Full text of "A compend on bacteriology : including animal parasites"

See other formats

ms^

3 RIO LOGY

PITFIELD

UNIVERSITY OF CALIFORNIA

SAN FRANCISCO

LIBRARY

»4TS.

:CS: IN-

By Rey-

; (Retired)

Consult-

-President

Students.

and Phar-

complete

ion and in

e U. S. P.

oth, $3.50

5 and Phy-
.1 Surgery,
ital; Out-
ions, 21 of
Bvised and
oth, $4.00

id Largely
American
d Director
IVith 1 182
b Volume,
oth, $6.50

part sold

3th, $1.50
System —
3th, $2. 50

3th, $2.00
Mammary
)th, $1.50
bth,$i.5o

BINNIEr \7rErKAriVK SURGERY. By John Fairbairn Binnie,
A.M., CM. (Aberdeen); Surgeon to the General Hospital, Kansas City,
Missouri; Fellow of the American Surgical Association; Membre de la
Societe Internationale de Chirurgie, etc. Seventh Edition. Thoroughly
Revised and Enlarged. 1597 Illustrations, many printed in colors.
8vo, 1377 pages. Cloth, $7.00

COPLIN. MANUAL OF PATHOLOGY. By W. M. Late Coplin,
M.D., Professor of Pathology and Bacteriology, Jefferson Medical Col-
lege; Pathologist to, and Director of the Clinical Laboratories in, Jeffer-
son Medical College Hospital, Philadelphia. Fifth Edition. With 612
Illustrations and 1 2 Plates, of which 1 1 are Colored. 8vo. Cloth, $4.50

P. BLAKISTON'S SON &, CO., Publishers Philadelphia

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 Illustra-
tions. Octavo. Fifth Edition, xii + 776 pages. Cloth, net, $3.50

POTTER. THERAPEUTICS, MATERIA MEDICA, AND
PHARMACY. By Samuel O. L. Potter, m.a., m.d., m.r.c.p.,
(Lond.), formerly Professor of the Principles and Practice of Medicine,
Cooper Medical College, San Francisco. Including the Physiological
Action of Drugs, Special Therapeutics of Diseases and Symptoms. The
Modern Materia Medica, Ofhcial and Practical Pharmacy, Minute
Directions for Prescription Writing, Incompatibility, etc. Also Antido-
tal and Antagonistic Treatment of Poisoning and over 650 Prescriptions
and Formulae. Twelfth Edition. In accordance with the latest reprint
U. S. Pharmacopoeia. 8vo. 956 pages. Thumb Index in each Copy.

Cloth, net, $5.00

KYLE. MANUAL OF DISEASES OF THE EAR, NOSE, AND
THROAT. By John Johnson Kyle, b.s., m.d., Professor of Otology,
Rhinology and Laryngology in the Indiana University School of
Medicine, Indianapolis, etc. Third Edition. With 176 Illustrations.
i2mo. xxxi + 642 pages.

Flexible Leather, Gilt Edges and Round Corners, net, $3.00

GORDON. DISEASES OF THE NERVOUS SYSTEM. FOR
THE GENERAL PRACTITIONER AND STUDENT. By

Alfred Gordon, m.d., formerly Associate in Mental Diseases, Jefferson
Medical College; Neurologist to Mount Sinai Hospital; Member Ameri-
can Neurological Association; late Examiner of the Insane at the Phila-
delphia Hospital, etc. Second Edition. With 169 Illustrations, a num-
ber of which are in colors. Octavo, xiv + 618 pages. Cloth, $4.00

LEWIS & STOHR. TEXT-BOOK OF HISTOLOGY. Arranged
upon an Embryological Basis. By Frederic T. Lewis, m.d., Assistant
Professor of Embryology at the Harvard Medical School. Second Edi-
tion, Revised, from the Fifteenth German Edition by Philipp Stohr,
m.d., Professor of Anatomy at the University of Wiirzburg. With 495
Illustrations, 51 in Colors. 8vo. xi + 539 pages. Cloth, fiet. $3.50

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 Dispensary, Philadel-
phia, etc. Octavo. 668 pages. Cloth, $5.00

STITT. PRACTICAL BACTERIOLOGY, BLOOD WORK AND
ANIMAL PARASITOLOGY. By E. R. Stitt, a.b., ph.g., m.d..
Medical Director U. S. Navy; Head of Department of Tropical Medi-
cine, U. S. Naval Medical School. i2mo. Fourth Edition, xvii +
497 pages. 106 Illustrations. Flexible Cloth, net,''$2.oo

MacNEAL. PATHOGENIC MICROORGANISMS. By Ward J.

MacNeal, m.d.. Professor of Pathology and Bacteriology, New York
Post-Graduate Medical School. (Based upon Williams' Bacteriology.)
With 213 Illustrations. i2mo. xxi + 462 pages.

Flexible Cloth, Round Comers, $2.50

P. BLAKISTON'S SON & CO., Publishers, Philadelphia

McMURRICH. THE DEVELOPMENT OF THE HUMAN
BODY. A Manual of Human Embryology. By J. Plavfair Mc-
MuRRiCH, A.M., PH.D., Profcssor of Anatomy, University of Toronto.
Fifth Edition, thoroughly Reyised. With 287 Illustrations. Octavo.
X + 493 pages. Flexible Cloth, Round Corners, $2.50

REESE. MEDICAL JURISPRUDENCE AND TOXICOLOGY.
Eighth Edition. A Text-Book for Medical and Legal Practitioners
and Students. By John J. Reese, m.d., formerly Professor of the
Principles and Practice of Medical Jurisprudence in the University of
Pennsylvania. Eighth Edition, Revised. By D. J. McCarthy, a.b.,
M.D., Professor of Medical Jurisprudence in the University of Pennsyl-
vania; Neurologist to the Philadelphia General and St. Agnes' Hos-
pitals, etc. i2mo. 669 pages. Cloth, $3.00

LONG. PHYSIOLOGICAL CHEMISTRY. By John Harper
Long, m.s., sc.d.. Professor of Chemistry and Director of the Chemical
Laboratories of the Northwestern University, Chicago. Second Edi-
tion, Revised. Illustrated. 8vo. viii -f 396 pages. Cloth, net, $2.50

SKEEL. MANUAL OF GYNECOLOGY. By R. E. Skeel, As-
sociate Clinical Professor of Gynecology, Western Reserve University,
Cleveland, Ohio. With 289 Illustrations. i2mo. xiv -f 680 pages.

Flexible Cloth, Round Comers, $3.00

SLUSS. MANUAL OF EMERGENCY SURGERY. By John
W. Sluss, M.D., Associate Professor of Surgery, Indiana University
School of Medicine, Indianapolis; Surgeon to the IndianapoUs City
Hospital, etc. Fourth Edition, Revised and Enlarged. 685 Illustra-
tions, some of which are Colored, xv + 848 pages.

Full Limp Leather, Gilt Edges, Round Corners, $4.00

O'REILLY. A MANUAL OF PHYSICAL DIAGNOSIS. By
Brefney Rolph O'Reilly, m.d., cm., f.t.m.c. (Toronto); m.r.c.s.
(England); l.r.c.p. (Lond.); Demonstrator in CHnical Medicine and in
Pathology, University of Toronto. With 6 Plates and 49 Text Figures.
i2mo. xix + 368 pages. Cloth, net, $2.00

HENDERSON. LESSONS OF THE EYE. By Frank L.
Henderson, m.d., Ophthalmic Surgeon, St. Mary's Infirmary; Con-
sulting Oculist to the St. Louis Hospital, the Wabash Railway, etc.
Fourth Edition. 138 Illustrations. i2mo. viii + 238 pages.

Cloth, net, $1.50

HAWK. PRACTICAL PHYSIOLOGICAL CHEMISTRY. A
Laboratory Handbook, designed for use in Courses in Practical Physio-
logical Chemistry in Schools of Medicine and Science. By P. B. Hawk,
M.S., PH.D., Professor of Medical Chemistry and Toxicology, Jefferson
Medical College. Fifth Edition, Revised. With 6 Full-page Plates
in Colors and 172 Text Figures, of which 12 are in Colors. Octavo,
xiv + 638 pages. Cloth, net, $3.00

THORINGTON. REFRACTION OF THE HUMAN EYE
AND METHODS OF ESTIMATING THE REFRACTION.
Including 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; Ophthalmic Surgeon, Presbyterian
Hospital; Ophthalmologist to the Elwyn, Vineland and New Jersey
State Training Schools for Feeble-minded Children, etc. With 343 Illus-
trations, 27 of which are Printed in Color. Octavo. Cloth, net, $2.50

P. BLAKISTON'S SON & CO., Publishers, Philadelphia

HEXRY I. FLEISSIG

BACTERIOLOGY

PITFIELD

HENRY I. FLEISSIG

From The Southern Clinic.

"We know of no series of books issued by any house that so fully meets our
approval as these 7Quiz-Compends?. They are well arranged, full, and concise,
and are really the best line of text-books that could be found for either student
or practitioner."

BLAKISTOIN'S 7QUIZ-C0MPEMDS?

The Best Series of Manuals for the Use of Students.

Price of each. Cloth, $1.00 net. Interleaved, for taking Notes, $1.35 net.

-BS^These 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.

tSTThe authors have had large experience as Quiz-Masters and attaches of
colleges, and are well acquainted with the wants of students.

4KS=|They are arranged in the most approved form, thorough and concise
containing over iioo fine illustrations, inserted wherever they could be used to
advantage.

4^Can be used by students of any college. They contain over 1 100 illustrations.

J>S"They contain information nowhere else collected in such a condensed, practical
shape.

Illustrated Circular Free.

POTTER'S ANATOMY. Seventh Revised and Enlarged Edition. Including

Visceral Anatomy, Can be used with either Morris' or Gray's Anatomy. 138

Illustrations and 16 Plates of Nerves and Arteries, with Explanatory Tables, etc.
BRUBAKER. PHYSIOLOGY. Thirteenth Edition, with Illustrations and a Table

of Physiological Constants. Enlarged and Revised.
LANDIS. OBSTETRICS. Eighth Edition. Revised and Edited by Wm. H.

Wells, m. d., Associate in Obstetrics, Jefferson Medical College, Philadelphia,

56 Illustrations.
POTTER. MATERIA, MEDICA THERAPEUTICS AND PRESCRIPTION WRIT-
ING. Seventh 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. Third Edition.
HORWITZ'S SURGERY, Minor Surgery, and Bandaging. Sixth Edition, Enlarged

and Improved. With 104 Formulae and 195 Illustrations.
LEFFMANN. CHEMISTRY, Inorganic and Organic. Fifth Edition. Including

Urinalysis, Animal Chemistry, Chemistry of Milk, Blood, Tissues, the Secretions,

etc.
STEWART. PHARMACY. Eighth Edition. Based upon Prof. Remington's

Text -book of Pharmacy.
BALLOU. VERTERINARY ANATOMY AND PHYSIOLOGY. With 29 graphic

Illustrations.
WARREN. DENTAL PATHOLOGY AND DENTAL MEDICINE. Fourth Edition,

Illustrated. Containing all the most noteworthy points of interest to the Dental

Student, and a Section on Emergencies.
HATFIELD. DISEASES OF CHILDREN. Colored Plate. Third Edition, Revised

and Enlarged.
ST. CLAIR. MEDICAL LATIN. Second Edition.
SCHAMBERG. DISEASES OF THE SKIN. Fourth Edition. Revised and Enlarged.

108 Illustrations.
RADASCH. HISTOLOGY. Third Edition. With 11 1 Illustrations.
HIRSCH. GENITO -URINARY AND VENEREAL DISEASES, AND SYPHILIS.

Second Edition. With 75 Illustrations.
PITFIELD. BACTERIOLOGY. Second Edition. 4 Plates and 85 other Illustrations.

BLAKISTON'S ?QUIZ-COMPENDS?

A COMPEND

BACTERIOLOGY

INCLUDING ANIMAL PARASITES
HENRY I. F-LSISSIO

ROBERT L.' PITFIELD, 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.

SECOND EDITION

WITH 4 PLATES AND 85 OTHER ILLUSTRATIONS

P(.6

PHILADELPHIA

P. BLAKISTON'S SON & CO.

1012 WALNUT STREET
1914

THE. MAPLE. PRESO.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
practitioner of medicine who desires to acquaint himself with the
principal 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 Kalle & Wasser-mann's
Atlas, Williams, McFarland, Tyson's Practice 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 rearrange-
ment of chapter, and the new matter that has been added
throughout the book.

To the firm of P. Blakiston's Son & Co. the writer is indebted
for valuable aid.

Robert. L. Pitfifxd.

Digitized by the Internet Archive

in 2007 with funding from

IVIicrosoft Corporation

http://www.archive.org/details/compendbaGteriaOOpitfrich

HENUVI.ILEISSIG

TABLE OF CONTENTS

Pagb
CHAPTER I.

The Classification, Morphology, and the Biology of Bacteria i

CHAPTER II.
Products of Bacterial Energy 21

CHAPTER III.
Infection 27

CHAPTER IV.
Immunity .• 41

CHAPTER V.
Study of Bacteria 83

CHAPTER VI.
Bacteriological Laboratory Technic 96

CHAPTER VII.
Antiseptics and Disinfectants 120

CHAPTER VIII.
Bacteria 127

CHAPTER IX.
Animal Parasites 211

CHAPTER X.

The Filterable Viruses 234

CHAPTER XI.
Bacteriology of Water, Soil, Air and Milk 261

Index 271

vii

HENRY I. FLEISSIO

COMPEND OF BACTERIOLOGY.

CHAPTER 1.

THE CLASSIFICATION, MORPHOLOGY, AND THE
BIOLOGY OF BACTERIA.

X BACTERIA (fission fungi or schizomycetes) may be defined
as very minute, unicellular vegetable organisms, almost always
devoid of chlorophyll, ^nd generally unbranchedjthat 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 bacteriol-
ogists. The following one is based somewhat upon Migula'Sy
and that adopted hy Lehmann and Neumann, which was compiled
from the systems of FlUgge, Fischer, Loffler, and Migula,

CLASSIFICATION .^Bacteria may be conveniently divided into
six families, according to their morphology or shape. j|

I. "Jt^COCCACEiE.^Sphencal or spheroidal bacteriaX (Globular
in free state but usually seen with one axis slightly larger.
They do not have parallel sides like the bacilJi^Xro mul-
tiply, the cell divides into halves, quarters, or eighths,
each of which grow again into perfect spheres. -^pndos-
pores and flagella are very rare. (Lehmann and Neumann.)
If mobile they are called Planococcus or Planosarcina.

2 BACTERIA

^ (a) Streptococcus. — Cells that divide 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

balelike packages, or blocks of eight are formed.JV-At

least one variety (Sarcina agilis) is motile, having flagella.

Plates of cocci, one thick in the plane, are called '^meris-

mopedia."

II.teACTERIACE^.— Rod bacteria are straight or slightly
curved. j( 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.

III. )LSPIRILLACEiE.— Spiral bacteria* Unicellular, more or
less elongated. Twisted more or less like a corkscrew.
Cells are sometimes united in short chains. j(^ 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.

CHLAMYDOBACTERIACE^ 3

(d) Spirochaeta. Cells with long flexible spiral threads,
without flagella. They move by means of an undulating
membrane. These have been thought to belong to the
bacteria but since we now know that most of them move
by an undulating membrane, they should be classified
with the protozoa.

IV. MYCOBACTERIACEiE.— 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
flagella, 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.
Not acid proof or acid fast, and frequently emit a musty

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

4 BACTERIA

whole branches, and by swarm spores or motile gonidia
having flagella.

(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 un-
branched threads by means of delicate sheaths, branching
threads are then formed. Division takes place in three
directions of space, producing sarcina-like groups of goni-
dia, which, when free, are spherical.

(d) Thiothrix. Are unbranched cells, sheathed, without
flagella, divided only in one direction, and contain sulphur
granules.

VI. BEGGIATOACEiE. Cells united to form threads that are
not sheathed: have scarcely visible septa; divide in one direc-
tion, 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 diiferent. The ulti-
mate differentiation of one species from another depends not only
on the morphology, which may be precisely similar, but on its biolog-
ical behavior in culture media and in the tissues of animals under
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 obser-
vation of the development of bacteria in culture media, and the re-
actions produced in it, and in the bodies of experiment animals, that
we can identify them positively from others of a foreign species. No
bacteriologist is able by a simple microscopical examination of a
given bacterium, to identify it absolutely at all times. -^

CLASSIFICATION

^ Bacteria that are globular in form are called cocci. J(
i/L Cocci that divide in one direction of space and grow in chains are
called streptococci. / (Fig. i.)

^ Cocci that divide irregularly and form pairs or fours, or irregular
groups, are called micrococci. Those of this class that form pairs

Fig. I. — Large and very large streptococci. (Kolle and Wassermann.)

are frequently called diplococci. When they form fours by division
in two directions, they are called tetrads. But when they divide
irregularly and form masses resembling bunches of grapes, they are
spoken of as staphylococci. X (Fig. 2.)

Fig. 2. — Staphylococci. Streptococci. Diplococci. Tetrads. Sarcinae. (.Williams.)

\ Cocci that divide in three directions are called sarcince. One
single coccus, by division in three directions, forms cubes of eight
or more, each of which becomes globular and equal in size to the
parent. 4

O BACTERIA

^ Motile micrococci are those that divide in two directions of space,
and have flagella. fsThey are known as planococci.

Micrococci that divide in three directions, and are motile, are
C2i\\ed piano sarcifhcd, (Fig. 3.)

fvBacteria 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. ^

Fig. 3. — ^Planosarcina ureae, showing very long flagella.
(Kolle and Wassermann.)

^Bacilli may grow singly or in chains; may be flagellated; contain
spores and gonidia; or, may be devoid of flagella. "j^

Members of the spirillaceae that resemble a curved rod, or are
comma shaped, are known as vibrios. (Fig. 4.) Those of the
same family that resemble a corkscrew, are called spirilla. (Fig. 5.)
When they are like long spiral threads they are called spirochcetce .

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
lanceolatus as the pneumococcus. As the organism appears in the
diseased lung, or in the sputum, one diameter of the coccus is invaria-
bly longer than another, and the rule of equal diameters cannot be

• CLASSIFICATION 7

applied to it. But in culture mediae the organism resembles a true
coccus, being globular and growing in chains. It is then called
the Streptococcus lanceolatus. It is common also to speak of
members of the family of Mycohacteriacece. as bacilli, as they are
more 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 belonging
to the sheathed group, or Chlamydobacteriacece, 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 it as pleo-

FiG. 4. — Cholera vibrios.
(Greene's Medical Diagnosis.)

Fig. 5. — Spirillum relapsing fever.
(Greene's Medical Diagnosis.)

morphic, or pleomorphism. To elucidate: Man is pleomorphic,
because among adult individuals some are tall or short, fat or thin.
-^ Involution or Degeneration forms. When the best or optimum
conditions for bacterial life (see page 17) are not found, bacteria
present appearances quite different from those of the young, active
or perfect adult type. These are called involution or degeneration
forms. For example: the diphtheria bacillus under good conditions
for life is a straight or slightly bent rod staining in a granular man-
ner. If living under unsuitable conditions it becomes quite short,
and stains solidly. Ag'ain bacilli that are^ accustomed to appear as
short elements may grow to long threads without dividing, or swell
into unrecognizable form. /

8 BACTERIA

y To measure bacteria, we use the thousandth part of a millimeter,
called the micromillimeter, or micron, as the unit?* (xhe Greek let-
ter // is the symbol for this unit)Av\ micron is about ywUu of an
inch, yet a bacterium one fi long, and a half ft 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 micro-micron, and is symbolized by n/i. The
shortest light-wave lengths are about 400 jujut, or .4 //, while chromatic
threads in cells of bacteria are often 100 juji in width. Then
again there are many things smaller than these threads. The thin-
nest part of a bursting soap bubble is but 7 /jl/i
in thickness. There are certain infectious
agents that are submicroscopic; that is, in-
visible even by the aid of Siedentopf's
ultraviolet microscope, which shows objects
smaller by half a light-wave length (.2 pL/x).
^ The structure of the bacterial cell is very
PiQ 6.— Diplococci simple. It consists of (i) a central nuclear
showing capsules \^Q^y ^iij^h can be stained like the nuclei of
(Greene's Medical , 1.1,,., ,

Diagnosis.) other vegetable and animal cells, with nuclear

or basic stains, such as haematoxylin, or
methylene-blue. \

< (2) A cytoplasm, or protoplasmic substance generally very thin.^l
J^(3) A cell wall, more or less thick, that stains with difficulty.
(Fig. 6.)

'Lin the nucleus we often see metachromatic bodies, called the
Babes-Ernst granules, and unstained spaces called vacuoles , both of
which are common to many bacteria. These are both probably
due to ingested food or fluid, -f^

> Through the cell wall the food of the bacterium passes by osmosis. -J-
4NThe cell wall of certain organisms, for example the 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

CLASSIFICATION 9

certain specific substances (ferment) found in the blood of immu-
nized animals, called hacteriolysins and agglutinins^ Where
great masses of bacteria are clumped in excessive mucilaginous
material we speak of this condition as zooglea. (Fig. 7.)
^ We sometimes find, as a prolongation of the cell wall, filamentous
organs of locomotion known as flagella. Not all bacteria possess

Fig. 7. — Zooglia formation. (Leuconostoc.) (Kolle and Wassermann.)

these^ut those that do, are called trichohaderia. Those that have
not/flagellaSare called gymnobacteria. Trichobacteria are classified
accoi^iw^^ the number and location of the flagella. When they
have one flagellum we call them monotrichous bacteria, and amphi-
trichous when there are two flagella, one at each pole. (Fig. 8.)
When the cell is surrounded by flagella, it is known as a peritrichous
bacterium, and lophotrichous when the flagella are arranged in tufts
of two or more. These are simple adjectives and not 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. (Fig. 9.)
♦ Flagella are not pseudopods, but distinct organs of locomotion.
In certain bacteria of the Beggiatoa, locomotion is accomplished

lO BACTERIA

by a peculiar amoeboid motion, or by an undulating membrane.
On looKng at bacteria known to have no powers of voluntary
motion, they are seen to oscillate, tremble or move slightly. Sus-
pensions 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. There are bacteria that are considered

Fig. 8. — Spirillum undula with polar Fig. 9 — Bacillus prolius vul-

flagella. (Kolle and Wassermann .) garis, showing peritrichous fla-

gella. (Kolle and Wassermann.)

non-motile, on which it is possible to demonstrate flagella. 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 or
bacteria varies from a simple rotatory, on one axis, to a swinging,

REPRODUCTION II

shaking, boring or serpentine action. The location of the flagella
has some influence upon their behavior. Flagella may be broken
off from the cell body by agitation. They are then clumped by
agglutinating sera.

Flagella may have other functions than locomotion. It is possible
that they may serve as organs for the absorption of nourishment
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 Zierler), but this is rare. Certain flagella have in their contin-
uity 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 progress by itself completely across the field
of the microscope.

t REPRODUCTION.— The process of direct cell division is the
commonest way by which bacteria multiply. Hence 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,

^nd by other granules called gonidia.J The spores appearing in the
lower bacteria, bacilli for example, are not reproduction forms but
states of high resistance.

The process of cell division or binary division is very simple, and
may be a matter of twenty minutes, or as long as six hours. Divi-
sion 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 fol-
lowed by a constriction or 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, u

12 BACTERIA

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 an hour becomes two, and at the end of a
second hour these two become four; at the end of another hour 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 re-
duced to simple gases that are quickly dissipated in the air.
•k 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

Fig. io. — The formation of Fig. ii. — Spores and their location in bacteria J
spores. (After Fischer from cells. (After Frost and McCampbell.)

Frost and McCampbell.)

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. 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.

SPORULATION 13

Endospores are produced under stress of circumstances, when
certain agencies' or conditions, such as absence of food, drying, and
heat, threaten the extinguishment of the organism. Spores are
bright, shining, oval, or round bodies, which do not take aniline
dyes readily, and which, when they are stained, retain the color
more tenaciously than the adult cells. They resist heat, often with-
standing a temperature of 150° C. dry heat for an hour. Steam
under pressure at a temperature of 150° C. will invariably kill them
after a short exposure.

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.

(1) Q f, I fe ^^ I k^^

0 1^

0 0 0

5 8

Fig. 12. — Spore germination, a, direct conversion of a spore into a bacillus
without the shedding of a spore- wall {B. leptosporus) ; h, polar germination of
Bad. anthracis; c, epuatorial germination of B. suhtilis; d, same of B. mega-
terium; e, same with "horse-shoe" presentation. (After Novy.)

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 diameter becomes greater in
one portion of the cell, and dust-like particles appear, then a bright
spot; a capsule then forms, the spore escapes, and the parent cell
dies, i

^ Certain spore bearing bacteria grown for a week at 42° C. 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. Qiie_spore only i<; pio-
duced by an adult cell. Some forms of bacteria can be diffejeiit^itQd

14 BACTERIA

from each other only by the way in which they sporulate, whether
from the poles or the equator. K

V The BacterlacecB are the prominent spore producers. Certain
round bodies found in bacteria of low thermal death-point, are
called by Heuppe arthrospores. It is believed that they are without
significance. A high thermal death-point in bacteria indicates
that the organism produces spores. Arthrospores are common

"^t:.

Fig. 13. — Capsules. Bad. pneumonicB (Friedlander) . (After Weichselbaum
from Frost and McCampbell.)

among the micrococci and may be associated with capsule forma-
tion and cell enlargement. The whole cell may stain more
intensely. They are also to be sought among the Strep tothrix
genus. >

"fiv 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.^
^ Sporulation is more apt to occur under poor nutritive conditions. Y
The anthrax bacillus thrives at 13° C. but cannot sporulate below

SPORULATION

18° C. Anthrax spores have been known to resist the germicidal
action of a 5 percent carbolic acid solution for forty days.

Babes-Ernst granules, or polar bodies are found in certain
bacteria (Mycobacteriaceae, etc.) after staining with special basic
stains. In the complex forms of bacteria, they evidently have an
important role in reproduction. The presence of such bodies in the
poles of diphtheria bacilli facilitates the recognition of these organ-
isms. (Fig. 14.)

Fig. 14. — ^Pest bacilli showing capsules. (Kolle and Wassermann.)

i Capsules. Certain well known pathogenic bacteria have thick
well marked capsules. The pneumococcus, pneumobacillus, and
Bacillus aero genes 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. JC
(Fjg^^

The^hi^her bacieria/2Lre those from the Mycohacteriacece up to the
[easts and moulds./ They are higher than the Bacteriacece 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 Strepto-

1 6 BACTERIA

thrix. 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 organisms of actinomycosis and Madura foot.
The Chlamydohacteriacece. and Beggiatoa are Saprophytes. These
require special technique for their laboratory culture.
Y^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 stalklcalled mycelia. ^They grow
well in the laboratory and may produce pig-
ments. They are much larger than the bac-
teria (ia-25 /x long). They multiply by bud-
ding 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 com-
PiQ_ 15, B a c i 1 1 i monest genus is Saccharomyces.

showing capsules xhe Moulds or .Hyphomycetes represent the
(Greene's Medical , . <r ^^~V^ 1 , r^^

Diagnosis.) next highest group of the plant algae. They

are characterized by a greater prominence of
the mycelium over simple segments or bodies. They are wide-
spread 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. Further 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.

^A Bodies^ of bacteria contain_water,_saItv .certain albumins, and
bodies that may be extracted with ethgrX Among the latter are
lecithin, cholesterin, and triolein.^ In the tubercle bacillus, fatty
acids and wax have been found. In others, xanthin bases, cellulose,
starch, chitin, iron salts, and sulphur grains have been discovered.
The essential albumin of the cell-body is highly nitrogenous and is
called mycoprotein. The salts in the ash are mostly composed of

BIOLOGICAL CONDITIONS 1 7

various phosphates. Intracellular toxins in combination with
the cytoplasm are found in certain groups of bacteria, e.g., B,
typhosus. pC

BIOLOGICAL CONDITIONS.

y( Bacteria are arbitrarily classed either 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
con6.\iioit{M ycohact. leprce) , or they may be capable of life on arti-
ficial culture media (tubercle bacillus), or of life in the body, on
culture media, or in the soil (B. tetani). fC

j( The bacteria of the soil, water, air, etc., that are incapable of
successful life in the body tissues are called saprophytes, j^
y( Certain biological conditions are essential for the growth of bac-
teria: water, oxygen, carbon, nitrogen, and salts are necessary.
For certain parasitic bacteria, highly complex substances are indis-
pensable: meat albumins, peptones, milk, egg albumin, blood
serum, and sugars are the ingredients of various culture media, y
)<. 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 prcjji^
duces its strongest toxins and grows best in alkaline media. Salt
free mkiia 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 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 namecfyoa*/-
tative anaerobes. Free oxygen is needed for spore formation by cer-
tain bacteria. Anaerobes obtain oxygen as they need it by breaking
up their food stuffs, y

>y Nutriment is most important for the growth of bacteria, nitrog-
enous compounds (albumins) particularly being required. Simple

l8 BACTERIA

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 MycobacteriacecB. Blood serum or whole blood is required by
some pathogenic organisms. The food stuffs 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 o° C. and
70° C, some varieties thrive at the one extreme, and otH^rs at the
other. j(

Psychrophilic bacterid are those that grow at 15° C, with a
maximum of 30° C. and a minimum of 0° C. Water bacteria
of the polar seas belong to this group.

Mesophilic grow best at 37° C. — the temperature of the body —
and thrive from 10° C. (minimum) to 45° C. (maximum). All
pathogenic bacteria belong to this group.

Thermophilic (min. temp. 40° C, max. 60-70° C.) are most
prolific at 50-55° 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. When thus associated, they sometimes
benefit each other. Such combination is called symbiosis. ]f^
V Certain anaerobic bacteria grow i^^ the presence of oxygen if
other particular varieties of aerobic bacteria are present./
\ Attenuated tetanus bacilli become virulent if cultivated with Bac-
terium vulgae. Again, complicated chemical changes, as the decom-
position of nitrites with the evolution of nitrogen cannot be accom-
plished by certain bacteria severally, but jointly, this is quickly
brought about. 'f'

^Pfeiffer has shown that certain chemical substances (foods, albu-
mins, etc.), attract bacteria (positive chemolaoHs), while other sub-
stances, as turpentine, repel them (negative chemotaxis).

Oxygen repels anaerobes and is particularly attractive to aerobes. J[^

AGENTS PREJUDICIAL TO BACTERIAL LIFE 1 9

FREE AGENTS PREJUDICIAL TO THE LIFE OF
BACTERIA.

High temperatures are surely germicidal: 60° C. coagulates
mycoprotein of bacteria and other common albumins. The degree
of temperature at which bacteria are killed is called the thermal
d^atit^oint. Most vegetative forms die after a short exposure at
60° C, 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 115° C. moist heat (steam),
from thirty to sixty minutes. Bacteria resist dry heat of 175° C.
from five to ten minutes. <

y Cold inhibits bacteria; destroys some; but is not a safe germicidal
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 ( — 312°
F.) 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. In each
instance, vegetative forms grew after the exposure.
/ Light is inimical to the life of bacteria; direct sunlight being the
most germicidal, as it destroys some, reduces the virulence of 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, especially lupus. Diffuse sun-
light, electric light, Rcentgen-rays, continuous and alternating
currents of electricity, are also more or less germicidal. Anti-

20 BACTERIA

septics, such as metallic salts, formalin, carbolic acid, cresol,
mineral acids, and essential oils, are powerful germicides; some
even in high dilution. /
"7^ 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 disinfectants.
Excessive amounts of sugar, salt, glycerine, and the pyroHgneous
acids act as destroyers, or inhibitors to bacterial growth in food stuffs^
r^Metals act as lethal agents in the presence of light and water, by
forming metallic peroxides, which either destroy the vitality of bac-
teria or hinder their growth. Silver, zinc, cadmium, bismuth, and
copper, have this action. Consequently silver wire, or foil, are
used in surgery because of their anti-septic action. Metallic
fillings in teeth prevent the growth of bacteria that cause caries,
w Certain cells in the bodies of animals (leucocytes) and some ele-
ments of the blood serum, being bactericidal, are a powerful means
4Bf internal defense against infection, j^

^ 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 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 smoking, prevents
bacterial growth, and consequently, decay of the food stuff. The
products of bacterial growths often inhibit, or destroy, the cells that
made them, as well as other bacteria. B. pyocyaneus and S.
cholercEf have this property of secreting autolytic ferments.y

CHAPTER II.

PRODUCTS OF BACTERIAL ENERGY.

"iiC According to their chemical activities, bacteria are arbitrarily
divided into the following classes:

)(^ Photogens Chromogens Zymogens

Saprogens Aero gens Pathogens y^

^MPhotogens 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,
notable 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. 7^
^ Zymogens are the bacteria of fermentation, which is the chemical
transformation of carbohydrates by the action of bacteria, with the
evolution of CO 2 CO & H. Such bacteria are useful in the in-
dustries 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. '^

C6H,P,= 2C2H«0+2C02

glucose 2 alcohol 2 carbonic acid

2 lactic acid

CqHi206=3 C2H4O2
2 acetic acid
21

22 PRODUCTS OF BACTERIAL ENERGY

>v 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 neces-
sarily a vital process. VThe fermentations of bacterial enzymes
may give acids, and also aldehydes, ketones, CO 2, CO, H, N, NH3,
marsh gas and H2S. The carbohydrate splitting powers are used
in determinative bacteriology. y<r^-<iXi^ V^viJX^-N^**' ' *^ u:i Aw cj /
")k Fermentation and putrefaction are bacterIar"eiT23rmic processes of
indispensable importance to life. Bacteria reduce excrementitious
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,
cultures of appropriate bacteria may be spread upon exhausted soil.
These are chiefly nitrifying bacteria. Manure contains the denitri-
fying organisms. Bacterial fermentations produce the flavor of
tobacco, opium and butter..

sC Enzyme Production by Bacteria. — ^Ferments of great variety
and power are formed by the zymogens, as proteolytic, which dissolve
proteids, such as casein; tryptic, gelatine liquefying; diastase, which

• converts starch into s\igaiT)s;invertase, which changes cane sugar into
grape sugar; ferments that curdle the casein of milk; and it may well
be tha^Rte activity of pathogenic bacteria in the body is due to
ferments of some kind. The hemolytic action of the golden staphy-
lococcus or the tetanus bacillus is thought, by some, to be of enzymic
nature. ^ <kiZ}f\/T>^ ^^ tWcK

^Organized ferments (bacteria/ yeasts) differ from the unorganized
(pepsin, diastase) . The latter " exercises 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 con-
stitution, which are soluble in water. The organized ones act differ-
ently. Highly complex molecules are split up, and numerous sub-
stances of a totally different character are formed with the evolu-

SAPROGENS AND PATHOGENS 23

tion 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.y^ According to the above named inves-
tigator, it is not possible to express such chemical changes by a sim-
ple equation. lExperiments have shown that B. typhosus and pyo-
cyaneus are able to split up olive oil or fat, and produce glycerine
and fatty acids, thus making them accessible to fermentation
(FischerjT] The action of the buttermilk organisms, while usually
very complex, may be represented by the following:

Ci2H220ii + H20 = C6Hi20g+C6Hi20g

lactose galactose dextrose

galactose lactic acid

i^^aprogens produce putrefaction which is the chemical trans-
formation of albuminous bodies with the evolution of nitrogen, and
of alkaloidal substances, known as ptomainey Aromatic elements
are also produced, such asindol, phenol, kresol, etc.

Pathogens. T^If the tissues are receptive to bacteria, and if the
latter, in any way, injure the tissues, then the invading organism is
called pathogenies (fflieoretically the tissues of the body are sterile.
But as a matter of fact, isolated pathogenic bacteria such as colon
and diphtheria bacilli, streptococci, and pneumococci, are often
found in the tissues and cavities of the body, and yet they cause
no pathogenic changes or symptoms. The blood during life is
sterile in health, y,

>/ Colon bacilli have been found encapsulated in the liver and
kidneys of nondiseased cadavers, shortly after death, which showed
that they had been there some time. 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, yc
^Bacteria may cause disease in several ways, mechanically: a

24 PRODUCTS OF BACTERIAL ENERGY

clump of bacteria may plug a capillary; or simply overwhelm the
tissues and absorb the oxygen (anthrax) ; they may cause new growths
(tubercle) ; or false membranes to form in the larynx causing suffo-
cation (diphtheria) ;i(ulceration of heart valves causing cardiac
insufficiency; thrombosis in the veins and arteries; pus formation; or,
by generating toxins that cause anaemias, or degeneration of im-
portant elements of the nervous system, parenchymatous organs and
the walls of the blood vessels.

J 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 patho-
genic for man, but not to swine, y
J As emphasizd 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, causing a peculiar
blood reaction and by refusing isolation both of which ptomaines
do not. 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 anything 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 medium in which organ-
isms are growing, the extracellular or soluble toxins, and those which

TOXINS 25

remain within the bacterial cells and are only liberated upon their
death and disintegration, 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 bacillus. In
diseases caused by these germs, bacteria do not enter the body fluids
but the general manifestations are due to absorbed soluble poisons.
Such toxins are soluble in water; they are rendered inert by heating,
sunlight and some chemicals. They dialyze very slowly and are not
crystallizable. They may be precipitated with the albumen frac-
tion 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 the toxins may be de-
stroyed by proteolytic 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 of the motor
system. 2) o<.ijjJl*^ /

l^ndotoxins. — These are exemplified by the poisons of the typnoid
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 than the extra-
cellular 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 80° C. and keep under
artificial conditions much longer than soluble toxinsj
j[j[he toxic bacterial proteins are best exemplified by tuberculin.
This is a complex mixture of the proximal principles of the tubercle
bacill us and is probably albuminose in character. These substances
are almost as specific for their own germs as the toxins and much
more so than the endotoxins. They are capable of producing a
reaction in animals similar to that which might be produced by the

26 PRODUCTS OF BACTERIAL ENERGY

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 reac-
tions from mallein and luetin {q.v.) injection are due to toxic pro-
teiBSfl They are thermostable, that is not destroyed at ioo° C.
TK^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. 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 seat of manufacture in the depths of a punctured wound.
They are harmless if swallowed. 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 determiding the nature of
certain germs. We may speak of these failures to react as fail-
ures of receptivity on the part both of the microbe and the injected
animal.

CHAPTER III.

INFECTION.

7 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 success-
fully 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 infection, be-
cause 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 gangrenous limbs may be in-
vaded by strict saprophytes incapable of life in living tissues, and
yet cause much harm by the absorption of their products.

Infestation is when bacteria, even pathogenic, are present in a
place without exciting a reaction. Matter carrying pathogenic
germs is called infective.

Depending upon their ability to grow in the body, bacteria may
be divided into (i) purely saprophitic; (2) occasionally parasitic;
and (3) purely parasitic. A host harbors a parasite.

Purely saprophitic germs cannot live in tissues at all; those that

INFECTION

are occasionally parasitic lead a saprophitic 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.

Kruse's Scheme Illustrating the Action of Various Parasitic

Bacteria.

Parasitic
Bacteria.

Occasionally paras-
itic. Such as the Tetan-
us bacillus.

Always parasitic only
found in the lesions of
disease. Such as the
B. tuberculosis.

1. Local infection due to the
ability of the organism to
take on unrestricted growth.

a. Surface inflammation ^
boils; staphylococci.

b. Surface inflammation
with extension of con-
tinuity; erysipelas
streptococci.

c Surface infection with
marked toxin products;
diphtheria.

d. Deep focal inflamma-
tion; tubercles.

2. General infection of un-
restricted growth.

a. By continuous infec-
tion; glanders.

b. Metastases, as in py-
comia.

c. By universal rapid
growth and invasion,
as in sepsis and an-
thrax.

koch's postulates 29

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 cul-
ture 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, y/

Some of the many organisms that certainly fulfil these conditions,
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 Oedema.

B. of Bubonic Plague. B. of Malta Fever.

B. of Typhoid. B. of Dysentery.

Spirillum cholerce. Meningococcus.

Pneumococcus {Pneumonia). B. of Leprosy.
SpirochcBta 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 fulfil the postulates.
Among these are:

The Organism of Scarlet Fever (Protozoans).

The Protozoa of Malarial Fever.

Amoeba Dysenterice.

3© INFECTION

In rheumatic fever, measles, whooping-cough, poliomyelitis,
mumps, yellow fever, typhus fever, chicken-pox, rabies, and dengue,
the specific cause has, thus far, eluded discovery. In the case of
yellow fever (Reed and Carrol) and hog cholera, 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 microscopic 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.

If the invading organism is a pure saprophyte the various forces
for internal defence immediately act upon and destroy it. If it is
pathogenic for other animals, their defensive agencies have no effect
upon it in their tissues, but in the human body the bacteriolysins
dissolve it, or the phagocytes devour it and carry it away. The
liver, according to Adami, destroys at once bacteria absorbed from
the intestines.

Bacteria are disposed of in divers ways, by means of the lymph
channels they are carried to the various mucous surfaces of the
body, intestinal and bronchial. During typhoid fever, the typhoid
bacilli are often found in the urine. The kidneys at least allow
the escape of some organisms from the blood. Pathogenic bacteria
are discharged from the body in feces, pus, sputum, and in scales
in the desquamating skin diseases.

To successfully inoculate a guinea pig with tuberculosis, the
tubercle bacilli should be injected beneath the skin.

In working with infections produced by the B. proteus vulgaris,
it was found by Watson Cheyne that 6,000,000 bacilli injected
under the skin, did not produce any lesion; 8,000,000 formed an
abscess; 56,000,000 gave rise to a phlegmon; and 225,000,000 were
necessary to cause death in two hours.

In experimenting with the staphylococcus aureus, it was found
that 250,000,000 were required to cause an abscess; and 1,000,000,-
000 were needed to cause death. The internal powers of defense

ATTENUATION OF BACTERIA 3 1

were able in each case to cope with or limit the action of a few mil-
lion to a certain locality, but could not withstand the injection of
overwhelming numbers, which caused the animal's death.

Bacteria to be successfully infectious must be virulent. Viru-
lence is best described as the power of a parasite to invade and grow
within the body by resisting its natural defenses, and gradations
depend upon the ratio of these two forces (Wolff Eisner). Pfeiffer's
explanation of virulence assumes that bacteria have binding posts
or receptors and the more of these a germ has, the 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° C. to 47° C; by drying; the exposure to light;
the action of chemicals; compressed oxygen; and by passing the or-
ganism through the bodies of non-susceptible animals. The atten-
uation or weakening of the pathogenic powers of bacteria is useful
for the production of various vaccines which are valuable in preven-
tive medicine.

By growing the anthrax bacillus at a high temperature, 42.5° C,
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 hydro-
phobia, but on the contrary, it, by repeated inoculation, caused
immunity. The inoculation of monkeys (which are non-suscept-
ible) 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 desiccation 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

32 INFECTION

effects on experimental animals and specify what attenuation is
meant when they are to be used as vaccine. A very interesting
virulent, yet attenuated, form of streptococcus is to be met in sub-
acute endocarditis. These organisms produce serious or even fatal
valvulitis, and yet have no effect upon other organs or upon lower
animals. They are extremely hard to remove from the body.
They have accustomed themselves to residence in the body, have
established a balance or 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
opposition of 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 meta-
bolic products of foreign bacteria. Cultures of pneumococci may
be made so virulent by the first means that only one pneumococcus
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 streptococcic infection
added to virulent diphtheria infection, greatly increases the severity
of the disease.

The secondary streptococcic infection in small-pox and in
phthisis complicates the primary infection and frequently causes
death of the individual affected. The hectic fever and sweats of
phthisis are due to this secondary infection. Combinations of
diphtheria bacilli and pneumococci increases the virulence of the
latter. The transference of infection 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 individ-

AVENUES OF INFECTION 33

ual. Two infections may occur simultaneously, each preserving
separate characteristics, and perhaps aggravating each other.

The avenue of infection and the tissues infected alter the type of
the disease exceedingly. Streptococci invading the tonsils cause
tonsillitis, but the same organisms entering the skin cause erysipelas
or phlegmons; or if the uterus is infected after the birth of a child
the disease is stil] 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 tubercular 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 exudates, tubercles, serum and
pus. The injection of pneumococci under the skin of a dog has a
more rapidly fatal effect than when they are injected into a vein,
according to Klemperer.

It seems practically proven now that tubercle bacilH may enter
the lung by way of the intestinal tract, but Ghon has lately shown
that tuberculosis in childhood usually starts as an infection directly
into the lung tissue by bacilli coming in with air.

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,
3

34 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 bacteria
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. 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 intestinal 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 ingested
with foods, particularly with milk, cheese, and over-ripe fruit.
These in the most part are quickly destroyed by the hydrochloric
acid. When the stomach is diseased and the contents become

AVENUES OF INFECTION 35

stagnant, as in stenosis of the pylorus, and in carcinoma, when HCl
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 safe-guard 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 organisms. 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). Cholera and typhoid
organisms thrive in intestinal contents, elaborating poisons which
greatly depress the individual.

The interior of the uterus, the bladder, urine, and deep urethra,
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.

The placenta is an avenue of infection in several diseases: notably
small-pox, anthrax, glanders, typhoid fever, and sometimes tuber-
culosis pass through the placenta from mother to foetus. Strep-
tococci may pass through the placenta of a woman with ante-deliv-
ery 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 pneu-
monia gave birth to a child, which died thirty-six hours afterward,
and autopsy revealed a consolidation of the lower left lung, and
microscopic examination discovered pneumococci. A hydrophobic
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 irrita-
tion.

36 INFECTION

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 is not
soluble the harm done is purely local, as in the formation of tubercles
by the toxin of the tubercle bacilli.

If the growth is 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 like the pneumococcus, which are generally
restricted to the lungs at the outset, but ultimately they 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.

Soluble products of bacterial activity which are alkaloidal (basic),
crystalline in character, and mostly poisonous, are known as pto-
maines, or putrefaction alkaloids. They are highly complex in
chemical structure, and are difficult to isolate.

Certain albuminoid bodies, products of bacterial activity, known
as toxins, are produced by several pathogenic bacteria.

Those that are essentially bound up in the protoplasm of the
bacteria itself, are known as intracellular toxins, and bacteria plas-
mins. The tubercle bacillus, and other members of the acid-fast
group, the colon and typhoid bacillus, and the cholera spirillum all
contain these. They may be extracted by freezing the organism
with liquid air, and grinding it while frozen and brittle, or by simply
grinding it with sterile sand and water. The new T.R. tuberculin
belongs to this group of toxins.

Bacterioprotein or plasmins are albuminous bodies produced
by bacteria that are not altered by heat, and which produce fever

TOXINS OR TOXALBUMINS 37

and inflammation. 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 bacterial products which are
removable by filtration from the bacteria, and which are thermola-
bile. The tetanus and diphtheria toxins belong to this class.

These various poisons produce many of the clinical pathological
entities and symptoms, known to physicians. Their highly com-
plex molecular structure enables a group of atoms in the toxic mole-
cule 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 (lat-
eral chains) which may unite with other toxic groups.

Various kinds of cells are attacked in infective processes. Leuco-
cytes may be degenerated, forming pus; red blood cells may be dis-
solved, causing anaemia; important nerves may be degenerated; 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 enveloped organs.
The heart orifices may be closed partially or emboli may form, or
false membranes block the air passages, and a hundred other patho-
logical changes may be wrought by these toxins.

If toxins are injected into the body with the specific organism pro-
ducing them, the effect of the latter is greatly increased. Tetanus
spores, washed free of toxins, if injected, are incapable of setting up
tetanus.

Most toxins are easily decomposed by sunlight, air, and heat.
Absolute alcohol separates the active principle from the bouillon
in which it grows. Ammonium sulphate also separates the toxins
of tetanus and diphtheria bacilli, which float on top of the fluid, from
which they may be collected, dried and powdered, and in this state'
may be kept much longer without deteriorating into inert substances.
Small quantities of bile and pancreatic juice destroy the toxic proper-
ties of diphtheria and tetanus toxin.

38 INFECTION

If toxin and anti-toxin (see immunity chapter) are mixed in rela-
tive proportions, chemical neutralization takes place. 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, one part toxin neutralizing one part of anti-toxin, and
ten parts of toxin neutralizing ten parts of anti-toxin. All this is in
accord with chemical laws. Toxins sometimes degenerate 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 sub-
stances, one of which is the toxin that causes the acute phenomena
of diphtheria intoxication. Another, toxon, causes cachexia and
paralysis some time after infection.

Tetanus toxin is composed of two substances; tetanospasmin and
tetanolysin. The first unites chemically with the motor elements
of the nervous system, producing degeneration and causing tremen-
dous contractions of the muscles governed by the nerves involved.
The second has the property of dissolving tissues, 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

AGGRESSINS 39

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, rapidly fatal tuberculosis is produced. If the exudate
produced in the peritoneum, consisting of lymphocytes, is 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 is injected no effect will follow; if bacilli alone are
injected, a tuberculous peritonitis will be produce 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 poly-
nuclear leucocytes, inhibiting their action as phagocytes.

By heating the exudate to 60° C. 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 thermo-
labile, which prevents rapid death, the other is thermostabile and
this is favorable to rapid death.

Bail assumes that a tubercular cavity in an animal contains a
great amount of the aggressin, which prevents chemotaxis of the
ploynuclear leucocytes, but not of the mononuclears or lymphocytes.

In the peritoneal cavity without aggressins, into which tubercle
bacilli have been injected, an active phagocytosis at once is begun
by the polynuclears, and the injected bacilli are in a great measure

40 INFECTION

destroyed, and those left develop more slowly, producing a tuber-
culosis in normal course of time. It is possible to immunize animals
against this aggressin producing an anti-aggressin, which substance
will not only neutralize the aggressin but also stimulate the leuco-
cytes to phagocytosis.

This aggressin theory has been apphed 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., bac-
teria, or such deleterious and toxic substances as toxins, drugs, com-
plex poisonous albumins, snake venom, foreign blood sera, etc. f^

The following tables will, perhaps, be helpful in the study of the
subject.

v..

V'<^AA.

I. Immunity

2. Immunity

Natural
Acquired

Racial immunity
Inherited immunil
Active immunity
Passive immunity

Anti-toxic ^t;A^.^o^

Anti-bacterial *^<^A^IU!j^,r^ & I

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. Ani-
mals cannot be infected with scarlet fever, malaria, and measles.

42 IMMUNITY

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. Certain diseases are known as chil-
dren's diseases, because adults rarely have them. Again, one
individual may contract a disease, while another exposed at the
same time will not.

Acquired Immunity. — Actively acquired by infection. One
attack of yellow fever immunizes the individual against subsequent
attacks. Vaccination actively immunizes against small-pox.

Passively acquired. Actually injecting protective substances
(anti-toxic sera) into the blood. The immunity against a 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.

Exhaustion Theory. — Pasteur conceived that bacteria as they
grow in the body, use up or exhaust something vitally necessary to
the subsequent growth of that particular kind of bacteria.

Retention Theory. — It was held that some noxious agent was
retained by the body which prevented the further growth of bacteria.

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 ingeni-
ous 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-
ytes which are white mobile cells of the blood, and other fixed cells,
defend the body against infection by devouring the invading agents
of disease. (Fig. i6.)

^^f::i.J^rsr-r^^^-f^-'^

PHAGOCYTOSIS 43

Metchnikoff considers the subject of phagocytosis under three
aspects: i. Nutritional. 2. Resorptive. 3. Protective.
7^ Nutritional. — Amceba and certain other unicellular vegetable
organisms belonging to the myxomycetes possessing amoeba proper-
ties and having the faculty of throwing out pseudopodia or proto-
plasmic arms, acquire their food by enveloping smaller organisms,

Fig. 16. — ^Phagocytosis. Gonococci in leucocytes in pus from gonorrhoea.
(KoUe and Wassermann.)

and other nutritious matter which they absorb. Certain intracellu-
lar ferments, which they possess, digest fibrin and gelatine, and con-
vert starch into sugar. These cells protect themselves against inim-
ical micro-organisms by enveloping and digesting them. \'
. They are attracted by food and moisture (called positive chemo-
taxis) and repelled by strong solution of salt, poisons, etc. {negative
chemotaxis). i

Higher in the animal scale among the multicellular organisms, the
cells of the intestines have the property of absorbing and digesting
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

44 IMMUNITY

into sugar. They are not directly concerned in the nutrition of the
organism. Cells of a protecting character in man are either micro-
phages, 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; micro-
cytase 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
chemotaxis. 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, as in the haemolysis of foreign red
blood cells in the peritoneum of a guinea pig. This phagolysis in
dissolving of the leucocyte is the cause of the chemical phenomena
(so he avers) which cannot be ascribed merely to phagocytosis.
Metchnikoff further claims that both phagocytosis and phagolysis,
either severally, or in combination, are responsible for natural or
acquired immunity.

In the case of acquired immunity, it is supposed that the leuco-
cytes become educated. Regarding the toxins against which

PHAGOCYTOSIS 45

animals can be immunized by gradually increased doses, it is held
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 enormous
outpouring of leucocytes at the site (positive chemotaxis), 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 the protection r6le
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 soulble and unorganized 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 bodies derived from phagocytes.

The cellulo-humeral theory claims the attention of most bacteri-
ologists, as the probable explanation of the phenomena of
immunity.

It is certain that cells, either sessile or mobile, and fluids, are im-
portant means of internal defense. In order that this theory may
be comprehended, certain well known properties of normal 'and
artificially immunized serum must be understood.

46 IMMUNITY

y^ Alexins. — It has been found by numerous observers, that normal
blood serum is germicidal for many bacteria, and the peculiarly
active substance that is contained in the serum, was called by Buch-
ner 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 hcemolytic.
It also is thermolabile, that is, its properties are destroyed by heat
(55° C). It is identical with the complement of Ehrlich, and the
cytase of Metchnikoff . K,

><^^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 in normal non-im-
mune sera. R. Pfeiffer found that if some serum from a guinea pig
immunized against cholera spirilla is injected into the peritoneal
cavity of a healthy non-immune guinea pig, with some cholera
spirilla, that the latter are agglutinated, and ultimatel;^ 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 am-
boceptor, are necessary to complete the solution of the bacteria.
If the complement is heated above 55° C. 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 heatT)

VJThe immune serum is not affected by heat, and is theretore called
thprmostabilej
Ijrhese various reactions may be expressed concretely thus:

Bacteria -h immune body = no solution.
B acteria + complement = no solution.

SERUM CHEMISTRY 47

Bacteria + immune body + complement = solution (Pfeiffer's re-
action).

Bacteria + immune body + complement (heated) = no solution.
Bacteria + immune body (heated) + complement = solution.

I The same phenomena in the blood of animals immunized against
the red blood corpuscles of another animal of foreign species have
been observedTj

Llfa rabbit is immunized with the blood of a dog by repeated and
increasing doses, the serum of that rabbit will become hcdmolytic to
the corpuscles of the dog's blood if they are mixed, provided 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 = solu-
tion.

Dog's erythrocytes + immune rabbit serum -f complement, heated
= no solution.

^he immune body acts as a preparer of the corpuscles, or bac-
teria, 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^

(i) Pepsin + fibrin = no solution or lysis.

(2) HCH- fibrin = no solution or lysis.

(3) Pepsin + HCl-f fibrin = solution or lysis.
The HCl corresponds to the immune body.

In the case of haemolysis, or bacteriolysis the action of the immune
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 ery-
throcytes prepare these of a pigj so that the complement may
act on it.

48 IMMUNITY

A loose chemical union takes place between the bacteria and the
immune body, but no such union occurs between the complement
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 different
from the other, and that their action is specific for the different kinds
of bacteria or cells with which an animal may be immunized. Bor-
det 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, epithe-
lial 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 caus-
ing certain cells to agglutinate. One of the earliest manifestations
of immunity of a certain serum to bacteria, or to blood cells, is this
peculiar action of the serum causing either the bacteria 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 dis-
solved by the complement and immune body.

If the serum of a typhoid fever patient is mixed, even in high dilu-
tions 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 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

PRECIPITINS 49

cells of a foreign species, its serum becomes agglutinative to these
cellsT^

[[Precipitins. — If a rabbit, or any other animal in fact, is immu-
nized by repeated injections of blood foreign to it, 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 blood against which the
animal was immunized. As soon as the immunized serum and the
specific blood are mixed, a precipitate forms. This is another
phenomenon of immunity, and is of more than theoretical import-
ance 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.
Naturalists also use this method for the differentiation of species.
By many, 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 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
4

50 IMMU.NITY

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 mix-
ture 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 anti-toxic
serum {reactivation) .

The production of bacteriolysins, cytolysins, agglutinins, precipi-
tins, and anti-toxins are manifestations of the activity of the immun-
ized 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 con-
sideration must be given to the study of the toxin molecule. Ehr-
lich 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 with-
out 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— C-^ "^C— H

II I

H— a ^C— H

BENZOL.

The benzol molecule C^Hg is here represented graphically as a

LATERAL CHAIN THEORY 5 1

ring with a central nucleus of Cg with lateral chains of H. con-
necting each atom of C.

If one of these lateral chains H. is supplanted by the acid radical
CO OH. the benzol is converted into benzoic acid and its formula is
represented thus:

O -

//
C— OH

H— C^ ^C— H

II I

H — Cv ^C — H

I
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.

//
C— O— Na

/\
H— C C— H

II I
H— C C— H

c :

BENZOATE OF SODA.

It is thought that as the soda is brought in contact with the central
nucleus of the benzol ring, so food stufiFs unite with the central body
of the cell molecule in the organism and nourish it.

52 IMMUNITY

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 recep-
tors 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 chemical affinity for the
nervous system and for its neural elements and not for liver or
spleen cellsj

Q]he poisons of snake venom seem incapable of uniting with
any cells of the pig; this animal is, therefore, immune to snake

venom.3

^Urow, as these toxins unite with the cells by means of the receptors,
the cell is stimulated to produce an excessive number of these recep-
tors, 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 pro-
duced^ Weigert first called attention to this phenomenon, which
has been called Weigert' s over-production theory, l^o 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 ceff\ Accordingly a great number of free receptors are gener-
ated, and floating in the blood, engage the haptophorous portion of
the toxin. Thus the toxophore is neutralized and rendered innocu-
ous before it can reach the cell. These free over-produced recep-
tors constitute the anti-toxin. This is the essence of Ehrlich's
theory. (Fig. 17.)

Through the process of time and oxygenation, the toxophorous
group in the toxin becomes innocuous, and only the haptophorous
group remains active; nevertheless the haptophorous group is able
to combine with the receptors and to stimulate the cell into generat-

RECEPTORS

ing 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 pro-
tection to the structure against lightning. This theory can be
applied to the production of other anti-bodies. If blood cell,
bacterial cell, or any animal fluid possessing a haptophore is capable
of combining with side-chains (receptors) of the cells of the immu-

FiG. 17. — a, receptor on cell; b, toxin molecule; c, haptophorous portion of
the molecule; J, toxophorous portion; e, receptor. (Williams.)

nized, just as a key fits a lock, then the cells are stimulated to pro-
duce excessive numbers of receptors, and these constitute the anti,
or immune body. It is possible to produce from rennet, egg-albu-
min, cow's milk, and from many other albuminous substances,
immune bodies by injecting these substances into animals. (Figs.
18, 19.)

IMMUNITY

Fig. i8.— EHRLICH'S LATERAL CHAIN THEORY. Cell with numer-
ous receptors of various kinds and shapes to which are united the toxin mole-
cule. Note the free receptors.

v!' rMi

;

Fig. 19.— EHRLICH'S LATERAL CHAIN THEORY. In one figure the
free receptors (anti-bodies) are united with the toxin molecule, the attached re-
ceptors have no haptophores united to cell.

IMMUNE BODIES

List of immune bodies and their anti-bodies (Ricketts) .
Antigens or Products of

Immunizing Immunization
Substances

Toxins

Anti-toxins

Complements

Anti-comple-
ments

Ferments

Anti-ferments

Precipitogenous

Precipitins

Substances

Agglutinogenous

Agglutinins

Substances

( Hemolysins

Opsinogenous

Opsinins

Bacteriolysins

Substances

Special cytotoxins

Consisting oj

Cytotoxin Produc-

Cytotoxins . .

Such as

two bodies

. ing Substances

Spermotoxin

Complement

Nephrotoxin

Ambocepter

Hepatotoxin, etc.

Syno

nyms.

Complement

Ambocepter.

Alexin.

Immunkorper.

Cytase.

Zwischenkorper.
Intermediary body.
Fixaleur.
Preparateur.
Copula.

Desmon.
Substance sensibilis

atrice.

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.

56 IMMUNITY

The production of bacteriolysins is explained by Ehrlich's lateral-
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, how-
ever, to confer passive immunity, as in the case of diphtheria, by
the injection of the serum of the immunized animal. The im-
mune body is alone present in the serum generally and some com-
plement must be added to effect bacteriolysis. The serums which
aid in the solution of bacteria are known as anti-bacterial ser-
ums, which, though not anti-toxic, may check invasions and aid in
recovery by destroying bacteria. It is possible to effect an in cor-
pore 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 figure 20 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 stimu-
lating 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, the normal thermolabile anti-body substance in
serum.

Zymophore, toxophore for agglutinins and precipitins.

Cytophile fraction is that part of anti-body which combines with
cell, while complementophile fraction joins with complement.

Immune body, the thermostabile anti-body against bacterial or
other cells.

By immunizing with complement or antibody we obtain respect-
ively anti-complement and anti-immune body which experimentally
will neutralize the action of these two substances. The comple-

58 IMMUNITY

ment 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 figure 20). First Order:
Simple union of toxins (soluble) and fixed or free receptors or anti-
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 produced the
agglutination or precipitation. No complement is needed.

Third Order: Concerns bacteriolysins, hemolysins or bacteroly-
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 accomplishes
through its zymophore.
-y^ Anaphylaxis. — Against protection, the opposite of prophylaxis;
also called Hypersusceptibility. This phenomenon, first described
by Theobald Smith, Portier and Richet, consists in a condition of
extreme sensitiveness of animals against foreign proteins. If a
guinea pig be injected into the peritoneum with a minute quantity,
say j^nnr ^^ ^ gi'^-ni, of horses' serum and eight to ten days later
receive a quantity of ^^ of a gram, the animal will become uneasy,
then depressed, have dyspnea, scratch itself violently about the
face and finally die after an intensification of these symptoms.
Similar symptoms have been observed in persons receiving diph-
theria anti-toxin therapeutically. The condition of high sensitivity
to this anti-toxin is called allergie and upon its degree depends the
reaction following anti-toxin administration. 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

ANAPHYLAXIS 59

are susceptible to the emanations from horses. This wilJ not
explain all cases however. The scientific world is beginning to
consider the contraction of any infectious disease as an evidence of
anaphylaxis on the patient's part to the causative agent.

In experimentally induced hypersusceptibility the reaction is
specific. The condition is transmissible from mother to fetus
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 bacterial
proteins from five to eight days. The sensitive period may last
for several years. In searching for the cause of this reaction it was
found that there are (i) a spastic distention of the pulmonary 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 con-
densed and compounded about as follows. The body is unprepared
to care for parenterally (otherwise than gastrointestinal tract)
introduced protein and must develop an 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 overproduction theory
this anti-substance is in large quantity when another 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. These protein toxins attack nervous and
parenchymatous tissues.

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.

Friedberger has used these facts to elaborate a theory of infection.
He believes that bacteria circulating in the body stimulate anti-

6o IMMUNITY

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 diatheses.

McKail divides anaphylaxis as follows:

Natural Anaphylaxis, depending upon

a. Species of animal, for example cholera in man, anthrax
in cattle, glanders in horses.

b. Age — diphtheria in children, erysipelas in the elderly.

c. Individual — to white of egg, or blood serum, even by inges-
tion ("one man's meat is another man's poison").

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.

/ 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 complement.
If, however, complement be absorbed in any way a solution of the
coloring matter of the red cells will not occur in this mixture.
Complement will combine with anti-body in the presence of antigen.
This fact has been taken advantage of in determining 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. This test is positive
from the initial lesions all during life unless the patient has been
successfully treated. Indeed the parasyphilitic states also give it.
The principles of the test are also used for determining the pres-
ence of tuberculous, leprous, typhoid and other anti-bodies.

WASSERMAN REACTION

The materials necessary in the Wassermann test are as follows:

I. Syphilitic antigen, extract from the syphilitic liver of a fetus,
in alcohol, ether or water; lipoids like lecithin or extracts from
guinea pig's heart are said to act as antigen.

2a. Serum from a known case of syphilis and containing therefore
syphilitic antibody.

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 washed sheeps' red blood cells.

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 56° C. for 30 minutes to destroy the native and inherent comple-
ment. 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 in 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 to observe. Tubes are
set as follows:

unit #1
unit #]

+ I unit #2a+ I unit #4.
+ I unit §3 + 1 unit §4.

C. I unit #1 + 1 unit #2b +

D. I unit #2a+ i unit §4.

E. I unit #2b-|- 1 unit #4.

F. I unit #3+1 unit §4.

G. I unit #4.
H. 1 unit #1.
J. I unit #2a.
K. I unit #2b.
L. •! unit #3.

. + I unit #5+1 unit #6 = No hemolysis.
+ I unit #5 + 1 unit #6 =

if #3 be Syphilitic, no hemolysis.

if #3 be Non-syphilitic hemolysis.
+ I unit #5 + 1 unit #6 »= Hemolysis.
-f- 1 unit #5 + 1 unit #6 = Hemolysis.
+ I unit #5 + 1 unit #6 = Hemolysis.
+ 1 unit #5 + 1 unit #6 = Hemolysis.
+ I unit #5 + 1 unit #6 = Hemolysis.
+ 1 unit #5+1 unit #6 = No hemolysis.
+ I unit #5+1 unit #6 = No hemolysis.
+ I unit #5+1 unit #6 = No hemolysis.
+ I unit #5+1 unit #6 = No hemolysis

62 IMMUNITY

The tubes receive first the solutions on the left and are placed
in the 37° C. incubator for 2 hours to allow union of their various
parts, particularly the complement with others. They then receive
the solutions on the right, are placed in the incubator for half an
hour and in the ice-box overnight, when they are examined 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 bottom,
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.

Cholera and typhoid organisms do not produce soluble toxins in
the body, but when they are disintegrated therein, soluble poisons
(intracellular) are liberated.

Bacteria may become accustomed to the fluids of the body by a
similar process and may elaborate free receptors for their own pro-
tection, 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.

MANUFACTURE OF ANTI-TOXINS 6^

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 poison 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-abrin for jequerity bean poison
Anti-robin for locust bean poison
Anti-crotin for croton-oil bean poison
Anti-pollen for pollen of plants that produce hay-fever.

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 arti-

64 IMMUNITY

ficially supplied to the animal unite with the haptophorous chains in
the toxin molecule, and neutralize, or bind, the toxophorous or pois-
onous chains in the molecule, and prevent toxophore from attacking
important vital cells belonging to the animal. But if the anti- toxin
and toxin, after being mixed in a test-tube, are injected into a sus-
ceptible animal, no harm results, if they are in proper proportions,
since the same thing has happened 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 several days in dextrose bouil-
lon at 37° C; as the bacilli grow they elaborate a very powerful
poison or toxin, which is highly complex in compositon. 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 or Berkefeld
filter, and is then stored in sterile bottles in an ice chest. Horses are
generally immunized, since they are susceptible to the action to the
toxin, and are easily managed. Before being used they are care-
fully tested with tuberculin for tuberculosis and with mallein for
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.

To make anti-toxin, a very virulent toxin is employed.* A horse,
previously examined for health, is injected with from .i to i. c.c. of
toxin. This is followed by a rise of temperature, local reaction, and
systemic disturbance. After waiting for all reactions to disappear
a second injection is given, which is followed by others larger in size,
until, after a few weeks or months, i,ooo c.c. of toxin are injected
at one time (enough to have killed a dozen horses that had not re-
ceived the smaller doses previously). The injection of the toxin is

ANTI-TOXINS 65

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 suf-
ficiently, his blood is drawn from the jugular vein, and after the clot
has formed the serum is drawn off and stored.

McFarland found that a horse was capable of producing enough
anti-toxin to protect 806 other horses against doses of toxin, each one
of which was equivalent to the total amount of toxin that the immun-
ized horse received. Thus there is evidently a tremendous over-
production 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 pro-
duction of anti-toxin. We measure the anti-toxic powers of the anti-
toxin with units arbitrarily devised. An anti-toxic unit is ten times
the least amount of anti-toxic serum that will protect a guinea pig weigh-
ing 300 grams {standard) against ten times the least certainly fatal
dose of diphtheria 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. A
unit dose of toxin is the smallest amount of toxin necessary to kill a
guinea pig weighing 300 grams, or the dose per 100 grams of guinea pig
necessary to kill.

Ehrlich's method of standardizing is to obtain an an ti- toxin of
known strength (anti-toxins do not deteriorate or vary as do toxins) .
A standard anti-toxin made by Ehrlich is now everywhere used, and
is furnished by him from his institute.

Against this standard anti-toxin a toxin of unknown strength is
measured by means of guinea pigs. The toxin unit thus founH is
then used to determine the anti-toxic unit of anti-toxins of unknown
power.
5

66 IMMUNITY

The power of anti- toxic sera varies; some contain from 200 to 300
units only per c.c; others may contain even 1,700 or 2,000 per c.c.

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. When used it is common to inject from 2,000
to 5,000 units.

It is not only of value as a curative agent, neutralizing the toxins
already formed, but is valuable as an immunizing one against infec-
tion. 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.

Tetanus Anti-toxin. — Tetanus anti-toxin is produced in a man-
ner similar to that of diphtheria anti-toxin. As the horse is exceed-
ingly sensitive to tetanus toxin, before the immunizing process is
begun, the toxin is attenuated by heat or iodine.

The anti-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 this guinea pig in about four days. This amount of toxin is
neutralized by ^^-^Vo^ of a unit of anti-tpxin, or one unit of anti-
toxin will protect 1,000 guinea pigs against the minimum fatal dose
of tetanus toxin. The United States unit of tetanus anti-toxin is
now the least quantity of anti-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.

There can be no doubt that tetanus anti-toxin, if given with the
toxin or soon afterwards, is a potent means of preventing lethal
action of the toxin. Tetanus toxin enters into such quick combina-
tion with the cells of the motor elements of the nervous system, and
the union is so permanent that it is difficult for the anti-toxin to form
any union with the combined toxin. If an immune animal whose
blood is powerfully anti-toxic received into his central nervous sys-
tem a dose of toxin he will succumb at once; the anti- toxin appar-

ANTI-TOXINS 67

ently has an inferior valency, or combining power. If however, it
meets the toxin before it reaches the nervous system, it, by its
receptors, binds the haptophores, and this prevents any combina-
tion of the toxophores with the receptors of the nervous system cells.

In general, anti-toxin is effectual if administered when acute
toxic manifestations of the disease are in evidence. It has been
found by Calmette and McFarland that if dried tetanus anti-toxin
is sprinkled over wounds infected with tetanus bacilli, or impreg-
nated with toxin, that it acts in a very prompt and effectual and anti-
dotal way.

If the toxic symptoms appear shortly after the infecting wound is
received it is well known that the prognosis is extremely grave. In
such cases, and in those that come to the care of the physician late
and after the toxic symptoms have appeared, the anti-toxin must
be used in large amounts directly about the wound to neutralize
the uncombined toxin, into the general circulation and directly into
the nervous tissues or into the ventricle of the brain, in the hope that
the excess of free receptors of the anti- toxin may engage the hapto-
phores and toxophores of the toxin molecule already attached to
the receptors of the nervous cells in the floor of the fourth ventricle.

Streptococcus Anti-toxin. — While there are at least several
strains of streptococci, it is a fact that the toxins produced by all
have the same charactertistics and properties. The toxin is of
the nature of a diastase, which is destroyed by a temperature of
70° C. In addition to the ferment of a diastatic nature others of
haemolytic power are formed. This is called streptococcolysin and
it is said by Reudiger to possess both haptophorous and toxophor-
ous chains in the toxin molecule. It is also destroyed at 70° C.
Jaundice and petechial rash is often found in streptococcic infec-
tions. It causes a blood stained oedema and exudate at the site of
infection in rabbits killed by the injection.

The anti-toxin is prepared by injecting horses with living cultures
of very virulent streptococci, beginning with small doses and increas-
ing them gradually. The last dose administered in the immunizing

68 IMMUNITY

process may be 600 c.c. of a virulent culture. Four weeks after the
last dose is given the serum is withdrawn. It is thought by Mar-
morek that the action of the serum is anti-bacterial, rather than anti-
toxic. It has been found that the use of streptococci from human
sources is the most efficient for the immunization of horses.

Anti-streptococcus serum is of some value in infection and
diseases caused by streptococci. Of these it has been used in puer-
peral fever, erysipelas, and septicaemia. It has by no means won an
undisputed place, like diphtheria anti-toxin.

The Anti-pneumococcus serum is prepared in the same way.
Horses are immunized by the injection of living cultures, and the
horse's blood, after a period of treatment by cultures, is drawn off,
preserved with tri-cresol and used in a manner similar to diphtheria
anti-toxin. Its use has not been attended with any marked results.
It is a curious phenomenon that pneumococci grow better in the
serum of a horse immunized against pneumococci than in noimal
horse serum. Autolysates of virulent pneumococci are now used
for immunizing animals. These seem to raise the anti-bodies
better than whole cocci.

There are anti-toxic sera for use against Botulism, or meat pois-
oning, pyocyaneus infection, hay-fever, staphylococcus infec-
tion, Malta fever, and typhoid, that have been used without much
success. They have a certain scientific interest, but are of no great
clinical 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. Later it was found that heat killed cultures were
just as effectual.

In 142 cases of plague treated with serum, 24 died, a mortality
of 14.78 percent, while in 72 cases untreated, 46 died, a mortality
rate of 63.72 percent.

The action of the serum is bactericidal, as well as anti-toxic. The

VACCINATION AGAINST SMALL-POX 69

dose varies with the stage of the disease; 5 c.c. is an effective pro-
phylactic dose, while from 20 to 300 c.c. have been used often as
curative doses.

VACCINATION.

By the use of attenuated, or killed micro-organisms, 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 sometimes
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.

Vaccination Against Small-pox.

There is now no doubt that vaccinia or cow-pox is but modified
small-pox in the cow. The causal agent of small-pox, through its
life in the tissues of the cow, becomes so modified that it does not
produce in man variola, but vaccinia. This causal agent is believed
to be a protozoan, called by its discoverers Cytoryctes variolce.

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 bodies, or anti-
body substances.

These various substances, as yet unknown, remain for along
period within the body of the vaccinated person and may protect it

70 IMMUNITY

•

for years against invasion and infection with the cytorcytes in viru-
lent form. A person who has variola cannot be vaccinated, sub-
sequently he is immunized against vaccinia by this attack of variola,
just as he can be immunized 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 anti-septic precaution. After a
week the virus is collected under similar anti-septic precautions by
scraping the base of the vesicle with a 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 hydrolytic action.
This virus is then rubbed into the skin of the individual to be vacci-
nated under strict aseptic precautions. At the end of a week, a
pearly white vesicle is formed, and it is then considered that vacci-
nation 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 diphtheria anti-toxin.

There can be no doubt about this being one of the greatest boons
that mankind has ever received. Vaccination is attended with some
risk. Septic infection with streptococci sometimes follows, likewise
tetanus infection. In both instances this may be due to the contami-
nation of the vesicle on the calf before the virus is lifted, to pirty
methods, or to contamination after vaccinations, probably the latter.

VACCINATION AGAINST CHOLERA 71

Vaccination Against Cholera.

By the injection of the bodies of dead bacteria, or attenuated Hve
ones, especially those containing in their cells insoluble poisons, it
is possible to create in the animals experimented upon a powerful
active immunity against the action of living virulent bacteria of the
same species.

By the attenuations of cholera spirilla, Haffkine has produced vac-
cines vi^hich effectively protect individuals against infection with
cholera, or if they become infected vdth the disease, it is so modified
that they can, and do, more easily recover. He employs two vac-
cines, 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, 39° C, 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. The best method is to use a culture that
kills a guinea pig in twenty-four hours by peritoneal injection. After
the am'mal is dead, the peritoneal exudate is collected, and grown at
35° C, the most favorable temperature for the organism to multiply.
This exudate is injected into a second guinea pig, and its exudate is,
after incubation, injected into guinea pig number three, and the
process is done repeatedly until the virulent virus that is lethal in
eight hours is obtained. This is called virus fixe. After cultivating
this virus on agar, the surface growth is washed off with sterile water
(8 c.c.) and J 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 injections
without an accident. . The following results were obtained by Haff-
kine who worked in India for the British Government:

IMMUNITY

Population

Cases— Cholera

Deaths

Total

Percent

Total

Percent

Non-inoculated, 1,735

Inoculated, 500

171
21

10.63

4.2

6.51
3.3

The immunity conferred by this mode of vaccination is not com-
plete until ten days after treatment. It is possible to vaccinate with
these relatively virulent bacteria because they are given under
the skin, a place where the 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 pos-
sible to create an immunity of a moderate kind against enteric fever.
The method has been perfected by Wright, and his mode of proce-
dure is to secure a virulent culture of typhoid, which is tested on
guinea pigs, and the minimum lethal dose for a 100 gram guinea pig
is used as the dose for man. This dose varies from .5 c.c. to 1.5 c.c
of an old culture sterilized by heat at 60° C, and preserved with
lysol. After the injection there is often redness and pain at the site
of inoculation, some fever and lymphangitis. The results obtained
in vaccinating the troops in South Africa are marked. Of the garri-
son of Ladysmith comprising nearly 12,000 troops, 1,705 were inoc-
ulated; 2 percent contracted typhoid afterward, and 4 percent of
these died of the disease. Among the non-inoculated, numbering
10,529, 14 percent contracted typhoid, and 3.12 percent of 10,529
died of the disease. It seems that this form of vaccination, in a
great measure, prevents the infection with typhoid, and modifies the
disease after infection occurs.

ANTI-TYPHOID VACCINATION 73

The results of Major 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 protected
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 present method is to give three injections six to ten days
apart of definite numbers of typhoid bacilli of a strain known to
produce a good quantity of agglutinins and other anti-bodies. The
injections usually number 100,000,000, 500,000,000 and 1,000,-
000,000.

Vaccination Against Plague.

Haffkine, in India, has vaccinated many 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

74 IMMUNITY

the bouillon, the culture is killed at 70° C. 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. In a village, Unhera, among
64 uninoculated people, there were 2 7 cases with 2 6 deaths. Among
71 inoculated persons under the same conditions, and of the same
families as the uninoculated, there were 8 cases and 3 deaths. The
fatalities among the unvaccinated exceeded those among the
inoculated by 89.65 percent.

The Indian Plague Commission reported that the measure was
valuable as a means of preventing infection; while it was not an abso-
lutely 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. Yersin and Wyssokowitsch have devised
an anti-toxic and bactericidal serum from injecting horses and
monkeys. This may be used as a remedy.

Vaccination Against Anthrax.

Of all forms of vaccination against disease with attenuated
bacteria this is the most successful. Its use is confined to domestic
animals, sheep, cattle, and horses, and has reduced the mortality
in the country where it is used from 10 percent to .5 percent.
The method requires the employment of two vaccines made of
attenuated anthrax bacilli. No. i is a culture of bacilli attenuated
by growing them at a high temperature, 42.5° C, 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 Govern-

VACCINATION AGAINST TUBERCULOSIS 75

ment of the Unites States supply the vaccine gratis to stock raisers
and others.

A valuable anti bacterial serum is used also as a therapeutic
measure-anthrax infection.

Vaccination Against Black-leg or Quarter-evil.

Quarter-evil, or Rauschbrand, is due to a specific bacillus. Vac-
cination against this disease may be accomplished by inoculating
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 temperature up to
93° C. These vaccines are given at a short time apart, and the
immunity is effective. The method is valuable to stockmen.

Vaccination Against Tuberculosis.

It is possible to vaccinate animals against tuberculosis by the
use of attenuated tubercle bacilli. To accomplish this, the sole
requisite is to so weaken the bacilli used to immunize, that there is
not any likelihood of causing any lesion. By long cultivation on
culture media bacilli are so attenuated that they cannot cause harm
to a guinea pig, even if repeatedly injected. Guinea pigs may,
when properly treated, live long after inoculation with virulent
bovine bacilli, but at no time do they become wholly immune;
cows may be immunized against bovine bacilli by inoculating them
with weak human cultures. Koch's new tuberculin, made by
grinding to a powder the dried bodies of tubercle bacilli, is also able
to set up an immunity in animals, and to a limited extent in man.
It is used in several large sanitariums devoted to the cure of tuber-
culosis, as a therapeutic agent. Those using it claim that it
immunizes the individual and thus increases his resisting powers.
Webb of Colorado claims to have produced immunity in monkeys
and children by injecting exceedingly small numbers of living
bacilli, I, 2, 4, 8, 12, 18, 25, etc.

76 IMMUNITY

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 -^ of its bulk, and to this a half of one
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, per-
haps, 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 peritubercular area with leucocytes. 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.

Tuberculin acts as a fever producer in an unknown way. It is
supposed, however, that the intense local reaction produces fever
through active tissue changes.

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 recom-
mend 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 dissolving 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

IMMUNIZATION AGAINST HYDROPHOBIA 77

reaction than the old tuberculin. Like the old, it is used in the
treatment of lung, bone, laryngeal, and skin tuberculosis. It
certainly causes a local reaction about tubercular foci, and no doubt
aids in the formation of an active immunity to the disease.

The dose of tuberculin for testing purposes varies from ^^ of a
milligram to 5 mgs. and in case the first dose does not produce a
reaction, it should be repeated. For therapeutic purposes one
begins with an injection of .0000001 grm. or smaller and increases
slowly according to the patient's condition. TubercuUn should
only be administered by experts.

Mallein.

Mallein is a preparation made from the toxin of the glanders
bacilli, and is prepared precisely as the old tuberculin. By increas-
ing 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 37° C. The resulting
fluid is sterilized by heat and filtered through a Pasteur filter. The
filtrate is evaporated to half its quantity, and to this a small amount
of carbolic acid is added in order to preserve it. Of the mallein thus
prepared i c.c. should kill a rabbit in one to two weeks.

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 infec-
tion 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 subsides. Mallein has been
used as a prophylactic agent against glanders with some success.

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

78 IMMUNITY

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 dry-
ing the spinal cords of rabid animals for two weeks, they become
totally avirulent. If the cord is dried but three or four days, the
virulence is but slightly modified. Immunity to rabies can be pro-
duced by injecting minute quantities of the poison, and then
gradually increasing the dose until virulent virus can be employed.

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 inocu-
lation 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 vaccination
must be rapid, so two injections per diem are given. In Berlin the
weakest injection used (the first) is made from a cord that has dried
but eight days, and the course is much quicker. 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 symp-
toms 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 injection 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. — In rabies the total mortality before the
introduction of vaccination was not less than lo percent. Among

COLEY'S FLUID 79

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 per
cent. Those cases in which the bites are on the head, are always
more serious, and the mortality 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 vary-
ing 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 disap-
pearance of malignant sarcomata. An attack of erysipelas (it has
long been observed) occurring in a patient with some malignant dis-
ease, has the effect of causing a disappearance, or retrogression, 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. Prodigiosus. The mixture after
a long period of incubation was sterilized by heat, and the fluid thus
obtained was injected into the tissues. Virulent strains of strep-
tococci are used and the dose of the dead culture is about half a drop
given under strict anti-septic precautions. Out of 200 cases many
were cured. In 35 cases treated by other surgeons 26 tumors dis-
appeared, and 14 of these cases were alive from two to four years
after. 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.

8o IMMUNITY

Opsonins and Opsonic Index.

Peculiar substances in blood serum have been called by Wright and Douglass
opsonins {Greek: prepare food for). If fresh blood is mixed with an emulsion 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
thein 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.

Phagocytosis then depends upon the action of some serum upon bacteria,
which are coped with in the body, first by the action of the serum, and then
by the leucocytes. This opsonic substance, like the amboceptors, sometimes
disappears from the blood. It is thermostabile.

The quantitative action of phagocytosis may be estimated by Lelshman's
method. He mixed blood and an emulsion of bacteria in salt solution in equal
quantities, and allowed them to stand for 30 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 so-
lution. 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 coagulation.
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 staphylococci
(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 15
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 polynuclear leucocytes
is determined and divided by 50 to obtain the average.

At the same time that this experiment is being performed, some norma 1

LOCAL REACTIONS 8 1

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.

To obtain the opsonic index, it is necessary to know the ratio of staphylococci
in the leucocytes treated with the furuncular serum, and in the normal. If the
normal serum leucocytes contained lo staphylococci, and the furuncular serum
contained 15, the index would be 1.5.

In the case of tubercle bacilli, the latter must be heated to 100° C. 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 powerful toward streptococci and pneumococci. It is possible 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.

Wright has treated tubercular and septic infections in this way with excellent
results, the opsonic index of the individual being very markedly raised. Others
have not had such convincing results with the opsonic index.

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 58). 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 or injected between the layers of the skin a red maculopapule or even vesicle
upon an inflamed base will appear within 24 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 anaphylactic state
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

82 IMMUNITY

conjunctiva can this reaction be obtained. These skin tests are called the von
Pirquet's cutaneous or Moro's percutaneous tests.

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 resulting
mass to 60 C. for an hour and preserving it with phenol. If this be instilled
into an abraded skin area a maculopapule or nodular eruption occurs in a
syphilitic. This positive outcome, hov^^ever, only appears in late cases, those
of tertiary stages and in treated cases. It therefore complements the
Wassermann reaction, being positive vi^here this is apt to fail.

Carriers. — After recovery from certain diseases, notably typhoid fever, diph-
theria 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 naso-
pharynx 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 carrier, "Typhoid Mary" a
cook, is known to have infected 26 persons.

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 protoplasm 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 is used and a slide with a con-
cavity ground in it. A drop of bacteria laden fluid is placed on
the cover-glass, and after the edges have been smeared with vaseline,
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
y^2 oil immersion objective. If the preparation is kept warm for
some time, various vital phenomena may be noted. Direct division,
sporulation, motility, agglutination, and bacteriolysis 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 so

84 STUDY OF BACTERIA

swiftly that the eye can hardly follow them, or they may merely
roll or waddle across the field slowly. Direct division, if proceeding
under the best conditions, requires but 15 to 40 minutes. It is best
observed in a warm stage or when working in a room kept at a
temperature of 35° C. Sporulation occurs differently 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 bouillon culture, mixed in proper proportions, is dropped
on a cover-slip, which is then placed, drop downwards, over the
cavity of the slide {hanging dropj fig. 21). (See Agglutination.)

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

Fig. 21. — Hanging drop, over hollow ground slide. (Williams.)

needle. The matter must be spread thinly and evenly. After the
water has evaporated and the preparation has become dry without
the use of heat, 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 accom-
plished, in the case of a slide, by holding it 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 needed to fix slide smears correctly.
The beginner would do well to use cover-slips. If a cover-slip is
used it must be passed through the flame three times rapidly. After

STAINING BACTERIA 85

fixing and thorough cooling, 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 differentiation 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, accord-
ing 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 too verstain with it, and it is
very easily applied. It has been found that certain physical and
chemical conditions are necessary for successful staining with ani-
line dyes. Alcoholic solution of dyes entirely devoid of water do
not stain, absolute alcohol does not decolorize bacteria after stain-
ing with aniline colors, while diluted alcohol decolorizes readily.
The more completely a dye is dissolved, the weaker is its staining
power. A dye stuff unites, as a whole, with the bacterial plasma.

86 STUDY OF BACTERIA

forming, as it were, a double salt between the two. Certain sub-
stances, 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 proto-
plasm 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 bacteria 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.

To 00^ solution caustic soda solution in water 100 c.c.

Mix.

This is the most useful of all the staining mixtures employed.

2. ZeihPs 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.

STAINS 87

4. Bismarck brown solution.

Water 100 ex.

Bismarck brown sufficient to saturate.
Filter and use as conlrast 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.Cc

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.

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 aniline
gentian violet is poured on, allov^^ed 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 repeat-
edly until the alcohol does not dissolve any more color. A contrast
stain of Bismarck brown or dilute fuchsin is 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."

88 STUDY OF BACTERIA

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. aerogenus capsulatu^ BvcL anthracis, Bad.
diphtherice, B. tetani, Bact. tuberculosis, Streptococcus pneumonicB,
Staph, pyogenes, Strep, pyogenes. Gram negative — B. coli, B.
dysenteric^, Bact. injiuenzce, Bact. mallei, Bact. pesth, B. pyocyaneus,
B. typhosus, Diplococcus intracellularis meningitidis, Micr. catarrh-
alis, Micr. gonorrhoece, Spirillum cholercB.

Thionin Blue, or Carbol Thionin.

This is a useful stain, prepared thus:

Thionin blue i gram.

Carbolic acid 2.5 gram.

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% solution of sodium bicarbonate 100 c.c.

Methylene blue i gram.

Mix and heat in sterilizer one hour at 100° C. Cool, filter, then mix ^(, 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.

This complex stain represents a type of which Jenner's, Leish-

SPECIAL STAINS 89

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, trypano-
somes, 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 newly discovered organ-
ism of syphilis — Treponema Pallidum {Spirochcete Pallida) 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.

1. Air dry the specimen.

2. Harden and fix in absolute alcohol.

3. Dilute stain with distilled water, using one drop of stain to each cubic
centimeter of water.

4. Cover preparation with dilute stain 15 minutes.

5. Wash in running water.

6. Blot and mount.

Capsule Staining.

Bacteria are often covered with capsules that are difficult to stain,
and special methods have been devised to demonstrate them.

Welch's Method.

I. Cover-glass preparations are made in the usual manner, and over the film
after fixing, glacial acetic acid is poured.

90 STUDY OF BACTERIA

2. Without washing oflf the acid, aniline water gentian violet is poured on.
Change the stain four or five times to remove the acid. Stain four minutes.
This demonstrates the capsule very well.

His's Method.

1. Make cover-glass preparation as usual. 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".

1. Dry and fix.

2. Heat and pour on the following stain:

a. Saturated alcoholic solution of gentian violet 5 c.c.

b. Water 95 c.c.

3. Wash in a 20 percent solution cupric sulphate.

4. 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 % 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 brilliant red, while the body of the bacilli are stained
blue.

FLAGELLA STAINING 9I

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, 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 precipi-
tating of the stain in the substance of the flagella or else a decom-
position 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 emulsion 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.

92 STUDY OF BACTERIA

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.

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 lo c.c.

B. Saturated watery solution alum (old) lo c.c.

Saturated alcoholic solution gentian violet i c.c.

Mix.

A heavy precipitate is formed by this process v^hich 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 flageUa are
on those bacteria nearest to the edges where the evaporation has been most
intense. If the preparation is not equally stained, Weigert's aniline gentian
violet can be run on for a minute to deepen the color.

LoflBler'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.

STAINING DIPHTHERIA BACILLI 93

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 demonstrate
flagella readily.

Fig. 22 — B. Diphtheria stained by Neisser's method. (Williams.)

Neisser*s method of staining the diphtheria bacillus.
Two stains are needed: (Fig. 22.)

A. Methylene blue i gram.

95 percent alcohol 20 c.c.

Water 950 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.

94 STUDY OF BACTERIA

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.

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 minutes.
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.

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 ultra microscope dark field illumination enables one to see
fiagella 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 method in one of its various
forms. Microscopic objects are measured by viewing with an
ocular fitted with a graduated glass disc. Their values are indi-
cated on the apparatus.

DARK BACKGROUND ILLUMINATION 95

Bacteria may be most beautifully studied by means of the dark
field method of illumination in which they appear luminious
against a black background. An arc light and an especial sub-
stage condenser are necessary. By mixing a bacterial emulsion in
fluid, such as blood in saliva, with a mixture of India ink and
water and drying it on a slide, in examination the bacteria are not
stained but aie sharply defined against the black ink in a beautiful
way. The various spirochaetas and trypanosomes may be studied
in these two ways very satisfactorily.

CHAPTER VII.

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
hanging drop, or block method, they must be cultivated either in
the bodies of experiment animals, or in culture media artifici-
ally prepared. The latter method is the most widely used in
laboratories. 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 these
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 overwhelmed
with a multitude of bacteria, these substances must be sterilized
too, in order that the media shall be free from any living organisms.

Glassware, such as pipettes, Petri dishes, flasks and test-tubes,
are sterilized best by dry heat in hot air sterilizers. The apparatus
is subjected to a temperature of 150° C. 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 prevents the ingress of
bacteria, while air can reach the media through it freely.

Sterilization of culture media is accomplished in steam sterilizers
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 auto-

STERILIZATION

clave after two quarts of water have been put in it. 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 one atmosphere (15 pounds) or 120° C. for twenty
minutes, all bacteria are destroyed, and the media can be safely
assumed to be sterilized. If media containing sugar or gelatine are

Fig. 23. — Autoclave.

to be sterilized, the temperature should not run above 110° C, since,
if this is done the gelatine will not solidify when cold, the sugar is
caramelized and the media blackened.

Potato tubes are harder to sterilize a times, and it is safer to
repeat the operation in twenty-four hours.

Fractional method of sterilization, or Tyndallization, is accom-
7

BACTERIOLOGICAL LABORATORY TECHNIC

plished by heating the media to ioo° C. on three successive days
in a Koch or Arnold sterilizer. By heating culture media to this
temperature, all the vegetative, or adult, forms are killed, v^hile the
spores are not affected; after the first sterilization, at room tempera-
ture, the spores vegetate and become adult bacteria, when on the
second sterilization they are non-resistant to ioo° C. and are killed.

Fig. 24. — Arnold sterilizer.

Spores remaining after this develop into adult forms again
and are killed on the third day, at the third sterilization. This
fractional sterilization is employed in many laboratories still, and
is certainly the best for media containing carbohydrates of any
kind. To be effective, the media must be exposed to a temperature
of 100° C. for thirty minutes, that is, thirty minutes after the steam
has begun to form. Over heating of sugars causes them to
caramelize and turn black.

BACTERIA CULTIVATION

Bacteria that grow best at a temperature of 37° C. (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 essential that the
interior of the incubator be kept at an even, unvarying temperature.

Fig. 25. — Incubator.

This is accomplished by using a small bunsen flame under the incu-
bator. The heat from the flame warms the outer air chamber or
jacket, and it in turn warms the water 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 diminishes the gas supply if the temper-
ature runs too high, or increases it if it runs too low. The Roux
regulator is the simplest and most efficient one.

lOO BACTERIOLOGICAL LABORATORY TECHNIC

A serum coagulating apparatus is needed in laboratories in order
to coagulate the tubes of blood serum. (Fig. 26.)

Serum tubes are coagulated in it at a temperature of about 70° C.
They are then sterilized by heating them for an hour at this tem-
perature, for five successive days.

The separation of bacteria from the bouillon in which they grow

Fig. 26. — Blood serum coagulating apparatus.

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 nepk of a 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. Bouil-
lon cultures are placed within the sacs, which are then inserted in
the abdomen of an animal and left there. The sac is made of
collodion because it is non-absorbent and allows the bacterial juices

NUTRIENT MEDIA

lOI

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 addition
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 meat 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 purpose of ferment-
ing the meat sugar. For this purpose the
inoculated juice is allowed to stand at
room temperature over night. Bring to a
boil and add

10 grams of Witte's peptone.
5 grams common salt.

Fig. 27. — Kitasato fil-
ter for filtering toxins.
(Williams.)
Weigh the saucepan and contents and heat

to 60° C. 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-

I02 BACTERIOLOGICAL LABORATORY TECHNIC

phthalein as an indicator, and after finding how much of a nor-
mal 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 re-
stored. After boiling, from .5 percent to 1.5 percent normal hydro-
chloric acid solution is added and the acidity thus produced is
spoken of as -I-.5 per-cent or + i-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.

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 over night squeeze
the beef and extract the juice.

b. Add I percent peptone, 5 percent salt, 10 percent to 15 per-
cent 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 coagu-
lates.

f. Cool to 60° C. and add an egg well beaten up in water.

g. Boil slowly till all the egg is coagulated. This clears the

AGAR-AGAR I03

medium 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 110° C.
Litmus, or lacmoid, or neutral red may be added to the gela-
tine as an indicator.

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 120° C. Then cool.

c. Add this to 500 c.c. of bouillon of double strength, making
1,000 c.c.

d. Neutralize.

e. Cool to 60° C.

f. Add an egg to the mixture, stir.

g. Boil till egg is coagulated thoroughly.

h. Titrate and adjust to desired acidity as given under bouillon,
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 6 percent of glycerine to the agar
before neutralizing. To make agar for tubercle bacilli, veal bouillon
must be employed, and glycerine must be added.

Litmus Milk.

Carefully skimmed milk, to which litmus has been added, is run
into tubes and sterilized. This is a valuable culture medium. It
is also a reagent.

I04

BACTERIOLOGICAL LABORATORY TECHNIC

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-io,ooo bichloride
of mercury 6 hours and running water over night.
Some laboratories soak their slices in sodium carbon-
ate solution. It is desirable to know the reaction
of the medium and each batch should be tested, then
marked whether faintly or strongly acid or alkalin.

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 lo grams

Salt 5 grams

Water i,ooo c.c.

Mix. Boil. Filter and store in tubes and sterilize.

This is used to demonstrate the production of

indol.

_ ^ ^ Dextrose and lactose culture media are often

Fig. 28. — Po- 1 rT.1 1,11-

tato in culture used. They are prepared by addmg i percent of

tube. (Wil- these sugars to the various media before neutraliza-
liams.) . "^

tion.

BLOOD AGAR.

Is prepared by adding to agar some defibrinated rabbit's blood
in varying proportions before the agar is tubed and hardened.

THE STUDY OF THE GROWTH OF BACTERIA I05

BLOOD SERUM.

The blood of a dog drawn under strictly aseptic precautions
from a vein of an anesthetized dog 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 harmful for the growth of the tubercle bacilli, because salts are
formed which interfere with the growth of the bacteria.

LOFFLER'S BLOOD SERUM MIXTURE.

Blood serum of an ox 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 65° C. or thereabouts.

After they are coagulated they are sterilized by heating an hour
each day at 65° C. five successive days, or at 95° C. for an hour on
three successive days. After sterilization the tubes should be sealed
carefully.

Egg 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 laboratory technic.

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 media

io6

BACTERIOLOGICAL LABORATORY TECHNIC

by simply inoculating the media with some pus or blood containing
them, by means of a sterile pipette or platinum needle. Bouillon
may be thus inoculated, as may any of the media, and other cultures
may be made from these by sterilized needles. But such cultures
are made up of colonies of different sorts of bacteria — some patho-
genic, some non-pathogenic, etc. To separate the various bacteria
so that they will grow in isolated groups, is a comparatively easy

Fig. 29, — Colonies in gelatine plate showing how they may be separated and
the organisms isolated. (Williams.)

matter, and is accomplished in several ways. The simplest is to
employ several tubes of agar or blood serum. Over the surface of
each of these, a platinum loop containing 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 confluently together, from which it is im-
possible to isolate any pure cultures. The second tube is less covered
with bacteria, while the third, instead of containing a mass of bac-

THE STUDY OF THE GROWTH OF BACTERIA I07

teria, exhibits tiny little dots, or colonies (pure cultures) growing
discretely isolated. By means of a sterilized platinum needle these
little colonies may be fished out and transplanted to fresh culture
tubes, and after a few hours' growth they become pure cultures.

Fig. 30 — Series of stab cultures in gelatine, showing modes of growth of different
species of bacteria. (Abbott.)

An old method employed in many laboratories, in breweries and
originated by Pasteur was what is known as the dilution method.
Numerous flasks are inoculated by matter containing bacteria
very highly diluted in bouillon and by means of a sterile

io8

BACTERIOLOGICAL LABORATORY TECHNIC

pipette drops of this highly attenuated mixture are dropped into
flasks of sterilized bouillon or wort. Among a great number of
flasks so inoculated, some will be found sterile, others will contain
two or three different forms of bacteria, while a few will, perhaps,
contain a pure colony of the kind of bacteria for which a search is
being made.

Another method is to inject some matter con-
taining pathogenic bacteria into a rabbit or
guinea pig. The various juices and the leuco-
cytes of the animal destroy the non-pathogenic
bacteria and a pure culture, often of a pathogenic
form, may be isolated from the blood or miliary
abscess or tubercle of the animal at autopsy and
transferred to culture media.

By far the most useful and ingenious method
of procedure is the Koch, or plate method. This
is used in many laboratories all over the world.

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

Fig. 31. Needles they have developed into isolated colonies they

used for inoculating jnay 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 multi-
tude 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.

THE STUDY OF THE GROWTH OF BACTERIA I09

To isolate a pure culture of bacteria, say the Bacillus pyocyaneus
from pus, the following procedure is adopted in this method.

Three sterilized petri dishes, and three tubes of gelatine melted
at 40° C. are used. A loopful of pus is taken up by a sterilized
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 position and
the cotton plug is removed, and held by its outside end between the
fingers of the left hand, care being taken to prevent the tubal part
of the plug touching anything and being contaminated. The plati-
num loop is then slowly and carefully introduced into the medium,
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

Fig. 32. — Method of inoculating culture media. (Williams.)

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 removed from number one and mixed with number
two tube. 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 steril-
ized and put aside. The petri dishes should now be laid on a
cold level slab, and the contents of the tubes run into the differ-
ent dishes. Tube number one is taken first; the lip of the tube is

110 BACTERIOLOGICAL LABORATORY TECHNIC

Fig. 33. — Dilution method of making cultures, i, Is first tube containing
great number of colonies; 2, contains less number; 3, relatively few.
(WUliams.)

ROLL CULTURE III

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 par-
tially lifted and the contents of the tube rapidly and evenly poured
over the bottom of the plate, and the lid quickly replaced.

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 other ways, before it is cer-
tain 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 often used since they have this advantage — they
do not melt at 37° C. incubator temperature. When agar is used it
must be melted at 100° C. and cooled below 45° C. and above 39° C.
Above 45° C. bacteria may be killed. Below 39° C. 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 hard-
ened, they then must be inverted 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 in a cool place.
It is often of advantage to cool the plates by means of ice, before
they are filled.

Roll Culture.

Instead of pouring out the contents of the inoculated tubes the
gelatine may be made to harden on the walls of the tubes by quickly

112

BACTERIOLOGICAL LABORATORY TECHNIC

rotating the tube in a groove melted in a block of ice. The centrifu-
gal 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. Bacteria differ in very many ways in cultures. Some
grow rapidly and luxuriantly; 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

Fig. 34. — Esmarchs' method of making roll cultures on ice. (Williams.)

by some; gas and acids produced by others. These various char-
acteristics 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

ROLL CULTURE II3

bacteriology. The principal characters to be noted are complete
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 temperature 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
milk culture. Milk is run into tubes, and sterilized tincture of
litmus is often added to act as an indicator. Before using the milk,
it must be skimmed and free from all fat.

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 identifying
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 in the closed arm are considered
anaerobes. By inoculating a gelatine tube with bacteria while it is
melted and then letting it solidify, previously shaking the tube
vigorously, gas formation will be speedily manifested by the presence
of bubbles. Acids are detected in cultures by the employment of
various indicators in the culture media. Litmus, lacmoid, and
neutral red are used for this purpose. By titrating bouillon of
previous known acidity with a decinormal soda solution, the amount
of acid produced by different bacteria can be estimated.
8

114 BACTERIOLOGICAL LABORATORY TECHNIC

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 culture
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

Fig. 35.— Fermentation tube. (Williams.)

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 solution
(.02 grams 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

ROLL CULTURE II5

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 (see other works for description).

Incubate for a week and then add one cubic centimeter each of
the following solutions:

a. Sulphuric acid . .5 grams.

Acetic acid. 150 c.c.

b. Amido naphthaline i gram.

Water 20 c.c.

Boil, filter, and add 180 c.c. of dilute acetic acid.

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 appa-
ratus are necessary.

The following methods are pursued in ordinary laboratory man-
ipulations.

1. Exclusion of oxygen.

2. Exhaustion of oxygen by means of an air pump.

3. Absorption of oxygen by means of chemicals that absorb oxy-
gen from the air. A mixture of pyrogallic acid and sodium hydrate
absorbs oxygen rapidly, leaving nitrogen only in the chamber.

4. Displacement of air by means of an air-pump and allowing
hydrogen to enter the vacuum.

Under the Jirst 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. 36), thus excluding air, or deep punctures
may be made in tubes half filled with gelatine or agar, for growths
often occur in the depths of the medium, especially if 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

Il6 BACTERIOLOGICAL LABORATORY TECHNIC

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 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.

Fig. 36. — ^A streak made in agar by a needle inoculated with anaerobic bacilli
and then covered at one spot with cover-glass. (Williams.)

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 withdrawn,
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

ANIMAL EXPERIMENTS

117

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 technic.

Fig. 37. — Novy jar.

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 intoduced 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

Il8 BACTERIOLOGICAL LABORATORY TECHNIC

culture is run into the peritoneal cavity, or under the skin. The
animal is carefully weighed, and it is watched from day to day.
If it dies an autopsy is made on it.

Other methods consist in injecting fluid culture into the veins of
the ear, or into the peritoneum, by means of a sterile hypodermic
syringe. The autopsy should be made carefully ; the animal should
be thoroughly wet with a solution of bichloride of mercury, 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 mer-
cury. 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. Sometimes it is neces-
sary 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. Often
it is of great importance to make smears on cover-slips as well
as cultures, from the heart cavities, liver, kidney, peritoneal cavity,
etc., and stain them directly with Jenner's 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.

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 absolute alcohol, and imbedded in
celloidin, then cut into sections and mounted in the following
different ways:

HISTOLOGICAL METHODS II 9

I. Loffler's Method.

a. Float section in alcohol.

b. 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.

f. Mount in balsam.

II. Weigert's Method.

a. Transfer section to alcohol.

b. Place in lithium carmine five minutes.

c. Then in acid alcohol fifteen seconds.

d. Wash in water.

e. Transfer to slide and dry with blotting paper.

f. Apply Ehrlich's gentian violet for three minutes.

g. Blot and place in Gram's solution for two minutes,
h. Wash and dehydrate in aniline oil.

i. Wash with xylol.

j. 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. This lat-
ter method is an admirable one. There are many other methods
of staining. Paraffine embedding methods may be employed, but
for these the student is referred to works solely devoted to technic.
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 chem-
ical union with the protoplasm of bacterial cells and so forming new
compounds, and often coagulating the protoplasm.

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 at a high rather 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.

It is often necessary to determine the exact minimum amount of
an antiseptic that will destroy a given organism or produce a com-
plete inhibition of growth; for this purpose small amounts of a dis-
infectant 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.

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.

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 most valuable chemical disinfectants are those that kill in
highly dilute solution in a short time.

I20

CHEMICAL DISINFECTANTS 121

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.

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 i6 parts of hot 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 watery 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 presence of the
albumins in the blood or bouillon, no doubt acts as a protecting
envelope about the bodies of the bacteria. Bichloride is then more
efficient outside the body than in it. It is also more useful and
powerful when it is acidulated with a . 5 percent of HCl, or when it
is mixed with common salt or ammonium chloride. In culture
1-1,000,000 solution prevents the growth of most pathogenic bac-
teria. Biniodide of mercury is said by some observers to be more
powerful than the bichloride. It is certainly less likely to be inter-
fered 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 sulphate in

122 ANTISEPTICS AND DISINFECTANTS

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 the preparations; it is about a fourth as efficient as bi-
chloride of mercury and is not nearly so toxic. Some of the albu-
minates 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 decomposi-
tion 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 mole-
cule of water combining with the hydrogen to form free HCl 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 1-5 minutes. Iodine preparations like chlorine ones
are very powerful. They are of great use in medicine; 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.

CHEMICAL DISINFECTANTS 1 23

Carbolic acid is valuable as a disinfectant because of its stability.
A 1-1,000 solution inhibits bacterial growth; a ,5 percent solution
kills spores in a few hours. A thorough solution should be made,
and to be very efficient, 5 percent HCl 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 germi-
cide sticks and continues its effects. Glycerin 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, CHjO, 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 combi-
nations with many organic bodies. When it unites with ammonia
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 apartments and ships,
actively destroying all bacteria. One percent of the vapor in the
air of a closed room, if the air is moist, destroys 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 genera-
tors which decompose the vapor of wood alcohol, when they
reach hot platinum sponges, salt, or hot copper; by vaporizing a

124 ANTISEPTICS AND DISINFECTANTS

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 apartment. 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 dilute 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 water 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 thick 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.

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

ANTISEPTIC VALUES 1 25

percent 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 albuminous
masses of the sputum or feces. Long contact with carbolic acid
acidulated with HCl is very efficient. Concentrated formaline 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 150° C. for one hour, or boil-
ing instruments for twenty to thirty minutes makes them certainly
sterile.

Disinfection of the skin is a difficult undertaking from a bacterio-
logical 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.

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

126 ANTISEPTICS AND DISINFECTANTS •

Ammonium chloride i to 9

Boric acid i to 143

Calcium chloride i to 25

Calcium hypochlorite i to 1,000

Carbolic acid i to 333

Chloral hydrate i to 107

Copper sulphate i to 2,000

Ferrous sulphate i to 200

Formaldehyde, 40 percent i to 10,000

Formaldehyde, pure i to 20,000

Hydrogen peroxide, fresh i to 14,300

Mercuric chloride i to 40,000

Mercuric iodide i to 25,000

Quinine sulphate i to 800

Silver nitrate i to 12,500

Zinc chloride i to 500

Zinc sulphate i to 20

CHAPTER V.

BACTERIA.

STREPTOCOCCUS PYOGENES.

Streptococcus Pyogenes.

Streptococcus Erysipelatis. Chain Coccus. (Fig. 38.)
Morphology and Stains. — Cocci grow in catenate form of from
4 to 40 individuals to a chain. Each coccus comprises two hemi-

FiG. 38. — Streptococcus pyogenes. (Kolle and Wassermann.)

spheres divided transversely. Some chains appear branched. The
cocci are not motile, and do not have spores. They can be stained
with all basic stains, and are not decolorized by Gram's method.

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 37° C. Will not grow at 47° C. Never vegetate

127

128 BACTERIA

luxuriantly on any culture media, but are most prolific on 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 with glycerine, 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, is totally coagulated in twenty-four
hours. Clotted casein may be digested. On potato the growth
is invisible and scanty.

Vital Resistance. — Thermal death-point is 54° C. in five
minutes. 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 dissolving
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.
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 feces. 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 aggrava-
tion of the original infection, and often death. It is especially active
in phthisis, scarlatina, small-pox, and diphtheria, in which diseases
it is often the cause of death. Many of the symptoms of phthisis

PNEUMOCOCCUS 1 29

are due to the toxins of the streptococcus; cavity formation and hectic
fever for example. Its virulence can be intensified by passing it
through a series of animals, until, finally, yxmr ^^ ^ cubic milli-
meter killed 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 sep-
ticaemia and in suppuration. The toxin of the streptococcus, if in-
jected under the skin, causes redness like erysipelas. 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. ")

PNEUMOCOCCUS.

Streptococcus lanceolatus, commonly known as the pneu-
mococcus, or Diplococcus lanceolatus. (Fig. 39.)

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 cap-
sule; in bouillon and other media, it loses the capsule and the lan-
ceolate 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 its 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
9

I30

BACTERIA

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 37.5° C; at 22° C. much
more slowly, often not at all. Grows better in the presence of
serum or hemoglobin.

Fig. 39. — Diplococcus pneumoniae, from the heart's blood of a rabbit. X 1000
(Frankel and Pfeiffer.)

Vital Resistance. — Easily killed at a temperature of 52° C, 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 sunlight and desiccation, for four
months.

Cultures. — On gelatine plate it produces very minute colonies

PNEUMOCOCCUS I3I

after quite a length of time. On glycerine agar it grows better,
but the colonies are small and difficult to see. In both, the colonies
are whitish, with a pearly lustre. On blood serum it grows in trans-
parent colonies. In bouillon it grows feebly, with a whitish sedi-
ment, 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 neu-
tralized by putting chalk into the bouillon the growth becomes
luxuriant and the bouillon becomes thick. On potato it will not
grow. It ferments some of the sugars, the most important being
inulin. No gas is formed.

Habitat. — Outside the human body it has not been found, but is
normally present in the mouth of about 30 percent of all people.
Even the alveoli of the lungs in health contain them. Human saliva
injected into animals often causes pneumococcic siepticaemia. They
are also 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 exudates on serous
membranes. All tissues of the body may be attacked. Some
strains of pneumococci are more neurotoxic than others.

In rabbits an injection (intravenous) of pneumococci very often
(33 percent) causes lobar pneumonia; certain strains cause lobular
pneumonia habitually among the susceptible animals (Eyre). 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

132 BACTERIA

abstracted from veins is mixed with 500 c.c. of milk and incubated.
In twenty-four hours pneumococci, if present, 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-pneumococcic 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
stimulates phagocytosis. It has been used therapeutically in man
for the cure of pneumonia with doubtful results. Oleate of soda
aids in bacteriolysis of pneumococci by sera, if added to the
various varieties of immune sera.

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.

Two intermediary streptococci are Str. viridans and Str. mucosus,
Str. viridans is like the Str. pyogenes but produces germ colonies.
It is most frequently met as the cause of valvular endocarditis.
Str. mucosus is a long chain former surrounded by a halo not stain-
able as a capsule and produces viscid exudate. In some ways it
resembles the pneumococcus.

The various streptococci from pus, saliva, feces, manure and sew-
age are differentiated by their action on blood, milk and the sugars.

COCCUS OF MENINGITIS.

Streptococcus Intracellularis.

Diplococcus intracellularis meningitidis.
Meningococcus. (Fig. 40.)

coccus OF MENINGITIS 1 33

This organism is the cause of cerebro-spinal meningitis.

Morphology and Stains. — Resembles the gonococcus closely,
because it grows in biscuit shaped pairs; is nearly always within pus
cells, and like the gonococcus it is decolorized by Gram's stain.

Fig. 40. — Meningococcus in spinal fluid. (From Hiss and Zinsser's Bacteri-
ology, Copyright by D. Appleton & Co.)

In reality it is a micrococcus, because it divides in two planes.
It has no spores or flagella; is not motile; grows in short chains at
times, and on ordinary media best at 37° C. It is Gram negative.

Relation to Oxygen. — It is an obligate aerobe.

Vital Resistance. — It is killed after 10 minutes' exposure to 65°
C. and is easily destroyed by drying, and by light. It dies out
rapidly on artificial culture media.

134 BACTERIA

Cultures. — On glycerine agar it grows, sparingly as white viscid
colonies; occasionally it develops on potato; thrives on blood
senun, especially if smeared with blood, and does not liquefy the
serum.

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 sub-dural
injection.

Chemical Activities. — ^Produces an endo-toxin buf no soluble
toxin. It is not chromogenic.

Agglutination is caused by immune serum.

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 sub-dural space.

Specific Therapy. — Flexner and Jobling have produced an anti-
serum for meningitis. It has anti-bacterial powers. Horses are
injected with bacterial suspensions until their serum possesses
curative properties. This anti-serum is injected directly into the
arachnoid space by lumbar puncture, after withdrawal of some
of the meningitic exudate. Little anti-serum will appear in the
cerebro-spinal fluid if it be injected subcutaneously. Therapeutic
results have been brilliant.

There is another important Gram negative diplococcus in the
nose called Micrococcus catarrhalis. It is differentiated from
the meningitis organism by its free yellow growth on agar and
absence of active pathogenic properties. It is thought to have
some relation to acute coryza.

STAPHYLOCOCCUS PYOGENES AUREUS

STAPHYLOCOCCUS PYOGENES AUREUS.

135

Staphylococcus Pyogenes Aureus. (Fig. 41.)

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. 41. — Staphylococcus aureus. (Williams.)

The Micrococcus pyogenes aureus only is here described.

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 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

136

BACTERIA

for over one hundred days in pus. Dry thermal death-point is 80°
C. for one hour. Moist heat 70° C, kills in ten to twenty minutes.
Resists freezing temperature 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 1 5 minutes; i percent HjOj in 30 minutes.
Chemical Activities. — Produces a golden
yellow pigment only under oxygen. Gen-
erates 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 abdominal cavity, it causes perito-
nitis. Subcutaneously it may produce sterile
abscess, or local necrosis. There is pro-
duced in cultures a toxin having a destruc-
tive action upon leucocytes and red blood
cells.

Cultures.T^In gelatine it rapidly forms
golden yellow colonies, that quickly liquefy
the gelatine. (Fig. 42.) Sterile products
of the growth also liquefy gelatine. On
gelatine plate, yellowish to orange colonies
Fig. 42.— Gelatine cul- are formed. On agar streak a luxuriant

rS'Sd.lwuila"™) "^-"g-^ g-^"-* 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.

GONOCOCCUS 137

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 previously infected
tissues (tubercles) and is active in small-pox and diphtheria. Ex-
perimental 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 disas-
trous. By growing it under anaerobic conditions its Virulence may
be intensified, and the activity 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. — Thus far it has been impossible to produce any
marked immunity either by anti-toxic sera, or by culture, living or
dead, but the bacterins 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 furunculo-
sis has established itself as most efficacious.

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 Gonorrhoeae (Neisser).
Diplococcus Gonorrhcece, commonly called the gonococcus. (Fig. 43.)

138 BACTERIA

Morphology and Stains. — The morphology of this organism is
peculiar and characteristic. Always found in pairs which are ce-
mented 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 pro-
toplasm of the leucocytes, 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. This point
is most important in differentiating it from
Fig. 43. — Gonococci other diplococci, except the meningococcus. A
ica^^iagnosL^^ ^ ^ 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 36° C. 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 withstand
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.

MICROCOCCUS TETRAGENUS 1 39

Pathogenic Virulence. — This organism does not infect any of
the lower animals. The " gonotoxin," if injected into small animals,
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 vio-
lent inflammation of the urethra, vagina, uterus, fallopian tubes,
and the peritoneum. It frequently affects the conjunctivae of the
newly born, and sometimes causes a pan-ophthalmia, which destroys
the sight. It is a common cause of suppurating arthritis gonor-
rhoeal rheumatism, endocarditis, pleuritis. In fact, any serous
membranes may be infected, and very serious results follow. Cysti-
tis 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.

I40 BACTERIA

Oxygen Requirements. — It grows very well in the presence of
oxygen, and poorly without it.

Cultures. — Grows well on all common cuKure 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 gelatine. In bouillon
it thrives well, depositing a heavy precipitate. In milk it causes
coagulation after four days. 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 H2S.

Habitat. — Has never been found outside the human body; is
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 infection
in phthisis.

BACILLUS OF MALTA FEVER.

Bacterium Melitensis.

Micrococcus 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 .5/1 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. Stains 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 37° C., is more rapid. The colonies are

INFLUENZA BACILLUS I4I

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
I- 1 00.

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 Influenzae.

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. Sometimes found chain-like. No flagella or
spores are formed. Stains weakly. Carbol fuchsin, diluted, 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° C. and 41° C. ; best at 37° C. Blood or haemoglobin is
required 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 Staphylococcus

142

BACTERIA

aureus, it increases more luxuriantly (symbiosis). It is probable
that the cocci, in some way, alter the blood of the culture media.

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.

Pathogenesis. — If pure cultures are placed on the mucous sur-
faces of monkeys, influenza results. Pure cultures injected into the
peritoneum of guinea pigs cause fatal peritonitis. In man, it causes
various affections of the upper respiratory tract — bronchitis, pneu-

FiG. 44. — ^Pest Bacilli from spleen of rat. (KoUe and Wassermann.)

monia, both croupous and catarrhal. Also conjunctivitis. It elab-
orates a powerful toxin, which produces strongly depressing effects
on certain organs, especially nervous tissues. It is an important
factor in abscess production in the middle ear, and elsewhere, and
complicates many pneumonia cases, seriously interfering with
recovery in young children, and the aged. Associated with the
pneumococcus, its toxic effect is increased. It has been found in the
blood.

KOCH-WEEKS BACILLUS 143

Influenzal meningitis is more frequent than formerly or at least
is more often diagnosed. It can be reproduced in monkeys.
Bacilli appear in the blood in influenzal meningitis.

By immunizing a goat with influenza bacilli Wallstein obtains a
serum which has a pronouncedly favorable effect upon the ex-
perimental disease in monkeys and promises some therapeutic
power for human beings. Its most important effect is to stimulate
phagocytosis in the cerebro- spiral fluid.

No immunity results from infection. No leucocytosis occurs
during infection. Serum from infected individuals agglutinates
bacilli even if diluted 1-500.

No artificial immunity can be produced but bacterins are some-
times used therapeutically.

Bordet-Gengou Bacillus of Whooping Cough. — This is a
very minute ovoid rod lying separately, varying from .8-1. 5/i long
and being .3// wide. No spores, no motility or flagella. Stains
poorly, best at ends; Gram negative. It may be cultivated from
expectoration early in the disease upon media containing glycer-
ine, potato, blood and agar. Aerobe, and grows best at 37° C.
There is an 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.

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.5/£X.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.

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
Kock- Weeks bacillus, measuring up to 2^.

144 BACTERIA

PLAGUE BACILLUS.

Bacterium Pestis.

Plague Bacillus. (Fig. 44.)

Morphology and Stains. — Short plump rods with rounded ends,
containing no spores and non-motile. Also surrounded by cap-
sule? Organisms from exudates, or blood, exhibit character-
istically peculiar polar staining. They are often found 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 J percent salt).

Relation to Oxygen. — Strict aerobe, the growth is stopped by
the exclusion of oxygen.

Vital Requirements. — Grows well at 22° C, but best at 37° C.;

Fig. 45. — Colonies of plague bacilli forty-eight hours old. (Kolle and

Wassermann.)

is killed after a short exposure to 55° C.-6o° C., 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

PLAGUE BACILLUS 145

days. Withstands freezing for months, but does not stand light
or chemicals very long.

Cultures. — Grows very well on culture media. In bouillon
it thrives abundantly, with a heavy pellicle which produces depend-
ent stalactites that drop to the bottom of the vessel. On gelatine
plates it grows in small flat colonies, which are gray and transpar-
ent, and which do not liquefy the gelatine. (Fig. 45.) In gelatine
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 is not coagulated,
and the growth is slight. Potato yields a slow whitish-yellow
growth that is sharply outlined.

Chemical Activities. — Does not produce HgS, enzyme, colors,
or odors, indol or nitrites. The toxin produced is not soluble and

Fig. 46. — B. Pestis in pus of bubo. (Jackson.)

the filtrate is non-poisonous. Old killed bouillon cultures can be
extracted and a highly poisonous substance precipitated therefrom
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
10

146 BACTERIA

and in the cutaneous pustules, lymphatics and in the lungs in plague
pneumonia; more rarely in the blood and other organs. In ani-
mals, 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 are 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.

Pathogenesis. — Highly pathogenic for man. Is the cause of
the bubonic or Oriental plague; bacilli gain entrance by way of the

Fig. 47. — Pest bacillus involution forms produced by growing on 3 per cent
salt agar. (KoUe and Wassermann.)

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. The mortality from this plague is from 50
percent to 80 percent. (Fig. 46.)

Almost all domestic animals — rats, mice, guinea pigs, rabbits and
squirrels are affected; horses and swine are very susceptible; cows

147

and dogs less so. Rats seem to be affected with a chronic form of the
malady, and by inhabiting ships and warehouses in foreign countries,
spread the disease. Post mortems on infected animals reveal haemor-
rhagic 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.

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 developes buboes and dies, and polar staining bacilli are
found, it is probable that the organism is the plague bacillus. Fur-
ther, if curious involution forms develop on heavily salted agar
(3 percent) the diagnosis is confirmed. (Fig. 47.)

The disease is spread by flies which, according to Yersin, are
susceptible to the disease, and spread it by depositing their feces
on the human skin, rather than through their bites.

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.

FRIEDLANDER'S BACILLUS.

Bacterium Pneumoniae.

Friedlander' s Pneumonia Bacillus. (Fig. 48.)

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, CO2 and H.
gas, alcohol, indol, ferment and HjS.

148 BACTERIA

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 yel-
lowish-white with a slimy lustre, and never liquefies the gelatine.
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 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 pneu-
monia in mice, also septicaemia. Ouinea
pigs and dogs are susceptible. It may be

. '^ k" 48--Friedland- ^^^^^ -^^ normal mouths. Friedlander's pneu-
er s bacillus and pneu- ^

mococci, showing cap- monia is much less frequent than that due to

kirDiagnosL"f ' ^^^' *« pneumococcus, but it is very fatal. Ag-
glutination takes place with immiine serum.

This is the most important representative of a group of organisms
of moderate pathogenic powers and importance called variously.
Bacterium aerogenes, Bacterium mucosus or Aerogenes mucosus
group. 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 carbodydrates
except saccharose, with gas.

These organisms are important members of the intestinal flora.

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 bacillis, but is non-motile.
It forms lactic acid among its principal products. The most

TYPHOID BACILLUS 149

important lactic acid producer is Bad. 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 bulgaricum 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 2/1
to 50/z in length. They grov^r with difficulty in the laboratory, best
on milk and whey. Optimum temperature 44° C. They form
branching filamentous colonies. Milk is coagulated in 18 hours
at 44° C. and in 36 hours at 37° C. The clot is not dissolved.
Gelatine is not liquefied.

TYPHOID BACILLUS.

Bacterium Typhi. Koch and Eberth.

Bacillus Typhosus.

Typhoid Bacillus. (Fig. 49.)

A most important pathogenic organism that
causes typhoid fever.

Morphology and Stains. — Generally short
plump rods i to 3 // 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 Fig. 49.— Typhoid
flageHated (peritrichous) ; contains no spores ; g^^^^/^ (j^oSrand Was-
exhibits pleomorphic and involution forms; is sermann.)
actively motile, and stains with all the basic
aniline dyes, but not by Gram's method.

Vital Resistance. — The thermal death-point is 60° C., ten to
fifteen minutes. Remains alive in ice for months; even the tempera-
ture of liquid air does not destroy it. In distilled water it lives for
months, but if other saprophytic bacteria are associated with it,

ISO

BACTERIA

however, it quickly dies. Does not resist drying or chemicals,
except carbolic acid, towards which it exhibits a tolerance. Sun-
light kills it in an hour.

Habitat. — It never exists in nature, except where water or soil
has been contaminated by feces or urine. It may multiply in po-
table waters, in milk, and the juices of oysters.

Chemical Activities. — Does not produce proteolytic enzymes;
forms HgS. but will not ferment the sugars with gas formation.
Does not yield indol or nitrites. Produces levorotatory lactic acid.

Fig. 50. — Seventy-two hour old culture of typhoid bacillus on gelatine. (Kolle
and Wassermann.)

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 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, 37° C. and more slowly at 20° C. On gela-
tine plate it produces at first small colonies, yellowish and punctate,
which become whitish, delicately notched and ridged. (Fig. 50.)

TYPHOID BACILLUS 151

In gelatine stab culture it grows in a thread-like granular line,
without producing gas. In neither case is the gelatine liquefied.
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 only detected by scratching with a needle. In bouillon
it grows uniformly, producing very little acid, and no demonstrable
amount of 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 points. It also
grows characteristically in Capaldi and the Drigalski and Conradi
media.

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.

Pathogenesis. — It is certainly the cause of typhoid fever. Is
found in the stools and urine of the patient, and may be recovered
from the blood. Also found in the spleen and gall bladder. It
produces well marked histological changes in the lymphoid struc-
tures, particularly in Peyer's patches, solitary follicles, and other
lymph glands. There is, according to Mallory, a massive endothe-
lial proliferation in the lymph glands. This causes occlusion of
the lymph vessels, and is followed by necrosis (ulceration) of the
Peyer's patches. The intense phagocytic action of the fixed lym-
phatic cells in the glands is manifest toward the red blood cells,
which are devoured in great numbers. The toxin causes degenera-
tion 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 bacteraemia. There is often a
mixed infection in which streptococcus pyogenes in the blood

152 BACTERIA

plays an active r6le. In the necrosis of bone and in subphrenic
abscess the typhoid bacilli may act as a pus former. Commonly
it 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 dis-
ease, and the bacilli rapidly die. In chimpanzees, however, it is
possible to produce typical typhoid lesions and symptoms.

Natural and Acquired Immunity. — Human blood serum is
strongly bactericidal toward the typhoid bacillus. Normal gastric
juice, with its hydrochloric acid, destroys the
bacillus when ingested and this forms the
natural means of protection. Immunity follow-
ing an attack of typhoid is generally of long
duration. If bacilli do reach the blood stream
of an immune individual, the amboceptors
originated by a previous infection, together
action. On7h!lfof tii^; "^^^^ the complement normally present, effect
field shows typhoid a solution of the invading organism. Artifi-
o ?h i V ' hX^^'Siows ^^^1 immunity has been effected against typhoid
clumping. (Greene's by vaccinating individuals with killed cultures.
Medical Diagnosis.) Anti-toxin 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 dis-
ease, 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

TYPHOID BACILLUS 1 53

if the reaction comes within an hour the 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 doubtful case of typhoid, and estabh'sh 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. The fetus of a woman suffering
from typhoid contains agglutinins in its blood. The milk, tears,
and other body fluids from an individual 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.

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. It is still uncertain whether the reaction
is merely a phenomenon of infection, or whether it has to do with
immunity. By many it is held that the two are distinct and separate
and that it is a phenomenon of infection. There are several reasons
for thinking so. i. The bactericidal action of serum is destroyed
at 56° C. The agglutinating power is not destroyed at 62° C. 2.
A serum may be bactericidal, but not agglutinative. 3. Bacteria
treated with bactericidal sera lose their virulence, and those that

154 BACTERIA

have been agglutinated do not do so. (Compare Friedberger's
idea of infection, page 59.)

Paratyphoid Bacillus. — A pathogenic organism producing all
the clinical symptoms of typhoid, only in milder form (at times) has
lately 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, even in
high dilution. Various varieties differ in growth upon litmus milk.
In every other respect it resembles the typhoid bacillus, and seems
to occupy a position betv^een it and the colon bacillus. Paratyphoid
endotoxin resists 60° C. from thirty to sixty minutes, so it is said.

The Paracolons are organisms like the paratyphoids, but some-
what closer to the colon bacillus. (For example, see page 156.)

Blood cultures are often employed in large hospitals for the diag-
nosis 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
vnthstand it. The bacilli may be easily isolated from the blood by
adding the latter to some bile and then incubating it. From the
bile, cultures are made in agar or in bouillon.

COLON BACILLUS.

Bacterium Coll.

Bacillus coli or Bacillus coli communis.

Colon Bacillus.

While not strictly a pathogenic organism, it plays such an impor-
tant 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-

COLON BACILLUS 1 55

phism; may grow in chains; and possesses vacuoles and polar
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 62° C. ; light and heat are destructive to it, and its resistance
to antiseptics is somewhat better than that of typhoid bacillus.

Cultures. — Thrives in all common culture media, especially if
sugar is present. It is restrained by excess of acids produced in

Fig. 52. — Colon bacillus showing flagella. (KoUe and Wassermann.)

culture media. On gelatine it grows like the typhoid bacillus (from
which it is difficult to differentiate, see page 263) in whitish raised
colonies that do not liquefy the media. Sometimes the growth is
thin and iridescent, and exhibits bizarie shapes — tadpole-like and
lobulated. Typhoid colonies show deep furrow-like r'dges 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 cloudiness, and forms gas

156 BACTERIA

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 coagu-
lation. 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, CO2 and some N. Some
varieties ferment cane sugar. Produces lactic, acetic and formic
acids, also indol abundantly, and H2 S. It decomposes urea. There
are a great many varities of colon bacilli having very different
chemical activities.

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
drinking water, the colon bacilli are easily found. If in abundance,
it indicates great fecal 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
it plays rather a subordinate pathogenic role, but it has been found
the causal agent of some cases of suppurative appendicitis, peri-
tonitis, 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 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.

DYSENTERY BACILLUS 1 57

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 transparent.
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 ngt.

5. Milk is coagulated by the colon, but not by the typhoid.

6. On potatoes colon grows much more luxuriantly than typhoid.

7. Typhoid reddens neutral red; colon changes it to bright yellow.

8. The most 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 infection,
but bacterins have been used with much benefit.

DYSENTERY BACILLUS.

Bacterium Dysenteriae.

Dysentery Bacillus of SJiiga and Flexner.

Supposed 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 various strains of this organism, the differentia-
tion of which depend upon their chemical activities, fermentation
of various carbohydrates being the most important, and agglutina-
tive properties with different sera.

Morphology and Stains. — The organism is, in many respects,
similar to the typhoid bacillus, but is plumper. It is said to be
flagellated, has no. spores, and exhibits pleomorphism. It stains
well with the common aniline dyes, but not by Gram's method.

158 BACTERIA

Vital Properties. — It is killed by i percent carbolic solution
in thirty minutes. Lives for twelve to seventeen days v^^hen dried.
Direct sunlight kills it in thirty minutes. Its thermal death-point
is 58° C. in thirty minutes. It is a facultative aerobe; grov^^s at
ordinary temperature, but better at 37° C.

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
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, 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 lymphoid
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 parenchymatous degeneration.
The bacilli being possessed of a powerful endo-toxin, so that dead
cultures, if injected under the skin cause marked local and general
reactions. Like the pyocyaneus bacillus, this organism undergoes

DYSETERY BACILLUS 159

auto-digestion in bouillon, which leaves the latter highly toxic owing
to the liberation of the toxins. Laboratory animals quickly suc-
cumb 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 disorders favor infection. Death may
be due to toxaemia or exhaustion. As a causal agent in the produc-
tion of summer diarrhoeas 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.
Nevertheless 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 shows
a strong bactericidal action. Anti-bodies are developed by infection
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 amboceptors act, but only
in the presence of a complement. Anti-toxic sera protected against
bacteria; and an anti-bacterial serum protected against toxin,
according to Rosenthal.

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

l6o BACTERIA

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 organ-
ism should be used. These types probably bear the same relation
to each other that typhoid and paratyphoid do.

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. It forms a slight capsule.

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. Superficial 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

PYOCYANEUS BACILLUS l6l

laboratory animals. Neither the bacilli or the toxin they elaborate
are destroyed by 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 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 8,000 parts of water
has produced the reaction.

No anti-serum or bacterin treatment is as yet possible.

PYOCYANEUS BACILLUS.

Bacterium Pyocyaneus.

Bacillus Pyocyaneus. (Fig. 53.)

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 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 pigments,
a water-soluble greenish bacteriofluorescein, and a chloroform-
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
the line of inoculation, coloring the gelatine greenish-blue, and a

1 62 BACTERIA

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 thick-
ened edges. The agar quickly turns a dark greenish-blue, and
in old cultures the growth changes from yellow to greenish-blue.

Fig. 53. — Bacillus pyocyaneus. (Kolle and Wassermann.)

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 de-
stroys 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 bacterial cells

BACILLUS OF SOFT CHANCRE 1 63

themselves. Old cultures are poisonous ; a haemolysin is produced —
an endo-toxin, and a soluble toxin. Against the endo-toxin and the
soluble toxin it is possible to prepare an anti-serum. This may pro-
tect laboratory animals. The last named toxin stands a tempera-
ture of 100° C.

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 pathogenic
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.

Bacterium Ulceris Chancrosi. (Ducrey.)

Streptohacillus of Soft Chancre.

Morphology and Stains. — A small thin bacterium .5// broad,
1.5/^ long, growing in chains with polar staining, which can be
demonstrated in sections of chancres without much difficulty.

This organism does not stain by Gram's method, but by Loffler's
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 in blood agar
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.

164 BACTERIA

ANTHRAX BACILLUS.

Bacillus Anthracis.

Anthrax Bacillus of Koch. (Fig. 54.)

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 }jl wide. Is often in pairs
or chains. In fresh specimens the ends of the rods are rounded;
when older, the ends become square or con-
cave. Often they have faint capsule sur-
rounding them. In culture media they ex-
hibit 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 cul-

Ko ^^ik'^'T! ^1 ^^A tures, bizarre involution forms are found. It
bacilli in blood. '

(Greene's Medical stains well with all the common basic dyes

Diagnosis.) ^^^ ^^ ^^^^,^ 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 14° C. and 45° C; best at 37° C.
Spores are formed, if oxygen is abundant, above 12° C. Sporu-
lationis more rapid at 37° C. Spores withstand high temperature
(dry) for a long time, 100° C. for one hour. The bacillus itself is
killed at 70° C, moist heat, in one minute. The thermal death-
point may be put down for the organism, at 100° C. moist, for
five minutes.

Vital Resistance. — Highly resistant to chemicals, light and dry-
ing. Spores resist 5 percent carbolic solution for days (Esmarch),

ANTHRAX BACILLUS 1 65

and I percent corrosive sublimate (aqueous) for three days. They
also resist formaldehyde and sulphur for a long time, and withstand
light. A 2 percent fresh solution of H^Oa kills spores in three
hours. Three and one-half hours' exposure to bright sunlight killed
the spores if oxygen was not excluded. (Dieudonnd.) (Fig. 55.)

Fig. 55. — Anthrax bacilli growing in a chain and exhibiting spores. (KoUe and

Wassermann.)

Sporulation Phenomena. — At 12° C. spores are formed if
oxygen is present. The most favorable temperature for sporula-
tion is that of the body (37° C). 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 42° C. for a long
time and frequently reinoculated, on fresh media, the ability to
form spores is lost even if grown again at 30° C. (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 HgS. 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

I 66 BACTERIA

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 magnified,
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
appears, lateral hair-Hke outgrowths are ^een 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
bacilH. (Fig. 56.)

In agar stab, the growth ismore 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 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 coagu-
lum 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 purely
toxic, as it causes, not a toxaemia, but a bacteraemia. It is especially

ANTHRAX BACILLUS 1 67

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 temperature of 30° C. they
become susceptible to infection. At their normal temperature they
are immune.i The disease produced by this organism is known
variously in different countries as Anthrax, Splenic fever. Wool-
sorter's disease. Malignant pustule, and Qiarbon. It frequently
devastates vast herds of sheep, cattle, and goats, and is often a
pestilence in European countries, China, and South America. It

Fig. 56. — Anthrax bacilli. Cover-glass has been pressed on a colony and then
fixed and stained. (KoUe and Wassermann.)

appears sporadically in the United States. Its origin in this country
can usually be traced to infection from hides or hair imported from
abroad. 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, invest-
ing all the organs and the blood. Pus is produced, and tissues are
degenerated. Infection is accompanied by a high leucocytosis and
fever. There is often congestion of the lungs; also an intense fria-

l68 BACTERIA

bility of the splenic pulp, and all the glands of the body become
enlarged, and, at times, many of them suppurate. In wool-sorter'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 42° C. for various lengths
of time to attenuate them. It is possible but impracticable to
produce an anthrax anti-toxin.

TETANUS BACILLUS.

Bacillus Tetani.

Tetanus Bacillus. (Fig. 57.)
Lockjaw Bacillus.
First seen by Nicolaier, and isolated in pure culture by Kitasato.

Fig. 57. — Tetanus bacilli showing end spores. (KoUe and Wassermann.)

Morphology and Stains. — Rod-shaped. Varying from 1.2 jj. in
length, to very long threads of 20 to 40/z. Sometimes grow in
chains; frequently appear like short drum-sticks with a spore at one

TETANUS BACILLUS 1 69

end, which is either round or oval. At times, the bacilli in chains
sporulate. The organism is motile; possesses numerous flagella
(from fifty to a hundred) peritrichously arranged; stains well with
all the common basic aniline dyes, and retains the color in Gram's
method. (Fig. 58.)

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 37° C. Below 14° C. not at all.

Vital Resistance. — Spores resist 80° C. for an hour. This fact
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 100° C.
for I hour. They are killed in 2 hours by 5 percent phenol +.5
percent HCl and in 30 minutes by i-iooo HgCl2 + .5 percent HCl.

Chemical Activities. — Ferments sugar; produces gas, indol,
alkali, and HgS. which gives to the culture an odor of burnt garlic or
onion; marsh gas, CO2, 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
feces of man and of animals. It is said to have been found in the
spinal cord of a man who did not die of tetanus. It has also 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 purulent and contains but few bacilli. Puer-
peral tetanus, and tetanus of the new-born, are but varieties of the
disease, dependent upon the site of infection, whether of the pla-
centa or umbilical cord. Tetanus sometimes occurs spontaneously,

lyo BACTERIA

without a sign of injury anywhere. Sheep and goats are suscep-
tible 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 is often due to infected
virus, the latter becoming infected from the
feces of the vaccine-producing cows but more
commonly is due to dirt getting into vacci-
nation wounds.

baSS- shlti^r'pS- Cultures.-This organism is difficult to
trichous flagella. (Kolle grow, and always requires an atmosphere of
and Wassermann.) hydrogen.

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
hne of the needle, eventually the gelatine becomes hquid, and a
bubble of gas, partly filled with whitish-cloudy Hquid gelatine,
appears. On agar plates the colonies are ragged, and are sur-
rounded 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 cultivate the organism under
mica covering, or paraffine poured upon freshly boiled media. If
sterile glass tubing is filled with agar or gelatine, and inoculated

TETANUS BACILLUS 171

with tetanus bacilli, then sealed, colonies will develop, as perfect
anaerobic conditions are thus obtained. Often the organism grows
best in the presence of saprophytic ones. Strongly pathogenic
organisms do not grow well in culture media, while comparatively
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
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 cause of tetanus. The toxin is
produced rapidly in wounds, or what is more likely, some is intro-
duced 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 production is as follows: —
The toxin is conveyed from the wound by means of the motor nerves
to the central nervous system affecting the motor elements. It
causes microscopic degeneration of the fibers and cells of the motor

172 BACTERIA

apparatus. Death is caused either by a spasm of the glottis or
diaphragm, or by cardiac failure and exhaustion. A local mani-
festation 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
from opisthotonos. The seizures may be constant or tonic; or
convulsive and violent, then they are designated as clonic.

Immunity. — Metchnikoff claims that the only natural immunity
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, estab-
lishes a means of natural defense. Acquired immunity is dependent
upon the formation of anti-toxin. The anti-toxin, formed by suscep-
tible animals injected with tetanus toxin, is chiefly useful and valu-
able as a prophylactic measure. An epidemic of puerperal tetanus
in a lying-in hospital was checked by its use. Sprinkling dry pow-
dered anti-toxic serum on wounds infected 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 cerebro-spinal 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.

Female mice immunized against tetanus toxin, transmit a great
amount of immunity to their off-spring. The milk of an immunized
mouse also causes a passive immunity in other young that are
suckled by her.

BACILLUS OF MALIGNANT CEDEMA 1 73

BACILLUS OF MALIGNANT (EDEMA.

Bacillus CEdematis Maligni.

Vibrion septiquh.

Bacillus of Malignant (Edema.

Morphology and Stains. — Thickish rods, resembling tetanus
and symptomatic anthrax baciUi, with a tendency to grow in long
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, but not soured, and
the reaction is amphoteric. Abundant alkaU is formed at times;
albumin is decomposed, forming fatty acids, leucin, an oil, and an
offensive odor. COjN. 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 gel-
atine 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 gela-
tine, 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 bacilH are appHed 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

174 BACTERIA

hours. In man, the clinical manifestation of infection with this
organism is known as maUgnant cedema. Infection has followed
penetrating wounds of the body, by dirty tools, nails, splinters,
bullets, etc. The disease is often quickly fatal. It produces, fre-
quently, rapid moist gangrene.

Bacilli have been found in the blood of dead animals. Infection
is very apt to follow contused wounds, especially if other bacteria,
like the Bacterium vulgaris, or Bad. prodigiosus, are present. A
mixed culture in vitro of this organism, and the Bacillus acidi para-
lactici produces butyl alcohol abundantly. Neither of these organ-
isms separately can do so. The organisms excrete a toxin and
animals can be immunized with it. One attack of the disease
confers immunity.

SYMPTOMATIC ANTHRAX BACILLUS.

Bacillus Chauvoei.

Bacillus of Symptomatic Anthrax.

Rauschhrand Bacillus. (Figs. 59 and 60.)

The cause of symptomatic anthrax, black-leg, or quarter-evil, in
cattle.

Morphology and Stains. — This is a large organism, .5/1 in width,
and 3 to 5// in length. It has rounded ends, and grows in pairs,
but not in strings or chains. It is motile, and has many peritrichous
flagella. When stained for spores, these bodies may be found dis-
tending the organism in the middle or at the end, and the bacillus
assumes a drum-stick, or spindle shape. Often chromophilic gran-
ules are present; involution forms also appear, and are of enormous
size. This organism stains with all the common 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 pastures,
soil, hay, etc.

SYMPTOMATIC ANTHRAX BACILLUS 1 75

Temperature Requirements. — It is best cultivated at body tem-
perature, but grows anywhere between i8° C. and 37° C.

Fig. 59. — Rauschbrand bacilli showing spores. (KoUe and Wassermann.)

Fig. 60. — 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 colonies
in a delicate tangled mass, with projecting filaments. The gelatine

176 BACTERIA

is liquefied, and bubbles of gas are formed in stab cultures. A sour
odor is emitted from cultures; i percent 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.

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 rab-
bits are not susceptible. Man is immune. It causes in animals
peculiar groups of emphysematous crepitating pustules, followed
by emaciation and death. These areas contain dark fluid, probably
broken-down blood. In guinea pigs inoculation is followed by
death within thirty-six hours. The site of inoculation is found to be
oedematous, and contains bloody fluid. The organs generally
are normal. The bacilli are mostly found at the site of the inocu-
lation, but later in the blood in every part of the body. The viru-
lence 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 organ-
ism, 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 bacilH treated
in this way is followed by a mild local reaction, which affords com-
plete 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 — 85° C. to 90° C. — afforded complete protec-
tion to the animal inoculated with them. It is best to use a weaker
vaccine muscle that has been heated to 100° C. 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

MEAT POISONING BACILLUS 1 77

that this means of preventative inoculation reduced the death rate
in unprotected animals from 5-20 percent 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.

Bacillus Botulinus. Van Ermengen.

Bacillus of Meat Poisoning, or Botulism. (Fig. 61.)

Morphology and Stains. — This bacillus resembles thick vigorous
rods, 4-9/jt long, and 9/1 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, but not by Gram's method.

Habitat. — Is found in raw meat, improperly cured hams, and in
sausages. It gains access to meat after the death of the animal.

Vital Characteristics. — Is an anaerobe. Its thermal death-
point, for a spore-bearing organism, is low, 80° C, for an hour.
Grows only in media that are alkaline, and is capable of growth at
from 18° C. to 35° C., though best below 35° C; 6 percent of
chloride of soda checks growth.

Chemical Activities. — Can produce toxin (which is soluble in
water) at a relatively low temperature. Milk is not coagulated,
grape sugar is fermented, and a foul, sour odor is produced in a
culture. It liquefies gelatine.

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 oedema.

Pathogenesis. — Its pathogenic action is marked, but only by its

1 78 BACTERIA

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 difiFers from the toxin of diphtheria and tetanus. If the
toxin is mixed with the emulsified nerve tissue, it becomes neutra-
lized. In fatal cases of infection, the ganglionic nerve cells are de-
generated. Man is very susceptible, while cats and dogs are more
or less non-susceptible. If bacilli are inoculated into animals, they

Fig. 6i. — Bacillus of botulism. (KoUe and Wassermann.)

do not proliferate. Animals that recover are found to have devel-
oped 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.

GASEOUS EDEMA BACILLUS.

Bacillus Capsulatus Aerogenes. — Welch.

Morphology and Stains. — A vigorous plump bacillus 3 to 4/i in
length, resembHng 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

GASEOUS EDEMA BACILLUS

179

are seen. Spores are generally equatorially situated. Is colored
by all the basic dyes, and holds the stain in Gram's method. Stain-
ing shows that it possesses a capsule.

Habitat. — The soil, the intestines, and, sometimes, the skin of
man.

Vital Characteristics. — ^Vital resistance is low, the thermal
death-point being 58° C. with ten minutes' exposure. It grows

r^-^

'^^n^

Fig. 62. — B. Aerogenes capsulatus of Welch, in smear. (Williams.)

best at body temperature. Has Hved for one hundred days on
culture media in the incubator. It is an anaerobe.

Chemical Activities. — ^Produces gas; does not usually hquefy
gelatine, but curdles milk. (Fig. 63.)

Cultures. — Grows best in neutral or alkaUne media, producing
abundant gas. Colonies appear grayish or brownish- white, and are
often surrounded by projections which are feathery or hair-like. On

i8o

BACTERIA

agar strict anaerobic conditions
are necessary for growth, gas bub-
bles appear in the media, and
the agar may be forced out of the
tube in stab cultures. In bouillon
it grows under anaerobic condi-
tions. The growth is rapid,
bouillon is clouded, and a froth
appears on the surface. After a
few days the media becomes clear,
owing to the sedimentation of the
bacilli. Growth occurs best in
sugar bouillon, which becomes
strongly acid. In milk the growth
is rapid and luxuriant; the pro-
teids are coagulated. 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 gase-
ous phlegmons in guinea pigs, and

injections are usually fatal to

Fig. 63. — B. Aerogenes capsulatis, , . , , • r x- x. r ^

agar culture showing gas formation, birds. In man mfection has fol-
(Williams.)

CHOLERA BACILLUS l8l

lowed 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 mat-
ter, feces 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 condi-
tions favorable to growth are obtained in the blood; from the latter
pure cultures are easily obtained.

Another spore forming anaerobe very close to Welch's bacillus
is called Bacillus enteritidis sporogenes. Its differentiation is
probably certain but difficult to make.

Vincents Angina is due to an anaerobic organism of two stages,
as Bacillus fusiformis and Spirochceta vincenti. The bacillus is a
fusiform irregularly staining pointed rod, 3-1 2/x 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, uni-
formly stained, flexible, pointed ended spirals. The bacillus forms
endospores chiefly at the end. ObHgate anaerobe, requiring
serum ascitic fluid or glycerin. Colonies delicate and whitish.
Gas in glucose media. Litmus milk only decolorized. Gives a
fetid odor on all cultures. No specific immunity reactions known.

SPIRILLACEiE.

CHOLERA BACILLUS.

Vibrio Cholerae. Koch.

Spirillum CholercB. , (Fig. 64.)

Cholera Bacillus.

Comma Bacilhis.

Morphology and Stains. — Curved or bent rods, the ends not
lying in the same plane. This bending varies greatly. Under
certain conditions of growth such as the presence of alcohol, or in-
sufficient albumin or oxygen in culture media, long spiral chains
are formed. It is motile, has one terminal flagellum, and like

I 82 BACTERIA

Other members of this family, has no spores. It stains well with
the common dyes but not by Gram's method. Dilute fuchsin
stains it best. Occasionally involution forms are developed, which
do not stain well. So-called arthrospores are formed, according to
Hiippe.

Habitat. — It is said to exist constantly in the waters of the Gan-
ges in India. Is frequently found in contaminated drinking water,
from rivers, lakes, and wells; also in human feces, which, used as

Fig. 64. — Cholera spirilla. (Kolle and Wassermann.)

manure, infests vegetables, and spreads the disease. It is found
in the intestines during cholera, and after death in other viscera.

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;
one percent carbolic kills rapidly. A 1-2,000,000 solution of cor-
rosive subHmate destroys in from five to ten minutes. Its thermal
death-point is 60° C. 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 sul-

CHOLERA BACILLUS 1 83

phuric acid. Dextrorotatory 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 HjS, 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 intra-peritoneal 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 37° C, but very well at 23° C.
Does not grow below 8° C.

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
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 a moist lustre. Deep colonies are whetstone shape.
Old agar colonies become yellowish-brown. Coagulated blood
serum is rapidly liquefied at 37° C. 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 alkahne, 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 pelHcle,
which in some cultures becomes wrinkled. In peptone, abundant
growth takes place, with the production of indol and nitrites. If a
few drops of H3SO4 are added, a beautiful red appears if nitrites are

184 BACTERIA

present. This is the "cholera red ".reaction. If the color does not
at once appear, nitrites must be added.

Pathogenesis. — Cholera spirilla are pathogenic for man and
guinea pigs. 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 suscep-
tible. 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 organ-
ism is intra-cellular (an endo-toxin). Old cultures become patho-
genic through a bacteriolytic action, by which the cells are dis-
solved, 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 patho-
genic process is mostly confined to the intestines. Toxic absorp-
tions, 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 cells found in the watery
discharges resemble rice grains. Peyer's patches may become
slightly swollen and reddened, and later, there may be a 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 liquefying colonies
are picked out, and bouillon cultures made. These are tested
by dried serum, from horses artificially immunized by injecting
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

GLANDERS BACILLUS 1 85

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 Pfeiffer 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 waters, 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
are: Vibrio Metchnikovii; Vibrio proteus; Vibrio tyrogenum; and
Vibrio sclmylkilliensis. 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// 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 37° C, in glycerine agar. Resists
drying but feebly. Its thermal death-point is 55° C, 10 minutes'
exposure.

Chemical Activities. — ^Produces a brown pigment on potato, also
mallein, and a httle indol in old bouillon cultures. It forms no gas.

I 86 BACTERIA

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 deHcate and ragged. The gel-
atine 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 co-
agulated. 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
glanders, or farcy, and the avenue of infection determines the clin-
ical 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 doubtftil 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 endotoxic^.

Agglutinations. — It has been shown by McFadyean that the
blood of infected horses exhibits markedly agglutinative properties
toward the glanders bacilli. A slight immunity is present after
an attack.

Mallein. — In old cultures a peculiar tuberculin-like substance

DIPHTHERIA BACILLUS 1 87

(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. Locally, if the horse
is glanderous, a hard swelHng is formed, and the temperature is
raised 1.5° C. to 2° C. in a few hours. This is considered a valuable
means of diagnosis by veterinarians.

DIPHTHERIA BACILLUS.

Corynebacterium Diphtherise. (Loffler.)
Bacillus Diphthericd.
Klehs-Loffler Bacillus.
Diphtheria Bacillus.

Morphology and Stains. — Long, bent, or curved bacilli of irreg-
ular contour, frequently clubbed or filiform at one or both ends;

Fig. 65. — Diphtheria bacilU in mucus of trachea stained with fuchsin. (Kolle
and Wassermann.)

which contain chromatophilic 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

I 88 BACTERIA

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
dyes, notably by Loffler'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 1.5/z to 3.5/^. It does not form chains. Bizarre,
or involution shapes predominate in old cultures. (Fig. 66.)

Fig. 66. — Diphtheria bacilli involution forms. (Kolle and Wassermann.)

Culture and Temperature Requirements. — It grows best at
body temperature, and on glycerine agar, or in LoflBler's blood
serum mixture of alkaline reaction.

Vital Characteristics. — It resists drying for a long time, and has
lived on culture media for eighteen months at room temperature;
also in silk threads for several months in a dried condition. Re-
mains alive in healthy throats for months. Formalin vapor kills
it speedily; corrosive subUmate solution, 1-10,000 destroys it in a
few minutes; light is lethal to it in from two to ten hours, and heat
at 58° C. in ten minutes.

DIPHTHERIA BACILLUS 1 89

Habitat. — It has not been found in sewage, or sewer gas, soil
or water, the disease therefore is never transmitted by these means.
Has been found in the throat, nose, and in the conjunctivae 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
systemically.

Cultures. — On gelatine plate the growth is scanty and raised.
This medium is never used for cultivating this organism. The gela-
tine is not liquefied. On glycerine agar plates the growth, though
moderate, is typically characteristic, but very slightly raised above
the medium, and is of duller lustre. Old colonies become yellowish-
brown, the center of which, under a magnification of sixty diameters,
appears darker, and with ravelled edges. On Loffler's blood
serum mixture, the organism grows rapidly and well. This and
ascites-glycerine-agar culture media are the best for it. Bouil-
lon 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 cul-
tures 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 ferments, but H2S, and indol, are produced.
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

I 90 BACTERIA

in the body and in culture, which is intensely poisonous. See
chapter on bacterial products.

Pathogenesis. — Diphtheria in man means generally an infection
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, vulva, or prepuce.
The toxin not only causes a local necrosis, with the formation 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 structures fragmentation of the
nuclei of the cells is noted. Guinea pigs, cats, horses, and cows,
may be infected artificially, but the disease never occurs spon-
taneously 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 empy-
emia, 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 during
an epidemic, is the healthy bacillus carrier who, wholly unaware
of his condition, is carrying virulent germs in his throat. This
further indicates that individual resistance or susceptibility plays
an important part in infection.

Immunity is natural, active, artificial, or passive. Active im-
munity, following infection, is generally a permanency, for, once
infected, the individual, if he recovers, may be considered immune
for a time, though some individuals are more susceptible, and suffer
several attacks. In active immunity anti-toxin is found in the blood,
and recovery, and subsequently, immunity is due to this fact. Anti-

DIPHTHERIA BACILLUS IQI

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. This is measured in units, i,ooo to 5,000 units forming
a dose. The earlier it is given, the better are the chances of recov-
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 prepared a serum that is
bactericidal, and is designed to destroy the
bacilli.

Pseudo-diphtheria bacilli, which mor-
phologically and culturally resemble the true
bacilli, have been described. They are not

pathogenic, in the sense of producing exuda- Fig. 67.— Diphtheria

J. , ^, . J 1 V J i. r ^^ bacilli stained with

tive diphthena, and are beheved to be atten- Loffler's blue. Striped.

uated diphtheria baciUi by many observers. (Greene's Medical
The diagnosis of diphtheria by culture is an ^^S^osis.
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 mem-
brane 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 cot-

192 BACTERIA

ton, or a stick, is rubbed over the false membrane, or throat, and
then over the serum; the latter is incubated, and the culture ex-
amined after eight or twelve hours, by staining with L6fl9er'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.

PSEUDO-DIPHTHERIA BACILLUS.

CorynebacteriumPseudo-diphtheriticum.

Pseudo-diphtheria Bacillus. (Hoffmann.)

Morphology and Stains. — This bacillus resembles the diph-
theria bacillus. The rods, however, are shorter and thicker; other-
wise, 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 true bacillus.

Habitat. — It is found in healthy throats and conjunctivae.

Pathogenesis. — It is non-pathogenic for guinea pigs. It can
produce abscesses, nasal sinusitis and otitis media, and even endo-
carditis in man.

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. 68.)
Tubercle bacillus.

TUBERCLE BACILLUS 1 93

Morphology and Stains. — Slender rods, generally unbranched,
I. .5/^ long, and .4/1 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
v^axy substance in the capsule which is more
than probable, because of the fact that stains
that are fat selective, such as Sudan III, color
it very v^rell. Boiling hot carbol-fuchsin gives
it the best stain. It keeps the color in spite
of the action of strong solutions of mineral acids Fig. 68. — Tubercle
in water, or dilute alcohol. So when tissues, gained ^with ^uchsiii
or secretions, are stained with hot carbol- and methylene blue,
fuchsin for a short time, or cold carbol-fuchsin i)iagnosis.)
for a long time, and then treated with a 25
percent solution of HNO3, or H2SO4, 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 37.5° C. It
grows slowly, is a strict parasite, and an obligate aerobe. In cul-
tures 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 minutes; can resist
60° C. for one hour, but succumbs to 95° C. in one minute. It is
13

194 BACTERIA

quickly killed by formaline and corrosive sublimate, but resists
3 percent solution of carbolic acid for hours. In sputum it with-
stands 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 tuber-
culins (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. Has also been observed in the tissues and secretions
of non- tuberculous persons. It is very widely distributed, being
found in all human communities.

Cultures. — Since the organism does not grow below 30° C, gela-
tine 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 ammal should be killed, and a gland removed under
strict aseptic precautions. It is then divided, and the halves con-
taining 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 per-
forations, which are stopped with asbestos fiber, 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 into clumps appear, which are lobulat^d and friable.

TUBERCLE BACILLUS

195

At first white, it later turns darker.
This medium is never liquefied by the
culture. On glycerine agar made of
veal broth containing 6 percent of
glycerine, the organism grows well
after isolation from the tissues, often
luxuriantly. (Fig. 69.)

A wrinkled film covers the surface
of the agar, from which it is removed
with ease. On bouillon, made of
veal and glycerinized, 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 albu-
mins mixed together, sterilized and
coagulated, this bacillus also develops
well.

Pathogenesis. — The discovery of
the tubercle bacillus, its methods of
cultivation 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 sometimes, but sheep, goats, and
horses seldom. It is easy to inoculate
guinea pigs or rabbits by injection or
feeding. The disease is widespread,

196 BACTERIA

but is much more common where human beings are huddled to-
gether in dark, badly ventilated rooms and shops. In tissues, the
characteristic lesion is a tubercle. This is a globular mass, about
the size of a very small shot, and grayish pearly white. Microscopi-
cally, in the center of the tubercle, are found several large multinu-
clear 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 the bacilli are at the other. About the giant cells epithe-
lioid cells are grouped, and about these leucocytes (phagocytes) 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 center of the tubercle frequently
undergoes caseous degeneration. If the lesion heals, the caseous
centers become calcareous, and the periphery changes into connec-
tive tissue. If the tubercles coalesce, great masses of caseous tissue
form. If the latter becomes infected with other pathogenic bacteria
(streptococci and pneumococci) rapid softening 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 tubercular diseases. They may, however, be found
in the urine, in kidney, or bladder tuberculosis. Milk from tuber-
culous cows, with infected udders, often contains bacilli, and is
certainly a means of transmitting the disease. Cerebro-spinal
fluid, in tubercular meningitis, often contains the bacilli. Bacilli
may penetrate mucous membranes, and not cause 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, or it may attack the larynx from the lungs. Infection
may spread by continuity, by the lymph stream, or by the blood.
Ingestion of bacilli may cause intestinal ulceration and invasion
of the peritoneum, also the tonsils. If the bacilli reach the blood
stream, the disease produced is generally acute miliary in type.

TUBERCLE BACILLUS 1 97

This is manifested by the formation of fine gray tubercles. In
tuberculosis of the lungs it is more than probable that the bacilh are
inhaled. Local tuberculosis has often followed skin inoculation,
either by accidental or intentional trauma. Tuberculous mothers
may have tuberculosis of the. genital tract, and fathers, having
tuberculous testes, discharge bacilli in the semen. Placental trans-
mission of the bacilli from mother to child occurs.

Fig. 70. — Tubercle bacilli showing involution forms. (Kolle and Wassermann.)

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 pathogenic for human
beings. Men have been infected on the hands, while performing
autopsies on tubercular cattle, and their skin lesions showed, histo-
logically, unmistakable tubercles. Cattle have been infected by

igS BACTERIA

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
neglible factor in adults. It very rarely causes pulmonary tuber-
culosis or phthisis, which disease causes the vast majority of deaths
from tuberculosis in man, and is the type of disease responsible for
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, temporary disable-
ment, discomfort and disfigurement. It causes a large percentage
of the rarer types of alimentary tuberculosis requiring operative
interference or causing the death of the child directly or as a con-
tributing cause in other diseases. In young children it becomes a
menace to life and causes from 6 J to 10 percent of the total fatal-
ities 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 bhnd
worm. Culturally, the difference is great. "Tuberculins" (using
the term as a convenience to describe extracts of cultures), of the
different acid-fast bacilli, if injected into animals already infected
with the same type of organism from which the extract was made,
cause the animal to react toward the "tuberculin." If a tuber-
cular cow was injected with a "tubercuHn" of a grass bacillus,
no reaction would occur, while a tubercle bacillus "tuberculin"

TUBERCLE BACILLUS 1 99

would cause the reaction. This shows that the grass bacilli and
the organism infecting the cow are not identical. We are able, in
this roundabout way, to differentiate the various acid-fasts (Moel-
ler.) By using carbol fuchsin as a stain, and a twenty-five percent
solution of H2SO4 as a decolorizer, and after allovdng the latter to
act for sixteen hours, it has been found that all of the "acid-fasts,"
except the tubercle bacilli, are decolorized, but this is not always
reliable. The tubercle bacillus resists this acid solution seventy-
two hours. By using a concentrated aqueous solution of methylene
blue as a stain for ten minutes, at room temperature, the tubercle
bacillus is not colored, while the smegma, timothy-hay, and lepra
bacilli are well stained. 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
other acid-fasts several methods 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 supension centrifuged. In the sediment many
more bacilli will be found than in the same bulk of the raw specimen.
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 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-

200 BACTERIA

ture of human tubercle bacilli that have been grown for some
time on culture media, and thus attenuated. The new tuberculins,
if injected into a person with chronic tuberculosis, stimulate the
development of anti-tuberculins, which act as a means of prevention
or defense against further infection. Thus far anti-tubercular
sera are not of a pronounced or certain therapeutic 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 various tubercuHns, some con-
taining endo-toxins, or plasmins, in solution, are capable of stimu-
lating 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.

BACILLUS OF LEPROSY.

Mycobacterium Lepra. Hansen.

Lepra Bacillus.

An acid-fast organism resembling the tubercle bacillus morpho-
logically when seen in secretions. The leprosy bacillus from
cultures presents a pleomorphic picture of short and long slender,
straight or slightly bent rods sometimes in filaments and possessing
deeply staining areas mixed with unstained ones. It is shorter
than the tubef cle bacillus, is non-motile, and probably has no spores.
In general it greatly resembles the tubercle bacillus, morphologic-
ally and tinctorially, though the granules are coarser and farther
apart in the B. lepra. Certain branched forms appear. The
morphology, at times, is Uke the diphtheria bacillus. It stains
by Gram's method, also by carbol-fuchsin. It is acid-fast, but
does not resist the action of acids nearly so well as the tubercle
bacillus.

Note. — ^Tubercle bacilli causing avian and fish tuberculosis, and other acid-
fast bacilli exist, but not being pathogenic for man, are not described here.

RAY FUNGUS 20I

Cultures have been made on serum and glycerine agar, which,
though resembHng the tubercle bacillus, are more delicate, and not
so luxuriant. To cultivate the leprosy 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. The growth is moist
and pale yellow or later pink. It is aerobic. The more recently
isolated strains grow very slowly. Variations in the media produce
various grades of pigmentations. Apparently leprosy bacilli cannot
break up complex protein molecules.

Pathogenesis. — It is highly pathogenic for man and monkeys,
producing in the former a slow chronic disease, which is, probably,
transmitted by more or less intimate personal contact. The bacillus
is seen in enormous numbers in lepra cells and elsewhere in diseased
tissues and has been found in the blood. The lepra cells are large
and vacuolated, and literally crammed full to bursting with bacilli.
In general the leprous lesion resembles a tubercle, as it consists of
giant cells, epithelial, and round cells.

Immunity. — There is very Uttle accurate knowlege as to immu-
nity against this organism; of late bacterins have been tried with
some success it is claimed.

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
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, from the ends of

202

BACTERIA

which they are discharged. The colonies in tissues are often i mm.
in diameter, and made up of many clubbed-shaped threads radi-
ally 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.

Fig. 71. — Actinomyces bo vis. (Williams.)

Vital Requirements. — It is a facultative aerobe, and grows best
in the presence of air, at 37° C. Resists drying for a long time,
and its thermal death-point is 80° C. after fifteen minutes exposure.
Chemical Activities. — Slowly liquefies gelatine, does not curdle
milk; and produces a mouldy odor. No gas or acids are formed,
nor is HgS developed.

Habitat. — It has been found in straw and hay, but never in a
healthy body.

FARCIN DU BOEUF 203

Cultures. — On gelatine plates it produces yellowish-gray colo-
nies that are very small. These grow into the gelatine, slowly lique-
fying it. The colonies are very tough and fibrous. In agar tubes it
grows very slowly, the first growth being hke dew-drops; later these
enlarge, turning yellow, and finally brown. The culture 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-Hke covering. On blood serum the colonies appear as dew-
drops, which later become brownish, then, yellowish-orange, or
brick-red. In bouillon the growth is at the bottom in ball-Hke
masses, that firmly cohere. Clubs do not form in this medium.
The supernatant bouillon is clear, with no surface 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. It is hard to inoculate
laboratory animals with the disease, though Wright succeeded in
so doing. 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.

FARCIN DU BOEUF.

Actinomyces Farcinicus.

Bacillus du farcin du Boeuf. No card.

Morphology and Stains. — Segmented threads with true branch-
ing, short and knotty, or long and delicate. Contains spores, is not
motile, and has no flagella. It stains with all the ordinary aniline
dyes, and by Weigert-Gram method. Ziehl's method stains it well.
It is often seen as tangled masses of threads.

204 BACTERIA

Vital Requirements. — Grows well at room temperature, and in
the incubator. Nocard kept a culture at 40° C. for four months
and it was still virulent.

Cultures. — It thrives well on all culture media. On bouillon the
growth is colorless, and in masses that float and then sink; or in a
fenestrated pellicle on the top. On Agar. — It appears in discrete
litde roundish yellowish-white masses that resemble lichens. On
blood serum its growth is like that on agar, only less luxuriant.
On potato it is scaly, wrinkled, yellowish and dry. In milk the
organism flourishes, without curdling the milk or altering its
reaction.

Pathogenesis. — This organism is pathogenic for all the laboratory
animals. Sheep, dogs, wild rabbits, horses, asses, and men are
immune. It produces an abscess, in those animals for which it is
pathogenic, that discharges, with subsequent induration, ulceration,
and sloughing. The disease in cattle resembles glanders.

If injected into the blood, miliary tubercles are found that
resemble tuberculosis.

ACTINOMYCES MADURA.

Actinomyces Madura.

Streptolhrix MadurcB, Vincent.

Morphology and Stains. — A non-motile, non- flagellated organ-
ism said to have spores. Its growth resembles that of Actinomyces
bovis. It consists of long threads that are clubbed. These stain
by all the basic aniline dyes and by Gram's method.

Vital Requirements. — It is a facultative aerobe. The thermal
death-point for the spores is 85° C. for three minutes, and 75° C.
for five minutes. Vegetative thread forms die at 60° C. Grows
best at 37° C., and scantily at room temperature.

Cultures. — Generates upon all culture media. In Bouillon. — It
appears in httle clumps which cHng to the glass, and are bright red
in color, eventually they sink to the bottom in pale masses. In
Gelatine. — It grows sparingly in clumps, slowly liquefying the

STREPTOTHRIX

205

medium. Upon Agar. — It forms shiny round colonies, that are first
devoid of color, then become deep red. They resemble an umbili-
cated 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.

Fig. 72. — Streptothrix hominis. (Kolle and Wassermann.)

Pathogenesis. — In man it produces madura foot, an affection
characterized by induration, ulceration, and fistulas formation with
pus.

STREPTOTHRIX (Eppinger).

The genus of truly branching mycelium- forming higher bacteria
(see page 3). The same genus includes the actinomyces. Kruse
has described nineteen different members of the streptothrix, some
pathogenic to man and animals.

Lately a number of cases of streptothrix (Streptothrix Hominis)
infection in man have been reported. The disease, in general,
resembles phthisis. In the pus, sputum, and stained sections xyi
these cases, strep to thricial threads have been found. (Fig. 72.)

2b6 BACTERIA

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
thick. When stained there is distinct beading and fragmentation
of the protoplasm.

Fig. 73. — Streptothrix Candida. (Kolle and Wassermann.)

There is true branching of an irregular type, which is best seen
in Hquid 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 60° 0.-70° C.
for an hour; moist heat, 60° C. however, kills them after an hour.
It is a strict aerobe.

Cultures. — On Loffler's blood serum, according to Tutde, 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-

OIDIOMYCOSIS 207

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 man, the disease picture is like that of tuberculosis. It causes
abscesses, adenitis, indurations of the skin, endocarditis, and pleuri-
tic inflammation. Many grayish tubercles were found that resem-
bled the lesions produced by the tubercle bacillus. Cavity for-
mation has been described.

This organism acts as a secondary infecting agent in tuberculosis
of the lungs. Tuttie reviews twelve cases, all of which were fatal.

In examining sputum from tubercular cases, in which the typical
bacilli are not found, it is well to look for the streptothrix by staining
with Gram's stain.

Leptothrix Buccalis. — Long unbranched threads that grow in
the walls of the pharynx, causing very sore throat. This organism
has not been cultivated, hence, very little is known of it. It is not a
member of the actinomyces, because it is not branched, nor is it a
streptothrix for the same reason.

Leptothrix Vaginalis. — Is another variety that has been found
growing in the vagina. Nothing is known of its pathogenicity, nor
of its cultural properties.

BLASTOMYCOSIS.
OIDIOMYCOSIS.

Oidium Albicans. Thrush, Soor. — A member of the higher
order of the fungi. This organism resembles both a yeast and a
mould, because it exhibits characteristics that are common to
both of these two forms. It exhibits budding yeast cells and
budding myceha. The yeast cell is 6jx long and i/£ wide, but the
cells vary very much in length and width.

2o8 BACTERIA

It Stains well in tissues and cultures by Gram's method, and by
the ordinary basic stains. It may be cultivated on bouillon, 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, however,
very susceptible to such antiseptics as phenol, salicyHc acid, sub-
limate, etc.

Fig. 74. — Thrush fungus. (Kolle and Wassermann.)

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 kidneys, or
nodules in the lungs. This organism may penetrate mucous tissues,
and fill the lumen of vessels (Virchow). By repeated injections of
cultures into rabbits anti-oidium serum may be prepared. This
serum exercises a bacteriolytic and an agglutinative action on the
oidium which normal serum does not have.

Oidium Coccidioides, Ophiils. Saccharomyces Busse. (Blas-
tomycetes). — In and near Chicago there have appeared parasitic
inflammations of the skin that have been termed blastomycetic
dermatitis. From the lesions of this disease fungi have been

MOULDS 209

cultivated which resemble closely the blastomycetes, but Ricketts
and Ophuls have placed this organism in the oidium genus. Not
only does it cause an infectious dermatitis, but it may invade the
deeper tissues and organs. The lungs may be primarily invaded,
setting up in them an oidiomycosis that resembles or imitates in its
general appearance pulmonary tuberculosis. The oidium may be
detected in the sputum, and exhibits budding. It is easily stained.
The diseases and organism described by Busse and Gilchrist are
probably closely related to Ophuls pictures. There seem to be
several species of pathogenic yeasts capable of a variety of influ-
ences. It is better to classify them all under Saccharomyces, as
there are no fundamental differences between Ophiils oidia and
Busse' s yeast. The character of the lesions depends upon the point
of entry. The yeast in the tissue presents doubly contoured,
highly refractive discs from which buds and short mycelia grow.
These so-called hyphae may intertwine. They may be obtained in
culture by injecting a guinea pig and culturing out. They grow in
a white, fluffy mass on agar and gelatine.

MOULDS OR HYPHOMYCETES

Aspergillus Niger, A. Fumigatus, and A. Flavus. — A poly-
cellular mycelial organism which produces spores and branched
threads, that are variously named from the macroscopic ap-
pearances of the growth. All thrive well as 37° C. and may be
cultivated on the usual culture media. In man, the external auditory
meatus is often infected with these orgnaisms, causing a trouble-
some disease. They may infect the lungs of weak anemic subjects
with wasting diseases, and may be pathogenic for cattle, horses,
and birds.

The author has found that the young hyphae, the sporangia,

and spores of some of these hyphomycetes (moulds) if treated with

hot or boihng alkaline solution of copper sulphate are stained by the

copper, which has an affinity for them, and appear a light lilac blue

2IO BACTERIA

under the microscope. If treated with a solution of ferro cyanide
of potash and acetic acid, these stained parts turn a dark brown,
showing that there is an actual absorption or perhaps 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 alkaUne copper others
are colored a bright yellow. Some moulds and bacteria have the
power of reducing copper in Fehhng's solution.

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 7-8//, of the latter 2-3//. They grow readily as dis-
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/^X3-4/^. 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 only invades the supeificial
layers of the skin.

CHAPTER IX.

ANIMAL PARASITES.

While numerous diseases are caused by vegetable parasites, such
as bacteria and moulds, there are others in which the etiological 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 knowledge that they are the
cause is lacking. Not all of the pathogens of the animal kingdom
will fulfil Koch's postulates but their number is increasing.
Within the past few years it has been found possible to cultivate
Trypanosomata, spirochaetae, amoebae, and hemosporidia with
completion 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
certain 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, though they will be described as such.

PROTOZOA.

The protozoa of importance as disease producers are to be found
in the classes, orders and families given as follows.

211

212 ANIMAL PARASITES

Protozoa.

Sarcodina.

Rhizopoda.

Gymnamoebida — Amoebae.
Mastigophora.
^ Flagellata.

Monadida, Cercomonas, Trypansosoma, Poly-
mastigida, Trichomonas.
Some authors separate a family Spirochaetidae to include
Spirochaeta and Treponema.
Sporozoa.

Gregarinida — gregarines.
Coccidia — coccidia.
Hemosporidia.

Plasmodium — malaria.
Infusoria.
Ciliata.

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 nucleolus. 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 organelles. The living
protoplasm is finely granular, is viscid, and exhibits a distinct
movement. The motility of protozoa is suppHed variously. In
the Rhizopoda progression takes place by pseudopods or false feet,
a phenomenon in which a section of the cell wall and protoplasm
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 the circum-

AMCEBA D YSENTERIiE 2 1 3

ferential 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 ectosarc, 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 fer-
ments, crystals, food particles (seen in the food vacuoles), 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 protozoa 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 be used
for reference to larger works, the various pathogenic protozoa are
given separately without direct reference to their systematic
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 DYSENTERIiE OR ENTOMCEBA

HISTOLYTICA.

•

This is a pear-shaped roundish body from .008 to .05 mm. in
diameter. The ectosarc is easily discernible in the pseudopodia,
but not in the round quiescent cell. In the endosarc, which is

214

ANIMAL PARASITES

®C N

%^-' ^ m

granular, vacuoles are easily seen; so are fragments of food, red
and white blood cells, bacteria, epithelial cells, and fecal 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
always pathogenic. It is now considered
Pig. 75.— Amoeba dysen- the cause of the protozoal form of dysen-
teriae (Greene's Medical tery. So far as known this particular
Diagnosis.) . , . , , . , .

variety exists only in the intestines of

affected 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 ob-
tained 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. In
over' 500 cases of dysentery the amoeba was
present in every instance.

Cats have been infected by pus from liver
abscesses devoid of bacteria (Kartulis).

Fig. 76.-

-Ameboid
The motion. (Greene's
r ,.^. ^ . 1 1 Medical Diagnosis.)

unne, in cases of cystitis, contained amoebae,

and it is believed to be the cause of the disease irj some rare
instances. In dysentery the amoebae are the cause of the necrosis
and ulceration, as they frequently become encysted in the submu-
cous tissues. From the Entomceba coHc the dysenteric amoeba is

FLAGELLATA 21 5

differentiated by the fact that it is larger, coarser in structure, and
takes up red blood cells, which the former does not. Differentia-
tion by Wright's stain Entomoehacoli ectoplasm lighj; blue, endoplasm
dark blue, nucleus red. Ent. histolytica ectoplasm dark blue, ento-
plasm light blue, nucleus pale red or pink.

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 in company with bacteria.
If a colony can be obtained free of bacteria, development will con-
tinue on agar smeared with organ extracts. The addition of dead
bacteria to culture media seems favorable to their development.
The poisin is not known. The free amoebae in the colon are easily
killed, but when encysted are more resistant. Quinine is fatal to
cultures in 10 minutes in strength of 1-2500. FormaUn is not
practicable.

The two varieties closely resembling Ent. histolytica sue Entamoeba
coli and Ent, tetragena. They vary in finer morphological details,
in their reproduction and their pathogenic properties. These two
varieties are not supposed to be pathogenic for man. According to
some authorities sulphur in some form is necessary for the growth of
amoebae.

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 in-
terest from a pathological view-point, are the
trypanosomes. Trypanosoma gambiense, Fig. 77.— -Flagellata.
transmitted by the tsetse-fly Glossina palpaUs i^iagnosi^.) ^
pathogenic for man (see page 218). The
Trypanosoma brucei, which causes the tsetse-fly disease (nagana)
in horses and cattle, is transmitted to cattle by the bite of the

2l6

ANIMAL PARASITES

tsetse- fly, glossinia morsitans. It can be grown on blood agar
(Novy).

Trypanosoma evansi causes surra, a disease of horses in Cetitral
Asia.

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.

Fig. 78. — Typanosome in rats' blood. (Williams.)

Trypanosoma noctuae. — A parasite of the Httle owl, which is
introduced into the bird through the bite of the mosquito Culex
pipiens.

Trypanosomas are elongated fusiform bodies pointed at both
ends, provided by a fin fold, or undulating membrane, running
along the dorsal edge and forming frill-Hke folds which terminate
in a whip-like extremity or flagellum.

TRYPANOSOMA GAMBIENSE 217

A large nucleus is always seen, also a centrosome, a small chro-
matic mass — likewise called a blepharophast — near one pole.

The flagellum is at the anterior extremity; the short pointed end
is the posterior extremity. Cell division begins in the nucleus, the
cell dividing longitudinally, the centrosome, flagellum, and the
protoplasm dividing last. Trypanosomes frequently 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 cUnging 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 pro-
toplasm. The microgametocytes arise from the asexual forms and
when mature, give rise to eight microgametes.

TRYPANOSOMA GAMBIENSE.

Castellani found that this trypanosome is the cause of sleeping
disease among the natives of South Africa. The organism has
been found in the cerebro-spinal fluid — in cases of sleeping-sickness
— quite uniformly. They have also been found in the blood. The
disease has a long period of incubation (months) , runs a long course
usually, and, at its full development, it is a meningo-encephalo-
myehtis. This is characterized by hebetude, somnolence, and
coma. 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.

2l8 ANIMAL PARASITES

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, mix-
tures such as Leishman'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.

Fig. 79. — Trypanosomes; showing ordinary structural appearance on left; in
middle a trypanosome undergoing division; on the right an agglutinated group.
(Tyson's Practice.)

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 sickness
of Africa. It is caused by Tr. cruzi, a parasite of 8 spores, develop-
ing in organs, serum or red cells. It is transmitted by Conorrhinus
megistus, a large insect.

In Dum Dum fever or Kala Azar, a disease occurring in India,
curious bodies, called Leishman-Donovan bodies have been found.
These resemble the malarial plasmodia roughly, and if cultivated
on blood agar elongated herpetomas-like bodies without undulating
membranes will develop. These bodies are evidently in the

TREPONEMA PALLIDUM 219

halteridium stage of trypanosome existence. They are to be found
in the juice obtained by splenic puncture. On the rare occasions
they have been met in the blood they were within a leucocyte.
The transmission is unknown.

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, as it does not possess an
undulating membrane, is flagellated, is of stiff and regular shape,
and multipHes by longitudinal division.

Morphology and Stains. — This organism is extremely delicate
in structure, from 4 to 14/^ in length and about .3/^ in width; has
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 vdth difficulty it requires a special one, that of
Giemsa yielding the best results. Aniline gentian violet, Romano w-
sky'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. — Nothing is known of its ability to withstand the action
of chemicals, Hght, heat, or other deleterious agencies. Glycerine
destroys its motility.

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
recultivated from them. In these experimental diseases it retains

220

ANIMAL PARASITES

the proper morphology. According to Noguchi there are two types,
a slender and a stout, which breed true to these characters and
correspond to slight pathogenic variations. Noguchi has succeeded

Fig. 8o. — ^The Spirochaeta refringens is the larger and more darkly stained
organism, while the lightly stained and more delicate parasite is the Spirochaeta
pallida {Treponema pallidum). P^om a chancre stained with Wright's blood
stain. (Hirsch — by Rosenberger.)

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

RELAPSING FEVER ORGANISM 221

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.

The Spirochceta refringens, which has been also cultivated by
Noguchi and thought by him to be a Treponema also, grows without
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 lym-
phatic glands. Investigators claim that it exists in smegma and
other foul secretions, but this has not been confirmed. Associated
with this organism, in nearly every case, is a coarser looking larger
spirochaete (Treponema), which stains deeper, and has been called
the Spirochaeta (Treponema) refringens.

In a series of experiments, Metchnikoff and Roux caused abortion
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 pro-
phylactic property.

It does not require any intermediate host for transmission as do
the recognized animal parasites of malaria and filariasis, etc.

RELAPSING FEVER ORGANISM.

European Relapsing Fever: — Caused by Spirochaeta obermeieri,
transmission not exactly known.

African Relapsing Fever: — Caused by Sp. duttoni, transmitted
by tick Ornithodorus moubata.

American Relapsing Fever: — Caused by Sp. Novii| transmission
not known.

Botnbay Relapsing Fever. — Caused by Sp. carteri, transmission
not known.

222 ANIMAL PARASITES

Morphology. — These are probably all transmitted by ticks or
related insects. They have lately been cultivated and retain some-
what of their virulence for monkeys and rodents. Close studies
have placed them among the Spirochetes, since they possess an
undulating membrane, some divide in longitudinal maimer and
since an insect is necessary for their transmission. They are
elongated, flexible, corkscrew-like, serpentine and vibratory in
motility, and do not form spores. They are stained with reasonable
ease by polychrome methods but not by Gram's method. They

Fig. 8i. — Spirilla of relapsing fever from blood of a man. (KoUe and

Wassermann.)

measure from io-40/i in length and about ifi in breadth. Coils
vary from 6-20. The American type is smaller than the rest.

Transmission. — The tick which transmits these organisms
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 relapsing fever with periods of
apyrexia in between. During the fever the spirochaetes are swim-
ming free in the blood and disappear in the afebrile interval.

MALARIAL PARASITES 223

Cultivation. — They are cultivated in the manner given for Trep.
pallidum by Noguchi, by adding citrated, therefore defibrinated,
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 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.

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
parasitic existence in the tissues, especially cells, of higher animals.
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 ex-
cepting the deserts, mountains, and arctic regions, this disease is
very widely distributed.

224 ANIMAL PARASITES

Three different parasites producing different clinical entities
are known. According to the time, frequency, and order of the
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.

I2I2I2I2. 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, leaving only a narrow
rim, which later on disappears. Six hours before the next paroxysm,
schizogony begins. The grains of melanin are arranged like the
spokes of a wheel, and then, leaving the radii, crowd about the cen-
ter (the rest of the cell being pigmendess) gradually dividing into

PLASMODIUM VI VAX 225

nine or twelve pear-shaped bodies, or merozoites. These separate
from each other and individually attack a fresh red cell, and this
attack brings about another paroxysm of fever seventy-two hours
after the previous one. The grains 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 stained. 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 malarice because of shorter period (forty-
eight hours) consumed in schizogony (or sporulation) , the much
greater activity of the amoeboid movement, and the affected corpus-
cles becoming enlarged; also by the fact that many of the melanin-
bearing stages are visible. The schizogony is rarely apparent
in the circulating blood, but in the spleen these stages are easily
seen. There are from fifteen to twenty merozoites (segmented
bodies or spores) which are arranged in an irregular heap, but not
radially hke wheel spokes. The merozoites are smaller than the
quartan variety and are more numerous. The flagellated form can
but rarely be seen in the freshly drawn blood. If some blood, con-
taining the large extra-corpuscular bodies, is put in a moist chamber,
they throw out flagella. These flagella are really microgametes and
are sexually active. The extra-corpuscular bodies are parUy mac-
rogameles, 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 extra-
15

226 ANIMAL PARASITES

cellular bodies and polymites if they develop flagella in the moist
chamber. There may be two infections in which schizogony occurs
every other day in alternate days 12121212.

PLASMODIUM FALCIPARUM.

The Plasmodium of aestivo-autumnal fever, or pernicious malarial
fever, also called tropical. The outbreaks of this occur irregularly.
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 polychromatophilic. 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 plasmodium in its schizogony divides into
7-25 merozoites or spores, and are arranged in a spore-like form.
The extra-corpuscular bodies may resemble a crescent or sickle; this
form is very characteristic 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 produced; by its great resistance to quinine; the fewer
number of merozoites; the finely granular appearance of the pig-
ment; the relatively small size of the young intra-corpuscular 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-cor-
puscular bodies become flagellated (polymites) in freshly drawn

PLASMODIUM FALCIPARUM 227

blood. The parasite may be studied in fresh film preparations,
and by staining dried films by methylene blue and eosin, Romanow-
sky's, or Jenner's methods. They are much more frequent in the
pyrexial period, and when quinine has not been given.

The various plasmodia are transmitted to man invariably by the
anopheles mosquito, in the bodies of M^hich 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., mosquito, 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 infection in man,
since the sporozoites of the various plasmodia are developed in the
salivary glands of these mosquitoes. In the act of biting, the sporo-
zoites reach the erythrocytes where they become the intra-corpus-
cular hyaline bodies beginning again their asexual cycle of develop-
ment 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 mosqui-
toes 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, filamentous micro gametes ^ penetrate the female ele-
ments, macro gametes (spheres), and after a time become mobile fusi-
form bodies, ookinets. These bore into the intestinal walls of the
mosquito and there remain. After a time they are converted into
round bodies, or oocysts. The nucleus of the oocysts divides rapidly
and other daughter nuclei are formed, and new cells called sporo-
blasts. 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 mos-

DESCRIPTION OF FIG. 82.

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; 9a, 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; 11, 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 sporo-
zoites; 15, older oocyst; 16, mature oocysts, containing sporozoites; 17, transverse
section of salivary gland of an Anopheles mosquito, showing sporozqites 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 Grassi and Leuckart, by permission of Dr. Carl Chun.)

MALARIAL PARASITE

229

Fig. 82.

230

ANIMAL PARASITES

quito. When a mosquito bites a human being they are introduced
into the blood where they are quickly transformed into the intracellu-
lar hyaline bodies and begin their asexual sporogony in the blood.
Each developmental cycle causing a febrile paroxysm either every
day or alternate days, or on every fourth day, etc., depending on the

Fig. 83. — Coccidium hominis, fron 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 — X1140. (From Railliet, in Tyson's
Practice.)

character of the organisms and the number of infections. To pre-
vent spread of malaria, mosquitoes must be prevented from reach-
ing individuals infected with malaria and those not infected. Screens
accomplish this best. The larva of the mosquito develops in stag-
nant water. To prevent the development of these young mosqui-

COCCIDIUM 231

toes oil should be poured on the water, thus cutting off the air and
means of respiration.

Boss, 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
40° C. The column of fluid is 1-2 inches high, the clear serum layer
being 1/2 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 all 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-intes-
tinal tract by preference, chiefly the liver and intestinal mucous mem-
branes. 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 numerous 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 microgametes that penetrate the
female granular cells, macrogametes, and fertilize them. These
fertilized macrogametes, or zygotes form capsules and become

232

ANIMAL PARASITES

oocysts which divide into numerous sporoblasts, changing into sic-
kle-shaped sporozoites upon liberation.

The coccidia are easily demonstrable in tissue and in feces. They
produce in man occasionally a fatal disease infecting the liver and
intestines. Cattle sometimes die from haemorrhagic dysentery due

Fig. 84. — 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, I, show-
ing development of spores; m, isolated spore, greatly magnified, showing the
two falciform bodies (pseudonavicella; sporozoites) in natural position; n, a spore
compressed so as to separate the two sporozoites, o, a sporozoite or falciform
body with y, its nucleus. (From Railliet after Balbiani — in Tyson's Practice.)

to one of the coccidia. The disease is transmitted by the ingestion
of food contaminated by feces containing the sporozoites.
Acid fuchsin stains the sporozoa.

BABESIA OR PIROPLASMA BIGEMINA.

A protozoon supposed to be the cause of spotted fever in the valley
of the Bitter Root river, Montana. This cattle disease is a febrile
one characterized by an irregular fever range, by muscular pains,

BABESIA OR PIROPLASMA BIGEMINA 233

arthritic involvement, petechia, and purpura in the skin. It is
supposedly infectious, but not contagious. Its cause is considered
by Wilson and Chowning to be the protozoon Pyroplasma, which
occurs in the blood of infected individuals. It appears within the
erythrocytes and they resemble hyaline bodies of malaria. They
are from i[i to 2// in length, sometimes from four to sixteen bodies
are found within a single cell. They grow gradually larger and then
exhibit amoeboid motion with pseudopodia formation.

By injecting blood from an infected man into rabbits, the latter
become infected, and the parasites are found in the blood. It is
believed by the discoverer that the parasites are conveyed from the
gopher Spermophilus columbianus to man by the means of ticks, the
Margaropus annulatus.

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 see.n 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 the spirochaetes when young will traverse
porcelain filters. The term will cover in this chapter those diseases
of importance to man whose causal agents cannot be morphologic-
ally described, but whose characters are more or less well known.
The list of diseases caused by sub microscopic agents is as follows:
African horse sickness, swamp fever of horses, catarrhal fever of
sheep, yellow fever. Dengue, three-day fever, typhus fever, polio-
myelitis, rabies, variola, with its congeners vaccinia and animal
pox, hog cholera, foot and mouth disease, fowl plague, fowl diph-
theria, 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 transmissible to human
beings are reviewed.

Hydrophobia. — This disease has long been considered to be an
infectious one, but the causal parasitic agent has never been discov-
ered. 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

234

HYDROPHOBIA 235

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 filtrate 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 sublimate
solution. Direct sunlight kills them quickly, so do radium emana-
tions. The latter have been used as a curative measure with
reputed success. A temperature from 52^-58° C. for one-half hour
destroys them, but they resist extreme cold of liquid air ( — 312°)
for many weeks. Pasteur found that desiccation attenuated 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 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.

Negri bodies, protozoon bodies discovered by Negri, are found in
the ganglionic cells of rabid animals. These bodies stain by eosin,
and are from one to twenty-seven microns in size, being generally

236 THE FILTERABLE VIRUSES

about five microns. 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. Histo-
logically, 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
finding 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.

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.

Like several of the protozoon parasites, this one is unquestionably
spread by mosquitoes, and it has been definitely determined by

YELLOW FEVER 237

Carrol and Reed that the female Stegomyia fasciata (also called
Steg. 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 con-
tinued existence that it undergoes alternate generation in man and in
the Stegomyia mosquito. This is peculiar to the sporozoa. 2. The
fact that two weeks must elapse before the mosquito is capable of
infecting 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 mos-
quito 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 temperature 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 mosquito 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 Berkefeld or porcelain filter is still capable of setting up the dis-
ease if injected, proving that the infection agent is submicroscopic.

6. An attack of yellow fever 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

238 THE FILTERABLE VIRUSES

only when there are present stegomyia capable of conveying the
parasite of the disease. 9. The spread of yellow fever may be
prevented by destroying the stegomyia and preventing egress and
ingress of the insects from yellow fever patients to the non-immune.
10. No insect, other than the stegomyia, has been found to be con-
cerned in the spread of yellow fever.

Yellow fever is a tropical or subtropical disease, because the
stegomyia 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 stego-
myia are then either killed or undergo hibernation. Many conclu-
sive experiments by Reed and Carrol, by Guiteras, and by the
French Commission have proved that the stegom3da is beyond
doubt the cause of the spread of the disease. No immunity, other
than the actively 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
micro-organism. i*

Certain protozoonoid bodies have been seen by numerous observ-
ers, notably by VanderLoeff, L. Peiffer, and Guarnieri. The latter
gave the name Cytoryctes vacciniae s. variolae. 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 proto-
plasm of the cells, these bodies are found. They are about the size
of a micrococcus and exhibit amoeboid movements in hanging drop
preparations. They are perfectly characteristic of the lesion pro-
duced in vaccinia and are not found in other diseased conditions.
Although championed by the great authority Prowaczek, their
protozoal nature is not accepted by all authorities.

In variola many different changes occur in the appearances of
these cytoryctes, suggesting developmental cycles.

In variola they are often intra-nuclear, while in vaccinia they are
never foimd within the nuclei.

SCARLET FEVER • 239

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 formation of the zygote can
also be discerned.

After the tenth day these bodies cannot be very v^^ell 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 inhalation.
It is certain that the disease can be produced by inoculation 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 (Monk-
man). The hypothetical organism above described, cytoryctes,
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
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 8-10 months. It is de-
stroyed at 57° C. in 15 minutes and easily by most disinfectants.
It has not been cultivated. Passive immunization has not been
achieved.

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

constantly in the skin of scarlet fever cases, also in the skin in cases
of measles and in anti-toxin rashes.

By several observers they 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.

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, trans-
mitted 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 diflftculty. It is found best
toward the end of the fever. It may be transmitted to monkeys.
It has not been cultivated. It is destroyed quickly at 52° C.
Brill's disease is a mild typhus fever.

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. 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

MEASLES 241

has not been cultivated. It resists glycerine, drying and autolysis.
It is destroyed at 50° C. in one-half hour. Hexner and Noguchi
have succeeded in staining a very minute spirochaete — the tissues
of monkeys affected with this disease.

Active artificial immunity and some passive immunity have been
obtained but these are not of therapeutic value.

Foot and Mouth Disease. — ^An acute infectious disease of
cattle, 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 communica-
ble. It has not been cultivated. It is filterable ; it is said to be due
to the Cytorrycetes. It is destroyed at 50° C. in 10 minutes, easily
by freezing and ordinary disinfectants. One attack gives immunity
and the blood is said to contain anti-bodies 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 after
the appearance of the eruption. It resists drying and freezing.
It is destroyed at 55° C. in 15 minutes; it has not been cultivated.
Immunity follows an attack but no passive immunity has been
reported.

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.
16

j-BS-B-j^Sng m
SBQ JO uoipnpojj

l+l+l+l+l 1+ + II 1+

Indol
Reaction

Slight.

Pronounced.
Very slight.

Slight.

Very
pronounced.
Faint with-
out nitrite.

uopBuiioj-gjods

1 1 i 1 II 1 1 1 II 1 1 + ++ !

1 u

Reaction

Acid.
Amphoteric.

Acid.

Acid.
+ Acid.

Acid.

Amphoteric,

later alkal.

Faintly alkal.

Faintly acid.

Faintly

alkaline.

Amphoteric.

Faintly
alkaline.
Alkaline.
+ Acid.

uop
-B|nSB03

1 1 +4- 1 + + + 1 + 1 0+ + 1

u
1
1

Cloudiness

Moderate.

Slight.
Moderate.

Marked.

Moderate.

Marked.

Very slight.
Moderate.

Moderate.

Slight.

Very

marked.

Moderate.

sPITPd

1 1 1 1 1 1 + 1 1 II 1 <1 1 + .

uijBpo
JO uoip^jgnbi^

1111+1+1+ 1+ + 1+ ++

Aerobic

and

Anaerobic

Growth

OTqojg-Buv

1 + + + + 1 + 1 O 0+ + ++ ++ '

Diqoi9V

+++++++++ ++ + ++ +1

UIBIS S.UIBO

1 1 1 1 1 1 + 1 + ++ + ++ +1

Flagella

Many.
A few.
Many.

A few.
+ 8
One.

Many.
Many.

Many.

Bact. influenzae.
Bact. pneumoniae.
Bact. typhosus.
Bact. coli.
Bact. prodigiosum.
Bact. dysenterias.
Bact. violaceum.
Bact. enteritidis.
Bact. pyocyaneum.

Bact. Zopfii.
Bact. vulgare.

Bact. vulgare /? mirabilis.

Bact. erysipelatos suum.
Bac. anthracis.

Bac. mycoides.
Bac. Botulinus.

jB3B-JBSns m
SBO P uopDnpoijj

+ \ III + + + 1 1 II 1 1 1 1 1 1

Indol
Reaction

"S^ . . .

<i^ 4J ^J *J y "5 aj -t-I "" -M* *^ ^J "" •"

1 1 1 |§§|§|-5§S.'IM s s

uopBuuoj-9Jods

++ ++++++ 1 1 III 1 1 1 1

Acid.
Faintly
alkaline.
Faintly alkal.
Strongly alk.
Faintly alkal.
Amphoteric.
Faintly acid.
Amphoteric.

Acid.

Acid.
Faintly alkal.

Acid.
Amphoteric.
Faintly acid.

Amphoteric.
Faintly alkal.

uop
-Bin3B03

+ ++<+l + l+ +l<i<l| II +

o
U

+
Very
marked.
Moderate.
Moderate.
Moderate.
Moderate.
Moderate .
Moderate.
Moderate.

Moderate.
Slight.

Moderate.
Very slight.
Almost clear.

Clear.

gpHFJ

++ ++ 1 1 1 + 1 1 1 1 1 + 1

m^Bpo
JO uopDBpnbiT

1 + + + + + + + + + 1 1 1 1 111 +

Aerobic

and

Anaerobic

Growth

3iqOJ9BUV

+ + + + + + + + + + + + + + +<]|0

Diqoj9v

1+ +++000+ +<+++ ++++

UTB^S S^UIBJO

+ -f + + + + + 1 1 1 + 1+4- +X + +

pj ^

. 1 J g^g^Sil-S^lg

Bac. cap. aerogenes.
Bac. subtilis.

Bac. megatherium.

Bac. vulgatus.

Bac. mesentericus.

Bac. tetani.

Bac. Chauvoei.

Bac. oedematis maligni.

Vibrio cholerae.

Vibrio proteus.
Spir. rubrum.
Corynebact. mallei.
Corynebact. diphtheriae.
Cory neb. pseudodiph-

theritic.
Corynebact. xerosis.
Mycobact. tuberculosis.
Mycobact. leprae.
Actinomyces bovis.

DESCRIPTION OF PLATE I.

Malarial Parasites.

1. Two tertian parasites about thirty-six hours old, attacked blood corpuscles
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, (^stivo-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 tertain ring about twelve hours old.

10. Tertian parasite about thirty-six hours old, so called amoeboid form,

11. Tertian parasite still showing ring fever, forty-two hours old.

12. Tertian parasite, two hours before febrile attack. The pigment is begin-
ning 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.

18. Three-fold infection with tertian parasite. The oval black granules
are the chromatin granules.

19. To the left, tertian parasite with large, sharply demarked, and deeply
colored chromatin granules. To the right, tertian parasite. Both thirty-
six hours old. Both probably gametes.

20. Tertian parasite thirty-six hours old, ring form.

21. Tertian parasite with beginning chromatin division, with eight chromatin
segments.

22. Tertian parasite chromatin division farther advanced with twelve chrom-
atin granules, in part triangular in form.

23. Completed division figure of a tertian parasite. Twenty-two chromatin
granules.

24. The young tertian parasites separating themselves from each other.
The pigment remains behind in the middle.

25. Quartan ring parasite, which is hard to differentiate from large tropical
ring or small tertian ring.

26. Quartan ring lengthening itself.

27. Small quartan ribbon form.

28. The quartan ribbon increases in width. The dark places consist almost
entirely of pigment.

PLATE II.

DESCRIPTION OF PLATE III.

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.

$;^. 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, pigment
in the middle.

40. Young parasites separated from one another.

41. Small and med'um 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 blood 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 (microgametocyte in sphere form), female.

48. An earlier quartan gamete (microgametocyte), male.

49. Tertian gamete, male form (microgametocyte).

50. Tertian gamete, female (microgamete) .

51. Tertian gamete (microgametocyte) still within a red blood corpuscle.

52. Microgamete tertian within a red blood corpuscle.

53. Tropical fever, (^stivo-autumnal) gamete, half moon (crescent) still
lying in a red blood corpuscle. In the middle is the pigment. The concave
side of the crescent is spanned by the border of the red blood corpuscle.

54. Gamete, tropical fever parasite.

55. Gamete of tropical fever parasite heavily pigmented.

56. Gamete of the tropical fever parasite (flagellated form), microgametocyte
sending out microgametes (flagella or spermatozoon) .

PLATE IV.

CHAPTER XI.

BACTERIOLOGY OF WATER, AIR, AND SOIL.

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 uncom-
mon to find it, at times, in muddy waters.

Bacteriological examinations of water are, in a measure, very
disappointing, because it is very diflficult, 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 feces 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 feces, and perhaps typhoid feces. The detec-
tion of the colon bacillus is therefore of prime importance in the ex-
amination of drinking water. Its detection is simple. Water must
be collected in sterile bottles, using every precaution against acci-
dental contamination. Fermentation tubes are employed, contain-

261

.262 BACTERIOLOGICAL EXAMINATIONS

ing bouillon with i percent of glucose. Into a series of these tubes,
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 cc, 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 bacillus. 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 quantities 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 cc. of raw water
half the pink growing colonies were those of B. coli and that the
water contained 10 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 streptococci of the
fecal type as pollution indicators. This is not absolutely reliable.

Typhoid bacilli have been found in water. One way that is
sometimes successful is to take 25 cc of a 4 percent peptone solution
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 media of Drigalski and Conradi. This media is made of 3

TO COUNT BACTERIA IN WATER 263

percent agar, to which has been added nutrose and crystal violet.
In the following order add two litres of water to three pounds of
beef, straining and boiling for an hour; after filtering, add twenty
grams each of nutrose and peptone, and ten grams of salt. Sixty
grams of agar are then added and the mixture boiled and filtered
after being rendered alkaline. Boil 300 c.c. litmus solution with
thirty grams of lactose, mix with the foregoing and alkalinize with a
soda solution, and then add to this 4 c.c. of a 10 percent soda
solution, and 20 c.c. 1-1,000 crystal violet (Hochst B.). Mix these
solutions together, tube, and pour on plates, spreading the feces
or water over the agar, dry and invert in the incubator twelve to
twenty-four hours.

The typhoid colonies in this medium appear less granular and
dark than do the colon colonies.

Typhoid colonies 1-3 mm. in size appear blue, colon colonies
red, all other bacteria are temporarily inhibited by the crystal violet.
Transfer the colonies to bouillon and test wdth a highly diluted
serum from a rabbit artificially immunized, by the agglutination test.

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 ^q- c.c. or h 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 American
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 -^^ 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 5 or 10 times before pouring plates.
A glass plate ruled into squares, known as a Wolffhiigel plate, should
be used for counting. The number of bacteria in potable waters

264 BACTERIOLOGICAL EXAMINATIONS

varies in many ways, according to the amount of pollution, or albu-
minous 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 i c.c.
is considered to be from a deep source, and uncontaminated 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 bacterial content.
Summer causes a diminution.

In identifying a certain water supply as the cause of an epidemic
of typhoid, the number of bacteria is of great value in locating the
place of infection.

The efficiency of filters in large municipal water supplies is only
known by the bacterial content of the effluent. In good sand and
mechanical (alum) filters, 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 one
metre thick; the upper half 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 filtei beds are quickly detected by
the rapid increase of the number of the bacteria 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

DISPOSAL OF SEWAGE 265

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, wholesome
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 entangles bac-
teria 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.
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 100 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 filters or it may be run out
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.

266 BACTERIOLOGICAL EXAMINATIONS

In the interior of closed tanks and in the depths of sand filters
anaerobic conditions prevail. On beds of coke, and on the surface
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 chemical
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 is 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.

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. 85.) Into one of these granulated
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 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

BACTERIOLOGY OF THE SOIL

267

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 violendy, 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 supposed. The
spread of organisms from sewage only extends 3-6 metres into the
atmosphere and then only by the bursting of bubbles in the presence
of gas under pressure. This is of course in the absence of extra-
neous air currents as far as possible.

Fig. 85. — Sedgwick-Tucker aerobioscope. (Williams.)

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 bacillus of malignant oedema has also been isolated from
soil. 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

268 BACTERIOLOGICAL EXAMINATIONS

first few inches of ordinary soil contain most of the bacteria, after
a depth of two 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 con-
cerned 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 nitrify-
ing 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 are found in cow's milk
include the tubercle bacillus, Streptococcus pyogenes, Staphylococ-
cus aureus, the colon bacillus, typhoid bacillus, the diphtheria bacil-

BACTERIOLOGY OF COW'S MILK 269

lus, 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 million per cubic centimeter
(sometimes 200,000,000).

In good milk the number of bacteria may increase when the tem-
perature is 90° F., from 5,200 originally in the milk immediately
after milking, to 654,000 in eight hours.

By exposing milk to a temperature of 165° F. for twenty to thirty
minutes and quickly cooling (Pasteurization) most of the non-spore
bearing bacteria are destroyed, so that the number may be reduced
99.999 percent by this process. The pasteurization of milk has
become an economic problem of great importance in large com-
munities and is not, as it should be, sufficiently supervised. That
method is best in which milk is held at 146° F. for 30 minutes.
No harm is done to the nutritional value of the milk. Some
authorities maintain that bacteria grow no better in pasteurized
than in unheated milk, while others claim the reverse. More
evidence is on the side of the second view. The practical im-
portance of the controversy is that milk whether heated or
not should be kept at a temperature at which bacteria will not
multiply, under 60° F. Pasteurized milk is safest in time of
typhoid epidemic.^.

Absolute cleanliness on the part of the milker, the use of sterilized
gloves and clothes, the absence of flies, dust, and the immediate
disposal of manure, the filtration 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

270 BACTERIOLOGICAL EXAMINATIONS

sources of typhoid infection through the 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 percent
antiformin) warming and examining the sediment after centrifu-
galization,

NDEX.

Abscesses, 137

Achorion Schoenleinii, 210

Acid, benzoic, 51

boric, 122

fast, 86

hydrochloric, 34, 122

lactic, 128, 149

production, 113
Acids, 122

mineral, 122
Acne, 137

Action, hydrolytic, 22
Actinomyces, 3

bovis, 201

farcinicus, 203

madura, 204
Acquired immunity, 41
Active immunity, 41
Aerobes, 17
Aerobioscope, 266
Aerogenes mucosus, 148
i^stivo-autumnal parasites, 226
Agar- agar, 103
Agar, blood, 104

glycerine, 103
Agglutinins, 9, 48, 55, 153
Aggressins, 39
Air, bacteria of, 266

liquid, 19
Alcohol, 124
Alexins, 46, 55
Allergic, 58

Alternate generation, 211, 237
Amboceptor, 46, 55
Ammonia, 114
Amoebae, 211, 212

Amoeba dysenteriae, 29, 213
Amoeboid motion, 10
Amphitrichous bacteria, 9
Anaerobes, 17
Anaerobic culture, 113, 115
Anaphylaxis, 58, 60
Aniline dyes, 85
Animal experiments, 117

parasites, 211
Anopheles maculipennis, 227
Anthrax bacillus, 14, 29, 31, 45, 88,
164

vaccine, 74, 168
Anti-aggressins, 40
Anti-bacteriolysins, 62
Antibody, 45, 56
Anti-complement, 56
Anti-ferments, 55
Antigens, 55, 56
Anti-immune body, 56
Anti-leucocidin, 63
Anti-plague serum, 68, 73, 147
Antiseptics, 120
Antiseptic values, relative, 125
Anti-toxins, 38, 49, 55

for animal toxins, 63
Anti- toxin for botulism, 63, 68, 178

for diphtheria, 63, 64, 191

for dysentery, 159

for Malta fever, 68

for plant toxins, 63

for pyocyaneus, 63, 68, 163
staphylococcus, 68
streptococcus, 67

for symptomatic anthrax, 63,
176

271

272

INDEX

Anti-toxin for tetanus, 63, 64, 66, 172

manufacture of, 63

standardization of, 65
Arethrospores, 14
Arnold sterilizer, 98
Artesian wells, 264
Aspergillus flavus, 209

fumigatus, 209

niger, 209
Attenuation of bacteria, 31
Autoclav, 97
Autopsies, animal, 118
Avenue of infection, 33, 151

Babes Ernst granules, 15

tubercles, 236
Babesia, 232
Bacillus, 2, 6

aerogenes capsulatus, 88, 1 78

of anthrax, 14, 29, 31, 45, 88, 164

of blue pus, 161

botulinus, 177

Chauvoei, 174

of cholera, 181

colon, 88, 154, 261, 267

comma, 181

of dysentery, 29, 88

of diphtheria, 29, 88, 187

Friedlander's, 147

fusiformis, 181

Gartner's, 160

of glanders, 29, 185

Koch Weeks, 143

lepra, 29, 198, 200

of lockjaw, 168

mallei, 29, 88, 185

of Malta fever, 29, 140

malignant oedema, 29, 173, 267

of malignant oedema, 29, 173,
267

Morax and Axenfeld, 143

Bacillus,of pseudo-diphtheria, 191,1

of plague, 29, 188

proteus vulgaris, 30

pyocyaneus, 88, 161

rauschbrand, 174

of soft chancre, 163

smegma, 198

of symptomatic anthrax, 1 74

of tuberculosis, 29, 30, 86, 88,
118, 192, 269, 270

typhosus, 29, 88, 149, 267

of tetanus, 29, 68, 88, 267

Xerosis, 192
Bacteria, attenuation of, 31

biological conditions of growth,

17
chemical composition of, 16
chromogenic, 21
definition of, i
disposal of, 30
. fixed strains of, 32
higher, 15

increasing malignancy of, 52
lophotrichous, 9
measuring of, 8
mesophilic, 18
of air, 266
of milk, 268
of mouth, 34
of skin, 34
of soil, 267
of stomach, 34
parasitic, 28
photogenic, 21
psychrophilic, 18
reproduction of, 11
staining of, 84
study of, 83, I OS
submicroscopic, 30
thermophilic, 18
Bacteriaceae, 2, 14

INDEX

273

Bacterial energy, 21

proteins, 25
Bacterins, 137, 139
Bacteriological diagnosis, 261
Bacteriolysins, 9, 39, 55, 56
Bacteriolysis, 56
Bacterium, 2

aerogenes, 148

Bulgaricum, 149

coli, 154

enteriditis, 98

influenzae, 88, 144

lactis aerogenes, 148

mucosus, 148

pestis, 88, 144

pneumoniae, 147

ulceris chancrosi, 163
Balantidium, 212
Beggiatoa, 4, 9
Beggiatoaceae, 4
Benzoate of soda, 51
Benzoic acid, 51
Benzol ring, 50
Biological conditions of growth of

bacteria, 17
Bismarck brown, 17
Black-leg vaccine, 75
Blastomycosis, 207
Bla£,toraycetes, 16
Blood agar, 104

serum, 100, 105
Blue, methylene, 86

pus bacillus, 161
Boils, 137

Bordet-Gengou bacillus of whooping-
cough, 143
Botulism, 177
Bouillon, loi
Bovine tuberculosis, 197
Bromine, 122
Bronchitis, 142

Brownian motion, 10, 83

Capsules, 8, 15
Capsule staining, 89
Carbol fuchsin, 86

thionin, 88
Carbolic acid, 123
Carbuncles, 137
Carriers, 82, 134
Cell division, 11
Cellulo-humeral theory, 45
Centrosome, 217
Cercomonas, 212
Chain coccus, 127
Chauvoie, bacillus of, 174
Chemo taxis, 18, 43, 45
Chlamydobacteriaceae, 3, 7
Chloride of lime, 122

of zinc, 1 24
Chlorine, 122
Cholera bacillus, 181
Cholera, vaccination against, 71
Chromogenic bacteria, 21
Ciliata, 212
Cladothrix, 3
Classification, i, 4
CO2, 21
Coccaceae, i
Cocci, 5

Coccidia, 212, 231
Coccidum hominis, 231
Coccus chain, 127
Coccus, Malta fever, 140

of meningitis, 132
Cold, influence of, 19
Coley's fluid, 79
Collodion sac, 100
Colon bacillus, 88, 154, 261, 267
Comma bacillus, 181
Complement, 45, 46, 48, 55, 56

fixation, 60

274

INDEX

Complement, deviation, 62
Complementophile, 56
Conjunctivitis, 139, 142, 143
Copper sulphate, 120, 121
Copula, 55
Cornybacterium diphtheriae, 187

pseudo-diphtheriae, 192
.Counting bacteria, 263
Crenothrix, 4
Creolin, 123
Cresol, 123

Culture media, 17, 96, 262
Cultures, 105

anaerobic, 113, 115

plate, 108
Cyclasterion Scarlatinale, 240
Cytase, 44, 46, 55
Cytolysins, 48
Cytolysis, 48
Cytophile, 56
Cytoplasm, 8

Cytoryctes variolae, 69, 238
Cytotoxins, 55

Direct division, 84
Disinfectants, 120
Drigalski-Conradi media, 262
Dum-dum fever, 218
Dyes, aniline, 85
Dysentery, amoeba, 213
bacillus, 157

Ectosarc, 213
Egg cultures, 105
Ehrlich's theory, 45, 50
Endocarditis, 128, 132, 137, 139, 161
Endosarc, 213
Endospores, 12
Endotoxins, 25-39
Entamoeba coli, 214

histolytica, 215

tetragena, 215
Enzymes, 22, 59
Erysipelas, 128
Esmarch's method, 112
Exhaustion theory, 42
Experiments, animal, 117

Dark field illumination, 95

Darkness, influence of, 18

Dengue fever, 240

Desmon, 55

Diarrhoea, 128, 159

Differentiation of B. typhosus and

B. coli, 263
Dilution method, 107
Diphtheria, 128, 137

an ti- toxin, 63, 64, 191

bacillus, 187

stain, 93

toxin, 25, 37
Diplococcus, 5

gonorrhoea, 137

lanceolatus, 129

meningitis, 88, 132

Farcin du Boeuf, 203
Favus, 210
Ferments, 55

diastic, 22

tryptic, 22
Fermentation tubes, 113
"Filters, 30, 100, 264

alum, 264

Kitasato, loi

Pasteur, 265

sand, 264
Fixateur, 55
Fixation, 85
Flagella, 9, 11

staining, 91
Flagellata, 212, 214
Fomites, 237

INDEX

275

Foot and mouth disease, 241
Formaldehyde, 123
Fractional sterilization, 97
Friedberger's theory, 59
Friedlander's bacillus, 147
Fuchsin solutions, 86

Ganglia, 235

Gartner's bacillus, 160

Gaseous edema bacillus, 178

Gastric juice, 34

Gelatine, 102

Generation, alternate, 211, 237

Giemsa's stain, 89

Glanders bacillus, 185

Glossina palpalis, 218

Gonidia, 11, 13

Gonococcus, 133, 137

Gonorrhoea, 139

Gram's method of staining, 87

Granules, chromophilic, 8

Babes Ernst, 15
Gregarinida, 212
Gregarines, 212
Gruber-Dunham reaction, 152
Gymnamoebida, 212
Gymnobacteria, 9

H, 21
H2S, 21

Haemolysins, 55
Haemolysis, 46, 60
Haemolytic serum, 46, 47
Hagmosporidia, 211, 212, 223
Haffkine, 71, 73
Halogens, 122
Hanging drop, 83
Haptophores, 52, 56
Heptotoxin, 55
Heterotrichida, 212

Hiss' capsule stain, 90
Histological methods, 118
Human tubercle bacilli, 197
Hydrogen peroxide, 123
Hydrochloric acid, 34, 122
Hydrophobia, 77, 234
Hyphomycetes, 16, 209
Hypersusceptibility, 58

Ice, bacteria in, 265

Immune body, 47, 53, 55, 56, 58

Immunkorper, 55

Immunity, 41, 152

acquired, 41, 152, 172

active, 41

anti-bacterial, 41

anti- toxic, 41

inherited, 41

local, ss

natural, 41, 152, 172

passive, 41

racial, 41
Incubator, 99
Index, opsonic, 80
Indol production, 114
Infection, 27

mixed, 45

phlogistic, 35

secondary, 32

terminal, 34

toxic, 35

septic, 35
Infestation, 27
Influenza bacillus, 141
infusoria, 212
Inoculating animals, 117
Inoculating media, 106
Insects, 34

Intermediary bodies, 55
Involution form, 7
Iodine, 122

276

INDEX

Jenner, 70
Jenner's stain, 88

Kidneys, excretion of bacteria by, 30
Kitasato filter, loi
Klebs-LofBer bacillus, 187
Koch's postulates, 29
Kruse's scheme, 28

Lactic acid, 128, 149
Laboratory technique, 96
Larva of mosquitos, 230
Lateral chain theory, 45, 50
Law of multiples, 49
Leischman's stain, 88
Leischman-Donovan bodies, 218
Lepra bacillus, 198, 200
Leptothrix Buccalis, 207

Vaginalis, 207
Leuococytosis, 44
Leutin, 26-82
Lightning rod theory, 53
Lime, 124

chlorinated, 122
Local immunity, S3
Lockjaw bacillus, 168
LofHer's blood serum, 105

blue, 86

flagella stain, 92

method of staining tissues, 119
Lophotrichous bacteria, 9
Lysis, 47
Lysol, 123

Macrogametes, 225, 227

Macrophages, 44

Madura foot, 205

Malarial parasites, 223

Malignant oedema, bacillus of, 173

Mallein, 26, 77, 186

Malta fever, bacillus of, 29, 140

Mannaberg's scheme, 224
Mastigophora, 212
Measles, 241
Measuring bacteria, 8
Meat poisoning bacillus, 177
Membrane, false, 24
Meningococcus, 132
Meningitis, 128, 131, 143, 161

anti-serum, 134
Mercury salts, 120
Merismopedia, 2
Merizoites, 225
Mesophilic bacteria, 18
Metals, influence of, 20
Micrococcus, 2, 5

catarrhalis, 88, 134

epidermidis alb us, 137

gonorrhoea, 88, 137

melitensis, 140

pyogenes, 135

tetragenus, 139
Microgametes, 225, 227
Microgametocytes, 225
Microphages, 44
Microspira, 2
Microsporon furfur, 210
Milk, bacteria of, 268

litmus, 103
Molecule, toxin, 52
Monadida, 212
Monkey injection, 239
Monotrichious bacteria, 9
Mordants, 86
Mosquitos, 227

anopheles, 227

larva, 230
Moulds, 16, 209
Muir-Pitfield flagella stain, 91
Multiplication of bacteria, 11
Mycelia, 16
Mycobacteriaceae, 3

INDEX

277

Mycobacterium, 3, 7

lepra, 200

tuberculosis, 192
Mycoprotein, 16
Myxomycetes, 43

Needles, inoculating, ic8

platinum, 106
Neisser's stain, 93
Negri bodies, 235
Nephrotoxin, 55
Neutralization of media, loi
Nitrites, 115

reduction, 22
Nitrifying bacteria, 268
Nitrogen, 22
Novy jars, 116
Nutriment of bacteria, 17

Oidium albicans, 207
Oidium coccidoides, 208
Oidiumycosis, 207
Oocysts, 227
Ookinets, 227
Opsonins, 55, 80, 132
Opsonic index, 80
Organelles, 212
Osteomyelitis, 128, 137

Paracolon bacillus, 154
Paratyphoid bacillus, 154
Parasites, animal, 17, 211
Pasteur filter, 265
Pasteurization of milk, 269
Pathogens, 23

Peptone solution (Dunhams), 104
Pericarditis, 131
Peritonitis, 128, 131
Peritrichous bacteria, 9
Peroxide of hydrogen, 1 23
Petrie dishes, 109

Pfeiffer's reaction, 46, 47, 48
Phagocytes, 43
Phagocytosis, 42, 44, 80
Phagolysis, 44
Phlogistic infection, 35
Photogenic bacteria, 21
Phragmidothrix, 4
Pink eye, 143
Piroplasma bigemina, 232
Pitfield's flagella stain, 92
Pityriasis versicolor, 210
Placenta, infection through, 35
Plague bacillus, 146

vaccination, 73, 147
Planococcus, i, 6
Planosarcina, i, 6
Plasmins, 25, 36
Plasmodium falciparum, 226

malariae, 212, 224, 231

vivex, 225, 231
Pleomorphism, 7
Pleuritis, 131, 139
Pneumococcus, 129
Pneumonia, 128, 131, 142, 147
Poliomyelitis, 240
Polymastigida, 212
Poly mites, 225
Porcelain filter, 100
Postulates, Koch's, 29
Potato, 104

Potassium permanganate, 124
Pragmidiothrix, 4
Preparateur, 55
Proteins, bacterial, 25
Precipitins, 49, 55
Protozoa, 211, 212

staining of, 94
Pseudomonas, 2
Psychrophilic bacteria, 18
Ptomaines, 23, 36
Puerperal fever, 128, 137

278

INDEX

Pus, 24, 37

Pyocyaneus, anti- toxin, 63, 68, 163

bacillus, 161
Pyroplasma humanis, 233

Quartan malarial parasite, 224

Racial immunity, 41
Rauschbrand bacillus, 174
Ravenel potato cutter, 104
Ray fungus, 201
Reactivation, 46, 50
Receptors, 52, 58
Relapsing fever, 221
Retention theory, 42
Rheumatic tetanus, 169, 171
Rhizopoda, 212
Ringworm, 210
Rod bacteria, 2
Roll culture, 1 1 1
Romanowsky's stain, 89
Roux regulator, 99

Sac, collodion, 100

Saccharomycetes, Bussi, 208

Sand-fly fever, 240

Sapraemia, 27

Saprogens, 23

Saprophytes, 17

Sarcina, 2, 5

Sarcode, 212

Sarcodina, 22

Scarlatina, 128

Scarlet fever, 239

Schizomycetes, i

Schizogony, 213, 227

Secondary infections, 32, 128, 137,154

Septic infections, 36

tank, 265
Septicaemia, 128, 137

pneumococci, 131

Serum, anti-plague, 68, 73, 147

anti-pneumonococcus, 68

anti-toxic, 65

hsemolytic, 60

reactivated, 46, 50
Sessile phagocytes, 43
Sewage disposal, 265
Silver salts, 122
Skin, disinfection of, 125
Sleeping sickness, 217
Small pox, 69, 137, 238
Smegma bacillus, 198
Soft chancre bacillus, 163
Soor, 207

Spermophilus Columbianus, 233
Spermo toxin, 55
Spirillaceae, 2, 181
Spirillum, 2, 6

cholera, 88, 181
Spirochasta, 3, 6, 211, 212

carteri, 221

duttoni, 221

Novi, 221

obermeieri, 221

pallida, 219

refringens, 219, 221

vincenti, 181
Spirosoma, 2
Sporangia, 16
Spore staining, 90
Spores, II, 98
Sporoblasts, 227
Sporogony, 213, 227
Sporozoa, 212, 223
Sporozoites, 227, 231
Sporulation, 12, 84
Spotted fever, 232
Stain, Bismarck brown, 87

Fuchsin solution, 89

Giemsa's, 89

Gram's, 87

INDEX

279

Stain, Hiss' capsule, 90 *

Leischman's, 88

Loffler's methylene blue, 86
flagella, 92

Neisser's diphtheria, 93

Pitfield's flagella, 92
modified by Muir, 91

spore, 90

thionin blue, 88

tubercle bacilli, 94

Weigert's, 87

Wright's, 88

Welsh's capsule, 89

Zeihl's carbol-fuchsin, 86
Staining bacteria, 84, 85
Standardization of anti-toxins, 65
Staphylococcus, 2, 5, 80, 81

albus, 135

aureus, 30, 135, 141

citreus, 135

pyogenes, 8S
Stegomyia Fasciata, 237
Sterilization, 96

culture media, 97

fractional, 97

glassware, 96
Sterilizer, Arnold, 98
Stomach, bacteria of, 34
Street virus, 235
Streptococcus, 2, 32, 267

anti- toxin, 67

erysipelas, 79

intraceUularis, 132

lanceolatus, 6, 129

mucosus, 132

pneumoniae, 88

pyogenes, 88, 127

viridans, 132
Strep to thrix, 3

hominis, 205 ^

madura, 204

Study of bacteria, 105

Substance sensibilisatrice, 55

Suctoria, 221

Sulphur dioxide, 1 24

Symptomatic anthrax anti-toxin,i76

bacillus, 174
Symbiosis, 18
Syphilis, 221

Table of characteristics of bacteria,

242
Temperature, influence on growth, 18
Terminal infection, 34
Tertian fever, 225
Test, tuberculin, 77
Tetanolysin, 38
Tetanospasmin, 38
Tetanus anti- toxin, 63, 64, 66, 172

bacillus, 29, 68, 88, 267

rheumatic, 168, 171

spore, 37

toxin, 26, 38, 45
Tetrads, 5

Theory, cellulo-humeral, 45
Thermolabile, 46
Thermostat, 99
Thionin, 88
Thio thrix, 4

Thrombosis formation, 24
Thrush, 207
Tonsillitis, 128
Toxalbumins, 37
Toxic infection, 36
Toxin, 24, 36, 55

molecule, 50
Toxoid, 38, 53
Toxons, 38
Toxophores, 52, 56
Trachoma, 241
Treponema, 212

pallida, 82, 219

28o

INDEX

Trichobacteria, 9
Trichomonas, 212
Trichophyton, 210
Trypanoma, 211, 212

brucei, 215, 218

cruzi, 218

equiperdum, 216

evansii, 216, 218

gambiense, 215, 217

lewisi, 216, 218

nocturna, 216
Tsetse fly, 215, 218
Tubercle bacillus, 198

stain, 94, 199
Tubercles, Babes, 236
Tuberculin, 25, 76, 80, 198

T.R., 76
Tuberculosis, 128
Turpentine, 124
Tyndallization, 97
Typhoid bacilli, 29, 88, 149, 267

in water, 261

vaccination against, 72
Typhus fever, 240

Udder, infection by, 261
Unit of anti-toxin, 65

toxin, 65
Uterus, bacteria in normal, 35

Vaccinia, 69, 238
Variola, 70, 238
Venom, 52
Virus fixe, 71, 235
Vibrio, 2, 6

cholera, 181

Metchnikovii, 185

protens, 185

Schulykilliensis, 185

septique, 173

tyrogenum, 185
Vincent's angina, 181
Virulency, 31

Wassermann's list of anti- toxins, 63

test, 61
Water, bacteria of, 261
Weigert's aniline gentian violet, 87

method of staining tissue, 119

theory, 52
Welch's capsule stain, 89

theory, 62
Wells, artesian, 264
Widal reaction, 48, 152
Woelffhiigle plate, 263
Wright, 80
Wright's stain, 88

Xerosis baciUi, 192

Vacuoles, 8
Vaccination, 69

for plague, 68
Vaccine, anthrax, 74, 168

black leg, 75

cholera, 71

plague, 68, ys, i47

small pox, 69

tuberculosis, 75

typhoid, 68, 72

Yeasts, 16
Yellow fever, 236

Zeihl's solution, 86
Zinc chloride, 124
Zooglia, 9
Zymase, 22

Zymogenic bacteria, 21
Zwischenkorper, 55
Zymophore, 56

FOR MEDICAL STUDENTS

WEBSTER'S DIAGNOSTIC

MIP T U /^ ri Q CHEMICAL, BACTERIOLOGICAL.
MIj K n\J MJ ^ AND MICROSCOPICAL

By Ralph W. Webster, m.d., ph.d., Asst. Professor of Pharmacologic
Therapeutics, and Instructor in Medicine Rush Medical College (University
of Chicago); Pathologic Chemist, Cook County Hospital. Fifth Edition,
Revised, Enlarged, xxxvii + 758 pages, with 37 Plates and 171 other
Illustrations. Cloth, $5.00

GOULD'S 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^ lbs. Bound in Handsome Flexible Cloth,
Marbled Edges, Round Corners, $2.75; with Thumb Index, $3.25. Also
Beautiful Flexible Leather, Marbled Edges, Round Corners, $4.00; with
Thumb Index, $4.50.

EDGAR'S OBSTETRICS

FIFTH EDITION

By J. Clifton Edgar, m.d.. Professor of Obstetrics and Clinical Mid-
wifery, Medical Department of Cornell University, New York City; Attending
Obstetrician to the New York Maternity Hospital, etc. Fifth Edition, Re-
written and Revised. 13 16 Illustrations. 38 Figures in Colors. 8vo. 1067
pages. Cloth, $6.00

GILE— THE NOSE. THROAT
AND EAR

By Ben Clark Gile, m.d., Instructor in Otology in the University of
Pennsylvania, and formerly Assistant in the Throat and Nose Dispensary of
the University Hospital; Fellow of the American Laryngological, Rhinological
and Otological Society, etc. With 131 Illustrations, 8 of which are ih Color.
Octavo, xvii + 450 pages. Cloth, $2.75

P. BLAKISTON'S SON 61 CO.. Publishers

PHILADELPHIA

Eleventh Revised Edition

Hughes*
Practice of Medicine

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 df
Clinical Medicine, Jefferson Medical College

Edited by

R. J. E. SCOTT, 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,

63 Illustrations. 12mo. xix + 785 Pages.
Flexible Leather, Gilt Edges, Round Corners

$3.00 Postpaid

The TREATMENT Is Specially full. 406 valuable prescriptions have been

included.

"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.

P. BLAKISTON'S SON 6. CO., Publishers

PHILADELPHIA

Gould

00513156

TIONARY

and Defined

1378 00513 1563 J of the Arteries, Mus-

Bacteria, Micrococci,
Spirilla, Thermometric Scales, Weights and
Measures, etc.; Dose Lists for Physicians and
Veterinarians in both English and Metric
Systems.

Seventh Edition. Thin 64mo; xx + 1005
pages. Full Limp Leather, Gilt Edges and
Round Corners, $1.25; with Thumb Index,
$1.50 ne/.

This is an extremely practical little
book, the definitions are plain and concise
and the systerp of pronunciation is clear,
simple and easily understood,

"The definitions are short, but we have not
noticed an instance in which accuracy has been
sacrificed to brevity. . . . Appears to include
nearly every word likely to be met with in current
medical literature." — British Medical Journal.
"It is neat, compact, and could not fail to be of practical value to any physician."
-Boston Medical and Surgical Journal.

"POCKET CYCLOPEDIA OF
MEDICINE AND SURGERV

By GEORGE M. GOULD, A.M., M.D., Author
of Gould's Medical Dictionaries, and WALTER L.
PYLE, A.M., M.D. Second Edition, Edited by
R. J. E. SCOTT, M.D. 728 pages. Bound in Full
Limp Leather, Rounded Corners, Gilt Edges, $1.25;
Thumb Indexed, $1.50 net.

UNIFORM WITH GOULD'S
"POCKET" DICTIONARY

Every subject, concerning which the stu-
dent may desire a brief and thorough descrip-
tion, supplementing the mention which may
be given in lectures or a general te.xt-book, is
taken up and treated thoroughly and concisely.
To the student desiring to refresh his 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.

BLAKISTON'S SON 6. CO., Publishers

1012 WALNUT STREET, PHILADELPHIA

i^iia

Internet Archive Audio

Featured

Top

Images

Featured

Top

Software

Featured

Top

Books

Featured

Top

Video

Featured

Top

Mobile Apps

Browser Extensions

Archive-It Subscription

Save Page Now

Full text of "A compend on bacteriology : including animal parasites"

See other formats