this they begin to
huddle together into clumps. In complete reaction they remain
immobile and tightly massed. In some cases bacteriolysis occurs,
and many of the bacteria are dissolved in the serum. The foetus
of a woman suffering from typhoid contains agglutinins in its
blood. The milk, tears, and other body fluids from an individial
with typhoid, agglutinate typhoid bacilli. Serum to perform the
test may be obtained by puncturing the skin, or by blistering it
and drawing off the serum, or else by abstracting blood from a
vein with a hypodermic syringe.
Agglutinin appears during typhoid, generally after the fifth day,
and persists for some time (several years?) after convalescence, 01
if the actual agglutinin titer be not high in an immune person, il
rises rapidly should typhoid bacilli gain entrance to the body.
In persons vaccinated against the germ, the agglutinin titei
remains partly high for at least six months but when the amounl
shall have returned to normal, it has the power to revive rapidb
should infection threaten. Agglutinins are in a measure ai
index of resistance, or at least their facility of action is 01
guarantee of protection.
PARATYPHOID BACILLUS 173
Paratyphoid Bacillus. A pathogenic organism producing all
the clinical symptoms of typhoid, only in milder form (at times)
has been discovered. It differs from the true bacillus because it
ferments dextrose and maltose producing gas and acid, and is not
agglutinated by the serum from a true typhoidal infection.
There are several closely related varieties differing in growth upon
litmus milk and in fermenting several sugars but in other respects
they resemble the typhoid bacillus, and seem to occupy a position
between it and the colon bacillus. Paratyphoid endotoxin resists
6oC. from thirty to sixty minutes and in the case of the organisms
of meat poisoning, paratyphoid beta, paracolon and the Gartner
bacillus, a short exposure to the boiling point does not seem to
destroy the toxin.
It is generally taught today that the foregoing organisms
produce infections of similar clinical characters in that they are
contracted in the same manner, have comparable pathology
and immunity reactions and are amenable to the same pro-
phylactic measures; they are designated "the typhoid fevers."
The Paracolons are organisms like the paratyphoids, but some-
what closer to the colon bacillus (for example, see page 177).
This term is best applied to organisms of the meat poisoning
group, as the Gartner bacillus, the hog cholera bacillus, so that
the varieties which cause typhoid fever in man can be recognized
under the term paratyphoid.
Blood cultures are often employed in large hospitals for the
diagnosis of typhoid fever. During the first week of the attack
bacilli may be recovered from the blood by withdrawing 10 c.c.
of blood from a vein and mixing it with 500 c.c. of bouillon. The
large amount of blood is necessary, because the bacilli are few in
number, and the bactericidal action of the serum outside the
body is powerful until mixed with the bouillon, after which the
bacilli are able to withstand it. The bacilli may be easily isolated
from the blood by adding the latter to some bile and then incubat-
ing it. From the bile, cultures are made in agar or in bouillon.
1 74 BACTERIA
COLON BACILLUS
Bacterium Coli.
Bacillus coll or Bacillus coll communis.
Colon Bacillus.
While not strictly a pathogenic organism, it plays such an im-
portant part in secondary infection, and resembles so closely the
typhoid bacillus, that it will be described here.
Morphology and Stains. Is not so motile as typhoid; has not
so many flagella; and is devoid of spores. It exhibits pleomor-
phism; may grow in chains; and possesses vacuoles and polar
FIG. 49. Colon bacillus showing flagella. (Kolle and Wassermann.)
bodies at times. Is readily stained by all the common basic
stains, but not by Gram's method.
Oxygen Requirements. It grows especially well in oxygen;
without oxygen its growth is not so good.
Temperature requirements, and vital resistance. It grows well
at room and incubator temperature. Its thermal death-point is
about 62C.; light and heat are destructive to it, and its resis-
tance to antiseptics is somewhat less than that of typhoid bacillus.
COLON BACILLUS 175
Cultures. Thrives in all common culture media, especially if
sugar is present. It is restrained by excess of acids produced in
culture media. On gelatine it grows like the typhoid bacillus
(from which it is difficult to differentiate, see page 176) in whitish
raised colonies that do not liquefy the media. Sometimes the
growth is thin and iridescent, and exhibits bizarre shapes
tadpole-like and lobulated. Typhoid colonies show deep furrow-
like ridges under the microscope. In the special semi-solid media
of Hiss, the typhoid produces uniform cloudiness, with thread-
like colonies. The colon does not so quickly cause this cloudi-
ness, and forms gas bubbles. On agar plates surface colonies are
like typhoid, only they are thicker and moister. If litmus is
added to this medium, a red zone forms about the colonies due
to the presence of lactic acid. In agar tubes the growth is more
luxuriant and resembles typhoid. In litmus bouillon it rapidly
reddens the litmus, clouds the medium, and deposits a slimy
sediment. In milk it always produces coagulation. On potato
it grows more rapidly and luxuriantly than typhoid, at first
yellowish-white, which later changes to yellowish-brown. It is
slimy.
Chemical Activities. Produces color on potato only. Sugars
are fermented with the production of H, COg and some N.
It ferments glucose, lactose, saccharose, maltose, dulcit and
some others with the production of gas. Produces lactic, acetic
and formic acids, also indol abundantly, and H 2 S. It decomposes
urea.
Habitat. Found always in the intestinal contents of most ani-
mals and man. Also in streams and rivers that run through farm
lands and by towns. While it is difficult to find typhoid bacilli
in contaminated drinking water, the colon bacilli are easily
found. If in abundance, it indicates great faecal pollution. In
milk it is often found, where it plays an important part in souring.
Pathogenesis. It is pathogenic to rabbits and guinea pigs,
causing peritonitis if injected into the peritoneal cavity. In man
176 BACTERIA
it plays rather a subordinate pathogenic role, but it has been
found the causal agent of some cases of suppurative appendicitis,
peritonitis, and cystitis. It may attack the lungs and meninges
of feeble children, and cause death by setting up a pneumonia or
meningitis. During the agonal period in wasting diseases it may
cause terminal infection and hasten death. Colon bacilli encysted
in the liver and kidney have been found by Adami in cirrhosis of
these organs, and it is believed by him to be partly the cause of
these diseases; chronic infections of the rectum are due to this
organism.
Agglutination. Animals immunized against colon bacilli by
repeated injections, exhibit agglutinins in their blood.
The differentiation of the typhoid from the colon bacillus is
largely accomplished by noting the chemical reactions of both
organisms in culture media. The chief differences are :
1. The typhoid bacillus has more flagella than the colon, and is
much more motile.
2. On gelatine culture plates, the typhoid colonies develop
more slowly than the colon, and are much more delicate and trans-
parent. If litmus is present the colon colonies are red, the
typhoid bluish.
3. In media containing dextrose, or lactose, gas is produced by
the colon, but not by typhoid.
4. In peptone solution the colon produces indol, while the
typhoid does not.
5. Milk is coagulated by the colon, but not by the typhoid.
6. Dn potatoes colon grows much more luxuriantly than typhoid.
7. Typhoid reddens neutral red; colon changes it to bright
yellow.
8. The m.ost important test is the agglutinative one. Typhoid
is clumped by anti-typhoid sera, highly diluted, while the colon
is not.
No anti-sera of value have been found for colon bacillus infec-
tion, but bacterins have been used with much benefit.
GARTNER'S BACILLUS 177
GARTNER'S BACILLUS
Bacillus Enteritidis.
Bacillus of Gartner.
The cause of one form of meat poisoning, and closely allied to
the paratyphoid bacillus in its morphological characteristics. It
gives a classical picture of the type "paracolon."
Morphology and Stains. This organism is a short plump
ovoid; is motile; has about eight flagella; does not form spores;
and stains well with all the common aniline dyes, but not with
Gram's method.
Vital Resistance. It is a facultative anaerobe. It is destroyed
by means outlined for the colon bacillus when in culture. In
meat it must be subjected to prolonged heating.
Cultures. Grows on all the common culture media. In
bouillon thrives well, producing gas in media containing dextrose.
It ferments without gas production lactose, galactose, maltose,
and cane sugar. Does not produce indol, which distinguishes
it from the colon bacillus, to which it is closely allied. In milk
it reduces litmus and coagulates the casein in a few days. On
potato it grows well, producing a yellowish shining layer. On
gelatine it multiplies without liquefying the medium. Super-
ficial colonies in plates are pale and gray, deep colonies yellow
and spherical.
Chemical Activities. Acid, gas and a powerful heat-resisting
toxin which is soluble, are found. Infected meat contains this
toxin, which is not destroyed by cooking.
Pathogenesis. It is pathogenic for man, horses, cattle, and
laboratory animals. Neither the bacilli nor the toxin they elabo-
rate are destroyed by moderate heat. Flesh is infected before
death, after which, both the bacilli and toxin increase. Mischief
follows the partaking (usually in the form of sausages, etc.) of
this meat, causing, in men, violent nausea and diarrhoea, skin
eruption, and in severe cases, pneumonia, nephritis, collapse and
12
178 BACTERIA
death. Mortality is from 2 percent to 15 percent. The post-
mortem findings are not specific. There may be evidence of an
enteritis with swollen lymph follicles, and an enlarged spleen.
Agglutination. The blood of infected individuals may agglu-
tinate bacilli. A dilution of such blood with 8, ooo parts of water
has produced the reaction.
No anti-serum or bacterin treatment is as yet possible.
DYSENTERY BACILLUS
Bacterium Dysenteriae.
Dysentery Bacillus.
The cause of one form of tropical dysentery. The group to
which this belongs comprises many closely related varieties some
of which are thought to be the cause of infant diarrhoea in this
country. There are numerous varieties of this organism, the
differentiation of which depend upon their chemical activities,
fermentation of various carbohydrates being the most important,
and agglutinative properties with different sera. The tropi-
cal form of dysentery is due to the type orginally described by
Shiga; this type is uncommon in temperate zones, the Flexner
variety being much more common. The Shiga variety is much
the more virulent.
Morphology and Stains. The organism is, in many respects,
similiar to the typhoid bacillus, but is plumper. It is non-motile,
has no spores, and exhibits pleomorphism. It stains well with
the common aniline dyes, but not by Gram's method.
Vital Properties. It is killed by i percent carbolic solution
in thirty minutes. Lives for twelve to seventeen days when
dried. Direct sunlight kills it in thirty minutes. Its thermal
death -point is 58C. in thirty minutes. It is a facultative aerobe;
grows at ordinary temperature, but better at 37C.
Cultures. Grows on all the common culture media, but more
slowly than the colon bacilli. Gelatine cultures resemble typhoid.
The growth in this media (which it does not liquefy) produces no
DYSENTERY BACILLUS 179
pellicle, but a sediment. Indol is not produced, and milk is first
mildly acid and then faintly alkaline, though not coagulated. On
potato it grows sparingly, often turning it brown. The Shiga
type ferments glucose, but no other sugar. The Flexner type
ferments glucose, dextrine, and mannite, but not lactose. The
latter type produces more acid than the former, and both are
best agglutinated with their corresponding serums.
Habitat. In living bodies the organism is found solely in
mucous discharges from the bowels. In the dead it is found in
the lymph-glands. If it reaches the circulation, it appears to be
rapidly destroyed by the blood. It has been discovered, however,
in the body of a foetus delivered from a woman with the disease.
The organism must have passed the placenta of the mother. The
disease is spread by water, food and personal contact and by
carriers, and it may become epidemic in large institutions.
Pathogenesis. The typical lesions caused by the organism
vary from a mere hyperaemia to a superficial necrosis of the lym-
phoid structures, which may be extensive. Peyer's patches are
slightly swollen but not ulcerated. The descending colon and
sigmoid are oftenest attacked. The necrotic masses separate,
leaving shallow ulcers. The lymph structures are engorged with
polynuclear leucocytes. No marked lesion is found in the
spleen. The liver and kidneys often undergo marked parenchy-
matous degeneration. The bacilli being possessed of a powerful
endotoxin, so that dead cultures, if injected under the skin cause
marked local and general reactions. Like the pyocyaneus bacillus,
this organism undergoes auto-digestion in bouillon, which leaves
the latter highly toxic owing to the liberation of the toxins.
Laboratory animals quickly succumb to injection of this organism,
injection producing a marked reaction in the colon, a phenomenon
suggesting that there is a predilection for the organ and that the
body uses it as an excretory organ for the poison. Dysentery
cannot be induced in animals by feeding cultures. Poorly
nourished subjects are easily infected and quickly die. Digestive
l8o BACTERIA
disorders favor infection. Death may be due to toxaemiaor ex
haustion. As a causal agent in the production of summer diarr-
hoeas of children, the dysentery bacillus plays a part, it has
been isolated from the stools of infants, with this disease, and
their sera have been found to agglutinate the bacilli. Never-
theless it is known that other bacteria (streptococci, etc.) cause
this disease, and Weaver found that "clinically twenty-four of
our ninety-seven cases of ileocolitis in which dysentery bacilli
were discovered did not differ from cases in which dysentery
bacilli were not found.
Immunity. The sera from convalescents from dysentery show
a strong bactericidal action. Anti-bodies are developed by in-
fection and by artificial inoculation with killed cultures. Kruse
obtained a serum from horses which strongly protected a guinea
pig against a lethal injection of bacilli. The protective property
of the serum is due to its bactericidal action. Here the ambo-
ceptors act, but only in the presence of a complement. It is
possible that a small amount of anti-toxin is present since there is
some reason to think that a modicum of free toxins is produced
by the bacilli.
Vaccination. Shiga tried to induce (i) passive and (2) active
immunity in many individuals by injecting both anti-toxic serum
and bacteria into them. This was not followed by a lowered
number of infections, but by a lowered mortality. A serum may
be produced by injecting horses with several dysentery strains,
called a polyvalent anti-serum. This has good therapeutic effects
but does not immunize prophylactically.
Agglutination. The serum from a patient suffering from either
dysentery or summer diarrhoea, will, after about a week's illness,
agglutinate bacilli. This property is not always present, and
its absence does not exclude the possibility of infection. In
performing the reaction, both Shiga's and Flexner's type of
organism should be used. These types probably bear the same
relation to each other that typhoid and paratyphoid do.
PYOCYANEUS BACILLUS l8l
PYOCYANEUS BACILLUS
Bacterium pyocyaneus.
Bacillus Pyocyaneus (Fig. 50).
Bacillus of Blue Pus. Also called Pseudomonas pyocyanea.
An organism of minor importance as a pathogenic agent, that
is often met with in groin or axilla.
Morphology and Stains. Slender rods, often growing into
thread-like forms. Exhibits pleomorphism. Sometimes is
rounded and cocci-like, is motile, has a polar flagellum, and
FIG. 50. Bacillus pyocyaneus. ^(Kolle and Wassermann.)
no spores. Stains with all the basic aniline dyes, but not with
Gram's method.
Oxygen Requirements. Usually a strict aerobe.
Cultures. Grows on all the common culture media luxuriantly,
at room and incubator temperatures. It elaborates two pig-
ments, a water-soluble greenish bacteriofluorescein, and a chloro-
form soluble pigment, a beautiful blue in color, called pyocyanin.
On gelatine plates it produces yellowish-white to greenish, yellow
colonies which liquefy the gelatine, causing crater-like excavations
about the colonies. Gelatine stab cultures rapidly liquefy along
l82 BACTERIA
the line of inoculation, coloring the gelatine greenish-blue, and a
white crumbly deposit forms in the bottom of the stab. On agar
plates it produces yellowish-white colonies, surrounded by a zone
of bluish-green fluorescence. It grows luxuriantly. In agar
tubes it multiplies rapidly, spreading over the medium, with
wavy thickened edges. The agar quickly turns a dark greenish-
blue, and in old cultures the growth changes from yellow to
greenish-blue.
In bouillon it is very dense and yellowish-green; a pellicle forms
on the surface, and a sediment is deposited. In old bouillon cul-
tures the bacilli undergo autolysis and disappear. In milk the
growth is luxuriant, the casein is coagulated, and the clot is ulti-
mately digested. The reaction is alkaline. On potato it varies in
luxuriance, often being slightly elevated, yellowish,turning to green.
The variance in growth is due to the kind of potato used. Drying
kills the organism speedily; four hours in sunlight also destroys it.
Chemical Activities. No gas is generated. Besides the pig-
ments (already specified) ammonia is produced, also a peculiar
enzyme called pyocyanase by Emmerich and Lowe, which not
only digests gelatine and milk curd, but its own and other bac-
terial cells as well. Old cultures are poisonous; a haemolysin is
produced an endo-toxin, and a soluble toxin. The last-named
toxin stands a temperature of iooC. Against the endo-toxin
and the soluble toxin it is possible to prepare an anti-serum. This
may protect laboratory animals.
Pathogenesis. Has been found a sole cause of meningitis and
vegetative endocarditis in man; is a pyogenic organism; can cause
suppuration anywhere in the body; produces blue pus; is patho-
genic to guinea pigs; and its virulence can be raised by passing
it through a series of animals.
Agglutination. The serum of infected and immunized animals
both in moderate dilution causes agglutination of bacilli. It is
possible to use bacterins of this germ. Bactericidal substances
develop by the use of killed cultures.
BACILLUS OF SOFT CHANCRE 183
BACILLUS OF SOFT CHANCRE
Bacterium Ulceris Chancrosi (Ducrey).
Streptobacillus of Soft Chancre.
Morphology and Stains. A small thin bacterium .5;* broad,
1.5/1 long, growing in chains with polar staining, which can be
demonstrated in sections of chancroids without much difficulty.
This organism does not stain by Gram's method, but by Lofflers
it is stained with ease.
Cultures are hard to make. It grows best in serum agar, and
blood agar in faint colonies that are not very characteristic. In
condensation water of agar it grows feebly.
In sections and in pus the organism is frequently found in the
interior of leucocytes.
By aspirating pus from buboes and planting it on old but moist
blood agar plates, cultures may be obtained.
Pathogenesis. From an old culture of over ten generations
typical ulcerations were produced in man. The organism is feeble
and quickly dies in culture media or in contact with mild
antiseptics.
ANTHRAX BACILLUS
Bacillus Anthracis.
Anthrax Bacillus of Koch (Fig. 51).
Practically the first pathogenic organism to be isolated. This
was accomplished by Dr. Robert Koch. It is the cause of a wide-
spread malignant disease, variously called Anthrax, Charbon, or
Splenic Fever. Animals and man are infected by it, and its action
is often rapidly fatal.
Morphology and Stains. In animal tissues this organism ap-
pears as a large rod 3-10^ long, and 1-1.2;* wide. Is of ten in pairs
or chains. In fresh specimens the ends of the rods are rounded;
when older, the ends become square or concave. Often they have
faint capsule surrounding them. In culture media they exhibit
1 84
BACTERIA
spores and grow in long threads, these threads form long spirally
twisted masses, like locks of wavy hair. No flagella are formed,
and the organism is not motile. In old cultures, bizarre involu-
tion forms are found. It stains well with
all the common basic dyes and by Gram's
method.
Oxygen Requirements. Is a facultative
anaerobe, but grows much better in the
presence of oxygen. If oxygen is excluded,
no liquefaction occurs.
Temperature. Grows between i4C. and
45C.; best at 37C. Spores are formed, if
oxygen is present, between i5C. and 4oC.
Sporulation is more rapid at 37C. Spores
withstand high temperature (dry) for a long time, iooC. for one
hour. The bacillus itself is killed at 7oC., moist heat, in one
. FIG. 51. Anth-
rax bacilli in blood.
(Greene's Medical
Diagnosis.)
FIG. 52. Anthrax bacilli growing in a chain and exhibiting spores,
and Wassermann.)
(Kolle
minute. The thermal death-point may be put down for the
organism, at iooC. steam, for five minutes.
ANTHRAX BACILLUS 185
Vital Resistance. Highly resistant to chemicals, light and dry-
ing. Spores resist 5 percent carbolic solution for days (Esmarch),
but i-iooo corrosive sublimate for only a few hours. They also
resist formaldehyde and sulphur for a long time, and withstand
light. A 2 percent fresh solution of H 2 O2 kills spores in three
hours. Three and one-half hours' exposure to bright sunlight
killed the spores if oxygen was not excluded (Dieudonne) (Fig. 52).
Sporulation Phenomena. At i2C. spores are formed if oxy-
gen is present. The most favorable temperature for sporulation
is that of the body (37C.). Spores are never found in the bodies
of living or dead animals if they remain unopened, and oxygen
is excluded. If bacilli are cultivated at 42C. for a long time and
frequently reinoculated, on fresh media, the ability to form spores
is lost even if grown again at 3oC. (Phisalix). If cultivated
upon media containing carbolic acid and hydrochloric acid, the
ability to sporulate may be lost.
Chemical Activities. Acetic acid is formed, as is H^S. Lique-
fying, milk coagulating, and milk digesting enzymes are formed.
Toxins have not been isolated, but may be produced.
Habitat. Only found where infected animals, hides, and hair
have been. Fields, hay, bristles, hides, manure, etc., have been
found to contain bacilli. Drinking water may be polluted by tan-
neries and the bodies of dead animals. Meadows and fields may
be contaminated for years. From the buried bodies of infected
animals anthrax spores may be brought to the top of the soil by
earth-worms.
Cultures. Grows exceedingly well on all culture media in the
air. On gelatine it grows in whitish round colonies, rapidly sink-
ing into the gelatine, due to the liquefaction. The liquid medium
is turbid. The interior of the colony is crumbly. When magni-
fied, the colonies seem to be made up of tangled waving bundles,
like locks of hair, especially about the periphery. In gelatine stab
cultures the growth is luxuriant and rapid; the medium is liquefied
more rapidly at the top, and finally a crater is formed; before this
1 86 BACTERIA
appears, lateral hair-like outgrowths are seen in the gelatine. At
the bottom of the crater a white crumbly mass is formed, but no
pellicle. On agar plates, small whitish colonies develop which
are elevated and round. When magnified, wavy hair-like growths
appear on the edge, caused by many twisted parallel chains of
bacilli (Fig. 53).
In agar stab, the growth is more luxuriant near the top; lateral
filamentous branches are seen along the stab line. In agar
streak the colonies are abundant, thick and fatty; have tangled
edges, and the water of condensation is cloudy. In bouillon, it
FIG. 53. Anthrax bacilli. Cover-glass has been pressed on a colony and
then fixed and stained. (Kolle and Wassermann.)
forms homogeneous flocculi, which precipitate, leaving the bouillon
clear. A fragile pellicle is formed. In milk, it multiplies rapidly,
the proteids are coagulated, generally rendered acid, and later the
coagulum is dissolved. Potato cultures are likewise luxuriant.
The growth is elevated, dull in lustre, and the outline is wavy.
Pathogenesis. The anthrax bacillus increases so rapidly, and
so luxuriantly, that it has been supposed to cause death merely
by mechanically overwhelming the animal : absorbing nutriment
and oxygen, and blocking capillaries. Its action is certainly not
ANTHRAX BACILLUS 187
purely toxic, as it causes, not a toxaemia, but a bacteriaemia. It is
especially virulent for man, sheep, cattle, goats, rabbits, guinea
pigs, mules, and horses. Rats rarely succumb. Pigeons,
chickens, and dogs are immune. If frogs are kept at a tempera-
ture of 3oC. they become susceptible to infection. At their
normal temperature they are immune. The disease produced by
this organism is known variously in different countries as Anthrax,
Splenic fever, Woolsorter's disease, Malignant pustule, and Char-
bon. It frequently devastates vast herds of sheep, cattle, and
goats, and is often a pestilence in European countries, China, and
South America. It appears sporadically in the United States.
Its origin in this country can usually be traced to infection from
hides or hair imported from abroad. The disease has been
contracted from using shaving brushes made of insufficiently
sterilized bristles. In man it is frequently fatal. The infection is
first manifest as* a small carbuncle or pustule, from this, rapid
general infection, as a rule, ensues. In man and animals anthrax
bacilli may be transmitted from mother to foetus via the placenta.
The organism is found in enormous numbers in infected bodies,
investing all the organs and the blood. Pus is produced by
necrosis of tissue. Infection is accompanied by a high leucocyto-
sis and fever. There is often congestion of the lungs; also an
intense friability of the splenic pulp, and all the glands of the body
become enlarged, and, at times, many of them suppurate. In
woolsorter's disease, the bacilli are inhaled, and lung lesions
result.
Immunity. It is possible to immunize animals against infection
with anthrax by means of vaccines. By this means the lives of
many thousands of domestic animals have been saved. The vac-
cines are made by growing the bacillus at 42C. for various lengths
of time to attenuate them. An anti-serum has been produced by
repeated injection of toxins and of sporeless rods. It is used
locally around a pustule, and in doses of 50-100 c.c. intravenously.
It is anti-toxic and seems to stimulate phagocytosis.
I 88 BACTERIA
TETANUS BACILLUS
Bacillus Tetani.
Tetanus Bacillus (Fig. 54).
Lockjaw Bacillus.
First seen by Nicolaier, and isolated in pure culture by Kitasato.
Morophology and Stains. Rod-shaped. Varying from i.2{j, in
length, to very long threads of 20 to 40;*. Sometimes grow in
FIG. 54. Tetanus bacilli showing end spores. (Kolle and Wassermann.)
chains; frequently appear like short drum-sticks with a spore at one
end, which is either round or oval. At times, the bacilli in chains
sporulate. The organism is motile; possesses numerous flagella
(from 50 to 100) peritrichously arranged; stains well with all the
common basic aniline dyes, and retains the color in Gram's
method (Fig. 55).
Oxygen Requirements. Strictly anaerobic when freshly iso-
lated from earth or wounds, but, after long cultivation on culture
media, it becomes more tolerant to small amounts of oxygen.
Temperature. Grows best at 37C. Below i4C. not at all.
Vital Resistance. Spores resist 8oC. for an hour. This fact
TETANUS BACILLUS 189
enabled Kitasato to kill all other organisms, except their spores, in
pus. Six days' exposure to direct sunlight is needed to kill the
spores. The thermal death-point is best considered as iooC.
for one hour. They are killed in two hours by 5 percent phenol
+ .5 percent HC1 and in thirty minutes by i-iooo HgCl 2
-f .5 percent HC1.
Chemical Activities. Ferments sugar; produces gas, indol,
alkali, and H^S. which gives to the culture an odor of burnt garlic
or onion; marsh gas, CO 2 , and nitrogen are produced. Gelatine is
liquefied. The most important product of growth is the highly
poisonous complex toxin, which is made up of tetanolysin, and
tetanospasmin; the latter has a great affinity for nerve tissues.
This toxin is soluble in water, and can be separated from it by
means of ammonia sulphate.
Habitat. Is found in garden soil, hay, manure, and dust.
Has been found in cobwebs, on weapons, in cartridges, and in the
faeces of man and of animals. It has been isolated from bronchi in
a case of rheumatic tetanus in which there was no lesion in the
body (Carbon and Perrors). In disease it is found in the infected
wound, generally in a deeply punctured one, which is usually puru-
lent and contains but few bacilli. Puerperal tetanus, and tetanus
of the new-born, are but varieties of the disease, dependent upon
the site of infection whether of the placenta or umbilical cord.
Tetanus sometimes occurs spontaneously, without a sign of injury
anywhere. Sheep and goats are susceptible to infection, so. are
guinea pigs and rabbits. Horses are peculiarly susceptible. Soil,
or manure, getting into wounds, is often a cause of tetanus. Cow-
dung poultices, mud dressings, or cobweb applications to stop
haemorrhages, have also caused the disease. Tetanus following
vaccination may be due to infected virus, the latter becoming in-
fected from the faeces of the vaccine-producing cows but more
commonly is due to dirt getting into vaccination wounds.
Cultures. This organism is difficult to grow, and always
requires an atmosphere of hydrogen.
I 90 BACTERIA
/
On gelatine plates, the colonies appear first as minute white
specks, which slowly liquefy the medium. As it grows, hair-like
threads branch out into the medium, and the colony resembles
the periphery of a chestnut burr; later, the white appearance
changes to yellow. In gelatine stab the growth is, at first,
whitish along the line of the needle, eventually the gelatine
becomes liquid, and a bubble of gas, partly filled with whitish-
cloudy liquid gelatine, appears. On agar plates the colonies are
ragged, and are surrounded by delicate out-spreading filaments.
In deep stab culture, down in the agar and remote from the top,
a spreading tree-like form appears, with spike-like growths in the
agar. Blood serum is sometimes liquefied. Bouillon is uniformly
clouded, gas is generated if sugar is present, and toxin is produced.
Milk is, generally, not coagulated.
All cultures of tetanus must be grown under an atmosphere of
hydrogen in media, from which all free
oxygen has been driven by boiling, or else
abstracted by a mixture of pyrogallic acid
and sodium hydrate. It is possible to culti-
vate the organism under mica covering, or
paraffine poured upon freshly boiled media.
If sterile glass tubing is filled, with agar or
JT IG rr Tetanus g e l atme > an d inoculated with tetanus bacilli,
bacilli showing peri- then sealed, colonies will develop, as perfect
ril?u. U S w gdla ' anaerobic conditions are thus obtained.
(Kolle and Wasser-
mann.) Often the organism grows best m the pres-
ence of saprophytic ones. Strongly patho-
genic organisms do not grow well in culture media, while com-
paratively non- virulent ones grow very well.
Pathogenesis. Tetanus may follow any wound, no matter how
insignificant, though deeply punctured ones, caused by nails or
splinters, are more often followed by tetanus infection, especially
if the puncture is sealed by blood clots or pus, and so creating an
anaerobic condition necessary for growth. If the wound is on the
TETANUS BACILLUS IQI
face or hand, tetanus symptoms more quickly supervene, while if
the wound is on the foot, these are apt to be delayed. The sooner
the symptoms appear after the reception of the injury, the more
likely will the disease be virulent and fatal. If spores are washed
free from toxin, according to Viallard and Rouget, and then injected
into a susceptible animal, they do not cause tetanus, but are taken
up by the phagocytes. In other words, the rods not the spores
produce toxin. Necrotic tissue in wounds favors infection with
tetanus, since it helps to fulfil anaerobic conditions, and in some
way hinders phagocytosis. Aerobic bacteria favor tetanus infec-
tion by absorbing the free oxygen which prevents the growth of
tetanus organisms. Free oxygen never kills the organism or its
spores, but merely prevents their development. Wounds that
have, apparently, healed, may be the origin of tetanus. The toxin
is produced rapidly in wounds, or what is more likely, some is
introduced with the bacilli and other dirt. Kitasato found, in the
case of mice, that if bacilli were introduced in the skin, near the
tail, and in an hour the whole area was excised, and the wound
cauterized, fatal tetanus nevertheless supervened. Rheumatic
tetanus follows pulmonary infection.
As related in the chapter on toxins, the mode of disease produc-
tion is as follows : The toxin is conveyed from the wound by
means of the motor nerves to the central nervous system affect-
ing the motor elements. It causes microscopic degeneration of
the fibers and cells of the motor apparatus. Death is caused
either by a spasm of the glottis or diaphragm, or by cardiac
failure and exhaustion. A local manifestation merely affecting
certain groups of muscles may occur. Laking of the blood by
tetanolysin found in the bodies dead from tetanus is a well-
known phenomenon. In fatal cases, toxin may be demonstrated
in the bladder by injecting the urine into mice, causing in them
tetanic symptoms. Various groups of muscles are affected in
tetanic seizures. The muscles of the jaw, if affected, cause
trismus; if those of the back are involved the individual suffers
I 92 BACTERIA
from opisthotonos. The seizures may be constant or tonic; or con-
vulsive and violent, then they are designated as clonic.
Immunity. Metchnikoff claims that the only natural immu-
nity possessed by man against tetanus resides in his leucocytic
powers of defense. Susceptibility of the natural receptors of the
nerve cells for the toxin, and the degree of affinity, constitutes the
cause of intoxication, its degree, and ultimate result. Affinity for
the receptors of other less vital organs, on the part of the toxin,
establishes a means of natural defense. Acquired immunity is
dependent upon the formation of anti-toxin. The anti-toxin,
formed by susceptible animals injected with tetanus toxin, is
chiefly useful and valuable as a prophylactic measure. An epi-
demic of puerperal tetanus in a lying-in hospital was checked by
its use. Sprinkling dry powdered anti-toxic serum on wounds in-
fected with tetanus bacilli, or toxin, prevented infection (Calmette
and McFarland). The anti- toxin may be injected either into the
substance of the brain in cases of well developed tetanus, or into
the cerebrospinal fluid, in the hope of neutralizing the toxin not
already in firm combination with the nervous elements. Large
nerves near the infecting wound may be injected with anti-toxin
in the hope of binding the toxin already in combination with the
nerve cells (see page 74) .
Female mice immunized against tetanus toxin, transmit a great
amount of immunity to their offspring. The milk of an immun-
ized mouse also causes a passive immunity in other young that are
suckled by her.
BACILLUS OF MALIGNANT (EDEMA
Bacillus (Edematis Maligni. .
Vibrion septique.
Bacillus of Malignant (Edema.
Morphology and Stains. Thickish rods, resembling tetanus
and symptomatic anthrax bacilli, with a tendency to grow in long
BACILLUS OF MALIGNANT (EDEMA 193
threads. It is actively motile, and is possessed of numerous peri-
trichous flagella. Spores are found which may be either equatori-
ally or polarly situated. This organism is readily stained by the
ordinary methods, but not by Gram's.
Chemical Activities. Milk is coagulated, digested, and the
reaction is amphoteric. Abundant alkali is formed at times;
albumin is decomposed, forming fatty acids, leucin, an oil, and an
offensive odor. CO2N. and marsh gas, are also formed.
Habitat. It is found in soil, dust, manure and dirty water and
is widely distributed.
Cultures. This organism is a strict anaerobic, and grows well
in most culture media, at incubator or room temperature. On
gelatine plates colonies develop on the surface (under hydrogen)
in tiny shining white bodies, which upon magnification are found
to be filled with a grayish-white substance composed of melted
gelatine, and long tangled filaments. The edges of the colonies
are fringed. In gelatine stab cultures (made in liquid gelatine,
which, after inoculation, is rapidly solidified in ice water) a
globular area of liquefaction occurred. If sugar is added, active
fermentation takes place, with the production of large amounts of
offensive gas. It grows well on agar, in bouillon, and in milk.
Pathogenesis. Is pathogenic for man, horses, sheep, dogs, rab-
bits, calves, pigs, goats, rats, mice, and guinea pigs. Cattle are
said to be immune. When bacilli are applied to a scratched sur-
face, infection is not likely to occur, as free oxygen seems to inhibit
the growth; if, however, the wound is deep, rapid infection follows,
young domestic, and laboratory animals dying within forty-eight
hours. The bacillus produces a moderate quantity of toxin and
has an antagonistic action on leucocytes. In man, the clinical
manifestation of infection with this organism is known as malig-
nant oedema. Infection has followed penetrating wounds of the
body, by dirty tools, nails, splinters, bullets, etc. The disease
is often quickly fatal. It produces, frequently, rapid moist
gangrene.
13
194
BACTERIA
The organisms and spores are not so very resistant as any
strong germicide or a temperature of 100 acting a few minutes
will kill them.
SYMPTOMATIC ANTHRAX BACILLUS
Bacillus Chauvoei.
Bacillus of Symptomatic Anthrax.
Rauschbrand Bacillus (Figs. 56 and 57).
FIG. 56. Rauschbrand bacilli showing spores. (Kolle and Wassermann.)
The cause of symptomatic anthrax, black-leg, or quarter-evil,
in cattle.
Morphology and Stains. This is a large organism, .5^ in width,
and 3 to 5/x in length. It has rounded ends, and grows in pairs,
but not in strings or chains. It is motile, and has many peritri-
chous flagella. When stained for spores, these bodies may be
found distending the organism in the middle or at the end, and the
bacillus assumes a drum-stick, or spindle shape. Often chromo-
philic granules are present; involution forms also appear, and are
of enormous size. This organism stains with all the common
SYMPTOMATIC ANTHRAX BACILLUS 1 95
stains, but not by Gram's method. They may be seen in an
unstained condition in blood or other fluids.
Habitat. This bacillus is found not only in the diseased tissues
and dead bodies of infected animals, but also in infected pas-
tures, soil, hay, etc.
Temperature Requirements. It is best cultivated at body
temperature, but grows anywhere between i8C. and 37C. The
spores resist boiling for half an hour but the vegetative rod is
killed by iooC.
FIG. 57. Rauschbrand bacillus showing flagella. (Kolle and Wassermann.)
Cultures. It is, like tetanus and malignant oedema organisms,
a strict anaerobe. On gelatine it grows in roundish whitish colo-
nies in a delicate tangled mass, with projecting filaments. The
gelatine is liquefied, and bubbles of gas are formed in stab cultures.
A sour odor is emitted from cultures; i to 2 percent of sugar is
required for successful cultivation; or 5 percent of glycerine will
answer. On agar the growth is marked; gas is produced, and
acidous odors evolved. In bouillon it grows rapidly. Large
masses of the organism sink to the bottom, gas is formed, and the
medium is clouded. Milk affords a good medium for the growth
of the organism, but the casein is not coagulated.
ig6 BACTERIA
Pathogenesis. Young cattle, six months to four years old,
sheep, goats, rats, mice, and more especially guinea pigs, are sus-
ceptible to it. Swine are immune, while dogs, cats, birds, and
rabbits are not susceptible. Man is immune. It causes in ani-
mals peculiar groups of emphysematous crepitating pustules, fol-
lowed by emaciation and death. These areas contain dark fluid,
probably broken-down blood. In guinea pigs inoculation is fol-
lowed by death within thirty-six hours. The site of inoculation
is found to be cedematous, and contains bloody fluid. The bacilli
are mostly found at the site of the inoculation, but later in the
blood in every part of the body. The virulence of this organism
in culture media is soon lost. The addition of lactic acid to the
cultures increases their virulence.
Immunity. It is possible to decrease the virulence of this
organism, and to use the weakened bacteria as a vaccine against
infection. To attenuate this bacillus, prolonged exposure to heat,
or to heat and drying together is necessary. Inoculation with
bacilli treated in this way is followed by a mild local reaction,
which affords complete immunity against infection with virulent
bacilli. It has been found by Kitt that the muscles of an infected
animal, if subjected to a high temperature 85C. to 9o c C.
afforded complete protection to the animal inoculated with them.
It is best to use a weaker vaccine muscle that has been heated to
iooC. for two hours, in order to protect against the active
vaccine. Before heating, the meat is ground. When used as an
injection, it is crushed and mixed in a mortar with sterile water.
Guillod and Simon found that this means of preventative inocula-
tion reduced the death rate in unprotected animals from 20 per-
cent to 5 percent. If this bacillus, and the prodigiosus bacillus
are injected into naturally immune animals, death will often
result.
There is a soluble toxin, anti-toxin against which appears in '
immunized animals. The toxin may be used for prophylaxis.
One attack confers immunity.
MEAT POISONING BACILLUS IQ7
MEAT POISONING BACILLUS
Bacillus Botulinus. Van Ermengen.
Bacillus of Meat Poisoning, or Botulism (Fig. 58).
Morphology and Stains. This bacillus resembles thick vigor-
ous rods, 4-9/4 long, and .9^ thick, is motile, has polar spores, and
from four to nine peritrichous flagella. It is strangely called a
saprophyte, because it is incapable of growth in the body, yet its
toxin is highly poisonous to man and other animals. It is stained
by all the usual basic aniline dyes, and by Gram's method.
FIG. 58. Bacillus of botulism. (Kolle and Wassermann.)
Habitat. It seems probable that this organism occurs in the
feces of animals, especially pigs, from which source it can gain
access to the ground, to vegetables, or to the meat of the animal
from which hams are cured or sausages made. While originally a
disease described as originating from improperly cured hams,
botulism has been known to follow the eating of tomatoes, beans
and olives.
Vital Characteristics. Is an anaerobe. Its thermal death-
point, for a spore-bearing organism, is low, 8oC., for an hour.
IQ8 BACTERIA
Grows only in media that are alkaline, and is capable of growth
at from i8C. to 35C., though best below 25C.; 10 percent
of chloride of soda checks growth.
Chemical Activities. It can produce, at room temperature,
a water-soluble toxin sufficiently stable to withstand drying of
meat if not exposed to sunlight, and not destroyed by the gastric
juice. It is destroyed by thorough cooking of meat. Milk is
not coagulated, grape sugar is fermented, and a foul, sour odor
is produced in a culture. It liquefies gelatine. There are two
varieties of the germ, A and B, differing in the quality of the
toxin produced, both having the same physical and pathogenic
properties but developing different anti-toxins.
Cultures. On gelatine plate, that contains sugar, colonies are
produced that are coarse and prickly in appearance. The lique-
faction of the gelatine is slow. Bouillon is rendered turbid. The
cultures resemble tetanus and malignant cedema.
Pathogenesis. Its pathogenic action is marked, but only by
its toxin, which has a decided affinity for nervous tissue. The
toxin is absorbed from the intestinal tract unchanged by the
gastric juice. In this it differs from the toxin of diphtheria and
tetanus. If the toxin is mixed with the emulsified nerve tissue,
it becomes neutralized. In fatal cases of infection, the gan-
glionic nerve cells are degenerated. Man is very susceptible,
while cats and dogs are more or less non-susceptible. If bacilli
are inoculated into animals, they do not proliferate. A men-
ingitic disease of horses and limberneck of fowl are believed to
be due to this intoxication. Animals that recover are found
to have developed strong anti-toxin in the blood serum.
Immunity. An artificially prepared anti-toxin has been found
to be active^ and is of use in treating cases of poisoning with meat.
The correctly typed anti-toxin should be used or that made by
artificial immunization with both varieties of bacilli. The latter
is now preferred since no rapid distinguishing clinical test of
types is available.
GASEOUS CEDEMA BACILLUS
GASEOUS (EDEMA BACILLUS
199
Bacillus Capsulatus Aerogenes. Welch.
This description covers those organisms sometimes described
under the title Bac. perfringens and Bac. enteriditis sporogenes;
they are in all probably but variants from this type. Other
types with slight variations in chemical action and toxin produc-
FIG. 59. B. Aerogenes capsulatus of Welch, in smear. (Williams.)
tion were noted during the war. It is this group which was
responsible for gas infection during the great European War.
Morphology and Stains. A vigorous plump bacillus 3 to 47* in
length, resembling the anthrax bacillus, and is usually straight.
It forms spores, is non-motile, and flagella have not been found.
It occurs in pairs, and in chains. In old cultures involution forms
are seen. Spores are generally equatorially situated. Is colored
200
BACTERIA
FIG. 60. B,
sulatus, agar
gas formation.
aerogenes cap-
culture showing
(Williams.)
by all the basic dyes, and holds the
stain in Gram's method. Staining
shows that it possesses a capsule.
Habitat. The soil, the intestines,
and, sometimes, the skin of man.
Vital Characteristics. Vital re-
sistance is low, the thermal death-
point being 58C. with ten minutes'
exposure, while spores require ten
minutes' boiling for killing. It
grows best at body temperature.
Has lived for one hundred days on
culture media in the incubator. It
is an anaerobe.
Chemical Activities. Produces
gas; does not usually liquefy gela-
tine, but curdles milk (Fig. 60).
Cultures. Grows best in neutral
or alkaline media, producing abund-
ant gas. Colonies appear grayish
or brownish-white, and are often
surrounded by projections which
are feathery or hair-like. On agar
strict anaerobic conditions are
necessary for growth, gas bubbles
appear in the media, and the agar
may be forced out of the tube in
stab cultures. In bouillon it grows
under anaerobic conditions. The
growth is rapid, bouillon is clouded,
and a froth appears on the surface.
After a few days the medium be-
comes clear, owing to the sedi-
mentation of the bacilli. Growth
GASEOUS (EDEMA BACILLUS 2OI
occurs best in sugar bouillon, which becomes strongly acid. In
milk the growth is rapid and luxuriant; the proteids are coagu-
lated. Anaerobic conditions must be observed. On potato it
grows well, producing bubbles in the water which may cover
the potato in the tube. The growth appears thin, moist, and
grayish-white.
Pathogenesis. The pathogenic properties of this organism are
limited. It is not able to endure the oxygen of the circulating
blood. Grows best in old clots, and in the uterus. It produces
gas rapidly in some cases of abortion and in peritonitis in man,
which is quickly followed by death. It causes gaseous phleg-
mons in guinea pigs, and injection are usually fatal to birds.
In man infection has followed wounds, and delivery of the child
in puerperal cases. It produces in fatal cases the condition
known as frothy organs " Schaumorgane. " It may be isolated
from infected matter, faeces, etc., by injecting the latter into a
rabbit's vein and then killing the animal. The carcass is then
placed in an incubator and an enormous growth of the organisms
follows; anaerobic conditions favorable to growth are obtained
in the body so that gas distention of the tissues results; from the
latter pure cultures are easily obtained.
Vincents Angina is due to an anaerobic organism of two stages,
as Bacillus fusiformis and Spirochata wncenti. The bacillus is a
fusiform irregularly staining pointed rod, 3-12/4 long by .3-.8/*
wide. Under cultivation it grows out into forms such as are seen
with it in smears from the diseased throat, that is, long, wavy,
uniformly stained, flexible, pointed ended spirals. The bacillus
forms endospores chiefly at the end. Obligate anaerobe, requir-
ing serum, ascitic fluid or glycerine. Colonies delicate and
whitish. Gas in glucose media. Litmus milk only decolorized.
Gives a foetid odor on all cultures. No specific immunity reac-
tions known.
These same spirals have been found in abundance in many
cases of ulcerative stomatitis, notably the variety which became
202 BACTERIA
well known during the late war. Their connection with this
so-called "Trench mouth" is not so generally accepted as is the
case with ulcerative angina but their discovery should suggest
remedies, salvarsan, silver nitrate, which have been useful in
Vincents Angina.
SPIRILLACE^E
CHOLERA BACILLUS
Vibrio Cholerse. Koch.
Spirillum Cholera (Fig. 61).
Cholera Bacillus.
Comma Bacillus.
Morphology and Stains. Curved or bent rods, the ends not
lying in the same plane. This bending varies greatly. Under
FIG. 61. Cholera spirilla. (Kolle and Wassermann.)
certain conditions of growth such as the presence of alcohol, or
insufficient albumin or oxygen in culture media, long spiral chains
are formed. It is motile, has one terminal flagellum, and like
other members of this family, has no spores. It stains well with
the common dyes but not by Gram's method. Dilute fuchsin
CHOLERA BACILLUS 203
stains it best. Occasionally involution forms are developed,
which do not stain well. So-called arthrospores are formed, ac-
cording to Hlippe.
Habitat. It is said to exist constantly in the waters of the
Ganges in India. Is found in contaminated drinking water,
from rivers, lakes, and wells; also in human faeces, which, used as
manure, infests vegetables, and spreads the disease. It is found
in the intestines during cholera, and after death in other viscera.
It can be disseminated by convalescent or healthy carriers;
chronic carriers are not known.
Vital Resistance. Is extremely sensitive to various deleterious
agencies. Minute quantities of mineral acids, and other chemical
disinfectants, as well as light, heat, and drying, quickly kill it;
i percent carbolic kills rapidly. A 1-2,000,000 solution of cor-
rosive sublimate destroys in from five to ten minutes. Its thermal
death-point is 6oC. for ten minutes (moist heat).
Chemical Activities. It creates indol in large quantities, and
may be detected in peptone cultures merely by the addition of
sulphuric acid. Laevorotatory lactic acid is produced from all the
sugars. Gases are not formed. Yields alkali in culture; causes
slight coloration of potato, and produces a disagreeable odor in
bouillon; also yields H 2 S, and ferments that liquefy gelatine.
Bacteriolysins and invertin are also produced, as well as a toxin
which is soluble in water. The most powerful toxin, by far, is
contained in the cells of the vibrio themselves. This causes death
after intraperitoneal injection in guinea pigs.
Oxygen Requirements. It is a facultative aerobe; its growth,
however, without oxygen is slow, while powerful toxins are
formed.
Temperature. Grows best at 37C., but very well at 23C.
Does not grow below 8C.
Cultures. On gelatine plates the growth is characteristic.
Small yellowish- white colonies, which rapidly liquefy the gelatine,
appear in twenty-four hours. As the colony increases in size it
204 BACTERIA
becomes more and more granular, and finally the whole medium
is liquefied. In gelatine tube stab culture, the growth, at first, is
not characteristic; but, after a few hours, a semi-spherical depres-
sion appears, which extends downward, and resembles a large
bubble of gas. As liquefaction progresses, the whole line of punc-
ture disappears, and the excavation looks cylindrical. This area
becomes cloudy. On agar plates the colonies are elevated, round
and white, with moist lustre. Deep colonies are whetstone shape.
Old agar colonies become yellowish-brown. Coagulated blood
serum is rapidly liquefied at 37C. Milk, at times, is coagulated.
No curdling ferment is formed; the acid produced is thought to be
sufficient. On potato the growth is slow, or not at all, if the
medium is acid. If the potato is rendered alkaline, growth occurs,
with a moist lustre, slightly elevated; white at first, later becoming
brown. On acid fruits it will not grow. In bouillon, after sixteen
hours, a diffuse cloudiness occurs, with the formation of a stiff
pellicle, which in some cultures becomes wrinkled. In peptone,
abundant growth takes place, with the production of indol and
nitrites. If a few drops of H 2 SO4 are added, a beautiful red
appears if nitrites are present. This is the " cholera red " reaction.
If the color does not at once appear, nitrites must be added. On
blood media certain strains produce distinct hemolysis (El Tor).
Pathogenesis. Cholera spirilla are pathogenic for man, but
only under experimental conditions, for lower animals for which
guinea pigs may be taken as an example. If the stomach of the
latter is rendered alkaline with bicarbonate of soda, and a bouillon
culture introduced, choleraic symptoms will follow and the animal
will die. If cholera spirilla are injected into the peritoneum, the
animal will quickly succumb to a general cholera peritonitis.
Young rabbits are equally susceptible. When cholera spirilla in
culture have been swallowed by man (laboratory workers), either
by design or accident, the disease has followed, sometimes with
fatal results. The toxin of this organism is intracellular (an
endo-toxin). Old cultures become pathogenic through a bac-
CHOLERA BACILLUS 2O$
teriolytic action, by which the cells are dissolved, and the toxin
liberated. Filtrates from young cultures are non-toxic. If
bouillon cultures are killed by chloroform, and then injected into
animals, toxic action follows. In cholera the pathogenic process
is mostly confined to the intestines. Toxic absorptions, due to the
liberation of toxic products by the bacteriolytic action of serum,
follow later. There is a desquamation of the epithelium of the
bowel, and epithelial flakes found in the watery discharges resem-
ble rice grains. Peyer's patches may become slightly swollen
and reddened, and later, there may be diphtheritic necrosis above
the iliocecal valve, and often a parenchymatous nephritis. The
vibrios do not enter the blood.
Diagnosis. Bacteriological diagnosis of cholera is accomplished
by examining the alvine discharges. A mucous flake is mixed
with some peptone solution, this is incubated, and the spirilla,
if present, rapidly grow on the surface; after a few hours, plates
are poured from this surface growth, and from the plates liquefy-
ing colonies are picked out, and bouillon cultures made. These
are tested by serum, from horses artificially immunized by inject-
ing cholera spirilla into them. If the organism under examination
(after serum mixed with 2,000 to 3,000 parts of water is added)
agglutinates, it is considered to be the cholera spirillum. Both
in early and fatal cases, the agglutinating reaction is not available,
since it takes some time for the agglutinins to form in the blood.
Under the chapter on immunity an account of the PfeifTer reaction
is given, also one on vaccination against cholera infection, by
means of killed cultures, under the chapter on vaccines.
Vibrios Allied to the Cholera Vibrio
Several other vibrios have been discovered that resemble the
cholera vibrio. These are mostly found in potable water, and
though in many respects identical with the cholera vibrio, they
differ in essential points, i.e., pathogenicity, and in their agglutina-
bility with specific sera. The most important of these organisms
206 BACTERIA
are: Vibrio Metchnikovii; Vibrio proteus; Vibrio tyrogenum; and
Vibrio schuylkilliensis. There are no important pathogenic mem-
bers of this group except the cholera vibrio.
GLANDERS BACILLUS
Bacterium Mallei.
Bacillus Mallei.
Glanders Bacillus.
Morphology and Stains. Slender rods 2 to 3/x. in length con-
taining no true spores, but shining chromatophilic bodies (Babes-
Ernst granules). In old culture, long club-like threads appear,
which exhibit true branching. This organism is not motile, and
has no flagella. It is stained with difficulty by ordinary methods,
and not at all by Gram's method.
Vital Activities. It is a facultative aerobe, growing feebly in
the absence of air, and best at 37C., in glycerine agar. Resists
drying but feebly. Its thermal death-point is 5SC., ten minutes'
exposure.
Chemical Activities. Produces a brown pigment on potato,
also mallein, and a little indol in old bouillon cultures. It forms
no gas.
Cultures. On gelatine it produces small punctiform colonies
that are white, and become, after a time, surrounded by a distinct
halo. The colonies are often very delicate and ragged. The gela-
tine is not liquefied. On agar the growth is best if glycerine is
present, but is not characteristic. Bouillon cultures cause an
abundant sediment, above which the medium is clear. Milk is
coagulated. On potato the growth is characteristic. The color
is, at first, yellowish-white like honey, becoming, finally, reddish-
brown. The potato is much darkened.
Pathogenesis. This organism is pathogenic for horses and
man; 50 percent of men succumb after infection. Horses, asses,
cats, dogs, sheep, and goats are susceptible in the order mentioned.
Cattle and birds are immune. In horses the disease is known as
DIPHTHERIA BACILLUS 207
glanders, or farcy, and the avenue of infection determines the
clinical form of the disease. The mucous membrane and the skin
are the chief places of infection. A primary ulcer is formed in the
mucous membrane of the nose, or in the skin. Subsequently, the
lymph-glands and the lungs may be infected. Guinea pigs are
easily infected. White and gray mice, and rats are immune. For
purposes of diagnosis guinea pigs are inoculated, but care must be
used, as several fatal cases have occurred in laboratory workers,
it being a treacherous organism with which to work. In infected
animals, it produces a rapid and marked inflammatory reaction,
with the formation of pus. Certain "buds," or nodules are
formed, which are between an abscess and a tubercle in structure.
The diagnosis of doubtful cases may be made by injecting the
material into the peritoneum of male guinea pigs. A violent
suppurative orchitis occurs from which the rods can be cultivated.
The poisons are endo-toxic.
Agglutinations. It has been shown by McFadyean that the
blood of infected horses exhibits markedly agglutinative properties
toward the glanders bacilli. Normal horse serum clumps often as
high as 1-400. Diagnostic reactions should be i-iooo, supported
by complement fixation and the Mallein test. A slight immunity
is present after an attack. Complement fixation may be attained
with the serum of an infected horse by using an antigen of glanders
bacilli grown on glycerine broth, killed and filtered.
Mallein. In old cultures a peculiar tuberculin-like substance
(mallein) is formed from the bodies of the bacilli themselves, and
in the bouillon. This is thermostabile and if injected into animals
having glanders, produces a marked reaction (see page 86).
DIPHTHERIA BACILLUS
Corynebacterium Diphtherias (Loftier).
Bacillus Diphtheria.
Klebs-Loffler Bacillus.
Diphtheria Bacillus.
208
BACTERIA
Morphology and Stains. Long, bent, or curved bacilli of
irregular contour, frequently clubbed or filiform at one or both
ends ; which contain chroma tophilic granules, and often exhibit true
branching ; have no spores or flagella, and are not motile. Accord-
ing to Wesbrook, stained bacilli are of three types; (i) granular
(containing the Babes-Ernst granules); (2) barred like a striped
stocking; or (3) solid, staining uniformly throughout. The pleo-
morphic differences of various bacilli are most characteristic, and
of diagnostic importance. This organism stains with all the basic
* B
"^
t
FIG. 62. Forms of B. diphtheria in cultures -on Loffler's serum. A,
Characteristic clubbed and irregular shapes with irregular staining of the
cell contents. X noo. B, Irregular shapes with even staining. X 1000.
(After Park and Williams.)
dyes, notably by LofHer's blue, or Neisser's special granule stain.
It is also stained by Gram's method. The length of the organism
differs much, according to the reaction of the medium in which it
grows. Alkaline media favor long forms, and acid the reverse.
Its length is from i-5/i to 3.5^- It does not form chains. Bizarre,
or involution shapes predominate in old cultures (Fig. 63).
Culture and Temperature Requirements. It grows best at
body temperature, and on glycerine agar, or in Loffler's blood
serum mixture of alkaline reaction.
Vital Characteristics It resists drying for a long time, and has
DIPHTHERIA BACILLUS 2OQ
lived on culture media for eighteen months at room temperature;
also in silk threads for several months in a dried condition.
Remains alive in healthy throats for months. Formalin vapor
kills it speedily; corrosive sublimate solution, i- 10,000, destroys
it in a few minutes; light is lethal to it in from two to ten hours,
and heat at 58C. in ten minutes.
Habitat. It has not been found in sewage, or sewer gas, soil
or water, the disease therefore is never transmitted by these means.
FIG. 63. Diphtheria bacilli involution forms. (Kolle and Wassermann.)
Has been found in the throat, nose, and in the conjunctivas of
healthy bodies. In disease, the organism is mostly found in the
throat, but has been isolated from all the organs in some fatal
cases. Sometimes it is discovered in the throats of animals.
Though its action is local, it elaborates a toxin which acts
systematically.
Cultures. On gelatine plate the growth is scanty and raised.
This medium is never for cultivating this organism. The
gelatine is not liquefied. On glycerine agar plates the growth,
though moderate, is typically characteristic, but very slightly
14
210 BACTERIA
raised above the medium, and is of duller lustre. Old colonies
become yellowish-brown, the center of which, under a magnifica-
tion of sixty diameters, appears darker, and with ravelled edges.
On Lb'ffler's blood serum mixture, the organism grows rapidly
and well. This and ascites-glycerine-agar culture media are
the best for it. Bouillon made from fresh meat is an excellent
medium for its growth. The bouillon, which must be alkaline
and freshly made, becomes first cloudy; then a fine precipitate
settles, and over the surface a delicate pellicle forms. The
reaction of the culture presents three types: A, is acid in the
beginning, and becomes progressively more acid. B, is alkaline
from the start, and progressively more alkaline; this is the most
toxic growth. C, acid at the start, becoming alkaline finally.
The growth is not so luxuriant as in B, nor is there as much
toxin produced. In milk, the growth is luxuriant, without
coagulation. The reaction is amphoteric, but in old cultures it
becomes alkaline. On potato, rendered alkaline, it will grow,
but not characteristically.
Chemical Activities. No gas is formed, or any curdling or
gelatine dissolving ferment. Acids are evolved from sugars;
even the sugar found in meat is converted, into lactic acid. In
the manufacture of toxin this muscle sugar must be removed. A
soluble toxalbumin is created, both in the body and in culture,
which is intensely poisonous. See chapter on bacterial products.
Pathogenesis. Diphtheria in man rneans generally an infec-
tion of the mucous membrane of the upper respiratory tract, with
the formation of false membranes. The latter may cause death
by suffocation. Infection may occur in the skin, vagina, or pre-
puce. The toxin not only causes a local necrosis, with the forma-
tion of an exudate, consisting of fibrin and leucocytes, but also
grave systemic action, with marked degeneration of important
nerves and nerve centers, and also of the parenchyma of the
kidneys, liver, and heart, paralysis following. In certain struc-
tures fragmentation of the nuclei of the cells is noted. Guinea
DIPHTHERIA BACILLUS 211
pigs, cats, horses, and cows, may be infected artificially, but the
disease never occurs spontaneously in these animals. Horses,
dogs, and cattle are susceptible to its toxin. Diphtheria bacilli
often have associated with them, streptococci, which add to their
virulence, and complicate the disease. Endocarditis, adenitis,
pneumonia, abscesses, and empyemia, may be caused by them.
There may be puerperal diphtheria, due to the infection of the
puerperal tract. Diphtheria is spread mostly by personal contact
with individuals suffering from the disease, or with convalescents,
in whose throats virulent bacilli linger, perhaps, for months. It
may originate from infected milk, contaminated from human
sources.
Perhaps the most important source of infection, especially dur-
ing an epidemic, is the healthy bacillus carrier who, wholly una-
ware of his condition, is carrying virulent germs in his throat.
This further indicates that individual resistance or susceptibility
plays an important part in infection. (See Schick Test.)
Immunity is natural, active, artificial, or passive. Active im-
munity, following infection, is seldom permanent for although the
individual, if he recovers, may be considered immune for a time,
some individuals are more susceptible, and suffer several attacks.
In active immunity anti-toxin is found in the blood, and recovery,
and subsequently, immunity are due to this fact. Anti-toxin
may be discovered in the blood, by mixing it with toxin of known
strength, and injecting it into guinea pigs. If these survive a
large lethal dose of the toxin, it is safely presumed that anti-toxin
was present in the serum abstracted.
Passive artificial immunity is induced by injecting anti-toxin in
the bodies of persons exposed to diphtheria. It is most effective
but is short-lived, lasting only a few weeks. Serum therapy (see
anti- toxin in previous chapter). If there is one natural specific
cure for any disease, it is diphtheritic anti- toxic serum, which is
prepared by immunizing horses with toxin, and abstracting their
blood. The earlier it is given, the better are the chances of recov-
212 BACTERIA
ery. As a prophylactic, from 600 to 1,000 units should be used.
As many as 100,000 units have been injected in a single patient.
No case is too trivial, or too far advanced in which to use it. The
serum is anti-toxic, and not bactericidal. Wassermann has pre-
pared a serum that is bactericidal, and is designed to destroy the
bacilli.
Pseudo-diphtheria bacilli, which morphologically and culturally
resemble the true bacilli, have been described. They are not
pathogenic, in the sense of producing exudative diphtheria, and
are believed to be attenuated diphtheria bacilli by many observ-
ers. The diagnosis of diphtheria by culture is an important
measure. It depends upon the rapid growth of the bacilli upon
Loffler's blood serum. Of all the various organisms found in the
throats of patients with diphtheria, the diphtheria bacilli outstrip
them in rapidity of growth. After eight to twelve hours, the
serum inoculated with the smear from the false membrane is
covered with fine granular colonies of pure diphtheria bacilli.
After twenty-four, or more hours, the other organisms present
overgrow the diphtheria colonies. A sterile swab of cotton, or a
stick, is rubbed over the false membrane, or throat, and then over
the serum; the latter is incubated, and the culture examined after
eight or twelve hours, by staining with LorHer's blue. If curved,
clubbed, irregularly stained bacilli are found, especially if they
contain dark polar granules, and are generally uneven in size and
bizarre, it may be safely considered that they are diphtheria
bacilli. Gram's stain may be needed to confirm the diagnosis
occasionally, or it may be necessary to inoculate guinea pigs.
This may be done by inoculating the whole throat culture and
plating out from the local inflammation, or the organisms may be
isolated directly by plating and then injected into animals.
It is well to check up the test by immunizing one animal with
200 units of anti-toxin before the culture is given, using another
guinea pig unprotected. This serves as a control upon toxin
production. Virulent bacilli will kill a guinea pig of 250 grams
PSEUDO-DIPHTHERIA BACILLUS 213
in three days if i c.c. of forty-eight has serum broth culture be
given subcutaneously.
Certain of the pseudo-diphtheria bacilli, or " diphtheroids " as
they are called, seem to have the power of increasing or continuing
inflammation after it has been started by other germs. They are
frequently found in sinusitis, prostatitis, bronchitis, etc. In a few
reported cases they seem to have been the only micro organismal
cause of disease but their pathogenic powers are usually not great.
PSEUDO-DIPHTHERIA BACILLUS
Corynebacterium Pseudo-diphtheriticum.
Pseudo-diphtheria Bacillus (Hoffmann).
Morphology and Stains. This bacillus resembles the diph-
theria bacillus. The rods, however, are shorter and thicker;
otherwise, it stains like the true bacillus, but not by Neisser's
method.
Culture. On glycerine agar the growth becomes diffuse,
spreading from the line of inoculation in a grayish-yellowish
pasty expanse. It grows well on gelatine. In bouillon it forms
a denser and more luxuriant growth than the bacillus.
Habitat. It is found in healthy throats and conjunctivas.
Pathogenesis. It is non-pathogenic for guinea pigs (see above).
Diagnosis. It can be differentiated from the true bacillus by:
1. Being non-pathogenic.
2. Not exhibiting polar granules with Neisser's stain.
3. Not producing acids in certain carbohydrate media.
Bacillus xerosis is a pseudo-diphtheria organism found on the
normal conjunctiva. It is not thought to possess any virulence.
TUBERCLE BACILLUS
Mycobacterium Tuberculosis.
Bacillus tuberculosis (Fig. 64).
Tubercle bacillus.
214 BACTERIA
Morphology and Stains. Slender rods, generally unbranched,
i~5ju long, and 4/4 thick, usually slightly bent; are non-motile,
and have no spores or flagella. In old cultures, and sometimes
in sputum, branching forms are seen, and, rarely, some that are
club-shape. On acid potato, thread forms are found. In the
continuity of most of the bacilli, unstained spaces are seen; in
others dense deep red granules are found
by fuchsin. As this bacillus is difficult to
stain, special methods have been devised to
demonstrate it, as the sheathing capsule
renders it extremely unsusceptible to the
ordinary methods of staining. The cause of
this resistance is supposed to be a fatty or
FIG. 64. Tubercle waxy substance in the capsule which is more
bacilli in sputum: , , , , , r i < , .,1 *
stained with fuchsin than probable, because of the fact that stains
and methylene blue, that are fat selective, such as Sudan III,
(Greene's Medical , t* -m i i i i r i
Diagnosis.) color it very well. Boiling hot carbol-fuchsm
gives it the best stain. It keeps the color
in spite of the action of strong solutions of mineral acids in
water, or dilute alcohol. So when tissues, or secretions, are
stained with hot carbol-fuchsin for a short time, or cold carbol-
fuchsm for a long time, and then treated with a 25 percent
solution of HNOa, or H 2 SO4, in water, everything is deprived of
the red color, except the tubercle bacilli. All such organisms
that are acid proof, are called " acid-fast." There are many
other bacilli that have this property. Aniline water and gentian
violet solution also stain it. Gram's method dyes the organism
violet. Sometimes very young bacilli do not stain at all.
Vital Requirements. This bacillus thrives best at 37.5C.
It grows slowly, is a strict parasite, and an obligate aerobe. In
cultures it dies quickly in sunlight, and in diffuse daylight it
dies in a few days. It resists drying and light in sputum for
months. Its thermal death-point (moist) is 80 C. for ten min-
utes; can resist 6oC. for one hour, but succumbs to 05C. in one
TUBERCLE BACILLUS 215
minute. It is quickly killed by formaline and corrosive subli-
mate, but resists 3 percent solution of carbolic acid for hours.
In sputum it withstands antiseptics for a long time.
Chemical Activities. It grows slowly, producing no coloring
matter; yields an aromatic sweetish odor, but no gas or acid. It
produces certain plasmins or endo-toxins, which are called
tuberculins (q.v.).
Chemically the tubercle bacillus contains two fatty matters,
one combined with an alcohol to form a wax. It has also a
protamin, a nucleic acid or an albumose. Various fatty acids
are to be derived from it by chemical treatment. The active
principle in tuberculin centers around its protein elements, but
is not exactly known.
Habitat. It is a strict parasite and never leads a saprophytic
existence. Is found wherever human beings live in crowded
quarters; in dust of rooms, vehicles, and streets; and often in
milk and butter. It is very widely distributed, being found in all
human communities.
Cultures. Since the organism does not grow below 3oC.,
gelatine is never used. On coagulated blood serum of cows,
horses, and dogs, this bacillus grows best. As it is very difficult to
isolate in pure cultures, the following procedure should be followed :
The suspected sputum, fluid, or tissue is injected into a guinea
pig, and when, in two weeks or more, large swollen glands can be
felt in the groin, the animal should be killed, and a gland removed
under strict aseptic precautions. It is then divided, and the
halves containing the bacilli are rubbed over the surface of
coagulated dog serum and allowed to remain in contact with it.
The serum should be coagulated in special tubes, with glass caps,
having small perforations, which are stopped with asbestos fibre,
or glass wool. The organism grows well in air, but too great
access thereto dries and kills it. After the tubes are incubated
for a week or two, little scales growing unto clumps appear,
which are lobulated and friable. At first white, it later turns
216
BACTERIA
darker. This medium is never liquefied
by the culture. On glycerine agar
made of veal broth containing 6 per-
cent of glycerine, the organism grows
well after isolation from the tissues,
often luxuriantly (Fig. 65). A wrinkled
film covers the surface of the agar,
from which it is removed with ease.
On bouillon, made of veal and glycer-
inized, it develops rapidly, covering
the medium with a dense white
wrinkled pellicle, which, though thick,
is friable. After a time it falls to the
bottom of the flask. It grows well on
glycerinized potato also, and milk
agar. On egg-albumins mixed together,
sterilized and coagulated, this bacillus
also develops well.
Pathogenesis. The discovery of the
tubercle bacillus, its methods of culti-
vation and differential staining, may
be ranked with the greatest of medical
discoveries. This organism causes in
man and cattle, chiefly, the disease
called tuberculosis. It rarely attacks
the carnivora, but has been found in
such animals when confined. Swine
are often infected; cats and dogs some-
times, but sheep, goats, and horses
seldom. It is easy to inoculate guinea
pigs or rabbits by injection or feeding.
FIG. 65. Bacillus tuberculosis; glycerine
agar-agar culture, several months old.
(Curtis.)
TUBERCLE BACILLUS 217
The disease is widespread, but is much more common where
human beings are huddled together in dark, badly ventilated
rooms and shops. In tissues, the characteristic lesion is a tuber-
cle. This is a globular mass, about the size of a very small shot,
and grayish pearly white. Microscopically, in the centre of the
tubercle, are found several large multinuclear cells, called giant
cells, which often contain thirty or more nuclei, and a number of
tubercle bacilli, the nuclei often being situated at one pole, while
FIG. 66. Tubercle bacilli showing involution forms. (Kolle and
Wassermann.)
the bacilli are at the other. About the giant cells epithelioid
cells are grouped, and about these small round cells are massed in
great numbers. No new blood-vessel formation is ever found
in the epithelial cell layers, or among the giant cells. Owing
to insufficient blood-supply the centre of the tubercle frequently
undergoes caseous degeneration. If the lesion heals, the caseous,
centres become calcareous, and the periphery changes into
connective tissue. If the tubercles coalesce, great masses of
caseous tissue form. If the latter becomes infected with other
2l8 BACTERIA
pathogenic bacteria (streptococci and pneumococci) rapid soften-
ing occurs, with cavity formation, etc. Tubercles may develop
in any organ or tissue of the body. The lungs, intestines,
peritoneum, glands, larynx, spleen, and bones become infected.
The liver and pancreas seem to resist invasion more than other
organs. Bacilli are rarely found in the blood in tuberculous
diseases. They may, however, be found in the urine, in kidney
or bladder tuberculosis. Milk from tuberculous cows, with,
infected udders, often contains bacilli, and is certainly a means
of transmitting the disease. Cerebro-spinal fluid, in tuberculous
meningitis contains the bacilli. Bacilli may penetrate mucous
membranes, and not causes any local lesions, but infect distant
organs. Tuberculosis may be spread in the body in four ways.
Sputum may be swallowed and infect the intestines, by continuity,
by the lymph-stream, or by the blood; this may cause intestinal
ulceration and invasion of the peritoneum. If the bacilli reach
the blood-stream, the disease produced is generally acute miliary
in type. This is manifested by the formation of fine gray
tubercles. In tuberculosis of the lungs it is more than probable
followed skin inoculation, either by accidental or intentional
trauma that the bacilli are inhaled. Local tuberculosis has often
Tuberculous mothers may have tuberculosis of the genital tract,
and fathers, having tuberculous testes, discharge bacilli in the
semen. Placental transmission of the bacilli from mother to child
occurs rarely.
Types of Tubercle Bacilli. It has been considered probable by
many observers that there are two types of bacilli, a human and a
bovine type. Theobald Smith was the first to advance this
theory. Koch has announced that the two types were totally
different, and that the human was incapable of infecting cattle,
and the bovine was not pathogenic for man. In view of the fact
that cattle are frequently tuberculous, and the bacilli are often'
found in the milk, it is important to know if the bovine type can
develop in man. Ravenel has shown that it is undoubtedly
TUBERCLE BACILLUS 2IQ
pathogenic for human beings. Men have been infected on the
hands, while performing autopsies on tuberculous cattle, and
their skin lesions showed, histologically, unmistakable tubercles.
Cattle have been infected by bacilli of the human type. The
bovine type of bacillus differs from the human in the following
ways:
1. It is much more pathogenic for guinea pigs and rabbits.
2. It produces more extensive lesions in cattle.
3. It is shorter than the human.
4. It produces more alkali in acid media.
5. It is more readily isolated from original lesions and does
not demand animal juices in culture media so emphatically.
The subject of the infectiousness of bovine tuberculosis for man
has lately been exhaustively studied by Park and Krumwiede.
Their conclusions are that bovine tuberculosis is practically a
negligible factor in adults. It very rarely causes pulmonary
tuberculosis or phthisis, which disease causes the vast majority
of deaths from the spread of virus from man to man. In children,
however, the bovine type of tubercle bacillus causes a marked
percentage of cases of cervical adenitis leading to operation, tern
porary disablement, discomfort and disfigurement. It causes a
large percentage of the rarer types to alimentary tuberculosis
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 6 L to 10
percent of the total fatalities from this disease.
It is not always easy to differentiate the tubercle bacillus from
other pathogenic and comparatively harmless acid-fast bacilli.
Among these are the B. lepra, the B. smegmatis, and a number
of organisms found in butter, milk, hay, grass, and in the blind
worm. Culturally, the difference is great. The surest way to
differentiate the tubercle bacillus from other acid-fast organisms
is by animal inoculations.
For the discovery of tubercle bacilli in materials apt to contain
220 BACTERIA
other acid-fasts several method are now employed. The material
to be examined may be stained in the ordinary manner and then
decolorized by Pappenheim solution or a saturated solution of
methylene blue in absolute alcohol. Preparations should be dried
thoroughly before using such solutions. For " enriching" in
organisms, the bulk of material, e.g., sputum, is suspended in 15
percent antiformin (the proprietary name for a mixture of Javelle
water and caustic soda), allowed to stand in the incubator for a
while and the suspension centrifuged. In the sediment many
more bacilli will be found than in the same bulk of the raw speci-
men. This antiformin seems to dissolve mucus, tissue and all
bacteria except tubercle bacilli. The method can be used to
procure cultures.
Even with this method organisms escape detection in some
certainly tuberculous lesions. This is said to be due to non-acid-
fast, but Gram-staining granules. They are said to be found by
a-modified Gram-Weigert staining, according to Much. Such
specimens should always be injected into guinea pigs for
corroboration.
Immunity. It is possible to immunize cattle against virulent
bovine tubercle bacilli by inoculating them previously with a cul-
ture of human tubercle bacilli that have been grown for some ,
time on culture media, and thus attenuated. The tuberculins, J;
if injected into a person with chronic tuberculosis, stimulate the
tissues to a slightly greater resistance to the disease. Thus far
anti-tuberculous sera are not of a pronounced or certain thera-
peutic value. By immunizing horses, Maragliano obtained a
serum that he claims is effective. The milk from immunized
cattle is used as a diet in tuberculous patients by him. The vari
ous tuberculins, some containing endo-toxins, or plasmins, in
solution, are capable of stimulating the formation of agglutinins in
the sera of man and animals. Blood from infected individuals
also contains these bodies. The agglutination test does not seem
to be of great practical diagnostic value, while the complement
t
BACILLUS .OF LEPROSY 221
fixation gives some information and is growing in favor as assist-
ance in obscure clinical cases.
BACILLUS OF LEPROSY
Mycobacterium Lepra. Hansen.
Lepra Bacillus.
The original description is of a pointed, curved, acid fast rod
occurring in groups within lepra cells. In recent years several
different organisms have been isolated on media containing trypto-
phan. They have a few features in common: frankly acid
fast or decolorized with difficulty. Gram positive, non-motile,
non-spore forming, staining shows beading or barring, polar
bodies, all capable of pleomorphism. They have been grouped
into 4, (i) acid fast bacilli varying from coccoid to filamentous
shapes, not easily isolated but growing well after once accustomed
to media; growth yellow or orange; (2) acid fast, non-chromogenic,
plump bipolar rods, short and long, growing with great sparsity
on laboratory media; (3) diphtheroid bacilli staining solidly, or
beaded, growing best at 37C. in a yellow- white manner on agar
and with a pellicle on broth; (4) anaerobic bacilli of more solid
staining character and growing feebly as a dry band on media.
Very marked variations in luxuriance and color production are
noted on different media.
To cultivate the leprosy organisms bits of tissue are stripped
off and allowed to digest with trypsin on blood serum or agar
plates. When the tissue has softened and the bacilli multiplied,
transfers are made to serum glycerine media or those containing
tryptophan. It is best alkaline in reaction.
Pathogenesis. The bacilli are seen in enormous numbers in
lepra cells and elsewhere in diseased tissues and have been found
in the blood. The lepra cells are large and vacuolated, and literally
crammed full to bursting with bacilli. In general the leprous
NOTE. Tubercle bacilli causing avian and fish tuberculosis, and other acid fast bacilli
exist, but not being pathogenic for man, are not described here.
222 BACTERIA
lesion resembles a tubercle, as it consists of giant cells, epithelial,
and round cells.
Immunity. There is very little accurate knowledge as to
immunity against this organism; of late bacterins have been tried
with some success it is claimed.
STREPTOTHRIX (Eppinger) OR NOCAKDIA
The genus of truly branching mycelium-forming higher bacteria
(see page 3), such as the actinomyces, belonging to the group
between bacteria proper and the moulds, called Trichomycetes.
Kruse has described nineteen different members of the strepto-
thrix, some pathogenic to man and animals.
FIG. 67. Streptothrix Candida. (Kolle and Wassermann.)
A number of cases of streptothrix (Streptothrix Hominis) infec-
tion in man have been reported. The disease, in general, resem-
bles phthisis. In the pus, sputum, and stained sections of these
cases, strep to thricial threads have been found (Figs. 67 and 70).
Morphology and Stains. Threads are thick and short, or long
and slender, depending upon the medium on which they grow. In
bouillon the threads are thin and long, on blood serum, short and
STREPTOTHRIX 223
thick. When stained there is distinct beading and fragmentation
of the protoplasm.
There is true branching of an irregular type, which is best seen
in liquid media. These threads often produce spores on culture
media. The threads often disappear in old cultures, leaving only
the spores, which stain with carbol-fuchsin and do not decolorize.
The threads stain by Gram's method, and Gram-Weigert method.
The threads are not acid-fast.
Vital Characteristics. These organisms live for years in cul-
ture media after it is dry. Spores resist dry heat at 6oC. to
7oC. for an hour; moist heat, 6oC. however, kills them after an
hour. It is a strict aerobe.
Cultures. On Loffler's blood serum, according to Tuttle, this
organism grows slowly in whitish colonies, which finally become
yellow. The adult colonies adhere to the serum. On agar it
grows rapidly and characteristically. The colonies are yellowish-
white and adhere to the agar. In Bouillon. It develops slowly
on the surface of the medium. Fluffy tufts, or balls, are formed,
that sink to the bottom of the tube. The growth is whitish.
Pathogenesis. For rabbits and guinea pigs this organism is
pathogenic, producing abscesses, tubercles, induration, etc. It is
a pus forming organism.
In animals the spontaneous disease appears, best known as
"farcin du boeuf," as ulcerative or infiltrative lesions of lungs
and skin.
In man, the disease picture is like that of tuberculosis. It
causes abscesses, adenitis, indurations of the skin, endocarditis,
and pleuritic inflammation. Many grayish tubercles were found
that resembled the lesions produced by the tubercle bacillus.
Cavity formation has been described.
This organism may act as a secondary infecting agent in tuber-
culosis of the lungs. Tuttle reviews twelve cases, all of which
were fatal.
In examining sputum from tuberculous cases, in which the typ-
224
BACTERIA
ical bacilli are not found, it is well to look for the streptothrix by
staining with Gram's stain.
RAY FUNGUS
Actinomyces Bovis.
Ray Fungus.
Morphology and Stains. This organism is called the ray fungus
because of the stellate arrangement of its threads in the colonies
FIG. 68. Actinomyces bovis. (Williams..)
found in tissues. It is of a more complex structure than the bac-
teria hitherto described. There are three elements found in every
colony: (i) long thread which may be branched or unbranched;
(2) threads that are clubbed, which may, or may not, be branched;
(3) spore-like bodies contained within the thread, which seem to
arise by breaking up of the threads. The colonies in tissues are
often i mm. in diameter, and made up of many clubbed-shaped
RAY FUNGUS
225
threads radially situated. Through the periphery and extending
beyond are other unclubbed threads, while scattered throughout
the colony and beyond it, and in the threads, may be seen many
spore-like bodies. The threads and spores stain by Gram's
method while the clubs do not. Basic stains also color all the
elements. The spores do not stain like bacterial endo-spores.
Vital Requirements. It is a facultative anaerobe, and grows
best in the absence of air, at 37C. Resists drying for a long time,
and its thermal death-point is 8oC. after fifteen minutes' exposure.
FIG. 69. Actinomyces. (Williams.)
Chemical Activities. Slowly liquefies gelatine, does not curdle
milk; and produces a mouldy odor. No gas or acids are formed,
nor is H 2 S developed.
Habitat. It has been found in straw and hay, but never in a
healthy body.
Cultures. On gelatine plates it produces yellowish-gray colo-
nies that are very small. These grow into the gelatine, slowly
liquefying it. The colonies are very tough and fibrous. In agar
tubes it grows very slowly, the first growth being like dewdrops;
later these enlarge, turning yellow, and finally brown. The cul-
15
226 BACTERIA
ture grows down into the agar, and the medium darkens. Old
cultures 'are dark and crumbly looking, adhere firmly to the agar,
and have a downy dust-like covering. On blood serum the colo-
nies appear as dewdrops, which later become brownish, then,
yellowish-orange, or brick-red. In bouillon the growth is at the
bottom in ball-like masses that cohere firmly. Clubs do not form
in this medium. The supernatant bouillon is clear, with no sur-
face growth. In milk it produces no chemical change. On
potato it grows in knot-like colonies.
Pathogenesis. Causes in cattle the disease known as "lumpy
jaw." The fungus reaches the jaw from the teeth and gums, the
latter first being injured by sharp spines in the food. In man, the
internal organs, lungs, intestines, and, rarely, the brain become
infected. The liver often is abscessed. In both cattle and man
universal actinomycosis sometimes occurs. The lesions produced
are rather massive at times; the nidus is often surrounded by
enormous numbers of polynuclear leucocytes, which, no doubt,
play a defensive role in the tissues. The disease is often fatal to
cattle and to man. It is hard to inoculate laboratory animals
with the disease, though Wright succeeded in so doing. No
useful immunity reactions seem to occur.- Vaccines have been
used in treatment with encouraging results but no cures. Potas-
sium iodide internally is always indicated.
ACTINOMYCES MADURA
Actinomyces Madura.
Streptothrix Madura, Vincent.
Morphology and Stains. A non-motile, non-flagellated organ-
ism said to have spores. Its growth resembles that of A ctinomyces
boms. It consists of long threads that are clubbed. These stain
by all the basic aniline dyes and by Gram's method. There are
three recognized forms of this organism, white, black and red,
which have been found in various cases but the interrelation of
ACTINOMYCES MADURA 227
which is not yet fully understood. The description given is
generally applicable only slight variations being noted.
Vital Requirements. It is a facultative aerobe. The thermal
death-point for the spores is 85C. for three minutes, and 75C.
for five minutes. Vegetative thread forms die at 6oC. Grows
best at 37C., and scantily at room temperature.
FIG. 70. Streptothrix hominis. (Kolle and Wassermann.)
Cultures. Generates upon all culture media. In Bouillon. It
appears in little clumps which cling to the glass, but eventually
sink to the bottom in masses. In Gelatine. It grows sparingly
in clumps, slowly liquefying the medium. Upon Agar. It forms
shiny round colonies, that are first devoid of color. They resem-
ble an umbilicated vaccine vesicle and adhere tightly to the agar.
In Milk. It grows without coagulating the medium. On
Potato. The culture is very slow, and without chromogenesis.
Old colonies are powdery, due to spores.
Pathogenesis. In man it produces madura foot, an affection
characterized by induration, ulceration, and fistulas formation
with pus.
228 BACTERIA
BLASTOMYCOSIS
OIDIOMYCOSIS
Oidium Albicans. Thrush, Soor. This organism resembles
both a yeast and a mould, because it exhibits characteristics that
are common to both of these forms. It exhibits budding yeast
cells and budding mycelia. The yeast cell is 6ju long and i/* wide,
but the cells vary very much in length and width.
It stains well in tissues and cultures by Gram's method, and by
the ordinary basic stains. It may be cultivated on bouillon,
FIG. 71. Thrush fungus. (Kolle and Wassermann.)
blood serum, agar, potato, etc., and it is rather indifferent to the ;
reaction of the media. It grows best if sugars are present. It is, j
however, very susceptible to such antiseptics as phenol, salicylic
acid, subMmate, etc.
Pathogenesis. Causes in man a condition known as oidio- ,
mycosis, and in young children a very troublesome . stomatitis, :
which, if the child is weak and illy nourished, may result seriously.
It may cause metastatic abscesses in the brain, spleen, and kid-
OIDIOMYCOSIS
229
neys, or nodules in the lungs. This organism may penetrate-
mucous tissues, and fill the lumen of vessels (Virchow).
Tropical spruce is held by some as due to a near relative
of the oidium, namely Monilia psilosis. The organism is to be
found all along the alimentary tract. It possesses many cultural
characters like oidium. Vaccines are said to be of practical use
in therapeutics.
FIG. 72. Doubly contoured organisms found in oidio mycosis (blastomyco-
sis). (From Buschko after Hyde and Montgomery.)
Sporothricosis is a subacute or chronic infection usually of the
skin, but at times involving the internal organs, caused by the
Sporothrix Schencki. The organisms grow as delicate mycelia
with very numerous spores, 2-4 X 3~6/i in size. They grow best
upon acid media at body temperature as white fluffy masses
which later become brown. The disease in man takes the form
of firm tumefactions under the skin which may ulcerate leaving
230 SACTERIA
indolent ulcers. Fever, prostration and emaciation follow. Rats
are susceptible. An agglutinin appears; this may assist in
diagnosis.
Saccharomycetes are sometimes pathogenic. They are bud-
ding fungi, multiplying by splitting off the bud when conditions
are favorable for active growth but capable of intracellular sporu-
lation, ascospores, when under adverse conditions. They usually
have a rather resistant capsule, sometimes double. Saccharo-
myces busse or hominis is capable of setting up in man a cutane-
ous and subcutaneous ulcerative and infiltrative or even suppura-
tive lesion which may last for a long time; involvement of internal
organs can occur. Transmission to animals is difficult. The
organisms are from 3 to 30^, round or elliptical, rarely forming
mycelia in the tissues. They stain well but not by Gram's
method. They are best seen by mixing the pus with a caustic
solution. They grow under aerobic conditions at 37C. upon acid
serum, dextrose or maltose agar as white plaques which later
become wrinkled and velvety. They are easily killed.
Coccidiosis or oidiomycosis is an infection very similar to the
foregoing but the causative organism, Coccidioides immitis, differs
from Sac. hominis in showing intracellular sporuiation and no
budding.
MOULDS OR HYPHOMYCETES
These are the next higher order of plant algae and consist of
cells which can elongate to threads, dividing by intracellular
sporuiation or by the development of reproductive organs which
in some varieties are bisexual in character. They are widely
distributed in nature living mostly as saprophytes. Diseases
due to these forms are practically confined to the skin although
extremely rare cases of dissemination are on record.
Ringworm of all kinds is due to the mould Trichophyton either
of the species megalosporon or microsporon. The spores of the
former are 7~8ju, of the latter 2-3 ju. They grow readily as dis-
MOULDS 231
crete mammillated fluffy colonies. They consist under the micro-
scope of slender septate hyphae.
Favus is due to the mould Achorion Schoenleinii. This fungus
gives off hyphae with knob-like reproductive organs. Spores are
oval 3~8/x X 3~4ju. This fungus grows as a "scutulum" on the
skin eruption. It can be cultivated on sugar agar, as a waxy, or
downy yellow or white round plate with a central mammillation.
Pityriasis versicolor is due to the mould Microsporon furfur.
It is similar to the Trichophyta, but invades only the superficial
layers of the skin.
Aspergillus Niger, A.Fumigatus, and A.Flavus. A polycellular
mycelial organism which produces spores and branched threads,
that are variously named from the macroscopic appearances of
the growth. All thrive well as 37C. and may be cultivated on
the usual culture media. In man, the external auditory meatus
is often infected with these organisms, causing a troublesome dis-
ease. They may infect the lungs of weak anaemic subjects with
wasting diseases, and may be pathogenic for cattle, horses, and
birds.
The author has found that the young hyphae, the sporangiar
and spores of some of these hyphomycetes (moulds) if treated with
hot or boiling alkaline solution of copper sulphate are stained be
the copper, which has an affinity for them, and appear a light lilac-
blue under the microscope. If treated with a solution of ferric
cyanide of potash and acetic acid, these stained parts turn a
dark brown, showing that there is an actual absorption or per-
haps chemical union of the protoplasm of the mould with the
copper. Some moulds are stained a deep blue, and are visible
to the naked eye in test-tubes, after treatment with the boiling
alkaline copper others are colored a bright yellow. Some moulds
and bacteria have the power of reducing copper in Fehling's
"solution.
CHAPTER IX
ANIMAL PARASITES
While numerous diseases are caused by vegetable parasites,
such as bacteria and moulds, there are others in which the etio-
logical role is played by minute microscopic organisms of the
animal kingdom. There are also infectious diseases that are
supposedly caused by animal parasites, and yet the exact knowl-
edge that they are the cause is lacking. Not all of the pathogens
of the animal kingdom will fulfil Koch's postulates but their num-
ber is increasing. Within the past few years it has been found
possible to cultivate Trypanosomata, spirochaetee, amoebae, and
hemosporidia with completipn of Koch's postulates in the first two.
In general, it may be said of animal parasites, particularly those
belonging to the protozoa, that an intermediate host, such as a
suctorial insect, is necessary for the transmission of the organism
to man or animal. This is called alternate generation and is a
very characteristic feature.
The protozoa, as parasites in man, are the cause of several well-
known diseases, namely: dysentery, malaria, sleeping-sickness,
and coccidiosis. In hydrophobia, scarlet fever, and small-pox cer-
tain peculiar bodies are constantly found that resemble protozoa,
but since it is not known whether they are animal bodies at all,
they cannot be classed as protozoa, and are discussed under
Chlamydozoa in the next chapter.
PROTOZOA
The protozoa of importance as disease producers are to be found
in the classes, orders and families given as follows:
232
PROTOZOA 233
Protozoa.
Sarcodina.
Rhizopoda.
Amoebina Amoebae.
Mastigophora.
Flagellata.
Monadida, Cercomonas, Trypanosoma, Poly-
mastigida, Trichomonas.
Some authors separate a family Spirochaetidae to in-
clude Spirochaeta and Treponema.
Sporozoa.
Gr egarinida gr egarines .
Coccidia coccidia.
Hemosporidia.
Plasmodium malaria.
Infusoria.
Cilia ta.
Heterotrichida Balantidium.
The protozoa are always, in every stage of development, primi-
tive unicellular bodies. They consist essentially of a cell body or
sarcode, a nucleus, and a nudeolus. All of the vital functions of
the cell are carried out by the cell body, the protoplasm of which
digests and assimilates food. Particular parts of the protoplasm
have special functions, these parts are called organdies. The
living protoplasm is finely granular, is viscid, and exhibits a dis-
tinct movement. The motility of protozoa is supplied variously.
In the Rhizopoda progression takes place by pseudopods or false
feet, a phenomenon in which a section of the cell wall and proto-
plasm are extended like a bud. Into this the latter then flows
with a shrinkage of the main body. At last the pseudopod is
large enough to- hold all the protoplasm and the former place of the
protozoon is vacated for the new. Motility is also supplied by
the lashing or vibratory action of flagella or the fine vibration of
234 ANIMAL PARASITES
circumferential cilia. In others a special muscular segment
of the body may exist. The suctorial tubes act also for motion
at times. In most protozoa two layers can be seen the ectosaro,
and endosarc. The ectosarc originates the movement, is concerned
in the ingestion and excretion of food, and the respiration. The
endosarc, which circulates slowly, is mainly for digestive purposes.
In it are ferments, crystals, food particles (seen in the food vacu-
oles), oil globules, gas, and pigment granules.
Flagella and suctorial tubes in protozoa that have them
belong to the ectosarc. Skeletal tissues, shells, etc., also belong
to this layer.
The food consists of bacteria, smaller animals, algae, and animal
waste.
Propagation is effected by direct cell division, beginning in the
nucleus, by cell budding or by a complicated course of sporulation
which may be sexual or asexual. Sometimes division, or budding,
occurs rapidly without the segments separating, leading to the
formation of protozoal colonies, or swarm spores.
In the case of the malarial plasmodia, asexual development,
(schizogony) takes place in man's blood, while the sexual develop-
ment (sporogony) takes place in the mosquito. Protozoa are
found in salt and fresh water, in damp places, and in animals as
parasites.
Since the zoological classification has been given and may
used for reference to larger works, the various pathogenic proto-
zoa are given separately without direct reference to their sys
tematic classification.
There are but two Rhizopods that are parasitic and pathogenic
to man. The only one of these of any import is the Amoeba.
AMCEBA DYSENTERIC OR ENTAMCEBA
HISTOLYTICA
This is a pear-shaped roundish body from .008 to .05 mm. ii
diameter. The ectosarc is easily discernible in the pseudopodia,
AMCEBA DYSENTERIC 235
but not in the round quiescent cell. In the endosarc, which is
granular, vacuoles are easily seen; so are fragments of food, red
and white blood cells, bacteria, eipthelial cells, and faecal matter.
The pseudopodia are broad and lobose; one or two are protruded
at a time. The motion of the organism depends upon the reaction
of the media, and the temperature. The vacuoles and nucleus
are always present. Propagation generally takes place by binary
division, the process beginning in the nucleus. When irritated,
the amoeba at once assumes a spherical form, the pseudopodia
being withdrawn.
Pathogenesis. Amoeba dysenteriae is the cause of the protozoal
form of dysentery. So far as known this particular variety exists
only in the intestines of effected persons. Lesions similar to those
of human dysentery have been produced in monkeys, dogs and
cats, and the amoebae recovered from them. Cultures consisting
only of amoebae have been obtained by special technique, but a so-
called pure mixed growth of colon bacilli and amoebae is cultivated
with little difficulty. In the lower gut of man and cats, in
dysentery cases, encysted amoebae are often found. They have
been seen in the liver (in old cases), also in the lungs and sputum.
Cats have been infected by pus from liver abscessed devoid of
bacteria (Kartulis). The urine, in cases of cystitis, contained
amoebae, and it is believed to be the cause of the disease in some
rare instances. In dysentery the amoebae are the cause of the
necrosis and ulceration, as they frequently become encysted in
the submucous tissues. From the Entamceba coli the dysenteric
amoebae is differentiated by the fact that it is larger, coarser in
structure, and takes up red blood cells, which the former does
not. Differentiation by Wright's stain Entamceba coli ectoplasm
light blue, endoplasm dark blue, nucleus red. Ent. histolytica
ectoplasm dark blue, entoplasm light blue, nucleus pale red or
pink.
Amoebae quickly loose their mobility as the surrounding tem-
perature leaves that of the normal body, although they are not
236
ANIMAL PARASITES
killed at low temperatures, and resist up to 6oC. when in the
encysted state. Quinine, permanganate of potash, weak acid and
silver nitrate are quickly fatal to vegetative forms. Emetin is
a useful drug since it kills all but encysted forms after a very
short exposure if direct.
Entamceba tetragena, formerly classified as a separate variety,
is now generally believed to be but one stage in the development
of Ent. histolytica.
In stools (from dysenteric cases) over a day old, amoebae are
not often found, as they undergo a rapid disintegration outside
the body.
Amoebae are cultivated upon stiff agar preferably with defibri-
nated blood and in company with bacteria. If a colony can be
obtained free of bacteria, development will continue on agar
FIG. 73. Entam&ba tetragena. The same living individual drawn at brief
intervals while moving. (From Doflein after Hartmann.)
smeared with organ extracts. The addition of dead bacteria to
culture media seems favorable to their development. The poisoi
is not known. The free amoebae in the colon are easily killed, bul
when encysted are more resistant. Quinine is fatal to cultures ii
ten minutes in strength of 1-2500. Formalin is not practicable.
Endamoeba buccalis is found in the mouth especially in carioi
FLAGELLATA
237
teeth and in inflamed gums; it may have some effect in con-
tinuing a gingivitis.
FLAGELLATA
The flagellata derive their name from the fact that all are pos-
sessed, at some time in their existence, of flagella, which are not
only organs of locomotion, but serve to apprehend food.
The principal members of this class of interest from a patho-
logical viewpoint, are the trypanosomes. Trypanosoma gam-
FIG. 74. Trypanosome in rats' blood. (Williams.)
biense, transmitted by the tsetse-fly Glossina palpalis, pathogenic
for man (see page 239). The Trypanosoma brucei, which causes
the tsetse-fly disease (nagana) in horses and cattle, is transmitted
to cattle by the bite of the tsetse-fly, Glossina morsitans. It can
be grown on blood agar (Novy).
Trypanosoma evansi causes surra, a disease of horses in Central
Asia transmitted by a fly of the genus Stomoxys.
238 ANIMAL PARASITES
Trypanosoma equiperdum causes a sexual disease in stallions
and mares called dourine; this is akin to syphilis in man.
Trypanosoma lewisi of rats is transmitted from animal to
animal by means of fleas.
Trypanosoma noctuae. A parasite of the little owl, which is
introduced into the bird through the bite of the mosquito Culex
pipiens.
Several other forms have been noted but these are perhaps
the most important and serve as examples for this family of
protozoa.
Trypanosomes are elongated fusiform bodies pointed at both
ends, provided by a fin fold, or undulating membrane, running
along the dorsal edge and forming frill-like folds which terminate
in a whip-like extremity or flagellum.
A large nucleus is always seen, also a centrosome, a small chro-
matic mass likewise called a blepharoplast near one pole.
The flagellum is at the anterior extremity; the blunt pointed
end is the posterior extremity. Cell division begins in the bleph-
aroplast, the cell dividing longitudinally, the nucleus, flagellum,
and the protoplasm dividing last. Dividing trypanosomes fre-
quently appear in clumps with the ends. united, resembling a
wheel.
The trypanosomes exist in two hosts one a suctorial insect
and have a sexual and an asexual existence (alternate generation).
In an infected owl the organism has been observed clinging fast
to the red cells, absorbing nutriment during the day, while at
night it swims about freely in the plasma.
In owl's blood the trypanosome assumes asexual forms, called
macro gametes. These macrogametes penetrate the erythrocytes,
accumulating the remnants of the red cells in the protoplasm.
The nucleus of the trypanosome^ may be seen in the interior of
the protoplasm. The microgametocytes arise from the asexual
forms and when mature, give rise to eight microgametes.
TREPANOSOMA GAMBIENSE 239
TRYPANOSOMA GAMBIENSE
Castellan! found that this trypanosome is the cause of sleeping-
sickness among the natives of South Africa, and the organism has
been found quite regularly in the blood, and also the cerebro-
spinal fluid sometimes as well, in this disease. The disease has a
long period of incubation (months), runs a long course usually,
and, at its full development, it is a meningo-encephalomyelitis.
This is characterized by hebetude, somnolence, and coma.
FIG. 75. Trypanosomes; showing ordinary structural appearance on left;
in middle a trypanosome undergoing division; on the right a group dividing
in radial manner. (Tyson's Practice.)
These symptoms are accompanied by disturbance of the motor
apparatus, oedema, irregular temperature, rapid pulse, emaciation,
skin eruptions, and death in coma. In these cases the parasites
may be seen in the blood slowly winding their way through the
corpuscles. The pathogenic action is due no doubt to some
toxin elaborated.
The disease is transmitted from man to man by the tsetse-fly
(Glossina palpalis). In the fly it exists as a true parasite in a
host, and not merely passively. It becomes infective within
three days of biting and remains so for four weeks.
The disease does not depend upon the age, sex of the individual,
nor upon drinking water, food, seasons, etc.
The organism may be stained by the ordinary blood stains,
240 ANIMAL PARASITES
mixtures such as Irishman's, Romano wsky's, etc., the nucleus,
centrosome and flagella, staining deepest. Thus far the T. gam-
biense has not been cultivated in artificial media.
Novy has succeeded in growing the T. lewisii and T. brucei on
agar mixed with defibrinated rabbit's blood. These are the first
animal parasites to be cultivated artificially.
Trypanosomiasis of South America is not unlike sleeping-sick-
ness of Africa. It is caused by Tr. cruzi, a parasite of eight spores
developing in organs, serum or red cells. It is transmitted by
Conorrhinus megistus, a large insect.
In Dum Dum fever or Kala Azar, a disease characterized by
wasting, anemia, fever and splenomegaly occurring in India,
curious bodies, called Leishmann-Donovan bodies, have been
found. These resemble the malarial plasmodia roughly, and if
cultivated on blood agar elongated herpetomas-like bodies with-
out undulating membranes will develop. They are to be found
in the juice obtained by splenic puncture lying within cells, espe-
cially endothelium and large lymphocytes; on rare occa-
sions they have been met in the blood. The transmission is
not certainly known but may be by the bed-bug or by fleas.
Trichomonas vaginalis and intestinalis. are flagellates which
are apparently able to set up some inflammatory irritation in the
places indicated by their special names.
TREPONEMA PALLIDUM (Schaudinn)
(Spirochaeta Pallida.)
Treponema Pallidum. There has been some discussion as to
the proper classification but now this organism is usually placed
among the Flagellata, genus Treponema. It does not possess an
undulating membrane, is flagellated, is of stiff and regular shape,
and multiplies by longitudinal division.
Morphology and Stains. This organism is extremely delicate
in structure, from 4 to 14/4 in length and about .3/4 in width; has
TREPONEMA PALLIDUM 241
from 3 to 12 turns or bends, and its ends are delicately pointed.
Its curves form a large arc of a small circle; the Sp. refringens
curves form a small arc, frequently irregular, of a larger circle.
It multiplies by both transverse and longitudinal division. As
this organism is stained with difficulty it requires a special one,
that of Giemsa yielding the best results. Aniline gentian violet,
Romanowsky's, and Leishman's stains also color it. It may be
stained in tissues by silver and pyrogallic acid methods.
Habitat. It has not been found in tissues of normal persons, or
those ill with carcinoma, tuberculosis, etc., but only in the tissues
of individuals suffering with syphilis. It is a strict parasite.
Vitality. The organism is readily destroyed by the ordinary
disinfectants and dies, after a few minutes' exposure to 5oC.
The Treponema pallidum has now fulfilled the postulates of
Koch. It can be cultivated from human lesions (with some
difficulty to be sure), it can be implanted in animals (monkeys
and rabbits) and there reproduce syphilitic lesions; and it can be
re-cultivated from them. In these experimental diseases it re-
tains the proper morphology. According to Noguchi there are
two types, a slender and a stout, which breed true to these charac-
ters and correspond to slight pathogenic variations. Noguchi
succeeded in cultivating the Tr. pall, in pure culture by using
the juice from human or monkey's lesions or from the syphilitic
orchitis of rabbits. This he grows in serum water or serum agar
to which has been added fresh tissue of rabbit. The organism
grows as fine fibrils in arborescent colonies. These can be selected
pure by cutting the tube and the agar column. Motion is of
screw and serpentine character. No odor or spores are produced.
This organism must be imagined and remembered as a corkscrew
and not a waving line. The Gram stain is negative.
From the cultures of this organism a toxic extract can be ob-
tained which, when rubbed into the skin of a syphilitic in the
late stages, gives a typical skin reaction, luetin and the luetin
reaction.
16
242 ANIMAL PARASITES
The Spiroch&ta refringens, which has been also cultivated by
Noguchi and thought by him to be a Treponema also, grows with-
out fresh animal tissue in a short time and produces no odor.
Pathogenesis. It has been found in chancre, condylomata, and
mucous patches in the early stages of syphilis; also in the blood,
blister-fluids, spleen, bone marrow, liver, thymus gland, and
FIG. 76. The Spirochaeta refringens is the larger and more darkly stained
organism, while the lightly stained and more delicate parasite is the Spiro-
chaeta pallida (Treponema pallidum). From a chancre stained with Wright's
blood stain. (Hirsch by Rosenberger.)
lymphatic glands, and in the brain and cord of taboparetics.
Associated with this organism, in nearly every case, is a coarse-
looking larger spirochaeta (Treponema), which stains deeper, and
has been called the Spirochaeta (Treponema) refringens (q.v.).
In a series of experiments, Metchnikoff and Roux caused abor-
tion of the chancre following inoculation of syphilitic virus on the
eyelid of a chimpanzee, by calomel inunction carried out less than
one hour after the infection; a solution of sublimate has not the
same prophylactic property.
RELAPSING FEVER ORGANISM 243
It does not require any intermediate host for transmission as
do the recognized animal parasites of malaria and filariasis, etc.
Treponema pertenue is the organismal cause of Frambcesia
or Yaws, a cutaneous and general infection of the tropics similar
to syphilis. It is about the size and shape of the syphilis spiro-
chete but is distributed differently in the human lesions, being
more in the outer skin and less in the vicinity of blood-vessels.
It has not been cultivated.
Spirochaeta nodosa (Huebner) or icterohemorrhagica (Inada)
is believed to be the cause of Weil's disease. The spiral, a tiny
organism about 5 micra long is found in the blood, liver and
kidneys. It is transmissible to guinea pigs. Rats are supposed
to be the means of transfer since several varieties probably
harbor the parasite. Excretion of the organism takes place via
the kidneys. Spirocheticidal substances occur in the blood in a
favorably progressing attack.
Spirochseta morsus muris and Sp. muris ratti are supposed
to be the cause of rat bite fever. These organisms, resembling
but grosser than the Treponema pallidum, enter with a rat bite
and can be found in swollen drainage lymph nodes.
RELAPSING FEVER ORGANISM
European Relapsing Fever. Caused by Treponema of Spiro-
chseta obermeieri.
African Relapsing Fever. Caused by Trep. or Sp. duttoni,
transmitted by tick Ornithodorus moubata.
American Relapsing Fever. Caused by Trep. or Sp. Novii.
Bombay Relapsing Fever. Caused by Trep. or Sp. carteri.
The transmission of the first, third and fourth, while not definitely
known, is probably by a louse; ticks may also be responsible.
Morphology. They have lately been cultivated and retain
somewhat of their virulence for monkeys and rodents. They are
elongated, flexible, corkscrew-like, serpentine and vibratory in
244 ANIMAL PARASITES
motility, and do not form spores. There is a single terminal
flagellum. They are stained with reasonable ease by plychrome
methods, especially Giemsa, but not by Gram's method. They
measure from 10 to 40/1 in length and about i/* in breadth.
Coils vary from 6 to 20. The American type is smaller than the
rest.
FIG. 77. Spirilla of relapsing fever from blood of a man. (Kolle and
Wassermann.)
Transmission. The known tick which transmits these organ-
isms becomes infective in one week after biting a patient and
remains so all its life; its young are also infective. The types of
disease vary but little. In all these is a relasping fever with
periods of apyrexia in between. During the fever the spirochsetes
are swimming free in the blood and disappear in the afebrile
interval.
Cultivation. They are cultivated in the manner given for
Trep. pallidum by Noguchi, by adding citrated, therefore de-
fibrinated, blood to serum or ascitic-fluid-fresh-tissue-agar. They
breed true to type. They remain alive several days under
favorable artificial conditions but cannot be cultivated after
SPOROZOA 245
they have left the body a few hours without being on suitable
culture media.
The periods of fever last from five to seven days, when a crisis
occurs. After an apyrexial period the fever recurs. The spiro-
chaetae are found in great numbers in every microscopical field.
In the apyrexial period the spleen becomes engorged and the
leucocytes devour the parasites. Monkeys with excised spleens
are more susceptible to infection than others.
Immunity. The blood from rats that have been immunized
by repeated injections of blood from spirochetal rats, if injected
into other rats, is capable of conferring an immunity on them by
causing spirochaetes to disappear from their blood. One attack
seems to confer immunity to the special form causing it but
probably not to the others.
SPOROZOA
The most important of this family are the malarial parasites
(which belong to the order Haemosporidia), and the Coccidia.
In general the sporozoa are unicellular organisms that lead a
parasite existence in the tissues, especially cells, of higher ani-
mals. They ingest liquid food, have no cilia in the adult stage,
and flagella are possessed only by the males. There may be one
or more nuclei. Propagation is effected by spores, but budding
and division do occur, though rarely. Alternate generation
takes place frequently.
MALARIAL PARASITES
Haemosporidia of Man. The most important disease caused
in human beings by the haemosporidia is malaria, or ague, and
excepting the deserts, mountains, and arctic regions, this disease
is very widely distributed.
Three different parasites producing different clinical entities
are known. According to the time, frequency, and order of the
246 ANIMAL PARASITES
outbreak of chills and fever, various clinical names have been
given to the manifestation of the disease. Mannaberg has
arranged the following scheme to show the different forms of
outbreaks. The numbers apply to the paroxysms. Each
developmental cycle is numbered alike:
i i i i i i i. Simple quotidian fever.
I o i o i o i Simple tertian fever.
looiooiooi. Simple quartan fever.
12121212. Double tertian fever. (Two infections.)
.123123123. Triple quartan fever. (Three infections.)
120120120. Double quartan fever. (Two infections.)
The figures refer to days on which paroxysms of fever occur.
The o represents the afebrile day.
PLASMODIUM MALARIA (Laveran)
This is the quartan parasite, and produces in man, in cases of
one infection, paroxysms of fever every fourth day.
It appears in the blood, after a paroxysm, as a small non-pig-
mented body on the bodies of the red blood cells. It has feeble
amoeboid motion; slowly penetrates the corpuscle, and specks of
melanin appear in its protoplasm. Forty-eight hours after the
attack the parasite measures from one-half to two-thirds the
size of the red cell. Sixty hours after the paroxysm twelve
before the next the parasite completely fills the red cell, leav-
ing x only a narrow rim, which later on disappears. Six hours
before the next paroxysm, shizogony begins. The grains of
melanin are arranged like the spokes of a wheel, and then, leaving
the radii, crowd above the centre (the rest of the cell being pig-
mentless) gradually dividing into 8 or 12 pear-shaped bodies, or
merozoites. These separate from each other and individually
attack a fresh red cell, and this attack brings another paroxysm
of fever seventy-two hours after the previous one. The grains
PLASMODIUM VIVAX 247
of pigment are taken up by the leucocytes and deposited in the
spleen and bone marrow.
The nucleus of the parasite may be seen if suitably strained.
The double or triple quartan is explained by the fact that there
are two or three groups of organisms that undergo sporogony at
periods separated from each by twenty-four hours.
PLASMODIUM VIVAX (Grassi)
The cause of tertian fever occurring in the spring. It differs
from the Plasmodium malaria because of shorter period (forty-
eight hours) consumed in schizogony (or sporulation), the much
greater activity of the amoeboid movement, and the affected cor-
puscles becoming enlarged; also by the fact that many of the
melanin-bearing stages are visible. The shizogony is rarely
apparent in the circulating blood, but in the spleen these stages
are easily seen. There are from 15 to 20 merozoites (segmented
bodies or spores) which are arranged in an irregular heap, but not
radially like wheel spokes. The merozoites are smaller than the
quartan variety and are more numero'us. The flagellated form
can but rarely be seen in the freshly drawn blood. If some blood,
containing the large extracorpuscular bodies, is put in a moist
chamber, they throw out flagella. These flagella are really
micro gametes and are sexually active. The extracorpuscular
bodies are partly macro gametes , and if they become flagellated
they are called polymites, and are the micro gametocytes. The
merozoites, or spores, finally burst forth from the erythrocytes,
starting again another cycle (attended with a paroxysm of fever) .
These spores appear in the freshly invaded corpuscles as hyaline
bodies with slight movement. As they grow in size, pigment
appears in the protoplasm. Certain of these do not break up
into merozoites, or spores, but become extracellular bodies
gametocytes of male or female character. There may be two
infections in which schizogony occurs every other day in alternate
days, 12121212.
248 ANIMAL PARASITES
PLASMODIUM FALCIPARUM
The plasmodium of aestivo-autumnal fever, or pernicious ma-
larial fever, also called tropical. The outbreaks of this occur ir-
regularly. The disease produced by them is very much more
malignant and is harder to cure. The young spore appears in
the corpuscle as a small hyaline body, smaller than the other
forms and much more active. The size and shape of the red cells
are little if any altered but they become granular and polychro-
matophilic. The pigment is very finely granular and the body
frequently presents the signet-ring appearance. There may be
more than one parasite to a red cell. The cycle of development
(schizogony) is twenty-four to forty-eight hours. The plas-
modium in its schizogony divided into 8 to 24 merozoites or spores,
and are arranged in a spore-like form. The extracorpuscular bod-
ies may resemble a crescent or sickle; this form is very character-
tic of aestivo-autumnal fever. There are two forms of these
crescents, one delicate, the male, and one larger and ovoid, the
female. They are very resistant to quinine and persist for a long
period in the blood. Plasmodia undergoing schizogony are often
found in the brain capillaries after death, which accounts for the
cerebral symptoms in such cases. This form can be differentiated
from the others by the irregular and pernicious type of fever pro-
duced; by its great resistance to quinine; the fewer number of
merozoites; the finely granular appearance of the pigment; the
relatively small size of the young intracorpuscular body; and,
by the ring shape of some of the young forms.
Often, in blood from malarial cases, pigmented leucocytes are
seen, and ghost, or shadow, red corpuscles from which the haemo-
globin has been dissolved are often met with. Spherical extra-
corpuscular bodies become flagellated (gametes) in freshly drawn
blood. The parasite may be studied in fresh film preparations
and by staining dried films by methylene blue and eosin, Roma-
nowsky's, or Jenner's methods. They are much more frequent
in the pyrexial period, and when quinine has not been given.
PLASMODIUM FALCIPARUM 249
The various plasmodia are transmitted to man invariably by
the anopheles mosquito, in the bodies of which they undergo a
different (sexual) existence. It has been positively demonstrated
that the various plasmodia undergo an alteration of generations
and require two different hosts for their development, i.e., mos-
quito, man.
The asexual development, or schizogony, takes place in the
blood of man, the sporogony, or sexual development, in the body
of the anopheles mosquitoes, the bite of which sets up an infec-
tion in man, since the sporozoites of the various plasmodia are
developed in the salivary glands of these mosquitoes. In the
act of biting, the sporozoites reach the erythrocytes where they
become the intracorpuscular hyaline bodies beginning again their
asexual cycle of development in the blood.
That the mosquito is the intermediate host of the malarial para-
site and that the infection in man follows bites by infected mos-
quitoes has been abundantly proven. The mosquitoes that act
in this way are the various Anopheles; the Anopheles maculipennis
being the offender most frequently. The freshly formed schizonts
in the blood of an infected man are conveyed into the intestines
of the mosquito. Here sexual reproduction of the parasite begins.
The male elements, flagellar microgametes penetrate the female
elements, macrogametes (cellular), and after a time there appear
intra-cellular fusiform bodies, ookinets. These bore into the
intestinal walls of the mosquito and there remain. After a time
they are converted into round bodies, or ob'cysts. The nucleus
of the oocysts divides rapidly and other daughter nuclei are formed
and new cells called sporoblasts. After about eight days these
form the sporozoites. The number of sporozoites in each oocyst
varies from hundreds to many thousands (often 10,000). These
oocysts burst and the- sporozoites in the circulation find their
way to the salivary glands of the mosquito. When a mosquito
bites a human being they are introduced into the blood where
they are quickly transformed into the intracellular hyaline bodies
DESCRIPTION OF FIG. 78
Life history of malaria parasite, Plasmodium. i, Sporozoite, introduced
by mosquito into human blood, the sporozoite becomes a schizont; 2, young
schizont; 3, young schizont in a red blood corpuscle; 4, full-grown schizont;
5, nuclear division; 6, spores, or merozoites, from a single mother-cell; 7, young
macrogamete (female), from a merozoite, and situated in a red blood cor-
puscle; 7a, young microgametoblast (male); 8, full-grown macrogamete; 8a,
full-grown microgametoblast; 9, mature macrogamete; ga, mature micro-
gametoblast; 96, resting cell, bearing six flagellate microgametes (male);
10, fertilization of a macrogamete by a motile microgamete; the macrogamete
next becomes an ookinete; n, ookinete, or wandering cell, which penetrates
into the wall of the stomach of the mosquito; 12, ookinete in the outer region
of the wall of the stomach, i.e., next to the body cavity; 13, young oocyst,
derived from the ookinete; 14, oocyst, containing sporoblasts, which develop
into sporozoites; 15, older oocyst; 16, mature oocysts, containing sporozoites;
17, transverse section of salivary gland of an Anopheles mosquito, showing
sporozoites of the malaria parasite in the gland cells surrounding the central
canal.
1-6 illustrate schizogony (asexual production of spores); 7-16, sporogony
(sexual production of spores).
(FOLSOM After GRASST and LEUCKART, by permission of Dr. Carl Chun.)
MALARIAL PARASITES
16
FIG. 78.
252
ANIMAL PARASITES
and begin their asexual sporogony in the blood. Each develop-
mental cycle causing a febrile paroxysm either every day or alter-
nate days, or on every fourth day, etc., depending on the character
of the organisms and the number of infections. To prevent
FIG. 79. Coccidium ho minis, from intestine of rabbit: i, a degenerate epi-
thelial cell containing two*coccidia; 2, free coccidium from intestinal contents;
3, coccidium with four spores and residual substances; 4, an isolated spore; 5,
spore showing the two falciform bodies X 1140. (From Railliet, in Tyson's
Practice.)
spread of malaria, mosquitoes must be prevented from reaching
individuals infected with malaria and those not infected. Screens
accomplish this best. The larva of the mosquito develops in
stagnant water. To prevent the development of these young
mosquitoes oil should be poured on the water, thus cutting off
the air and means of respiration.
COCCIDIUM 253
Bass, of New Orleans, claims to have successfully cultivated
malarial plasmodia of the species vivax and falciparum by the use
of human blood. He has also succeeded when using Locke's fluid
minus calcium chloride plus ascitic fluid. One-half percent dex-
trose is usually added. The blood is drawn, so that it can be
defibrinated, into small flat-bottom tubes. These are incubated
at 4oC. The column of fluids is 1-2 inches high, the clear serum
layer being % inch at least. The parasites grow in the upper
layer of the cellular sediment. Undiluted serum and leucocytes
are lytic for plasmodia. For renewed cultures these must be
removed but uninjured red cells must be added. Only the asexual
division has been observed. Leucocytes phagocyte pll free
parasites under artificial conditions.
COCCIDIUM
Coccidium hominis is another member of the sporozoa that occa-
sionally infects man. Coccidia are infectious also for horses,
goats, oxen, sheep, pigs, guinea pigs, weasels and rabbits. The
organism is essentially a cell parasite inhabiting the cells of the
gastro-intestinal tract by preference, chiefly the liver and intes-
tinal mucous membranes. They lead a sexual and asexual
existence like the malarial parasites (alternate generation). The
young sickle-shaped nucleated sporozoite penetrates an epithelial
cell, where it gradually develops, ultimately dividing into numer-
ous sporozoites. This is the asexual stage of development
(schizogony) , the sexual stage being called sporogony.
The sporozoites are differentiated into the two sex elements.
These are large granular appearing cells; the male being smaller,
divides into numerous flagellated microgamates that penetrate the
female granular cells, macrogametes, and fertilize them. These
fertilized macrogametes, or zygotes, form capsules and become
oocysts which divide into numerous sporoblasts, changing into
sickle-shaped sporozoites upon liberation.
254
ANIMAL PARASITES
The coccidia are easily demonstrable in tissue and in faeces.
They produce in man occasionally a fatal disease infecting the
liver and intestines. Cattle sometimes die from haemorrhagic
FIG. 80. Development of coccidium cuniculi: a, b, c, young coccidia in epi-
thelial cells of gall duct; d, e,f, fully grown encysted coccidia; g, h, i, k, /,
showing development of spores; m, isolated spore, greatly magnified, showing
the two falciform bodies (pseudonawcdla; sporozoites} in natural position; n, a
spore compressed so as to separate the two srjorozoites, o, a sporozoite or
falciform body with y, its nucleus. (From Railliet after Balbiani in Tyson's
Practice.)
dysentery due to one of the coccidia. The disease is transmitted
by the ingestion of food contaminated by faeces containing the
sporozoites.
Acid f uchsin stains the sporozoa.
CHAPTER X
THE FILTERABLE VIRUSES
This general term means that the virus of a disease can pass
through a porcelain filter and usually that it cannot be seen by the
microscope. It, however, does not mean that it is invisible at all
stages since in one case at least we have been able by means of the
ultramicroscope to see what is almost certainly the particular
causal agent. Again it is said that spirochaetes when young will
traverse porcelain niters. The term will cover in this chapter
those diseases of importance to man whose causal agents cannot
be morphologically described, but whose characters are more or less
well known. The list of diseases caused by submicroscopic agents
is as follows: African horse sickness, swamp fever of horses, catar-
rhal fever of sheep, yellow fever, Dengue, three-day fever, typhus
fever, poliomyelitis, rabies, variola, with its congeners vaccinia
and animal pox, hog cholera, foot and mouth disease, fowl plague,
fowl diphtheria, transplantable sarcoma and leukemia of fowls,
cattle plague, trachoma, pleuropneumonia of cattle, molluscum
contagiosum, measles, scarlet fever, guinea-pigs epizootic and
some diseases of plants. As said above, only the diseases trans-
missible to human beings are reviewed.
Some of the above diseases, notably rabies, scarlatina and tra-
choma, show in the leucocytes and epidermal cells certain struc-
tures or inclusion bodies to which the name Chlamydozoa was given
by Prowaczek, a term implying that a parasitic body is growing in
a mantle. They start as tiny specks in the cytoplasm shortly
found to be surrounded by a clear, sharply outlined halo. They
seem to grow at the expense of the host cell. Their exact char-
acter is not understood; they are probably evidences of cellular
degeneration under the influence of some noxa.
255
256 THE FILTERABLE VIRUSES
Hydrophobia. This disease has long been considered to be an
infectious one, but the causal parasitic agent has never been dis-
covered. It is commonly found in dogs, cats, wolves, rabbits, etc.,
but other domestic animals, and man may become infected. It is
a disease of the central nervous system, highly infectious, always
following a bite or other injury in which the skin is broken, and
in which lesion the virus may be deposited. Infection may be
caused by injecting emulsified infected nerve tissue (brain) into
susceptible animals (rabbits or monkeys). The disease is always
fatal after it is well established. Well-marked histological lesions
of the central nerve tissues, particularly the large ganglia, have
been found by Van Gehutchen and Nelis, and Ravenel and Mc-
Carthy. If emulsified brain tissue from an animal that has died of
hydrophobia is filtered through a "germ-proof " filter the nitrate is
capable of setting up the disease in a healthy animal if it is injected
into it. By long centrifugation of emulsified infected brain tissue,
the supernatant fluid loses its power of reproducing the disease on
injection. Virus may also be found in mammary and lacrymal
secretions, pancreas, cerebro-spinal fluid and aqueous humor.
The organism is toxic in character, since filtrates sometimes fail
to produce transmissible disease, but emaciation, paralysis, and
death are caused by their injection into rabbits, the tissues of
which, in turn, are not infectious.
The unknown organisms are rather resistant to agents that are
germicidal. They are destroyed in fifty minutes by a 5 percent
carbolic solution, and in three hours by a 1-1,000 corrosive subli-
mate solution. Direct sunlight kills them quickly, as do radium
emanations. The latter have been used -as curative measure
with reputed success. A temperature from 52 to 58C. for one-
half hour destroys them, but they resist extreme cold of liquid air
(312) for many weeks. Pasteur found that desiccation attenu-
ated the virus. Chlorine kills it quickly, while glycerine does not.
The virus may be increased in virulence by passing the " street
virus" of dogs through a series of rabbits. Here the period of
HYDROPHOBIA
257
incubation decreases from three weeks to six days, but beyond
this the period does not become less, and the degree of virulence
from the virus lead Pasteur to name it virus fixe (fixed virus).
Passing the virus through foxes, cats, and wolves also intensifies
the virulence, while monkeys and chickens attenuate it.
FIG. 81. Section through the cornu ammonis of brain of a rabid dog;
lined by the method of Lentz. Five Negri bodies of different sizes are
>wn, enclosed within the ganglion cells. The smallest contains only three
tiute granules. (After Lentz, Centralbl f. Bakt., 1907, Abt. I, Vol.XLIV,
> 378.)
Negri bodies, intracellular bodies discovered by Negri, are
>und in the ganglionic cells of rabid animals. These bodies stain
>y eosin, and are from i to 27;* in size, being generally about s/x.
258 THE FILTERABLE VIRUSES
They are found particularly in the cornu of Ammon; in Purkinje's
cells in the cerebellum; and in the larger cells of the cortex of the
cerebrum. These may be the cause of the disease, but there are
several objections to this hypothesis. Their distribution does not
correspond to the parts of the nervous system that are most
intensely affected in hydrophobia, i.e., medulla and pons. In the
latter locality these bodies are rarely encountered. They are not
found invariably in animals dead from rabies, and are considered
to be too large to pass through a Berkefeld filter; this latter view
may not be a correct one. The finding of these bodies has been
considered by Negri to be good grounds for considering the case
to be hydrophobia. The rapid diagnosis of the disease in animals
can only be effected by killing them and examining the nervous
tissues, or inoculating other animals with them. Histologically,
three marked changes may be noted: (i) The finding of the Negri
bodies. (2) The finding of the degeneration of the cells of the
larger ganglia with the proliferation of the endothelial cells lining
the ganglionic spaces (Van Gehutchen and Nelis). (3) The find-
ing of certain tubercles in the medulla, which are called Babes
tubercles, though these are not wholly characteristic, as they are
found in other diseases. Hydrophobia is transmitted from the
site of the wound to the central nervous tissues by the nerves, and
the incubation period varies with the distance of the wound from
the central nervous system; the majority of cases occur between
twenty to sixty days after a bite.
Immunity against infection and the development of the disease
after the reception of an infected wound, may be accomplished by
Pasteur's method (see chapter on Vaccine).
Yellow Fever
That this disease is caused by a parasite there can be no doubt.
It is highly infectious and largely confined to the tropical regions'
of the western hemisphere and in parts of Africa. Resembling
diseases established as due to protozoa, this one is unquestion-
YELLOW FEVER 2 59
ably spread by mosquitoes, and it has been definitely determined
by Carrol and Reed that the female Aedes capolus (formerly
called Stegomyia fasciata and St. calopus) is the means of its
propagation. Carrol believes that the undiscovered parasite of
yellow fever is of the animal kingdom, for the following reasons:
(i) It is absolutely necessary for its continued existence that it
undergoes alternate generation in man and in the Stegomyia
mosquito. This is peculiar to the sporozoa. (2) The fact that
twelve days must elapse before the mosquito is capable of infect-
ing man is evidence that a cycle of development of the unknown
parasite is taking place in the mosquito. (3) The limitation of the
cycle of development of the parasites to a single genus of the
mosquito and to a single vertebrate (man) conforms to a natural
zoologic law, and this does not conform to our knowledge of the
life history of bacteria. (4) The effects of climate and tempera-
ture on the life history of the Stegomyia, and on the rate of
development of the parasites in the bodies of the mosquitoes are
exactly the same as the effects of the same conditions on
the anopheles mosquito and the malarial parasite. Without the
Stegomyia there can be no yellow fever. Infection requires the
fulfilling of the following conditions: (i) By the bite of the mos-
quito providing the insect has fed on the blood of a yellow fever
patient within the first three days of the fever. (2) The disease
is not transferred immediately, but a definite incubative period
of more than eleven days must elapse before the mosquito can
transfer the disease. After twelve days the mosquito has been
found to be infected for at least fifty-seven days. (3) Yellow
fever cannot be carried by fomites. (4) Yellow fever may be
produced in a healthy man by the subcutaneous injection of blood
from a yellow fever case (parasites in the blood). (5) The serum
of a yellow fever patient filtered through a very fine Berkef eld or
porcelain filter is still capable of setting up the disease if injected,
proving that the infection agent is capable at some stage of its
life to pass through filter pores. (6) An attack of yellow fever
260 THE FILTERABLE VIRUSES
produced by the bite of a mosquito confers immunity against
subsequent infection. (7) The period of infection is usually
three days but may be from two to six days. (8) A house or ship
may be said to be infected with yellow fever only when there are
present mosquitoes capable of conveying the parasite of the
disease. (9) The spread of yellow fever may be prevented by
destroying the aedes and preventing egress and ingress of the
insects from yellow fever patients to the non-immune. (10) No
insect, other than the aedes, has been found to be concerned in
the spread of yellow fever.
Noguchi reports the discovery of a tiny spirochaete, 4-9/4 long
and .2;u wide, in the blood of yellow fever patients. The organism
may be transferred to guinea pigs in which it produces lesions
comparable to those of human yellow fever. It may be cultivated
in anaerobic serum water tubes and it will pass through a filter.
During an attack, either human or artificial in a guinea pig,
protective anti-bodies are formed. Spirals are only found in the
blood in the first few days of an attack. They do not stain well
and can best be seen by aid of the dark field microscope. It is
claimed that vaccination with these spirals produces some resist-
ance to yellow fever.
Yellow fever is a tropical or subtropical disease, because the
aedes is confined to these regions, and the disease is found
in low moist localities rather than those that are drier and higher,
from the fact that the mosquito inhabits the former and not the
latter. Yellow fever dies out after the first sharp frost, because
the, aedes are then either killed or undergo hibernation. Many
conclusive experiments by Reed and Carrol, by Guiteras, and
by the French Commission have proved that the aedes is beyond
doubt the cause of the spread of the disease. No_immunity, other
than the activity acquired one, is known.
Small-pox and Vaccinia. These two diseases must be consid-
ered to be but two clinical activities of one unknown specific
microorganism.
SMALL POX 26l
Certain protozoonoid bodies have been seen by numerous ob-
servers, notably by VanderLoeff, L. Peiffer, and Guarnieri. The
latter gave the name Cytoryctes vaccinias s. variolas. In the
deep layers of the epithelial cells of the pustules of vaccinia and
small-pox, in the experimental lesions on the corneae of rabbits,
and in the protoplasm of the cells, these bodies are found. They
are about the size of a micrococcus and exhibit amoeboid move-
ments in hanging-drop preparations. They are perfectly char-
acteristic of the lesion produced in vaccinia and are not found in
other diseased conditions.
In variola many different changes occur in the appearances of
these cytoryctes, suggesting developmental cycles.
In variola they are often intranuclear, while in vaccine they are
never found within the nuclei.
The cycle of development is suggestive of the development
of many of the protozoa. Stages of development exhibiting
fusiform amoeboid shapes can be seen, and pseudopodia can be
detected in the process of developmental stages suggestive of
gametocytes; the union of the gametes and the ultimate forma-
tion of the zygote can also be discerned.
After the tenth day these bodies cannot be very well discerned
in the tissues.
There is reason to think that the parasites circulate in the blood
in variola. The contagion in variola is thought to be by inhala-
tion. It is certain that the disease can be produced by inocula-
tion with virus from a case of small-pox. The contagion exists
in the scales, pus cells, and excretions of patients ill with small-pox.
If the virus of small-pox is introduced into a monkey, and then
into a cow the disease produced is not variola, but vaccinia
(Monkman). The hypothetical organism above described, cyto-
ryctes, becomes attenuated in the cow, so that it is incapable of
producing variola, but vaccinia.
Rabbits, horses, and sheep are susceptible of inoculation with
the virus of vaccinia (see Vaccination). Virus may be tested by
262 THE FILTERABLE VIRUSES
rubbing over the shaven bellies of rabbits, setting up minute
vesicles and finally crusts (Calmette).
The two viruses, that of variola and that of vaccinia, are now
thought to be identical. In a diluted condition it is filterable.
It resists drying for weeks and glycerine eight to ten months. It
is destroyed at S7C. in fifteen minutes and easily by most dis-
infectants. Passive immunization has not been achieved. No-
guchi has succeeded in growing the virus in the testes of rabbits;
one of his objects in so doing was to afford a sterile cultivation of
virus which might be used for preparing vaccine.
Scarlet Fever
Mallory in 1903 found certain bodies in the skin of scarlet fever
cases. These bodies, he assumed, were protozoan in character
and were the etiological cause of the disease. He named them
Cyclasterion Scarlatinale. They have been found rather con-
stantly in the skin of scarlet fever cases, also in the skin in cases
of measles and in anti-toxin rashes. Dohle has also described
an inclusion body within the polynuclear leucocytes. By several
observers these bodies have been considered to be artefacts or
degeneration products in the epithelial cells.
The virus of scarlatina is now considered to be filterable and
transmissible to monkeys. Mallory has lately found diphtheroid
bacilli in great numbers in the pharynx in scarlatina and believes
they may be its cause. Virulent streptococci, usually of hemoly-
tic quality, are so frequently encountered in the respiratory tract
and in complications during scarlet fever that many persons look
upon them as the cause; this cannot be proven as the disease
cannot be successfully transmitted to lower animals by use of these
organisms.
Dengue Fever. This is an acute infectious disease of the
tropics, characterized by fever, skin eruptions, rheumatoid pains,
an afebrile remission and a febrile end, due to a filterable virus,
SCARLET FEVER 263
transmitted by the mosquito, Culex fagitans. The virus is in
the blood-stream. One attack gives immunity; little is known
of the virus.
Three-day or Sand-fly Fever. A mild infectious disease
chiefly of southeastern Europe, due to a virus which will pass
through a bacteria-proof filter and is transmitted by the sand-fly,
Phlebotomus pappatacii. Cultures have not been obtained.
Typhus Fever or Spotted Fever. An acute epidemic disease
with prolonged course, prostration, a macular eruption, ending by
crisis, transmitted by the body louse, Pediculus vestamenti. The
virus is filterable but is obtained with difficulty. It is found best
toward the end of the fever. It may be transmitted to monkeys.
It is destroyed quickly at 52C. Brill's disease is a mild typhus
fever. A Gram-positive anaerobic non-motile bacillus .2-.6 X
.9-2/1, has been described as inhabiting the blood in typhus. As
claims to being the cause of the disease one finds that injection
causes a distinct febrile reaction in guinea pigs and the culture
may be used as antigen in a complement fixation test.
Poliomyelitis. An acute infectious disease, chiefly of children
characterized by a short febrile attack, followed by a rapidly
appearing paralysis in various muscles. Means of transmission
from child to child is unknown, but it has lately been shown that
the stable fly, Stomoxys calcitrans, can transmit it from monkey
to monkey. Greater weight is, however, laid upon transmission
from person to person by contact and the emanations from the
upper respiratory; this idea gains in value because monkeys can be
infected by nasal washings from patients during the disease. The
virus is in the central nervous system, lymphatic system, blood,
succus entericus, nasal mucous and various organs. It is said to
be constantly in the nasal mucosa of not only patients but of the
well in their vicinity. This is supposed to be its portal of entry to
the body. It is transmitted to monkeys by injecting emulsions
of the virus-containing parts into the brain, blood-stream or
peritoneum. It can be filtered through porcelain. It resists
264 THE FILTERABLE VIRUSES
glycerine, drying and autolysis. It is destroyed at 5oC. in one-
half hour. The virus can be cultivated by growing bits of brain
and cord from a case dead of the disease in unheated ascitic fluid
to which bits of sterile tissue have been added; the growth is
anaerobic. It appears as tiny globoid bodies, singly, in pairs or
short chains. Appropriate staining will demonstrate them also
in the nervous tissues.
Active artificial immunity and some passive immunity have
been obtained but these are not of therapeutic value. One
attack probably confers immunity. In the therapeutics of the
disease it is practicable to inject into the spinal column, the blood
serum of human cases that have recovered from the disease; this
indicates that virus-neutralizing bodies are formed during an
attack.
Foot and Mouth Disease. An acute infectious disease of cat-
tle, characterized by a vesicular eruption in the mouth and around
the crown of the hoof. It may be transmitted to man by the
use of milk from infected cows. It is also directly communicable.
It has not been cultivated. It is filterable; it is said to be due
to the Cytorrycetes. It is destroyed at 5oC. in ten minutes,
easily by freezing and ordinary disinfectants. One attack gives
no lasting immunity but the blood is said to contain anti-bodies
immediately after the attack, which will be protective to other
animals.
Trachoma. An infectious inflammation of the conjunctiva
with the production of minute but visible nodules on the under
sides of the lids. By some it is said to be due to a form of the
influenza bacillus, by others to an invisible virus. It is directly
communicable, filterable and transmissible to monkeys. It has
not been cultivated.
Measles. An acute eruptive fever due to a filterable virus
which is found in the blood, buccal and nasal secretions. It is
transmissible to monkeys by inoculations of patient's blood, even
before the Koplik spots appear. It persists in the blood until
MUMPS 265
after the appearance of the eruption. It resists drying and freez-
ing. It is destroyed at 55C, in fifteen minutes; it has not been
cultivated. Immunity follows an attack but no passive immunity
has been reported.
Mumps. This disease seems also to be due to a filterable virus,
resident within the parotid gland, capable of transmission to
monkeys. Many organisms have been described but none is
probably specific.
It must be said of both the hypothetical organisms of variola
and scarlatina, that if they are the cause of these two diseases
they differ from all other known protozoan parasites, because
the latter require an intermediate host for the transmission of
the parasite from individual to individual while these certainly
do not.
Rocky Mountain Fever is not due to a filterable virus but its
agent resides in the blood. It is a disease of the Rocky Mountain
States characterized by general pains; macular eruption and con-
stitutional symptoms of infection, it is transmitted by means of the
tick, Dermacentor venustus. The female tick obtains the agent
by blood sucking and can transmit it to her young. The virus
is destroyed by heating and drying. A minute coccoid body has
been found in the tick but it has not been cultivated. One
artificial attack in the guinea pig leaves immunity, as does the
spontaneous disease in man.
Trench Fever is a disease which became known in the Great
War characterized by fever, pains in the legs, back and head, with
marked dizziness, due to a resistant filterable virus, in all proba-
bility transmitted by lice; the virus is found in the blood. The
entrance is effected either by the bite of a louse or its f eces may be
scratched into tiny wounds on the skin. The virus seems to be
excreted in the urine and sputum. It is not killed at 8oC. The
louse is not infective for seven days after biting a patient and
then remains infective for three weeks. No immunity follows an
attack.
266 TTHE FILTERABLE VIRUSES-
Encephalitis lethargica is a disease alleged to be due to a
virus. It appears sometimes in epidemic form and was observed
after the last pandemic of influenza; some persons believe the two
are related. Some students of the subject report successful trans-
mission to monkeys.
SBo1o B uo:, n o S npoa d
1 1 1 1 1 1 1 II
!J
1
1 1 1^ 1 |
1 ^
1
001^0,^8
1 1 1 1 1 1 1 1 1 1
i i
g - |
5 ' "8
"3 5
^j ^
=3 ' .
rs."H 33 ^ "3
-< < _, 3
I'? i
++<,+ !<.!!
1
31 1
I* 5
u^
* 5
8 **
2 i
5 .8
1 1
1 G
'13
*->
"8 5
1 Kl-g 1 3 +-o|
s " a
3
IJ *
"fit ^ ^ 8
3
II 14-1 1 14-
1 5 35 35 SI
c ^ g 2
p- cL< E.
aui+Bpo
1 1 + 1 + + 4- 1 1 1
lltSu
5 i ss
2 ^^ ojqoaaBUV
Mil
++ I++ + 4- 1
I s PI
o 7 i <i
s-ss
<; CO oiqojay
++4-4-+ + ++++
I +
fl tn
0) C
UtB^g S.UIBJQ
++ 1 ++ + +111
rt W)
* CO
^3
1
1 1 1 1 1 1 1 1 1 1
i
-.2 ^ 3 ;
Sl2 ^
"8 .S 3 S G ^ 8
III II ! ||1,
imVitll!
a a a. . . . Z . . <*
J+l+lll+l I++ I I 1+
5
1 1 1 1 1 1 1 1 1 1 I 1 1
UOI?
I I ++ I + + + I + I C + +
a^a
H 43 i-i
0> " <U 0) V
1+1 l! I i, bll
| + l &| I S ||
f^ *^.ft ;- fQ ^
Illlll + ll II I < I +
jo uopoepnbrj
1111 + 1 + 14- 1 + + I
oiqcuaeuv
otqojay
+ + 1
UIB^g S.UIBJQ
Illlll+ll ++++++I
I I I +
.-*J O
-J +J
a : s
i-S g.2 s
fill I
0.-5T g a
i
pq pq pq pq pq pq pq pq pq
00
rtci
pqpq
2 a
tj '5 tj s o
M W
mycoide
Botulinu
+ I III +++ I
I I I I I II II
8
o
I o:
fi t; 8 -H * t;
o^'C MS to
CH 4-J ."*""* i*"") +"* *" !.
a
I II I II II
Re
^ 'o
P-13.S g.S S' S
$3%
< u,
o
I
^i
a |
+0+ I + I
+ I 1 II
<U <U <U (U <U 4) <U
1 1 1 1 1 1 I
I
3
^^1^
fe-ss^
S B 2I
+ + 1 1 1
^
III I I + I
jo
i +
+ 1 I I I II I +
1+ +++000
!
+++++ I I I + I + + +X +
I II II
5.2
.! .1
fit * *5
d
a
<u &
?T3 h
J8
I1SSS
pq cqpqpqpqpqpq
Q< *"* QJ
s^-sllssi 1
lisillll
|H||1.I4
' O Ug
M
Ac
DESCRIPTION OF PLATE I
Malarial Parasites
i. Two tertian parasites about thirty-six hours old, within blood cor-
puscles magnified.
2 Tertian parasite about thirty-six hours old; stained by Romanowsky's
method The black granule in the parasite is not pigment but chromatin.
Next to it and to the left is a large lymphocyte, and under it the black spot
is a blood plate.
3. Tertian parasite division form nearby is a polynuclear leucocyte.
4. Quartan parasite ribbon form.
5. Quartan parasite, undergoing division.
6. Tropical fever parasite (aestivo-autumnal). In one blood corpuscle
may be seen a smaller, medium, and large tropical fever-ring parasite.
7. Tropical fever parasite. Gametes half-moon spherical form. Smear
from bone marrow
8. Tropical fever parasite which is preparing for division heaped up in the
blood capillaries of the brain.
Asexual Forms
9. Smaller tertian ring about twelve hours old.
10. Tertian parasite about thirty-six hours old, so-called amoeboid form,
n. Tertian parasite still showing ring form, forty-two hours old.
12. Tertian parasite, two hours before febrile attack. The pigment is
beginning to arrange itself in streaks or lines.
13. Tertian parasite further advanced in division. Pigment collected in
large quantities.
14. Further advanced in the division (tertian parasite).
PLATE I
DESCRIPTION OF PLATE II
Malarial Parasites
15. Complete division of the parasite. Typical mulberry form.
16. To the left is the completed division form, an almost developed gamete
which is to be recognized by its dispersed pigment.
17. A tertian ring parasite, small size broken up.
1 8. Three-fold infection with tertian parasite. The oval black granules
are the chromatin granules.
19. To the left, tertian parasite with large, sharply demarked, and deeply
colored chromatin granules. To the right, tertian parasite. Both thirty-
six hours old. Both probably gametes.
20. Tertian parasite thirty-six hours old, ring form.
21. Tertian parasite with beginning chromatin division, with eight chrom-
atin segments.
22. Tertian parasite chromatin division farther advanced with twelve
chromatin granules, in part triangular in form.
23. Completed division figure of a tertian parasite. Twenty-two chrom-
atin granules.
24. The young tertian parasites separating themselves from each other.
The pigment remains behind in the middle.
25. Quartan ring parasite, which is hard to differentiate from large tropical
ring or small tertian ring.
26. Quartan ring lengthening itself.
27. Small quartan ribbon form.
28. The quartan ribbon increases in width. The dark places consist almost
entirely of pigment.
PLATE II
\ A
DESCRIPTION OF PLATE HI
Malarial Parasite
29, 30, 31. The quartan ribbon increases in width. The dark places
consist almost entirely of pigment.
32. Beginning division of the quartan parasite and the black spot in the
middle is the collected pigment.
33. Quartan ring.
34. Double infection with quartan parasites.
35. Wide quartan band. The fine black stippling in the upper half of the
parasite is pigment.
36. Beginning division of the quartan parasite. The chromatin (black
fleck) is split into four parts.
37. Division advanced, quartan parasites.
38. Typical division figure of the quartan parasite.
39. Finished division of the quartan parasite. Ten young parasites, pig-
ment in the middle.
40. Young parasites separated from one another/
41. Small and medium tropical ring, the latter in a transition stage to a
large tropical ring.
42. Small, medium and large tropical ring, together in one corpuscle.
PLATE III
DESCRIPTION OF PLATE IV
Malarial Parasite
43. To the left a young (spore) tropical parasite. To the right a medium
and large tropical parasite.
44. An almost fully developed tropical parasite. The black granules are
pigment heaps.
45. Young parasites separated from one another. Broken up division
forms twenty-one new parasites.
46. To the left a red corpuscle with basophilic, karyochromatophilic
granules. Prototype of malarial parasite. On the right a red blood corpuscle
with remains of nucleus.
Sexual Forms or Gametes
47. An earlier quartan gamete (macrogametocyte in sphere form), female.
48. An earlier quartan gamete (microgametocyte), male.
49. Tertian gamete, male form (microgametocyte).
50. Tertian gamete, female (macrogamete).
51. Tertian gamete (macrogametocyte still within a red blood corpuscle.
52. Microgamete tertian within a red blood corpuscle.
53. Tropical fever. (^Estivo-autumnal) gamete, half moon (crescent) still
lying in a red blood corpuscle. In the middle is the pigment. The concave
side of the crescent is spanned by the border of the red blood corpuscle.
54. Gamete, tropical fever parasite.
55. Gamete of tropical fever parasite heavily pigmented.
56. Gamete of the tropical fever parasite (flagellated form), microgameto-
cyte sending out microgametes (flagella or spermatozoon).
PLATE IV
CHAPTER XI
BACTERIOLOGY OF WATER, AIR, SOIL, AND MILK
Bacteriological examination of water is of importance for the
determination of the presence of pathogenic bacteria, and for the
enumeration of the total number of all bacteria contained therein,
the latter being considered an index of the purity of the water.
Several well-known pathogenic bacteria have been found in
water; among these are the typhoid, anthrax, cholera, plague, and
colon bacilli, also the pus cocci. Since the tetanus bacillus is a
normal inhabitant of the cultivated soil and manure, it is not at
all uncommon to find it, at times, in muddy waters.
Bacteriological examinations of water are, in a measure, very
disappointing, because it is difficulty and at times impossible to
determine the presence of the typhoid bacillus, even when it is
certain that it is present; having been added to water to be ex-
amined it is even then difficult to isolate.
The fact that the colon bacillus is always found in water con-
taminated by faeces is a great help in the recognition of polluted
water. In the case of typhoid contamination the typhoid bacillus
may elude detection, but the colon bacillus is easily found; we
may then assume that, since it is impossible for typhoid bacilli
to reach water without the colon bacilli that water having no
colon bacilli is also free from typhoid bacilli. Also water having
colon bacilli in great numbers is contaminated with faeces, and
perhaps typhoid faeces. The detection of the colon bacillus is
therefore of prime importance in the examination of drink-
ing water. Its detection is simple. Water must be collected
in sterile bottles, using every precaution against accidental
contamination. Fermentation tubes are employed, containing
bouillon with i percent of lactose. Into a series of these tubes,
278
BACTERIOLOGY OF WATER 279
varying amounts of water are run by means of a sterile pipette,
2 c.c., i c.c., .5 c.c., .1 c.c., .01 c.c., of water being used. After a
stay of twenty-four hours in the incubator, if gas appears, the
bouillon should be examined by plate cultures for the colon bac-
illus. Lactose litmus agar is used, and where colonies appear that
redden the litmus and resemble the colon colonies in appearance,
they are planted in milk, fermentation tubes, peptone solution,
neutral red agar, nitrate solution, and gelatine, and the various
reactions in the various media noted. Some idea of the numerical
presence of colon bacilli can also be obtained. Definite quanti-
ties of the raw water, similar to those used in the fermentation
tubes, may be plated directly without previous incubation. A
deeply tinted litmus lactose agar is used and upon this medium
colon bacillus colonies appear, small, pink, round or whetstone-
shaped surrounded by a pink zone or halo. Such pink colonies
are fished out into the different media as above. If there were
twenty pink colonies of the colon type upon a plate of litmus
lactose agar that had been seeded with i c.c. of water and of
these eight were fished and determined, with the discovery that
four only were true B. coli, we would assume that in i c.c. of
raw water half the pink growing colonies were those of B. coli
and that the water contained ten B. coli per cubic centimeter.
The significance of the colon bacilli is often overestimated.
They are found in all rivers, and often reach streams, wells, and
even springs by contamination from the barnyard, or manured
fields. Attempts to separate colon bacilli from human and animal
sources have been unsuccessful. Some authorities use strepto-
cocci of the faecal type as pollution indictors. This is not abso-
lutely reliable.
Typhoid bacilli have been found in water. One way that is
sometimes successful is to take 25 c.c. of a 4 percent peptone solu-
tion and add this to a litre of the water to be examined; from this,
after twenty-four hours in an incubator, plates may be prepared
with the agar medium of Endo as already given on page 120.
280 BACTERIOLOGICAL EXAMINATIONS
To Count Bacteria in Water
The sample must be collected in a sterile bottle, and the plates
poured immediately, since bacteria multiply enormously after a
few hours.
Take ^fo c.c. or J^j c.c. or i c.c. of the water in sterile pipettes
and mix with a tube of melted gelatine or agar, pour quickly into
cool sterile petri dishes and place in a cool dry place. The Ameri-
can Public Health Association also recommends the use of + i
percent agar plates grown both at room and body temperature.
The counts for the two are averaged. After forty-eight hours
count the colonies and the result (after multiplication where 3^0
or J^ c.c. of water was used) will be the number of bacteria per
cubic centimeter. It may be necessary to dilute the water five
or ten times before pouring plates. A glass plate ruled into
squares, known as a WolrThiigel plate, should be used for counting.
The number of bacteria in potable waters varies in many ways,
according to the amount of pollution, or albuminous matter in
the water, while depth, and the swiftness with which it flows are
conditions that modify bacterial contents. The water in a
reservoir becomes almost free from bacteria during the first ten
days. The number of bacteria diminishes- 10 percent per day
for the first five or eight days, due no doubt to gravitation of
the bacteria to the bottom, also in part to the action of light,
which plays an important role in the destruction of the bacteria
of water supplies.
In general, water containing less than 100 bacteria per cubic
centimeter is considered to be from a deep source, and uncon-
taminated by drainage. Deep artesian wells often contain but
from 5 to 15 bacteria per cubic centimeter, water from rivers
often contain 12,000 or 20,000 depending somewhat upon the
season of the year. Rains cause an augmentation of the bac-
terial content. Summer causes a diminution.
In identifying a certain water supply as the cause of an epi-
TO COUNT BACTERIA IN WATER 281
demic of typhoid, the number of bacteria is of great value in
locating the place of infection.
The efficiency of niters in large municipal water supplies is
known only by the bacterial content of the effluent. In good
sand and mechanical (alum) niters, the reduction in the number
of bacteria is often over 95 percent (sometimes 99 percent).
Plate cultures should be made daily from every filter in order to
determine how each filter is performing. Sand filters should not
filter more than 1,000,000 gallons per acre a day. They should be
at least i metre thick; the upper Y^ inch of the sand performs over
90 percent of the filtration, due to a certain zooglea, or growth
of bacteria. Cracks, or imperfections in the filter beds are
quickly detected by the rapid increase of the number of the bac-
teria in the effluent. It is supposed that not only are bacteria
filtered by the sand but that destructive changes occur in the
filter which greatly diminish the number of bacteria. A filter
must be used for a few days before it becomes efficient or "ripe. "
After a time it becomes inefficient and it must then be scraped,
finally the sand must be removed and washed.
A sand filter is a highly efficient means of water purification.
It often converts a foul dirty water into a bright, clean, whole-
some water of low bacterial content.
Mechanical filters depend for their efficiency upon the addition
of aluminum sulphate to the water. This is decomposed by the
carbonates and aluminum hydroxide is produced, which is a
white jelly-like flocculent precipitate, which mechanically en-
tangles bacteria and removes them from the water. Mechanical
filters, as a rule, are highly efficient. Domestic filters, even the
Pasteur, are often unreliable.
In time of epidemics of cholera and typhoid even filtered water
should be boiled before use, as it was found by experiments in the
Medico-Chirurgical Laboratories that typhoid bacilli live longer
in filtered water than in bouillon; they may even live three months.
282 BACTERIOLOGICAL EXAMINATIONS
The fewer the number of other bacteria the longer will typhoid
live. They can live many days in ordinary river water.
Ice may contain great numbers of bacteria; it is well known
that freezing does not destroy pathogenic bacteria, such as the
typhoid bacillus. Prudden found typhoid bacilli in ice after one
hundred days, although the number was greatly reduced over that
placed in the ice originally. Many are squeezed out by contraction
of the water. The greatest danger from ice is in dirty handling.
Disposal of sewage is a bacteriological process in many cases;
either the sewage may be treated in sand niters or it may be run
put on land where over 200,000 gallons may be disposed of on an
acre of land a day. As far as possible nature should be imitated
in every way and the breaking up of masses of matter in sewage
may be accomplished in the septic-tank process in which active
oxidization of the matter is accomplished by bacteria. It appears
from the observations of many sanitarians that both aerobic and
anaerobic bacteria are necessary to finally reduce sewage to the
elementary gases and pure water.
In the interior of closed tanks and in the depths of sand niters
anaerobic conditions prevail. On beds of coke, and on the sur-
face of sand filters, aerobic conditions obtain. The effluent from
a septic-tank sewage-disposal plant is very often pure water from
both chemical and bacteriological standpoints, due to the chem-
ical action of the bacteria.
Bacteriology of the Air
That the lower layers of the earth's atmosphere contain many
bacteria is well known. The air over the sea and over mountain
ranges is freer from bacteria than the air over arable lands and
large cities.
When air is still and confined, all bacteria, according to Tyndall,
gravitate to the ground, and the air above becomes quite sterile.
The atmosphere of sick rooms, hospitals, public conveyances,
theatres, etc., contains many bacteria and often pathogenic ones.
BACTERIOLOGY OF THE AIR 283
The pus cocci, tubercle bacilli, and the organisms causing small-
pox, scarlet fever, and measles, all may contaminate the air.
The number of bacteria in a given quantity of air may be accu-
rately measured by means of a Sedgwick-Tucker aerobioscope;
this consists of a large cylindrical glass vessel opening at either
end into various tubulations (Fig. 82). Into one of these granu-
lated sugar may be packed; the ends are then plugged with cotton
and the apparatus sterilized. To examine the air, a litre or more
is drawn through the sugar and the latter is then shaken into the
large cylinder where it is dissolved in melted gelatine culture
FIG. 82. Sedgwick-Tucker aerobioscope. (Williams.)
media. The latter is distributed over the interior of the glass
and allowed to harden. All the bacteria that were in a litre of
air having been mixed with gelatine and those that are not strict
anaerobes grow in the gelatine and a number of colonies can
then be counted.
The dust of dwellings and streets contains most of the bacteria.
Dried sputum is ground under foot and swept up in gusts of wind,
and the contained bacteria are thus inhaled and do harm. The
air coming quietly from the lungs is pure and sterile. Even in
active disease processes of the throat this is true. In case the
breath comes violently, as in speaking, coughing, and sneezing, the
reverse is the case. In general it may be put down as an axiom
that disease germs cannot rise from a fluid, such as sewage. If
they could it would mean that they are lighter than air, which is
not the case. Sewer gas, as a rule, is a bearer of some pathogenic
bacteria chiefly cocci but in reality it is purer than generally sup-
posed. The spread of organisms from sewage only extends 3-6
metres into the atmosphere and then only the the bursting of bub-
284 BACTERIOLOGICAL EXAMINATIONS
bles in the presence of gas under pressure; air currents may of
course carry germs so freed a much longer distance.
Bacteriology of the Soil
At least two forms of pathogenic bacteria are habitually found
in the soil. The tetanus bacillus, it is well known, exists in garden
earth, manure, and top soil generally. Dirt getting into wounds
is the most frequent cause of tetanus. Drinking water laden with
soil has been known to have in it tetanus bacilli, and if used in
an unsterilized condition in wounds or when a comparatively feeble
antiseptic, such as creolin, has been added, it may cause tetanus.
The gaseous edema group, the bacilli of malignant edema,
symptomatic and parasitic anthrax are frequently found in soil.
The highly tilled soil of the battlefields in France was heavily
laden with the first, hence the great incidence of infection after
wounds in the late war. Streptococci and colon bacilli, too, have
been found in garden soil. Typhoid bacilli may contaminate soil,
but do not multiply in it. In sandy soil 100,000 bacteria per
gram have been found, in garden soil 1,500,000 bacteria per gram,
and in sewage-polluted soil 115,000,000 bacteria per gram have
been determined. The first few inches of ordinary soil contain
most of the bacteria, after a depth of 2 metres no bacteria at all
are found and the earth is sterile.
Soil may be collected in sterile sharp-pointed iron tubes, and
diluted with sterile water of given quantity and plates poured
from it.
Arable lands may be enriched very much by inoculating them
with 'certain nitrifying bacteria, some of which convert ammonia
into nitrous acid, which form in them nitrites; others change
nitrites into nitrates (nitrosomonas) . Certain of these bacteria
are concerned in the assimilation of nitrogen from the atmosphere
and adding to the nitrogen content of the soil, thus enriching it.
On the roots of some plants, alfalfa, beans, peas, and clover,
minute tubercles develop. These little growths are caused by the
BACTERIOLOGY OF COW'S MILK 285
nitrifying bacteria, and add to the nutrition of the plant by adding
to it ammonia.
Bacteriology of Cow's Milk
Theoretically the milk in the interior of the breasts of nursing
women and the udders of cows is sterile. So soon as it leaves the
nipple it becomes contaminated with bacteria, and by the time it
reaches the pail, in the case of cow's milk, it is far from sterile.
Bacteria of the air, and dust from the cattle and bedding, at
every movement of the cow, and by the agency of flies, find their
way into milk and contaminate it. The number of bacteria that
develops in the milk depends upon the number that reach it in the
first place, the temperature of the air, and the length of time milk
is kept at a temperature favorable for their multiplication. Two
hundred and thirty-nine different varieties of bacteria have been
isolated from milk at different times.
Pathogenic varieties of bacteria that have been found in cow's
milk include- the tubercle bacillus, Streptococcus pyogenes, Staphy-
lococcus aureus, the colon bacillus, typhoid bacillus, the diphtheria
bacillus, and a whole host of bacteria that sour or ferment the
milk and render it unwholesome or poisonous for young children.
Cattle may be tuberculous, and the tubercle bacilli may reach
the milk in this way. There may be abscesses of the udder and
the streptococci from the pus may cause infection in those that
use it. Ordinary follicular tonsillitis may be caused in this way.
Bacteria may develop rapidly in milk, which is a good culture
medium, until they number many millions per cubic centimeter
(sometimes 200,000,000).
In good milk the number of bacteria may increase when the
temperature is goF., from 5,200 originally in the milk imme-
diately after milking, to 654,000 in eight hours.
By exposing milk to a temperature of i65F. for twenty to
thirty minutes and quickly cooling (Pasteurization) most of the
non-spore-bearing bacteria are destroyed, so that the number may
286 BACTERIOLOGICAL EXAMINATIONS
be reduced 99.999 percent by this process. The Pasteurization
of milk has become an economic problem of great importance in
large communities and is not, as it should be, sufficiently super-
vised. That method is best in which milk is held at i46F. for
thirty minutes. No harm is done to the nutritional value of the
milk. One of the dangers of the method is that the commercial
Pasteurizing machines are not always thoroughly clean and them-
selves contaminate the milk when discharging it after heating.
More evidence is on the side of the second view. The practical
importance of the controversy is that milk whether heated or not
should be kept at a temperature at which bacteria will not mul-
tiply, under 6oF. Pasteurized milk is safest in time of typhoid
epidemics.
Absolute cleanliness on the part of the milker, the use of steril-
ized gloves and clothes, the absence of flies, dust, and the imme-
diate disposal of manure, the nitration of the milk after collection,
the immediate cooling of it, the uses of sterilized milk cans and
bottles, all lessen the bacterial content of milk. It then keeps
better, and is a wholesomer and safer food for infants, especially
in hot weather.
By drinking water containing typhoid bacilli cows cannot
be sources of typhoid infection through trie milk. The typhoid
bacilli are not transmitted through the bodies and udders of the
animals.
A bacteriologic examination of milk comprises a total count,
the presence of colon bacilli, streptococci in pus cells, tubercle
bacilli and special species as the case suggests. The first is done
as given for water, as is the second. The discovery of streptococci
is made by centrifugalizing a definite quantity and examining the
sediment for chains, particularly in relation to leucocytes, the pus
cells. Tubercle bacilli are found by injecting guinea pigs or by
dissolving the milk in antiformin (i part milk and i part 15 per-
cent antiformin) warming and examining the sediment after
centrifugalization.
INDEX
Abscesses, 154
Achorion Schoenleinii, 231
Acid, benzoic, 56
boric, 137
fast, 1 06
hydrochloric, 39, 137
lactic, 167
production, 128
Acids. 137
mineral, 137
Acne, 154
Acquired immunity, 47
Actinomyces, 3
bovis, 224
farcinicus, 223
madura, 226
Action, hydrolytic, 25
Active immunity, 47
Aedes calopus, 259
Aerobes, 19
Aerobioscope, 283
Aerogenes mucosus, 166
^Estivo-autumnal parasites, 248
Agar-agar, 117
Agar, blood, 119
glycerine, 116
Agglutinins, 53, 60, 92, 171, 172
Aggressins, 44
Air, bacteria of, 280
borne infection, 36
liquid, 20
Alcohol, 141
Alexins, 51, 60
Allergic, 63, 66
Alternate generation 245
Amboceptor, 51. 60
Ammonia, 129
Amoeba dysenterise, 32, 235
Amoebae, 234
Amoeboid motion, 233
Amphitrichous bacteria, 211
Anaerobes, 19
Anaerobic culture, 130
Anaphylaxis, 63
Andrade indicator, 118
Aniline dyes, 97
Animal experiments, 132
carriers, 37
parasites, 232
Anopheles maculipennis, 249
Anthrax bacillus, 16, 31, 33, 50, 100,
183
anti-serum, 187
vaccine, 83, 187
Anti-aggressins, 45
Antibiosis, 19
Antibody, 49, 60
Anti-complement, 61
Anti-ferments, 60
Antigens, 60, 61
Anti-immune body, 61
Anti-leucocidin, 70
Anti-plague serum, 77, 83, 165
Antisepsis, 135
Antiseptic values, relative, 142
Antiseptics, 135
Anti-toxin for botulism, 70, 198
for diphtheria, 71, 194, 211
287
288
INDEX
Anti-toxin, dosage, 74
for dysentery, 180
for plant toxins, 70
for pyocyaneus, 70
staphylococcus, 70, 154
streptococcus, 75
for symptomatic anthrax, 70,
196
for tetanus, 70, 74, 192
manufacture of, 71
standardization of, 73
Anti-toxins, 43, 54, 60, 70
for animal toxins, 70
standard, 73
Arnold sterilizer, no
Artesian Wells, 280
Arthrospores, 16
Aspergillus, 5
flavus, 231
fumigatus, 231
niger, 231
Attenuation of bacteria, 34, 135
Autoclav, 109
Autopsies, animal, 133
Avenue of infection, 35
Babes Ernst granules, 9
tubercles, 258
Bacillus, 2, 7
aerogenes capsulatus, 100, 199
of anthrax, 16, 31, 34, 50, 100,
3
of blue pus, 181
botulinus, 197
Chauvoei, 194
of cholera, 202
colon, 100, 174, 278, 284, 286
comma, 202
of diphtheria, 31, 100, 207
of dysentery, 100, 178
Bacillus, enteriditis sporogenes, 199
Friedlander's, 166
fusiformis, 201
Gartner's, 177
of glanders, 31, 206
Koch Weeks, 162
lepra, 31, 221
of lockjaw, 1 88
malignant oedema, 31, 192, 264,
284
mallei, 31, 100, 206
of Malta fever, 31, 158
Morax and Axenfeld, 162
perfringens, 199
of plague, 31, 162
pseudo-diphtheria, 213
pyocyaneus, 100, 181
rauschbrand, 194
smegma, 219
of soft chancre, 183
of symptomatic anthrax, 194
of tetanus, 31, 74, 100, 188, 284
of tuberculosis, 31, 33, 98, 100,
213, 286
typhosus, 31, 100, 168, 278, 279,
285
Xerosis, 213
Bacteria, attenuation of, 34
of air, 282
biological conditions of growth,
18
chemical composition of, 17
chromogenic, 23
definition of, i
disposal of, 32
fixed strains of, 35
higher, 4, 5, 17
increasing malignancy of, 35
lophotrichous, n
measuring of, 9
mesophilic, 19
INDEX
289
Bacteria of milk, 285
of mouth, 38
parasitic, 31
photogenic, 23
psychrophilic, 19
reproduction of, 1 2
of skin, 38
of soil, 284
staining of, 96
of stomach, 38
study of, 95, 120
submicroscopic, 255
thermophilic, 19
Bacteriaceae, 2
Bacterial energy, 23
proteins, 28
Bacterins, 77, 140-154
sensitized, 77
Bacteriological diagnosis, 108, 123
Bacteriolysins, 10, 41, 49, 57, 60
Bacteriolysis, 61
Bacterium ; 2
aerogenes, 166
Bulgaricum, 167
coli, 174
enteriditis, 177
influenzas, 100, 154
lactis aerogenes, 166
mucosus, 1 66
pestis, 100, 162
pneumonias, 166
ulceris chancrosi, 183
Balantidium, 233
Beggiatoa, 4
Beggiatoaceae, 4
Benzoate of soda. 56
Benzoic acid, 56
Benzol ring, 51, 55
Biological conditions of growth of
bacteria, 18
Bismarck brown, 99
19
Black-leg vaccine, 84
Blastomycetes, 5, 17, 228
Blastomycosis, 228
Blood agar, 119
cultures, 170
serum, 112, 119
Blue, methylene, 98
pus bacillus, 181
Boils, 154
Bordet-Gengou bacillus of whoop-
ing-cough, 161
Botulism, 197
Bouillon, 113, 198
Bovine tuberculosis, 219
Bromine, 137
Bronchitis, 160
Brownian motion, 12, 95
Capsule staining, 101
Capsules, 10, 15, 16
Carbol fuchsin, 98
thionin. 100
Carbolic acid, 139
Carbuncles, 154
Carriers, 37, 38, 151. 170
Cell division, 13
Cellulo-humeral theory, 50
Centrosome, 238
Cercomonas, 233
Chain coccus, 143
Chauvoei, bacillus of, 194
Chemotaxis, 20,44, 46, 47, 50
Chlamydobacteriaceae, 3, 8
Chlamydozoa, 255
Chloramin. 138
Chloride of lime, 138
of zinc, 141
Chlorinated lime, 138
Chlorine, 137
Cholera bacillus, 202
Cholera, vaccination against, 79
290
INDEX
Chromogenic bacteria, 23
Ciliata, 233
Cladothrix, 3
Classification, i, 5
CO,,. 23
Coccaceae, i
Cocci, 5
Coccidia, 233, 235, 253
Coccidioides, 5
immitis, 230
Coccidiosis, 230
Coccidum hominis, 253
Coccus chain, 143
Coccus, Malta fever, 158
of meningitis, 149
Cold, influence of, 20
Coley's fluid, 88
Collodion sac, 113
Colon bacillus, 90, 174, 279, 286
Comma bacillus, 202
Complement, 49, 51, 56, 57, 60, 61
deviation, 69
fixation, 67
Complementophile, 61
Conjunctivitis, 146, 156. 162
Copper sulphate, 137
Copula, 60
Corenybacterium diphtheriae, 207
pseudo-diphtherias, 273
Counting bacteria, 280
Crenothrix, 4
Creolin, 139
Cresol/ 139
Culture media, 19, 102, 113
Cultures, 120
anaerobic, 130
plate, 123
Cyclasterion Scarlatinale, 262
Cytase, 49, 60
Cytolysins, 53, 60
Cytolysis, 53, 60
Cytophile, 61
Cytoplasm, 9
Cytoryctes variolae, 7}
Cytotoxins, 60
261
Dakin's solution, 138
Dark field illumination, 106
Darkness, influence of, 20
Dengue fever, 262
Desensitization, 65
Desmon, 60
Diarrhoea, 170
Dichloramin T, 138
Differentiation of B. typhosus and
B. coli, 176
Dilution method, 122
Diphtheria, 144, 207
anti-toxin, 54, 70, 192
bacillus, 207
stain, 105
toxin, 43, 71, 210
toxin-antitoxin injections, 82
virulence test, 212
Diplococcus, 5
gonorrhoea, 155
lanceolatus, 146
meningitis, 100, 149
Direct division, 13
Disinfectants, 135
Disinfection, 135
Dum-dum fever, 240
Dust, 36
Dyes, aniline, 97
Dysentery, amoeba, 234
bacillus, 178
Ectosarc, 234
Egg cultures, 120
Ehrlich's theory, 50
Encephalitis lethargica, 266
Endo medium, 120
INDEX
2QI
Endocarditis, 144, 146, 156
Endosarc, 234
Endospores, 13
Endotoxins, 28, 41, 50, 56, 57, 60
Entamceba coli, 235
buccalis, 236
histolytica, 234
tetragena, 219, 236
Enzymes, 24
Erysipelas, 144
Exhaustion theory, 47
Experiments, animal, 132
Farcin du Boeuf, 223
Favus, 231
Fermentation, 25
tubes, 128
Ferments, 24, 60
diastatic, 24
tryptic, 24
Filters, 32, 101, 113, 263, 281
alum, 281
Pasteur, 113
sand, 281
Fixateur, 60
Fixation, 96
Flagella, 10, 12
staining, 102
Flagellata, 219, 233, 237
Focal infection, 41
Fomites, 259
Foot and mouth disease, 264
Formaldehyde, 139
Fractional sterilization, no
Frambcesia, 243
Friedberger's theory, 65
Friedlander's bacillus, 166
Fuchsin solutions, 98
Gabbet's solution, 106
Ganglia, 259
Gartner's bacillus, 177
Gaseous edema bacillus, 199
Gastric juice, 39
Gelatine, 116
Generation, alternate, 215, 232, 241,
259
Giemsa's stain, 101
Glanders bacillus", 206
Glossina palpalis, 239
Glyco-nucleo-protein, 18
Gonidia, 12, 17
Gonococcus, 155
Gonorrhoea, 156
Gram's method of staining, 99
Granules, chromophilic, 9
Babes Ernst, 9
Gregarines, 233
Gregarinida, 233
Gruber-Dunham reaction, 96, 155,
171
Gymnobacteria, 10
H, 23
H 2 S, 24
Haemolysins, 60, 92
Haemolysis, 52, 63
Haemolytic serum, 51, 53
Haemosporidia, 228, 233, 245
Haffkine, 79
Halogens, 137
Hanging drop, 96
Haptophores, 57, 6 1
Hepatotoxin, 60
Hemoglobinophilic, 161
Heterotrichida, 233
Hiss' capsule stain, 102
Histological methods, 133
Human tubercle bacilli, 219
transmission, 39, 218
Hydrogen ion concentration, 114
media method, 114
peroxide, 139
INDEX
Hydrochloric acid, 36, 137
Hydrophobia, 256
Hyphomycetes, 5, 17, 230
Hypersensitivity, 66
Hypersusceptibility, 63
Ice, bacteria in, 282
Immune body, 51, 59, 60, 61, 63
Immunkorper, 60
Immunity, 46
acquired, 46
active, 46
anti-bacterial, 46
anti-toxic, 46
inherited, 47
local, 38
natural, 46
passive, 46
racial, 46
Incubator, in
Index, opsonic, 89
Indicators, 115, 118
Andrade, 118
Indol production, 129
Infection, 30
focal, 41
mixed, 35, 50,
phlogistic, 40
secondary, 35
septic, 40
terminal, 39
toxic, 40
Infestation, 30
Influenza bacillus, 159
Infusoria, 233
Inoculating animals, 132
media, 121
Insects, 37
Intermediary bodies, 60
Involution form, 8
Iodine, 137
Isoagglutination, 92
Isohemolysin, 92
Jenner, 78
Jenner's stain, 101
Kidneys, excretion of bacteria by, 33
Klebs-Loffler bacillus, 207
Koch's postulates, 31
Laboratory technique, 108
Lactic acid, 25, 167
Larva of mosquitos, 251
Lateral chain theory, 50, 55
Law of multiples, 55
Leishman-Donovan bodies, 240
Leishman's stain, 101
Lepra bacillus, 221
Leucocytosis, 49
Leutin, 28, 91, 241
Lightning rod theory, 58
Lime, 140
chlorinated, 138
Litmus milk, 117
tincture, 120
Local immunity, 38
Lockjaw bacillus, 188
Loffler's blood serum, 119
blue, 98
flagella stain, 104
method of staining tissues, 134
Lophotrichous bacteria, n
Lysis, 52
Lysol, 139
Macrogametes, 247, 249, 251
Macrophages, 49
Madura foot, 226
Malarial parasites, 244
Malignant oedema, bacillus of, 192
Mallein, 28, 86
Malta fever, bacillus of, 31, 158
INDEX
293
Mannaberg's scheme, 246
Mastigophora, 233
Measles, 264
Measuring bacteria, 9
Meat poisoning bacillus, 197
Membrane, false, 26
Meningitis, 144, 148, 149, 160, 182
anti-serum, 76, 151, 161
Meningococcus, 149
Mercury salts, 136
Merismopedia, 2
Merizoites, 247
Mesophilic bacteria, 19
Metals, influence of, 21
Micrococcus, 2, 6
catarrhalis, 100, 151
epidermidis albus, 155
gonorrhoea, 100, 155
melitensis, 158
pyogenes, 152
tetragenus, 157
Microgametes, 247, 249
Microgametocytes, 247, 249
Microphages, 49
Microspira, 2
Microsporon, 5
furfur, 231
Milk, bacteria of, 285
borne infection, 37
litmus, 117
Mixed infection, 35
Molecule, toxin, 56
Monadida, 233
Monilia, 5
Monkey injection, 262, 263
Monotrichous bacteria, n
Mordants, 98
Mosquitos, 249, 259
aedes, 259
anopheles, 249
larva, 251
Moulds, 5, 17, 230
Muir-Pitfield flagella stain, 103
Multiplication of bacteria, 1 2
Mycelia, 5, 17
Mycobacteriaceae, 3
Mycobacterium, 3, 7
lepra, 221
tuberculosis, 213
Mycomycetes, 5
Mycoprotein, 18
Needles, inoculating, 123
Negri bodies, 257
Neisser's stain, 105
Nephrotoxin, 60
Neutralization of media, 114-116
Nitrates, 129
Nitrifying bacteria, 24, 284
Nitrites, 129
reduction, 24
Nitrogen, 24
Nocardia, 222
Novy jars, 131
Nutriment of bacteria, 19
Oidia, 5
Oidium albicans, 228
coccidoides, 228
Oidiumycosis, 210, 228
Oocysts, 251
Ookinets, 251
Opsonic index, 89
Opsonins, 60. 89
Organelles, 235
Osteomyelitis, 144, 154
Paracolon bacillus, 173, 177
Paratyphoid bacillus, 173
Parasites, animal, 30, 232
Park diphtheria, treatment, 74
Pasteur filter, 112
294
INDEX
Pasteurization of milk, 285
Pathogenicity, 33
Pathogens, 25
Peptone solution (Dunham's), 118
Pericarditis, 148
Peritonitis, 144, 148
Peritrichous bacteria, n
Peroxide of hydrogen, 139
Petrie dishes, 123
Pfeiffer's reaction, 51
Phagocytes, 47, 89
Phagocytosis, 44, 47, 82, 89
Phagolysis, 49
Phlogistic infection, 40
Photogenic bacteria, 23
Phragmidothrix, 4
Phycomycetes, 5
Pink eye, 162
Pitfield's flagella stain, 103
Pityriasis versicolor, 231
Placenta, infection through, 39, 218
Plague bacillus, 162
vaccination, 83, 165
Planococcus, r, 6
Planosarcina, i, 6
Plasmins, 27
Plasmodium falciparum, 248
malarice, 246
vivax, 247
Pleomorphism, 8
Pleuritis, 148, 154
Pneumococcus, 146
types, 76
vaccination, 82
Pneumonia, 144, 148, 154, 160, 167
Poliomyelitis,. 263
virus of, 263
Polymastigida, 233
Polymites, 248
Porcelain filter, 112
Postulates, Koch's, 31
Potassium permanganate, 140
Potato, 117
Pragmidiothrix, 4
Precipitins, 54, 60
Preparateur, 60
Proteins, bacterial, 28
Protozoa, 232
staining of, 106
Pseudomonas, 2
Psychrophilic bacteria, 19
Ptomaines, 25, 41
Puerperal fever, 144, 154
Pus, 26, 40
Putrefaction, 24
Pyocyaneus, anti-toxin, 70
bacillus, 181
Quartan malarial parasite, 246
Racial immunity, 46
Rat bite fever, 243
Rauschbrand bacillus, 194
Ravenel potato cutter, 117
Ray fungus, 224
Reactivation, 51, 55
Receptors, 57, 63
Relapsing fever, 243
Retention theory, 47
Rheumatic tetanus, 191
Rhizopoda, 233
Ringworm, 231
Rocky mountain fever, 265
Rod bacteria, 2
Roll culture, 126
Romano wsky's stain, 101
Roux regulator, 112
Russell's medium, 120
Sac, collodion, 113
Saccharomyces, 5
INDEX
295
Saccharomycetes, Busse, 230
Sand-fly fever, 263
Sapraemia, 30
Saprogens, 25
Saprophytes, 18
Sarcina, 2, 6
Sarcode, 233
Sarcodina, 233
Scarlatina, 144, 262
Scarlet fever, 262
Schizomycetes, i
Schizogony, 234, 246, 247, 251
Secondary infections, 35, 144, 154
Septic infections, 40
tank, 282
Septicaemia, 144, 148, 154, 162
pneumococci, 148
Serum, anti-plague, 77, 165
anti-pneumococcus, 75
anti-toxic, 70
haemolytic, 52
reactivated, 51, 55
shock, 66
water, 119
Sessile phagocytes, 48
Sewage disposal, 282
Silver salts, 137
Skin, disinfection of, 141
Sleeping sickness, 237
Small pox, 78, 154, 260
Smegma bacillus, 219
Soft chancre bacillus, 183
Soil, bacteria in, 284
borne infection, 37
Soor, 228
Sparing action, 25
Spermo toxin, 60
Spirillaceae, 2, 202
Spirillum, 2, 7
cholera, 100, 202
Spirochaeta, 3, 7, 233
Spirochaeta, carteri, 243
duttoni, 243
icterohemorrhagicae, 243
morsus muris, 243
muris ratti, 243
nodosa, 243
Novi, 243
obermeieri, 243
pallida, 240
refringens, 240
vincenti, 201
Spirosoma, 2
Sporangia, 17
Spore staining, 102
Spores, 13, no
Sporoblasts, 251
Sporogony, 234, 246, 2,51
Sporothrices, 5, 249
Sporothrix schenki, 249
Sporozoa, 233, 245
Sporozoites, 246, 251
Sporulation, 13, 96
Spotted fever, 263
Stain, Bismarck brown, 99
Fuchsin solution, 98
Giemsa's, 101
Gram's, 99
Hiss' capsule, 102
Leishman's, 101
Loffler's methylene blue, 98-
flagella, 104
Neisser's diphtheria, 105
Pitfield's flagella, 104
modified by Muir, 103
spore, 102
thionin blue, 100
tubercle bacilli, 106
Weigert's, 134
Welsh's capsule, 101
Wright's, 100
Zeihl's carbol-fuchsin, 98
296
INDEX
Staining bacteria, 96
Standardization of anti-toxins, 73
of media, 114
Staphylococcus, 2, 6
albus, 152
aureus, 152
citreus, 152
pyogenes, 152
Stegomyia calopus, 259
Fasciata, 251
Sterilization, 108, 135
culture media, 109
fractional, no
glassware, 108
Sterilizer, Arnold, no
Sterling's solution, 99
Stern's method for spirochetes, 106
Stomach, bacteria of, 39
Street virus, 256
Streptococcus, 2, 34, 143, 286
antiserum, 75
intracellularis, 149
lanceolatus, 146
mucosus, 149
pneumonias, 100, 146
pyogenes, 100, 143
viridans, 146
Streptothrix, 3
hominis, 222
madura, 226
Study of bacteria, 120
Substance sensibilisatrice, 60
Sulphur dioxide, 140
Symbiosis, 19
Symptomatic anthrax anti-toxin,
196.
bacillus, 177, 193
Syphilis, 241
Table of characteristics of bacteria,
267-269
Temperature, influence on growth,
19
Terminal infection, 39
Tertian fever, 247
Test, tuberculin, 85, 91
Schick, 91
Tetanolysin, 44, 189
Tetanospasmin, 44, 189
Tetanus anti-toxin, 74, 192
bacillus, 31, 100, 188, 284
rheumatic, 191
spore, 1 88
toxin, 28, 43, 44, 189
Tetrads, 5
Theory, cellulo-humeral, 50
Thermolabile, 51
Thermostat, in
Thionin, 100
Thiothrix, 4
Thrombosis formation. 26
Thrush, 228
Tonsillitis, 144
Toxalbumins, 42
Toxic infection, 40
Toxin, 28, 43, 60
molecule, 55
Toxoid, 43, 58
Toxons, 43
Toxophores, 57, 6 1
Trachoma, 264
Trench fever, 265
mouth, 202
Treponema, 233
pallidum, 91, 240
pertenue, 243
Trichobacteria, n
Trichomonas, 233, 240
Trichomyces, 222
Trichophyton, 5, 230
Trypanosoma, 233, 237
brucei, 237
INDEX
297
Trypanosoma, cruzi, 238
equiperdum, 220, 238
evansii, 237
gambiense, 237, 239
lewisi, 238
noctuae, 220, 238
Tsetse fly, 237, 239
Tubercle bacillus, 213
stain, 106
Tubercles, Babes, 258
Tuberculin, 28, 85, 91, 220
T.R., 85
Turpentine, 140
Tyndallization, no
Typhoid bacilli, 32, 35, 100, 168, 279
284
in water, 278
vaccination against, 80
Typhus fever, 263
Udder, infection by, 285
Unit of anti-toxin, 73
toxin, 73
Uterus, bacteria in normal, 39
Vaccination, 77
diphtheria, 82
for plague, 83
Vaccine, anthrax, 83
black leg, 84
cholera, 79
diphtheria, 82
paratyphoid, 80
plague, 83
pneumonia, 82
small pox, 78
tuberculosis, 85
typhoid, 80
Vaccinia, 78, 260
Vaccinoid, 79
Vacuoles. 10
Variola, 78, 260
Venom, 57
Vibrio, 2, 7
cholera, 202
Metchnikovii, 206
proteus, 206
Schuylkilliensis, 206
septique, 192
tyrogenum, 206
Vincent's angina, 201
Virulence, 33
Virus fixe, 257
Wassermann's list of anti-toxins, 64
test, 70
Water, bacteria of, 278
borne infection, 36
Weigert's aniline gentian violet, 99
method of staining tissue, 134
theory, 57
Weil's disease, 243
Welch's capsule stain, 101
Wells, artesian, 280
Widal reaction, 53, 171
Wolffhiigel plate, 280
Wright's stain, 100
Xerosis bacilli, 213
Yaws, 243
Yeasts, 5, 17, 228
Yellow fever, 258
Zeihl's solution, 98
Zinc chloride, 141
Zooglea, 10
Zwischenkorper, 60
Zymase, 24
Zymogenic bacteria, 23
Zymophore, 61
UNIVEKSITY OF CALIFORNIA LIBRARY,
BERKELEY
THIS BOOK IS DUE ON THE LAST DATE
STAMPED BELOW
Books not returned on time are subject to a fine of
50c per volume after the third day overdue, increasing
to $1.00 per volume after the sixth day. Books not in
demand may be renewed if application is made before
expiration of loan period.
192?
20m-l,'22
Diagnostic Methods
CHEMICAL, BACTERIOLOGICAL AND MICROSCOPICAL
6th Edition, Revised and Enlarged. 207 Illustrations, including
37 Colored Plates. 883 Pages. 88 More Pages than Previous
Edition. Cloth $9.00 Postpaid.
By RALPH W. WEBSTER, M.D., Ph.D.
Asst. Prof. Pharmacolog^c Therapeutics, Instructor in Medicine,
Rush Medical College, ( University of Chicago. )
For all who think in the problems of present-day medicine, there
is hardly another book that can be so useful, both to the general
practitioner and to the man who is devoting his time to laboratory
work. The various sections are divided so as to present the problems
to be met, details of methods available, and the practical significance
of positive and negative findings in the chosen method. It points
out beforehand likely objects that may interfere with results. Simple
tests as well as the more complicated are carefully detailed so that
anyone following directions can obtain good results.
Medical Diagnosis
4th Edition, Revised and Enlarged. 548 Text Illustrations, 14
Colored Plates. 8vo. xix -f- 1302 pages. Cloth $13.00 Postpaid.
Contains 577 more Pages and 307 more Illustrations than
previous edition.
By CHARLES LYMAN GREENE, M.D.
Formerly Professor of Medicine and Chief of the Department of
Medicine and Medical Clinic, College of Medicine, University
of Minnesota.
"It consists of a dictionary of diagnosis, a textbook of medicine and a
treatise on clinical methods rolled into one. . . . There is a vast
amount of information in the volume and it is readily accessible by means
of a good index." British Medical Journal.
"Includes both physical diagnostic methods and laboratory procedures.
Considerable space is given to practical advice on case taking, relative
values of various observations, and other points culled by the author from
his experience of many years." Jour. Am. Med. Assoc.
5-8-22
WILCOX. MATERxA MED1CA AND THERAPEUTICS: INCLUD-
ING PHARMACY AND PHARMACOLOGY. By REYNOLD WEBB
WILCOX, M.D., LL.D., Professor of Medicine (Retired) at the
New York Post-Graduate Medical School and Hospital. This
work is divided into two distinct parts: Materia Medica and
Pharmacy; and Pharmacology and Therapeutics. It offers a
very complete presentation of the subjects treated with its
natural separation and in logical order. Tenth Edition Revised
in accordance with the U. S. P. IX. xii 4- 860 pages.
Cloth, $4.75
STEWART. A MANUAL OF SURGERY. For Students and Phy-
sicians. By FEANCIS T. STEWART, M.D., late Professor of Clinical
Surgery, Jefferson Medical College. 580 Illustrations, 21 printed
in colors. Fifth -Edition. Thoroughly Revised and Enlarged.
Octavo. Cloth, $10.00
LANG. GERMAN-ENGLISH MEDICAL DICTIONARY. By the
late DR. HUGO LANG, B.A. (Munich). Second Edition, Edited
and Revised by MILTON K. MEYERS, M.D., Neurologist to the
Jewish Hospital Dispensary and to St. Agnes Hospital Dis-
pensary, Philadelphia, etc. Octavo. 668 pages. Cloth, $6.00
McGUIGAN. AN INTRODUCTION TO CHEMICAL PHARMA-
COLOGY. Pharmacodynamics. In Relation to Chemistry. 8vo;
xii-f418 pp. By HUGH MCGUIGAN, PH.D., M.D., Professor of
Pharmacology, University of Illinois, College of Medicine.
Cloth, $4.00
MAcNEAL. PATHOGENIC MICROORGANISMS. 2d Edition,
Revised. 221 Illustrations. 12mo; xx-f488 pp. By WARD J.
MACNEAL, M.D., Professor of Pathology and Bacteriology, New
York Post-Graduate Medical School. Cloth, $4.00
BRUBAKER. A TEXT-BOOK OF HUMAN PHYSIOLOGY. By
ALBERT P. BRUBAKER, A.M., M.D., Professor of ' Physiology and
Hygiene, Jefferson Medical College. Colored Plate and 359
Illustrations. Octavo. Sixth Edition, xii -|- 794 pages.
Cloth, $4.75
DAVIS. PLASTIC SURGERY. ITS PRINCIPLES AND PRAC
TICE. By JOHN STAIGE DAVIS, PH.B., M.D., F.A.C.S., formerly
Captain U. S. Army Medical Corps, Instructor in Clinical
Surgery, Johns Hopkins University. 864 Illustrations, con-
taining 1637 figures. 8vo. Cloth, $12.00
POTTER. THERAPEUTICS, MATERIA MEDICA, AND PHAR-
MACY. By SAMUEL O. L. POTTER, M.A., M.D., M.R.C.P., (Lond.)
Including the Physiological Action of Drugs, Special Therapeutics
of Diseases and Symptoms. The Mbdern Materia Medica,
Official and Practical Pharmacy, Minute Directions for Pre-
scription Writing, Incompatibility, etc. Also Antidotal and
Antagonistic Treatment of Poisoning and over 650 Prescriptions
and Formulae. Thirteenth Edition. Revised by ELMER H.
FUNCK, M.D., Associate in Medicine, Jefferson Medical College,
Philadelphia. In accordance with the Ninth Revision U. S.
Pharmacopoeia. 8vo. xvi -J- 960 pages. Thumb Index in Each
Copy. Cloth, $8.50
XB 65642
lition. Thoroughly
BINNIE. OPERATIVE SURGERY. 8th Edition. Thoroughly Re-
vised. 1628 Illustrations. By JOHN FAIRBAIRN BINNIE, A.M.,
C.M. (Aberdeen) ; Surgeon to the Christian Church, the Research
and the General Hospitals, Kansas City, Missouri. Cloth, $12.00
STITT. PRACTICAL BACTERIOLOGY, BLOOD WORK AND
ANIMAL PARASITOLOGY. By E. R. STITT, A.B., PH.G., M.D.,
Medical Director U. S. Navy. 12mo. 6lh Edition. Illustrated.
Flexible Cloth,$4.00
STITT. DIAGNOSTICS AND TREATMENT OF TROPICAL DIS-
EASES. Third Edition. 119 Illustrations and Several Refer-
ence Tables in the Text. 8vo. xiii -f- 534 pages. Cloth, $3.25
McMURRICH. THE DEVELOPMENT OF THE HUMAN BODY.
A Manual of Human Embryology. By J. PL-AYFAIR McMuRRiCH,
A.M., PH.D., Professor of Anatomy, University of Toronto. Sixth
Edition, thoroughly Revised. Illustrated. Cloth $3.25
DERCUM. REST, SUGGESTION AND OTHER THERAPEUTIC
MEASURES IN NERVOUS AND MENTAL DISEASES. By
FR^jf^g^lfe. DERCUM, M.D., PH.D., Professor of Nervous
LOO
SKE1
C
c
SLUS
S:
SI
E
RODD
A
C<
488853
P6
UBRARY
G
ate
1.75
W.
sity
ind
L75
cal
res.
:.00
HA o1 ' do-
ogy, Jefferson Medical College. Sixth Edition, Revised. 6 Full-
page Plates in Cblors and 185 Text Figures. Octavo. Cloth, $5.00
THORINGTON. REFRACTION OF THE HUMAN EYE AND
METHODS OF ESTIMATING THE REFRACTION. In-
cluding Section on the Fitting of Spectacles and Eye-Glasses,
etc. By JAMES THORINGTON, A.M., M.D., Emeritus Professor of
Diseases of the Eye in the Philadelphia .Polyclinic. 343 Illus-
trations, 27 Printed in Color. Octavo. Cloth, $3.00
SWANZY AND WERNER. A HANDBOOK OF THE DISEASES OF
THE EYE AND THEIR TREATMENT. 12th Edition. 9
Colored Plates and 274 other Illustrations. Octavo. By SIR
HENRY R. SWANZY, M.D., Surgeon to the Royal Victoria Eye and
Ear Hospital, and Ophthalmic Surgeon to the Adelaide Hospital,
Dublin, and Louis WERNER, M.D., F.R.C.S.I., Professor of Ophthal-
mology, University College, Dublin. Cloth, $6.50
1R811
rl ;A/V^V^>ViV>'' >Vswttw